sincalide and calmidazolium

The role of protein kinase C (PKC) in sustained contraction was examined in intestinal circular and longitudinal muscle cells. Initial contraction induced by agonists (CCK-8 and neuromedin C) was abolished by 1) inhibitors of Ca(2+) mobilization (neomycin and dimethyleicosadienoic acid), 2) calmidazolium, and 3) myosin light chain (MLC) kinase (MLCK) inhibitor KT-5926. In contrast, sustained contraction was not affected by these inhibitors but was abolished by 1) the PKC inhibitors chelerythrine and calphostin C, 2) PKC-epsilon antibody, and 3) a pseudosubstrate PKC-epsilon inhibitor. GDPbetaS abolished both initial and sustained contraction, whereas a Galpha(q/11) antibody inhibited only initial contraction, implying that sustained contraction was dependent on activation of a distinct G protein. Sustained contraction induced by epidermal growth factor was inhibited by calphostin C, PKC-alpha,beta,gamma antibody, and a pseudosubstrate PKC-alpha inhibitor. Ca(2+) (0.4 microM) induced an initial contraction in permeabilized muscle cells that was blocked by calmodulin and MLCK inhibitors and a sustained contraction that was blocked by calphostin C and a PKC-alpha,beta,gamma antibody. Thus initial contraction induced by Ca(2+), agonists, and growth factors is mediated by MLCK, whereas sustained contraction is mediated by specific Ca(2+)-dependent and -independent PKC isozymes. G protein-coupled receptors are linked to PKC activation via distinct G proteins.

Topics: Alkaloids; Animals; Antibodies; Benzophenanthridines; Bombesin; Calcium; Carbazoles; Enzyme Inhibitors; Epidermal Growth Factor; Fatty Acids, Unsaturated; Guinea Pigs; Imidazoles; In Vitro Techniques; Indoles; Intestines; Isoenzymes; Muscle Contraction; Muscle, Smooth; Myosin-Light-Chain Kinase; Naphthalenes; Neomycin; Peptide Fragments; Phenanthridines; Phosphodiesterase Inhibitors; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Protein Kinase C-epsilon; Protein Synthesis Inhibitors; Sincalide

Agents like carbachol and cholecystokinin (CCK), that activate chief cell phosphoinositidase C, thereby increasing cell calcium concentration, increase cAMP levels in cholera toxin-treated, but not control, gastric chief cells. In the present study, we found that phorbol esters, like PMA, that activate protein kinase C, also cause augmentation of chief cell cAMP levels. The maximal effect with PMA (100 nM) was about 50% of the maximal response with CCK (10 nM) or carbachol (100 microM). Because protein kinase C is a calcium-dependent enzyme, we examined the effect of modulating cell calcium levels with the ionophore A23187. The ionophore alone caused a dose-dependent augmentation of cAMP levels. Adding 100 nM PMA caused an additive response, such that a maximal cAMP response, equal to that seen with 100 microM carbachol, was observed with 30 nM A23187. Carbachol-, A23187-, and PMA-induced augmentation of cAMP levels was progressively reduced by increasing concentrations of calmidazolium, a calmodulin inhibitor. Combination of phorbol esters that activate protein kinase C with ionophores that increase cell calcium mimics the actions of CCK and carbachol on cAMP levels in cholera toxin-treated chief cells.

Topics: Animals; Calcimycin; Calmodulin; Carbachol; Cholera Toxin; Gastric Mucosa; Guinea Pigs; Imidazoles; In Vitro Techniques; Kinetics; Male; Phorbol 12,13-Dibutyrate; Phorbols; Signal Transduction; Sincalide; Tetradecanoylphorbol Acetate