PLCγ1-PKCε-IP3R1 signaling plays an important role in hypoxia-induced calcium response in pulmonary artery smooth muscle cells

Abstract

Hypoxia-induced pulmonary vasoconstriction (HPV) is attributed to an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in pulmonary artery smooth muscle cells (PASMCs). We have reported that phospholipase C-γ1 (PLCγ1) plays a significant role in the hypoxia-induced increase in [Ca²⁺]_i in PASMCs and attendant HPV. In this study, we intended to determine molecular mechanisms for hypoxic Ca²⁺ and contractile responses in PASMCs. Our data reveal that hypoxic vasoconstriction occurs in pulmonary arteries, but not in mesenteric arteries. Hypoxia caused a large increase in [Ca²⁺]_i in PASMCs, which is diminished by the PLC inhibitor U73122 and not by its inactive analog U73433 . Hypoxia augments PLCγ1-dependent inositol 1,4,5-trisphosphate (IP₃) generation. Exogenous ROS, hydrogen peroxide (H₂O₂), increases PLCγ1 phosphorylation at tyrosine-783 and IP₃ production. IP₃ receptor-1 (IP₃R1) knock-down remarkably diminishes hypoxia- or H₂O₂-induced increase in [Ca²⁺]_i. Hypoxia or H₂O₂ increases the activity of IP₃Rs, which is significantly reduced in protein kinase C-ε (PKCε) knockout PASMCs. A higher PLCγ1 expression, activity, and basal [Ca²⁺]_i are found in PASMCs, but not in mesenteric artery smooth muscle cells from mice exposed to chronic hypoxia (CH) for 21 days. CH enhances H₂O₂- and ATP-induced increase in [Ca²⁺]_i in PASMCs and PLC-dependent, norepinephrine-evoked pulmonary vasoconstriction. In conclusion, acute hypoxia uniquely causes ROS-dependent PLCγ1 activation, IP₃ production, PKCε activation, IP₃R1 opening, Ca²⁺ release, and contraction in mouse PASMCs; CH enhances PASM PLCγ1 expression, activity, and function, playing an essential role in pulmonary hypertension in mice.

Keywords: calcium; hypoxia; hypoxia-induced pulmonary vasoconstriction; inositol 1,4,5-trisphosphate receptor-1; phospholipase C-γ1; protein kinase C-ε; reactive oxygen species.

Fig. 1.

Phospholipase C-γ1 (PLCγ1) play an important role in hypoxia-induced pulmonary vasoconstriction (HPV). A: pulmonary artery (PA) and mesenteric artery (MA) rings were exposed to a physiological saline solution (PSS) bubbled with a normoxic gas (21% O₂, 5% CO₂, and the balance with N₂) for 10 min and then a hypoxic gas (1% O₂, 5% CO₂, and the balance with N₂) for 30 min. B: graph summarizes the tension generated in response to hypoxia in PAs and MAs (n = 4; *P < 0.05 compared with PA). C: representative Western blots of PLCγ1 expression in pulmonary artery smooth muscle cells (PASMCs) and mesenteric artery smooth muscle cells (MASMCs). D: graph summarizes the quantification of Western blots of PLCγ1 from three independent experiments. *P < 0.05 compared with MASMCs. E: representative traces show a hypoxia-induced increase in [Ca²⁺]_i in PASMCs preincubated without or with U73122 (1 µM) or U73433 (1 µM) for 30 min. F: quantification of the hypoxic increase in [Ca²⁺]_i in PASMCs untreated and treated with U73122 or U73433. Data were obtained from at least four different animals. *P < 0.05 compared with control.

Fig. 2.

Hypoxia and H₂O₂ both increase PLCγ1 activity and inositol monophosphate-1 (IP₁) production in PASMCs. A: Western blots of PLCγ1 phosphorylated at tyrosine 783 (pTyr-783) and total PLCγ1 in PASMCs exposed to normoxia, hypoxia, untreated control, and H₂O₂ (500 µM, 10 min). B: bar graph summarizes the ratio of pTyr-783 expression level relative to total PLCγ1 protein expression level from three separate experiments. *P < 0.05 compared with respective control. C: IP₁ production in cells untreated and treated with H₂O₂ in various concentrations for 10 min. Data were obtained from three separate experiments. *P < 0.05 compared with untreated group.

Fig. 3.

PLCγ1 knock-down (KD) abolishes hypoxia- and H₂O₂-induced increase in IP₁ production in pulmonary artery smooth muscle cells PASMCs. A: summary of the hypoxia-induced IP₁ production in PASMCs. Cells were uninfected (control) or infected with lentiviral particles containing nonsilencing (NS) short hairpin (sh)RNAs or shRNAs for PLCγ1. Data were obtained from three different experiments. *P < 0.05 compared with respective normoxia group. #P < 0.05 compared with normoxia. ^ωP < 0.05 compared with normoxia. B: H₂O₂ (500 µM) induced IP₁ production in PASMCs infected with NS shRNA or PLCγ1 shRNAs. Data were obtained from three different experiments *P < 0.05 compared with NS shRNA. #P < 0.05 compared with NS shRNA.

Fig. 4.

Ca²⁺ release from IP₃ receptor 1 (R1) is involved in hypoxia- and H₂O₂-induced increase in [Ca²⁺]_i in PASMCs. A: Western blot of IP₃R1 in PASMCs infected with lentiviral particles containing shRNAs targeting IP₃R1 and NS shRNAs. B: graph summarizes the quantification of Western blots of IP₃R1 from three independent experiments. C: Western blots of the expression of IP₃R2 and IP₃R3 in PASMCs infected with NS shRNA and IP₃R1 shRNA. D: summary of the quantification of IP₃R2 protein expression. E: graph summarizes the average IP₃R3 expression. F: effect of IP₃R1 gene KD on hypoxia-induced induced increase in [Ca²⁺]_i. G: effect of IP₃R1 KD on H₂O₂ (500 µM)-induced increase in [Ca²⁺]_i. Cells were uninfected (control) or infected with lentiviral particles containing NS shRNAs and shRNAs specific for IP₃R1. *P < 0.05 compared with control.

Fig. 5.

PKCε regulates hypoxia- and H₂O₂-induced increase in IP₃R binding affinity or activity in PASMCs. Radiolabeled [³H] IP₃ binding assay was conducted in lysates of PAMSCs to test the activity of IP₃Rs. A: effect of acute hypoxia on the binding of IP₃Rs in control (wild-type, WT) and PKCε^−/− PASMCs. B: effect of H₂O₂ (500 μM) on [³H] IP₃ binding of IP₃Rs in control and PKCε^−/− PASMCs. Data were obtained from three independent experiments. *P < 0.05 compared with control.

Fig. 6.

Mice exposed to chronic hypoxia (CH) show higher basal PLC activity and [Ca²⁺]_i in PASMCs than that in MASMCs. A: lysates from PAs and MAs from mice exposed to CH for 3 wk were subjected to PLC activity assay. Data were obtained from four independent experiments. The data were obtained by taking the average of the test values in the normoxia group, dividing the individual value from each test in the normoxia and hypoxia groups by the averaged value from the normoxia group, and finally taking the average of the calculated values in the normoxia and hypoxia groups (B) to provide basal intracellular [Ca²⁺]_i in PASMCs and MASMCs. Data were obtained from four animals. *P < 0.05 compared with normoxia.

Fig. 7.

Mice exposed CH showed higher expression and basal activity of PLCγ1 in PASMCs than in MASMCs. A: lysates from PASMCs of mice exposed to normoxia and CH were subjected to Western blot analysis. A, top: representative blots of PLCγ1 and GAPDH. A, bottom: quantification of PLCγ1 expression levels in PASMCs from five animals exposed to normoxia and three animals to CH. B, top: representative Western blots of protein expression of PLCγ1 phosphorylated at tyrosine 783 (pTyr-783) and total PLCγ1 in PASMCs from normoxia and CH mice. B, bottom: summary of the fold increase in the ratio of pTyr-783 expression level relative to total PLCγ1 protein expression level in PASMCs. Data were obtained from four mice exposed to normoxia and three mice exposed to CH. *P < 0.05 compared with normoxia.

Fig. 8.

ATP and H₂O₂-induced increase in [Ca²⁺]_i in PASMCs and PLC-dependent norepinephrine (NE)-induced contraction in PAs are greatly enhanced in mice exposed to CH. A: effect of ATP (10 µM) on [Ca²⁺]_i in PASMCs from normoxic and CH mice. B: effect of H₂O₂ (500 µM) on [Ca²⁺]_i in PASMCs from normoxic and CH mice. Cells were obtained from at least four different animals. *P < 0.05 compared with normoxia. C: summary of NE-induced contraction in PA rings from normoxia and CH mice treated with and without U73122 (10 µM). Data were obtained from three normoxic and four CH mice. *P < 0.05 compared with normoxia, and #P < 0.05 compared with CH.

Fig. 9.

A schematic diagram of the signaling mechanisms for hypoxia-induced PLCγ1- and PKCε-dependent increase in [Ca²⁺]_i in PASMCs and HPV. Hypoxia causes an increase in RISP-dependent mitochondrial ROS production, which causes PLCγ1 activation, IP₃ production, IP₃R1 opening, and Ca²⁺ release. RISP-dependent mitochondrial ROS may also activate PKCε, phosphorylate IP₃R1, enhance IP₃ binding, induce Ca²⁺ release in PASMCs. Taken together, PLCγ1 and PKCε can synergistically contribute to the hypoxic increase in [Ca²⁺]_i in PASMCs, HPV, and pulmonary hypertension.