1. The regulatory effects of cyclic AMP and cyclic GMP on ADP- and thrombin-induced increases in [Ca2+]i were studied in mouse bone marrow megakaryocytes. Changes in [Ca2+]i were continuously monitored in single Fura-2-loaded cells using microspectrofluorometry, and cyclic nucleotides were directly introduced into the single cells using the whole-cell patch-clamp technique. 2. ADP increased [Ca2+]i in a concentration-dependent fashion, and its threshold concentration was in the order of 0.01 microM. A low dose of ADP (below 0.1 microM) induced a transient response of [Ca2+]i which recovered to original levels during the stimulation. A high dose of ADP (0.3-10 microM) induced a biphasic response of [Ca2+]i with an initial peak and a plateau lasting until the end of the stimulation. Repeated stimulation with the same dose of ADP induced a reduced response, probably as a result of desensitization. 3. Thrombin increased [Ca2+]i in a concentration-dependent manner. The time courses of the responses were different from those caused by ADP. Thrombin-induced responses lacked the initial sharp peak observed in ADP-induced responses, and caused a sustained response. 4. The ADP-induced increase in [Ca2+]i was antagonized by the presence of prostaglandin E1 (PGE1, 100-1000 nM), in the medium, and by direct injection of cyclic AMP (100-500 microM) or cyclic GMP (500 microM) into the megakaryocyte. When 500 microM-cyclic AMP was injected into the cells, the rise of [Ca2+]i induced by ADP was reduced by 85%. Effects of these antagonists were inhibited by treatment with a protein kinase inhibitor, H-8. Thrombin-induced increases in [Ca2+]i were reduced by direct injection of cyclic AMP or cyclic GMP. 5. ADP could induce an increase in [Ca2+]i in the absence of external Ca2+. The time course of the response was essentially similar to that observed in the normal condition (1 mM-CaCl2), but the size of the response was reduced by 33%. Thus, 67% of the rise in [Ca2+]i induced by ADP could be accounted for by calcium mobilization from internal storage pools. The presence of NiCl2 (5 mM) duplicated the effects of external Ca2+ removal, suggesting the involvement of a Ca2+ influx pathway, which could be inhibited by Ni2+ in ADP stimulation. 6. Injection of cyclic AMP or cyclic GMP reduced ADP-induced increases in [Ca2+]i under conditions of inhibited Ca2+ influx by NiCl2 (5 mM).(ABSTRACT TRUNCATED AT 400 WORDS)
展开▼
