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PubMed1998

Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that epsilon is the isoform involved.

Brief ischemic episodes confer marked protection against myocardial stunning 1-3 d later (late preconditioning [PC] against stunning). The mechanism of this powerful protective effect is poorly understood. Although protein kinase C (PKC) has been implicated in PC against infarction, it is unknown whether it triggers late PC against stunning. In addition, the entire PKC hypothesis of ischemic PC remains controversial, possibly because the effects of PKC inhibitors on PC protection have not been correlated with their effects on PKC activity and/or translocation in vivo. Thus, conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles for three consecutive days (days 1, 2, and 3). In the control group (group I, n = 7), the recovery of systolic wall thickening after the six O/R cycles was markedly improved on days 2 and 3 compared with day 1, indicating the development of late PC against stunning. Administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg before the first O on day 1 (group II, n = 10) abrogated the late PC effect against stunning, whereas a 10-fold lower dose (0.5 mg/kg; group III, n = 7) did not. Administration of 5 mg/kg of chelerythrine 10 min after the sixth reperfusion on day 1 (group IV, n = 6) failed to block late PC against stunning. When rabbits were given 5 mg/kg of chelerythrine in the absence of O/R (group V, n = 5), the severity of myocardial stunning 24 h later was not modified. Pretreatment with phorbol 12-myristate 13-acetate (4 microg/kg) on day 1 without ischemia (group VI, n = 11) induced late PC against stunning on day 2 and the magnitude of this effect was equivalent to that observed after ischemic PC. In vehicle-treated rabbits (group VIII, n = 5), the six O/R cycles caused translocation of PKC isoforms epsilon and eta from the cytosolic to the particulate fraction without significant changes in total PKC activity, in the subcellular distribution of total PKC activity, or in the subcellular distribution of the alpha, beta1, beta2, gamma, delta, zeta, iota, lambda, and mu isoforms. The higher dose of chelerythrine (5 mg/kg; group X, n = 5) prevented the translocation of both PKC epsilon and eta induced by ischemic PC, whereas the lower dose (0.5 mg/kg; group XI, n = 5) prevented the translocation of PKC eta but not that of epsilon, indicating that the activation of epsilon is necessary for late PC to occur whereas that of eta is not. To our knowledge, this is the first demonstration that a PKC inhibitor actually prevents the translocation of PKC induced by ischemic PC in vivo, and that this inhibition of PKC translocation results in loss of PC protection. Taken together, the results demonstrate that the mechanism of late PC against myocardial stunning in conscious rabbits involves a PKC-mediated signaling pathway, and implicate epsilon as the specific PKC isoform responsible for the development of this cardioprotective phenomenon.

Alkaloids↗

PubMed2003

Activation of Mst1 causes dilated cardiomyopathy by stimulating apoptosis without compensatory ventricular myocyte hypertrophy.

Activation of mammalian sterile 20-like kinase 1 (Mst1) by genotoxic compounds is known to stimulate apoptosis in some cell types. The importance of Mst1 in cell death caused by clinically relevant pathologic stimuli is unknown, however. In this study, we show that Mst1 is a prominent myelin basic protein kinase activated by proapoptotic stimuli in cardiac myocytes and that Mst1 causes cardiac myocyte apoptosis in vitro in a kinase activity-dependent manner. In vivo, cardiac-specific overexpression of Mst1 in transgenic mice results in activation of caspases, increased apoptosis, and dilated cardiomyopathy. Surprisingly, however, Mst1 prevents compensatory cardiac myocyte elongation or hypertrophy despite increased wall stress, thereby obscuring the use of the Frank-Starling mechanism, a fundamental mechanism by which the heart maintains cardiac output in response to increased mechanical load at the single myocyte level. Furthermore, Mst1 is activated by ischemia/reperfusion in the mouse heart in vivo. Suppression of endogenous Mst1 by cardiac-specific overexpression of dominant-negative Mst1 in transgenic mice prevents myocyte death by pathologic insults. These results show that Mst1 works as both an essential initiator of apoptosis and an inhibitor of hypertrophy in cardiac myocytes, resulting in a previously unrecognized form of cardiomyopathy.

Alkaloids↗

PubMed1996

The inhibiting effect of aqueous Azadirachta indica (Neem) extract upon bacterial properties influencing in vitro plaque formation.

The purpose of this investigation was to examine the inhibitory effects of aqueous extracts derived from the bark-containing sticks (Neem stick) of Azadirachta indica upon bacterial aggregation, growth, adhesion to hydroxyapatite, and production of insoluble glucan, which may affect in vitro plaque formation. Neem stick extracts were screened for minimal bacterial growth inhibition (MIC) against a panel of streptococci by means of a broth dilution assay. Initial bacterial attachment was quantified by the measurement of the adhesion of 3H-labeled Streptococcus sanguis to saliva-conditioned synthetic hydroxyapatite. The effect of the Neem stick extract upon insoluble glucan synthesis was measured by the uptake of radiolabeled glucose from 14C-sucrose. Aggregating activity of the Neem stick extracts upon a panel of streptococci was also examined. No inhibition of bacterial growth was observed among the streptococcal strains tested in the presence of < or = 320 micrograms/mL of the Neem stick extract. The pre-treatment of S. sanguis with the Neem stick extract or the gallotannin-enriched extract from Melaphis chinensis at 250 micrograms/mL resulted in a significant inhibition of the bacterial adhesion to saliva-conditioned hydroxyapatite. Pre-treatment of saliva-conditioned hydroxyapatite with the Neem stick or gallotannin-rich extract prior to exposure to bacteria yielded significant reductions in bacterial adhesion. The Neem stick extract and the gallotannin-enriched extract from Melaphis chinensis inhibited insoluble glucan synthesis. Incubation of oral streptococci with the Neem stick extract resulted in a microscopically observable bacteria aggregation. These data suggest that Neem stick extract can reduce the ability of some streptococci to colonize tooth surfaces.

Alkaloids↗

PubMed2003

Immunocytochemical detection of GLUT2 at the rat intestinal brush-border membrane.

We have proposed a new model of intestinal sugar absorption in which high sugar concentrations promote rapid insertion of the facilitative transporter GLUT2 into the brush-border membrane so that absorptive capacity is precisely regulated to match dietary intake during the assimilation of a meal. However, location of GLUT2 at the brush border by immunocytochemistry has been problematical. We report that control of rapid GLUT2 trafficking and the use of an antibody to a sequence within the large extracellular loop of GLUT2 permits localization of GLUT2 at the brush border. To reveal brush-border GLUT2 fully, it is necessary to digest the sugar chain at the glycosylation site close to the antigenic site. In this way, we have demonstrated by immunocytochemistry PKC-dependent changes in the regulation of brush-border GLUT2 in rat jejunum that correspond to those seen by Western blotting. The functional and immunocytochemical data are now reconciled.

Alkaloids↗

PubMed2003

Vasospasm following subarachnoid hemorrhage: evidence against functional upregulation of protein kinase C constrictor pathway.

This study tested the hypothesis that vasospasm due to subarachnoid hemorrhage involves the functional upregulation of protein kinase C. Spasm of the rabbit basilar artery was achieved using a double hemorrhage model, which we previously demonstrated was endothelin-1 dependent. In situ effects of agents were determined by direct measurement of vessel diameter following their suffusion in a cranial window. Chelerythrine, a protein kinase C inhibitor, relaxed the spasm. However, relaxations to chelerythrine were not significantly greater in endothelin-1 constricted spastic vessels initially relaxed with the endothelin converting enzyme inhibitor, phosphoramidon, as compared to endothelin-1 constricted control vessels. These results suggest that subarachnoid hemorrhage induced vasospasm does not involve functional upregulation of protein kinase C.

Alkaloids↗

PubMed2002

Phorbol ester stimulates a protein kinase C-mediated agatoxin-TK-sensitive calcium permeability pathway in human red blood cells.

Calcium entry into mature erythrocytes (red blood cells; RBCs) is associated with multiple changes in cell properties. At low intracellular Ca(2+), efflux of potassium and water predominates, leading to changes in erythrocyte rheology. At higher Ca(2+) content, activation of kinases and phosphatases, rupture of membrane-to-skeleton bridges, stimulation of a phospholipid scramblase and phospholipase C, and induction of transglutaminase-mediated protein cross-linking are also observed. Because the physiologic relevance of these latter responses depends partially on whether Ca(2+) entry involves a regulated channel or nonspecific leak, we explored mechanisms that initiate controlled Ca(2+) influx. Protein kinase C (PKC) was considered a prime candidate for the pathway regulator, and phorbol-12 myristate-13 acetate (PMA), a stimulator of PKC, was examined for its influence on erythrocyte Ca(2+). PMA was found to stimulate a rapid, dose-dependent influx of calcium, as demonstrated by the increased fluorescence of an entrapped Ca(2+)-sensitive dye, Fluo-3/AM. The PMA-induced entry was inhibited by staurosporine and the PKC-selective inhibitor chelerythrine chloride, but was activated by the phosphatase inhibitors okadaic acid and calyculin A. The PMA-promoted calcium influx was also inhibited by omega-agatoxin-TK, a calcium channel blocker specific for Ca(v)2.1 channels. To confirm that a Ca(v)2.1-like calcium channel exists in the mature erythrocyte membrane, RBC membrane preparations were immunoblotted with antiserum against the alpha(1A) subunit of the channel. A polypeptide of the expected molecular weight (190 kDa) was visualized. These studies indicate that an omega-agatoxin-TK-sensitive, Ca(v)2.1-like calcium permeability pathway is present in the RBC membrane and that it may function under the control of kinases and phosphatases.

Adult↗

PubMed2001

Role of protein kinase C zeta isoform in Fas resistance of immature myeloid KG1a leukemic cells.

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.

Alkaloids↗

PubMed2006

Disruption of nucleocytoplasmic trafficking of cyclin D1 and topoisomerase II by sanguinarine.

BACKGROUND: The quaternary isoquinoline alkaloid sanguinarine is receiving increasing attention as a potential chemotherapeutic agent in the treatment of cancer. Previous studies have shown that this DNA-binding phytochemical can arrest a number of different types of transformed cells in G0/G1, and upregulate the CKIs p21 and p27 while downregulating multiple cyclins and CDKs. To more closely examine the responses of some of these cell cycle regulatory molecules to sanguinarine, we used immunocytochemical methods to visualize cyclin D1 and topoisomerase II behavior in MCF-7 breast cancer cells. RESULTS: 5-10 microM sanguinarine effectively inhibits MCF-7 proliferation after a single application of drug. This growth inhibition is accompanied by a striking relocalization of cyclin D1 and topoisomerase II from the nucleus to the cytoplasm, and this effect persists for at least three days after drug addition. DNA synthesis is transiently inhibited by sanguinarine, but cells recover their ability to synthesize DNA within 24 hours. Taking advantage of the fluorescence characteristics of sanguinarine to follow its uptake and distribution suggests that these effects arise from a window of activity of a few hours immediately after drug addition, when sanguinarine is concentrated in the nucleus. These effects occur in morphologically healthy-looking cells, and thus do not simply represent part of an apoptotic response. CONCLUSION: It appears that sub-apoptotic concentrations of sanguinarine can suppress breast cancer cell proliferation for extended lengths of time, and that this effect results from a relatively brief period of activity when the drug is concentrated in the nucleus. Sanguinarine transiently inhibits DNA synthesis, but a novel mechanism of action appears to involve disrupting the trafficking of a number of molecules involved in cell cycle regulation and progression. The ability of sub-apoptotic concentrations of sanguinarine to inhibit cell growth may be a useful feature for potential chemotherapeutic applications; however, a narrow effective range for these effects may exist.

Active Transport, Cell Nucleus↗

PubMed2001

High calcium and dobutamine positive inotropy in the perfused mouse heart: myofilament calcium responsiveness, energetic economy, and effects of protein kinase C inhibition.

BACKGROUND: In perfused hearts, high calcium-induced inotropy results in less developed pressure relative to myocardial oxygen consumption compared to the beta-adrenergic agonist dobutamine. Calcium handling is an important determinant of myocardial oxygen consumption. Therefore, we hypothesized that this phenomenon was due to reduced myofilament responsiveness to calcium, related to protein kinase C activation. RESULTS: Developed pressure was significantly higher with dobutamine compared to high perfusate calcium of 3.5 mM (73 +/- 10 vs 63 +/- 10 mmHg, p < 0.05), though peak systolic intracellular calcium was not significantly different, suggesting reduced myofilament responsiveness to intracellular calcium with high perfusate calcium. The ratio of developed pressure to myocardial oxygen consumption, an index of economy of contraction, was significantly increased with dobutamine compared to high perfusate calcium (1.35 +/- 0.15 vs 1.15 +/- 0.15 mmHg/micromoles/min/g dry wt, p < 0.05), suggesting energetic inefficiency with high perfusate calcium. The specific protein kinase C inhibitor, chelerythrine, significantly attenuated the expected increase in developed pressure when increasing perfusate calcium from 2.5 to 3.5 mM (3.5 mM: 64 +/- 8 vs 3.5 mM + chelerythrine: 55 +/- 5 mmHg, p < 0.05), though had no effects on dobutamine, or lower levels of perfusate calcium (1.5 to 2.5 mM). CONCLUSIONS: By measuring intracellular calcium, developed pressures and myocardial oxygen consumption in perfused mouse hearts, these results demonstrate that high perfusate calcium positive inotropy compared to dobutamine results in reduced myofilament responsiveness to intracellular calcium, which is associated with energetic inefficiency and evidence of protein kinase C activation.

Actin Cytoskeleton↗

PubMed2026

Sanguinarine as a multi-target therapeutic candidate for laryngeal cancer: insights from network pharmacology, molecular dynamics and in vitro validation.

OBJECTIVE: To identify the core targets and elucidate the potential molecular mechanisms of sanguinarine (SA) against laryngeal squamous cell carcinoma (LSCC), and to validate its antitumor effects in vitro. METHODS: Potential targets of SA were predicted using SwissTargetPrediction, TargetNet, and SuperPred and intersected with LSCC-related targets obtained from the GeneCards, OMIM, and DISEASES databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. A protein-protein interaction (PPI) network was constructed using the STRING database (combined score > 0.900), and topological parameters including degree centrality (DC), betweenness centrality (BC), closeness centrality (CC), eigenvector centrality (EC), and local average connectivity (LAC) were calculated in Cytoscape to identify core genes based on median thresholds. Molecular docking and 100-ns molecular dynamics (MD) simulations were conducted for epidermal growth factor receptor (EGFR), Phosphatidylinositide-3-kinase catalytic subunit alpha (PIK3CA), phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit β (PIK3CB), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD), and Non-Receptor Tyrosine Kinase (SRC). The effects of SA on LSCC were evaluated using CCK-8, colony formation, Transwell migration, and wound-healing assays in TU177 cells and TU212. RESULTS: A total of 213 common targets were identified, which were significantly enriched in PI3K-Akt signaling, EGFR tyrosine kinase inhibitor resistance, and adhesion- and migration-related pathways. The PPI network comprised 259 nodes and 259 edges, from which five core genes-PIK3CA, PIK3CB, PIK3CD, EGFR, and SRC-were identified. Molecular docking revealed strong binding affinities between SA and the PI3K family proteins (- 9.79 to - 10.96 kcal/mol), as well as EGFR (- 8.58 kcal/mol) and SRC (- 6.77 kcal/mol). MD simulations indicated greater stability of SA complexes with EGFR and PI3K family members compared with SRC. In vitro assays demonstrated that SA significantly inhibited TU177 cell and TU212 cell proliferation, colony formation, and migration. CONCLUSION: SA may exert anti-laryngeal cancer effects through synergistic multi-target inhibition centered on the EGFR/SRC/PI3K signaling axis, highlighting its potential as a promising therapeutic candidate for LSCC.

Humans↗

PubMed2003

Natriuretic peptides suppress protein kinase C activity to reduce evoked dopamine efflux from pheochromocytoma (PC12) cells.

The observation that natriuretic peptides and protein kinase C activators influence evoked neurotransmitter efflux by diametrically opposed mechanisms prompted an investigation of the influence of natriuretic peptides on protein kinase C activity and the potential involvement of this pathway in neuromodulatory responses to natriuretic peptides. C-Type natriuretic peptide attenuated both evoked dopamine efflux and protein kinase C activity in a concentration-dependent manner consistent with a 10% diminution in protein kinase C activity producing a 4.6-6.2% reduction in evoked dopamine efflux. The ability of C-type natriuretic peptide to suppress evoked dopamine efflux was abolished by treatment with the protein kinase C inhibitors chelerythrine (10 micro M) and staurosporine (10 nM). Both chelerythrine and staurosporine attenuated protein kinase C activity at the concentrations used. The natriuretic peptide C receptor (NPR-C) appeared to mediate the attenuation of protein kinase C activity, because the effect was mimicked by a pentadecapeptide fragment of the NPR-C, and the effect of C-type natriuretic peptide was attenuated by an antibody generated against the same region of the receptor. These data suggest that C-type natriuretic peptide attenuates neurotransmitter efflux by a mechanism involving suppression of neuronal protein kinase C activity via an interaction with the NPR-C.

Alkaloids↗

PubMed2005

Palmitate induces tumor necrosis factor-alpha expression in C2C12 skeletal muscle cells by a mechanism involving protein kinase C and nuclear factor-kappaB activation.

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.

Alkaloids↗

PubMed1995

Differential involvement of protein kinase C in the regulation of transforming growth factor-beta (TGF-beta) secretion by porcine theca and granulosa cells in vitro.

Transforming growth factor-beta (TGF-beta) has been implicated in the regulation of ovarian follicle development. Little, however, is known regarding the regulation of TGF-beta expression within the follicle. To investigate this, granulosa and theca cells were isolated from small antral follicles of prepubertal porcine ovaries, maintained in monolayer culture, and treated with gonadotropins or intracellular activators of the protein kinase A and C pathways. TGF-beta secreted into the medium was measured using a proliferation inhibition bioassay with MvLu1 epithelial cells. Over a broad dose range, FSH and LH were ineffective in stimulating TGF-beta secretion relative to controls in granulosa and theca cells, respectively. Additionally, 8-bromo-cAMP, a direct activator of protein kinase A, was ineffective in stimulating TGF-beta secretion in either cell type. In marked contrast, PMA, a stimulator of protein kinase C, dose-dependently stimulated TGF-beta secretion in theca cells. Interestingly, however, PMA had virtually no effect upon granulosa cells. The stimulatory effect of PMA on theca cell TGF-beta secretion was not observed with the inactive derivitive 4 alpha-PMA, and the PMA effect was inhibited by chelerythrine chloride, an inhibitor with high specificity toward protein kinase C. Taken together, these results argue against a direct role of the protein kinase A pathway in the regulation of TGF-beta expression in porcine follicle cells and support direct involvement of the protein kinase C pathway. Moreover, there appears to be marked differences in the regulation of this growth factor between theca and granulosa cells.

Alkaloids↗

PubMed1996

Lysophosphatidylcholine stimulates interleukin-6 release from rat anterior pituitary cells in vitro.

Interleukin-6 (IL-6) is a B-cell differentiation-inducing cytokine that affects the secretion of several neuroendocrine hormones. Normal rat anterior pituitary (AP) cells synthesize and release IL-6, suggesting a paracrine role for the stimulation of AP hormone release by this cytokine. We have previously reported that IL-1 beta enhances IL-6 release and phospholipase A2 (PLA2)-mediated hydrolysis of phosphatidylcholine (PC) in AP cells. Because lysophosphatidylcholine (LPC) may function as a second messenger for IL-1 beta, we have investigated the effects of exogenous LPC on IL-6 release from AP cells in vitro. AP cells from male Long-Evans rats were dispersed and cultured for 5-6 days in 96-well (100,000 cells/well) culture plates. Cells were rinsed and incubated in the absence or presence of 1.25-40 microM LPC 18:0 (stearoyl) for 6 h, and IL-6 concentrations determined using the 7-TD1 cell bioassay. LPC 18:0 significantly (P < 0.01) stimulated IL-6 release up to 10-fold in a concentration-related manner. In contrast, LPC 18:0 did not affect PRL release. LPC species substituted with progressively shorter saturated 1-acyl chains (16:0-10:0) were less effective for IL-6 induction. Examination of structurally related glycerophospholipid species revealed the specificity of the LPC stimulation of IL-6 release. Thus, 1.25-40 microM lysophosphatidylethanolamine (LPE; 18:0) and lysophosphatidic acid (LPA; 18:0) were without significant effect on AP IL-6 release, demonstrating the specific functional requirement for the phosphorylcholine headgroup. Hydrolysis of the structurally related choline-linked phospholipid sphingomyelin (SM) has been implicated in IL-1 beta action in certain cell types. Similarly, 1.25-20 microM lysosphingomyelin (sphingosylphosphorylcholine; SPC) also significantly (P < or = 0.01) stimulated IL-6 release from AP cells, although SPC exhibited discernibly lower potency and efficacy than LPC. An acyl analog of platelet-activation factor (PAF), i.e. 18:0-2:0 PC (1-stearoyl-2-acetoyl-sn-glycero-3-phosphorylcholine), differs from LPC by an acetyl group in the sn-2 position; PAF was at least as effective as LPC for the stimulation of IL-6 release from AP cells in vitro. Stimulation of IL-6 release by LPC 18:0 was completely suppressed by pharmacological inhibitors of protein kinase C such as H7 (20 microM) and chelerythrine (5 microM). In addition, H7 (20 microM) abolished the stimulation of IL-6 release by IL-1 beta (0.16-100 ng/mL). These findings demonstrate that LPC, acyl PAF, or SPC (but not other lysophospholipids) stimulate IL-6 release from AP cells in vitro. We conclude that LPC-mediated activation of protein kinase C is involved in the stimulatory actions of IL-1 beta in AP cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Transforming growth factor-beta1 inhibits membrane association of protein kinase C alpha in a human prostate cancer cell line, PC3.

The postreceptor signaling pathway(s) that mediates the effects of transforming growth factor-beta1 (TGF-beta1) is incompletely understood. The present study investigated the involvement of protein kinase C (PKC) in the growth-inhibitory action of TGF-beta1 in PC3, a human prostate cancer cell line. PKC alpha, the only conventional PKC isoform detected in PC3 cells, appeared to be constitutively active based on its presence in both Triton-soluble membrane fraction and cytosol. However, levels of membrane-associated PKC alpha were decreased by a growth-inhibitory dose of TGF-beta1. The response to TGF-beta1 was rapid (within 5 min), time dependent, isoform specific, and occurred without apparent changes in levels of total PKC alpha protein. TGF-beta1 also decreased the levels of membrane-associated PKC activity coincident with its inhibitory effect on PKC alpha's membrane association. Inhibition of PKC activity appeared to be associated with growth inhibition in PC3 cells, because chelerythrine (a specific PKC inhibitor) likewise decreased cell proliferation. Taken together, our data suggest that inhibition of PKC activity, at least in part due to inactivation of PKC alpha, is an early event associated with TGF-beta1 postreceptor signaling that might mediate suppression of cell proliferation.

Alkaloids↗

PubMed1997

Melatonin action and signal transduction in the rat suprachiasmatic circadian clock: activation of protein kinase C at dusk and dawn.

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.

Alkaloids↗

PubMed1997

Activation of phospholipase D in FRTL-5 thyroid cells by forskolin and dibutyryl-cyclic adenosine monophosphate.

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 in the FRTL-5 thyroid cell (PKA and PKC) appear to converge on PLD activation. Stimulation of both of these pathways by TSH may be required for optimal physiological activation of PLD.

Alkaloids↗

PubMed2004

Activation of protein kinase C contributes to the isoflurane-induced improvement of functional and metabolic recovery in isolated ischemic rat hearts.

Isoflurane enhances myocardial functional recovery and improves energy levels after ischemia. We sought to determine whether isoflurane-induced cardioprotection is mediated by protein kinase C (PKC). The Langendorff model was used, and isolated perfused rat hearts were separated into untreated, isoflurane, chelerythrine (PKC inhibitor) plus isoflurane, and chelerythrine groups. All hearts were subjected to treatment before ischemia, followed by 30 min of ischemia and 60 min of reperfusion. We recorded hemodynamic variables, measured metabolites by high-performance liquid chromatography, and analyzed subcellular localization of PKC isoforms by Western blot analysis. Isoflurane significantly improved the recovery of left ventricular developed pressure, attenuated the depletion of myocardial adenosine triphosphate (ATP) and creatine phosphate at 15 min of ischemia, enhanced the recovery of myocardial ATP and creatine phosphate concentrations after ischemia, and was associated with the translocation of PKC-delta and -epsilon to the membrane. Chelerythrine suppressed the translocation of PKC-delta and -epsilon and blocked the improvement of cardiac function and ATP. We conclude that isoflurane delays the decrease in ATP during ischemia and improves the recovery of mechanical function and the energy state 60 min after ischemia. These effects of isoflurane are dependent on the activation of PKC.

Adenosine Triphosphate↗