Calcium pools in Ehrlich carcinoma cells. A major, high affinity Ca 2+ pool is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin

Abstract

To investigate the presence and the size of different non-mitochondria) Ca 2+ pools of Ehrlich ascites tumor cells (EATCs) , digitonin-permeabilized cells were allowed to accumulate Ca 2+ in the presence of mitochondrial inhibitors and treated with the reticular Ca 2+-ATPase inhibitor thapsigargin, IP 3 and the Ca 2+ ionophore A23187. Emptying of thapsigargin-sensitive Ca 2+ stores prevented any Ca 2+ release by IP 3, and, after IP 3 addition, little or no Ca 2+ was released by thapsigargin. In both instances, a further Ca 2+ release was accomplished by A23187. The IP 3-thapsigargin-sensitive pool and the residual A23187-sensitive one corresponded to approximately 60 and 37% of non-mitochondria) stored Ca 2+, respectively. In intact EATCs, IP 3-dependent agonists and thapsigargin discharged Ca 2+ pools almost completely overlapping, and A32187 released a minor residual Ca 2+ pool. The IP 3-insensitive pool appeared to have a relatively low affinity for Ca 2+ (below 600 nM). The high affinity, IP 3-sensitive Ca 2+ pool was discharged in a ‘quantal’ manner following step additions of sub maximal [IP 3], and the IP 3-induced fractional Ca 2+ release was more marked at higher concentrations of stored (luminal) Ca 2+, The IP 3-sensitive Ca 2+ pool appeared to be devoid of the Ca 2+-activated Ca 2+ release channel since caffeine did not released any Ca 2+ in intact and permeabilized EATCs, and Western blot analyses of EATC microsomal membranes failed to detect any known ryanodine receptor isoform.