Regulation of RAW264 macrophage morphology and spreading: studies with protein kinase C activators, inhibitors and a cyclic AMP analog

Abstract

Protein kinases C and A probably play important roles in membrane signal transduction. To test the role of protein kinases in macrophage spreading, we have measured cell perimeters in the absence and presence of protein kinase C activators, inhibitors and a cAMP analog. Scanning electron microscopy indicated that macrophages spread extensively in the presence of protein kinase C activators. In contrast, protein kinase C inhibitor and dbcAMP (N6-2'-O-di-butyryladenosine 3':5'-cyclic monophosphate AMP) promote a round cell morphology with many surface folds. Quantitative optical microscopy experiments showed that the maximal effects of these reagents were achieved within 30 min. The protein kinase C activators dioctonylglycerol (3 microM), phenylephrine (1 microM), and phorbol myristate acetate (1 micrograms/ml) increased macrophage spreading. Similarly, the calcium ionophore A23187 (1 microgram) increased spreading. In contrast, the protein kinase C inhibitors chlorpromazine (30 microM), sphingosine (10 microM), trifluoroperazine (10 microM), and H-7 (10 microM) significantly reduce macrophage spreading. The analog dibutyryl cAMP (30 microM) abrogates the effects of protein kinase C activators. These data suggest that protein kinase C participates in the regulation of macrophage spreading. Furthermore, the protein kinase A activator dibutyryl cAMP can inhibit the effects of protein kinase C activators.