epidermal-growth-factor 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

Epidermal growth factor (EGF) stimulates prolactin (PRL) gene expression in GH3 cells in a Ca2+-dependent manner (White, B. A., and Bancroft, F. C. (1983) J. Biol. Chem. 258, 4618-4622). The present report shows that the phenothiazine, calmidazolium (compound R 24571), blocks the ability of EGF plus Ca2+ to increase levels of PRL mRNA. Calmidazolium inhibition of this response is dose dependent in the range of 0.05-1.00 microM. Total inhibition of the response was consistently obtained at a level of calmidazolium (0.5 microM) that had no effect on total cytoplasmic RNA synthesis, total cytoplasmic protein synthesis, cell viability, or extent of EGF plus Ca2+-induced cell aggregation. The drug inhibited the increase in PRL mRNA when given immediately before or 48 h after treatment with EGF plus Ca2+. Another calmodulin inhibitor, W13, similarly blocked the ability of EGF plus Ca2+ to stimulate PRL mRNA, whereas the less active analog, W12, had little effect. These results implicate Ca2+-binding proteins such as calmodulin in the mechanism of action of EGF in GH3 cells, and, therefore, provide further evidence for a role of intracellular Ca2+ in the regulation of the expression of a specific eukaryotic gene, the PRL gene.

Topics: Animals; Calcium; Calmodulin; Cell Line; Epidermal Growth Factor; Gene Expression Regulation; Imidazoles; Kinetics; Pituitary Neoplasms; Prolactin; Rats; RNA, Messenger; Sulfonamides; Thyrotropin-Releasing Hormone

A single exposure to a wide range of concentrations (10(-15)-10(-4) mol/l) of the tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phenobarbital (PB) significantly stimulated the flow into DNA synthesis and mitosis of neonatal rat hepatocytes in 4-day-old primary cultures kept in high-calcium (1.8 mmol/l) Eagle's FBS-MEM. Maximal effects were observed in the dose range 10(-12)-10(-6) mol/l. Moreover, both xenobiotics retained their full mitogenic effectiveness when given to hepatocytes incubated in calcium-deficient (0.01 mmol/l) FBS-MEM, thereby evoking a neoplastic phenotype in otherwise normal (i.e., non-initiated) cells. Proliferation kinetic studies showed that TPA and PB acted according to the actual cell cycle setting of the cells: they committed a fraction of the quiescent (GO) hepatocytes to grow, and enabled hepatocytes previously poised at the G1/S and G2/M boundaries to start cycling again, but exerted no influence on liver cells already engaged by themselves in active cycling. These diverse activities of TPA and PB were independent of serum (growth) factors; they were also fully elicited in hepatocytes grown in synthetic media (Eagle's MEM or HiWoBa2000), and were not changed by the addition of a mitogenically effective dose (10(-10) mol/l) of epidermal growth factor/urogastrone (EGF) with or without FBS. Conversely, the addition of the specific plasmalemmal calcium chelator, EGTA, or displacer, La3+, or of the anticalmodulin drugs, W-13 and calmidazolium (or R24571: an agent hardly entering cells) fully inhibited the mitogenic activities of tumour promoters in quiescent and intra-cycle-blocked hepatocytes, while having no effect on untreated or xenobiotic-treated liver cells already cycling by themselves. Such inhibitory effects were in all instances independent of the actual extracellular calcium concentration and took place according to time-related double kinetics. Hence, xenobiotic-activated processes taking place at the plasmalemmal level and involving the activation of calcicalmodulin-dependent enzymes were of critical importance for both the G0/G1 and G1/S transitions in primary hepatocytes.

Topics: Animals; Calmodulin; Carcinogens; Cell Division; Egtazic Acid; Epidermal Growth Factor; Female; Imidazoles; In Vitro Techniques; Lanthanum; Liver; Male; Phenobarbital; Phorbols; Rats; Rats, Inbred Strains; Stimulation, Chemical; Sulfonamides; Tetradecanoylphorbol Acetate