Nitric oxide augments signaling for contraction in hypoxic pulmonary arterial smooth muscle-Implications for hypoxic pulmonary hypertension

Abstract

Introduction: Hypoxic persistent pulmonary hypertension in the newborn (PPHN) is usually treated with oxygen and inhaled nitric oxide (NO), both pulmonary arterial relaxants. But treatment failure with NO occurs in 25% of cases. We previously demonstrated that 72 h exposure to hypoxia, modeling PPHN, sensitized pulmonary artery smooth muscle cells (PASMC) to the contractile agonist thromboxane and inhibited relaxant adenylyl cyclase (AC) activity. Methods: In this study, we examined the effects of sodium nitroprusside (SNP), as NO donor, on the thromboxane-mediated contraction and NO-independent relaxation pathways and on reactive oxygen species (ROS) accumulation in PASMC. In addition, we examined the effect of the peroxynitrite scavenger 5,10,15,20-Tetrakis (4-sulfonatophenyl)porphyrinato Iron (III) (FeTPPS) on these processes. Results: Exposure of PASMC to 72 h hypoxia increased total intracellular ROS compared to normoxic control cells and this was mitigated by treatment of cells with either SNP or FeTPPS. Total protein nitrosylation was increased in hypoxic PASMC compared to controls. Both normoxic and hypoxic cells treated with SNP exhibited increased total protein nitrosylation and intracellular nitrite; this was reduced by treatment with FeTPPS. While cell viability and mitochondrial number were unchanged by hypoxia, mitochondrial activity was decreased compared to controls; addition of FeTPPS did not alter this. Basal and maximal mitochondrial metabolism and ATP turnover were reduced in hypoxic PASMC compared to controls. Hypoxic PASMC had higher basal Ca2+, and a heightened peak Ca2+ response to thromboxane challenge compared to controls. Addition of SNP further elevated the peak Ca2+ response, while addition of FeTPPS brought peak Ca2+ response down to control levels. AC mediated relaxation was impaired in hypoxic PASMC compared to controls but was normalized following treatment with FeTPPS. Addition of SNP inhibited adenylyl cyclase activity in both normoxic and hypoxic PASMC. Moreover, addition of the Ca2+ chelator BAPTA improved AC activity, but the effect was minimal. Discussion: We conclude that NO independently augments contraction and inhibits relaxation pathways in hypoxic PASMC, in part by a mechanism involving nitrogen radical formation and protein nitrosylation. These observations may partially explain impaired effectiveness of NO when treating hypoxic pulmonary hypertension.

Keywords: adenylyl cyclase; calcium; hypoxia; nitric oxide; persistent pulmonary hypertension of the newborn; smooth muscle; thromboxane.

FIGURE 1

Effects of hypoxia or nitrosylating stress on reactive oxygen and nitrogen species. PASMC were exposed to 72 h of normoxia (N; 21%O2) or hypoxia (H; 10%O2) with or without daily addition of 1uM FeTPPS (F) or 1uM SNP (S). Reactive O2 species (A); DCF; N = 3, n = 10), superoxide (B); DHE; N = 4, n = 8–16), total nitrosylation (C); biotin switch assay; N = 3, n = 6–8). Conditioned media was collected and analyzed for nitrate concentration (D); N = 4, n = 8). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to N. ••p < 0.01, •••p < 0.001 compared to H. # 0 < 0.05, ##p < 0.01, ####p < 0.0001 compared between FeTPPS and SNP treatments within similar O2 environments.

FIGURE 2

Effect of hypoxia on mitochondria abundance and cellular respiration. PASMC were exposed to 72 h s of normoxia (N; 21%O2) or hypoxia (H; 10%O2) and fixed for EM evaluation of mitochondria (red arrows) density by EM (A), (B); N = 3, n = 22–31). Oxygen consumption was evaluated using a Seahorse analyzer (N = 3, n = 20). Non-mitochondrial (C), basal mitochondrial (D) maximal mitochondrial (E) respiration were compared, as well as ATP turnover (F) and proton leak (G). Cell viability (H); calcein assay; N = 5, 6, n = 20–25) and mitochondrial activity assay (I); MTT; N = 3, n = 18) were also measured with and without daily addition of 1uM FeTPPS (F) or 1uM SNP (S). Apoptosis was compared by comparison of cleaved caspase-3 (J); N = 5, n = 5; (K); representative blot), *p < 0.05, **p < 0.01.

FIGURE 3

Effect of hypoxia or nitrosylating stress on PASMC contractile mediators. Basal Ca2+ (A); N = 7–10, n = 54–182) and peak Ca2+ mobilization to 1uM U46619 (B); N = 7–19, n = 53–213) in fura-2AM loaded PASMC after exposure to 72 h s of normoxia (N; 21%O2) or hypoxia (H; 10%O2) with or without daily addition of 1uM FeTPPS F) or 1uM SNP (S) were compared (C); representative traces). The effect of daily addition of both 1uM FeTPPS and 1uM SNP (F + S) during the same 72h period on U46619-induced Ca2+ mobilization was also studied (D); N = 3, n = 14–24). Abundance of contractile Ca2+- cascade enzyme, phospholipase C β (PLCβ; (E(i)), N = 4; (F(i)), representative blot), contractile protein filaments, smooth muscle α-actin (sm-α-actin; E(ii), N = 4; F(ii) representative blot) and desmin (E(iii)), N = 4; (F(ii)), representative blot) were compared and normalized to β-actin (F(iii)), representative blots shown; N = 4). Similarly, basal (G); N = 3, 4, n = 14–16 and agonist-stimulated (H); N = 3, n = 8) mitochondrial membrane potential was measured in JC-1 loaded PASMC. *p < 0.05, **p < 0.01 compared to N. •p < 0.05, ••p < 0.01 compared to H. ####p < 0.0001 compared between FeTPPS and SNP treatments within similar O2 environments.

FIGURE 4

Effect of hypoxia or nitrosylating stress on PASMC relaxation. Adenylyl cyclase (AC) activity was measured in lysates from PASMC exposed to 72 h of normoxia (N; 21%O2) or hypoxia (H; 10%O2) with or without daily addition of 1uM FeTPPS (F) or 1uM SNP (S) (A); N = 5–8, n = 5–14). Intracellular Ca2+ was normalized with chelator BAPTA (2uM, 24 h) prior to lysate collection and AC activity assay (B); N = 2–4, n = 3–4). AC6 abundance was compared by Western blot, normalized to β-actin (C); N = 4, n = 4; (D); representative blot shown). *p < 0.05, **p < 0.01, ***p < 0.001compared to N. •p < 0.05, • p < 0.01 compared to H. ###p < 0.001 compared between FeTPPS and SNP treatments within similar O2 environments.