Activation of glucose-6-phosphate dehydrogenase promotes acute hypoxic pulmonary artery contraction

Abstract

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to a decrease in airway O(2) tension, but the underlying mechanism is incompletely understood. We studied the contribution of glucose-6-phosphate dehydrogenase (Glc-6-PD), an important regulator of NADPH redox and production of reactive oxygen species, to the development of HPV. We found that hypoxia (95% N(2), 5% CO(2)) increased contraction of bovine pulmonary artery (PA) precontracted with KCl or serotonin. Depletion of extracellular glucose reduced NADPH, NADH, and HPV, substantiating the idea that glucose metabolism and Glc-6-PD play roles in the response of PA to hypoxia. Our data also show that inhibition of glycolysis and mitochondrial respiration (indicated by an increase in NAD(+) and decrease in the ATP-to-ADP ratio) by hypoxia, or by inhibitors of pyruvate dehydrogenase or electron transport chain complexes I or III, increased generation of reactive oxygen species, which in turn activated Glc-6-PD. Inhibition of Glc-6-PD decreased Ca(2+) sensitivity to the myofilaments and diminished Ca(2+)-independent and -dependent myosin light chain phosphorylation otherwise increased by hypoxia. Silencing Glc-6-PD expression in PA using a targeted small interfering RNA abolished HPV and decreased extracellular Ca(2+)-dependent PA contraction increased by hypoxia. Similarly, Glc-6-PD expression and activity were significantly reduced in lungs from Glc-6-PD(mut(-/-)) mice, and there was a corresponding reduction in HPV. Finally, regression analysis relating Glc-6-PD activity and the NADPH-to-NADP(+) ratio to the HPV response clearly indicated a positive linear relationship between Glc-6-PD activity and HPV. Based on these findings, we propose that Glc-6-PD and NADPH redox are crucially involved in the mechanism of HPV and, in turn, may play a key role in increasing pulmonary arterial pressure, which is involved in the development of pulmonary hypertension.

FIGURE 1.

Bovine PA is contracted upon reduction of pO₂. A, representative trace illustrating the typical response of adult bovine PA to hypoxia (pO₂ = 10–20 torr). PA precontracted with KCl (30 mmol/liter; n = 20) was exposed to hypoxia (20 min) followed by reoxygenation (ReO) and then washout (WO) with normal buffer. *, p < 0.05. B and C, incubating PA in glucose-free Krebs buffer containing KCl (30 mmol/liter; n = 6) significantly reduced NAD(P)H levels (B) and suppressed HPV (C).

FIGURE 2.

NADPH redox status is altered in PA by hypoxia. A, time course of hypoxia-induced PA contraction (n = 5). B, estimated NADP⁺ and NADPH levels from PA pre-contracted with KCl (30 mmol/liter; n = 5) at the indicated time points during the course of HPV. A reduction in NADP⁺ levels from base line coincided with initiation of the HPV response, and a corresponding increase in NADPH was sustained for up to 20 min. C and D, in PA exposed to hypoxia for 10-min Glc-6-PD (G6PD) activity assay (C, n = 5), Michaelis-Menten graph (D, average of three experiments) shows Glc-6-PD activity was increased in PA exposed to hypoxia for 10 min. *, p < 0.05.

FIGURE 3.

Effect of hypoxia on the PA metabolic status. A and B, NAD⁺, ADP, and ATP estimated in PA pre-contracted with KCl (30 mmol/liter; n = 5) and exposed to hypoxia for 5 min. Hypoxia increased NAD⁺ levels (A) and decreased ATP-to-ADP ratios (B), as compared with normoxia. C, NAD(P)H levels in homogenates of PA exposed to arsenic (10 μmol/liter; n = 5), rotenone (10 μmol/liter; n = 5), or antimycin (10 μmol/liter; n = 5). *, p < 0.05. D, effect of antimycin (10 μmol/liter; n = 5) on Glc-6-PD (G6PD) activity in PA.

FIGURE 4.

ROS generation increases NADPH levels in PA. A and B, ROS production in PA under normoxic and hypoxic conditions was determined using DHE and MitoSOX probes. PA rings were loaded with either DHE (5 μmol/liter; n = 5) or MitoSOX (5 μmol/liter; n = 5) and contracted with KCl (30 mmol/liter) for 10 min in 21% O₂ (Norm+30K), 0% O₂ (Hypo+30K), or Tempol (10 μmol/liter) + 0% O₂ (Hypo+30K), after which the rings were quickly embedded in OCT media. Untreated PA rings served as time-matched controls (Control). B immunofluorescence (left panels) and bright field (right panels) of control are shown. C, ebselen (Eb) (100 μmol/liter; n = 8) treatment reduced NADPH levels in normoxic (Nor) and hypoxic (Hypo) PA rings pre-treated with KCl (30 mmol/liter). *, p < 0.05 versus normoxia; #, p < 0.05 versus hypoxia.

FIGURE 5.

Effect of reduced PA Glc-6-PD expression on HPV. A, representative Western blot showing knockdown of Glc-6-PD (G6PD) expression by siRNA targeting Glc-6-PD but not by scrambled (NT) siRNA. Expression of α-actin, a cytoskeletal protein, was unaffected by Glc-6-PD siRNA. B, traces showing a typical response to hypoxia by PA transfected with NT (blue line) and Glc-6-PD (yellow line) siRNA. ReO, reoxygenation; WO, washout. C, group data showing the effects of NT and Glc-6-PD siRNA on HPV (n = 10). *, p < 0.05. D, Glc-6-PD activity was significantly lower in lungs from Glc-6-PD^mut(−/−) mice than Glc-6-PD^mut(+/−) or Glc-6-PD^mut(+/+) mice (n = 6). E, hypoxia-induced PA contraction was significantly weaker in Glc-6-PD^mut(−/−) than Glc-6-PD^mut(+/−) mice (n = 6).

FIGURE 6.

Hypoxia augments Ca²⁺-independent and -dependent contraction of PA. A, hypoxia (Hypo) augmented PA contractions evoked by KCl (30 mmol/liter; n = 25) in Ca²⁺-free solution and after addition of Ca²⁺ to the tissue bath. Nor, normoxia. B, hypoxia enhanced contraction evoked by 5-HT (10 μmol/liter; n = 20) in Ca²⁺-free solution and after addition of Ca²⁺ to the tissue bath. C, addition of Ca²⁺ to the tissue bath 20 min after the initial 5-HT-evoked PA contraction in Ca²⁺-free buffer elicited a robust contraction in normoxia and hypoxia. D, representative concentration-response curves showing Ca²⁺-induced contraction of permeabilized PA. 6-AN (5 mmol/liter; green trace) significantly reduced the Ca²⁺ sensitivity of the myofilaments, which was otherwise increased by hypoxia (blue trace). The red trace is a normoxic control. E, hypoxia reduced the Ca²⁺ concentrations required to contract skinned PA (n = 5) by 4-fold, as compared with normoxia. F, time course of PA contractions (n = 5) evoked by pCa 4 were accelerated by hypoxia. G, 6-AN completely blocked contraction of skinned PA (n = 5) by decreasing the sensitization to Ca²⁺, which was otherwise enhanced by hypoxia.

FIGURE 7.

Hypoxia augments Ca²⁺-independent and -dependent phosphorylation of MLC PA. A, representative Western blot of phospho-CPI-17 (top blot) and phospho-MLC (2nd blot) is shown. Levels of phospho-CPI-17 and phospho-MLC normalized by α-actin were increased (80 ± 16 and 55 ± 6%, respectively; n = 5) by hypoxia in a Ca²⁺-independent manner (1st lane, normoxia and hypoxia) but were further enhanced (40 ± 2% and 20 ± 1%, respectively) by addition of Ca²⁺ (0.375 mmol/liter; 2nd lane, normoxia and hypoxia). 6-AN (5 mmol/liter; n = 5) blocked the hypoxia-induced increase in phospho-MLC in the presence of Ca²⁺ by 50–60% but did not prevent the increase in phospho-CPI-17 (3rd and 4th lanes, normoxia and hypoxia). B, representative Western blot of phospho-CPI-17 (top blot) and phospho-MLC (2nd blot) is shown. Conventions are the same as in A (n = 5).

FIGURE 8.

Hypoxia-induced, Ca²⁺-independent and -dependent PA contraction is dependent on Glc-6-PD. A, KCl (30 mmol/liter)-induced contraction of PA treated with scrambled siRNA (NT; n = 8) or Glc-6-PD siRNA (Glc-6-PD⁻; n = 8) after addition of Ca²⁺ to the tissue bath under normoxic (Nor) and hypoxic (Hypo) conditions. Glc-6-PD (G6PD) knockdown completely abolished the hypoxia-induced augmentation of contractions at all concentrations of Ca²⁺. B, 5-HT-induced contraction of PA treated with NT (n = 6) or Glc-6-PD (n = 6) siRNA in the absence of extracellular Ca²⁺ was significantly increased by hypoxia. C, knocking down Glc-6-PD blocked hypoxia-induced augmentation of PA contractions elicited by addition of extracellular Ca²⁺. *, p < 0.05.

FIGURE 9.

Hypoxia-induced, Ca²⁺-independent and -dependent phosphorylation of MLC in PA is dependent on Glc-6-PD. A, representative Western blot showing that knocking down Glc-6-PD (G6PD) significantly reduced levels (normalized by α-actin) of KCl (30 mmol/liter)-induced phospho-CPI-17 (70 ± 12% of NT) and phospho-MLC (48 ± 6% of NT) in PA exposed to hypoxia. B, representative Western blot showing that knocking down Glc-6-PD significantly reduced (56 ± 3% of NT) levels of 5-HT (10 μmol/liter)-induced phospho-MLC in PAs exposed to hypoxia. The experiment was repeated four times with similar results.

FIGURE 10.

Glc-6-PD activity is positively correlated with HPV. A and B, regression analysis relating Glc-6-PD (G6PD) activity (A) and NADPH-to-NADP⁺ ratios (B) to peak contractions elicited by exposure to hypoxia for 10 min.

FIGURE 11.

Schematic diagram illustrating the potential role played by Glc-6-PD during the development of HPV. Hypoxia increases glucose uptake (via glucose transporter (GLUT)) and inhibits glycolysis and mitochondrial ETC. As a result, glucose 6-phosphate (G-6-P) is shunted into the PPP, due to simultaneous activation of glucose-6-phosphate dehydrogenase (G6PD) and inhibition of e⁻ flow-through the mitochondrial ETC complex III, in the PASMC. Increase in Glc-6-PD activity elevates intracellular NADPH, which is a reducing co-factor for numerous enzymes, including NADPH oxidases (NOX). NADPH enhances reactive oxygen species (ROS) generation. Elevated hydrogen peroxide (H₂O₂) stimulates Glc-6-PD in a feed-forward reaction. Superoxide () anion and/or H₂O₂ activates RhoA-ROCK and inhibits myosin light chain phosphate (MYPT1), enhances intracellular [Ca²⁺]_i via influx and release, and increases myosin light chain phosphorylation (MLC-P). Increase in MLC phosphorylation causes contraction of PASMC and promotes hypoxic pulmonary vasoconstriction.