INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

CHRONIC HYPOXIA (CH) occurs during extended residence at high altitude or when gas exchange within the lung is impaired because of pathological conditions. Prolonged exposure to hypoxia results in attenuated constrictor responsiveness of the systemic vasculature that persists for several hours following restoration of normoxia (2, 9-11, 15, 18, 23). Our laboratory (10, 11) recently reported that blunted agonist-induced and myogenic responsiveness after hypoxic exposure is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and diminished [Ca²⁺]. These studies also demonstrated that inhibition of nitric oxide (NO) synthase (NOS) activity does not reverse VSM cell hyperpolarization associated with CH exposure, suggesting that NO is not responsible for altered VSM cell resting membrane potential (E_m) (10). However, recent studies indicate that that NO may influence vascular tone by reducing the sensitivity of the VSM cell contractile apparatus to Ca²⁺ (7, 26). These findings suggest that potentially increased vascular NO production following CH could contribute to attenuated vasoconstrictor responsiveness by diminishing Ca²⁺ sensitivity independent of alterations in VSM cell E_m.

Investigations of the effects of CH on endothelial NOS expression and NO-dependent vasodilatory responses in systemic vasculatures have yielded conflicting findings. For example, increased vascular endothelial NOS (eNOS) expression (33) and elevated NO-dependent vasodilatory responses have been demonstrated in sheep uterine arteries (33) and fetal guinea pig hearts after CH (30). In contrast, eNOS protein levels and NO-dependent vasodilatation were decreased after prolonged hypoxic exposure in the rat aorta (31). Consistent with this report, elevated contractility of guinea pig middle cerebral arteries associated with CH has been attributed to decreased production of NO (28). These divergent findings may reflect the unique nature of some of these vascular beds or different properties between conduit and resistance vessels. Our previous studies have shown that mesenteric resistance arteries demonstrate similar responses to CH as those observed in the intact animal (15) and that this bed may thus be representative of the predominant effects of CH on the peripheral vasculature. Therefore, we chose to examine the hypothesis that increased production of NO contributes to blunted systemic vasoconstriction following CH in arteries isolated from this bed. Our findings suggest that elevated production of NO after CH attenuates vasoconstrictor responsiveness by decreasing Ca²⁺ sensitivity. Furthermore, we demonstrate that increased production of NO in small arteries isolated from CH rats is likely associated with increased endothelial cell [Ca²⁺].

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals. Male Sprague-Dawley rats (250-400 g; Harlan Industries) were provided with fresh bedding, food, and drinking water, and a 12-h:12-h light-dark cycle was maintained. CH rats were exposed to hypobaric hypoxia at a barometric pressure of 380 Torr for 48 h, whereas control rats were housed in identical cages at ambient barometric pressure (~630 Torr). Our laboratory has previously reported that arterial PO₂ for rats under identical control conditions was 73 ± 1 Torr compared with 44 ± 2 Torr under hypoxic conditions (15). The duration of hypoxic exposure employed for this study was selected based on previous reports demonstrating attenuated vascular reactivity following 48-h hypoxic exposure for the renal and mesenteric circulations in vivo (15, 18). In addition, attenuated reactivity of small mesenteric and diaphragmatic arteries isolated from rats exposed to hypoxia for 48 h has also been reported (10, 11, 15, 32). Before the experimentation, rats were deeply anesthetized with pentobarbital sodium (32.5 mg ip) and euthanized by exsanguination after vessels were harvested according to a protocol approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine.

Isolated vessel preparation. Mesenteric resistance arteries from control and CH rats were isolated and pressurized. The chest and abdomen of anesthetized rats were opened, and heparin (100 units in 0.1 ml) was injected into the heart to prevent clotting. The mesenteric arcade was excised and placed in ice-cold physiological saline solution (PSS) aerated with the normoxic gas mixture. The arcade was secured in a Silastic-coated petri dish containing cold, aerated PSS. Veins were removed and resistance artery branches were cleaned of adipose tissue and transferred to a beaker of cold, aerated PSS. Vessel segments (100-200 µm passive ID) were dissected from the cleaned branches, transferred to a vessel chamber (Living Systems), cannulated with glass micropipettes, and secured with ligatures. Vessels were slowly pressurized to 60 Torr by using a column filled with PSS and superfused (5 ml/min) with aerated PSS warmed to 37°C. After a 30-min equilibration period, phenylephrine (PE) (10 µM) was administered to demonstrate the viability of the preparation. Vessels were superfused with PSS for an additional 30 min after PE administration before further manipulation.

Endothelium disruption. For experiments involving endothelium-denuded resistance vessels, endothelial cell integrity was verified before disruption by demonstrating intact responses after the administration of the endothelium-dependent vasodilator acetylcholine (ACh) (1 µM) in the presence of PE (10 µM). Disruption of the endothelium was accomplished by passage of an air bubble (1 ml) through the vessel lumen. Endothelium-denuded arteries were then perfused and superfused with warmed, aerated PSS for 30 min to wash out endothelial-derived factors. Vessels were repressurized to 60 Torr and, after a 30-min equilibration period, were constricted with PE (10 µM), and ACh (1 µM) was administered to assess the efficacy of endothelium disruption procedures. The endothelium was considered to be functionally inactivated if ACh administration reversed PE-induced vasoconstriction by <10%.

Agonist-induced vasoconstrictor and VSM Cell Ca²+ responses. Pressurized, fura-loaded resistance arteries were superfused with PSS containing increasing concentrations of the purine-pyrimidine receptor agonist UTP (1 µM to 1 mM) or the ₁-receptor agonist PE (10 nM to 10 µM). UTP was employed as the primary vasoconstrictor agonist for this study because in pilot experiments UTP-induced vasoconstrictor and vessel wall [Ca²⁺] responses were found to be more stable than PE-induced responses. Ratiometric images were collected by using a Nikon Diaphot 300 microscope equipped with a ×20 Nikon Fluor objective (numerical aperature = 0.75). The vessel preparation was alternately excited at 340 and 380 nm, and images of the respective 510-nm emissions were collected at a rate of ~0.3 Hz by using MetaFluor software (version 4.5; Universal Imaging). Vessel images were collected for 3 min for each concentration of vasoconstrictor that was administered, and the mean fluorescence (F₃₄₀/F₃₈₀) ratio for the recording period was calculated and expressed as VSM cell [Ca²⁺]. The F₃₄₀/F₃₈₀ ratio is linearly related to the true molar [Ca²⁺] assuming that the dissociation constant of fura 2 does not differ between treatment groups (16). Experiments were performed by using endothelium-intact (n = 5 for both control and CH groups), endothelium-disrupted (n = 5 for both groups), and endothelium-intact vessels in the presence of N-nitro-L-arginine (L-NNA, 100 µM) (n = 5 for control, n = 6 for CH). After completion of the vasoconstrictor response curves, vessels were maximally dilated with papaverine (100 µM), and the outer diameters were measured by using Metamorph software (version 4.5). Some vessels were further treated with the NO donor S-nitroso-N-acetyl-dl-penacillamine (SNAP, 10 µM), the voltage-dependent Ca²⁺ channel (VDCC) blocker nifedipine (1 µM), or superfused with Ca²⁺-free PSS (in mM: 129.8 NaCl, 5.4 KCl, 0.83 MgSO₄, 19 NaHCO₃, 5.5 glucose, and 3 EGTA) to validate maximal dilation after papaverine administration. Little or no further increase in diameter of endothelium-intact resistance arteries was observed after these treatments. However, the diameter of endothelium-denuded vessels treated with papaverine further increased after SNAP administration. Therefore, for these vessels, the diameter obtained after SNAP treatment was considered as the maximum diameter. Vessel outer diameters were measured for every fifth image frame for each vasoconstrictor concentration and averaged and expressed as the percent change from the maximally dilated state.

NO production by small mesenteric arteries. NO production by small mesenteric arteries was measured by using an NO-sensitive amperometric electrode (ISO-NOP30, World Precision Instruments). Before each experiment, electrodes were calibrated with the NO donor SNAP by using a procedure described by the manufacturer and previously employed by other investigators (14). Linear response (r²  0.98) was routinely obtained by using a five-point calibration between 6 and 200 nM of NO. Baseline current was established by placing the electrode in a glass tube containing PSS (in mM: 129.8 NaCl, 5.4 KCl, 0.83 MgSO₄, 19 NaHCO₃, 1.8 CaCl₂, and 5.5 glucose) maintained at 37°C and bubbled with a normoxic gas mixture consisting of 21% O₂-6% CO₂, balance N₂. Baseline current was recorded for 30 min, and the electrode was then immediately transferred to an identical tube containing PSS and 13 third-order mesenteric arteries and the associated fourth- and fifth-order branches that had been isolated from a single rat. The tube containing the vessels was bubbled with normoxic gas mixture, the temperature was maintained at 37°C, and the current was recorded for 30 min. Basal NO production was calculated via linear regression from the standard curve by using the difference in current observed in the presence of vessels compared with baseline (n = 5 for both CH and control groups). These experiments were repeated in the presence of the NOS inhibitor L-NNA (100 µM) (n = 5 for both groups).

Western blotting for NOS isoforms. Mesenteric resistance arteries were isolated from control and CH rats and snap-frozen in liquid nitrogen. Vessels were homogenized on ice in 10 mM Tris · HCl buffer (pH 7.4) containing 255 mM sucrose, 2 mM EDTA, 12 µM leupeptin, 4 µM pepstatin A, 1 µM aprotinin, and 2 mM phenylmethylenlfonyl fluoride (all from Sigma). Homogenates were then centrifuged at 13,400 g at 4°C for 10 min to remove tissue debris, and protein concentrations of samples were determined by the Bradford method (Bio-Rad protein assay). Whole cell proteins (n = 4 for both groups) were resolved by SDS-PAGE. In addition to the samples, each gel included both molecular mass (Bio-Rad) and specific protein standards. The separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) and blocked overnight at 4°C with 5% nonfat milk, 3% BSA (Sigma), and 0.05% Tween 20 (Bio-Rad) in a Tris-buffered saline solution (TBS) containing 10 mM Tris · HCl and 50 mM NaC1 (pH 7.5). Blots were incubated for 4 h at room temperature with antibodies raised against eNOS, inducible NOS (iNOS), or neuronal NOS (nNOS) (all from Transduction Labs) at an appropriate dilution in TBS. Immunochemical labeling was achieved by incubation for 1 h at room temperature with a horseradish peroxidase-conjugated goat anti-mouse IgG (1:5,000; Bio-Rad) in TBS followed by chemiluminescence labeling (Amersham ECL). Protein bands were detected by exposure to chemiluminescence-sensitive film (Kodak). Densitometric analysis of protein bands was performed by using Scion Image software (version 4.0.2, Scion). Membranes were stained with Coomassie brilliant blue to confirm equal protein loading per lane.

Endothelial cell [Ca²+]. Endothelial cell [Ca²⁺] was measured as described by others (19). Fura solution (2 µM fura 2-AM and 0.05% pluronic in PSS) was administered to the lumen of pressurized mesenteric arteries by using a servo-controlled peristaltic pump (Living Systems), which allowed intraluminal pressure to remain nearly constant (60 Torr) during loading procedures. After a 5-min loading period, the lumen was perfused with PSS for 20 min to wash out excess fura solution. The vessel preparation was then superfused with warmed, aerated PSS for 20 min. Visual inspection of arteries loaded via the lumen by using 380 nm illumination revealed a cobblestone morphology, distinct from the appearance of VSM cell-loaded vessels. Ratiometric images of unstimulated vessels were collected for ~3 min, and ACh (1 µM) was administered to demonstrate selective endothelial loading. Previous studies have demonstrated that ACh induces an increase in endothelial cell [Ca²⁺] (8), whereas it causes a reduction in VSM cell [Ca²⁺] when administered to endothelium-intact vessels (4). Therefore, an increase in F₃₄₀/F₃₈₀ after ACh administration demonstrated selective loading of the endothelium.

Calculations and statistics. All data are expressed as means ± SE. Values of n refer to the number of animals in each group. For experiments employing the pressurized vessel preparation, one artery was isolated per animal. Two-way ANOVA followed by Student-Newman-Keuls post hoc test was used to compare NO production between vessels isolated from control and CH rats in the presence of L-NNA or vehicle. A two-way repeated measures ANOVA followed by Student-Newman-Keuls post hoc test was used to compare UTP- and PE-induced vasoconstrictor and VSM cell [Ca²⁺] responses and endothelial cell [Ca²⁺]. Vasoconstriction, expressed as a fraction of passive diameter, was normalized by arcsine square root transformation before analysis. Unpaired t-tests were used to compare eNOS Western blot densitometric analysis between control and CH groups. A probability of P 0.05 was accepted as statistically significant for all comparisons.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Agonist-induced vasoconstrictor and VSM cell [Ca²+] responses. Vasoconstriction in response to the purinergic receptor agonist UTP was attenuated for vessels isolated from CH rats compared with controls (Fig. 1A). Consistently, increases in VSM cell [Ca²⁺] resulting from UTP administration were also blunted for arteries from CH rats compared with controls (Fig. 1B). UTP-induced vasoconstriction (Fig. 2A) and changes in VSM cell [Ca²⁺] (Fig. 2B) of endothelium-disrupted arteries were not different between groups. In the presence of the NOS inhibitor L-NNA, vasoconstriction in response to UTP was not different between arteries isolated from control and CH rats (Fig. 3A). In contrast, UTP-induced increases in VSM cell [Ca²⁺] of L-NNA-treated vessels from CH rats remained blunted compared with L-NNA-treated controls at all but the largest concentration of agonist administered (Fig. 3B). UTP-induced vasoconstriction did not differ among endothelium-intact, endothelium-denuded, and L-NNA-treated vessels from the control rats, whereas constriction of endothelium-denuded and L-NNA-treated arteries from the CH rats was greater than that of endothelium-intact vessels from this group at 10, 100, and 1,000 µM UTP. Changes in VSM cell [Ca²⁺] after UTP administration were not different among endothelium-intact, endothelium-denuded, and L-NNA-treated arteries isolated from either the control or CH rats.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Vasoconstriction (A) and vascular smooth muscle (VSM) cell [Ca2+] (B) after UTP administration for endothelium-intact small mesenteric arteries isolated from control or chronic hypoxia (CH) rats (n = 5 for both groups). F340/F380, mean fluorescence. *P  0.05 vs. control.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Vasoconstriction (A) and VSM cell [Ca2+] (B) after UTP administration for endothelium-disrupted small mesenteric arteries isolated from control or CH rats (n = 5 for both groups). There were no significant differences.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Vasoconstriction (A) and VSM cell [Ca2+] (B) after UTP administration in the presence of N-nitro-L-arginine (L-NNA, 100 µM) for endothelium-intact small mesenteric arteries isolated from control (n = 5) or CH rats (n = 6). *P  0.05 vs. control.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Vasoconstriction (A) and VSM cell [Ca2+] (B) after phenylephrine (PE) administration in the presence of L-NNA (100 µM) for endothelium-intact small mesenteric arteries isolated from control (n = 5) or CH rats (n = 6). *P  0.05 vs. control.

NO production by small mesenteric arteries. Consistent with our hypothesis, basal production of NO was greater in small mesenteric arteries isolated from CH rats compared with controls (Fig. 5). In addition, these experiments demonstrated that NO production was essentially abolished by administration of the NOS inhibitor L-NNA (100 µM) (Fig. 5).

View larger version (9K): [in this window] [in a new window]   Fig. 5.   Nitric oxide (NO) production of small mesenteric arteries isolated from control or CH rats in the presence of L-NNA (100 µM) or vehicle (n = 5 for all groups). *P  0.05 vs. control vehicle, #P  0.05 vs. CH vehicle.

Western blots for NOS isoforms. To test the hypothesis that increased production of NO after CH results from elevated levels of NOS protein, we performed Western blots for eNOS, iNOS, and nNOS by using protein homogenates prepared from small mesenteric arteries isolated from control and CH rats. Gel lanes loaded with positive controls for iNOS and nNOS produced visible bands. However, these proteins were not detected in lanes loaded with extracts of mesenteric arteries isolated from either group (not shown). In contrast, eNOS bands were present in lanes loaded with mesenteric artery homogenates from both groups of rats (Fig. 6, top). No differences in eNOS band density between lanes loaded with proteins from the control and CH rats were detected (Fig. 6, bottom).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Results from Western blot experiments (top) for endothelial NO synthase (eNOS) in small mesenteric arteries from control and CH rats (n = 4 for both groups). Bottom, mean densitometric data for eNOS bands from each group. eNOS was identified as a single band at ~135 kDa. S, eNOS standard. There were no significant differences.

Endothelial cell [Ca²+]. Production of NO by eNOS is Ca²⁺/calmodulin dependent (5). Therefore, selective loading of endothelial cells with the Ca²⁺ indicator fura 2-AM was employed to test the hypothesis that increased production of NO after CH results from elevated endothelial cell [Ca²⁺]. The endothelium-dependent vasodilator ACh induced an increase in F₃₄₀/F₃₈₀ of vessels that had been administered fura via the lumen, suggesting that endothelial cells had been selectively loaded by this procedure (Fig. 7). Furthermore, these experiments demonstrate that basal endothelial cell [Ca²⁺] is greater in mesenteric arteries from CH rats compared with control rats (Fig. 7).

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Endothelial cell [Ca2+] of small mesenteric arteries isolated from control and CH rats under baseline conditions and after acetylcholine (ACh) administration (n = 5 for both groups). *P  0.05 vs. control, #P  0.05 vs. CH.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study examined the contribution of NO to attenuated agonist-induced vasoconstrictor responsiveness after prolonged hypoxic exposure. The major findings of this study are the following: 1) NOS inhibition normalizes agonist-induced vasoconstriction, but not VSM cell [Ca²⁺], between vessels isolated from control and CH animals; 2) NO production is greater in small mesenteric arteries isolated from CH rats compared with controls; 3) NOS protein levels do not differ between small mesenteric arteries isolated from control and CH rats; and 4) endothelial cell [Ca²⁺] is greater in vessels from CH rats compared with controls. These findings support the hypothesis that increased production of NO contributes to blunted vasoconstriction after CH by diminishing the Ca²⁺ sensitivity of the VSM cell contractile apparatus. In addition, these data suggest that enhanced NOS activity resulting from elevated endothelial cell Ca²⁺, rather than increased NOS protein levels, is responsible for greater NO production after hypoxic exposure.

Conflicting accounts of the effects of CH on NOS expression and NO-dependent vasodilatory responses in both pulmonary (6, 13, 24) and systemic (28, 30, 31, 33) vessels have been reported. For example, Toporsian et al. (31) demonstrated that eNOS mRNA and protein expression, ACh-induced relaxation, and ACh-induced increases in cGMP and nitrate levels were diminished in the aortic tissue from CH rats compared with control rats. In agreement with these findings, Sillau and co-workers (28) demonstrated that elevated U-46619-induced contractile responses of middle cerebral arteries isolated from CH guinea pigs resulted from decreased NO production or diminished sensitivity to its effects. In contrast, Zhang et al. (34) reported that plasma nitrate levels were greater in pregnant sheep and fetuses exposed to CH compared with control rats. Consistent with this observation, Thompson et al. (30) demonstrated that NO-dependent vasodilation resulting from ACh administration was increased in fetal guinea pig hearts isolated from CH animals compared with control rats. Furthermore, eNOS gene expression and NO production of uterine arteries from CH sheep were reported elevated when compared with controls (33). However, a number of these studies could be complicated by their examination of vascular beds that are not representative of the predominant effect of CH to diminish vasoconstrictor reactivity in vivo and by differences in the duration of hypoxic exposure employed. The study of isolated mesenteric arteries allowed the examination of a vascular bed shown previously to contribute to this response following 48 h of CH (15). The findings of the current study demonstrate that NO production is increased in small mesenteric arteries isolated from CH rats compared with control rats. In addition, our data show that attenuated agonist-induced vasoconstriction is relieved by disruption of the endothelium or by NOS inhibition. Therefore, we conclude that increased endothelial production of NO after CH contributes to blunted systemic vasoconstrictor responsiveness.

Alteration of VSM cell E_m influences vascular tone by modulation of Ca²⁺ entry through VDCCs (22). Previous reports from our laboratory (10, 11) demonstrated that attenuated vasoconstriction following CH is associated with endothelium-dependent VSM cell hyperpolarization and decreased [Ca²⁺]. These findings suggest that decreased VSM cell Ca²⁺ influx via VDCCs contributes to blunted systemic vasoconstriction following CH. Although NO has been shown to elicit VSM hyperpolarization in some experimental settings (29), NOS inhibition does not normalize VSM E_m between vessels isolated from control and CH rats (10), indicating that other endothelium-derived factors are responsible for altered E_m after prolonged hypoxic exposure. In agreement with these findings, recent reports suggest that diminished VSM cell [Ca²⁺] and hyperpolarization result from increased endothelial heme oxygenase (10) and cytochrome P450 activity (12). Interestingly, the current study demonstrates that inhibition of NO production normalizes UTP- and PE-induced vasoconstriction between groups, whereas VSM cell [Ca²⁺] in arteries from CH rats remains less than that of controls. This dissociation between vasoconstrictor and VSM cell [Ca²⁺] responses after NOS inhibition supports the hypothesis that increased NO production after CH results in attenuated vasoconstriction via diminished sensitivity of the contractile apparatus to Ca²⁺, rather than through altered VSM cell Ca²⁺ mobilization. These findings are consistent with recent reports demonstrating that NO can elicit decreased Ca²⁺ sensitivity through inhibition of the Rho/Rho kinase pathway (7, 26). We conclude that the current study suggests that diminished Ca²⁺ sensitivity, rather than altered Ca²⁺ mobilization, is the predominant influence of elevated NO production following CH.

The caliber of small arteries is influenced by both direct agonist stimulation as well as pressure induced, or myogenic, vasoconstriction (3). Myogenic vasoconstriction of diaphragmatic (32) and mesenteric resistance arteries (10, 11) is attenuated following CH. In addition, reduced myogenic responsiveness of mesenteric arteries isolated from CH rats is associated with endothelium-dependent VSM cell hyperpolarization and decreased VSM cell [Ca²⁺] (11). Myogenic responsiveness of small arteries from CH rats remains diminished compared with controls during NOS inhibition (11), whereas the current study demonstrates that agonist-induced vasoconstriction is normalized between groups when NO production is blocked. These findings suggest that increased NO production after CH preferentially blunts agonist-induced, but not myogenic, vasoconstriction. Myogenic vasoconstriction primarily results from stretch-induced VSM cell depolarization and Ca²⁺ influx via VDCC (17, 20). In contrast, agonist-induced vasoconstriction is more complex and involves both VSM cell depolarization and Ca²⁺ influx (22) as well as increases in Ca²⁺ sensitivity (1, 27). Our findings are consistent with the hypothesis that increased NO production following CH blunts vasoconstriction by attenuating Ca²⁺ sensitization associated with agonist stimulation.

Increased production of NO following CH could result from elevated NOS expression levels, increased activity, or from a combination of these factors. Although previous reports demonstrate both increased (33) and decreased (31) eNOS expression in systemic vessels after CH, our findings show that eNOS expression in small mesenteric arteries is not altered by CH exposure. Because eNOS activity is Ca²⁺/calmodulin dependent (5), increased endothelial cell [Ca²⁺] could result in enhanced NO production. In experiments employing luminal administration of a Ca²⁺ indicator, we found that endothelial cell [Ca²⁺] was greater in vessels isolated from CH rats compared with controls. A recent report suggests that increased endothelial cell [Ca²⁺] may be a consequence of VSM cell hyperpolarization associated with CH. VSM and endothelial cells appear to have the same E_m in the mesenteric circulation, possibly due to electrical communication via myoendothelial gap junctions (25). Endothelial cell hyperpolarization increases the electrical gradient for Ca²⁺ influx via nonselective cation channels and results in elevated endothelial cell [Ca²⁺] (21). Therefore, endothelial cell hyperpolarization associated with altered VSM cell E_m after CH may be responsible for increased endothelial cell [Ca²⁺]. These findings support the hypothesis that increased production of NO after CH results from enhanced NOS activity due to elevated endothelial cell [Ca²⁺]; however, additional studies are required to elucidate the mechanism of this effect of CH exposure.

In conclusion, this study demonstrates that increased NO production following CH diminishes vasoconstriction of small mesenteric arteries. Blunted reactivity may be the result of attenuated agonist-induced VSM Ca²⁺ sensitization. In addition, our findings suggest that increased NO production following CH results from elevated endothelial cell [Ca²⁺] rather than from enhanced NOS gene expression.