PHARMACOLOGICAL CHARACTERIZATION OF REFILLING INOSITOL 1,4,5-TRISPHOSPHATE-SENSITIVE CA2-15 CELLS( STORES IN NG108)

Following mobilization with the inositol 1,4,fi-trisphosphate (IP3)-ge nerating agonist bradykinin, Ca2+ stores in neuroblastoma X glioma hyb rid, NG108-15 cells require extracellular Ca2+ to refill. The process by which this store refills with Ca2+ was characterized by recording b radykinin-induced intracellular free Ca2+ concentration transients as an index of the degree of refilling of the store. Cyclopiazonic acid, a microsomal Ca2+ ATPase inhibitor, reversibly depleted intracellular Ca2+ stores in these cells, but did not recruit detectable Ca2+ influx , suggesting that these cells lack substantial capacitative Ca2+ entry . The paucity of voltage-sensitive Ca2+ channels in undifferentiated N G108-15 cells, suggested that a channel analogous to that proposed to mediate capacitative Ca2+ entry in nonexcitable cells might assist ref illing IP3-sensitive Ca2+ stores in these cells. The possibility that compounds shown previously to inhibit capacitative Ca2+ entry in nonex citable cells might inhibit the refilling of the IP3-sensitive store i n NG108-15 cells was explored. The IP3-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 evaluat e the degree of refilling of the store. The imidazole derivatives, eco nazole (10 mu M), L-651582 (10 mu M) and SKF 95365 (20 mu M), all comp letely blocked the bradykinin-induced Ca2+ response. Calmodulin antago nists, W-7 (100 mu M) and trifluoperazine (10 mu M), were also effecti ve, although at concentrations well above those required to inhibit ca lmodulin. Because of the high concentrations required to inhibit brady kinin responses, the possibility that these agents might have addition al effects was explored. Compounds were tested in a paradigm in which the store was preloaded with Ca2+ before treatment. All of these agent s depleted, at least partially, the preloaded store. Econazole was the least effective of the compounds tested for releasing stores, althoug h it was comparable to the other compounds for inhibition of refilling . Although NG108-15 cells refill intracellular Ca2+ stores by a plasma lemmal Ca2+ leak, this leak shares a pharmacology similar to the capac itative Ca2+ entry pathway described for nonexcitable cells.