A widespread occurrence of Ca +-dependent protein kinase was shown in various tissues and phyla of the animal kingdom. Phosphatidylserine appeared to be more effective than calmodulin in supporting the Ca2+-dependent phosphotransferase activity. The phospholipid-sensitive Ca2+-dependent protein kinase activity, distributed in both the cytosolic and particulate fractions, was not inhibited by trifluoperazine, a specific inhibitor of calmodulin-sensitive, Ca2+_ dependent reactions or processes. The enzyme activity levels, compared to those of cyclic AMP-dependent and cyclic GMPdependent protein kinases, were exceedingly high in certain tissues (such as brain and spleen) and exhibited a much-greater disparity among tissues. The Ka for Ca2+ was about 100 gM in the presence of phosphatidylserine; the value was as low as 2

Activated macrophages (M phi s) are important participants in host defense, but their uncontrolled activation leads rapidly to septic shock and death. One mechanism for regulating other dangerous cells in the immune system is programmed cell death, or apoptosis. Monocytes are known to undergo spontaneous apoptosis upon leaving the circulation unless provided with specific survival signals, but mature tissue M phi s are more robust cells, and it was not clear that they could be similarly regulated by apoptosis. We now show that during differentiation monocytes rapidly lose their sensitivity to apoptosis triggered by passive cytokine withdrawal, but they may retain a novel pathway which initiates apoptosis after activation with specific stimuli (zymosan and phorbol esters). Sensitivity to activation-induced apoptosis was developmentally determined, being downregulated by the maturation-promoting cytokine macrophage colony-stimulating factor but stably upregulated by even transient exposure to the proinflammatory cytokine interferon gamma (IFN-gamma). Apoptosis began within 2-4 h of activation, occurred in > 95% of susceptible cells, and in mixed cocultures selectively affected only those M phi s with a history of IFN-gamma priming. Consistent with a possible role for protein kinase C in the signaling pathway leading to cell death, the kinase inhibitor staurosporine was protective against both phorbol ester- and zymosan-induced apoptosis. Our studies describe a novel form of activation-induced M phi apoptosis which is developmentally regulated by two physiologically relevant cytokines. We speculate that apoptosis may serve to restrict the destructive potential of inflammatory M phi s.

Hyperhomocysteinemia has been recognized as an independent risk factor for cerebral, coronary, and peripheral atherosclerosis. To examine the contribution of homocysteine (H[cys]) in the pathogenesis of vascular diseases, we sought to determine whether the H[cys] effect on vascular smooth muscle (VSMC) proliferation is mediated by a specific receptor/transporter or is due to an interaction with growth factors or cytokines. We show that H[cys] induced c-fos and c-myb and increased DNA synthesis and cell proliferation 12-fold in neural crest-derived VSMC (N-VSMC). The H[cys] effect on N-VSMC proliferation is inhibited by Mk-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, a glutamate-gated calcium ion channel receptor, and CGS 19755, a competitive antagonist of NMDA-type glutamate receptor. H[cys] stimulates the synthesis of mass amounts of sn-1,2 diacylglycerol, and activates protein kinase C translocation from the nucleus and cytoplasm to cell membranes. Furthermore, protein kinase C inhibitors block the growth effect mediated by H[cys]. These findings indicate that H[cys]-mediated responses are coupled to diacylglycerol-dependent protein kinase C activation. Our results suggest that homocysteine activates a receptor/transporter-like factor in neural crest derived smooth muscle.

MCF-7 cells growing in culture were used to study the mechanism of the antiproliferative activity of the antiprogestin mifepristone, as compared with the antiestrogen 4-hydroxytamoxifen or the combination of both. These steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl2, and induction of TGFbeta1 protein. Abrogation of the mifepristone- and/or 4-hydroxytamoxifen-induced cytotoxicity by TGFbeta1 neutralizing antibody confirms the correlation between induction of active TGFbeta1 and subsequent cell death. The effect of a combination of mifepristone and 4-hydroxytamoxifen on cell growth inhibition, on the increase in DNA fragmentation, bcl2 downregulation, and induction of TGFbeta1 protein was additive and significantly different (P < 0.05) from the effect of monotherapy. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive in cells treated with a combination of both 4-hydroxytamoxifen and mifepristone. These results suggest that the mechanism of the additive antiproliferative activity of mifepristone and tamoxifen could be explained at least in part by an additive induction of apoptosis in both estrogen and progesterone receptor positive MCF-7 breast cancer cells. A bcl2 downregulation, the PKC transduction pathway, and TGFbeta1 expression seem to be involved in this additive mechanism of action. Our data further suggest that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of human breast cancer.

Modifications in characteristics and activities of beta-adrenergic receptors and certain parameters of the cyclic nucleotide systems were observed in the hypertrophied heart of the rat chronically treated with T4. These include: 1) an increased number of beta-adrenergic receptors without a change in their affinity, as determined by binding of (-)-[3H]dihydroalprenolol to the membrane; 2) increased sensitivity and magnitude of stimulation of adenylate cyclase in homogenates by isoproterenol, without a change in the basal or NaF-stimulated (total) enzyme activity; 3) decreased formation of cAMP and decreased activation of cAMP-dependent protein kinase in the minced heart stimulated by isoproterenol, probably due to decreased myocardial ATP concentration; 4) decreased activity of cAMP phosphodiesterase in the particulate fraction; 5) decreased activity of cGMP-dependent protein kinase in both the soluble and particulate fractions, accompanied by decreased activity of cAMP-dependent protein kinase in the particulate fraction; 6) decreased activity of the stimulatory modulator of cGMP-dependent protein kinase and, conversely, increased activity of the inhibitory modulator of cAMP-dependent protein kinase; and 7) increased sensitivity accompanied by decreased maximum tension development of the ventricular strip to contract in response to isoproterenol. These alterations largely disappeared upon regression of the hyperthyroid state. It is suggested that the above changes, many of which were the opposite of those reported earlier for the desensitized and hypertrophied rat heart caused by isoproterenol, may in part consitute the molecular basis for the reputed catecholamine supersensitivity of the heart in the hyperthyroid state.