CALCIUM ENTRY-DEPENDENT OSCILLATIONS OF CYTOPLASMIC CALCIUM-CONCENTRATION IN CULTURED ENDOTHELIAL-CELL MONOLAYERS

Bovine endothelial cell monolayers grown to confluence and stimulated with bradykinin responded with periodic fluctuations in intracellular Ca2+ concentration ([Ca2+]i) when exposed to K+-free Hepes-buffered saline. The fluctuations in [Ca2+]i measured with fura-2 were synchronized among the population of cells observed and were sensitive to extracellular Ca2+ concentration ([Ca2+]o). Thapsigargin, which inhibits the endoplasmic reticular Ca2+-ATPase, did not inhibit the [Ca2+]i oscillations. Removal of extracellular Ca2+ or inhibition of Ca2+ entry by using La3+ or 1-{beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SKF 96365) abolished the [Ca2+]i oscillations in endothelial cell monolayers. The fluctuations in [Ca2+]i were therefore dependent on Ca2+ influx rather than Ca2+ mobilization from intracellular stores. Simultaneous measurements of membrane potential (E(m)) using the potential-sensitive bisoxonol dye bis(1,3-dibutylbarbituric acid)trimethine oxonol [Di-BAC4(3)] and [Ca2+]i using fura-2 showed that E(m) oscillated at the same frequency as the fluctuations in [Ca2+]i. The peak depolarization signal coincided with the maximum rate of increase in the [Ca2+]i signal. Oscillations in the E(m) signal were inhibited by removal of Ca2+ or by addition of 1 mM Ni2+ to the external solution. Taken together, these observations suggest that the change in E(m) is the consequence of oscillatory changes in a membrane conductance that also allows Ca2+ to enter the cell. Oscillations in the DiBAC4(3) signal may reflect a rhythmic entry of Ca2+ through nonselective cation channels.