EFFECT OF ANTID‘IURETIC HORMONE
0N RENAL MEDULLARY cvcuc AMP
IN THE NEWBQRN

Thesis for the Degree of M. S,
MICHIGAN STATE UNIVERSITY
LEE=TZE LU
1972

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ABSTRACT
EFFECT OF ANTIDIURETIC HORMONE ON RENAL
MEDULLARY CYCLIC AMP IN THE NEWBORN
By

Lee-tze Lu

In the mammalian kidney, the antidiuretic hormone
(ADH) prevents the excretion of excess water in a dilute
urine and affects conservation of water by permitting full
operation of the concentrating mechanism. Cyclic AMP is
recognized as the intracellular mediator in the action of
ADH. The purpose of this study was to determine the effect
of ADH on the concentration of cyclic AMP in renal medullary
tissue of new-born and adult animals and to elucidate the
possible mechanisms responsible for differences in urinary
concentrating capacity between the new-born and the adult.
Cyclic AMP concentration was measured in vitrg using
a kidney slice technique. The method involves incubation of
medullary slices with ADH in a buffered medium under a gas
phase of 100% oxygen. Cyclic AMP concentration was determined
by a method based on competition for protein binding of the
nucleotide to a cyclic AMP—dependent protein kinase. Cyclic
AMP concentration was expressed as picomoles per mg wet medul~

lary tissue.

Lee-tze Lu

Adult Spraque—Dawley rats, New Zealand white rabbits
and mongrel dogs were used for verifying the method, eluci—
dating the time course, dose—response relationship and for
comparing cyclic AMP concentration to the respective young
animals. An increase in cyclic AMP concentration in renal
medullary slices from three species supports the hypothesis
that the action of ADH on epithelial structures are mediated
by cyclic AMP.

Cyclic AMP concentration in renal medullary slices
from adult rats, rabbits and dogs increased with increased
incubation time up to 30 minutes. This increase was seen,
however, only when 10 mM theophylline was included in the
incubation mixture. ADH increased the concentration of
cyclic AMP in renal medullary slices in a dose-dependent
manner in the presence of 10 mM theophylline. In the ab-
sence of theophylline cyclic AMP concentration was slightly
increased but the effect was not dose—related. All the values
obtained in the absence of theophylline were significantly
lower than in its presence. This suggested that theophylline
potentiated the action of ADH by inhibiting the activity of
phosphodiesterase in renal medullary tissues.

A developmental pattern of cyclic AMP concentration
in response to ADH was observed in dogs from 1 day to 2 weeks
of age. Body weight, kidney weight, protein concentration and
Uosm/Posm ratio were low in the new-born period. It is sug-
gested that lower cyclic AMP concentration may play some role
in the immature concentrating capacity in the young of this

species. Data obtained from rabbits did not show a similar

Lee-tze Lu

developmental pattern suggesting that factors limiting the
concentrating capacity in young animals vary from species
to species.

Treatment of 1 day, 3 day and 5 day old dogs with
ADH did not increase cyclic AMP concentration or urine os-
molar concentration demonstrating both biochemical and
physiological insensitivity of young animals to ADH. This
may result from a less receptive adenyl cyclase system, or
development of resistance to ADH, or difference in the en-
vironment at cyclic AMP generation sites.

These observations elucidated a possible role of

cyclic AMP in the development of concentrating capacity in

the new-born animals. However, these experiments do not ex-

clude the importance of other limiting factors such as short

loops of Henle, low urea excretion rate, permeability char—

acteristics of the nephron or limited capacity of the sodium

pump.

EFFECT OF ANTIDIURETIC HORMONE ON RENAL

MEDULLARY CYCLIC AMP IN THE NEWBORN

BY

Lee-tze Lu

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of
MASTER OF SCIENCE
Department of Pharmacology

1972

 

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C, I

ACKNOWLEDGMENTS

I would like to express my sincere appreciation
to Dr. J. B. Hook for his valuable assistance, interest,
encouragement and constructive criticism throughout the
course of this investigation. I would also like to thank
Dr. T. M. Brody, Dr. J. E. Gibson, Dr. M. D. Bailie, and
Dr. L. D. Muschek for their helpful assistance in the prep-

aration of this thesis.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . .

LIST OF FIGURES . . . . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . . . . . .

MATERIALS AND METHODS

1. Materials . . . . . . . . . . . . . . . .
2. Animals . . . . . . . . . . . . . . . . .
3. In Vitro Slice Technique . . . . . . . . .
4. Incubations and Extractions . . . . . . .
5. Measurement of Cyclic AMP . . . . . . . .
6. Time course . . . . . . . . . . . . . . .
7. Dose-response . . . . . . . . . . . . . .
8. Urine osmolality gs plasma osmolality
(U/P ratio) . . . . . . . . . . . . . . .
9. Protein determination . . . . . . . . .
10. Statistical analyses . . . . . . . . . .
RESULTS
1. Effect of Incubation Time . . . . . . . .
2. Effect of Varying ADH Concentration . . .
3. Comparison of Cyclic AMP Concentration
in Renal Medullary Tissue of Young and
Adult Animals . . . . . . . . . . .
a. 1 Day Old Rabbit vs Adult . . . . .
b. 1 Week Old, 2 Week Old and
Adult Dogs . . . . . . . . . . . . .
4. Comparison of Body Weight, Kidney Weight,
Protein Concentration and Uosm/Posm Ratio
from 1 Day Old, 3 Day Old, 5 Day Old and
Adult Dogs . . . . . . . . . . . . . . . .
5. Comparison of Cyclic AMP Concentration in

Response to ADH Among 1 Day Old, 3 Day Old,

5 Day Old and Adult Dogs . . . . . . . . .

DISCUSSION . . . . . . . . . . . . . . . . . . .

SUMMARY . . . . . . . . . . . . . . . . . . . .

BIBLIOGRAPHY . . . . . . . . . . . . . .

iii

Page

iv

16
16
17
18
19
20
20

20
21

22
23
24
24

24

26

26
28
40

57

Table

LIST OF

Relationship between

TABLES

Page

incubation

time and cyclic AMP concentration
in adult rat renal medulla . . . . . . . . . . . 42

Effect of various doses of ADH on
cyclic AMP concentration in renal
medullary slices from adult rats
in the absence and presence of

10 mM theophylline .

. . . . . . . . . . . . . . 43

Effect of various doses of ADH
on cyclic AMP concentration in

renal medullary slice
rabbit in the absence
of 10 mM theophylline

Comparison of body we

5 from adult
and presence
0 O O O O O O O O O O O O 44

ight, kidney

weight, and protein concentration
in medullary slices and Uosm/Posm
ratios from 1 day old, 3 day old,
5 day old and adult dogs . . . . . . . . . . . . 45

Comparison of effect of ADH on
c clic AMP concentration (picomoles/

mg wet weight) among

1 day old, 3

day old, 5 day old and adult dogs
in the absence and presence of

10 mM theOphylline

. . . . . . . . . . . . . . 46

iv

LIST OF FIGURES

Figure Page

1. Effect of ADH on cyclic AMP
concentration in medullary
slices from adult rabbits . . . . . . . . . . 48

2. Cyclic AMP concentration in
renal medullary slices in
response to various doses of
ADH from 1 day old and adult
rabbits . . . . . . . . . . . . . . . . . . . 50

 

3. Effect of varying incubation
time on cyclic AMP concentra-
tion in renal medullary tissues
from adult dog . . . . . . . . . . . . . . . 52

4. Comparison of cyclic AMP con-
centration in response to
various concentrations of ADH
in renal medullary slices from
1 week old, 2 week old and
adult dogs on the basis of wet
medullary tissue . . . . . . . . . . . . . . 54

5. Comparison of cyclic AMP con-
centration in response to ADH
in renal medullary slices from
1 week, 2 week and adult dogs
on the basis of protein concen-
tration in the medullary tissue . . . . . . . 56

INTRODUCTION

Cyclic AMP (adenosine 3',5'-monophosphate), is
recognized as a versatile regulatory agent acting to con—
trol the rate of a number of cellular processes. Sutherland
and Rall discovered cyclic AMP (1) during a continuation of
studies of the mechanism by which epinephrine and glucagon
promoted glucose release in the liver. In earlier studies,
Sutherland had demonstrated that the rate-limiting enzyme
in the conversion of glycogen to glucose was liver phosphorylase.
This enzyme existed in two forms, inactive and active, and both
epinephrine and glucagon caused the activation of phosphorylase.
They also identified an enzyme, dephosphorylase kinase, which
activated phosphorylase in the presence of ATP and Mg++, and a
phosphatase that inactivated the enzyme. Although they could
elicit effects of epinephrine and glucagon on phosphorylase
activation in whole homogenates fortified with ATP and Mg++,
supernatant fractions containing the phosphorylase system were
entirely unresponsive to the hormones. Experiments in which
particulate and supernatant fractions were recombined showed
that the hormones acted on a component of the particulate frac—
tions to increase the accumulation of a heat-stable factor-
cyclic AMP, which, in turn, accelerated the rate of conversion
of inactive to active phosphorylase. The enzyme from the par—
ticulate fraction responsible for the formation of cyclic AMP

was named adenyl cyclase (2).

Adenyl cyclase is widely distributed in nature and
has been identified in every mammalian tissue studied with
the exception of the mature mammalian erythrocyte (2). In
almost all tissues adenyl cyclase is in the particulate frac—
tion, indicating an association with cell membranes (2,3,4).
The interactions of several hormones with their target cells
have been shown to result in the activation of adenyl cyclase,
which catalyzes the transformation of adenosine triphosphate
to cyclic AMP and inorganic pyrophosphate. The increased
cyclic AMP, acting intracellularly, then carries out the work
of the hormone by affecting the activities of enzymes, per-
meability processes etc. Subsequent studies have shown that
cyclic AMP mimics the actions of many hormones (5,6).

The neurohypophyseal hormone, vasopressin (antidiuretic
hormone, ADH) and certain of its analogues enhance the perme-
ability of a number of epithelial membranes to water, sodium
and certain small molecules. Considerable effort has been ex-
pended in searching for an adequate description of these pro—
cesses and although progress has been achieved, the goal is not
in sight. Orloff and Handler (7) proposed that cyclic AMP is
the intracellular mediator of the physiological effects of
vasopressin. A number of experimental observations are now
available which support this proposal and define an integral
role of cyclic AMP in the action of vasopressin.

In the mammalian kidney, the antidiuretic hormone
prevents the excretion of excess water in a dilute urine and

effects further the conservation of water by permitting full

operation of the urinary concentrating mechanism. There is
general agreement on the overall nature of the process in-
volved in the production of urine hypertonic to body fluids.
Almost all have accepted the interpretation, originally pro-
posed by Wirz, Hargetay and Kuhn (8), that the urine becomes
concentrated as it flows through the medullary collecting
ducts by passive outward movement of water into an inter—
stitium that has been rendered hyperosmotic by the accumula-
tion of a high concentration of sodium chloride. Most workers
would accept the following account of the events involved in
the conversion of a large volume of isosmotic glomerular fil—
trate to a small volume of hyperosmotic urine: In the proxi-
mal tubule, as a consequence of the active removal of salt in
a highly water—permeable segment, the volume of fluid is mark—
edly reduced without change in its osmotic pressure. A vari-
able fraction of the glomerular filtrate enters the medulla

in the thin-walled descending limb of the loop of Henle. As
it flows into the hyperosmotic medulla, the fluid becomes pro-
gressively concentrated by: (1) loss of water to its highly
concentrated surroundings; (2) inward diffusion of urea from
the urea-rich interstitium; and (3) possibly some entry of
sodium chloride. Somewhere between the bend of the loop and
re-entry of the nephron into the cortex the permeability of
the loop to water diminishes and outward transport of sodium
and chloride dilutes the fluid that remains in the tubule
lumen. Thus, when the fluid arrives in the distal convoluted
tubule, it is hypotonic to plasma. The salt that has been

removed in this dilution process remains behind to raise the

osmolality of the interstitial fluid and blood in the medulla.
In the presence of antidiuretic hormone, the fluid flowing
through the distal nephron loses its excess water by passive
re-equilibration with its surroundings. Before the tubular
fluid re-enters the medulla in the collecting ducts, it gives
up water to reach the same hyperosmotic state as the surround-
ing medullary interstitial fluid. It is certain that the ma-
jor effect of ADH in the concentrating mechanism is exerted
through its effects on the permeability to water of the distal
nephron.

The View that vasopressin increases the permeability
of the distal nephron to water derives mainly from studies
utilizing amphibian skin and bladder. Much of the early in-
formation stems from the work of Ussing and his collaborators
(9). Their conclusions concerning the action of vasopressin
have been confirmed and extended by Bentley (10,11), Sawyer
(12) and Leaf (13) and their co-workers on the basis of simi—
lar studies on amphibian bladder. Their studies demonstrated
two characteristic effects of vasopressin, an increase in the
permeability of certain epithelial structures to water, and
an associated stimulation of sodium transport.

Tischer, et a1. (14) studied the morphological changes
of renal medulla of rats with hereditary hypothalamic diabetes
insipidus during vasopressin-induced antidiuresis. They found
that vaSOpressin-induced antidiuresis in diabetes insipidus
rats was associated with: expansion of the medullary inter-

stitium, and widening of the lateral intercellular spaces of

medullary collecting ducts. These findings were not present
in untreated diabetes insipidus rats or in normal rats of the
same strain, with or without vasopressin administration. The
results suggested that fluid reabsorption from collecting
ducts during vasopressin—induced antidiuresis occurs at least
in part via lateral intercellular channels. How this occurs
or what kind of a process is involved is not understood.
There are, however, two hypotheses regarding the cellular
mode of action of vasopressin. The first is attributable to
Ginetzinsky (15), who suggested that vasopressin stimulates
the secretion of a depolymerizing enzyme, hyaluronidase, by
the renal tubule. The second, that of Schwartz, Fong,
Rasmussen and their associates (16,17,18), involves a mechan—
ical system, not metabolic in nature, in which interaction of
vasopressin with specific tissue receptors directly initiates
structural changes within the membranes.

According to Ginetzinsky (15), antidiuretic hormone
has no direct effect on the permeability of the tubule cells,
but instead stimulates the secretion by these cells of
hyaluronidase into urine. Subsequent depolymerization of
mucopolysaccharide complexes of the intercellular spaces and
basement membrane by the enzyme is thought to result in the
characteristic increase in the permeability of the collecting
duct to water. The following evidence has been presented in
support of the theory (15): Hyaluronidase activity in urine
is constant and independent of urine flow in osmotic diuresis

in the rat at a time when antidiuretic hormone secretion is

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assumed to be maximal, but this is not the case in the animal
undergoing water diuresis. In the latter situation, when
antidiuretic hormone secretion is absent, urinary hyaluroni-
dase activity is inversely related to urine flow. This is

as would be expected if the development of antidiuresis re-
quires secretion of the depolymerizing enzymes. Furthermore,
on the basis of histochemical studies, Ginetzinsky reported
that a marked reduction in mucopolysaccharide material occurs
in the papilla of rats in antidiuresis as compared to that in
the papilla of animals following 20 to 60 minutes of water
diuresis. Additional support for the theory was supplied by
Dicker and Eggleton (19) who, in agreement with Ginetzinsky's
animal studies, reported that the rate of excretion of hyal—
uronidase in urine of human subjects undergoing water diuresis
varied inversely with urine flow.

The hyaluronidase theory has been severely criticized
by Berlyne (20). The reliability and specificity of the hyal-
uronidase assay used by Ginetzinsky have been questioned, and
when Berlyne used an assay which he considered superior he
was unable to find any relationship between rate of excretion
of the enzyme and urine flow either in water diuresis or in
osmotic diuresis. The histochemical observations were not
confirmed by Breddy gt il- (21), who suggested that the pat-
tern observed by Ginetzinsky in rat papilla may have been the
result of sampling at different depths of the medulla in di—
uretic and non-diuretic animals. Also, Thorn (22) reported

that after injection of a large amount of hyaluronidase, the

urinary response was delayed for approximately 20 minutes,
whereas that evoked by exogenous hormones was virtually
immediate. Rosenfeld at 31. (23) were unable to demonstrate
any effect of leave in testicular hyaluronidase into the
renal artery of the dog which could not be ascribed to con-
current changes in hemodynamics. Finally, neither Leaf (13)
nor Bentley (24) observed any effect of hyaluronidase on
water permeability of toad or frog urinary bladder. It now
seems reasonable to disregard the hyaluronidase thesis since
none of the data presented in its support has withstood
critical evaluation.

Fong, Rasmussen and Schwartz and their co-workers
(16,17,18) proposed that vasopressin binds to toad bladder
and kidney at a minimum of two sites, the most important of
which involves a covalent linkage between the disulfide
bridge of the octapeptide and free sulfhydryl groups on the
membrane. Their suggestion was that the hormone-induced in-
crease in permeability is initiated by a series of disulfide—
sulfhydryl interchanges which induce separation of fibrillar
elements in a protein diffusion barrier (17), or in some
other fashion mechanically opens aqueous channels through
which water may flow. They initially excluded a metabolic
basis for the effect, since they found that none of a series
of potent metabolic inhibitors eliminated the water permeabil-
ity response of the toad bladder to hormone. The thesis is
based largely on the experimental observation that tritiated

vasopressin binds to kidney and toad bladder, as evidenced by

 

 

 

Elcgczumulation of radioactivity with kidney and bladder tissue
£11161 its partial release from fixed tissue preparations by
cysteine. The latter is a sulfhydryl—containing compound
which presumably ruptures the disulfide bond between the
hormone and tissue. The extent of binding was said to be
related to the physiologic response since the degree of
accumulation of radioactivity in kidney protein in anti-
diuretic rats was greater than in rats excreting larger
volumes of urine during recovery from the effects of the
hormone. Acidification of the inner bathing solution,
which Bentley (10) first demonstrated, eliminates the per—
meability response of toad bladder to hormone, also reduces
the accumulation of radioactivity in the tissue. This is
consistent with their hypothesis, since depression of the
dissociation of sulfhydryl groups by hydrogen ion should
reduce the affinity of the tissue receptor sites for the
hormone. They also reported that certain sulfhydryl in-
hibitors, such as n-ethyl maleimide and p-chlormercuriben-
zoate, interfere with both the binding of the hormone and
its physiological effects.
Although the foregoing hypothesis is attractive,

in that it affords an explanation for the cellular action
Of vasopressin, the results of interest with respect to the
Possible nature of a linkage between vasopressin and a re—
Ceptor provide no information concerning the proposed struc-
tural alterations within the tissue which are said to be

responsible for the hormone induced permeability effects.

'mua :reported effects of n-ethyl maleimide are not neces-
sarrily pertinent. Simple incubation of the intact bladder
witli this agent alone is followed by progressive deterior-
ation of the tissue as evidenced by a decline in net sodium
transport and oxygen consumption, as well as unresponsive—
ness to other agents, such as theophylline and cyclic AMP
(7). These latter compounds although not containing disulfide
bridges, effectively mimic vasopressin in toad bladder.

In view of all of these arguments, it would seem more
reasonable to View the results of Fong, Schwartz and Rasmussen
as providing information concerning the nature of a possible
linkage between the hormone and its receptor, rather than as
evidence for the proposed mechanism for the opening of aqueous
channels.

Regardless of the details of the reaction between vaso-
pressin and tissue, there is now a considerable body of evi-
dence in support of the proposal that: (1) this reaction re-
sults in an increase in the rate of production of cyclic AMP
by pertinent epithelial cells and (2) cyclic AMP is the intra-
Icellular mediator of the permeability response to vasopressin.

The two characteristic effects of vasopressin, an in-
cxrease in the permeability of certain epithelial cells to
waster, and stimulation of sodium transport have been demon—
ggtrated in the amphibian skin and bladder (9,12,13). A simi-
lar effect on the permeability to water of isolated rabbit
(xgllecting tubule has also been reported (25). If cyclic AMP

jig-the intracellular mediator of the action of vasopressin it

10

smmlld mimic the hormone in all respects. In toad bladder
this has been shown to be the case. The similarity of ef—
fects of the two agents, the hormone and its postulated in-
tracellular mediator, on net water flow in isolated rabbit
collecting tubule has been reported by Grantham and Burg
(25). Their demonstration that cyclic AMP also augments the
water permeability of the rabbit collecting tubule and that
the biochemical degradation product 5'-AMP does not, firmly
establishes the role of the cyclic 3'.5'-AMP system in the
renal tubule.

Direct support of the cyclic AMP hypothesis was
achieved by the demonstration that the concentration of the
nucleotide is markedly increased in renal tissue and in toad
bladder following incubation with vasopressin (26,27). In
toad bladder Handler 33 a1. (27) were able to show that pur-
ified arginine vasopressin and theophylline increase the
concentration of cyclic AMP in the tissue whereas no incre-
ment in cyclic AMP concentration was effected by either in-
sulin or angiotensin, polypeptides without physiological
effects on permeability in this tissue.

The kidneys of most new-born mammals are immature
(28,29,30,3l). The capacity to concentrate the urine and
to conserve water is low due to short loops of Henle and
consequent undeveloped countercurrent system in the medulla.
Histological investigations of the kidney of the new-born
mammal mention the absence of lack of differentiation of the

loops of Henle (31,32). The kidney of the new-born rat

11

resembles that of the foetus of other mammals and man (33) .

The study of kidney functions of the new-born rat may,

therefore, throw some light on renal functions of the foetus

of other mammals and man.

Bogomolova (32) studied the cytological and cyto-

chemical change in the rat kidney from birth to old age (30

111<3nths). At birth, the rat kidney is not yet fully formed.

The tubular system is not fully formed, the loops of Henle
are weakly differentiated, the renal papilla is short and

the collecting ducts are few in number. Concentrating ca-

pacity is low. An increase in urea excretion produces a

marked increase in the concentrating capacity. A further

possibility contributing to the immature concentrating ca—

pacity is a limitation in the capacity of the cells to pump

sodium against a gradient. In experiments on rats, Yunibhand

and Held (34) were able to demonstrate a significant increase
in medullary sodium concentration from 1 to 30 days of age.
This may reflect an increase in the length of the loop of

Henle or an increased capacity for sodium reabsorption, or

b0th. It is unlikely that reduced urine concentration in

the neonate can be attributed to an insufficient amount of

ar11:idiuretic hormone. The amount of antidiuretic activity

has been estimated in glands of new—born rats (35) , puppies

(36) and new-born infants (37) . And although the amount

Present is small when compared with that found in adult

animals, it is sufficient for physiological measurement.

12

The neurohypophysis acts as a store, the quantity
of vasopressin in it represents the difference between the
rate at which it is synthesized and that at which it is
released. Increasing evidence is accumulating against the
hypothesis that this might constitute a limiting factor in
concentrating capacity at that age. The osmoregulatory
apparatus in the neonate responds to the stimulation of
dehydration and relatively large amounts of antidiuretic
hormone are released (38). In contrast to the absence of
antidiuretic hormone in the adult, the urine of fully hy—
drated new-born animals contained a substantial amount of
antidiuretic activity, presumably of neurohypophyseal ori—
gin (38).

When compared with the effect of similar doses of
antidiuretic hormone in adults, it was found that kidneys
of new-born humans were highly insensitive to antidiuretic
hormone (29). Similar results have been shown in other new-
born animals. Though these experiments illustrate the marked
insensitivity of the kidney of new—born mammals to the anti—
diuretic hormone, they fail to indicate the reason for it.
This raises the question: Does antidiuretic hormone act on
the collecting ducts of new-born mammals in the same way as
it acts in the adult?

Studies of enzymes in new-born kidneys have indicated
that many enzymes are immature (39,40). Accordingly, adenyl
cyclase may be immature in new—born kidneys. Since the kidneys

of most neonates are immature, it might be that the protein

13

receptor of the target site is not yet fully receptive. It
was of interest, therefore, to determine the activity of
adenyl cyclase and hormone responsiveness in the new-born
kidney.

The objective of this investigation originally was
to study the adenyl cyclase activity of broken cell prepara-
tions. These studies are useful for several reasons, one
of which is that the environment in the vicinity of the adenyl
cyclase can be greatly simplified and therefore better con-
trolled than in more organized systems (6). In most mammalian
tissues which have been studied, using conventional homogeni-
zation techniques, adenyl cyclase has been located in the low
speed or nuclear fraction, which contains fragments of the
cell membranes (2). Often these preparations can be washed
repeatedly, thus eliminating many metabolites and soluble
enzymes, and in some cases can be taken through additional
purification steps with the retention of hormonal sensitivity
(2). The ultimate goal here would be to obtain a preparation
containing only adenyl cyclase, but to date it has not been
possible to purify adenyl cyclase from mammalian sources be-
yond a certain point without destroying its sensitivity to
hormonal stimulation.

A major factor complicating the quantitative inter—
pretation of most adenyl cyclase measurements is contamina-
tion by other enzymes, including ATPase, pyrophosphatase and
phosphodiesterase. In this study, cyclic AMP produced from

ATP in broken cell preparations was measured by the rate of

14

conversion of labeled ATP to cyclic AMP (41). Chase and
Aurbach (42) found that the medulla was the anatomically
specific site at which ADH increase renal adenyl cyclase
activity. I was unable to obtain the specific increase

in production of cyclic AMP by ADH. Further study of the
cyclic AMP fraction by paper chromatography showed that
there were contaminations by other nucleotides in this
fraction. The validity of the method was then questioned
because of failure to separate the pure cyclic AMP from
other nucleotides. This also indicated that other enzymes
which catalyze the breakdown of ATP or cyclic AMP were not
separable from adenyl cyclase in the broken cell prepara-
tions. Thus the method was inappropriate.

The results from studies with broken cell prepara—
tions are not always directly applicable to events occurr-
ing in more highly organized systems. Studies with in-
tact tissues may be especially important from this point
of View because they can often be carried out under con-
ditions where the physiological response and the change in
cyclic AMP can be measured simultaneously. The information
obtained is, therefore, more directly relevant to physio-
logical and clinical situations. Many of the points which
can be tested with broken cell preparations can also be
studied in intact tissues. In addition, the change in
cyclic AMP and the physiological response can be compared
as functions of the dose of the hormone needed to elicit
the response and the time required for the response to be

manifested. The rate or magnitude of some cellular processes

15

may be directly related to the level of cyclic AMP. There-
fore, it was the purpose of this study to compare the change
in the concentration of cyclic AMP in the intact tissue of
new-born and adult animals in response to antidiuretic hor-
mone and to elucidate the possible mechanisms responsible

for the differences in urine concentration capacity between

the new-born and the adult.

MATERIALS AND METHODS

1. Materials:

Arginine vasopressin (synthetic) (150 U/ml) was
purchased from Nutritional Biochemicals Corporation.
Tritiated 3',5'-AMP [8-3H] (20.8 Ci/mMole) was purchased
from Schwarz/Mann. Non-radioactive 3',5'-AMP was ob—
tained from P. L. Biochemicals, Inc. Bovine serum al-
bumin (Fraction V) was purchased from General Biochemicals.
Other reagents and chemicals were obtained from standard

sources .

2. Animals:

Rats of the Sprague-Dawley Strain, New Zealand
white rabbits, purebred beagles and mongrel dogs were
used. These were housed in the departmental animal quar-
ters under controlled conditions of temperature and hu-
midity appropriate for each species. They received stan—
dard laboratory diet and water. Young animals were left
with their mothers until sacrificed. One day, 3 day and
5 day old beagles received 0.1 ml (0.5 Unit) ADH, intra-

muscularly, 2 hrs before sacrifice.

16

17

Methods

3. In Vitro Slice Technique

 

Animals were killed by a blow on the head or by
decapitation and the kidneys removed immediately, weighed
and placed in ice—cold Krebs-Ringer Tris HCl buffer solu—
,tion composed of the following: NaCl, 120 mM; KCL, 4.8 mM;
CaC12,2.6 mM; MgSo4, 1.2 mM; glucose, 2 mg/ml; and Tris—
HCl, pH 7.4, 15 mM. Slices of renal medulla were prepared
freehand and pooled in ice—cold buffer solution for each
experiment. A 40-60 mg quantity was gently blotted and
weighed into 10 ml beakers. A 1.0 ml quantity of ice-cold
buffer solution was added and the mixture gently swirled
until the slices were separated. All beakers were stored

in ice until incubations were performed.

4. Incubations and Extractions:

Incubations were carried out in a Dubnoff metabolic
shaker at 37° C under a gas phase of 100% oxygen. Arginine
vasopressin prepared in 0.25% acetic acid was added to each
sample in a volume of 10 ul. Control samples received 10 ul
of 0.25% acetic acid. Incubation time varied from 5-60 min-
utes. After incubation the slices were rapidly removed and
homogenized with 1.0 ml of cold 5% trichloroacetic acid in
a Dounce ball homogenizer. The homogenizer was rinsed with
an additional 1.0 ml of TCA and the rinse added to the orig-

inal homogenate. The homogenate was centrifuged at 800 x g

 

Alb

Q“

I.“
IVI‘

18

for 10 minutes. A 0.1 ml quantity of 1N HCl was added to
each supernatant which was then extracted 5 times with 2
volumes of ether. The extracts were lyophilized and re-

dissolved in 0.5 m1 of 50 mM sodium acetate at pH 4.0.

5. Measurement of cyclic AMP:

Cyclic AMP was determined by the method of Gilman
(43). The assay is based on competition for protein bind-
ing of the nucleotide to a cyclic AMP—dependent protein
kinase. The nucleotide protein complex is absorbed on a

cellulose ester filter.

Procedure:

The cyclic AMP binding reaction was conducted in
a total volume of 50 pl in 50 mM sodium acetate, pH 4.0

composed of the following:

H3~cyclic AMP 20 pl (1.0 pmoles/20 pl) non-
radioactive standard cyclic AMP (1-20 pmole)
10 pl or unknown sample 10 pl

kinase inhibitor 10 pl (1.24 mg/ml)*

protein kinase 10 pl (0.1 mg/ml)*

The reactions were initiated by addition of binding protein-
protein kinase and incubated for 1 hr to 2 hrs at 0° C. At
equilibrium, the mixtures were diluted to 1 ml with cold
20 mM potassium phosphate, pH 6.0, then passed through a

24 mm cellulose ester (millipore) filter (0.45 pm pore size)

19

previously rinsed with the same buffer. The filter was
then washed with 10 ml of this buffer and placed in a
counting vial with 1 ml of CellosolveR, in which the
filter readily dissolves. A scintillation mixture of
toluene (750 ml) Cellosolve (ethylene glycol monoethyl-
ether) (250 ml), PPQ_(2,5—Diphenyloxazole) (5.0 gm/l)
and dimethyl PQPQP-l,4-bis—[2-(4—methyl-5-phenyloxazolyl)]-
Benzene (100 mg/l) was utilized. Samples were then counted
on Beckman LS. 100 liquid scintillation counter.

For the assay of cyclic AMP [3H] C~AMP was utilized
at saturating concentration, and the effect of added unknown
or standard cyclic AMP solutions could thus be evaluated

from a linear decrease in the total bound [3H] cyclic AMP.

*Kinase inhibitor and protein kinase were gifts from
Dr. Lawrence Muschek. These were prepared by the

method of Miyamoto gt g£.(65) and Appleman 33,3l-(66)-

6. Time course:

Adult rats, rabbits and dogs were used for this
study. Incubation times varied from 5-60 minutes. Krebs-
Ringer Tris HCl buffer solution containing 10 mM theophylline
was used as the incubating medium. ADH concentration used
for all incubation times was 100 mU/ml for rats and rabbits
and 1 U/ml for dogs. Control samples which received 10 pl

of 0.25% acetic acid were used for each incubation time.

20

7. Dose-response:

The effect of various concentrations of ADH on
cyclic AMP concentration in renal medullary slices from
adult rats, 1 day old rabbits, adult rabbits, 1 week old,
2 week old, adult mongrel dogs, 1 day old, 3 day old and
5 day old beagles was investigated. Concentrations of
ADH ranging from 1 mU/ml to lU/ml were added to the in-
cubation medium. Experiments were done in the absence
and presence of 10 mM theophylline in all species and all
ages except 1 day old rabbits. Because of an insufficient
amount of medullary tissues in 1 day old rabbit, this study
was only carried out in the presence of theophylline. In—
cubations were carried out at 37° C for 30 minutes under

100% oxygen.

8. Urine osmolality Ki plasma osmolality (U/P ratio):

Urine and plasma samples were collected from 1 day
old, 3 day old, 5 day old, 1 week old, 2 week old and adult
dogs. Osmolalities of both urine and plasma of each age
were estimated by freezing point depression with an Advanced

Osmometer.

9. Protein determination:

Protein concentration in the slice was determined by
the method of Lowry et 31. (44) after digestion of samples

of slices for 1 hour with l N NaOH.

21

10. Statistical analyses:

Analysis of variance using a completely randomized
design was used (45). Treatment means were compared to
control using the least significant difference test. (47).
Student's t test for paired data was used where appropriate.

The QLQE level of probability was used as the cri-

terion of significance in all statistical tests.

RESULTS

Arginine-vasopressin (ADH) increased the concen—
tration of cyclic AMP in rat, rabbit and dog renal medul-
lary slices in a dose-dependent manner in the presence of
10 mM theophylline. In the absence of theOphylline cyclic
AMP was slightly increased by ADH but this was not dose
related. Cyclic AMP concentrations increased with in-
creasing incubation time up to 30 minutes in both control
and ADH-stimulated renal medullary tissue in the presence
of 10 mM theophylline. A difference in cyclic AMP concen-
tration in tissue between 1 day old and adult rabbits was
not detected while the results obtained from 1 week old,

2 week old and adult dogs showed a definite pattern of de-

velopment in response to ADH.

1. Effect of Incubation Time

 

The concentration of cyclic AMP in renal medullary
slices from adult rats increased with increased incubation
time up to 30 minutes (Table 1). This increase was seen,
however, only when theophylline (10 mM) was added to the
incubation mixture. Optimal incubation time of 30 minutes
was observed in both control and ADH—containing beakers.
Cyclic AMP concentration in slices in the presence of ADH

at each incubation time was greater than the respective

22

23

control. At 30 min cyclic AMP concentration increased
from 11.7 i 1.5 to 24.4 i 2.7 (p moles/mg wet weight :
S.E).

The time course for slices from adult dogs (Fig.
3) and adult rabbits (Fig. l) were also examined. These
tissues displayed the same pattern as was seen with tissue
from adult rats. The basal and ADH-stimulated cyclic AMP
concentration was lower for both species than that observed
in rats. For instance, basal cyclic AMP concentration at
optimal incubation time of 30 minutes was 11.7 i 1.5 p
moles/mg wet weight for rats, 2.0 i 0.4 for rabbits and 2.0
in a single experiment with dog tissue. With ADH stimula—
tion, values increased to 24.4 i 2.7, 4.3 i 0.9 and 3.2,

respectively.

2. Effect of Varying_ADH Concentration

 

ADH increased the concentration of cyclic AMP in
renal medullary slices in a dose-dependent manner. In the
absence of theophylline cyclic AMP concentration was slightly
increased by ADH over a concentration range of l mU/ml to
l U/ml. When theophylline (10 mM) was included in the in—
cubations higher concentrations of cyclic AMP were detected
from all species and all age groups studied (Table 2, 3, 5,
Fig. 4, 5). Maximal concentration of cyclic AMP in rat and
rabbit tissue was reached in 30 minutes with 100 mU/ml of
ADH. Cyclic AMP concentration at l U/ml of ADH (22.7 i 3.1

Picomoles/mg wet weight for rats, 2.91 i 0.40 Picomoles/mg

24

wet weight for rabbits) was lower than that at 100 mU/ml
(26.1 i 2.8 Picomoles/mg wet weight for rats, 4.02 i 0.40
Picomoles/mg wet weight for rabbits). In adult dog tissue
maximal concentration was reached at l U/ml of ADH (3.93 i

0.60).

3. Comparison of Cyclic AMP Concentration in Renal

 

Medullary Tissue of Young and Adult Animals

 

a. 1 Day Old Rabbit vs Adult

Because the quantity of medullary tissue from 1 day
old rabbits was limited, 10 mM theophylline was included
in all incubations. All rabbits from a single litter
were employed to obtain one series of incubation beakers
for the 1 day animals. These experiments were conducted
to determine the response to ADH under optimal conditions,
that is, under maximal inhibition of phosphodiesterase.
The results from this study are shown in Figure 2. Neither
the basal concentration of cyclic AMP in the slices nor
the concentration of cyclic AMP after maximal ADH-stimula-
tion showed any age-related differences. In adult rabbit
tissue the optimal incubation time was found to be 30 min—

utes and the same time was used in the newborn animals.
b. 1 Week old, 2 Week old and Adult Dogs

The concentration of cyclic AMP/mg wet weight in renal
medullary slices from dogs at all ages studied was enhanced

by ADH in the presence of theophylline but the effect was

 

,
mm
in“! ‘-

a;

va-

‘1'

\I-

If)

'(7

25

most pronounced in the tissue from the adult (Fig. 4). In
the absence of theophylline ADH produced a small increase

in the concentration of cyclic AMP in the adult (increased
from 1.6 i 0.3 to 2.3 i 0.3 Picomoles per mg wet weight at
maximal response). There appeared to be some small effect

in the newborn as well but this was not statistically signif-
icant. When theophylline was added to the beakers there was
a statistically significant increase in cyclic AMP in tissue
from all age animals in the absence of any antidiuretic hor-
mone (Fig. 4). The cyclic AMP concentration increased from
1.3 i 0.1 to 1.8 i 0.2 in 1 week old, 1.4 i 0.1 to 2.3 t 0.1
in 2 week old, and 1.6 i 0.3 to 2.3 i 0.4 in adult. In the
presence of theophylline there was a significant increase in
cyclic AMP concentration in response to ADH in tissue from
all ages though the response in the young animals (increased
from 1.8 t 0.2 to 2.3 t 0.2 at 1 week; 2.3 i 0.1 to 2.9 i 0.3
at 2 weeks) was significantly less than that observed in the
adult (increase from 2.3 i 0.4 to 3.9 t 0.6). Furthermore,
the response in the 1 week animal (1.3 i 0.2 at l U/ml of
ADH) was less than that seen in the 2 week animal (1.9 i 0.3).
When the above data were factored by protein concentration
rather than wet weight the magnitude of these differences was
reduced (Fig. 5) (29 i 2.5 to 37 i 0.3, 30.5 t 2.0 to 38.5 i
4.0 and 25.5 i 4.5 to 42 i 6.5 respectively). Whereas the
difference between the control beakers and the theophylline
beakers was still apparent. Age related differences in re-

sponse to antidiuretic hormone were no longer apparent.

26

4. Comparison of Body Weight, Kidney Weight, Protein

 

Concentration and Uosm/Posm Ratio from 1 Day Old,

 

3 Day Old, 5 Day Old and Adult Dogs

 

As shown in Table 4 body weight, kidney weight,
protein concentration and U/P ratios were low in the
immediate newborn period. The osmolar U/P ratio was less
in the 1 day old animals (1.8 i 0.1) than in the 3 day
(2.8 i 0.6) and 5 day (2.9 i 0.5) animals. Protein con-
centration in the medullary tissue was also lower in the
newborn animals and was lowest at 1 day. These young
animals were all pretreated with ADH 2 hours before sac-
rifice yet the U/P ratios for osmolality were still rel—

atively low compared to the adult values (6.1 i 0.6).

5. Comparison of Cyclic AMP Concentration in Response

 

to ADH Among 1 Day Old, 3 Day Old, 5 Day Old and

 

Adult Dogs

 

When the young animals were pretreated with ADH
2 hours prior to sacrifice cyclic AMP concentration in
response to various concentrations of ADH was still below
the adult values (Table 5). Though these tissues were
capable of responding to theophylline and to antidiuretic
hormone the values were still significantly less than that
observed in the 1 week animals illustrated in Fig. 4 and 5.
For instance, at maximal dose (1 U/ml) of ADH and in the

presence of theophylline values obtained for 1 day old, 3

27

day old, 5 day old and 1 week old animals are 2.55 i 0.17,
2.27 i 0.31, 2.59 t 0.34 and 2.30 t 0.20 p moles/mg wet

weight, respectively.

DISCUSSION

Incubation of renal medullary slices with anti-
diuretic hormone or with theophylline produced an increase
in the concentration of cyclic AMP in the tissue. These
results were obtained in tissue from three species: rats
(Table 1, 2), rabbits (Figure 1, Table 3) and dogs (Figure
3, 4). A similar effect of ADH has been demonstrated for
the toad bladder (7, 27), dog kidney (26) and rabbit col-
lecting tubules (25). The present study is consistent with
the hypothesis of Orloff and Handler that the physiological
actions of ADH on responsive epithelial structures are me-
diated by cyclic AMP (7).

The major problem at this time is how cyclic AMP
elicits the permeability changes characteristic of the re-
sponse to ADH. The mechanism is so far still unknown.
Cyclic AMP is known to influence a variety of enzymes, in-
cluding phosphorylase, in other tissues (1). Phosphorylase
activity in toad bladder and kidney is increased by ADH and
cyclic AMP (46,47). The known metabolic effects of ADH in-
clude an increase in oxygen consumption and glycogen break-
down (48). These changes are not evident when the bladder
is incubated in a solution free of sodium, although the hor-

mone is still capable of eliciting the characteristic effect

28

29

on water movement under these circumstances. It is apparent
that stimulation of oxygen consumption and glycogenolysis
reflects a metabolic requirement for sodium transport (48).
The effects of cyclic AMP on oxygen consumption and glyco—
genolysis resemble those of ADH and are similarly dependent
on the presence of sodium ion in the bathing medium (48).
However, an increase in phosphorylase activity is observed
in toad bladder incubated in sodium free solution, a situ—
ation in which ADH has no observed effect on glycogenolysis.
Therefore, phosphorylase activation is not responsible for
glycogenolysis in this tissue (48). Although its activation
parallels the water permeability response of toad bladder to
hormone its function in permeability is also unknown.

Handler gt gt, (49) observed that the physiological
effects of ADH and cyclic AMP on water and sodium permeability
of toad bladder are inhibited by certain metabolic inhibitors
either in the presence or absence of sodium in the medium.
Dinitrophenol, nitrogen,azide, and iodoacetic acid all inter-
fere with the characteristic response to hormone. Apparently
interruption of energy production from either glycolysis or
oxidative metabolism reduces the capacity of the tissue to
alter its permeability. Examination of the reaction of toad
bladder to metabolic inhibitors has yielded conflicting re—
sults. Rasmussen gt gt. (1?) reported no effect of metabolic
inhibitors on the hydro-osmotic response to ADH and inferred
from this that the hormone-induced increase in permeability

does not require energy. Stimulation of glycogenolysis and

30

oxygen consumption may reflect increased sodium transport
and not represent an integral response to the hormone. It
should be apparent that the mechanism of action of ADH is
not completely understood. It is likely that the presence
of sodium in the media exerts a critical role in elicitating
metabolic changes. ADH has two characteristic effects, an
increase in permeability of certain epithelial structures

to water and associated stimulation of the sodium pump.
Possibly different chemical and physical processes are in—
volved in the regulation of these effects, even though each
is influenced by cyclic AMP. Petersen and Edelman (50) pro-
posed that ADH stimulates the production of cyclic AMP at

2 separate sites within the cell: one calcium-sensitive,
related to the regulation of water movement, the other in—
sensitive to calcium and related to sodium transport. They
observed that an increase in the concentration of calcium

in the bathing medium reduced the hydro-osmotic effect of
ADH without altering its capacity to stimulate sodium trans-
port. This result was later confirmed by Bourguet gt gt.
(51). These reports support the contention that two separate
receptors of differing affinity determine the physiological
responses to the hormone. Each receptor is involved in the
generation of cyclic AMP at an independent site. Orloff

gt gt. (52) favored the proposal that two independent sites
of cyclic AMP generation are present in the basal membrane.
(It is essential that no exchange of cyclic AMP occurs between

the two sites, for without this assumption it would be difficult

31

to account for the calcium studies.) The View of Orloff

et a1. (52) was that cyclic AMP in the site related to water
movement results in the formation of specific metabolic pro-
ducts which differ from those formed in the site controlling
sodium transport. The products may then diffuse to their
respective effectors in the apical membrane or in some other
fashion induce the characteristic alterations in permeability
to water and sodium.

An alternative possibility suggested by Orloff and
Handler (52) was that the effect of ADH on sodium transport
might be mediated by a different cyclic nucleotide, the pro-
duction of which is unaffected by calcium. Cyclic AMP could
mimic its effect on sodium transport but it could not mimic
the effect of cyclic AMP on water permeability. Although it
seemed unlikely for a time, the probability that this hypoth-
esis is correct was greatly increased by the demonstration
that cyclic GMP shares the ability of cyclic AMP to stimulate
sodium transport but has no effect on water permeability (53).
It remains to be seen whether ADH can actually stimulate the
formation of cyclic GMP in the toad bladder.

Cyclic AMP concentration in renal medullary slices
was shown to increase with time up to 30 minutes, after which
the concentration of nucleotide declined (Table 1, Figure l,
3). Studies of adenyl cyclase activity with broken cell prep-
arations or cyclic AMP concentration from other tissue in re—
sponse to hormones such as histamine and norepinephrine often

show that the incubation time required to reach maximal response

32

was shorter than that required in this study. The explana-
tion for this could be that each tissue has a specific
affinity for its stimulating hormone. It might take longer
for ADH to bind to the receptor in order to change the con-
figuration of the receptor or to elicit the response. Among
the studies of cyclic AMP concentrations in the toad bladder
the time taken for measurement of cyclic AMP concentration
after addition of hormone has ranged from 20—30 minutes (7,
27,54). The decline in concentration of cyclic AMP after

30 minutes can be explained by exhaustion of substrate or by
lability of the enzyme. Inactivation of ADH in isolated
tissues has been studied by several investigators. Smith and
Sachs (55) demonstrated inactivation of ADH in rat kidney
slices. Since the enzymes were not released into the medium
from the slices, it was concluded that the kidney slices
transformed ADH into inactive molecules. Intracellular mech-
anisms involve reduction of the disulfide bond and enzymatic
attack on some groups on the ADH molecule (56).

Cyclic AMP concentration in renal medullary tissue
in response to ADH displayed a dose-dependent pattern (Table
2, 3, Figure 2, 4). Cyclic AMP concentrations from rat
tissue were much higher than that observed from rabbits and
dogs. This may reflect a true species difference. Cyclic
AMP concentrations in this study are comparable to those re—
ported by Beck gt gt. (57) and Senft gt gt. (58). Slightly
lower values found in their work could be due to shorter in-

cubation times (15 minutes). The maximal effect observed in

33

rat and rabbit tissue was seen with 100 mU/ml of ADH. When
the concentration of hormone was increased to l unit/m1 the
response declined. It seems unlikely that pH changes in
the media could account for this difference since control
samples also received the same volume of acid used for ADH
preparation. In many enzyme studies it is not uncommon to
find that while the Michaelis law is obeyed at low substrate
concentrations the velocity falls off at high concentrations.
In order to explain the inhibitory effect of high
concentrations of hormone it is necessary to introduce the
receptor concept and consider how receptors and hormones are
related to the enzyme adenyl cyclase. The hypothetical model
for adenyl cyclase consists of at least two types of subunits,
a regulatory subunit, facing the extracellular side, and a
catalytic subunit with its active center directed toward the
interior of the cell. The receptor is here regarded as part
of the regulatory subunit. Interaction with the hormone leads
to a conformational change which is extended through the reg—
ulatory subunit to the catalytic subunit, thereby altering
activity of the latter (6). In the effective receptor-hormone
complex one hormone molecule is combined entirely with a re-
ceptor. At high hormone concentrations, where the hormone
molecules tend to crowd onto the receptor, the chance of for—
mation of ineffective complexes with 2 or more hormone mole-
cules combined with a specific receptor increases. Therefore,
high concentrations of the hormone lead to inhibition of

enzyme activity. Although hormones might not serve as enzyme

34

substrates it is logical to suggest that an excess of hor-
mone has inhibited the reaction in a manner analogous to
substrate inhibition.

The ability of theophylline to potentiate the ef—
fects of ADH on cyclic AMP is demonstrated in this study
(Table 2, 3, Figure 2, 4). Theophylline interferes with
the action of phosphodiesterase, the enzyme which catalyzes
the degradation of cyclic AMP to its physiologically in-
active product, 5'-monophosphate (59). In the absence of
theophylline cyclic AMP concentrations in the tissue were
significantly lower than in its presence. Both ADH and
theophylline increased the permeability of tissue to water
when added individually to the isolated perfused rabbit
collecting tubule (46,25), but the possibility that the two
agents might act synergistically in the system has not been
tested. It could perhaps be assumed that this would occur
if ADH stimulated cyclic AMP formation and theophylline pre—
vented its breakdown. Nevertheless, the finding has been
viewed as not necessarily supportive of the hypothesis that
cyclic AMP mediates the action of theophylline, since in the
mammal organism theophylline is a potent diuretic. It is
conceivable, however, that theophylline exerts its diuretic
effect on the proximal tubule of the mammalian nephron by
interfering with sodium reabsorption to such a degree that
any simultaneous effect on water reabsorption elsewhere in
the nephron is masked.

Renal medullary tissue from rabbits and dogs did

not show similar developmental patterns in terms of cyclic

35

AMP concentration (Figure 2, 4). In rabbit tissues cyclic
AMP concentration was as high in 1 day old tissue as in '

that of adult. In dogs cyclic AMP concentrations were low

at one week and increased to maximal values in adults. It

is not known if rabbit kidneys are relatively more mature
shortly after birth than are dog kidneys. This finding does
not indicate that one day rabbit necessarily has a urinary
concentrating capacity equivalent to the adult. Concentrat—
ing performance in young animals is limited by several other
factors: metabolically low rate of excretion of urea, short-
ness of the loops of Henle and limitation in the capacity of
the cells to pump sodium against a concentration gradient.

It is possible that the osmotic gradient between the medulla
and the urine is low compared to the adult. Therefore, in
this species even if ADH is capable of promoting water move-
ment through the cells of the collecting ducts the final urine
concentration could be low. In the case of dogs cyclic AMP
concentration was lower in the young animal tissue than in
that of adults. Hommes et al. recently studied the develop-
ment of adenyl cyclase in rat liver, kidney, brain and skeletal
muscle. The adenyl cyclase activity in kidney cortex was found
to increase gradually to 30 days of age (60). The same de-
velopmental pattern may occur in dog kidney. The low activity
of adenyl cyclase would well be related to the low tissue con—
centration of cyclic AMP. Since the action of ADH is mediated
Inf<3yclic AMP it might be assumed that the low concentrating

CaPaCity in young dogs is partly due to low cyclic AMP

36

concentration in the tissues. In this study cyclic AMP
concentration paralleled the Uosm/Posm between young and
adult dogs (Table 4, 5). After injection of ADH in one

day old, 3 day old and 5 day old dogs cyclic AMP concen—
tration and urinary osmolar concentration still remained
significantly lower than adult values (Table 4, 5). This
reflects the insensitivity of the young tissue to ADH
administration. There are several possible explanations

for this insensitivity. First, based on the developmental
pattern of a number of kidney enzymes (39,40,60), adenyl
cyclase activity during the period of morphological change
of the kidney may be low. Thus, adenyl cyclase could be
less receptive to hormone stimulation. Secondly, the en-
vironment at the site of cyclic AMP generation, with respect
to enzyme, substrate and other constituents may be very
different between young and adult animals. If the proposal
that cyclic AMP-induced permeability changes occur through
metabolic products is valid (52), the difference in environ—
ment may determine a difference in metabolic response to
cyclic AMP. Thirdly, there is evidence indicating that in
the kidneys of mammals ADH must be bound to tissue before it
exerts its antidiuretic action (61). Only tissues which bind
ADH inactivate it (62). Dicker gt gt. (62) showed that in-
activation of ADH by the particle-free supernatant of kidney
homogenates resulted from an enzyme, possibly acting on the
disulfide link of the hormone reducing it to sulfhydryl.

Since the enzyme would be activated by compounds like cysteine

37

or glutathione containing an sulfhydryl group, they con-
cluded that the enzyme is an sulfhydryl enzyme. Early
studies in newborn dogs demonstrated no limitation in the
availability of antidiuretic hormone. Studies in the urine
of the newborn animal suggested that these animals contain
a substantial amount of antidiuretic activity (38). Martinek
and associates (63) suggested that resistance to antidiuretic
hormone is demonstrable in early infancy. Possibly in new-
born, the affinity of ADH binding sites is reduced. The
antidiuretic activity found in newborn urine suggests that
in the newborn none or little of the ADH that reaches the
kidneys has been inactivated. Following prolonged and re-
peated exposure to ADH newborn kidneys may eventually become
resistant to the hormone. As kidney function increases bind—
ing sites may be subjected to new conformational changes and
become more receptive and sensitive to the hormone. The hor—
mone could be bound, elicit its effect and then be inactivated
by the tissue enzyme. This does not exclude the possibility
that the mature kidney cannot develop resistance. Because of
rapid inactivation of the hormone after binding to the re-
ceptor exposure time of hormone to the receptor could be shorter.
Therefore, it may have less opportunity to induce resistance.
Another explanation for the finding that cyclic AMP
concentration in medullary slices in young dogs was lower than
adult could be related to medullary tissue water content.
Horster gt gt. (64) demonstrated that medullary tissue water

content decreases with age in very young dogs. This suggested

38

that lower cyclic AMP concentration in medullary slices from
young animals could be attributed to the higher water content.
When cyclic AMP concentration was factored by tissue protein
the difference between young and adult was abolished (Fig. 5).
This could be interpreted to mean that production of cyclic
AMP in tissue from newborn is the same as the adult. Clarif-
ication of this would require kinetic analysis of adenyl
cyclase activity. However, expressing the data in terms of
concentration (i.e., per mg wet weight) more closely repre—
sents conditions in the functioning kidney. When expressed

in this manner, cyclic AMP concentration (and presumably
physiological action) was less in tissue from newborn. Cyclic
AMP after formation would be diluted by medullary tissue water.
The possibility that young animals are unable to respond to
ADH to the same degree as the adult may be due to this diluting
effect.

These observations support a role for cyclic AMP in
the action of ADH in young and adult animals. They do not
exclude, however, the importance of the length of the loops
of Henle in the immature concentrating mechanism. It has
been shown that a significant portion of the limitation in
concentrating capacity in infants results from the low rate
of excretion of urea. This is due to the strongly anabolic
state that prevails during that period of life. When the
infant is fed with urea on a high protein diet a marked in-
crease in concentrating capacity is observed owing entirely

to the additional urea. Permeability characteristics of

39

various segments of the nephron and rate of excretion of
electrolytes are all important in the concentrating mech—
anism. Too little of the renal physiology of the neonate
is known to be able to draw conclusions as to which factors
are more responsible for the poor ability of the kidney of

new-born animals to concentrate their urine.

S UMMA RY

Cyclic AMP concentration was measured in renal
medullary tissue from rats, rabbits and dogs. Increased
concentration of cyclic AMP in response to ADH supports
the hypothesis that the actions of ADH on epithelial struc-
tures are mediated by cyclic AMP.

The pattern of development of the concentrating
ability was determined in 1 day old, and adult rabbits and
dogs from age 1 week to adult by measuring the cyclic AMP
concentration in renal medullary slices in response to anti-
diuretic hormone. Cyclic AMP concentration increased with
age in dogs. Body weight, kidney weight, protein concentra-
tion and Uosm/Posm ratio from dogs were low in the new-born
period. It is suggested that lower cyclic AMP concentration
may play some role in the low concentrating capacity in young
animals. Data obtained from rabbits did not show a similar
developmental pattern, suggesting that other limiting factors
in the concentrating mechanism may play major roles in this
species.

Treatment of 1 day old, 3 day old and 5 day old dogs
with ADH did not increase cyclic AMP concentration nor urine
osmolar concentration demonstrating the insensitivity of
young animals to ADH. This may result from a less receptive

adenyl cyclase system, or development of resistance to ADH,

40

41

or difference in the environment at the cyclic AMP genera—
tion site.

Cyclic AMP concentration in renal medullary slices
from adult rats, rabbits and dogs increased with incubation
time up to 30 minutes. This increase was seen only when
10 mM theophylline was added to the incubation mixture. ADH
increased the concentration of cyclic AMP in renal medullary
slices in the presence of 10 mM theophylline. In the ab-
sence of theophylline cyclic AMP concentration was slightly
increased but was not dose-related. Values obtained here
were also significantly lower than those when theophylline
was present.

This study demonstrated the possible role of cyclic
AMP in the development of urinary concentrating capacity in
new—born animals. However, due to the species difference
observed here, cyclic AMP may only play one of several roles
in limiting the concentration performance in new-born animals.
The importance of other factors such as short loops of Henle,
low rate of urea excretion, permeability characteristic of
various segments of the nephron, rate of excretion of electro-
lytes and limitation of capacity for sodium pump should not

be overlooked.

42

Table 1. Relationship between incubation time and cyclic

AMP concentration in adult rat renal medulla.a

 

Cyclic AMP Concentration
(picomoles/mg wet wt.)

 

Incubation Time (min) Control ADHb
5 6.6:0.2‘ ll.3:0.7
10 7.3:0.4 13.7il.0
20 10.5i0.9 17.3i0.7
30 ll.7il.5 24.4:2.7
60 ll.6i0.6 l7.7il.1

 

aEach value represents the mean cyclic AMP concentration i

S.E. obtained in 3 experiments.

Renal medullary slices

from 6 adult rats were pooled for each experiment. In—
cubations were performed in the presence of 10 mM

theophylline.

bThe concentration of ADH used for each incubation time

was 100 mU/ml.

43

 

 

Table 2. Effect of various doses of ADH on cyclic AMP
concentration in renal medullary slices from
adult rats in the absence and presence of
10 mM theophylline.a

Cyclic AMP Concentration
(picomoles/mg wet wt.)
ADH Dose (mU/ml) Without Theophylline With Theophylline

Control 4.9il.3 9.7il.6

l 6.5:l.5 l4.8il.4

10 5.8:0.8 l7.2:l.3

100 6.811.2 26.1:2.8

1000 6.6:0.6 22.7:3.l

 

aEach value represents mean:tS.E. of 3 experiments. In
each experiment the medullary slices from 8 adult rats
were used and randomly distributed among the incuba—
tion beakers.

 

Table 3.

44

10 mM theophylline.a

Effect of various doses of ADH on cyclic AMP
concentration in renal medullary slices from

adult rabbit in the absence and presence of

 

ADH Dose (mU/ml)

Cyclic AMP Concentration
(picomoles/mg wet wt.)

Without Theophylline

With Theophylline

 

Control

1
10
100

1000

0.98:0.11
1.11:0.10
1.15:0.12
1.62:0.12

1.47:0.18

2.16:0.24
2.32:0.23
2.45:0.20
4.02:0.40

2.91:0.40

 

aEach value represents mean : S.E. obtained from duplicate

determinations in tissue from 4 adult rabbits.

45

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