ACUTE-HYPOXIA CAUSES MEMBRANE DEPOLARIZATION AND CALCIUM INFLUX IN FETAL PULMONARY-ARTERY SMOOTH-MUSCLE CELLS

Changes in oxygen tension in the perinatal period contribute to high pulmonary vascular tone in the fetus and the decline in resistance that occurs at birth. Distal pulmonary artery smooth muscle cells (PASMC) isolated from late-gestation ovine fetuses respond to acute hypoxia with an increase in cytosolic calcium concentration ([Ca2+](i)) dependent on Ca2+ entry. The purpose of this study is to determine 1) whether acute hypoxia results in PASMC membrane depolarization, 2) whether Ca2+ entry was through voltage-operated calcium channels (VOCC), 3) the contribution of Ca2+-induced Ca2+ release (CICR) to the hypoxic response, and 4) whether a subset of K+ channels might serve as oxygen sensors in fetal PASMC. We used microfluorimetry on subconfluent monolayers of PASMC in primary culture loaded with either a membrane potential-sensitive dye, bis(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4); DPASMC), to estimate membrane potential, or the Ca2+-sensitive fluorophore, fura 2, to measure [Ca2+](i). Hypoxia increased fluorescence from PASMC loaded with DiBAC(4), consistent with membrane depolarization. Verapamil (an inhibitor of VOCC) attenuated, and BAY K 8644 (a VOCC facilitator) potentiated, the hypoxia-induced increase in [Ca2+](i), respectively. The hypoxic response was transient after treatment with ryanodine (10(-7) M), a blocker of calcium release from intracellular stores. Charybdotoxin (10(-7) M), an inhibitor of Ca2+-activated K+ channels, almost doubled [Ca2+](i), whereas glibenclamide (10(-5) M), an ATP-sensitive K+-channel antagonist, had no effect. We conclude that hypoxia causes membrane depolarization in fetal PASMC. The hypoxia-induced increase in fetal DPASMC [Ca2+](i) is mediated by entry of Ca2+ through VOCC causing CICR and sustained increase in [Ca2+](i). We speculate that hypoxia may induce DPASMC membrane depolarization through inactivation of charybdotoxin-sensitive K+ channels and facilitate subsequent Ca2+ entry through VOCC.