CYCLIC NUCLEOTIDE-DEPENDENT REGULATION OF AGONIST-INDUCED CALCIUM INCREASES IN MOUSE MEGAKARYOCYTES
CYCLIC NUCLEOTIDE-DEPENDENT REGULATION OF AGONIST-INDUCED CALCIUM INCREASES IN MOUSE MEGAKARYOCYTES
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DOI：
10.1113/jphysiol.1992.sp019025
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发表时间：
1992-02-01
影响因子：
5.5
通讯作者：
MARUYAMA, Y
中科院分区：
文献类型：
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作者：
IKEDA, M;KUROKAWA, K;MARUYAMA, Y
1. 在小鼠骨髓巨核细胞中研究了cAMP和cGMP对ADP和凝血酶诱导的[Ca ~（2+）]i升高的调节作用。 采用显微荧光法连续监测单个Fura-2负载细胞内[Ca 2 +]i的变化，并采用全细胞膜片钳技术将环核苷酸直接导入单个细胞. ADP浓度依赖性地增加[Ca ~（2+）]i，其阈值浓度为0.01 μ M。 低剂量的ADP（低于0.1 μ M）引起[Ca ~（2+）]i的瞬时反应，在刺激过程中恢复到原始水平。 高剂量ADP（0.3-10 μ M）可引起[Ca ~（2+）]i的双相反应，有一个起始峰和一个持续到刺激结束的平台。 相同剂量的ADP重复刺激引起的反应降低，可能是脱敏的结果。 凝血酶以浓度依赖性方式增加[Ca 2 +]i。 反应的时程与ADP不同。 凝血酶诱导的反应缺乏ADP诱导反应中观察到的初始尖峰，并且引起持续的反应. 在培养基中加入前列腺素E1（PGE 1，100-1000 nM）和直接向巨核细胞内注射环磷酸腺苷（100-500 μ M）或环磷酸鸟苷（500 μ M）可拮抗ADP诱导的[Ca ~（2+）]i增加。 当500 μ M cAMP注入细胞时，ADP引起的[Ca ~（2+）]i升高降低了85%。 这些拮抗剂的作用被抑制治疗与蛋白激酶抑制剂，H-8。 凝血酶诱导的[Ca 2 +]i的增加通过直接注射环AMP或环GMP而减少。 ADP可诱导[Ca ~（2+）]i增加。 反应的时间过程基本上类似于在正常条件下（1 mM-CaCl 2）观察到的，但反应的大小减少了33%。 因此，67%的ADP诱导的[Ca 2 +]i的上升可以解释从内部存储池的钙动员。 NiCl 2（5 mM）的存在复制了外部Ca 2+去除的效果，表明涉及Ca 2+内流途径，在ADP刺激中Ni 2+可以抑制该途径. 在NiCl 2（5 mM）抑制Ca 2+内流的条件下，注射环AMP或环GMP可降低ADP诱导的[Ca 2 +]i增加。 两者合计，这些结果表明，ADP和/或凝血酶诱导的[Ca 2 +]i的增加是负调控的环AMP和/或环GMP依赖性蛋白激酶，推测作用于Ca 2+动员，在小鼠巨核细胞。
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-mu-M. A low dose of ADP (below 0.1-mu-M) induced a transient response of [Ca2+]i which recovered to original levels during the stimulation. A high dose of ADP (0.3-10-mu-M) 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-mu-M) or cyclic GMP (500-mu-M) into the megakaryocyte. When 500-mu-M-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).7. Taken together, these results indicate that the ADP- and/or thrombin-induced increases in [Ca2+]i are negatively regulated by cyclic AMP- and/or cyclic GMP-dependent protein kinases, presumably acting on Ca2+ mobilization, in mouse megakaryocytes.