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Treatment with 300 nM phorbol 12-myristate 13-acetate (PMA) transforms polygonal-shaped cultured astrocytes into process-bearing cells and produces a shift in protein kinase C (PK-C) from the cytosol to the membrane. Exposure to PMA also produces increases in the phosphorylation of several proteins including vimentin, glial fibrillary acidic protein (GFAP), an acidic 80,000 molecular weight protein, and two 30,000 molecular weight proteins (pI 5.5 and 5.7). The effects of PMA on the translocation of PK-C and on protein phosphorylation precede the PMA-induced changes in astrocyte morphology, and a close correlation exists between the concentration of PMA necessary to elicit half-maximal and maximal effects on the shift of PK-C to the membrane and on protein phosphorylation. In addition, the PMA-induced alterations in cell morphology are not permanent, and within 24 hr after PMA treatment the cells have reverted almost to their original morphology. A second exposure to PMA at this time fails to elicit further change in cell shape and is also incapable of producing increases in the phosphorylation of proteins. It was determined that there is little, if any, PK-C present in these PMA-pretreated cells. The morphological responsiveness to PMA gradually returns in 5 to 8 days after the initial treatment with PMA, and this is accompanied by the recovery of PK-C activity and the phosphorylation response. Therefore, these studies suggest that the effect of PMA on astrocyte morphology is mediated by the activation of PK-C and subsequent protein phosphorylation.

Analogues of clozapine, some prepared by a novel, shorter synthesis than those described previously, were evaluated as potential antipsychotic agents using clozapine binding sites in rat forebrain that are nonmuscarinic and nondopaminergic in nature and from which [3H]clozapine is displaced by known antipsychotic agents. The binding of clozapine to muscarinic sites is inhibited in the presence of atropine. Displacement of [3H]clozapine by an analogue of clozapine in the presence of atropine represents nonmuscarinic binding, while displacement in the absence of atropine represents muscarinic (cholinergic) plus nonmuscarinic binding. The relative affinity of the analogues for dopamine binding sites was determined by their ability to displace [3H]spiroperidol from binding sites in rat caudate nuclei. To the extent which binding affinity for nonmuscarinic clozapine sites in rat forebrain reflects the antipsychotic potential of a particular drug, dibenzo-5H-cycloheptene analogues of clozapine are as effective as clozapine itself. Strong binding to nonmuscarinic clozapine sites is not dependent on the presence of a chlorine atom on th tricyclic system. One or both of the nitrogen atoms in the dibenzo-5H-[1,4]diazepine ring of clozapine appear to be necessary for the strong inhibition of clozapine binding to spiroperidol sites in rat caudate nuclei. Anticholinergic activity is substantially higher for clozapine and its dibenz[1,4]oxazepine analogue than for its benzo-5H-cycloheptene analogue.

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.