Protein Kinase C Inhibitor Chelerythrine Research Articles

Ecstasy and methamphetamine elicit action potential bursts via different mechanisms in a central snail neuron

This study sought to determine the effects of (+) methamphetamine (METH) and its ring-substituted analog (±)3,4-methylenedioxymethamphetamine (MDMA; ecstasy) on electrophysiological behavior and their relationships to second messenger systems in an identifiable RP4 neuron of the African snail, Achatina fulica Ferussac. Extracellular application of MDMA at 1 mM and METH at 3 mM elicited action potential bursts that were not blocked after immersing the neurons in Ca 2+-free solution. Notably, MDMA- (1 mM) elicited action potential bursts were blocked by pretreatment with the protein kinase C (PKC) inhibitors chelerythrine (20 μM) and Ro 31-8220 (20 μM), but not by the PKA inhibitors KT-5720 (10 μM) and H89 (10 μM). The PKC activator phorbol 12,13-dibutyrate (PDBu; 3 μM), but not the PKA activator forskolin (50 μM), facilitated the induction of bursts elicited by MDMA at a lower concentration (0.3 mM). In contrast, METH- (3 mM) elicited action potential bursts were blocked by pretreatment with KT-5720 (10 μM) and H89 (10 μM), but not by chelerythrine (20 μM) and Ro 31-8220 (20 μM). Forskolin (50 μM), but not PDBu (3 μM) facilitated the induction of bursts elicited by METH at a lower concentration (1 mM). Tetraethylammonium chloride (TEA), a blocker of the delayed rectifying K + current ( I KD), did not elicit bursts at a concentration of 5 mM but did facilitate the induction of action potential bursts elicited by both METH and MDMA. Voltage clamp studies revealed that both METH and MDMA decreased the TEA-sensitive I KD of the RP4 neuron. Forskolin (50 μM) or dibutyryl cAMP (1 mM), a membrane-permeable cAMP analog, alone did not elicit action potential bursts. However, co-administration with forskolin (50 μM) and TEA (5 mM) or co-administration with dibutyryl cAMP (1 mM) and TEA (50 mM) elicited action potential bursts in the presence of the PKC inhibitor chelerythrine (20 μM). Similarly, PDBu (10 μM) or phorbol 12-myristate 13-acetate (PMA; 3 μM) alone did not elicit action potential bursts. However, co-administration with PDBu (10 μM) and TEA (5 mM) or co-administration with PMA (3 μM) and TEA (5 mM) elicited action potential bursts in the presence of the PKA inhibitor KT-5720 (10 μM). These data suggest that action potential bursts in the RP4 neuron were not due to Ca 2+-dependent synaptic effects. Rather, action potential bursts may be elicited through (1) combined activation of the cAMP-PKA signaling pathway and inhibition of the I KD and (2) combined activation of PKC and inhibition of the I KD.

Regulation of K–Cl cotransport in erythrocytes of frog Rana temporaria by commonly used protein kinase and protein phosphatase inhibitors

Recently (Agalakova and Gusev in J Comp Physiol 179:443-450, 2009), we demonstrated that the activity of K-Cl cotransport (KCC) in frog red blood cells is inhibited under stimulation of protein kinase C (PKC) with phorbol ester PMA (12-myristate-13-acetate). Present work was performed to uncover possible implication of protein kinases and protein phosphatases (PPs) in the regulation of baseline and volume-dependent KCC activity in these cells. K+ influx was estimated as 86Rb uptake by the cells in isotonic or hypotonic media in the presence of ouabain, K+ efflux was determined as the difference between K+ loss by the cells incubated in parallel in isotonic or hypotonic K(+)-free Cl(-)- and NO(3)(-)-media. Swelling of the cells in hypotonic medium was accompanied by approximately 50% activation of Cl-dependent K+ influx and efflux. Protein tyrosine kinase (PTK) inhibitor genistein (0.1 mM) stably and considerably (up to 89%) suppressed both baseline and volume-dependent KCC activity in each direction. Other PTK blockers (tyrphostin 23 and quercetin) had no influence on KCC activity in frog erythrocytes. PKC inhibitor chelerythrine (20 microM) and both PP inhibitors, fluoride (5 mM) and okadaic acid (1 microM), reduced KCC activity by 25-70%. Neither basal nor swelling-activated KCC in frog erythrocytes was affected by PKC inhibitor staurosporine (1 microM). Based on the previous and present results, we can suggest that the main role in the maintenance of basal and volume-dependent KCC activity in frog erythrocytes belongs to PTKs and PPs, whereas PKC is a negative regulator of this ion system.

Anesthetic-induced Preconditioning Delays Opening of Mitochondrial Permeability Transition Pore via Protein Kinase C-ϵ–mediated Pathway

Cardioprotection by volatile anesthetic-induced preconditioning (APC) involves activation of protein kinase C (PKC). This study investigated the importance of APC-activated PKC in delaying mitochondrial permeability transition pore (mPTP) opening. Rat ventricular myocytes were exposed to isoflurane in the presence or absence of nonselective PKC inhibitor chelerythrine or isoform-specific inhibitors of PKC-delta (rottlerin) and PKC-epsilon (myristoylated PKC-epsilon V1-2 peptide), and the mPTP opening time was measured by using confocal microscopy. Ca-induced mPTP opening was measured in mitochondria isolated from rats exposed to isoflurane in the presence and absence of chelerythrine or in mitochondria directly treated with isoflurane after isolation. Translocation of PKC-epsilon was assessed in APC and control cardiomyocytes by Western blotting. In cardiomyocytes, APC prolonged time necessary to induce mPTP opening (261 +/- 26 s APC vs. 216 +/- 27 s control; P < 0.05), and chelerythrine abolished this delay to 213 +/- 22 s. The effect of isoflurane was also abolished when PKC-epsilon inhibitor was applied (210 +/- 22 s) but not in the presence of PKC-delta inhibitor (269 +/- 31 s). Western blotting revealed translocation of PKC-epsilon toward mitochondria in APC cells. The Ca concentration required for mPTP opening was significantly higher in mitochondria from APC rats (45 +/- 8 microM x mg control vs. 64 +/- 8 microM x mg APC), and APC effect was reversed with chelerythrine. In contrast, isoflurane did not protect directly treated mitochondria. APC induces delay of mPTP opening through PKC-epsilon mediated inhibition of mPTP opening, but not through PKC-delta. These results point to the connection between cytosolic and mitochondrial components of cardioprotection by isoflurane.

Pharmacological Preconditioning in Type 2 Diabetic Rat Hearts: The Roles of Mitochondrial ATP-Sensitive Potassium Channels and the Phosphatidylinositol 3-Kinase-Akt Pathway

The authors examined whether olprinone, a phosphodiesterase type 3 inhibitor, or isoflurane, a volatile anesthetic, could protect the heart against myocardial infarction in type 2 diabetic rats and whether the underlying mechanisms involve protein kinase C (PKC), mitochondrial ATP-sensitive potassium (m-K(ATP)) channels, or the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. All rats underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion. Wistar rats received isoflurane or olprinone before ischemia with or without the PKC inhibitor chelerythrine (CHE), the m-K(ATP) channel blocker 5-hydroxydecanoic acid (5HD), or the PI3K-Akt inhibitor LY294002 (LY). Goto-Kakizaki (GK) rats were randomly assigned to receive isoflurane or olprinone. In another group, GK rats received LY before the olprinone. In the Wistar rats, both isoflurane (38 +/- 11%) and olprinone (40 +/- 11%) reduced infarct size as compared to the control group (59 +/- 8%). In the GK rats, olprinone (41 +/- 9%) but not isoflurane (53 +/- 11%) reduced infarct size as compared to the GK control group (58 +/- 14%). The beneficial effects of olprinone were blocked by LY (58 +/- 14%). In the Wistar rats, CHE, 5HD, and LY prevented isoflurane-induced reductions of infarct size. On the other hand, LY but not CHE or 5HD prevented olprinone-induced reductions of infarct size. Olprinone but not isoflurane protects the heart against myocardial infarction in type 2 diabetic rats. The olprinone-induced cardioprotective effect is mediated by the PI3K-Akt pathway but not PKC or m-K(ATP) channels.

Involvement of the ornithine decarboxylase/polyamine system in precondition-induced cardioprotection through an interaction with PKC in rat hearts

Polyamines (putrescine, spermidine, and spermine) play an essential role in cell growth, differentiation, and apoptosis. Protein kinase C (PKC) stimulates polyamine biosynthesis through the induction of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis. Activation of PKC mediates ischemic preconditioning to reduce necrosis and apoptosis in intact hearts and in isolated culture cardiomyocytes. In this study, we examined whether the ODC/polyamine system is involved in the ischemic preconditioning signaling pathway and whether this system interacts with PKC in preconditioning-induced cardioprotection. Hearts were preconditioned with three cycles of 5-min ischemia and 5-min reflow, which caused an increase of ODC expression and spermidine, spermine, and total polyamine pool levels. alpha-Difluoromethylornithine (DFMO) and ethylglyoxal bis (guanylhydrazone) (EGBG) inhibited the key enzymes involved in polyamine biosynthesis, and abolished the preconditioning-induced reduction in infarct size and improvement in postischemic heart contractility function. They also increased cell apoptosis extent and aggravated myocardium ultrastructure damage. Inhibition also attenuated the preconditioning-induced translocation and activation of the PKC-delta, -epsilon isoforms from the cytosol to the particulate. Conversely, activation of PKC by phorbol 12-myristate 13-acetate (PMA) upregulated the ODC/polyamine system, whereas the PKC inhibitor chelerythrine (Che) downregulated the ODC/polyamine system. These findings suggest that upregulation of the polyamine synthesis metabolism occurs in response to preconditioning and mediates preconditioning-induced cardioprotection. The ODC/polyamine system and PKC signals may "cross-talk" in preconditioned hearts such that inhibiting one pathway leads to a reduction in the activity of the other pathway and vice versa.

Titanium implants alter endothelial function and vasoconstriction via a protein kinase C-regulated pathway

The application of titanium (Ti) alloy in joint prostheses is a good choice in orthopedic reconstruction. An elevated serum concentration of Ti has been shown in the patients with loosened knee prostheses. The precise actions of elevated Ti on the circulation remain unclear. In this study the maximal contractile responses elicited by phenylephrine in the aortas of rats 4 weeks after Ti alloy implantation and in cultured rat aortas treated with a soluble form of Ti for a period of 18 h were significantly decreased as compared with controls. Aortas isolated from rats with Ti alloy implants or aortas treated with a soluble form of Ti had enhanced protein expression of endothelial nitric oxide synthase (eNOS) and protein kinase C (PKC)-α and enhanced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Treatment of human umbilical vein endothelial cells (HUVECs) with a soluble form of Ti for 24 h dose-dependently increased eNOS protein expression. Short-term treatment of HUVECs with Ti for 1 h effectively enhanced the phosphorylation of eNOS, PKC (pan) and ERK1/2. PKC inhibitors RO320432 and chelerythrine effectively inhibited Ti-enhanced phosphorylation of eNOS and PKC (pan). These results indicate that Ti in the circulation may alter endothelial function and reduce vasoconstriction.

Activation of peripheral delta‐2 opioid receptors increases tolerance to ischemia/reperfusion injury

We investigated the role of peripheral δ2 opioid receptors (OR) in cardiac tolerance to ischemia/reperfusion injury and examined the contribution of Protein Kinase C (PKC), tyrosine kinase (TK), potassium ATP (KATP) channels and the autonomic nervous system in δ2 cardioprotection. Deltorphin II and inhibitors were administered prior to coronary artery occlusion and reperfusion in a rat model. Rats were monitored for the development of arrhythmias and infarct development. Pretreatment with δ2 specific OR antagonist naltriben completely abolished the cardioprotective effects of Deltorphin II. In contrast the selective δ1 OR antagonist 7‐Benzylidenenaltrexone had no effect. The PKC inhibitor chelerythrine and the nitric oxide synthase (NOS) inhibitor L‐NAME (N‐nitro‐L‐arginine methyl ester) also reversed both Deltorphin II effects. The nonselective KATP channel inhibitor glibenclamide and the selective mitochondrial KATP channel inhibitor 5‐hydroxydecanoic acid abolished the infarct‐sparing effect of Deltorphin II. Inhibition of TK with genistein, the ganglion blocker hexamethonium and the depletion of endogenous catecholamine storage with guanethidine reversed the antiarrhythmic action of deltorphin II, but did not change its infarct sparing action. The cardioprotective mechanism of Deltorphin II is mediated via stimulation of peripheral δ2 opioid receptors.

Inhibition of voltage-gated L-type calcium channels by labedipinedilol-A involves protein kinase C in rat cerebrovascular smooth muscle cells

Labedipinedilol-A, a novel calcium channel blocker with α/β-adrenoceptor blockade properties, inhibits L-type calcium channels (LTCCs) in rat cerebrovascular smooth muscle cells (CSMCs). We used conventional whole cell patch-clamp electrophysiology to investigate Ba 2+ currents ( I Ba) through LTCCs in rat CSMCs enzymatically dissociated from rat cerebral arteries. Labedipinedilol-A (1, 10 µM) reversibly inhibited I Ba in a voltage-dependent manner without modifying the I Ba current–voltage relationship. The I Ba was also abolished by the LTCC blocker nifedipine (1 µM), but enhanced by the LTCC activator Bay K8644 (100 nM). Labedipinedilol-A shifted the steady-state inactivation curve of I Ba to more negative potentials. Additionally, labedipinedilol-A had greater inhibitory activity on I Ba holding at − 40 mV than at − 80 mV. This might contribute to labedipinedilol-A's more selective effect on vascular muscles compared to cardiac muscles. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and norepinephrine-enhanced I Ba were also inhibited by labedipinedilol-A. Pretreatment with the PKC inhibitor chelerythrine (5 µM) attenuated labedipinedilol-A-mediated I Ba inhibition. However, the Rho kinase inhibitor Y-27632 (30 µM) had little effect on labedipinedilol-A inhibition of I Ba. Labedipinedilol-A inhibition of voltage-dependent LTCCs may be, at least in part, due to its modulation of the PKC pathway.

Obligatory role for phosphatidylinositol 4,5‐bisphosphate in activation of native TRPC1 store‐operated channels in vascular myocytes

In the present study the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) was studied on a native TRPC1 store-operated channel (SOC) in freshly dispersed rabbit portal vein myocytes. Application of diC8-PIP2, a water soluble form of PIP2, to quiescent inside-out patches evoked single channel currents with a unitary conductance of 1.9 pS. DiC8-PIP2-evoked channel currents were inhibited by anti-TRPC1 antibodies and these characteristics are identical to SOCs evoked by cyclopiazonic acid (CPA) and BAPTA-AM. SOCs stimulated by CPA, BAPTA-AM and the phorbol ester phorbol 12,13-dibutyrate (PDBu) were reduced by anti-PIP2 antibodies and by depletion of tissue PIP2 levels by pre-treatment of preparations with wortmannin and LY294002. However, these reagents did not alter the ability of PIP2 to activate SOCs in inside-out patches. Co-immunoprecipitation techniques demonstrated association between TRPC1 and PIP2 at rest, which was greatly decreased by wortmannin and LY294002. Pre-treatment of cells with PDBu, which activates protein kinase C (PKC), augmented SOC activation by PIP2 whereas the PKC inhibitor chelerythrine decreased SOC stimulation by PIP2. Co-immunoprecipitation experiments provide evidence that PKC-dependent phosphorylation of TRPC1 occurs constitutively and was increased by CPA and PDBu but decreased by chelerythrine. These novel results show that PIP2 can activate TRPC1 SOCs in native vascular myocytes and plays an important role in SOC activation by CPA, BAPTA-AM and PDBu. Moreover, the permissive role of PIP2 in SOC activation requires PKC-dependent phosphorylation of TRPC1.

Angiotensin II stimulates water and NaCl intake through separate cell signalling pathways in rats

Angiotensin II (AngII) stimulation of water and NaCl intake is a classic model of the behavioural effects of hormones. In vitro studies indicate that the AngII type 1 (AT(1)) receptor stimulates intracellular pathways that include protein kinase C (PKC) and mitogen-activated protein (MAP) kinase activation. Previous studies support the hypotheses that PKC is involved in AngII-induced water, but not NaCl intake and that MAP kinase plays a role in NaCl consumption, but not water intake, after injection of AngII. The present experiments test these hypotheses in rats using central injections of AngII in the presence or absence of a PKC inhibitor or a MAP kinase inhibitor. Pretreatment with the PKC inhibitor chelerythrine attenuated AngII-induced water intake, but NaCl intake was unaffected. In contrast, pretreatment with U0126, a MAP kinase inhibitor, had no effect on AngII-induced water intake, but attenuated NaCl intake. These data support the working hypotheses and significantly extend our earlier findings and those of others. Perhaps more importantly, these experiments demonstrate the remarkable diversity of peptide receptor systems and add support for the surprising finding that intracellular signalling pathways can have divergent behavioural relevance.

Cholinergic excitation of dopaminergic cells depends on sequential activation of protein kinase C and the L-type calcium channel in ventral tegmental area slices

Dopaminergic projections from the ventral tegmental area (VTA) constitute the mesolimbocortical system that underlies addiction and psychosis primarily as the result of increased dopaminergic transmission. Dopaminergic neurons in the VTA receive glutamatergic and cholinergic innervations that regulate their firing activities. Both transmitter systems can activate protein kinase C (PKC) by increasing intracellular calcium and lipid second messengers, however, whether PKC mediates increased firing following glutamatergic and cholinergic activation remains unknown. This paper examined the effects of acute PKC inhibition on firing responses to carbachol, NMDA or AMPA using patch clamp recordings from brain slices. The three ligands all induced a reversible increase in firing, however, only carbachol-induced increase in firing was attenuated by the PKC inhibitors chelerythrine or GF 109203X. The L-type calcium channel blocker nifedipine partially blocked carbachol-induced excitation similar to PKC inhibitors. PKC inhibition and L-type channel blockade did not significantly alter NMDA- or AMPA-induced excitation. Concurrent blockade of PKC and L-type channels with chelerythrine and nifedipine did not additively suppress carbachol-induced excitation indicating they were sequential events in the same signaling pathway. Furthermore, preincubation with the PKC inhibitor GF 109203X reduced the carbachol-induced increase in nifedipine-sensitive high-voltage gated calcium currents. These results indicate that cholinergic activation enhances PKC activity, which in turn facilitates L-type channel opening to excite dopaminergic cells, a finding that is in line with reports of increased PKC in the VTA in animals displaying addictive behavior.

Impact of protein kinase C activation on epileptiform activity in the hippocampal slice

There is evidence suggesting that protein kinase C (PKC) activation can prevent the enhanced network excitability associated with status epilepticus and group I metabotropic glutamate receptor (mGluR)-induced epileptogenesis. However, we observed no suppression of mGluR-induced burst prolongation in the guinea pig hippocampal slice when applied in the presence of the PKC activator phorbol-12,13-dibutyrate (PDBu). Furthermore, PDBu alone converted picrotoxin-induced interictal bursts into ictal-length discharges ranging from 2 to 6s in length. This effect could not be elicited by the inactive analog 4-alpha-PDBu and was suppressed with the PKC inhibitor chelerythrine, indicating PKC dependence. PKC activation can enhance neurotransmitter release, and both glutamate and acetylcholine are capable of eliciting similar prolonged synchronized discharges. However, neither mGluR1 nor NMDA receptor antagonist suppressed PDBu-driven burst prolongation, suggesting that increased glutamate release alone is unlikely to account for the PKC-induced expression of ictaform discharges. Similarly, atropine, a broad-spectrum muscarinic receptor antagonist, had no effect on PKC-induced burst prolongation. By contrast, AMPA/kainate receptor antagonist abolished PKC-induced burst prolongation, and mGluR5 antagonist significantly blunted the maximum burst length induced by PKC. These data suggest that PKC-induced prolongation of epileptiform bursts is dependent on changes specific to mGluR5 and AMPA/kainate receptors and not mediated simply by a generalized increase in transmitter release.

Myocardial Hsp70 phosphorylation and PKC-mediated cardioprotection following exercise

Both protein kinase C (PKC) activation and Hsp70 expression have been shown to be key components for exercise-mediated myocardial protection during ischemia-reperfusion injury. Given that Hsp70 has been shown to undergo inducible phosphorylation in striated muscle and liver, we hypothesized that PKC may regulate myocardial Hsp70 function and subsequent exercise-conferred cardioprotection through this phosphorylation. Hence, acute exercise of male Sprague-Dawley rats (30 m/min for 60 min at 2% grade) was employed to assess the role of PKC and its selected isoforms in phosphorylation of Hsp70 and protection of the myocardium during ischemia-reperfusion injury. It was observed that administration of the PKC inhibitor chelerythrine chloride (5 mg/kg) suppressed the activation of three exercise-induced PKC isoforms (PKCalpha, PKCdelta, and PKCepsilon) and attenuated the exercise-mediated reduction of myocardial infarct size during ischemia-reperfusion injury. While this study also demonstrated that exercise led to an alteration in the phosphorylation status of Hsp70, this posttranslational modification appeared to be dissociated from PKC activation, as exercise-induced phosphorylation of Hsp70 was unchanged following inhibition of PKC. Taken together, these results indicate that selected isoforms of PKC play an important role in exercise-mediated protection of the myocardium during ischemia-reperfusion injury. However, exercise-induced phosphorylation of Hsp70 does not appear to be a mechanism by which PKC induces this cardioprotective effect.

Delayed anti-arrhythmic effect of nitroglycerin in anesthetized rats: Involvement of CGRP, PKC and mK ATP channels

Delayed anti-infarct and anti-stunning effects of nitroglycerin (NTG) have well been established in some animal models. The main goals of this study in anesthetized rats were to determine whether NTG has a delayed anti-arrhythmic effect and if so, whether calcitonin gene-related peptide (CGRP), protein kinase C (PKC) and mitochondrial K ATP channels (mK ATP) are involved in triggering this response. For this purpose, on day 0, male Wistar rats received NTG (120 μg/kg, iv) with or without pre-administration of PKC inhibitor chelerythrine (CHE), capsaicin (CAP) to deplete CGRP from sensory nerves or mK ATP channel blocker 5-hydroxydecaonic acid (5HD). On day 1, their hearts were subjected to 30 min ischemia and 120 min reperfusion. In rats pretreated with NTG, the incidence of ventricular tachycardia and ventricular fibrillation and the mortality rate significantly reduced (from 100%, 61% and 18.1% in the control group to 45.4%, 10% and 0% in the NTG group, respectively). Infarct size also reduced from 58 ± 4.7% in the control group to 31 ± 3.7% in the NTG group. These effects were abolished by CHE, CAP and 5HD, which none of them alone had any effect on infarct size or the incidence of myocardial arrhythmias. These results show that a low dose of NTG has a delayed anti-arrhythmic effect and this effect may share a common mechanism with anti-infarct effects of this drug, involving CGRP release and PKC and mK ATP activation.

Epidermal growth factor receptor activation by protein kinase C is necessary for FSH-induced meiotic resumption in porcine cumulus–oocyte complexes

It is proved that epidermal growth factor (EGF)-like factors mediate gonadotropin-induced rodent oocyte maturation via EGF receptor (EGFR). However, the detail kinetics and signal pathway between FSH and EGF/EGFR is not clear in large animals. In the present study, we investigated the roles of EGFR and protein kinase C (PKC) in FSH-induced porcine oocyte meiotic resumption. Porcine cumulus-oocyte complexes were cultured in NCSU37 medium containing 10% porcine follicular fluid and germinal vesicle breakdown (meiotic resumption) was detected after different treatments. The results showed that EGF-like factor amphiregulin (AR) and EGFR mRNA were expressed in porcine cumulus cells, but not oocytes. FSH significantly induced AR mRNA expression with maximum at 4 h and activated EGFR phosphorylation at 8 h. AR (1-100 ng/ml) dose-dependently induced meiosis resumption of porcine oocyte. The specific EGFR inhibitor, AG1478, but not AG43 (the inactive analog of AG1478), completely blocked FSH, EGF, and AR-induced oocyte meiotic resumption; the inhibitory effect of AG1478 on FSH action gradually decreased when the inhibitor was added at 6 h or later and disappeared when it was added at 11 h; EGF reversed the inhibitory effect on FSH when AG1478 was added within 6 h. FSH triggered porcine oocyte meiotic resumption (at 20 h) later than that of EGF and AR (at 18 h). All these results supported that endogenously produced EGFR activator(s), possibly AR (maximum at 4 h) and EGFR activation (began at 6 h and finished within 11 h), in cumulus cells is necessary for FSH-induced porcine oocyte meiotic resumption (began at 18 h). Furthermore, PKC activator PMA mimicked but PKC inhibitor chelerythrine chloride inhibited FSH action, and AG1478 also suppressed PMA-induced porcine oocyte meiotic resumption. These data together suggested that EGFR activation, by PKC signal pathway, participates in FSH-induced porcine oocyte meiotic resumption.

Isoflurane Preconditioning Delays Opening of Mitochondrial Permeability Transition Pore via Protein Kinase C Signaling Pathway

Isoflurane protects the myocardium from ischemia and reperfusion (I/R) injury via protein kinase C (PKC) signaling pathway. It has been suggested that the mitochondrial permeability transition pore (mPTP) is the end effector of I/R injury, whereas PKC has a protective effect by blocking mPTP opening. Here we investigated whether anesthetic preconditioning (APC) with isoflurane delays mPTP opening and whether the PKC plays a role in isoflurane‐induced delay in mPTP opening. Isoflurane preconditioned rat cardiomyocytes were loaded with fluorescent probe tetramethylrhodamine ethyl ester (TMRE) and subjected to a simulated I/R injury by laser‐induced ROS production. Using the laser‐scanning confocal microscopy, we measured the time to 50% decrease in TMRE fluorescence, which coincides with depolarization of the mitochondria and represents an opening of mPTP. To confirm the role of mPTP, we used cyclosporine A (CsA). The role of PKC was tested using the non‐specific PKC inhibitor chelerythrine (CHEL). APC with isoflurane prolonged the mean depolarization time of the control fluorescence of 216±8 s, to 261±16 s. A similar time delay of 279±22 s was found in cells pretreated with CsA. Isoflurane‐induced delay in an mPTP opening was abrogated to 219±7 s in cells treated with CHEL. In conclusion, our results indicate that APC with isoflurane delays the opening of mPTP in a PKC‐dependent manner.Supported by NIH (HL 034708 and GM 066730)

Chelerythrine treatment influences the balance of pro- and anti-apoptotic signaling pathways in the remote myocardium after infarction

Apoptotic processes may be implicated in the molecular pathomechanisms of ventricular remodeling after myocardial infarction (MI). The modulation of apoptosis by pro- and anti-apoptotic pathways in the myocardium remote from the infarction, including its link to protein kinase C (PKC), was focus of the present study. Rats were subjected to MI by LAD ligation in situ. Some animals were pretreated with the PKC inhibitor chelerythrine. After 1 h up to 28 days, pro- and anti-apoptotic signals (caspase-3, Bcl-2/Bax ratio, Akt, Bad), and marker of apoptosis execution (DNA laddering, TUNEL) were quantified in the myocardium remote from the infarction. Activation of caspase-3, a pro-apoptotic shift of the Bcl-2/Bax ratio, and DNA fragmentation were observed as early as 3 h after infarction and persisted up to 28 days. Akt- and Bad-phosphorylation was unchanged. Chelerythrine markedly reduced DNA fragmentation. Caspase-3 activation was unchanged. Surprisingly, Bad and Akt phosphorylation were highly increased (180% and 750% of control). Chelerythrine influences the balance of pro- and anti-apoptotic pathways in the remote myocardium after infarction, with an inhibition of proapoptotic and an activation of anti-apoptotic signals.

H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes

The present study was aimed to investigate the regulatory effect of protein kinase C (PKC) on intracellular Ca(2+) handling in hydrogen sulfide (H(2)S)-preconditioned cardiomyocytes and its consequent effects on ischemia challenge. Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (an H(2)S donor, 10(-4) M) preconditioning (SP, 30 min). Intracellular Ca(2+) was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia-reperfusion insults to indicate the extent of hypercontracture. SP motivated translocation of PKCalpha, PKCepsilon, and PKCdelta to membrane fraction but only translocation of PKCepsilon and PKCdelta was abolished by an ATP-sensitive potassium channel blocker glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [Ca(2+)] transients, which were reversed by a selective PKC inhibitor chelerythrine. These data suggest that SP facilitated Ca(2+) removal via both accelerating uptake of Ca(2+) into sarcoplasmic reticulum and enhancing Ca(2+) extrusion through Na(+)/Ca(2+) exchanger in a PKC-dependent manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca(2+) overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. We demonstrate for the first time that SP activates PKCalpha, PKCepsilon, and PKCdelta in cardiomyocytes via different signaling mechanisms. Such PKC activation, in turn, protects the heart against ischemia-reperfusion insults at least partly by ameliorating intracellular Ca(2+) handling.

Insulin causes renal dopamine D1 receptor desensitization via GRK2-mediated receptor phosphorylation involving phosphatidylinositol 3-kinase and protein kinase C

The renal dopamine system plays an important role in sodium homeostasis and a defect in dopamine D1 receptor (D1R) function is present in hypertension, diabetes, and aging. Our previous studies in hyperinsulinemic animals and in renal cell cultures treated with insulin showed decrease in D1R number and defective coupling to G proteins; however, the exact mechanisms remained unknown. Therefore, we investigated insulin-mediated D1R desensitization and underlying molecular mechanism in opossum kidney (OK) cells. Chronic exposure (24 h) of OK cells to 10 nM insulin caused significant decrease in D1R number and agonist affinity. The D1R was hyperserine phosphorylated, uncoupled from G proteins and SKF38393, a D1R agonist, failed to stimulate G proteins and inhibit Na-K-ATPase activity. Insulin increased protein kinase C (PKC) activity and caused G protein-coupled receptor kinase 2 (GRK2) translocation to the membranes. Tyrosine kinase inhibitor genistein and phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin blocked insulin-mediated PKC activation and GRK2 membranous translocation. In addition to genistein and wortmannin, GRK2 membranous tranlocation was also blocked by PKC inhibitor chelerythrine chloride and GRK2-specific siRNA. Genistein, wortmannin, chelerythrine chloride, and GRK2 siRNA abrogated D1R serine phosphorylation and normalized D1R expression and affinity in insulin-treated cells. Furthermore, these inhibitors and siRNA restored D1R G protein coupling and ability of SKF38393 to inhibit Na-K-ATPase activity. In conclusion, insulin-induced D1R desensitization involves PI3K, PKC, and GRK2. Insulin activates PI3K-PKC-GRK2 cascade, causing D1R serine phosphorylation, which leads to D1R downregulation and uncoupling from G proteins, and results in the failure of SKF38393 to stimulate G proteins and inhibit Na-K-ATPase activity.

Protein kinase C protects preconditioned rabbit hearts by increasing sensitivity of adenosine A 2b-dependent signaling during early reperfusion

Although protein kinase C (PKC) plays a key role in ischemic preconditioning (IPC), the actual mechanism of that protection is unknown. We recently found that protection from IPC requires activation of adenosine receptors during early reperfusion. We, therefore, hypothesized that PKC might act to increase the heart's sensitivity to adenosine. IPC limited infarct size in isolated rabbit hearts subjected to 30-min regional ischemia/2-h reperfusion and IPC's protection was blocked by the PKC inhibitor chelerythrine given during early reperfusion revealing involvement of PKC at reperfusion. Similarly chelerythrine infused in the early reperfusion period blocked the increased phosphorylation of the protective kinases Akt and ERK1/2 observed after IPC. Infusing phorbol 12-myristate 13-acetate (PMA), a PKC activator, during early reperfusion mimicked IPC's protection. As expected, the protection triggered by PMA at reperfusion was blocked by chelerythrine, but surprisingly it was also blocked by MRS1754, an adenosine A 2b receptor-selective antagonist, suggesting that PKC was somehow facilitating signaling from the A 2b receptors. NECA [5′-( N-ethylcarboxamido) adenosine], a potent but not selective A 2b receptor agonist, increased phosphorylation of Akt and ERK1/2 in a dose-dependent manner. Pretreating hearts with PMA or brief preconditioning ischemia had no effect on phosphorylation of Akt or ERK1/2 per se but markedly lowered the threshold for NECA to induce their phosphorylation. BAY 60-6583, a highly selective A 2b agonist, also caused phosphorylation of ERK1/2 and Akt. MRS1754 prevented phosphorylation induced by BAY 60-6583. BAY 60-6583 limited infarct size when given to ischemic hearts at reperfusion. These results suggest that activation of cardiac A 2b receptors at reperfusion is protective, but because of the very low affinity of the receptors endogenous cardiac adenosine is unable to trigger their signaling. We propose that the key protective event in IPC occurs when PKC increases the heart's sensitivity to adenosine so that endogenous adenosine can activate A 2b-dependent signaling.