PERWHERM. VASCULAR MANIFESTATEOKS

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mm THE CARME FORELIMB

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GARY FRANK MERmLL

1973

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ABSTRACT
PERIPHERAL VASCULAR MANIFESTATIONS INDUCED BY INTRA-ARTERIAL

INFUSION 0F SEROTONIN (5-HYDROXYTRYPTAMINE)
INTO THE CANINE FORELIMB

By

Gary Frank Merrill

The collateral-free, innervated canine forelimb perfused either
naturally or at constant inflow (pump perfusion) was employed to examine
the effects of local, intra-arterial infusions of low (l5 pg base/min)
and high (lSO pg base/min) doses of serotonin on forelimb weight, vascular
pressures, blood flow. and segmental vascular resistances in both cutan-
eous and skeletal muscle vasculature. Net transvascular fluid fluxes were
inferred from changes in segmental vascular resistances and forelimb
weight. In naturally perfused forelimbs, neither dose of serotonin pro-
duced a significant alteration in forelimb weight with respect to control.
In forelimbs perfused at constant inflow, both the high dose and the low
dose produced significant weight increases of 8 gm and 14 gm respectively
during the infusion period (0-15 min. for the high dose and 0—30 min. for
the low dose). These weight gains were associated with increases in total
forelimb resistance, manifested almost exclusively as an increase in skin
large vessel (large artery and large vein) resistances, There was some
elevation in resistance in the skin small vessel segment during infusion

of the high dose at natural flow. Small vein pressure, which represents

Gary Frank Merrill

the minimum for capillary hydrostatic pressure. was significantly in-
creased in both skin and skeletal muscle during the infusion period with
the greatest increase found in the forelimbs perfused at constant inflow.
This finding suggests that at constant inflow extravascular fluid volume
increased subsequent to a rise in microvascular pressure. Lymph flow
rate and lymph protein concentration were measured to provide an index
of alterations in microvascular fluid filtration. Upon examination we
found that the low dose of serotonin regularly increased the rate of
lymph flow without significantly altering the protein concentration of
the lymph. Small vein pressure was substantially elevated throughout
the entire infusion period and returned toward control values after sero-
tonin administration was terminated.

Although serotonin produces marked hemodynamic alterations, the
present data suggest that in the canine forelimb, neither locally infused
high doses nor low doses of serotonin generate edema formation. Hence,
the local effects of serotonin in the canine forelimb contrast markedly
from those of histamine and bradykinin which can cause edema under

similar conditions.

PERIPHERAL VASCULAR MANIFESTATIONS INDUCED BY INTRA-ARTERIAL
INFUSION OF SEROTONIN (5-HYDROXYTRYPTAMINE)
INTO THE CANINE FORELIMB

By

Gary Frank Merrill

A THESIS

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

MASTER OF SCIENCE

Department of Physiology

1973

'2! ACKNOWLEDGMENTS

I would like to express my appreciation and gratitude to those who
have been instrumental in my being able to complete this thesis. To my
wonderful wife Marlene and three sons, Rory, Justin, and Travis I wish
to express deepest appreciation. Marlene's unwavering support and en-
couragement not only have made my efforts possible but rewarding as well.
I wish to thank her for the lack of selfishness she possesses as an
individual.

A special thanks I would like to offer to those members of the
Graduate Acceptance Committee who have made possible the continuance of
my education.

To my major professor. Dr. George J. Grega, I wish to extend my
appreciation. His patience concerning my inadequacies has many times
bridged the gap that exists between discouragement and desire.

I am indebted to Dr. Robert L. Kline for his continual willingness
to share with me gems of thought which are essential to a student's
progress.

To Edward Gersabeck I express gratitude for the assistance he has
offered at both the technical and general levels. The gathering of lymph
data reported in this study would have been greatly hampered without
his aid.

ii

TABLE OF CONTENTS

INTRODUCTION...........0....000000000
LITERATURESURVEY.......................

Biochemistry. . . . . . .,. . . . . . . . . . . . . .

Premeability: Serotonin and Other Mediators of Perme-
ability.................p...

General Systemic Effects. . . . . . . . . . . . . . .

Pulmonary Vasculature . . .

Renal Vasculature . . . . .

Coronary Vasculature. . . .

Splanchnic Vasculature. . .

Cerebral Vasculature. . . . . . . . .

Dog Forelimb and Hindlimb Vasculature

METHODS O O O O O O O O O O O O O O 0 O O O O O O Q 0 O O O 0 O

Hemodynamic Measurements. . . . . . . . . . . . . . . . .
Lymph StUdies O O I O O O O O O O O O O O O O O O O O O 0

RESULTS 0 O O O O C O O O O O O C O O O O 0 O O O . O O O O O O

Forelimb Weight (A Ht. 1, Tables 1 and 2 .
Mean Arterial Pressure (Pa),T Tables l and
Perfusion Pressure (Pba), Tables 1 and 2..
Forelimb Vascular Pressures, Tables 1 and 2 .
Forelimb Blood Flows, Tables 1 and 2. . . .
Skin Segmental Vascular Resistances, Tables 3 and
Skeletal Muscle Segmental Vascular Resistances, Tables 3

an 0 O O 0 O O O O O O O O O O O O O O O O O 0
Total Vascular Resistance, Tables 3 and 4 . . . . . . . .
Lymph Protein and Flow Rate, Table 5. . . . . . . . . . .

N.
.

h.....

DISCUSSION 0 O O Q 0 O O O 0 O O O O O O O O O O O O O O O O O
SUMMRY O O O O O O O O O O O O O O O O O O O O O O O O O O O O
BIBLIOGRAPHY O O O O O O O O O O O O O O O O O O O O O O O O 0

iii

Page

TABLE

1.

LIST OF TABLES

Effects of Serotonin Base Infused Intra-arterially Into
Collateral-free, Innervated, Naturally-perfused Canine
Fora] imb. O O O O O O O O O O O I O O O O Q 0 O O O O 0

Effects of Serotonin Base Infused Intra-arterially Into
Collateral-free, Innervated, Constantly-perfused Canine
Fore] imb. 0 I O O O I O O O O O O O O O O O O O O C C Q

Effects of Serotonin Base Infused Intra-arterially Into
Collateral-free, Innervated, Naturally-perfused Canine-
Fore] 1mb. 0 O O C O O O D O O O O O O O O Q 0 O O O O 9

Effects of Serotonin Base Infused Intra-arterially Into
Collateral-free, Innervated, Constantly-perfused Canine
Fore-l imb. O O O O O O O O O O O O O O O O O O O O O O 0

Effects of Low-dose (15 ug/min) Serotonin Base Infused

Intra-arterially Into the Collateral-free, Innervated,
Naturally-perfused Canine Forelimb. . . . . . . . . . .

iv

Page

32

33

34

35

36

LIST OF FIGURES

FIGURE Page

l. Forelimb preparation showing the location and placement of
catheters for the measurement of vascular pressures and
f10ws......0.................... 23

INTRODUCTION

In preparing the foreword to the book Serotonin, authored by
S. Garattini and L. Valzelli (69), I. H. Page, made the following declara-
tion, "serotonin in walnuts and bananas and in jelly-fish and in car-
cinoids and in brain leaves me with a sense of bewilderment and fear that
some will ask me what serotonin does." Perhaps few statements can better
characterize an agent whose diversity of actions are as broad and numerous
as the biological tissues in which it has been identified and upon which
it acts.

Not yet have three decades expired since Erspamer made the first
serotonin-containing extracts which led to its subsequent purification,
identification and synthesis, yet so universally explosive was the genera-
tion of an interest in serotonin's biological implications that to date
a wealth of knowledge is to be found in the almost innumerable articles
that deal specifically with this agent. Erspamer himself, in a recent
review chronicles some five thousand papers dealing with serotonin.

Hence, by taking part in the examination of an agent whose actions are
associated with the acute inflammation which accompanies the sting of the
hornet, as well as with the rampant hyperplasia associated with enterologi-
cal carcinoid, I feel compelled to make the admission that my understanding
of serotonin's activities is microscopic in volume and much in need of

nourishment.

The literature contains conflicting and inadequate information deal-
ing with serotonin's possible role(s) and mechanism(s) of edemogenic
action that have been shown to be associated with the inflammatory re-
sponse. Other mediators of vascular permeability such as histamine and
bradykinin have been shown to be directly responsible for edema formation
in various species, including man and dog. The edemogenic effect of
serotonin. although well-established in the rat, is questionable in
dog and man. Our current investigation was made in an attempt to further
clarify reports in the literature dealing with these findings. This
thesis represents the results of that undertaking. Data were gathered
from two well-defined vascular beds, cutaneous and skeletal muscle.
Justification for an examination of this nature may be granted if one
accepts evidence in the literature suggesting that vasoactive agents
have a physiological and a pathological role in the maintenance of such
hemodynamic parameters as resistance, pressures, transvascular fluid

fluxes and blood volume distribution.

LITERATURE SURVEY

Biochemistry

Serotonin (5-hydroxytryptamine, 5-HT), first known as enteramine
(indicative of the tissue in which this vasoactive agent first became
-SUSpect for research) was originally extracted by Erspamer and Asero
(1952) from the posterior salivary gland of the Octopus vulgaris and
from the skin of Discoglossus pictus (3). Since that time, the wide-
spread distribution of this substance in biological tissues both of the
plant and animal kingdoms has been extensively investigated (3,67,72,7,
52). Although histochemists and cytochemists have been at variance for
some time as to whether the active substance of the mammalian entero-
chromaffin cells was indeed serotonin or possibly a resorcinol derivative
or a harmaline-like compound, there seems now to be general agreement
that the agent is in fact serotonin.

The chemical substrate required for the endogenous synthesis of
serotonin is the amino acid tryptophan. Exogenous supplies of tryptophan
are carried to and transported across the cellular membrane of the mam-
malian intestinal enterochromaffin cells where they are subjected to the
enzymatic machinery required for 5-HT synthesis. The enzyme tryptophan
S-hydroxylase transforms tryptophan into 5-hydroxytryptophan which is
then decarboxylated by 5-hydroxytryptophan decarboxylase to yield
5-hydroxytryptamine. A monoamine oxidase (MAO) then catabolizes 5-HT to

yield 5-hydroxyindoleacetaldehyde which in the presence of low concentra-
tions of erythrocytes is reduced to 5-hydroxytryptophol (5-HT'ol), and

in the presence of high erythrocyte concentrations is oxidized to yield
5-hydroxyindoleacetic acid (5-HIAA) (5l). This metabolic pathway for the
synthesis and degradation of serotonin represents only the major pathway
involved; there are other minor pathways for both synthesis and degrada-
tion and these biochemical pathways are adequately illustrated by Valzelli
and Garattini (69).

Perhaps the largest endogenous supply of mammalian (human, dog, rat)
serotonin is to be found in the circulating platelets (51,74,34).
Humphrey and Toh (34), Zucker and Borrelli (74), and Hardisty and Stacey
(32) have shown that circulating platelets not only take up 5-HT but are
capable of doing so against a concentration gradient, implying perhaps
the existence of an active, i.e., energy-requiring mechanism. Pletcher's
findings that both KCN and ouabain inhibit uptake of serotonin by plate-
lets adds strength to this hypothesis (51). The dual reports that mega-
karyocytes, the mother cell of circulating platelets, do not possess
serotonin-containing granules, and the fact that platelets lack one of
the enzymes necessary for S-HT synthesis, yield sound evidence that
platelets do not synthesize serotonin but merely act as storage sites.
The question then arises, why do platelets store such large quantities of
serotonin? Humphrey and Toh (34) propose that the platelets may serve as
a means for rendering free plasma serotonin almost negligible in an
attempt to minimize its vasoconstrictor activity. Rand and Reid (52)
suggest that the local release of serotonin from damaged platelets may

play a part in hemostasis by virtue of its vasoconstrictor action.

Like bradykinin, 5-HT is chiefly removed from the circulation by
the lungs (l7,l2). Thomas and Vane (unpublished work) found that 90 to
98 per cent of an intravenous injection of serotonin disappears in one
passage through the pulmonary circulation. Davis and Yang (l2)
report that 33 per cent of an injection of 5-HT into the pulmonary

artery is removed within twelve to fifteen seconds.

Permeability: .Serotonin and Other Mediators
ofgjermeability

Little more than a century has passed since the introduction of the
theory which proposed that associated with the acute inflammatory re-
sponse was an increase in vascular permeability (9). Time has since
definitely established this thesis (36,38,39,46,58), yet relatively little
is known concerning the actual mechanism(s) by which vessels become more
permeable. Mediators of vascular permeability (histamine, serotonin,
acetylcholine, bradykinin) appear to act via one of two, or a combina-
tion of two, mechanisms; however, the actual biochemical mechanism in-
volved at the cellular level is still uncertain. The first proposed of
these two mechanisms evolves around an increased microvascular hydrostatic
pressure with subsequent "stretching" of pores, i.e., the separation of
intercellular attachments which exist between adjacent endothelial cells
(58.59.65). The second mechanism involves the direct action of the
mediator on subcellular (endothelia and smooth muscle) structures of the
microvasculature which alters the ability of these vessels to maintain a
steady state with regard to fluid filtration and plasma protein efflux

(40).

 

Regardless of the mechanism of increased permeability, it has been
the general concensus that the increased transudation of vascular filtrate
occurs primarily at the level of the capillary exchange vessels. There
are those investigators, however, who feel that in such species as the
rat (40,58), the rabbit (8,40), and the guinea pig (l2), the majority
of the vascular filtrate is moved across the postcapillary venular wall,
thereby "partially sparing' the capillaries. The electronmicrosc0pic
identification of subcellular contractile filaments (6,8,13,41), within
the endothelia of the vascular wall has led some investigators to propose
that the agents which mediate alterations in vessel permeability do so
via an energy-requiring mechanism. Diamond and Brody (13) have shown that
5-HT stimulates both activation and motility of phosphorylase b kinase
in rat smooth muscle strips. Cater and his associates (8) have demon—
strated a reduction in muscle and cutaneous tissue oxygen tension in
response to intravenously administered serotonin. Some workers who were
interested in factors other than the subcellular biochemical actions of
the histamine-mediators have attempted a more indirect line of investi-
gation, such as determining alterations in tissue weight, observing
"vascular labelling" in response to administration of radio-opaque carbon
particles, and optic examination of the microvasculature in such beds as
the sclera of the eye. These varied techniques have led to a wealth of
knowledge concerning actions of the histamine-mediators, serotonin being
no exception. Only that work which is of some significance to our cur-
rent study with serotonin will be presented here.

Buckley and Ryan (6) working with rats attempted to determine if

serotonin could increase vascular permeability and if so what its

mechanism of action is. After isolating a loop of the distal ileum,
colloidal carbon black was injected into the rat's lateral tail vein
followed by adding dropwise to the exposed major ileal vessels a solu-
tion containing 0.01 mg/ml of serotonin. Both ileal artery and the
accompanying vein contracted within fifteen to sixty seconds after appli-
cation of the solution. The major action on the microvasculature was
dilatation of the arteriolar resistance vessels. When serotonin was
applied to the ileal artery, carbon labelling of the vessel walls always
failed to develop. However, when serotonin was administered so as to
come in contact with only the small blood vessels, carbon labelling of
the vessel wall occurred as with histamine.. This increased perme-
ability resulted in the absence of an increase in microVascular hydro-
static pressure. Buckley thus proposed that in the rat, venous con-
striction and venular dilatation are not necessary steps in the genesis
of serotonin-induced edema formation. Majno and associates (38,39,40)
have further investigated the role played by serotonin in mediating an
alteration in vascular permeability. Employing as a test tissue the

rat cremaster muscle, colloidal suspensions of carbon black were adminis-
tered intravenously. Test solutions of serotonin creatinine sulfate,
0.018 mg/ml, were then injected subcutaneously. Carbon labelling occurred
primarily in those venules having an outer diameter of 20 to 30 micra.
The carbon deposition ended, often very abruptly, at the junction of the
venule with a larger vessel. The deposition in vessels smaller than
venules occurred in uneven patches, and was unobserved in those vessels
having an outer diameter of 4 to 5 micra. These studies indicated that

quantitatively serotonin was much more powerful on a mole-to-mole basis

than was histamine in generating an increase in vascular permeability.
On the basis of the size of the carbon-labelled lesions, Majno et al.
conclude that histamine is only 1/100 as potent as serotonin in generat-
ing edema in the rat cremaster. Rowley (59) has produced

evidence in the rat which supports findings indicating that serotonin
alters vascular permeability, however, Rowley suggests that the
mechanism by which serotonin mediates this alteration is that of an
increased hydrostatic pressure. Rats were injected intravenously with

2 mg of Evans blue or with 15 mg of radio-opaque carbon particles prior
to subcutaneous administration of serotonin. Thus the blue staining

of tissues was indicative of an increase in vascular permeability to
protein. Following serotonin, the paw skin over the injected site
appeared dark blue, while the subcutaneous areolar tissue was diffusely
blue. Direct observation of tissue with a dissecting microscope indi-
cated that carbon particles penetrated the walls of the injured venules.
Serotonin was also injected into the dorsal skin of the hind paw of rats
previously treated with Evanis blue. A dose of 2 ug/ml of serotonin
produced intense swelling and bluing of the tissue within 5 minutes, and
this persisted for several hours. A dose of 400 pg/ml of histamine was
required to elicit the same response. Direct observation of abdominal
skin vasculature revealed that the large veins were intensely constricted
by serotonin within 5 to 15 seconds, thus greatly distending smaller
veins and venules. Blood flow in the venules was markedly slowed and
sometimes stopped in response to serotonin. After treatment with an anti-
S-lfr drug, large vein constriction was inhibited and vascular permea-

bility failed to increase. By elevating venous pressures mechanically an

increase in venular permeability identical to that produced by serotonin
was seen. Although Rowley's work does not exclude the possibility of
direct action by serotonin on the microvasculature, it indicates that
such action is not necessary to explain the permeability effects.
Alth0ugh serotonin's ability to increase vascular permeability in
the rat has been well documented, the possibility of this same action
existing in other species including the dog and man seems to be unclear.
Ebert et al. (14) have examined the effects of serotonin on the micro-
vasculature of the rabbit ear chamber. Changes in vessel caliber were
witnessed under direct microscopic observation. Unlike histamine, which
produces a marked increase in permeability in this species, serotonin
failed to generate the formation of edema. The arteriolar dilatation
that was observed in response to serotinin was partially accounted for
by the formation of multiple venular platelet-leukocyte thromboemboli.
Investigation suggests that locally administered serotonin elevates micro-
vascular hydrostatic pressure in the dog and in man (11,28,48), an action
which alters transvascular movement of fluids, but little can be definitely
established regarding the possible fluxes of protein or even the total
volume of fluid accumulation in the interstitium. Roddie and associates
(56) have shown that intra-arterial administration of low doses of
serotonin into the human forearm is associated with a reduction in flow
to both forearm and hand. They also observed an increase in hand and
forearm total volume, a finding which suggests the possibility of an
increase in vascular volume or an increase in fluid accumulation in the

interstitium. The sensitivity of their experimental techniques did not

To

allow for a clear determination as to which of the above possibilities
played the major role in increasing forearm volume; however, the reduced
blood flow would seem to favor the latter. Haddy and co-workers have
produced evidence that in the dog forelimb locally infused serotonin
regularly increases small vein pressure (26.28.30) and the rate of
weight gain in the limb (28). Serotonin was infused via a side branch

of the brachial artery while pressures and weight were continuously
monitored. In several of the experiments in which small doses (5 ug/min)
were infused. the limbs became edematous to inspection near the end of
the infusion period. That these weight increases were not due to an
increased vascular volume is evidenced by the fact that there was an
increase in calculated total forelimb resistance, with much of this in-
crease occurring in large veins (capacitance vessels). Haddy proposed
that the weight gain was the result of large vein constriction with a
subsequent increase in microvascular filtration. Small vein pressure.

an index of minimal capillary hydrostatic pressure, was markedly elevated
throughout the infusion period, reaching levels as high as 30 mm Hg.

This suggests that the hydrostatic pressure may have exceeded colloid
osmotic pressure along the entire length of the capillary exchange vessel.
thereby promoting fluid filtration. If the capacity of the lymphatics

to remove this fluid was surpassed. there would result an increase in limb
weight from the accumulation of fluids in the interstitium. Haddy's work
indicated also that the rate of weight gain was directly proportional to
the magnitude of the small vein pressure increase, Abboud (1) and

Daugherty et al. (11) working with both the dog forelimb and hindlimb have

11

published data concerning the effects of local infusion of serotonin on
pressures and resistances. These findings in the dog and those produced
by Roddie and his associates in the human forearm suggest that the
increase in forelimb weight and forearm total volume are primarily the
result of an increase in fluid filtration secondary to an elevation of
capillary hydrostatic pressure. There are apparently no reports in the
literature of work that has been done with radio-opaque carbon particles
and serotonin in the dog or man. This probably stems from the difficulty
of locating in these species a vascular bed conducive to such a study.
Recently. Grega and his associates (24) and Haddy et al. (29) have
published data from studies in the dog forelimb suggesting that histamine
(high doses) produces a pressure-independent increase in vascular permea-
bility which is of greater significance than is the proposed hydrostatic
pressure mechanism. Grega et al. (24) found that local infusion of
histamine increased forelimb weight during both natural and constant flow
perfusion. only the former being associated with an increased blood flow
and small vein pressure. Their data suggested that the weight gain
observed at constant inflow was independent of an increase in microvascu-
lar pressures since small artery pressures which represent a maximum for
capillary hydrostatic pressure in both skin and skeletal muscle were
less than or equal to 30 mm Hg. To further test this hypothesis. fore-
limbs were perfused at constant inflow at a pressure of 30 mm Hg.
Histamine infusion reduced this pressure to 20 mm Hg but there was still
a significant increase in forelimb weight despite the fact that capillary
hydrostatic pressure had to be less than normal plasma colloid osmotic

pressure (at25 mm Hg).

12

If serotonin and the other mediators of permeability are able to
generate a pressure-dependent increase in the transvascular movement of
fluids and protein (25.59) as well as a pressure-independent alteration
in permeability (6.25.38). perhaps the magnitude of the importance of
these two mechanisms in the genesis of edema could be ascertained by
sampling the rate of lymphatic drainage of the interstitium (lymph flow
rate) as well as the lymph protein content in response to these mediators.
Rankin and Garlick (20,55) artificially elevated venous pressure by 35
to 40 mm Hg by placing an inflatable cuff around the dog's thigh. Under
these conditions both lymph flow and interstitial volume were markedly
elevated but there was no change in the transvascular movement of plasma
proteins. In another series of experiments, a section of electric heat-
ing tape was wrapped around the dog's paw in an attempt to change the
above parameters by producing vasodilatation. Again they recorded only
a marked increase in lymph flow rate. Haddy et al. (29) mechanically
elevated the venous pressure in the dog forelimb to 30 mm Hg and were
able to record a significant increase in lymph protein content. When
blood flow and venous pressure were elevated, there resulted a significant
elevation in both lymph flow rate and lymph protein content. The most
marked increases in these variables were seen however in response to local
infusion of histamine (40 ug/min) while blood flow and venous pressure
were maintained at control levels. Their results suggested that at low
rates of histamine infusion, both the pressure-dependent and the pressure-
independent mechanisms were important in the genesis of edema. However.

with high-dose infusates of histamine. the ability of the lymphatics to

l3

drain the excess fluid and protein accumulation in the interstitium was
overwhelmed with the resultant formation of edema. To date. investiga-
tion concerning serotonin's effects on vascular permeability has
established several facts: (1) although it has been reliably demon-
strated that this agent does increase vascular permeability in the rat
(18.40.58), data concerning this activity in such species as the rabbit.
guinea pig. dog and man appears to be less certain, (2) the mechanism
of serotonin-increased permeability. whether pressure dependent or pres-
sure independent, is controversial. (3) even though investigation has
shown that locally administered serotonin elevates microvascular hydro-
static pressure in the dog (28,48). little can be definitively estab-
lished regarding the total volume of fluid accumulation in the interstitium.
and (4) serotonin's effects on such variables as lymph flow rate and

lymph protein content in the dog was. until this investigation, unknown.

General Systemic Effects

Rudolph and Paul (60,61) have examined the systemic responses in dogs
to varying rates of continuously infused solutions of serotonin. Maximal
effects were produced with an infusion rate of 150 ug/kg/min. They
observed a 300 per cent increase in mean pulmonary pressure, a 28 per
cent decrease in mean systemic arterial pressure. and a 60 per cent rise
in cardiac output. Calculated pulmonary resistance increased markedly to
500 per cent of control values, whereas systemic peripheral resistance
decreased by 55 per cent. The reduction in total peripheral resistance

can be accounted for by the facts that intravenously administered

l4

serotonin acts as a dilator in those peripheral beds receiving the great-
est per cent of the cardiac output (Splanchnic and skeletal muscle) and
displaying the greatest resistance per unit weight ratio (skeletal
muscle). This reduction in resistance is seen despite the fact that
intravenous infusion of serotonin produces marked constriction in cutaneous
vasculature. The renal responses to intravenous serotonin in the dog are
not as well defined as are the responses in other peripheral beds. Page
and McCubbin (47) reported that this response ranges from a slight reduc-
tion to a very marked reduction in renal arterial pressure. Likewise,
Reid's reports (54) indicate that the renal response is variable; usually
the blood flow falls. increases during the rise in arterial pressure,
and then decreases again.

Working with dogs. Page (49) found the response to intravenous infu-
sion of 0.06 to 0.12 mg of serotonin to be triphasic. consisting of
(1) an initial reduction in pressure associated with bradycardia. This
response was immediately followed by (2) a sustained pressor response of
from 20 to 60 mm Hg. succeeded by (3) a prolonged decline in pressure.
Schneider and Yonkman (62) with the dog verified Page's findings while
infusing 50 ug/kg of serotonin intravenously. However, the initial
depressor response was not observed as frequently as in Page's reports.
Reid (54) has also reported this triphasic response to be present in the
cat. The above reports indicate that the initial fall in systemic
arterial pressure is the result of a left ventricular von Bezold-type
reflex (reduction in left ventricular output) subsequent to marked con-

striction of the pulmonary vasculature. The pressor response is due then

15

to an increase in reflex-mediated sympathetic discharge to the periphery.
while the secondary depressor response seems to be the result of an
active reduction in total peripheral resistance accompanied by a possible
reduction in the mechanical efficiency of the left ventricle.

Findings that are reported in the literature indicate that the re-
sponse to intravenous infusion of serotonin is dependent among other
things upon the extent of pre-existing neurogenic constriction (26.48.61).
Page has demonstrated in all species studied. that when neurogenic vaso-
constrictor tone is reduced or absent. the response to serotonin is always
pressor; when neurogenic tone is high the response is depressor. When
chronically neurogenic hypertensive dogs were subjected to intravenous
serotinin infusion, Page (48) found that the arterial pressure responses
were always depressor and more sustained than depressor responses in con-
trol animals. Further contrasting these findings with those in the norm-
otensive dog. Page discovered that increasing the dose of serotonin pro-
duced a further drop in total peripheral resistance in the hypertensive
animals.

Of special interest are the observations which have been made con-
cerning the effects of sympathetic nerve stimulation on skin and skeletal
muscle venous and arterial pressures. blood flow, and volume of distribu-
tion (21.35.57). Kelly and Visscher (35) found that electrical stimula-
tion of the lumbar sympathetic chain in dogs increased small vein pres-
sure from an average value of 17 mm Hg to a value of 30 mm Hg. Haddy
et al. (26). and Abboud (1.2) have proposed that serotonin's ability to

generate actions similar to the above may be explained on the basis of

16

the pre-existing neurogenic tone in the various vessel segments. Local
infusion of serotonin into the dog's forelimb selectively constricts the
arterial segment upstream from the metacarpal artery and the venous seg-
ment downstream from the metacarpal vein. The small vessel segment
usually dilates. At low sympathetic tone, local administration of sero-
tonin would produce constriction in the relaxed arterial and venous seg-
ments. thus producing an increase in total resistance within that particu-
lar bed. This proposal of the relation between serotonin's actions and
the existing degree of sympathetic vasoconStriction seems to be in agree-
ment with Page's findings (43.48) in the hypotensive, normotensive and

hypertensive dogs.

Pulmonary Vasculature

Vasoactive agents as a rule have displayed much weaker effects on
the pulmonary circulation than on the systemic circulation. Serotonin,
however. may be considered the exception to the rule because it produces
a distinctive rise in pulmonary vascular resistance both in intact
animals (37,60) and in 'nonintact' perfused lungs (19,22). Young et al.
(73) working with the anesthetised dog measured pulmonary capillary blood
volume (Vc) during intravenous infusion of serotonin and found increases
as high as 133 per cent above preinfusion control values. Venous re-
sistance was markedly elevated resulting in passive congestion of the
pulmonary capillary bed. Rudolph and Paul (61) noted a marked increase
in pulmonary arterial pressure when serotonin (20 ug/kg/min) was infused

into the venous circulation of the intact dog. Maximal effects were

17

noted at an infusion rate of 150 ug/kg/min. Calculated pulmonary
resistance increased to 500 per cent of control levels. Gilbert and his
associates (22) have published evidence indicating that local administra-
tion of serotonin produces marked constriction of large arteries and
veins in the pulmonary vascular bed of the dog. MacCanon et al. (37)
have suggested that this marked increase in resistance upon local admin-
istration of serotonin is in part the result of massive platelet aggrega-
tion. Tone, hence resistance, in pulmonary arterioles and veins is
neurogenically low. thus the predicted response to serotonin would be

constriction.

Renal Vasculature

The renal vasculature is a relatively low tone bed and the only
responses observed after local administration of serotonin are constrictor
(15.25.47). Page and McCubbin (47) working with the dog found that in-
fusion of serotonin (27 ug/kg/min) via a renal artery produced a marked
increase in renal vascular resistance. Equipressor doses of serotonin,
although not as strong as epinephrine and norepinephrine produced a more
sustained action than did either of these agents. Page observed that the
responses to local administration of serotonin into the canine kidney
are in contrast to the partial or occasional entirely depressor response
seen upon intravenous administration of serotonin. Reid's reports (54)
indicate that the renal vascular responses to intravenous serotonin
administration are variable, but usually the blood flow falls during the

initial depressor response. rises during the elevation of arterial

18

pressure and then decreases again. Flow changes accompanied by changes

in renal arterial pressure indicated changes in resistance.

Coronary_Vasculature

Serotonin when infused into the canine coronary artery produces
potent vasodilatation (63.71). Schofield and Walker (63) employing '
heart-lung preparations from 'nonintact' animals reported that injection
of 0.1 to 12 pg of serotonin into a cannulated coronary artery resulted
in a marked elevation of coronary blood flow. Injection of serotonin
into the coronary artery does not produce the increased cardiac frequency
seen with intravenous infusion (31). Maxwell and his associates (42)
examined the effects produced by infusing 20 ug/kg/min of serotonin into
the right atrium of the intact dog. They repbrted-that coronary blood
flow increased by 79 per cent while the mechanical efficiency of the

left ventricle was reduced by 3 per cent.

Splanchnic Vasculature
With the use of serial photography McCubbin and co-workers (43)

examined the effects on the different segments of the dog mesenteric bed
produced by local infusion of serotonin. They found that resistance to
flow in small arteries. small veins, and to a lesser extent large veins
was reduced while that in large arteries was elevated. The effects on
the small vessels was much shorter lived than that on the large vessels.
Scott and Dabney (64) have shown that local infusion of serotonin into a

naturally-perfused gut artery produces very little effect on intestinal

19

blood flow. They attributed this finding to the ability of the intestinal
vasculature to autoregulate its blood supply. Demonstrating that variable
responses to serotonin within this bed do exist. Texter et al. (67) have
cited evidence suggesting that serotonin can produce a decrease. an in-
crease or no change in total gastrointestinal flow when administered
locally. Resistance to flow is increased in both the hepatic and splenic
vasculature in the dog. Working with rats, Bohr et al. (5) found that
intravenous injection of serotonin induced constriction in the terminal

arterioles of the mesoappendix.

Cerebral Vasculature

Relatively little is known concerning serotonin's actions in the
dog cerebral bed as evidenced by the scanty number of reports that com—
prise the literature. Berkinshaw and associates (4) examined the effects
of serotonin on cerebral vascular permeability in the guinea pig and
found that massive doses (500 to 2000 ug/kg) injected intra-arterially
failed to affect vascular permeability. In dogs, local administration
may elicit first a short decrease and then a sustained increase in

cerebral flow possibly due to local vasodilatation (10).

Dog_Forelimb and Hindlimb Vasculature

The contribution of segmental vascular resistance to distribution
of blood volume and to microvascular filtration-reabsorption capacity
within these two vascular beds has been well documented (1.11.25.26.48,

57). Abboud (l) and Daugherty et al. (11) have shown that intra-arterial

20

administration of serotonin produces marked constriction of cutaneous
vessels. The arterial segment upstream from the paw and the venous seg-
ment downstream from the paw experienced the greatest increases in
resistance. Because total forelimb blood flow.was increased during the
infusion period, these investigators concluded that serotonin possibly
dilates skeletal muscle vasculature. In comparing with these effects.
the effects produced by intravenously administered serotonin, Daugherty
found minimal changes in total forelimb blood flow. With infusion rates
greater than 206 ug/min.. cephalic venous outflow decreased slightly
while brachial outflow and systemic arterial pressure were unaffected.
To further explore the effect of serotonin on skeletal muscle resistance
the gracilis muscle of the hindlimb was infused with serotonin. The
hindlimb preparation offers better flow separation between skin and
skeletal muscle, thus enabling a more critical analysis of responses.
Intra-arterial infusion of 2 to 40 ug/min had little or no affect on
muscle vascular resistance. Haddy et al. (26) found that local infusion
of serotonin into the forelimb antagonizes extremes of neurogenic vascu-
lar tone. i.e., serotonin produces net dilatation when tone is high and
net constriction when tone is low. They concluded that the 'bidirection-
alism' is accounted for by the fact that nervous activity alters small
vessel caliber without greatly affecting large vessel caliber, and that
serotonin produces small vessel dilatation at the same time that it pro-
duces large vessel constriction.

In conclusion, the responses to intra-arterial or intravenous

administration of serotonin are dependent upon numerous observable

21

considerations (11.26.27.28). For instance, Daugherty et al. (11) found
that the effects of serotonin and other vasoactive agents on the hemo-
dynamics of a localized vascular bed vary not only according to the

rate of blood flow perfusing that limb. but also according to the route
of administration of an agent. Hence. the responses of skin and skeletal
muscle blood flow in the face of intravenously administered serotonin
are generally much weaker than when serotonin is given intra-arterially.
Responses to serotonin vary also according to the species (10.33.16.44,
73) as well as to the particular vascular beds being investigated (33.37.
42.47.71). A consideration at least as important as those previously
mentioned is that dealing with the particular vessel segment within a
given bed that one may wish to examine. Thus intra-arterial serotonin
constricts large veins (11.27.48), large arteries (16.25.26) and appears
to dilate the small vessel segment (arterioles, capillaries, venules)
(16.25.27). Finally, Haddy (25) has concluded that because of serotonin's
uniqueness amongst the vasoactive agents, it can be classified neither

as a pressor nor a depressor agent.

METHODS

Mongrel male dogs having an average weight of 20 kg (range 17-24 kg)
were anesthetized with sodium pentobarbital (30 mg/kg) and ventilated

with room air using a Harvard respiratory pump.

Hemodynamic Measurements

The skin of the right forelimb was circumferentially sectioned 3-4
cm above the elbow. The right brachial artery, forelimb nerves. brachial
and cephalic veins were isolated and the muscles and remaining connective
tissue sectioned by electrocautery. The humerus was cut and the ends of
the marrow cavity packed with bone wax to prevent blood loss. Blood
entered the limb only through the brachial artery and exited only through
the brachial and cephalic veins. The forelimb nerves (median, ulnar,
radial, and musculocutaneous) were left intact and coated with an inert
silicone spray to prevent desiccation, Heparin (10: mg/kg) was administered
intravenously to prevent clotting.

Intravascular pressures were measured with small-bore polyethylene
tubing inserted into the following sites: (1) skin small artery from the
third superficial volar- metacarpal artery on the ventral surface of the
paw; (2) muscle small artery from a vessel supplying a flexor muscle in
the upper portion of the forelimb; (3) skin small vein from the second
superficial dorsal metacarpal vein on the dorsal surface of the paw;

(4) muscle small vein from one of the deep vessels draining a flexor

22

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muscle in the middle portion of the forelimb; (5) skin large vein from
the cephalic vein via a side branch; (6) muscle large vein from the
brachial vein via a side branch. The small artery catheters were in-
serted in a downstream direction while the small vein catheters were
directed upstream from the site of insertion. The cannulated small
vessel acts as an extension of the catheter and thus a pressure is
measured in the vessel or vessels to which the cannulated vessel con-
nects. This pressure is a true lateral pressure as long as the cannu4
lated vessel is patent and without valves (verified by the ability to
freely withdraw blood from and to flush saline into the cannulated
vessel). The presence of the catheter does not measurably alter the
pressure in the arterial or venous system because in the canine forelimb
the cannulated vessel is a negligible fraction of the total cross-
sectional area of the arterial or venous bed and there are abundant
artery to artery and vein to vein anastomoses (24.28.45). Pressures were
measured with low volume displacement Statham transducers and recorded on
a Sanborn direct-writing oscillograph.

' The brachial and cephalic veins were partially transsected 3-5 cm
downstream from the sites of large vein pressure measurement and the end
of each vessel was cannulated with a short section of polyethylene tubing
(P. E. 320). Outflow from both veins was directed into a reservoir main-
tained at constant volume with a variable speed pump which continuously
returned blood to the animal via a cannulated jugular vein. Blood flow
was determined by timed collections of the two venous outflows. In this

preparation the median cubital vein represents the major anastomotic

25

channel between the brachial and cephalic veins. This vessel was ligated
in all experiments to insure that brachial venous flow was predominantly
from muscle whereas cephalic flow came primarily from skin. Although
this approach does not accomplish complete anatomical isolation of skin
and muscle, the degree of flow separation is sufficient to permit com-
parison of resistance changes in the two parallel-coupled beds (2.11.50).
When all cannulae were in position, the limb was suspended on a wire
mesh platform attached to a strain guage balance which could be cali-
brated by adding known weights to the platform. The addition of a 2 gm
weight usually produced a pen deflection of 5415 mm paper. Mean systemic
arterial pressure was continuously monitored from a catheter in the lower
abdominal aorta. High doses (150 pg base/min) of serotonin were infused
for 15 minutes while the low dose preparation (15 pg base/min) was infused
for 30 minutes into the brachial artery via a side branch. The volume of
the infusate was 0.2 ml/minute. Limb weight was continuously monitored
and all pressures and flows were recorded twice during the preinfusion
control period and at 2. 5, 10 and 15 minutes after initiating the high
dose infusate. and at 2. 5. 10. 15 and 30 minutes after introducing the
low dose. Postinfusion control values were recorded 15 minutes after
terminating both doses of the infusate. Total and segmental (large artery.
small vessel, large vein) vascular resistances in muscle and in skin
were calculated by dividing brachial or cephalic blood flows into corre-
sponding pressure gradients. In addition. total forelimb resistance
and combined muscle and skin segmental vascular resistances were calcu-

lated as follows:

26

Total Forelimb Resistance = -RL§—é—§Lm-
ts tm
R . R
Total Forelimb Large Artery Resistance = §§9—;—ng-
sa ma

= RsLs-v) ' Rm(s-v)

Total Forelimb Small Vessel Resistance
Rs(s-v) + Rm(s-v)

Total Forelimb Large Vein Resistance

where R resistance in mm Hg/ml/min/lOO gm; t total; 5 = skin; m =
muscle; a = large artery; s-v = small vessels; v = large vein.

In a second series of animals. forelimb inflow was held constant
throughout the experiment at a rate which initially produced a perfusion
pressure similar to systemic pressure. This was accomplished by pumping
arterial blood, obtained from a femoral artery into the brachial artery
with a Sigmamotor pump. Perfusion pressure was continuously monitored
throughout the experiment. In all other respects, the preparation and

experimental protocol was as described above.

Lymph Studies

Small incisions were made over the brachial artery, cephalic vein
(above the elbow) and second superficial dorsal metacarpal vein in the
right forelimb. A side branch of the brachial artery. a lymph vessel.
and the vein were isolated. After administering heparin intravenously.
these vessels were cannulated in an upstream direction with polyethylene

tubing for drug administration. lymph collection, and pressure measurement

27

respectively. The lymph vessels in the area of the cephalic vein above
the elbow drain forelimb skin and paw (45). Several vessels were usually
tied centrally and one of them was cannulated distally with a 10 cm
length of PE 10 tubing which had been beveled at the cannulating end.
Lymph was collected in miniature 0.5 ml graduated cylinders. The
cylinders, constructed from plastic pipettes. were very narrow. thus
minimizing evaporation. Flow rate was measured in ml/lO minutes.

A Harvard infusion pump was employed for infusion of the drug into the
brachial artery. Lymph was collected during each of two 10 minute pre-
infusion control periods. After measuring small vein pressure, serotonin
(15 pg base/min) was infused intra-arterially for a period of 30 minutes
via the side branch of the brachial artery. The volume of the infusate
was 0.2 ml/minute. Lymph samples were then collected at 10 minute
intervals throughout the 30 minute infusion period. Serotonin infusion
was then terminated. lymph was collected and small vein pressure was
measured during a 10 minute postinfusion period.

Total protein concentration in the lymph was measured by the spectro-
photometric (Beckman DB spectrophotometer) method of Waddell (70).
Albumin and globulin fractions were determined by electrophoresis
(Photovolt Corp.. Broadway, New York). All data from the hemodynamic and
lymph studies were analyzed with Student's t-test modified for paired

replicates.

RESULTS

Data presented in this section represents information gathered from

42 mongrel dogs. Eight animals were used at each of two dose levels
during natural and constant blood perfusion of the dog's right fore-
limb. An additional ten animals were used for collection of lymph pro-
tein and for determination of lymph flow rate. All data are presented
as the mean value. Statistical evaluation of the data was accomplished
using Student's t-test for comparison between groups (paired replica).

A statistically significant difference at the P < 0.05 level was con-

sidered as a true difference between pairs.

Forelimb‘Weight (A Wt.). Tables 1 and 2

During the natural perfusion experiments. neither dose of serotonin
produced a statistically significant alteration in forelimb weight.
While forelimb perfusion was maintained constant all limbs gained weight

slightly but significantly.

Mean Arterial Pressure (Pa); Tables 1 and 2

High dose infusion of serotonin at natural inflow failed to alter
significantly mean: arterial pressure relative to control. The response
to the low dose infusate was a slight reduction in pressure which was of

statistical significance from minute 10 to minute 30. Neither dose

28

29

generated a significant alteration in arterial pressure when perfusion

was held constant.

Perfusion Pressure (Pba), Tables 1 and 2

Both the low dose and the high dose infusates generated a signifi-
cant increase in brachial artery perfusion pressure when inflow was
held constant. Perfusion pressure remained markedly elevated throughout
the infusion period with the greatest response being produced by the

high dose.

Forelimb Vascular Pressures,_Tables l and 2

Small vein pressures were markedly elevated throughout the infusion
period by both doses of serotonin. The only change in skin large vein
pressure was a slight but significant reduction at constant inflow in
response to the low dose infusate. Muscle large vein pressure was ele-
vatedito a significant level at both natural and constant inflow and in
response to both doses. Small artery pressures in skin and skeletal
muscle were reduced markedly in response to both doses during natural
perfusion. During constant inflow skin and skeletal muscle small artery
pressures followed this same pattern in response to the low dose. Muscle

small artery pressure was markedly elevated by the high dose.

Forelimb Blood Flows._Tables l and 2
There was a net reduction in total forelimb flow during natural

inflow preparations. Skin blood flow was reduced in response to both doses

30

of serotonin during natural and constant inflows. The most marked re-
duction was seen at natural flow during the high dose infusion. Muscle
blood flow increased in response to both doses in the naturally and

constantly perfused forelimbs.

Skin_§egmental Vascular Resistances, Tables 3 and 4

Calculated resistance (mm Hg/min/ml/lOO g forelimb weight) in skin
large artery increased dramatically in response to high and low dose
infusates during natural and constant flow experiments. Skin small vessel
(small artery. capillary, small vein) resistance failed to change sig-
nificantly in response to the low dose. Skin large vein resistance in-

creased markedly in all cases.

Skeletaluflgscle Segmental-VascularResistances,
Tables 3 and 4

Muscle large artery resistance remained relatively unaltered through-
out the infusion period in both preparations, whereas muscle small
vessel resistance was significantly reduced relative to control in all
cases. Muscle large vein reSistance did not change significantly relative
to control during low dose infusion. The high dose infusate produced a
small but significant reduction in muscle large vein resistance during

both flow rates.

Total Vascular Resistance. Tables 3 and 4
There was a marked increase in total forelimb resistance at natural

and constant inflow in response to both doses of serotonin. However, the

31

increased total resistance produced by the low dose at natural inflow
was not significant. Skin total resistance was significantly elevated
by both doses during natural and constant inflow. Total skeletal

muscle resistance was unaffected by the low dose while the high dose at
both natural and constant flow produced a slight but significant reduc-
tion. Both doses produced a marked increase in total large artery re-
sistance under all conditions. while total small vessel resistance was
markedly reduced or unaffected (high dose. natural inflow) by both doses
at both flow rates. Total large vein resistance was significantly ele-
vated by both doses during natural inflow, but unaffected by both doses

during constant inflow.

Lymph Protein and Flow Rate, Table 5

Lymph flow rate was markedly elevated in response to the low dose
infusate although total protein concentration did not change signifi-
cantly relative to control. Small vein pressure was significantly

elevated throughout the infusion period.

32

Table 1. Effects of Serotonin Base Infused Intra-arterially Into the
Collateral-free, Innervated, Naturally-perfused Canine Forelimb.

 

 

 

Serotonin - Control Infusion Period Postcontrol
pg/min - 0 ~ 2' 5' 10' 15' 30' 30' 45'
A Wt. 15 (n68 120.0; oiov‘ 0.1 0.9 2.0 2.4 3.7 5.7
gms 150 n=8 0.0 0.2 -2.0 0.4 1.8 2.0 3.3
Pa 15 (n=8) 123 121 121 117 114* 113* 115* 119
mmHg 150 (n=8) 131 131 131 126 124 125 128
Pmsa 15 (n=8; 96 94 86* "78* 74* 73* 74* 88
mmHg 150 n=8 97 98 86* 75* 74* 75* 85*
Pssa 15 n=8 101 100 68* 58* 53* 51* 54* 86
mmHg 150 n=8 99 100 41* 46* 43* 41* 82
Pmsv 15 (n=8) 11 11 13 16* 15* 14 13 11
mmHg 150 n=8) 8 8 12* 15* 14* 14* 9
Pssv 15 (n=8 10 10 19* 20* 16* 14* 14* 11
mmHg 150 (n=8 1o 10 16* 21* 19* 18* 10
Pmlv 15 (n=8) 8 8 10 12* 11* 11* 10* 8
mmHg 150 (n=8) 4 4 7* 11* 10* 10* 6*
Pslv 15 (n=8) 6 6 8 8 8 6 6 7
mmHg 150 (n=8) 4 4 2 2 2 2 3
Fm/ 15 (n=8 7 7 8 10 9 9 9 8
1009 150 n=8 7 7 9* 11* 11* 11* 8
Fs/ 15 (n=8 11 11 8 6* 7 7 8 14
1009 150 (n=8 12 12 3* 1* 1* 2* 9*
Ft/ 15 (n=8) 19 18 17 17 16 15 18 22
1009 150 (n=8) 20 19 12* 12* 12* 13* 17

 

—r——

A Wt. gms. = change in forelimb weight

Pa = aortic pressure

Pmsa = muscle small artery pressure
Pssa = skin small artery pressure
Pmsv = muscle small vein pressure
Pssv = skin small vein pressure
Pmlv = muscle large vein pressure
Pslv = skin large vein pressure

Fm/lOO g.= muscle venousfloutflow (ml/min/lOO g forelimb tissue)
Fs/lOO g * skin venous outflow (ml/min/lOO g forelimb tissue)
Ft/lOO g = total forelimb outflow ml/min/lOO g forelimb tissue
* = P < 0.05 when compared to control

33

Table 2. Effects of Serotonin Base Infused Intra-arterially Into the
Collateral-free. Innervated, Constantly-perfused Canine Forelimb.

 

' v fi

 

 

Serotonin Control Infusion Period Postcontrol
pg/min -5 0 2' 5' 10' 15' 130'~ 30' 45'
£79.. 15 (n=8) 0.0 0.0 -0.3 3.2* 6.5* 8.7* 13.7* 16.1
gms 150 (n=8) 0.0 0.2 3.1* 6.5* 8.0* 8.4* 10.5*
Pa 15 (n=8) 125 121 124 124 125 126 128 130
mmHg 150 (n=8) 128 128 128 127. 129 127 127
Pba 15 n=8} 112 113 163* 156* 145* 144* 139* 97
mmHg 150 n=8 119 120 245* 223* 209* 212* 139*
Pmsa 15 (n=8) 87 88 83 76 63* 61* 53* 62*
mmHg 150 (n=8) 84 87 133* 124* 126* 133* 96*
Pssa 15 (n=8) 74 77 56 50* 40* 33* 31* 52*
mmHg 150 (n=8) 83 84 62* 47* 45* 43* 77
Pmsv 15 (n=8) 14 13 20* 24* 21* 18* 19* 13
mmHg 150 (n=8) 10 10 32* 28* 24* 23* 13*
Pssv 15 (n=8) 16 16 33* 35* 26* 23* 21* 17
mmHg 150 n=8) 16 16 38* 33* 30* 26* 19*
Pmlv 15 (n=8) 10 10 16* 18* 16* 15* 14* 11*
mmHg 150 (n=8) 7 7 28* 26* 21* 20* 11*
Pslv 15 (n=8) 9 9 8 7* 6* 6* 6* 8
mmHg 150 (n=8) 6 6 4 4 4 4 6
Fm/ 15 (n=8; 10 9 16* 16* 16* 16* 15* 10
100g 150 n=8 9 9 17* 17* 16* 16* 10
Fs/ 15 (n=8) 12 12 6* 5* 6* 6* 6* 12
1009 150 (n=8) 18 19 9* 11* 11* 11* 18
Ft/ 15 (n=8) 22 22 22 22 22 22 22 22
1009 150 n=8) 27 27 27 27 27 27 27

 

A Wt. gms. = change in forelimb weight
P8 = aortic pressure
Pba = brachial artery perfusion pressure

Pmsa = muscle small artery pressure

Pssa = skin small artery pressure

Pmsu = muscle small vein pressure

Pssu = skin small vein pressure

Pmlv = muscle~large vein pressure

Pslv = skin large vein pressure

Fm/lOOg = muscle venous outflow (ml/min/lOOg forelimb tissue)
Fs/lOOg = skin venous outflow (ml/min/lOOg forelimb tissue)
Ft/100g = total forelimb outflow ml/min/lOOg forelimb tissue

* = P < 0.05 when compared to control

34

' Table 3.

Effects of Serotonin Base Infused Intra-arterially Into the

Collateral-free. Innervated, Naturally-perfused Canine Forelimb.

 

__.-

 

 

 

Serotonin Control Infusion Period. ‘flPostcontrol
pg/min -5 O 2' 5' 10' 15' 30' 30' 45'
RSa 15 (n=8) 2 2 10* 15* 13* 13* 11* 2
150 (n=8) 3 3 34* 60* 61* 47* 5*
Rs(s_v) 15 (n=8) 8 8 7 8 6 6 6 6
150 (n=8) 8 8 9 21* 20* 13* 10
Rsv 15 (n=8) 0.3 0.3 2.0* 2.4* 1.4* 1.3* 1.2* 0.3
150 (n=8) 0.5 0.5 6.7* 14.4* 14.0* 9.2 1.0*
Rt 15 (n=8) 10 11 19 25* 21* 21* 18 8
S 150 (n=8) 11 11 50* 95* 94* 68* 16*
R 15 (n=8) 4 4 5 7 7 7 8 4
ma 150 (n=8) 5 5 6 5 5 5 6
Rm(S-v) 15 (n=8) 13e 13 12 9* 9* 10 13 10
150 (n=8) 13 14 11* 6* 6* 6* 10*
R v 15 (n=8) 0.5 0.4 0.4 0.5 0.7 0.4 0.5 0.3
m 150 (n=8) 0.6 0.6 0.5 0.4* 0.4* 0.4* 0.4*
Rtm 15 (n=8) 18 18 17 16 17 18 22 15
150 (n=8) 19 20 17* 12* 11* 12* 16*
Rt 15 (n=8) 1 1 3* 3* 4* 4* 3* 1
a 150 (n=8) 2 2 5* 5* 5* 4* 3
Rt(sav) 15 (n=8) 5 5 4 3* 3* 4 4 3*
150 (n=8) 5 5 4 4 4 3 4
Rt 15 (n=8) 0.2 0.2 0.3* 0.3* 0.3* 0.3* 0.3* 0.2
v 150 (n=8) 0.2 0.2 0.4* 0.4* 0.4* 0.3* 0.2
Rt 15 (n=8) 6 6 8 8 8 8 8 5
150 (n=8) 7 7 11* 10* 10* 10* 7
R = resistance (mmHg/ml/min/lOOg forelimb tissue)
Rsa = skin large artery resistance
Rs(s-v) = skin small vessel resistance
Rsv = skin large vein resistance
Rts = total skin resistance
Rma = muscle large artery resistance

Rm(s-v) = muscle small vessel resistance

Rmv = muscle large vein resistance

Rtm = total muscle resistance

Rta = total forelimb large artery resistance
Rt(s-v) = total forelimb small vessel resistance
Rtv = total forelimb large vein resistance

Rt = total forelimb resistance

* = P < 0.05 when compared to control

Table 4.

Effects of Serotonin Base Infused Intra-arterially Into the

35

Collateral-free. Innervated, Constantly-perfused Canine Forelimb.

 

 

 

Serotonin Control Infusion Period Postcontrol
pg/min -5 o 2' 5' 10' 15' 30' 30' 45'
R53 15 (n=8) 3 0 22* 25* 20* 20* 19* 4
150 (n=8) 2 2 25* 21* 17* 18* 3
R (S_ ) 15 (n=8) 5 5 5 4 3 2* 2* 4
S V 150 (n58) 4 4 4 - 2* 2* 2* 3
15 (n=8) 0.6 0.6 5.4* 6.0* 3.6* 2.7* 2.5* 0.8
SV 150 (n=8) 0.6 0.5 5.2* 4.1* 3.0* 2.5* 0.8*
Rts 15 (n=8) 9 9 33* 35* 27* 24* 23* 8
150 (n=8) 6 6 34* 27* 22* 23* 7
Rma 15 (n=8) 3 3 5* 5* 5* 6* 6* 4
150 (n=8) 4 4 6 6 5 5 4
Rm(s_v) 15 (n=8) 9 9 5 4* 5* 5* 4* 6*
150 (n=8) 9 10 6* 6* 6* 7* 9
Rmv 15 (n=8) 0.4 0.4 0.4 0.5 0.4 0.2* 0.3* 0.2*
150 (n=8) 0.4 0.4 0.2* 0.2* 0.2* 0.2* 0.3
Rtm 15 (n=8) 12 12 10 10 10 10 10 9*
150 (n=8) 14 14 13 12* 11* 12* 14
Rt 15 (n=8) 1 1 4* 4* 4* 4* 4* 2
a 150 (n=8) 1 1 5* 4* 4* 4* 2
Rt(s_v) 15 (n=8) 3 3 2* 2* 1* 1* 1* 2*
150 (n=8) 3 3 2 1* 1* 1* 2
Rt 15 (n=8) 0.2 0.2 0.3 0.4* 0.3 0.2 0.2 0.2
V 150 (n=8) 0.2 0.2 0.2 0.1 0.2 0.2 0.2
Rt 15 (n=8) 5 5 2* 7* 7* 6 6 4
150 (n=8) 4 4 9* 8* 7* 8* 5

 

R = resistance (mmHg/ml/min/lOOg forelimb tissue)

Rsa = skin large artery resistance

Rs(s-v)
Rsv
Rts
Rma
Rm(s-v)

Rta = total forelimb large artery resistance

= skin small vessel resistance
skin large vein resistance
total skin resistance

muscle large artery resistance

= muscle small vessel resistance
Rmv = muscle large vein resistance
Rtm = total muscle resistance

Rt(s-v) = total forelimb small vessel resistance
Rtv = total forelimb large vein resistance
Rt = total forelimb resistance
* = P < 0.05 when compared to control

36

Table 5. Effects of Low-dose (15 pg/min) Serotonin Base Infused Intra-
arterially Into the Collateral-free. Innervated, Naturally-perfused
Canine Forelimb. (n = 10)

 

 

 

 

Control - Infusion Period Postcontrol

C.l C2 ~ 0-10' 10-30' 20'-30' 30'-40'
FL '"0.03 0.03 0.10* 0.15* 0.23* 0.10*
TPL 2.2 2.2 2.4 2.4 2.4 2.4
AL 1.1 1.0 1.1 1.1 1.1 1.1
GL 1.1 1.2 1.3 1.3 1.3 1.3

* * 'k 'k

PSSV 12 12 22 22 22 14
FL = lymph flow (ml/10 min)
TPL = lymph total protein concentration (gm %)
AL = lymph albumin Concentration (gm %)
GL = lymph globulin concentration (gm%)
PSSV = skin small vein pressure
* = P < 0.05 when compared to control

DISCUSSION

Combined findings from this study reliably demonstrate that neither
low doses nor high doses of locally administered serotonin create sig-
nificant edema formation in the canine forelimb perfused at natural
inflow. Small vein pressure in both skin and skeletal muscle was
markedly elevated throughout the infusion period suggesting an increase
in capillary hydrostatic pressure. This finding would suggest a net
increase in the total volume of filtrate moved across the capillary wall.
However. the fact that total forelimb weight did not increase signifi-
cantly under these conditions suggests that other factors must have pre-1
vented extravascular fluid volume from rising substantially. Hence, in
the dog this agent differs markedly from bradykinin and histamine which
have been shown to produce marked edema formation under these same con-
ditions. Our findings however. differ qualitatively from those of Haddy
et al. (28). This discrepancy may be the result of differences in experi~
mental techniques. Forelimbs in Haddy's study were denervated while
those in the current study were left intact. It is conceivable that
forelimb blood flow in Haddy's preparations could have been somewhat ele-
vated as the result of a decrease in arteriolar neurogenic tone. This in
turn may have increased capillary hydrostatic pressure and the rate of
weight gain.

Although observable edema does not develop when this agent is

infused for 30 minutes into naturally-perfused forelimbs. fluid filtration

37

38

is apparently increased. Two observations support this conclusion:

(1) small vein pressure. an index of minimum capillary hydrostatic
pressure. was markedly elevated throughout the infusion period, suggesting
an increase in microvascular pressure. (2) In addition, the rate of

lymph flow increased greatly without a disproportionate increase in
protein efflux. The failure of edema to develop despite evidence for an
increased fluid filtration may be attributed to the effectiveness of the
lymphatic system to drain the excess interstitial fluid.

Clearly, serotonin does not greatly increase microvascular permea-
bility to plasma proteins in a pressure-independent manner as does
bradykinin and histamine. The latter two agents markedly increase both
lymph flow and lymph protein concentration. However, there may have
been an increase in protein efflux by serotonin. If. for example, fluid
filtration increased without an increase in protein efflux an increased
lymph flow would be predicted while lymph protein would be expected to
decrease. Since data from this study provide indirect evidence for an
increased fluid filtration resulting in an increased lymph flow with no
change in lymph protein concentration. it must be assumed that the rate
of protein efflux was increased proportionately. This would simply
indicate that the increased fluid filtrate, owing to the rise in micro-
vascular pressure, contained some protein.

In contrast to the above findings in the dog. Rowley (59),.
and Majno and his associates (4.38.39) have shown in the rat that the
magnitude of serotonin-induced edema formation is directly related to

the increase in microvascular hydrostatic pressure. Rowley found that

39

intravenous infusion of Evans blue or radio-opaque carbon particles
followed by subcutaneous administration of serotonin induced an intense
bluing of the paw skin with a lesser degree of bluing being observed in
the subcutaneous areolar tissue. Direct microscopic observation of
tissue infused with carbon particles indicated that these particles
penetrated the venular walls. Majno's group observed the same serotonin-
induced permeability changes using as a test organ the rat cremaster.

0n the basis of the size of the carbon-labelled lesions, Majno concluded
that serotonin in this test tissue increases vascular permeability much
more than does histamine. Ebert and Graham (l4)working with

serotonin in the rabbit ear chamber failed to see the formation of edema
in response to this agent. 0n the other hand. it has been shown that
histamine produces marked edema formation in this species. Roddie and
his associates (56) have shown that intra-arterial administration of
serotonin (low doses) into the human forearm produces an increase in hand
and forearm total volume. They were unable to determine if this was

due to increased vascular volume or increased fluid accumulation in the
interstitium.

Data from constant-flow studies in the dog forelimb support the
conclusions that serotonin increases microvascular hydrostatic pressures
and fluid filtration without generating noticeable edema formation.
However. there was a rise in extravascular fluid volume under this con—
dition. Small vein pressures increased much more markedly at constant
inflow than they did at natural inflow, largely due to a better main-'

tained flow. This finding again demonstrates the differences between

40

serotonin and histamine and bradykinin, both of which under similar
conditions fail to alter small vein pressure.

Daugherty et al. (11) have examined the resistance alterations pro-
duced by both intravenous as well as intra—arterial infusion of serotonin
(low doses). Their findings suggest that at constant flow total forelimb
resistance increased due to a marked increase in total skin resistance
despite a fall in total muscle resistance. Most of the increase in skin
resistance was the result of arterial constriction. The reduction in
skeletal muscle resistance was likely the result of a passive increase
in vessel caliber subsequent to an increase in the transmural pressure
gradient. This finding is supported by the fact that flow shifted from
skin to skeletal muscle when inflow was held constant.

In the current study. total resistance increased relative to control
in both the naturally-perfused and the constantly-perfused forelimbs in
response to both low and high doses of intra-arterially administered
serotonin (15 and 150 pg base/min). This response was primarily the
result of intense constriction in the skin vasculature. Total skin
resistance in skeletal muscle vasculature was somewhat reduced at the
same time. The marked increase in total skin resistance is the result of
a proportionately greater constriction of the large artery and large vein
segments directly mediated by serotonin. At natural flow. the high dose
infusate also produced a marked increase in skin small vessel resistance.
This increase was likely passive, the result of a reduction in the trans-
mural pressure gradient in this segment related to a greatly attenuated

blood flow.

41

The degree of dilatation in the muscle small vessel and large venous
segments was nearly proportional in response to the high-dose infusate at
both natural and constant flows. The large artery segment of skeletal
muscle was little affected by either dose. The passive reduction in
skeletal muscle total resistance is due to equally proportionate decreases
in small vessel and large venous resistances subsequent to a shift in
flow from skin to skeletal muscle.

Skin and skeletal muscle resistance responses to local infusion of
serotonin into the innervated canine forelimb indicate that this agent
mediates directionally-opposite effects in these two parallel-coupled
vascular beds when inflow is held constant. The reduction in skin blood
flow was almost directly proportional to the increase in skeletal muscle
blood flow. Our findings lend support to reports by both Haddy (25.30)
and Daugherty (ll) concerning the redistribution of blood flow in response
to local infusion of serotonin into the dog forelimb and hindlimb.

In conclusion. data gathered from this investigation suggest that
although serotonin may produce marked hemodynamic responses when infused
locally into the dog forelimb, it does not produce significant edema
formation in this species. This finding is evidenced by two facts:

(1) forelimb weight failed to increase significantly in response to sero-
tonin. and (2) the concentration of protein in the lymph failed to change

significantly relative to control values.

SUMMARY

Intra-arterial administration of serotonin (15 and 150 pg base/min)
infused into the naturally-perfused canine forelimb. decreased blood
flow. increased the rate of lymph flow, increased small vein pressure
in skin and skeletal muscle, produced varied effects on skin and skeletal
muscle total and segmental vascular resistances, and failed to increase
vascular permeability to plasma proteins and total forelimb weight.

The reduction in forelimb blood flow was the result of an increase in
total forelimb vascular resistance. manifested primarily as an increase
in skin total and segmental resistances, despite no change or a slight
reduction in muscle total and segmental resistances. Marked increases

in skin and skeletal muscle small vein pressure accompanied by a very
significant increase in the rate of lymph flow support the conclusion
that net fluid filtration did occur in response to serotonin. Therefore.
the failure of the limb to gain weight must be ascribed in part to the
fact that the transudate was not allowed to accumulate in the inter~
stitium but rather was removed via the lymphatics.

In the face of a better maintained blood flow (constant perfusion)
there was a slight but significant increase in forelimb weight. This
weight gain cannot be ascribed to an increase in vascular volume even
though total flow was not allowed to diminish. This conclusion is

supported by the fact that at constant flow total forelimb resistance was

42

43

markedly elevated and total segmental vascular resistances were either
elevated (total large artery). reduced (total small vessel). or un-
affected (total large vein). Again, the marked increase in microvascular
hydrostatic pressure suggests that the weight gain was due to net fluid
filtration. These same doses of serotonin appear to shift blood flow
from skin to skeletal muscle when inflow is held constant. This observa-
tion can be explained on the basis that skin resistance was markedly
increased while skeletal muscle resistance remained unchanged.

At no time was there any visible evidence of edema formation. nor
could it be inferred from palpation of the forelimb.

These data clearly suggest that serotonin. unlike histamine and
bradykinin, does not generate the formation of edema in the canine fore-
limb even though it may produce hemodynamic changes which are compatible
with edemogenesis, i.e.. an increase in capillary hydrostatic pressure

and an increase in net fluid filtration.

' ...a

10.

11.

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