ACUTE REDUCTIONS IN PO-2 DEPOLARIZE PULMONARY-ARTERY ENDOTHELIAL-CELLS AND DECREASE [CA2+](I)

Whereas pulmonary artery endothelial cells (PAECs) are sensitive to oxygen, neither the effect of an acute reduction in PO2 on PAEC membrane potential nor its effect on intracellular free Ca2+ ([Ca2+](i)) is known. We hypothesized that in confluent primary cultures of PAECs, an acute decrease in PO2 would depolarize the cell membrane, inhibit Ca2+ influx, and reduce [Ca2+](i). To test this hypothesis, the membrane-sensitive fluorophore bis(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC(4), 1 mu M) and [Ca2+](i)-sensitive probe fura 2 (3 mu M) were used. A decrease in PO2 from 125 to 35 mmHg caused membrane depolarization and a 60 +/- 8% (data are means +/- SE) reduction in Ca2+ influx, estimated by manganese quenching of fura 2 fluorescence. While basal [Ca2+](i) was 79 +/- 5 nM in normoxic cells, it decreased to 31 +/- 2 nM after 15 min of hypoxia. Decreasing the electrochemical gradient for Ca2+ entry with either low extracellular Ca2+, the K+ channel blockers tetraethylammonium or charybdotoxin, or blockade of Ca2+ entry with lanthanum decreased [Ca2+](i) by 54-71% of that observed during an acute reduction in PO2. These results demonstrate that an acute reduction in PO2 1) depolarizes PAECs, 2) reduces Ca2+ influx, and 3) decreases [Ca2+](i), and that a similar reduction in [Ca2+](i) was observed with interventions designed to reduce the electrochemical driving force for Ca2+ entry.