© 1996 American Heart Association, Inc.
Articles |
Differential Sensitivity of Venular and Arteriolar 
-Adrenergic Receptor Constriction to Inhibition by Hypoxia
Role of Receptor Subtype and Coupling Heterogeneity
From the Department of Physiology, University of North Carolina, Chapel Hill.
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Abstract |
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Abstract Reflex adrenergic constriction of the venous circulation is considerably less sensitive than the arterial circulation to local metabolic inhibition, but the basis for this difference remains unclear. The purpose of the present study was to determine whether
-adrenergic receptor (AR) constriction of venular smooth muscle
is in fact protected against inhibition by hypoxia, per se, and
to examine possible mechanisms for this protection. An intermediate
level of 1-AR (norepinephrine+rauwolscine)
or 2-AR (UK 14,304+prazosin) tone was induced in rat
cremaster skeletal muscle arterioles and venules (control lumen
diameter, 134 and 194 µm, respectively), and tissue bath
PO2 was lowered from the control value (30
mm Hg). Arteriolar 2-AR tone was inhibited by 29% at 5
mm Hg PO2 (P<.05), whereas
arteriolar 1-, venular 1-, and venular
2-AR constrictions were unaffected. Like these findings
obtained for in situ vessels with normal blood flow,
1-AR tone induced in vascularly "isolated" venules
and basal diameter were again unaffected by hypoxia, whereas
2-AR tone was actually enhanced by 19%
(P<.05). This constriction was prevented by
indomethacin but not by endothelin or nitric oxide
blockade; importantly, however, venular 2- and
1-AR tone still remained insensitive to inhibition by
hypoxia. ATP-sensitive K+ (KATP)
channels, which are known to participate in hypoxic inhibition of
arteriolar smooth muscle, were examined for a role in this differential
arteriolar versus venular sensitivity to hypoxia. Use of the
KATP antagonists glibenclamide and U-37883A and
the KATP channel opener cromakalim suggested that venular,
unlike arteriolar, smooth muscle had no detectable basal or inducible
KATP activity. Also, unlike arteriolar 2-AR
constriction, venular 2-AR tone did not depend on
KATP activity. Finally, venular 2-AR tone
was unaffected by nifedipine (0.06 to 3 µmol/L), whereas
venular 1-AR tone was inhibited by 50%
(P<.05), findings opposite those found for arteriolar
1 and 2 tone. These data demonstrate that
venular 1- and 2-AR constrictions are
insensitive to inhibition by hypoxia and suggest that this may
be due to a paucity of KATP channels on venular smooth
muscle. In addition, venular 1- but not
2-ARs appear to couple to
dihydropyridine-sensitive voltage-operated
Ca2+ channels.
Key Words: venules • hypoxia • -adrenergic receptors
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Introduction |
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It is well established that reflex adrenergic constriction of the venous circulation is maintained and the arterial circulation is antagonized during increased tissue metabolic activity.1 2 The attenuation of arterial tone, expressed most prominently in arterioles of the microcirculation, ensures adequate tissue perfusion, whereas relative venous insensitivity to metabolic inhibition allows maintained reflex control of venous return to the heart to support cardiac preload. For example, neither local nor systemic hypoxia inhibits sympathetic venoconstriction in exercising men.3 In cat skeletal muscle, venous adrenergic constriction is maintained during reduction in arterial inflow when precapillary responses are reduced to <25% of the control value.4 In addition, increased metabolites during exercise potently antagonize arteriolar tone at levels well below those required to inhibit venous tone in cat gastrocnemius muscle.5 Moreover, adrenergic constriction of isolated canine saphenous vein is maintained during severe hypoxia (PO2, <1 mm Hg).6 Thus, venous adrenergic constriction appears to be relatively insensitive to hypoxic and metabolic inhibition, compared with arterial tone, which is potently antagonized by these same signals. However, the direct effects of reduced O2 on the adrenergic constriction of venules have not been examined.
The cellular mechanisms for hypoxic inhibition of arterial tone and apparent resistance to inhibition of venous tone are not fully understood; however, several possible components have been identified. One proposed "sensor" of reduced O2 is the KATP channel. KATP modulation of basal arterial tone and mediation of hypoxic vasodilation have been demonstrated in a number of arteries and arterioles, including the rat cremaster arterioles studied herein.7 8 9 10 11 In addition, KATP channels are found in rat portal, human saphenous, and canine saphenous vein smooth muscle and, in the latter vessel, mediate ß-AR dilation.12 13 14 Besides KATP channel involvement, there is also limited evidence that acidosis and hypoxia can inhibit VOC activity in arterial VSM,15 16 indicating that VOCs themselves might respond to changes in PO2. However, we have found that hypoxia and acidosis do not directly affect VOC-mediated constriction of rat skeletal muscle arterioles studied in vitro.17 Whether a difference exists in KATP or VOC activity between resistance and capacitance vessels is not known.
Basal vascular tone is dependent on adrenergic activity and intrinsic
mechanisms, with adrenergic tone exhibiting a much greater sensitivity
than intrinsic tone to metabolic
inhibition.11 17 18 19 20 21 We and others have demonstrated that
constriction of resistance and capacitance vessels (unlike large
arteries, which generally rely on 1-ARs) is mediated by
both 1- and
2-ARs.18 19 21 22 23 In fact,
2-AR–mediated constriction may be dominant in terminal
arterioles and venous vessels (References 21, 22, and 2421 22 24 and references
therein). In rat skeletal muscle, arteriolar 2 tone is
selectively inhibited by metabolic factors, including
hypoxia, whereas arteriolar 1, venular
1, and venular 2 constrictions are
unaffected.11 17 18 19 20 21 Different 2-AR subtypes
(of which three have been cloned in the rat and other species) on
arterioles and venules could underlie this difference in sensitivity to
hypoxic metabolic inhibition. However, in rat skeletal
muscle, the same 2-AR subtype, the
2D, appears to mediate constriction of both
vessel types, whereas different 1-AR subtypes mediate
constriction of arterioles (1D) and venules
(1B).23 Therefore, at least in this
tissue/species, different 2-AR subtypes do not appear to
explain the insensitivity of venular 2-AR constriction
to inhibition by metabolic signals. However, differences in
coupling of the same 2-AR between arterioles and venules
could confer differences in sensitivity to hypoxic inhibition.
The purpose of the present study was first to determine whether
-AR constriction of venular smooth muscle in rat skeletal muscle is
in fact protected from inhibition by hypoxia, per se. Although
the effect of hypoxia on arteriolar -AR constriction has
been examined previously,11 17 venules have received
little attention. A second goal was to determine whether
KATP channels modulate venular
contractility and interact with -ARs.
KATP channels appear to be coupled with arteriolar
2-AR constriction and to mediate hypoxic inhibition of
2 tone in cremaster muscle arterioles.11
Finally, we determined whether venular -AR constriction depends on
VOCs, since Ca2+ influx via VOCs may be inhibited by
hypoxia.15 16 In many cell types,
1-ARs generally stimulate intracellular Ca2+
release, and 2-ARs rely on extracellular
Ca2+ influx.25 In VSM in particular, -AR
contraction has been shown to depend on VOCs, receptor-operated
Ca2+ channels, and the release of intracellular
Ca2+, depending on receptor subtype and
vessel.26 27 28 However, little is known about the
postreceptor coupling of 1- and
2-AR–mediated constriction in venules and small veins,
the major regulators of venous capacitance.
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Materials and Methods |
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In Vivo Skeletal Muscle Microvascular Preparation
Sixty-two 6- to 7-week-old male Sprague-Dawley rats (187± 2 g) were anesthetized with urethane and
-chloralose (425 and 100 mg/kg IM, respectively). The right
cremaster muscle was acutely denervated to prevent release of
endogenous NE and prepared for in situ microvascular
analysis, as described in detail previously.18 21
The muscle with intact circulation was suspended over an optical port
in a 50-mL tissue bath, which was filled from a stock reservoir
containing a modified Krebs' solution. Nitrogen and CO2
were bubbled through both the tissue bath and the reservoir to provide
mixing and to maintain normal tissue PO2 (30
mm Hg),29 PCO2 (40 mm Hg), and
pH (7.4), as measured with a blood-gas analyzer. Tissue
bath and reservoir solution pH and temperatures were continuously
monitored, and tissue bath temperature was maintained at the normal
cremaster in situ temperature (34°C). The microcirculation was viewed
at x400 to x1220 magnification, and vessel wall inner diameter was
measured with a videomicroscopic digital image analysis
system.21 Unless otherwise noted, the first-order
arteriole and paired venule that provide the major inflow and outflow
to the cremaster were chosen for measurement at a point approximately
one third of their length into the muscle. Vessels exposed to
nifedipine (below) were illuminated with low-intensity
(420- to 600-nm) light in a darkened room. Twenty to 30 minutes was
allowed to pass after suspension of the cremaster in the tissue bath to
ensure equilibration. The preparation was examined before the start of
each protocol and was judged to be acceptable if (1) mean
arterial pressure was stable and 
80 mm Hg, (2) terminal
arterioles in the area of study exhibited vasomotion, and (3) no venous
stasis, leukocyte adhesion, or petechial hemorrhages existed in
the area of study. Experiments were terminated if these criteria could
not be maintained.
Venular Stop-Flow Preparation
To assess the direct effect of interventions applied to the
tissue bath on venular diameter, it was necessary to prevent indirect
changes in diameter caused by alterations in venular flow and pressure
induced by simultaneous actions on arteriolar diameter. A
glass micro-occluder was positioned with a hydraulic
micromanipulator on the first-order venule several hundred microns
upstream from the measurement site to stop flow in the venule. This
allowed venular pressure below the occlusion to be held relatively
constant by pressure in the pudic-epigastric vein located outside
of the tissue bath downstream from the first-order venule in the
rat perineum. Flow stoppage and constancy of pressure in the
first-order venule were aided by ligation of second-order
venules (which were occasionally present downstream of the
measurement site) with 7.0 silk suture. In all stop-flow
experiments, bath washes between concentration-response curves were
performed after removal of the first-order venular occlusion to
facilitate elimination of pharmacological agents from the tissue and
the venule. All experiments, with the exception of the data shown in
Fig 1
, were conducted using the stop-flow preparation.
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Effect of Hypoxia on Venular -AR
Constriction
To examine the effect of hypoxia on venular adrenergic
constriction, an intermediate amount of 1- or
2-AR tone was induced in the venule in either the
blood-perfused "free-flowing" cremaster or in the above
stop-flow preparation (protocol depicted in Fig 1A
). Selective
1-AR stimulation at an intermediate level was achieved
using NE (0.3 to 0.9 µmol/L) plus 1 µmol/L rauwolscine to
antagonize 2-mediated responses, whereas intermediate
2-AR stimulation was achieved with UK 14,304
(2-AR agonist, 0.08 to 0.5 µmol/L) plus 0.01 µmol/L
prazosin to block 1-ARs. Propranolol (1
µmol/L) was present in all -AR experiments to prevent
activation of ß-ARs. These concentrations of propranolol
and of NE and UK 14,304 in the presence of rauwolscine and prazosin,
respectively, have been shown previously to achieve ß-AR blockade and
selective activation of 2- and 1-ARs in
this preparation.21 All antagonists identified
here and elsewhere were present a minimum of 20 minutes before the
application of agonists. After reaching steady intermediate
constriction (5 to 10 minutes), -AR tone was maintained for 10
minutes at the control bath PO2 of 30 mm Hg
(approximately normal cremaster tissue O229 ).
Bath PO2 was then lowered to 15 mm Hg for 10
minutes by increasing N2 bubbling. After a bath change to
prevent buildup of metabolites and breakdown of -AR agonists
(antagonists, agonists, and PO2
were maintained at previous concentrations),
PO2 was lowered further to 5 mm Hg for 10
minutes. The tissue bath was covered with plastic wrap (Saran Wrap),
and bath PO2 was continuously monitored with a
Clark-type polarographic O2 electrode (Cameron
Instruments). A 30- to 40-minute wash period was then observed, during
which venular occlusion was released, and the bath was exchanged four
times, with PO2 maintained at 30 mm Hg. After
reestablishing venular occlusion, the same concentration of
1 or 2 agonist was retested as a control
for changes in preparation sensitivity with time, followed by maximal
constriction with 30 µmol/L NE or UK 14,304 for use in data
normalization and assessment of -AR efficacy among preparations. The
bath was then washed twice, and maximal vessel diameter was obtained
with 0.1 mmol/L nitroprusside, first in the presence and then after
removal of venular occlusion. This was done to test the adequacy of
vascular isolation of the venule during flow stoppage and also to
determine the amount of intrinsic tone present in the vessels
studied. Intrinsic tone is defined as tone present in this acutely
denervated preparation during control conditions, as revealed by
maximal smooth muscle relaxation with nitroprusside.
To test whether the unexpected hypoxia-induced constriction
of venules in the presence of 2-AR tone might be masking
hypoxic inhibition of 2 tone (and also to identify the
nature of the constriction), an additional group of experiments
examined blockade of endothelin receptors and inhibition of nitric
oxide and prostanoid synthesis, since hypoxia-induced
changes in the activity of these factors have been reported
previously.30 31 32 The nonpeptide endothelin receptor
antagonist SB 209670 (0.5 µmol/L) was used to antagonize
both endothelin A and B receptors.33
Indomethacin (3 µmol/L) was administered to inhibit
cyclooxygenase17 and L-NMMA (300
µmol/L) to inhibit nitric oxide synthesis.34
Contribution of KATP Channel Activity to Venular
Constriction
To examine whether the failure of hypoxia to inhibit
venular -AR tone, uncovered in the preceding experiments, is
possibly due to the absence of KATP channel activity in
venular smooth muscle, experiments were conducted with the
KATP antagonists GLB and U-37883A (0.01 to 10
µmol/L).35 For these experiments, an intermediate amount
of tone was induced with 40 to 50 mmol/L KCl (with equimolar
reductions in NaCl), and a cumulative concentration-response curve
was obtained with one of the KATP antagonists.
After a bath wash, a second curve was obtained with the other
KATP antagonist in the presence of KCl tone.
Maximal KCl constriction (100 mmol/L KCl+equimolar decrease in NaCl)
was then induced, and the effect of nifedipine (0.3 and 0.6
µmol/L) on this constriction was determined, followed by a bath wash
and nitroprusside dilation. A second experiment examined the effect of
the KATP agonist cromakalim (0.001 to 1 µmol/L) on
intermediate 1- or 2-AR stimulation (as
discussed above). The specificity of any cromakalim effect was
determined with subsequent exposure to GLB. After a bath wash, -AR
agonists were retested, and the maximal responses to 1
or 2 stimulation were obtained. Maximal vessel diameter
was then determined with 0.1 mmol/L nitroprusside before and
after the removal of venular occlusion.
Role of VOC Activity in Venular Constriction
To determine if venular 1- and
2-ARs couple to VOCs, intermediate 1-AR
constriction of venules (0.1 to 0.9 µmol/L NE+1 µmol/L
rauwolscine) was induced, and a cumulative nifedipine
concentration-response curve was then obtained (0.06 to 3
µmol/L). After a 40-minute wash period, the nifedipine
curve was repeated in the presence of an intermediate amount of
2-AR tone (0.08 to 0.5 µmol/L UK 14,304+0.01 µmol/L
prazosin). The bath was washed twice, and maximal 2-AR
constriction (30 µmol/L UK 14,304+prazosin) was determined. This was
followed by two additional bath washes and determination of maximal
1-AR constriction (30 µmol/L NE+rauwolscine). After a
final bath wash, maximal vessel diameter was determined with 0.1 mmol/L
nitroprusside before and after removal of venular occlusion.
Nifedipine response curves against both 1-
and 2-AR tone were obtained in each animal, and the
order of the curves was randomized.
Data Analysis
Unless otherwise indicated, average values reported for control
diameters represent the arithmetic mean diameter during the
last 3 minutes of the control periods. Agonist responses are the mean
diameter during the last 2 minutes before an intervention or a change
in drug concentration. Inhibition curves are expressed as a percentage
of maximal inhibition:
response=(Dx-Dag)/(Dc-Dag)x100,
where Dx is the diameter produced by the intervention
(either hypoxia or a drug), Dag is the diameter in
the presence of an approximate EC50 concentration of the
agonist, and Dc is the diameter before constriction with
the agonist. Data were analyzed with parametric or
nonparametric (Kruskal-Wallis) single-factor ANOVA,
with Bonferroni a posteriori tests, or with paired or unpaired
Student's t tests where appropriate. Results are expressed
as the mean+SEM for n vessels (one per animal), with a value
of P<.05 representing significance.
Drugs
Stock solutions of NE were prepared daily in 1 mmol/L ascorbate
saline. UK 14,304 (kindly provided by Pfizer Pharmaceuticals, Groton,
Conn) was dissolved in Krebs' solution and stored frozen. Prazosin
(Sigma Chemical Co), rauwolscine (Atomergic Chemical Co), SB 209670
(kindly provided by E. Ohlstein and Smith Kline and Beecham
Pharmaceuticals), U-37883A (kindly provided by K. Meisheri and the
Upjohn Company), phentolamine (ICN Biochemicals), and L-NMMA
(Calbiochem) were dissolved in water. Propranolol and
nitroprusside (Sigma) were dissolved in saline. GLB and cromakalim
(Sigma) were dissolved in dimethyl sulfoxide; the maximal bath
concentration of dimethyl sulfoxide was <0.1%, which we have shown
has no effect on vessel tone.11 23 Nifedipine
(Sigma) was dissolved in 100% ethanol and protected from light
exposure; the maximal bath concentration of ethanol (0.054%) has no
effect on vessel tone (M. Ohyanagi and J.E. Faber, unpublished data,
1990). Indomethacin (Sigma) was dissolved in 1 mg/mL
sodium carbonate and diluted with water. Concentrated stock solutions
of all drugs were stored frozen at -20°C for no more than 6
weeks before use.
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Results |
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Effect of Hypoxia
The first experiment was conducted with the blood-perfused free-flowing cremaster preparation to examine the effect of hypoxia on venular
-AR tone. This preparation allowed the
simultaneously obtained responses of the adjacent,
parallel, first-order arteriole to be used as a positive control,
since we have shown previously that arteriolar 2- but
not 1-AR constriction is inhibited by
hypoxia.11 17 Fig 1 shows the
protocol used in this experiment; similar protocols were used in
subsequent experiments. Arteriolar 2-AR constriction
(2D)23 was inhibited by 26±14% when bath
oxygen was decreased to 15 mm Hg and by 29±13% at 5 mm Hg
(P<.05), whereas arteriolar 1 tone
(1D)23 was unaffected (Fig 1B). In
contrast, venular 1-AR
(1B)23 and venular 2-AR
(2D)23 responses were unaffected by
hypoxia (Fig 1C).
Analysis of control data for this experiment indicated that
arteriolar and venular diameters did not differ between the first
(control 1, Fig 1) and second (control 2) control periods. Intermediate
(baseline) 2 and 1 constrictions of
arterioles were 73±5% and 76±5% of the maximal arteriolar
2 and 1 responses obtained at the end of
the experiment. Baseline 2 constriction of venules was
45±9% of the maximal 2 response, whereas baseline
1 constriction was 64±6% of the maximal
1 response. Arteriolar 2 and
1 sensitivities were maintained over the course of the
experiment, because the retested intermediate 2
(69±14% of maximal response) and 1 (82±5%)
constrictions were not significantly different from the initial
baseline arteriolar constrictions. Similarly, retested 2
constriction of venules was 42±8% and 1 constriction
was 39±7% of the maximal 2 and 1
response; neither was significantly different from the baseline
constriction. Venular maximal 2-AR constriction to UK
14,304 (10 µmol/L) decreased the diameter to 139±4 µm, whereas
maximal 1 (10 µmol/L NE) constriction reduced the
diameter to 82±10 µm. Maximal arteriolar 2-AR
constriction was 78±4 µm, and 1-AR constriction was
56±4 µm. The sensitivity and efficacy of these 2 and
1 responses are similar to those found in previous
studies.21 23 Venular diameter during complete relaxation
by nitroprusside (185±9 µm) was not significantly different from
control 1 diameter (183±10 µm), indicating that these venules (n=13)
had no significant intrinsic tone and that arteriolar dilation in the
presence of nitroprusside did not induce passive pressure-dependent
venular distension. This is consistent with similar findings
reported previously.18 19 21 23 34 Arteriolar control
diameter (134±7 µm) was 15% less than nitroprusside diameter
(157±3 µm, P<.05), indicating the amount of basal
intrinsic tone.18 19 21 23 34 These control data
demonstrate the stability of the preparation and validate the various
comparisons made herein and below.
In a second experiment, the stop-flow preparation was used to
confirm the above hypoxia findings in the
hemodynamically isolated venule. Use of the same
protocol as in the first experiment (Fig 1A) confirmed that venular
1 and 2 tone were again not inhibited by
hypoxia (Fig 2A). However, during
2 tone, constriction was actually increased by
hypoxia, causing a further 19±6% and 19±8% reduction in
diameter at 15 and 5 mm Hg bath oxygen, respectively (Fig 2A,
P<.05). In the absence of -AR tone, hypoxia had
no effect on venular diameter (Fig 2A). Responses of the paired
first-order arteriole, which was exposed to disturbances in
flow and pressure by venular occlusion, were not measured, since a
positive control for hypoxic inhibition of arteriolar
2-AR tone had been obtained in the Fig 1 experiment.
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Control data for this experiment, as well as the subsequent experiments
using the stop-flow preparation (below), are given in Tables 1 and 2. For all experiments (Table 1),
the initial stop-flow period did not alter venular diameter;
however, diameter did decrease slightly during the second period of
venular occlusion, possibly because of a decrease in venular pressure
below the point of occlusion. Control diameters were comparable at
different times during the experiments (control 1 versus control 2).
Also, comparison of venular diameter with nitroprusside during stop
flow and free flow indicated additional slight dilation when venular
occlusion was removed, presumably because of increased venular pressure
and/or inhibition of a small amount of intrinsic tone. Venular
stop-flow diameter during nitroprusside was not greater than the
diameter during the second period of stop flow (stop-flow 2). This
suggests that good vascular isolation of the venule from changes in
pressure secondary to upstream arteriolar effects was achieved by the
venular occluder. Arteriolar dilation with nitroprusside otherwise
favors passive pressure-dependent venular distension in the normal
free-flow condition (ie, nitroprusside induced significant dilation
during free flow, Table 1). Table 1 also gives control data for
arterioles.
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These data, as expected from hemodynamic
considerations, suggest that venular pressure at the point of diameter
measurement downstream from the occlusion is largely isolated from the
upstream effects of vasoactive agents acting on arterioles. As in
previous studies, nitroprusside dilation indicated that these
arterioles possess intrinsic tone during control conditions. Arteriolar
diameter tended to decrease during stop flow, when both myogenic
vasoconstrictor (resulting from increased arteriolar pressure) and
metabolic (resulting from decreased flow) dilator signals
would be placed in competition. Because of these complications,
arteriolar diameter in most stop-flow experiments was not measured.
Venular control data for individual experimental groups (Table 2)
indicate that control diameters across groups were comparable, as were
the magnitudes of baseline 1- and 2-AR
tone and maximal -AR responses.
To determine the nature of the hypoxic venular constriction in the
presence of 2 tone identified in the preceding
experiment and, more importantly, whether it might be masking a
separate direct inhibitory effect of hypoxia on
2 tone, three constrictor mechanisms that may be induced
by hypoxia were examined: release of constrictor prostanoids,
release of endothelin, and inhibition of nitric oxide
production.30 31 32 Indomethacin (3
µmol/L) was used to prevent production of prostanoids; L-NMMA
(300 µmol/L), to block production of nitric oxide; and SB
209670 (0.5 µmol/L), to inhibit endothelin A and B receptors (see
"Materials and Methods" for selection of concentrations). A
protocol similar to that depicted in Fig 1A was used, except that two
consecutive PO2 concentration-response
curves were obtained in each animal, with a different
antagonist present for each curve, and only the effect
of 5 mm Hg PO2 was measured.
Antagonists were present a minimum of 20 minutes before
-AR stimulation and hypoxia. Venular diameter in the absence
versus the presence of indomethacin (199±7 versus
194±6 µm), SB 209670 (200±6 versus 200±7 µm), and L-NMMA (202±6
versus 196±4 µm) were not different (n=8 or 9). SB 209670 and L-NMMA
had no significant effect on hypoxic constriction during
2 tone, whereas indomethacin abolished
the response (Fig 2B). These data indicate that the hypoxic
constriction during 2-AR tone, which was possibly due to
release of a constrictor prostanoid, is not masking an otherwise
inhibitory action of hypoxia on venular
2 tone. There were no differences among
indomethacin, SB 209670, and L-NMMA groups in control
diameter (values listed above), intermediate 2-AR
constriction (in order, 153±8, 161±11, and 146±3 µm; n=5), and
nitroprusside diameters (in order, 205±6, 205±8, and 212±5 µm; n=8
or 9), justifying comparisons among these groups. These agents also had
no effect on 1 tone during hypoxia (5 mm Hg)
and likewise did not unmask any hypoxic dilation of 1-AR
tone (n=3 or 4). Venular diameters during 1-AR tone in
the absence and presence of hypoxia were not different for
indomethacin (132±11 and 128±13 µm), SB 209670
(160±14 and 164±16 µm), and L-NMMA (148±16 and 152±12 µm).
It is possible that countercurrent diffusion of oxygen from the
first-order arteriole to the adjacent venule could lessen the
reduction in venular PO2 as bath oxygen is
lowered, perhaps contributing to the absence of sensitivity of venular
-AR tone to hypoxia. To test this hypothesis, in a separate
experiment second-order venules (one per animal), which did not
have a paired arteriole, were selected, and the effect of
hypoxia on intermediate 1- and
2-AR tone was examined. In these experiments,
second-order venular responses were measured at a site 
200 µm
upstream from their bifurcation from the first-order venule, with
the micro-occluder positioned several hundred microns further
upstream on the second-order venule. This approach maintains
stop-flow conditions in the second-order venule but allows
lumen pressure to vary with pressure in the free-flowing
first-order venule. A protocol similar to that depicted in Fig 1A
was used, except that two consecutive PO2
concentration-response curves were obtained to examine
1 and 2 sensitivity to hypoxia in
the same vessel. Indomethacin (3 µmol/L) was
present during 2-AR stimulation to prevent the
hypoxic vasoconstriction seen in the first-order venules in the
preceding experiment. Neither 1- nor 2-AR
constriction of second-order venules was affected when bath oxygen
was decreased to 5 mm Hg (Fig 3). These data, taken
together with the results for first-order venules, indicate that
venular 1- and 2-AR constrictions are not
inhibited by tissue hypoxia at levels that inhibit arteriolar
2-AR tone. Control data for these experiments are given
in Table 2.
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Contribution of KATP Activity
In previous studies, we demonstrated that arteriolar
2-AR, but not 1-AR, tone is inhibited by
hypoxia through an antagonism between hypoxia, which
increases KATP channel activity, and 2
stimulation, which inhibits KATP channel
activity.11 To determine if the failure of hypoxia
to inhibit venular 2-AR tone could extend from a
relative paucity of KATP channel activity on venular smooth
muscle, the ability of KATP antagonists (GLB
and U-37883A) to augment an intermediate amount of KCl constriction was
examined (Fig 4A). In preliminary studies, venular tone
showed no increase to 0.01 to 1 µmol/L GLB. Since, unlike arterioles,
venules lack intrinsic tone and their smooth muscle cells are more
hyperpolarized36 in this denervated preparation, KCl was
used to induce partial depolarization and constriction and to favor
detection of KATP channels. If KATP channels on
smooth muscle cells are active, their blockade (closure) by
KATP antagonists should induce additional
constriction.11 KCl (40 to 50 mmol/L) reduced venular
diameter by 29±3 µm from control diameter (48% of maximum, Table 2
and Fig 4C). However, the KATP antagonists
produced no additional venular constriction (Fig 4B and 4C). In
contrast, the paired first-order arteriole in the same preparation
evidenced significant constriction to GLB or U-37883A
(P<.05). This arteriolar constriction was similar in
magnitude to that obtained in these same arterioles studied in vitro in
the absence of KCl (Fig 4B).11 One hypothesis suggested by
these data is that, unlike arterioles, venules exhibit little or no
KATP activity.
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Maximal constriction of arterioles and venules with KCl (100 mmol/L)
was significantly inhibited by nifedipine (0.3 and 0.6
µmol/L), indicating that both venules and arterioles in this tissue
possess dihydropyridine-sensitive VOCs (Fig 4C). The magnitudes of 1,
2, and maximal KCl constriction of venules and
arterioles (Table 2) are similar to those obtained in previous
experiments.21 Unlike the venules (and arterioles during
free-flow conditions21 ), increasing KCl to 40 to 50
mmol/L did not induce significant arteriolar constriction (Fig 4C). It
is possible in the stop-flow preparation that myogenic,
metabolic, and flow-mediated alterations in arteriolar
reactivity could attenuate sensitivity to KCl, although the maximal
response was unaffected.21
An experiment (n=4) preliminary to the above study was conducted as a
control for a possible -AR–mediated component of KCl constriction
of vessels in the cremaster muscle preparation that was due to release
of NE from nerve endings. Intermediate vessel constriction was induced
with 20 to 50 mmol/L KCl, followed by 0.5 µmol/L phentolamine
for 10 minutes. This concentration of phentolamine is selective
for -AR blockade and lower than the concentration (5 to 50 µmol/L)
that has been found to block KATP channels.37
Diameters during KCl versus KCl+phentolamine were not different
for arterioles (132±26 versus 128±28 µm) or venules (151±8 versus
160±7 µm). Thus, KCl constriction of arterioles and venules in the
acutely denervated rat cremaster muscle does not have a significant
-AR–mediated component; therefore, phentolamine was not
present during the subsequent KCl experiments (Fig 4).
Among other interpretations (see "Discussion"), the above failure
of KATP antagonists to cause venular
constriction (Fig. 4) might arise either because KATP
channels are sparse or absent or because they are inactive (closed). To
differentiate between these possibilities, the ability of a
KATP agonist to dilate venules was examined. An
intermediate amount of 1- or 2-AR tone
was induced, and a cumulative concentration-response curve for the
KATP agonist cromakalim was then obtained (Fig 5A). Cromakalim is selective for KATP
channels at concentrations <1 µmol/L.38 After obtaining
the response at the highest cromakalim concentration (1 µmol/L), the
bath was changed (maintaining the final concentrations of -AR
agonist, antagonists, and cromakalim), and a cumulative GLB
response curve was obtained to test for the specificity of any
cromakalim dilation. In contrast to the effect of cromakalim on
arteriolar 2 tone, examined in vitro
previously11 and shown here for comparison (Fig 5B),
cromakalim had no effect on venular 1 or
2 tone at concentrations selective for KATP
(<1 µmol/L). The highest concentration of cromakalim (1 µmol/L)
caused similar inhibition of both 1- and
2-AR venular tone, which was not reversed by GLB (10
nmol/L to 10 µmol/L). The possibility that 1 µmol/L cromakalim is
acting through channels other than KATP (eg, delayed
rectifier and Ca2+-activated K+
channels38 ) to produce dilation is supported by the
similar dilation of both 1 and 2 venular
tone and by the inability of GLB to reverse this dilation. Since
venular adrenergic and KCl constrictions in this preparation are
known to be accompanied by smooth muscle cell
depolarization,36 which should then render the
constriction sensitive to stimuli that are known to open
(hypoxia and cromakalim) or close (GLB and U-37883A)
KATP channels, the data in the previous figures, taken
together with those in Fig 5, suggest that these venules may lack
significant KATP channels.
|
VOC Activity
Intermediate 1- or 2-AR tone was
induced in venules and examined for concentration-dependent
inhibition by nifedipine. Nifedipine produced
dose-dependent inhibition of 1 tone, whereas
2 tone was unaffected (Fig 6A and 6B).
Thus, venular smooth muscle appears to possess
dihydropyridine-sensitive VOCs, to which
1-ARs, but not 2-ARs, couple. Moreover,
the presence or absence of -AR dependence on VOCs does not appear to
correlate with the protection of venular -AR tone from hypoxic
inhibition.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previous studies from this and other laboratories have demonstrated that
2-AR constriction of arterioles is
antagonized by metabolic disturbances, whereas
arteriolar 1, venular 1, and
venular 2 constriction exhibits little or no inhibition
(References 11 and 1711 17 through 2118 19 20 21 and references therein).
Hypoxia selectively inhibits arteriolar 2 tone
but not 1, intrinsic, and KCl
tone.17 However, before the present study, the effect
of hypoxia itself on venular -AR constriction and the
mechanisms responsible for this difference in sensitivity had not been
studied directly. The major findings of the present study were that
arteriolar 2, but not 1,
constriction was inhibited by hypoxia, confirming previous
studies11 17 ; neither venular 1- nor
2-AR constriction was affected. Since KATP
channels have been shown to be important in hypoxic inhibition of
resistance vessel tone, we examined whether venules might have a
deficit in basal or inducible KATP activity to explain our
findings. In support of this hypothesis, the structurally dissimilar
KATP antagonists GLB and U-37883A, which act
through different binding sites,35 had no significant
effect on venular tone at concentrations as high as 10 µmol/L in the
absence or presence of prior KCl constriction. In addition, the
K+ channel agonist cromakalim, at
KATP-selective concentrations (1 to 100
nmol/L),8 9 38 did not inhibit intermediate
1 or 2 venular constriction. The
inhibition by 1 µmol/L cromakalim was not selective for
1 or 2 tone and was not reversed by GLB,
which suggests that the dilation in this tissue is due to actions at
non-KATP K+ channels. The findings for venules
herein are opposite the findings obtained for arterioles in the
present and previous11 17 studies. Finally, the
dihydropyridine Ca2+ channel
antagonist nifedipine (up to 3 µmol/L) had no
effect on 2-mediated venular tone but inhibited
1 tone by 50%, suggesting that venular
1- and 2-ARs are coupled to different
sources of contractile Ca2+. Therefore, for cremaster
venules, we speculate that 2-AR (and
1-AR) constriction is insensitive to hypoxia
because of a paucity of KATP channels and that the coupling
of 1-ARs to VOCs does not confer sensitivity to
hypoxia.
Instead of inhibiting -AR constriction, hypoxia (5 mm Hg)
caused a modest additional venular constriction in the presence of
2-AR tone (cremaster arterioles do not exhibit this
hypoxic constriction17 ) (Fig 2). Although hypoxia
augments NE constriction of rabbit mesenteric veins via
endothelial cell production of
endothelin,30 in the present study, hypoxic
venoconstriction was unaffected by the endothelin-A and -B receptor
antagonist SB 209670.33 L-NMMA also had no
effect, making it unlikely that this response is due to a decreased
release of nitric oxide during hypoxia.31 Venular
hypoxic constriction was, however, inhibited by
indomethacin, consistent with
hypoxia-induced release of a constrictor prostanoid
reported for human and monkey coronary arteries.32
It is possible that venular hypoxic constriction during
2-AR, but not 1-AR, tone is due to
coupling of 2-AR stimulation to liberation of
arachidonic acid, as demonstrated in rabbit
aorta.39 Importantly, however, inhibition of the hypoxic
constriction with indomethacin did not unmask a hypoxic
dilation. Hypoxic venoconstriction was only apparent in the
stop-flow preparation, suggesting that either flow-mediated
dilator signals or washout of a prostanoid constrictor could be masking
hypoxic vasoconstriction in the free-flowing cremaster preparation.
Interestingly, during metabolic inhibition of precapillary
2 tone and increased venular pressure and flow, this
prostanoid-dependent increase in venular tone would provide an
additional mechanism, besides the absence of hypoxic inhibition of
venular -AR tone, to optimize maintenance of reflex venous
return by capacitance vessels.
The differential sensitivity of arteriolar versus venular -AR
constriction to hypoxia could be due to different -AR
subtypes on these vessels, since at least five -AR subtypes may be
expressed by rat VSM (1D,
1B, 1A,
2D, and 2B).40 41 On
the basis of pharmacological studies, in rat cremaster skeletal muscle
the 1B- and the 1D-AR subtypes appear to
mediate constriction of venules and arterioles, respectively, whereas
arterioles and venules both use the 2D-AR for
constriction.23 However arteriolar, but not venular,
2D tone is inhibited by hypoxia. Therefore, the
subtype of -AR does not seem to determine adrenergic sensitivity to
hypoxic inhibition. Coupling of the 2D-AR to
constriction in venules and arterioles could also differ. It has been
suggested that L-type VOCs may mediate hypoxic vasodilation, since
Ca2+ influx via VOCs is reduced by severe hypoxia
and acidosis.15 16 However, we have found that venular
1 constriction, rather than 2
constriction, is inhibited by the VOC antagonist
nifedipine but that neither constriction is sensitive to
hypoxia, suggesting that VOC-dependent venular constriction is
not sensitive to the level of hypoxia examined here. In
addition, a previous study found that constriction of in vitro
cremaster arterioles induced by KCl and by the VOC agonist SDZ-202-791
were unaffected by a pH of 7.1 or a PO2 of 10
mm Hg.17 Arteriolar constriction to the partial
1 agonist ST-587, which is strongly inhibited by
nifedipine, was also unaffected by hypoxia or
acidosis in that study.17 Therefore, it appears that
hypoxia does not act through VOCs to inhibit -AR tone in
cremaster muscle arterioles or venules.
Recent work suggests that the cremaster arteriolar 2-AR,
but not the 1-AR, induces constriction via closure of
KATP channels, presumably leading to depolarization and
influx of Ca2+ across VOCs, upon which arteriolar
2-AR constriction depends.11 17 Moreover,
hypoxia appears to selectively inhibit arteriolar
2 constriction by potentiating KATP
activity.11 On the basis of the albeit indirect data
presented herein, we speculate that -AR constriction of
venules does not depend on an interaction with KATP
channels, nor is it sensitive to hypoxia, possibly because
venular smooth muscle has a paucity of KATP
channels. It is also possible that KATP
channels are present but that a KATP-associated
protein(s) that binds GLB, U-37883A, and cromakalim and couples low
O2 to increased channel activity is limiting.
Alternatively, KATP channels in the venules could be under
tonic inhibition or in a low-open-probability state so that
activation by agonists (cromakalim and low O2) and
antagonists (and the consequential changes in membrane
conductance) would have less effect on membrane potential and
contractile state.42
Membrane potential for rat cremaster venules (-55 mV) is more
hyperpolarized than for arterioles (-51 mV) in the presence of
-AR blockade with phentolamine.36 This may
limit the ability of antagonists that close
KATP channels to exceed VOC threshold and cause venular
constriction. Moreover, unlike arterioles, cremaster venules have
little or no intrinsic tone. Therefore, in the GLB and U-37883A
experiments, we used an intermediate concentration of KCl (40 to 50
mmol/L) to depolarize the venular smooth muscle and maintain membrane
potential near the threshold for activation of VOCs (-32 mV for
rat vena cava VOCs43 ). The KCl depolarization of venules
used in the present study should then increase the ability of
KATP antagonists, if KATP channels
are present and open, to block KATP channels,
depolarize the membrane, and activate VOCs. GLB constricted the
arterioles in a concentration-dependent manner, confirming our
previous in vitro studies of these vessels.11 However,
unlike arterioles, there was no venular response to KATP
antagonists in the absence or presence of KCl tone.
It is commonly accepted that 2-ARs rely greatly on the
influx of extracellular Ca2+ via VOCs to mediate
contraction of VSM, whereas 1-ARs use intracellular and
extracellular stores.25 For example, the initial transient
1-AR contraction of rabbit saphenous, renal, and
plantaris veins relies on intracellular Ca2+
release.26 In addition, 1-mediated
constriction of rat cremaster muscle arterioles is dependent on release
of intracellular Ca2+, whereas 2-ARs
rely entirely on extracellular Ca2+.17 In
contrast, like our present findings, NE contraction of dog
saphenous vein via 2-ARs is not antagonized by the
Ca2+ channel antagonists nifedipine
and verapamil.27 However,
2-mediated contraction of this vein still depends on
extracellular Ca2+, indicating that
2-ARs may couple to receptor-operated
Ca2+ channels.27 It is possible that
2-ARs in some veins may also induce intracellular
Ca2+ release, since 2-ARs can induce
contraction of rabbit ear vein in low-Ca2+ EGTA-buffered
solution,26 and 2-ARs may couple to
intracellular Ca2+ release in human resistance
arteries.28 In addition, Aburto et al44 found
that stimulation of 2-ARs on rabbit saphenous vein
sensitizes the contractile apparatus by a mechanism that
does not involve increased myosin light chain
phosphorylation, suggesting that 2-ARs
can modulate smooth muscle contractility independent of
effects on Ca2+. In the present study, we were unable
to consistently achieve zero Ca2+ conditions in the
in vivo cremaster muscle preparation. This precluded an
analysis of whether venular 2-ARs rely on
receptor-operated channels and/or intracellular Ca2+
stores for constriction. However, we did find that 2-AR
constriction is insensitive to inhibition by nifedipine,
whereas venular 1-ARs appear to rely on the influx of
extracellular Ca2+ via nifedipine-sensitive
VOCs.
In conclusion, the present studies demonstrate that venular
1- and 2-AR constrictions are insensitive
to hypoxia but that arteriolar 2-AR constriction
is inhibited and suggest that this protection of venular -AR
constriction from hypoxic inhibition may be due to a paucity of
functional KATP channels on venules. In contrast,
hypoxia appears to selectively inhibit arteriolar
2-AR constriction because of the reliance of the
arteriolar 2D-AR constriction on KATP
channels.11 If these findings are
representative of arterioles and venules in general,
they suggest that a relative lack of KATP channels may
explain how sympathetic constriction of the venous circuit and, hence,
reflex venous return are maintained during hypoxia and
decreased tissue blood flow, whereas arterial sympathetic
tone is antagonized to provide for autoregulation of tissue blood flow
and interstitial oxygen levels.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by National Institutes of Health grants HL-38783 and the American Heart Association, North Carolina Affiliate, Inc.
|
Footnotes |
|---|
Reprint requests to James E. Faber, PhD, Department of Physiology, 474 MSRB, CB 7545, University of North Carolina, Chapel Hill, NC 27599-7545. E-mail jefaber@med.unc.edu.
Received September 13, 1995; accepted February 28, 1996.
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 H. Sako, S. A. Green, E. G. Kranias, and A. Yatani Modulation of cardiac Ca2+ channels by isoproterenol studied in transgenic mice with altered SR Ca2+ content Am J Physiol Cell Physiol, November 1, 1997; 273(5): C1666 - C1672. [Abstract] [Full Text] [PDF]
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 A. D. Eckhart, N. Yang, X. Xin, and J. E. Faber Characterization of the alpha 1B-adrenergic receptor gene promoter region and hypoxia regulatory elements in vascular smooth muscle PNAS, August 19, 1997; 94(17): 9487 - 9492. [Abstract] [Full Text] [PDF]
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 J. P. Slack, I. L. Grupp, D. G. Ferguson, N. Rosenthal, and E. G. Kranias Ectopic Expression of Phospholamban in Fast-Twitch Skeletal Muscle Alters Sarcoplasmic Reticulum Ca2+ Transport and Muscle Relaxation J. Biol. Chem., July 25, 1997; 272(30): 18862 - 18868. [Abstract] [Full Text] [PDF]
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 M. L. Clements, A. J. Banes, and J. E. Faber Effect of Mechanical Loading on Vascular {alpha}1D- and {alpha}1B-Adrenergic Receptor Expression Hypertension, May 1, 1997; 29(5): 1156 - 1164. [Abstract] [Full Text]
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Y. Ji, M. J. Lalli, G. J. Babu, Y. Xu, D. L. Kirkpatrick, L. H. Liu, N. Chiamvimonvat, R. A. Walsh, G. E. Shull, and M. Periasamy Disruption of a Single Copy of the SERCA2 Gene Results in Altered Ca2+ Homeostasis and Cardiomyocyte Function J. Biol. Chem., November 22, 2000; 275(48): 38073 - 38080. [Abstract] [Full Text] [PDF]
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Y. Li, E. G. Kranias, G. A. Mignery, and D. M. Bers Protein Kinase A Phosphorylation of the Ryanodine Receptor Does Not Affect Calcium Sparks in Mouse Ventricular Myocytes Circ. Res., February 22, 2002; 90(3): 309 - 316. [Abstract] [Full Text] [PDF]
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