VAR w '0»,— “‘3: —- m.

.T . HATE, T .r.. ,.. Ty“ ..,T. Em,” ”HE RTE . .
I WT '\l\lv‘T( «\T E, .T “A“ THE , N E
H‘.‘ ELL-"Al‘s all»! ,m \g; .T'T|du_¥,‘<.-_T' mm,“ My. , .“r‘flwf'fl: 1‘ L ., T,“ H T

 

 

5..

- . THE SPECTF Tc BINDTNG 0E OP ATE AGONIST AND§:f-‘;Tf'ii‘i;;}_,‘
, ANTAGONIST To IRATN TTssuE TN VITRO ITS
., _ . ['ERELAT ONSHTPTQ QRIATE RECEPTORS AND THE _ E_
; < ;:  TNFLUENGE OF CHRONTC MORPHINE TREATMENT-effjf__;;T';;j_}‘::g:; .

Dassertahon for the Degree of Ph D
MICHIGAN STATE UNIVERSITY
CHENG Yl LEE
1975

 

 

 

't'M/»-,--,/rv.t_g_> p Hey-'- !

This is to certify that the

THE SPECIFIC BINDING OF OPIATE AGONIST AND ANTAGONIST T0
BRAIN TISSUE IN VITRO: ITS RELATIONSHIP TO OPIATE

thesis entitled
RECEPTORS AND THE INFLUENCE OF CHRONIC MORPHINE TREATMENT. A

presented by
Cheng—YT Lee

has been accepted towards fulfillment
of the requirements for

Ph.D. Pharmacology

degree in

01744: flaw

Major professor

 

0-7639 \

 

 

 

 

 

 

 

 

 

 

. ABSTRACT
GKK THE SPECIFIC BINDING OF OPIATE AGONIST AND ANTAGONIST
‘ TO BRAIN TISSUE IN VITRO: ITS RELATIONSHIP
FL TO OPIATE RECEPTORS AND THE INFLUENCE
”/ OF CHRONIC MORPHINE TREATMENT

By
Cheng-Yi Lee

EThe primary aims of this investigation were to further
characterize the properties of specific binding sites for an
opiate agonist dihydromorphine and an opiate antagonist nal—
exone in_vitro and to demonstrate that these specific bind—
ing sites are consistent with what is known about the phar—

macologic receptor. Studies also were performed to determine

 

if the development of tolerance and physical dependence
during chronic morphine treatment is associated with changes
in the concentration or affinity of specific binding sites
for dihydromorphine and naloxone.

The binding in 11339 of (3H)-dihydromorphine was
studied using particulate fraction obtained from rat brain
homogenates and compared with that of (3H)-naloxone. Tissue
preparations were incubated with or without 10 uM levorpha—
nol, unless otherwise indicated, at 35°C for 5 minutes in
50 mM Tris—HCl buffer (pH 7.4), artificial cerebrospinal
fluid (CSF), or simulated intracellular fluid (ICF). Sub—

sequently, (3H)-dihydromorphine or (3H)—naloxone was added

 

 

: v

 

Cheng-Yi Lee - g
-to the incubation mixture in final concentrations of 2 to
40 nM and incubated for an additional 15—minute period at
I35°C. Bound drug was collected on Millipore filters (pore
size, 0.8 pm) and washed immediately with 18 ml of ice—cold"
Tris-HCl buffer, CSF or ICF. Radioactivity of bound drug
was assayed using liquid scintillation counting method.
Levorphanol as well as its pharmacologically inactive
stereo—enantiomer dextrorphan inhibited (3H)-dihydromorphine
binding but dextrorphan was approximately three orders of
magnitude less potent than levorphanol. The binding of
(3H)—dihydromorphine may be separated into two components:
one saturable and stereospecific and the other non-saturable.
The saturable, stereospecific binding may be calculated from
the difference in binding assayed in the absence and presence

of high concentrations of levorphanol. The use of dextror-

 

phan resulted in an artifactual separation of the saturable
binding component. The apparent Km value of the saturable,
stereospecific binding sites for dihydromorphine in brain-
stem, estimated from Scatchard plot, was 7.9 i 1.2 nM in

50 mM Tris—HCl buffer. The maximal specific binding was
0.25 i 0.01 pmoles/mg protein. Based on Ki values estimated
from Dixon plots of specific (3H)—dihydromorphine binding
in the presence of several non-labelled opiate analogs,
levorphanol had the highest affinity for the specific di-
hydromorphine binding sites, followed by naloxone, morphine
and d,l-methadone. Dextrorphan had an affinity 2000 times

lower than that for levorphanol. Codeine and thebaine had

 

 

Cheng—Yi Lee
the lowest affinities. Apomorphine, dopamine, chlorpromazine,
xylazine (Bayer 1470) and N-methylnicotinamide did not affect
specific (3H}dihydromorphine binding at concentrations up to
lO-SM. SKF—SZSA inhibited specific dihydromorphine binding
but this inhibition appeared to be resulted from the non-
specific effects of this compound on the membranes. Thus,
the saturable dihydromorphine binding sites appear to be
specific for active opiate analogs.

Specific (3H)-naloxone binding, assayed under the same
conditions, also appeared to have two components. The
apparent Km value of saturable, stereospecific binding sites
in brain—stem for naloxone was 24.0 i 6.6 nM in 50 mM

Tris-HCl buffer and the maximal specific binding was

 

0.57 i 0.01 pmoles/mg protein.

In CSF, as well as in ICF, the apparent affinity of
specific binding sites for dihydromorphine was decreased
while that for naloxone was increased as compared to those
in Tris—HCl buffer. In CSF and ICF, apparent affinity of
the specific binding sites for naloxone was significantly
higher than that for dihydromorphine. The maximal specific
binding for dihydromorphine and naloxone were both decreased
in CSF and ICF as compared to those in Tris-HCl buffer.

There were marked regional differences in the distri—
bution of specific (3H)-dihydromorphine binding in the brain.
It appeared that the specific binding sites in various brain
regions had similar affinities for dihydromorphine except

those binding sites in the cerebral cortex which had higher

 

Cheng—Yi Lee
affinity. In contrast, specific binding sites for naloxone
in varous brain regions had different affinities. It
appeared that naloxone has at least two types of specific
binding sites, one of which is not available to dihydromor;
phine. This is based on observations that (l) the total
concentration of sepcific binding sites for naloxone was
greater than those for dihydromorphine in each brain region
studied, except in the striatum, irrespective of the assay
medium used and that (2) non—labelled dihydromorphine inhi-
bited the specific (3H)—naloxone binding in the striatum
but failed to alter it significantly in the cerebellum
whereas non—labelled naloxone reduced specific (3H)—naloxone
binding significantly in both brain regions. The differences
in total binding sites for naloxone and dihydromorphine were
relatively small in the striatum but large in the cerebellum,
indicating that the specific binding sites in the cerebellum
are predominantly naloxone—specific whereas those in the
striatum are capable of binding both naloxone and dihydro—
morphine.

Two weeks after an intraventricular injection of 75 ug
of 5,7—dihydroxytryptamine creatinine sulfate, specific
(3H)—dihydromorphine binding to preparations obtained from
diencephalon and midbrain-low brain—stem of treated animals
were not significantly different from specific binding to
comparable preparations obtained from control animals.
Similarly, pretreatment of rats with two intraventricular

injections of 250 ug of 6—hydroxydopamine HBr also failed to

 

 

 

Cheng—Yi Lee
significantly alter specific (3H)~naloxone binding to
preparations obtained from cerebral cortex and brain—stem.

Chronic morphine treatment of rats or subsequent with—
drawal failed to alter the concentration of either specific
dihydromorphine or naloxone binding sites in the brain—stem
when binding was assayed in 50 mM Tris—HCl buffer or in CSF.
Chronic morphine treatment also failed to alter the affini—
ties of the specific binding sites for naloxone and dihydro—
morphine. During withdrawal from morphine, there was a ten-
dency toward a reduced affinity of specific binding sites
for dihydromorphine, which returned toward control level
upon the dissipation of the withdrawal syndrome.

It was concluded that the specific binding sites for

dihydromorphine and naloxone could be demonstrated using low

 

concentrations of radiolabelled opiate analogs. These bind—
ing sites appear to be saturable, stereospecific, specific

' for active opiate analogs and closely related to the phar—
macologic receptors. Naloxone appears to have at least two
types of specific binding sites, one of which is not avail—
able to dihydromorphine. It appears that these specific
binding sites are not associated with central monoaminergic
pre—terminal axons and nerve terminals which have been
postulated to play an important role in the pharmacologic
actions of opiate analgesics and the development of narcotic
tolerance and physical dependence. Chronic morphine
treatment failed to alter the concentration and affinity of
specific binding sites for dihydromorphine and naloxone in

particulate fraction obtained from brain-stem.

———.—#

 

IIIIIIIIIIIIIIIII:7____________________________________________‘—TTTTTTTTTTTTTT:§1"

_THE SPECIFIC BINDING OF OPIATE AGONIST AND ANTAGONIST
TO BRAIN TISSUE IN VITRO: ITS RELATIONSHIP
TO OPIATE RECEPTORS AND THE INFLUENCE
OF CHRONIC MORPHINE TREATMENT

By

Cheng—Yi Lee

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Pharmacology

 

1975

 

 

 

 

ACKNOWLEDGMENTS

The author would like to express his gratitude to
Dr. Tai Akera for his interest and encouragement throughout
this werk. His expert guidance and valuable criticism made
the completion of this thesis possible.

To Dr. Theodore M. Brody, Chairman of the Department
of Pharmacology, Dr. Richard H. Rech, Dr. James R. Weeks and
Dr. Sheldon Stolman the author extends particular thanks
for their Constructive assistance at various stages and on

various aspects of this work.

ii

 

TABLE OF CONTENTS

INTRODUCTION.

A.

C.
MATERIALS

WUOW>

F.

Opiate receptors.

A—l. Properties of opiate receptors. .
A—2. Attempts to localize opiate receptors

in the central nervous system . . . .
A-3. In_vitro studies of opiate receptors.

Narcotic tolerance and physical dependence.

B—l. Development of narcotic tolerance and
physical dependence . . . . . . .

2. Dual action hypothesis. . . . . . . .

-3. Altered metabolism and distribution of

opiate analgesics . . . . . . . . .

B 4 Redundancy hypothesis . . . . . . . .

B-S. Specific proteins and immune mechanisms

B 6 Alterations in central synaptic

transmission. . . . . . . . . . .

B—7. Pharmacological supersensitivity
hypothesis. . . . . . . . . . . .

B—8. Receptor occupation hypothesis.

B—9. Alterations in opiate receptors

Summary and objectives.
AND METHODS

Materials . . .
Tissue preparation.

Binding assays. . . . . . . . . . .

Chronic morphine treatments of rats . . . I .
6—Hydroxydopamine and 5,7—dihydroxytryptam1ne
treatment of rats . . . . . . . . . . . . . .
Statistical analysis.

EXPERIMENTS AND RESULTS

A.

. . 3 .
Characterization of specrfic ( H)-d1hydro:
morphine binding to particulate fraction in
50 mM Tris-HCl buffer (pH 7.4). . . . . ,

Page

61

 

 

 

TABLE OF CONTENTS (Continued . . . .)

Page

B. Relative potency of opiate analogs to
inhibit specific (3H)-dihydromorphine
binding to brain—stem particulate fraction
assayed in 50 mM Tris-HCl buffer (pH 7.4) . . 69

C. Specific (3H)—dihydromorphine binding to
particulate fraction obtained from various
brain regions assayed in 50 mM Tris-HCl
buffer (pH 7.4) . . . . . . . . . . . . . . .

D. Specific (3H)-naloxone binding to particulate
fraction obtained from various brain regions
assayed in 50 mM Tris—HCl buffer (pH 7.4) . . 81

E. Comparison of specific (3H)—dihydromorphine
and (3H)~naloxone binding aSSayed in 50 mM
Tris—HCl buffer, cerebrospinal fluid (CSF)
and simulated intracellular fluid (ICF) . . . 87

F. Effect of dihydromorphine and naloxone on
( H)-naloxone binding to particulate fraction
obtained from cerebellum and striatum assayed
in CSF. . . . . . . . . . . . . . . . . . . .

G. Effect of 5,7—dihydroxytryptamine retreat—
ment of rats in vivo on specific ( H)—
dihydromorphifig Hifiding assayed in CSF in
vitro . . . . . . . . . . . . . . . . . . .

H. EffEEt of 6—hydroxydopaming pretreatment of
rats in vivo on specific ( H)-naloxone
bindifig assayed in CSF. . . . . . . . .

I. Effect of chronic morphine treatment on
specific (3H)—dihydromorphine binding to
brain—stem particulate fraction assayed in
50 mM Tris—HCl buffer (pH 7.4). . . . .

J. Effect of ghronic morphine treatment on
specific ( H)—naloxone binding to brain-stem
particulate fraction assayed in 50 mM Tris—
HCl buffer (pH 7.4) . . . . . . . . . . . .

K. Effect of chronic morphine treatment on
specific (3H)-dihydromorphine and (PH)—
naloxone binding to brain-stem particulate
fraction assayed in CSF . . . . . . . .

77

96

 

104

109

116

DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . 122

A. Specific binding and uptake (transport) of
Opiate analogs. . . . . . . : '.' . . . . 122
B. Components of the specific binding of
opiate analogs. . . . . . . . . . . 2 : .
C. Affinity and specificity of the spec1f1c
binding sites for opiate analogs.' _. .
D. Regional distribution of the spec1f1c
binding sites for dihydromorphine and
naloxone in rat brain . . . . . .

iv 444447 ;‘III

125
127

132

 

 

 

TABLE OF CONTENTS (Continued . . . .)

E. The specific binding sites for
opiate analogs and the central mono—
aminergic preterminal axons and nerve
endings . . . . . . . . . . . . . . . .

F. Specific binding of dihydromorphine and
naloxone in vitro and hypotheses of
narcotic f51Ef§EEe and physical
dependence . . . . . . . .

SUMMARY AND CONCLUSION.

BIBLIOGRAPHY.

Page

137

139
145

151

 

 

Table

 

LIST OF TABLES

Inhibition constant (Ki )of several opiate
analogs for specific (3H)—dihydromorphine
binding. . . . . . . . . . . . . . . . .

ID50 of several opiate analogs for specific
binding of (3H)—dihydromorphine, (3H)—naloxone
( 3H-NLX ), and (3H)-etorphine . . . . . . .

Inhibitory effect of several non~opiate drugs
on specific (3H)*dihydromorphine binding
assayed in 50 mM Tris—HCl buffer ( pH 7.4 ).

Maximal binding and apparent Km for3

specific (3H)—dihydromorphine and ( H)—naloxone
binding to particulate fraction obtained

from various brain regions assayed in 50 mM
Tris—HCl buffer ( pH 7.4 ) ... . . . . .

Maximal binding and apparent Km for specific
( H)-naloxone binding to brain-stem particulate
fraction assayed in different media. . . . . .

Maximal binding and apparent Km for specific
(3H)—dihydromorphine binding to particulate
fraction obtained from control and

5,7—dihydroxytryptamine ( 5,7—DHT )—treated
rats assayed in CSF. . . . . . . . . . . . .

Maximal binding and apparent Km for specific
(3H)—naloxone binding to particulate fraction
obtained from control and 6—hydroxydopamine
( 6—OHDA )—treated rats assayed in CSF . . .

Maximal binding and apparent Km for specific
(3H)-dihydromorphine and (3H)—naloxone binding
to brain—stem particulate fraction obtained
from control, chronically morphine-treated

and subsequently morphine-withdrawn rats
assayed in 50 mM Tris-HCl buffer ( pH 7.4 ).

vi

-..—<- f

Page

72

74.

76

82

90

 

103

106

115

LIST OF FIGURES
Figure Page

1. Levorphanol and dextrorphan inhibition of
dihydromorphine binding assayed with 6 nM
(3H)—dihydromorphine ( 3H-DHM ) in 50 mM
Tris—HCl buffer ( pH 7.4 ). . . . . . . . 62

2. Levorphanol and dextrorphan inhibition of
dihydromorphine binding assayed with 20 nM
(3H)-dihydromorphine in 50 mM Tris—HCl
buffer ( pH 7.4 ) . . . . . . . . . . . . 65

3. Binding of (3H)—dihydromorphine ( 3H—DHM ) in
the absence and presence of 10 uM levorphanol
assayed in 50 mM Tris-HCl buffer ( pH 7.4 ) . . 67

4. Scatchard plot of the specific (3H)—dihydro—
morphine binding to brain—stem particulate
fraction assayed in 50 mM Tris—HCl buffer
( pH 7.4 ). . . . . . . . . . . . . . . . 68

5. Dixon plot: Dextrorphan inhibition of
specific (3H)—dihydromorphine binding assayed
in 50 mM Tris—HCl buffer ( pH 7.4 ) . . . . . 71

6. Specific (3H)—dihydromorphine binding to
particulate fraction obtained from various
brain regions assayed in 50 mM Tris—HCl

buffer ( pH 7.4 ) 79

 

7. Scatchard plots of specific (3H)—dihydro—
morphine binding to particulate fraction
obtained from various brain regions assayed
in 50 mM Tris—HCl buffer ( pH 7.4 ) . . . . 80

8. Specific (3H)—naloxone ( 3H—NLX ) binding to .
particulate fraction obtained from various brain
regions assayed in 50 mM Tris—HCl buffer
( pH 7.4 ). . . . . . . . . . . . . . . 84

9. Scatchard plots of specific (3H)-na1oxone
binding to particulate fraction obtained
from various brain regions assayed in
50 mM Tris—HCl buffer ( pH 7.4 ) . . . . . . . 85

 

 

LIST OF FIGURES (Continued . . . .)
Figure Page

10. Comparison of specific (3H)-dihydromorphine
binding assayed in 50 mM Tris— HCl buffer,
aftificial cerebrospinal fluid ( CSF ) and
simulated intracellular fluid ( ICF ). . . . 89

 

11. Binding of (3 H)—naloxone ( 3H-NLX ) in the
absence and presence of 10 uM levorphanol
assayed in 50 mM Tris— HCl buffer, CSF and
ICF. . . . . . . . . . . . 92

12. Comparison of specific (3H)—naloxone binding
assayed in 50 mM Tris—HCl buffer, CSF and
ICF. . . . . . . . . . . . . . . . . . . . . . 93

13. Effect of dihydromorphine and naloxone
on (3H) naloxone binding to cerebellum
particulate fraction assayed in CSF. . . . . . 95

14. Effect of dihydromorphine and naloxone
on (3 H)- -naloxone binding to striatum
particulate fraction assayed in CSF. . . . . . 97

15. Effect of 5,7-dihydroxytryptamine
pretreatment of rats on specific ( H)—
dihydromorphine binding to particulate
fraction obtained from diencephalon and mid—
brain—low brain—stem assayed in CSF. . . . . . 101

 

l6. Double—reciprocal plot of specific (3H)-
dihydromorphine binding to midbrain-low
brain-stem particulate fraction obtained
from control rats assayed in CSF . . . . . . . 102

17. Effect of 6-hydroxydopamine pretreatment of
rats on specific (3H)-naloxone binding to
particulate fraction obtained from brain—
stem and cerebral cortex assayed in CSF. . . . 105

18. Effect of chronic morphine treatment on (3H)—
dihydromorphine binding to brain— stem
particulate fraction assayed in 50 mM Tris- HCl
buffer ( pH 7. 4 ). . . . . . . . . . . . . . 110

19. Effect of chronic morphine treatment and
subsequent morphine withdrawal on specific
( H)-dihydromorphine binding to brain—stem
particulate fraction assayed in 50 mM
Tris-HCl buffer ( pH 7.4 ) . . . . . . . . . . 113

viii

 

LIST OF FIGURES (Continued . . . . )

Figure

20.

21.

22.

23.

Doble—reciprocal plot of specific (3H)—
dihydromorphine binding to brain—stem

.partiCulate fraction obtained from control,

chronically morphine—treated and subsequently
morphine—withdrawn rats assayed in 50 mM
Tris-HCl buffer ( pH 7.4 ). .

Effect of chronic morphine treatment on
specific (3H)—naloxone binding to brain—stem
particulate fraction assayed in 50 mM Tris—
HCl buffer ( pH 7.4 ) . . . . . . . . .

Effect of chronic morphine treatment on
specific (3H)—dihydromorphine binding to
brain—stem particulate fraction assayed
in CSF. . . . . . .

Effect of chronic morphine treatment on

specific (3H)-naloxone binding to brain—stem
particulate fraction assayed in CSF .

ix

Page

114

117

119

121

 

 

 

a [‘[I'lrllt [I’llf‘
[If [[I[

 

 

 

 

INTRODUCTION

A. Opiate receptors
A—l. Properties of opiate receptors

It has long been believed by many investigators that
there are specific receptors for opiate analgesics. Infer—
ential data about opiate receptor were first derived by
Beckett and Casy (1954) from studies on structure-activity
relationships in several series of opiate analgesics. A
receptor model has been formulated, containing a flat sur-
face, a cavity and an anionic group in the proper spatial
relationship to accommodate the active compounds. These
studies called attention to the stereochemical requirements
for analgesic activity. Portoghese and his colleagues [see
Portoghese, 1965, 1966] have made major advances in under-
standing the stereospecificity inherent in the analgesic and
addiction-producing actions of opiate analgesics.’ For mor—
phine and for the various natural and synthetic morphine-

type analgesics, it is always the D(—)-stereoisomer which is

 

active, while the L(+)—isomer is essentially devoid of
activity.

Further evidence for the existence of receptors is the
fact that minor structural changes can result in the forma—
tion of potent and specific antagonists of many of the
actions of morphine and its congeners. Thus, the

1

 

 

fi. _ -j‘r.‘,=___‘_ 11-

2
replacement of the methyl group on the tertiary nitrogen atom
of morphine molecule by a large group, e.g., an allyl group,
results in a potent morphine antagonist, nalOrphine. How—
ever, this drug, as well as many other morphine antagonists,

retains some analgesic potency and physical dependence-

 

producing potential. Also they have some other undesirable
psychotomimetic effects [see Casy, 1971]. Recently, it has
been found that naloxone, an allyl analog of a potent opiate
analgesic, oxymorphone, is a potent antagonist which is de-
void of measurable agonistic properties [Blumberg et al.,
1965; Harris and Dewey, 1966].

Changes in substituents on the nitrogen atom, however,
are not consistently correlated with changes in analgesic

activity. For example, N—allyl analogs of methadone and

 

meperidine do not possess opiate antagonistic action [Costa
and Bonnycastle, 1955; Portoghese, 1966]. Furthermore, as
more compounds have been synthesized, a large variety of
structurally-unrelated compounds have been found to possess
morphine—like activity. Therefore, Portoghese introduced a
new concept on the mode of interaction of opiate analgesics
with their receptor in order to accommodate all the data ob-
tained from extensive structure-activity relationship

studies. The possibility of induced fit [Belleau, 1964] as

 

a factor contributing to receptor binding of diverse analge—
sics was also recognized [Portoghese, 1965, 1966].
One of the possible modes of interaction is that

different analgesics may interact with different sites on

 

 

 

3

the same receptor macromolecule. In this theory, it is
assumed that the steric environment required for different
analgesic molecules in different binding positions on a
receptor are not identical. Smits and Takemori [1970],
based on studies of pA2 values (the negative logarithm of the
molar dose of the injected antagonist which reduce the
effect of a double dose of an agonist to that of a single
dose) of naloxone for a series of opiate agonists and
agonist-antagonistsl, concluded that opiate agonists and
agonist-antagonists have different apparent pAzs with nal-
oxone. Takemori and his associates further showed that the
apparent pA2 value changed significantly from 6.96 in con-
trol mice to 7.30 in mice 2 hours after morphine treatment
and further changed to 7.80 in morphine tolerant and depen—
dent mice [see Takemori, 1974]. Takemori [1974] then postu-
,lated an opiate receptor similar to that suggested by Porto-
ghese. The postulated receptor has different binding sites
for opiate agonists, agonist-antagonists, and antagonists
(naloxone); with a common site of attachment for the proto-
nated nitrogen of these drug molecules. It is assumed that
naloxone also interact at agonist site competitively and
agonist-antagonists also interact at naloxone binding site.
An opiate agonist is assumed to be able to induce a ”better

fit" at naloxone binding site. It is also assumed that

 

lAgonist-antagonists are drugs with morphine-like ac-
tion that have capacity to counteract the morphine-like ac-
tion of other drugs under certain circumstances [see WHO
Technical Report Series 49§211-12, 1972].

 

 

 

IIIIIIIIIIIIIIIIIIII7_____________________________—"TTTTTTTTTTTTVTTTI" "'*::4444i7w

4

drugs interacting with different sites on the same receptor
macromolecule could trigger different sequences of biochemi-
cal events and thus produce different pharmacologic effects.

Portoghese also pointed out that two or more species
of receptors might mediate a similar analgesic response.
Different receptors might interact with a single opiate
analgesic, with somewhat different steric requirements. Also,
different receptors could interact specifically and exclu-
sively with different opiate analgesics. The last possibi—
lity is compatible with Martin’s hypothesis [Martin, 1967].

Interaction studies between nalorphine and morphine on
analgesia in man indicated that the dose-response curve was

biphasic, with increasing antagonism as the dose of nal—

 

orphine was increased to a certain level and then reemergence
of analgesia as the dose was further increased [Houde and
Wallenstein, 1956; Houde et 31., 1960]. Pentazocine also

had similar effects [Jasinski 33 al., 1970]. When cyclazo-
cine was chronically administered to man, tolerance developed
to its sedative, psychotomimetic and ataxia-producing pro—
perties. Cross tolerance to these actions by morphine was
observed [Martin et a1., 1965]. Patients tolerant to either
cyclazocine or nalorphine were refractory to the effects of
morphine [Martin and Gorodetzky, 1965]. However, tolerance

had not developed to the antagonistic properties of cyclazo—

 

cine or nalorphine [Martin et al., 1965, 1966]. Patients
tolerant to morphine were not cross tolerant to the psychoto-

mimetic effects of cyclazocine. Physical dependence of

E ;___‘J

 

 

 

 

nalorphine-type drugs has not been associated with drug-
seeking behavior [see Martin and Jasinski, 1972]. Thus,
Martin hypothesized that both morphine—type agents and nal—
orphine-type agents act as agonists and that their agonistic
actions are responsible not only for the desirable therapeu-
tic effects but for dependence and tolerance. He further
hypothesized that there are at least two types of receptors,'
the "morphine" and "nalorphine" type [see Martin, 1967].
These two receptors are responsible for two distinguishable
types of agonistic activity and two types of dependence.
Nalorphine binds to nalorphine-type receptors, that are not
available to morphine, in addition to morphine—type

receptors.

 

It has been argued that pharmacologic effects produced
by morphine—type andnalorphine-type drugs can also be ex-
plained by postulating that different sequences of bio-
chemical events could be triggered after the drug-receptor
interaction. This possibility was discussed by Dole [1970]
who has suggested that the pharmacological activity of
opiate analgesics is an expression of an nllostcric inter-
action lKoshlund, 1958; Monod gt NI., 1903] in which a
change in configuration of the receptor is essential to the
biological action if the receptor occupancy theory [Clark,
1933] is to be retained. Both potency of the drug and the
nature of its effect would be determined, not by the good-
ness of fit, but by the biochemical effects of the drug to

cause a deformation of receptor molecules-

____,_J

 

 

 

 

 

IIIIIIIIIIIIIIIIIIIIT_________________________———Iiiiiiiiiiiiiiiiiiiinfggifggfiéigg

6

So far, there is no way of critically differentiating
these hypotheses. However, it appears that there are some
problems in the one receptor hypothesis. The basic point is
the assumption that this receptor species has a common site
of attachment for protonated nitrogen. According to this
assumption, one receptor can only interact with one drug
molecule at a time. Thus, the action of the drug molecule
would depend entirely on its affinity with its receptor.
However, this assumption could not explain the biphasic
actions of nalorphine-type drugs. Presumably, one has to
assume that nalorphine-type drugs have higher affinities
for naloxone binding site than their affinities for agonist—
antagonist binding site because these drugs produce opiate

antagonistic effects at low concentrations. Following this

 

assumption, then, one has to postulate that there is another
species of receptor with a low affinity binding site for

nalorphine-type drugs in order to explain the agonistic

effects which are produced at high concentrations. If there

Vis only one type of receptor, nalorphine-type drugs have no

opportunity at all to interact with a low affinity binding

'site on the same receptor species. Since psychotomimetic

effects and analgesic effects of nalorphine—type drugs can

 

be separated, more types of receptors may have to be postu-
lated. It is possible that different types of receptors are
located close to one another and that allosteric effects
could be induced by opiate agonists. Since naloxone can

antagonize the analgesic effects of both the morphine—type

E 4_L,_J

 

 

 

 

 

 

 

7

and nalorphine—type opiate drugs [Blumberg gg g1., 1966;
McClane and Martin, 1967a; Jasinski gg_gl,, 1968], naloxone
appears to have a high affinity for several types of receptors.

In addition to these anti-analgesic effects, naloxone
also antagonizes the depressant effects of cyclazocine on
the flexor reflex [McClane and Martin, 1967b] as well as the
respiratory depressant and psychotomimetic effects of cycla—
zocine in man [Martin g3 g1., 1966]. These data would indi-
cate that the receptors responsible for the analgesic, res-
piratory depressant and psychotomimetic effects could be
stereochemically quite similar and in some instances identi-
cal to the morphine—type receptor [Martin, 1967]. Alterna-
tively, naloxone may have its own specific receptor(s) in

various brain regions.

A-Z. Attempts to localize opiate receptors in the central
nervous system

In earlier studies, many investigators studied the
selective distribution of various opiate analgesics in the
central nervous system of laboratory animals in an attempt
to correlate the physiological disposition of the drug with
the localization of pharmacologic receptors. Efforts to
achieve this, however, have largely been unsuccessful. No
selective localization of labelled opiate analgesics has been
found in any region of the central nervous systenr[Miller and
Elliott, 1955;Muléand Woods, 1962; Chernov and Woods, 1965].

Generally, the cerebral cortical gray matter contained.higher

 

 

 

 

 

 

 

 

 

8

concentrations of free morphine. In cerebral and cerebellar
white matter, levels of free morphine were lower than those
in gray matter when sampled at early time intervals. This re—’
lationship tended to reverse itself when tissues were Sampled
at later time periods. It is interesting to note that while
the overt response to morphine in the cat is stimulation in
contrast to depression in the dog, comparative disposition
studies have yielded no clue to explain the difference in
responses between the cat and thedog [Chernov and Woods, 1965}
Studies of the intracellular distribution of (3H)—di-
hydromorphine in the brain have shown that most of the
radioactivity was in the soluble fraction. The neuclear
fraction, which contains some cell membranes in addition to
nuclei, contained 10 to 20% of the radioactivity of the
homogenate. However, the lack of effect of non—labelled
nalorphine administration on the radioactivity found in the
nuclear fraction made it unlikely that the (3H)-dihydro—
morphine found in the nuclear fraction represented the bind—
ing of the drug to pharmacologically active sites [Van Praag
and Simon, 1966]. A high dose of dihydromorphine (100 mg/kg)
was used in this study using high concentrations of non—
labelled carrier dihydromorphine. Therefore, it is possible
that the pattern of distribution demonstrated by these inves—
tigators presented non—specific binding (see Section A-3).
Ingoglia and Dole [1970] were the first to use the
principle of stereospecificity in an attempt to identify

opiate receptor sites. They studied the localization of

 

 

 

 

 

 

9

l4C—labelled g- and T-methadone after intraventricular

injection into rat brain. Radioactivity was higher in the
ipsilateral lateral ventricle and hypothalamus but there was
no significant difference in the accumulation of the two
isomers in the hypothalamus and in the other brain regions
they studied. In experiments of this kind, most of the drug
that diffuses into the tissue appears to be present unbound.
in tissue water or dissolved in tissue lipid. The amount of
drug bound to receptors could Only have been a minute
fraction of the total drug. Seeman gg g1. [1972] also
failed to observe any stereospecific binding, or selective
distribution, with d— and 1—methadone. In these investiga—
tions [Ingoglia and Dole, 1970; Seeman g3 g1., 1972], the

failure to demonstrate stereospecific binding may have been

 

partly due to a poor choice of drugs, 1.6., the isomers of
methadone, since differences in pharmacologic effective

doses of d- and l—methadone are not so great as with other
stereoisomeric pairs. This probably reflects a higher degree
of conformational flexibility of the methadone molecule than
may occur with other pairs of stereoisomers [Portoghese,
1966].

Clouet and Williams [1973] have studied the localiza-
tion of radio—labelled dihydromorphine, morphine, l—methadone,
levorphanol, naloxone and nalorphine administered intra—
cisternally to rats. In general, the levels of morphine and
dihydromorphine were higher in regions rich in cell bodies

such as cerebellum and hypothalamus, and lower in regions

4—4

 

 

 

 

 

 

—: ~ “1’-

10
containing many lipid structures such as midbrain and medulla
Concentrations of l-methadone and meperidine, on the other
hand, were higher in midbrain. There seemed to be a positive
correlation between the relative lipid solubility of the
drugs and their abundance in anatomical and subcellular
lipid-rich regions of brain. Subcellular studies indicated
that these drugs were localized in the synaptosomal as well
as in the soluble fractions. The administration of inactive
isomers dextrorphan and d—methadone, had no effect on the
amount of (3H)—1evorphanol and (3H)—T—methadone distributed
to the synaptosomal fraction. It should be pointed out that
pharmacologically active doses of these drugs (equianalgesic
with 60 mg/kg morphine, i.p.) were used and thus the concen-

trations of these drugs in the brain [Sanner and Woods,

 

1965] were much higher than the Km values (IO-QM to 10'7M)
of specific binding sites for these drugs estimated recently
with ig_ygggg_studies [Lee g3 g1., 1973; Pert and Snyder,
1973b; Wong and Horng, 1973]. Thus, the major portion of
binding they observed was probably nonspecific and would
have masked the relatively small stereospecific binding.-
Diffusion of drug throughout the brain water would produce
apparent localization in synaptosomes, because drug molecules
dissolved in the acqueous interior of the nerve terminals
would be trapped there when synaptosomes were formed during
homogenization, whereas molecules in the axons and perikarya
would be freed into the surrounding medium. This is demon-

strated by the observation that about 70% of the radioactivity

 

 

 

 

11.
in the synaptosomes could be released after osmotic lysis
[Clouet and Williams, 1973].

Foster gt a1. [1967] observed that microinjections of
morphine into the periventricular gray matter of the rostral
hypothalmus caused a marked analgesia in a majority of the
rats studied. Buxbaum gt al. [1970] demonstrated that anal—
gesic dose—response relationships could be observed in rats
if micrOinjections were made into the anterior thalamic
nuclei. They also noted analgesic effects when morphine was
injected into other thalamic and hypothalamic areas. Mere
recently, Jacquet and Lajtha [1973] injected morphine via
fine-guage cannulas permanently implanted in various sub-

cortical sites in the rat brain and obServed that 10 pg of

 

morphine injected into the posterior hypothalamus resulted in
a significant analgesia, while the same dose injected into
the medial septum, the caudate, or the periaqueductal gray
matter yielded hyperalgesia. Thus, in rats, the main site

of analgesic action of morphine appears to be in the peri-
ventricular structures of the third ventricle.

Tsou and Jang [1964], based on their investigation of
analgesic effects after microinjections of morphine into
various parts of rabbit brain, have concluded that the main
site of morphine resides in the periventricular gray matter
of the third ventricle. Herz et al. [1970] developed a
method by which they were able to inject drugs into various
specific and limited portions of the ventricular system in

rabbits. Using this technique, the authors concluded that

+__J

 

 

 

 

 

 

12
the main sites of analgesic action of morphine were located
in the periventricular gray matter surrounding the aqueduct
and structures on the floor of the fourth ventricle.' Whether \
these different conclusions are due to the techniques
utilized remains to be investigated.

It is relevant to note from the above discussion that
the intraventricular injections of morphine have been shown
to produce tolerance and physical dependence in rats
[Watanabe, 1971] and rabbits [Herz and Teschemacher, 1973].

Sites of action of opiate antagonists also have been
studied. It was shown that the concurrent injection of
nalorphine into the periventricular gray matter or into the
aqueduct and the fourth ventricle in rabbits was effective
in antagonizing the analgesic effects of morphine [Tsou and
Jang, 1964; Albus et al., 1970]. In tolerant animals, the
intraventricular injection of an opiate antagonist also
precipitated withdrawal signs [Watanabe, 1971; Herz and
TeschemaCher, 1973]. Using a more elegant stereotaxic
approach, Wei 3: a1. [1972, 1973] introduced crystals of
naloxone into various parts of the brain in morphine—depen-
dent rats through concisely placed cannulas. Withdrawal
signs were most frequently observed when the naloxone was
placed in the medial thalamus and medial areas of the
diencephalic-mesencephalic junctures. These are, therefbre,
presumed to be the primary sites of naloxone action.

[These data would suggest that receptors responsible

[for the analgesia differ from that responsible for

 

 

 

 

 

 

——

13
precipitation of withdrawal syndrome or the primary sites of
action of morphine-type analgesics and naloxone are located

in different brain regions.

A-3. in xitgg studies of opiate receptors

Recently, Goldstein et a1. [1971] demonstrated in vigpg
that approximately 2% of radioactive levorphanol binding to
mouse brain homogenate was saturable and stereospecific.
The stereospecific binding was defined as the difference in
labelled levorphanol binding observed in the presence of 100-
fold excess of non—labelled levorphanol and its pharmacologi—
cally inactive enantiomer, dextrorphan. The fact that
levorphanol is pharmacologically active whereas the L(+)
isomer, dextrorphan is inactive may not require that the
receptors be stereospecific. It is possible that both com—
pounds combine with the receptors with the same affinity but
only levorphanol makes the right molecular interaction to
produce a pharmacologic effect. If the latter statement is
true, however, it would be predicted that dextrorphan should
be an antagonist; yet this is not so. Moreover, it is known
that L(+) enantiomers of allyl—substituted antagonists are
inert, having neither agonistic nor antagonistic effects.
Therefore, it seems reasonalbe to conclude that only, D(-)
enantiomers of opiate analogs can bind to the opiate recep-
tors [Goldstein, 1974].

More recently, Pert and Snyder [1973a, 1973b] have

demonstrated that more than 70% of (3H)-naloxone binding is

 

 

 

 

 

 

 

14
saturable, stereospecific and can be displaced by other
opiate agonists or antagonists. Moreover, the opiate
receptor was found only in neuronal tissues. The quantita-
tive differences observed by these two groups of investiga-
tors appears to result from differences in the concentration
of radio—labelled compounds rather than from the specific
compounds used, namely agonist or antagonist. The low con—
centration (4 x 10‘9M) employed by Pert and Snyder has been
found to reduce the nonsaturable binding drastically and to
affect the high affinity specific binding toda lesser extent
[Lee gg g1., 1973]. The percentage of the specific binding,
therefore, was dependent on the concentration of the labelled

compound used in the study. Similar specific binding was

 

observed with (3H)-dihydromorphine [Terenius, 1973; Lee
g3 g1., 1973; Wong and Horng, 1973] and with (SHJ—etorphine
[Simon gg'g13, 1973] using low concentration of labelled
compounds and a combination of either 1evorphanol~dextrorphan
or 1— and d-methadone to determine stereospecificity.

It was shown that specific naloxone binding had a
Q10 (change in the reaction rate caused by a 10°C change in
temperature) value of 1.5 between 25°C and 35°C, and about
70% of the specific binding was totally eliminated at 4°C
[Pert and Snyder, 1973a, 1973b]. However, specific (3H)-
etorphine binding was not affected by high concentrations of
sodium azide or sodium fluoride [Simon gg gl., 1973].
Specific (3H)—dihydromorphine binding was not affected by
S

10- M ouabain [Wong and Horng, 1973]. These data would

 

 

 

 

 

 

 

 

 

15

suggest that the specific binding of naloxone, etorphine
and dihydromorphine is not dependent upon energy from
oxidative metabolism or glycolysis.

Specific (3H)-naloxone binding had a sharp pH optimum
at 7.4 [Pert and Snyder, 1973a, 1973b]. Specific (3H)-
etorphine binding, on the other hand, had a broad pH optimum
between 6.5 and 8. Calcium and magnesium had no effect on
specific naloxone binding [Pert and Snyder, 1973b]. Sodium
decreased specific binding of (3H)—etorphine and other opiate
agonists [Simon gg gl., 1973; Pert gg gl., 1973] while it
enhanced specific binding of (3H)—naloxone and (3H)-1evallor-
phan [Pert gg gl., 1973]. Pert gg gl. [1973] have concluded
that sodium increases the number of binding sites with no
change in affinity for naloxone. No data was provided to
support this conclusion.

Valinomycin and monensin, which can function as
mobile carriers for monovalent cations in biological mem-
branes in general, had no effect on the binding of (3H)-
dihydromorphine [Wong and Horng, 1973]. Since ouabain also
has no effect on (3H)-dihydromorphine binding, the authors
interpreted their data as the absence of coupling between
sodium transport and the uptake of the opiate analgesics.

Specific binding of (3H)—naloxone and (3H)-etorphine
was proportional to the amount of protein over the range of
0.2 - 4.0 mg of protein. Specific binding of (3H3-naloxone,
(3H)-etorphine and (3H)—dihydromorphine was most rapid at

37°C and reached equilibrium in 15 minutes [Pert and Snyder,

 

 

 

 

 

16
1973a; Simon gg_gl., 1973; Wong and Horng, 1973]. The
specific binding of these agents were also shown to be
reversible and the specific binding sites were saturable.
For specific (3H)-naloxone binding, the association constant

6M_1 sec_1 and the dissociation

constant (K2) was 1.16 a 0.24 x 10'2 Sec—l, at 25°C

(K1) was 1.15 i 0.34 x 10

[Pert and Snyder, 1973b].

Specific binding of (SHz-naloxone, (3H)-etorphine and
(3H)—dihydromorphine could be competitively inhibited by
opiate analogs but not by putative neurotransmitters, prostar
gladin E1 or E2, acetylsalicylic acid, phenobarbital or A9—
tetrahydrocannabinol [Pert and Snyder, 1973a, 1973b; Simon
g3 g1., 1973; Wong and Horng, 1973].

Pert and Snyder [1973a, 1973b] were the first to study
the relationship between the affinity of the specific nal—

oxone binding sites for opiate analogs and the pharmacolo—

 

gic potency of these compounds. Based on the IDSO (the ’
concentration of drug that reduces specific (3H)—naloxone

binding by_50%) of various opiate analogs to inhibit the

specific bihding of 8 nM (3H)-naloxone, they demonstrated

that etorphine has the greatest potency, the 1DSO being about

1/20 of morphine. Levorphanol had 4000 times the potency of,

dextrorphan; Similarly, gfileVallorphan was 5000.times as

potent as its d—enantiomer. However, lfmethadone was only

~about 10 times as potent as.d;methadone, perhaps because it

has greater conformational mobility than levorphanol [Porto-

ghese, 1966]. Codeine, which is analgesically about

 

 

 

—l—'.—fifl’_

117'
1/4 — 1/10 as potent as morphine, displayed less than 1/3000
'of the potency of morphine. Because codeine is o—demethy-
lated by liver microsomal enzyme to morphine, this drug may
exert analgesic activity only after metabolism to morphine
[Johannesson and Schou, 1963]. Naloxone was slightly less
potent than morphine. On the specific etorphine binding
observed in the presence of 3 nM (3H)—etorphine,‘etorphine
was about 60 times more potent than morphine. Dextrorphan
was 4000 times less potent than morphine. However, naloxone
was 6 times more effective than morphine [Simon gg gl., 1973L
On the specific (3H)~dihydromorphine binding observed with

2 nM (3H)-dihydromorphine; dihydromorphine, l—morhpine and

levorphanol had about similar potency. Naloxone and l-metha-

 

done were slightly less effective. d-Methadone was about

50 times less potent than l-methadone while dextrorphan was
4000 times less effective than levorphanol [Wong and Horng,
1973]. Thus, these authors concluded that the affinity
(IDSO) of various opiate agonists and antagonists generally
parallels the known pharmacologic potency of these drugs.
Opiate agonists and their antagonists compete for the same
receptor sites. However, in these studies, a low concentra—

tion of labelled compounds were used. It should be noted

 

that drug binding at a certain concentration is determined

by both affinity and maximal binding capacity of binding
sites. Moreover, it has been demonstrated that the Ki is

not the same as the 1DSO when competitive inhibition kinetics

apply [Cheng and Prusoff, 1973]. Therefore, it is not

 

 

 

 

18
appropriate to assess the affinity of opiate analogs from a
comparison of their IDSOS.

Goldstein $3.21? [1971] reported that the major regions
of mouse brain (cerebrum, cerebellum, medulla, pons, dience-
phalon) did not differ greatly in their capacity for specific
(14C)—levorphanol binding. On the other hand, Pert and
Snyder [1973a] reported that specific (3H)—naloxone binding
in mouse brain homogenates was high in striatum and low.in
midbrain, cortex, and brain-stem. According to these inves—
tigators, no specific (3H)-naloxone binding was detectable
in the cerebellum. In further studies, Kuhar gg g1. [1973]
have demonstrated that the limbic system, thalamus and hypo-

thalamus in monkey and human brain had highest specific [3H)-

 

dihydromorphine binding. Extrapyramidal areas, midbrain,

and cerebral cortical white areas had lower specific binding.
No specific (3H)-dihydromorphine binding was detectable in
the cerebellum-lower brain-stem and spinal cord (thoracic).
Using (3H)-etorphine similar results have been reported by
Hiller gg g1. [1973]. They grouped the specific [3H)—etor-
phine binding levels into four categories. The specific
binding to most structures of human limbic system was grouped
as the highest binding (0.44 - 0.23 pmole/mg protein).

Caudate nucleus, putamen, hypothalamus, periaquaductal gray

 

matter, etc., had moderate binding while hippocampus, globus
pallidus, colliculi, substantia nigra, area postrema, cere—
bellar cortex, etc., had low binding. Cerebral white matter,

dentate nucleus of cerebellum, pineal gland, pituitary gland,

 

 

 

 

 

19

etc., had very low binding. Kuhar gg 31- [1973] have con—
cluded that regional differences in stereospecific (3H)—
dihydromorphine binding_reflected variations in total number
of receptor sites (Vmax) rather than in affinity (Km). No
kinetic data was provided to support their conclusions.
Whether the properties of specific (14C)—levorphanol binding
are different from the properties of specific (3H)-etorphine
and (3H)—dihydromorphine binding remains to be studied.

Pert and Snyder [1973a] suggested that the regional
differences in acetylcholine concentration paralleled the
observed regional differences in specific naloxone binding
and proposed a relationship with the action of opiates in
diminishing acetylcholine release [see Weinstock, 1971].

In a subsequent study [Kuhar gg gl., 1973], electrolytic
lesions resulting in the destruction of cholinergic, norad-

renergic or S-hydroxytryptaminergic pathways did not affect

 

specific (3H)-dihydromorphine binding in regions where the
lesioned pathways terminate. The authors thus concluded ]
that the opiate receptor is not a unique component of axons
or nerve endings of any one of these neuronal tracts. Both
Snyder and his associates and Simon and his associates have
emphasized the importance of the limbic system in the mode
of action of opiate analgesics.

Studies with subcellular particles indicated that spe—
cific (14C)-levorphanol binding was high in the crude mito—
chondrial/cytoplasmic membrane fraction and not in the

soluble supernatant [Goldstein gg gl., 1971]. Studies of

 

 

 

 

20
specific_binding of (3H)—naloxone to subcellular fractions
gave similar results but the crude microsomal fraction
appeared to have relatively higher specific binding [Pert
and Snyder, 1973a]. Specific (3H)—dihydromorphine binding

was high in synaptosomes and low in mitochondria and micro-

 

somes [Wong and Horng, 1973].

Goldstein gg g1. [1971] demonstrated that nearly all
stereospecific (14C)-1evorphanol binding in the crude
nuclear fraction was accounted for in nuclear membranes.
Terenius [1973] also demonstrated specific dihydromorphine
binding in the synaptic plasma membrane fraction of rat cere—
bral cortex. Goldstein gg g1. [1971] reported that the
membranes retained their stereospecific binding capacity for
(14C)—levorphanol after extraction of 70% of the protein by
Triton X—100 or sodium dodecyl sulfate, providing that deter—
gent was removed by dialysis. This binding in such prepara-
tions was largely abolished by treating with neuraminidase
or pronase but not by trypsin. p—Chloromercuribenzoate,
mercaptoethanol and iodoacetic acid failed to affect specific
binding capacity. The binding capacity was retained nearly
quantitatively in material extracted into chloroform—methanol.
Simon gg g1. [1973] reported that specific (3H)-etorphine
binding was sensitive to trypsin and pronase and to N-ethyl—
maleimide, p-hydroxymercuribenzoate or iodoacetamide treat-
ment. It was unaffected by phospholipase A and C. More
recently, Pasternak and Snyder [1974] reported that specific

(3H)-naloxone binding was reduced by low concentrations of

 

 

 

 

— 7

21
trypsin and Chymotrypsin, low concentrations of phospholi—
pase A, high concentrations of phospholipase C and relatively
insensitive to phospholipase D and neuraminidase. It should
be pointed out that in these three studies, the specific
binding was assayed under different experimental conditions.
A series of studies on the effects of various treatments on
the specific binding of a radiolabelled compound under the
various experimental conditions utilized in studies cited
above would be necessary before evaluating the results of
the above studies.

1 In summary, the interaction of opiate analgesics with
receptor may be interpreted in several ways. Takemori, based
on pA2 studies, favored the one receptor proposal while Mar—
tin favored the multi—receptor proposal. Attempts to corre-

late the localization of opiate receptors with selective

 

physiological disposition of opiate analgesics have largely

been unsuccessful. Using stereotaxic techniques, opiate *
receptors have been shown to be associated with periventri-

cular structures of the third ventricle. Naloxone appears

to have its primary sites of action in the medial thalamus

and medial areas of the diencephalic—mesencephalic junctures.

 

Specific binding 13 KiEES of opiate agonists and
antagonists have been demonstrated with low concentrations of
radiolabelled compounds. The specific binding is reversible
and saturable at relatively low concentrations. In general,
the ID50 of various opiate agonists and antagonists for I

specific opiate binding parallels the known pharmacologic

 

 

 

‘22'
potency of these drugs. Specific binding assayed with a
low concentratibn of radiolabelled opiate analog was differ—
ent in various brain regions of laboratory animals as well
as in man. This regional variation in specific opiate bind—
ing does not correlate with the regional distribution of
any known neurotransmitter or its neuronal axons or nerve
endings. It should be pointed out that the Ki rather than
the IDSO should be estimated in order to assess the affinity
of various opiate analogs for specific binding sites.
Specific binding observed at a certain_concentration of an
opiate analog is determined by two independent variables,
namely, maximal binding capacity and affinity. Thus, one
cannot ascertain whether the regional differences in specific
binding observed in previous studies are due to the differ-
ences in the concentration of specific binding sites,
differences in affinity of specific binding sites for an
Opiate analog, or both. Therefore, in order to further
understand the properties of the specific opiate binding
sites, maximal binding and affinity of the specific binding

sites should be studied.

B. Narcotic Tolerance and Physical Dependence

 

Development of tolerance and physical dependence are
well known consequences of frequent, repeated administration
of morphine and various natural and synthetic opiate anal-
gesics. After repeated dosage, these drugs lose depressant

activities while retaining stimulant potency [Seevers and

 

 

 

 

 

23
Woods, 1953]. The sedative, analgesic and respiratory
effects become so attenuated that doses fatal for a normal in-
dividual can be taken without consequences. When an appro- I
priate level of opiate analgesic is maintained, subjects ap~

pear functionally normal. ‘However, an abrupt cessation of

 

drug input or interruption of its action with an antagonist
precipitates a set of excitatory abstinence (withdrawal) syn—
drome. Thus a physical dependence can be developed. In sus~
ceptible persons, opiate analgesics produce both physical and
psychological dependence on the drug of such an intensity that
the drive for drugs displaces all other desires. In many 1a-
boratory animals such as chimpanzee, monkey, dog, rat, mouse,
cat,rabbitand guinea pig,toleranceto,and physicaldependence

on,opiate analgesics can alsoixadeveloped toa.greater orless—

 

er degree [see Seevers and Deneau, 1963]. The riddle of the
biochemical nature of such unique and hazardous effects of
opiate analgesics has fascinated many researchers and many

hypotheses have been advanced to explain these phenomena.

B—l. Development of narcotic tolerance and physical
dependence

A majority of the investigators of opiate analgesics
have assumed that tolerance and physical dependence are inse—
parable parts of a common mechanism since both syndromes
develop and disappear concurrently [see Way gg_gl., 1969].
The intimate relationship of tolerance with physical depen—

dence is indicated by the fact that, as the animals become

 

 

 

 

24
more tolerant to morphine, the dose of naloxone required to
precipitate the withdrawal syndrome becomes progressively
less, and, Conversely, more naloxone is required after physi-
cal dependence to morphine has largely subsided [Way gg gl.,

1969]. The finding that tolerance and physical dependence

 

can both be prevented by an antagonist also supports this
concept. Nalorphine, administered either systemically or
directly into the anterior hypothalamus, blocked the develop-
ment of tolerance to the hypothermic and the analgesic
effects of morphine in rats [Orahovats gg gl., 1953; Lomax
and Kirkpatrick, 1967]. The development of physical depen—
dence and tolerance to morphine could be prevented by a

simultaneous administration of levallorphan in monkeys

 

[Seevers and Deneau, 1968]. Furthermore, a pure narcotic
antagonist naloxone, which lacks physical dependence liabi—
lity, failed to produce tolerance [Jasinski and Martin,
1967], whereas an agonist-antagonist, like cyclazocine or
nalorphine produces very mild physical dependence and weak
tolerance [Martin gg gl., 1965; Martin and Gorodetzky, 1965].

Other investigators believe that narcotic tolerance
and physical dependence may originate via different
mechanisms. Cochin and Kornetsky [1964] demonstrated a per-
sistence of morphine tolerance during a 15 month period
without a manifestation of withdrawal syndrome following a
single injection of the analgesic.

Nevertheless, it is generally believed that narcotic

tolerance and physical dependence are inseparable parts of a

 

 

 

 

common mechanism and many hypotheses have been postulated to

explain both phenomena simultaneously.

B—2. Dual action hypothesis

Tatum, Seevers and Collins [1929], based on classic
observations of acute and chronic effects of morphine in
several laboratory animals, have concluded that morphine
simultaneously stimulates certain parts of the central
nervous system and depresses others. Irritability increases
with repeated administration of morphine because of the in—
crement of stimulant effects. The increased nervous irrita-
bility thus required a larger dose of morphine to counteract
and produce depressant effects. This increased dosage fur—
ther augments the nervous excitability; hence a vicious
Cycle is developed. Addiction is largely a question of phy-
siological balance between stimulation and depression at
any given level of irritability of the integrated nervous
system. Abstinence syndrome may result when increased irri—
tability outlasts the depression. This concept has been
termed the "dual action" theory because it visualizes a
simultaneous existence of depression and stimulation in
different parts of the nervous system. Twenty five years
later, Seevers and Woods [1953] further postulated that the
action of merphine is either depressant or stimulant
depending on the‘locatibn of the receptor. They postulated
that the binding of morphine to certain sites on or near the

surface of axons results in central depression, while that

 

 

 

 

 

 

26
to other intracellular sites in the cell body of the same
or other neurons results in central stimulation. Morphine
binding on the axon is visualized to be essentially a Surface
phenomenon dependent upon physicochemical forces, the pharma—

cologic response occurring only at the time of receptor

 

occupation by the drug (see Section B-7). Morphine binding
in the cell body is visualized to require intracellular
penetration, to be slow in onset, firm in combination and
long—lasting, the action being proportional (within limits)
to the quantity present.

As time passed by, evidence has accumulated which
apparently renders this hypothesis untenable as the sole
explanation of the mechanism of morphine tolerance and phy-

sical dependence.

 

(1) In the first place, morphine must be present in
nervous tissue during the entire period of withdrawal to
elicit an excitatory response. However, it is known that
free morphine disappears from the brain within 48 hours of a
single injection [Mulé and Woods, 1962; Misra g3 g1., 1971].
Thus, at the time of the maximal intensity of abstinence,

48 to 72 hours, only traces of morphine remain in the body.
4

 

Several conjugated forms of 1 C—N-methyl morphine, on the
other hand, could be detected in brain 3 weeks after a

single subcutaneous injection of 10 mg/kg of 14C-N-methyl
morphine [Misra gg gl., 1971]; These investigators suggested

that repeated administration of morphine could lead to a

‘cumulative deposition of conjugated morphine in brain and

 

 

 

 

 

 

27

could bring about a biochemical alteration of a specific-
neuronal structure in the central nervous system and produce
hyperexcitability if the site happens to be one where a
receptor~neurotransmitter interaction occurs. It Should be
noted, however, that the small quantities of conjugated
morphine reported by these investigators was within the range
of error (110%) and it has been shown that there was no sige
nigicant difference in the quantity of conjugated morphine
in brains of non-tolerant and tolerant dogs [Woods, 1954;
Richter and Goldstein, 1970]. Furthermore, one of the con—
jugated morphine derivatives, morphine glucuronide has been
shown to be pharmacologically inactive [Woods, 1954; Schulz
and Goldstein, 1972]. Two contradictory reports [Hosoya and
Oka, 1970; Sasajima, 1970] which claimed that the intracere-
bral injection of morhpine—3—glucuronide produced analgesia
in mice, have been criticized on the basis that the observed
action was caused by the free base resulting from hydrolysis
of the conjugate [Schulz and Goldstein, 1972].

(2) According to this hypothesis the syndrome elicited
by the direct stimulant action of morphine and morphine—like

analgesics must he qualitatively similar to the abstinence

 

syndrome. Although similarities are obvious in that both in-
volve increases in reflex hyperexcitability, the two syn—
dromes are by no means identical [see Seevers and Deneau,
1968].

(3) If the stimulant phase of morphine action is iden—

tical with the abstinence syndrome, a drug such as thebaine

 

 

 

 

IIIIIIIIIIIIIIlIII:____________________________________________________Fw'j=“fi ggfififiifi

28
which posseSses only stimulant properties should either
inddce a high degree of physical dependence following chronic
administration or it should produce the classical abstinence
syndrome following acute administration. No physical depen—
dence of any kind is developed to thebaine and the signs of
drug action are not similar to those of abstinence. '

(4) Shuster gg g1. [1963] and Goldstein gg g1. [1968]
have demonstrated tolerance development to the stimulatory
effects of morphine in mice, a finding that makes it very
difficult to accept the Concept that imbalance from the stic
mulatory effects elicited by morphine could account for the
abstinence syndrome. However, depression of an inhibitory
pathway may produce similar results to the unOpposed stimu-

lation of an excitatory pathway, and hence the classifica-

 

tion of depressant and stimulatory actions of opiate anal-

gesics becomes ambiguous.

B-3. Altered metabolism and distribution<xfopiate analgesics
The most obvious explanation for tolerance phenomena to

many drugs is to propose the protection of susceptible

tissue by the diminished absorption of the drugs, accelerated

metabolism, or exclusion of drugs from the region of sensi-

tive cells. However, in an extensive review of studies on

distribution and the fate of morphine and its surrogates,

 

Way and Adler [1960] concluded that any changes in the in-
activation processes of morphine and its surrogates 13 vivo

were disproportionately small when compared to the magnitude

 

—-’
- 29

of the loss of analgesic activity. The differences in the
physiological disposition of morphine between nontolerant
and tolerant dogs did not appear to be of sufficient magni-
tude to account for tolerance development [Mulé and Woods,
1962]. Similarly brain concentration of morphine after
intraperitoneal injection in tolerant and nontolerant rats
were not significantly different [Johannesson and Schou,
1963]. :

N-Dealkylation is the metabolic pathway common to most,
if not all, opiate analogs [see Axelrod, 1968]. An interest~
ing theory of cellular tolerance was based on the diminution
of N—demethylating enzyme activity in the liver with repeated
doses of opiate analgesics [Cochin and Axelrod, 1959]. This

enzyme with its specific protein-drug interaction has been

 

proposed to be an analog of the opiate receptor within brain
tissue. If receptors in the brain are also decreased with
chronic exposure to opiate analgesics, the neurons might
diminish in reactivity to opiate analgesics and so the animal
becomes tolerant. However, there was a lack of consistency
and specificity with respect to the loss of demethylating
ability by the liver microsomal enzymes and development of
telerance to opiate analgesics [see Way and Adler, 1960].

Tolerance to morphine and codeine analgesia and decreased

 

drug—metabolizing activities in the liver microsomes were
coincidental phenomena [Johannesson gg gl., 1965]. Various
agents which altered demethylase activity did not always

alter the rate of development of tolerance in the rat in a

 

 

 

30
similar fashion [Clouet and Ratner, 1964]. Although this
hypothesis is now only of historical interest, Goldstein
gg g1. [1973] reported recently that although the principal
basis of tolerance of levorphanol in mouse running activity
was a loss of sensitivity to levorphanol at the cellular
level in brain, metabolic tolerance was also present. They
indicated that this is due to increased conjugation and
excretion of levorphanol.

More recently, Wang and Takemori [1972] have demon-
strated that morphine is actively transported into the ven-
tricular system via the choroid plexus and that the concen—
trations of morphine in cerebrospinal fluid can be two to
three times higher than those in plasma. Since analgesic

receptors appear to be readily accessible to cerebrospinal

 

fluid [Tsou and Jang, 1964; Herz gg gl., 1970; Herz and
Teschemacher, 1973], the concentration ofopiate analgesics
in the cerebrospinal fluid may be more important than
average brain concentrations in determining the response to
opiate analgesics, and hence changes in the capabilities

of the active transport mechanism may play a role in toler—
ance development. However, the active transport mechanism
dose not appear to be altered during chronic morphine

treatment [Craig gg gl., 1971].

B—4. Redundancy hypothesis
Basically, this hypothesis states that there are two

or more alternative pathways for mediating a physiological

 

 

 

 

 

 

 

 

31
function and that these pathways differ from the other in
that they have different spectra of vulnerability to opiate
analgesics [Martin, 1968]. It is assumed that the opiate
analgesic interrupts one of the redundant pathways (pathway
A), but not the other (pathway B). The next assumption is
that pathway B will eventually hypertrophy with the conti-
nuous presence of opiate analgesic and takes over all or an
increasingly large portion of the function mediated by
pathway A. Thus, tolerance that developed to the opiate
analgesic is a consequence of hypertrophy of the redundant
and opiate~insensitive pathway B, not a decrement in effect
on pathway A. When the opiate analgesic is withdrawn, path-
way A returns to its normal level of excitability. However,
because pathway B is now functioning at a higher level than
prior to the chronic administration of the opiate analgesic
the total system functions at much higher levels than it did
in the pre—drug state. This exaggerated function is the
hypersensitivity seen during abstinence [Andrews, 1943; Mulé
et al., 1968; Kayan and Mitchell, 1968; Kayan et 31., 1971;
Tilson ct El-’ 1973]. According to this hypothesis, the
nature of the agonistic action of the opiate analgesic on
a given functional system, as well as the rate at, and
degree to which tolerance and physical dependence develops,
depends on at least two factors: (1) the importance of the
opiate-sensitive pathway in mediating the physiological
response, and (2) the capacity of the opiate-insensitive

pathway to hypertrophy. Thus, for some functions the

 

 

 

 

 

32

opiate—sensitive subpathway may represent only a small part
of the total pathway, whereas in other functional systems
it may represent a major portion of the pathway. Further,
the opiate‘insensitive pathway‘s capacity for hypertrophy
may vary among the different functional systems independent
of its role in mediating the function. Therefore, this
hypothesis not only provides an explanation for both toler—
ance and physical dependence but also for the coexistence
of partial and complete tolerance, as well as acute and
chronic tolerance [Martin, 1968].

This attractive hypothesis, however, lacks supporting
evidence. It was initially formulated to explain some of

the actions of atropine on the ascending activating system

 

and the descending vasomotor pathways emanating from the
midbrain reticular formation [see Martin, 1968]. In these
studies it was hypothesized that there is an alternative
pathway mediating these physiological responses that did not
contain muscarine synapses. Neurophysiological studies

have shown that there are indeed atropine— sensitive pathways
in parallel with atropine— insensitive pathways in the
descending vasomotor and ascending activating systems. 'It
has also been shown that there are independent cholinergic
and adrenergic pathways that are facilitatory to the flexor
reflex of the chronic spinal dog and whose activation pro—
duces spinal cord signs that are similar to those seen in
abstinence [see Martin, 1968]. HoweVer, the hypothetical

redundant pathways responsible for narcotic tolerance and

4____'4

 

 

 

 

33

dependence have not been identified.

Another controversial point relates to the changes in
the nervous system occurring during the chronic exposure
to an opiate analgesic. Martin suggested that nervous
pathway will hypertrophy when the pathway is "exercised”;
on the other hand, Jaffe and Sharpless [1968] suggested
that ”disuse", not use, will strengthen the nervous pathway
(see Section B—7). No direct evidence supports either

suggestion.-

B—S. Specific proteins and immune mechanisms

Apart from the previous hypotheses, it is possible
that metabolic processes are involved in the development
of tolerance. Actinomycin D given with repeated doses of
morphine, impairs the development of tolerance in mice and
rats [Cohen E£.§l” 1965; Cox et at., 1968]. When given
to animals not previously exposed to morphine, actinomycin
D does not diminish the analgesic effect of morphine, and
when given for a short period to animals with established
tolerance, it does not lessen the tolerance. Thus, actino—
mycin D appears to affect a process which occurs only while
tolerance is developing. Similarly, 8-azaguanine [Spoerlein
and Scrafani, 1967; Yamamoto et 31., 1967], puromycin [Smith
gt at., 1966] and cycloheximide [Way et at., 1968; Loh et at-,
1969] also have been shown to inhibit the development of
narcotic tolerance without blocking the action of the opiate

analgesics in non—tolerant mice. In such experiments, it is

 

 

 

 

———

34
essential to establish that the opiate is indeed preventing
the t3 gttg_incorporation of radioactive precursors into
brain proteins or nucleic acids. The half-life of at least
some mammalian messenger RNAs are such that there is little
change in proteins for several days after an injection of
actinomycin D [Appleman and Kemp, 1966]. Most brain proteins‘
that have been examined turn over slowly. Ha1f~life for
various fractions of rat brain proteins range from 12 ted:
22 days [Lajtha and Toth, 1966; von Hungen gt gt., 1968].
By contrast, the half-life of many liver proteins is a
few days or, in some cases, a few hours [Schimke gt_gt.,
1968]. Because narcotic tolerance and physical dependence

can be induced within a day or less [Lotti EE.§l-’ 1965;

 

Cox gt gt., 1968; Cheney and Goldstein, 1971], brain proteins
that may increase in this process should have a rapid turn—
over. On the other hand, increases in proteins with a long
half-life may account for the long persistence of some
forms of narcotic tolerance.’ In this case, there may be
permanent alterations produced in some manner by the opiate
analgesics.

The above findings have been interpreted as evidence

that synthesis of some specific proteins is required for

 

development of tolerance. Since tolerance to opiate analge—
sics may represent a rise in the threshold to a chemical
stimulus that acts to depress specific nervous pathways,
Smith [1971] suggested that introduction of specific protein

molecules somewhere in specific pathways may reduce the

 

 

 

35
sensitivity of synapses or neurons involved in transmission
of the impulse in question.

Supporting the involvement of specific proteins in the
development of morphine tolerance and dependence, Spoerlein
and Scrafani [1967] noted an increase in the microSomal
protein fraction from morphine tolerant mice. Clouet and
Ratner [1968] also demonstrated a slight enhancement of 13
KEEEE ribosomal protein synthesis from rats receiving 5 daily
injections of 30 mg/kg morphine sulfate. However, in an
earlier study [1967], they observed a greater inhibition
of leucine incorporation into brain proteins tg_tttg after
the fifth daily injection of morphine than after the first,
although there was no analgesic response on the fifth day.
More recently, efforts to detect increased amounts or rates
of synthesis of brain proteins using radioactive amino acid
precursors and acrylamide gel electrophoresis also have
failed [Hahn and Goldstein, 1971; Franklin and Cox, 1972].
Perhaps present methods are not sensitive enough to detect
small changes which may occur in a specific protein in the
brain of tolerant-dependent animals.

Not all inhibitors of protein synthesis block tolerance.
Failure of ethionine to impair development of narcotic
tolerance has been reported [Kato, 1967], although in the
same experiments ethionine blocked the development of
tolerance to phenobarbital and meprobamate, presumably by
impairing synthesis of the hepatic drug-metabolizing enzymes.

The lack of ethionine effect on morphine tolerance may be

 

 

 

 

IIIIIIIIIIIIIIIIT________________________________________________”“ffi444fit44i gfifffif*

36
due to the inability of this drug to alter protein synthesis
in the brain.

*‘Some of the protein synthesis inhibitors are also‘
potent immunosuppressants. Cochin and Kornetsky [1964] have
found that narcotic tolerance in rats can persist as long
as 15 months after the termination of morphine treatments.
Pretreatment of cycloheximide blocked the development of such’
tolerance [Feinberg and Cochin, 1969]. Since tolerance can
be extremely persistent and since it does take a finite time
to develop, it was proposed that tolerance might involve
immune mechanisms. Morphine and its surrogates may stimulate
the formation of antibody-like substances. Cycloheximide,
therefore, should block the development of tolerance by

blocking the synthesis of protein which can sequester or

 

antagonize opiate analgesics. The possible immune mechanisms
in the development of narcotic tolerance have been tested

by experiments designed to transfer the tolerance factor from
tolerant to non-tolerant animals. Successful transfer of
narcotic tolerance by cell—free extracts had been claimed by
Cochin and Kornetsky [1968] who reported a transfer of
tolerance by blood serum from morphine—treated rabbits to
naive mice. Kiplinger and Clift [1964], however, have
reported that serum from tolerant humans and dogs potentiated,
rather than inhibited, the analgesic effect of morphine in

mice. Ungar and Cohen [1966] reported that extracts of

 

brain from morphine tolerant rats and dogs conferred toler-

ance on mice. Other investigators, however, do not find

 

 

 

 

 

37

support for this hypothesis [Tirri, 1967; Smits and Takamori,
1968; Tilson gt gt., 1972].

Since it has been shown that brain levels of morphine
are not different in tolerant and non-tolerant animals (see
Section B—3), sequestering morpine in the blood cannot be
the mechanism of tolerance. However, an antibody-like
substance may be located near the opiate reCeptor sites and
thus divert morphine from opiate receptors without altering
the brain level of morphine. The specific antibody-like
substance may be specific to opiate analgesics and may be
common to different species of animals, but it would be
overly optimistic to expect that the antibody-like substance
administered systemically would reach the desired site
within the central nervous system.

As pointed out earlier, efforts to detect new proteins
in the brains of acutely morphine-treated and morphine toler—
ant and dependent mice and rats.were unsuccessful [Hahn and
Goldstein, 1971; Franklin and Cox, 1972]. Since morphine
tolerance and physical dependence can be induced within a
day or less [Lotti gt gt., 1965; Cox gt gt., 1968; Chenny
and Goldstein, 1971] and since antibody formation in peri-
pheral system would take about a week to develop, acute
morphine tolerance and dependence appears not to involve

immune mechanisms.

 

 

 

 

 

38

B—6. Alterations in central synaptic transmission

Since synapses are likely sites of the action of many
drugs, attempts have been made to explain narcotic tolerance
and physical dependence by alterations in synaptic trans—
mission. Theories involving feedback control of neurotrans-
mitter concentration have been put forward to explain both
narcotic tolerance and physical dependence [Goldstein and
Goldstein, 1961]. Opiate analgesics are assumed to inhibit
an enzyme E that catalyzes the formation of product C, an
enzyme reaction essential to a neuronal function or synaptic
transmission. If C mediates an excitatory function, the
response of the central nervous system to an opiate analgesic
is a depression. If at the same time the level of C controls
the synthesis and/or the breakdown of enzyme E, a decrease
in the concentration of C will increase the synthesis or
‘depress the breakdown of enzyme E. With continued exposure
to the Opiate analgesic, the quantity of active enzyme
will be restored by an increase in synthesis and/or a stabi—
lization of the enzyme E, resulting in tolerance development.
If the opiate analgesic is suddenly withdrawn, the excess
quantity of the enzyme, no longer inhibited, produces an
excess of substance C which leads to excitation and an absti-
nence syndrome. A similar hypothesis was proposed indepen—
dently by Shuster [1961]. According to this hypothesis, the
steady state cencentration of some central neurOtransmitters
would not change or would rather decrease during the tolerant

state and would increase after the withdrawal of opiate

 

 

 

 

 

39

I analgesics. This concept however, has not been supported by
evidence. 9

Chronic administration of morphine has been shown to
increase, rather than decrease, brain norepinephrine levels
[Freedman gt_gt:, 1961; Maynert and Klingman, 1962; Sloan
gt_gt., 1963; Gunné, 1963; Akera and Brody, 1968]. Chronic
morphine treatment failed to alter brain dopamine levels in
monkeys [Segal and Deneau, 1962] and in dogs [Gunné, 1963].
Although Segal and Deneau [1962] have reported an increase
in dopamine levels in caudate nucleus 24-48 hours after
morphine withdrawal or after nalorphine precipitated with—
drawal in monkeys, Gunné [1963] reported a decrease in brain
dopamine levels 72 hours following morphine withdrawal in
dogs when the animals were exhibiting moderate to severe
withdrawal symptoms. Acute or chronic morphine treatment
failed to alter brain 5—hydroxytryptamine (5—HT) levels in
several animal species. Withdrawal of morphine in chroni—
cally treated animals failed to affect brain S-HT levels
[see Way and Shen, 1971]. Brain acetylcholine (ACh) levels‘
increased after a single morphine administration [Maynert,
1967; Large and Milton, 1970]. Although an increase in brain
ACh levels has been observed during morphine withdrawal
[Large and Milton, 1970], brain ACh levels were either
unchanged [Large and MiltOn, 1970] or increased [Hano gt gt.,
1964; Maynert, 1967] during chronic morphine treatment. Thus,
Changes in brain levels of known neurotransmitters during

the cycle of narcotic addiction do not follow the pattern

 

 

 

 

40

predicted from this hypothesis.
As pointed out earlier a depression of an excitatory
pathway may produce similar results to the unopposed stimu—
lation of an excitatory pathway, and hence the classification
of depressant and stimulatory functions of neurotransmitters
is ambiguous.

The hypothesis also predicts that the development of
narcotic tolerance may be blocked by treatment with inhibi—
tors of protein or nucleic acid synthesis. As pointed out
previously, there have been claims that the development of
narcotic tolerance can be blocked by treatment with actino—
mycin D, azaguanine, puromycin and cycloheximide. However,
as discussed previously (see SeCtion B-S) efforts to detect
increased amount or rates of synthesis of brain proteins
were not successful.

In recent attempts to elucidate mechanisms involved in
the genesis of morphine—induced analgesic tolerance, Shen
gt gt. [1970] directed their attention to the effects
of morphine on S—HT turnover. Mice rendered tolerant by a
morphine—pellet implantation procedure exhibited a signifi—
cant increase in S—HT turnover compared to placebo-implanted
controls. Attenuation of these augmented responses upon the
concurrent administration of either cycloheximide or p-chlo-
rophenylalanine (PCPA) with morphine [Loh gt gt., 1969;

Shen gt gt., 1970; Ho gt gt., 1972] suggested a plausible
relationship between the synthesis of biologic macromolecules

and morphine tolerance and physical dependence. Although an

 

 

 

 

41

intimate relationship between tolerance or physical depen-
dence and brain S-HT metabolism has been reported by several
investigators to support this hypothesis [Tennen, 1968;
Haubrich and Blake, 1969; Fennessy and Lee, 1970; Burks
and Ducharme, 1971; Iwamoto gt gt., 1971; Thornburg gt.gt.,
1971; Bower and Kleber, 1971], other investigators have
failed to observe an increased S—HT turnover in morphine
tolerant animals [Marshall and Grahame-Smith, 1970;
Maruyama gt gt., 1971; Cheney gt gt., 1971; Algeri and Costa,
1971; Schechter gt gt., 1972]. 9
Recently, the involvement of biogenic amines in the
analgesic action of morphine has been approached by inducing
degeneration of central monoaminergic nerve terminals with
drugs such as 6~hydroxydopamine and 5,6-dihydroxytryptamine.
6-Hydroxydopamine, which produces a prominent and long-
1asting depletion of catecholamines in the brain [Uretsky
and Iverson, 1969] without significantly affecting the
brain S-hydroxytryptamine level, has been shown to decrease
morphine analgesia [Ayhan, 1972; Blasig gt gt., 1973].
5,6—Dihydroxytryptamine, which produces a prominent and long—
lasting depletion of brain S-hydroxytryptamine [Baumgarten
_t gt., 1972; Daly gt gt., 1973] did not affect morphine
analgesia [Blasig gt gt., 1973].
Midbrain raphé lesions, which induced a great reduction
of forebrain S-hydroxytryptamine, also did not affect mor-
phine analgesia [Blasig gt gt., 1973]. Samanin gt gt. [1973]

did observe decreased morphine analgesia after raphé lesions,

 

 

 

 

—-f

42

but analgesia prodUced by methadone, meperidine, codeine or
propoxyphene was not affected. These results thus suggested
that catecholamines play a more important role in morphine
analgesia. Whether the involvement of catecholamines sug-
gested for the analgesic effect of morphine can be general—
ized to other opiate analgesics remains to be investigated.

It has been shown that 5,6—dihydroxytryptamine, but
not 6—hydroxydopamine, inhibited the development of tolerance
to and physical dependence on morphine [Frielder gt gt.,
1972; Ho gt gt., 1973]. These results would suggest that
S—HT plays a more important role in the development of
narcotic tolerance and physical dependence. Since there

is evidence suggesting that the analgesic effect produced by

 

morphine is closely related with the development of narcotic
tolerance and physical dependence (see Section A-1 and B—8)
this conclusion appears not to be consistent with previous
conclusion which stated that catecholamines play a more
important role in morphine analgesia. Further supporting
evidence, therefore, is necessary before evaluating the

results of the above studies.

B-7. Pharmacological supersensitivity hypothesis
Pharmacological or ”disuse” supersensitivity of the

central cholinergic system has been described by Friedman

gt gt. [1969]. These authors treated mice with scopolamine,

a centrally acting anticholinergic drug. After several days

or weeks, scopolamine was withdrawn and mice were tested for

 

 

 

 

 

 

43

the hypothermic effect of pilocarpine, a cholinergic drug
that can act on the peripheral and central nervous systems.
The'scopolamine pretreatment increased the degree and dura—
tion of the hypothermic response to pilocarpine. Moreover,
development of tolerance was observed to the pilocarpine—
blocking action of scopolamine. The development of toler-
ance followed the same time course as the development of
-supersensitivity. Both phenomena were ascribed to an in:
crease in the number of central cholinergic receptors. This
is supported indirectly by the finding that either surgical
or pharmacological denervation (produced by botulinum.toxin)
increases the concentration of cholinergic receptors on the'
membrane of a striated muscle cell [Axelsson and Thesleff,

1959; Thesleff, 1960].

 

Jaffe and Sharpless [1968] suggested that most clini—
cally important narcotic withdrawal syndrome represent some
form of rebound hyperexcitability in central nervous path-
ways. According to this suggestion and based on the findings
of Friedman gt gt. [1969] on central cholinergic systems,
Jaffe and Sharpless [1968] postulated that the primary
cause of the rebound hyperexcitability occurring during
narcotic withdrawal is the result of disuse or depression
of nervous pathways rather than the presence of the drug
entity itself. In identifying the drug-induced depression
rather than the opiate analgesic itself as the primary
causative mechanism, they further suggested that drugs

that act on different sites or occupy different receptors

 

 

 

 

44

might still produce the same abstinence syndrome by causing,
directly or indirectly, a diminution in the flow of impulses
along the same nervous pathway.' On the other hand, classes
of drUgs that directly produce patterns of depression on
different systems might be expected to produce distinct
patterns of withdrawal hyperexcitability.

The primary controversy is the postulated central
supersensitivity itself. Stolk and Rech [1968] have demon-
strated that chronic treatment with reserpine made rats more
sensitive to locomotor stimulation by gfamphetamine. This
would be another example of pharmacological supersensitivity
in the central nervous system. However, events observed
in animals are an integrated activity of the total nervous
system. Pharmacological effects of drugs such as scopola—
mine or reserpine on the central nervous system are undoubted—
ly a summation of complex interactions of the drug with
different neuronal components, and hence the interpretation
of results obtained with these agents may be multivariant.
Moreover, supersensitivity with morphine or other opiate

analgesics still has not been clearly demonstrated.

B—8. Receptor occupation hypothesis

Based on classic observations of acute tolerance to
the vascular effects of morphine, Schmidt and Livingston
[1933] have suggested that tolerance is the result of a
change which occurs in depressible cells as soon as the

concentration of morphine in contact with them has reached

 

 

 

 

45

a certain critical level, and that the change is reversed
as soon_as the concentration falls below this level. Absti—
nence syndromes may be the external manifestations of the
reversal of the cell tolerance reaction when morphine is
withheld. This hypOthesis is compatible with the hypothesis
which states that the tolerance is the result of receptor
occupation [Clark, 1933], and_the rate theory postulated by.
Paton [1961]. These hypotheses postulated that drug mole-
cules exert their action at the time of initial attachment
'to the receptor sites and that, while the receptor sites
are occupied or the dissociation rate of drug from receptor
is very slow, the original molecules-exert no further

effect, but do prevent the initiation of response by recep~

 

tor interaction with additional molecules of the same or
similar drugs. Although many aspects of morphine action
could be explained successfully by this theory, it would

not explain the long-term persistence of tolerance shown

by Cochin and Kornestsky [1964]. There is a lack of evidence
that a significant amount of morphine residue could persist
in brain for several weeks after a single injection (see
Section B—Z). Moreover, it has been shown that the chronic
administration of naloxone, a potent narcotic antagonist,
does not produce physical dependence or tolerance develop-
ment to its antagonistic action [Jasinski and Martin, 1967].
These findings would indicate that receptor occupation it-
self is not sufficient to produce narcotic tolerance and phy—

sical dependence. Agonistic action of opiate analgesics

 

 

 

 

 

46 ,i j

appears to be necessary for their development.

B-9. Alterations in the receptor

Induction of protein synthesis also is a central
feature of the theory proposed by Collier [1965]. ACcording
to this theory, it is not necessary for the neurotransmitter
concentration to be altered during the development of physi—
cal dependence to an opiate analgesic. Instead, it was
proposed that there is an increase in the amount of protein
which binds opiate analgesics.' The opiate—induced synthesis
of "silent receptors," i.e., macromolecules that interact
with drugs but do not produce any detectable pharmacological
effect, would reduce the amount of the drug bound by "active
receptors" and hence result in tolerance development. How-
ever, efforts to detect such an increase in the amount of
brain proteins have failed (see Section B-S).

The possibility remains that narcotic tolerance may
be related to a change in the quality rather than quantity
of certain brain proteins. In this case, opiate analgesics
need not alter the overall rate of turnover of brain proteins
but the development of tolerance still could be blocked by
inhibitors of protein synthesis. For example, opiate anal—
gesics may cause ambiguity in genetic coding. If the result-
ing altered proteins cause a central stimulatiOn that could
antagonize the depressant effects of an opiate analgesic, the
result would be tolerance. 'Upon withdrawal of the opiate

analgesic, the unopposed action of the altered proteins_

 

 

 

 

 

47

could give rise to a withdrawal syndrome [Shuster, 1971]..

If opiate analgesic did produce miscoding, they may also act
[as inhibitors of protein synthesis. Morphine and related
compounds in high concentrations do inhibit protein synthe-
sis in mammalian cells. However, there is no correlation
between the analgesic activity of various derivatives or
their potency to produce narcotic tolerance and their potency
as inhibitors of protein synthesis [see Shuster, 1971].

A novel approach to detect anomalous protein content
in brain homogenate of morphine-tolerant mice, however,
was unsuccessful. Antibodies to naive brain homogenates
were used to precipitate unaltered protein in homogenates
obtained from brains of morphine-tolerant animals. After
a precipitation of normal protein by antibodies no protein
remained (Hosoya, personal communication). The failure to
detect anomalous protein would indicate several possibili-
ties: (1) no altered protein exists in the tolerant brain
(change is quantitative), (2) altered protein is a part of
larger particles and precipitates with other proteins, (3)
the quantities of altered protein are too small to be detec—
ted, or (4) the altered protein is still recognizable by
antibodies.

In summary, the biochemical basis for the development
of narcotic tolerance and physical dependence appears to
involve protein synthesis mechanisms in the central nervous
system. Tolerance to and physical dependence on opiate

analgesics may develop as a result of quantitative changes

 

 

 

 

48
in brain proteins if chronic morphine treatment induces
changes in concentratiOn-of opiate receptors, changes in
amounts of enzymes related to central neurotransmitters, or]
the synthesis of new proteins which could affect the opiate—
receptor binding or.affect the neurotransmitter-receptor
.interactions which are involved in pharmacologic manifesta-
tion of opiate analgesics. Tolerance and physical dependence
may also develop as a result of qualitative changes in brain
proteins if chronic opiate treatment alters the binding
affinity of the existing receptors for morphine and its
congeners. A number of inveStigators have failed to detect
quantitative changes in brain proteins associated with the
development of narcotic tolerance and physical dependence.
The failure to detect increased amounts or alterations in the
rate of synthesis of brain proteins could be due to the
fact that currently available techniques do not allow the
detection and accurate measurement of the turnover of minor

constituents.

C. Summary and objectives

 

The elucidation of the site of action of drugs is one
of the main concerns of pharmacology. Despite extensive
efforts by numerous investigators, we have little positive
knowledge about the site of action of opiate analgesics. It
has long been believed by many investigators of opiate anal—
gesics that receptors for these drugs do exist in the central

nervous system. Based on the results of extensive structure-

 

 

 

 

 

 

 

49

activity relationship studies, Portoghese [1966] suggested

the existence of three types of interaction of opiate anal—

gesics with their receptor. ~Although Takemori [1974] favored

the one receptor hypothesis, Martin's multi—receptor hypothe-

sis [1967] in general appears to be more adequate for explain—

ing the mode of actions of various opiate analgesics (see
Section A—l). Attempts to correlate the localization of
pharmacologic receptors with selective physiological dispo-
sition of opiate analgesics were unsuccessful (see Section
A-Z). Recently, it was suggested that the receptor for the
analgesic action of opiates might be distributed within

periventricular gray matter. Using elegant stereotaxic

techniques, Jacquet and Lajtha [1973] concluded that the main

site of morphine analgesia was in the posterior hypothalamus
of the rat brain. Wei gt gt, [1972, 1973] concluded that
the primary sites of action of naloxone, an opiate antago—
nist, are in the medial thalamus and the medial area of the
diencephalic—mesencephalic junctures of rat brain. The
first successful demonstration of opiate receptors tg ttttg
was reported by Goldstein and his associates [1971] Using
radiolabelled levorphanol. In last two years, specific}
binding of (3H)-naloxone, (3H)-dihydromorphine and (3H)-
etorphine to brain tissue of laboratory animals and man has
also been demonstrated (see Section A-3). However, in most
of these studies, only one concentration of radiolabelled
compound was utilized. Since specific binding observed at

a certain concentration of an opiate analog is determined

 

 

 

 

 

 

 

 

50 V 7 *3" .
by affinity and maximal binding capacity of specific binding
sites for the particular opiate, it is apparent that these
two kinetic parameters should be determined in order to
understand the properties of the specific binding sites
observed t2 ttttg. The demonstration of specific opiate
binding tg_ttttg requires (3H)-labelled compounds with high
specific radioactivity. Since the only radiolabelled com-
pounds available are dihydromorphine, etorphine, morphine

and naloxone, the ID has been used to assess the affinity

50
of other opiate analogs for the specific binding sites.

However, the IDSO, particularly assayed in a simple buffer
solution, is not an appropirate assessment of affinity for
opiate analogs. the inhibiton constant (Ki) should be used

for this purpose.

Narcotic tolerance and dependence, both physical and

 

psychological, are well known consequences of frequent,
repeated administration of morphine and various natural and
synthetic opiate analgesics. Because of these undesirable
effects, morphine and its congeners have been abused widely
around the world. Many hypotheses have been proposed to

explain these phenomena but none of them has been generally

 

accepted. In general, some of the hypotheses postulated
'that a direct adaptation takes place in which the concentra-
tion and/or the affinity of receptor for the opiate analogs
areCis) altered after repeated administration of opiates. 1
Since the specific binding sites for several Opiate agonists

and antagonists in brain tissue asSayed 12 vitro could be

, ,

 

 

 

 

51

candidates of pharmacologic opiate receptor, it is of
interest to teSt these possibilities by examining changes in
the maximal binding and the Km of specific binding sites for
(3H)—dihydromorphine and for (SH)—naloxone tg_ttttg_using
chronically morphine-treated animals. I .

The primary aims of this investigation were to further
characterize the properties of specific dihydromorphine and
naloxone binding sites tg ttttg and to demonstrate that these
specific binding Sites are consistent_with what is known"
about the pharmacologic receptor. Studies also were perform-
ed to determine if the development of tolerance and physical
dependence during chronic morphine treatment is associated
with changes in the concentration or affinity of specific

binding sites for dihydromorphine and naloxone.

 

 

 

 

 

 

.MATERIALS AND METHODS

A. Materials

3H labelled) was purchased from

Dihydromorphine (7, 8—
New England Nuclear, Boston, Mass. This compound was sup-
plied with a specific radioactivity of 46 Curies/m mole and
radiochemical purity of 98.5%. Naloxone (3H, randomly
labelled) was also purchased from New England Nuclear.
This compound was obtained with a specific radioactivity of
23.6 Curies/m mole and a radiochemical purity of 98.5%.
These two compounds were used without further purification.
(SHJ-dihydromorphine was supplied as an ethanol solution
(1 mCi; 6.4 ug/ml) and (3H)—naloxone was supplied as a
methanol solution (1 mCi; 1.39 ug/ml). Both compounds were
stored in the refrigerator or in the cold room at 0-5°C.
Fresh solutions of both compounds at the appropriate concen—
tration were prepared for each experiment by diluting the
supplied solution with ice-cold buffer solution, pH 7.4
(usually more than a 200 times dilution) in the cold room.

Morphine sulfate USP, was purchased from Mallinckrodt
Chemical Works (St. Louis, Mo.). Levorphanol tartrate and
dextrorphan tartrate were obtained from HoffmaneLaRoche
Inc. (Nutley, N.J.). Naloxone HCl was purchased from Endo
Laboratories Inc. (Garden City, N.Y.). Dihydromorphine HCl,’

g,t-methadone HCl were generous gift of Dr. J. H. Woods,

52

 

 

 

 

IIIIIIIIIIIEIII___________________________—_gE_________________________fiivfigflfiEEfirfiflfifi:fl

53 : "
'(Department of Pharmacology, University of Michigan, Ann.
Arbor). Morphine base pellets were supplied by Dr. E. L.
Way, (Department of Pharmacology, School of Medicine,
University of California, San Francisco). 6‘Hydroxydopamine
HBr was purchased from Sigma Chemical Company (St. Louis,
Mo.). 5,7—Dihydroxytryptamine creatinine sulfate was a
generous gift of Dr. A. A. Manian, (NIMH, Rockville, Md.).

.Other chemicals were analytical reagent grade.

B. Tissue preparation

 

 

Male, Sprague—Dawley rats weighing 200 to 300 grams :1
[were used. The animals were decapitated and the brains

were rapidly excised. Meninges and choroid plexuses were
removed with a dissecting forceps. The brain—stem, that is
the brain without cerebral cortex and cerebellum, was sepa-
rated as will be described later, weighed and homogenized
in 19 volumes of 50 mM Tris—HCl buffer (pH 7.4) using a
motor—driven Teflon—pestle homogenizer (Potter type: A. H.
Thomas Company, Philadelphia, Pa; type A, BB or B; clearance
0.10—0.18 mm at 25°C). Pestle was driven with a Tri—R
motor at approximately 900 rpm and the tissue was homoge-
nized with 5 passes in 30 seconds.

When regional studies of the specific binding of di-
hydromorphine or naloxone were performed, brains were dis-
sected as essentially described by Nyback and Sedvall [1969].
First, the cerebellum was removed with a forceps. Then,

with the aid of the central fissure, an incision to the

 

 

 

 

54

corpus callosum was made with a small spatula. The cerebral

cortices were separated from subcortical structures using
the lateral ventricles as a guide. The caudate nuclei
(striatum) were carefully separated from the cortex using
the exposed upper surface of caudate nuclei (external
capsule) as a guide and then separated from the rest of
diencephalic structures by cutting through the internal
capsule. The diencephalon (thalamus-hypothalamus) and
mesencephalon (mid-brain) were separated by cutting through
the anterior border of the anterior colliculi. Pens and
medulla oblongata were separated from midbrain by cutting
through the posterior border of the posterior Colliculi.
The pens and medulla oblongata were combined. The striatum
and midbrain were pooled from four rats and cerebral cortex,
thalamus—hypothalamus, pens—medulla oblongata and cerebellum
were pooled from two rats.

The homogenate was centrifuged in a Beckman L3-50
ultracentrifuge using a type 40 rotor at 100,000 x g (40,000
rpm) for 25 minutes at 1°C. The supernatants were discarded
and the pellets were rehomogenized in Tris—HCl buffer
and recentrifuged as described above. Pellets were finally
homogenized in Tris-HCl buffer as described above and then
diluted with 9 volumes of Tris-HCl buffer, i.e., to the 200
volumes of the original tissue. The homogenate of the parti-
culate fraction of indicated brain regions were assayed
immediately for (3H)-dihydromorphine or (3H)-naloxone bind—

ing as described later.

 

 

 

 

 

 

55
All preparative procedures_were performed in a cold
room in which the temperature is kept below 5°C.
I In some experiments, tissue preparations were prepared
and incubated with (3H)—dihydromorphine or (3H)-naloxone in
artificial cerebrospinal fluid (CSF) or in simulated intra—
cellular fluid (ICF). The artificial cerebrospinal fluid

contained 125 mM NaCl, 3 mM KCl, 1 mM MgCl 1.2 mM CaCl

2’ 2
and 25 mM sodium bicarbonate. The simulated intracellular
fluid contained 22 mM NaCl, 100 mM KCl, 1 mM MgC12 and
25 mM sodium bicarbonate [Davson, 1967]. Salts were dis-
solved in double—distilled water and the solution was
bubbled with 95% OZ‘5% CO2 gas for 60 minutes. The solutions
were kept in the refrigerator or cold room and bubbled with
95% 02-5% CO2 gas for 20 minutes prior to use. Both CSF and
ICF solutions prepared as described above had a pH of 7.4.
Protein concentrations of homogenates were assayed by
the method of Lowry gt gt. [1951]. One ml aliquot of the
homogenate was taken at the beginning and the end of each
experiment and diluted with 3 ml of double—distilled water.
Two 0.3 ml samples of the diluted homogenates were stored in
the freezer until assayed. Bovine serum albumin (crystal—
lized and lyophilized, Sigma Chemical Company, St. Louis,
Mo.) was used as the protein standard. It was found that
Tris-HCl buffer increased the background absorbance whereas
CSF and ICF had no such effect. Therefore, when the homo—‘
genate was prepared in 50 mM Tris—HCl buffer, bovine serum

albumin was dissolved and diluted in 12.5 mM Tris—HCl buffer.

 

 

 

 

 

 

 

56

Frozen samples of tissue homogenates were thawed to room

temperature prior to assay.

C. Binding assays

Tissue preparations (0.4 mg of protein in a final
volume of 2 ml) were incubated with or without 10 uM levor-
phanol, unless otherwise indicated, at 35°C for 5 minutes in
the presence of 50 mM Tris-HCl buffer (pH 7.4). Subsequently,
(3H)—dihydromorphine or (3H)—naloxone was added to the
reaction mixture and incubated for an additional 15-minute
period at 35°C. Each experiment was performed in triplicate.
Bound drug was collected on Millipore filters (type AA,
pore size, 0.8 um) and washed immediately with 18 m1 of
ice—cold Tris—HCl buffer (pH 7.4). Each filter was dissolved
in 1.0 m1 of ethyleneglycol monomethylether and assayed for
radioactivity in a liquid scintillation counter [Beckman,
L—100]. Counting efficiency was monitored with the external
standard channel-ratio which was calibrated with internal
standards. For (3H)-labelled compounds, counting efficiency
was approximately 32%. The amount of tissue-bound drug
assayed in the presende of 10 MM levorphanol (non—saturable
binding) was subtracted from that assayed in the absence of
levorphanol (total binding) to calculate the saturable,
stereospecific binding. Km value and maximal specific bind-
ing were determined using doub1e~reciproca1 [see Webb, 1963]
or Scatchard plots [Scatchard, 1949]. Ki value for various
opiate agonists and antagonists on specific (3H)-dihydromor-

phine binding were determined using Dixon plots

 

é—__J

 

 

 

 

 

57

[see Webb, 1963].

D. Chronic morphine treatments of rats

 

Rats weighing about 200 grams were rendered tolerant
to and physically dependent on morphine by subcutaneous.
injections of morphine sulfate solution at 9 a.m. and again
at 5 p.m. The initial dose of 10 mg morphine sulfate per
Kg of body weight per injection was increased every other
day over a period of 12 days to a final dose of 100 mg mor-
phine sulfate per Kg of body weight per injection. Control
animals were given a comparable volume of isotonic saline
solution subcutaneously. Compared to control animals, the
growth rate of morphine treated animals was greatly retarded.
At the beginning of treatment, morphine induced catalepsy,
rigidity of the muscles of body and limbs and constipation.
After 5—6 days of chronic treatment, the catalepsy become
less apparent and hyperactivity and gnawing became prominent.
When morphine injections were terminated after the chronic
treatment, severe diarrhea and acute body weight losses
were observed and the animals were hypersensitive to handling
and to external stimuli. These signs would indicate that
the animals had become physically dependent on morphine
following chronic morphine treatement [Akera and Brody,

1968; Nozaki gt gt, 1974].

Rats treated with morphine as described above had a

high mortality rate (1—2 out of 6 rats) when the dose of

morphine sulfate was increased.from 10 to 20 or from 20 to

 

 

 

 

IIIIIIIIIIIIEIIT______________________________—_________ ““J"

58

40 mg/Kg/injection. Therefore a log scale doses of morphine
sulfate starting from 10 mg/Kg/injection to 100 mg/Kg/injec-
tion was adopted in later experiments. No rat died during
this dosage schedule and tolerance to 100 mg morphine sulfate
can be achieved in a shorter time period.

In a series of experiments (results are shown in Figure
19 and Table 5) rats were injected with morphine solution
(morphine base dissolved in 0.01 N HCl) subcutaneously at 8—
hour intervals (6 a.m., 2 p.m. and 10 p.m.). The initial
dose of 1.67 mg/Kg/injection was increased to 10 mg/Kg/injec-
tion over a period of 16 days and this dose was maintained
for at least 6 days prior to the sacrifice. Control animals
received comparable volumes of 0.01 N HCl solution.

In another series of experiments (results are shown in

 

Figure 18) rats were rendered tolerant to and physically
dependent on morphine by implanting two 75 mg morphine baSe
pellets subcutaneously 36 hours apart as described by Way
gt gt. [1969]. These investigators and Cicero and Meyer
[1973] have demonstrated that rats treated by this procedure
developed morphine tolerance and physical dependence.
Animals were sacrificed at about 9 a.m. in the morning

16 hours after the last injection or 36 hours after the

 

second morphine base pellet implantation unless otherwise

indicated.

 

 

 

 

59

E. 6-Hydroxydopamine and 5,7-dihydroxytryptamine treatment

 

of rats

6—Hydroxydopamine HBr and 5,7-dihydroxytryptamine
creatinine sulfate solutions were freshly prepared in isoto-
nic saline solution containing ascorbic acid (1 mg/ml). As—
corbic acid was used to prevent the oxidation of these two
drugs in the aqueous solution. Rats were anesthetized with
sodium pentobarbital (50 mg/Kg, i.p.). Ten ul of a solution
containing 250 ug of 6-hydroxydopamine HBr was then injected
into the left lateral ventricle of the brain over a period of
one minute by the method essentially described by Noble gtgt,
[1967]. Briefly, rat was placed in a stereotaxic apparatus
and a mid—sagittal incision was made from eyes to the ears
and the bregma was exposed. A small hole, made with a den-
tal drill, was placed 1.5-2.0 mm lateral to the crossing of
the sagittal and coronal sutures. A 23-gauge needle, con—
nected to a 100 pl Hamilton syringe with a polyethylene
tube, was lowered 3.5-4.0 mm into the brain through the hole
and 10 ul of drug solution or saline was injected, Seven
days later, the same dose of 6-hydroxydopamine HBr was in—
jected into the right lateral ventricle. Control animals
were similarly treated with 10 ul injections of isotonic sa-
line solution containing ascorbic acid (1 mg/ml). By the
same method other groups of rats were treated with a single
dose of 75 ug of 5,7-dihydroxytryptamine creatinine sulfate.

The drug solution was injected into the leftlateral

ventricle.

 

 

 

 

 

 

60
All animals were sacrificed 14 to 21 days after the
first drug treatment. The dose of both drugs chosen in the
present studies has been shown to produce prominent damage
of either catecholaminergic or Sshydroxytryptaminergic nerve
terminal structures [Uretsky and Iversen, 1970; Baumgarten

and Lachenmayer, 1972].

F. Statistical analysis

 

Statistical evaluations of the data were performed
by Student's t—test. The level of significance was selected

as a probability of less than 5%.

 

 

 

 

 

EXPERIMENTS AND RESULTS

A. Characterization of specific (3H)-dihydromorphine binding
to brain-stem particulate fraction assayed in 50 mM Tris—HCl
buffer (pH 7.4)

Since levorphanol and dextrorphan have been used to
differentiate stereospecific and non—specific binding
[Goldstein gt gt., 1971; Pert and Snyder, 1973a, 1973b], the
effects of these agents on (3H)-dihydromorphine binding were
investigated.

The binding of (3H)-dihydromorphine to brain—stem
particulate fraction in the absence of levorphanol or dex-
trorphan (total binding) was 0.24 i 0.01 pmole/mg protein
(mean 1 standard error of 5 experiments) with 6 nM (3H)-
dihydromorphine present in the incubation mixture. Both
levorphanol and dextrorphan inhibited (3H)-dihydromorphine
binding (Figure l). Levorphanol significantly inhibited
(3H)-dihydromorphine binding when its concentration was
higher than 5 x 10‘10M and inhibited 50% of the total

9M. On the other

dihydromorphine binding (IDSO) at 3 x 10-
hand, dextrorphan did not inhibit dihydromorphine binding
significantly until its concentration was higher than

6 6

5 x 10- M. However, both levor—

M and its IDSO was 9 x 10'
phanol and dextrorphan produced a similar maximal inhibi-

tion: approximately 75% of total (3H)—dihydromorphine

61

 

 

 

 

 

62

 

0.25b/I {I l I I I I I ‘ I
Dextrorphan

A _

0.15 —

Levorphanol

0.10 — i\

§\§_

Bound 3H—DHM (pmoles/mg protein)

 

 

 

_._______._i._ __._.v:\§
0.05 e i _
:I II
0 ”Hi i 1 I i i l L J

 

010-‘1 10'9 10‘7 10'5 10'3

Drug conceniralion (M)

 

Figure 1. Levorphanol and dextrorphan inhibition of dihydro-
morphine binding assayed with 6 nM (3H)-dihydromorphine(3H-DHM) in
50 mM Tris-HC1 buffer(pH 7.4).

Brain-stem particulate fraction was incubated for 5 minutes
with indicated concentrations of either levorphanol or dextrorphan.
Subsequently, 6 nM (3H)-dihydromorphine was added to the incubation
mixture and the binding was assayed. Each point represents the mean
of 5 experiments. Vertical lines indicate standard errors.

 

 

 

 

63
binding (Figure 1, horizontal broken line). Thus, although
the magnitude of maximal inhibition obtained were similar
with both compounds, dextrorphan was markedly less potent'
than levorphanol, indicating that the affinity of dextrorphan
for the binding sites was approximately three orders of
magnitude lower than that of levorphanol. Under this
experimental condition, the difference in (3H)edihydromor—
phine binding assayed in the presence of 10—7M dextrorphan
and in the presence of 10—7M levorphanol would represent the
saturable binding which is relatively stereospecific
(Figure 1, line I). This observation is consistent with
the data shown by Pert and Snyder-[1973a]. The use of higher
conCentrations of dextrorphan.and levorphanol, for example

10'4

M as employed by Goldstein gt gt. [1971], would artifi—
cially separate the saturable binding into two components:
saturable, non-stereospecific binding (Figure 1, line II A)
and saturable, stereospecific binding (Figure 1, line 11 B).
In addition to the difference in potency of inhibiting
the (3H)—dihydromorphine binding, levorphanol and dextrorphan
also appeared to inhibit the dihydromorphine binding dif—
ferently. While levorphanol had a sigmoid inhibition curve
with a dose range of more than 5 log units, dextrorphan had
a steeper inhibition curve with a dose range of 3 log units.
A steeper asymmetric curve suggests that dextrorphan might
inhibit dihydromorphine binding irreversibly. Therefore, in
several preliminary experiments, tissue preparations were

incubated with (3H)-dihydromorphine first and then

 

 

 

 

,i--- fig ”I.
it

64
levorphanol or dextrorphan was added to the incubation mix—
ture and incubated for an additional 15—minute period.
Results similar to that depicted in Figure l were obtained.
Thus, both (3H)-dihydromorphine binding and dextrorphan
binding was reversible. Preliminary studies also indicated
that specific dihydromorphine binding was greatly reduced
when homogenates stored in a freezer overnight were used
for the assay.

In the presence of 20 nM (3H)-dihydromorphine both
total and non-saturable binding were significantly higher
(Figure 2). The concentrations of levorphanol and dextror—
phan required to inhibit the saturable binding were also
higher. Approximately 10 uM levorphanol was required to
inhibit the saturable binding (Figure 2, A+B). Dextrorphan
remained approximately three orders of magnitude less

effective than levorphanol. Ten uM dextrorphan thus inhi-

 

bited only portions of the saturable binding (Figure 2, A). I
At 10 4M, the inhibitory effect of both drugs appeared to

be resulted from nonspecific changes in membrane properties.

Therefore, the difference in (3H)—dihydromorphine binding

assayed in the absence and presence of 10 uM levorphanol

 

was used as an estimate of the saturable, stereospecific
binding in the following studies. The inhibition curve of
levorphanol became more shallow with a dose range of more
than 6 log units whereas the inhibition curve of dextrorphan
was steeper with a dose range of 2 log units. The saturable,

relatively stereospecific component was approximately 50%

+1

 

 

 

 

 

    
     
   
 

 

 

 

 

 

u I I l 1 I l
I Dextleorphan
’2 ..
ii ()A1 A. T
g Levorphanol
a
a:
E
\
In
2
o _
E :
e E -
2 0.3 5
I :
° 5
z:
n
'0
v:
3
o
a:
0.2 ' "
/
0“‘~{£1 l I 1 I
0 10-10 10-8 10-6 10-4 .

Drug Concentration (M) I

Figure 2. Levorphanol and dextrorphan inhibition of dihydro-
morphine binding assayed with 20 nM (3H)-dihydromorphine in 50 mM
Tris—HCl buffer(pH 7.4).

I
Brain-stem particulate fraction was incubated for 5 minutes
with indicated concentrations of either levorphanol or dextrorphan.
Subsequently, 20 nM (3H)-dihydromorphine was added to the incubation ‘
mixture and the binding was assayed. Each point represents the mean I
of 5 experiments. Vertical lines indicate standard errors. ‘

 

 

 

——i—_— 7 “

66
of the total binding assayed with 20 nM (3H)—dihydromorphine
compared to approximately 75% of the total binding assayed
with 6 nM (3H)-dihydromorphine.

The binding of (3H)-dihydromorphine increased with
higher (3H)-dihydromorphine concentrations in the medium
(Figure 3). Binding in the presence of 10 “M levorphanol
was proportional to the (3H)-dihydromorphine concentration,
indicating that this fraction is non-saturable. When this'
fraction was subtracted from the (3H)—dihydromorphine
binding in the absence of levorphanol (total binding) to
calculate the levorphanol-inhibitable binding, a typical
absorption isotherm curve was observed (Figure 3, broken
line). Figure 4 shows a Scatchard plotof the data (saturable,
stereospecific component) shown in Figure 3. The regression

line was calculated by the non-weighted, least squares

 

method. In this type of plot, the slope of the regression
line indicates the Km value whereas the intercept of the
regression line at the ordinate represents the maximal
binding. In this study, a Scatchard plot of data from each
independent experiment was performed in order to calculate

the apparent Km and maximal binding. Then the mean and the

 

standard error of the apparent Km and maximal binding was
calculated. The apparent Km value of the saturable, stereo-
specific binding site for (3H)-dihydromorphine was 7.9 i

1.2 nM and the maximal binding was 0.25 i 0.01 pmole/mg

protein.

 

 

 

 

67

 

 

 

 

 

05 I I I I
o non-specific
I specific ~-
E 0 total
.2 0:3 - _
8
O.
0')
E
U)
2
50.2 - ..
3
i i
,1"
9 T ,,./1'
:1:
6) 0 l - I,r”/ -
-u ,,I
5 ,II'
8 /
z
.o I I l I
OI 2 5 IO 20

 

Dihydromorphine ( n M )

Figure 3. Binding of (3H)—dihydromorphine(3H-DHM) in the
absence and presence of 10 uM levorphanol assayed in 50 mM Tris-HCl
buffer(pH 7.4).

Brain—stem particulate fraction was incubated for 5 minutes
with or without 10 uM levorphanol. Subsequently, various concentra-
tions of (3H)-dihydromorphine were added to the incubation mixture
and the binding was assayed. Substracting the non-specific binding
(binding assayed in the presence of 10 uM levorphanol) from the
total binding(binding assayed in the absence of 10 MM levorphanol)
gives the specific binding. Each point represents the mean of 5
experiments. Vertical lines indicate standard errors.

 

 

 

 

 

\

      

0.25 —- lntorcopHMaxlmal binding): __

‘./ 0.25: 0.01

 

 

 

E
2 0.20 - ._
2
D.
2’
E /Slope(Km ):7.911.2
3 0.15 - _
E
3;
U
E
U
E 0.10 " —
I)
I:
II
9
I:
m
.2 0.05 — -<
15
O
Q
U)
o l l l
0 0.01 0.02 0.03

Specific binding ( pmoles
DHM) mg protein - nM)

Figure 4. Scatchard plot of the specific (3H)-dihydromorphine
binding to brain—stem particulate fraction assayed in 50 mM Tris-
HCl buffer(pH 7.4).

Brain—stem particulate fraction was incubated for 5 minutes
with or without 10 uM levorphanol. Subsequently, various concen-
trations of (3H)-dihydromorphine were added to the incubation
mixture and the binding was assayed. The saturable, stereospecific
(3H)-dihydromorphine binding was plotted in this figure. Each point
represents the mean of 5 experiments. Vertical and horizontal lines
indicate standard errors. The regression line is calculated by the
least squares method fitted to the means.

 

 

 

 

69

 

B. Relative potency of opiate analogs to inhibit specific

(3H)7dihydromorphine binding to brain—stem particulate

 

fraction assayed in 50 mM Tris-HCl buffer (pH 7.4)

 

Since the consequences of the drug-receptor interaction
cannot be observed with 13 11339 studies, the pharmacologic
significance of the specific binding can only be explored
from a comparison of the characteristics of the 12 31339
receptor binding with the known characteristics of pharmaco-
logic receptor. Thus the affinity of several opiate analogs
for the specific (3H)-dihydromorphine binding sites were
studied and compared to their analgesic potency for each
drug.

In previous studies [Pert and Snyder, 1973a, 1973b;
Simon 23 31., 1973; Wong and Horng, 1973], the concentra—
tions of various opiate analogs to inhibit 50% of the
specific binding of a given concentration of a radiolabelled
opiate analog (IDSO) have been determined and used to
assess the affinity of these opiate analogs for the
specific binding sites. As discussed earlier in Introduc—
tion, Section A-3, the specific binding of a certain compound
observed at one concentration is not only determined by the
affinity of specific binding site for that particular com-
pound but also by its maximal binding. Therefore, determi-
nation of the inhibition constant, Ki, is a more appropriate

assessment of the affinity of opiate analogs for the specific

binding site. Ki values of several opiate analogs for speci—I

fic dihydromorphine binding were determined graphically

 

 

 

 

 

 

70
from Dixon plots. Brain-stem particulate fraction,
prepared in 50 mM Tris-HCl buffer, pH 7.4, were incubated
with various concentrations of opiate analogs for 5 minutes.
Subsequently, the specific binding was assayed using 0.6
and 6 nM (3H)-dihydromorphine. Figure 5 shows a Dixon
plot of specific dihydromorphine binding inhibited by
dextrorphan. The regression line was calculated by the non—
weighted, least squares method fitted to the data obtained
from 4 independent experiments. The concentration range of
dextrorphan shown in Figure 5 correspond to that producing
a linear inhibition of specific (3H)—dihydromorphine binding.
Table 1 shows the results of such studies for several
opiate analogs. Levorphanol had the highest affinity for
the specific (3H)-dihydromorphine binding site. Naloxone,
morphine and d,l;-methadone had moderate affinities.
Dextrorphan and codeine had markedly lower affinities and
thebain had the lowest affinity. Compared to IDSO studies,
the ranking order of affinity for these opiate analogs is
similar and is generally correlated with their analgesic
potency as determined by the systemic injection of these
drugs. However, the Ki values are 3 to 4 times lower than

the ID values of corresponding compounds for specific

50
(3H)-naloxone binding reported earlier [Pert and Snyder,
1973a], 40 to 100 times lower than the ID50 values of
corresponding compounds for specific (3H)-etorphine binding

[Simon e; al., 1973] and 2 to 4 times lower than the ID50

values of corresponding compounds for specific

 

 

 

 

 

 

71

 

180 ', . . . r

100 P

50L

0 1 J L '

o 0.1 0.3 0.5 1.0

 

1/Specitic3H-DHM Binding
(p moles/mg proteIn)

 

 

 

Dextrorphan (x iO'SM)

Figure 5. Dixon plot: Dextrorphan inhibition of specific
(3H)-dihydromorphine binding assayed in 50 mM Tris-Hcl buffer(pH 7.4).
Brain-stem particulate fraction was incubated with various
concentrations of dextrorphan for 5 minutes. Subsequently, the
specific dihydromorphine binding was assayed with 0.6 and 6 nM
(3H)-dihydromorphine. Each point represents a result of an
independent experiment performed in triplicate. The regression line
is calculated by the least squares method fitted to the data from
4 experiments.

 

 

 

 

 

 

 

 

72

Table 3.. Inhibition constant< Ki ) of several opiate analogs

 

for spfcific (3H)- ~dihydromorphine binding.

 

 

, Opiate Analogs K1 ( nM )
Levorphanol tartrate 0.52
Naloxone hydrochloride 1.74

, Morphine sulfate 2.22
d,l-Methadone hydrochloride 2.25
Dextrorphan tartrate 1300
Codeine phosphate 2020
Thebaine hydrochloride 2520

 

Brain-stem particulate fraction
concentrations of opiate analogs for
specific dihydromorphine binding was
H)— dihydromorphine. Ki values were

was incubated with various
5 minutes. Subsequently, the
assayed with 0.6 and 6 nM
determined from Dixon plots.

One of the Dixon plots is shown in Figure 5.

 

 

 

 

 

 

 

73
(3H)-dihydromorphine binding [Wong and Horng, 1973]. The
‘IIDSO of opiate analogs determined in the present studies
are shown in Table 2. It is clearly demonstrated that the
ID50 values increased as the concentration of (3H)—dihy-
dromorphine increased. The ID50 values for 6 nM (3H)-dihy-
dromorphine are similar to those determined by Pert and
Snyder [1973a] using 5 nM (3H)-naloxone. Since etorphine
is much more potent than dihydromorphine [Blane 2: al.,
1967] and thussupposedlyhas a higher affinity for the
specific binding sites, the ID50 values of other opiate
analogs for the specific (3H)-etorphine binding are expected I
to be higher than the ID50 values for the specific binding
of (3H)-dihydromorphine and (3H)-naloxone. However, the
difference was about 100 times lower than that expected.
Simon pp al. [1973] argued that the affinity for receptors
is only one of several factors responsible for the enormous
potency of etorphine.

As pointed out earlier, dextrorphan and levorphanol

 

appeared to inhibit specific (3H)-dihydromorphine binding
by different mechanisms. This speculation, however, was not
substantiated by the Ki determination of dextrorphan. A

Dixon plot of the specific dihydromorphine binding inhibited

 

by dextrorphan (Figure 5) indicated that the two inhibitory
regression lines did not intercept on the abscissa. IThat is,
dextrorphan did not appear to exhibit non-competitive inhi-
bition on specific dihydromorphine binding as speculated.

All Opiate analogs tested in the preSent studies, therefore,

 

 

 

 

 

74“

 

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75
exhibited competitive inhibition of the specific binding of
(3H)—dihydromorphine. I
Effects of several non-opiate drugs on the specific-
binding of 3.33 nM (SHl—dihydromorphine have also been

studied. Apomorphine, dopamine, chlorpromazine, xylazing

 

[Bayer l470]'and N-methylnicotinamide did not affect specific

5M.

(3H)—dihydromorphine binding at concentrations up to 10-
SKF—SZSA inhibited approximately 81% of specific (3H)~
dihydromorphine binding at lO-SM (Table 3). Such an inhibi—

6

tion, however, was greatly reduced at 10_ M. Thus, it ap—

peared that the inhibition of (3H)-dihydromorphine binding
by SKF—SZSA is not a competitive type. Since lO—SM SKF-SZSA
has been shown to alter the properties of biological

membrane, the effect of SKF-SZSA on (3H)-dihydromorphine

binding could be nonspecific. Apomorphine is a relatively

 

Specific stimulator of dopaminergic receptors at low concen—
trations [Ernst, 1967] while chlorpromazine is ablocker of _ ,
dOpaminergic receptors [Carlsson and Lindqvist, 1963]. It
has been suggested that central dopaminergic receptors are
important in morphine analgesia and tolerance development
[Vander Wende and Spoerlein, 1972, 1973]. Xylazine (Bayer
1470) is a potent, non-narcotic analgesic [Kroneberg 33 al.,
1967]. N—methylnicotinamide and SKF—SZSA have been shown to.
inhibit the active transport of morphine from the systemic
circulation into the CSF [Wang and Takemori, 1972]. The
present data would indicate that the stereospecific,

saturable binding of dihydromorphine is neither a nonspecific

. .

 

 

 

 

 

76

Table 3. Inhibitory effect of several non-opiate
drugs on specific (3H)-dihydromorphine binding assayed in
50 mM Tris-801 buffer(pH 7.4).

 

 

 

Drugs ' % Inhibition of Specific Binding
at 10‘5M, at 10‘6 M
Apomorphine 8.8 n=2
Dopamine - n=2
Chlorpromazine ~ n=2
Xylazine(Bayer 1470) - n=2
N—Methylnicotinamide - n=6
SKF-SZSA 80.9 10.5 n=4

 

Brain-stem particulate fraction was incubated for 5
minutes with various concentrations of drugs. Subsequently,
the specific dihydromorphine binding was assayed with 3.33 nM
(3H)-dihydromorphine. Inhibition was calculated and expressed
in this table as percent. short horizontal bars indicate no
siggificant inhibition was observed at concentrations up to
10‘ M.

 

 

 

 

 

, «‘7‘;ng

77
binding phenomenon nor a transport phenomenon.

C. Specific (3H)~dihydromorphine binding to particulate

 

fraction obtained from various brain regions assayed in

 

50 mM Tris-HCl buffer (pH 7.4)

 

 

Goldstein 33 El- [1971] have shown that there were no
regional variations of specific (14C)-levorphanol binding
in the mouse brain. On the other hand, other investigators
[Pert and Snyder, 19733; Kuhar e£_al,, 1973; Hiller g3 al.,
1973] have demonstrated regional differences of specific
binding of (3H)-naloxone, (3H)—dihydromorphine and (3H)—etor-
phine. It should be pointed out that in all these studies
specific binding was assayed with only a low concentration

of radiolabelled compound. Since the magnitude of binding

 

observed with a given concentration of compound is determined
by the maximal binding and the affinity, one cannot ascertain
whether the regional differences in specific binding observed
in the previous studies are due to the differences in the
concentration of specific binding sites, differences in affi—
nity of specific binding sites for the.opiate analogscn‘both.
Therefore, these two kinetic parameters were determined

using Scatchard or double-reciprocal plots of the data in

order to delineate whether specific opiate binding site

 

distribute differently in various regions and to determine
whether characteristics of specific opiate binding site are
different in various brain regions.

The particulate fraction obtained from striatum had

 

 

 

 

 

the highest specific (3H)-dihydromorphine binding (Figure 6),

followed by those obtained from midbrain, cerebral cortex,
thalamus—hypothalamus and pons—medulla, in decreasing order.
Cerebellum exhibited only minimal specific dihydromorphine
binding. There was more than a 20—fold differences in
specific dihydromorphine binding between the striatum and
cerebellum. The nonspecific binding assayed with 10 nM
_(3H)—dihydromorphine was quite similar in various brain
regions, ranging from 0.101 pmole/mg protein in the cere~
bellum to 0.135 pmole/mg protein in the poms-medulla. The
specific binding was only about 10% of the total binding in
the cerebellum whereas it was about 57% in the striatum.
Figure 7 shows Scatchard plots of specific dihydro—
morphine binding to particulate fraction obtained from 5
brain regions. Scatchard plot of the specific dihydromor-
phine binding to particulate fraction obtained from cerebral
cortex appeared to have two regression lines with different
slopes. Thus, the data suggest that there are two types of
specific binding sites for dihydromorphine in the cerebral
cortex. Because of the limitation of the specific radio—
activity of (3H)-dihydromorphine, the specific dihydromor—
phine binding assayed with low concentrations of (3H)-dihy—
dromorphine tends to show large variability. Therefore,
in the present studies, the apparent Km value and the maximal
binding were determined from a regression line calculated
by the least squares method fitted to all data in each

experiment. Scatchard plots of the specific dihydromorphine

 

 

 

 

79

 

0.20

0.1 5

 

0.10 - /_ \,« l —
i 0
/;\~ I

/1 l
0.05 9' {/1 _

Specific 3H—DHM Binding (p moles/mg protein)

 

 

 

CAL
D Cerebeilum
913—? l l
0 2 5 10 2O

Di hydromorphine (nM)

Figure 6. Specific (3H)-dihydromorphine binding to particulate
fraction obtained from various brain regions assayed in 50 mM Tris-
HCl buffer(pH 7.4).

Particulate fraction was incubated for 5 minutes with or without
10 uM levorphanol. Subsequently, variOus concentrations of (3H)-dihy—
dromorphine were added to the incubation mixture and the binding was
assayed. Values shown are differences in (3H)-dihydromorphine binding
assayed in the absence and presence of 10 uM levorphanol(saturable,
stereospecific binding). Brain tissues were pooled from 2 or 4 rats
for each preparation. Each point represents the mean of 5 experiments.
Vertical lines indicate standard errors.

 

 

 

 

 

 

0.25

0.20 -

O
.
d
U!
I

O

L:

O
I

9

o

u-
I

 

Speci ic 3H-DHM Binding (p moles/mg protein)

 

 

 

0 I l
0 0.01 0.02 0.03 0.04

Specific binding p moles )
(DHM) (mg protein-nM

Figure 7. Scatchard plots of specific (3H)-dihydromorphine
binding to particulate fraction obtained from various brain regions
assayed in 50 mM Tris-HCl buffer(pH 7.4) .

(H) :Cerebral cortex; (A—-¢) :Thalamus-Hypothalamus;
(H) :Striatum; (o—o) :Midbrain; (H) :Pons-Medulla.
Eacf point represents the mean of triplicate determinations.
Regression lines are calculated by the least squares method fitted
to the means in each experiment.

 

 

 

 

IIIIIIIIII:_____________________________________________________________________'" "' "t';a

81

binding to other brain regions appeared to have only one
regression line and thus suggests that there is only one
major type of specific binding site for dihydromorphine in
these brain regions. I

Table 4 shows the maximal binding and apparent Km for
dihydromorphine calculated from Scatchard plots of the data
as shown in Figure 7. The maximal binding for dihydromor—
phine was significantly higher in the striatum, midbrain
and thalamus-hypothalamus than in cerebral cortex and pons—
medulla. The affinity, which can be expressed as the reci—
procal of the apparent Km value, was similar in all brain
regions studied except the cerebral cortex where the affinity
for dihydromorpine was significantly higher than for other
brain regions. The apparent Km and maximal binding for dihy-
dromorphine in the cerebellum could not be determined because
the Specific dihydromorphine binding in this region was

only minimal under these experimental conditions.

 

D. Specific (3H)-naloxone binding to particulate fraction

 

obtained from various brain regions assayed in 50 mM Tris-

 

HCl buffer (pH 7.4)

 

Pert and Snyder [1973a] have demonstrated that the
corpus striatum had the highest Specific naloxone binding
followed by midbrain, cerebral cortex and brain—stem. In
their study, the cerebellum had no detectable specific
naloxone binding. These investigators, however, employed

only one concentration of (3H)-naloxone(5 nM). Moreover,

 

 

 

82

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83
there is evidence suggesting that naloxone may bind to
another site in addition to the morphine binding site
(see Introduction, Section A-l). Therefore, regional
differences for specific (3H)-naloxone binding were also
studied and compared with that for dihydromorphine. Speci—
fic naloxone binding was defined as the difference in bind-
ing observed in the absence and presence of 10 uM levorphanol.

The particulate fraction obtained from the striatum
had the highest specific (3H)tnaloxone binding (Figure 8),
followed by midbrain, thalamus—hypothalamus, cerebral
cortex, pons~medulla and cerebellum. Thus, a somewhat
similar pattern of regional differences in specific binding
were observed with both dihydromorphine and naloxone. The
regional differences in specific (3H)—naloxone binding,
however, were rather small; there was only a two-fold
difference in specific naloxone binding between striatum
and cerebellum. This was primarily due to the higher
specific naloxone binding in cerebellum. This particular
result is not in agreement with that reported by Pert and
Snyder [1973a].

Figure 9 shows several Scatchard plots of data shown
in Figure 8.‘ In contrast to the specific dihydromorphine
binding, the specific (3H)—naloxone binding to particulate
fraction obtained from cerebral cortex, striatum, thalamus—
hypothalamus and midbrain appeared to have two regression
lines. Only the specific binding of naloxone to the pons—

medulla and cerebellum appeared to have a single regression

 

 

 

 

 

84

 

Specific 3H-NLx Binding(pmoies/mg protein)

 

 

.0 I ‘ I I I
0 2 5 10 20

Naloxone (nM)

 

Figure 8. Specific (3H)-naloxone(3H-NLX) binding to particulate
fraction obtained from various brain regions assayed in 50 mM Tris-
HCl buffer(pH 7.4).

Particulate fraction was incubated for 5 minutes with or without
10 uM levorphanol. Susequently, various concentrations of (3H)-nalo-
xone were added to the incubation mixture and the binding was assayed.
values shown are differences in (3H)-naloxone binding assayed in the
absence and presence of 10 uM levorphanol(saturable, stereospecific
binding). Brain tissues were pooled from 2 or 4 rats for each
preparation. Each point represents the mean of 5 experiments. Vertical
lines indicate standard errors.

 

 

 

 

 

 

 

 

 

 

 

85
T l l
0.30 r
0.25 t .
T5
o
2
0' 0.20 L
o:
E
> n
o
8 ° '
E _ _ ‘1
a_ 0.15 A i
c) .
.E
E 0 o
'55 0.10 - -
><
.1
z ,
i: a
M
o
E 0.05 - ~
g o
a
(D D
o l 4 l
0 001 002 003 004
specific binding p moles )
(NLX ) ( mg protein-nM

Figure 9. Scatchard plots of specific (3H)-naloxone binding to
particulate fraction obtained from various brain regions assayed in
50 mM Tris-HCl buffer(pH 7.4).

(l———l):Cerebral cortex; Qk———A):Thalamus-Hypothalamus;
(A—-—A):Striatum; (o——«3):Midbrain; (o———O):Pons—Medulla;
(D———D):Cerebellum. Each point represents the mean of triplicate
determinations. Regression lines are calculated by the least squares
method fitted to the means in each experiment.

 

 

 

r7 , I’un’v'fl‘ 1,_ - ~77

2...... .' .

86
line. Thus, it appears that there are two types of specific
binding sites for naloxone in brain regions such as the
striatum, thalamus-hypothalamus, midbrain and cerebral
cortex. However, as pointed out earlier, the spec1f1c
binding assayed with low concentrations of (3HJ—naloxone
tends to show large variability. Therefore, more specific
binding studies, particularly in the lower concentration
range, are necessary in order to substantiate the two
specific binding sites for naloxone. In the present studies,
the apparent Km values and maximal binding were determined
from a regression line calculated by the least squares
methods fitted to all data in each experiment. The Scatchard
plot of the specific naloxone binding to cerebellar parti-
culate fraction appeared to indicate that there is only
one type of specific binding site for naloxone. However,
the slope is steep and the specific naloxone binding assayed
with the low concentration of (3H)-naloxone is quite small,
and neither the Scatchard plot nor the double—reciprocal
plot could provide an unequivocal answer. Nevertheless,
the present studies would suggest that the specific binding
sites in cerebellum could bind naloxone but not
dihydromorphine.

Maximal binding and the apparent Km value for specific
naloxone binding to various other brain regions are also
shown in Table 4. In contrast to dihydromorphine binding,
regional differences in the concentration of naloxone bind-

ing sites (maximal binding) were rather small. It was

 

 

 

IIIIIIIIIIIIII7______________________________——aE_____________________“_“_-______“ '"V"}

 

87
relatively higher in thalamus-hypothalamus and lower in
midbrain. Apparent affinities, on the other hand, were
.different among various brain regions.
The apparent Km for naloxone was significantly higher
than that for dihydromorphine in each brain region, i.e.,
the apparent affinity for naloxone was lower than that for
dihydromorphine in 50 mM Tris~HCl buffer. The maximal
binding for naloxone was higher than that for dihydromor—
phine in each brain region. Since naloxone, a competitive
antagoniSt for dihydromorphine binding, should bind to dihy—
- dromorphine binding sites, these data appear to substantiate
the findings revealed in Scatchard plots that naloxone has
two or more types of specific binding sites, only one of

which is available to dihydromorphine.

E. Comparison of specific (3H)-dihydromorphine and (3H)-

 

 

naloxone binding assayed in 50 mM Tris-HCl buffer, cerebro-

 

spinal fluid (CSF) and simulated intracellular fluid (ICF)
The finding that naloxone had a lower affinity than

dihydromorphine for specific binding sites is not compatible

with the pharmacologic potency of these two drugs. Moreover,

most of the opiate receptor binding studies were performed

 

using relatively simple buffer solution, 50 mM Tris-HCl
buffer, as the incubationinedium. It has been shown that
sodium decreases specific (3H)—etorphine and other opiate
agonists binding [Simon 3; al., 1973; Pert 33 al., 1973] but

it enhanCes specific (3H)-naloxone and opiate agonist—

 

 

 

 

88
antagonist binding [Pert 33 al., 1973]. Potassium, on the
other hand, decreases binding of both agonists and antago-
nists [Pert g3 31., 1973]. Calcium and magnesium also inhi—
‘bit specific (3H)-naloxone binding at 5 mM but has no effect
at physiological concentrations of these ions [Pert and
‘_Snyder, 1973a, 1973b]. Since it appears reasonable to
assume that the environment surrounding opiate receptors is
either extracellular fluid (cerebrospinal fluid) or intra—
cellular fluid which contains various concentrations of
sodium, potassium and calcium, it was of interest to study
the binding of dihydromorphine and naloxone in simulated
cerebrospinal fluid (CSF) and intracellular fluid (ICF)
and compare it to the binding assayed in 50 mM Tris—HCl
buffer.

Total (3H)-dihydromorphine binding was greatly reduced
in CSF and ICF. The specific dihydromorphine binding in CSF
or in ICF was approximately 50% of that in Tris-HCl buffer
(Figure 10). Scatchard plots of these data indicate that
the maximal binding is 0.25 r 0.01, 0.15 r 0.01 and 0.17 i
0.01 pmoles/mg protein in Tris—HCl buffer, CSF and ICF,
respectively (Table 5). Apparent Km values, however, were
significantly higher in CSF and in ICF than in Tris-HCl
buffer. Thus, the reduced specific dihydromorphine binding
assayed in CSF or ICF is due both to reduced maximal binding
capacity and reduced affinity of binding sites for
dihydromorphine. ‘

In contrast to (3H)—dihydromorphine binding, total

 

 

 

 

89

 

. . r ,' I
O 2" T i
O
l
" T 50mM Tris—HCI ‘
O
l

0.1- _

 

 

Specific 3H—DHM Binding (p moles/mg protein)

 

 

'0 I 1% L I J
02 5 10 2O 40

Dihydromorphine (nM)

Figure 10. Comparison of specific (3H)-dihydromorphine binding
assayed in 50 mM Tris-HCl buffer, artificial cerebrospinal fluid(CSF)
and simulated intracellular fluid(ICF).

(3H)-Dihydromorphine binding to brain-stem particulate fraction
was assayed in 50 mM Tris-HCl buffer(pH 7.4), CSF or ICF. Values

I shown are the differences in dihydromorphine binding assayed in the

absence and presence of 10 uM levorphanol. Each point represents the

mean of 4 experiments. Vertical lines indicate standard errors.

 

 

 

 

 

90

 

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91

(3H)—naloxone binding was increased in CSF and in ICF

(Figure 11). This increase in total naloxone binding was
primarily due to an increase in the nonspecific binding
(binding in the presence of 10 uM levorphanol). Specific
naloxone binding was also slightly higher in CSF at low
naloxone concentrations (Figure 12). The specific naloxone
binding sites, however, saturated at lower concentrations in
both CSF and ICF as compared to those in Tris-HCl buffer.
Thus, maximal binding was significantly lower in CSF and ICF
than in Tris—HCl buffer, wereas affinities for naloxone

were significantly higher in CSF and ICF as indicated by
lower apparent Km values in these media than in Tris-HCl
buffer (Table 5). Thus, it would appear that the maximal
binding for both dihydromorphine and naloxone was reduced

in CSF or in ICF compared to that in Tris—HCl buffer, whereas
the apparent affinity for dihydromorphine was decreased and
that for naloxone was increased. In any media, however, the
maximal binding for naloxone was increased. In any media,
however, the maximal binding for naloxone was significantly
greater than that for dihydromorphine. In CSF or ICF, but
not in Tris—HCl buffer, naloxone had a significantly higher
apparent affinity than did dihydromorphine indicating that
the binding observed in CSF or ICF may be closer to the phar—
macologic properties of these compounds. Thus, while some
experiments, such as the comparison of the binding site
population, may be performed using a simple buffer solution,

in vitro studies may be pharmacologically more relavant if

 

 

 

 

92

 

1.0 t

 
 
 
    

I).
l|>.

non—specific binding

total binding

 

 

Bound 3H—NLX (p moieS/mg protein)

 

 

I
0 2 5 10 20

Naloxone ( nM)

Figure 11. Binding of (3H)—naloxone(3H-NLX) in the absence
and presence of 10 uM levorphanol assayed in 50 mM Tris-HCl buffer,
CSF and ICF.

Brain-stem particulate fraction was incubated for 5 minutes
with or without 10 oM levorphanol. Subsequently, various concen—
trations of (3H)-naloxone were added to the incubation mixture
and the binding was assayed. Binding assayed in the absence of 10
uM levorphanol is total binding. Binding assayed in the presence of
10 uM levorphanol is non-specific binding. Each point represents
the mean of triplicate determinations. Since binding assayed in
CSF was similar to binding assayed in ICF, they are represented
by a single line.

 

 

 

 

 

 

  

 

 

 

93
I i T _ .1.
. o
’E 025“ 50mMTris—HCI "
E
2
G.
g 0.20- g -
E l
% CSF I
g ICF
o. 0.15- X "
O)
.E T
“g 0
ii 0.10r / ‘
x '6
5 _/://€
. X
I:
‘1, 0.05- E/ -
'6
0
O.
U)
.0 l l I g
o 2 5 10 2o

Naloxone ( nM )

Figure 12. Comparison of specific (3H)-naloxone binding assayed
in 50 mM Tris-HCl buffer, CSF and ICF.

(3H)-Naloxone binding to brain-stem particulate fraction was
assayed in different media. Values shown are the differences in
binding assayed in the absence and presence of 10 uM levorphanol.
Each point represents the mean of 4 experiments. Vertical lines
indicate standard errors. '

 

 

 

 

 

 

94
performed using CSF or ICF.

F. Effect of dihydromorphine and naloxone on (3H)-naloxone
binding to particulate fraction obtained from cerebellum ”
and striatum assayed in CSF

In earlier studies presented here, kinetic analyses_
have indicated that naloxone binds to dihydromorphine binding
site and to another type of binding site that has a different
affinity for naloxone and is not available to dihydromorphine.
If this is true, then dihydromorphine may inhibit naloxone
binding poorly in the cerebellum, a region where dihydromor—
phine binding sites are minimal compared to the naloxone
binding sites. In striatum, however, a significant portion
of naloxone should be bound to binding sites that are capable
of binding both dihydromorphine and naloxone, and therefore
a significant portion of naloxone binding should be inhibited
by dihydromorphine. Thus, the effects of non—labelled
dihydromorphine and naloxone on (3H)-nalox0ne binding were
compared in both the cerebellum and striatum using CSF as
an incubation medium. In cerebellum, non-labelled dihydro-
morphine failed to inhibit total (3H)—naloxone binding sig—
nificantly at all concentrations tested whereas non-labelled ‘
naloxone inhibited total (3H)—naloxone binding (Figure 13).
At 10 5M, non—labelled naloxone inhibited approximately 10%
of the total (3H)—naloxone binding, that is, approximately
10% of the total naloxone binding is saturable. The remain—

ing portion, approximately 90% of the total naloxone binding,

 

 

 

 

 

 

95

 

I 1
”1'1 I I r I I

    
    
 

 

Cerebeilum
0.20 *- _
‘5'
3. 0.19 _ Dihydromorphine I _
eI
. I ii --a
E 0.18 — / -
E
o.
0 0.17 '*
C
2
,3 0.16 ._ Naloxone
2.
,3: 0.15 )-
1:
g I.
o 0.14
00 ~

 

—/

0L/ 1 l I l i I
0 10'910'8 10-7 10'5 10'5 10'4

Concentration (M)

Figure 13. Effect of dihydromorphine and naloxone on
(3H)-naloxone binding to cerebellum particulate fraction assayed
in CSF.

Particulate fraction obtained from cerebellum was incubated
for 5 minutes with indicated conentrations of either dihydromorphine
or naloxone. Subsequently, 3.33 nM ( H)-naloxone was added to the
incubation mixture and the binding was assayed. Each point represents
the mea of 5 experiments. Vertical lines indicate standard errors.
Note: ( H)-naloxone binding in the absence of inhibitor represents
the total binding, which includes non-specific binding.

 

 

 

 

 

96'
therefore, is non-saturable binding in cerebellum. Since
non-labelled dihydromorphine did not inhibit the total
(3H)~naloxone binding at 10-SM, or even with concentrations
as high as 10'4M, these data would suggest that the specific
binding sites for dihydromorphine is negligible in the
cerebellum. In striatum, both non—labelled naloxone and
dihydromorphine inhibited total (3H)-naloxone binding (Figure
14). Naloxone appeared to have a higher affinity than
dihydromorphine for specific binding sites in striatum when
assays were performed using CSF. Naloxone also tended to
inhibit a greater portion of the total (3H)-naloxone binding.
than that inhibited by dihydromorphine but the difference
was not statistically significant. These data clearly indi—
cate that naloxone binds to at least two distinct specific
binding sites, one of which is not available to

dihydromorphine.

G. Effect of 5,7-dihydroxytryptamine pretreatment of rats
in vivo on the specific (3H)—dihydromorphine binding assayed
in CSF in vitro

The specific (3H)-naloxone binding has been shown to
be restricted to neuronal tissue [Pert and Snyder, 1973a].
These authors have concluded that regional differences in
acetylcholine concentration within the brain parallel the
observed regional differences in specific naloxone binding.
In a subsequent study [Kuhar at al., 1973}, electrolytic

lesions resulting in the destruction of cholinergic,

 

 

 

 

 

 

 

.-. r. .._.—w..

97

  
   
   

 

’IT’_I T I 1'!

Striatum

     
 

 

 

:3 25 'I’ Dihydromorphine
a .
g I
> .23
o
E
fj- .21 H --
0
I:
g .19 - —
E Naloxone
z
E .17 L— ——
‘2,
E
3 .15 —— ——4
o
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.13 *" -L

OL/lljljllj

o 10'1010-9 10-8 10-7 10'6 10-5 10-4

 

Concentration (M)

Figure 14. Effect of dihydromorphine and naloxone on
(3H)~naloxone binding to striatum particulate fraction assayed
in CSF.

Particulate fraction obtained from striatum was used in these
studies. The binding of 3.33 nM ( H)—naloxone was assayed in the
absence and presence of either dihydromorphine or naloxone. Each
point represents the mean of 5 experiments. Vertical lines indicate
standard errors.

i..:.‘-

 

 

 

 

 

 

98

noradrenergic or 54hydroxytryptaminergic pathways failed to
affect specific (3H)-dihydromorphine binding in regions
where the lesioned pathways terminate. However, the failure
of electrolytic lesions to affect specific dihydromorphine
binding could be due to an incomplete destruction of the par—
ticular nerve pathways. Dilution by non—lesioned cells may
mask the alterations caused by the electrolytic lesions.
Chemical destruction induced by drugs such as 5,7-dihydroxy—
tryptamine or 6-hydroxydopamine may be a better method, since
these agents selectively destroy the terminals associated
with the particular neurotransmitter and this destruction
is not limited to a focal region. The main sites of mor-
phine analgesia have been shown to be associated with peri—
ventricular structures (see Introduction, Section A—Z).
Therefore, lesions of S-hydroxytryptaminergic or catechola—
minergic nerve endings in brain were induced by injecting
5,7-dihydroxytryptamine or 6—hydroxydopamine solution into
the lateral ventricles of rats and the specific binding of
(3H)-dihydromorphine and (3H)-naloxone was monitored in
several brain regions. _

5,6-Dihydroxytryptamine, administered intraventricular—
ly, has been shown to produce prominent, long-lasting dege—
nerative damage to S-hydroxytryptaminergic neurons in the
rat brain [Baumgarten e£_al,, 1972; Daly e3 a1}, 1973].
Recently, an isomer, 5,7-dihydroxytryptamine has been shown
to produce similar toxic damage to central tryptaminergic

neurons [Baumgarten and Lachenmayer, 1972]. Since

 

 

 

 

 

 

'99
5,7-dihydroxytryptamine is claimed to have lower toxicity
[Baumgarten at 31,, 1973],-this compound was used. 5,7-Di-
hydroxytryptamine creatinine sulfate, 75 ug in 10 ul normal
saline solution containing ascorbic acid (1 mg/ml), was
injected into the lateral ventricle of rats by lowering the
needle stereotaxically through a small hole made 1.5-2.0 mm
lateral to the bregma and 3.5-4.0 mm down into the brain
tissue. Rats became hypersensitive in first two days but
behaved quite normally thereafter. The body weight of_
treated animals was generally slightly less than that of
control animals. The brains of treated animals appeared
normal. The behavioral response in treated animals were
apparently smaller than that previously reported. For exam-
ple, no incidence of convulsion was observed following 5,7—
dihydroxytryptamine injection. This may be due to the pre-L
treatment of animals with sodium pentobarbital. Sodium pen—
tobarbital has been used to treat convulsions developed in
rats treated with high doses of 5,7-dihydroxytryptamine
[Baumgarten and Lachenmayer, 1972]. All of the 24 rats
survived following the treatment in the present studies.

Methoxyflurane and ether have been used to anesthetize

 

rats in preliminary studies and previous studies cited above.

However, anesthesia induced by these two anesthetics was
generally not deep enough for the surgery and animals were
very frequently overdosed and be killed by these two.
anesthetics. In the present studies, 5,7—dihydroxytrypta—

mine was administered in a smaller volume (10 ul) and

 

 

 

 

—1——_‘fi-‘r fifirfi!‘

100

injected slowly over a period of 1 minute, therefore, may
produce less non-specific damage [Rech, 1968] compared to
most of previous studies in which 20 pl or more of drug
solution was injected rapidly into the lateral ventricle.

Two weeks after the injection, rats were decapitated.
At this time treated animals appeared to have no spinal
reflexes. Particulate fraction of diencephalon and mid—
brain—low brain—stem regions were prepared in CSF and assayed
immediately for specific (3H)—dihydromorphine binding.
Since the forebrain region has been examined previously
[Kuhar 33 al., 1973], the diencephalon, which is rich in
S-hydroxytryptaminergic nerve terminals, and midbrain-low
brain-stem, which is rich in cell bodies and axons of
S—hydroxytryptaminergic neurons-[Ungerstedt, 1971a], were
used in the present studies. In both brain regions studied,
5,7-dihydroxytryptamine pretreatment of rats did not signifi—
cantly alter the total or specific (3H)—dihydromorphine

binding (Figure 15). A Scatchard plot was found to be

 

inadequate for analyzing these data, particularly the
specific (3H)-dihydromorphine binding to particulate
fraction obtained from midbrain—low brain—stem. Therefore,

a double—reciprocal plot was used to determine the maximal

 

 

binding and the apparent Km for specific (3H)-dihydromorphine
binding (Figure 16). Table 6 shows that there are no

i remarkable changes occurred in these two kinetic parameters
of specific binding sites for dihydromorphine in either

brain region after 5,7—dihydroxytryptamine treatment.

 

 

 

 

 

 

 

   

Dloncephalon

  
  

_o.oa -

0.06 —

   

Mid brain

0.04 - -lowbnln-stem

Specific 3H—DHM Binding
(pmoles/mg protern)

H" Control -

8:8 Treated

0 1| I L J
0 2 5 10 20

Dihydromorphine (nM)

0.02 ‘-

 

 

 

Figure 15. Effect of 5,7—dihydroxytryptamine pretreatment of
rats on specific (3H)-dihydromorphine binding to particulate
fraction obtained from diencephalon and midbrain—low brain—stem
assayed in CSF.

Rats were treated with an intraventricular injection of 75 pg
5,7—dihydroxytryptamine creatinine sulfate(dissolved in 10 ul
isotonic saline solution containing ascorbic acid; 1 mg/ml). Control
animals were similarly injected with the same volume of vehicle.
Animals were sacrificed 2 weeks later. Particulate fraction of
indicated brain regions was prepared in CSF and assayed for specific
(3H)-dihydromorphine binding. Each point represents the mean of 6
experiments. Vertical lines indicate standard errors.

 

 

 

 

 

 

 

 

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H. Effect of 6-hydroxydopamine pretreatment of rats in
vivo on the specific (3H)—naloxone binding assayed in CSF
_ NEE—‘-

67Hydroxydopamine, given intraventricularly, has been
shown to produce a long lasting damage to catecholaminergic
neurons [Uretsky and Iversen, 1969]. Therefore, this drug

was used to produce degeneration of central catecholaminergic

. neurons .

Rats were treated with two intraventricular injections
of 250 ug of 6-hydroxydopamine HBr given 7 days apart. Rats
treated with 6-hydroxydopamine became hypersensitive in the
first few days but behaved normally thereafter. One week
after the last injection, these rats appeared normal although
their body weights were slightly less than those of control
animals. There was apparent damage to periventricular
‘structures of brains of 6—hydroxydopamine treated rats at
the time of sacrifice. 7

Rats were decapitated 7 days after the second 6—hydro-
xydopamine injection, and particulate fraction of cerebral
cortex and brain-stem were prepared in CSF and assayed
immediately for (3H)-naloxone binding. In both brain regions
studied, pretreatment with 6-hydroxyd0pamine did not alter
the total or specific (3H)—naloxone binding significantly
(Figure 17). The maximal binding and the apparent Km value
for naloxone as calculated from Scatchard plots also indi-
cated no significant difference (Table 7).

' It should be noted that, in this study, the specific

(3H)-naloxone binding was defined as the difference in the

 

 

 

 

 

 

 

 

 

10‘s
1 I I I
0.3 - -i
m Brain-stem
: A
3 r:
a; . ..
a o I" _.
O u
3 a
t a
m: } Cortex
.2 o
r: 3 o < _L. n _
0 . .
a E H Control
w v
0:0 Treated
o I l l l
0 2 5 10 20

Naloxone (nM)

Figure 17. Effect of 6-hydroxydopamine pretreatment of rats
on specific (3H)-naloxone binding to particulate fraction obtained
from brain—stem and cerebral cortex assayed in CSF.

Rats were treated with two intraventricular injections of 250
ug of 6-hydroxydopamine HBr(dissolved in 10 ul isotonic saline
solution containing ascorbic acid; 1 mg/ml) given 7 days apart.
Control animals were similarly injected twice with the same volume
of vehicle. Particulate fraction of indicated brain regions was
prepared in CSF and incubated with or without 10 uM non-labelled
naloxone for 5 minutes. Subsequently, various concentrations of
(3H)-naloxone were added to the incubation mixture and the binding
was assayed. Values shown are differences in (3H)—naloxone binding
assayed in the absence and presence of 10 uM non-labelled naloxone
(saturable, stereospecific binding). Each point represents the
mean of 6 experiments. Vertical lines indicate standard errors.

 

 

 

 

 

 

 

 

 

 

106

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ocoonmcIAva camaoomm How EM ecommmmm can maaccan HmEonz .e magma

 

 

 

 

 

 

107
(3H)-naloxone binding observed in the absence and in the
presence of 10 uM non-labelled naloxone. A previous study
present here has shown that the affinity of the specific
binding sites for naloxone was increased whereas that for
dihydromorphine was decreased in CSF and ICF (see Table 5).
Pert e3 al. [1973] also demonstrated that sodium enhanced
the specific binding of naloxone but reduced the specific
binding of dihydromorphine and levorphanol. Therefore, non-
labelled naloxone, rather than levorphanol, is a more appro-
priate opiate analog for the inhibition of the saturable,
Stereospecific component of (3H)-naloxone binding assayed
in CSF. Compared to an earlier experiment in which 10 uM
levorphanol was used to define the specific binding, the
specific (3H)—naloxone binding observed in this study was
slightly higher, the maximal binding was increased and
.affinity was also increased as indicated by the lower appa-
rent Km value.

A similar study was also performed using the striatum
obtained from 6-hydroxydopamine treated mice. Twelve male
albino mice were injected with 16 pg of 6-hydroxydopamine
HBr into the left striatum.2 It has been shown that after
the unilateral injection of 6—hydroxydopamine into the
striatum, mice exhibited a marked ipsilateral reduction in
forebrain dopamine concentration (reduced to 17% of the

Opposite non-lesioned side) and turned preferentially toward

 

2Work performed collaboratively with Dr. John E.
Thornburg.

 

 

 

 

 

 

e w , 7.7 . 1.72:}. , ‘ , ,
— , --' if"?
I.

108

the side of lesion. iApomorphine, in doses too low to produce
stimulation of,5pontaneous motor activity,.caused contra-
1atera1 turning [Von Voigtlander and Moore, 1973]. All
mice exhibited marked contralateral turning after apomorphine
injectiOn. Mice were decapitated and the striatum were
dissected. Left striata were pooled and used as 6-hydro‘
xydopamine-treated tissue whereas right striata (not directly
injected with 6-hydroxyd0pamine) were pooled and used as the
control tissue.

Particulate fraction of treated and control striata
were prepared with 50 mM Tris-HCl buffer and immediately
assayed for (3H)—dihydromorphine binding as described

previously. Neither total nor specific (3H)—dihydromorphine

 

binding to particulate fraction obtained from 6-hydroxydo-
pamine-treated and control striata were significantly
different. The maximal binding and the apparent Km values
calculated from double-reciprocal plots also indicated no
significant difference. The maximal binding was 0.13
pmoles/mg protein and the apparent Km was 3.47 nM for both
6—hydroxydopamine—treated and control striatum. I
These data would suggest that the specific binding
sites for dihydromorphine and naloxone are not specifically
associated with central monoaminergic nerve terminal

elements.

 

 

 

 

 

— 7” “ “7: ' .. "

109

I. Effect of chronic morphine treatment on (3H)-dihydromor-

 

phine binding to brain-stem particulate fraction assayed in

 

50 mM Tris-HCl buffer (pH 7.4)

 

Effects of levorphanol and dextrorphan on (3H)—dihydro-
morphine binding were first studied. Rats were rendered
tolerant to and dependent on morphine by implanting two 75
mg morphine base pellets 36 hours apart. Animals were
sacrificed 36 hours after the implantation of the second
pellet. In withdrawn animals, morphine base pellets were
removed 36 hours after the implantation of the second pellet
and animals were sacrificed 24 hours later. Withdrawn
animals were hypersensitive, had severe diarrhea and lost
weight sharply for 24 hours. About 48 hours after with—

drawal, animals started to regain their body weight gradually

 

and returned to their normal body weight 4-5 days following
withdrawal. Particulate fraction of brain-stem were prepared .‘
in 50 mM Tris—HCl buffer and assayed for dihydromorphine
binding immediately by incubating the preparation with 6 nM
(3H)—dihydromorphine.

Levorphanol and dextrorphan appeared to be similarly
effective in inhibiting (3H)-dihydromorphine binding to
brain—stem particulate fraction obtained from control,
tolerant and withdrawn animals (Figure 18). Dextrorphan
remained to be about three orders of magnitude less potent
than levorphanol. Both the total and the nonspecific
(3H)-dihydromorphine binding to the three preparations were

not significantly different and thus the specific binding

 

 

 

 

 

 

 

110

 

 

 

 

 

 

 

o. 25 l r l V I I 1 I
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.5 \ Dextrorphan
“
2
a. 0.20 ' B -
2’
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2 \
o 0.15 ‘ ‘
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3 0.05 ' o 0 control
8 I I tolerant
0 a withdrawal
0 4 n J_ l n 4 _L 3
1o"11 10‘9 10'7 10‘5 10’

Drug concentration ( M)

Figure 18. Effect of chronic morphine treatment on (3H)—dihydro-
morphine binding to brain—stem particulate fraction assayed in 50 mM
Tris-HCl buffer(pH 7.4).

Rats were implanted with two 75 mg morphine base pellets 36
hours apart. Tolerant animals were sacrificed 36 hours after the
implantation of the second pellet. In withdrawn animals, morphine
base pellets were removed 36 hours after the implantation of the
second pellet and these animals were sacrificed 24 hours later.
Control'animals had sham operation. Particulate fraction of brain-stem
was prepared in 50 mM Tris-HCl buffer(pH 7.4) and assayed for
dihydromorphine binding with 6 nM (3H)-dihydromorphine. Each point
represents the mean of 5 experiments.

 

 

 

 

 

 

111

component was not altered after chronic morphine treatment
or 24 hours after the withdrawal under these experimentalr
conditions.

Since the specific binding was assayed with 6 nM
I (3H)—dihydromorphine, a concentration of dihydromorphine
close to its Km value, the magnitude of specific dihydromor-
phine binding is dependent on two factors, i.e., the affinity
and the maximal binding capacity of specific binding sites
for (3H)-dihydromorphine. This study indicated that the
apparent affinity for dihydromorphine, levorphanol and dexa
trorphan was not altered by chronic morphine treatment or
during morphine abstinence. Alternatively, the apparent
affinity for these three drugs was changed by the same
magnitude in the same direction. The concentration of (3H)-
dihydromorphine was well below saturable levels, therefore,
the alteration of the maximal binding may not be great
enough to be detected by this assay.

In further studies, specific dihydromorphine binding
to brain—stem preparation obtained from control, chronically
morphine-treated and morphine-withdrawn rats was assayed
with various concentrations of (3H)-dihydromorphine and the
maximal binding and the apparent Km values were determined
separately in order to detect possible changes of these
kinetic parameters induced by chronic morphine treatment
or subsequent morphine withdrawal. Rats were rendered
tolerant to and dependent on morphine by the subcutaneous

injections of morphine solution (morphine base dissolved in

 

 

 

 

 

 

.112 _
0.01 N HCl) at 8-hour intervals. The initial dose of 1.67Ing/
Kg/injection was increased to 10 mg/Kg/injection over a
period of 16 days and this dose was maintained for at least
6 days. Control animals received comparable volumes of

0.01 N HCl solution. Withdrawal syndrome such as hypersensi-
tivity, severe diarrhea and weight loss appeared within 24
hours after termination of morphine injections in animals
tolerant to high dose of morphine. These syndromes
indicated that morphine-treated rats were physically depen—
dent to morphine.

Chronic morphine treatment and subsequent morphine
withdrawal failed to alter total and specific (3H)—dihydro—
morphine binding significantly (Figure 19). The maximal
binding and the apparent Km value for (3H)—dihydromorphine,
determined by a double-reciprocal plot of data in each
independent experiment (Figure 20), also indicated no signi—
ficant changes occurred after chronic morphine treatment
or during morphine withdrawal (Table 8). During the with—
drawal period, there was an apparent increase in the Km value
for dihydromorphine indicating that the apparent affinity
for dihydromorphine may be decreased at the peak of the with—
drawal syndrome (35 hours). The apparent Km value returned
toward control levels as withdrawal syndrome subsided

(7 days).'

 

 

 

 

 

113

 

 

 

 

on r, 1 , 1
g .
2 0.16 — _
2
o.
O) 0.14 — —l
E
a
.2 0J2 - _
- o
E
3 0.10 I- _
a:
.E
:g (108 F -
m
E: (106 r _
9
AF (’04 —
'3 ' I 7days withdrawal
'3 e 35hours withdrawal
3 0.02 L 0 Control a
m A Tolerant

O in l I

 

0 I 2 5 I0 ‘ 20
Dihydromorphlne (nM)

Figure 19. Effect of chronic morphine treatment and subsequent
morphine withdrawal on specific (3H)—dihydromorphine binding to
brain—stem particulate fraction assayed in 50 mM Tris-HCl buffer
(pH 7.4 ).

Rats were injected with morphine solution(morphine base dissolved
in 0.01 N HCl solution) subcutaneously at 8-hour intervals. The
initial dose of 1.67 mg/Kg/injection was increased to 10 mg/Kg/injec-
tion over a period of 16 days and this dose was maintained for at
least 6 days prior to the sacrifice. Control animals received
comparable volumes of 0.01 N HCl solution. Particulate fraction of
brain-stem obtained from control, chronically morphine-treated and
subsequently morphine-withdrawn rats was prepared in 50 mM Tris-HCl
buffer(pH 7.4) and assayed for specific ( H)-dihydromorphine binding.
Each point represents the mean of 5 experiments. Vertical lines
indicate standard errors.

 

 

 

 

 

114

 

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J. Effect of chronic morphine treatment on Specific (3H)—
naloxone binding to brain—stem particulate fraction assayed

in 50 mM Tris-HCl buffer (pH 7.4)

 

Since the dose of naloxone required to precipitate
the withdrawal syndrome become progressively smaller as the
animals become more tolerant to morphine [Way 33 al., 1969],
it was of interest to determine whether chronic morphine
treatment would increase the affinity of specific binding
sites for naloxone. Rats were rendered tolerant to and
dependent on morphine by the subcutaneous injections of
morphine sulfate solution twice per day. The initial dose
of 10 mg/Kg/injection was increased to 100 mg/Kg/injection
over a period of 12 days. Control animals received compa-
rable volumes of isotonic saline solution.

Specific naloxone binding to brain—stem particulate
fraction obtained from morphine tolerant and dependent
rats was not significantly different from that of control
preparations at all (3H)-naloxone concentrations studied'
(Figure 21). The maximal binding and the apparent Km for
(3H)—naloxone, calculated from double-reciprocal plots, also
indicated that no remarkable changes occurred after chronic

morphine treatment (Table 8).

K. Effect of chronic morphine treatment on the specific
(3H)—dihydromorphine and (3H)—naloxone binding to brain-stem
particulate fraction assayed in CSF

It is well known that tolerance develops to morphine

and to various natural and synthetic opiate analgesics after

 

 

 

 

 

 

117

 

 

 

 

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Figure 21. Effect of chronic morphine treatment on specific
(3H)-naloxone binding to brain-stem particulate fraction assayed
in 50 mM Tris-HCl buffer(pH 7.4).

Rats were injected with morphine sulfate solution, twice per
day, starting with 10 mg/Kg/injection. The dose was increased to
100 mg/Kg/injection over a period of 12 days. Control animals
received comparable volumes of isotonic saline solution. Specific
(3H)-naloxone binding to brain-stem particulate fraction obtained
from control and chronically morphine-treated rats was assayed in
50 mM Tris~HCl buffer(pH 7.4). Each point represents the mean of
5 experiments. Vertical lines indicate standard errors.

 

 

 

 

 

118

repeated doses of these drugs. At the same time as animals
become more tolerant to morphine, the dose of nalox0ne
required to precipitate the withdrawal syndrome becomes
progressively less [Way 9: al;, 1969]. It was of interest
to note that.sodium reduced the specific binding of several
opiate agonists whereas the specific binding of naloxone
and opiate agonist—antagonist were enhanced [Pert e:_al.,
1973]. Thus changes in properties of opiate receptors after
chronic morphine treatment may be related to the change in
the sodium-sensitivity of the binding site. Therefore, the
effect of chronic morphine treatment on the specific binding
of (3H)—dihydromorphine and (3H)—naloxone were reinvestigated
using CSF, which contains a high concentration of sodium,
rather than using Tris—HCl buffer.

Under these experimental conditions, total and non—
specific (SH)—dihydromorphine binding were greatly reduced
as observed in_the earlier study. Chronic morphine treatment
did not alter the total and the specific (3H)-dihydromorphine
binding significantly although the specific binding to pre—
parations obtained from tolerant animals was generally
slightly higher than that of control preparations (Figure 22)
The maximal binding for dihydromorphine of brain—stem parti—
culate fraction obtained from control and chronically mor—
phine-treated animals, calculated from double-reciprocal
plot of the data in each independent experiment, was 0.19 i
0.01 and 0.16 i 0.02 pmoles/mg protein (mean 1 standard of

5 experiments) respectively. The apparent Km values for

 

 

 

 

 

 

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r I I 1 I 1
‘1—
Tolerant
Control
I I l 4
2 5 10 20

Dihydromorphine ( nM)

Figure 22. Effect of chronic morphine treatment on specific
(3H)-dihydromorphine binding to brain-stem particulate fraction

assayed in CSF.

Brain-stem particulate fraction obtained from control and
chronically morphine-treated rats was prepared in CSF and then'
assayed for specific (3H)-dihydromorphine binding. Each point
represents the mean of 5 experiments. Vertical lines indicate

standard errors.

 

 

 

 

 

 

120

(3H)—dihydromorphine binding in control and tolerant prepara-
tions were 33.5 i 4.2 and 27.2 1 3.0 nM, (mean 1 standard of
5 experiments) respectively. The maximal binding and
apparent affinity for (3H)-dihydromorphine in preparations
obtained from tolerant animals were not significantly
different from those of control preparations.

'Both the total and the nonspecific (3H)-naloxone
binding were increased in CSF as observed in previous study.
The specific (3H)-naloxone binding, defined as the difference
between the (3H)—naloxone binding observed in the absence
and in the presence of 10 uM non-labelled naloxone, was not
significantly altered after chronic morphine treatment
(Figure 23). In this experiment, the maximal binding and
the apparent Km for (3H)-naloxone were determined from Scat~p
chard plots of data in each independent experiment and the
mean and the standard error were calculated. The maximal-
binding for naloxone to brain-stem particulate fraction of
control and chronically morphine—treated animals was 0.36 r
10.02 and 0.38 i 0.02 pmoles/mg protein, respectively, and
they were not statistically different. The apparent Km for
naloxone was 5.5 i 0.47 and 6.4 1 0.51 nM in control and
chronically morphine-treated animals, respectively. These
were not statistically different.

The present data thus indicate that the maximal bind-
ing and the apparent affinity for (3H)-dihydromorphine and

(3H)—naloxone of brain-stem particulate fraction are not

altered by chronic morphine treatment.

 

 

 

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121
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a a. 0.1 — ‘
a, V e—e Tolerant
o L l l l
O 2 5 10 20

'Naloxone (nM)

Figure 23. Effect of chronic morphine treatment on specific
(3H)-naloxone binding to brain—stem particulate fraction assayed
in CSF.

Brain-stem particulate fraction obtained from control and
chronically morphine-treated rats was prepared in CSF and then
assayed for specific (3H)-naloxone binding. Each point represents
the mean of 5 experiments. Vertical lines indicate standard errors.

 

 

 

i m-— 3-

DISCUSSION

A. Specific binding and uptake(transport) of opiate analogs
The present studies and several previous reports demon-
strated that it is possible to observe the specific binding
of several opiate analogs to brain homogenates or particulate
fraction obtained from brain homogenates. However, it
should be noted that tissue binding and active transport of
drugs have many features in common: saturability, chemical
specificity and competition among structurally related
analogs. This can be expected since the initial phase of an
active transport mechanism is the binding of the drug to a

carrier of the transport system and thus it resembles the

 

binding of a drug to a specific receptor site. Since the
existence of active transport of opiate analgesics has been
reported [see Hug, 1971; Wang and Takemori, 1972; Vasko and
Hug, 1973], it is important to differentiate specific bind—
ing from an active transport mechanism which accumulates
opiate analogs into synaptosomes or other organelles.

One of the generally recognized features of active
transport is an energy requirement. It was shown that spe-
cific naloxone binding was temperature dependent with a
maximal binding at 35°C and a Q10 (change in the reaction
rate for each 10°C change in temperature) value of 1.5 when

measured between 25°C and 35°C and 1.3 when measured between

122

 

 

 

 

123
15°C and 25°C after 15 minute incubation.v At 4°C, binding
was reduced to 25% of the values at 35°C [Pert and Snyder,
1973a, 1973b]. The specific naloxone binding could reach
a steady state within 15 minutes at 33°C and 25°C [Pert and
Snyder, 1973a; Pasternack and Snyder, 1974]. Generally
speaking Q10 value should be discussed in relation to the
rate of reacion. Therefore, the Q10 value could be small
at the steady state, as reported for specific naloxone bind—
ing, if both binding rate and dissociation rate were affected
to the same degree by a temperature change. The Q10 value
for specific naloxone binding may be large if a shorter in—
cubation period is employed or at lower temperatures because
the binding may not reach the steady state under these con—
ditions. Thus, the reported small Q10 values per se may not

support the conclusion that the observed uptake is not an

 

active transpOrt process.

The specific (3H)-etorphine binding was not affected
by high concentrations of sodium azide or sodium fluoride
[Simon et al., 1973]. Thus, the specific etorphine binding
is not dependent upon energy from oxidative metabolism or
glycolysis. Specific (3H)-dihydromorphine binding was not
affected by 10-5M ouabain [Wong and Horng, 1973]. The same
concentration of ouabain has been shown to inhibit approxi-
mately 60% of the active accumulation of S—hydroxytryptamine
and norepinephrine by synaptosomes [Tissari 3: al., 1969].
Wong and Horng [1973] have further reported that valinomycin

and monensin, which can function as mobile carriers for

 

 

 

124
monovalent cations in biological membranes in general, had
no effect on the binding of (3H)—dihydromorphine. Therefore,
the specific (3H)—dihydromorphine binding is not coupled
with sodium transport.

One of the features which clearly distinguishes active
transport from binding is the storage of the transported
drug. For instance, the uptake of norepinephrine by synap-
tosomes in Krebs bicarbonate buffer was linear for 50 minutes
and the amine was concentrated well above 200 times-the con-
centration in the incubation media [Colburn e£_al., 1967].
The specific binding of (3H)—naloxone, (3H)-etorphine and
(3H)-dihydromorphine to brain homogenates was linear for

only 1 to 3 minutes and a steady state was reached in 10

 

minutes [Pert and Snyder, 1973b; Simon gt al., 1973; Wong
and Horng, 1973]. On the other hand, when brain slices
were used, (3H)—etorphine, at low concentrations, was accu—
mulated into slices and a steady state was not reached until
30 minutes later [Huang and Takemori, 1974]. However, it
was also found that (3H)-etorphine accumulation into brain
slices was not affected by metabolic inhibitors such as
dinitrophenol, fluoride, azide or iodoacetamide. Thus,
this accumulation phenomenon may be just a redistribution
of (3H)—etorphine between the incubation medium and the
brain slice which has a high lipid content.

Organic basic compounds such as N-methylnicotinamide
and SKF—SZSA have been shown to inhibit the active trans-

port of morphine from the systemic circulation into the

 

 

 

125
CSF [Wang and Takemori, 1972]. However, the present studies
have shown that N—methylnicotinamide did not affect specific
(Sm-dihydromorphine binding (see Table 3). SKF-SZSA did
inhibit the specific binding of 3.33 nM (3H)-dihydromorphine

at concentrations higher than 10—6

M. However, the inhibition
of specific dihydromorphine binding by SKF—525A appears not
to be a competitive type and may be resulted from nonspecific
changes in membrane properties. (3H)eEtorphine accumulation
into brain slices also was not affected by N-methylnicotina-
mide and hexamethonium [Huang and Takemori, 1974]. These
data would suggest that in brain tissue there is no active

transport mechanism for opiate analogs in contrast to that

demonstrated in choroid plexus. The active transport of

 

opiates by cerebral cortical slices demonstrated by Hug and
his associates [see Hug, 1971; Vasko and Hug, 1973] may not
be involved in the present binding studies because active
transport was demonstrated only with high concentrations

of opiates. Moreover, it has been shown that at concentra-
tions above 15 nM, the accumulation of (3H)—etorphine into
brain slices appeared to be nonsaturable [Huang and Takemori,
1974]. Therefore, the overall evidence appears to indicate
that the specific binding of opiate analogs to brain homo-

genates assayed in vitro is not a transport phenomenon.

B. Components of the binding of opiate analogs
Goldstein gt El' [1971] first demonstrated that appro—

ximately 2% of the (14C)—levorphanol binding to mouse brain

 

 

 

 

 

126

homogenates (assayed with 1.95 uM (14CJ—levorphanol) was
saturable and stereospecific binding, 53% of the binding
was saturable, nonspecific binding and 46% of the binding
was non—saturable, nonspecific binding. The saturable,
stereospecific binding was defined as the difference in the
(14C)—levorphanol binding observed in the presence of 100
times excess of dextrorphan, a pharmacologically inactive
.enantiomer of levorphanol, and in the presence of 100 times
excess of non-labelled levorphanol. The saturable, non—
specific binding was defined as the difference in the (14C)—
levorphanol binding observed in the absence and in the pre-
sence of 100 times excess of dextrorphan. Recently, Pert
and Snyder [1973] have shown that approximately 70% of the
(3H)-naloxone binding (assayed with 5 nM (3H)-naloxone) was
saturable and stereospecific.

The present studies clearly demonstrated that when
dihydromorphine binding was studied with low concentrations
of (SHJ-dihydromorphine, the bulk of the binding is saturable
(Figure 1; Vertical line I). This is in agreement with the
finding of Pert and Snyder [1973a] for naloxone binding.
Since the affinity for dextrorphan and levorphanol differed
by more than three orders of magnitude, the saturable bind-
ing may be called stereospecific. As the concentration of
(3H)—dihydromorphine increased, the non—saturable binding
increased proportionally whereas the saturable binding was
close to the maximal binding which is achieved at approxi—

mately 80 nM. Thus, the proportion of this component

 

 

 

 

I7——fi‘—m_1?" 7"

127
decreased as the concentration of (3H)-dihydromorphine was
increased. These findings are therefore also in agreement
with the finding of Goldstein gt gt. [1971]. In the presence
of high concentrations of levorphanol and dextrorphan, the
saturable binding may be further separated into saturable,
non—stereospecific and saturable, stereospecific components
(Figure l and Figure 2, A and B, respectively) as postulated
by Goldstein gt gt. [1971]. However, this separation appears
to be due-to the use of a concentration of dextrorphan that
inhibits the saturable binding only partially and is also
due to the relative stereospecificity of the binding sites
and hence may be artifactual. Therefore, the binding of
opiate analogs to brain tissue 13 ytttg_can be separated

into two components; one is saturable, relatively stereo-

 

specific and the other is non—saturable, non-stereospecific.

C. Affinity and specificitygof the specific binding sites

 

for gpiate analogs

 

According to Ki values of several opiate analogs for
the specific (3H)—dihydromorphine binding, the affinity for
levorphanol was approximately 4 times higher than that for
morphine. Naloxone, morphine and d,t-methadone had similar,
affinities. Dextrorphan, codeine and thebaine had much
lower affinities. These data and previous studies of IDSO's
Of several opiate analogs [Pert and Snyder, 1973a, 1973b;
Simon gt gt., 1973; Wong and Horng, 1973] all indicate

that, in general, the affinity of opiate analogs parallel

 

 

 

 

 

128

their analgesic potency as determined by the systemic
injection of these drugs. The low potency of codeine

13 ttttg may be due to the lack of metabolic activation
under these experimental conditions. It has been proposed.
previously that the demethylation of codeine is required
for its pharmacologic activity [Johannesson and Schou,
1963].

In the present studies, the affinity of levorphanol
was approximately 2000 times higher than that of dextrorphan.
Based on IDSO‘s, the affinity of levorphanol is about 4000
times higher than that of dextrorphan. The affinity of i
‘t-levallorphan was 5000 times higher than that of its d-I
enantiomer [Pert and Snyder, 1973a; Simon gt gt., 1973].

The affinity of t-methadone was 10 to 50 times higher than
that of d—methadone [Pert and Snyder, 1973a; Wong and Horng,
1973]. These data thus clearly demonstrate that the specific
binding sites for etorphine, dihydromorphine and naloxone
are relatively stereospecific. The less impressive differ—
ence in stereospecificity between tfmethadone and d—metha—
done may be due to that the methadone molecule has greater
conformational mobility than the other opiate analogs
[Portoghese, 1966].

A serious problem in comparative studies of analgesic
potency of various opiate analgesics lg yttg_and tg_ytttg
is that the assessment of tg_ttxg potency are not based on
drug concentrations at receptor sites. It is well known

that the blood-brain barrier impedes or prevents the

 

 

 

 

 

129
‘penetration of drugs with low lipid solubility into the
brain [See Schanker, 1962]. iThus, the pharmacokinetic
differences will probably be of considerable magnitude for-
the chemically heterogeneous opiates and related synthetic
analgesics. For instance, heptane/water partition coeffi-
cients range from less than 0.0001 for the more hydrophilic
morphine and normorphine to 100 for the lipophilic methadone
and fentanyl [von Cube gt gt., 1970]. One would expect
methadone and fentanyl to cross the blood—brain-barrier
more easily than morphine and normorphine. Therefore, it
may not be appropriate to estimate the relative analgesic
potency of various opiate analgesics from a comparison of
the dosages which produce equal analgesic effects when
administered systemically.

The comparative analgesic potency of various opiate
analogs according to their brain concentrations has been
reported by Herz and Teschemacher [1971]. In these studies,
morphine has been shown to be 8 times more potent than
levorphanol and 30 times more potent than d,t—methadone.
Thus, the relative affinities for various opiate analogs,
estimated from their Ki or ID50 values, do not correlate
well with their analgesic potencies as estimated from their
brain concentrations [Terenius, 1974; Takemori, 1974].
However, some cautions must be taken before making this
conclusion. Herz and Teschemacher [1971] have concluded
that drugs with lower lipid solubilities reach their maximal

activity much later when the drug is administered

 

 

 

 

 

130
intraventricularly. With intravenous application, the
differences are much less pronounced. It has been shown
that intraventricularly applied morphine produces its I
effects very slowly and does not reach its maximal effect
until 1.5 hours later, while fentanyl, a highly lipid
soluble opiate analgesic, produced its maximal effect
within minutes. No mere than 10% of intraventricularly
applied fentanyl and etorphine were found in brain 15 minutes
after the application, while 40-60% of morphine and dihydro-
morphine were found at that time. More than 10% of morphine
and dihydromorphine were present in the CSF even after one
hour. Autoradiographic studies have shown that various
opiate analgesics had different distribution patterns.
Morphine and dihydromorphine preferentially diffused into
gray matter, whereas fentanyl exhibited a pronounced pre-
ference for white matter. Whereas morphine and dihydromor-
phine continue to penetrate further into the brain, fentanyl
does not. Fentanyl can scarcely be detected in a small
zone at the periventricular wall 120 minutes after intraven-
tricular injection [see Herz and Teschemacher, 1971]. Thus
a depot of morphine and dihydromorphine is maintained in I
the ventricular system for a good length of time, which
insures a steep concentration gradient within the brain
tissue whereas the concentration of fentanyl and etorphine
in ventricular system falls at a rapid rate. In the pres—
sence of a steep concentration gradient, the concentration

of morphine or dihydromorphine at receptor sites, which is

 

 

 

 

131
presumably located a few mm from the ventricular wall,
increases with time. The concentration of fentanyl or
etorphine at receptor sites, on the other hand, reaches a
peak earlier but then declines rapidly.

In studies in which the comparative analgesic potency
of Various analgesics was estimated from the drug concentra-
tion in brain tissue [Herz and Teschemacher, 1971; Terenius,
1974], neither the time of onset nor the grade of analgesia
was taken into consideration. Therefore, more careful
studies should be performed in order to estimate the true
potency of various opiate analogs.

The present studies show that the affinity of
specific binding sites for naloxone was higher whereas that
for dihydromorphine was lower in CSF and ICF compared to
those in 50 mM Tris-HCl buffer. Therefore, if affinities
for various opiate analogs are monitored in CSF or ICF, the
absolute value of Km or Ki would be different. The ranking
order of affinity for opiate agonists in CSF or ICF may be
similar to that in 50 mM Tris—HCl buffer. However, the
absolute affinity for opiate antagonists or agonist-anta—
gonists such as naloxone, nalorphine, levallorphan, cycla—
zocine and pentazocine would be higher in CSF or ICF and the
ranking order may be affected by the incubation media.
Since it is reasonable to assume that the opiate receptors
are exposed either to extracellular fluid or intracellular.
fluid, the affinities ofopiateagonists or antagonists

assayed in CSF or ICF may be more relevant to the study of

 

 

 

132
opiate receptors. Thus, studies of affinity and specifi-
city of the specific binding sites for opiate analogs need

to be carefully reevaluated.

D. Regional distribution of the specific binding sites
for dihydromorphine and naloxone in rat brain _
The present data confirm and extend the marked regional

differences in (3H)-dihydromorphine and (3H)-etorphine bind-
ing reported earlier [Kuhar gt_gt:, 1973; Hiller gt gt.,
1973]. The differences in dihydromorphine binding
observed in various brain regions appear to be primarily
due to the difference in the concentration of binding sites.
Binding sites for dihydromorphine from various brain regions
would appear to have a similar apparent Km value except

[ for those from cerebral cortex, which has a significantly
higher apparent affinity. This would suggest a similarity
in binding sites in different brain areas but does not rule
out the possibility that multiple binding sites exist. It
is possible, for example, that dihydromorphine bound to
low affinity binding sites may be lost during the washing
of the Millipore filters if the complex of dihydromorphine
with such a low affinity binding sites has a high dissocia—
tion rate.

Kuhar gt gt. [1973] and Hiller gt gt, [1973] have

demonstrated that the specific binding of (3H)-dihydromor-
phine and (3H)-etorphine were high in the limbic cortex

such as amygdala, temporal lobe, parahippocampal gyrus and

 

 

 

 

 

133

cingulate_gyrus but low in the hippocampus and very low in
cortical white matter areas. The specific binding in these
studies was assayed with l or 3 nM of tritium—labelled com;
pounds. Present data show that cerebral cortex has a high_
affinity specific (3H)-dihydromorphine binding site and thus
would suggest that the high specific (3H)-dihydromorphine or
(3H)—etorphine binding observed in the previous two studies
may be due to the high affinity of specific binding sites
in those brain regions. The maximal binding for (3H)—dihy—
dromorphine was relatively low in the whole cerebral cortex.
Whether the specific agonist binding sites are mainly located
in certain cerebrocortical regions such as those structures
categorized as the limbic cortex remains to be elucidated.
A Scatchard plot of the specific (3H)~dihydromorphine bind—
ing to particulate fraction of rat cerebral cortex (Figure
7) indicates that there are probably two types of specific
binding sites with different affinities for dihydromorphine.
This observation is compatible with the finding that whereas
several cortical limbic structures have high specific bind—
ing, other cortical limbic structures such as the hippo—
campus and cortical white matter have low specific binding
when assayed at one low concentration of (3H)—dihydromor—
phine or (3H)-etorphine.

The binding sites for naloxone distribute differential—
ly in various brain regions not only in quantity but also in
quality(apparent Km values). Differences in affinities in—

dicate the presence of different binding sites for the

 

 

 

 

 

134

antagonist. Since naloxone is capable of displacing dihy—
dromorphine from its binding site, it appears that naloxone
interacts with dihydromorphine binding sites and with other
type of binding sites which have different affinities and V
are not available to dihydromorphine. This hypothesis is
supported by the finding that in each brain region tested,
maximal binding, i.e., the concentration of binding sites
accessible under the particular experimental condition, is
greater for naloxone than for dihydromorphine. Similarly,
greater values for maximal naloxone binding than that for
dihydromorphine binding were observed in artificial cere—
brospinal fluid or in simulated intracellular fluid. Final—

ly, experiments shown in Figure 13 and 14 clearly indicate

 

that a part of the (3H)—naloxone binding cannot be displaced
by dihydromorphine, although it can be displaced by non-
labelled naloxone. The naloxone binding that cannot be
displaced by dihydromorphine was the primary fraction of
(3H)-naloxone binding in the cerebellum but a relatively
small portion in striatum. These data are consistent with
results shown in Figure 6 and 8, which indicate that the
levels of dihydromorphine binding are only small fractions
of the naloxone binding in the cerebellum but a substantial
portion in the striatum. Thus, it would appear that the
observed regional differences in the apparent Km values for
naloxone binding represent the presence of at least two
different populations of binding sites with different Km

values, and that the observed Km value depends upon the

 

 

 

 

 

 

135

relative abundance of each type of binding site.

The present data also clearly indicate that naloxone
but not dihydromorphine binds to cerebellar tissue. This is
in contrast with a previous report by Pert and Snyder [1973a]
who claim that cerebellar tissue does not contain naloxone
binding sites. The discrepancy appears to depend on differ—
ences in the concentrations of (3H)-naloxone used and those
of levorphanol employed to dilute specific naloxone binding.
The binding of (3H)—naloxone to cerebellar tissue was indeed
small at low (3H)—na10xone concentrations and may be regarded
as insignificant if higher concentrations are not examined
(Figure 8). Additionally, (3H)—naloxone binding to cere-
bellar tissue is primarily due to a site which is not
available to dihydromorphine. Inhibition of (3H)-naloxone
binding to such a site may require a higher concentration
of levorphanol, such as the 10 uM concentration used in the
present study, rather than that used by Pert and Snyder
(0.1 uM). Thus, the successful demonstration, in the present
study, of a naloxone binding site which is not available to
dihydromorphine may partly depend on the use of higher
concentrations of levorphanol. With high concentrations,
dextrorphan was capable of inhibiting the specific opiate
binding indicating that the stereospecificity of the binding
site is only relative.

In CSF or ICF, the affinity for dihydromorphine was
decreased while the affinity for naloxone was increased as

compared to those in Tris—HCl buffer. Moreover, the present

 

 

.136
data indicate that naloxone has more binding sites than
dihydromorphine. Therefore, when specific (3H)—naloxone
binding is to be studied in CSF or ICF, non—labelled naloxone,
' rather than levorphanol, should be used to define the
I saturable, specific naloxone binding. Results of regional
studies of specific naloxone binding would not be affected
by this change in design because these studies were performed
in 50 mM Tris-HCl buffer and levorphanol appears to have a
higher affinity than naloxone for agonist sites in 50 mM

Tris—HCl buffer (Table 1).

 

Previous studies have shown that the main sites of
morphine analgesia in rats are periventricular structures
of the third ventricle (see Introduction, Section A-Z),
whereas the primary sites of naloxone action appears to be
located in the medial thalamus and/medial areas of dience-
phalic-mesencephalic junctures. However, Martin [1967] has
suggested that the receptors responsible for the analgesic,
respiratory depressant and psychotomimetic effects could be
stereochemically quite similar since all of these pharmaco-

logic effects could be antagonized by naloxone in non-toxic

 

doses. Thus, it is not possible to correlate a specific
pharmac010gic receptor, namely the analgesic receptor with
the results of present regional studies. Studies with more
delicately dissected brain regions may yield a relevant
answer to this question. 0n the other hand, Martin's
suggestion gives the support for the attempts to correlate

the affinity of the specific binding site for various opiate

 

137
analogs with their analgesic potency in order to demonstrate
the pharmacologic significance of the specific binding site

observed tg vitro.

E.. The specific binding sites for_gpiate analogs and the

 

central monoaminergic preterminal axons and nerve endings

 

The central nervous system has been categorized
biochemically as containing several types of neuronal
systems according to the neurotransmitter utilized for
nerve transmission. If the specific binding sites for opiate
analogs were associated with certain presynaptic terminals,
such an association may be demonstrated by destroying

those presynaptic terminals and monitoring the change in the

 

specific binding of opiate analogs. This would be an impor—
tant finding since the functional role of the binding
macromolecules is not known.

The present studies show that specific dihydromorphine
and naloxone binding to tissue preparations obtained from
rats treated with 5,7-dihydroxytryptamine creatinine sulfate
and 6—hydroxydopamine HBr were not significantly different
from the specific binding of dihydromorphine and naloxone in
control preparations. This would suggest that the specific
binding sites for dihydromorphine and naloxone are not a
unique component of preterminal axons or nerve endings of
central monoaminergic neurons.

Ungerstedt [1971b] concluded that intraventricularly

injected 6-hydroxydopamine produces a two stage effect with

 

138
a large dose (200 ng/ZO ul). The first stage involved an
area limited to a periventricular zone, being about 2.0 mm-

wide. The second stage involved the noradrenergic axons in

the lateral hypothalamus, tegmentum, pons and medulla oblon-

gata and the dopaminergic cell bodies in the substantia
nigra and the ascending dopaminergic axons in the hypothala-
mus. Noradrenergic terminals in the cerebral and cerebellar
cortices and dopaminergic terminals in the caudate nucleus
were also affected. Therefore, the failure of 6—hydroxydo-
pamine to alter Opiate binding in the present studies is

not due to the route of administration which may limit the
drug to the periventricular structures.

Richards [1971] has pointed out that structurally-
damaged axon profiles, after two intraventricular injections
of 200 ug 6-hydroxydopamine, were not more than 1-3% of the
total number of nerve terminals observed in survey micro—
graphs. The present experimental method cannot detect such
a small change if the specific opiate binding is not
unique to catecholaminergic preterminal axons and nerve
endings.

In previous studies, the specific binding of opiate
analogs has been proposed to be primarily associated with
synaptosomal membranes (see Introduction, Section A-3). In
a subsequent detailed study, Pert gt gt. [1974] demonstrated
that the specific binding of 1 nM (3H)-dihydromorphine was
primarily found in synaptosomal fractions. Within synapto-

somal fractions, the specific dihydromorphine binding was

 

 

 

 

 

139
highly restricted to the membrane fraction- Since Kuhar
gt gt. [1973] have failed to correlate the specific dihydro~
morphine binding with central noradrenergic, S-hydroxytrypta-
minergic or cholinergic nerve terminal elements, Pert gt gt.
[1974] proposed that specific dihydromorphine binding might
be associated with post-synaptic thickening which
could persist after electrolytic lesions of axons in pre-
vious studies [Kuhar gt gti, 1973]. Alternatively, the
specific binding sites for Opiate analogs may be associated
with other pathways which are not yet clearly understood;
such as the GABA and histamine pathways or other pathways
which are not demonstrated yet. It is also possible that
the specific binding sites are not specifically associated
with nerve terminal elements of any particular nerve

pathway.

F. Specific binding of dihydromotphine and naloxone in

 

vitro and hypotheses of narcotic tolerance andtphysical

 

dependence

Tolerance to and physical dependence on opiate
analgesics may develop as a result of quantitative changes
in macromolecules (proteins) associated with the binding
of opiates (receptors) or with the expression of drug-
receptor interaction. In attempts to explain the develop-
ment of narcotic tolerance and physical dependence, it has
been postulated that chronic morphine treatment induces

changes in the concentration of opiate receptors (Collier,

 

 

 

 

140

1965; Martin, 1968; Jaffe and Sharpless, 1968], changes in
amounts of opiate sensitive enzymes that synthesize central
neurotransmitters [Goldstein and Goldstein, 1961; Shuster,
1961], the synthesis of new proteins which could affect
the morphine—receptor binding (see Introduction, Section
B-S; Cochin and Kornetsky, 1964] or the synthesis of new
proteins which affect the neurotransmitter-receptor inter—
actions inVolved in pharmacologic manifestations of opiate
effects [Smith, 1971]. Narcotic tolerance and physical

dependence may also develop as a result of qualitative

 

changes in opiate sensitive brain proteins if chronic mor—
phine treatment alters the affinity of the existing receptors
for opiate analogs.

However, the present studies indicate that neither
the maximal binding nor the apparent Km of the specific
binding sites for dihydromorphine and naloxone were signifi—
cantly altered after chronic morphine treatment of rats. A
significant decrease in the apparent affinity of specific
binding sites for dihydromorphine was observed during the
morphine withdrawal in rats chronically treated with morphine.
This phenomenon, however, does not appear to correlate with
the development of tolerance since no significant change in
the apparent affinity of specific dihydromorphine binding
sites was observed shortly after the termination of morphine
injections. It appears that this phenomenon is a result
rather than the cause of morphine withdrawal. These data

thus suggest that morphine tolerance and physical dependence

 

 

 

141

is not the result of alteration in the number or the affinity
‘of the specific receptor sites in the rat brain. The finding
that there is no specific binding of levorphanol and naloxone
in the soluble supernatant [Goldstein gt gt., 1971; Pert and
Snyder, 1973] would rule out the possibility that opiate
analogs could specifically interact with certain soluble
lenzymes which may be important in neurotransmitter synthesis;
The possibility that chronic morphine treatment could induce
the synthesis of new soluble proteins which may regulate

the opiate-receptor or neurotransmitter-receptor interactioni'
remains to be elucidated.

Klee and Streaty [1974] argued that if narcotic toler-
ance and dependence were simply the result of an increased
number or a decreased affinity of receptor sites.much large
doses of naloxone or nalorphine, not decreased doses as
documented, should be required to precipitate withdrawal
syndrome. Since the present studies clearly indicate that
naloxone has at least two types of specific binding sites,
the assumption which states that naloxone binds only to i
the morphine receptors is too simple.

It should be pointed out, however, that rat brain
regions not including the cerebral cortex and cerebellum
were utilized in the present studies. Relatively large
regions such as cerebral cortex, brain without the cerebellum
or the whole brain were used in other studies [Terenius,
1973; Klee and Streaty, 1974; Hitzemann gt gt., 1974].

Thus, it is not possible to rule out the possibility that

 

 

 

 

142
changes in properties of the specific opiate binding site
occurs only in a very small brain region; for inStance,
in the hypothalamus or medulla oblongata, and therefore
would not be detected in the present studies.

Recently, Ahtee [1974] has shown that 2 hours after
methadone injection (10 mg/Kg), the striatal homovanillic
acid (HVA) concentration of rats receiving methadone for
8 weeks was increased to about the same degree as in control
(saline) rats receiving the same dose of methadone as an
acute single injection. However, 19 hours after the last
injection of methadone the striatal HVA concentration of
rats receiving methadone for 8 weeks was decreased to 55%

of that of untreated control rats. Since it is generally

 

accepted that the distribution and metabolism of opiates
and synthetic analgesics is not significantly altered after
chronic treatment of these drugs (see Introduction, Section
B—3), Ahtee's observations may be interpreted as indicating
that the affinity of methadone receptors on dopaminergic
neurons which innervate striatal neurons is decreased.
Neither the concentration nor the efficacy of receptors are
altered after chronic methadone treatment because HVA in-
creased to about the same degree in control rats 2 hours
after the last injection and only decreased thereafter.

If dopaminergic neurons are not the primary neurons affected
by methadone, the same interpretation would apply to the
unknown primary sites of the action of methadone. Thus

this study would substantiate the hypothesis that the

 

 

 

IIIIII___I____________________________EIuEa_______________________________—_—“I—ET

143

affinity of opiate receptors was altered after chronic
‘treatment by opiate analogs. It also suggests that the use
of brain preparations obtained from a small, particular
brain region may yield positive data.
Alternatively, the effectiveness of the system with
which opiate-receptor interaction is translated into the
primary pharmacologic response or the function of a system
which regulates such processes may be greatly altered in
the tolerant state. A successful reversal of morphine to-
lerance by medial thalamic lesions in the rat [Teitelbaum -» ,
gt gt., 1974] appears to support the latter hypothesis. If [
chronic morphine treatment causes a proliferation of an .
inhibitory pathway which modulates the expression of the
drug-receptor interaction, then lesions of such an inhibi—
tory pathway could restore the sensitivity to opiate
analgesics. However, this study was performed using the EEG
response at cortical and subcortical recording sites as
the criterion for the development of morphine tolerance.
A further study of morphine analgesia in animals with or
without medial thalamic lesions might substantiate this
hypothesis.
Since the functions of the central nervous system
are mediated through neurotransmitters released from nerve
terminals, if the events which follow the opiate—receptor
interaction are altered in the tolerant state, then one
would predict that neurotransmitter released from the neuron

which possesses specific opiate receptors is either greatly

 

 

144

increased or decreased. As discussed in the Introduction,
Section B—6, no such phenomenon could be detected. The_
evidence, thus, appears to encourage further studies of the
properties of the specific binding sites for various opiate
analogs. The use of preparations obtained from delicately
dissected small brain regions or purified preparations as
described by Lowney gt gt. [1974] and Loh gt gt. [1974] may

be helpful in such studies.

 

 

 

 

 

SUMMARY AND CONCLUSION

In the present studies, the binding tg ttttg of an
opiate agonist, (3H)—dihydromorphine was studied using par—
ticulate fraction obtained from rat brain homogenates and
compared with that of an opiate antagonist, (3H)-naloxone.
The significant observations and conclusive remarks of the
present investigations are as follows:

A. Levorphanol as well as its stereo-enantiomer, dex—
trorphan inhibited (3H)—dihydromorphine binding. Although
the magnitude of maximal inhibition was similar with both
compounds, dextrorphan was approximately three orders of
magnitude less potent than levorphanol. The binding of (3H)-
dihydromorphine in the presence of 10 uM levorphanol was
proportional to the (SH)—dihydromorphine concentration in
the medium, indicating that this fraction is non-saturable.
When this fraction was subtracted from the (3H)—dihydromor—
phine binding observed in the absence of levorphanol (total
[binding), the levorphanol—inhibitable binding followed a
typical absorption isotherm curve. Thus, the binding of
(3H)—dihydromorphine may be separated into two components:
one saturable and stereospecific and the other non-saturable.
The use of dextrorphan results in an artifactual separation
of the saturable component. The apparent Km value of the

saturable, stereospecific binding sites for dihydromorphine

145-

 

 

 

 

146
in brain—stem was 7.9 i 1.2 nM. The maximal specific bind—

ing was 0.25 i 0.01 pmoles/mg protein. Specific (3H)-

 

naloxone binding assayed under same conditions also appeared
to have two components. The apparent Km value of saturable,
stereospecific binding sites for naloxone in brain—stem was
24.0 i 6.6 nM and the maximal specific binding was 0.57 i
0.10 pmoles/mg protein.

B. Based on Ki values estimated from Dixon plots of
specific (3H)~dihydromorphine binding in the presence of
several non—labelled opiate analogs, levorphanol had the

highest affinity for the specific dihydromorphine binding

 

site, followed by naloxone, morphine and g,t-medhadone.
Dextrorphan had an affinity 2000 times lower than that of
levorphanol. Codeine and thebaine had the lowest affinities.
lApomorphine, dopamine, chlorpromazine, xylazine [Bayer

1470] and N—methylnicotinamide did not affect specific
(3H)-dihydromorphine binding at concentrations up to 10_5M.
SKF-525A inhibited specific dihydromorphine binding. Such
inhibition, however, appeared to depend on the irreversible
alteration of membranes and hence to be nonspecific to

opiate binding sites. Thus, the specific dihydromorphine

binding site appeared to be specific for other active opiate

 

analogs.

C. There were marked regional differences in the dis—
tribution of specific (3H)-dihydromorphine binding in the
brain. These were primarily due to differences in the

concentration of the binding sites within various brain

 

 

 

 

147
regions. It appeared that_specific binding sites in various
brain regions had similar affinities for dihydromorphine,
except for those binding sites in cerebral cortex which
had high affinity. In contrast, specific binding sites

for naloxone in various brain regions had different affini—

 

ties for anloxone. It appeared that naloxone has at least
two types of binding sites, one of which is not available
to dihydromorphine. This is based on observations that (l)
the total concentration of specific binding sites for
naloxone was greater than that for dihydromorphine in each
brain region studied irrespective of the assay medium used

and (2) unlabelled dihydromorphine inhibited the specific

 

(3H)—naloxone binding in striatum but failed to alter it
significantly in cerebellum whereas unlabelled naloxone
reduced (3H)-naloxone binding significantly in both brain
regions. The difference in total binding sites for naloxone
and dihydromorphine was relatively small in striatum but
large in cerebellum,indicating that the binding sites in
cerebellum are predominantly naloxone—specific whereas those
in striatum are capable of binding both naloxone and
dihydromorphine.

D. In CSF as well as in ICF, the apparent affinity of
specific binding sites for dihydromorphine was decreased
while that for naloxone was increased as compared to those
in 50 mM Tris-HCl buffer. The maximal specific binding
for dihydromorphine and naloxone were both decreased in CSF

and ICF. The apparent affinity for naloxone was significantly

 

 

148
higher than that for dihydromorphine in CSF and ICF in con—
trast to those observed in Tris—HCl buffer. This is consis—
tent with the pharmacologic properties of these two drugs.
Thus, while some experiments, such as the comparison of

the binding site populations, may be performed using a simple

 

buffer solution, tg ttttg studies may be pharmacologically
more relevant if performed using CSF and ICF. When specific
(3H)-naloxone binding is to be studied in CSF or ICF, non-
labelled naloxone, rather than levorphanol, should be used
to define the saturable, specific naloxone binding because
naloxone appeared to bind to other sites in addition to
agonist binding sites and the affinity of naloxone binding

sites for naloxone in CSF and ICF is higher than that for

 

opiate agonists.

E. Two weeks after an intraventricular injection of
75 pg of 5,7—dihydroxytryptamine creatinine sulfate, speci—
fic (3H)-dihydromorphine binding to preparations obtained
from diencephalon and midbrain—low brain-stem of these
treated animals was not significantly different from the
specific binding to comparable control preparations. Simi—
larly, pretreatment of rats with two intraventricular
injections of 250 ug of 6-hydroxydopamine HBr also failed to
alter significantly the specific (3H)-naloxone binding to
particulate fraction obtained from cerebral cortex and
brain—stem. These data appear to indicate that the specific
binding sites for dihydromorphine and naloxone are not a

unique component of preterminal axons and nerve endings of

 

A

 

 

 

 

 

149
central monoaminergic neurons.

F. Chronic morphine treatment of rats or subsequent
withdrawal failed to alter the concentration of either
dihydromorphine or naloxone specific binding sites in the
brain-stem when binding was assayed in 50 mM Tris—HCl buffer
as well as in CSF. Chronic morphine treatment also failed
to alter the affinity of these specific binding sites.
During withdrawal from morphine there was a tendency toward
a reduced affinity of specific binding sites for dihydromor—
phine, which returned toward control level upon the diSsi?
pation of the withdrawal syndrome.

G. It was concluded that the specific binding sites
for dihydromorphine and naloxone could be demonstrated using
low concentrations of radiolabelled compounds. These bind-
ing sites appear to be saturable, stereospecific, specific
to active opiate analogs and closely related to the pharma—
cologic receptors. Naloxone appears to have at least two
types of specific binding sites, one of which is not avail—
able to dihydromorphine. It appears that these specific.
binding sites are not associated with central monoaminergic
preterminal axons and nerve terminals which have been
emphasized to play an important role in pharmacologic
actions of opiate analgesics and the development of narcotic
tolerance and dependence. Chronic morphine treatment does
not alter the concentration and affinity of specific binding
sites for dihydromorphine and naloxone in brain-stem parti—

culate fraction. Since a relatively large brain region was

 

 

 

 

 

150

utilized in the present studies, it cannot be ruled out that
changes in the properties of the specific opiate binding
sites may occur only in certain small brain region(s).

H. The present data failed to positively support
one of many hypotheses which have been proposed in attempts
to explain the develOpment of narcotic tolerance and physical
dependence. Several hypotheses, however, are inconsistent

with the present data and may be ruled out.

 

 

 

 

 

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