In human embryonic kidney cells stably expressing the human m3 muscarinic acetylcholine receptor (mAChR) subtype, agonist (carbachol) activation stimulated phospholipase C, increased cytoplasmic calcium concentration, induced tyrosine phosphorylation of various cellular proteins and activated phospholipase D. Bypassing membrane receptors, phospholipase D was activated in these cells by direct activation of protein kinase C by phorbol esters, by direct activation of GTPbinding proteins by AlFy and a stable GTP analogue (in permeabilized cells), by increasing cytoplasmic calcium concentration with the calcium ionophore A23187 and also apparently by tyrosine phosphorylation. In order to identify possible mechanisms by which the m3 mAChR couples to phospholipase D, various inhibitors of protein kinase C, tyrosine kinases and calcium-dependent events were studied. Prevention of an agonist-induced increase in cytoplasmic calcium concentration did not alter the mAChR-induced phospholipase D stimulation. The protein kinase C inhibitors, calphostin C and staurosporine, efficiently prevented phospholipase D activation by phorbol 12-myristate 13-acetate but only partially inhibited the activation induced by the mAChR agonist. Additionally, down-regulation of protein kinase C by prolonged exposure to phorbol 12-myristate 13-acetate abrogated phospholipase D activation by this effector but had only minor or no effects on the response to the mAChR agonist and direct activators of GTP-binding proteins. In contrast, the tyrosine kinase inhibitor genistein abolished the carbachol-induced and A1F;-induced phospholipase D activation but had no effect on enzyme activation by phorbol 12-myristate 13-acetate. The data indicate that phospholipase D in m3 mAChR-expressing human embryonic kidney cells can be activated by various different mechanisms, i.e. receptor agonists, GTP-binding proteins, protein kinase C-dependent and calcium-dependent events and tyrosine phosphorylation. The coupling of m3 mAChR to phospholipase D appears to be largely independent of concomitant phospholipase C activation with subsequent increase in cytoplasmic calcium concentration and protein kinase C activity. The data instead suggest the involvement of an essential protein tyrosine phosphorylation mechanism in phospholipase D activation by the m3 mAChR and heterotrimeric GTP-binding proteins. Abbreviations. mAChR, muscarinic acetylcholine receptor; BAPTA/AM, bis-(0-aminophenoxy)-ethane N, N, N' , "-tetraacetic acid tetra(acetoxymethy1ester); Fura-2/AM, Enzymes. Phospholipase C (EC 3.1.4.3); phospholipase D (EC 3.1.4.4); protein kinase C (EC 2.7.1.37).gers, inositol 1,4,5-trisphosphate, which releases sequestered calcium from a subpopulation of the endoplasmic reticulum, and diacylglycerol, which activates protein kinases C [l, 21. These two signals apparently also participate in the transduction of mitogenic signals across the plasma membrane into the nucleus leading to the stimulation of cell growth, DNA synthesis and cell division [2]. Phosphatidylcholine...