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Hypoxia promotes relaxation of bovine coronary arteries through lowering cytosolic NADPH

Department of Physiology, New York Medical College, Valhalla, New York

Submitted 9 June 2005 ; accepted in final form 9 January 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Hypoxia relaxes endothelium-denuded bovine coronary arteries (BCA) through mechanisms that do not appear to involve reactive oxygen species, prostaglandins, or nitric oxide. Because of similarities in the relaxation of BCA to hypoxia (PO₂ = 8–10 Torr) and inhibitors of the pentose phosphate pathway (PPP) including 6-aminonicotinamide and epiandrosterone, we measured NADPH and NADP and found that hypoxia caused NADPH oxidation (decreased NADPH/NADP). The relaxation to hypoxia was similar to previously reported properties of relaxation to PPP inhibitors in that both responses were associated with glutathione oxidation and depressed intracellular calcium release and calcium influx-mediated contractile responses. Inhibitors of potassium channels had minimal effects on these relaxation responses. Relaxation to hypoxia and PPP inhibitors were attenuated by a thiol reductant (3 mM dithiothreitol) and by eliciting contraction with an activator of protein kinase C (phorbol 12,13-dibutyrate). In the presence of contraction to U-46619, relaxation to hypoxia and PPP inhibitors were attenuated by the sarco(endo)plasmic reticulum Ca²⁺-ATPase pump inhibitor 200 µM cyclopiazonic acid and by 10 mM pyruvate. Hypoxia decreased BCA levels of glucose-6-phosphate but not ATP. Pyruvate prevented the hypoxia-elicited decrease in glucose-6-phosphate and glutathione oxidation, and it increased NADPH levels under hypoxia to levels observed under normoxia. Thus hypoxia causes a metabolic stress on the PPP that promotes BCA relaxation through processes controlled by lowering the levels of cytosolic NADPH.

calcium; oxygen sensor; pentose phosphate pathway; pyruvate; sarco(endo)plasmic reticulum calcium-adenosinetriphosphatase pump

Our laboratory previously showed (22, 23) that endothelium-denuded bovine coronary arteries (BCA) relax to hypoxia through unknown mechanisms that do not appear to involve changes in superoxide or peroxide and endothelium-derived mediators including prostaglandins or NO. While studying the mechanisms involved in relaxation of precontracted rat aorta and BCA to inhibitors of the pentose phosphate pathway (PPP) (9, 11), we identified a vasodilator mechanism in BCA associated with decreases in [Ca²⁺]_i that are controlled by the oxidation of cytosolic NADPH and glutathione (GSH) (9). Thus we investigated whether hypoxia could oxidize NADPH and/or GSH and activate relaxing mechanisms controlled by these redox cofactors. On the basis of preliminary studies that detected that hypoxia promoted a relaxation associated with oxidation of these cofactors, we developed the current study to examine whether hypoxia was causing relaxation through a metabolic process causing NADPH oxidation resembling inhibition of the PPP. Because pyruvate can potentially stabilize or enhance tissue levels of NADPH through a metabolic effect potentially associated with inhibition of phosphofructokinase (20), we examined whether pyruvate could be used as a probe to prevent NADPH oxidation and the BCA response to hypoxia.

	MATERIALS AND METHODS

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Materials. Many of the reagents used in the present study were obtained from sources previously described (9, 10, 16). DTT, reducing and protective agent for sulfydryl groups; tetraethylammonium chloride (TEA), nonspecific blocker of potassium channels; iberiotoxin (IBTX), antagonist of big-conductance calcium-activated potassium (K_Ca) channels; apamine (Apa), antagonist of small-conductance K_Ca channels; glibenclamide (Gli), blocker of K_ATP channels; 4-aminopyridine (4-AP), blocker of voltage-sensitive potassium (K_V) channels; phorbol 12,13-dibutyrate (PDBu), protein kinase C activator; EGTA, calcium chelator, pyruvate, and cyclopiazonic acid (CPA) were from Sigma. U-46619, a thromboxane A₂ analog, was from Cayman Chemical (Ann Arbor, MI), and all salts were purchased from J. T. Baker (Phillisburg, NJ).

Measurement of changes in force in BCA. Isolated endothelium-denuded arterial rings 4 mm in diameter and length were prepared from the left anterior descending artery and circumflex coronary artery of bovine calf hearts obtained from the slaughterhouse immediately after death and studied by an adaptation of previously described methods (22, 23). Briefly, arterial rings were mounted on wire hooks attached to force displacement transducers (model FT-03, Grass) for measurements of changes in isometric force on a polygraph (model 7, Grass). Arteries were incubated for 1 h at an optimal passive tension of 5 g in individually thermostated (37°C) 10-ml baths (Metro Scientific). These studies were conducted by using Krebs bicarbonate buffer (pH 7.4), gassed with 21% O₂-5% CO₂ (balance N₂). The Krebs bicarbonate buffer contained the following (in mM): 118 NaCl, 4.7 KCl, 1.5 CaCl₂, 25 NaHCO₃, 1.1 MgSO₄, 1.2 KH₂PO₄, and 5.6 glucose. After a 1-h equilibration period, the vessels were depolarized with Krebs bicarbonate containing KCl in place of NaCl. This treatment produces maximal contraction and enhances the reproducibility of subsequent contractions. The arteries were then reequilibrated with Krebs solution for 30 min before the experiments were conducted. The functional removal of the endothelium by gently rubbing the lumen of the vessel was confirmed by examining the effect of 10^–8–10^–6 M acetylcholine on arteries precontracted with 10^–7 M serotonin. Endothelium-denuded arteries did not show relaxation to acetylcholine and usually contracted to the 10^–6 M dose. After a 30-min equilibration period, experiments were conducted. In these experiments, arteries were typically precontracted to 60% of maximal force (7 g) with KCl (30 mM) or other contractile agents. Once a steady-state level of force was observed, the tissue was exposed either to a vasoactive agent or to hypoxia (PO₂ 8–10 Torr) produced by use of a gas mixture of 95% N₂-5% CO₂. After a 20-min exposure to hypoxia, arteries were reoxygenated with 21% O₂. The study of PO₂-elicited responses was designed to minimize the influence of O₂ gradients in the vessel wall on metabolism by examining a change in PO₂ from 150 Torr, which is well above the PO₂ range in which mitochondrial metabolism would be altered, to 8–10 Torr, which should elicit a hypoxic metabolic response in the vessel wall. In mechanistic probing experiments, coronary arterial rings were preincubated for 30 min with various drugs (see RESULTS). None of these probes significantly altered the level of force generation to KCl.

Measurement of NADPH, NADP⁺, and ATP in BCA. The levels of NADPH and ATP in BCA were determined by previously published HPLC methods (9, 10). Briefly, BCA were pretreated with hypoxia in a manner similar to the studies on vascular force and immediately frozen in liquid nitrogen. The frozen tissues were crushed and homogenized in an extraction medium consisting of 0.02 N NaOH containing 0.5 mM cysteine at 0°C for measurement of NADPH. The extracts were then heated at 60°C for 10 min and neutralized with 2 ml of 0.25 M glycylglycine buffer, pH 7.6. Acidic extracts for measurements of NADP⁺ and ATP were prepared by homogenizing the tissues in hot 0.1 N HCl. The neutralized extracts were centrifuged at 10,000 g for 10 min, the supernatants were passed through 0.45-µm Millipore filters, and the filtered solutions were used for measurement of NADP(H) and ATP by HPLC. NADP(H) and ATP were eluted on a reverse-phase HPLC column (4.6 x 250 mm; Bondapak C₁₈, Shiseido) at room temperature with an HP 1100 Series (Aligent Technologies) with a previously reported buffer system consisting of 100 mM potassium phosphate, pH 6.0 (buffer A) and 100 mM potassium phosphate, pH 6.0, containing 5% methanol (buffer B). The column was eluted with 100% buffer A from 0 to 8.5 min, 80% buffer A plus 20% buffer B from 8.5 to 14.5 min, and 100% buffer B from 14.5 to 40 min. The flow rate was 1.0 ml/min, and the ultraviolet absorbance was monitored at 260 nm. NADPH standards were used to calibrate the HPLC. Internal standards containing 2 nmol of NADP(H) and ATP were used to verify the quantitative recovery of the extraction procedure and HPLC retention time in the presence and absence of tissue samples.

Measurement of glutathione levels in BCA. Frozen BCA pretreated with 30 mM KCl in the absence or presence of hypoxia were crushed in liquid nitrogen, homogenized in 50 mM potassium phosphate buffer, pH 6–7, 1 mM EDTA, and then centrifuged at 10,000 g for 15 min at 4°C. The supernatant was used for measurement of glutathione (GSH) and oxidized glutathione (GSSG) after deproteination of the sample with equal amounts of 5 g of metaphosphoric acid in 50 ml of distilled water. The samples were centrifuged at 2,000 g for at least 2 min, and the supernatant was used for GSH measurement after neutralization of the samples with 4 M triethanolamine. To estimate GSSG after separation by centrifugation and neutralization, the samples were treated with 1 M 2-vinylpyridine. GSH and GSSG were estimated with a kit purchased from Cayman Chemical.

Statistical analysis. An ANOVA statistical analysis employing a post hoc Fisher protected t-test was used for all studies on vascular contractility. All data were analyzed by Student’s t-tests employing a Bonferroni correction for multiple comparisons. The acceptable level of significance was P < 0.05. The number of experimental determinations (n) in all cases is equal to the number of animals from which an arterial ring was used for a treatment or a control group in contractile studies. Values are means ± SE.

	RESULTS

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Effects of hypoxia on NADPH, NADP⁺, and ATP levels in BCA. To investigate whether hypoxia affects NADPH redox and ATP levels in vascular smooth muscle, endothelium-denuded BCA were incubated under normoxia or hypoxia in the presence of 30 mM KCl. At the end of 20-min incubation, BCA exposed to normoxia and hypoxia were homogenized, and NADPH, NADP⁺, and ATP were measured after extraction by HPLC methods. As shown in Fig. 1, NADPH levels decreased to 30% of the normoxic value, and NADP⁺ levels accumulated in the BCA. Under these conditions, ATP levels were not altered by hypoxia. Thus hypoxia appears to promote a conversion of NADPH to NADP⁺ or an oxidation of NADPH.

View larger version (7K): [in this window] [in a new window]   Fig. 1. Effects of hypoxia on bovine coronary artery (BCA) NADP(H) redox and ATP levels. In the presence of 30 mM KCl, hypoxia significantly decreased NADPH levels (A; n = 10–18). In contrast, NADP+ levels (B; n = 5–10) were increased by 3-fold. However, ATP levels (C; n = 5–10) remained unaltered by the 20-min hypoxic treatment. Con, control.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Effects of hypoxia on force generation by BCA in the presence of contractile agents and in the absence and presence of inhibitors of cyclooxygenase, nitric oxide synthase, and K+ channels. A: BCA precontracted with 30 mM KCl (n = 29) and 100 nM U-46619 (n = 25) relaxed to hypoxia in a time-dependent manner. However, BCA precontracted with 10 µM phorbol 12,13-dibutyrate (PDBu; n = 33) did not relax under hypoxia. B: response to hypoxia in BCA precontracted with 30 mM KCl in the absence or presence of either 10 µM indomethacin (Indo; n = 10) or 100 µM nitro-L-arginine (L-NNA; n = 9). Indo and L-NNA did not modulate the relaxation to hypoxia. C: response to hypoxia in BCA precontracted with 100 nM U-46619 in the absence or presence of various specific and nonspecific K+ channel blockers. As shown, tetraethylammonium chloride (TEA, 10 mM; n = 27), a nonspecific K+ channel blocker, glibenclamide (Gli, 10 µM; n = 8), an ATP-sensitive K+ channel blocker, 4-aminopyridine (4-AP, 10 mM; n = 9), a voltage-sensitive K+ channel blocker, iberiotoxin (IBTX, 23 µM; n = 10), a big-conductance calcium-sensitive K+ (KCa) channel blocker, and apamine (Apa, 100 nM; n = 8), a small-conductance KCa channel blocker, did not decrease the relaxation to hypoxia.

Effects of indomethacin and nitro-L-arginine on hypoxia-elicited BCA relaxation. The effects of indomethacin (10 µM), a cyclooxygenase inhibitor, and nitro-L-arginine (100 µM), a NO synthase inhibitor, on hypoxic relaxation were examined to detect whether prostaglandins and NO contributed to the observed relaxation. These agents did not affect force generation in the endothelium-denuded arteries studied (not shown). Consistent with our previous reports (22, 23) in which we showed that prostaglandins and NO do not mediate hypoxic relaxation in endothelium-intact BCA, pretreatment with indomethacin and nitro-L-arginine did not significantly alter the relaxation of endothelium-denuded BCA to hypoxia (see Fig. 2B).

Effects of K⁺ channel blockers on hypoxia-elicited BCA relaxation. Because it is possible that opening of K_ATP or K_Ca channels due to hypoxia may hyperpolarize vascular smooth muscle and elicit relaxation, we investigated the effects of K⁺ channel blockers on the relaxation elicited by hypoxia (Fig. 2C). As previously reported (9, 16), the K⁺ channel inhibitors examined did not significantly affect force generation in BCA (not shown). The nonspecific K⁺ channel blocker TEA (10 mM; Ref. 24) did not antagonize the BCA relaxation caused by hypoxia. In contrast, it appeared to slightly enhance the relaxation of BCA under hypoxia, but this effect was not statistically significant. Moreover, the K_ATP, K_V, big-conductance K_Ca, and small-conductance K_Ca channel antagonists Gli (10 µM), 4-AP (10 mM), IBTX (23 µM), and Apa (100 nM), respectively, also did not decrease the relaxation of BCA induced by hypoxia. Furthermore, we did not notice any effects of these drugs on the transient relaxation during posthypoxic reoxygenation (not shown).

Effects of hypoxia on contraction to different types of stimuli elicited in Ca²⁺-free solution followed by addition of CaCl₂. In this series of experiments, vessels were first contracted with KCl (30 mM) in normal Krebs solution, followed by washout, and subsequent contractile responses produced by various contractile agents were studied with the protocols described below. BCA were then placed in Ca²⁺-free Krebs solution containing EGTA (0.1 mM), and the contractile effects of U-46619 (100 nM), 5-hydroxytryptamine (5-HT; 10 µM), and KCl (30 mM) were examined in the absence or presence of hypoxia, followed by addition of CaCl₂ (1.5 mM) to detect contractile responses originating from the initial release of intracellular Ca²⁺ and the subsequent influx of extracellular Ca²⁺, respectively. Hypoxia was induced 10–15 min before the application of contractile agents, and the initial transient increase in force on addition of contractile agents was observed for 15 min before the addition of CaCl₂, when force had returned to a steady state at close to baseline levels. A typical experimental recording for responses to 5-HT is shown in Fig. 3A. The data in Fig. 3B show BCA responses to U-46619 (100 nM) and 5-HT (10 µM) in Ca²⁺-free solution and the subsequent contraction elicited by the addition of CaCl₂ (1.5 mM) either in normoxia or hypoxia (Fig. 3C). In normoxia, U-46619 and 5-HT caused rapid transient contraction, and sustained increases in force were observed on readdition of CaCl₂. In the presence of hypoxia, the initial transient contraction was significantly decreased, and subsequent contraction elicited by CaCl₂ was also inhibited (Fig. 3B). The addition of KCl (30 mM) to Ca²⁺-free Krebs buffer did not elicit a contraction. Subsequent addition of CaCl₂ (1.5 mM) caused a sustained contraction in normoxia; however, this sustained contraction was markedly inhibited by hypoxia (Fig. 3C).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Effects of hypoxia on the BCA force generation to contractile agents under Ca2+-free conditions and on readdition of CaCl2. A: experiment showing the inhibitory effects of hypoxia (bottom) on the transient contraction to 10 µM 5-hydroxytryptamine (5-HT) calcium-free conditions, followed by force generation elicited by the readdition of calcium seen under normoxic conditions (top). B: summary data for the response of BCA treated with 30 mM KCl (n = 9–11), 100 nM U-46619 (n = 7), and 10 µM 5-HT (n = 4) in Ca2+-free solution. U-46619 and 5-HT caused a transient contraction, and this contraction was attenuated by hypoxia. In these experiments, after the initial contraction decayed to a near-baseline level of steady-state force (15 min), 1.5 mM CaCl2 was readded to the bath solution. C: summary data showing that the contraction caused by readdition of Ca2+ in the presence of KCl, U-46619, and 5-HT was reduced by hypoxia.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Effects of a sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) inhibitor on the relaxation of BCA precontracted with 100 nM U-46619 to hypoxia and pentose phosphate pathway (PPP) inhibitors. A: hypoxia-elicited relaxation was almost completely inhibited by SERCA inhibitor 200 µM cyclopiazonic acid (CPA; n = 10). Force generation to 100 nM U-46619 (11 ± 2 g, n = 25) was not altered by the presence of CPA (11 ± 5 g). B: the relaxation induced by PPP inhibitors 1 mM 6-aminonicotinamide (6-AN; n = 4) and 100 µM epiandrosterone (Epi; n = 5) was attenuated by CPA.

View larger version (11K): [in this window] [in a new window]   Fig. 5. Effects of a SERCA inhibitor on BCA force generation to 100 nM U-46619 under Ca2+-free conditions and on readdition of CaCl2 in the absence and presence of hypoxia. A: under Ca2+-free conditions, the SERCA inhibitor 200 µM CPA (n = 8) did not alter force generation to U-46619 under normoxia and hypoxia. B: the contraction caused by readdition of 1.5 mM Ca2+ in the presence of U-46619 was increased by CPA in the presence of normoxia and hypoxia, preventing the detection of a decrease in force by hypoxia.

View larger version (12K): [in this window] [in a new window]   Fig. 6. Effects of hypoxia on BCA GSH redox and attenuation of BCA relaxation to hypoxia by DTT. A: GSH levels (n = 7) were significantly decreased under hypoxia. In contrast, the GSSG levels (n = 7) were increased by the 20-min hypoxic treatment. B: BCA were precontracted with 100 nM U-46619 (n = 10) in the absence and presence of 3 mM DTT and then exposed to hypoxia. DTT pretreatment significantly attenuated the relaxation of BCA to hypoxia. Force generation to 100 nM U-46619 (11 ± 2 g) was not altered by the presence of DTT (12 ± 4 g).

Effects of glucose removal on influence of hypoxia on NADPH levels and force in BCA. In vascular smooth muscle, glucose is classically metabolized through glycolysis and PPP. Therefore, the effects of removal of glucose from the extracellular solution on NADPH generation were examined in the presence of 100 nM U-46619 (see Fig. 7). Under normoxic conditions, incubation of BCA in glucose-free Krebs appeared to decrease NADPH levels [glucose(+) 3.47 ± 0.79 nmol/g, glucose(–) 2.61 ± 0.61 nmol/g; n = 14], but this decrease was not statistically significant. However, under hypoxic conditions NADPH levels were significantly decreased by incubating BCA in glucose-free Krebs solution for 20 min [glucose(–): 0.28 ± 0.19 nmol/g] vs. glucose-containing Krebs solution [glucose(+): 1.61 ± 0.75 nmol/g]. Exposure of BCA incubated in glucose-free solution to hypoxia significantly enhanced hypoxia-elicited relaxation. Although glucose-free Krebs appeared to decrease force generation to 100 nM U-46619 (see Fig. 7A), this decrease was not statistically significant. Hypoxia did not alter ATP levels in glucose-free treated tissues (Fig. 7C). In addition, in these experiments, the contractile agent used did not influence NADPH levels, because the NADPH levels reported in these experiments in the presence of glucose and 100 nM U-46619 under normoxia were not significantly different from BCA treated with 30 mM KCl under similar conditions (see Fig. 1A).

View larger version (9K): [in this window] [in a new window]   Fig. 7. Effect of glucose-free conditions on hypoxia-elicited relaxation and NADPH and ATP levels. A: hypoxic relaxation was enhanced in glucose-free [glucose (–)] solution (n = 10). Force generation to 100 nM U-46619 (11 ± 2 g) was not altered by the absence of glucose (9 ± 3 g). B: incubation of BCA (n = 14) with U-46619 in glucose-free Krebs solution under hypoxic conditions for 20 min decreased NADPH levels. C: ATP levels were not altered by 20 min of hypoxia under the glucose-free conditions examined.

View larger version (12K): [in this window] [in a new window]   Fig. 8. Effect of sodium cyanide (NaCN) and deoxyglucose on hypoxic dilation of BCA. Mitochondrial inhibitor 10 µM NaCN (n = 4) and glycolytic pathway inhibitor 2 mM 2-deoxyglucose (2-DG; n = 5) did not significantly change relaxation of BCA to hypoxia.

View larger version (10K): [in this window] [in a new window]   Fig. 9. Effect of pyruvate on NADPH and glucose-6-phosphate (G-6-P) levels in the absence and presence of hypoxia. Pretreatment of BCA with 10 mM pyruvate (n = 5) for 1 h increased NADPH (A) and G-6-P (B) under normoxic and hypoxic conditions and prevented the decrease in G-6-P caused by hypoxia.

View larger version (11K): [in this window] [in a new window]   Fig. 10. Effect of pyruvate on reduced and oxidized GSH (GSSG) levels in the absence and presence of PPP inhibitor and hypoxia. Pretreatment of BCA with 10 mM pyruvate (n = 5) for 1 h prevented increase in oxidized GSH (GSSG) elicited by inhibition of the PPP with 6-AN (A) and by hypoxia (B). Measurements of GSH and GSSG were made in the presence of 100 nM U-46619 after exposure to 5 mM 6-AN or hypoxia for 20 min.

View larger version (12K): [in this window] [in a new window]   Fig. 11. Effect of pyruvate on relaxation of BCA induced by hypoxia and PPP inhibitors. A: pretreatment of BCA with 10 mM pyruvate suppressed relaxation to hypoxia. Force generation to 100 nM U-46619 (11 ± 2 g) was not altered by the presence of 10 mM pyruvate (10 ± 2 g). B: pretreatment of BCA with 10 mM pyruvate suppressed relaxation of BCA contracted with U-46619 to the PPP inhibitors 6-AN and Epi (n = 15–20).

View larger version (11K): [in this window] [in a new window]   Fig. 12. Effects of hypoxia on the BCA force generation to contractile agents under Ca2+-free conditions and on readdition of CaCl2 after pyruvate pretreatment. A: summary data for force generation under Ca2+-free conditions in the presence of pyruvate under normoxic and hypoxic conditions resulting from the transient contractions elicited by the addition of 30 mM KCl (n = 10), 100 nM U-46619 (n = 5), and 10 µM 5-HT (n = 5). In the presence of 10 mM pyruvate, KCl did not elicit a contraction, and force generation during the transient contractions to U-46619 and 5-HT were partially attenuated by hypoxia. B: summary data for force generation in the presence of pyruvate under normoxic and hypoxic conditions resulting from the readdition of 1.5 mM CaCl2 to Ca2+-free Krebs after the transient contractions decayed to near-baseline conditions. Hypoxia partially inhibited force generation in the presence of KCl, but it did not significantly inhibit force generation caused by readdition of Ca2+ in the presence of U-46619 and 5-HT in the presence of pyruvate. C: summary data for the effects of pyruvate on force generation by the readdition of Ca2+ under hypoxic conditions. Pyruvate significantly enhanced force generation to the readdition of Ca2+ in the presence of KCl, U-46619, and 5-HT.

	DISCUSSION

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View larger version (10K): [in this window] [in a new window]   Fig. 13. Model showing regulatory mechanisms that appear to influence the relaxation of BCA to hypoxia. Hypoxia was observed to decrease G-6-P and oxidize NADPH and GSH in a manner reversed by pyruvate. The effects of hypoxia appeared to mimic the effects of inhibitors of the PPP of G-6-P metabolism in causing an oxidation of NADPH and GSH associated with coordinating a depression of contractions related to the release of intracellular Ca2+ and the influx of extracellular Ca2+. The actions of the SERCA inhibitor CPA suggest that stimulation of Ca2+ reuptake is one of the mechanisms contributing to the actions of these redox changes elicited by hypoxia. Although the actual sensor for hypoxia remains to be identified, it appears to be associated with processes controlling the maintenance of G-6-P by glycolysis and perhaps systems independent of mitochondrial respiration and the generation of reactive oxygen species, which remain to be identified, that could promote NADPH and/or GSH oxidation under hypoxia. [Ca2+]i, intracellular Ca2+ concentration. SR, sarcoplasmic reticulum.

Relaxation of BCA to hypoxia shows many mechanistic similarities to the response elicited by inhibition of the G-6-P dehydrogenase reaction in the PPP with the 6-AN and epiandrosterone pharmacological probes in BCA and rat aorta and pulmonary arteries (9, 11). Both hypoxia and PPP inhibition decrease the contraction mediated by KCl and U-46619, without markedly attenuating the force generated by activation of protein kinase C with PDBu. These relaxation responses of BCA were associated with NADPH and GSH oxidation, and treatment with the thiol reductant DTT attenuates relaxation to both PPP inhibitors (9) and hypoxia. Relaxation of BCA to hypoxia and PPP inhibitors (9) does not seem to be mediated through mechanisms dominated by the opening of K⁺ channels, because none of the K⁺ channel antagonists was observed to have prominent effects on the responses that were observed. Although a previous study reported that hypoxia produces minimal (18%) relaxation of porcine coronary artery by opening K_V channels (28), we could not detect a significant role for the involvement of K⁺ channels in mediating the relaxation of BCA to hypoxia under the conditions examined in the present study. Both hypoxia and PPP inhibitors (9) attenuate the contraction of BCA elicited by U-46619 and 5-HT in Ca²⁺-free Krebs solution and the recovery of force on readdition of 1.5 mM Ca²⁺ with KCl, U-46619, and 5-HT. Hypoxia appeared to inhibit Ca²⁺ influx because it attenuated contractile responses associated with Ca²⁺ addition to BCA exposed to contractile agents under Ca²⁺-free conditions in a manner similar to PPP inhibitors. It was previously reported that PPP inhibition (9) and a thiol oxidant diamide (16) attenuate contractile responses mediated by Ca²⁺ influx through both voltage-dependent and receptor-regulated calcium channels and that PPP inhibitors suppress L-type Ca²⁺ currents (12). Data in the present study also show that the SERCA pump inhibitor CPA attenuated relaxation to hypoxia and PPP inhibition with 6-AN and epiandrosterone in BCA precontracted with U-46619. Because the SERCA pump is a major site of redox regulation of Ca²⁺ reuptake by the sarcoplasmic reticulum (SR) (1), accelerated SR Ca²⁺ uptake by hypoxia and perhaps control of the influx of Ca²⁺ by the Ca²⁺ storage capacitive properties of the SR may be important factors contributing to the regulation of force generation by controlling the availability of intracellular Ca²⁺ in BCA contracted with agents such as U-46619. Thus redox regulation of Ca²⁺-dependent contractile mechanisms by hypoxia is similar to those controlled by NADPH generation by the PPP, and hypoxia may coordinate these NADPH redox-controlled mechanisms associated with lowering intracellular Ca²⁺ through processes included in the model shown in Fig. 13.

The effects of altering aspects of glucose metabolism suggest that hypoxia is controlling the maintenance of cytosolic NADPH by the PPP. Hypoxia was observed to cause an oxidation of NADPH in BCA that was enhanced by glucose-free conditions and inhibited by pyruvate, suggesting that glucose metabolism was controlling the ability of hypoxia to regulate NADPH. Because glucose-free conditions and pyruvate enhanced and inhibited, respectively, the relaxation to hypoxia, the oxidation of NADPH appears to be closely associated with the decrease in force that is observed. The inhibitor of glycolysis 2-deoxyglucose would not be expected to deplete NADPH because its key metabolite 2-deoxyglucose-6-phosphate is a substrate for NADPH generation by G-6-P dehydrogenase. Hypoxia decreased G-6-P levels, suggesting that a decreased availability of substrate for the PPP could be an important factor associated with NADPH oxidation. Pyruvate increased G-6-P levels and prevented the decrease caused by hypoxia. Although pyruvate could have multiple metabolic actions that affect tissue NADPH levels (20), it appears to be functioning in BCA by increasing the availability of G-6-P for the generation of NADPH by the G-6-P dehydrogenase reaction of the PPP and inhibiting processes involved in the response to hypoxia (see Fig. 13) through maintaining increased levels of cytosolic NADPH. Thus hypoxia appears to be causing relaxation through a metabolic effect on the maintenance of NADPH by the PPP.

Metabolic inhibitors influence force generation in BCA and the response to hypoxia in a manner that provides information on how hypoxia is promoting relaxation. Because mitochondrial inhibitors do not cause a depression of force in a manner similar to hypoxia in BCA (9), the PO₂ requirement for mitochondrial energy metabolism is not a primary factor in eliciting the relaxation to hypoxia. When the PPP was discovered (32), it was initially thought to be a direct oxidation pathway for the metabolism of carbohydrate (15). Although the initial investigation of the oxidative dependence of this pathway focused on identifying a cytochrome-containing electron transport chain that transferred electrons from NADPH to oxygen, studies that evolved focused on the intermediates in the PPP and how they functioned in cellular metabolism (15). The PO₂ dependence of a system oxidizing NADPH by molecular oxygen would be an attractive mechanism for explaining the relaxation to hypoxia; however, there is little evidence for a system that oxidizes NADPH as PO₂ decreases. Our study has detected evidence that GSH oxidation occurs under hypoxia and that reactive oxygen species (ROS) levels in BCA decrease either under hypoxia (22, 23) or as NADPH levels decrease when the PPP is inhibited (9, 11). Thus a ROS-independent mechanism of oxidizing NADPH and GSH may be involved in the mechanism of sensing hypoxia in BCA. It is well established that hypoxia can promote oxidative conditions that have been thought to be mediated through the generation of ROS. However, our studies may be detecting evidence of an alternative mechanism for hypoxia producing oxidative conditions originating from the oxidation of NADPH, which may be functioning as a PO₂ sensor in BCA.

Relaxation of BCA to hypoxia appears to be mediated through a novel mechanism independent of mitochondrial energy metabolism and changes in the generation of ROS, involving a process through which decreased PO₂ promotes cytosolic NADPH oxidation. Lower levels of G-6-P dehydrogenase in BCA compared with bovine pulmonary arteries (which do not relax to hypoxia) (10) and the decrease in G-6-P by hypoxia provide evidence for a decrease in the flux of glucose metabolism through the PPP as a key component in the mechanism of NADPH oxidation and the relaxation of BCA to hypoxia that is observed. Because stabilization of G-6-P levels by pyruvate maintains NADPH levels and attenuates relaxation to hypoxia, the control of G-6-P by glycolysis seems to be a major influential factor in the processes controlled by decreased PO₂. However, GSH oxidation observed under hypoxia and NADPH still shows a small oxidation in the presence of pyruvate-elicited increases in G-6-P, suggesting that an oxygen-sensitive process promoting NADPH oxidation under hypoxia is also a potential contributing factor to the PO₂ sensor for hypoxia in BCA. Because pyruvate has been observed to attenuate functional increases in blood flow associated with the detection of redox changes linked to tissue hypoxia (33), the vascular smooth muscle PO₂-sensing mechanism reported in the present study associated with promoting cytosolic NADPH oxidation may be a fundamental mechanism of controlling blood flow and sensing cellular hypoxia.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported by AHA Grant 0435070N and by National Heart, Lung, and Blood Institute Grants HL-31069, HL-43023, and HL-66331.

	ACKNOWLEDGMENTS

 
A part of this study was presented at the American Heart Association (AHA) Scientific Sessions at Chicago, IL, in 2002 (Circulation 106: II-495, 2002) and the Experimental Biology meeting at San Diego, CA, in 2005 (FASEB J 19: A1279, 2005).

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. S. Wolin, Dept. of Physiology, Basic Sciences Bldg., Rm. 604, New York Medical College, Valhalla, NY 10595 (e-mail: mike_wolin{at}nymc.edu)

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	REFERENCES

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