In order to investigate the action mechanism of protein kinase C on K+ channel in osteoblastic cell, effects of phorbol 12,13-dibutyrate on human osteoblast-like cells (G292) were studied by patch clamp technique with cell-attached configuration. In this experiment, 45pS ion channel was dominant in G292 cell line according to their approximate conductances in symmetrical 140mM KCl saline at holding potential of 60mV. In current-voltage relationship, reversal potential was 5.5mV at the condition of potassium enriched saline in the pipette and -27 mV at the condition of standard extracellular saline in the pipette. Phorbol 12, 13-dibutyrate 10 nM increased the open probability of 45 pS channel and staurosporine, an inhibitor of protein kinase C, suppressed this effect. Phorbol 12,13-dibutyrate moved the reversal potential of 45pS channel to more negative potential and increased the single channel current at the same membrame potential. In order to check the activation of protein kinase C in G292 cell by phorbol 12,13-dibutyrate, western blot of protein kinase C was performed. Phorbol 12,13-dibutyrate 0.1 micrometer translocated protein kinase C from cellular compartment to membrane compartment of the cell. These findings suggest that phorbol 12,13-dibutyrate, one of phorbol esters, activate 45pS channel in G292 cell and affect cell membrane potential, that regulate cellular function.
Blotting, Western ; Cell Line ; Cell Membrane ; Humans ; Ion Channels ; Membranes ; Osteoblasts ; Phorbol 12,13-Dibutyrate ; Phorbol Esters ; Potassium ; Protein Kinase C ; Staurosporine

BACKGROUND AND OBJECTIVES: The aim of this study was to determine the effect of ethanol on the regulation of vascular tone. MATERIAL AND METHODS: Using rat aorta ring, isometric contraction and 45Ca uptake were measured. Phorbol 12,13-dibutyrate (PDBu), phenylephrine, KCl were used for the regulation of smooth muscle tone. RESULTS: Ethanol induced transient contraction in rat aorta ring by dose-dependent manner. Ethanol suppressed the dose dependent contractile responses of vascular strip by phenylephrine, KCl and PDBu. Endothelium-dependent relaxation by acetylcholine was inhibited by ethanol. Ethanol depressed 45Ca uptake by high KCl but not by phenylephrine or PDBu in rat aorta. n-butanol selectively suppressed tonic contraction by high KCl, but t-butanol did not at the same concentration of butanol in rat aorta. PDBu-induced contraction was selectively suppressed by n-butanol but not by t-butanol. CONCLUSIONS: These findings suggest that the action of ethanol on phospholipase D is involved in the decreased response of rat aorta strip by vasoconstrictors.
1-Butanol ; Acetylcholine ; Animals ; Aorta* ; Ethanol* ; Isometric Contraction ; Muscle, Smooth* ; Phenylephrine ; Phorbol 12,13-Dibutyrate ; Phospholipase D ; Protein Kinase C ; Rats* ; Relaxation ; tert-Butyl Alcohol ; Vasoconstrictor Agents

Drinking excessive alcohol has been recognized as a risk factor for hypertension. However, the mechanism by which alcohol intake causes hypertension still remains elusive. We tested the hypothesis that ethanol itself acts as a stress factor on vasculature and indirectly modulates vascular contractility. After end of exposure to 1, 2.5 and 5% ethanol for 45 min, rat aortic strips were subjected to contractile responses, immunoblot for Hsp70 and the measurement of levels of myosin light chain phosphorylation. Exposure to 5% ethanol not only augmented contractions to KCl or phenylephrine, but also increased expression of Hsp70 and the levels of myosin light chain phosphorylation. There were no significant differences in contractions produced by 1 micromol/L phorbol 12,13-dibutyrate, a protein kinase C activator, whether the tissues were exposed to 5% ethanol or not. This is the first report to show that even short exposure to ethanol has a delayed effect to increase vascular smooth muscle contractility through a modulation of thick filament regulation. It may be a mechanism by which ingestion of alcohol induces hypertension.
Animals ; Drinking ; Eating ; Ethanol* ; Hypertension ; Muscle, Smooth, Vascular ; Myosin Light Chains ; Phenylephrine ; Phorbol 12,13-Dibutyrate ; Phosphorylation ; Protein Kinase C ; Rats ; Risk Factors

The characteristics of Na+/-dependent cycloleucine uptake was investigated in OK cells with regard to substrate specificity and regulation by protein kinase C (PKC). Inhibition studies with different synthetic and natural amino acids showed a broad spectrum affinity to neutral amino acids regardless of their different side chains including branched or aromatic, indicating that the Na+/-dependent cycloleucine uptake in OK cells is mediated by System B-o or System B degree -like transporter rather than the classical System A or ASC. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate, but not 4 alpha-PMA elicited a time-dependent biphasic stimulation of Na+/-dependent cycloleucine uptake, which produced early transient peak at 30 min and late sustained peak at 180 min. Both the early and late stimulations by PMA were due to an increase in Vmax and not due to a change in Km. PKC inhibitors blocked both the early and late stimulation by PMA, while protein synthesis inhibitors blocked the late stimulation only. These results suggest the existence and regulation by PKC of System B degree or System B degree -like broad spectrum transport system for neutral amino acids in OK cells.
Amino Acid Transport Systems* ; Amino Acids ; Amino Acids, Neutral ; Cycloleucine ; Kidney* ; Opossums* ; Phorbol 12,13-Dibutyrate ; Protein Kinase C ; Protein Synthesis Inhibitors ; Substrate Specificity

BACKGROUND: The goal of this in vitro study was to investigate the effect of lipid emulsion on vasodilation caused by toxic doses of bupivacaine and mepivacaine during contraction induced by a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), in an isolated endothelium-denuded rat aorta. METHODS: The effects of lipid emulsion on the dose-response curves induced by bupivacaine or mepivacaine in an isolated aorta precontracted with PDBu were assessed. In addition, the effects of bupivacaine on the increased intracellular calcium concentration ([Ca²⁺]ᵢ) and contraction induced by PDBu were investigated using fura-2 loaded aortic strips. Further, the effects of bupivacaine, the PKC inhibitor GF109203X and lipid emulsion, alone or in combination, on PDBu-induced PKC and phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) was examined by western blotting. RESULTS: Lipid emulsion attenuated the vasodilation induced by bupivacaine, whereas it had no effect on that induced by mepivacaine. Lipid emulsion had no effect on PDBu-induced contraction. The magnitude of bupivacaine-induced vasodilation was higher than that of the bupivacaine-induced decrease in [Ca²⁺]ᵢ. PDBu promoted PKC and CPI-17 phosphorylation in aortic VSMCs. Bupivacaine and GF109203X attenuated PDBu-induced PKC and CPI-17 phosphorylation, whereas lipid emulsion attenuated bupivacaine-mediated inhibition of PDBu-induced PKC and CPI-17 phosphorylation. CONCLUSIONS: These results suggest that lipid emulsion attenuates the vasodilation induced by a toxic dose of bupivacaine via inhibition of bupivacaine-induced PKC and CPI-17 dephosphorylation. This lipid emulsion-mediated inhibition of vasodilation may be partly associated with the lipid solubility of local anesthetics.
Anesthetics, Local ; Animals ; Aorta ; Blotting, Western ; Bupivacaine* ; Calcium ; Fura-2 ; In Vitro Techniques ; Mepivacaine* ; Muscle, Smooth, Vascular ; Myosin-Light-Chain Phosphatase ; Phorbol 12,13-Dibutyrate ; Phosphorylation ; Protein Kinase C ; Rats ; Solubility ; Vasodilation*

BACKGROUND: The goal of this in vitro study was to investigate the effect of lipid emulsion on vasodilation caused by toxic doses of bupivacaine and mepivacaine during contraction induced by a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), in an isolated endothelium-denuded rat aorta. METHODS: The effects of lipid emulsion on the dose-response curves induced by bupivacaine or mepivacaine in an isolated aorta precontracted with PDBu were assessed. In addition, the effects of bupivacaine on the increased intracellular calcium concentration ([Ca²⁺]ᵢ) and contraction induced by PDBu were investigated using fura-2 loaded aortic strips. Further, the effects of bupivacaine, the PKC inhibitor GF109203X and lipid emulsion, alone or in combination, on PDBu-induced PKC and phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) was examined by western blotting. RESULTS: Lipid emulsion attenuated the vasodilation induced by bupivacaine, whereas it had no effect on that induced by mepivacaine. Lipid emulsion had no effect on PDBu-induced contraction. The magnitude of bupivacaine-induced vasodilation was higher than that of the bupivacaine-induced decrease in [Ca²⁺]ᵢ. PDBu promoted PKC and CPI-17 phosphorylation in aortic VSMCs. Bupivacaine and GF109203X attenuated PDBu-induced PKC and CPI-17 phosphorylation, whereas lipid emulsion attenuated bupivacaine-mediated inhibition of PDBu-induced PKC and CPI-17 phosphorylation. CONCLUSIONS: These results suggest that lipid emulsion attenuates the vasodilation induced by a toxic dose of bupivacaine via inhibition of bupivacaine-induced PKC and CPI-17 dephosphorylation. This lipid emulsion-mediated inhibition of vasodilation may be partly associated with the lipid solubility of local anesthetics.
Anesthetics, Local ; Animals ; Aorta ; Blotting, Western ; Bupivacaine* ; Calcium ; Fura-2 ; In Vitro Techniques ; Mepivacaine* ; Muscle, Smooth, Vascular ; Myosin-Light-Chain Phosphatase ; Phorbol 12,13-Dibutyrate ; Phosphorylation ; Protein Kinase C ; Rats ; Solubility ; Vasodilation*

L-type Ca2+ channels play an important role in regulating cytosolic Ca2+ and thereby regulating hormone secretions in neuroendocrine cells. Since hormone secretions are also regulated by various kinds of protein kinases, we investigated the role of some kinase activators and inhibitors in the regulation of the L-type Ca2+ channel currents in rat pituitary GH3 cells using the patch-clamp technique. Phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, and vanadate, a protein tyrosine phosphatase (PTP) inhibitor, increased the Ba2+ current through the L-type Ca2+ channels. In contrast, bisindolylmaleimide I (BIM I), a PKC inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, suppressed the Ba2+ currents. Forskolin, an adenylate cyclase activator, and isobutyl methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, reduced Ba2+ currents. The above results show that the L-type Ca2+ channels are activated by PKC and PTK, and inhibited by elevation of cyclic nucleotides such as cAMP. From these results, it is suggested that the regulation of hormone secretion by various kinase activity in GH3 cells may be attributable, at least in part, to their effect on L-type Ca2+ channels.
Adenylyl Cyclases ; Animals ; Cell Line* ; Colforsin ; Cytosol ; Genistein ; Neuroendocrine Cells ; Nucleotides, Cyclic ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; Phosphotransferases ; Protein Kinase C ; Protein Kinases* ; Protein Tyrosine Phosphatases ; Protein-Tyrosine Kinases ; Rats* ; Vanadates

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism

The present study was attempted to examine the effect of staurosporine (STS) on secretion of catecholamines (CA) evoked by cholinergic stimulation and membrane depolarization from the isolated perfused rat adrenal gland and to establish its mechanism of action. The perfusion of STS (3 X 10(-7) ~3 X 10(-8) M) into an adrenal vein for 20 min produced a dose-dependent inhibition in CA secretion evoked by ACh (5.32 X 10(-3) M), high K+ (5.6 X 10(-2) M), DMPP (10(-4) M for 2 min), McN-A-343 (10(-4) M for 2 min), cyclopiazonic acid (10(-5) M for 4 min) and Bay-K-8644 (10(-5) M for 4 min). Also, in the presence of tamoxifen (2 X 10(-6) M), which is known to be a protein kinase inhibitor, CA secretory responses evoked by ACh, high K+, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly depressed. However, in adrenal glands preloaded with STS (10(-7) M) under the presence of phorbol-12,13-dibutyrate (10(-7) M), a specific activator of protein kinases (for 20 min), the inhibitory effect of STS on CA secretory responses evoked by ACh, high K+, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid was greatly recovered to the extent of the control release as compared to those in the presence of STS only. These results demonstrate that STS causes the marked inhibition of CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as by membrane depolarization, indicating strongly that this effect may be mediated by inhibiting influx of extracellular calcium and release in intracellular calcium in the rat adrenomedullary chromaffin cells through preventing activation of protein kinases. Furthermore, these findings also suggest that these STS-sensitive protein kinases play a modulatory role partly in regulating the rat adrenomedullary CA secretion.
(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride ; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; Adrenal Glands* ; Animals ; Calcium ; Catecholamines ; Chromaffin Cells ; Dimethylphenylpiperazinium Iodide ; Membranes* ; Perfusion ; Phorbol 12,13-Dibutyrate ; Protein Kinases ; Rats* ; Staurosporine* ; Tamoxifen ; Veins

An environmental pollutant, tetrachloro dibenzo dioxin (TCDD) is known to illicit the cognitive disability and motor dysfunction in the developing brain. TCDD induced effects leading to neurodevelopmental and neurobehavioral deficit may have been defined, however underlying molecular mechanism and possible intracellular targets remain to be elucidated. In this study, we attempted to analyze TCDD-induced neurotoxic effects in the granule cells from cerebellum where certain cognitive abilities and motor function command are known to be excuted. [3H]PDBu, (phorbol 12,13-dibutyrate) binding assay indicated that TCDD induced a dose-dependent increase of total PKC activity and its induction was the aryl hydrocarbon receptor (AhR) dependent and N-methyl-D-aspartate receptor (NMDAR) independent. TCDD also caused the translocation of both PKC-alpha and -epsilon in a dose-dependent manner but associated with different receptors; PKC-alpha via AhR but not PKC-epsilon indicating an isozyme-specific pattern of the induction. Increase of the ROS formation was also observed in the cells treated with TCDD in a dose-dependent and an AhR-dependent manner. The treatment of the cells with the diamino dicyano-bis(2-aminophenylthio) butadiene (U0126, MEK-1/2 inhibitor), dizocilpine maleate (MK-801, non-competitive N-methyl-D-aspartate glutamate receptor antagonist) and vitamin E attenuated the TCDD-induced ROS production indicating that TCDD-induced ROS formation may be associated with activation of ERK-1/2 in the MAP kinase pathway or the NMDA receptor. TCDD also increased [Ca2+]i, which is associated with ROS formation and PKC activation in the cerebellar granule cells. It is suggested that TCDD activates the NMDA receptor, which may induce a sustained increase of [Ca2+]i in neurons followed by the ROS formation. Our findings may contribute to understanding the mechanism of TCDD-related neurotoxicity, thereby improving the health risk assessment of neurotoxic compounds in humans.
Animals ; Binding, Competitive ; Butadienes/pharmacology ; Carcinogens/pharmacology ; Cerebellum/cytology/*drug effects/enzymology ; Dizocilpine Maleate/pharmacology ; Environmental Pollutants/*toxicity ; Enzyme Activation/drug effects ; Enzyme Inhibitors/pharmacology ; Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism ; Neuroprotective Agents/pharmacology ; Nitriles/pharmacology ; Phorbol 12,13-Dibutyrate/pharmacology ; Protein Kinase C/*metabolism ; Protein Transport ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Receptors, Aryl Hydrocarbon/metabolism ; Receptors, N-Methyl-D-Aspartate/metabolism ; Research Support, Non-U.S. Gov't ; Tetrachlorodibenzodioxin/*toxicity