ROLE or METABDUCALLY LINKED f "
cumms m LOCAL REQUMTION . ,i A _; -—
OF BLOOD nownmmcnou or ,

w OXYGEN AND (mason 010mm ‘3
‘susmmm ‘EXERCiSE .HYPEREMIA OF-

THE CAE‘UNE moms MUSCLE .. v ,  f¢ 1 h

Dissertation for the Degree of PM.

MECHEGM STATE UNWERS-[TY'V ' ‘

' NERD. FRANCE3 VSTGWE
1974 -

 

  

”L
LIBRARY 1
Michigan State
University

     

This is to certify that the

thesis entitled

Role of Metabolically Linked Chemicals in Local Regulation
of Blood Flow: Interaction of Oxygen and Carbon Dioxide
in Sustained Exercise Hyperemia of the Canine Gracilis Muscle

presented by

David Francis Stowe

has been accepted towards fulfillment
of the requirements for

Ph.D. Physiology

degree in

 

 

mg- a?

M r professor

Date 7/26/74

0-7539

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and an inCI

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trations 0.
hYdY‘Ogen a,
venous eff'
shows Erlhar
other VaSCt
the Concen'
SWoundm.
the blood .
metaboliswz
fairly Con

Th
”5'99" and

exerClSe h

ABSTRACT
ROLE OF METABOLICALLY LINKED CHEMICALS IN LOCAL
REGULATION OF BLOOD FLOW: INTERACTION OF
OXYGEN AND CARBON DIOXIDE IN SUSTAINED

EXERCISE HYPEREMIA OF THE CANINE
GRACILIS MUSCLE

By

David Francis Stowe

Exercise of skeletal muscle is associated with vascular dilation
and an increase in muscle blood flow. The exact mechanism(s) responsi-
ble for the vasodilation is uncertain but it is known that the concen-
trations of certain metabolically linked chemicals (e.g., oxygen,
hydrogen and potassium ions, and osmotically active particles) in the
venous effluent from exercising muscle are altered and that this blood
shows enhanced vasodilator properties when bioassayed in muscle.and
other vascular beds. The metabolic theory proposes that changes in
the concentration of one or more of these chemicals in the tissue fluid
surrounding the resistance vessels result in active vasomotion so that
the blood flow is adjusted to a level more appropriate to the rate of
metabolism. In this way the ratio of blood flow to metabolism remains
fairly constant and local tissue homeostasis is insured.

The purpose of this study was to evaluate the contributions of
oxygen and hydrogen ions singly and in combination, to steady-state

exercise hyperemia in canine gracilis muscle. Studies were carried

 

 

 

 

out on th
anesths“
what exte'
ing the e:
pH of the
muscle ser
the venous
rection of
by alterin
placed in
In
tension an.
While moni‘
mixture of
the arteria
Ironitored.
decreased ,3
during vari
POIe 0f 56v!
Steady-stat.
and the C0”
the blood (1

The

of venous bl

n07? David Francis Stowe
(/00

out on the collateral free, denervated muscle with the animal
anesthetized. One series of experiments was designed to determine to
what extent the enhanced vasodilator activity of the venous blood drain-
ing the exercising muscle is attenuated when the oxygen tension and/or
pH of the blood are selectively corrected. The contralateral gracilis
muscle served as the bioassay organ to test the vasodilator activity of
the venous effluent before, and during exercise, without and.with cor-
rection of the P02 and pH of the effluent. The latter was accomplished
by altering the mixture of gas ventilating a gas exchange permeator
placed in the venous effluent.

In another series, single and simultaneous reductions in oxygen
tension and pH were produced in blood perfusing resting gracilis muscle
while monitoring resistance. This was accomplished by altering the
mixture of gas ventilating a lung (taken from another dog) placed in
the arterial supply. Both arterial and venous effluent P02 and pH were
monitored. In another series the degree of vasodilation produced by
decreased P02 and pH in resting muscle was compared to that observed
during various degrees of exercise. Finally, to assess the relative
role of several metabolically linked factors in the maintenance of
steady-state exercise hyperemia, the blood flow, gas tensions, pH,
and the concentrations of potassium ions and water, were measured in
the blood draining skeletal muscle during 60 minutes of sustained
stimulation and during step increases in the level of stimulation.

The findings indicate that (a) the enhanced vasodilator activity

of venous blood seen during steady state exercise can be completely

 

 

 

 

abolishec

 

ore-exert;
pH in the i
the venous
produces c
equal that
concentra:
sustained
the only n1
cise is a

better wit

 

The
activity 0-
decreases .
OXYQEH and
taining Bx:
involved,
the bIOOd
ured durin
that SEen
miner F016

hypflemj d I

David Francis Stowe

abolished by simultaneously returning the oxygen tension and pH to
pre-exercise levels, (b) simultaneous reduction of oxygen tension and
pH in the blood perfusing resting muscle, so as to produce levels in
the venous effluent similar to those observed during heavy exercise,
produces marked vasodilation. The dilation, however, does not quite
equal that seen during heavy exercise, (c) the increased potassium
concentration and osmolality in the venous effluent disappear during
sustained exercise but the changes in flow, pH and P02 do not, and (d)
the only measured change in the venous effluent during mild steady exer-
cise is a fall in P02; during graded exercise, resistance correlates
better with P02 than with pH, potassium, or osmolality.

These studies indicate that in the steady state the vasodilator
activity of venous blood from exercising muscle is due to simultaneous
decreases in oxygen tension and pH. Therefore, the concentrations of
oxygen and hydrogen ions may be the most important determinants main-
taining exercise hyperemia in skeletal muscle. Other factors may be
involved, however, since induced reduction of oxygen tension and pH in
the blood draining resting muscle to levels comparable to those meas-
ured during heavy exercise, failed to produce dilation comparable to
that seen during heavy exercise. Finally, these studies suggest only
minor roles for potassium and osmolality in steady-state active

hyperemia of skeletal muscle.

ROLE OF METABOLICALLY LINKED CHEMICALS IN LOCAL
REGULATION OF BLOOD FLOW: INTERACTION OF
OXYGEN AND CARBON DIOXIDE IN SUSTAINED
EXERICSE HYPEREMIA OF THE CANINE
GRACILIS MUSCLE

By

David Francis Stowe

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Physiology

1974

DEDICATION

To my fluent“ who 6ha/LQ. my duper/Lott]

and to those who am; not hephoachfiul.

ii

 

 

 

 

Q
I

sincere a,
his suppo
about the
Research I
course of
other meri
Burnell H
Bowdler,
S
his stlmu
T
of Mrs. J
Chief Sur
MTS- Marg
T
a Specie]

adminlstr

ACKNOWLEDGMENTS

The author wishes to take this opportunity to express his
sincere appreciation to Professor Joe M. Dabney, Academic Advisor, for
his support and encouragement and for the many stimulating discussions
about the teaching of physiology, and to Professor Jerry B. Scott,
Research Advisor, for his invaluable direction and advice during the
course of these investigations. Appreciation is extended also to the
other members of the writer's Guidance Committee, Drs. Francis J. Haddy,
Burnell H. Selleck, Donald K. Anderson, Theodore M. Brody and Anthony J.
Bowdler.

Special thanks are extended to my colleague, Dr. T. Lon Owen for
his stimulating ideas and assistance during all phases of this study.

The author also acknowledges the skillful technical assistance
of Mrs. Josephine Johnston, Laboratory Supervisor, Mr. Booker Swindall,
Chief Surgical Technician, Mr. George Gamble, Surgical Technician and
Mrs. Margo Smith, Laboratory Technician.

To Mrs. Amylou Davis, Graduate Affairs Secretary, is extended
a special debt of gratitude for assistance in overcoming awesome

administrative details.

 

 

 

 

 

LIST OF F

INTRODUCTI

Genera
Local
The Me

MUS”-

   

 

SURVEY OF

STATEMENT .
METHODS .

Bloasse
Exercis

RESULTS ,

 

Bioass.
EXercis

DISCUSSION
sunnn Ans

BIBUOGRAPH

TABLE OF CONTENTS

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

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

General Description of Vascular Control Systems .......
Local Vascular Control Systems ................
The Metabolic Theory for Active Hyperemia in Skeletal

Muscle ...........................

SURVEY OF LITERATURE .......................

Local Blood Flow Regulation: Early Studies and Theories . . .
More Recent Evidence for Participation of Specific
Metabolically Linked Chemicals in Exercise Hyperemia . . . .
Vasoactivity of Metabolically Linked Chemicals
Proposed as Mediators . . ............
Recent Chemical and Biological Evidence Supporting
a Role for Metabolically Linked Chemicals

Proposed as Mediators .................

STATEMENT OF PURPOSE .......................
METHODS .............................
Bioassay Studies: Bilateral Gracilis ............
Exercise vs. Mimicked Exercise: Unilateral Gracilis .....
RESULTS .............................
Bioassay Studies: Bilateral Gracilis ............
Exercise vs. Mimicked Exercise: Unilateral Gracilis .....
DISCUSSION ............................
SUMMARY AND CONCLUSIONS .....................
BIBLIOGRAPHY ...........................

iv

 

 

 

 

 

Figure

 

 

Figure

LIST OF FIGURES

Remote and local mechanisms believed to control
vascular smooth muscle tone. Modified from
Folkow (46) ......................

Preparation for constant flow perfusion of

regulatory (RG) and assay (AG) gracilis muscles.

PRG and PAG = perfusion pressures of RG and AG.

VRG and AAG = R6 effluent blood before and after
passage through permeator ...............

Preparation for natural flow perfusion of RG and
constant flow perfusion of AG. Psys = systemic

arterial blood pressure. FRGV = RG vein flow.

VAG = AG effluent blood. PAG: VRG and AAG as

in Figure 2 ......................

Preparation for constant flow perfusion of a single
gracilis (G) muscle. P9 = G perfusion pressure.
AG = Blood perfusing G after passage through donor

lung. VG = G effluent blood ................

Representative tracing from the preparation
illustrated in Figure 2 showing the effects of
ventilating the permeator with various gas mixtures
on PAG and AAG parameters during sustained exercise

of R6. Arrows refer to time of gas exchange .......

Average effects of ventilating the permeator with
various gas mixtures (randomized) on AG resistance

and AAG parameters during sustaineg_exercise (l to

6 Hz, l.6 msec., 6 volts) of R6. X t S.E.M. represents
the mean i standard error of the group mean. P values
relative to pre-exercise controls. Preparation

illustrated in Figure 2 .................

Average of the l2 experiments shown in Figure 6 together
with 5 additional experiments in which the permeator was
ventilated with only one gas mixture. P values relative

to pre-exercise controls .................

Page

38

41

43

47

49

ST

 

-.Ll

Figure

 

 

l2.

 

l0.

ll.

 

Figure Page

8. Average effects of exercise on RG resistance, flow
and effluent blood parameters as a function of
time. Preparation illustrated in Figure 3. P
values relative to pre-exercise controls ......... 52

9. Average effects of ventilating the permeator with
various gas mixtures (randomized) on AG resistance
and AAG parameters during exercise of RG (see Figure 8).
Preparation illustrated in Figure 3. Statistical evalua-
tion employed a 2-way analysis of variance with multiple
comparison among means after the Students-Newman-Keuls
test (145). Any row mean is significantly different
from all other row means unless marked by a common
symbol .............. . ........... 54

lO. Representative data from the preparation illustrated
in Figure 4 showing the effects of ventilating the
donor lung with various gas mixtures on P5 and Ye
parameters. Arrows refer to the onset of vascular
response ......................... 56

ll. Average effects of ventilating the donor lung with
various gas mixtures (randomized) on P5 and V9
parameters. Preparation shown in Figure 4.
P values relative to pre-ventilatory change control . . . 58

12. Average effects of step increases in contraction
frequency and ventilation of the donor lung with
hypoxic, hypercapnic gas on G resistance and V3
parameters. Preparation shown in Figure 4. P
values relative to pre-exercise controls ......... 59

vi

 

 

 

 

Tr
is largel_
According
of local a
various va

ARd arteri

 

Systems, t
Continuall‘
Vascular b

No
conseQuent
demenstrat
pressure .
nerve trar
UNEquiVOCE
0T blood 1
into 5” al

Fi

beheVEd t.

INTRODUCTION

General Description of Vascular
fbhtrol Systems

 

The distribution of blood flow in the peripheral circulation
is largely regulated by alterations of vascular smooth muscle tone.
According to current concepts, synergistic or antagonistic interactions
of local and remote control systems regulate the blood flow to the
various vascular beds by altering the radius of precapillary sphincters
and arterioles. As a result of the balance of local and remote control
systems, the vascular calibers in the different systemic circuits change
continually, depending in part on the function of the particular
vascular bed.

No one explanation for the phenomenon of vasomotion (and
consequently the maintenance of local tissue homeostasis) has been
demonstrated. The remote control mechanisms directed at controlling
pressure are best defined. Several of their specific mediators, the
nerve transmitters and the blood-born vasoactive hormones, have been
unequivocally identified. The mechanisms responsible for local control
of blood flow are, in general, more hypothetical and have been clumped
into an all-inclusive multiple factor theory.

Figure 1 summarizes the remote and local mechanisms which are

[Delieved to control vascular smooth muscle tone. Vasoconstrictor fibers

 

 

 

 

Remote c

 

a. Vosocon
fibr

b. Blood-bc
excitotor
inhibitory
mfluence

 

 

 

c. Vosodilot

Figure 1 .

Remote control LOCGI control

 

a. Myogenically active "pacemaker"

  
 
 
  
 
 

 

a. Vasoconstrictor cells
fibres
’c Local
b. Blood-borne ivosodiloior
excuioiory and metabolites

inhibitory v.

influences

c. Vosodiloior fibres

Figure 1. Remote and local mechanisms believed to control vascular

smooth muscle tone. Modified from Folkow (46).

 

 

 

from t:
catechl
vascule
release
appears
other t
or at t
chemica'
catechol
hormones

bradykin

l
demonstra
Skeletal
TiOW thrc
VdSCUiar
Ter”-POl‘ary
associate
Value UPOI
hierenia_
varied 0V6
DIOportiOH‘

”Veil cons

from the adrenergic sympathetic nerve supply are well known to release
catecholamines locally which act mainly on alpha receptors. In a few
vascular beds, namely skeletal muscle, cholinergic sympathetic fibers
release acetylcholine which causes vasodilatation. The latter response
appears to be mediated through higher brain centers and may serve among
other things to increase blood flow to skeletal muscle slightly before,
or at the onset of, exercise. Blood-born vasoactive hormones and
chemicals elaborated outside the vascular bed, such as the circulating
catecholamines (norepinephrine and epinephrine), some polypeptide
hormones (angiotensin and vasopressin), and the kinins (kallidin and

bradykinin), are also well known to affect vascular smooth muscle tone.
Local Vascular Control Systems

Several forms of local regulation of blood flow have been
demonstrated. Increased metabolic rate produced by activation of
skeletal or cardiac muscle, for example, produces an increase in blood
flow through the activated organ at a constant perfusion pressure; this
vascular response is called active (exercise, functional) hyperemia.
Temporary occlusion of arterial inflow to a vascular bed is usually
associated with a transient increase in blood flow above the basal
value upon release of the occlusion; this response is known as reactive
hyperemia. When perfusion pressure to most systemic vascular beds is
varfied over the approximate range 70 to 200 mm Hg, there is a less than
proportionate change in blood flow. This ability to maintain a rela-

‘tively constant blood flow in face of a varying inflow perfusion pressure

is ca

react

 

regul
bolic

the l

these

necha

 

actiV'
ciatec

(73).

 

 

blood

contin
Propag
tranSm
actiVe
hrex
decree:
Sinila‘

result

 

 

Occlus'
l inW or
i Centrat
35 subs

Changes

is called autoregulation. These local responses, i.e., active and
reactive hyperemia, and autoregulation, are manifestations of the local
regulatory mechanisms to maintain a relatively constant ratio of meta-
bolic rate to blood flow. Thus, like the remote controlling mechanisms,
the local mechanisms act to maintain cardiovascular homeostasis.
Numerous theories or hypotheses have been advanced to explain
these local regulatory responses; the exact nature of the mechanism or
mechanisms responsible remain controversial. Vascular smooth muscle
activity is thought to be controlled locally by chemical factors asso-
ciated with tissue metabolism and myogenic reactions related to stretch
(73). Blood viscosity and tissue pressure may also participate in local
blood flow control (67). It is thought that the larger arterioles with
continuous smooth muscle coats exhibit myogenic activity by cell to cell
propagation of impulses originating in pacemaker cells. A change in
transmural pressure across vascular smooth muscle cells may initiate
active vasomotion as a sort of positive or negative stretch reflex.
For example, exercise may increase tissue pressure which would cause a
decrease in transmural pressure and subsequently produce vasodilation.
Sinfilarly, partial occlusion of the major artery to a vascular bed would
result in a fall in transmural pressure in blood vessels distal to the
occlusion and hence produce vasodilation. Finally, a change in blood
f1<nv or metabolism in a vascular bed is followed by changes in the con-
centrations of substances delivered to the tissue by the blood, as well
as substances produced or released by the tissue. These chemical

changes reportedly result in an adjustment of blood flow to a level

 

 

 

 

the
tic

pY‘E

red

more appropriate to the rate of metabolism. It is now well established
that muscular contraction reduces or abolishes sympathetic vasoconstric-
tion in the vascular bed of skeletal muscle, i.e., local mechanisms
predominate over remote mechanisms during exercise (24, 84, 142-144).
Although the myogenic response to stretch probably contributes
in varying degrees to local blood flow regulation and tissue homeostasis
in different vascular beds (44, l07), a very attractive explanation for
many types of local control is based upon the metabolic hypothesis (63,
65). It is proposed that changes in metabolism or blood flow are
followed by alterations in the concentration of oxygen and vasodilator
metabolites in the tissue fluid surrounding the arterioles; these
alterations result in active vasomotion that adjusts flow to a level
more appropriate to the rate of metabolism. For example, during a
reduction in flow to skeletal muscle, caused by a fall in perfusion
pressure or occlusion of the arterial supply, substrate concentrations
(particularly oxygen) are thought to fall and the concentrations of
certain metabolites to rise in the tissue fluids surrounding the
arterioles and precapillary sphincters. Following a reduction of the
inflow pressure the flow increases to approximately the initial level.
Presumably, the accumulated metabolites or lack of metabolic substrates
act by inhibiting or attenuating the basal contractile state of vascular
smooth muscle cells. Thus, the vascular radius increases and the blood
flow returns to near the control level. In this form of local regula-
tion (autoregulation) the ratio of blood flow to metabolism remains

approximately constant and local tissue homeostasis is assured.

 

 

 

Lil
cor
inc

an:

SD
th.
va:
one
Chi
vat

en!

dor
the
an i
conc
durh
Subst
well a

Likewise, a local increase in metabolism as a result of muscular
contraction, for example, also causes vascular dilation with an
increase in flow (active hyperemia). Again, changes in substrate
and certain metabolite concentrations are believed to be importantly
involved in this response.

The Metabolic Theory_for ActivegHyperemia
in SkeletaTTMUSCTe

 

 

The metabolic hypothesis receives strong support from bioassay
studies and chemical analysis of venous effluent. Bioassay studies show
that the blood draining exercising skeletal muscle exhibits enhanced
vasodilator activity. For example, if active hyperemia is induced in
one muscle vascular bed by stimulating the muscle to contract, thereby
changing its flow-to-metabolism ratio, similar directional changes in
vascular resistance are promptly seen in a bioassay organ (65). This
enhanced vasoactivity of the venous blood undoubtedly reflects to a
large degree alterations in the vasoactivity of the tissue fluids in the
donor organ, suggesting that the concentration of vasoactive agents in
the tissue fluid changes sufficiently during local regulation to have
an effect on blood vessels. Such bioassay studies clearly show that the
concentration of a vasoactive substance(s) is altered in venous blood
during exercise hyperemia of the regulatory (donor) muscle and that the
substance(s) acts on the resistance vessels of the bioassay organ as
\uell as on the regulatory muscle. However, it may be possible for a

substance to escape this criteria if it acts on the vascular smooth

 

 

 

nu:
di‘
re'
the
st'
ves
Pa'
do

to
in
may
as
sex
tre

TEE

IiOn

muscle to effect vasodilation, but does not appear in the effluent in
dilator concentrations due to rapid inactivation, reuptake or limited
release. Also, it must be assumed, with some supporting evidence, that
the effluent concentration of a substance closely reflects the inter-
stitial fluid concentration in the vicinity of the affected resistance
vessels. Although bioassay studies provide compelling evidence for
participation of chemicals in local blood flow regulation, they alone
do not firmly identify any specific mediator.

Chemical analysis of blood draining exercising muscle shows
alterations in the concentration of certain vasoactive chemicals related
to tissue metabolism. Many metabolically linked chemicals have been
implicated in the hyperemia of exercise but the concentration of some
may change coincidentally and not be vasoactive. In order to qualify
as a mediator of active hyperemia any proposed chemical must satisfy
several criteria: (l) The chemical must be found in altered concen-
trations in the tissue fluid or venous effluent during the vascular
response. For example, it is well known that the oxygen tension and pH
of blood draining exercising muscle drops, concomitant with increased
blood flow (65). (2) The substance must be vasoactive within the range
of plasma effluent or tissue concentrations found during local regula-
tion when its concentration is artificially altered in the blood or
fluid perfusing the resting organ (65). For example, the observed
increase in hydrogen ion concentration of the venous effluent during
exercise hyperemia, when mimicked in the venous effluent of the muscle

durfing rest, must elicit a vasodilator response. (3) There must be a

 

be
ini
whi

EXE

 

COT

 

reasonable temporal relationship between the concentration of the agent
in the tissue fluid or venous blood and the change in resistance (65).
For example, the concentration of the substance in the effluent must

be rapidly altered at the onset of exercise, if it is involved in the
initiation of exercise hyperemia, and/or must be continually altered
while the exercise continues, if it is involved in the maintenance of
exercise hyperemia. Similarly, if the hyperemia or vasodilation
continues unabated, while the concentration of the substance is
artificially altered in the blood perfusing the resting organ, then
the substance may be involved in the maintenance of hyperemia.

To be an actual effector the substance must be shown to act
directly on the vascular smooth muscle affecting resistance and not
through release of intermediary substance(s) (1). It has been postu-
lated, for example, that oxygen lack may have either a direct effect on
vascular smooth muscle cells to attenuate oxidative phosphorylation and
thus the contractile apparatus (31, 34), or that it stimulates the
breakdown and release of various metabolites (16) such as adenosine
or'lactic and pyruvic acid which are dilators.

Several factors recently proposed in local regulation of blood
'flow which have not withstood the test of these criteria, at least in
actfive hyperemia, are inorganic phosphate (6, 72) and prostaglandins
(llDS). Several metabolically linked chemicals, however, remain as being
nKyre or less important factors in the regulation of blood flow during
exercise. Currently, the most important of these appear to be oxygen,

hydrogen ions, carbon dioxide, potassium ions, adenosine and its

 

 

 

TlU
TC
CE‘.

in\

Sir
the
res

fac

vasl
SOD:

EXEI

nucleotides, and osmolality (see reviews l, 7, 17, 64, 65, 67, 68,
l07, 108, 127, 151). Although all of these chemical factors satisfy
certain of the outlined criteria, and thus may be directly or indirectly
involved in exercise hyperemia, it seems highly unlikely that any one
of these accounts completely for active hyperemia in skeletal muscle.
Since the tissue and blood concentrations of several chemical factors
change during the hyperemia, it has been postulated that the vascular
responses result from the combined actions of two or more chemical
factors (65). Some studies have shown that simultaneous changes in
the concentration of vasoactive chemicals in the blood perfusing skel-
etal muscle interrelate in such a way as to reinforce their individual
vascular effects (l39-l44). Also, recent studies have suggested that
some factors may be important dilators during the initial phase of
exercise, but not during later phases, and vice-versa (68, refs.).

The relative importance of the various dilator factors may
differ depending on the type of skeletal muscle and the intensity of
work performed. For example, comparative studies of blood flow in red
(slow, tonic) soleus and white (fast, phasic) gastrocnemius muscles in
“theecat have shown that the soleus has a resting flow twice that of the
gastrocnemi us but exhibits a smaller exercise hyperemia (45, 75, 76,
125). During maximal dilation soleus flow increases about three times
resting flow, whereas gastrocnemius flow increases about 10 to 15 fold.
Red muscle contains more myoglobin and mitochondria and gets most of its
energy from oxidative phosphorylation. Apparently, the smaller increase

in “flow provides an oxygen delivery and exchange in red muscle sufficient

 

 

 

 

for n
high!
would
other
muscle
concen

higher

several
hyperer
evaluao
ions in
Of the c
the enha
contrala
natural .
Oxygen tr
ievels,
muscle) t
DH were m
Evaluate
exercise i
extent FEC
”Sting gr

ID

for maintaining a predominately aerobic metabolism even in situations of
high work loads, whereas in white muscle, even low rates of contraction
would lead to "oxygen debt" and a greater release of lactic acid and
other metabolites. Perhaps the vascular bed of predominately white
muscles dilates to a greater extent during exercise because the tissue
concentration of oxygen is lower and the concentration of metabolites
higher.

The purpose of this study was to elucidate further the role of
several of the metabolically linked chemicals believed to mediate the
hyperemia (or vasodilation) of exercise. Experiments were devised to
evaluate the relative and combined contributions of oxygen and hydrogen
ions in the metabolic control of blood flow during steady-state exercise
of the canine gracilis muscle. Two series were designed to determine if
the enhanced vasodilator activity of venous blood (by bioassay in the
contralateral muscle) from exercising gracilis muscle (perfused at
natural or constant flow) can be attenuated or abolished when the
oxygen tension and/or pH of the blood are returned to the pre-exercise
levels. In the second series (natural flow perfusion of the exercising
Inuscle) the blood flow and the tensions of oxygen and carbon dioxide and
pH were measured during a prolonged period of exercise (60 minutes) to
evaluate the role of these factors in the maintenance of steady-state
exercise hyperemia. A third series was designed to determine to what
extent reduction of oxygen tension and/or pH of the blood perfusing
resting gracilis muscle produce vascular dilation. In a fourth series

simultaneous reductions in oxygen tension and pH were made in resting

ll

gracilis muscle in order to mimic venous levels like those observed in
blood from exercising muscle. The degree of vascular dilation produced
in the resting muscle was compared to that produced in the exercising
muscle at various levels of nerve stimulation. The potassium and
osmolal concentrations were measured in gracilis muscle venous blood
during these series to evaluate their roles in the genesis of the

resistance changes observed.

SURVEY OF LITERATURE

Local Blood Flow Regulation: Early
Studies and Theories

Gaskell (1877) made the first quantitative description of the
flow changes that follow skeletal muscle contraction and suggested that
the blood supply to active tissues could be regulated by chemical alter-
ations affecting vascular tone (49). His further suggestion in 1880
that intermediary metabolites play a role in local regulation of blood
flow probably originated from his observation that lactic acid painted
on blood vessels produced dilation (50). Roy and Brown (1879) found
evidence that the blood vessels carrying nutrients to the tissues con-
tract or expand in accordance with the blood flow requirements of the
tissues (130). They suggested that the quantity of oxygen or dilators
determine the degree of vascular diameter. In 1901, Bayliss (9) first
hinted at a role for pH in local regulation. He observed an increase
in blood flow in the lower extremities of the frog when the perfusate
was changed from a Ringer's solution with normal carbon dioxide to one
saturated with carbon dioxide. But Bayliss' greatest contributions in
the area of local regulation were his studies on reactive hyperemia,
which formed the basis of the myogenic hypothesis (10, 11). He
proposed that the vasodilation in the dog leg following relief of

circulatory arrest produced by compression of the aorta for 8 seconds

T2

 

 

 

 

 

Vi

T3

was due to collapse of the arterial tree and lack of the stimulus of
stretch acting upon the smooth muscle of the vessel wall. He did not
think that deprivation of blood flow for 8 seconds could cause an
appreciable accumulation of metabolites or lack of substrates. Lewis
and Grant reported in 1925 that local vasodilation occurred in response
to occlusion of vessels in the forearm and that this effect was inde-
pendent of local nervous reflexes because the dilation was unaltered
after nerve degeneration (96). They concluded that the vasodilation
took place during the period of occlusion and that it was due to the
action of metabolites, continuously formed and accumulating; the longer
the occlusion, the greater the accumulation of metabolites, and the
larger the vasodilation after release. Since the flush was bright red
they thought that the dilation was not due to anoxia, but suggested
histamine as a cause. Markwalder and Starling in 1913 (102), Dale (26),
and Krogh (95) in 1929 also suggested that vasodilator metabolites accu-
mulate in tissues and cause dilation. Krogh (95) found that decreasing
the oxygen saturation and increasing carbon dioxide content caused
vasodilation in rabbit ears, but failed to suggest that oxygen lack
might have an effect on the circulation.

More reports on the possible roles for oxygen lack and increased
hydrogen ion concentration in local regulation began to appear around
the turn of the century. Verzar (1912) reported that decreasing the
flow to resting cat skeletal muscle decreased oxygen uptake, although
venous blood from that muscle still contained substantial amounts of

0XVgen (153). He suggested that oxygen consumption is limited by the

 

14

blood flow. Fleisch _t_gl,, in 1932, observed that local hypoxemia

did not produce dilation in the cat hindlimb until the hypoxemia was
severe but that a small increase in carbon dioxide concentration pro-
duced dilation (42). Therefore, they suggested that carbon dioxide,

but not oxygen, was important in local regulation. In 1925, Hilton

and Eichholtz (69) and in 1926, Gremels and Starling (58) using heart-
lung preparations, found that perfusion of the coronaries with de-
oxygenated blood (about 40% saturation) produced a large increase in
coronary blood flow. Flow also increased when lactic acid or carbonic
acid was added to the blood perfusing the coronaries (58). Anrep and
Saalfeld, in 1935, first used a bioassay technique to study active
hyperemia (3). They perfused a dog gastrocnemius muscle with blood
either from the arterial circulation or with its own venous blood
collected during rest or rhythmic muscular contraction. Marked hyper-
emia accompanied the contraction. Neither venous blood re-perfused from
the resting muscle nor blood collected during hyperemia and later re-
perfused had vasodilator activity; but blood collected during vasodi-
lation (restricted arterial inflow) and later re-perfused was a powerful
vasodilator. They concluded that the vasodilator action must have been
due to non-labile metabolites, but not oxygen since blood "reoxygenated"
after collection during exercise at constant flow was still a good vaso-
dilator. Kramer gt_gl, (1939) noted that during steady-state exercise
of the dog gastrocnemius muscle, the muscle blood flow was directly
proportional to its rate of oxygen consumption (94). This was confirmed

by Pappenheimer (1941) in the isolated perfused dog hindlimb (119).

15

Jalavisto gt_gl,, in 1948, measured the venous oxygen saturation and

the blood flow to the gastrocnemius muscle of the dog during and follow-
ing activity. They found that blood flow increased during the recovery
period roughly in proportion to the oxygen deficit incurred during the
period of muscular activity (78). This was similarly observed by
Hilton (70).

In a typical experiment, Gollwitzer-Meier (1950) observed a
0.06 unit fall in pH of venous blood and a rise in flow to 370% of the
control value with stimulation of the canine gastrocnemius for 3 minutes
(54). Her study showed no correlation between venous blood pH during
exercise and the magnitude of the hyperemia, but nevertheless suggested
a possible minor role for hydrogen ions in active hyperemia. Kester gt
21, (1952) observed a 100% rise in blood flow when the pH of the dog
hindlimb blood was locally reduced from 7.4 to 6.5 with buffered solu-
tions (81). Deal and Green (1954) found that intra-arterial injections
of solutions ranging in pH from 7.4 to 2.0 increased blood flow in the
dog hindlimb (30). Diji (1959) observed dilation in hand vessels with
high carbon dioxide application (32).

Studies implicating a role for potassium in active hyperemia
began to appear in the thirties. Baetjer, in 1934, observed an increase
in the concentration of potassium in the blood draining cat skeletal
muscle~during activation by motor nerve stimulation and during cross
clamping of the aorta (4). Fenn (1937) reported a loss of potassium
from cat, rat and frog skeletal muscle during exercise (41). In 1938,

Katz and Linder found that increasing the potassium ion concentration

16

in blood perfusing the coronary circulation from 1.1 to 1.7 times the
normal concentration produced a 52% increase in coronary blood flow,
while increases from 1.5 to 2.5 times normal decreased coronary blood
flow 63% (80). Dawes (1941) was the first to suggest a role of potas-
sium in exercise hyperemia; he observed that small amounts of KCl (up
to 10 mg in a 1% solution) injected locally increased canine or feline
hindlimb flow (29).

With regard to osmolality, Barcroft and Kato, in 1915, suggested
that the increase in metabolism during skeletal muscle exercise leads to
an increased number of osmotically active particles in the tissue (8).
Years later, Marshall and Shepard (1959) found that rapid local injec-
tions of 2 m1 of 10-20% NaCl or 25—50% dextrose solutions into the
femoral artery of the dog produced a prolonged two or three fold
increase in flow through the hindlimb (103). Read gt_gl,, in 1960,
observed that rapid intravenous injections of 1500 mOSm/kg solutions,
produced peripheral vasodilation in dogs (124). Overbeck g£_gl,
demonstrated in 1961 that locally increasing forelimb osmolality by
100 mOSm/kg decreased forelimb vascular resistance to 71% of the control
when flow was held constant (117). A direct physiological role for
tissue osmolality in vascular control was first suggested by Mellander

in 1967, when he presented evidence that venous osmolality was causally

linked to the hyperemia of exercise (106)-

17

More Recent Evidence for Participation of
Specific Metabolically Linked Chemicals
in ExerCise Hyperemia

 

 

 

During the past 25 years a more systematic approach has been
made to identify some of the metabolically linked vasoactive chemicals
responsible for the genesis and maintenance of the hyperemia of exercise.
From evidence based on the criteria outlined in the introduction for
involvement of any particular factor, three general conclusions have
gradually emerged over the past few years (65, 67, 68). First, no
single metabolic factor appears to be able to explain exercise hyperemia;
i.e., probably a combination of factors are involved. Second, the con-
tribution of the various factors appears to change with the duration and
intensity of exercise. Third, the relative contribution of the various
factors may vary depending on the type of skeletal muscle (red, white or
mixed) and the species.

The following two sections review evidence for the roles of
oxygen, carbon dioxide, hydrogen ions and potassium ions and osmolality
in exercise hyperemia. The first section relates to studies which demon-
strate that vasodilation occurs when the concentrations of these vaso-
active chemicals are altered in the arterial inflow of resting muscle
vascular beds. The second section reviews evidence for participation
(If these factors in exercise hyperemia based on bioassay and chemical

analysis of the tissue fluid or venous effluent during muscle contraction.

l8

Vasoactivity_of Metabolically Linked
Chemicals Propgsed as Mediators

Qxyggg, Investigations by Fairchild e;_gl, (40), Crawford.;t.;L.
(25) and Ross g5_gl, (128) added substantial support to the theory that
moderate oxygen lack can cause vascular dilation. Ross g; 31, (128)
showed that perfusing the resting dog hindlimb with blood in which the
oxygen saturation was reduced stepwise to 0% caused up to a 3 to 4 fold
increase in blood flow. The vasodilator effects of hypoxemia were
verified in the dog forelimb by Daugherty gt_gl, (28) and Scott g§_gl,
(134). It soon became evident that oxygen lack was importantly involved
in, but could not wholly explain the vasodilation of exercise. Altera-
tions of oxygen content over the range of normal arteriovenous differ-
ences had little effect on the dog forelimb and hindlimb vascular beds
as reported by Molnar §t_gl, (113). Stainsby and Otis (149) found that
oxygen uptake by resting dog skeletal muscle was not altered by changes
in blood flow except when the blood flow was reduced below a critical
value. Daugherty e§_gl, (28) observed that dog forelimb vascular
resistance was unchanged when the oxygen tension in the perfusate was
decreased from 120 to 30 mm Hg, but decreased 19% when the perfusate
oxygen tension was reduced to 8 mm Hg.

More definitive studies on the relative role of oxygen lack in
exercise hyperemia have appeared. Ross gt_gl, (126) showed that the
hyperemia accompanying contraction of the canine gastrocnemius was
greater than that occurring at rest during perfusion with hypoxic blood,

even though the effluent blood oxygen tensions were the same. They

19

found that during heavy exercise the increase in flow averaged 173%
with a venous oxygen tension of 25 mm Hg, whereas venous blood perfused
during rest resulted only in a 56% increase in blood flow with a venous
oxygen tension of 23 mm Hg. Moreover, Scott g§.gl, (136) demonstrated
that constant flow perfusion of the dog hindlimb with hypoxic blood
produced a moderate reduction in vascular resistance, but that the drop
in resistance during exercise was larger even though the venous oxygen
tension was not permitted to fall below the level immediately preceding
exercise by the addition of oxygen via a donor lung interposed in the
arterial supply. They found that the resting hindlimb showed a 17%
drop in resistance when it was perfused with hypoxic blood so that the
venous oxygen tension was 10 mm Hg. Increasing the oxygen tension of
the perfusing blood during exercise (6 Hz) did not allow the venous
oxygen tension to fall below 17 mm Hg; this procedure produced a 36%
drop in resistance. In a series of papers, Skinner and his colleagues
(142-144), reported that perfusion of the resting dog gracilis with
hypoxic blood produced only a slight drop in vascular resistance, but
perfusion with hypoxic, hyperkalemic, hyperosmotic blood increased both
the rate and magnitude of the vasodilation. A consideration of these
studies suggests, therefore, that oxygen contributes substantially in,
but is not a complete explanation for exercise hyperemia.

The mechanism by which hypoxia alters vascular smooth muscle
tone is not known. That oxygen acts causally through a direct effect
on vascular smooth muscle to alter tone has been supported by many

investigators (21, 31, 40, 69, 128, 141). The studies of Carrier t 1.

20

(21) and Detar and Bohr (31) indicate good correlation between
mechanical tension of the smooth muscle of isolated arterial segments
and oxygen tension. Honig (75) has suggested that the oxygen sensitiv-
ity of vascular smooth muscle may be the result of an inhibition of
smooth muscle myosin ATPase by inorganic phosphate and AMP. On the
other hand, others have proposed that oxygen lack exerts an effect
secondary to altered parenchymal tissue metabolism and release of
vasodilator substances (14, 15, 34, 35). Berne (14, 15) observed
breakdown products of adenine nucleotides in coronary sinus blood during
cardiac hypoxia and proposed the hypothesis that oxygen lack leads to
myocardial release of adenosine, a potent vasodilator in muscle. He
suggested a similar hypothesis for skeletal muscle vascular beds. More
recently, Berne gt_gl, (16) found that the tissue concentrations of ADP,
AMP, IMP, adenosine, inosine and hypoxanthine in rat skeletal muscle
rose during exercise, especially when performed under ischemic Condi-
tions. Dobson §t_al, (33) examined the effluent draining dog hindlimb
during severe, ischemic exercise and found adenosine but not adenine
nucleotides.

Hydrogen ion. A causal relationship between pH and blood flow
has been often demonstrated by locally altering the pH of blood perfus-
ing resting muscle and noting the change in vasoactivity. Local
increases in hydrogen ion concentration have been produced by either
locally infusing acidic solutions or by increasing the carbon dioxide
tension or content of the blood. Molnar gt_al, (114) found that intra-

brachial infusion of various acids, which produced a fall in forelimb

21

venous pH on the average from 7.39 to 6.85, decreased small vessel
resistance about 43%. Zsoter _t_al, (154) observed that a reduction

of femoral venous blood pH from 7.4 to 7.2 by local infusion of acid
solutions was associated with a 20% increase in canine hindlimb flow.
Daugherty gt_al, (28) altered the pH of blood perfusing the dog forelimb
by changing the ventilatory gas mixture of an extracorporeal lung placed
in series with the forelimb. They observed that severe, local hyper-
capnic acidosis reduced pH from 7.58 to 7.19 and decreased forelimb
resistance 24%. In a study by Kontos gt_§1, (88), human forearm
resistance decreased 43% on increasing venous carbon dioxide tension
from 40 to 48 mm Hg by intrabrachial infusion of a saline solution with
a carbon dioxide tension greater than 600 mm Hg. In another study by
Kontos gt_gl, (89), intra-arterial infusion of acid phosphate buffer
solutions produced a fall in forearm venous blood pH from 7.34 to 7.24
and a rise in carbon dioxide tension from 42 to 52 mm Hg. This was
accompanied by a 170% increase in forearm blood flow. Kontos (90) and
Kontos gt_al, (91) more recently reported that hypercapnic acidosis
produced a more pronounced vasodilation in human forearm than in the
canine gastrocnemius muscle and that in man it was dependent on
increased carbon dioxide tension, rather than decreased pH; this
suggested the possibility that its action was brought about by decreased
pH within vascular smooth muscle cells. Radawski gt_al, (123) compared
skin and muscle vascular responses to various carbon dioxide tensions

in the dog forelimb and reported that large reductions in arterial pH

produce only a small dr0p in muscle vascular resistance and a moderate

 

22

dr0p in skin vascular resistance. Emerson gt_al, (39) recently
demonstrated that large stepwise decreases in pH in blood perfusing

dog gracilis muscle by intra-arterial infusion of isotonic lactic acid
solutions produced only small stepwise decreases in muscle vascular
resistance. The above studies indicate, therefore, that the hydrogen
ion or carbon dioxide is locally vasoactive, high concentrations causing
vasodilation, but that the induced change in pH must be quite large to
produce substantial changes in resistance.

Potassium ion. Since Katz and Linder (80) and Dawes (29) early
suggested that potassium is a vasodilator, many studies have shown sub-
sequently that the resistance to flow decreases when potassium salts are
infused into the arterial supply of the resting muscle, so that the
venous plasma potassium ion concentration is elevated over the approx-
imate range of 4 to 10 mEquiv/l (20, 23, 38, 48, 53, 60, 83, 85, 98,
117, 129, 132, 135, 139-144). Furthermore, a decrease in the local
plasma potassium concentration below approximately 4 mEquiv/1 (2, 23,
‘60, 62, 140) and an increase above approximately 10 mEquiv/l (29, 38,
‘48, 86) causes vascular constriction. Emanuel gt_al, (38) found that
local infusion of 0.25 to 1.75 mEquiv K+/min, as the chloride salt,
produced dilation mainly in the small vessels of the dog forelimb.
Overbeck gt_gl, (117) observed a 22% fall in forelimb resistance during
intrararterial infusion of isotonic KCl into the dog forelimb; potassium
iCNI concentration was estimated to have increased 1.4 mEquiv/1 in the
periHJSing blood. Studies by Glover gt_gl, (53) and Lowe and Thompson

(98) showed that local intra-arterial infusion of KCl increased human

 

23

forearm blood flow. Kjellmer (83, 85) reported that local infusion
of isotonic KCl, in amounts sufficient to raise feline calf muscle
venous effluent potassium by 100%, produced a 40% fall in vascular
resistance.

Using the isolated dog gracilis muscle preparation, Skinner
and his group observed that moderate hyperkalemia (inflow potassium
ion concentration of 6.5 mEquiv/1) produced vasodilation (77% of
control), but to a lesser degree than that produced by muscular
contraction (35% of control) (140, 141). However, for any degree
of hypoxia, the degree and rate of vasodilation was greater or less
depending on the concomitant potassium ion concentration and level of
osmolality (143, 144). They found that blood, altered in a reservoir
to make it oxygen deficient (41% saturated), hyperosmotic (352 mOSm/kg),
and hyperkalemic (6.9 mEquiv/1), and perfused into a gracilis muscle,
produced greater vasodilation (30% of control) than any one of the
combination of any two of these. Brace gt_gl, (20) reported that
hyperkalemic (7.1 mEquiv/l) perfusion of the isolated dog forelimb
initially decreased resistance, but that this response was followed
by'a gradual increase in resistance to a level above the control value
vrithin 5 minutes. Chen gt_al, (23) suggested that the vasodilation
induced by potassium over the range 4 to 12 mEquiv/l results from
stimulation of the electrogenic Na/K pump in the sarcoplasma of the
smooth muscle cell since it is blocked by ouabain, a potent inhibitor
of Na/K ATPase. Ouabain, however, had little effect on the magnitude

0f exercise dilation but did delay its onset and increase the time to

24

the steady-state response. These local infusion studies indicate
that potassium is vasodilatory, but not to a great degree, and that

the effect wanes with time.

Osmolality, Injection or infusion of hyperosmotic solutions

 

into the vascular bed of resting muscle produces vasodilation (51, 55,
56, 59, 99-101, 102, 106, 109, 117, 118, 124, 136, 137, 142-144, 149.
150). Mellander §t_al, (106) have proposed that increased osmolality
is a dominant factor in active hyperemia of skeletal muscle. They
reported that local intra-arterial infusion of hypertonic glucose

or xylose (about 330 to 340 m05m/kg) into the feline calf muscles
produces nearly the same degrees of venous osmolality and vasodilation
as observed during heavy short-term (less than 5 minutes) exercise.
Mellander, Lundvall and their co-workers have since expanded their
studies to include man (99-101, 109). In the resting human forearm

and leg, intra-arterial infusion of hyperosmotic solutions were found
to increase venous osmolality by 15 to 30 mOSm/kg and blood flow 3 to

5 fold. Similar studies by Overbeck and Grega (118) showed that intra-
brachial infusions of hypertonic dextrose or hypertonic NaCl increased
cephalic venous plasma osmolality 10 to 23 mOSm/kg and reduced forearm
vascular resistance 4 to 14%. They also found a positive linear corre-
latfion between the level of initial vascular resistance and magnitude of
response to all hypertonic solutions. Scott _e__t_ _a_l_. (136) and Scott and
Radawski (137) showed that intra-arterial infusion of hyperosmotic
solIJtions of NaCl and dextrose in the gracilis muscle (perfused at

nattrral flow) produced about a 10% decrease in vascular resistance

25

corresponding to a 10 mOSm/kg increase in venous plasma osmolality;
however, exercise dropped resistance 85% with an increase of venous
osmolality of only 10 mosm/kg. Gazitua gt_al, (51) found that a 40

to 50 mOSm/kg increase in venous plasma osmolality, produced by intra-
arterial infusion of hyperosmotic solutions of dextrose, urea and NaCl
into the dog forelimb, initially (at 2 minutes) decreased vascular
resistance an average of 30%, but that continued infusion of any of
these substances for 10 minutes resulted in a waning of the resistance
toward control values and a complete return in the case of urea.
Stainsby and Barclay (150) also reported that the initial rise in
muscle and hindlimb flow during close arterial infusion of hyperosmotic
urea waned toward the control level within 5 minutes. Skinner and
Costin (142—144) observed that infusion of hyperosmotic glucose into
the dog gracilis muscle increased venous plasma osmolality to 343
m05m/kg and decreased resistance 25%. However, when the hyperosmolality
was combined with oxygen deficiency (27% saturation) and hyperkalemia

(7.6 mEquiv/l) resistance decreased 53%.
Recent Chemical and Biological Evidence
Su ortin a R01e for Metabolically Linked
Chemicals Proposed as Mediators

Additional evidence for the participation of various meta-

 

bolically linked vasoactive chemicals in exercise hyperemia has been
(Matained from bioassay studies and studies showing increased levels of
:nrtassium and hydrogen ions, carbon dioxide and osmolality and decreased

levels of oxygen in the tissues or venous effluent during exercise

hype remi a .

 

26

Bioassay studies on the enhanced vasoactivity of venous blood
draining exercising muscle provide compelling evidence that chemicals
linked to metabolism play a role in exercise hyperemia. The early
bioassay technique of Anrup and Saalfeld (3) was described on page 14.
More recently, Ross gt_al, (126), using an improved reservoir-free
system, found that perfusion of one resting canine gastrocnemius muscle
(bioassay organ) with blood from the contralateral contracting muscle
(donor or regulatory organ), produced a hyperemic response in the rest-
ing muscle. Similarly, Scott gt_gl, (133) observed that perfusion of
the dog forelimb with blood from the contracting hindlimb promptly
lowered resistance in the forelimb. As shown by Scott gt 31, (138)
and Radawski gt_al, (122), the same holds true when the donor organ
is the gracilis muscle and the assay organ the hindlimb or contra-
lateral gracilis. Thus, an immediate fall in resistance in the down-
stream bioassay organ indicates that the concentration of vasoactive
chemicals from the upstream exercising muscle has changed sufficiently
to affect the caliber of blood vessels in the bioassay organ. Bioassay
studies alone, however, do not firmly establish any specific mediator
of exercise hyperemia. Chemical analysis of the tissue fluid or venous
effluent during exercise hyperemia is necessary to identify any specific
mediator and permits a better evaluation of the relative role of these
factors. Oxygen lack will be considered first.

Qxyggg, During exercise, the oxygen saturation, content and
tension of blood draining skeletal muscle decreases, and the blood flow

or degree of vasodilation increases (5, 76, 78, 94, 112, 113, 115, 119,

27

126, 136, 141, 147, 148). Ross §t_al, (126) found that during
stimulation of the sciatic nerve (1-4 Hz, 1 msec., 7-10 volts) the
increase in blood flow averaged 173% with a venous oxygen tension of

25 mm Hg. Scott gt_gl, (136) observed that faradic stimulation of the
gracilis nerve (6 Hz, 0.6 msec., 6 volts) produced a 36% drop in
resistance (constant flow preparation) when the venous oxygen tension
was not permitted to fall below 17 mm Hg. Using a similar preparation,
Skinner and Powell (141) found that at 5 minutes of stimulation (0.4 Hz),
vascular resistance had dropped 60% while venous oxygen tension had
dropped from 70 to 15 mm Hg.

Recent studies by Mohrman gt_al, (111) and Morhman and Sparks

(112) indicate that the vasodilator response following a brief tetanus
(32 Hz for one second) or sinusoidal exercise (0.5 to 1.0 Hz) of dog
calf muscles parallels a fall in venous oxygen saturation, but that the
response is probably too rapid during natural flow or high constant flow
perfusion to be explained by changes in tissue oxygen content related to
oxidative metabolism. Honig (76) reported that the time course of the
hyperemia in dog gracilis muscle following contraction (2 Hz for 10
seconds) was poorly correlated with that of the oxygen tension in venous
blood, since oxygen tension returned to control levels while post con-
striction hyperemia continued. Morganroth gt.gl, (115) reported similar
results for venous oxygen saturation following 20 and 60 minutes of
exercise (4 Hz) of dog calf muscles. They observed that venous oxygen
saturation and vascular conductance remained elevated during exercise,

but 'that following exercise oxygen saturation returned to control with

28

a half-time five fold faster than vascular conductance. The two
latter studies suggest that oxygen is not a mediator of prolonged
vasodilation following exercise.

Hydrogen ion. Studies by Gollwitzer-Meier (54), Ross §t_gl,

 

(126), Kontos gt_al, (87), Kilburn (82), Scott gt_al, (137), Radawski
§t_al, (122), Gebert and Friedman (52) and others (65) demonstrate

that moderate to severe exercise causes an increase in blood flow and,
in the steady-state, an increase in the hydrogen ion concentration of
the venous blood. Ross §t_al, (126) showed that active hyperemia of
the dog gastrocnemius muscle produced a rise in flow to 173% of the
control level with only a 0.05 unit fall in pH and a 4 mm Hg rise in
carbon dioxide tension in the effluent blood. Kontos gt_al, (87) found
that exercise hyperemia of the human forearm could triple flow despite
only a 0.03 unit fall in pH and a 5 mm Hg rise in carbon dioxide tension.
Scott gt_al, (137) reported that skeletal muscle contraction of the dog
hindlimb produced a rise in hindlimb flow to 270% of the control level
with only a 0.07 unit fall in effluent blood pH. A comparison of these
studies with those in which the hydrogen ion concentration was increased
locally in resting muscle, shows that changes in venous blood pH corre-
late well with the direction but not the magnitude of the hyperemia.

The importance of hydrogen ions during the initiation and
maintenance of exercise hyperemia has only recently been investigated.
Gebert and Friedman (52) implanted hydrogen ion sensitive electrodes
in the tissue of the rat gastrocnemius muscle. Contractions greater

that) one Hertz first transiently decreased hydrogen ion activity for

29

about one minute before increasing hydrogen ion activity. At lower
contraction frequencies, hydrogen ion activity only decreased. Their
findings, and those of Ross gt_al, (126) and Scott gt_gl, (136), showing
that the flow rises maximally within 30 seconds upon motor nerve stim-
ulation, suggest that the hydrogen ion does not participate in the
initiation of the dilation and that pre-existing hydrogen ions are

first washed out before being added as exercise continues. Radawski

gt al, (122) suggested that the hydrogen ion may not be so important

in the maintenance of prolonged exercise of the gracilis muscle, since
the pH arteriovenous difference at the end of a 2 hour exercise (6 Hz,
1.6 msec., 3 volts) period was only 0.04 units. Morganroth gt_al, (115)
reported that venous lactate concentration initially rose and then
returned slowly to control values within a one hour exercise (4 Hz)
period of dog calf muscles. Moreover, Tobin and Coleman (152) showed
that marked hyperemia still occurs under conditions where effluent pH
does not decrease (in part because lactic acid is not produced).

Potassium ion. The concentration of potassium in the blood

 

draining skeletal muscle increases during exercise hyperemia (2, 82,
83, 85, 93, 110, 122, 135-137, 140, 141, 146). Kjellmer (83, 85)
studied the efflux of potassium from cat limb skeletal muscle at
various levels of somamotor nerve stimulation; increased contraction
frequency was found to be correlated with increased venous plasma
potassium ion concentration. He found that during maximum exercise
dilation (constant flow) the venous plasma potassium ion concentration

rose to a level twice that at rest and that infusion of potassium salt

30

solutions to resting muscle, at a level which produced venous

potassium ion concentrations similar to that observed during graded
exercise, produced vasodilation which was 25 to 65% of that seen during
active hyperemia. Kilburn (82) showed that the exercising human forearm
venous plasma contained 0.7 mEquiv/1 more potassium than arterial blood.
Skinner and Powell (140, 141) reported that active contraction of canine
gracilis muscle produced increases in blood flow of 170% and 350% corre-
sponding, respectively, with venous potassium ion concentrations of 0.8
and 0.95 mEquiv/1 above the control level. Using a dog hindlimb prepa-
ration, Scott gt_gl, (136) demonstrated a 270% increase in flow asso-
ciated with a 0.8 mEquiv/l rise in potassium ion concentration in the
venous effluent.

Sreter and Friedman (146) showed that the potassium content of
rat gastrocnemius muscle decreased fast at first and then more slowly
during a 60 minute exercise period. Scott gt_al, (136) showed that the
source of increased potassium appearing in venous effluent during exer-
cise was predominantly from skeletal muscle, because during perfusion of
the canine gracilis with cell free solutions exercise caused venous
potassium ion concentration to rise an average of 0.39 mEquiv/l above
the control level. Regarding the temporal relationship between venous
plasma potassium ion concentration changes during exercise, Scott gt_al,
(136) and Scott and Radawski (137) showed that stimulation of the
gracilis nerve (6 Hz, 0.06 msec., 6 volts) produced an 8 fold increase
in gracilis muscle blood flow and a 1.2 mEq/l increase in potassium ion

concentration within 60 and 10 seconds, respectively, of the onset of

31

exercise. With continued exercise (5 minutes) the arteriovenous
difference for potassium waned toward the control level while the
hyperemia continued unabated. Radawski et_al, (122) observed that
during a 2 hour period of gracilis muscle exercise (6 Hz, 1.6 msec.,

6 volts, natural flow), venous potassium ion concentration waned toward
the control level and was only 0.2 mEquiv/1 higher at the end of the
exercise period; vascular resistance fell 80% and remained at that level
throughout exercise. Anderson §t_gl, (2) demonstrated that reducing the
potassium ion concentration (of the blood perfusing gracilis muscle with
a hemodialyzer interposed in the arterial supply) from normal to hypo-
kalemic during the dilation brought on by simulated exercise. did not
change vascular resistance. Mohrman and Abbrecht (110) found that a

one second tetanus produced dilation lasting 30 seconds and calculated
that this could produce an increase in interstitial potassium within 10
seconds, which was sufficiently rapid to cause the accompanying vascular
response.

A consideration of these studies on the role of potassium in
exercise hyperemia suggests that potassium cannot contribute suffi-
ciently to totally explain hyperemia since exercise is associated with
up to a 10 fold increase in flow and only a small increase (about 1
mEquiv/l) in effluent plasma potassium ion concentration. Furthermore,
these studies suggest that potassium may participate to a greater extent

during the initiation than during the maintenance of exercise.

Osmolality. Mellander _t_gl, (106) reported that active
hyperemia (for 5 to 15 minutes) in the lower leg muscles of the cat
results in a sustained 80% drop in vascular resistance associated with
a 38 mOSm/kg rise in effluent plasma osmolality. Generally, resistance
decreased and osmolality increased as a function of sciatic nerve stim-
ulation. Lundvall (100) and Lundvall gt_gl, (99) showed that exercise
of the human forearm and leg produces an 85% decrease in resistance
accompanied by a 17 mOSm/kg increase in venous plasma osmolality. They
also showed a positive correlation between the degree of hyperosmolality
and the magnitude of the dilation. In the studies of Mellander and
Lundvall on cat and man the extent of vasodilation and venous plasma
hyperosmolality induced by arterial infusion was only slightly lower
than that observed in exercise.

Scott gt 31, (136) and Scott and Radawski (137) confirmed in the
dog that exercise hyperemia of the gracilis muscle was associated with
an increase in venous osmolality. However, they reported that at the
end of one minute of exercise, an 8 fold increase in flow was associated
with only a 9 mOSm/kg rise in osmolality. Furthermore, by the 5th min-
ute of exercise, osmolality returned to pre-exercise levels, while flow
remained elevated. Radawski gt_al, (122), using a similar preparation,
found no arteriovenous difference in osmolality, but sustained hyperemia,
at the end of a two hour exercise period. Similarly, Murray gt_gl,
(116) and Morganroth gt_al, (115) recently observed that exercise (4 Hz)
of dog calf muscles (perfused at constant flow) for 60 minutes produced

a sustained vasodilation, a 17 m05m/kg increase in venous osmolality at

33

2 minutes, but no change by the 55th minute of exercise. Following
exercise osmolality decreased below control levels while the flow was
still elevated. Hilton gt 31, (74) noted that graded exercise of the
cat gastrocnemius and soleus muscles produced changes in osmolality
that did not correlate well with the degree of hyperemia.

Mellander gt_al, (106) suggested that the increase in tissue
and venous osmolality during exercise is most likely due to parenchymal
cell fbrmation and release of osmotically active metabolites (such as
hydrogen ions, phosphates, lactate and other Kreb's cycle intermediates)
(121)). Hyperosmotic solutions appear to dilate primarily arteriolar
and precapillary resistance vessels when applied t0pica11y to rat
muscle, as shown by Gray (56). The studies of Johansson and Jonsson
(82) suggest hyperosmolality may cause active vasodilation due to
hyperpolarization of the vascular smooth muscle cell. Those of Gray
(56) suggest passive vasodilation due to cellular dehydration, whereas
those of Maxwell gt_al, (104) and Braasch (19) suggest decreased acto-
myosin ATPase activity and decreased blood viscosity, respectively.

Although the vasodilation and increased venous osmolality which
occurs subsequent to skeletal muscle exercise or local infusion of
hyperosmotic substances clearly indicate that hyperosmolality is a
factor in exercise hyperemia, many of the studies suggest that hyper-
osmolality cannot completely explain the magnitude or time course of the
vasodilation of exercise. As indicated above, Scott gt_gl, (136) and
Scott and Radawski (137) showed in the dog gracilis muscle that venous

osmolality rises to a maximum in one minute whereas flow increases much

34

faster. The reports by Scott gt_al, (136), Scott and Radawski (137),
Radawski gt_al, (122), Murray gt_al, (118), and Morganroth et_al, (115)
demonstrate that prolonged exercise of dog skeletal muscle is associated
with a waning of the arteriovenous osmolality difference to control
levels, while blood flow remains elevated. Scott et al, (136), Skinner
and Costin (144) and Gazitua gt_al, (51) showed that exercise of canine
skeletal muscle causes only a small increase in venous plasma osmolality
(about 9 mOSm/kg) and a large increase in blood flow (up to 8 fold),
whereas infusion of hyperosmolal solutions at rates which produce larger
increases in venous osmolality (approximately 40 mOSm/kg), induces
smaller increases in flow (2 fold). Furthermore, Gazitua et_al, (51)
and Stainsby and Barclay (150) found that the decrease in vascular
resistance produced by close arterial infusion of some hyperosmotic

solutions wanes toward the control values within several minutes.

 

STATEMENT OF PURPOSE

It is evident from this review that there is a paucity of
information on the combined effects of chemical factors during exercise
hyperemia. In particular, there appears to be no jn_vjvg_data on the
possible interaction of oxygen lack and increased concentrations of
hydrogen ions and/or carbon dioxide in the metabolic control of blood
flow during exercise. In addition, the relative contribution of the
various metabolites during exercise of different duration and intensity
has only recently received attention. The studies described in this
thesis were designed to evaluate the relative contribution of several
of these metabolically linked factors (in particular oxygen and hydrogen
ions or carbon dioxide), but also potassium ions and osmolality during
moderate to heavy long-term exercise. Evidence for participation and
interaction of these factors was obtained by bioassay of the vasodilator
properties and chemical analysis of the blood draining exercising
skeletal muscle perfused at natural and constant flow, and by chemical

alteration of the blood perfusing resting skeletal muscle.

35

 

METHODS

All experiments were performed on mongrel dogs (15-20 Kg) of
either sex, sedated with morphine sulfate (30 mg, subcutaneously), and
anesthetized twenty minutes later by intravenous injection of a mixture
of urethane (0.5 g/Kg) and alpha-chloralose (75 mg/Kg). The animals
were artificially ventilated with a positive pressure respirator
(Harvard Apparatus Co., Model 607, Dover, Mass.) at a volume and rate
sufficient to maintain systemic arterial pH between 7.35 and 7.45.
Following surgical procedures, heparin sodium (600 units/Kg) was
administered intravenously to prevent blood coagulation. Supplemental
doses of urethane-chloralose and heparin were given during the course
of the experiment. Blood volume was maintained with dextran (6% w/v;
average molecular weight, 75,000) in saline. All blood pressures men-
tioned were continuously monitored with pressure transducers (Statham
Laboratories, low volume displacement model P23Gb, Hato Rey, Puerto
Rico) and recorded on a direct writing oscillograph (Hewlett-Packard,
model 7796A, Boston, Mass.).

Data were evaluated by the Student's t-test modified for paired
replicates (145) except in one series of experiments where a 2-way
analysis of variance was used (145). Initial non-experimental values
served as statistical controls. In averaging data only the standard
error of group means are displayed. The experiments were conducted

in several series.

36

37

Bioassay Studies: Bilateral_Gracilis

 

In each of 17 animals one gracilis muscle (regulatory gracilis,
RG), was exposed and freed of connective tissue (Figure 2). All blood
vessels communicating with this muscle except the major artery and
vein were ligated. The gracilis nerve was severed and the distal stump
carefully fixed under oil on platinum stimulating electrodes. Extra-
corporeal blood circuits were designed using polyethylene tubing, P.E.
190-220 (Intramedic, Becton, Dickinson and Co., Parsippany, N.J.), so
that the RG artery was perfused at constant flow by diverting femoral
artery blood through a pulsatile pump (Sigmamotor, Inc., model TM-5,
Middleport, N.Y.). The RG vein was cannulated and the venous blood
pumped at constant flow through a hollow fiber gas exchange permeator
(type b/HFG-l, capillary tube surface area =500 cmz, Dow Chemical Co.,
Midland, Mich.) to the contralateral gracilis artery. The permeator
rather than a lung was used in these experiments because bioassay (66,
133, 138) and chemical analysis (18, 22, 33, 47, 120) of venous blood
suggest that one or more of the vasoactive adenyl compounds are released
during exercise hyperemia. These compounds are rapidly degraded on
passage through the lung, but presumably not on passage through the
permeator. A bypass shunt permitted excess effluent blood to be
diverted to the femoral vein. The contralateral muscle (assay gracilis,
AG), was surgically isolated, including denervation, exceptfor the
najor artery and vein. Isolation included occlusion of vessels at the

sites of muscle origin and insertion.

 

38

CONSTANT
FLMN 02

002
villi l TPermeator
L_J

_\_/_}l L©I\ Are

1

Pump Pump

\/\

I, Stimulator
RG

\

 

 

 

 

 

 

  
   

 

 

Femoral emoral
Artery Artery
Femoral Femoral
Vein ,'&, Vein
14%71' ///a
,r,‘ Regulatory
' v Gracilis Gracilis

Figure 2. Preparation for constant flow perfusion of regulatory (RG)
and assay (AG) gracilis muscles. PRG and PAG = perfusion
pressures of RG and AG. VRG and AAG = RG effluent blood

before and after passage through permeator.

be

CC

51'

 

 

 

39

Systemic blood pressure, regulatory gracilis vein pressure and
regulatory and assay gracilis perfusion pressures (PRG and PAG) were
monitored and recorded. The perfusion pressure of each muscle was
adjusted initially to approximate systemic blood pressure by adjusting
pump flows. RG flow was set at a rate equal to or slightly greater than
that of the AG to assure that the regulatory muscle furnished the total
blood supply to the assay muscle. The transit time for blood flowing
between the two muscles varied between 1 and 3 minutes.

To produce muscle exercise the electrodes were connected to a
square wave stimulator (Grass Instruments, model S-5, Quincy, Mass.)
and the sectioned gracilis nerve was stimulated at a voltage of 6,
duration of 1.6 milliseconds and a frequency of 1, 3 or 6 Hertz.
Before, after and initially during exercise gas exchange across the
permeator was prevented by occluding the inflow and outflow ports;
thus, the resting AG was perfused with unaltered venous blood from the
exercising RG. During exercise the gas permeator was ventilated ran-
domly with selected oxygen, carbon dioxide, nitrogen mixtures at 37°C.
Appropriate selection of the mixtures permitted selective corrections
of gas tensions and pH of blood perfusing the AG to levels observed
before exercise of the RG. In 12 animals, P02 and P002 (pH) were
corrected singly and simultaneously. In 5 additional animals only
simultaneous corrections of P02 and PC02 (pH) were made.

Blood samples (1 to 3 ml) were drawn anaerobically upstream
(VRG) and downstream (AAG) to the permeator during rest and during

exercise with and without gas exchange for determination of P02, PC02

40

and pH, potassium ion concentration and osmolality. PC02 and pH
determinations were made using potentiometric methods (Radiometer—
C0penhagen, blood micro system, acid base analyzer, Copenhagen,
Denmark). A calomel electrode served as the comnon reference electrode.

P02 was determined by reduction of 02 to H202 and measuring the result-

‘s

ing current flow. The P02 electrode was made of a platinum cathode and
a silver-silver chloride anode placed in an electrolytic solution behind
a polypropylene membrane. The Pco2 and pH electrodes utilized a combina-
tion of a glass electrode and a silver-silver chloride electrode. The
separate electrodes, encased in a common thermostatically controlled
water filled jacket, allowed simultaneous measurement of these param-
eters at body temperature (38°C). Oxygen tension was also monitored
continuously with a macroelectrode (Beckman Instruments, Inc., physi-
ological gas analyzer, model 160, Fullerton, Cal.) interposed between
the permeator and the assay muscle. Potassium ion concentration was
determined by flame photometry (Beckman Instruments, Inc., model 105,
Fullerton, Cal.) and plasma osmolality by the freezing point depression
method (Advanced Instruments Osmometer, model 31L, Watertown, Mass.).
RG vascular resistance (RRG) was calculated by dividing the difference
of PRG and gracilis venous pressure by the flow and is expressed as
mm Hg/ml/min/lOO grams of wet tissue. AG resistance was calculated
by dividing PAG by flow, since AG venous pressure was not monitored.

In another series of animals (N =12) the regulatory muscle
artery was not pump perfused, but retained its natural connection with

the circulation so that RG blood flow was allowed to vary (Figure 3).

41

NATURAL
FlDMl 02
‘I 002
Pump RG 1.1T Permeat or

        

L62)“ Ans

Rs

     
  
 

Stimulator

PsTs

 
      
 
  

/,

 

Femoral amoral
Artery——» Artery

Femoral Femoral
Vein Vein

 
 

Regulatory
Gracilis

    

Gracilis

    

|\ r
\'1

 

Figure 3. Preparation for natural flow perfusion of R6 and constant
flow perfusion of AG. PSYS = systemic arterial blood
pressure. FRGV = RG vein flow. VAG = AG effluent blood.
PAG’ VRG and AAG as in Figure 2.

 

42

RG venous outflow was measured with an electromagnetic flowmeter

(Biotronex Laboratory, model 610, BLC-2048-E04 probe, Silver Spring,

Md.) and expressed in ml/min/100 grams. The AG was pump perfused at

constant flow with RG venous blood after passing through the permeator.
During rest, a small fraction of the blood supply to the AG was obtained

lay retrograde flow of systemic venous blood through a shunt connecting

'the femoral vein. The AG vein was cannulated in order to obtain venous

ssamples (VAG). To determine the relationship between chemical changes
‘in venous blood and resistance to blood flow during prolonged exercise,
the regulatory muscle was stimulated (6 Hz, 1.6 msec., 6 volts) for 60

rrfinutes and its effluent blood was sampled at timed intervals (2, 5, 15,

£30, 45, 60 minutes) for analysis as described above. The contralateral

111uscle (AG) in the same 12 animals, prepared as described above, was

used again as a bioassay organ to test the vasoactivity of the RG venous

ee'Fffiuent during exercise at natural flow and to determine to what extent

czcarwection of blood gases and pH influence its vasoactivity.

Exercise vs. Mimicked Exercise:
Unilateral Gracilis
In this series the resistance response to reduced P02 and
'i hcreased PC02 during rest was compared to that seen during exercise.
I:'1 each of 17 animals a single gracilis muscle was surgically isolated
except for the major artery and vein (Figure 4) as described above.
The gracilis artery was perfused by diverting blood from a femoral

aY‘tery through the pulmonary circulation of an isolated lung obtained

‘13!"cnn a 5-7 Kg dog. This procedure has been described in detail

 

43

       

gilung Pump

  

 

 

 

l to Femoral Vein
Stimulator

 

Figure 4. Preparation for constant flow perfusion of a single
gracilis (G) muscle. PG = G perfusion pressure.
AG = Blood perfusing G after passage through donor
lung. VG = G effluent blood.

44

previously (27, 134). In brief, femoral artery blood was pumped
(Sigmamotor, model T-GSH) into the pulmonary artery of the donor lung.
The pulmonary effluent flowed through a cannula tied in the partially
preserved left atrium. Pulmonary artery and vein pressures were
monitored in the isolated lung and maintained similar to the normal
jn.yixg_values by varying the rate at which femoral arterial blood was
pumped into the lung. Excess pulmonary blood flow was diverted to a
femoral vein. The lung was substituted for the permeator in this series
because the permeator was not capable of producing the desired large
reduction in blood P02 during a single pass. An extracorporeal circuit
was also interposed in the gracilis venous outflow (VG) to facilitate
blood sampling.

In 8 animals the donor lung was ventilated (Harvard Apparatus
Co., Dual Phase Control Respirator Pump, Millis, Ma.) in random order
with mixtures of oxygen, carbon dioxide and nitrogen which produced gas
changes in the blood perfusing the resting muscle such that the P02,
Pco2 and pH of the venous blood resembled those in venous blood from
heavily exercising muscle. Gracilis artery (AG) and gracilis vein (VG)
blood was sampled before, during and after gas changes and analyzed as
described above.

In 9 other experiments, the degree of vasodilation occurring
during graded steady-state exercise was compared to that produced in
the same resting muscle during perfusion with hypoxic-hypercapnic blood.
To produce graded exercise the sectioned gracilis nerve was continuously

stimulated at 6 volts, a duration of 1.6 milliseconds with step increases

45

in frequency every eight minutes as follows: 0.2, 0.4, 0.8, 1.6, 3.0,
6.0 Hz. During exercise, as during rest, the donor lung was ventilated
with a control gas mixture (20% 02, 5% C02, 75% N2). Samples of
gracilis venous blood (VG) were taken just before each change in
contraction frequency. With the muscle at rest, the ventilatory gas
mixture was changed from the control mixture to 0% 02, 20% C02, 80% N2
in order to reduce arterial and thus, venous blood P02 and pH. This
maneuver was conducted randomly either before or after graded exercise.
The resistance and venous blood composition during mimicked exercise

were compared to those during steady-state exercise.

 

RESULTS

Bioassay Studies: Bilateral Gracilis

 

Figure 5 presents representative data from one experiment.
Exercise of the RG produced, within 2 minutes, a 52% decrease in RG
perfusion pressure which was maintained throughout the 92 minute
exercise period. The drop in R0 perfusion pressure was associated
with decreased P02 and pH and increased potassium ion concentration
and osmolality in the RG venous effluent perfusing AG and a 45% decrease
in AG perfusion pressure (panel 2). Since RG blood flow (14 m1/min) was
greater than AG flow (12 ml/min), only RG effluent blood perfused AG.
Exercise continuing, ventilation of the permeator with 100% 02 returned
P02 and pH of the blood perfusing AG and the AG perfusion pressure to
approximately pre-exercise values (panel 3). At this time of exercise
(46 minutes), RG venous plasma potassium ion concentration had stabil-
ized slightly above control and osmolality had returned to the control
level (permeator ventilation has no effect on these factors). Permeator
ventilation was then terminated and RG venous blood P02 and pH and AG
perfusion pressure again fell to approximately pre-ventilatory values
(panel 4). Ventilation with 20% 02 and 0% C02 increased pH slightly
above control but did not affect P02; concomitantly AG perfusion pres-
sure rose slightly (panel 5). Ventilation with 95% 02 and 5% C02

elevated P02 to the pre-exercise level and reduced pH to near the

46

47

(EKERCISE REGULATORY GRACIllS(3Hz,l.6ms.6v) STOP EXERCISE,

20

P116 100

mmHg
Flow to AG: l4ml/min

4

 

no 0% co2 5% co2 no

no exchange /100% O2 Kexchange /20% O2 ,95% O, Kexcnange

20
P“; roo

mmllg
Flow to AG=12ml/min

0541‘“ 315
milsm/Kg

Figure 5.

A —\ ——-’-

 

f

V

20 2'5 46 4'8 5'8 6'0 7'1 74 8'0 83 9'1 9'4 1138 1'10
TiME(min)

7.20 7.41 7.29 7.46 7.24 7.29 7.35
4.2 3.8 3.8 3.8 3.8 4.5 3.7
326 316 317 316 317 317 311

Representative tracing from the preparation illustrated in
Figure 2 showing the effects of ventilating the permeator
with various gas mixtures on PAG and AAG parameters during
sustained exercise of RG. Arrows refer to time of gas

exchange.

 

48

exercise level. In this animal AG perfusion pressure returned to the
pre-exercise value (panel 6). Cessation of ventilation was again
associated with reductions in P02 and pH of the blood perfusing AG
and AG perfusion pressure returned to the initial exercise level
(panel 7). All variables returned to or approached control values
soon after exercise was terminated (panel 8).

Figure 6 presents average data from 12 such constant flow
experiments. RG exercise produced a maintained 50% drop in R8 resist-
ance and a 45% drop in AG resistance associated with decreased P02 (49
to 19 mm Hg) and pH (7.37 to 7.20) and increased potassium ion concen-
tration (3.6 to 4.5 mEq/l) and osmolality (306 to 316 mOSm/Kg) of R6
venous blood perfusing AG. When the pH of the blood perfusing AG was
returned to the pre-exercise level (7.39 vs. 7.37), while P02 remained
at the exercise level (21 vs. 19 mm Hg), AG resistance increased signif-
icantly (P‘<.025) 13% above the exercise level. Correction of P02
(54 vs. 49 mm Hg), but not pH (7.23 vs. 7.37) increased AG resistance
29% (P <.025) above the exercise level. Simultaneous correction of both
P02 and pH (44 vs. 7.37, respectively), however, completely abolished
the drop in AG resistance (5th set of columns). At the time of simul-
taneous gas correction, RG venous blood potassium ion concentration and
osmolality were also at pre-exercise levels (4.1 vs. 3.6 mEq/l and
306 vs. 306 mOSm/Kg, respectively). As will be shown below, this
probably occurred spontaneously with time and independently of per-
meator ventilation. It is important to note, however, that AG resist-

ance again dropped when permeator ventilation was terminated and this

 

Figure 6.

49

       

L EXERCISE RE I
l

 

d
A
Y

(T$s.E.u.)

 

PH“ units 7.37 7.20' 7.39 7.23' 7.38 7.29' 7.36
$.014 $.008 $.016 $.014 $.016 $.009 $.010

P02mmllg43919. 21' 54 44 21' 40°
32 $2.1 $1.9 $5.2 $3.5 $1.9 $1.6

Ah———————————————

K+mEo/Li 3.6 4.5 4.2 4.2. 4.1 4.1 3.8
-a 0.13 $0.06 $0.10 $0.10 $0.11 $0.13 $0.14

03" mOsm/Kg3306 316 310 310 306 309 306
3.0 $3.5 $3.0 $3.1 $2.9 $3.3 $3.2

Average effects of ventilating the permeator with various

gas mixtures (randomized) 0n AG resistance and AAG parameters
during sustained exercise (1 t0 6 Hz, 1.6 msec., 6 volts) of
RG. 7 t S.E.M. represents the mean 1 standard error of the
group mean. P values relative to pre—exercise controls.

Preparation illustrated in Figure 2.

 

50

was unassociated with large changes in potassium ion concentration and
osmolality (6th set of columns). Following exercise the average AG
resistance was 28% higher than the pre-exercise control. This gradual
increase in vascular constriction during the course of the experiment
may be due to sympatho-adrenal discharge of catecholamines or release
of other pressor substances.

Figure 7 summarizes selected data from the 12 animals in Fig:
ure 6 plus 5 additional animals in which only P02 and pH were corrected
simultaneously in the effluent blood from the exercising gracilis. Cor—
rection of both P02 and pH were found to completely reverse the fall in
AG resistance associated with exercise of RG. In 14 of 17 experiments
AG resistance completely returned to, or rose slightly higher than, pre-
exercise values during simultaneous gas changes. RG venous potassium
ion concentration was significantly elevated during the exercise period,
but waned in the direction of the control level as exercise continued.
The osmolality was significantly altered during the exercise periods
before and after gas exchange, but not during correction of P02 and pH.
Again the dilation in the assay muscle reappeared when the changes in
P02 and pH were allowed to reoccur (4th set of columns).

Figure 8 shows data from a series of experiments (N =12) in
which the regulatory gracilis was naturally perfused. It is evident
that steady-state exercise of the RG was quickly associated with a large
initial (7 fold) increase in blood flow and reduction in resistance
(90%) and that these changes waned only slightly during the 60 minute

exercise period. Measured at 2 minutes of exercise, venous blood (VRG)

Figure 7.

51

    

 

 

 

 

121- i EXERCISE no
114- If
R 10‘
ES 9..
1 8"
i 7"
A 6"
u .
c 5
E 4L
mmll 3
ml min 24
1002
trssue
H . Z37 Z35um
P Imlts$.01o $012

4 18' 49 20° 39° .
P02PPHE$2I.9 $1.5 $3.9 $1.5 $1.4 ""7
59. 31 46. 38 .

M;P(:02 "While $3.2 $2.5 $1.4 $2.2 "'5

+ O . .
3.7 4.5 4.0 4.2 3.8 .
K ”514$.09 $.09 $.11 $.11 $.16 ""2

313' 308 10‘ 306.
Osmmosm/Kzgg? $2.5 $2.7 $2.6 $2.4“2

Average of the 12 experiments shown in Figure 6 together
with 5 additional experiments in which the permeator was
ventilated with only one gas mixture. P values relative

to pre-exercise controls.

 

52

. EXERCISE oi REGULATORY GRACILIS
(6111.1.61ns,6V)

R“ :8 -=:<.os /}_

db

N

 

 

 

 

 

 

 

 

 

 

 

 

 

mmllg/mMuin/lOOg 2 L :1 i E 51/
8 o 1’. I; .
FIG 4 1 l i n1\
mllminllOOg o i—
36 /I—
P02 “ e s 51/
mmllg ‘ ° '
7.3 o o
n . . 5 r ”—
p 72 .
units 1.16
v“ ' s 4
mmllg \I—
l‘+ ; 4
J- ; 0
min/1 \I—
i e 5.. $._
Mosul/K230 : : 4 : : c 5 5 :fif—o—r
20 30 40 5O 60 ”gm

TIRE minutes

Figure 8. Average effects of exercise on RG resistance, flow and
effluent blood parameters as a function of time.
Preparation illustrated in Figure 3. P values

relative to pre-exercise controls.

53

P02 and pH were markedly reduced and PC02: osmolality and potassium ion
concentration were elevated. The P02, Pco2 and pH changes tended to
wane during exercise but did not attain pre—exercise levels after 60
minutes of exercise; P02 increased from 10 to 19 mm Hg, pH increased
from 7.18 to 7.29 and P502 decreased from 72 to 55 mm Hg. In contrast,
the initial increase in potassium ion concentration (1.25 mEq/l) and
osmolality (l3 m05m/Kg) tended to return to control more rapidly and
neither were significantly different from pre-exercise levels after
60 minutes of exercise. At the end of the exercise period RG blood
flow was still 5 times greater than before exercise and RG vascular
resistance had increased only 4%. Following exercise, effluent plasma
potassium ion concentration was 0.8 mEq/l lower than before exercise.
Figure 9 presents the findings on bioassay of the venous blood
from the naturally perfused RG. The 12 animals used in this series
were the same as those depicted in Figure 8. Since flow to the RG was
allowed to increase it is unlikely that products of metabolism accumu-
lated to the same extent in these studies as in the constant flow
studies. As before, during those exercise periods when the permeator
was not ventilated (columns 1, 2, 6 and 7), any changes in gas tensions
and pH of the blood perfusing the AG were caused by the exercising up-
stream muscle (RG). In Figure 8 it was shown that all the blood param-
eters studied tended to wane to or toward control levels during the
exercise period. This is reflected in columns 2 and 6 of Figure 9,
which also shows that AG resistance rose 40% from the beginning to end

of the exercise of RG. Column 6 of Figure 9 shows that AAG"pH’ PC02

54

l EXERCISE oi REGULATORY GRACILIS '
r

ASSAV
GRAGILIS
NM"
| .
lOOg

. 21-
USS"!

 

 

01:31.11.)

H mm, 7.32' 122° 124° 7.41- 7.37- 7.31- 7.32-
P .009 .016 .013 .019 .020 .009 .006
36‘ 15° 46' ° 3 o .
P02111111"! 1.7 1.7 3.2 21? 4,3, 213 31%
46 61° 5 ° v v . .
[16021-1111. 3.34 3.0 371 32:32 379 51 45

41.

3.6 2.9
+ 3J7' 4. ° . . 3 . ' . . '

K ”ml .10 .0?! 4.0%9 4.3 4.0'9 4.1%8 :11:
308; 312° 310' z ' z -

05" ”MM“ 1.9 2.0 1.9 3899 31987 3'23 3'26

Figure 9. Average effects of ventilating the permeator with various
gas mixtures (randomized) 0n AG resistance and AAG param-
eters during exercise of R6 (see Figure 8). Preparation
illustrated in Figure 3. Statistical evaluation employed
a 2-way analysis of variance with multiple comparison among
means after the Students-Newman-Keuls test (145). Any row
mean is significantly different from all other row means

unless marked by a common symbol.

 

55

and osmolality returned to control levels during exercise of RG. The
vascular response on changing both P02 and pH of the RG venous blood
before it entered the AG artery were similar to those shown in Figure 6;
induced changes of P02 and pH to levels slightly higher than non-
exercising levels caused a rise in AG resistance which was 13% higher
than the pre-exercise level and 5% lower than the post-exercise level
(column 5). Single changes in either Po2 (column 3) or pH (column 4)
did not increase AG resistance to the extent as a simultaneous change
in P02 and pH. The potassium ion concentration and osmolality were
elevated during the pre-ventilatory exercise period (column 2). During
the post-ventilatory exercise period (column 6) potassium ion concen-
tration was slightly elevated and osmolality had returned to the non-
exercise level.

Exercise vs. Mimicked Exercise:
Unilateral Gracilis

 

 

Figure 10 shows data obtained from one representative experiment
in which gas tensions and pH were altered sequentially and simultane-
ously in a single resting gracilis muscle. The arrows refer to the
onset of the vascular reSponse. The steady-state response to each
change is shown on the left half of the subsequent panel. The tracings
show that the perfusion pressure (PG) dropped when either the inflow and
thus, gracilis venous (VG) blood P0z or pH was selectively reduced (left
half of panels 3 and 4), but that the pressure drop was greater when pH

and P02 were decreased simultaneously (panel 5). As has been shown

 

(5

.11....
mall: 0. ii'
I I V

25%002

control 20%02 0%‘17‘02
017115716.

1001‘

..

56

T—cmu BLOOD GAS rErrsrorun—I2o

 

   

control
“'61 .

  

fi2 39:50 gfl¥x

 
 

 

 

wig/L
05M

mOsnr/llg H20

Figure 10.

7.43

20

3.8

300

 

 

. . 4 5:
TIME (min)

21 55 29 52
7.38 7.17 7.06 7.38
28 59 88 28
3.7 4.1 4.1 4.0

295 302 302 291

Representative data from the preparation illustrated

in Figure 4 showing the effects of ventilating the

donor lung with various gas mixtures on PG and VG

parameters.

response.

Arrows refer to the onset of vascular

 

57

previously (42), the steady-state resistance response to carbon dioxide
was preceded by a transient response of the opposite sign (panels 3 and
4). Induced changes in gas tensions of the blood perfusing the muscle
did not appear to affect potassium ion concentration or osmolality in
the effluent blood (VG)'

Figure 11 summarizes the data from 8 such experiments.
Reduction of effluent blood pH (7.38 to 7.11) and a slight rise in P02
(52 to 69 mm Hg) produced an 18% fall in resistance. Reduction of P02
(52 to 21 mm Hg) but not pH (7.38 to 7.36) caused a 30% drop in
resistance. When both pH (7.38 to 7.10) and P02 (52 to 23 mm Hg) were
simultaneously reduced to levels observed during heavy steady-state
exercise (see Figures 6, 7 and 12) vascular resistance dropped to 53%
of the control level. The vasodilation induced by hypoxia alone was
not significantly different than that produced by hypercapnia alone.
However, hypercapnic-hypoxic blood perfusion produced a greater drop
in resistance (P‘<0.05) than perfusion with either hypercapnic or
hypoxic blood. In 1 of 8 dogs, reduction of pH alone caused a small
steady-state increase in resistance. Reduction of blood P02 alone
produced vasodilation in all 8 animals. Effluent blood potassium ion
concentration increased slightly from 3.3 to 3.8 mEq/l during the course
of the experiments. Effluent blood osmolality increased slightly during
hypercapnic-hypoxic perfusion.

Figure 12 shows the average effects of graded nerve stimulation
on vascular resistance and on various vasoactive factors in the effluent

blood (VG). The last column shows the effects of simultaneous reduction

 

58

     
    

 

 

 

cum: sLuoo us msmu
20— I - I
N=8
smms‘s" .
ME: 10- I
ml/min
100:
Nssue 5‘
(itsu)
- 7.38 7.11 ' 7.36 7.10’ . 4'
PH ""5 .027 .022 .026 .019 7.0322
P02"'”"“ 3% ‘33 '11 i2 3%
V. P0021“! 3.72 3.1 3% 23 3.3
+ 3.3 3.6' 3.4 . ° . '
K "EVL 0.18 0.18 0.12 3272 2188

‘3(72 ‘3 ‘4 ‘4
own/«z ,9. =22 3.28 =33

Figure ll. Average effects of ventilating the donor lung with
various gas mixtures (randomized) on PG and VG
parameters. Preparation shown in Figure 4. P

values relative to pre-ventilatory change control.

59

I EXERCISE GRACILIS (l.8msec.8volts)

2.1
N=9

201

RGMCILIS
m '51
Ill/min
100g ‘0‘
tissue

(1151.11,)

m can re

P" ""3 7.37 7.35 7.32' 7.27' 7.20“ 7.16'
.017 .012 .013 .019 .015 .007

 

 

     

no: con 10

7.31' 7.12'
.018 .018

E334
dd '
o-l.

poz'""' 51 41' 35' 30' 26' 23' 24' 49 23'
3.3 2.4 2.4 2.2 1.8 1.8 2.0 3.0 1.3
v6 co ""136 39 42‘ 52° 66' 72' 83° 41 2‘
P 2 42 43 40 49 00 55 72 40 30
K+mE1/l3.5 3.6 3.8’ 4.0' 4.5' 4.9' 5.1' 3.9’ 3.0
0.14 0.13 0.12 0.12 0.11 0.11 0.09 0.11 0.17
oSIMSI'a/laoo 309 309 313' 317' 319' 319' 309 309
2.1 3.0 2.8 2.4 2.8 2.4 3.7 2.4 2 4

Figure 12. Average effects of step increases in contraction frequency
and ventilation of the donor lung with hypoxic, hypercapnic
gas on G resistance and VG parameters. Preparation shown

in Figure 4. P values relative to pre-exercise controls.

60

in pH and P02 during rest. Resistance and P02 decreased and osmolality
increased as a function of stimulation frequency over the range 0.2 to
1.6 Hz and remained constant over the range l.6 to 6.0 Hz; effluent
blood pH decreased and PCO2 and potassium ion concentration increased
over the entire range of frequencies. Stimulation at 0.2 Hz produced
a 24% decrease in resistance while the only significant change in
effluent blood was a slight drop in P02. During rest, reduction of
the effluent blood pH and P02 to 7.l2 and 23 mm Hg, respectively,
reduced resistance only to the extent observed during stimulation at
0.8 Hz (P02--30 mm Hg, pH--7.27) and not to the extent seen during
stimulation at 6.0 Hz where the venous P02 (24 mm Hg) and pH (7.ll)
were like those produced by gas changes during rest. Osmolality and
potassium ion concentration were Unaffected during gas changes at rest

but were elevated during stimulation above 0.4 Hz.

DISCUSSION

In brief, these studies show that the enhanced vasodilator
activity of venous blood from the constant flow or naturally perfused
canine gracilis muscle during steady-state exercise can be completely
abolished by simultaneously returning the P02 and pH to pre-exercise
levels. They also show that simultaneously induced changes in P02 and
pH in the blood perfusing a resting gracilis muscle, to levels observed
in venous blood from heavily exercising muscle, produce a fall in
resistance which does not quite equal that seen during heavy exercise.
Assuming that the enhanced vasodilator activity of the effluent blood
during exercise is due to changes in the concentrations of metabolically
linked vasoactive chemicals, these studies suggest that oxygen and hydro-
gen ions are important factors in the maintenance of active hyperemia of
skeletal muscle.

It has been previously demonstrated by bioassay that the venous
blood draining contracting skeletal muscle exhibits greater vasodilator
activity than that draining resting skeletal muscle (3, 66, l22, l26,
l33, l36, l38). Anrep and Saalfeld (3) found that venous blood col-
lected from the dog gastrocnemius muscle during muscular activity and
restricted arterial inflow was a powerful dilator when re-perfused later
in the same muscle at rest. They concluded that the vasodilator action

was due to a stable metabolite present in venous blood during exercise

61

62

but not during rest. Oxygenation of this blood did not abolish the
vasodilator property, so they concluded further that oxygen lack was
not a cause of the vasodilation. It may well be, however, that oxygen
lack was a stimulus that caused vasodilation, particularly in blood
collected from exercising muscle that was not re—oxygenated. Since
they collected and only later re-perfused blood from exercising muscle,
the vasodilatory property of the blood could have been due to potassium
release from blood cells, independent of oxygenation. Using improved
bioassay techniques investigators have more recently confirmed the
marked vasodilator property of venous blood draining exercising muscle.
The gastrocnemius (126), hindlimb (133) or gracilis (122) muscles of
the dog have been used as the regulatory organ and the gastrocnemius
(126), forelimb (133), hindlimb (138) or gracilis (122) muscles as the
bioassay organ with similar results. Similarly, this study shows that
exercise of the dog gracilis muscle produced a 50% fall in R0 resistance
when perfused at constant flow and a 90% fall in R0 resistance when per-
fused at natural flow; this vasodilation in the regulatory gracilis was
accompanied by a 45% and a 33% drop in resistance, respectively, in the
assay gracilis perfused at constant flow. Clearly dilator factors are
present in the effluent blood but the bioassay technique alone cannot
identify the specific factor or factors eliciting this response.

As pointed out in several recent reviews (1, 7, 17, 64, 65, 67,
68, 107, 108, 127, 151), no single metabolic factor has been found which
can entirely explain the hyperemia of exercise. The participation of
oxygen in local regulation has been considered for many years and is

considered here first.

 

63

Oxygen content, saturation and tension of blood draining
contracting skeletal muscle has been shown to fall together with
vascular resistance (5, 57, 76, 78, 94, 97, 111, 112, 115, 119, 126,
136, 141, 147) and oxygen lack has often been shown to produce vaso-
dilation (28, 40, 42, 58, 61, 69, 95, 126, 134, 142-144). An evalua—
tion of these studies, however, indicates that the magnitude of the
dilation produced by oxygen lack is often considerably less than that
produced by muscular exercise. In particular, the study by Ross gt_al,
(126) showed that induced reduction of venous blood P02 (to 23 mm Hg)
from resting muscle to levels obtained during exercise (25 mm Hg) pro-
duces only 31% of the flow increase observed with exercise. A related
study by Scott gt 31, (136) demonstrated that perfusion of resting
skeletal muscle with hypoxic blood produced a larger drop in venous
oxygen tension (10 mm Hg) and a smaller drop in resistance (17%) than
that produced by exercise (6 Hertz) when the venous oxygen tension was
not allowed to fall to exercise levels by increasing the oxygen tension
of the perfusing blood (oxygen tension: 17 mm Hg, resistance: 37%
decrease). The present study supports the conclusion that oxygen is
not wholly responsible for, but contributes significantly in, exercise
hyperemia. It was observed that the enhanced vasodilatory activity of
venous blood from exercising muscle is attenuated (29%) when the efflu-
ent blood P02 is singly corrected to pre-exercise levels (Figures 6 and
9) and that selective reduction of resting muscle effluent blood P02 to
levels seen during heavy eXercise produces a significant drop in

resistance (30%) (Figure 11). In addition, effluent blood P02 remains

64

decreased during 60 minutes of exercise hyperemia (Figure 8) and is the
only measured factor altered concomitant with exercise dilation at a
low contraction frequency (Figure 12). On the other hand, correction
of the P02 in the venous blood did not completely abolish its enhanced
vasodilator activity (AG resistance increased 29% above the exercise
level, see Figure 6), and reduction of P02 in resting muscle venous
blood did not lower resistance to levels observed during heavy exercise
(30 and 53% of pre-ventilatory or pre-exercise control, respectively.
Compare Figures 11 and 12).

The participation of carbon dioxide or pH in local regulation
has been also considered for many years. The pH of venous blood drain—
ing skeletal muscle dr0ps during moderate to heavy exercise (52, 54, 66,
82, 87, 122, 126, 136, 137). A comparison of these studies with those
in which the blood perfusing resting skeletal muscle was exposed locally
to high CO2 tensions or acids shows a good correlation between the
hydrogen ion concentration and the direction but not the magnitude of
the dilation. As the blood flow increases up to three times resting
values during exercise, the pH decreases only .03 to .07 units (66, 122,
126, 136, 137). Induced changes in venous blood pH during rest must be
very large to produce measurable changes in resistance (28, 30, 39, 54,
81, 88-91, 114, 123, 154). The present study concurs with these pre-
vious reports that pH or P002 changes alone cannot account for most of
the vascular response to exercise. We found that correction of the pH
of the blood draining exercising muscle to pre-exercise levels only

partially reduced (13% increase above the exercise level) the

F7 -

65

vasodilation of the assay muscle (Figure 6). Reduction of the venous
blood pH in resting muscle to the levels observed during heavy exercise
produced a significant, but much smaller drop in resistance (18% of pre-
ventilatory control) than during heavy exercise (53% of pre-exercise
control, compare Figures 11 and 12). However, the pH, like P02,
remained depressed and the P002 remained elevated during 60 minutes

of steady-state exercise hyperemia (Figure 8).

The basis for the transient rise in vascular resistance during
hypercapnic blood perfusion (Figure 10) is not known. Fleish gt_gl,
(42), who first reported it, suggested that carbon dioxide might have
a double effect on vascular resistance; increased intravascular carbon
dioxide concentrations might constrict arterial blood vessels, whereas
increased tissue carbon dioxide concentrations might be vasodilatory.
Hypercapnia might also produce a temporary increase in red blood cell
size (chloride shift) and thus increase viscosity or increase the
availability of molecular oxygen through the Bohr effect. Increased
intravascular Pco2 has been shown to increase perivascular Po2 of the
resistance vessels (37). Kontos gt_gl, (92) have attributed the initial
vasodilation of hypocapnic alkalosis to release of histamine but offered
no eXplanation for the initial vasoconstriction of hypercapnic acidosis.

This study indicates that oxygen and hydrogen ions interact to
play a major role in the maintenance of exercise hyperemia. Correction
of both P02 and pH in the venous effluent of exercising muscle com-
pletely abolished its enhanced vasodilator activity (Figures 6, 7 and 9).
Reduction of both P02 and pH in the blood perfusing resting muscle to

66

venous blood levels observed during steady-state exercise decreased
resistance almost to the same degree as exercise itself which produced
the same P02 and pH (Figure 12). Also, P02, P002 and pH levels were
still significantly different from control levels during one hour of
exercise hyperemia (Figure 8).

0n the other hand, other studies suggest that oxygen and
hydrogen ions may not participate in the initiation of exercise hyper-
emia. Hydrogen ion sensitive electrodes, implanted in rat gastrocnemius
muscle, indicate that hydrogen ion activity transiently decreases before
it increases with contractions in excess of one per second and only
decreases with a contraction frequency of one per second (146). This
suggests that the pre-existing hydrogen ions are first washed out before
being added as exercise begins. Because flow rises very rapidly upon
motor nerve stimulation (126, 136), the hydrogen ion may not participate
in the genesis of exercise hyperemia (68). The time course of the change
in oxygen saturation following a brief tetanus or "sinusoidal" exercise
parallels the change in blood flow, but the vasodilator response
appears to be too rapid during free flow and high constant flow per-
fusion to be explained by changes in tissue oxygen content (11, 112).
In addition, the time course of the flow recovery following short term
(10 seconds) (76) and long term (20-60 minutes) (115) exercise
correlates poorly with the oxygen tension in the effluent blood.

A necessary assumption underlying any conclusions about this
study is that the effluent blood P02 and P002 or pH, whether altered

by muscle exercise or by induced gas changes in the blood perfusing

67

resting muscle, closely reflect interstitial (perivascular) fluid gas
tensions and pH. This assumption is supported by several studies which
indicate that perivascular P02 is not much different from intravascular
P02 at the level of the resistance vessels; i.e., oxygen diffuses not
only from capillaries but also from pre-capillary vessels (sphincters
and terminal arterioles), and that the diameter of these vessels is
altered when perivascular P02 is changed (34-37, 77). It has also been
demonstrated that increased intravascular P002 produces significant
dilation of large arterioles when P02 is held low and increases peri-
vascular P02 of the resistance vessels, possibly due to the Bohr shift
(37).

Potassium has been proposed to participate significantly in the
genesis of exercise hyperemia because many studies have shown that exer-
cise causes an initial loss of potassium from skeletal muscle (l36-l46)
associated with increased venous potassium ion concentration (2, 4, 41,
82, 83, 85, 110, 122, 135, 137, 140, 141, 146) and because potassium
itself is vasodilatory (23, 29, 38, 48, 53, 60, 80, 83, 85, 86, 98, 117.
135, 139-144). Several findings, however, indicate that potassium does
not totally explain exercise hyperemia. Exercise can cause up to a 10
f01d increase in flow and this is associated with only a small increase
in effluent plasma potassium ion concentration (122, 135-137, 141).
Although potassium is vasoactive when infused into resting muscle, the
magnitude of the response is much less than that observed during exer-
cise (83, 86, 136, 134-144). Moreover, the initial increase in the

effluent potassium ion concentration wanes toward the control level

 

68

during prolonged exercise hyperemia (115, 122, 137). Tissue analysis
shows that the total potassium content of skeletal muscle decreases
rapidly in the first few minutes of exercise but then much more slowly
during the next hour (146). Moreover, the initial dilation of skeletal
muscle during hyperkalemic perfusion is replaced by a mild constriction
within 5 minutes of continued perfusion (20). Finally, reduction of the
inflow potassium ion concentration to a hypokalemic level during skele—
tal muscle exercise does not affect the magnitude of the exercise
dilation (2).

This study confirms and strengthens previous reports suggesting
that potassium cannot totally explain exercise hyperemia and that potas-
sium plays only a relatively minor role during its maintenance. It was
found that the enhanced vasoactivity of venous blood from exercising
muscle can be abolished by simply correcting the P02 and pH (Figures 6,
7 and 9) and that induced large reductions of both P02 and pH in resting
muscle can cause dilation nearly as great as heavy exercise itself (Fig-
ure 12). Also, during steady-state exercise hyperemia the potassium ion
concentration, although initially elevated, rapidly and then slowly
decreases to the pre-exercise level (Figure 8). However, as observed
by Kjellmer (85) in the cat, increased contraction frequency was found
to be correlated with increased venous plasma potassium concentration
(Figure 12).

Osmolality has been proposed more recently as a mediator of
active hyperemia (106) since an increase in osmolality occurs early

during skeletal muscle exercise (76, 99-101, 106, 115, 116, 122, 136,

 

69

137) and perfusion of hyperosmotic blood to skeletal muscle decreases
the resistance to flow (51, 55, 56, 59, 99-101, 103, 106, 109, 117, 118,
124, 136, 137, 142-144, 149, 150). However, like potassium, osmolality
does not appear to participate importantly in steady-state exercise
hyperemia. Infusion of hyperosmotic solutions into the arterial supply
of resting skeletal muscle in the dog does not cause pronounced vasodi-
lation, even when perfused in the highest concentration ranges seen in
effluent blood during heavy exercise (51, 136, 137, 142-144, 149, 150).
This is in contrast to that found in cat skeletal muscle (101, 106, 109)
and man (99-101), perhaps reflecting differences in the type of muscle
(mixture of red and white) and species. As with potassium, the venous
effluent osmolal concentration progressively wanes during the course of
exercise (115, 116, 122, 136, 137) and the vasodilation resulting from
close arterial infusion of some hyperosmotic solutions wanes toward the
control level within several minutes (51, 151).

The present study gives additional support to suggestions that
osmolality is not greatly involved in the magnitude of active hyperemia,
particularly during prolonged exercise. Again, the enhanced vasodilator
activity of venous blood can be totally abolishing by correcting the P02
and pH without the necessity of correcting the osmolality (Figures 6, 7
and 9) and dilation of the resting muscle vascular bed to nearly the
level seen during heavy exercise can be achieved by simultaneously
reducing P02 and pH to levels seen during heavy exercise levels (Figure
12). Furthermore, osmolality, like potassium, rapidly and then more
slowly decreases to the pre-exercise level while the hyperemia continues

(Figure 8).

70

A number of recent observations show that increases in
potassium ion concentration and osmolality occur rapidly at the onset
of exercise and thus suggest that these factors are involved in the
initiation of exercise dilatation. The potassium concentration in the
venous plasma increases within the first 10 seconds of exercise (136).

A one second tetanus (32 Hz) of dog calf muscles has been estimated to
increase,interstitial potassium levels within 10 seconds, which is
sufficiently rapid to cause the accompanying vascular dilation lasting
30 seconds (110). In addition, Chen gt_a1, (23) found that ouabain,
which blocks the responsiveness of the vessels to potassium ions, has
little effect on the magnitude of the exercise dilatation but delays the
onset and increases the time to steady-state response. The increase in
venous blood osmolality appears within one minute following the onset of
exercise (136, 137). Although tissue fluid osmolality has not yet been
determined during exercise hyperemia, it appears that muscle contraction
is accompanied by vasodilation before the venous osmolality begins to
rise. Once osmolality is elevated it may contribute with potassium in
the genesis of exercise hyperemia.

In the past several years increased attention has been directed
toward evaluating the interrelationships among the various chemical
factors proposed to operate in active hyperemia. The studies by Skinner
and his colleagues (139-144) suggest that exercise hyperemia may result
may result from a combination of several factors which decrease vascular
smooth muscle tone. They showed that perfusion of the resting canine

gracilis muscle with hyperosmotic, hyperkalemic, hypoxic blood at levels

71

found in muscle venous blood during heavy short-term exercise increases
both the rate of deve10pment and magnitude of the vasodilatation above
that found when these factors are altered individually (142-144). In
the present study experiments were designed to determine a possible
interaction between oxygen and hydrogen ions or carbon dioxide in local
control of blood flow during graded and heavy exercise of skeletal
muscle. The data suggest that during long term exercise the concen-
trations of these substances are the most important determinants of
blood flow regulation.

The mechanism of action of oxygen on peripheral resistance is
not known. Oxygen has been proposed to regulate blood flow through a
direct action on vascular smooth muscle cells in the arteriole wall
and indirectly by the production and release of vasodilator metabolites
from parenchymal cells (34). For example, Berne gt_gl, (13-16) and
Rubio gt_al, (131) have shown that oxygen lack leads to myocardial
release of adenosine, which is a potent vasodilator. The vasodilator
activity of adenyl compounds has been long known (12, 43).

Bioassay studies from this laboratory suggest the presence of
AMP and/or adenosine in the blood draining skeletal muscle during
ischemic exercise (66, 133, 138). On chemical analysis of this effluent
blood, some investigators have shown increased levels of ATP (18, 22, 47,
120) and AMP (22). Dobson 35 31, (33) found higher levels of adenosine,
but not its nucleotides. These findings seem to disagree with the
present experiments showing that the enhanced vasodilator activity

of venous blood from exercising muscle can be totally abolished by

72

correcting the P02 and pH. Two possibilities are offered which may
explain the apparent absence of adenyl compounds in the blood perfusing
the assay muscle: (a) These agents may be released only in severe
ischemic exercise. Perhaps vasodilator concentrations of these agents
were not produced under the conditions of exercise in this study. (b)
The long circuit lag (l to 3 minutes) for blood flowing between the two
muscles may have allowed for a lessening of potency of these agents due
to degradation in the blood. The possibility also remains that ischemia
may cause release of these agents in vasodilator concentration in the
bioassay as well as in the exercising muscle. Thus, correction of both
P02 and pH could abolish the vasodilation in the bioassay muscle by
preventing the local release of adenyl compounds in the bioassay muscle
rather than by, or in addition to, the removal of the direct dilator
effects of oxygen lack and high carbon dioxide concentration.

Factors other than oxygen and hydrogen ions are probably
involved to a lesser extent in the maintenance of exercise hyperemia.
Theoretically, the myogenic mechanism would be operative throughout the
exercise period. Also, substances may act on vascular smooth muscle to
contribute to vasodilation but not appear in the effluent in dilator
concentrations due to rapid inactivation, reuptake or limited release.
Finally, the roles of the potassium ion and osmolality in the mainte-
nance of active hyperemia appear to be very minor.

With regard to the metabolic hypothesis the present study, as

well as other recent studies, suggest that the vascular changes during

 

73

exercise hyperemia may be produced by several metabolites. The
initial vasodilation with the onset of exercise may be due to combined
increases in potassium ion concentration and osmolality. The mainte-
nance of the dilation during steady-state exercise may result from
increased hydrogen ion concentration and from decreased oxygen tension

when oxygen consumption exceeds oxygen delivery.

 

SUMMARY AND CONCLUSIONS

Exercise of skeletal muscle is associated with a local increase
in blood flow and changes in tissue metabolism and effluent blood
chemistry. This hyperemia has been shown to override the vasocon-
strictor effects of neural and humoral factors regulating vascular
smooth muscle tone during rest. The purpose of this study was to
elucidate further the relative contribution of oxygen and carbon dioxide
(or hydrogen ions) in the metabolic control of blood flow during steady-
state exercise of the denervated gracilis muscle in the anesthetized dog.
In addition, the potassium and osmolal concentrations of effluent blood
were measured to further evaluate their roles in the hyperemic response.
Evidence for participation and interaction of these metabolically linked
factors was obtained by bioassay and chemical analysis of the blood
draining exercising muscle perfused at natural and constant flow, and
by altering the gas tensions in the blood perfusing resting muscle by
means of a gas exchange permeator or donor lung placed in the arterial
supply.

These studies show: (a) The enhanced vasodilator activity of
venous blood from the constant flow or naturally perfused muscle during
steady-state exercise can be completely abolished by simultaneously
returning the oxygen tension and pH to pre-exercise levels. Correction

of potassium ion concentration and osmolality is not necessary. This

74

75

vasodilator activity is attenuated but not abolished when the effluent
blood oxygen tension or pH is singly corrected to pre-exercise levels.
(b) Simultaneously induced reductions in oxygen tension and pH in the
blood perfusing resting muscle, to levels observed in venous blood from
heavily exercising muscle, produce a vaSodilatation which does not quite
equal that seen during heavy exercise, but which is greater than that
produced by reduction of oxygen tension or pH alone. (c) During exer-
cise at constant stimulation frequency for 60 minutes, effluent blood
oxygen tension and pH initially fall and then rise slowly but remain
significantly lower than pre-exercise levels while the hyperemia remains
unabated. 0n the other hand, potassium ion concentration and osmolality,
although initially elevated, rapidly approach pre-exercise levels. (d)
Mild exercise (0.3 Hz) at constant flow causes moderate vascular dila-
tion (24% reduction in resistance) and is associated with only a slight
drop in effluent blood oxygen tension. Resistance and oxygen tension
continue to decrease as a function of contraction frequency during
moderate exercise (0.2 to 1.6 Hz) but remain constant during heavy
exercise (1.6 to 6.0 Hz). pH decreases and carbon dioxide tension and
plasma potassium ion concentration increases as a function of frequency
over the entire range. Osmolality is unchanged during mild exercise
(0.2 to 0.8 Hz) but slightly elevated during moderate to heavy exercise
(0.8 to 6 Hz).

These results suggest that the concentrations of oxygen and
hydrogen ions (the classical metabolic factors) are the most important

chemical determinants maintaining exercise hyperemia, and that potassium

 

76

ions and osmolality play only relatively minor roles in exercise
hyperemia of long duration. It has been known for at least one

hundred years that muscle exercise is associated with local hyperemia.
Later researchers found this to be accompanied by changes in the
concentration of metabolites in the tissue and venous effluent. The
metabolic hypothesis was formulated and proposes that these changes
result in an increase in blood flow so that the exercising muscle is
better able to receive an adequate supply of nutrients and to eliminate
products of metabolism. Today, we are still not clear on the mechanisms
involved nor the relative contributions of these mechanisms. Knowledge
of the basic mechanisms underlying exercise hyperemia not only contri-
butes to a better understanding of the normal physiological state, but
also establishes a foundation on which to treat rationally diseases of

the cardiovascular system.

 

 

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10.

11.

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