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Elevation of Intracellular Calcium Ion by Prostaglandin E₁ and Its Inhibition by Protein Kinase C in a Human Megakaryocyte Leukemia Cell Line

Departments of Laboratory Medicine [M. A., A. Y., K. T., N. Y.] and Biochemistry [M. H., Y. T.], Kobe University School of Medicine and Blood Transfusion Service, Kobe University Hospital [R. R.], Kobe 650; and Department of Pediatrics, Chiba University School of Medicine, Chiba 280 [T. S.], Japan

In the present study, we examined the effect of prostaglandin E₁ (PGE₁) on Ca²⁺ mobilization in a human megakaryocyte (the progenitor of platelets) leukemia cell line, designated as CMK. PGE₁ caused a rapid and dose-dependent increase in the intracellular free calcium level ([Ca²⁺]_i) associated with the elevation of cyclic AMP. The PGE₁-induced elevation of [Ca²⁺]_i was decreased by the prior addition of ethylene glycol bis(2-aminoethylether)tetraacetic acid to the medium by approximately 25% of the control. This result indicates that the PGE₁-induced elevation of [Ca²⁺]_i is due to influx of Ca²⁺ from the external medium and to mobilization of Ca²⁺ from intracellular stores. Pretreatment of CMK cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), a stimulus for protein kinase C, further enhanced the PGE₁-induced increase in the cellular cyclic AMP level. Inversely, pretreatment of CMK cells with TPA (10 nM), prior to the addition of PGE₁, inhibited the PGE₁-induced elevation of [Ca²⁺]_i. Dibutyryl cyclic AMP and forskolin did not elevate [Ca²⁺]_i or affect the PGE₁-induced Ca²⁺ mobilization. The inhibitory action of TPA in the PGE₁-induced elevation of [Ca²⁺]_i was mimicked by other protein kinase C-activating agents, such as 1-oleoyl-2-acetylglycerol and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, and was selectively restored by protein kinase C inhibitors, such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride. Thus, the inhibitory modulation of TPA on the PGE₁-induced elevation of [Ca²⁺]_i is mediated through protein kinase C activation. PGE₁ had no inductional effect of megakaryocytic phenotypic changes in CMK cells. The biological role of PGE₁, which increased [Ca²⁺]_i and cyclic AMP levels in the CMK cells, remains to be determined.

¹ To whom requests for reprints should be addressed.

Received 12/ 7/88. Revised 3/20/89. Accepted 4/12/89.