ucn-1028-c and methoctramine

This study investigated regulation of L-type calcium channels (Cav1.2b) by acetylcholine (ACh) in rabbit portal vein myocytes. Whole-cell currents were recorded using 5 mmol/L barium as charge carrier. ACh (10 micromol/L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4-DAMP (100 nmol/L) but was blocked by the M2 receptor antagonist methoctramine (5 micromol/L). The classical and novel protein kinase C (PKC) antagonist calphostin C (50 nmol/L) abolished ACh responses, whereas the classical PKC antagonist Gö6976 (200 nmol/L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 micromol/L), by the c-Src inhibitor PP2 (10 micromol/L) (but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G-protein subunit Gbetagamma. Cells dialyzed with c-Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c-Src is downstream of PI3K. Phorbol 12,13-dibutyrate (PDBu, 0.1 micromol/L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Gö6976. The PDBu response was also reduced by PP2, and the PP2-insensitive component was blocked by Gö6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to Gbetagamma, PI3K, a novel PKC, and c-Src. PDBu stimulates the novel PKC/c-Src pathway along with a second pathway that is independent of c-Src and involves a classical PKC.

Topics: Acetylcholine; Animals; Barium; Calcium Channels, L-Type; Carbazoles; Cells, Cultured; Chromones; Class Ib Phosphatidylinositol 3-Kinase; Diamines; Enzyme Inhibitors; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; Indoles; Ion Channel Gating; Ion Transport; Isoenzymes; Male; Morpholines; Muscarinic Agonists; Muscarinic Antagonists; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Naphthalenes; Patch-Clamp Techniques; Phorbol 12,13-Dibutyrate; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Piperidines; Portal Vein; Protein Kinase C; Proto-Oncogene Proteins pp60(c-src); Pyrimidines; Rabbits; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Signal Transduction