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PubMed2002

Remote preconditioning protects the heart by activating myocardial PKCepsilon-isoform.

OBJECTIVE: Myocardial protection can be achieved by brief ischemia-reperfusion of remote organs, a phenomenon described as remote preconditioning (RPC). Since the intracellular mechanisms of RPC are not known, we tested the hypothesis that RPC might activate myocardial PKCepsilon, an essential mediator of classical ischemic preconditioning. Furthermore, we tried to delineate the mechanisms by which RPC is transduced to the heart with respect to the possible contribution of kinins and neuronal reflexes. METHODS: Anesthetized rats were randomised to undergo either 30 min of waiting (controls) or RPC (brief mesenteric artery occlusion followed by reperfusion) in the absence or presence of chelerythrine (5 mg kg(-1)), a specific PKC inhibitor. Myocardial infarct size was measured by TTC staining after 30 min of coronary artery occlusion followed by 150 min of reperfusion. In separate sets of experiments RPC was performed with or without pretreatment with HOE140, a selective B(2)-antagonist or hexamethonium was used to explore the influence of ganglion blockade on RPC. Translocation of PKCepsilon from cytosol to the particulate fraction was measured by quantitative immunoblotting. RESULTS: RPC significantly reduced infarct size which was completely blocked by the PKC inhibitor. RPC shifted the ratio between cytosolic and particulate PKCepsilon, an indicator for PKC-activation, from 0.95+/-0.06 in controls to 0.41+/-0.09 (P<0.05), and this effect was abolished by HOE140. Activation of PKCepsilon could not be achieved after pretreatment with HEX (0.69+/-0.06 in HEX vs. 0.78+/-0.06 in HEX+RPC). CONCLUSIONS: RPC activates myocardial PKCepsilon through a neuronal and bradykinin-dependent pathway. We assume that activation of PKCepsilon is an important step in cardioprotection induced by remote preconditioning.

Adrenergic beta-Antagonists↗
PubMed2003

Adenosine-mediated early preconditioning in mouse: protective signaling and concentration dependent effects.

OBJECTIVES: Signaling in adenosine-mediated preconditioning is controversial. We examined roles of mitochondrial (mito) K(ATP) channels, protein kinase C (PKC) and nitric oxide (NO). METHODS: Langendorff perfused C57/Bl6 mouse hearts were subjected to 20 min ischemia and 45 min reperfusion. Effects of adenosine-mediated preconditioning were assessed in the absence and presence of signaling inhibitors. RESULTS: Control hearts recovered 70+/-2 mmHg ventricular pressure, and released 18.1+/-2.0 IU/g lactate dehydrogenase (LDH). Preconditioning with 10 microM adenosine limited necrosis (10.6+/-1.4 IU/g) without modifying contractility (72+/-2 mmHg) whereas 50 microM adenosine reduced necrosis (10.3+/-1.6 IU/g) and contractile dysfunction (91+/-2 mmHg). All protective effects of 10 and 50 microM adenosine were abrogated by mito K(ATP) channel blockade with 100 microM 5-hydroxydecanoate (5-HD) during the 'trigger' phase, but unaltered by PKC or NO synthase inhibition with 3 microM chelerythrine or 100 microM N(G)-nitro-L-arginine methyl ester (L-NAME), respectively. Protection against necrosis was eliminated by 5-HD but unaltered by chelerythrine or L-NAME during the 'mediation' phase (ischemia-reperfusion). Reduced contractile dysfunction with 50 microM adenosine was partially sensitive to 5-HD and chelerythrine, and only eliminated by co-infusion of the inhibitors. CONCLUSIONS: Adenosine-mediated preconditioning is dose-dependent with high level stimulation reducing contractile dysfunction in addition to necrosis. Preconditioning is triggered by a mito K(ATP) channel dependent process independently of PKC and NO. Subsequent protection against necrosis is also mediated by a mito K(ATP) channel dependent process independent of PKC and NO. In contrast, functional protection may be mediated by parallel mito K(ATP) and PKC dependent paths.

Adenosine↗
PubMed2003

Diverse regulation of atrial natriuretic peptide secretion by serotonin receptor subtypes.

OBJECTIVE: Serotonin (5-hydroxytryptamine [5-HT]) receptors are located in peripheral tissues as well as in the central nervous system. Serotonin receptors mediate positive inotropic and chronotropic effects in atria. The aim of this study was to investigate physiological role of endogenous serotonin on the regulation of atrial natriuretic peptide (ANP) secretion from the atria. METHODS: An isolated perfused nonbeating rat atrial model was used. Changes in atrial volume induced by increasing intra-atrial pressure were measured. The concentration of ANP was measured by radioimmunoassay and the translocation of ECF was measured by [3H]-inulin clearance. RESULTS: Serotonin, an endogenous 5-HT receptor agonist, caused concentration-dependent suppressions of stretch-induced ANP secretion, which were less pronounced than those caused by alpha-methyl-5-HT maleate, a 5-HT(2) receptor selective agonist. The suppression of stretch-induced ANP secretion due to serotonin and alpha-methyl-5-HT maleate was attenuated by ketanserin, a 5-HT(2) receptor antagonist, and accentuated by RS23597-190, a 5-HT(4) receptor antagonist. The suppressive effect of serotonin on ANP secretion was attenuated by neomycin, staurosporine, and chelerythrine. In contrast, 2-[1-(4-piperonyl)piperazinyl]benzothiazole, a 5-HT(4) receptor selective agonist, caused an accentuation of stretch-induced ANP secretion, which was completely blocked by RS23597-190 and SB203186 HCl but not by ketanserin. This effect was not affected by MDL12330, KT-5720, or H-89. The intracellular Ca(2+) concentration in single atrial myocytes was not changed by serotonin and agonist for either 5-HT(2) or 5-HT(4) receptor. CONCLUSIONS: These results suggest that atrial 5-HT(2) and 5-HT(4) receptor agonists have opposite actions on the regulation of ANP secretion and the suppressive effect of serotonin on the ANP secretion may act through 5-HT(2) receptor and phospholipase C pathway.

Adenylyl Cyclase Inhibitors↗
PubMed1998

Oxidant stress with hydrogen peroxide attenuates calcium paradox injury: role of protein kinase C and ATP-sensitive potassium channel.

OBJECTIVE: We tested the hypotheses that low concentration of H2O2 attenuates the Ca2+ paradox (Ca2+ PD) injury, and that activation of protein kinase C (PKC) and/or ATP-sensitive potassium channel (KATP) are involved in the protective effects of H2O2. METHODS: Langendorff-perfused rat hearts were subjected to the Ca2+ PD (10 min of Ca2+ depletion followed by 10 min of Ca2+ repletion). Functional and biochemical effects of H2O2 and other interventions on the cell injury induced by the Ca2+ PD were assessed. RESULTS: In the Ca2+ PD hearts pretreated with 20 mumol/l H2O2, left ventricular end-diastolic pressure and coronary flow were significantly preserved. Furthermore, peak lactate dehydrogenase release was significantly decreased and ATP contents were more preserved, compared with non-treated Ca2+ PD hearts. H2O2-treated hearts also showed remarkable preservation of cell structure. Addition of a specific PKC inhibitor, chelerythrine during H2O2 treatment completely abolished the beneficial effects of H2O2 on the Ca2+ PD. Similarly, an activator of PKC. Phorbol 12-myristate 13 acetate mimicked the protection by H2O2. Furthermore, pretreatment with a KATP opener, cromakalim also provided protection similar to H2O2 against the Ca2+ PD injury. However, a specific KATP inhibitor, glibenclamide was not able to completely block the effects of H2O2. CONCLUSIONS: These findings suggest that pretreatment with low concentration of H2O2 provides significant protection against the lethal injury of Ca2+ PD in rat hearts. PKC-mediated signaling pathways appear to play a crucial role in the protection against the Ca2+ PD injury.

Adenosine Triphosphate↗
PubMed1998

Phenylephrine-induced stimulation of Na+/Ca2+ exchange in rat ventricular myocytes.

OBJECTIVE: The effect of an alpha-adrenergic agonist, phenylephrine, on the Na+/Ca2+ exchange current in rat ventricular myocytes was investigated. METHODS: The Na+/Ca2+ exchange current was measured at room temperature in rat ventricular myocytes as the whole-cell current induced by addition of extracellular Na+ and Ca2+, while blocking Na+ current by setting the holding potential at -30 mV, K+ currents by intracellular Cs+, TEA+ and by extracellular Ba2+, Ca2+ current by nifedipine and Na+ pump current by ouabain or by 0 extracellular K+. RESULTS: Under these experimental conditions, application of external Na+ and Ca2+ induced a current which was further increased by phenylephrine. Phenylephrine (80 microM) increased the current by up to 31.0 +/- 5.4% of control at all membrane potentials tested both below and above the reversal potential. The reversal potential (+21.0 +/- 3.2 mV), which corresponded with the theoretical reversal potential for the Na+/Ca2+ exchange current under our ionic conditions (+21.3 mV), was not changed by phenylephrine (+23.2 +/- 4.1 mV). Applying phenylephrine in the absence of Na+/Ca2+ exchange (0 Na+e, 0 Ca2+e) did not change the current. The effect was resistant to propranolol, a beta-adrenergic blocker, but prevented by prazosin, an alpha-receptor antagonist, by neomycin, an inhibitor of phospholipase C, and by chelerythrine, a selective inhibitor of protein kinase C. Phorbol 12-myristate 13-acetate failed to stimulate the current. The effect remained similar under conditions of high (HEPESi = 5 mM) and low (HEPESi = 0.5 mM) intracellular pH buffering. CONCLUSION: Our data indicate that phenylephrine stimulates the Na+/Ca2+ exchange, both in the forward and the reverse modes, probably via a protein kinase C-dependent pathway.

Adrenergic alpha-Agonists↗
PubMed1999

Signal transduction in spontaneous myogenic tone in isolated arterioles from rat skeletal muscle.

OBJECTIVE: The mechanism of spontaneous myogenic tone was investigated in isolated arteriolar segments. METHODS: Arterioles were isolated from rat cremaster muscle. Segments were endothelium-denuded and mounted in a pressure myograph at 75 mmHg. Under this condition, segments spontaneously constricted from a passive diameter of 167 +/- 3 to 82 +/- 4 microns (n = 41). The effects of several inhibitors were tested on the maintenance of myogenic tone. RESULTS: Gadolinium (10(-6)-10(-4) M), a putative inhibitor of stretch-activated cation channels, was ineffective. The phospholipase C (PLC) inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) induced a dose-dependent inhibition of tone. NCDC inhibited phenylephrine- (10(-6) M), but not potassium buffer-induced (100 mM) constriction. The protein kinase C (PKC) inhibitors staurosporine, chelerythrine and calphostin C inhibited myogenic tone in a concentration-dependent manner. At an intermediate concentration, calphostin C selectively inhibited phenylephrine-induced constriction. However, all PKC inhibitors abolished responses to phenylephrine and potassium buffer at higher concentrations. The cytochrome P450 inhibitor 17-ODYA (0.3-3 x 10(-6) M) did not inhibit myogenic tone. CONCLUSIONS: No evidence was found for a role of gadolinium-sensitive, stretch-activated cation channels or cytochrome P450 metabolites. On the other hand, both PLC and PKC contribute to the maintenance of myogenic tone.

Adrenergic alpha-Agonists↗
PubMed1998

Morphine preconditioning attenuates neutrophil activation in rat models of myocardial infarction.

Previous results from our laboratory have suggested that morphine can attenuate neutrophil activation in patients with acute myocardial infarction. To elucidate if morphine preconditioning (PC) has the same effects via activation of neutrophil endopeptidase 24.11 (NEP), we measured serum levels of intercellular adhesion molecule-1 (ICAM-1), gp100MEL14 and NEP in adult Wistar rats subjected to ten different protocols (n = 10 for each) at baseline, immediately after and 2 h after morphine PC. All groups were subjected to 30 min of occlusion and 2 h of reperfusion. Similarly, morphine-induced PC was elicited by 3-min drug infusions (100 micrograms/kg) interspersed with 5-min drug-free periods before the prolonged 30-min occlusion. Infarct size (IS), as a percentage of the area at risk (AAR), was determined by triphenyltetrazolium staining. Pretreatment with morphine increased NEP activities (9.86 +/- 1.98 vs. 5.12 +/- 1.10 nmol/mg protein in control group; p < 0.001). Naloxone (mu-opioid receptor antagonist) (4.82 +/- 1.02 nmol/mg protein) and phosphoramidon (NEP inhibitor) (4.66 +/- 1.00 nmol/mg protein) inhibited morphine-activated NEP, whereas glibenclamide (ATP-sensitive potassium channel antagonist) and chelerythrine (protein kinase C inhibitor) had no effects. The ICAM-1 and gp100MEL14 of the third sampling were lowest for those with morphine PC (280 +/- 30 ng/ml and 2.2 +/- 0.7 micrograms/ml; p < 0.001), but naloxone (372 +/- 38 ng/ml and 3.8 +/- 0.9 micrograms/ml) and phosphoramidon (382 +/- 40 ng/ml and 4.2 +/- 1.1 micrograms/ml) abolished the above phenomenon. IS/AAR were definitely lowest for those with morphine PC (24 +/- 7%; p < 0.05). Morphine preconditioning increases NEP activities to attenuate shedding of gp100MEL14 and to ICAM-1 and, thus, provides myocardial protection.

Alkaloids↗
PubMed1998

Effects of prostaglandin F2 alpha on intracellular pH, intracellular calcium, cell shortening and L-type calcium currents in rat myocytes.

OBJECTIVE: We have studied the mechanisms underlying the positive inotropic action of prostaglandin F2 alpha (PGF2 alpha) by monitoring intracellular calcium transients, intracellular pH, L-type calcium currents and cell shortening in isolated ventricular myocytes. METHODS: Rat myocytes were loaded with fura-2AM for intracellular calcium measurements, or BCECF-AM for pH measurements. Cell shortening was recorded using an edge detection system, and L-type calcium currents measured using whole cell patch clamping. RESULTS: PGF2 alpha (3 nmol l-1-3 mumol l-1 increased single myocyte shortening and reduced resting cell length in a concentration-dependent manner. While myocyte shortening was increased by PGF2 alpha, this was not associated with any change in the amplitude of intracellular calcium transients, diastolic calcium, or L-type calcium currents. However, the same myocytes were capable of responding to catecholamines with increases in calcium transient amplitude and L-type calcium currents. PGF2 alpha (3 mumol l-1 caused a reversible rise in intracellular pH of 0.08 +/- 0.01 pH units (n = 5, p < 0.05). The Na(+)-H+ exchanger inhibitor, HOE 694 (10 mumol l-1, abolished the PGF2 alpha-induced rise in pH and the increase in cell shortening. PGF2 alpha-induced increases in cell shortening and intracellular pH were also attenuated by the protein kinase C (PKC) inhibitor, chelerythrine (2 mumol l-1. CONCLUSION: The positive inotropic action of PGF2 alpha appears to be mediated via activation of the Na(+)-H+ exchanger with the possible involvement of PKC. This suggests that PGF2 alpha-produces intracellular alkalosis, which then sensitizes cardiac myofilaments to calcium.

Alkaloids↗
PubMed2000

Participation of prostaglandin E2 in the endothelial protective effect of ischaemic preconditioning in isolated rat heart.

OBJECTIVE: To assess whether the protective effect of ischaemic preconditioning (IPC) on endothelial function in coronary arteries of the rat involves prostaglandins. METHODS: Isolated rat hearts perfused under constant flow conditions were exposed to 30 min of partial ischaemia (flow-rate 1 ml/min) followed by 20 min of reperfusion, after which coronaries were precontracted with U-46619 0.1 microM, and the coronary response to the endothelium-dependent vasodilator, serotonin (5-HT, 10 microM), was compared to that of the endothelium-independent vasodilator, sodium nitroprusside (SNP, 3 microM). Prostaglandin production was blocked with a perfusion of indomethacin 10 microM started 15 min before IPC or a corresponding sham period and stopped just before the 20-min reperfusion period. RESULTS: In untreated hearts, ischaemia diminished selectively 5-HT-induced vasodilatation, compared to sham hearts. The vasodilatation by SNP was unaffected after ischaemia and reperfusion. IPC (5 min of zero-flow ischaemia followed by 10 min reperfusion before the 30-min partial ischaemia) preserved the vasodilatation produced by 5-HT. Enzymeimmunoassays showed an increased production of PGE2 in the IPC group. Treatment of hearts with indomethacin blocked the protective effect of IPC on the vasodilatation produced by 5-HT and decreased the production of PGE2. A 5-min perfusion with 3 nM PGE2 started 15 min before the partial ischaemia, protected the endothelium. This was blocked by 1 microM chelerythrine, but not by 0.3 microM glibenclamide. CONCLUSIONS: These results suggest that IPC affords protection to endothelial function in coronary arteries of the rat partially via the release of PGE2. Under our experimental conditions, the protective effect of PGE2 is mediated by PKC.

6-Ketoprostaglandin F1 alpha↗
PubMed2002

DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the 32P-postlabeling technique.

Using the 32P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with beta-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by 32P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C11-C12 bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.

Alkaloids↗
PubMed2001

Use of protein kinase C inhibitors results in rapid [Mg(2+)](i) mobilization in primary cultured rat aortic smooth muscle cells: are certain kinase C isoforms natural homeostatic regulators of cystolic free Mg(2+).

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.

Alkaloids↗
PubMed2001

Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology.

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.

Alkaloids↗
PubMed2001

Capsaicin potentiates 1,25-dihydoxyvitamin D3- and all-trans retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells.

Human promyelocytic leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or all-trans retinoic acid, respectively. In this study, the effect of capsaicin, an active component of the red pepper of the genus Capsocum, on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 5-30 microg/ml capsaicin for 72 h inhibited cell proliferation and induced a small increase in cell differentiation. Interestingly, synergistic induction of HL-60 cell differentiation was observed when capsaicin was combined with either 5 nM 1,25-(OH)2D3 or 50 nM all-trans retinoic acid. Flow cytometric analysis indicated that combinations of 1,25-(OH)2D3 and capsaicin stimulated differentiation predominantly to monocytes whereas combinations of all-trans retinoic acid and capsaicin stimulated differentiation predominantly to granulocytes. Capsaicin enhanced protein kinase C activity in 1,25-(OH)2D3- and all-trans retinoic acid-treated HL-60 cells. In addition, inhibitors for protein kinase C [bisindolylmaleimide (GF-109203X), chelerythrine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7)] and an inhibitor for extracellular signal-regulated kinase [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD-098059)] significantly inhibited HL-60 cell differentiation induced by capsaicin in combination with either 1,25-(OH)2D3 or all-trans retinoic acid. These results indicate that capsaicin potentiates 1,25-(OH)2D3- or all-trans retinoic acid-induced HL-60 cell differentiation and that both protein kinase C and extracellular signal-regulated kinase are involved in the cell differentiation synergistically enhanced by capsaicin.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2001

Chelerythrine and genistein inhibit the endothelin-1-induced increase in myofilament Ca(2+) sensitivity in rabbit ventricular myocytes.

We performed experiments to elucidate the cellular mechanism for the biphasic inotropic response to endothelin-1 of single rabbit ventricular myocytes loaded with a fluorescent dye, acetoxymethylester of indo-1. Endothelin-1 at 10 nM elicited a biphasic inotropic effect: a transient decrease in cell shortening and Ca(2+) transients followed by an increase in cell shortening without significant elevation of peak Ca(2+) transients. The selective endothelin ET(A) receptor antagonist FR139317 (2(R)-[2(R)-[2(S)-[(1-hexahydro-1H-azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-[3-(1-methyl-1H-indolyl)propionyl]amino-3-(2-pyridyl)propionic acid) at 1 microM abolished the biphasic effect of endothelin-1 on cell shortening and Ca(2+) transients. The selective protein kinase C inhibitor chelerythrine at 1 microM and the tyrosine kinase inhibitor genistein at 5 microM inhibited the endothelin-1-induced increase in cell shortening without significantly affecting Ca(2+) transients and the transient decrease in cell shortening and Ca(2+) transients. The present results indicate that both protein kinase C and tyrosine kinase may contribute to the increase in myofilament Ca(2+) sensitivity induced by endothelin-1, whereas the decrease in Ca(2+) transients induced by endothelin-1 may be mediated by a signalling pathway different from that involved in the increase in cardiac contractility in rabbit ventricular myocytes.

Actin Cytoskeleton↗
PubMed2001

Inhibition of acetylcholine-activated K(+) current by chelerythrine and bisindolylmaleimide I in atrial myocytes from mice.

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.

Acetylcholine↗
PubMed2001

Protein kinase C regulates dopamine D4 receptor-mediated phospholipid methylation.

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.

Alkaloids↗
PubMed2001

Role of protein kinase C in opioid modulation of glycine-gated Cl(-) current in rat periaqueductal gray neuron.

The Role of protein kinase C in the modulatory effect of a mu-opioid receptor agonist, [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), on the glycine-gated Cl(-) current was examined in acutely dissociated rat periaqueductal gray neurons. Using the nystatin-perforated patch-clamp technique, the neurons were voltage-clamped at -60 mV. The glycine-gated Cl(-) current (I(Gly)) was sensitive to strychnine. On pretreatment with 1 microM DAMGO, the 30-microM glycine response increased with time and showed a maximum amplitude of 209+/-37% of control. After a protein kinase C activator, phorbol-12-myristate-13-acetate (PMA, 0.1 microM) as pretreatment, I(Gly) increased to 138+/-6% of control. The DAMGO potentiation of I(Gly) was not altered by coapplication with PMA. Although protein kinase C inhibitors, chelerythrine (3 microM) and 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X, 1 microM), did not alter I(Gly), the DAMGO-induced potentiation of I(Gly) was reduced to 161+/-21% or 164+/-31% of the control after coapplication with chelerythrine or GF109203X, respectively. These results indicate that the potentiation of I(Gly) by a mu-opioid receptor agonist is partly mediated by activation of protein kinase C.

Alkaloids↗
PubMed2002

Human urotensin II-induced aorta ring contractions are mediated by protein kinase C, tyrosine kinases and Rho-kinase: inhibition by somatostatin receptor antagonists.

Human urotensin II-(1-11) and its N-terminally shortened analogues, human urotensin II-(4-11)-OH and human urotensin II-(4-11)-NH2 are potent vasoconstrictor peptides in isolated rat thoracic aorta. Human urotensin II-induced tonic aorta ring contractions are inhibited by the Ca2+ channel antagonists, verapamil, nitrendipine and diltiazem; D609 (Tricyclodecan-9-yl-xanthogenate, K), selective inhibitor of phosphatidylcholine-specific phospholipase C and partially by phospholipase C inhibitor U-73122 [1-[6-((17ss-3 Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-25-dione] and a selective inhibitor of phosphatidyl-inositol-specific phospholipase C-ET-18-OCH3 (Edelfosine,1-O-octadecyl-2O-methyl-rac-glycero-3-phosphorylcholine); protein kinase C inhibitors, chelerythrine and NPC-15437 [S-2,6-diamino-N-[[1-(1-oxotridecyl)-2-piperidinyl]methyl]-hexanamide dihydrochloride]; tyrosine kinase inhibitors, genistein and tyrphostin B42 and Rho-kinase inhibitor HA-1077 [1-(5-isoquinolinylsulfonyl)-homopiperazine dihydrochloride]. This indicates that human urotensin II-induced tonic contractions of the rat aorta are mediated by phospholipase C, protein kinase C, tyrosine kinases and Rho-kinase related pathways. In the high K+ medium, human urotensin II induces dose-dependent phasic oscillations of aortic rings. These are inhibited by Ca2+ channel antagonists, the phospholipase C inhibitor, U-73122 and protein kinase C inhibitors, chelerythrine and NPC-15437, indicating that human urotensin II-induced phasic oscillations of the rat aorta are mediated by phospholipase C and protein kinase C-dependent pathways. Given their close structural similarity, several somatostatin analogues, importantly containing DCys5 and DTrp7 and expressing different degrees of somatostatin receptor antagonist activity, were tested for possible inhibitory effects on human urotensin II-induced contractions of the rat aorta rings. Pre-incubation of rat aorta rings in the presence of somatostatin analogues, which are preferentially sst2 specific binders: PRL-2882; PRL-2903 and PRL-2915 at micro-molar concentrations significantly blocked the development of human urotensin II-induced tonic contractions. Somatostatin receptor antagonists dose-dependently inhibited human urotensin II-induced Ca2+ transients in rat thoracic aorta rings. These somatostatin receptor antagonists displayed moderate affinities for recombinant rat and human urotensin II receptor binding sites. The data support the suggestion that urotensin II receptor and somatostatin type 2/5 receptors display similar surface topologies and that analogues of somatostatin could provide useful lead compounds for the development of more potent urotensin II receptor antagonists.

Alkaloids↗