Acute reductions in PO2 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 (DiBAC4, 1 microM) and [Ca2+]i-sensitive probe fura 2 (3 microM) 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.
Stevens
T
T
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Cornfield
D N
DN
McMurtry
I F
IF
Rodman
D M
DM
D000818:Animals; D001463:Barbiturates; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism* / Q000502:physiology*; D005456:Fluorescent Dyes; D007425:Intracellular Membranes / Q000378:metabolism*; D007555:Isoxazoles; D008345:Manganese / Q000378:metabolism; D009994:Osmolar Concentration; D010100:Oxygen / Q000378:metabolism*; D010313:Partial Pressure; D026902:Potassium Channel Blockers; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism* / Q000502:physiology*; D013997:Time Factors
D001463:Barbiturates; D005456:Fluorescent Dyes; D007555:Isoxazoles; D026902:Potassium Channel Blockers; C052795:bis(1,3-dibutylbarbiturate)trimethine oxonol; D008345:Manganese; D010100:Oxygen; D002118:Calcium