Stimulated calcium efflux from fura-2-loaded human platelets.
1. The reduction of cytoplasmic free calcium, [Ca2+]i following stimulation, has been investigated in fura-2-loaded human platelets in the presence of low extracellular calcium concentration. Thrombin produced a rapid rise in [Ca2+]i which then fell back to the basal level within 2 min. 2. Ionomycin produced a rapid elevation in [Ca2+]i which then declined to a plateau well above the basal calcium level. The addition of thrombin after ionomycin accelerated the decline in [Ca2+]i back towards basal levels, an action mimicked by phorbol myristate acetate (PMA). 3. Thrombin promoted the efflux of 45Ca2+ from cells co-loaded with fura-2 and the isotope. Ionomycin also promoted an efflux of 45Ca2+ which was increased by the subsequent addition of thrombin or PMA. These results confirm the ability of thrombin and PMA to stimulate Ca2+ removal from the cells. 4. The complete substitution of extracellular Na+ with N-methyl-D-glucamine (NMDG) did not alter the time course of the return of [Ca2+]i to basal following stimulation by thrombin, nor the ability of thrombin or PMA to promote Ca2+ efflux after elevation of [Ca2+]i by ionomycin. 5. The insensitivity to external Na+ suggests that the stimulated Ca2+ efflux is mediated by a Ca2+-ATPase rather than Na+-Ca2+ exchange. This pump does not appear to be activated by Ca2+-calmodulin since [Ca2+]i remains high when elevated by ionomycin. The ability of PMA to stimulate removal suggests that its known target, protein kinase C, can stimulate the Ca2+ pump. Forskolin, which stimulates adenylate cyclase, did not stimulate a fall in [Ca2+]i in the presence of ionomycin, indicating that cyclic AMP-dependent protein kinase does not stimulate Ca2+ extrusion.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001572:Benzofurans; D001792:Blood Platelets / Q000187:drug effects*; D002118:Calcium / Q000097:blood*; D002132:Calcium Radioisotopes; D005576:Colforsin / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D016257:Fura-2; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors
D000900:Anti-Bacterial Agents; D001572:Benzofurans; D002132:Calcium Radioisotopes; D004987:Ethers; D007473:Ion Channels; D005576:Colforsin; D015759:Ionomycin; D013917:Thrombin; D004072:Digitonin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2