INTRODUCTION

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EXPOSURE TO CHRONIC HYPOXIA (CH) elicits reduced systemic responsiveness to vasoconstrictors (3, 4, 7, 10, 19, 20), which is not restored upon acute return to normoxia (3, 7). Several mechanisms have been proposed to account for this effect of long-term hypoxic exposure on vasoreactivity. For example, ₁-adrenoreceptor density and agonist-binding affinity (15, 42) as well as calcium mobilization and myofilament sensitivity to calcium may be altered in vascular smooth muscle cells (43) from CH animals. Alternatively, constrictor reactivity may be impaired due to enhanced release of vasodilatory compounds, most notably carbon monoxide (CO) (4, 29). CO is the product of heme degradation by heme oxygenase (HO) and causes vasorelaxation by stimulation of vascular smooth muscle cell (VSMC) soluble guanylyl cyclase (5, 23) and by putative effects on potassium channel activity (40). Both of these actions are similar to those elicited by nitric oxide (NO) in the VSMC. Vascular expression of the inducible isoform of HO (HO-1) is enhanced by hypoxia (26), and recent in vivo studies (29) and experiments on aortic rings from rats exposed to CH for 4 wk (4) strongly suggest the involvement of endogenous CO in regulating vascular tone in this setting. However, the time course of onset for this effect has not been determined. Attenuated vasoconstrictor responsiveness in rat aortic segments has been observed with 48 h of hypoxic exposure (3). In addition, aortic (11, 26) and lung (6) HO-1 mRNA levels are elevated within several hours of hypoxic exposure but may return to control levels within 48 h (6). However, it is unclear whether HO activity accounts for diminished reactivity with shorter-term hypoxia. Thus one goal of the present study was to establish the onset of reliance of vascular reactivity on HO activity during hypoxic exposure.

Vascular HO-1 expression may be enhanced by a direct effect of hypoxia, although the polycythemia that accompanies CH could also potentially affect expression. The HO-1 promoter contains a hypoxia response element for binding the hypoxia-inducible transcription factor (HIF-1). Hypoxic exposure stabilizes HIF-1 in vitro, and thus the rapid increase in vascular expression of HO-1 could be regulated directly by hypoxia (26). Alternatively, CH induces polycythemia, which increases blood viscosity and thus shear stress on the vascular wall (14). Shear stress has been shown to enhance HO-1 gene expression in cultured VSMC (39). However, under in vivo conditions, endothelial cells represent the vascular surface exposed to shear forces. Because we have earlier observed that the endothelium appears to be the source of enhanced CO release in aortic rings from 4-wk CH rats (4), HO-1 expression in endothelial cells could also be elevated by shear stress during CH. In addition, it has also been suggested that HO enzyme activity could be stimulated by additional heme availability due to increased free hemoglobin during polycythemia (31). Because polycythemia is a slowly developing characteristic of hypoxic exposure (14, 34), observation of an early onset of hyporeactivity and HO expression would support hypoxia per se as a likely stimulus for these responses. Similarly, polycythemia is sustained upon restoration of normoxia after CH. Therefore, during posthypoxia (PH), a rapid restoration of vasoreactivity would support the direct role of hypoxia and a minimal influence of polycythemia on HO gene expression.

Thus the current study defines the time course of development and reversal of hypoxia-induced alterations in vascular reactivity and its correlation with HO-1 expression. These experiments suggest that such a relationship exists and that HO-1 expression appears to result from hypoxic induction of the enzyme rather than from secondary effects related to polycythemia.

	METHODS

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All protocols employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine.

Experimental groups. Male Sprague-Dawley rats (250-300 g; Harlan) were divided into control and hypoxia-exposed groups. The hypoxic rats were housed in a hypobaric chamber (380 ± 10 mmHg) that was briefly opened three times each week to clean cages and replenish food and water. All control rats were housed under ambient barometric pressure (~630 mmHg).

Surgical procedures for conscious animal studies. Rats were chronically instrumented with renal pulsed Doppler flow probes. Flow probes were assembled by forming Silastic cuffs around a piezoelectric crystal (20 MHz; diameter, 1 mm; Crystal Biotech). Rats were anesthetized with a mixture of ketamine (91 mg/kg im) and acepromazine (9 mg/kg im). With the use of sterile techniques, a midline laparotomy was performed to expose the left kidney. The left kidney was denervated by severing visible renal nerves and swabbing the renal artery with 20% phenol in ethanol. This method has been shown to produce effective renal denervation, as exhibited by a significant reduction of tissue catecholamine content (22), and was performed to eliminate the influence of reflex alterations in vascular tone (32) at the time of study. A pulsed Doppler flow probe was positioned around the artery and secured into place without affecting flow. In addition, some animals were instrumented with an inflatable occluder cuff positioned on the descending aorta just cephalad to the left renal artery to permit restoration of renal perfusion pressure during infusion of vasoconstrictors as previously described (9). The probe leads and occluder tubing were then subcutaneously routed to the back of the head, where they were exteriorized and placed in a protective plastic cap sutured to the skin. Five days were allowed for recovery, and a second surgery was then performed to implant femoral arterial and venous catheters. The rats were anesthetized as previously described. The left femoral artery and vein were isolated, and polyethylene catheters were advanced into the abdominal aorta and vena cava, respectively. The catheters were secured and routed as with the probe leads. CH rats were returned to the hypobaric chamber upon recovery from anesthesia. Two days were allowed for recovery before experimentation. After each surgery, the rats received 30,000 units im penicillin G benzathine-penicillin G procaine and topical Triple Antibiotic Ointment at the site of the incision (Fougera).

Conscious animal experiments. On the day of the experiment, rats were placed in a study chamber (23 × 14 × 10 cm) with fresh bedding. The chamber was flushed with either room air or 12% O₂. The catheters and Doppler probe leads were fed out the top of the chamber, and the catheters were opened and flushed with heparinized saline. Rats were allowed 30-60 min to adjust to their environment, and experiments were initiated on demonstration of stable blood pressure and heart rate (HR). Mean arterial blood pressure (MABP) and pulsatile arterial pressure were measured via the arterial catheter by a pressure transducer (Statham-Gould P23 Gb) with the output amplified by a Gould Universal amplifier. HR was monitored with a Biotach amplifer. Renal blood flow (RBF) was assessed by a directional pulsed Doppler flowmeter (VF-1, Crystal Biotech) and recorded as kilohertz of Doppler shift. All data were recorded on a Gould RS 3800 chart recorder and a computer-based data-acquisition system (Dataq Instruments).

Renal vasoconstrictor responses of 24-h and 48-h CH rats to phenylephrine. Previous experiments have shown that rats exposed to 4-wk CH demonstrate an attenuated renal vasoconstrictor response to the ₁-adrenoceptor agonist phenylephrine (PE) (29). However, the precise onset of this attenuated vasoconstriction during hypoxic exposure is unknown. Therefore, experiments were performed to determine the renal vasoconstrictor responses to increasing doses of PE, first under control conditions and then after 24 and 48 h of CH (n = 5). After equilibration, CH rats were returned to normoxia for 20 min before vasoreactivity was assessed. Under maintained normoxia, 5 min of baseline data were collected, followed by a graded intravenous infusion of PE at 3, 6, and 9 µg · kg¹ · min¹ for 5 min each. Steady-state renal hemodynamic responses were achieved at each rate. At the end of each experiment, hematocrit was measured. Blood gases were determined under normoxic conditions in some experiments with an ABL-5 analyzer (Radiometer; Copenhagen, Denmark).

Effect of HO inhibition on renal hemodynamics in control and 48-h CH rats. We previously demonstrated that HO inhibition with zinc protoporphyrin IX (ZnPPIX) increases basal renal vascular resistance (RVR) in 4-wk CH rats but not in control animals (29). A similar assessment of the tonic in vivo contribution of HO activity was performed in control and 48-h CH rats. Control (n = 5) and 48-h CH rats (n = 5) were surgically instrumented as above. At the time of study, 48-h CH rats were maintained under hypoxic conditions for the duration of the experiment, whereas normoxic control rats breathed room air. After baseline data were collected for 5 min, ZnPPIX (11 µmol/kg) or its vehicle was infused intravenously for 5 min. Hemodynamic data were collected for an additional 25 min. This time course was shown in earlier experiments to produce stable hemodynamic responses to the inhibitor (29). Because ZnPPIX is photosensitive, all experiments were performed in reduced light.

Renal vasoconstrictor responses to PE in 4-wk CH and PH rats. Earlier experiments have shown that attenuated renal vasoconstrictor reactivity persists on acute return to a normoxic environment after 4 wk of hypoxic exposure (29). However, the duration of this attenuation is unknown. To determine the time for reversal of this response, experiments were performed on control (n = 6) and 4-wk CH rats (n = 6) to determine the renal vasoconstrictor response to increasing doses of PE as above. Four-week CH rats were then tested sequentially at 1-, 5-, 24-, and 96-h PH.

Agonist vs. autoregulatory components of PE-induced renal vasoconstriction. Autoregulation plays an important role in the modulation of regional perfusion upon rapid changes in blood pressure, which occurs during PE infusion. A prior study (9) from our laboratory has shown that attenuation of agonist-induced renal vasoconstriction in acutely hypoxic rats is not caused by diminished autoregulatory mechanisms. Conscious animal aortic occlusion studies were performed to elucidate the autoregulatory versus agonist-induced vasoconstriction in rats (n = 4) studied under normoxic conditions both before and after 48 h of CH. After stable baseline hemodynamics were achieved, PE (9 µg · kg¹ · min¹) was administered for 5 min. After steady PE-induced vasoconstriction had been established, the occluder cuff was inflated until renal arterial pressure (RAP) was returned to baseline levels (±2 mmHg) and maintained for 5 min. Increases in RVR above the baseline value during the uncontrolled period were composed of both a direct PE-induced vasoconstriction and a pressure-dependent or autoregulatory component. Similarly, increases in RVR during PE infusion where RAP was controlled were caused by direct actions of PE in the absence of autoregulatory influence on vascular tone. From this assessment, the percent contribution of the autoregulatory versus the direct agonist effects of PE infusion were calculated.

Renal and aortic Western blot analysis in 48-h CH, 4-wk CH, and PH rats. Western blots were performed to determine the level of HO-1 protein in the kidneys and aortas from control and CH rats. Each blot consisted of one of the following experimental groups and their parallel control group: 48-h CH, 4-wk CH, 5-h PH, and 96-h PH. Rats were anesthetized with 32.5 mg pentobarbital sodium, and both kidneys and aortas were removed and snap-frozen in liquid N₂. A microsomal fraction was prepared using a 10 mM Tris · HCl homogenization buffer containing protease inhibitors (Preparation of experimental solutions). Samples were centrifuged at 10,000 g for 10 min at 4°C to remove cellular debris. The supernatant was collected and centrifuged at 100,000 g for 60 min at 4°C. The microsomal pellet from the high-speed spin was resuspended in homogenization buffer, and sample protein concentrations were determined by the Bradford method (Bio-Rad Protein Assay). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Each gel included a molecular weight standard (Bio-Rad) and a HO-1 standard (Stressgen) as a positive control. After being blocked overnight at 4°C, the blots were incubated for 2 h at room temperature with a mouse monoclonal antibody for HO-1 (1:600 dilution; Stressgen). For immunochemical labeling, blots were incubated for 1 h at room temperature with goat anti-mouse IgG-HRP (1:5,000 dilution; Stressgen). After enhanced chemiluminescence labeling (ECL, Amersham), HO-1 bands were detected by exposing the blots to chemiluminescence-sensitive film (Kodak). Membranes were stained with Coomassie brilliant blue to confirm equal protein loading in all lanes. Quantification of the bands was accomplished by densitometric analysis of scanned images (SigmaGel software, Jandel). Bands were normalized to the amount of protein loaded.

Preparation of experimental solutions. PE (Sigma) was dissolved in 0.9% saline, and aliquots were prepared and frozen until use. ZnPPIX (Porphyrin Products) solutions were prepared by solubilizing 50 mg ZnPPIX in 500 µl of 10% ethanolamine. Two milliliters of 0.9% NaCl were slowly added, and 1 M HCl was then used to bring the pH to 7.6-8.0. The solution was brought to a final volume of 5 ml with H₂O and sterile filtered with a 0.2-µm filter. Spectrophotometric measurements were performed to verify the final ZnPPIX concentration. Solutions were prepared in subdued light on the day of the experiment and stored at 4°C until use. The Western blot homogenization buffer contained 255 mM sucrose, 2 mM EDTA, 12 µM leupeptin, 1 µM pepstatin A, 0.3 µM aprotinin, and 1 mM phenylmethylsulfonyl fluoride. The blocking solution contained 5% nonfat milk, 3% bovine serum albumin, 5% goat serum, 5% rabbit serum, and 0.05% Tween 20 in Tris-buffered saline (TBS) containing 10 mM Tris · HCl and 50 mM NaCl (pH 7.5). HO-1 primary antibodies were diluted with TBS containing 5% nonfat milk and 0.05% Tween 20. HO-1 secondary antibodies were diluted with TBS containing 0.05% Tween 20. All reagents were purchased from Sigma unless otherwise noted.

Calculation and statistics. RVR was calculated by dividing MABP by RBF. RBF was measured as kiloHertz of Doppler shift because flow probes were not calibrated in situ for volume flow. However, kilohertz of Doppler shift and blood flow are linearly related when probe placement is fixed, as in chronic studies. Because actual blood flow was not measured, all RBF and RVR data are expressed as a percentage of initial values for a given experiment. In studies involving assessment of the autoregulatory contribution to PE-induced vasoconstriction, the fractional compensation (G) of the renal bed was calculated as an index of the degree of autoregulation as previously described (9) using the following equation

(1)

where G = 0 would indicate an absence of autoregulation and G = 1 would suggest complete autoregulation.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Renal vasoconstrictor responses of 24- and 48-h CH rats to PE. MABP responses to the higher doses of PE were significantly attenuated in 48-h CH rats compared with control conditions, whereas there was no difference between control and the 24 h time point (Fig. 1A). Similarly, RVR responses to PE were attenuated after 48 h of CH; however, there was no significant difference between control and 24 h of CH (Fig. 1B). Baseline HR tended to be greater after 24 and 48 h of CH compared with the initial normoxic control (Table 1); however, reflex bradycardia was observed in response to PE at each time point (Table 1). Arterial blood gases were obtained for three CH rats at the end of each PE experiment while breathing room air. These rats demonstrated evidence of hyperventilation secondary to renal compensation in response to 24 and 48 h of CH. Consistent with this interpretation, calculated plasma [HCO] was diminished after hypoxic exposure (Table 1). Interestingly, no change in hematocrit was observed after exposure to 24 or 48 h of CH in these studies (Table 1). Parallel experiments repeated on normoxic control rats showed no differences in pressor or vasoconstrictor responses over time (Fig. 2). Similarly, no differences were noted in any other experimental variables between time points in the parallel normoxic experiments (Table 2).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Mean arterial blood pressure (MABP; A) and renal vascular resistance (RVR; B) responses to a graded infusion of phenylephrine (PE) in rats (n = 5) under control conditions and after 24 and 48 h of chronic hypoxia (CH). Data are expressed as means ± SE. *P  0.05 vs. control.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   MABP (A) and RVR (B) responses to a graded infusion of PE in rats (n = 5) under control conditions and after 24 and 48 h of maintained normoxia. Data are expressed as means ± SE. There were no differences from the initial control experiment.

Effect of HO inhibition on renal hemodynamics in control and 48-h CH rats. Administration of ZnPPIX resulted in a dramatic fall in RBF and rise in RVR in the 48-h CH group but not in the normoxic controls (Fig. 3). In contrast, neither MABP nor HR was affected by administration of the HO inhibitor (Table 3). Vehicle treatment was without effect on any of the measured hemodynamic variables in either group (Fig. 3 and Table 3), although baseline HR was significantly higher in CH rats compared with controls, as observed above. In contrast to the preceding protocol, hematocrit was slightly elevated in 48-h CH rats in these studies. However, the increase in hematocrit was modest compared with 4-wk CH rats. Arterial pH did not differ between groups, but both PO₂, PCO₂, and plasma [HCO] were decreased in the hypoxic rats, indicative of hyperventilation in response to the sustained hypoxic stimulus (Table 3).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Renal blood flow (RBF; A) and RVR (B) responses 30 min after infusion of zinc protoporphyrin IX (ZnPPIX; 11 µmol/kg) or its vehicle in control (n = 5) and 48-h CH rats (n = 5). Data are expressed as means ± SE. *P  0.05 vs. control; #P  0.05 vs. vehicle.

Renal vasoconstrictor responses to PE in 4-wk CH and PH rats. Four-week hypoxic and 1- and 5-h PH groups showed significantly attenuated MABP responses to PE compared with controls; however, there were no differences in blood pressure responses between 24- or 96-h PH rats and controls (Fig. 4). RVR responses also returned toward control values after return to normoxia; however, there was still a slight difference between the RVR response to the highest dose of PE at 96-h PH (Fig. 5). Hematocrit remained elevated during the PH period (Table 4). As seen previously, baseline HR was elevated in 4-wk CH but after return to normoxia was not different from control rats (Table 4).

View larger version (47K): [in this window] [in a new window]   Fig. 4.   MABP responses to PE in control rats (n = 6) and rats (n = 6) studied successively at 4-wk CH and 1-, 5-, 24-, and 96-h posthypoxic (PH). Data are expressed as means ± SE. *P  0.05 vs. control.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   RVR responses to PE in control rats (n = 6) and rats (n = 6) studied successively at 4-wk CH and 1-, 5-, 24-, and 96-h PH. Data are expressed as means ± SE. *P  0.05 vs. control.

Autoregulatory component of PE-induced renal vasoconstriction. Inflation of the aortic occluder cuff during PE infusion successfully returned renal perfusion pressure to control levels (Table 5). The degree of reflex bradycardia associated with PE infusion was not altered by occlusion, suggesting that return of RAP to control did not significantly affect arterial pressure at the level of the arterial baroreceptors as previously observed (9). RVR responses to PE were significantly attenuated after 48 h of CH compared with normoxic controls both when perfusion pressure was uncontrolled and when RAP was returned to preinfusion levels (Fig. 6A). Furthermore, the relative contribution of pressure-dependent autoregulation does not appear to be altered in 48-h CH rats compared with normoxic rats (Fig. 6B). The calculated fractional compensation is an indicator of the autoregulatory nature of a bed and was unaltered by CH. Values for this variable were similar to those previously reported for PE infusion in conscious rats (9). Thus attenuated vasoreactivity during PE infusion after 48 h of CH exposure is not due exclusively to decreased autoregulatory responsiveness. Moreover, PE-induced vasoreactivity during 48 h of CH was still significantly attenuated in the absence the autoregulatory component.

View this table: [in this window] [in a new window]   Table 5.   RAP, HR, and autoregulatory fractional compensation in response to PE (9 µg · kg1 · min1) in normoxic and 48-h CH rats under conditions where RAP was uncontrolled and controlled

View larger version (14K): [in this window] [in a new window]   Fig. 6.   A: RVR responses to 9 µg · kg1 · min1 PE with perfusion pressure (renal arterial pressure) uncontrolled or returned to preinfusion levels by inflation of a suprarenal occluder in rats under control conditions and after 48 h of CH. B: relative agonist-induced and myogenic contributions to the RVR response to PE in control and 48-h CH rats. HO, heme oxygenase. *P  0.05 vs. control.

Western blot analysis of tissue from 48-h CH, 4-wk CH, and PH rats. HO-1 renal protein levels were elevated in 4-wk CH and 5-h PH groups compared with parallel controls (Fig. 7). HO-1 densitometry values corrected for protein loading were ~10-fold greater in the 4-wk CH group compared with its parallel control. Although the densitometric values for the 5-h PH group appear even greater, this is a function of variability between gels because this group was approximately sixfold greater than its parallel normoxic control run on the same gel. Interestingly, there was a large variation in the level of HO-1 increase in response to hypoxia. Whereas all 4-wk CH and 5-h PH samples demonstrated greater HO-1 levels than their controls, some samples displayed a dramatically greater increase, accounting for the variability evident in Fig. 7. HO-1 levels in 48-h CH and 96-h PH groups were not significantly increased from controls (Fig. 7). Indeed, samples from 48-h CH tissue demonstrated slightly less HO-1 than their control. In contrast, aortic tissue from 48-h CH rats did display greater HO-1 levels than control aortas (Fig. 8). Overall, these data suggest an inverse correlation between observed renal vasoreactivity to PE and tissue HO-1 concentrations in CH and PH rats.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Renal HO-1 protein levels in 48-h CH (n = 6), 4-wk CH (n = 6), 5-h PH (n = 6), and 96-h PH rats (n = 6) compared with the parallel normoxic controls run for each group (n = 6 in each). Top: Western blot showing protein expression in the respective groups. Data are normalized to the amount of protein loaded and expressed as means ± SE. *P  0.05 vs. parallel control.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Aortic HO-1 protein levels in 48-h CH rats (n = 5) compared with normoxic controls (n = 5). Inset: Western blot showing protein expression in control and 48-h CH rats. Data are normalized to the amount of protein loaded and expressed as means ± SE. *P  0.05 vs. control.

	DISCUSSION

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This study investigated the onset and offset of attenuated systemic vasoconstrictor reactivity induced by hypoxia as well as the possible regulatory role of CO in this phenomenon. Our findings indicate the following: 1) persistent attenuation of renal vasoconstrictor reactivity occurs within 48 h of CH exposure; 2) similar to results with longer-term hypoxia, HO inhibition causes renal vasoconstriction in 48-h CH rats but not in controls; 3) vasoreactivity largely returns toward control levels after 24-h PH but is still slightly blunted at 96-h PH; and 4) renal HO-1 protein levels are elevated at 4-wk CH and 5-h PH but return to control levels by 96-h PH. These results suggest that both induction and reversibility of hypoxia-induced vascular hyporeactivity occur within 2-3 days and elevated HO-1 protein levels and/or activity correspond to attenuated vasoconstrictor reactivity.

Many groups have demonstrated diminished systemic vasoconstrictor responsiveness to a variety of pressor agents in animals exposed to CH (7, 10, 19, 20, 29) and that this attenuated vasoconstriction is maintained on acute return to normoxia (7, 29). Furthermore, several studies (3, 4, 7, 10, 37) have documented diminished contractility in isolated vascular preparations from CH rats compared with controls. A recent study (29) in our laboratory has shown a reduction in the RVR response to the vasoconstrictor PE in 4-wk CH rats, which suggests that the renal bed is an important contributor to diminished systemic vasoconstrictor reactivity under these conditions. Moreover, the presence of reduced responsiveness in the renal bed after sympathetic denervation indicates that CH-induced attenuation of systemic vasoconstrictor reactivity is induced by factors present at the level of the vasculature and is not a reflex response. The present study also establishes that the reduced renal vasoconstrictor response after CH is not due to impaired renal autoregulation, because agonist-induced vasoconstriction was similarly blunted under controlled pressure conditions and the relative autoregulatory contribution to PE-induced vasoconstriction was unaltered by CH. Previous experiments in our laboratory have only examined diminished vasoconstrictor responses of rats exposed to 4 wk of hypoxia. However, Auer and Ward (3) observed that attenuated vasoconstrictor responsiveness is present within 48 h of hypoxia in isolated vessels. Our findings demonstrate the in vivo significance of this earlier observation.

Although several studies (15-17, 38, 42, 43) suggest altered VSMC signaling responses to vasoconstrictors after CH, our work and that of others provide evidence that diminished reactivity may also be influenced by the enhanced local release of a vasodilator. For example, Harrison et al. (10) described the involvement of a cyclooxygenase product in the reduced vasoconstrictor responsiveness in CH guinea pigs. In addition, expression of endothelial NO synthase (NOS) is augmented in rat pulmonary circulation during CH (18, 25, 32), although the effect of hypoxia on expression of this enzyme in the systemic vasculature is not established. Studies from our laboratory have illustrated that blockade of NOS activity in isolated aortic rings from CH rats does not normalize contractile responses compared with similarly treated control groups. However, in NOS-inhibited aortic rings, further inhibition of HO results in equal contractile responses in vessels from CH and control rats (4). Although a recent study (36) has postulated that CO elicits vasodilation in renal resistance vessels by the release of NO from a preexisting intracellular heme-bound pool, our data suggest that endogenous CO acts as a tonic vasodilator after CH independent from NO.

Considerable recent evidence supports a role for CO as an endogenous vasodilator. In addition to CH, a vasodilatory influence of enhanced HO gene expression is likely in other pathophysiological settings such as hypertension (35) and endotoxic shock (41). Other investigators have described vasodilator actions of HO activity in control rats (21, 27) and have suggested that CO may modulate myogenic responses in resistance vessels (24). In contrast to these latter findings, we observed no hemodynamic response to ZnPPIX in control animals, whereas the HO inhibitor caused profound renal vasoconstriction in 48-h CH rats. These results are nearly identical to those observed in 4-wk CH rats in an earlier study (29). At higher concentrations, ZnPPIX has been shown to inhibit soluble guanylyl cyclase and NOS as well as HO (2). The dose utilized in the present study was chosen because it resulted in an estimated vascular concentration well below the range associated with nonspecific actions (2). Indeed, the lack of an effect of ZnPPIX on hemodynamics in control rats strongly suggests specificity of HO inhibition, because inhibition of soluble guanylyl cyclase or NOS would be expected to increase blood pressure in both groups of animals. Nevertheless, the exact circulating concentration of the inhibitor is difficult to assess and we inferred specificity through the selectivity of the response to CH rats.

Despite this evidence for functional upregulation of HO within the vasculature after 48 h of hypoxia, we were not able to detect elevated renal HO-1 levels using a Western blot at this time point. One possible explanation for this discrepancy could be insufficient sensitivity of a whole kidney assessment to detect vascular upregulation of HO-1. Because of this finding, we performed a Western blot for HO-1 on aortas from 48-h CH rats and observed elevated HO-1 levels after hypoxia compared with controls (Fig. 8). These data, and the effectiveness of ZnPPIX in eliciting vasoconstriction, suggest that vascular HO-1 expression is elevated after 48 h of hypoxia. These results are in contrast to recent studies that demonstrated a return of HO-1 mRNA levels to baseline in the lung by 48 h of sustained hypoxic exposure and a restoration of HO-1 protein levels to control levels by 7 days (6). Thus it is possible that the profile of HO expression in response to hypoxia may differ between tissues.

Consistent with earlier studies (4, 7, 29), we observed attenuated reactivity to PE in 4-wk CH rats. This duration of hypoxic exposure has been previously shown to be associated with elevated renal HO enzyme activity and development of renal vasoconstriction on administration of ZnPPIX (29). Our current finding of elevated renal HO-1 protein in 4-wk CH rats suggests that the previously observed augmentation of HO enzyme activity and reliance of basal tone on HO are functions of greater enzyme levels in this tissue. The present study does not distinguish the cell type within the kidney associated with increased HO-1 expression. Indeed, recent data suggest that the site of greatest expression of HO-1 in response to angiotensin II may reside in the renal tubules in control rats (1, 13). Although it is presently unclear whether vascular or tubular expression is elevated by CH, CO produced at either site could be effective as a vasodilator due to the relative stability of CO compared with NO as a gaseous mediator. We also observed that vasoreactivity to PE largely returned to control by 96 h of restoration of normoxia and that this corresponded to a return of renal HO-1 to a level not different from the control level. Because polycythemia was persistent on return to normoxia, these data strongly suggest that HO-1 expression is not increased in response to increased shear stress or elevated free hemoglobin associated with greater hematocrit. Conversely, the rapid reversal of HO-1 protein levels suggests that expression of the enzyme is most likely regulated by hypoxia per se. Indeed, HO-1 mRNA expression is increased within hours of hypoxic exposure, and this increase is dependent on the presence of HIF-1 DNA-binding activity (26). At 96-h PH, there was still a slight reduction in the RVR response to the highest dose of PE compared with controls. This small effect could be due to an effect of polycythemia on another vasodilatory pathway. For example, in a preliminary study (30), we recently reported that a comparable degree of polycythemia induced by erythropoietin administration results in elevated aortic endothelial NOS levels and increased vasorelaxant responsiveness to acetylcholine. However, the primary factor associated with reduced constrictor reactivity after CH is likely increased HO expression.

Hypoxic induction of HO-1 has been linked to the transcription factors HIF-1 and activator protein-1 and may further depend on cell type (12, 26). Additionally, other studies suggest that NO may stimulate increased HO activity in endothelial cells (28) and VSMC (8, 11). However, the upregulation of NO production after CH appears to be modest and likely persists along with polycythemia after restoration of normoxia. Thus NO regulation of HO-1 expression seems unlikely considering the rapid reversal of expression in PH rats. A final possibility is that the CH-induced increase in HO activity is not regulated by increased gene transcription but rather by enhanced enzyme stability or rate of translation. Additional experiments assessing HO mRNA will be required to delineate this point.

In conclusion, the present studies have determined the rate of onset and reversal of attenuated vasoconstrictor reactivity on prolonged hypoxic exposure. The results suggest that this reduced reactivity largely correlates with the expression and/or activity of HO. Furthermore, the dissociation of polycythemia and HO-1 expression supports a direct role of hypoxia on expression of this enzyme. These studies support the concept that endogenous CO acts as a tonically released vasodilator under prolonged hypoxic conditions.