Ca2+ influx induced by the Ca2+-ATPase inhibitors 2,5-di-(t-butyl)-1,4-benzohydroquinone and thapsigargin in bovine adrenal chromaffin cells

We have characterized the effect of the Ca(2+)-ATPase inhibitors 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ) and thapsigargin on the concentration of cytosolic Ca2+ in single bovine adrenal chromaffin cells by video-imaging of fura-2-loaded cells. Addition of either inhibitor released Ca2+ from internal stores in the absence of external Ca2+. tBHQ was unable to stimulate further Ca2+ release after addition of thapsigargin, but thapsigargin could do so after release by tBHQ, indicating that the tBHQ-sensitive stores are a sub-set of those sensitive to thapsigargin. Angiotensin II was able to elicit Ca2+ release after application of tBHQ, indicating that at least part of the tBHQ-sensitive stores were distinct from those discharged by Ins(1,4,5)P3. In the presence of external Ca2+, both Ca(2+)-ATPase inhibitors produced a more prolonged rise in cytosolic Ca2+ consistent with stimulated Ca2+ entry. The ability of the inhibitors to activate a Ca(2+)-entry pathway was confirmed by monitoring quenching of fura-2 after stimulated entry of the Ca2+ surrogate Mn2+. These findings indicate that bovine adrenal chromaffin cells possess a mechanism by which Ca2+ entry can be activated, following emptying of certain internal stores, independently of receptor occupation.