Activation Of Protein Kinase C Pathway Research Articles

Crosstalk between PKC and MAPK pathway activation in cardiac fibroblasts in a rat model of atrial fibrillation.

Atrial fibrillation (AF) is the most frequent form of cardiac arrhythmia and major cause of cardiac ischemia. Defective calcium homeostasis due to anomalous expression of ryanodine receptor type 2 (RyR2) or its hyperactivation by phosphorylation by serine threonine kinases has been implicated as a central mechanism of AF pathogenesis. Given the role of protein kinase C (PKC) isoforms in cardiac function we investigated role of PKC in AF using a rat model. PMA induced global increase in protein synthesis in cardiac fibroblasts isolated from AF rats, but not healthy controls, and the increase was inhibited by PKC inhibition. PMA mediated activation of both PKC and ERK and either inhibition of PKC by Go6983 or ERK by the MEK inhibitor Trametinib attenuated both P-ERK and P-PKC in both cardiac fibroblasts isolated from AF rats or from healthy rats but transduced with PKC-delta. The PKC and ERK mediated induction of global protein synthesis was found to be mediated by increased phosphorylation of the ribosomal protein S6. Our findings provide a foundation for future testing of PKC and MEK inhibitors to treat AF in pre-clinical models. It also needs to be determined if PKC and MAPK pathway activation is functioning via RyR2 or some yet undefined substrates.

Ascorbic Acid Induces Necrosis in Human Laryngeal Squamous Cell Carcinoma via ROS, PKC, and Calcium Signaling.

Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 232: 417-425, 2017. © 2016 Wiley Periodicals, Inc.

Recent advances in understanding the role of oxidative stress in diabetic neuropathy

Diabetic neuropathy (DN) is one of the most common and severe manifestations of diabetes mellitus. The mechanisms underlying the structural, functional and metabolic changes in diabetic neuropathy have been under study for a long time. In this review the biochemistry and implications of the four pathways responsible for the development of DN, polyol pathway; increased AGEs (advanced glycation end-products) formation; activation of PKC (protein kinase C) and hexosamine pathway have been discussed. Experimental and clinical evidences suggest a close link between neurodegeneration and oxidative stress which serves as a unifying mechanism, thus linking the four pathways. Recent studies indicate that oxidative stress mediated DNA damage causes poly(ADP-ribose) polymerase (PARP) overactivation and reduced activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a factor common to all the four pathways. The exact mechanism of PARP mediated cell death in DN needs further investigation. Based on current studies neuroprotective and antioxidant therapy have been suggested as potential treatment and preventive solutions for DN.

Membrane associated RGC‐32 promoted macrophage phagocytosis via direct and specific activation of novel PKC pathway (802.24)

Phagocytosis of bacterial pathogens by macrophages requires the activation of protein kinase C (PKC), a family of protein serine/threonine kinases. Previous publications have demonstrated that both classical and novel PKCs were required for macrophage phagocytosis. However recruitment of classical PKCs or novel PKCs or both is macrophage linage specific, which prompt us to hyposize that there exist PKC isozyme specific regulators in macrophage. Here we found that response gene to complement 32 (RGC32) is a direct and specific activator for novel PKC pathway in macrophage phagocytosis. RGC32 was induced and specifically expressed in macrophage lineages in differentiation. While bone marrow progenitor cells from RGC32 knockout mice did not have significant defects in macrophage differentiation, both peritoneal macrophages (PMs) and bone marrow derived macrophages (BMDMs) presented dramatic defects in their phagocytosis activities. Moreover, macrophages over expressed RGC32 showed promoted phagocytosis activity. During phagocytosis, RGC32 was recruited to cell membrane and co‐localized with F‐actin to forming phagocytic cups and macrophages from RGC32 knockout mice showed impaired F‐actin assembling and phagocytic cup formation. Mechanistically, RGC32 participated in phagocytosis through activation of PKC induced alanine‐rich C‐kinase substrate (MARCKS) phosphorylation. Recombinant RGC32 increased PKC activity in vitro. Knocking down RGC32 in macrophage suppressed MARCKS phosphorylation while over expression RGC32 increased MARCKS phosphorylation. Interestingly, RGC32 directly interacted with novel but not classical PKC isozymes and RGC32 induced MARCKS can only be suppressed by novel PKC inhibitor. Finally, RGC32 knockout mice showed defects in pathogens clearance to peritoneal bacterial infection. In summary, for the first time, we identified RGC32 as an membrane associated novel PKC activator during macrophage phagocytosis.

High-density lipoprotein induces cyclooxygenase-2 expression and prostaglandin I-2 release in endothelial cells through sphingosine kinase-2

High-density lipoprotein (HDL) has a significant cardioprotective effects. HDL induces cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in vascular endothelial cells, which contributes to its anti-atherogenic effects. However, the underlying mechanisms are not fully understood. In the present study, we observed that HDL-stimulated COX-2 expression and PGI-2 production in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. These effects triggered by HDL were inhibited by pertussis toxin (PTX), protein kinase C (PKC) inhibitor GF109203X, and ERK inhibitor PD98059, suggesting that Gαi/Gαo-coupled GPCR, PKC, and ERK pathways are involved in HDL-induced COX-2/PGI-2 activation. More importantly, we found that silencing of sphingosine kinase 2 (SphK-2) also blocked HDL-induced COX-2/PGI-2 activation. In addition, HDL-activated SphK-2 phosphorylation accompanied by increased S1P level in the nucleus. Our ChIP data demonstrated that SphK-2 is associated with CREB at the COX-2 promoter region. Collectively, these results indicate that HDL induces COX-2 expression and PGI-2 release in endothelial cells through activation of PKC, ERK1/2, and SphK-2 pathways. These findings implicate a novel mechanism underlying anti-atherothrombotic effects of HDL.

Effect of Phorbol 12-Myristate 13-Acetate on Function and Gene Expression of P-Glycoprotein in Adriamycin-Resistant K562/ADM Cells

Background/Aims: Multidrug resistance (MDR) is a critical issue during chemotherapy of cancers. Phorbol 12-myristate 13-acetate (PMA), a diester of phorbol, is a typical activator of protein kinase C (PKC). In the present study, we investigated the effect of PMA on MDR and P-glycoprotein (P-gp) gene expression in K562/ADM cells. Methods: 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay was used to assess adriamycin (Adr)-induced cytotoxicity towards K562/ADM cells in the absence or presence of PMA. The intracellular accumulation of Adr was measured by determining the mean fluorescence intensity. The effect of PMA on P-gp activity was investigated by rhodamine-123 accumulation and efflux experiment. Protein expression and mRNA expression of P-gp in K562/ADM cells were determined by Western blot analysis and real-time qPCR, respectively. Results: Adr-induced cytotoxicity towards K562/ADM cells was significantly decreased by PMA at 5 μmol/l. Furthermore, intracellular Adr-associated mean fluorescence intensity was attenuated by 53.8% 1 h after exposure to PMA at 5 μmol/l compared with the control group (p < 0.05). A dose-dependent decrease of intracellular rhodamine-123 and increase of efflux activity of P-gp were also observed in K562/ADM cells incubation with PMA. In addition, P-gp mRNA and protein expression were significantly induced by PMA. Conclusion: Activation of PKC pathway by PMA can significantly induce expression and activity of P-gp, and thus decrease intracellular Adr level and strengthen MDR in K562/ADM cells.

Inflammation and the pathogenesis of diabetic nephropathy

The most problematic issue in clinical nephrology is the relentless and progressive increase in patients with ESRD (end-stage renal disease) worldwide. The impact of diabetic nephropathy on the increasing population with CKD (chronic kidney disease) and ESRD is enormous. Three major pathways showing abnormality of intracellular metabolism have been identified in the development of diabetic nephropathy: (i) the activation of polyol and PKC (protein kinase C) pathways; (ii) the formation of advanced glycation end-products; and (iii) intraglomerular hypertension induced by glomerular hyperfiltration. Upstream of these three major pathways, hyperglycaemia is the major driving force of the progression to ESRD from diabetic nephropathy. Downstream of the three pathways, microinflammation and subsequent extracellular matrix expansion are common pathways for the progression of diabetic nephropathy. In recent years, many researchers have been convinced that the inflammation pathways play central roles in the progression of diabetic nephropathy, and the identification of new inflammatory molecules may link to the development of new therapeutic strategies. Various molecules related to the inflammation pathways in diabetic nephropathy include transcription factors, pro-inflammatory cytokines, chemokines, adhesion molecules, Toll-like receptors, adipokines and nuclear receptors, which are candidates for the new molecular targets for the treatment of diabetic nephropathy. Understanding of these molecular pathways of inflammation would translate into the development of anti-inflammation therapeutic strategies.

Rat Pial Microvascular Responses to Transient Bilateral Common Carotid Artery Occlusion and Reperfusion: Quercetin’s Mechanism of Action

The aim of the present study was to assess quercetin’s mechanism of action in rat pial microvessels during transient bilateral common carotid artery occlusion (BCCAO) and reperfusion. Rat pial microcirculation was visualized using fluorescence microscopy through a closed cranial window. Pial arterioles were classified in five orders of branchings. In ischemic rats, 30 min BCCAO and 60 min reperfusion caused arteriolar diameter decrease, microvascular leakage, leukocyte adhesion in venules, and reduction of capillary perfusion. Quercetin highest dose determined dilation in all arteriolar orders, by 40 ± 4% of baseline in order 2 vessels, and prevented microvascular permeability [0.15 ± 0.02 normalized gray levels (NGL)], leukocyte adhesion, and capillary failure. Protein kinase C (PKC) inhibition exerted by chelerythrine prior to quercetin attenuated quercetin-induced effects: order 2 arterioles dilated by 19.0 ± 2.4% baseline, while there was an increase in permeability (0.40 ± 0.05 NGL) and leukocyte adhesion with a marked decrease in capillary perfusion. Tyrosine kinase (TK) inhibition by tyrphostin 47 prior to quercetin lessened smaller pial arterioles responses, dilating by 20.7 ± 2.5% of baseline, while leakage increased (0.39 ± 0.04 NGL) sustained by slight leukocyte adhesion and ameliorated capillary perfusion. Inhibition of endothelium nitric oxide synthase (eNOS) by NG-nitro-L-arginine-methyl ester (L-NAME) prior to PKC or TK reduced the quercetin’s effects on pial arteriolar diameter and leakage. eNOS inhibition by L-NAME reduced quercetin effects on pial arteriolar diameter and leakage. Finally, combined inhibition of PKC and TK prior to quercetin abolished quercetin-induced effects, decreasing eNOS expression, while blocking ATP-sensitive potassium (KATP) channels by glibenclamide suppressed arteriolar dilation. In conclusion, the protective effects of quercetin could be due to different mechanisms resulting in NO release throughout PKC and TK intracellular signaling pathway activation.

Effects of Fluvastatin on the Expression of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells

Abstract 2253 OBJECTIVE:3-Hydroxyl-3-methyl coenzyme A reductase inhibitors (statins) inhibit the production of mevalonate and other isoprenoid intermediates of the cholesterol biosynthetic pathway, such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). Statins can protect the vasculature from inflammation and atherosclerosis caused by cholesterol-dependent and cholesterol-independent mechanisms. The latest investigations show that statins modulate the expression of genes related to inflammation, blood coagulation and fibrinolysis in cultured endothelial cells. Tissue factor pathway inhibitor (TFPI) which is expressed by endothelial cells plays a crucial role in hemostasis by regulating TF-induced initiation of coagulation. The aim of this study was to elucidate the effects of fluvastatin, lipophilic statin, on expressions of TFPI in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were incubated for 24 h in culture medium including fluvastatin (0.1, 1.0, 10.0 μM). The expression of TFPI mRNA and protein was evaluated by western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. To identify which product of statin reaction is necessary for the effect of fluvastatin, HUVECs were incubated for 24h with fluvastatin with mavalonate, FPP, or GGPP. On the other hand, it is known that fluvastatin increase nitric oxide (NO) bioavailability. To determine whether fluvastatin induced NO affects TFPI mRNA and protein expression, HUVECs were incubated for 24h with fluvastatin with NG-Nitro-L-arginine methyl ester, hydrochloride (L-NAME: specific inhibitor of NO synthase). Additionally, to determine whether fluvastatin affects p38MAPK, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), and protein kinase C (PKC) pathways, HUVECs were incubated for 24h with fluvastatin with the inhibitors of p38MAPK (SB203580), JNK (SP600125), MEK (U0126), PI3K (LY294002), and PKC (GF109203). The expression of TFPI mRNA and protein was evaluated by western blot. RESULTS: Fluvastatin increased TFPI mRNA and protein expression (1μM: p<0.01, 10μM: p<0.05; Figure 1). This fluvastatin-dependent up-regulation of TFPI was prevented by mevalonate and geranylgeranylphosphate (GG-PP). In contrast, the addition of L-NAME did not alter induction of TFPI expression by fluvastatin. Similarly, Y-27632 (Rho kinase inhibitor) and NSC23766 (Rac1 inhibitor) were ineffective. Additionally, the inhibitors of p38MAPK, PI3K, and PKC prevented fluvastatin-dependent up-regulation. On the other hand, the inhibitors of JNK and MEK were ineffective. CONCLUSIONS: This study suggests that fluvastatin significantly increases TFPI mRNA and protein expression, and this effect of fluvastatin is accompanied by the activation of p38 MAPK, PI3K, and PKC pathways. Therefore, this effect may play an important role in preventing cardiovascular events. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Requirement of phospholipase C and protein kinase C in cholecystokinin‐mediated facilitation of NMDA channel function and anxiety‐like behavior

Although cholecystokinin (CCK) has long been known to exert anxiogenic effects in both animal anxiety models and humans, the underlying cellular and molecular mechanisms are ill-defined. CCK interacts with CCK-1 and CCK-2 receptors resulting in up-regulation of phospholipase C (PLC) and protein kinase C (PKC). However, the roles of PLC and PKC in CCK-mediated anxiogenic effects have not been determined. We have shown previously that CCK facilitates glutamate release in the hippocampus especially at the synapses formed by the perforant path and dentate gyrus granule cells via activations of PLC and PKC. Here we further demonstrated that CCK enhanced NMDA receptor function in dentate gyrus granule cells via activation of PLC and PKC pathway. At the single-channel level, CCK increased NMDA single-channel open probability and mean open time, reduced the mean close time, and had no effects on the conductance of NMDA channels. Because elevation of glutamatergic functions results in anxiety, we explored the roles of PLC and PKC in CCK-induced anxiogenic actions using the Vogel Conflict Test (VCT). Our results from both pharmacological approach and knockout mice demonstrated that microinjection of CCK into the dentate gyrus concentration-dependently increased anxiety-like behavior via activation of PLC and PKC. Our results provide a novel unidentified signaling mechanism whereby CCK increases anxiety.

Effects of cholecystokinin octapeptide on the contractile activity of guinea-pig colonic smooth muscles, L-type calcium currents and membrane potentials of myocytes

To investigate the effects and mechanism of cholecystokinin octapeptide (CCK-8S) on the contractile activity of smooth muscles, L-type calcium current and membrane potentials of proximal colon myocytes in guinea pig. (1) Strips of proximal colon were obtained from adult guinea pigs. The contraction of these stripes was measured by a RM6240 multi-channel physiological signal system. (2) Suspension of single smooth muscle cells (SMCs) were obtained from proximal colon and isolated by enzymatic digestion. The effect of CCK-8S on intracellular calcium concentration ([Ca(2+)]i) of SMCs was examined by fura-2-loaded microfluorimetric measurement. (3) Resting potential (RP), action potential (AP) and L-type calcium current (I(Ca-L)) were recorded by patch-clamp technique. (1) The contractile amplitude and frequency of muscle stripes enhanced by CCK-8S (10(-7) mol/L) were (149 ± 12)% and (132 ± 13)% respectively of those of control group (all P < 0.05). They were significantly attenuated by pretreating strips with CCK1 receptor antagonist devazepide (10(-7) mol/L), L-type calcium channel blocker nifedipine (10(-5) mol/L), Ca(2+)-ATPase inhibitor TG (thapsigargin) (10(-5) mol/L) and BA (boric acid) (10(-5) mol/L) respectively. (2) [Ca(2+)]i of SMCs intensified by CCK-8S was (738 ± 24)% of that of control group. And it was inhibited by pretreating SMCs with devazepide (all P < 0.05). (3) After the superfusion of CCK-8S, RP depolarized to (52 ± 9)%, the exogenously stimulated peak values of AP rose to (140 ± 4)% and fast repolarization time of AP decreased to (61 ± 13)% (all P < 0.05). They were significantly inhibited when these cells were pretreated with devazepide and/or nifedipine (n = 8, P < 0.05 for each group) whereas CI 988 had little effect. (4) The CCK-8S-evoked I(Ca-L) of SMCs at the voltage of + 10 mV was boosted to (138 ± 7)%. Such an effect was suppressed by a pretreatment with nifedipine, devazepide, TG and BA respectively. In the presence of an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptors, heparin (10(-6) mol/L) and an protein kinase C (PKC) inhibitor, saurosporine (10(-6) mol/L), CCK-8S did not significantly intensify I(Ca-L) (all P > 0.05). CCK-8S promotes proximal colon contraction by CCK1 receptors through the activation of IP3-mediated PKC pathway.

Akt and PKC are involved not only in upregulation of telomerase activity but also in cell differentiation-related function via mTORC2 in leukemia cells

We have shown previously that PI3K/Akt pathway is active after cell differentiation in HL60 cells. In the present study, we have investigated whether additional molecules, such as protein kinase C (PKC), are involved in the regulation, not only of telomerase, but also of leukemia cell differentiation. We show that PKC activates telomerase and is, itself, activated following VD3- or ATRA-induced differentiation of HL60 cells, as was observed for PI3K/Akt. To clarify the significance of PI3K/Akt and PKC pathway activation in leukemia cell differentiation, we examined the active proteins in either the downstream or upstream regulation of these pathways. In conjunction with the activation of Akt or PKC, mTOR and S6K were phosphorylated and the protein expression levels of Rictor were increased, compared with Raptor, following cell differentiation. Silencing by Rictor siRNA resulted in the attenuation of Akt phosphorylation on Ser473 and PKCα/βII phosphorylation, as well as the inhibition of Rictor itself, suggesting that Rictor is an upstream regulator of both Akt and PKC. In addition, in cells induced to differentiate by ATRA or VD3, Nitroblue-tetrazolium (NBT) reduction and esterase activity, were blocked either by LY294002, a PI3K inhibitor, or by BIM, a PKC inhibitor, without affecting cell surface markers such as CD11b or CD14. Intriguingly, the silencing of Rictor by its siRNA also suppressed the reducing ability of NBT following VD3-induced cell differentiation. Taken together, our results show that Rictor associated with mTOR (mTORC2) regulates the activity of both Akt and PKC that are involved in cell functions such as NBT reduction and esterase activity induced by leukemia cell differentiation.

Genomic organization and regulation of the human orexin (hypocretin) receptor 2 gene: identification of alternative promoters

Orexins (hypocretins), acting via their receptors, are involved in the control of feeding behaviour, sleep, arousal and energy homoeostasis. However, regulation of the human orexin receptor 2 (hOX2R) gene remains unknown. We have identified four transcripts arising from alternative splicing from three exons. These exon 1 variants were designated exons 1A, 1B and 1C on the basis of their 5'-3' order. RT (reverse transcription)-PCR demonstrates the differential expression in various human tissues. The alternative 5'-UTRs (untranslated regions) possessed by these isoforms have different translational efficiencies, which regulate the level of protein expression. In the present study, we have demonstrated that the hOX2R gene is regulated by two promoters and the novel transcripts are regulated by the distal promoter located upstream of exon 1A. We have demonstrated that the AP-1 (activator protein 1) motif is critical for sustaining the basal activity of distal promoter. Analysis of the proximal promoter revealed the region regulating promoter activity contained putative binding elements including those for CREB (cAMP-response-element-binding protein), GATA-2 and Oct-1. Using the chromatin immunoprecipitation assay, we demonstrated that CREB, GATA-2 and Oct-1 transcription factors bind to these critical regulatory promoter elements. Mutational studies suggested that these motifs functioned independently, but have a compound effect regulating hOX2R gene transcription. Furthermore, proximal promoter activity is enhanced by both PKA (protein kinase A) and PKC (protein kinase C) pathway activation, via binding of CREB and GATA-2 transcription factors. In conclusion, we have demonstrated that expression of hOX2R is regulated by a complex involving a proximal PKA/PKC-regulated promoter and a distal promoter regulating tissue-specific expression of alternative transcripts which in turn post-transcriptionally regulate receptor levels.

Induction of ANGPTL4 expression in human airway smooth muscle cells by PMA through activation of PKC and MAPK pathways

In this study, we demonstrate that protein kinase C (PKC) activators, including phorbol-12-myristate-13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DOG), and platelet-derived growth factor alpha are potent inducers of angiopoietin-like protein 4 (ANGPTL4) expression in several normal lung cell types and carcinoma cell lines. In human airway smooth muscle (HASM) cells induction of ANGPTL4 expression is observed as early as 2 h after the addition of PMA. PMA also increases the level of ANGPTL4 protein released in the medium. PKC inhibitors Ro31-8820 and Gö6983 greatly inhibit the induction of ANGPTL4 mRNA by PMA suggesting that this up-regulation involves activation of PKC. Knockdown of several PKCs by corresponding siRNAs suggest a role for PKCalpha. PMA does not activate MAPK p38 and p38 inhibitors have little effect on the induction of ANGPTL4 indicating that p38 is not involved in the regulation of ANGPTL4 by PMA. In contrast, treatment of HASM by PMA induces phosphorylation and activation of Ra, MEK1/2, ERK1/2, JNK, Elk-1, and c-Jun. The Ras inhibitor manumycin A, the MEK1/2 inhibitor U0126, and the JNK inhibitor SP600125, greatly reduce the increase in ANGPTL4 expression by PMA. Knockdown of MEK1/2 and JNK1/2 expression by corresponding siRNAs inhibits the induction of ANGPTL4. Our observations suggest that the induction of ANGPTL4 by PMA in HASM involves the activation of PKC, ERK, and JNK pathways. This induction may play a role in tissue remodeling during lung injury and be implicated in several lung pathologies.

Facilitation of glutamate release from rat cerebral cortex nerve terminal by subanesthetic concentration propofol

Propofol is now the most commonly used intravenous anesthetic-for general anesthesia and sedation because of its rapid onset and recovery. Besides the well-known adverse effects of cardiovascular and respiratory depression, recent studies indicate that propofol may cause excitatory phenomena such as myoclonus, opisthotonus, and even seizure. However, the mechanisms of these excitatory effects of propofol have not been elucidated. Considering glutamate as the principle excitatory neurotransmitter in the central nervous system and excessive glutamatergic synaptic transmission can cause seizure, we examined the effect of propofol on the release of glutamate from rat cerebral cortex nerve terminals (synaptosomes). Results showed that subanesthetic concentration propofol facilitated 4-aminopyridine (4-AP), but not KCl- or ionomycin-evoked glutamate release from nerve terminals. The facilitation of 4-AP-evoked glutamate release by propofol also occurred in the calcium chelation and significantly attenuated by glutamate transporter inhibitors, DL-threo-beta-benzyloxyaspartic acid (DL-TBOA) and L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC). In addition, propofol increased 4-AP-evoked depolarization of the plasma membrane potential. Furthermore, protein kinase C (PKC) inhibition suppressed propofol-mediated facilitation of glutamate release. These results suggest that subanesthetic concentration propofol facilitates glutamate release from rat cerebrocortical glutamatergic terminals by increasing nerve terminal excitability, likely through the activation of PKC pathway. This finding may provide an explanation for propofol-induced excitatory phenomena.

Urocortin induces positive inotropic effect in rat heart

The aim of this study is to evaluate the positive inotropic effect of urocortin (Ucn) and to characterize its signalling pathways. Contractility was measured in ex vivo Langendorff-perfused hearts isolated from Wistar rats. Isolated ventricular cardiomyocytes were used to analyse intracellular calcium ([Ca(2+)](i)) transients evoked by electrical stimulation and L-type Ca(2+) current by confocal microscopy and whole-cell patch-clamping, respectively. The application of Ucn to perfused hearts induced progressive, sustained, and potent inotropic and lusitropic effects that were dose-dependent with an EC(50) of approximately 8 nM. Ucn effects were independent of protein kinase A (PKA) activation but were significantly reduced by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors and by brefeldin A, an antagonist of guanine nucleotide exchange factor, suggested to be an inhibitor of exchange protein activated by cAMP (Epac). These whole-organ effects were correlated with the inotropic effects observed in isolated cells: Ucn increased I(CaL) density, [Ca(2+)](i) transients, cell shortening and Ca(2+) content of sarcoplasmic reticulum. Our results show that Ucn evokes potent positive inotropic and lusitropic effects mediated, at least in part, by an increase in I(CaL) and [Ca(2+)](i) transient amplitude. These effects may involve the activation of Epac, PKC, and MAPK signalling pathways.

Improved spatial learning in aged rats by genetic activation of protein kinase C in small groups of hippocampal neurons.

Age-related decline in human cognition is well known, and there are correlative changes in the function of neocortical and hippocampal neurons. Similarly, age-related decline in learning has been observed in rodents, including deficits in a hippocampal-dependent learning paradigm, the Morris water maze. Furthermore, there are correlative deficits in specific signaling pathways, including protein kinase C (PKC) pathways, in cerebellar, hippocampal, or neocortical neurons. PKC pathways are strong candidates for mediating the molecular changes that underlie spatial learning, as they play critical roles in neurotransmitter release and synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), and deletion of specific PKC genes results in deficits in learning. Conversely, genetic activation of PKC pathways in small groups of hippocampal or cortical neurons enhances learning in specific paradigms. In this study, the authors delivered a constitutively active PKC into small groups of hippocampal dentate granule neurons in aged rats (using a herpes simplex virus-1 vector). Aged 2-year-old rats that received the constitutively active PKC displayed improved performance in the Morris water maze relative to controls in three different measures. These results indicate that PKC pathways play an important role in mediating spatial learning in aged rats. Additionally, these results represent a system for studying the neural mechanisms underlying aging-related learning deficits, and potentially developing gene therapies for cognitive and age-related deficits.

Negative Regulation of Inducible Nitric-oxide Synthase Expression Mediated through Transforming Growth Factor-β-dependent Modulation of Transcription Factor TCF11

Inducible nitric-oxide synthase (iNOS) plays a central role in the regulation of vascular function and response to injury. A central mediator controlling iNOS expression is transforming growth factor-beta (TGF-beta), which represses its expression through a mechanism that is poorly understood. We have identified a binding site in the iNOS promoter that interacts with the nuclear heterodimer TCF11/MafG using chromatin immunoprecipitation and mutation analyses. We demonstrate that binding at this site acts to repress the induction of iNOS gene expression by cytokines. We show that this repressor is induced by TGF-beta1 and by Smad6-short, which enhances TGF-beta signaling. In contrast, the up-regulation of TCF11/MafG binding could be suppressed by overexpression of the TGF-beta inhibitor Smad7, and a small interfering RNA to TCF11 blocked the suppression of iNOS by TGF-beta. The binding of TCF11/MafG to the iNOS promoter could be enhanced by phorbol 12-myristate 13-acetate and suppressed by the protein kinase C inhibitor staurosporine. Moreover, the induction of TCF11/MafG binding by TGF-beta and Smad6-short could be blocked by staurosporine, and the effect of TGF-beta was blocked by the selective protein kinase C inhibitor calphostin C. Consistent with the in vitro data, we found suppression of TCF11 coincident with iNOS up-regulation in a rat model of endotoxemia, and we observed a highly significant negative correlation between TCF11 and nitric oxide production. Furthermore, treatment with activated protein C, a serine protease effective in septic shock, blocked the down-regulation of TCF11 and suppressed endotoxin-induced iNOS. Overall, our results demonstrate a novel mechanism by which iNOS expression is regulated in the context of inflammatory activation.

Caffeine facilitation of glutamate release from rat cerebral cortex nerve terminals (synaptosomes) through activation protein kinase C pathway: An interaction with presynaptic adenosine A1 receptors

The present study used nerve terminals (synaptosomes) isolated from rat cerebral cortex to investigate the relationship between caffeine and 4-aminopyridine (4AP)-evoked endogenous excitatory neurotransmitter glutamate release. Micromolar concentrations of caffeine facilitated 4AP, but not KCl or ionomycin-evoked glutamate release from synaptosomes. This release facilitation resulted from an enhancement of vesicular and nonvesicular release and associated with an increase both in 4AP-evoked depolarization of the synaptosomal plasma membrane potential and in 4AP-evoked increase in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](C)). In addition, the release facilitation by caffeine was significantly reduced in synaptosomes pretreated with a wide spectrum blocker of N- and P/Q-type Ca(2+) channels, omega-conotoxin MVIIC. Furthermore, protein kinase C (PKC) activator and inhibitor, respectively, superseding or suppressing the caffeine-mediated facilitation of glutamate release. These results concluded that caffeine exerts their presynaptic facilitatory effect, likely through the activation of PKC pathway, which subsequently enhances terminal excitability and Ca(2+) entry to cause an increase in evoked glutamate release from rat cerebrocortical nerve terminals. Additionally, this release facilitation may involve an interaction of caffeine with presynaptic adenosine A1 receptors as adenosine A1 receptor inhibition abolished the caffeine-mediated facilitation of evoked glutamate release.

Involvement of phosphatidylinositol-phospholipase C in immune response to Salmonella lipopolysacharide in chicken macrophage cells (HD11)

The activation of phospholipases is one of the earliest key events in receptor-mediated cellular responses to a number of extracellular signaling molecules. Lipopolysaccharide (LPS) is a principle component of the outer membrane of Gram-negative bacteria and a prime target for recognition by the innate immune system. In the present study, we evaluated the role of specific phospholipase in the activation of a chicken macrophage cell line HD11 by LPS. Activation of HD11 cells by LPS results in induction of nitric oxide (NO). Using selective inhibitors, we have identified that phosphatidylinositol (PI)-phospholipase C (PI-PLC), but not phosphatidylcholine (PC)-phospholipase C (PC-PLC) nor PC-phospholipase D (PC-PLD), was required for LPS-induced NO production. Preincubation with PI-PLC selective inhibitors (U-73122 and ET-18-OCH 3) abrogated LPS-induced NO production in HD11 cells, whereas PC-PLC inhibitor (D609), phosphatide phosphohydrolase inhibitor (propranolol), and PC-PLD inhibitor ( n-butanol) had no inhibitory effects. We also showed that inhibition of protein kinase C (PKC) by selective inhibitors Ro 31-8220 and calphostin C and chelating intracellular Ca 2+ by BAPTA-AM significantly reduced NO production in LPS-stimulated HD11 cells. Our results demonstrate that PI-PLC plays a critical role, most likely through activation of PKC pathway, in TLR4 mediated immune responses of avian macrophage cells to LPS.