Refilling state of internal Ca 2+ stores is not the only intracellular signal stimulating Ca 2+ influx in human endothelial cells

Abstract

To further analyse the role of the refilling state of internal Ca 2+ pools in the stimulation of Ca 2+ influx in human endothelial cells, we investigated the combined effect of thapsigargin (TG) and histamine on cytosolic Ca 2+ concentration ([Ca 2+] i) and inositol polyphosphate production. At normal extracellular Ca 2+ levels, TG induced a progressive and sustained elevation in [Ca 2+] i which was dose-dependently prevented by pretreatment with 1–10 μM histamine. Similarly, pretreatment with 0.1 and 1 μM TG suppressed histamine-induced Ca 2+ transients partially and totally, respectively. TG pretreatment did not alter the inositol triphosphate (IP 3) level liberated by histamine, but modified IP 3 metabolism by decreasing inositol biphosphate (IP 2) and increasing inositol monophosphate (IP i) contents. In the absence of Ca 2+ influx, 1 μM TG only induced a small transient increase in [Ca 2+] i whereas the Ca 2+ mobilization evoked by 10 μM histamine was unchanged. In both cases, the absence of any additional effect of either TG, histamine or 2 μM ionomycin indicated the complete depletion of Ca 2+ stores. The re-establishment of the transmembrane Ca 2+ gradient induced a transient rise in [Ca 2+] i. Its amplitude differed between histamine- and TG-treated cells. It was imposed by cell pretreatment and was selectively affected by changes in the membrane potential. At 5 mM external K +, the transient rise in [Ca 2+] i was more marked in histamine- than in TG-stimulated cells; this difference was suppressed by TG pretreatment. The presence of 130 mM external K + increased Ca 2+ entry in TG-treated cells but reduced it in histamine-stimulated cells. These results indicate that the refilling state of internal Ca 2+ stores does not constitute the single regulator of Ca 2+ influx. TG and histamine seem to activate Ca 2+ influx through distinct but interdependent pathways regulated by membrane potential.