REFILLING STATE OF INTERNAL CA2-CELLS( STORES IS NOT THE ONLY INTRACELLULAR SIGNAL STIMULATING CA2+ INFLUX IN HUMAN ENDOTHELIAL)

To further analyse the role of the refilling state of internal Ca2+ po ols in the stimulation of Ca2+ influx in human endothelial cells, we i nvestigated the combined effect of thapsigargin (TG) and histamine on cytosolic Ca2+ concentration ([Ca2+](i)) and inositol polyphosphate pr oduction. At normal extracellular Ca2+ levels, TG induced a progressiv e and sustained elevation in [Ca2+](i) which was dose-dependently prev ented by pretreatment with 1-10 mu M histamine. Similarly, pretreatmen t with 0.1 and 1 mu M TG suppressed histamine-induced Ca2+ transients partially and totally, respectively. TG pretreatment did not alter the inositol triphosphate (IP3) level liberated by histamine, but modifie d IP3 metabolism by decreasing inositol biphosphate (IP2) and increasi ng inositol monophosphate (IP1) contents. In the absence of Ca2+ influ x, 1 mu M TG only induced a small transient increase in [Ca2+](i) wher eas the Ca2+ mobilization evoked by 10 mu M histamine was unchanged. I n both cases, the absence of any additional effect of either TG, hista mine or 2 mu M ionomycin indicated the complete depletion of Ca2+ stor es. The re-establishment of the transmembrane Ca2+ gradient induced a transient rise in [Ca2+](i). Its amplitude differed between histamine- and TG-treated cells. It was imposed by cell pretreatment and was sel ectively affected by changes in the membrane potential. At 5 mM extern al K+, the transient rise in [Ca2+](i) was more marked in histamine- t han in TG-stimulated cells; this difference was suppressed by TG pretr eatment. The presence of 130 mM external K+ increased Ca2+ entry in TG -treated cells but reduced it in histamine-stimulated cells. These res ults indicate that the refilling state of internal Ca2+ stores does no t constitute the single regulator of Ca2+ influx. TG and histamine see m to activate Ca2+ influx through distinct but interdependent pathways regulated by membrane potential.