Pharmacologic characterization of refilling inositol 1,4,5-trisphosphate-sensitive Ca 2+ stores in NG108-15 cells

Abstract

Following mobilization with the inositol 1,4,5-trisphosphate (IP 3)-generating agonist bradykinin, Ca 2+ stores in neuroblastoma × glioma hybrid, NG108-15 cells require extracellular Ca 2+ to refill. The process by which this store refills with Ca 2+ was characterized by recording bradykinin-induced intracellular free Ca 2+ concentration transients as an index of the degree of refilling of the store. Cyclopiazonic acid, a microsomal Ca 2+ ATPase inhibitor, reversibly depleted intracellular Ca 2+ stores in these cells, but did not recruit detectable Ca 2+ influx, suggesting that these cells lack substantial capacitative Ca 2+ entry. The paucity of voltage-sensitive Ca 2+ channels in undifferentiated NG108-15 cells, suggested that a channel analogous to that proposed to mediate capacitative Ca 2+ entry in nonexcitable cells might assist refilling IP 3-sensitive Ca 2+ stores in these cells. The possibility that compounds shown previously to inhibit capacitative Ca 2+ entry in nonexcitable cells might inhibit the refilling of the IP 3-sensitive store in NG108-15 cells was explored. The IP 3-sensitive store was depleted by exposure to bradykinin, allowed to refill briefly in the presence of the test compound and then challenged again with bradykinin to evaluate the degree of refilling of the store. The imidazole derivatives, econazole (10 μM), L-651582 (10 μM)and SKF 96365 (20 μM), all completely blocked the bradykinin-induced Ca 2+ response. Calmodulin antagonists, W-7 (100 μM)and trifluoperazine (10 μM), were also effective, although at concentrations well above those required to inhibit calmodulin. Because of the high concentrations required to inhibit bradykinin responses, the possibility that these agents might have additional effects was explored. Compounds were tested in a paradigm in which the store was preloaded with Ca 2+ before treatment. All of these agents depleted, at least partially, the preloaded store. Econazole was the least effective of the compounds tested for releasing stores, although it was comparable to the other compounds for inhibition of refilling. Although NG108-15 cells refill intracellular Ca 2+ stores by a plasmalemmal Ca 2+ leak, this leak shares a pharmacology similar to the capacitative Ca 2+ entry pathway described for nonexcitable cells.