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Department of Physiology, New York Medical College, Valhalla, New York 10595
Submitted 20 March 2003 ; accepted in final form 31 July 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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calcium; redox signaling; vasodilator mechanisms
It is now well established that NADPH derived from the pentose phosphate pathway (PPP) is a key system involved in maintaining the function of several important redox and antioxidant defense mechanisms, through processes such as reducing oxidized GSH (GSSG), and other thiol and heme-linked systems. Recent studies have begun to detect potentially important roles for the PPP and NADP(H) redox in the control of signaling processes. For example, PPP-derived NADPH is an essential cofactor for NO production by NO synthase (8, 12) and for controlling NO-induced relaxation in bovine arteries through a NADPH-dependent methemoprotein reductase, which maintains the heme of sGC in its NO-binding ferrous form (7). In these studies on the actions of NO and heme oxidants on sGC, the PPP inhibitors 6-aminonicotinamide (6-AN) and epiandrosterone (Epi) were employed to inhibit the generation of NADPH. An early hypothesis on the mechanism of hypoxic vasoconstriction proposed a role for the loss of a relaxing mechanism mediated through the lowering of oxidant generation and NAD(P)+ and GSSG levels (22), which potentially open VSM K+ channels through processes such as regulating the redox states of thiols (22, 25). PPP inhibitors have recently been reported to contribute to the relaxation in the rat pulmonary artery and aorta through hyperpolarizing VSM by opening of Kv channels (5) and reduction of myocardial contractility by inhibition of L-type Ca2+ channels in cardiac myocytes (8). Thus PPP-derived NADPH could be important in regulating the signaling mechanisms that control vasomotor function.
The present study was developed based on the observation that the PPP inhibitors 6-AN and Epi caused relaxation of BCAs. Because inhibition of the PPP could potentially cause oxidation of the cytosolic NADP(H) and GSH redox systems and increase H2O2 levels in VSM due to the loss of metabolic control mechanisms, components of each of these systems could be involved in producing relaxation of coronary arteries. On the basis of previous observations, roles for modulating the production of cGMP by sGC, K+ channel opening-induced hyperpolarization, and processes that modulate intracellular Ca2+ concentration ([Ca2+]i) needed to be considered in the evaluation of the mechanisms through which PPP inhibition could cause relaxation of VSM. Thus this study focuses on providing evidence that PPP inhibition increases NADP+-to-NADPH and GSSG-to-GSH ratios associated with activating vascular relaxation through processes that suppress [Ca2+]i.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Measurement of changes of force generation in BCAs. Isolated, endothelium-removed left anterior descending coronary arterial rings were prepared from slaughterhouse-derived bovine hearts and studied for changes in isometric force, as previously described (6, 7, 9, 14, 15). The rings were incubated in individually thermostated (37°C) 10-ml baths (Metro Scientific) for 2 h at an optimal passive tension of 5 g in Krebs bicarbonate buffer (pH 7.4) containing the following (in mM): 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.1 MgSO4, 1.2 KH2PO4, and 5.6 glucose, gassed with 21% O2-5% CO2-balance N2. After a 2-h equilibration and a brief depolarization with 123 mM KCl, BCAs were reequilibrated in Krebs buffer for 30 min before the experiments described in RESULTS were conducted. In studies examining the actions of mechanistic inhibitors on the response to 6-AN and Epi, probes were usually added at least 15 min before exposure to increasing cumulative concentrations of 6-AN, Epi, or other relaxing agents. None of the probes or treatments, except those mentioned in RESULTS, had a statistically significant effect on force generation.
Measurement of NADPH and NADP+ in BCAs. The levels of NAD(P)H in BCAs were determined by HPLC using adaptations of previously published methods (5, 7). Briefly, BCAs were pretreated with and without 6-AN or Epi 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. 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 were prepared by homogenizing the tissues in hot 0.1 N HCl, followed by neutralization. NAD(P)H was eluted on a reverse-phase HPLC column (4.6 x 250 mm, Bondapak C18, Shiseido) at room temperature using a HP 1100 Series (Agilent Technologies) and 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.
Measurement of glutathione levels in BCAs. Frozen BCAs pretreated with or without 6-AN or Epi were crushed in liquid nitrogen, homogenized in 50 mM potassium phosphate buffer (pH 6–7) and 1 mM EDTA, and then centrifuged at 10,000 g for 15 min at 4°C. The supernatant was used for measurement of GSH and GSSG after the samples were deproteinated. The GSH and GSSG levels were determined by employing a kit purchased from Cayman Chemical.
Measurement of superoxide and hydrogen peroxide levels in BCAs. Employing previously described methods (14, 15), we prepared BCAs as described for force measurement, but the rings pretreated with or without drugs in the tissue bath were placed in plastic scintillation minivials containing 5 µM lucigenin for the detection of superoxide and 10 µM luminol plus 1 µM horseradish peroxidase for the detection of H2O2 and other additions in a final volume of 1 ml of air-equilibrated Krebs solution buffered with 10 mM HEPES-NaOH (pH 7.4). Chemiluminescence was measured in a liquid scintillation counter (LS6000IC, Beckman Instruments) at 
37°C, and data are reported as counts per minute per gram of BCA after background subtraction. The inhibition of chemiluminescence by the addition of 1 µM catalase was used for measurement of H2O2 released from BCAs, which occurs when GSH peroxidase activity is inhibited (15).
Estimation of smooth muscle [Ca2+]i by fura-2 fluorescence ratios. In separate experiments, the smooth muscle of isolated coronary arteries was loaded with fura-2 (2 µM fura-2 AM, 30 min at 25°C). Changes in background-corrected fura-2 fluorescence ratios (F340/F380; the ratio of 510-nm emission intensity resulting from excitation at 340 nm divided by the emission intensity from excitation at 380 nm) and estimates of smooth muscle [Ca2+]i were measured by the ratiometric fluorescence method using the Ionoptix Microfluorimeter System (Ionoptix) as previously described in detail (23, 24). VSM was depolarized with 30 mM KCl, and changes in smooth muscle [Ca2+]i to 6-AN (0.010–1 mM), Epi (0.001–0.100 mM), and nifedipine (10 µM) were obtained. In other experiments, the time course of changes in smooth muscle [Ca2+]i to 5-hydroxytryptamine (5-HT; 10 µM) were assessed in the absence and presence of 1 mM 6-AN or 100 µM Epi. Decreases in F340/F380 were normalized to the maximal nifedipine-induced responses, whereas 5-HT-induced increases in F340/F380 were normalized to the maximal response to 5-HT in the absence of 6-AN and Epi.
Statistical analysis. ANOVA statistical analysis employing a post hoc Fisher's protected t-test was used for all studies on vascular contractility. All chemiluminescence and fura-2 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 animals from which an arterial ring was employed for a treatment or a control group in all studies. Values are means ± SE.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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6-AN and Epi inhibit BCA contraction to K+ and U-46619. Endothelium-denuded BCA rings precontracted to 70% of maximal force by depolarization- and receptor-dependent mechanisms with KCl (30 mM) and the thromboxane A2 analog U-46619 (100 nM) or endothelin-1 (100 nM), respectively, relaxed in a dose-dependent manner when treated with 6-AN or Epi (Fig. 1, C and D). The PPP inhibitors 6-AN and Epi elicited relaxation of BCAs (Fig. 1, C and D). In contrast, the BCA precontracted with a PKC activator, phorbol 12,13-dibutyrate (PDBu; 10 µM), through a mechanism that is essentially Ca2+ independent (9), did not relax to 6-AN or Epi (Fig. 1, C and D). Interestingly, inhibition of either glycolysis with 2-deoxyglucose or mitochondrial electron transport by cyanide or antimycin did not cause relaxation of BCAs precontracted with 30 mM KCl (Fig. 1E).
Effect of 6-AN and Epi on GSH and GSSG levels in BCAs. The effect of PPP inhibition on GSH and GSSG levels were measured in BCAs in the absence and presence of 6-AN and Epi. Both 6-AN (5 mM) and Epi (0.5 mM) decreased GSH (control: 270 ± 17 µg/g, 6-AN: 193 ± 15 µg/g, Epi: 200 ± 26 µg/g) and increased GSSG (control: 26 ± 7 µg/g, 6-AN: 79 ± 17 µg/g; Epi: 40 ± 6 µg/g) levels in BCAs (Fig. 2, A and B). These values were converted into molar amounts to enable the calculation of redox ratios (GSH: control 878.6 ± 55.3 nM/g, 6-AN 628.1 ± 48.8 nM/g, and Epi 650.8 ± 84.6 nM/g; GSSG: control 42.4 ± 11.4 nM/g, 6-AN 128.9 ± 27.8 nM/g, and Epi 65.3 ± 9.7 nM/g). GSH-to-GSSG ratios were decreased from 20.7 (control) to 4.9 and 9.9 in the presence of 6-AN and Epi, respectively, indicating that marked changes in GSH redox were occurring in the presence of these PPP inhibitors.
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Effect of thiol reduction with DTT on relaxation elicited by PPP inhibition. Because inhibition of the PPP decreased GSH and elevated GSSG levels in BCAs, the effects of the thiol reductant DTT (3 mM), which reduces GSSG or S-thiolated proteins, on relaxation to 6-AN and Epi were examined. BCA rings contracted with KCl (30 mM; Fig. 2, C and D) or U-46619 (100 nM; data not shown) that were treated with DTT (30 min) showed a significantly inhibited relaxation to 6-AN (Fig. 2C) and EPI (Fig. 2D) compared with responses to PPP inhibition in the absence of DTT. Under these conditions, DTT treatment did not change the force generated by either KCl or U-46619, and it did not significantly effect relaxation to isoproterenol (Fig. 2E).
Effect of 6-AN and Epi on 
and H2O2 levels in BCAs. Inhibition of the PPP surprisingly decreased the release from BCAs of H2O2, which was detected as catalase-inhibitable luminol chemiluminescence. Further investigation indicated that inhibition of the PPP attenuated generation of 
, a precursor to H2O2 (Fig. 3, A and B). To further probe for an absence of a role for these O2 species, the actions of PPP inhibitors were investigated under severely hypoxic conditions (95% N2-5% CO2) and in the presence of the H2O2 scavenger ebselen. BCAs were relaxed by 100% and 60% by 6-AN (5 mM) and Epi (0.5 mM) under hypoxia (n = 5), and ebselen (n = 11; Fig. 3C) did not inhibit the relaxation elicited by PPP inhibitors under aerobic conditions.
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Effect of inhibition of sGC, adenylate cyclase, and sarco(endo)plasmic reticulum Ca2+-ATPase on relaxation of BCAs to 6-AN. Because redox systems modulate the activity of sGC and adenylate cyclase, and these systems regulate many of the other systems reported to be redox regulated (e.g., K+ and Ca2+ channels, sarcoplasmic reticulum Ca2+ uptake, etc.) in the VSM (16, 22, 25, 27, 28), inhibitors of sGC [LY83583 (10 µM)], adenylate cyclase [3',5'-dideoxyadenosine (DDA; 4 µM)], and sarco(endo)plasmic reticulum Ca2+-ATPase [cyclopiazonic acid (CPA; 200 µM)] were examined. LY83583 (Fig. 4A), DDA (Fig. 4B), and CPA did not significantly inhibit the relaxation to 6-AN. In BCAs precontracted with 100 nM U-46619, the relaxation induced by 6-AN (79 ± 6% at 1 mM, n = 5) or Epi (75 ± 6% at 100 µM, n = 5) was not significantly altered by CPA (6-AN + CPA: 76 ± 9% relaxation and Epi + CPA: 69 ± 9% relaxation).
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Effect of K+ channel antagonists on relaxation elicited by PPP inhibition. To further examine whether the opening of K+ channels by PPP inhibition participates in the observed relaxation, the arteries were pretreated with different K+ channel blockers before they were exposed to PPP inhibitors. Pretreatment of BCAs with a nonspecific K+ channel blocker, tetraethylammonium chloride (TEA; 10 mM), did not significantly increase force generation by U-46619; however, it caused a small, statistically significant reduction of relaxation to 6-AN (Fig. 5A). Opening of ATP-sensitive K+ (KATP) channels did not mediate relaxation, because glibenclamide (10 µM, n = 12), a KATP channel blocker (16), did not effect the relaxation to 6-AN (Fig. 5B). Roles for small- and large-conductance KCa channels were probed by blockade with apamin (Apa) and iberiotoxin (IbTX), respectively (16). Pretreatment of BCAs with either IbTX (23 nM) or Apa (100 nM) caused small decreases in the relaxation to 6-AN, suggesting that relaxation appears to be influenced by the function of both small- and large-conductance KCa channels (Fig. 5, C and D). A high dose of the Kv channel blocker 4-aminopyridine (4-AP; 10 mM) was used to examine whether the opening of this type of channel contributes to the relaxation elicited by 6-AN. Pretreatment with 4-AP did not significantly alter the contraction to U-46619, whereas it caused a partial inhibition (P < 0.05) of BCA relaxation to 6-AN (Fig. 5D). The combination of both 4-AP and IbTX further reduced the relaxation of BCAs to 6-AN (Fig. 5D), consistent with BCA relaxation being modulated by the function of both Kv and KCa channels. Similar experiments were done using Epi, and we found that inhibition of PPP by Epi also partially caused relaxation of BCAs by regulating the function of K+ channels.
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Effect of 6-AN and Epi on force generation by 5-HT, U-46619, and KCl in Ca2+-free buffer and the subsequent contraction to Ca2+ addition. In this series of experiments, vessels were first contracted with 30 mM KCl in normal Krebs solution, followed by washout, and subsequent contractile responses were studied in Ca2+-free Krebs solution containing 0.1 mM EGTA, as previously described (9). The contractile effects of 100 nM U-46619, 10 µM 5-HT, and 30 mM KCl were examined in the absence or presence of a 10-min pretreatment with 6-AN and Epi to detect contractile responses originating from the initial release of intracellular Ca2+. This was followed 10 min later by the addition of 1.5 mM Ca2+, after the initial transient contractions decayed to baseline force, to examine the actions of PPP inhibition on the subsequent influx of extracellular Ca2+. Figure 6A shows summary data for the initial BCA control response to 30 mM KCl in the presence of extracellular Ca2+ and rapid transient contraction responses to 10 µM 5-HT in Ca2+-free solution in the absence or presence of 6-AN and Epi. Subsequently sustained tonic contraction was elicited by the addition of 1.5 mM Ca2+. In the presence of 6-AN and Epi, the initial transient contraction was significantly decreased (Fig. 6A), and subsequent contraction elicited by Ca2+ was markedly inhibited (Fig. 6B). Similar results were obtained when U-46619 (100 nM) was used as a contractile agent. 6-AN (n = 11) and Epi (n = 6) depressed the force generated by U-46619 in the Ca2+-free condition (U-46619: 7.9 ± 1.2 g vs. 6-AN: 3.1 ± 1.1 g and Epi: 2.4 ± 0.8 g) and after the addition of 1.5 mM Ca2+ (U-46619: 14.9 ± 1.5 g vs. 6-AN: 6.1 ± 2.1 g and Epi: 2.4 ± 0.8 g). There was a complete absence of BCA contraction to 30 mM KCl in Ca2+-free solution (data not shown). Subsequent addition of 1.5 mM Ca2+ caused a sustained contraction in the absence of PPP inhibitors, and this sustained contraction was markedly inhibited by 6-AN and Epi (Fig. 6, C and D). Contraction elicited by the addition of Ca2+ was eliminated by 6-AN (Fig. 6C). The effects of Epi on the contraction-dependent contraction to Ca2+ addition are shown in Fig. 6D.
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Effect of PPP inhibitors on changes in [Ca2+]i detected by fura-2. KCl depolarization increased [Ca2+]i, as indicated by the increase in F340/F380 by 50%, and 6-AN and Epi elicited a significant decrease in F340/F380 (Fig. 7, A and B) over the concentration ranges that caused BCA relaxation (Fig. 1, C and D). Nifedipine (10 µM) also decreased F340/F380 by 60%. 5-HT elicited rapid peak increases in smooth muscle [Ca2+]i, which were followed by a plateau phase (Fig. 7C). In the presence of 6-AN or Epi, both phases of 5-HT-induced calcium signals were significantly diminished (Fig. 7, C and D).
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Effect of TEA and DTT on the attenuation by PPP inhibitors of contractions to 5-HT and KCl in Ca2+-free solution and after Ca2+ addition. To examine whether K+ channel and thiol redox mechanisms contribute to the inhibitory actions of 6-AN and Epi on contractile responses originating from the release of intracellular Ca2+ and Ca2+ influx, the effects of combinations of the thiol reductant DTT (3 mM) and the nonspecific K+ channel antagonist TEA (10 mM) on contractile responses of BCAs elicited by 10 µM 5-HT (Fig. 8, A and B) and 30 mM KCl (Fig. 8C) in Ca2+-free solution, followed by the addition of Ca2+, were studied in the absence or presence of 6-AN (5 mM). Blocking of K+ channels by TEA significantly (P < 0.005) increased the 5-HT-elicited contraction of BCAs in Ca2+-free solution, whereas this contraction was not altered by DTT (Fig. 8A). However, pretreatment with TEA and/or DTT did not alter the inhibitory actions of 6-AN on this transient contraction initiated as a result of intracellular Ca2+ release. Both DTT and TEA significantly increased the contraction of BCAs elicited by the addition of 1.5 mM Ca2+ in the presence of 5-HT. In addition, DTT and/or TEA partly reversed the inhibition of contraction to Ca2+ addition caused by 6-AN (Fig. 8B). Similar studies were performed in the presence of 30 mM KCl (Fig. 8C). TEA and DTT increased the force generated by Ca2+ addition in the presence of 30 mM KCl in the absence of 6-AN (Fig. 8C) and partially reversed the inhibition of 30 mM KCl-elicited force by 6-AN. TEA and DTT did not have additive effects on force generation under the conditions examined.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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In BCAs, it was observed that 6-AN (a competitive inhibitor of glucose-6-phosphate dehydrogenase) and Epi (a noncompetitive inhibitor of glucose-6-phosphate dehydrogenase) lowered NADPH and elevated NADP+ levels (Fig. 1). These changes were associated with decreased GSH and increased GSSG, presumably due to a loss of redox control by systems including the NADPH-dependent GSH reductase reaction. The reduction of GSH levels (Fig. 2) theoretically could also result in an increase in H2O2 levels; however, peroxide release appeared to be decreased in PPP-inhibited BCAs (Fig. 3). While this could occur through either decreased generation or increased degradation of H2O2, PPP inhibition also caused a reduction in the detection of (Fig. 3), the major source of H2O2 generation in BCAs (15). We speculate that due to elevation of GSSG and/or a loss of NADPH, the major source of , NAD(P)H oxidase (15), may have been inhibited, resulting in lowered and H2O2 levels. Because two structurally different PPP inhibitors (6-AN and Epi) caused decreases in tissue NADPH levels and vasodilation, it seems likely that changes in NADP+/NADPH levels and GSH redox were, at least in part, responsible for the vasodilation induced by the PPP inhibitors.
Glycolysis and oxidative phosphorylation pathways do not seem to be involved in mediating smooth muscle relaxation induced by PPP inhibition, because in separate experiments, the glycolysis inhibitor 2-deoxyglucose and the mitochondrial electron transport inhibitors cyanide and antimycin did not have any effect on the force generated by 30 mM K+. Consistent with this finding, it was also observed that the KATP channel blocker glibebclamide did not have any blocking effect on the BCA relaxation elicited by 6-AN. The opening of K+ channels (Kv and small- and large-conductance K+ channels) seems to modulate BCA relaxation resulting from PPP inhibition, because antagonists for these channels partially suppressed the 6-AN-elicited relaxation. In contrast, the K+ channel inhibitors did not alter relaxation in BCAs to the thiol oxidant diamide under the conditions examined in the present study (9). Free radical generation and changes in the ratio of cellular reducing cofactors, including NAD+/NADH, NADP+/NADPH, and GSH/GSSG in VSM, and oxidizing agents have been proposed or reported to regulate the activity of KCa, KATP, and Kv channels and to influence force generation through modulating membrane potential (22, 25). Kv channels are generally known to regulate resting membrane potential in coronary VSM (16), and therefore changes in Kv channel activity may be critical in modulating smooth muscle contractility. Oxidizing agents activate K+ currents, presumably by modifying a channel protein or by regulating the redox state of a cysteine residue that appears to regulate opening of the ion channel (11, 17, 18, 22, 25). Oxidizing agents activate both KCa and Kv channels of isolated VSM cells from the rabbit pulmonary and ear artery (11, 17, 18), and opening of Kv channels partially contributes to the relaxation of the adult rat pulmonary artery and aorta induced by PPP inhibitors (5). Thus it seems that while the redox changes caused by PPP inhibition may have opened both Kv and KCa channels, their role was relatively minor and other mechanisms appear to be more important in eliciting relaxation of BCAs. The differences between previous reports (5, 11, 17, 18) and the present study may reflect the impact of species, age, vascular segments examined, baseline membrane potential, and/or experimental conditions on the redox-controlled systems being studied.
A potential mechanism responsible for the relaxation caused by PPP inhibition could be a lowering of intracellular Ca2+ through inhibition of Ca2+ release and influx mechanisms, by systems that are dependent and independent of control by K+ channels. PPP inhibitors did not cause relaxation in the presence of PDBu, an agent that causes a Ca2+-independent contraction of BCAs by activation of protein kinase C (9). Measurements with fura-2 ratios suggest that the initial 5-HT-induced transient elevation of [Ca2+]i and the sustained increased levels of [Ca2+]i, which are thought to increase the release of Ca2+ from the sarcoplasmic reticulum followed by the influx of extracellular Ca2+ through opening of receptor-operated channels (21), respectively, were both attenuated by PPP inhibition. However, the actions of CPA suggest that attenuation of Ca2+ uptake by sarco(endo)plasmic reticulum Ca2+-ATPase does not appear to be an important mechanism in regulating BCA relaxation to PPP inhibition. The PPP inhibitors also significantly attenuated contraction and increases in [Ca2+]i caused by 30 mM KCl, thereby suggesting that PPP inhibition also suppressed the entry of extracellular Ca2+ initiated by depolarization-dependent mechanisms. Potentiation of contraction to 5-HT by TEA in the absence and after the addition of Ca2+ suggests that K+ channels may also influence sarcoplasmic reticulum Ca2+ release and entry of extracellular Ca2+. DTT enhanced contractions to Ca2+ addition to Ca2+-free Krebs solution containing 5-HT or KCl, and it attenuated the near-complete inhibition of these responses by 6-AN. These findings suggest that thiol-reducing conditions enhance the entry of extracellular Ca2+ through receptor- and voltage-regulated channels and that a thiol redox site regulated by PPP inhibition appears to control Ca2+ influx through these channels. These thiol redox-associated actions of PPP inhibition are similar to the previously reported actions of the thiol oxidant diamide in BCAs (9). The absence of effects of DTT and TEA on the transient contraction to 5-HT under Ca2+-free conditions is consistent with the idea that PPP inhibition also activates relaxation through processes that control [Ca2+]i release and/or force generation by Ca2+ through mechanisms that are not influenced by thiol redox and the function of K+ channels under the conditions examined. Thiol oxidation-regulated processes appear to inhibit L-type Ca2+ channels and nonselective cation channel transmembrane Ca2+ influx in VSM and cardiac myocytes (2, 10), and L-type Ca2+ inhibition by Epi is associated with inhibition of myocardial contractility (8). Although opening of K+ channels seems to be a contributing mechanism to BCA relaxation elicited by PPP inhibitors, mechanisms controlling Ca2+ release and influx appear to be the primary processes that mediate relaxation induced by 6-AN and Epi.
It seems logical to assume that signaling mechanisms mediated through cAMP and cGMP (16, 28) could contribute to the vasoactive actions of PPP inhibitors. However, the guanylate and adenylate cyclase inhibitors LY83583 and DDA, respectively, did not significantly affect the relaxation induced by PPP inhibition (Fig. 3). These observations are consistent with previous studies reporting that Epi and 6-AN open K+ channels of VSM and/or cause relaxation in the rat pulmonary artery and aorta, rabbit coronary artery, and isolated ferret and rat lungs in an endothelium-, receptor-, cAMP-, cGMP-, prostaglandin-, and NO-independent manner (3, 5, 29). Because PPP inhibition suppressed H2O2 generation, and neither the cell-permeable H2O2 scavenger ebselen nor hypoxia affected the BCA relaxation induced by 6-AN, it is unlikely that peroxide has a role in relaxation caused by PPP inhibition. Nonetheless, the thiol reductant DTT partly suppressed the 6-AN- and Epi-induced BCA relaxation and facilitated force generation by Ca2+ addition to Ca2+-free conditions. Therefore, it can be suggested that signaling processes controlled by increases in GSSG and/or S-thiolation, and processes directly controlled by NADP(H) redox, regulate channel activity and ionic homeostasis. For example, there is evidence for NADPH-controlled oxidoreductases on the 
-subunit of Kv channels (13, 19) and the skeletal muscle ryanodine-sensitive Ca2+ release channel (1), which could potentially regulate relaxation through a direct action of cytosolic NADPH redox on these ion channels.
This study provides evidence for the novel concept of an important role for the PPP, a key redox regulating pathway, and a loss of cytosolic NADPH in controlling the relaxation of BCAs through the coordination of processes including potentially minor roles for the opening of Kv and KCa channels and major roles for processes that control the inhibition of Ca2+ release and influx. Because the mechanisms are activated by lowering of NADPH levels, they may compensate for the potential loss of cGMP-dependent relaxation due to an impairment of NO biosynthesis and reduction of the sGC heme to the Fe2+ form required for NO activation, which are NADPH-dependent processes (7, 12). A deficiency of glucose-6-phosphate dehydrogenase activity in the African-American population is associated with endothelial oxidant stress, decreased NO bioavailability, and an increased risk for vascular disease in the absence of hypertension (4, 12). In addition, inhibition of the PPP almost completely abolishes acute hypoxic pulmonary vasoconstriction in isolate rat lungs (5). The activity of the PPP and the redox status of cytosolic NADP(H) are fundamental cellular metabolic systems that could be influenced by many physiological processes that regulate vascular function. Thus the PPP and cytosolic NADP(H) redox may be important additional systems that control vascular function in response to physiological, pathophysiological, metabolic, and oxidative stress conditions.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 R. Matsui, S. Xu, K. A. Maitland, R. Mastroianni, J. A. Leopold, D. E. Handy, J. Loscalzo, and R. A. Cohen Glucose-6-Phosphate Dehydrogenase Deficiency Decreases Vascular Superoxide and Atherosclerotic Lesions in Apolipoprotein E-/- Mice Arterioscler. Thromb. Vasc. Biol., April 1, 2006; 26(4): 910 - 916. [Abstract] [Full Text] [PDF]
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 C. J. Mingone, S. A. Gupte, N. Ali, R. A. Oeckler, and M. S. Wolin Thiol oxidation inhibits nitric oxide-mediated pulmonary artery relaxation and guanylate cyclase stimulation Am J Physiol Lung Cell Mol Physiol, March 1, 2006; 290(3): L549 - L557. [Abstract] [Full Text] [PDF]
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M. S. Wolin, M. Ahmad, and S. A. Gupte Oxidant and redox signaling in vascular oxygen sensing mechanisms: basic concepts, current controversies, and potential importance of cytosolic NADPH Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L159 - L173. [Abstract] [Full Text] [PDF]
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R. A. Oeckler, E. Arcuino, M. Ahmad, S. C. Olson, and M. S. Wolin Cytosolic NADH redox and thiol oxidation regulate pulmonary arterial force through ERK MAP kinase Am J Physiol Lung Cell Mol Physiol, June 1, 2005; 288(6): L1017 - L1025. [Abstract] [Full Text] [PDF]
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C. S. Wilcox and D. Gutterman Focus on oxidative stress in the cardiovascular and renal systems Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H3 - H6. [Full Text] [PDF]
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S. A. Gupte, P. M. Kaminski, B. Floyd, R. Agarwal, N. Ali, M. Ahmad, J. Edwards, and M. S. Wolin Cytosolic NADPH may regulate differences in basal Nox oxidase-derived superoxide generation in bovine coronary and pulmonary arteries Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H13 - H21. [Abstract] [Full Text] [PDF]
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 C.J Zuurbier, O Eerbeek, P.T Goedhart, E.A Struys, N.M Verhoeven, C Jakobs, and C Ince Inhibition of the pentose phosphate pathway decreases ischemia-reperfusion-induced creatine kinase release in the heart Cardiovasc Res, April 1, 2004; 62(1): 145 - 153. [Abstract] [Full Text] [PDF]
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B. Kalyanaraman and D. D. Gutterman Prologue: Vascular effects of free radicals Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2253 - H2254. [Full Text] [PDF]
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