Human peripheral blood lymphocytes incubated with the tumor promoter and T-cell mitogen phorbol-12-myristate-13-acetate (PMA) had elevated cyclic guanosine 3′:5′-monophosphate (cyclic GMP) levels and activities of guanylate cyclase and cyclic GMP phosphodiesterase and reduced activity of adenylate cyclase. No changes occurred in cyclic adenosine 3′:5′-monophosphate levels or cyclic adenosine 3′:5′-monophosphate phosphodiesterase. Cyclic GMP levels increased in a bimodal fashion corresponding to the brisk rise at 1 min in both membrane and soluble guanylate cyclases and to a second increase at 20 to 60 min in soluble guanylate cyclase. Decreases in adenylate cyclase were similar for basal and isoproterenol- or NaF-stimulated activity. The magnitude of these changes increased with increasing concentrations of PMA from 0.01 to 10 µg/ml, while the effects of PMA on lymphocyte proliferation progressively increase from 0.001 to 1 µg/ml. The effects of a series of phorbol diesters on guanylate cyclase were consistent with their relative activities for lymphocyte mitogenesis and for tumor promotion. Because the mononuclear cell populations are heterogeneous, cause and effect relationships between enzyme changes and biological responses cannot now be ascertained. None of the PMA-induced changes were significantly affected by inhibition of macromolecular synthesis or by omitting Ca2+ from the lymphocyte preincubation medium. PMA-induced increases in both membrane and soluble guanylate cyclase were markedly inhibited by the intracellular Ca2+ antagonist methoxyverapamil but not by [ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. PMA stimulation of the membrane enzyme was prevented by the arachidonic acid lipoxygenase inhibitors 15-hydroxyeicosatetraenoic acid and nordihydroguaiaretic acid. Increases in soluble guanylate cyclase and cyclic GMP phosphodiesterase and reduction of adenylate cyclase by PMA were less susceptible to the lipoxygenase inhibitors. These results suggest that PMA stimulates cyclic GMP synthesis by a Ca2+-dependent process which, in the membrane, involves one or more lipoxygenase metabolites and that different mechanisms may account for the alterations in the other cyclic nucleotide-metabolizing enzyme activities.


This study was supported in part by Grants BC 216B from the American Cancer Society, CA 08748 from the NIH, and by Newport Pharmaceuticals. Newport, Calif. A preliminary report of some of this work was presented at the 65th Annual Meeting of the Federation of American Societies for Experimental Biology, St. Louis, Mo., 1981 (10).

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