Protein Kinase C Inhibitor Chelerythrine Chloride Research Articles

Kisspeptin increases intracellular calcium concentration by protein kinase C-mediated signaling in the primary cultured neurons from rat hippocampus

In addition to the fact that kisspeptin and its receptor GPR54 are well known to be abundantly expressed in the hypothalamus with suggestive roles in the initiation of puberty and similar reproductive system properties, there is also proof showing that kisspeptin might have influences on hippocampal functions. In our previous study, it was shown that kisspeptin increased free intracellular Ca2+ values ([Ca2+]i) through protein kinase C (PKC) activation in GT1-7 cells. For this reason, we examined the influences of kisspeptin on [Ca2+]i in hippocampal neurons to determine if kisspeptin shows its effects on hippocampus through the same mechanism. Hippocampal neurons were excised from the brains of fetuses on 17th embryonic day from maternal rats. The influences of kisspeptin on [Ca2+]i in hippocampal neurons were examined through in vitro calcium imaging system. The responses of [Ca2+]i to kisspeptin were quantified by the changes in 340nm/380nm ratio. Kisspeptin-10 caused [Ca2+]i transients in hippocampal neurons. The change in [Ca2+]i by 100 nM kisspeptin was prevented by pre-treating the cells in PKC inhibitor chelerythrine chloride. According to the results, kisspeptin activates intracellular calcium signaling in hippocampal neurons via the pathway that depends on PKC. The results of this study suggest that kisspeptin may have a role in hippocampal neuron functions.

Stress peptide PACAP engages multiple signaling pathways within the carotid body to initiate excitatory responses in respiratory and sympathetic chemosensory afferents

Consistent with a critical role in respiratory and autonomic stress responses, the carotid bodies are strongly excited by pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide implicated in stress responses throughout the sympathetic nervous system. PACAP excites isolated carotid body glomus cells via activation of PAC1 receptors, with one study suggesting PAC1-induced excitation is due entirely to protein kinase A (PKA)-mediated inhibition of TASK channels. However, in other systems, PAC1 is known to be coupled to multiple intracellular signaling pathways, including PKA, phospholipase C (PLC), phospholipase D (PLD), and protein kinase C (PKC), that trigger multiple downstream effectors including increased Ca²⁺ mobilization, inhibition of various K⁺ channels, and activation of nonselective cation channels. This study tests if non-PKA/TASK channel signaling helps mediate the stimulatory effects of PACAP on the carotid body. Using an ex vivo arterially perfused rat carotid body preparation, we show that PACAP-38 stimulates carotid sinus nerve activity in a biphasic manner (peak response, falling to plateau). PKA blocker H-89 only reduced the plateau response (~41%), whereas the TASK-1-like K⁺ channel blocker/transient receptor potential vanilloid 1 channel agonist anandamide only inhibited the peak response (~48%), suggesting involvement of additional pathways. The PLD blocker CAY10594 significantly inhibited both peak and plateau responses. The PLC blocker U73122 decimated both peak and plateau responses. Brefeldin A, a blocker of Epac (cAMP-activated guanine exchange factor, reported to link Gs-coupled receptors with PLC/PLD), also reduced both phases of the response, as did blocking signaling downstream of PLC/PLD with the PKC inhibitors chelerythrine chloride and GF109203X. Suggesting the involvement of non-TASK ion channels in the effects of PACAP, the A-type K⁺ channel blocker 4-aminopyridine, and the putative transient receptor potential channel (TRPC)/T-type calcium channel blocker SKF96365 each significantly inhibited the peak and steady-state responses. These data suggest the stimulatory effect of PACAP-38 on carotid body sensory activity is mediated through multiple signaling pathways: the PLC-PKC pathways predominates, with TRPC and/or T-type channel activation and Kv channel inactivation; only partial involvement is attributable to PKA and PLD activation.

Interaction between nitric oxide and superoxide in the macula densa in aldosterone-induced alterations of tubuloglomerular feedback

Tubuloglomerular feedback (TGF)-mediated constriction of the afferent arteriole is modulated by a balance between release of superoxide (O(2)(-)) and nitric oxide (NO) in macula densa (MD) cells. Aldosterone activates mineralocorticoid receptors that are expressed in the MD and induces both NO and O(2)(-) generation. We hypothesize that aldosterone enhances O(2)(-) production in the MD mediated by protein kinase C (PKC), which buffers the effect of NO in control of TGF response. Studies were performed in microdissected and perfused MD and in a MD cell line, MMDD1 cells. Aldosterone significantly enhanced O(2)(-) generation both in perfused MD and in MMDD1 cells. When aldosterone (10(-7) mol/l) was added in the tubular perfusate, TGF response was reduced from 2.4 ± 0.3 μm to 1.4 ± 0.2 μm in isolated perfused MD. In the presence of tempol, a O(2)(-) scavenger, TGF response was 1.5 ± 0.2 μm. In the presence of both tempol and aldosterone in the tubular perfusate, TGF response was further reduced to 0.4 ± 0.2 μm. To determine if PKC is involved in aldosterone-induced O(2)(-) production, we exposed the O(2)(-) cells to a nonselective PKC inhibitor chelerythrine chloride, a specific PKCα inhibitor Go6976, or a PKCα siRNA, and the aldosterone-induced increase in O(2)(-) production was blocked. These data indicate that aldosterone-stimulated O(2)(-) production in the MD buffers the effect of NO in control of TGF response, an effect that was mediated by PKCα.

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.

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.

Effects of protein kinase C inhibitor,chelerythrine chloride,on drug-sensitivity of NSCLC cell lines

Protein kinase C(PKC) is a potentially important target for can-cer therapeutics due to its potential role in carcinogenesis.Abnormal expression and increasing activity of PKC-α are present in non-small cell lung cancer(NSCLC).PKC inhibitor can show anti-tumor effects through inducing tumor cell apoptosis,enhancing cytotoxic effects and down-regulating expressions of multidrug resistance gene.By observing the effects of PKC inhibitor chelerythrine chloride(CH) on drug-sensitivity to cisplatin of four NSCLC cell lines its mechanism of effect initially is explored. NSCLC cell lines(H1299,H460,A549 and cisplatin-resistant A549) were dealed with PKC inhibitor CH respectively.The expressions of PKC-α mRNA and protein in NSCLC cell lines were examined by reverse transcription polymerase chain reaction(RT-PCR) and Western blot.The apoptosis rates of NSCLC cells lines were detected by flow cytometry.The drug-sensitivity to cisplatin of NSCLC cells lines was measured by methabenzthiazuron(MTT) assay. The expression levels of PKC-α mRNA and protein in cisplatin-resistant A549 cell lines were significantly higher than H1299,H460 and parent A549 cell lines(P < 0.05).The expression levels of PKC-α mRNA and protein in four NSCLC cell lines decreased at different extent.The apoptosis rates of cisplatin-resistant A549 cell lines increased obviously after treating with CH for 4 and 24 hours,but it was not seen in H1299,H460 and parent A549 cell lines.The IC50 value of cisplatin of NSCLC cell lines decreased at different degree after treating with CH and it was more obvious in cisplatin-resistant A549 cell lines(P < 0.05). High expressions of PKC-α mRNA and protein exist in all four NSCLC cell lines.PKC inhibitor CH can enhance the drug-sensitivity of NSCLC cell lines to cisplatin by inhibiting their expression of PKC-α mRNA and protein.When compared with parent A549 cell lines,cisplatin-resistant A549 cell line's drug-sensitivity to cisplatin is increasing more efficiently by PKC inhibitor CH through inhibition of PKC-α protein's expression and elevation of tumor cell apoptosis rates.

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.

Implication of protein kinase C in the orexin‐induced elevation of extracellular dopamine levels and its rewarding effect

In the present study, we investigated the role of orexinergic systems in the activation of midbrain dopamine neurons. In an in vitro study, exposure to either orexin A or orexin B under superfusion conditions produced a transient increase in the intracellular Ca(2+) concentration through the phospholipase C (PLC)/protein kinase C (PKC) pathway via G(q11)alpha or Gbetagamma subunits in midbrain cultured neurons, which were shown to be tyrosine hydroxylase (TH)-positive cells, but not in purified midbrain astrocytes. Here we show that in vivo injection with a selective PKC inhibitor chelerythrine chloride or 2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-1-methyl-1H-indol-3-ylmaleimide HCl (Ro-32-0432) into the ventral tegmental area (VTA) significantly suppressed the place preference and increased levels of dopamine in the nucleus accumbens (NAcc) induced by intra-VTA injection of orexins. These results strongly support the idea that activation of the orexin-containing neuron in the VTA leads to the direct activation of mesolimbic dopamine neurons through the activation of the PLC/PKC pathway via G(q11)alpha or Gbetagamma-subunit activation, which could be associated with the development of its rewarding effect.

Transcriptional stimulation of the human NHE3 promoter activity by PMA: PKC independence and involvement of the transcription factor EGR-1

NHE3 (Na+/H+ exchanger 3) is essential for Na+ absorption in the ileum and is expressed in a cell-specific manner in the apical membrane of the intestinal epithelial cells. In the present study, we report the stimulatory effect of PMA on the hNHE3 (human NHE3) transcription. Pretreatment with actinomycin D or cycloheximide blocked the up-regulation of the NHE3 mRNA by PMA, indicating that the increased level of NHE3 mRNA expression is regulated by transcriptional activation and is dependent on de novo protein synthesis. 5'-Deletion of the promoter region and transfection analysis in C2BBe1 cells revealed that the PMA effect is mediated through a GC-rich DNA region between nt -88 and -69. Gel mobility-shift assays demonstrated that in nuclear extracts from C2BBe1 cells grown under the basal growth conditions, Sp1 (stimulating protein-1) and Sp3 interact with this GC-rich DNA region, while, in PMA-treated nuclear extracts, PMA-induced EGR-1 (early growth response gene product 1) transcription factor binds to the same site. Binding of EGR-1 diminished the Sp1 and Sp3 interactions with this promoter region significantly. Co-transfection of Sp1 or Sp3 into SL2 cells activated the NHE3-reporter constructs, suggesting that Sp1 and Sp3 act as positive regulators of the NHE3 expression. In addition, overexpression of EGR-1 was sufficient to transactivate the NHE3-reporter gene activity, and knockdown of EGR-1 with gene-specific small interfering RNA resulted in inhibition of the PMA-induced up-regulation of the endogenous NHE3 mRNA expression. Furthermore, the PKC (protein kinase C) inhibitor chelerythrine chloride did not affect PMA-induced NHE3 promoter activity, suggesting that PMA stimulation of the hNHE3 gene expression may be PKC-independent.

Inhibition of Experimental Proliferative Vitreoretinopathy with Protein Kinase C Inhibitor (Chelerythrine Chloride) and Melatonin

Purpose: To investigate whether a protein kinase C (PKC) inhibitor and melatonin prevent proliferative vitreoretinopathy (PVR). Methods: Twentypigmented rabbits were used in this study. All rabbits except controls received an intravitreal injection of 0.15 ml (75,000 units) of platelet-rich plasma into their left eye. The animals were divided into four groups: group I was treated with intravitreal injection of 0.1 ml (100 µmol/ml) of PKC inhibitor (chelerythrine chloride), group II received 1 ml (4 mg/kg) of intraperitoneal melatonin for 3 days, group III received nothing (blank group), and group IV (control group) received only 0.5 ml of 1% ethanol intraperitoneally for 3 days. Proliferative changes were graded in a masked fashion by indirect ophthalmoscopy for a 15-day follow-up period. The malondialdehyde (MDA), reduced glutathione (GSH) and total nitrite levels were measured in the vitreous humor. Results: The grades of PVR were A and B in group I and II, treated with PKC inhibitor and melatonin, respectively. The PVR grade in the blank group was C-D. The mean MDA level in group I (4.2 ± 0.9 µmol/l) was significantly lower than in the blank group (6.0 ± 1.0 µmol/l; p < 0.05). The mean GSH level in group I (66.3 ± 8.8 µmol/l) was not significantly different from that in the blank group (p > 0.05). The MDA and GSH levels in group II were 3.2 ± 0.7 and 70.1 ± 13.3 µmol/l, respectively. Both these levels were significantly different from those of the blank group (p < 0.05). The NO levels in both treatment groups were significantly lower than in the blank group (p < 0.001). Conclusion: These findings suggest an inhibitory effect of PKC inhibitor and melatonin on PVR. The inhibition of PVR development was associated with lower MDA and NO levels with higher GSH levels in the treatment groups.

Regulation of myocardial heat shock protein 70 gene expression following exercise

Post-exercise induction of myocardial heat shock protein (Hsp70) gene expression involves the activation of the heat shock transcription factor (HSF1). While the exact mechanisms governing the regulation of HSF1 are unclear, activation is believed to occur subsequent to hyperphosphorylation of specific serine residues. As two important serine kinases, protein kinase A (PKA) and protein kinase C (PKC), have been implicated in many phosphorylative events in myocardial cells, we examined the role of these kinases in the activation of Hsp70 gene expression following exercise. In this report, we show that prior administration of a PKA inhibitor, N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide 2HCl (H-89; 0.36 mg/kg), significantly suppressed the elevation in Hsp70 mRNA ( P < 0.05) and protein synthesis ( P < 0.05) in male Sprague–Dawley rats following a single bout of exercise. In contrast, this post-exercise elevation in Hsp70 mRNA and protein synthesis was not suppressed following the administration of a PKC inhibitor chelerythrine chloride (CHEL; 5 mg/kg) ( P < 0.05). Of note, inhibition of PKA did not alter the nuclear localization and binding affinity of HSF1 to the promotor region of the Hsp70 gene. These data indicate that PKA, and not PKC, plays a necessary role in the early exercise-induced regulation of Hsp70 gene expression, downstream of DNA-binding acquisition. However, the current study does not support previous observations regarding major changes in HSF1 phosphorylation and suggests that other PKA-related mechanisms mediate the activation of Hsp70 gene expression following exercise.

Epidermal Growth Factor Induces Fibroblast Contractility and Motility via a Protein Kinase C δ-dependent Pathway

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCdelta to promote the acute phase of EGF-induced MLC activation.

Implications of protein kinase C in the nucleus accumbens in the development of sensitization to methamphetamine in rats

Repeated treatment with methamphetamine leads to an enhancement in the methamphetamine-induced dopamine release and its related behaviors. This phenomenon is called sensitization or reverse tolerance. Protein kinase C (PKC) controls numerous signaling cascades by virtue of its ability to phosphorylate target proteins that include other kinases. The purpose of study was then to investigate the implication of PKC in the development of sensitization to the rewarding effect and to the extracellular dopamine release induced by methamphetamine in rats. The conditioned place preference paradigm and in vivo microdialysis assay were performed in the present study. An intra-nucleus accumbens injection of a selective PKC inhibitor chelerythrine chloride abolished the enhancement of the methamphetamine-induced place preference following repeated treatment with methamphetamine. Furthermore, intra-nucleus accumbens injection of chelerythrine chloride blocked the development of sensitization to dopamine release and to the decrease in the major dopamine metabolites, 3′4-dihydroxyphenylacetic acid and homovanillic acid, in the nucleus accumbens induced by repeated methamphetamine treatment. Under these conditions, the immunoreactivity of the cytosolic phosphorylated conventional- or classic-type PKC in the limbic forebrain region including the nucleus accumbens was slightly, but significantly increased in methamphetamine-sensitized rats. The present data provide evidence for the implication of PKC in the nucleus accumbens in the development of sensitization to the methamphetamine-induced rewarding effect, dopamine release and inhibition of dopamine metabolism/re-uptake in rats.

Involvement of signaling through protein kinase C and phosphatidylinositol 3-kinase in the excystation and metacystic development of Entamoeba invadens.

Using an axenic excystation system in vitro, we examined the effect of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), which are signaling molecules responsible for numerous cellular responses, on the excystation and metacystic development of Entamoeba invadens. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by the PKC inhibitors staurosporine, chelerythrine chloride and calphostin C in a concentration-dependent manner during incubation, compared with the controls. As cyst viability was not affected by these inhibitors, reduced excystation was not due to their direct toxic effects on cysts. Metacystic development, when determined by the number of nuclei in the amoebae, was delayed by these PKC inhibitors, because the percentage of 1-nucleate amoebae was lower than in controls at day 3 of incubation. Wortmannin, a potent inhibitor of PI3K, also inhibited excystation and metacystic development of E. invadens in a concentration-dependent manner, compared with the controls. These results indicate that signaling through PKC and PI3K contributes to the excystation and metacystic development of E. invadens.

Various inotropic effects of angiotensin II in post-ischaemic rat hearts depending on ischaemic time with possible involvement of protein kinase C.

The present study investigated if the inotropic effect of angiotensin II (AngII) is altered during post-ischaemic reperfusion in hearts subjected to mild and severe ischaemia. The possible involvement of protein kinase C (PKC) in the change in the inotropic effect was also investigated. Isolated Langendorff-perfused rat hearts were perfused under constant flow with oxygenated Krebs-Henseleit buffer and paced at 360 beats min(-1). A saline-filled balloon catheter inserted into the left ventricle was used for measurement of contractile force. In the first series of experiments, hearts were subjected to continuous perfusion, 15- or 25-min global ischaemia followed by 45-min reperfusion. At the end of reperfusion, 0.1 micromol L(-1) AngII was infused for 5 min. In a second series of experiments, AngII was infused in hearts subjected to 25-min ischaemia followed by 45-min reperfusion in the absence or presence of the PKC inhibitor chelerythrine chloride (5 micromol L(-1)). The current study demonstrates that AngII exerts a positive inotropic effect in normoxic hearts with an increase of left ventricular developed pressure (LVDP) by 11% (P<0.05 vs. prior to AngII infusion). In post-ischaemic hearts subjected to 15-min ischaemia no effect of AngII was observed. In hearts subjected to 25 min of ischaemia, however, AngII evoked a negative inotropic response with a decrease of LVDP by 18% (P<0.05 vs. prior to AngII infusion). The negative inotropic effect of AngII was inhibited by the PKC inhibitor chelerythrine chloride. AngII exerts negative inotropic effect in severely injured post-ischaemic heart, possibly through the PKC pathway.

Drinking behavior elicited by central injection of angiotensin II: roles for protein kinase C and Ca2+/calmodulin-dependent protein kinase II.

Prior studies utilizing neurons cultured from the hypothalamus and brain stem of newborn rats have demonstrated that ANG II-induced modulation of neuronal firing involves activation of both protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). The present studies were performed to determine whether these signaling molecules are also involved in physiological responses elicited by ANG II in the brain in vivo. Central injection of ANG II (10 ng/2 microl) into the lateral cerebroventricle (icv) of Sprague-Dawley rats increased water intake in a time-dependent manner. This ANG II-mediated dipsogenic response was attenuated by central injection of the PKC inhibitors chelerythrine chloride (0.5-50 microM, 2 microl) and Go-6976 (2.3 nM, 2 microl) and by the CaMKII inhibitor KN-93 (10 microM, 2 microl). Conversely, icv injection of chelerythrine chloride (50 microM, 2 microl) and KN-93 (10 microM, 2 microl) had no effect on the dipsogenic response elicited by central injection of carbachol (200 ng/2 microl). Furthermore, injection of ANG II (10 ng/2 microl) icv increases the activity of both PKC-alpha and CaMKII in rat septum and hypothalamus. These data suggest that signaling molecules involved in ANG II-induced responses in vitro are also relevant in physiological responses elicited by ANG II in the whole animal model.

Expression of calcitonin gene-related peptide, substance P and protein kinase C in cultured dorsal root ganglion neurons following chronic exposure to mu, delta and kappa opiates

The mechanisms involved in morphine tolerance are poorly understood. It was reported by our group that calcitonin gene-related peptide (CGRP)-like immunoreactivity (IR) was increased in the spinal dorsal horn during morphine tolerance [Ménard et al. (1996) J. Neurosci. 16, 2342–2351]. More recently, we observed that it was possible to mimic these results in cultured dorsal root ganglion (DRG) neurons allowing for more detailed mechanistic studies [Ma et al. (2000) Neuroscience 99, 529–539]. The aim of the present series of experiments was to further validate the DRG cell culture model by establishing which subtypes of opioid receptors are involved in the induction of CGRP in cultured rat DRG neurons, and to examine the signaling pathway possibly involved in the induction of CGRP-like IR following repeated opiate treatments. Other neuropeptides known to be expressed in DRG neurons, such as substance P (SP), neuropeptide Y (NPY) and galanin, were investigated to assess specificity. Following treatment with any of the three opioid agonists (μ, DAMGO; δ, DPDPE; κ, U50488H), the number of CGRP- and SP-IR cultured DRG neurons increased significantly, and in a concentration-dependent manner, with the effects of κ agonist being less pronounced. NPY and galanin were not affected. Double-immunofluorescence staining showed that the three opioid receptors were co-localized with both CGRP- and SP-like IR. Protein kinase C (PKC)-like IR was found to be significantly increased following a repetitive treatment with DAMGO. Double-immunofluorescence staining showed the co-localization of PKCα with CGRP- and SP-IR in cultured DRG neurons. Moreover, a combined treatment with DAMGO and a PKC inhibitor (chelerythrine chloride or Gö 6976) was able to block the effects of the opioid on increased CGRP-like IR. These data suggest that the three opioid receptors may be involved in the induction of CGRP and SP observed following chronic exposure to opiates, and that PKC probably plays a role in the signaling pathway leading to the up-regulation of these neuropeptides. These findings further validate the DRG cell culture as a suitable model to study intracellular pathways that govern changes seen following repeated opioid treatments possibly leading to opioid tolerance.

Role of protein kinase C in control of ethanol-modulated beta-endorphin release from hypothalamic neurons in primary cultures.

We have previously shown that short-term exposure to ethanol stimulates immunoreactive beta-endorphin (IR-beta-EP) release from hypothalamic neurons and that chronic ethanol exposure decreases the IR-beta-EP release from these neurons. The role of protein kinase C (PKC) in the ethanol-regulated beta-EP release from hypothalamic neurons has not been established. In this study, by using the primary cultures of hypothalamic neurons, we tested the effects of PKC stimulator phorbol ester 4 beta-phorbol 12-myristate-13-acetate (PMA) and PKC inhibitor chelerythrine chloride on ethanol-induced IR-beta-EP release. Additionally, the effects of ethanol with or without PMA on expression and translocation of various PKC isoenzymes from cytosolic to membrane fraction were determined. PMA treatment increased IR-beta-EP release in a time- and dose-dependent manner. Acute ethanol treatment (3 h) increased, while chronic ethanol treatment (24 h) reduced, the magnitude of PMA-induced IR-beta-EP release. The stimulatory effect of acute ethanol on IR-beta-EP release was reduced by chelerythrine chloride. Determination of the effects of ethanol with or without PMA on seven different PKC isoenzymes (PKC-alpha, -beta I, -beta II, -gamma, -delta, -epsilon, and -zeta) revealed that the expression and translocation of only two PKC isoenzymes, PKC-delta and PKC-epsilon, were stimulated by acute treatment with ethanol. Acute ethanol also increased PMA-stimulated expression of these two isoenzymes. Chronic ethanol treatment reduced both basal and PMA-induced increase of PKC-delta and PKC-epsilon expression and translocation. These data provide evidence for the first time that ethanol-regulated IR-beta-EP secretion is controlled by the PKC system, possibly involving PKC-delta and PKC-epsilon isoenzymes.

Effect of modulators of protein kinase C activity on Ca2+ transport in retinal rod microsomes.

The effect of modulators of protein kinase C (PKC) activity on Ca2+ translocation in retinal rod microsomes was studied. It is shown that PKC activators (phorbol 12-myristate-13-acetate (PMA) and diacylglycerol (DAG)) and inhibitors (chelerythrine chloride, polymyxin B, and phloretin) stimulate and inhibit ATP-dependent Ca2+ uptake in retinal rod microsomes, respectively. This effect is apparently due to an influence of PKC on Ca-ATPase contained in these vesicular structures. It was found that PKC inhibitors (chelerythrine chloride, polymyxin B, and phloretin) and activators (PMA and DAG) potentiate Ca2+ release from Ca2+-loaded retinal rod microsomes. Specific and nonspecific mechanisms of Ca-release stimulation by the modulators of PKC activity are discussed.

Effects of mercuric chloride exposure on the glutamate uptake by cultured retinal pigment epithelial cells

The cytotoxicity of mercuric chloride and the effects of mercuric chloride on glutamate and calcium uptake and the factors regulating glutamate uptake were studied in retinal pigment epithelium (RPE) cell cultures. RPE cells isolated from pig eyes and human RPE cell line (D407) cells were cultured to confluency and further subcultured according to the test protocol in question. The cytotoxicity caused by 15 min of exposure to mercuric chloride (0.01–1000 μ m) was evaluated by WST-1 assay based on the activity of mitochondrial dehydrogenases. [ 3H]Glutamate uptake was measured after the cells were exposed to 0.1–100 μ m mercuric chloride and the selected regulators of protein kinase C (PKC) pathway: PKC activator SC10, PKC inhibitor chelerythrine chloride, phospholipase A 2/C inhibitor manoalide, tyrosine kinase inhibitor lavendustin A, competitive NMDA receptor antagonist AP7 and IP 3 receptor antagonist heparin. Intracellular calcium was monitored with Fluo-3 probe starting immediately after the exposure to 1–1000 μ m mercuric chloride. Mercuric chloride showed concentration-dependent effects on cell viability, on glutamate uptake and on intracellular calcium concentration. The results give some support to the concept that glutamate uptake is affected by PKC. The PKC inhibitor chelerythrine chloride decreased glutamate uptake by 25%, but the PKC activator SC10 could partly prevent the inhibitory effect of mercuric chloride. Lavendustin A, manoalide and heparin had smaller, but statistically significant, effects. All these substances act on mediators which can regulate the activity of PKC. However, PKC is not likely to be the only regulator of glutamate uptake. The rise observed in [Ca 2+] i may initiate various cellular events during mercury intoxication.