Mechanisms of hemolysate-induced [Ca²⁺](i) elevation in cerebral smooth muscle cells

The effects of hemolysate on free cytosolic [Ca²⁺] ([Ca²⁺](i)) homeostasis were studied in freshly isolated rat basilar artery smooth muscle cells using fura 2 and dual excitation wave-length microfluorimetry. Hemolysate reversibly produced a transient [Ca²⁺](i) peak followed by a slowly decaying plateau which was absent in Ca²⁺-free solution. This effect of hemolysate was attenuated by 1) the sarcoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin and cyclopiazonic acid, 2) the Ca²⁺ release- blocking agents ryanodine and dantrolene, 3) the cytochrome P-450 inhibitor econazole, and 4) the inorganic Ca²⁺ channel blocker lanthanum but was not significantly attenuated by 1) the receptor-regulated Ca²⁺ channel blocker SKF-96365 or 2) the voltage-dependent Ca²⁺ channel blocker nimodipine. Fractionation of hemolysate using membranes with specific pore sizes (0.5, 1, and 12-14 kDa) indicated that a component(s) >0.5 but <1 kDa could produce a similar [Ca²⁺](i) peak and plateau while fractions > 1 and > 12-14 kDa produced a small and slow [Ca²⁺](i) rise without a significant peak. ATP, which was found in hemolysate, produced a [Ca²⁺](i) response similar to that of hemolysate. P₂-purinoceptor antagonists significantly attenuated the effect of ATP, hemolysate, and the fractions < 1 and < 12-14 kDa. We conclude that hemolysate elevates [Ca²⁺](i) by both releasing Ca²⁺ from internal stores and triggering Ca²⁺ entry, possibly from a voltage-independent Ca²⁺ influx pathway, an effect apparently identical to that of ATP.