Cyclic nucleotide‐dependent regulation of agonist‐induced calcium increases in mouse megakaryocytes.

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)