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Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131-5218
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Chronic hypoxia (CH) is associated with a persistent reduction in systemic vasoconstrictor reactivity. Experiments on aortic ring segments isolated from CH rats suggest that enhanced vascular expression of heme oxygenase (HO) and resultant production of the vasodilator carbon monoxide (CO) may underlie this attenuated vasoreactivity after hypoxia. Similar to the aorta, small arteries from CH rats exhibit blunted reactivity; however, the regulatory role of CO in the resistance vasculature has not been established. Therefore, we examined the significance of HO activity on responsiveness to phenylephrine (PE) in the mesenteric circulation of control and CH rats. To document that the mesenteric bed demonstrates reduced reactivity after CH, we determined the vasoconstrictor responses of conscious, chronically instrumented male Sprague-Dawley rats to PE under control conditions and then immediately after exposure to 48 h CH (0.5 atm). All rats showed reduced mesenteric vasoconstriction to PE after CH. To examine the role of CO in reduced reactivity, small mesenteric arteries (100-200 µm intraluminal diameter) from control and 48-h CH rats were isolated and mounted on glass cannulas, pressurized to 60 mmHg and superfused with increasing concentrations of PE under normoxic conditions. Similar to the intact circulation, vessels from CH rats exhibited reduced vasoconstrictor sensitivity to PE compared with controls that persisted in the presence of nitric oxide synthase inhibition. The HO inhibitor, zinc protoporphyrin IX (5 µM) enhanced reactivity only in CH vessels. Additionally, a range of concentrations of the HO substrate heme-L-lysinate caused vasodilation in CH vessels but not in controls. Thus we conclude that CO contributes a significant vasodilator influence in resistance vessels after CH that may account for diminished vasoconstrictor responsiveness under these conditions.
rat; heme oxygenase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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SEVERAL RECENT STUDIES (3,4) have demonstrated that exposure to prolonged environmental hypoxia results in reduced systemic vasoconstrictor reactivity that persists on restoration of normal oxygenation. This effect has been observed not only in vivo, but also in vascular tissue isolated from hypoxia-exposed animals, suggesting that altered sensitivity is a local vascular phenomenon (18). Mechanisms associated with the regulation of altered responsiveness in the vasculature during hypoxia are not well understood. However, experiments on aortic tissue isolated from chronically hypoxic (CH) rats suggest that enhanced vascular expression of heme oxygenase (HO) and resultant production of the vasodilator carbon monoxide (CO) may be responsible for the attenuated vasoreactivity after long-term hypoxia (18).
Expression of HO has been reported (6, 16, 17, 20) in a
variety of cell types including vascular smooth muscle (VSM) and
endothelial cells. In the endothelium the production of CO, much like
nitric oxide (NO), elicits dilation by diffusing across the endothelial
membrane and interacting with the underlying VSM activating soluble
guanylyl cyclase, leading to the subsequent production of cGMP
(7, 20). Some studies have suggested that during hypoxia
NO synthase (NOS) and HO expression are increased and each may
contribute to altered regulation of vascular tone. Thus a portion of
the attenuated vasoconstrictor reactivity to CH may in part be due to
increases in NO and CO production. Experiments on aortic ring segments
isolated from CH rats demonstrate that contractility to
1-agonist stimulation is blunted and that this response
persists with NOS inhibition (3). Although the aorta from
CH rats exhibits a reduction in vascular reactivity, the regulatory
role of CO in the resistance vasculature has not been established.
Therefore, one goal of the current study was to determine if mesenteric
resistance arteries from animals exposed to CH exhibit an attenuated
vasoconstrictor response to phenylephrine (PE). In addition, the
current study was designed to test the hypothesis that enhanced
endogenous production of CO contributes to reduced vasoconstrictor
reactivity in the resistance vasculature after CH exposure.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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All of the protocols and animal handling in this study were approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine.
Demonstration of Attenuated Mesenteric Vasoconstrictor Responses After CH in Conscious Rats
Previous experiments from our laboratory have demonstrated that CH exposure results in diminished vasoconstrictor responses to the1-adrenergic agonist PE in the renal vascular bed of
conscious rats (18) that is independent of altered
autoregulation (10). Because subsequent mechanistic
studies were planned for isolated mesenteric resistance arteries,
experiments were first performed to assess whether the mesenteric
vascular bed demonstrates similar diminished vasoconstrictor
responsiveness after hypoxia. Thus we determined the mesenteric
vasoconstrictor responses of conscious chronically instrumented rats to
PE first under control conditions and then after 48-h exposure to
hypobaric hypoxia.
Surgical Preparation of Rats for Chronic Study
Male Sprague-Dawley rats (270-330 g; Harlan) were anesthetized with a mixture of ketamine (91 mg/kg im) and acepromazine (9 mg/kg im) and prepared for chronic instrumentation with a pulsed Doppler flow probe. Flow probes were assembled using a Silastic cuff formed around a piezoelectric crystal (20 mHz, 1-mm diameter, Crystal Biotech). With the use of a midline laparotomy, the superior mesenteric artery was isolated and the cuff containing the flow probe was positioned around the artery and secured with a silk suture. Probe leads were passed through the abdominal wall and routed subcutaneously to the base of the neck and exteriorized. The leads were housed in a small plastic protective container sutured to the scalp and covered with a rubber serum stopper to prevent the animals from disturbing the leads. After surgical closure, the animals were treated with systemic antibiotics (30,000 U; penicillin G benzathine and penicillin G procaine) and topical (triple antibiotic ointment) antibiotics. After a 5-day recovery period, animals were instrumented with abdominal arterial and venous catheters (polyethylene; PE-50 and PE-10) via the femoral artery and vein. Like the probe leads, catheters were routed subcutaneously and stored in the protective plastic container. Animals were given an additional 2-3 days to recover before experiments were performed.Mesenteric Vascular Response to PE in Control and CH Conscious Rats
On the day of experiments, rats were placed in a Plexiglas chamber (23 × 14 × 10 cm) containing fresh bedding and drinking water. Before the experiments were performed, all animals were food restricted for 4 h and allowed time to acclimate to the testing chamber. After acclimation, probe leads were connected to a Crystal Biotech Doppler flowmeter for measurement of mesenteric blood flow (MBF). Catheters were flushed with heparinized saline and the arterial line was connected to a pressure transducer, the output of which was fed to a Gould Universal amplifier. After equilibration in the chamber, 5 min of baseline data were collected, followed by a graded infusion of PE at 3, 6, and 9 µg · kg
1 · min1 iv for 5 min at each dose. Mean arterial blood pressure (MAP), heart rate, and
mean MBF Doppler signals (KHz of Doppler shift) were continuously
recorded using a chart recorder (model RS 3800, Gould). Analog signals
from the transducer and the Doppler mainframe were digitized and stored
on computer for analysis. At the end of the experiment, blood samples
were collected for hematocrit and blood gas analysis. After this
initial normoxic (control) experiment, the rats were placed in a
hypobaric chamber maintained at 380 ± 5 Torr for 48 h or
remained at ambient barometric pressure (~630 Torr) and then were
restudied in an identical fashion with the following exceptions. First,
during the equilibration period before experimentation, hypoxic rats
were maintained in an environment (12% O2) mimicking the
PO2 of the hypobaric chamber. Second, hypoxic rats were switched to room air for the final 15 min of equilibration and during the infusion of PE. The normoxic control group breathed room
air throughout equilibration and study. Finally, blood gas samples were
collected under normoxic conditions for both groups and then the
hypoxia-exposed rats were switched back to 12% O2 and
another blood sample was drawn.
Pressurized Mesenteric Resistance Artery Preparation
Vessels were isolated from male Sprague-Dawley rats (250-350 g wt; Harlan) either housed under hypoxic conditions as above or their age-matched controls housed at ambient barometric pressure (~630 Torr). On the day of the experiment, animals were anesthetized with pentobarbital sodium (50 mg/kg ip), and the thoracic and abdominal cavity was exposed. Heparin (100 U) was immediately injected into the right ventricle, and a small volume of blood was collected for hematocrit measurement before animals were exsanguinated. A portion of the small intestine (jejunum) was then removed and pinned in a Sylgard-coated dissection dish containing ice-cold physiological saline solution (PSS) composed of (in mmol/l) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 0.4 NaH2PO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose bubbled with 21% O2-6% CO2-balance N2. Small segments of third-order arteries (100-200 µm diameter) were carefully dissected and placed in a vessel chamber (Living Systems; Burlington, VT) containing ice-cold PSS. The vessel was mounted on glass cannulas (60-80 µm diameter), rinsed free of blood, and secured in place with nylon ties. A stopcock distal to the artery was closed and the vessel pressurized to 60 mmHg and maintained constant with a servo control unit (Living Systems). The vessel was superfused (5 ml/min) with PSS (37°C), aerated with 21% O2-6% CO2-balance N2, and the preparation was viewed on an inverted microscope (model TMS, Nikon) equipped with a video camera and monitor. A video dimension analyzer (Living Systems; Burlington, VT) was used to measure intraluminal diameter (ID). Changes in ID were recorded on a chart recorder (model RS 3600, Gould) and simultaneously digitized from analog signal using a computer-based data analysis acquisition system (CODAS; Dataq Instruments).Vasoreactivity of Mesenteric Resistance Arteries to PE
Vessels were equilibrated for 60 min. During that time, the vessels were constricted with PE (105 mol/l) and were
then administered acetylcholine (106 mol/l) to test for
constrictor and endothelial viability, respectively. After
equilibration, a complete concentration-response curve to PE
(108-104 mol/l) was generated.
Effect of NO Synthesis Inhibition on Reactivity of Pressurized Mesenteric Resistance Vessels
To test for the potential involvement of NO in attenuated constrictor responsiveness of arteries from CH rats, experiments were performed as in the preceding protocol, except that the NOS inhibitor N
-nitro-L-arginine
(L-NNA; 100 µmol/l) was added to the superfusate at the
beginning of the equilibration period and remained present throughout
the experiment.
Effect of HO Blockade on Reactivity of Pressurized Mesenteric Resistance Vessels
To determine whether endogenously produced CO contributes to the reduced constrictor responsiveness after CH, isolated pressurized mesenteric resistance arteries from CH or control rats were challenged with a PE concentration response curve (108-104 mol/l) in the presence of the HO
inhibitor, zinc protoporphrin IX (ZnPPIX, 5 µmol/l) or vehicle. These
experiments were performed in the presence of L-NNA (100 µmol/l) to eliminate the contribution of NO. Because ZnPPIX is
photosensitive, all experiments were conducted in reduced light.
Vasodilator Responses of Mesenteric Resistance Arteries to the HO Substrate Heme-L-Lysinate
This study was designed to contrast the responses to heme-L-lysinate, an HO substrate, between arteries from animals exposed to control or CH conditions. Heme-L-lysinate (38 mmol/l) was prepared using the methods described by Tenhunen et al. (21). At the time of experimentation, dilutions of heme-L-lysinate (107-104 mol/l) were prepared using PSS
and stored on ice. Pressurized vessels were equilibrated for 60 min in
PSS containing L-NNA (100 µmol/l). After equilibration, a
concentration-response curve to heme-L-lysinate was
generated in vessels partially constricted with a half-maximal
constrictor dose of PE (hypoxic 106.5; normoxic
106 mol/l). Vehicle for heme-L-lysinate did
not affect artery diameter (data not shown).
Data Analysis and Statistics
Conscious rat experiments.
Data are reported as means ± SE. Mesenteric vascular resistance
(MVR) was calculated by dividing MAP (mmHg) by MBF (kilohertz of
Doppler shift). Blood flow and vascular resistance are expressed as a
percentage of baseline values. Arcsine transformation was used to
successfully normalize the distribution of the percentage data before
being statistically analyzed. Data were compared between day
1 and 48 h for both normoxic and hypoxic groups using
Student's paired t-test or analysis of variance (ANOVA) for
repeated measures as appropriate. Multiple comparisons were made with
the Student-Newman-Keuls test when ANOVA indicated that differences
existed. P 
0.05 was considered statistically significant.
Isolated vessel experiments.
Groups were compared at each point of the concentration-response curves
using Student's unpaired t-test, with P 0.05 considered statistically significant. The n value
represents the number of animals used in each group. Changes in ID from
the heme-L-lysinate microvessel experiments are expressed
as a percentage reversal of constriction to PE. Again, percentage data
values were normally distributed by arcsine transformation before
statistical comparison.
Drugs and Chemical Solutions
PE stock solutions (1 mol/l) were dissolved in ddH2O and aliquots stored at
4°C until use. For isolated vessel studies,
PSS and PSS containing L-NNA (100 µmol/l) were prepared
on the day of experiments. Heme-L-lysinate was prepared as
described by Tehunen et al. (21). Briefly, hemin (3.85 mmol) was added to a solution containing ethanol (10 g), 1,2-proanediol
(40 g), lysine (11.5 mmol), and ddH2O to bring the total
volume of solution to 100 mL. The solution was filtered (0.2-µm pore
size), and aliquots were stored at 4°C. ZnPPIX was prepared as an
adaptation of methods described by Vreman et al. (23) on
the day of each experiment. Specifically, ZnPPIX (50 mg) was solublized
in 10% ethanolamine (500 µl) and 0.9% saline (2 ml) was slowly
added and mixed thoroughly. HCl (1 M) was then used to adjust the pH to
8.0. Heme-L-lysinate and ZnPPIX are photosensitive
compounds, and solutions containing these drugs were protected from
light. ZnPPIX was purchased from Frontier Scientific and all other
reagents were purchased from Sigma.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Conscious Animal Experiments
Table 1 presents hematocrit and blood gas values obtained from chronically instrumented rats. Exposure to CH caused a significant increase in hematocrit that was unaffected by acute normoxic exposure during the testing phase. Additionally, arterial PO2 and PCO2 were significantly lower, and pH was elevated after CH compared with control. During acute return to normoxic conditions, PO2 and PCO2 levels increased with a slight decrease in pH; however, PO2 was elevated and PCO2 decreased compared with blood gas values before 48-h hypoxic exposure, indicative of adaptation to sustained hypoxia. No differences in blood gases were observed in animals that were maintained normoxic for the entire 48-h period. Similarly, baseline MAP and heart rate were unaffected by 48-h normoxic exposure; however, heart rate was significantly elevated after 48 h of hypoxia as previously observed (10) (Table 2). Baseline MAP was slightly reduced during hypoxia but returned to prehypoxic control on restoration of normoxia before PE infusion (Table 2).
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MVR Response to PE in Control and CH Conscious Rats
Figure 1 shows successive MAP, MBF, and MVR responses to graded infusions of PE in control rats on day 1 and after 48 h of continued normoxic exposure. Infusion of PE elicited a dose-dependent decrease in MBF and an increase in MAP and MVR that was not different between experiments. In contrast, animals exposed to hypoxia for 48 h and returned acutely to normoxia demonstrated significantly blunted blood flow and vasoconstrictor responses to PE compared with responses generated before exposure (Fig. 2). Additionally, the change in MAP was significantly blunted in response to increasing doses of PE.
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Vasoreactivity of Mesenteric Resistance Arteries to PE
Changes in ID in response to PE in pressurized (60 mmHg) mesenteric resistance arteries are shown in Fig. 3. PE elicited a dose-dependent decrease in ID of arteries isolated from control (n = 5) and CH (n = 5) rats. However, vasoconstrictor responses to PE were significantly attenuated in vessels isolated from CH rats compared with control.
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Effect of NO Synthesis Inhibition on Reactivity of Pressurized Mesenteric Resistance Vessels
In the presence of the NOS inhibitor L-NNA, vasoconstrictor responses to PE were greater in vessels from control and CH rats compared with untreated preparations (Fig. 4). However, L-NNA-treated vessels from CH rats exhibited persistent attenuated vasoreactivity to PE compared with similarly treated control tissue.
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Effect of HO Blockade on Reactivity of Pressurized Mesenteric Resistance Vessels
Because blunted vasoreactivity to PE was sustained after NOS blockade, the effect of HO inhibition in the presence of L-NNA on reactivity was assessed. Figure 5 presents changes in ID to PE in resistance arteries isolated from control rats treated with ZnPPIX or vehicle in the presence of L-NNA. Vehicle treatment did not affect reactivity to PE in vessels from either control or CH animals. However, ZnPPIX significantly attenuated reactivity to low concentrations of PE in control arteries. Conversely, PE vasoreactivity was significantly augmented at higher doses after ZnPPIX treatment in arteries from CH rats compared with vehicle (Fig. 6), suggesting a role for HO in diminished reactivity to vasoconstrictors during CH.
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Vasodilator Responses of Mesenteric Resistance Arteries to the HO Substrate Heme-L-Lysinate
Figure 7 presents the responses to heme-L-lysinate in pressurized mesenteric resistance arteries constricted to a similar degree with PE (control, 106.5 mol/l; hypoxic, 106 mol/l). Change in
vessel diameter is expressed as a percent reversal of PE-induced
vasoconstriction. Heme-L-lysinate resulted in a significant
dose-dependent increase in ID in resistance arteries from CH rats. In
contrast, this substrate for HO had a vasodilator effect only at the
highest dose in vessels isolated from control rats. Vehicle did not
affect diameter in vessels from either control or CH rats (data not
shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The current study investigated the contribution of enhanced
endogenous CO production to reduced vasoconstrictor reactivity in
mesenteric resistance arteries after CH exposure. The results of this
study indicate that: 1) rats exposed to CH exhibit a blunted response to 1-agonist stimulation in the mesenteric
vasculature compared with animals exposed to normoxic (control)
conditions; 2) reactivity to PE is significantly blunted
compared with control in isolated, pressurized mesenteric resistance
arteries from CH rats; 3) the HO inhibitor ZnPPIX reversed
blunted vasoreactivity to PE in CH vessels; and 4)
heme-L-lysinate, an HO substrate, elicited vasodilator
responses selectively in vessels isolated from hypoxic rats compared
with controls. Together, these results suggest that the attenuated
hypoxia-induced vasoconstrictor reactivity to PE after CH may be due in
part to enhanced CO production within the resistance vasculature.
Several studies have provided evidence for altered vascular control
after prolonged hypoxia. For example, earlier experiments from our
laboratory (4) demonstrated that total peripheral resistance responses to PE, angiotensin II, and arginine vasopressin were blunted in conscious rats exposed to 4 wk of CH. More recently, it
was observed that renal vasoconstriction to 1-adrenergic
stimulation was diminished in conscious rats after 4 wk
(18) and 48 h (10) exposure to hypoxia.
Interestingly, acute return to normoxia did not restore the blunted
vasoconstrictor responses to that observed in control groups in any of
these studies. In the latter studies, the reduced renal vascular
responsiveness in the conscious animal persisted after renal
denervation, suggesting that CH-induced attenuation of systemic
vasoconstrictor reactivity is influenced by endogenous factors within
the vasculature independent of sympathetic reflexes. In the present
study, it was not possible to denervate the mesenteric bed; however, as
in the renal vasculature, we observed attenuated MVR responses to PE in
conscious rats exposed to 48-h CH that then acutely returned to
normoxia. Nevertheless, we did note a significant increase in HR after
CH that could be indicative of elevated sympathetic tone. Indeed, it is
possible that increased sympathetic activity may represent a
compensatory mechanism to maintain blood pressure in the presence of
enhanced local release of vasodilator CO in vivo. However, based on
studies conducted in the denervated renal vasculature and on isolated
vessels devoid of sympathetic influences, we conclude that the
mesenteric vascular bed demonstrates CH-induced attenuation of systemic
vasoconstrictor reactivity independent of altered reflexes. In
addition, the reduced vasoreactivity to PE does not appear to be due to
altered hematocrit or the partially compensated respiratory alkalosis
seen after CH, because similar blunted vasoconstrictor responses were
observed in isolated mesenteric resistance vessels studied under
controlled conditions. Thus CH appears to elicit a widespread
persistent reduction in vasoconstrictor sensitivity in several vascular
beds that appears to be due to vascular effects of increased local CO production.
Although the above in vivo data suggest alterations in the responses of
the resistance vasculature after CH, most studies examining the
potential mechanism of this altered reactivity have employed large
conduit arteries. Similar to conscious animal studies, experiments on
conduit vessels have shown that CH exposure attenuates responsiveness
to various vasoconstrictors. For example, maximum tension responses of
abdominal aortic rings to arginine vasopressin are diminished after CH
exposure (4). Also, in thoracic aorta segments,
contractility to 1-agonist stimulation is blunted over a
wide range of doses of PE (2, 3). Although these data support the in vivo observations, the study of isolated resistance vessels represents a better assessment of the physiological adaptations that are relevant in vascular control. Therefore, we extended our
observations of the intact mesenteric circulation to resistance arteries isolated from this bed. In isolated, pressurized resistance vessels from CH rats, vasoconstrictor responses to PE were attenuated compared with controls. These data are in agreement with earlier studies by Toporsian and Ward (22), who observed similar
diminished PE-induced reactivity in diaphragmatic resistance arteries
from 48-h CH rats. Together, these results provide evidence for altered responsiveness in resistance arteries after CH. However, the mechanism responsible for the persistent reduction of vascular responsiveness after CH exposure has not been conclusively established.
Several mechanisms have been proposed for the persistent alteration in
vascular control elicited by prolonged hypoxic exposure. Some
investigators (15) have hypothesized that hypoxia
adversely affects vascular autoregulation. For example, isolated
pressurized diaphragmatic arterioles from CH rats demonstrate decreased
basal tone compared with arterioles from control animals
(22). This decrease in basal tone is again consistent with
the hypothesis that CH induces a sustained release of a local
vasodilator, which may affect the sensitivity to all vasoconstrictor
stimuli. In addition, differences in intraluminal pressure during PE
infusion between control and CH rats could potentially influence our in vivo assessments of reactivity because several studies (5, 24) have shown that wall tension or intraluminal pressure may influence reactivity to -agonist stimulation. However, a recent study from our laboratory (10) suggests that reduced
reactivity of the renal vasculature persists in CH rats when perfusion
pressure is normalized. Specifically, when the autoregulatory portion
of the response to PE is eliminated by the control of perfusion
pressure in conscious rats, the direct agonist-induced component of
renal vasoconstriction remains diminished in CH animals compared with controls. In support of these in vivo results, the present study demonstrated reduced constrictor responsiveness in vessels from CH rats
held at constant intraluminal pressure, thereby eliminating any
myogenic contribution to the response to PE. Thus diminished autoregulatory responsiveness does not likely seem to account for
reduced vasoconstrictor sensitivity after CH. Alternatively, other
studies propose that CH leads to alterations in VSM signaling in
response to vasoconstrictor agonists. For example, investigations (8, 26) in ovine uterine arteries demonstrate decreased
1-adrenergic receptor number and affinity after hypoxia
that may contribute to diminished vasoreactivity. In addition, it has
been suggested that altered calcium sensitivity and/or handling in VSM
may contribute to diminished vasoreactivity after long-term hypoxia
(25). Although alterations in VSM function per se could
contribute to altered vasoconstriction in this setting, additional
evidence supports a role for a locally released vasodilator in this response.
Several endothelial factors promote vasodilation, and their enhanced release could explain the blunted responses to vasoconstrictors. Whereas NO production and expression of endothelial NOS have been shown to be elevated in the pulmonary arterial circulation of CH rats (9, 19), the role of NO in attenuated systemic vasoreactivity after CH is unclear. In the present study, we observed a sustained reduction in responsiveness to PE in CH resistance vessels compared with controls after NOS inhibition. These data are consistent with earlier experiments on isolated aortic rings from CH rats (3), and suggest the involvement of a different vasodilator endothelial factor in the blunted constrictor reactivity after hypoxia.
Several lines of evidence suggest that enhanced production of endothelial-derived CO may underlie diminished vasoconstriction after CH. The expression of HO, the enzyme responsible for CO production, has been reported in VSM and endothelial cells (6, 16, 17, 20). Lee et al. (13) demonstrated that HO-1 mRNA levels are elevated in aortic tissue after exposure to hypoxia. In addition, recent studies (10) from our laboratory have reported increased expression of HO-1 protein in aortas isolated from 48-h CH rats compared with controls. Thus these experiments suggest that the expression of HO-1 is upregulated in vascular tissue after hypoxic exposure.
Similar to NO, CO may elicit VSM dilation both by activating soluble guanylyl cyclase (6) and by direct effects on large conductance calcium-activated potassium channels (24). Furthermore, it has been observed that HO inhibition augments contractile responses to PE in L-NNA-treated aortic rings from CH rats but not from controls (3) and this response is dependent upon an intact endothelium. Furthermore, we have previously observed that renal HO enzyme activity is increased after CH and that the HO inhibitor ZnPPIX causes renal vasoconstriction selectively in CH rats compared with controls. Thus at least a portion of the attenuated vasoconstrictor reactivity after CH may be due to an increase in CO production. Because the previous studies testing this hypothesis had been conducted either on conduit vessels or in intact animals where other complicating influences affecting vascular control may exist, we examined the role of HO activity on vasoreactivity directly in resistance vessels from control and CH rats. We found that the HO inhibitor ZnPPIX enhanced PE constrictor reactivity only in arteries from CH rats. Conversely, this inhibitor had the opposite effect on PE responsiveness in control vessels, which perhaps may represent nonselective actions of the drug. However, previous studies in our laboratory have demonstrated that ZnPPIX selectively enhances PE reactivity in aortic rings isolated from CH rats without affecting PE responses in control tissue. Furthermore, ZnPPIX did not alter vasorelaxant responses to the NO donor S-nitroso-N-acetyl-penicillamine (3), suggesting a lack of nonspecific actions on soluble guanylyl cyclase. Additionally, Appleton et al. (1) demonstrated that ZnPPIX elicited inhibition of HO activity without affecting NOS and soluble guanylyl cyclase activity at concentrations similar to those employed in our studies. To provide additional support for the hypothesis that HO activity is elevated in CH arteries, experiments were designed to examine the vascular responses to the HO substrate heme-L-lysinate. Indeed, we observed that heme-L-lysinate reversed PE-induced constriction in CH vessels but had minimal effect on control tissue. The lack of effect of either HO inhibition or addition of substrate on constriction of control arteries differs from the results of others who have provided evidence for significant HO activity in other microvascular preparations (11, 12, 14). The reasons for these discrepancies are unclear; however, our data provide support that the vasodilator role of HO-derived CO is enhanced after CH and may be responsible for diminished in vivo sensitivity to vasoconstrictor stimuli in this setting.
In summary, we have demonstrated that, like the renal vasculature, the
mesenteric vascular bed displays diminished vasoconstrictor responses
after prolonged hypoxia. In conscious animal studies we documented that
long-term hypoxia causes a blunted mesenteric vasoconstrictor response
to 1-agonist stimulation. Resistance vessels from this
bed of CH rats exhibited similar reduced vasoconstrictor sensitivity to
PE, and this response persisted in the presence of NOS inhibition. In
addition, HO inhibition by ZnPPIX enhanced reactivity to PE, and the HO
substrate heme-L-lysinate augmented vasodilation in
mesenteric resistance arteries from CH rats. We conclude that
diminished vasoconstrictor responsiveness after CH likely results from
augmented production of CO in resistance vessels.
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ACKNOWLEDGEMENTS |
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The authors thank Minerva Murphy and Nikki Jernigan for technical assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-58124 and HL-63207 (to B. R. Walker) and HL-07736 (to R. J. Gonzales).
Address for reprint requests and other correspondence: B. R. Walker, Vascular Physiology Group, Dept. of Cell Biology and Physiology, Univ. of New Mexico Health Sciences Center, 915 Camino de Salud NE, Albuquerque, NM 87131-5218 (E-mail: bwalker{at}salud.unm.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 June 2001; accepted in final form 11 September 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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