ucn-1028-c and chelerythrine

In previous publications we were able to demonstrate the exposure of phosphatidylserine (PS) in the outer membrane leaflet after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA), phorbol-12 myristate-13acetate (PMA), or 4-bromo-A23187 (A23187). It has been concluded that three different mechanisms are responsible for the PS exposure in human RBCs: (i) Ca2+-stimulated scramblase activation (and flippase inhibition) by A23187, LPA, and PMA; (ii) PKCα activation by LPA and PMA; and (iii) enhanced lipid flip flop caused by LPA. Further studies aimed to elucidate interconnections between the increased Ca2+ content, scramblase- and PKCα-activation. In addition, the role of the Ca2+-activated K+ channel (Gardos channel) activity in the process of PS exposure needs to be investigated.. The intracellular Ca2+ content and the PS exposure of RBCs have been investigated after treatment with LPA (2.5 µM), PMA (6 µM), or A23187 (2 µM). Fluo-4 and annexin V-FITC has been used to detect intracellular Ca2+ content and PS exposure, respectively. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry. Inhibitors of the scramblase, the PKCα, and the Gardos channel have been applied.. The percentage of RBCs showing PS exposure after activation with LPA, PMA, or A23187 is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 as well as after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced when the scramblase and the PKCα are inhibited simultaneously. Additionally, the inhibition of the Gardos channel using charybdotoxin resulted in a significant reduction of the percentage of RBCs showing PS exposure under all conditions measured. Similar results were obtained when the Gardos channel activity was suppressed by increased extracellular K+ content.. PS exposure is mediated by the Ca2+-dependent scramblase but also by PKCα activated by LPA and PMA in a Ca2+-dependent and a Ca2+-independent manner. Furthermore, we hypothesize that a hyperpolarisation of RBCs caused by the opening of the Gardos channel is essential for the scramblase activity as well as for a fraction of the LPA-induced Ca2+ entry.

Topics: Annexin A5; Benzophenanthridines; Calcimycin; Calcium; Cells, Cultured; Charybdotoxin; Erythrocyte Count; Erythrocytes; Gene Expression Regulation; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Lysophospholipids; Methomyl; Naphthalenes; Phosphatidylserines; Phospholipid Transfer Proteins; Protein Kinase C-alpha; Signal Transduction; Tetradecanoylphorbol Acetate

Mitogen-activated protein (MAP) and tyrosine kinases play an important role in regulating smooth muscle contraction of the gastrointestinal (GI) tract. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. Thus, the role of MAP and tyrosine kinases on the pacemaker potentials of colonic ICCs was investigated.. Cultured ICCs were prepared from mice colons, and their pacemaker potentials were recorded using whole-cell patch clamping.. In current-clamping mode, colonic ICCs displayed spontaneous pacemaker potentials. SB203580 (a p38 MAP kinase inhibitor), SP600125 (a c-jun NH2-terminal kinase (JNK) inhibitor), genistein and herbimycin A (tyrosine kinase inhibitors) blocked the generation of pacemaker potentials. However, PD98059 (a p42/44 MAP kinase inhibitor) had no effects on pacemaker potentials. LY-294002 (phosphoinositide 3-kinase inhibitor) also reduced the pacemaker potential frequency but calphostin C and chelerythrine (protein kinase C inhibitors) had no effects. However, PD98059, SB203589, SP600125, genistein, herbimycin A, LY-294002, and calphostin C had no effect on normal pacemaker activity in small intestinal ICCs.. Endogenous p38 MAP kinases, JNKs, tyrosine kinases, and PI3-kinases participate in the generation of pacemaker potentials in colonic ICCs but not in ICCs of the small intestine.

Topics: Animals; Anthracenes; Benzophenanthridines; Cells, Cultured; Chromones; Colon; Flavonoids; Genistein; Imidazoles; Interstitial Cells of Cajal; Intestine, Small; Membrane Potentials; Mice; Mitogen-Activated Protein Kinases; Morpholines; Naphthalenes; Protein-Tyrosine Kinases; Pyridines; Rifabutin

Salmonid fish sperm motility is known to be suppressed in millimolar concentrations of extracellular K(+), and dilution of K(+) upon spawning triggers cAMP-dependent signaling for motility initiation. In a previous study, however, we demonstrated that suspending sperm in a 10% glycerol solution and subsequent dilution into a low-osmotic solution induced motility independently of extracellular K(+) and cAMP. In the present study, we further investigated the glycerol-induced motility mechanism. We found that treatment with solutions consisting of organic or inorganic ions, as well as glycerol, induced sperm motility in an osmolarity-dependent manner. Elimination of intracellular Ca(2+) by BAPTA-AM significantly inhibited glycerol-treated sperm motility, whereas removal of extracellular Ca(2+) by EGTA did not. Monitoring intracellular Ca(2+), using fluo-4, revealed that intracellular Ca(2+) increased when sperm were suspended in hypertonic solutions, and a subsequent dilution into a hypotonic solution led to a decrease in intracellular Ca(2+) concomitant with motility initiation. In addition, upon dilution of sperm into a hypertonic glycerol solution prior to demembranation, the motility of demembranated sperm was reactivated in the absence of cAMP. The motility recovery suggests that completion of axonemal maturation occurred during exposure to a hypertonic environment. As a result, it is likely that glycerol treatment of sperm undergoing hypertonic shock causes mobilization of intracellular Ca(2+) from the intracellular Ca(2+) store and also causes maturation of axonemal proteins for motility initiation. The subsequent dilution into a hypotonic solution induces a decrease in intracellular Ca(2+) and flagellar movement. This novel mechanism of sperm motility initiation seems to act in a salvaging manner for the well-known K(+)-dependent pathway.

Topics: Animals; Benzophenanthridines; Calcium; Egtazic Acid; Estrenes; Flagella; Glycerol; Hypertonic Solutions; Isoquinolines; Male; Naphthalenes; Osmotic Pressure; Pyrrolidinones; Salmonidae; Sperm Motility; Sulfonamides

Exposure to drugs of abuse or stress results in adaptation in the brain involving changes in gene expression and transcription factors. Morphine withdrawal modulates gene expression through various second-messenger signal transduction systems. Here, we investigated changes in activation of the transcription factor, cAMP-response element binding protein (CREB), in the hypothalamic paraventricular nucleus (PVN) and the kinases that may mediate the morphine withdrawal-triggered activation of CREB and the response of the hypothalamic-pituitary-adrenocortical (HPA) axis after naloxone-induced morphine withdrawal.. The effects of morphine dependence and withdrawal, phosphorylated CREB (pCREB), corticotrophin-releasing factor (CRF) expression in the PVN and HPA axis activity were measured using immunoblotting, immunohistochemistry and radioimmunoassay in controls and in morphine-dependent rats, withdrawn with naloxone and pretreated with vehicle, calphostin C, chelerythrine (inhibitors of protein kinase C (PKC) or SL-327 [inhibitor of extracellular signal regulated kinase (ERK) kinase]. In addition, changes in PKCα and PKCγ immunoreactivity were measured after 60 min of withdrawal.. In morphine-withdrawn rats, pCREB immunoreactivity was increased within CRF immunoreactive neurons in the PVN and plasma corticosterone levels were raised. SL-327, at doses that reduced the augmented pERK levels in the PVN, did not attenuate the rise in pCREB immunoreactivity or plasma corticosterone secretion. In contrast, PKC inhibition reduced the withdrawal-triggered rise in pCREB, pERK1/2 and corticosterone secretion.. PKC mediated, in part, both CREB activation and the HPA response to morphine withdrawal. The ERK kinase/ERK pathway might not be necessary for either activation of CREB or HPA axis hyperactivity.

Topics: Animals; Benzophenanthridines; Corticotropin-Releasing Hormone; Cyclic AMP Response Element-Binding Protein; Extracellular Signal-Regulated MAP Kinases; Hypothalamo-Hypophyseal System; Male; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase 3; Morphine; Morphine Dependence; Naloxone; Naphthalenes; Paraventricular Hypothalamic Nucleus; Phosphorylation; Pituitary-Adrenal System; Protein Kinase C; Rats; Rats, Sprague-Dawley; Substance Withdrawal Syndrome

The proinflammatory marker C-reactive protein has been demonstrated to play a role in the development of atherosclerosis. Endothelin-1 and nitric oxide homeostasis is crucial for normal vasomotor function, limiting inflammatory activation and maintaining a nonthrombogenic endothelial surface. In addition to its vasoactive properties, endothelin-1 is also an inflammatory cytokine. We have previously demonstrated that C-reactive protein impairs endothelial cell nitric oxide production. Protein kinase C, an important signal transducer within the cell, is involved in several cellular responses to external stimuli. We therefore sought to determine whether endothelin-1 exposure modulates C-reactive protein's effects on nitric oxide production via protein kinase C.. Endothelial cells were incubated with C-reactive protein (200 microg), endothelin-1 (100 nM), C-reactive protein + endothelin-1, or phosphate-buffered saline solution (control) for 24 hours. After exposure, endothelial nitric oxide synthase expression was determined in addition to total nitric oxide production and protein kinase C translocation and activity.. Endothelial nitric oxide synthase protein expression was reduced following incubation with C-reactive protein and endothelin-1 treatment compared with baseline by 40% and 45%, respectively (P = .04); however, no additive effects were seen with coincubation. C-reactive protein produced a 47% decrease in nitric oxide production compared with control. Coincubation with endothelin-1 resulted in a synergistic 70% reduction in nitric oxide production (P = .001). C-reactive protein exposure inhibited translocation of protein kinase C lambda compared with control (P = .01). Furthermore, coincubation of C-reactive protein with endothelin-1 led to a synergistic inhibition of protein kinase C lambda translocation (P = .01). C-reactive protein exposure reduced protein kinase C activity by 40% compared with control (P = .02), although coincubation with endothelin-1 had a synergistic reduction in activity (P = .02).. Our results indicate that endothelin-1 exposure accentuated C-reactive protein's impairment of endothelial nitric oxide production via synergistic inhibition of protein kinase C lambda translocation and activity. Our investigations suggest that endothelin-1 inhibition and protein kinase C stimulation may provide a novel therapeutic strategy to improve vascular nitric oxide homeostasis and mitigate the proatherosclerotic effects of C-reactive protein.

Topics: Alkaloids; Atherosclerosis; Benzophenanthridines; Bosentan; C-Reactive Protein; Cells, Cultured; Cytokines; Endothelin-1; Endothelium, Vascular; Homeostasis; Humans; Naphthalenes; Nitric Oxide; Nitric Oxide Synthase Type III; Protein Kinase C; Saphenous Vein; Sulfonamides

The mechanisms responsible for increased expression of TNF-alpha in skeletal muscle cells in diabetic states are not well understood. We examined the effects of the saturated acid palmitate on TNF-alpha expression. Exposure of C2C12 skeletal muscle cells to 0.75 mm palmitate enhanced mRNA (25-fold induction, P < 0.001) and protein (2.5-fold induction) expression of the proinflammatory cytokine TNF-alpha. This induction was inversely correlated with a fall in GLUT4 mRNA levels (57% reduction, P < 0.001) and glucose uptake (34% reduction, P < 0.001). PD98059 and U0126, inhibitors of the ERK-MAPK cascade, partially prevented the palmitate-induced TNF-alpha expression. Palmitate increased nuclear factor (NF)-kappaB activation and incubation of the cells with the NF-kappaB inhibitors pyrrolidine dithiocarbamate and parthenolide partially prevented TNF-alpha expression. Incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C (PKC), abolished palmitate-induced TNF-alpha expression, and restored GLUT4 mRNA levels. Palmitate treatment enhanced the expression of phospho-PKCtheta, suggesting that this PKC isoform was involved in the changes reported, and coincubation of palmitate-treated cells with the PKC inhibitor chelerythrine prevented the palmitate-induced reduction in the expression of IkappaBalpha and insulin-stimulated Akt activation. These findings suggest that enhanced TNF-alpha expression and GLUT4 down-regulation caused by palmitate are mediated through the PKC activation, confirming that this enzyme may be a target for either the prevention or the treatment of fatty acid-induced insulin resistance.

Topics: Alkaloids; Animals; Benzophenanthridines; Biological Transport; Cell Line; Enzyme Inhibitors; Glucose; Mice; Muscle, Skeletal; Naphthalenes; NF-kappa B; Palmitic Acid; Phenanthridines; Protein Kinase C; Pyrrolidines; Sesquiterpenes; Thiocarbamates; Tumor Necrosis Factor-alpha

Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone and pathological states such as ischemia/reperfusion (I/R) injury, coronary vasospasm, and cardiac allograft vasculopathy. We assessed the effects of elevated ET-1 levels as seen after I/R to determine if ET-1 modulates nitric oxide (NO) production via the translocation of specific protein kinase C (PKC) isoforms.. Human saphenous vein endothelial cells (HSVECs) (n=8) were incubated with ET-1 or phosphate-buffered saline (PBS) for 24 hours. NO production was determined in the supernatant by measuring nitrate/nitrite levels. Protein expression of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), caveolin-1 and PKC were determined. Lastly, PKC translocation and activity were assessed after exposure to the drug of interest. HSVECs exposed to ET-1 displayed decreased NO production. PKC inhibition reduced NO production, whereas PKC activation increased production. NO production was maintained when HSVECs exposed to ET-1 were treated with the PKC agonist, PMA. eNOS protein expression was reduced after ET-1 treatment. PKC inhibition also downregulated eNOS protein expression, whereas PMA upregulated expression. ET-1 exposure led to a significant increase in PKCdelta and PKCalpha translocation compared with control, whereas translocation of PKClambda was inhibited. ET-1 exposure significantly reduced overall PKC activity compared with control.. Our study demonstrates that high levels of ET-1 impair endothelial NO production via an isoform-specific PKC-mediated inhibition of eNOS expression. ET-1 antagonism with bosentan stimulates translocation of PKClambda and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.

Topics: Alkaloids; Benzophenanthridines; Bosentan; Caveolin 1; Cell Membrane; Cells, Cultured; Endothelial Cells; Endothelin-1; Endothelium, Vascular; Enzyme Activation; Enzyme Induction; Homeostasis; Isoenzymes; Naphthalenes; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Phenanthridines; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Protein Transport; Saphenous Vein; Sulfonamides; Tetradecanoylphorbol Acetate

The main objectives of this study were to investigate the effects of deoxycorticosterone acetate (DOCA)-induced hypertension on the aortic and mesenteric vascular responses to vasodilator and vasoconstrictor agents and also to elucidate whether protein kinase C (PKC) was involved in these responses, by using chelerythrine and calphostin C, the inhibitors of protein kinase C. Hypertension was induced in male Sprague-Dawley rats (200-250 g) by DOCA-salt injection [20 mg/kg, twice weekly for 5 weeks, subcutaneously (s.c.)] and NaCl (1%) was added to their drinking water. Control rats received a saline injection (0.5 ml/kg, twice weekly for 5 weeks, s.c.), then the animals were anaesthetised [thiopental, 30 mg/kg, intraperitoneally (i.p.)] and the arterial blood pressure was measured. Mean arterial blood pressure in control and hypertensive rats were 98+/-7.5 and 163+/-3.5 mmHg, respectively (P<0.0001). In the in vitro studies, rings of descending aorta and mesenteric beds were precontracted with phenylephrine and then concentration-response curves to acetylcholine and sodium nitroprusside were constructed. In the tissue removed from hypertensive rats, the responses to acetylcholine, but not to sodium nitroprusside, were significantly reduced. However, addition of chelerythrine (10 microM) or calphostin C (100 nM) to the organ bath significantly restored these impaired responses. Our data suggest that protein kinase C plays a crucial role in the endothelial dysfunction induced by hypertension.

Topics: Acetylcholine; Alkaloids; Anesthesia; Animals; Aorta, Thoracic; Benzophenanthridines; Blood Pressure; Body Weight; Desoxycorticosterone; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Heart; Heart Rate; Hypertension; In Vitro Techniques; Male; Naphthalenes; Nitroprusside; Organ Size; Phenanthridines; Phenylephrine; Protein Kinase C; Rats; Rats, Sprague-Dawley; Splanchnic Circulation; Vasodilator Agents

Supersensitivity to muscarinic, kappa- and mu-opioid agents modulating cholinergic neurons in the guinea pig colon develops after chronic sympathetic denervation. A possible role for protein kinase C (PKC) in contributing to development of these sensitivity changes was investigated. The PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced acetylcholine (ACh) overflow in preparations obtained from normal animals. The facilitatory effect of PMA was significantly reduced after prolonged exposure to the phorbol ester and by the PKC inhibitors, chelerythrine and calphostin C. Subsensitivity to the facilitatory effect of PMA developed after chronic sympathetic denervation. In this experimental condition, immunoblot analysis revealed reduced levels of PKC in myenteric plexus synaptosomes. The facilitatory effect of the muscarininc antagonist, scopolamine, on ACh overflow was significantly reduced by the phospolipase C (PLC) inhibitor, U73122, chelerythrine and calphostin C, both in normal and denervated animals. However, in both experimental groups, PLC antagonists and PKC antagonists did not affect the inhibitory effect of the muscarinic agonist, oxotremorine-M on ACh overflow. The inhibitory effects of U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on ACh overflow significantly increased in the presence of U73122, chelerythrine and calphostin C in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. These results indicate that activation of PKC enhances ACh release in the myenteric plexus of the guinea pig colon. At this level, chronic sympathetic denervation entails a reduced efficiency of the enzyme. In addition, PKC is involved in the inhibitory modulation of ACh release mediated by muscarinic-, kappa- and mu-opioid receptors, although with different modalities. Muscarinic receptors inhibit PKC activity, whereas kappa- and mu-opioid receptors increase PKC activity. Both the inhibitory and the facilitatory effect on PKC involve modulation of PLC activity. The possibility that the change in PKC activity represents one of the biochemical mechanisms at the basis of development of sensitivity changes to opioid and muscarinic agents after chronic sympathetic denervation is discussed.

Topics: Acetylcholine; Adaptation, Physiological; Alkaloids; Analgesics, Opioid; Animals; Benzophenanthridines; Colon; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Guinea Pigs; Immunoblotting; In Vitro Techniques; Muscarinic Agonists; Muscarinic Antagonists; Myenteric Plexus; Naphthalenes; Neurons; Oxotremorine; Parasympathetic Nervous System; Phenanthridines; Protein Kinase C; Receptors, Opioid, kappa; Receptors, Opioid, mu; Scopolamine; Sympathectomy; Tubulin

We examined the involvement of the oxidative stress in high glucose-induced suppression of human aortic endothelial cell proliferation. Chronic glucose treatment for 72 h concentration-dependently (5.6-22.2 mol/l) inhibited human coronary endothelial cell proliferation. Temocaprilat, an angiotensin-converting enzyme inhibitor, at 10 nmol/l to 1 micromol/l inhibited high glucose (22.2 mmol/l)-mediated suppression of human aortic endothelial cell proliferation. Temocaprilat at 1 micromol/l inhibited high glucose-induced membrane-bound protein kinase C activity in human aortic endothelial cells. The protein kinase C inhibitors calphostin C 100 nmol/l or chelerythrine 1 micromol/l inhibited high glucose-mediated suppression of human aortic endothelial cell proliferation. Chronic high glucose treatment for 72 h increased intracellular oxidative stress, directly measured by flow cytometry using carboxydichlorofluorescein diacetate bis-acetoxymethyl ester, and this increase was significantly suppressed by temocaprilat 10 nmol/l to 1 micromol/l. Bradykinin B2 receptor antagonist icatibant 100 nmol/l significantly reduced the action of temocaprilat; whereas bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin 100 nmol/l had no effect. These findings suggest that high glucose inhibits human aortic endothelial cell proliferation and that the angiotensin-converting enzyme inhibitor temocaprilat inhibits high glucose-mediated suppression of human aortic endothelial cell proliferation, possibly through suppression of protein kinase C, bradykinin B2 receptors and oxidative stress.

Topics: Alkaloids; Angiotensin-Converting Enzyme Inhibitors; Aorta; Benzophenanthridines; Bradykinin; Bradykinin B2 Receptor Antagonists; Cell Division; Cell Membrane; Cells, Cultured; Coronary Vessels; Dose-Response Relationship, Drug; Endothelial Cells; Glucose; Humans; Naphthalenes; Oxidative Stress; Phenanthridines; Protein Kinase C; Receptor, Bradykinin B1; Receptor, Bradykinin B2; Thiazepines; Time Factors

The effect of noradrenaline on the volume-sensitive chloride current (I(Cl(swell))) was studied with conventional whole-cell recording techniques in freshly dispersed isolated smooth muscle cells of the rabbit portal vein. In the absence of receptor antagonists, noradrenaline produced an increase in the amplitude of I(Cl(swell)) in some cells and a decrease in others. In the presence of the beta-adrenoceptor antagonist propranolol, noradrenaline increased I(Cl(swell)) and in the presence of the alpha(1)-adrenoceptor antagonist prazosin, noradrenaline reduced I(Cl(swell).) The phospholipase C (PLC) inhibitor U73122 reduced the amplitude of I(Cl(swell)) whereas the inactive analogue U73343 had no effect. The phorbol esters phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu) increased the amplitude of I(Cl(swell)) by approximately 60 and 100 %, respectively, in a voltage-independent fashion. Inhibitors of protein kinase C (PKC) chelerythrine and calphostin-C decreased the amplitude of I(Cl(swell)) in a concentration-dependent but voltage-independent manner. Bath application of 8-Br-cAMP decreased I(Cl(swell)) by about 60 % whereas the inhibitor of protein kinase A (PKA) KT5720 increased the amplitude of I(Cl(swell)) by approximately 80-90 %. In the presence of propranolol, chelerythrine prevented the increase of I(Cl(swell)) by noradrenaline; in the presence of prazosin, KT5720 blocked the inhibitory action of noradrenaline. The results show that in rabbit portal vein myocytes noradrenaline enhances I(Cl(swell)) by acting on alpha(1)-adrenoceptors and reduces I(Cl(swell)) by stimulating beta-adrenoceptors. The data suggest that the potentiating and inhibitory effects of noradrenaline are mediated, respectively, by PKC and PKA.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Alkaloids; Animals; Benzophenanthridines; Carbazoles; Chloride Channels; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Estrenes; In Vitro Techniques; Indoles; Membrane Potentials; Muscle, Smooth, Vascular; Naphthalenes; Norepinephrine; Phenanthridines; Portal Vein; Prazosin; Propranolol; Pyrroles; Pyrrolidinones; Rabbits; Tetradecanoylphorbol Acetate

We have shown previously that red blood cells (RBCs) can be induced to influx Ca(2+) when treated with lipid mediators, such as lysophosphatidic acid and prostaglandin E(2), that are released during clot formation. Since calcium loading of RBCs can lead to both protein kinase C (PKC) activation and phosphatidylserine (PS) exposure, we decided to investigate the possible linkage between PKC activation and membrane PS scrambling using phorbol 12-myristate-13-acetate (PMA), a commonly used activator of PKC. Treatment of RBCs with PMA in a calcium-containing buffer caused immediate PS exposure in an RBC subpopulation. The size of the subpopulation did not change upon further incubation, indicating that not all RBCs are equally susceptible to this treatment. Using a fluorescent indicator, we found a subpopulation of RBCs with elevated intracellular calcium levels. In the absence of extracellular calcium, no PS exposure was found. However, we did find cells with high levels of calcium that did not expose PS, and a variable percentage of PS-exposing cells that did not show elevated calcium concentrations. Inhibition of PKC with either calphostin C, a blocker of the PMA binding site, or chelerythrine chloride, an inhibitor of the active site, diminished the level of formation of PS-exposing cells. However, the inhibitors had different effects on calcium internalization, indicating that a high calcium concentration alone was not responsible for inducing PS exposure in the absence of PKC activity. Moreover, PKC inhibition could prevent PS exposure induced by calcium and ionophore treatment of RBCs. We conclude that PKC is implicated in the mechanism of membrane phospholipid scrambling.

Topics: Alkaloids; Benzophenanthridines; Enzyme Activation; Enzyme Inhibitors; Erythrocytes; Humans; Naphthalenes; Phenanthridines; Phosphatidylserines; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate

Many excitatory neurotransmitters and neuropeptides regulate the activity of neuronal and endocrine cells by modulating voltage-operated Ca2+ channels. Paradoxically, however, excitatory neuromediators that provoke mobilization of intracellular calcium from inositol trisphosphate (IP3)-sensitive stores usually inhibit voltage-gated Ca2+ currents. We have recently demonstrated that neurotensin (NT) stimulates the electrical and secretory activities of frog pituitary melanotrophs, and increases intracellular calcium concentration in these cells. In the present study, we have investigated the effects of NT on Ca2+ currents in cultured frog melanotrophs by using the perforated patch-clamp technique. Frog neurotensin (f NT) reduced the amplitude and facilitated the inactivation of both L- and N-type Ca2+ currents. Application of the membrane-permeant Ca2+ chelator BAPTA-AM, the sarcoendoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, or the IP3 receptor antagonist 2-APB suppressed the reduction of Ca2+ currents induced by f NT. Incubation of melanotrophs with the diacylglycerol analogue PMA, which causes desensitization of protein kinase C (PKC), or with the PKC inhibitors chelerythrine and calphostin C, reduced the inhibitory effect of f NT. The NT-induced action potential waveforms, applied as voltage-clamp commands, decreased the amplitude of Ca2+ currents, and enhanced Ca2+ influx by increasing the Ca2+ spike frequency. Altogether, these data indicate that the inhibitory effect of f NT on Ca2+ currents results from activation of the IP3/PKC pathway. The observation that NT controls Ca2+ signalling through both amplitude and frequency modulations of Ca2+ currents suggests that NT might induce spacial and temporal changes of intracellular Ca2+ concentration leading to stimulation of exocytosis.

Topics: Alkaloids; Animals; Benzophenanthridines; Boron Compounds; Calcium Channels; Calcium Channels, L-Type; Calcium Channels, N-Type; Cell Culture Techniques; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Inositol 1,4,5-Trisphosphate Receptors; Male; Naphthalenes; Neurotensin; Patch-Clamp Techniques; Phenanthridines; Pituitary Gland; Protein Kinase C; Rana ridibunda; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Thapsigargin

The transcription factor nuclear factor-kappaB (NF-kappaB) regulates expression of a variety of genes involved in immune responses, inflammation, proliferation, and programmed cell death (apoptosis). Here, we show that in rat neonatal ventricular cardiomyocytes, activation of NF-kappaB is involved in the hypertrophic response induced by myotrophin, a hypertrophic activator identified from spontaneously hypertensive rat heart and cardiomyopathic human hearts. Myotrophin treatment stimulated NF-kappaB nuclear translocation and transcriptional activity, accompanied by IkappaB-alpha phosphorylation and degradation. Consistently, myotrophin-induced NF-kappaB activation was enhanced by wild-type IkappaB kinase (IKK) beta and abolished by the dominant-negative IKKbeta or a general PKC inhibitor, calphostin C. Importantly, myotrophin-induced expression of two hypertrophic genes (atrial natriuretic factor [ANF] and c-myc) and also enhanced protein synthesis were partially inhibited by a potent NF-kappaB inhibitor, pyrrolidine dithio-carbamate (PDTC), and calphostin C. Expression of the dominant-negative form of IkappaB-alpha or IKKbeta also partially inhibited the transcriptional activity of ANF induced by myotrophin. These findings suggest that the PKC-IKK-NF-kappaB pathway may play a critical role in mediating the myotrophin-induced hypertrophic response in cardiomyocytes.

Topics: Alkaloids; Animals; Animals, Newborn; Benzophenanthridines; Blotting, Northern; Blotting, Western; Cell Nucleus; Cells, Cultured; Cytoplasm; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Activation; Genes, Dominant; Growth Substances; Hypertrophy; I-kappa B Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Luciferases; Microscopy, Confocal; Microscopy, Fluorescence; Myocardium; Naphthalenes; NF-kappa B; NF-KappaB Inhibitor alpha; Phenanthridines; Phosphorylation; Protein Binding; Protein Kinase C; Protein Transport; Rats; Rats, Sprague-Dawley; Rats, Wistar; RNA, Messenger; Time Factors; Transcription, Genetic; Transfection

Our previous study demonstrated that endothelin-1 induced a phosphorylation of GATA-4 transcription factor, which plays important roles in cardiac hypertrophy and failure. The goal of the present study was to determine whether protein kinase C (PKC) is involved in the signaling pathway, and, if so, whether alpha-tocopherol inhibits the GATA-4 phosphorylation. Treatment of HL-1 adult mouse cardiac muscle cells with PMA, a known activator of PKC, induced a transient phosphorylation of GATA-4. PMA also phosphorylated MEK and ERK, and PMA-induced GATA-4 phosphorylation was blocked by an MEK inhibitor, PD98059, suggesting that PMA phosphorylates GATA-4 via the MEK-ERK pathway. Treatment of HL-1 cells with 1 microM PMA for 24 h resulted in a downregulation of PKC. In PKC-downregulated cells, PMA- or ET-1-induced GATA-4 phosphorylation was suppressed, suggesting the role of PKC in GATA-4 phosphorylation. However, alpha-tocopherol (5--100 microM) did not inhibit the phosphorylation of GATA-4 or ERK in HL-1 cells. In contrast, alpha-tocopherol potently inhibited the PMA-induced ERK activation in smooth muscle cells. Our studies in HL-1 cells showed that PKC inhibitors, such as calphostin C and chelerythrin, failed to inhibit the PMA signaling. Furthermore, HL-1 cells appear to possess a unique PKC-signaling mechanism as PKC is constitutively phosphorylated and PMA did not cause further phosphorylation. Thus, in HL-1 cardiac muscle cells, PMA activates the MEK-ERK-GATA-4 pathway, apparently via a PKC-independent mechanism.

Topics: Alkaloids; alpha-Tocopherol; Animals; Benzophenanthridines; Blotting, Western; Cell Line; Cells, Cultured; DNA-Binding Proteins; Down-Regulation; Enzyme Inhibitors; Free Radicals; GATA4 Transcription Factor; Mice; Mitogen-Activated Protein Kinases; Myocardium; Naphthalenes; Phenanthridines; Phosphorylation; Protein Kinase C; Signal Transduction; Time Factors; Transcription Factors

The effects of five different protein kinase C inhibitors--calphostin C, chelerythrine, bisindolylmaleimide I, staurosporine and Gö6979--on intracellular free magnesium ([Mg(2+)](i)) content and mobilization were investigated in primary, cultured rat aortic smooth muscle cells. All these protein kinase C inhibitors significantly and rapidly increased [Mg(2+)](i) both in normal media (1.2 mM Mg(2+)) and in Mg(2+) free media. These data suggest that the increments of [Mg(2+)](i), induced by the diverse protein kinase C inhibitors, are derived from the release of bound intracellular Mg(2+) and that activation of protein kinase C isozymes are normally responsible for helping to maintain basal levels of [Mg(2+)](i) in rat aortic smooth muscle cells.

Topics: Alkaloids; Animals; Aorta; Benzophenanthridines; Carbazoles; Cells, Cultured; Enzyme Inhibitors; Indoles; Magnesium; Maleimides; Muscle, Smooth, Vascular; Naphthalenes; Phenanthridines; Protein Isoforms; Protein Kinase C; Rats; Staurosporine

Angiotensin II (ANG II) evokes positive inotropic responses in various species. However, the effects of this peptide on L-type Ca(2+) currents (I(Ca)) are still controversial. We report in this study that the effects of ANG II on I(Ca) differ depending on the mode of patch-clamp technique used, standard whole cell (WC) or perforated patch (PP). No significant effects of ANG II (0.5 microM) were observed when WC in cells dialyzed with high EGTA was used. However, when the intracellular milieu was preserved using PP, ANG II induced a significant 77 +/- 6% increase in I(Ca) (-2.2 +/- 0.3 in control and -3.9 +/- 0.6 pA/pF in ANG II, n = 8, P < 0.05). When WC was used in cells dialyzed with low Ca(2+) buffer capacity (EGTA 0.1 mM), ANG II was able to induce an increase in I(Ca) (-3.5 +/- 0.3 in control vs. -4.8 +/- 0.4 pA/pF in ANG II, n = 13, P < 0.05). This increase was prevented when the cells were also dialyzed with the protein kinase C (PKC) inhibitor chelerythrine (50 microM) or calphostin C (1 microM). The above results allow us to conclude that strong intracellular Ca(2+) buffering prevents the physiological actions of ANG II on cardiac I(Ca), which are also dependent on activation of PKC.

Topics: Alkaloids; Angiotensin II; Animals; Benzophenanthridines; Calcium; Calcium Channels, L-Type; Cats; Egtazic Acid; Enzyme Inhibitors; Heart; In Vitro Techniques; Losartan; Membrane Potentials; Myocardium; Naphthalenes; Patch-Clamp Techniques; Phenanthridines; Protein Kinase C

We investigated the regulation of ATP-sensitive K(+) (K(ATP)) currents in murine colonic myocytes with patch-clamp techniques. Pinacidil (10(-5) M) activated inward currents in the presence of high external K(+) (90 mM) at a holding potential of -80 mV in dialyzed cells. Glibenclamide (10(-5) M) suppressed pinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 x 10(-7) M) inhibited pinacidil-activated current. 4-alpha-Phorbol ester (5 x 10(-7) M), an inactive form of PDBu, had no effect on pinacidil-activated current. In cell-attached patches, the open probability of K(ATP) channels was increased by pinacidil, and PDBu suppressed openings of K(ATP) channels. When cells were pretreated with chelerythrine (10(-6) M) or calphostin C (10(-7) M), inhibition of the pinacidil-activated whole cell currents by PDBu was significantly reduced. In cells studied with the perforated patch technique, PDBu also inhibited pinacidil-activated current, and this inhibition was reduced by chelerythrine (10(-6) M). Acetylcholine (ACh; 10(-5) M) inhibited pinacidil-activated currents, and preincubation of cells with calphostin C (10(-7) M) decreased the effect of ACh. Cells dialyzed with protein kinase C epsilon-isoform (PKCepsilon) antibody had normal responses to pinacidil, but the effects of PDBu and ACh on K(ATP) were blocked in these cells. Immunofluorescence and Western blots showed expression of PKCepsilon in intact muscles and isolated smooth muscle cells of the murine proximal colon. These data suggest that PKC regulates K(ATP) in colonic muscle cells and that the effects of ACh on K(ATP) are largely mediated by PKC. PKCepsilon appears to be the major isozyme that regulates K(ATP) in murine colonic myocytes.

Topics: Acetylcholine; Adenosine Triphosphate; Alkaloids; Animals; Benzophenanthridines; Colon; Enzyme Activation; Enzyme Inhibitors; Female; Glyburide; In Vitro Techniques; Isoenzymes; Kinetics; Male; Membrane Potentials; Mice; Mice, Inbred BALB C; Muscle, Smooth; Naphthalenes; Patch-Clamp Techniques; Phenanthridines; Phorbol 12,13-Dibutyrate; Pinacidil; Potassium Channels; Protein Kinase C; Protein Kinase C-epsilon

Dopamine D4 receptors (D4 receptors) mediate dopamine-stimulated, folate-dependent phospholipid methylation. To investigate possible regulation of this multi-step D4 receptor-mediated phospholipid methylation cycle by protein kinases, specific kinase activators and inhibitors were studied in SK-N-MC human neuroblastoma cells, using [14C] formate to label folate-derived single-carbon groups. Phorbol dibutyrate (PDB), an activator of protein kinase C, stimulated basal phospholipid methylation and also shifted the dose-response curve for dopamine-stimulated phospholipid methylation to the right by more than an order of magnitude. Calphostin C, an inhibitor of protein kinase C, had little effect on basal phospholipid methylation but significantly inhibited dopamine-stimulated phospholipid methylation and also blocked the stimulatory response to PDB. Chelerythrine, which inhibits protein kinase C and other kinases, strongly inhibited both basal and dopamine-stimulated phospholipid methylation. Forskolin, an activator of protein kinase A, inhibited basal and dopamine-stimulated phospholipid methylation, but only at high concentrations while Rp-cAMP, an inhibitor of protein kinase A, did not block this effect. Inhibition of protein kinase G produced a modest decrease in dopamine-stimulated phospholipid methylation, but neither sodium nitroprusside, which increases nitric oxide (NO) production and activates protein kinase G, nor the NO synthase inhibitor N-nitro-L-arginine had any effect on basal or dopamine-stimulated phospholipid methylation. These observations indicate that protein kinase C is an important regulator of basal and D4 receptor-mediated folate-dependent phospholipid methylation, whereas protein kinase A and protein kinase G have a lesser or minimal role.

Topics: Alkaloids; Benzophenanthridines; Carbazoles; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP-Dependent Protein Kinases; Dopamine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Indoles; Methylation; Naphthalenes; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Phenanthridines; Phorbol 12,13-Dibutyrate; Phospholipids; Protein Kinase C; Receptors, Dopamine D2; Receptors, Dopamine D4; Thionucleotides; Tumor Cells, Cultured

Cellular adhesion and spreading are critical components involved in the processes of cell and tissue development, and immune responses in molluscs, but at present, little is known regarding the signaling pathways involved in these basic cellular functions. In the present study, the molluscan Biomphalaria glabrata embryonic (Bge) cell line was used as an in vitro model to study the signal transduction pathways regulating molluscan cell adhesion and spreading behavior. Western blot analysis using antibodies specific to mitogen-activated protein kinase (MAPK) revealed the presence of an MAPK-like immunoreactive protein in Bge cells, that was phosphorylated upon exposure to phorbol myristate acetate (PMA). Moreover, Bge cell treatment with inhibitors of protein kinase C (PKC), Ras and MAPK kinase (Mek) suppressed PMA-induced expression of activated MAPK, suggesting that PKC-, Ras- and Mek-like molecules may be acting upstream of MAPK. Similarly, in vitro Bge cell-spreading assays were performed in conjunction with the same panel of inhibitors to determine the potential involvement of PKC, Ras and Mek in cellular adhesion/spreading. Results revealed a similar pattern of inhibition of cell-spreading behavior strongly implying that the Bge cell spreading also may be regulated through a MAPK-associated signal transduction pathway(s) involving proteins similar to PKC, Ras and Mek.

Topics: Alkaloids; Animals; Benzophenanthridines; Biomphalaria; Cell Line; Enzyme Activation; Mitogen-Activated Protein Kinases; Naphthalenes; Phenanthridines; Protein Kinase C; Signal Transduction

The effects of the protein kinase C inhibitors chelerythrine and bisindolylmaleimide I on acetylcholine-activated K+ currents (I(KACh)) were examined in atrial myocytes of mice, using the patch clamp technique. Chelerythrine and bisindolylmaleimide I inhibited I(KACh) in a reversible and dose-dependent manner. Half-maximal effective concentrations were 0.49+/-0.01 microM for chelerythrine and 98.69+/-12.68 nM for bisindolylmaleimide I. However, I(KACh) was not affected either by calphostin C, which is also known as a protein kinase C inhibitor, or by a protein kinase C activator, phorbol 12,13-dibutyrate. When K(ACh) channels were activated directly by adding 1 mM GTPgammaS to the bath solution in inside-out patches, chelerythrine (10 microM) decreased the open probability from 0.043+/-0.01 to 0.014+/-0.007 (n=5), but bisindolylmaleimide I did not affect the channel activity. From these results, it is concluded that both chelerythrine and bisindolylmaleimide I inhibit K(ACh) channels independently of protein kinase C inhibition, but the level of inhibition is different.

Topics: Acetylcholine; Alkaloids; Animals; Atrial Function; Benzophenanthridines; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanosine 5'-O-(3-Thiotriphosphate); Heart Atria; Indoles; Maleimides; Membrane Potentials; Mice; Naphthalenes; Patch-Clamp Techniques; Phenanthridines; Phorbol 12,13-Dibutyrate; Potassium Channels; Protein Kinase C

Leukemic CD34(+) immature acute myeloid leukemia (AML) cells express Fas receptor but are frequently resistant to Fas agonistic reagents. Fas plays an important role in T-cell-mediated cytotoxicity, and recently it has been suggested that altered Fas signaling may contribute to drug resistance. Therefore, Fas resistance could be one of the mechanisms by which AML progenitors escape chemotherapy or T-cell-based immune intervention. However, the molecular mechanism of Fas resistance in AML cells has not been identified. Fas signaling can be interrupted at 3 mains levels: Fas clustering, alteration of death-inducing-signaling-complex (DISC) formation, and effector caspase inhibition of downstream caspase-8. This study shows that in the Fas-resistant CD34(+)CD38(-) KG1a cells, Fas agonists resulted in Fas aggregation but not in caspase-8 activation, related to a defect in DISC formation. However, pretreatment with chelerythrin, but not with calphostin C, resulted in the restoration of Fas-induced caspase-8 activation and cytotoxicity, suggesting that some atypical protein kinase C (PKC) isoforms contributed to the lack of DISC formation. Indeed, treatment with antisense oligonucleotides directed against PKC zeta and enforced expression of Par-4, a negative regulator of PKC zeta activity, restored Fas-induced caspase-8 activity and apoptosis. Moreover, it was found that PKC zeta interacts with FADD and that PKC zeta immunoextracts prepared from KG1a cells are able to phosphorylate FADD in vitro, whereas this phosphorylation is dramatically reduced in Par-4 transfectant cells. In conclusion, it is suggested that in AML cells, PKC zeta plays an important role in Fas resistance by inhibiting DISC formation, possibly by phosphorylating FADD.

Topics: Alkaloids; Antibodies, Monoclonal; Apoptosis; Apoptosis Regulatory Proteins; Benzophenanthridines; Carrier Proteins; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cytotoxicity, Immunologic; Enzyme Activation; Enzyme Inhibitors; fas Receptor; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Isoenzymes; Jurkat Cells; Leukemia, Myeloid, Acute; Microscopy, Confocal; Naphthalenes; Oligonucleotides, Antisense; Phenanthridines; Protein Kinase C; Signal Transduction; Transfection; Tumor Cells, Cultured

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

Influx of Ca2+ via Ca2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage-gated Ca2+ and K+ channels, the primary determinants of the influx of Ca2+, are regulated by GH-releasing hormone (GHRH) through G-protein-coupled intracellular signalling systems. Using whole-cell patch-clamp techniques, the changes of the Ca2+ and K+ currents in primary cultured ovine and human somatotropes were recorded. Growth hormone-releasing hormone (10 nmol/L) increased both L- and T-type voltage-gated Ca2+ currents. Inhibition of the cAMP/protein kinase A (PKA) pathway by either Rp-cAMP or H89 blocked this increase in both L- and T-type Ca2+ currents. Growth hormone-releasing hormone also decreased voltage-gated transient (IA) and delayed rectified (IK) K+ currents. Protein kinase C (PKC) inhibitors, such as calphostin C, chelerythrine or downregulation of PKC, blocked the effect of GHRH on K+ currents, whereas an acute activation of PKC by phorbol 12, 13-dibutyrate (1 micromol/L) mimicked the effect of GHRH. Intracellular dialysis of a specific PKC inhibitor (PKC19-36) also prevented the reduction in K+ currents by GHRH. It is therefore concluded that GHRH increases voltage-gated Ca2+ currents via cAMP/PKA, but decreases voltage-gated K+ currents via the PKC signalling system. The GHRH-induced alteration of Ca2+ and K+ currents augments the influx of Ca2+, leading to an increase in [Ca2+]i and the GH secretion.

Topics: Alkaloids; Animals; Benzophenanthridines; Calcium Channels; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Growth Hormone-Releasing Hormone; Humans; Isoquinolines; Naphthalenes; Patch-Clamp Techniques; Phenanthridines; Phorbol 12,13-Dibutyrate; Pituitary Gland; Potassium Channels; Protein Kinase C; Sheep; Signal Transduction; Sulfonamides

Vascular smooth muscle cell (VSMC) proliferation is a key event in the development and progression of atherosclerotic lesions. Accumulating evidence suggests that lipoprotein lipase (LPL) produced in the vascular wall may exert proatherogenic effects. The aim of the present study was to examine the effect of LPL on VSMC proliferation. Incubation of growth-arrested human VSMCs with purified endotoxin-free bovine LPL for 48 and 72 hours, in the absence of any added exogenous lipoproteins, resulted in a dose-dependent increase in VSMC growth. Addition of VLDLs to the culture media did not further enhance the LPL effect. Treatment of growth-arrested VSMCs with purified human or murine LPL (1 microg/mL) led to a similar increase in cell proliferation. Neutralization of bovine LPL by the monoclonal 5D2 antibody, irreversible inhibition, or heat inactivation of the lipase suppressed the LPL stimulatory effect on VSMC growth. Moreover, preincubation of VSMCs with the specific protein kinase C inhibitors calphostin C and chelerythrine totally abolished LPL-induced VSMC proliferation. In LPL-treated VSMCs, a significant increase in protein kinase C activity was observed. Treatment of VSMCs with heparinase III (1 U/mL) totally inhibited LPL-induced human VSMC proliferation. Taken together, these data indicate that LPL stimulates VSMC proliferation. LPL enzymatic activity, protein kinase C activation, and LPL binding to heparan sulfate proteoglycans expressed on VSMC surfaces are required for this effect. The stimulatory effect of LPL on VSMC proliferation may represent an additional mechanism through which the enzyme contributes to the progression of atherosclerosis.

Topics: Alkaloids; Animals; Antibodies, Monoclonal; Arteriosclerosis; Benzophenanthridines; Cattle; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Hot Temperature; Humans; Lipoprotein Lipase; Mice; Muscle, Smooth, Vascular; Naphthalenes; Neutralization Tests; Phenanthridines; Polysaccharide-Lyases; Protein Kinase C; Time Factors; Up-Regulation

We examined the effects of several protein kinase C (PKC) inhibitors on the murine 5-hydroxytryptamine3 (5-HT3) receptor to determine whether they acted directly on the receptor. The 5-HT-evoked currents in Xenopus laevis oocytes expressing the recombinant 5-HT3 receptor were measured with the two-electrode voltage-clamp technique. The PKC inhibitors bisindolylmaleimide I (BIM, GF109203x) and staurosporine, but not calphostin C or chelerythrine, decreased the 5-HT3 receptor-mediated currents when coapplied with 5-HT. BIM blocked 0.5 microM 5-HT-elicited currents with an IC50 value of 7 nM, whereas in the presence of 5 microM staurosporine, 42% inhibition of 0.5 microM 5-HT-mediated currents was observed. Increasing concentrations of BIM resulted in a rightward shift of the 5-HT concentration-response curve, without altering efficacy. A Schild plot was generated, which had a slope of -1.01, suggesting competitive antagonism. The Ki value of BIM was determined to be 29 nM. To confirm competitive antagonism, a competitive binding assay was performed on Sf21 insect cells infected with the mouse 5-HT3 receptor cDNA in a baculovirus expression vector. BIM completely displaced binding of the selective 5-HT3 receptor antagonist [3H]GR65630. BIM bound to the 5-HT3 receptor with a Ki value of 61 nM, which was slightly less potent than that of the selective 5-HT3 receptor antagonist MDL72222 (27 nM). The PKC inhibitor BIM is a potent competitive antagonist at the 5-HT3 receptor.

Topics: Alkaloids; Animals; Benzophenanthridines; Binding, Competitive; Cell Membrane; DNA; Enzyme Inhibitors; In Vitro Techniques; Indoles; Maleimides; Mice; Naphthalenes; Oocytes; Patch-Clamp Techniques; Phenanthridines; Protein Kinase C; Receptors, Serotonin; Receptors, Serotonin, 5-HT3; RNA; Serotonin Antagonists; Staurosporine; Xenopus laevis

Previous studies indicate that a zinc- and phorbol ester-binding factor is necessary for in vitro endosome fusion and for the effect of Rab5 on endosome fusion. Rab5 is a small GTPase that regulates membrane fusion between early endosomes derived from either receptor-mediated endocytosis or fluid-phase endocytosis. In its GTP-bound form, Rab5 promotes endocytosis and enhances fusion among early endosomes. To determine if PMA stimulates endocytosis by activating a factor required for endosome fusion, we overexpressed wild-type Rab5, a dominant negative mutant (Rab5:S34N), and a GTPase deficient mutant (Rab5:Q79L) in BHK-21 cells. The phorbol ester PMA stimulates endocytosis and increases the number and the size of endocytic vesicles, even in the presence of Rab5:S34N. Zinc depletion with N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and addition of calphostin C (CPC), an inhibitor of PKC that interacts with zinc and phorbol ester binding motifs, inhibited both basal and Rab5-stimulated fluid phase endocytosis. These two reagents also inhibited the size and number of endocytic vesicles promoted by Rab5. These results suggest that PMA stimulates endocytosis by regulating the dynamics of the early endosome compartment.

Topics: Alkaloids; Animals; Benzophenanthridines; Cell Line; Cell Membrane; Chelating Agents; CHO Cells; Cricetinae; Endocytosis; Endosomes; Enzyme Inhibitors; Ethylenediamines; GTP Phosphohydrolases; GTP-Binding Proteins; Membrane Fusion; Mutagenesis, Site-Directed; Naphthalenes; Phenanthridines; Protein Kinase C; rab5 GTP-Binding Proteins; Recombinant Proteins; Sindbis Virus; Tetradecanoylphorbol Acetate; Transfection; Zinc

As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which we show to be a substrate for PKC in N. crassa.

Topics: Adaptation, Physiological; Alkaloids; Alkyl and Aryl Transferases; Benzophenanthridines; Diglycerides; DNA-Binding Proteins; Enzyme Inhibitors; Fungal Proteins; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Light; Naphthalenes; Neurospora crassa; Oxidoreductases; Phenanthridines; Phosphorylation; Protein Kinase C; Substrate Specificity; Transcription Factors

The stimulant drug amphetamine is postulated to enhance dopamine release through the plasmalemmal dopamine transporter by an exchange diffusion with synaptosomal dopamine. Because protein kinase C has been shown to have an effect on dopamine transporter activity, we examined the effect of protein kinase C inhibitors on endogenous dopamine release stimulated by amphetamine in perfused rat striatal slices. At concentrations of 1 microM, the selective protein kinase C inhibitors chelerythrine, Ro31-8220 and calphostin C nearly completely inhibited endogenous dopamine release elicited by 1 microM amphetamine. The inactive analog bisindoylmaleimide V had no effect. Extracellular Ca++ was not required for the effect of the inhibitors. The importance of vesicular dopamine release was examined by determining inhibitor activity in reserpine-treated rats. Dopamine release elicited by 1 microM amphetamine was not significantly altered in reserpine-treated rats compared with control animals. Ro31-8220 at 1 microM completely blocked amphetamine-induced dopamine release in reserpine-treated rats. Activation of protein kinase C with 250 nM of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate increased dopamine release, and the release was not additive with 1 microM amphetamine. Both chelerythrine and Ro31-8220 at 1 microM increased [3H]dopamine uptake by 17% and 30%, respectively, whereas a brief exposure to 12-O-tetradecanoylphorbol 13-acetate slightly inhibited [3H]dopamine uptake. Our results suggest that amphetamine-mediated dopamine release through the plasmalemmal transporter is highly dependent on protein kinase C activity.

Topics: Alkaloids; Amphetamine; Animals; Benzophenanthridines; Biological Transport; Calcium; Corpus Striatum; Dopamine; Enzyme Activation; Enzyme Inhibitors; Female; Indoles; Naphthalenes; Phenanthridines; Protein Kinase C; Rats; Reserpine; Tetradecanoylphorbol Acetate

Dopamine inhibits Na+ and water reabsorption in the rat cortical collecting duct (CCD) in the presence of arginine vasopressin (AVP). This inhibition appears to involve the D4 dopamine receptor isoform, which inhibits cAMP production; however, the D1A receptor, which stimulates cAMP production, is also expressed in the CCD. To discriminate between these opposing effects, we measured cAMP production in intact CCD segments. The basal rate of cAMP production ranged from 6.5 to 10 fmol/mm of tubule length over a 7-min incubation period, and it was unaffected by either dopamine or the D1A-specific agonist fenoldopam. AVP increased cAMP production to the range of 85-153 fmol . mm-1 . 7 min-1. Whereas neither 0.1 nor 1.0 microM fenoldopam affected AVP-dependent cAMP production, dopamine reduced it in a dose-dependent manner, achieving a maximum inhibition of 50% at 10 microM. This effect was reversed by the D4 receptor antagonist clozapine but not by pimozide or spiperone (antagonists of D2 and D3 receptors) or by calphostin C or chelerythrine (inhibitors of protein kinase C). We conclude that dopamine inhibits transepithelial Na+ transport and osmotic water permeability in the presence of AVP by inhibition of cAMP production, which is mediated by the D4 receptor isoform linked via the inhibitory G protein Gi.

Topics: Alkaloids; Animals; Arginine Vasopressin; Benzophenanthridines; Clozapine; Cyclic AMP; Dopamine; Dopamine Antagonists; Dopamine D2 Receptor Antagonists; Enzyme Inhibitors; Epinephrine; Fenoldopam; GTP-Binding Proteins; In Vitro Techniques; Kidney Cortex; Kidney Tubules, Collecting; Kinetics; Male; Naphthalenes; Phenanthridines; Pimozide; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D1; Receptors, Dopamine D3; Receptors, Dopamine D4; Spiperone; Time Factors; Yohimbine

Microglia are activated by amyloid beta (Abeta) in vivo and in vitro, and Abeta-activated microglia may be involved in the pathogenesis of Alzheimer's disease (AD). We investigated the mechanism of microglial chemotaxis induced by Abeta (25-35), an active fragment of Abeta. Abeta (25-35) 0.1 and 1 nM stimulated microglial chemotaxis. The protein kinase C (PKC) inhibitors chelerythrine (0.5 and 2 microM), calphostin C (1 microM) and staurospine (10 nM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). The chemotactic effect of Abeta (25-35) on microglia was desensitized by pretreatment of microglia with 1 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA). Pretreatment of cells with Abeta (25-35) (1 nM) also desensitized the chemotactic effect by Abeta (25-35) (1 nM). The desensitization by TPA or Abeta (25-35) was inhibited when staurosporine was present in the pretreatment media. The tyrosine kinase inhibitor herbimycin A (0.1 and 1 microM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). Based on these observations, it seems likely that PKC and tyrosine kinase are involved in the Abeta-induced chemotaxis of microglia.

Topics: Alkaloids; Alzheimer Disease; Amyloid beta-Peptides; Animals; Benzophenanthridines; Benzoquinones; Brain; Carcinogens; Cells, Cultured; Chemotaxis; Dose-Response Relationship, Drug; Enzyme Inhibitors; Lactams, Macrocyclic; Microglia; Naphthalenes; Peptide Fragments; Phenanthridines; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Wistar; Rifabutin; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate

Signal transduction of a human follicular thyroid cancer cell line (FTC133) was investigated. The protein kinase C (PKC)-agonist TPA enhanced invasion by 15%, whereas its antagonists staurosporine, chelerythrine and calphostin C were inhibiting by up to 62%. TSH and EGF stimulated invasion of FTC133. Antagonism of PKC reversed TSH-mediated stimulation, whereas it had no effect on EGF-stimulation. Our data provide evidence for an essential role of PKC in signal transduction of invasive thyroid cancer.

Topics: Benzophenanthridines; Cell Line, Tumor; Enzyme Inhibitors; Humans; Naphthalenes; Neoplasm Invasiveness; Protein Kinase C; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate; Thyroid Neoplasms; Thyrotropin

Nocturnal synthesis of the pineal hormone melatonin (MEL) is regulated by the circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. We examined the hypothesis that MEL can feed back to regulate the SCN using a brain slice preparation from rat. We monitored the SCN ensemble firing rate and found that MEL advanced the time of peak firing rate by more than 3 h at restricted circadian times (CTs) near subjective dusk [CT 10-14 (10-14 h after lights on)] and dawn (CT 23-0) on days 2 and 3 after treatment. The effect of MEL at CT 10 was blocked by pertussis toxin. The protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate, reset the SCN firing rate rhythm with a profile of temporal sensitivity congruent with that of MEL. Two specific PKC inhibitors, calphostin C and chelerythrine chloride, independently blocked MEL-induced phase advances at each sensitive period. Furthermore, MEL administration increased PKC phosphotransferase activity transiently to 200% at CT 10 and CT 23, but not at CT 6. These data demonstrate that 1) MEL can directly modulate the circadian timing of the SCN within two windows of sensitivity corresponding to dusk and dawn; and 2) MEL alters SCN cellular function via a pertussis toxin-sensitive G protein pathway that activates PKC.

Topics: Alkaloids; Animals; Benzophenanthridines; Circadian Rhythm; Enzyme Activation; Enzyme Inhibitors; Male; Melatonin; Naphthalenes; Pertussis Toxin; Phenanthridines; Protein Kinase C; Rats; Signal Transduction; Suprachiasmatic Nucleus; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella

A rapid loss of protein kinase C (PKC) activity is a prognostic feature of the lethal damage inflicted on neurons by cerebral ischemia in vivo and by hypoxic and excitotoxic insults in vitro. However, it is not known if this inactivation of PKC is incidental or is an essential part of the neurodegenerative process driven by such insults. To address this issue, the effects of glutamate on PKC activity and neurotoxicity were studied in immature [8 days in vitro (DIV)] and mature (15-20 DIV) embryonic day 18 rat cortical neuronal cultures. Exposing 16 DIV neurons to as little as 20-50 microM glutamate for 15 min was neurotoxic and induced a rapid (approximately 1-2 h) Ca(2+)-dependent inactivation of membrane PKC. By contrast, neurons 8 DIV were resistant to > 800 microM glutamate, and no evidence of PKC inactivation was observed. Reverse transcription-polymerase chain reaction analysis of NMDA and AMPA receptor subtypes and fluorometric intracellular Ca2- concentration measurements of the effects of NMDA, AMPA, kainate, and metabotropic glutamate receptor activation demonstrated that this striking difference in vulnerability was not due to an absence of functional glutamate receptors on neurons 8 DIV. However, 8 DIV neurons became highly vulnerable to low (< 20 microM) concentrations of glutamate when PKC activity was inhibited by 50 nM staurosporine, 1 microM calphostin C, 5 microM chelerythrine, or chronic exposure to 100 nM PMA. A 15-min coapplication of 50 nM staurosporine with glutamate, NMDA, AMPA, or kainate killed between 50 and 80% of 8 DIV cells within the ensuing 24 h. Moreover, cell death was observed in these cells even when PKC inactivation was delayed up to 4 h after glutamate removal. The evidence indicates that a loss of PKC activity is an essential element of the excitotoxic death of neurons 8 DIV and that cellular event(s) responsible for linking glutamate-mediated Ca2+ influx to PKC inactivation in vulnerable neurons 16 DIV are undeveloped in resistant cells 8 DIV. These results also suggest that the loss of neuronal PKC activity observed in cerebral ischemia may indeed be an important part of the neurodegenerative process. The 8 DIV/16 DIV cortical cell model may prove to be valuable in discerning those intracellular signaling events critical to glutamate-mediated neuronal death.

Topics: Alkaloids; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzophenanthridines; Calcium; Carcinogens; Cell Death; Cells, Cultured; Cerebral Cortex; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Female; Fetus; Gene Expression; Glutamic Acid; Kainic Acid; Membrane Proteins; N-Methylaspartate; Naphthalenes; Neurons; Phenanthridines; Potassium Chloride; Pregnancy; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Staurosporine; Tetradecanoylphorbol Acetate

Induction of the p40/46 and p69/71 isoforms of the 2',5'-oligoadenylate (2-5A) synthetase by interferon-alpha (IFN-alpha) is variable among six different Burkitt lymphoma cell lines with Ramos cells expressing among the highest levels of these enzymes. Inhibitors of protein kinase C (PKC) block induction of mRNAs encoding both isoforms; however, induction of the p69/71 isoform is more sensitive to these inhibitors. Down-regulation of PKC by prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) also blocks induction of 2-5A synthetase mRNAs and decreases both constitutive and IFN-alpha-induced enzymatic activity. Cotreatment of cells with TPA and IFN-alpha increases induction of 2-5A synthetase mRNAs above that seen in cells treated with IFN-alpha alone. IFN-alpha does not directly activate PKC-alpha or PKC-delta, the two most abundant PKC isoforms present in Ramos cells, suggesting that PKC activation by another signaling pathway is necessary for maximal induction of 2-5A synthetases by IFN-alpha.

Topics: 2',5'-Oligoadenylate Synthetase; Alkaloids; Benzophenanthridines; Burkitt Lymphoma; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; Interferon-alpha; Isoenzymes; Naphthalenes; Phenanthridines; Protein Kinase C; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

We demonstrated previously that TSH activates phospholipase D (PLD) via stimulation of protein kinase C (PKC) in Fischer rat thyroid line (FRTL)-5 thyroid cells. To examine the role of the cAMP pathway in the regulation of PLD, we studied the effects of forskolin (0-100 microM; 30 min) and dibutyryl cAMP (dbcAMP; 0-1 mM; 30 min) on PLD activation. FRTL-5 thyroid cells were labeled mainly in phosphatidylcholine with [3H]myristate followed by incubation with 200 mM ethanol before the addition of agonist. PLD was assessed by the measurement of [3H]phosphatidylethanol. Forskolin (100 nM to 100 microM) and dbcAMP (100 pM to 100 microM) increased PLD activity significantly. Maximal responses to forskolin and dbcAMP exceed the PLD responses produced by 100 microU/ml of TSH. To determine whether the effects of forskolin and dbcAMP on PLD occurred as a consequence of PKC activation, FRTL-5 thyroid cells were preincubated for 10 min with the PKC inhibitors, chelerythrine (1 microM) or calphostin C (1 microM), or they were pretreated for 24 h with phorbol myristate acetate (100 nM) to down-regulate PKC. Unlike TSH-mediated PLD activation, these treatments had no effect on PLD activation by cAMP agonists. Forskolin (10 microM; 30 min) had no effect on the subcellular distribution of PKC alpha-, epsilon-, or zeta-isoforms, confirming the lack of involvement of PKC. The protein kinase A (PKA) inhibitors, H-89 (10 microM; 30 min) and dideoxyadenosine (5 nM; 10 min) significantly decreased the forskolin- and dbcAMP-mediated PLD activation without any effect on the phorbol ester-mediated PLD response. Following pretreatment with H-89 or dideoxyadenosine, the TSH-mediated PLD response was also significantly reduced. These studies indicate that forskolin and dbcAMP stimulate PLD in FRTL-5 thyroid cells directly via PKA without involvement of PKC. Studies of cells in the presence and absence of ethanol revealed approximately 60% of the phosphatidate plus diacylglycerol produced via TSH occurs via PLD activation. Although TSH-mediated inositol phosphate generation occurred with similar concentrations of TSH that led to PLD activation, 10-fold higher TSH concentrations were required to increase intracellular Ca2+. These results and the lack of a rapid Ca2+ transient following physiological TSH concentrations suggest that alternatives to conventional hydrolysis of phosphatidylinositol 4,5-bisphosphate may initiate PKC activation. Thus, the two major signal transduction systems

Topics: Alkaloids; Animals; Benzophenanthridines; Bucladesine; Calcium; Cell Line; Colforsin; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Glycerophospholipids; Humans; Naphthalenes; Phenanthridines; Phosphatidic Acids; Phospholipase D; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Thyroid Gland; Thyrotropin

An inhibitory effect of several zinc chelators on endosome fusion reconstituted in an in vitro system has been recently reported (A. Aballay et al., 1995, Biochem. J. 312, 919-923). The factor that requires zinc for its activity is still unknown. Since the regulatory domain of protein kinase C (PKC) contains cysteine-rich motifs which coordinate zinc, we suspected that PKC or a PKC-like protein might be that factor. To test this hypothesis, we studied the effect of calphostin C, a specific inhibitor of PKC that interacts with the cysteine-rich motif, and PMA (phorbol 12-myristate 13-acetate), an activator of several PKC isoforms that bind to the same region, on endosome fusion. Calphostin C inhibited endosome fusion in a zinc-regulated manner, whereas PMA enhanced endosome fusion. Moreover, fusion was strongly stimulated when both PMA and zinc were added together to zinc-depleted fusion reactions. Inhibitors of the catalytic domain of PKC had no effect on the assay suggesting that the kinase activity is not required. In contrast, a glutathione S-transferase fusion protein containing a cysteine-rich region of the regulatory domain of PKCgamma inhibited endosome fusion in a PMA-dependent manner. Western blot analysis demonstrated the presence of proteins containing PKC-like cysteine-rich regions that are released from endosomal fractions by zinc chelators. These results indicate that the previously proposed zinc-dependent factor required for endosome fusion could be either a PKC isoform or a protein containing the phorbol ester-binding domain of PKC.

Topics: Alkaloids; Animals; Benzophenanthridines; Binding Sites; Cell Line; Cysteine; Endosomes; Enzyme Inhibitors; Isoenzymes; Kinetics; Macrophages; Membrane Fusion; Naphthalenes; Peptide Fragments; Phenanthridines; Protein Kinase C; Recombinant Fusion Proteins; Staurosporine; Tetradecanoylphorbol Acetate; Zinc

Cardiomyocytes isolated from rabbit hearts were preconditioned in vitro by 10 min of ischemia or treatment with 100 microM adenosine. Protection was assessed as average integrated mortality following osmotic swelling and determination of viability by trypan blue exclusion over 60-180 min ischemia. Repetitive sub-maximal stimulations with 1 microM adenosine amplified the protective response. Treatment with adenosine only at the onset of prolonged ischemia afforded a dose-dependent protection. The PKC inhibitor calphostin C (500 nm) blocked preconditioning and, when added during ischemic incubation of non-preconditioned cells, significantly increased injury. The memory of adenosine-induced preconditioning decayed over a 60 min post-incubation period. Light activation of calphostin C initially added to preconditioned ischemic cells in the dark indicated that a 10 min period of PKC activity at the onset of ischemia affords full protection. The reversible PKC inhibitors chelerythrine (5 microM) or staurosporine (100 nM) added only to bracket induction of ischemia, reduced but did not abolish protection. Protection was abolished when either drug was present during induction and a subsequent 30 min post-incubation period. Staurosporine included during initiation and post-incubation but washed out in the final 5 min of post-incubation allowed significant protection to occur. It is concluded that a single adenosine receptor-stimulation induces protection as it preconditions, and PKC activity appears to be required for both induction and protection. Memory may reside in post-receptor amplification of an initial protective response.

Topics: Adenosine; Alkaloids; Animals; Benzophenanthridines; Cardiovascular Agents; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Heart; Ischemic Preconditioning, Myocardial; Myocardial Ischemia; Myocardium; Naphthalenes; Phenanthridines; Protein Kinase C; Rabbits; Staurosporine

The involvement of protein kinase C (PKC) in the action of GH-releasing factor (GRF) and synthetic GH-releasing peptides (GHRP-2 and GHRP-6) was investigated in ovine somatotrophs in primary culture. In partially purified sheep somatotrophs, GRF and GHRP-2 caused translocation of PKC activity from the cytosol to the cell membranes and caused GH release in a dose- and time-dependent manner. GHRP-6 did not cause PKC translocation. The PKC inhibitors, calphostin C, staurosporine and chelerythrine, partially reduced GH release in response to GRF and GHRP-2 at doses which selectively inhibit PKC activity. These inhibitors totally abolished GH release caused by phorbol 12-myristate 13-acetate (PMA). Down-regulation of PKC by the treatment of cells with phorbol 12,13-dibutyrate for 16 h caused a significant (P < 0.001) reduction in total PKC activity and totally abolished PKC translocation in response to a challenge with GRF, GHRP-2 or PMA. In addition, down-regulation abolished GH release in response to GRF, GHRP-2 or GHRP-6. Treatment of cells with H89, a selective PKA inhibitor, totally blocked GH release caused by either GRF or GHRP-2 and partially reduced PMA-induced GH release. H89 had no effect on PKC translocation caused by GRF, GHRP-2 or PMA and did not affect GH release caused by GHRP-6. These data suggest that GHRP-2 and GRF activate PKC in addition to stimulating adenylyl cyclase activity. Although the cAMP-protein kinase A (PKA) pathway is the major signalling pathway employed by GRF and GHRP-2, the activation of PKC may potentiate signalling via the cAMP-PKA pathway in ovine GH secretion. Importantly, the effect of PMA in increasing the secretion of GH from ovine somatotrophs is effected, in part, by up-regulation of the cAMP-PKA pathway. We conclude that there is cross-talk between the PKC pathway and the cAMP-PKA pathway in ovine somatotrophs during the action of GRF or GHRP.

Topics: Alkaloids; Animals; Benzophenanthridines; Carcinogens; Cell Membrane; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytosol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Growth Hormone; Growth Hormone-Releasing Hormone; Isoquinolines; Naphthalenes; Oligopeptides; Phenanthridines; Pituitary Gland; Protein Kinase C; Second Messenger Systems; Sheep; Staurosporine; Sulfonamides; Tetradecanoylphorbol Acetate; Time Factors

Expression of certain mammalian protein kinase C (PKC) isoforms inhibits the proliferation of Schizosaccharomyces pombe (Goode et al., Mol. Biol. Cell 5 (1994) 907-920). We have taken advantage of this fact to determine the in vivo isoform preference of a number of PKC inhibitors, using a microtitre plate assay which allows rapid screening. This in vivo model has revealed previously unreported preferences; calphostin C is a more efficient inhibitor of the novel PKCS than chelerythrine chloride whereas the efficiencies are reversed for inhibition of the classical PKCgamma. We have also shown that the anti-leukaemic agent bryostatin 1 inhibits or activates in vivo in an isoform-specific manner.

Topics: Acetophenones; Alkaloids; Benzophenanthridines; Benzopyrans; Bryostatins; Carbazoles; Enzyme Activation; Enzyme Inhibitors; Indoles; Isoenzymes; Lactones; Macrolides; Maleimides; Naphthalenes; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; Recombinant Proteins; Schizosaccharomyces; Sphingosine; Tetradecanoylphorbol Acetate; Transformation, Genetic

Muscarinic receptor stimulation increases Ca2+ sensitivity, i.e., the amount of force produced at a constant submaximal cytosolic Ca2+ concentration ([Ca2+]i), in permeabilized smooth muscle preparations. It is controversial whether this increase in Ca2+ sensitivity is in part mediated by protein kinase C (PKC). With the use of a beta-escin permeabilized canine tracheal smooth muscle (CTSM) preparation, the effect of four putative PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC [PKC peptide-(19-31)], and staurosporine] on Ca2+ sensitization induced by acetylcholine (ACh) plus GTP was determined. Preincubation with each of the inhibitors did not affect subsequent Ca2+ sensitization induced by muscarinic receptor stimulation in the presence of a constant submaximal [Ca2+]i, neither did any of these compounds reverse the increase in Ca2+ sensitivity induced by ACh plus GTP. Administration of a 1,2-diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, did not induce Ca2+ sensitization at a constant submaximal [Ca2+]i. Thus we found no evidence that PKC mediates increases in Ca2+ sensitivity produced by muscarinic receptor stimulation in permeabilized CTSM.

Topics: Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Calcium; Cell Membrane Permeability; Cytosol; Diglycerides; Dogs; Enzyme Inhibitors; Escin; Female; Guanosine Triphosphate; In Vitro Techniques; Kinetics; Male; Muscle Contraction; Muscle, Smooth; Naphthalenes; Peptide Fragments; Phenanthridines; Protein Kinase C; Receptors, Muscarinic; Staurosporine; Trachea

The mechanism of endothelin-1 (ET-1)-induced atrial natriuretic peptide (ANP) release was studied in neonatal rat ventricular cardiomyocytes. These cells expressed a single high-affinity class of ETA receptor (dissociation constant = 54 +/- 18 pM, n = 3), but no ETB receptors. Incubation of cardiomyocytes with ET-1 led to concentration-dependent ANP release and prostacyclin production. ET-1-induced ANP release was affected by neither protein kinase C (PKC) inhibition or downregulation nor by cyclooxygenase inhibition, indicating that ET-1-stimulated ANP secretion is not a PKC-mediated, prostaglandin-dependent process. Furthermore, ET-1 significantly stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production and increased cytosolic calcium concentration in these preparations. Both ET-1-induced calcium influx and ANP release were decreased by the cAMP antagonist Rp-cAMPS, the Rp diastereoisomer of cAMP. Moreover, ET-1-induced ANP secretion was strongly inhibited in the presence of nifedipine as well as in the absence of extracellular calcium. Thus our results suggest that ET-1 stimulates ANP release in ventricular cardiomyocytes via an ETA receptor-mediated pathway involving cAMP formation and activation of a nifedipine-sensitive calcium channel.

Topics: Alkaloids; Animals; Animals, Newborn; Atrial Natriuretic Factor; Benzophenanthridines; Calcium; Cells, Cultured; Cyclic AMP; Cyclooxygenase Inhibitors; Egtazic Acid; Endothelin-1; Enzyme Inhibitors; Epoprostenol; Heart; Heart Ventricles; Kinetics; Myocardium; Naphthalenes; Nifedipine; Phenanthridines; Protein Kinase C; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Staurosporine; Thionucleotides

Intracellular Ca2+ and pH are potent modulators of growth factor-induced mitogenesis and contraction. This study examined platelet-derived growth factor-(PDGF-BB) and insulin-like growth factor (IGF-1)-mediated signal transduction in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric arteries of Sprague-Dawley rats. Intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) were measured by fluorescence digital imaging using fura-2 AM and 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Characteristics of [Ca2+]i transients were determined by pre-exposing cells to Ca2+-free buffer, and involvement of the Na+/Ca2+ exchanger was assessed by withdrawal of extracellular Na+ and by exposure to dimethylbenzamil (Na+/Ca2+ exchange blocker). To determine whether pHi responses were mediated via the Na+/H+ exchanger, cells were preincubated with 10(-5) mol/L 5-(N-ethyl-N-isopropyl)amiloride (a selective Na+/H+ exchange blocker). The role of protein kinase C (PKC) and tyrosine kinases in growth factor signaling was assessed by pre-exposing cells to calphostin C and chelerythrine chloride (selective PKC inhibitors; 10(-5) mol/L) and tyrphostin A23 (a selective tyrosine kinase inhibitor; 10(-5) mol/L). PDGF-BB and IGF-1 (1 to 10 ng/mL) increased [Ca2+]i and pHi in a dose-dependent manner. At concentrations greater than 1 ng/mL both growth factors induced a biphasic [Ca2+]i response with an initial transient peak followed by a sustained elevation. At 5 ng/mL PDGF-BB and IGF-1 significantly increased [Ca2+]i from 95+/-3 nmol/L to 328+/-28 and 251+/-18 nmol/L, respectively. Ca2+ withdrawal abolished the second phase of [Ca2+]i elevation. Agonist-induced [Ca2+]i responses were similarly altered by Na+ withdrawal, by Na+/ Ca2+ exchange blockade, and by PKC inhibition; latency, the period from stimulus application to the first [Ca2+]i peak, was increased, the initial [Ca2+]i peak was attenuated, and the sustained phase was prolonged. PDGF-BB and IGF-1 (10 ng/mL) significantly increased pHi from 6.89+/-0.04 nmol/L to 7.11+/-0.01 and 7.09+/-0.02 nmol/L, respectively. EIPA and calphostin C completely inhibited agonist-elicited alkalinization. Tyrphostin A-23 abolished second-messenger responses to PDGF-BB and IGF-1, whose receptors have tyrosine kinase activity. In conclusion, PDGF-BB and IGF-1 elicit significant [Ca2+]i and pHi responses in VSMC. The underlying pathways that mediate these responses are partially depe

Topics: Alkaloids; Animals; Becaplermin; Benzophenanthridines; Calcium; Cells, Cultured; Hydrogen-Ion Concentration; Insulin-Like Growth Factor I; Kinetics; Male; Mesenteric Arteries; Muscle, Smooth, Vascular; Naphthalenes; Phenanthridines; Platelet-Derived Growth Factor; Protein Kinase C; Proto-Oncogene Proteins c-sis; Rats; Rats, Sprague-Dawley; Signal Transduction; Sodium

Topics: Alkaloids; Animals; Benzophenanthridines; Cloning, Molecular; Enzyme Inhibitors; Isoenzymes; Kinetics; Mammals; Naphthalenes; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; Recombinant Proteins; Schizosaccharomyces; Sphingosine; Structure-Activity Relationship; Tetradecanoylphorbol Acetate

Recent study results suggest that protein kinase C [PKC (EC 3.1.4.3)] -dependent up-regulation of c-jun may be involved in leukemic cell programmed cell death, or apoptosis, occurring in response to various chemotherapeutic agents. The current study was undertaken to further evaluate the contribution of c-jun in apoptosis with the use of two highly specific pharmacological inhibitors of PKC (calphostin C and chelerythrine). To address this issue, two human leukemic cell lines, HL-60 and U937, and a U937 subline stably expressing a dominant negative c-jun mutant (TAM67) were exposed to calphostin C and chelerythrine, and c-jun expression was monitored at both the mRNA and protein levels. Both PKC inhibitors induced the classic morphological features of apoptosis as well as internucleosomal DNA degradation in a concentration- and schedule-dependent manner. Concomitant with these changes, unequivocal increases were observed in c-jun mRNA (U937 and HL-60) and protein (U937). In contrast, up-regulation of c-jun mRNA and protein in TAM67-expressing cells exposed to both PKC inhibitors was markedly attenuated relative to effects observed in parental U937 cells. Importantly, despite impaired up-regulation of c-jun at both the message and protein levels, TAM67-expressing cells were equally susceptible to PKC inhibitor-induced apoptosis as parental and empty vector U937 cells. Collectively, these findings raise the possibility that c-jun up-regulation in human myeloid leukemia cells undergoing PKC inhibitor-associated apoptosis represents a response to, rather than a cause of, apoptotic events. They further suggest that this phenomenon involves pathways that do not require PKC activation.

Topics: Alkaloids; Apoptosis; Benzophenanthridines; DNA Damage; Enzyme Inhibitors; Gene Expression Regulation; Genes, jun; HL-60 Cells; Humans; Naphthalenes; Phenanthridines; Protein Kinase C; Proto-Oncogene Proteins c-jun; RNA, Messenger; Up-Regulation

Activation of protein kinase C-delta (PKC-delta) by 12-O-tetradecanoylphorbol-13-acetate (TPA) is followed by a gradual decrease in detectable protein 12-24 h later in the mouse B lymphoma cell line A20. Down-regulation is associated with TPA-induced proteolysis and a 50-86% decrease in PKC-delta mRNA 0.5-24 h post-treatment which is due to both a 50% decrease in transcription and accelerated degradation of PKC-delta mRNA as determined using the pulse-chase method. Destabilization of PKC-delta mRNA is also observed when actinomycin D is added to cells pretreated with TPA for 2 h; however, addition of actinomycin D or cycloheximide prior to TPA treatment blocks destabilization. Addition of PKC inhibitors to TPA-treated cells also blocks destabilization of PKC-delta mRNA. Cells treated with TPA for 4 h contain an activity not found in control cells which destabilizes PKC-delta mRNA but not glyceraldehyde-3-phosphate dehydrogenase mRNA in vitro. Addition of TPA to control extracts fails to increase degradation of PKC-delta mRNA in vitro, suggesting that treatment of intact cells is required to induce the synthesis of a factor(s) that destabilizes PKC-delta mRNA. This factor(s) then acts along with transcriptional and post-translational regulatory mechanisms to down-regulate PKC-delta.

Topics: Alkaloids; Animals; Benzophenanthridines; Blotting, Northern; Cell Line; Cell Membrane; Cell Nucleus; Cytosol; Dactinomycin; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Isoenzymes; Kinetics; Lymphoma, B-Cell; Mice; Naphthalenes; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; RNA Processing, Post-Transcriptional; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

We report that WEHI-231 undergo apoptosis following exposure to the protein kinase C inhibitors chelerythrine chloride and calphostin C. Following the addition of chelerythrine or calphostin C to WEHI-231 cells, ceramide production increased over baseline levels with a concurrent decrease in sphingomyelin. More detailed examinations determined that the ceramide accumulation resulted from activation of neutral, but not acidic, sphingomyelinase. These results suggest an antagonistic relationship between protein kinase C activity and ceramide in the signaling events preceding apoptosis.

Topics: Alkaloids; Apoptosis; Benzophenanthridines; Cells, Cultured; Ceramides; Hydrolysis; Naphthalenes; Phenanthridines; Protein Kinase C; Sphingomyelin Phosphodiesterase; Sphingomyelins

1. The effect of angiotension II (Ang) on delayed rectifier K+ current (IK(V)) was studied in isolated rabbit portal vein smooth muscle cells using standard whole-cell voltage clamp technique. The effect of 100 nM Ang on macroscopic, whole-cell IK(V) was assessed in myocytes dialysed with 10 mM BAPTA, 5 mM ATP and 1 mM GTP either at room temperature or at 30 degrees C. 2. Application of Ang caused a decline in IK(V) which was reversed upon washout of the drug. Tail current recorded after 250 ms pulses to +30 mV and repolarization to -40 mV was reduced from 3.9 +/- 0.7 to 2.5 +/- 0.5 pA pF-1 at 20 degrees C (n = 6) and from 4.5 +/- 0.5 to 3.13 +/- 0.4 pA pF-1 at 30 degrees C(n = 17). 3. Ang had no effect on outward current in the presence of an AT1 selective antagonist, losartan (1 microM), which alone had no direct effect on the amplitude of IK(V). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 microM intracellular BAPTA did not affect the suppression of IK(V) by Ang. 4. Ang induced a decrease in time constant for the rapid phase of inactivation of the macroscopic current (tau 1 reduced from 377 +/- 32 to 245 +/- 11 ms; tau 2 unchanged, n = 17). Neither the voltage dependence of activation nor inactivation were affected by Ang. 5. The inhibition of IK(V) by Ang was abolished by intracellular dialysis with the selective PKC inhibitors, calphostin C (1 microM) and chelerythrine (50 microM). These data provide strong evidence that the decline in IK(V) due to Ang treatment is due to PKC activation. 6. The pattern of expression of PKC isoforms was examined in rabbit portal vein using isoenzyme-specific antibodies: alpha, epsilon and zeta isoenzymes were detected, but beta, gamma, delta and eta isoenzymes were not. 7. The lack of requirement for Ca2+, as well as the sensitivity of the Ang response to chelerythrine, suggest the involvement of the Ca(2+)-independent PKC isoenzyme epsilon in the signal transduction pathway responsible for IK(V) inhibition by Ang.

Topics: Alkaloids; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Benzophenanthridines; Biphenyl Compounds; Calcium; Enzyme Activation; Enzyme Inhibitors; Imidazoles; In Vitro Techniques; Ion Transport; Isoenzymes; Kinetics; Losartan; Membrane Potentials; Muscle, Smooth, Vascular; Naphthalenes; Phenanthridines; Portal Vein; Potassium; Protein Kinase C; Rabbits; Signal Transduction; Tetrazoles

Lysophosphatidylcholine (lyso PC) mediates multiple potentially atherogenic effects on endothelial cells, although the cellular mechanism of these effects remains unclear. Phospholipase D (PLD) has been recognized as a novel second-messenger system that may regulate cellular function. The purpose of this study was to determine the effect of lyso PC on PLD activity in human coronary artery endothelial cells (HCAEC) by measuring [3H]phosphatidylethanol production in cells labeled with [3H]myristic acid. After incubation with lyso PC (20 microM) for 40 min, PLD activity was markedly stimulated from five- to sixfold. Stimulation of PLD activity by lyso PC was concentration dependent (half-maximum effective concentration of 7.6 microM) and was not mimicked by phosphatidylcholine (20 microM). Because PLD can be regulated by protein kinases, the effect of several protein kinase inhibitors on lyso PC-stimulated PLD activity was tested. The protein kinase A inhibitor H-89 (300 nM) and the tyrosine kinase inhibitors genistein (30 microM) and tyrphostin A25 (100 microM) had no effect on the stimulation of PLD by lyso PC (20 microM). The protein kinase C (PKC) inhibitor calphostin C (10-300 nM) affected neither lyso PC (20 microM)-nor 4 beta-phorbol 12,13-dibutyrate (PDBu, 300 nM)-stimulated PLD activity, suggesting that this agent may not inhibit PKC in these cells. In contrast, the selective PKC inhibitors GF-109203X (0.3-10 microM) and chelerythrine (1-30 microM) concentration dependently inhibited lyso PC (20 microM)-stimulated PLD activity and blocked PDBu (300 nM)-stimulated PLD activity. Together, these data document that lyso PC stimulated PLD in human endothelial cells, possibly by a PKC-dependent mechanism, and provide evidence that PLD activation in human endothelium is a novel and important mechanism by which lyso PC mediates its cellular and possibly atherogenic effects.

Topics: Alkaloids; Benzophenanthridines; Cells, Cultured; Coronary Vessels; Endothelium, Vascular; Enzyme Inhibitors; Humans; Indoles; Lysophosphatidylcholines; Maleimides; Naphthalenes; Phenanthridines; Phorbol 12,13-Dibutyrate; Phospholipase D; Protein Kinase C

The involvement of protein kinase C (PKC) in exocytosis of the mammalian sperm acrosome is still a controversial issue. Work carried out thus far has failed to provide direct evidence for the activation of this enzyme upon stimulation with natural agonists of acrosomal exocytosis. We have therefore used progesterone stimulation of the acrosome reaction in human spermatozoa to clarify this issue. In spermatozoa preincubated under conditions known to support capacitation and fertilization in vitro, treatment with progesterone caused a time-dependent stimulation of phosphorylation of at least eight proteins ranging in size from approximately 20-220 kDa. The inclusion of the PKC inhibitors chelerythrine chloride or calphostin C reduced the observed phosphorylation in a concentration-dependent manner. Exogenously supplied phorbol 12-myristate-13-acetate (PMA) or the permeant diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (OAG), synthetic activators of PKC, also stimulated phosphorylation in preincubated spermatozoa, but inclusion of calphostin C diminished the response. Furthermore, the prior inclusion of the 1,4-dihydropyridine Ca2+ channel antagonist nifedipine also inhibited phosphorylation, suggesting that PKC is activated downstream of Ca2+ channel opening. Exocytosis triggered by progesterone was significantly inhibited by chelerythrine chloride or calphostin C. Both PMA and OAG triggered exocytosis, but the inclusion of chelerythrine chloride significantly inhibited the response; exocytotic responses were seen only in capacitated cells. These results provide the first direct evidence that PKC activation plays a role in the signal transduction pathway underlying acrosomal exocytosis in progesterone-stimulated capacitated spermatozoa.

Topics: Acrosome; Alkaloids; Benzophenanthridines; Calcium Channel Blockers; Calcium Channels; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Exocytosis; Humans; In Vitro Techniques; Male; Naphthalenes; Nifedipine; Phenanthridines; Phosphorylation; Progesterone; Protein Kinase C; Signal Transduction; Sperm Capacitation; Spermatozoa; Tetradecanoylphorbol Acetate

Topics: Alkaloids; Alkylating Agents; Anthracenes; Benzophenanthridines; Binding Sites; Carcinogens; Cysteine; Enzyme Inhibitors; Hydrogen Peroxide; Isoenzymes; Isoquinolines; Naphthalenes; Nitric Oxide; Oxidants; Periodic Acid; Perylene; Phenanthridines; Phorbol Esters; Phosphotransferases; Protein Kinase C

An immobilized hepatocyte preparation was used to show that both vasopressin and glucagon could desensitize the ability of glucagon to increase intracellular cyclic AMP concentrations. This process was not dependent on any influx of extracellular Ca2+ and was not mediated by any rise in the intracellular level of Ca2+. The protein kinase C-selective inhibitors chelerythrine, staurosporine and calphostin C acted as potent inhibitors of the desensitization process but with various degrees of selectivity regarding their ability to inhibit the desensitizing actions of glucagon and vasopressin. The protein phosphatase inhibitor okadaic acid was just as potent as vasopressin and glucagon in causing desensitization. Treatment of hepatocyte membranes with alkaline phosphatase restored to near control levels the ability of glucagon to stimulate adenylate cyclase activity in membranes from both glucagon- and vasopressin-treated (desensitized) hepatocytes. It is suggested that the desensitization of glucagon-stimulated adenylate cyclase activity involves a reversible phosphorylation reaction with the likely target being the glucagon receptor itself.

Topics: Adenylyl Cyclases; Alkaline Phosphatase; Alkaloids; Animals; Arginine Vasopressin; Benzophenanthridines; Cell Adhesion; Cells, Cultured; Collagen; Cyclic AMP; Drug Tolerance; Ethers, Cyclic; Glucagon; Liver; Male; Naphthalenes; Okadaic Acid; Phenanthridines; Phosphorylation; Polycyclic Compounds; Protein Kinase C; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Signal Transduction; Staurosporine

We have previously demonstrated that muscarinic and alpha-adrenergic receptors regulated a phospholipase D (PLD) activity in parotid glands. Since phorbol 12-myristate, 13-acetate (PMA) induced production of phosphatidylethanol (PEt), a stable metabolite widely accepted as marker of PLD activation, we have investigated the role of protein kinase C (PKC) in PLD stimulation in parotid acini. We tested PKC inhibitors on PEt formation elicited by PMA, by muscarinic and adrenergic agents. Staurosporine and chelerythrine, which act on the catalytic domain of PKC, did not allow the attribution of a role for PKC in PLD activation. Indeed, staurosporine did not affect PMA-mediated PLD activity and chelerythrine showed an important non-specific effect, independent of PKC inhibition. On the other hand, calphostin C, which acts on the regulatory domain of PKC, affected PMA- and receptor-mediated PLD stimulation. We attributed this effect to PKC inhibition and we suggested PKC involvement in PLD regulation in parotid gland. Since only PKC inhibitor acting on the regulatory part of the enzyme affected PLD activity, we also suggested that PKC could be involved in PLD activation through a pathway independent of the phosphorylation mechanism.

Topics: Alkaloids; Animals; Benzophenanthridines; Enzyme Activation; Glycerophospholipids; Male; Naphthalenes; Parotid Gland; Phenanthridines; Phosphatidic Acids; Phospholipase D; Polycyclic Compounds; Protein Kinase C; Rats; Rats, Sprague-Dawley; Staurosporine; Tetradecanoylphorbol Acetate

We have recently demonstrated the existence of an ATP-activated phospholipase D (PLD) in the nuclei of MDCK-D1 cells (Balboa, M. A., Balsinde, J., Dennis, E. A., and Insel, P. A. (1995) J. Biol. Chem. 270, 11738-11740). We have now found that nuclear PLD is synergistically activated by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and ATP in a time- and concentration-dependent manner, but these compounds do not alter the sensitivity of the enzyme to activation by Ca2+. The synergistic stimulation of PLD activity could be blocked by addition of the protein kinase C inhibitors chelerythrine and calphostin C. Stimulation by GTP gamma S was abolished by guanosine 5'-O-(2-thiodiphosphate). Incubation of isolated nuclei with Clostridium botulinum C3 exoenzyme inhibited the potentiating effect of GTP gamma S on ATP-dependent nuclear PLD activity. Moreover, use of the Rho GDP dissociation inhibitor to extract Rho family G proteins from cell nuclei also inhibits PLD activity. Western blot analyses of isolated nuclei revealed the presence of the small G protein RhoA, but not of RhoB or the ADP-ribosylation factor. GTP gamma S-stimulated ATP-dependent PLD activity could be reconstituted in Rho GDP dissociation inhibitor-washed nuclei by addition of recombinant prenylated RhoA, but not by addition of non-prenylated RhoA. Taken together, these results indicate that nuclear PLD activity is modulated via a RhoA-dependent activation that occurs downstream of protein kinase C. Nuclear PLD, which appears to be a previously unrecognized effector regulated by protein kinase C and G proteins, may be involved in the regulation of nuclear function or structure.

Topics: Adenosine Triphosphate; ADP Ribose Transferases; Alkaloids; Animals; Benzophenanthridines; Botulinum Toxins; Calcium; Cell Fractionation; Cell Line; Cell Nucleus; Clostridium botulinum; Dogs; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Kidney; Kinetics; Naphthalenes; Phenanthridines; Phospholipase D; Protein Kinase C; rhoA GTP-Binding Protein

The present studies were undertaken to characterize further the potential role of protein kinase C (PKC) in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. The capacity of acute exposure to specific and nonspecific pharmacological inhibitors of PKC to promote apoptotic DNA fragmentation was examined both quantitatively and qualitatively and correlated with effects on cellular differentiation and proliferation. Incubation of HL-60 cells for 6 h with chelerythrine and calphostin C (highly specific inhibitors that act at the regulatory domain) or H7 and gossypol (nonspecific inhibitors that act at the PKC catalytic domain) produced concentration-dependent increases in DNA fragmentation. Induction of DNA fragmentation by chelerythrine, calphostin C, and gossypol was biphasic, resulting in a sharp decline in effect at concentrations above 5 microM, 0.1 microM, and 100 microM, respectively, whereas maximal and more stable effects were observed in response to H7 (100 microM). A 6-h exposure to staurosporine, a nonspecific but potent PKC inhibitor, failed to induce DNA fragmentation at concentrations generally used to achieve maximal inhibition of enzyme activity (e.g., 50 nM) but promoted fragmentation at considerably higher concentrations (e.g., > or = 200 nM). In contrast, 6-h exposures to the nonspecific protein kinase inhibitor hypericin (0.1 to 100 microM) or to the nonspecific inhibitor of protein kinase A, HA1004 (50 microM), were without effect on DNA fragmentation. DNA obtained from cells exposed to chelerythrine (5 microM), calphostin C (100 nM), H7 (50 microM), gossypol (50 microM), and staurosporine (200 nM)--but not hypericin (25 microM)--exhibited clear evidence of internucleosomal DNA cleavage on agarose gel electrophoresis; moreover, these cells exhibited the classical morphological features of apoptosis (cell shrinkage, nuclear condensation, and the formation of apoptotic bodies). All of the PKC inhibitors that induced apoptosis, and one of the inhibitors that did not (hypericin), substantially inhibited HL-60 cell clonogenicity at the concentrations evaluated. None of the agents tested induced cellular maturation as assessed by nonspecific esterase and nitro-blue tetrazolium positivity. DNA fragments obtained from cells exposed to specific and nonspecific PKC inhibitors possessed predominantly 5'-phosphate termini, consistent with the action of a Ca(2+)-/Mg(2+)-dependent endonuclease. Finally, Northern blot analysis revealed

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Anthracenes; Antineoplastic Agents; Apoptosis; Benzophenanthridines; Cell Differentiation; Cell Line; Cyclic AMP-Dependent Protein Kinases; DNA Damage; DNA, Neoplasm; Enzyme Inhibitors; Gossypol; Humans; Isoquinolines; Leukemia, Promyelocytic, Acute; Naphthalenes; Perylene; Phenanthridines; Piperazines; Polycyclic Compounds; Protein Kinase C; Staurosporine; Time Factors; Tumor Cells, Cultured

Previously, we observed that long-term treatment of distal nerve fibers of rat sympathetic neurons in compartmented cultures with phorbol 12-myristate 13-acetate (PMA) caused a reduction in the rate of neurite elongation by > 50%. In the present report we show that protein kinase C (PKC) activity could be measured in extracts of distal neurites by an assay of the Ca(2+)-dependent phosphorylation of a PKC-specific octapeptide substrate. We found that local application of 1 microM PMA for 24 h to distal neurites caused nearly complete down-regulation of Ca(2+)-dependent PKC activity measured in this manner. We determined that the inhibition of neurite elongation by PMA was mediated by local mechanisms in the neurites because local application of PMA to center compartments containing cell bodies and proximal neurites did not inhibit the rate of elongation of distal neurites. We then investigated the effects of the recently available PKC inhibitors, calphostin C and chelerythrine, finding that, like PMA, these inhibited the growth of distal neurites when applied locally to them, and had no effect when applied to cell bodies and proximal neurites. However, the inhibition of neurite growth by calphostin C occurred at a concentration far below its IC50 value for protein kinase inhibition, and both calphostin C and chelerythrine inhibited distal neurite growth even in neurons pretreated with PMA. Thus, it appears that these agents do not all inhibit neurite growth through the same mechanisms. Although the PKC activities involved in neurite elongation in sympathetic neurons have not been precisely defined, these data presented in this study indicate that protein kinases localized to growth cones play a complex and important role in regulating axonal growth.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Antibodies; Benzophenanthridines; Calcium; Cells, Cultured; Isoquinolines; Naphthalenes; Nerve Growth Factors; Neurites; Phenanthridines; Phosphorylation; Piperazines; Polycyclic Compounds; Protein Kinase C; Rats; Rats, Sprague-Dawley; Superior Cervical Ganglion; Tetradecanoylphorbol Acetate

The efficacy of two structurally and functionally unrelated protein kinase C (PKC) inhibitors, chelerythrine and calphostin C, was assessed in intact human platelets by studying platelet aggregation in response to stimulation with phorbol 12-myristate 13-acetate (PMA) or the thromboxane-A2 mimetic, U46619. Surprisingly, both inhibitors increased aggregation in response to PMA, but decreased aggregation in response to U46619. To further explore this phenomenon, gel electrophoresis of 32P-labelled proteins from PMA- or U46619-stimulated platelets in the presence and absence of the two putative PKC inhibitors was performed. Although neither chelerythrine nor calphostin C proved to be effective PKC inhibitors in intact human platelets, a strong correlation between the dephosphorylation of a 68 kDa protein and the rate of platelet aggregation was observed. From these results, the indiscriminate use of PKC inhibitors in whole platelets is questioned and attention is drawn to the role of protein dephosphorylation in platelet activation. The 68 kDa protein was the major phosphorylated substrate in resting platelets. Okadaic acid increased phosphorylation of this band, indicating active phosphate group turnover under resting conditions.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alkaloids; Benzophenanthridines; Blood Proteins; Humans; Male; Molecular Weight; Naphthalenes; Phenanthridines; Phosphorus Radioisotopes; Phosphorylation; Platelet Aggregation; Platelet Aggregation Inhibitors; Polycyclic Compounds; Prostaglandin Endoperoxides, Synthetic; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Thromboxane A2