Role of pentose phosphate pathway-derived NADPH in hypoxic pulmonary vasoconstriction

Pulmonary Pharmacology & Therapeutics 19 (2006) 303–309 www.elsevier.com/locate/ypupt

Role of pentose phosphate pathway-derived NADPH in hypoxic pulmonary vasoconstriction Sachin A. Gupte a,1,*, Takao Okada a, Ivan F. McMurtry b, Masahiko Oka a,1 a

Departments of Physiology and Respiratory Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113, Japan b CVP Research Laboratory, University of Colorado Health Sciences Center, Denver, CO, USA Received 19 January 2005; revised 5 May 2005; accepted 18 August 2005

Abstract We have previously shown that pentose phosphate pathway (PPP) inhibitors, 6-aminonicotimade (6-AN) and epiandrosterone (EPI), markedly reduce hypoxic pulmonary vasoconstriction (HPV). Although it has been suggested that changes in the NADPH/NADPC ratio and redox status are involved in the mechanism of HPV, the role of PPP-derived NADPH in this phenomenon is not known. The aim of this study, therefore, was to investigate the role of PPP-derived NADPH in HPV using isolated rat pulmonary arteries (PA) and perfused rat lungs. The NADPH/NADPC ratio and NADPH levels in PA and lungs exposed to hypoxia increased 2-fold and 7-fold, respectively, compared to time-matched normoxic controls. Both hypoxia-induced increases in lung NADPH levels and lung perfusion pressure were inhibited by 6-AN (500 mM) or EPI (300 mM). The chemical inhibitors of PPP and hypoxia similarly decreased lung tissue NOx levels by approximately 50%. In contrast, hypoxia increased the lung soluble guanylate cyclase (sGC) activity (from 22.9G6.3 to 57.1G7.6 pmol/min/g), which was prevented by PPP inhibitors. ODQ, a sGC inhibitor, potentiated HPV. These results suggest that while PPP-derived NADPH may play a significant role in HPV, it may also moderate the magnitude of HPV through activation of the NO-sGC-cGMP vasodilation pathway. q 2005 Elsevier Ltd. All rights reserved. Keywords: 6-Aminonicotinamide; Epiandrosterone; Nitric Oxide; Guanylate Cyclase

Pulmonary arteries (PA) constrict in response to hypoxia, low partial oxygen tension (Po2), to help maintain a balance between perfusion and ventilation ratio in the lungs. The cellular and molecular mechanisms involved in this phenomenon, hypoxic pulmonary vasoconstriction (HPV), have been extensively investigated, but the exact mechanisms responsible for the response are not yet well understood. In 1986, Archer and Weir proposed that mitochondria in pulmonary artery smooth muscle cells (PASMC) were the primary PO2 sensor. Subsequently, these authors suggested that hypoxic inhibition of free radical production and changes in the ratios of cytosolic reducing co-factors, such as NADPH/NADPC, inactivate voltage-gated KC (Kv) channels * Corresponding author Address. Rm # 604, Department of Physiology, Basic Sciences Building, New York Medical College, Valhalla, NY 10595, USA. Tel: C1 914 594 4103/4094; fax: C1 914 594 4826. E-mail address: [email protected] (S.A. Gupte). 1 SAG is currently at Department of Physiology, New York Medical College, Valhalla, NY U.S.A.; and MO is at the CVP Research Laboratory, University of Colorado Health Sciences Center, Denver Co, U.S.A.

1094-5539/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2005.08.002

in PASMC, resulting in membrane depolarization, Ca2C influx, and vasoconstriction [1,27]. Although it now appears that hypoxic inhibition of the Kv channel may contribute to the initiation of HPV, the precise sequence of events that leads to the blockade of KC currents is still unclear. It also remains controversial whether reactive oxygen species production is decreased or increased during hypoxia [1,16,25–28]. Recently, we have demonstrated that inhibition of the pentose phosphate pathway (PPP) by 6-aminonicotinamide (6-AN) increases Kv currents in PASMC [5], epiandrosterone (EPI) attenuates L-type Ca2C channels in cardiac myocytes, and 6-AN & EPI reduce Ca2C release and influx thus eliciting dilatation of coronary artery [4,8]. Furthermore, studies indicate that NAD(P)H, NAD(PC) and glutathione regulate rabbit PASMC voltage-gated and calcium-sensitive KC channels [12,19,20]. These channels are opened by the oxidizing agents and are inhibited by the reducing co-factors, respectively, either by directly binding to the channel protein or by regulating the redox state of residue cysteine residue on the protein [12,27]. These studies suggest that NADPH redox regulates ion channel activity and Ca2C release mechanisms in vascular smooth muscle cells.

304

S.A. Gupte et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 303–309

The PPP generates most of the major reducing co-factor NADPH and co-ordinates multiple redox reactions in cells. We have recently demonstrated that PPP inhibitors reduce lung tissue NADPH production under normoxic conditions and attenuate HPV [5]. Although it seems likely that changes in the NADPH/NADPC ratio and redox status may be important in HPV [28], the role of PPP-derived NADPH has not been well investigated. Therefore, one aim of this study was to test if PPP-derived NADPH production is increased during hypoxia and plays a role in HPV. In addition, it is well established that NADPH is an essential co-factor for nitric oxide (NO) synthase [13]. Furthermore a NADPH-dependent reductase also appears to be required to prevent the oxidation of iron in the heme group of soluble guanylate cyclase (sGC) and the inhibition of sGC activity by oxidizing agents and oxygen in bovine PA and coronary artery [7,9]. The NO-sGC pathway is regarded as an important modulator of HPV [10]. Activity of this pathway could be impaired under hypoxic conditions due to a decrease of NO synthesis resulting from suppression of oxygen-dependent NO synthase activity [10,13]. Hence, a second objective of this study was to investigate the roles of PPP-derived NADPH on the NO level and sGC activity during hypoxia. 1. Material and methods The Institutional Animal Use Committee of Juntendo University School of Medicine (Tokyo, Japan) approved all protocols and surgical procedures, which were in accordance with National Institutes of Health and American Physiological Society guidelines. 6-AN, EPI, angiotensin II (A-II), and other salts were purchased from Sigma (St. Louis, MO). The stock solutions of steroids were made either in ethanol (Sigma) or dimethyl sulfoxide (DMSO; Sigma), and final 1:1000 dilutions in buffered physiological salt solution were used in the study. 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Cayman Chemical, MI, USA) was dissolved in DMSO. 1.1. Animals Experiments were performed with adult male SpragueDawley (SD) rats (250–350 g). The rats were housed at the ambient barometric pressure. All rats were exposed to a 12:12h light-dark cycle and allowed free access to standard rat food and water. 1.2. Determination of pulmonary arterial pressure of isolated perfused rat lungs Isolated lungs were prepared as previously described [17] with minor modifications. The lungs were isolated from rats after intraperitoneal administration of 30 mg of pentobarbital sodium and intracardiac injection of 100 U of heparin. Isolated lungs were ventilated with a humid mixture of 21% O2–5% CO2–74% N2 at 60 breaths/min, with an inspiratory pressure of

9-cm H2O and an end-expiratory pressure of 2.5 cm H2O. They were perfused through a main pulmonary arterial cannula with a peristaltic pump at a constant flow of 0.04 ml/g body wt/min. The perfusate was a physiological salt solution (PSS) containing (in mM) 116.3 NaCl, 5.4 KCl, 0.83 MgSO4, 19.0 NaHCO3, 1.04 NaH2PO4, 1.8 CaCl2$2H2O, and 5.5 D-glucose (Earle’s balanced salt solution; Sigma). Ficoll (4 g/100 ml, type 70; Pharmacia, Uppsala, Sweden) was included as a colloid and meclofenamate (3.1 mM) was added to inhibit cyclooxygenase and production of vasodilator prostaglandins. After blood was flushed out of the lungs with 20 ml of the PSS, the lungs were perfused with a re-circulated volume of 30 ml. Effluent perfusate drained from a left ventricular cannula into a perfusate reservoir. Lung and perfusate temperatures were maintained at 37 8C, and the pH of the perfusate was kept between 7.3 and 7.4. The pulmonary perfusion pressure was measured continuously with a transducer (Nihon Kohden; RMP-6004, Tokyo, Japan) and a pen recorder, and the lungs were equilibrated for 20 min before vascular responses were elicited. After equilibration, the lungs were challenged four times with alternating arterial injection of 0.05 mg A-II and 5-min periods of hypoxic ventilation (0% O2–5% CO2–95% N2). 6-AN, EPI, or their vehicle (ethanolCDMSO) was added to perfusate 15 min prior to the fourth injection of A-II. The maximal increase in perfusion pressure over baseline in response to a given stimulus was measured as the pressor response. 1.3. Determination of changes in force of isolated rat PA PA (lobar branch) was prepared as previously described [5] with minor modifications. The rings were mounted on wire hooks attached to force displacement transducers (TB 611T; Nihon Kohden, Tokyo, Japan) for measurement of changes in the isometric force. Resting passive force was adjusted to a previously determined optimum (determined by maximum response to 80 mM KCl: 750 mg for PA), and vessels were equilibrated for 1 h in muscle baths containing Earle’s balanced salt solution, gassed with 21% O2/5% CO2/74% N2. In the endothelium-intact phenylephrine pre-constricted PA rings, acetylcholine elicited O80% relaxation. Subsequently, PA was contracted with U46619 (10–50 nM) to study hypoxic vasoconstriction. After arteries reached a stable steady state contraction with U46619, they were exposed to 5 min hypoxia and then re-oxygenated for 5–10 min. All values for vasoconstriction and vasodilation are expressed as percentage change of the precontracted force. 1.4. Determination of NADPH levels in isolated lungs The levels of NAD(P)H in isolated lungs were determined by HPLC after slight modification of previously published methods [7]. Briefly, isolated, normoxia and hypoxia-ventilated lungs were treated with 500 mM 6-AN, 300 mM EPI, or vehicle for 30 min and then freeze-clamped in liquid nitrogen. The frozen tissues were homogenized in an extraction medium consisting of 2 ml of 0.02 N NaOH containing 0.5 mM

S.A. Gupte et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 303–309

The levels of NOx in isolated lungs were determined by modified Griess method using a kit (catalogue no.1756281) from Boehringer Mannheim (Mannheim, Germany). Briefly, isolated normoxia-ventilated lungs were treated with 500 mM 6-AN, 300 mM EPI, 50 mM ODQ or vehicle and hypoxiaventilated lungs were freeze-clamped in liquid nitrogen and collected for estimating NOx levels. The frozen tissues were homogenized in 1 ml 50 mM Tris–HCl pH 7.4 after centrifuging the samples at 14,000 rpm at 4 8C the supernatant was used to estimate NOx by the procedure provided in the kit. 1.6. Determination of sGC activity in isolated lungs The activity of sGC in isolated lungs was determined by previously published method [6]. Briefly, isolated, normoxiaventilated lungs treated with vehicle and then freeze-clamped in liquid nitrogen. Hypoxia-ventilated lungs were treated with 500 mM 6-AN, 300 mM EPI, 50 mM ODQ were also collected for estimating sGC activity. The frozen tissues were homogenized in of 1 ml 50 mM Tris–HCl pH 7.4. Reaction mixture (0.2 ml final volume) contained 50 mM Tris–HCl pH 7.4, 0.1 mM GTP, 2 mM MgCl2, 0.3 mM of the phosphodiesterase inhibitor IBMX, a GTP-regenerating system consisting of 10 mM phosphocreatine and 150 U/ml creatine phosphokinase, and 0.1 ml of homogenate. Assays of sGC activity were initiated by the addition of homogenate protein. Incubations were conducted for 10 min at 37 8C, and they were terminated by the addition of 0.1 ml of preheated 12 mM EDTA. This was followed by boiling the assay mixtures for 10–15 min. Each tube was centrifuged at 15,000 rpm, and the supernatant, which was subsequently used to estimate cGMP by enzyme immunoassay. The 10-min incubation for the assay of sGC activity was chosen to optimize the detection of cGMP

1.7. Statistical analysis Values are meansGSEM and the sample size, n, refers to number of rats. Comparisons between groups were made with Student’s t-test or analysis of variance (ANOVA) with Scheffe´’s post-hoc test for multiple comparisons. Differences were considered significant at P!0.05. 2. Results NADPH levels in lungs were estimated by HPLC. As demonstrated in Fig. 1A, NADPH levels were markedly A

500

Rat Lung NADPH (nmol/g)

1.5. Determination of NOx levels in isolated lungs

under the wide variety of conditions examined. After ether extraction cGMP was estimated by the EIA kit (catalogue no.581021) purchased from Cayman Chemical Co. (Ann Arbor, MI, USA).

400

P<0.0001

300

200

100

0

Baseline

B

Hypoxic Pressor Response (mmHg)

cysteine at 0 8C. Acidic extracts were prepared by homogenizing the tissues in hot 0.1 N HCl. The extracts were then heated at 60 8C for 10 min and neutralized with 0.2–0.4 ml of 0.25 M glycylglycine buffer, pH 7.6. The neutralized extracts were centrifuged at 10,000 g for 10 min, the supernatants were passed through 0.45 mm Millipore filters, and the filtered solutions were used for measurement of NAD(P)H by HPLC. NAD(P)H was eluted on a reverse-phase HPLC column (4.6! 250 mm; Bondapak C18, Shiseido) at 40 8C by the CMA HPLC system with slight modifications of previously reported [7] 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 absorbance was monitored at 260 nm (Ex). NADPH standards were used to calibrate the HPLC. Internal standards containing 2 nM of NADPC/NADPH were used to verify the quantitative recovery of the extraction procedure and HPLC retention time in the presence and absence of tissue samples.

305

Hypoxia

8

6

4

* 2

* 0

VEH

EPI

6AN

Fig. 1. Panel A: Estimation of NADPH in rat lungs during normoxia (Baseline: nZ6) and hypoxia (nZ6). Panel B: Hypoxia increased pulmonary artery perfusion pressure in isolated lungs (Hypoxia) from baseline (nZ9), which was inhibited by EPI (300 mM; nZ3) and 6-AN (500 mM; nZ3).

S.A. Gupte et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 303–309

A

2.0 *P<0.0241

1.5

NADPH/NADP +

higher (7.2-fold) in lungs during hypoxic ventilation than in those during normoxic ventilation (nZ6). Pretreatment of lungs with two structurally different inhibitors of glucose-6phosphate dehydrogenase (a rate limiting enzyme of PPP), EPI (300 mM; nZ5) and 6-AN (500 mM; nZ4), blocked the elevation of NADPH levels induced by hypoxia. NADPH levels were 22.8G19.2 and 59.3G18.5 nmol/g in hypoxiaventilated lungs by treatment with PPP inhibitors as compared to normoxia-ventilated lungs. Additionally, since it is difficult to determine G-6-PD activity under hypoxia due to technical limitations, we quantified glucose-6phosphate (G-6-P), a substrate for G-6-PD, levels in normoxic and hypoxic condition. The G-6-P levels increased during hypoxia (1312.0G377.1 nmol/g) by 3.7G0.9 fold from normoxia (420.2G137.1 nmol/g). Consistent with our previous studies [5,17], acute hypoxia increased the perfusion pressure in vehicle-pretreated lungs (Fig. 1B). This hypoxic pressor response was attenuated by EPI and 6-AN (untreated: 6.5G0.2 mmHg; EPI: 1.6G0.2 mmHg; and 6-AN treated: 2.3G0.4 mmHg; P!0.05). EPI or 6-AN did not change the baseline perfusion pressure (in mmHg; Control: 6.2G0.5; EPI: 6.2G0.5; 6-AN: 6.0G0.6), but did reduce the pressor responses to A-II (from 2.9G0.4 to 1.3G1.3 and 3.0G0.5 to 2.3G0.3 mmHg by EPI and 6-AN, respectively, P!0.05), although the inhibition was less compared to that of HPV. To examine whether hypoxia elicited changes in NADPH redox in PA, we incubated U46619 treated lobar PA branches in normoxia (time-match control) or hypoxia (5 min) and determined NADPH and NADPC levels. The ratio of NADPH/NADPC was increased by hypoxia and decreased after re-oxygenation (Fig. 2A). In control PA, U46619 (10 nM) elicited 52.6G4.8% contraction as compared to 80 mM KCl. In EPI and 6-AN treated PA, U46619 (30–50 nM), which was used to match the size of the contraction in the absence of the PPP inhibitors, caused 44.7G3.8 and 48.3G6.7% of 80 mM KCl-induced contraction, respectively. In addition, PA precontracted with U46619 also contracted by hypoxia and relaxed upon reoxygenation (Fig. 2B). EPI and 6-AN, suppressed the elevation of NADPH/NADPC ratio induced by hypoxia (Fig. 3A). Contraction of PA induced by hypoxia was also abolished by EPI and 6-AN (Fig. 3B). Lung tissue NOx levels were decreased during hypoxia by approximately 50% (Fig. 4). During normoxic ventilation, EPI and 6-AN significantly reduced lung tissue NOx levels, while ODQ (50 mM), a sGC inhibitor, did not. NOx was also inhibited under hypoxia by 25–30% by PPP inhibitors. Soluble GC activity was estimated in the lungs exposed to normoxia or hypoxia. As shown in Fig. 5, hypoxia increased lung tissue sGC activity. This hypoxia-induced increase in sGC activity was prevented by PPP inhibitors, EPI and 6-AN (Fig. 5). ODQ pretreatment also blocked the increased sGC activity during hypoxia, but in contrast to effects of EPI and 6AN, which inhibited HPV (Fig. 3), inhibition of sGC with ODQ enhanced HPV (Fig. 6).

1.0

0.5

0.0 Baseline

Hypoxia

Re-Ox

B 200

150

Contraction (% 30 K+)

306

P<0.0001

100

50

0 Baseline

Hypoxia

Re-Ox

Fig. 2. Panel A: Estimated ratio of NADPH/NADP in PA treated with U46619 during normoxia (Baseline; nZ6), hypoxia (nZ6), and reoxygenation (ReOx; nZ6). Panel B: Pulmonary artery pretreated with U46619 in normoxia (Baseline) further contracted by hypoxia and returned to baseline after re-oxygenation (nZ6). C

3. Discussion The major findings of this study in isolated perfused rat lungs and PA were, (1) hypoxia caused increases in lung tissue NADPH levels, PA NADPH/NADPC ratio, and lung and PA vascular tone, which were inhibited by PPP inhibitors; (2) elevated NADPH/NADPC ratio and vascular tone returned to baseline after re-oxygenation; (3) hypoxia reduced lung tissue NOx levels but, in contrast, it increased sGC activity; (4) PPP inhibitors reduced NOx levels in normoxia-ventilated lungs; (5) the hypoxia-induced increase in sGC activity was prevented by PPP inhibitors; (6) ODQ reduced sGC activity and potentiated HPV. These results suggest that NADPH derived

S.A. Gupte et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 303–309

200

A 2.0

1.5

150

NOx (microM/g)

NADPH/NADP +

307

1.0

* 100

*

*

0.5 50 0.0 Baseline

EPI

6-AN 0

Hypoxia

Baseline

Contraction (% 30 K+)

B 200

VEH +HYPO

EPI

6AN

ODQ

Fig. 4. NOx levels in lungs treated with vehicle (Baseline; nZ5), EPI (300 mM, nZ5), 6-AN (500 mM, nZ5) or ODQ (10 mM; nZ4) during normoxic ventilation and in lungs treated with vehicle during hypoxic ventilation (HYPO; 0% O2, nZ4). * P!0.05 vs. O2.

150

PA exposed to hypoxia [24], which may have been due to the methods they employed in extracting pyridine nucleotides for HPLC measurements [24]. We further observed that the marked increases in NADPH and NADPH/NADPC ratio were completely blocked by EPI or 6-AN, two structurally distinct PPP inhibitors [4,5], suggesting that the PPP is the source of the increased NADPH production in lungs and PA exposed to hypoxia. The mechanism by which hypoxia

100

50

0 Baseline

EPI

70

6-AN

*

Hypoxia

from PPP may have multiple effects on the signaling pathways that regulate PA vasomotor tone and HPV. We found that exposure of isolated rat lungs and PA to acute hypoxia markedly increased lung tissue NADPH levels and PA NADPH/NADPC ratio, respectively. The latter decreased almost to the pre-hypoxic levels upon reoxygenation. Consistent with these findings are reports by Leach et al. [11] and by White et al. [30]. Leach et al. showed that hypoxia caused a rapid, sustained increase in NAD(P)H fluorescence which returned to baseline immediately after re-oxygenation in isolated rat PA, although these authors found no relationship between increased levels of NAD(P)H fluorescence and magnitude of HPV. White et al. reported that isolated perfused rat lungs pre-exposed to hypoxia were resistant to oxidative stress-induced lung injury due to increased production of NADPH and reduced glutathione (GSH). In contrast, Shigemori et al. have reported that NADPH redox is unchanged in

60

sGC activity (pmol/min/g)

Fig. 3. Panel A: Ratio of NADPH/NADPC in PA with U46619 and EPI and 6AN during normoxia and hypoxia (nZ5). Panel B: Contraction of PA by hypoxia was almost completely blocked by EPI and 6-AN pretreatment (nZ5).

50

40

30

20

10

0

Baseline

VEH

EPI

6AN

ODQ

Hypoxia Fig. 5. sGC activity in lungs treated with vehicle during normoxic ventilation (Baseline; nZ5), and lungs treated with vehicle (VEH; nZ4), EPI (300 mM: nZ4), 6-AN (500 mM: nZ5) or ODQ (nZ4) during hypoxic ventilation. * P! 0.05 vs. O2.

308

S.A. Gupte et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 303–309

Hypoxic Pressor Response (mmHg)

25

*

20

15

10

5

0

VEH

ODQ

Fig. 6. The NO-specific sGC inhibitor, ODQ (10 mM; nZ5), significantly potentiated the pressor response to acute hypoxia in isolated perfused lungs. * P!0.05 vs. VEH (nZ5).

increases PPP activity and NADPH production is uncertain. However, oxidative stress activates PPP [30], and recent evidence suggests that hypoxia paradoxically increases production of superoxide and hydrogen peroxide [25,26]. Thus, one possibility is that increased oxidative stress during hypoxia stimulates PPP and increases NADPH synthesis. Although still controversial [1–3,11,16,18,22,31], a likely mechanism by which acute hypoxia elicits pulmonary vasoconstriction is through inhibition of voltage-gated KC (Kv) channels, which leads to smooth muscle cell (SMC) membrane depolarization, increased Ca2C influx through L-type Ca2C channels, and contraction of SMC [3,11,18,22]. It is suggested that the cellular redox status regulates KC currents. Studies indicate that reducing agents such as dithiothreitol, GSH, and NAD(P)H inhibits KC current, while oxidizing agents such as diamide and oxidized glutathione (GSSG) have the opposite effect [12,19,20,27,28]. Recently, we have demonstrated that PPP inhibitors decrease NADPH, relax PA and inhibit HPV, plausibly by opening KV channels and increasing outward KC current [5]. Moreover, recent studies show that redox sensitive aldo–keto reductase on the b-subunit of Kv1.4 and Kv1.5 channels regulates outward KC currents and is inhibited by increased cytosolic NADPH levels [14,21]. Results of our study provide evidence that hypoxia increases lung tissue NADPH levels by activation of PPP. Thus, PPP-derived NADPH may play a role in mediating HPV via inhibition of Kv channels, although other mechanisms in the initiation and maintenance of HPV are likely to be involved. In fact, while pretreatment with PPP inhibitors resulted in complete inhibition of the hypoxia-elicited increased NADPH production and NADPH/NADPC ratio, it markedly reduced but did not completely block the HPV and hypoxia-induced PA contraction. As pretreatment of lungs and PA with the PPP

inhibitors reduced NADPH levels and baseline tone, which was partially corrected in PA but not in lungs, could be responsible for the partial inhibition of HPV and hypoxia-induced PA contraction. Activation of Kv channels and membrane hyperpolariztion after inhibition of NADPH production by 6AN or EPI could account for the blunting of the pulmonary vasoconstriction responses to A-II and U46619, which are partly dependent on voltage-gated Ca2C influx [15]. Interestingly, Gupte et al. preliminarily found that hypoxia had an opposite effect on PPP activity in the systemic circulation compared to the pulmonary circulation, i.e. hypoxia inhibited PPP activity and decreased NADPH/NADPC ratio, which caused relaxation of bovine coronary artery [4]. This may shed new light on the unsolved question of how hypoxia has opposite vasoactive effects on the pulmonary and systemic circulations. In addition to its role in regulation of Kv channel activity [13, 19], NADPH is also a co-factor for nitric oxide synthase (NOS) in endothelial cells [13], and for an unidentified NADPHhemoprotein reductase, which appears to reduce the iron in the heme group of sGC and increase basal as well as NO-dependent sGC activity in bovine PASMC [7]. Consistent with NADPH being a co-factor for NOS, inhibition of PPP reduced NOx levels in normoxic lungs, suggesting that PPP-derived NADPH maintains NOS activity during normoxia. We also found that lung NOx levels were significantly decreased under hypoxic conditions. This is consistent with reports that exhaled and perfusate NOx levels decrease immediately after switching ventilation from normoxia to hypoxia in isolated lungs [10,29]. Since NOS is an oxygen-dependent enzyme, a likely interpretation of our observation is that due to low Po2 lung NOS activity decreased despite an increase in NADPH levels during hypoxia. Although NO production may be decreased during hypoxia, the residual NO is important in suppressing HPV, since NOS inhibition potentiates HPV [10,23,29]. We also assessed the lung sGC activity, and found that hypoxia increased it by 2-fold. This increased sGC activity, and presumably increased production of cGMP, moderates HPV, since ODQ, a sGC inhibitor, potentiated HPV. EPI or 6-AN was as effective as ODQ in inhibiting the increased sGC activity during hypoxia, suggesting that activation of PPP by hypoxia may be responsible for the increased activity in sGC. Since, as stated earlier, NADPH plays a role as a co-factor for an unidentified NADPH-hemoprotein reductase, which can increase basal as well as NO-dependent sGC activity [7], our results can be interpreted that increased production of NADPH through PPP enhanced the sGC activity during hypoxia despite the decreased production of NO. It is also possible that some other unidentified activator of sGC is produced during hypoxia. In summary, we have demonstrated in isolated rat lungs and PA that hypoxia increases NADPH production and the NADPH/NADPC ratio, and that inhibitors of PPP markedly reduce both hypoxia-induced NADPH production and HPV. In addition, our results also suggest that PPP-derived NADPH sustains both lung tissue NOS and sGC activities. Based on these observations, we propose that PPP-derived NADPH may play multiple, complex roles in regulating the magnitude of

S.A. Gupte et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 303–309

HPV, i.e. whereas NADPH-mediated inhibition of Kv channels would promote HPV, this effect would be opposed by NADPHdependent NO production and activation of sGC. Acknowledgements Authors gratefully acknowledge Juntendo University School of Medicine, Tokyo, Japan for supporting SAG with grant-in-aid in 2000–2001 and Kyotristu International Foundation, Tokyo, Japan for giving SAG a fellowship from 2000–2002. A part of this work was presented at Experimental Biology 2005 Meeting, San Diego, CA, USA, April 2–6, 2005.

[14]

[15]

[16]

[17]

[18]

References [19] [1] Archer SL, Will JA, Weir EK. Redox status in the control of pulmonary vascular tone. Herz 1986;11:127–41. [2] Evans AM, Dipp M. Hypoxic pulmonary vasoconstriction: cyclic adenosine diphosphate-ribose, smooth muscle Ca(2C) stores and the endothelium. Respir Physiol Neurobiol 2002;132:3–15. [3] Franco-Obregon A, Lopez-Barneo J. Differential oxygen sensitivity of calcium channels in rabbit smooth muscle cells of conduit and resistance pulmonary arteries. J Physiol 1996;491(Pt 2):511–8. [4] Gupte SA, Arshad M, Viola S, Kaminski PM, Ungvari Z, Rabbani G, et al. Pentose phosphate pathway coordinates multiple redox-controlled relaxing mechanisms in bovine coronary arteries. Am J Physiol 2003. [5] Gupte SA, Li KX, Okada T, Sato K, Oka M. Inhibitors of pentose phosphate pathway cause vasodilation: involvement of voltage-gated potassium channels. J Pharmacol Exp Ther 2002;301:299–305. [6] Gupte SA, Rupawalla T, Mohazzab HKM, Wolin MS. Regulation of NOelicited pulmonary artery relaxation and guanylate cyclase activation by NADH oxidase and SOD. Am J Physiol 1999;276:H1535–H42. [7] Gupte SA, Rupawalla T, Phillibert Jr D, Wolin MS. NADPH and heme redox modulate pulmonary artery relaxation and guanylate cyclase activation by NO. Am J Physiol 1999;277:L1124–L32. [8] Gupte SA, Tateyama M, Okada T, Oka M, Ochi R. Epiandrosterone, a metabolite of testosterone precursor, blocks L-type calcium channels of ventricular myocytes and inhibits myocardial contractility. J Mol Cell Cardiol 2002;34:679–88. [9] Iesaki T, Gupte SA, Wolin MS. A flavoprotein mechanism appears to prevent an oxygen-dependent inhibition of cGMP-associated nitric oxideelicited relaxation of bovine coronary arteries. Circ Res 1999;85:1027–31. [10] Le Cras TD, McMurtry IF. Nitric oxide production in the hypoxic lung. Am J Physiol 2001;280:L575–L82. [11] Leach RM, Hill HM, Snetkov VA, Robertson TP, Ward JP. Divergent roles of glycolysis and the mitochondrial electron transport chain in hypoxic pulmonary vasoconstriction of the rat: identity of the hypoxic sensor. J Physiol 2001;536:211–24. [12] Lee S, Park M, So I, Earm YE. NADH and NAD modulates Ca(2C)activated KC channels in small pulmonary arterial smooth muscle cells of the rabbit. Pflugers Arch 1994;427:378–80. [13] Leopold JA, Cap A, Scribner AW, Stanton RC, Loscalzo J. Glucose-6phosphate dehydrogenase deficiency promotes endothelial oxidant stress

[20]

[21] [22]

[23]

[24]

[25]

[26]

[27] [28] [29]

[30]

[31]

309

and decreases endothelial nitric oxide bioavailability. Faseb J 2001;15: 1771–3. Liu SQ, Jin H, Zacarias A, Srivastava S, Bhatnagar A. Binding of pyridine nucleotide coenzymes to the beta-subunit of the voltage-sensitive KC channel. J Biol Chem 2001;276:11812–20. McMurtry IF, Davidson AB, Reeves JT, Grover RF. Inhibition of hypoxic pulmonary vasoconstriction by calcium antagonists in isolated rat lungs. Circ Res 1976;38:99–104. Michelakis ED, Hampl V, Nsair A, Wu X, Harry G, Haromy A, et al. Diversity in mitochondrial function explains differences in vascular oxygen sensing. Circ Res 2002;90:1307–15. Oka M, Hasunuma K, Webb SA, Stelzner TJ, Rodman DM, McMurtry IF. EDRF suppresses an unidentified vasoconstrictor mechanism in hypertensive rat lungs. Am J Physiol 1993;264:L587–L97. Olschewski A, Hong Z, Nelson DP, Weir EK. Graded response of KC current, membrane potential, and [Ca2C]i to hypoxia in pulmonary arterial smooth muscle. Am J Physiol 2002;283:L1143–L50. Park MK, Bae YM, Lee SH, Ho WK, Earm YE. Modulation of voltagedependent KC channel by redox potential in pulmonary and ear arterial smooth muscle cells of the rabbit. Pflugers Arch 1997;434:764–71. Park MK, Lee SH, Lee SJ, Ho WK, Earm YE. Different modulation of Caactivated K channels by the intracellular redox potential in pulmonary and ear arterial smooth muscle cells of the rabbit. Pflugers Arch 1995;430: 308–14. Peri R, Wible BA, Brown AM. Mutations in the Kv beta 2 binding site for NADPH and their effects on Kv1.4. J Biol Chem 2001;276:738–41. Robertson TP, Hague D, Aaronson PI, Ward JP. Voltage-independent calcium entry in hypoxic pulmonary vasoconstriction of intrapulmonary arteries of the rat. J Physiol 2000;525(Pt 3):669–80. Rodman DM, Yamaguchi T, Hasunuma K, O’Brien RF, McMurtry IF. Effects of hypoxia on endothelium-dependent relaxation of rat pulmonary artery. Am J Physiol 1990;258:L207–L14. Shigemori K, Ishizaki T, Matsukawa S, Sakai A, Nakai T, Miyabo S. Adenine nucleotides via activation of ATP-sensitive KC channels modulate hypoxic response in rat pulmonary artery. Am J Physiol 1996;270:L803–L9. Waypa GB, Chandel NS, Schumacker PT. Model for hypoxic pulmonary vasoconstriction involving mitochondrial oxygen sensing. Circ Res 2001; 88:1259–66. Waypa GB, Marks JD, Mack MM, Boriboun C, Mungai PT, Schumacker PT. Mitochondrial reactive oxygen species trigger calcium increases during hypoxia in pulmonary arterial myocytes. Circ Res 2002; 91:719–26. Weir EK, Archer SL. The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. Faseb J 1995;9:183–9. Weir EK, Hong Z, Porter VA, Reeve HL. Redox signaling in oxygen sensing by vessels. Respir Physiol Neurobiol 2002;132:121–30. Weissmann N, Winterhalder S, Nollen M, Voswinckel R, Quanz K, Ghofrani HA, et al. NO and reactive oxygen species are involved in biphasic hypoxic vasoconstriction of isolated rabbit lungs. Am J Physiol 2001;280:L638–L45. White CW, Jackson JH, McMurtry IF, Repine JE. Hypoxia increases glutathione redox cycle and protects rat lungs against oxidants. J Appl Physiol 1988;65:2607–16. Yuan JX. Oxygen-sensitive K(C) channel(s): where and what? Am J Physiol 2001;281:L1345–L9.