ucn-1028-c and calmidazolium

Superficial gastric mucosal injury is rapidly repaired by epithelial cell migration. This study aims to characterize the intracellular signal transduction pathways underlying the repair process. Primary monolayer cultures of rabbit gastric epithelial cells were wounded. The measured spontaneous cell migration speed at the edge of the wound was 457+/-89 microm/24 hr. Epidermal growth factor stimulated and genistein (receptor tyrosine protein kinase inhibitor) inhibited cell migration significantly. Down-regulation of protein Kinase C (PKC) with long-term phorbol 12-myristate 13-acsetate or inhibition with calphostin-C significantly inhibited cell migration. Blocking of Ca2+ channels with verapamil and endogenous Ca2+ release with TMB-8 or inhibition of the Ca2+/calmodulin complex with calmidazolium likewise significantly inhibited migration speed and also abolished the rise of [Ca2+]i, which was measured in migrating cells. Modulation of the cAMP-PKA pathway or prostaglandin synthesis had no influence on cell migration. Gastric epithelial cell migration implies activation of receptor tyrosine kinase. It is associated with increased [Ca2+]i and requires an intact Ca2+/calmodulin complex. Intact PKC activity also is needed.

Topics: Animals; Calcium; Calcium Channels; Calmodulin; Cell Movement; Cells, Cultured; Down-Regulation; Epithelial Cells; Gallic Acid; Gastric Mucosa; Imidazoles; Male; Naphthalenes; Prostaglandins; Protein Kinase C; Rabbits; Tetradecanoylphorbol Acetate

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

Gap junctional communication during the progression of cell cycle from quiescent G0 to S phase was examined in cultured clone 9 rat liver cells. The transfer of scrape-loaded fluorescent dye was suppressed immediately after the stimulation of cell cycle progression in a synchronized cell population. Northern blot analysis showed that the temporal disturbance of gap junctional communication in cells passing from G0 to S phase did not result from transcriptional down-regulation of connexin 43. It was also found that the PKC inhibitor, calphostin C, was able to restore intercellular communication in serum stimulated cells. Data suggest a control mechanism by PKC mediated phosphorylation in the regulation of gap junction function which is vulnerable to cell cycling. The loss of gap junctional communication correlated with the increased phosphorylation of connexin 43 on serine residues in clone 9 cells.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cell Communication; Cells, Cultured; Clone Cells; Connexin 43; Enzyme Inhibitors; Fluorescent Dyes; Gap Junctions; Imidazoles; Indoles; Liver; Naphthalenes; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C; Pyrroles; Rats; Resting Phase, Cell Cycle; S Phase; Transcription, Genetic

Calcium/calmodulin-dependent protein kinase-II (CamK-II) is a major neuronal protein which plays a significant role in the cellular process of long-term potentiation (LTP), and vesicular release of neurotransmitters. Here, we show that KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine, a specific cell-permeable inhibitor of CamK-II substantially protected neurons from (1) acute NMDA toxicity and (2) hypoxia/hypoglycemia-induced neuronal injury in fetal rat cortical cultures. KN-62 did not directly inhibit glutamate, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), glycine, or [piperidyl-3,4-(N)]-(N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine) (TCP) binding to rat brain membranes. Finally, KN-62 significantly reduced cellular calcium accumulation following either NMDA challenge or hypoxia/hypoglycemia insult. Our results show that CamK-II plays a key role in mediating some of the biochemical events leading to cell death following an acute excitotoxic insult.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cell Death; Cell Hypoxia; Cell Survival; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fetus; Genistein; Imidazoles; Indole Alkaloids; Isoflavones; Isoquinolines; Kinetics; N-Methylaspartate; Naphthalenes; Neurons; Piperazines; Polycyclic Compounds; Protein Kinase C; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Spectrin

A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN62 (inhibitor of Ca(2+)-calmodulin-dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-acetate (an activator of protein kinase C) and 4 alpha-phorbol 12,13-didecanoate (an inactive phorbol ester) were also tested. At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4 alpha-phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron, and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every parameter that was measured (including viability). These results suggest that these three protein kinases selectively modulate different aspects of neurite development. The university of effects caused by calmodulin inhibition make it impossible to determine if there are specific targets of calmodulin action involved in neurite development. Finally, our data indicate that some superficially similar characteristics of neuronal differentiation, such as neurite initiation and branching, may be control

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Axons; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Carbazoles; Cells, Cultured; Dendrites; Female; Hippocampus; Imidazoles; Indoles; Isoquinolines; Naphthalenes; Neurites; Piperazines; Polycyclic Compounds; Pregnancy; Protein Kinase C; Protein Kinase Inhibitors; Pyramidal Tracts; Pyrroles; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate