Role Of Hypoxia On Intracellular Ca²⁺ Concentration In Pulmonary Venous Smooth Muscle Cells (PVSMC) Isolated From Rat Distal Pulmonary Veins

ObjectiveHypoxia causes remodeling and alters contractile responses in both pulmonary arteries and pulmonary veins (PV). Although pulmonary arteries have been studied extensively in this disorder, the mechanisms by which pulmonary veins respond to hypoxia and whether these responses contribute to chronic hypoxic pulmonary hypertension (CHPH) remain poorly understood. In pulmonary arterial smooth muscle, influx of Ca²⁺ through store-operated calcium channels thought to be composed of canonical transient receptor potential (TRPC) proteins is likely to play an important role in development of CHPH. The aim of this study is to determine whether this mechanism could also be operative in pulmonary venous smooth muscle.MethodsThe rat distal PVSMC were isolated and cultured by collagenase digestion and characterized by morphological activity, by immunoblotting and immunofluorescence of smooth muscle -actin and by response of intracellular Ca²⁺ concentration ([Ca²⁺]_i) to KCL.The effect of acute hypoxia on [Ca²⁺]_i in PVSMC and the inhibition of nifedipine were measured by Incyte intracellular Ca²⁺ concentration system. The expression of TRPC in distal pulmonary venous smooth muscle or PVSMC was detected by real-time PCR and western blotting. Store-operated Ca²⁺ entry (SOCE) and the effect of acute hypoxia on SOCE were assessed by measurement of increase in [Ca²⁺]_i caused by restoration of extracellular Ca²⁺ and the rate at which extracellular Mn²⁺ quenched fura-2 fluorescence.CHPH rats were obtained using a hypoxic (10 % O2) chamber for 21 days. PVSMC were isolated from CHPH rats or normal rats. The effect of chronic hypoxia on [Ca²⁺]_i in PVSMC was assessed by comparison of [Ca²⁺]_i in PVSMC from CHPH rats with that from normal rats.The effect of chronic hypoxia on TRPC expression was measured by comparison of TRPC mRNA and protein expression in pulmonary venous smooth muscle from CHPH rats with that from normal rats using real-time PCR and western blotting. PVSMC from CHPH rats or normal rats were isolated. The mechanisms by which [Ca²⁺]_i in PVSMC respond to chronic hypoxia were assessed by comparison of CPA-induced Ca²⁺ influx and Mn²⁺ quenching in PVSMC isolated from CHPH rats with that from normal rats and by comparison of OAG-induced Ca²⁺ influx and Mn²⁺ quenching in PVSMC isolated from CHPH rats with that from normal rats.ResultsCells isolated from rat distal pulmonary veins displayed morphological characteristic of vascular smooth muscle cells and expressed smooth muscle -actin and the functional voltage-depended Ca²⁺ channels (VDCC). Acute hypoxia could increase [Ca²⁺]_i in rat distal PVSMC.VDCC antagonist nifedipine could block KCL-induced increase of [Ca²⁺]_i in rat distal PVSMC. However, nifedipine partially prevented the effect of acute hypoxia on [Ca²⁺]_i , indicating acute hypoxia actived influx of Ca²⁺ through VDCC and another independent pathway. Real-time PCR and western blotting both indicated that TRPC were expressed in rat distal pulmonary venous smooth muscle or PVSMC with most abundant expression of TRPC1, TRPC6 and TRPC4.Restoration of extracellular Ca²⁺ caused a large increase of [Ca²⁺]_i in PVSMC perfused with Ca²⁺-free KRB solution containing CPA and nifedipine. In the presence of CPA, fura-2 fluorescence decreased more than that in the absence of CPA after administration of Mn²⁺ in PVSMC perfused with Ca²⁺-free KRB solution containing nifedipine. The store-operated Ca²⁺ channels (SOCC) antagonists SKF-96365 and NiCl2 both inhibited CPA-induced Ca²⁺ influx and Mn²⁺ quenching, but had no effect on KCL-induced increase of [Ca²⁺]_i in PVSMC. These data indicated SOCE in rat distal PVSMC.Acute hypoxia caused enhancement of CPA-induced increase in [Ca²⁺]_i elicited by Ca²⁺ restoration and the rate of Mn²⁺ quenching. SKF-96365 and NiCl2 blocked hypoxic enhancement of both the [Ca²⁺]_i response to extracellular Ca²⁺ restoration and the rate of Mn²⁺ quenching. In normoxic PVSMC treated with nifedipine but not CPA, Mn²⁺ quenching did not differ from the spontaneous decline of fluorescence in cells not exposed to Mn²⁺. During acute hypoxia, however, the rate of Mn²⁺ quenching in the absence of CPA increased, and this increase was blocked by SKF-96365 or NiCl2.The MPAP, RVSP and RV/(LV+S) were all increased in rats exposed to chronic hypoxia. The pulmonary vessels from CHPH rats were thicker than that from normal rats. [Ca²⁺]_i in PVSMC from CHPH rats was higher than that from normal rats, indicating chronic hypoxia could increase [Ca²⁺]_i in rat distal PVSMC.Both TRPC6 mRNA and protein expression in pulmonary venous smooth muscle from CHPH rats were stronger than that from normal rats. But neither TRPC1 nor TRPC4 mRNA or protein expression in pulmonary venous smooth muscle from CHPH rats differed from that from normal rats.In the presence of nifedipine and CPA, neither the increase of [Ca²⁺]_i elicited by Ca²⁺ restoration nor the rate of Mn²⁺ quenching in PVSMC from CHPH rats differed from that from normal rats. In the presence of nifedipine and OAG, however, restoration of extracellular Ca²⁺ caused a larger increase of [Ca²⁺]_i in PVSMC from CHPH rats than that from normal rats. Otherwise, in the presence of nifedipine and OAG, the rate of Mn²⁺ quenching in PVSMC from CHPH rats was also larger than that from normal rats.Conclusion1. Both acute and chronic hypoxia could increase [Ca²⁺]_i in rat distal PVSMC.2. SOCE and TRPC proteins were expressed in rat distal pulmonary venous smooth muscle with most abundant expression of TRPC1, TRPC6 and TRPC4.3. Acute hypoxia was able to evoke SOCE in rat distal PVSMC on its own, as well as amplify SOCE.4. Chronic hypoxia could increase Ca²⁺ influx through receptor-operated Ca²⁺ channels (ROCC) thought to be composed of TRPC6 protein by upregulation of TRPC6 expression in rat distal PVSMC.