None of the channel blockers reduced the release of NE thus demon strating that the decrease of SMD is mediated by postjunc tional smooth muscle mechanisms. The EFS induced SMD remained unaffected by the InsP3 receptor inhibitors 2APB and xestospongin C, ruling out a substantial role of InsP3 receptor mediated Ca2 release in this response. Therefore, smooth muscle p53/MDM2 interaction cell chloride channels and/or NCSS appear to be the primary activities that mediate the SMD in the canine mesenteric vein. Furthermore, we identified a novel signal transduction mechanism governing the slow membrane depolariza tion, which involves enzymes of the PKC family. First, we used two PKC inhibitors with distinct PKC isozymes selec tivity.

Chelerythrine, for example, acts on the conserved catalytic domain of PKC as a competitive inhibitor with respect to the phosphate acceptor and a non competitive inhibitor with respect to ATP. Therefore, cheleryth rine can inhibit PKCs of all classes and effects mediated by them, including the slow depolarization in mesenteric veins. In contrast, calphostin C interacts with the regula tory domain of PKC by competing for the binding site of diacylglycerol and phorbol esters, but not Ca2 and phos pholipids. Thus, calphostin C is more specific inhibitor for classical and novel, and less specific for atypical PKC. In the present study, chelerythrine reduced, while calphostin C had no effect on the SMD. The lack of effect of calphostin C indicates that the EFS induced membrane depolarization is most likely mediated by atypical PKC.

We provided experimental support of this possibility by showing a distinct activation of the atypical PKC? in mesenteric veins incubated with exogenous NE and cloni dine. Although further studies are needed to identify the precise PKC isoform contributing to the ?2 adrenocep tor mediated SMD, the present study clearly suggests a PKC isozyme specific regulation of the EFS induced slow membrane depolarization in canine mesenteric vein. The observation that atypical PKCs are involved in the SMC allowed us to speculate about the identity of some specific signaling molecules that function upstream of PKC?, and could mediate the SMD as well. PI3Ks, and par ticularly PI3K, was an obvious candidate for several rea sons. Firstly, PI3K is essential for activation of various ion transporters including L type Ca2 channels and metabotropic nonselective cation channels. Sec ondly, PI3K activates the phosphoinositide dependent protein kinase 1, and although the latter can modulate classical and novel PKC iso form, activation Drug_discovery of atypical PKCs, and particularly PKC?, is a highly specific event downstream of PI3K PDK1.