Protein Kinase C Inhibitor Chelerythrine Research Articles

Glutathione Peroxidase-1 Knockout Facilitates Memory Impairment Induced by β-Amyloid (1-42) in Mice via Inhibition of PKC βII-Mediated ERK Signaling; Application with Glutathione Peroxidase-1 Gene-Encoded Adenovirus Vector.

A growing body evidence suggests that selenium (Se) deficiency is associated with an increased risk of developing Alzheimer's disease (AD). Se-dependent glutathione peroxidase-1 (GPx-1) of a major antioxidant enzyme, and the most abundant isoform of GPx in the brain. In the present study, we investigated whether GPx-1 is protective against memory impairments induced by beta-amyloid (Aβ) (1-42) in mice. As the alteration of protein kinase C (PKC)-mediated ERK activation was recognized in the early stage of AD, we examined whether the GPx-1 gene modulates Aβ (1-42)-induced changes in PKC and ERK levels. We observed that Aβ (1-42) treatment (400pmol, i.c.v.) significantly decreased PKC βII expression in the hippocampus of mice. Aβ (1-42)-induced neurotoxic changes [i.e., oxidative stress (i.e., reactive oxygen species, 4-hydroxy-2-noneal, and protein carbonyl), reduced PKC βII and phospho-ERK expressions, and memory impairment under Y-maze and passive avoidance test] were more pronounced in GPx-1 knockout than in wild type mice. Importantly, exposure to a GPx-1 gene-encoded adenovirus vector (Adv-GPx-1) significantly increased GPx-1 mRNA and GPx activity in the hippocampus of GPx-1 knockout mice. Adv-GPx-1 exposure also significantly blocked the neurotoxic changes induced by Aβ (1-42) in GPx-1 knockout mice. Treatment with ERK inhibitor U0126 did not significantly change Adv-GPx-1-mediated attenuation in PKC βII expression. In contrast, treatment with PKC inhibitor chelerythrine (CHE) reversed Adv-GPx-1-mediated attenuation in ERK phosphorylation, suggesting that PKC βII-mediated ERK signaling is important for Adv-GPx-1-mediated potentials against Aβ (1-42) insult. Our results suggest that treatment with the antioxidant gene GPx-1 rescues Aβ (1-42)-induced memory impairment via activating PKC βII-mediated ERK signaling.

Guanosine prevents oxidative damage and glutamate uptake impairment induced by oxygen/glucose deprivation in cortical astrocyte cultures: involvement of A1 and A2A adenosine receptors and PI3K, MEK, and PKC pathways.

Glial cells are involved in multiple cerebral functions that profoundly influence brain tissue viability during ischemia, and astrocytes are the main source of extracellular purines as adenosine and guanosine. The endogenous guanine-based nucleoside guanosine is a neuromodulator implicated in important processes in the brain, such as modulation of glutamatergic transmission and protection against oxidative and inflammatory damage. We evaluated if the neuroprotective effect of guanosine is also observed in cultured cortical astrocytes subjected to oxygen/glucose deprivation (OGD) and reoxygenation. We also assessed the involvement of A1 and A2A adenosine receptors and phosphatidylinositol-3 kinase (PI3K), MAPK, and protein kinase C (PKC) signaling pathways on the guanosine effects. OGD/reoxygenation decreased cell viability and glutamate uptake and increased reactive oxygen species (ROS) production in cultured astrocytes. Guanosine treatment prevented these OGD-induced damaging effects. Dipropyl-cyclopentyl-xanthine (an adenosine A1 receptor antagonist) and 4-[2-[[6-amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride (an adenosine A2A receptor agonist) abolished guanosine-induced protective effects on ROS production, glutamate uptake, and cell viability. The PI3K pathway inhibitor 2-morpholin-4-yl-8-phenylchromen-4-one, the extracellular-signal regulated kinase kinase (MEK) inhibitor 2'-amino-3'-methoxyflavone, or the PKC inhibitor chelerythrine abolished the guanosine effect of preventing OGD-induced cells viability reduction. PI3K inhibition partially prevented the guanosine effect of reducing ROS production, whereas MEK and PKC inhibitions prevented the guanosine effect of restoring glutamate uptake. The total immunocontent of the main astrocytic glutamate transporter glutamate transporter-1 (GLT-1) was not altered by OGD and guanosine. However, MEK and PKC inhibitions also abolished the guanosine effect of increasing cell-surface expression of GLT-1 in astrocytes subjected to OGD. Then, guanosine prevents oxidative damage and stimulates astrocytic glutamate uptake during ischemic events via adenosine A1 and A2A receptors and modulation of survival signaling pathways, contributing to microenvironment homeostasis that culminates in neuroprotection.

Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts.

Hypertonicity increases water permeability, independently of vasopressin, in the inner medullary collecting duct (IMCD) by increasing aquaporin-2 (AQP2) membrane accumulation. We investigated whether protein kinase C (PKC) and adenosine monophosphate kinase (AMPK) are involved in hypertonicity-regulated water permeability. Increasing perfusate osmolality from 150 to 290 mosmol/kgH2O and bath osmolality from 290 to 430 mosmol/kgH2O significantly stimulated osmotic water permeability. The PKC inhibitors chelerythrine (10 µM) and rottlerin (50 µM) significantly reversed the increase in osmotic water permeability stimulated by hypertonicity in perfused rat terminal IMCDs. Chelerythrine significantly increased phosphorylation of AQP2 at S261 but not at S256. Previous studies show that AMPK is stimulated by osmotic stress. We tested AMPK phosphorylation under hypertonic conditions. Hypertonicity significantly increased AMPK phosphorylation in inner medullary tissues. Blockade of AMPK with Compound C decreased hypertonicity-stimulated water permeability but did not alter phosphorylation of AQP2 at S256 and S261. AICAR, an AMPK stimulator, caused a transient increase in osmotic water permeability and increased phosphorylation of AQP2 at S256. When inner medullary tissue was treated with the PKC activator phorbol dibutyrate (PDBu), the AMPK activator metformin, or both, AQP2 phosphorylation at S261 was decreased with PDBu or metformin alone, but there was no additive effect on phosphorylation with PDBu and metformin together. In conclusion, hypertonicity regulates water reabsorption by activating PKC. Hypertonicity-stimulated water reabsorption by PKC may be related to the decrease in endocytosis of AQP2. AMPK activation promotes water reabsorption, but the mechanism remains to be determined. PKC and AMPK do not appear to act synergistically to regulate water reabsorption.

Hepatitis B core antigen regulates dendritic cell proliferation and apoptosis through regulation of PKC/NF‑κB signaling pathway.

Hepatitis B core antigen (HBcAg) possesses unusual immunologic features. However, the biological roles and mechanisms of HBcAg in dendritic cell proliferation and apoptosis remain to be elucidated. In the present study, DC2.4 cells were treated with different concentrations of HBcAg (10, 20 and 30µg/ml). MTT assay and flow cytometry (AnnexinV/propidium iodide analysis) were performed to investigate changes in cell proliferation and apoptosis. Western blot analysis was conducted to examine the changes in nuclear factor (NF)‑κB and protein kinase C (PKC) signaling pathways. NF‑κB inhibitor pyrrolidine dithiocarbamate (PDTC) and PKC inhibitor Chelerythrine were used to block these two signaling pathways. It was identified that HBcAg increased proliferation and decreased apoptosis in a dose‑dependent manner. Western blotting results demonstrated that HBcAg upregulated p‑PKC, p‑IκB, p‑P65, tumor necrosis factor‑α and B‑cell lymphoma2 (Bcl‑2) levels, and downregulated cleaved caspase3, demonstrating that HBcAg activated the PKC and NF‑κB signaling pathways. NF‑κB inhibitor PDTC reduced the effects of HBcAg on DC2.4 proliferation (0.6 fold vs.0.25 fold) and apoptosis (0.43 fold vs. 0.17 fold), and on Bcl‑2 expression levels. PKC inhibitor Chelerythrine reduced the biological effects of HBcAg; it reduced proliferation (0.67 fold vs. 0.23 fold) and upregulated apoptosis (0.43 fold vs. 0.13 fold). Chelerythrine also blocked NF‑κB activity and the HBcAg‑induced Bcl‑2 increase, suggesting the effect on Bcl‑2 from HBcAg was dependent on the PKC/NF‑κB signaling pathway. In conclusion, HBcAg promoted proliferation and inhibited apoptosis through the PKC/NF‑κB/Bcl‑2 signaling pathway in DC2.4 cells.

Effects of lipopolysaccharide-induced septic shock on rat isolated kidney, possible role of nitric oxide and protein kinase C pathways.

Objective(s):Pathophysiology of sepsis-associated renal failure (one of the most common cause of death in intensive care units) had not been fully determined. The effect of nitric oxide and protein kinase C (PKC) pathways in isolated kidney of Lipopolysaccharide-treated (LPS) rats were investigated in this study. Materials and Methods:Vascular responsiveness to phenylephrine and acetylcholine in the presence and absence of a potent PKC inhibitor (chelerythrine) and nonspecific NO inhibitor (L-NAME) as well as responses to acetylcholine and sodium nitroprusside (SNP) were examined.Results:LPS (10 mg/kg, IP) treatment resulted in a lower systemic pressure and reduction of responses to vasoconstrictor and vasodilator agents (P<0.05 to P<0.01). The contractile response to phenylephrine and the relaxation response to acetylcholine were significantly blunted in isolated kidneys removed from LPS-treated rats. L-NAME (10 µM) preincubation modified the responses to acetylcholine in isolated kidneys of control animal (P<0.001) but not in LPS-treated rats. While, chelerythrine (10 µM) preincubation partially restored response to phenylephrine in LPS-treated tissues. Conclusion:Present study highlighted that five hours of intraperitoneal endotoxin injection is adequate to reduce renal basal perfusion pressure. These results also suggest that PKC inhibition may have a beneficial role in vascular hyporesponsiveness induced by LPS. Although our study partly elaborated on the effects of LPS on isolated renal vascular responses to vasoactive agents, further studies are required to explain how LPS exerts its renal vascular effects.

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.

Doxazosin Stimulates Galectin-3 Expression and Collagen Synthesis in HL-1 Cardiomyocytes Independent of Protein Kinase C Pathway.

Doxazosin, a drug commonly prescribed for hypertension and prostate disease, increases heart failure risk. However, the underlying mechanism remains unclear. Galectin-3 is an important mediator that plays a pathogenic role in cardiac hypertrophy and heart failure. In the present study, we investigated whether doxazosin could stimulate galectin-3 expression and collagen synthesis in cultured HL-1 cardiomyocytes. We found that doxazosin dose-dependently induced galectin-3 protein expression, with a statistically significant increase in expression with a dose as low as 0.01 μM. Doxazosin upregulated collagen I and α-smooth muscle actin (α-SMA) protein levels and also induced apoptotic protein caspase-3 in HL-1 cardiomyocytes. Although we previously reported that activation of protein kinase C (PKC) stimulates galectin-3 expression, blocking the PKC pathway with the PKC inhibitor chelerythrine did not prevent doxazosin-induced galectin-3 and collagen expression. Consistently, doxazosin treatment did not alter total and phosphorylated PKC. These results suggest that doxazosin-stimulated galectin-3 is independent of PKC pathway. To determine if the α1-adrenergic pathway is involved, we pretreated the cells with the irreversible α-adrenergic receptor blocker phenoxybenzamine and found that doxazosin-stimulated galectin-3 and collagen expression was similar to controls, suggesting that doxazosin acts independently of α1-adrenergic receptor blockade. Collectively, we show a novel effect of doxazosin on cardiomycytes by stimulating heart fibrosis factor galectin-3 expression. The mechanism of action of doxazosin is not mediated through either activation of the PKC pathway or antagonism of α1-adrenergic receptors.

Modulation of kidney urea transporter UT-A3 activity by alpha2,6-sialylation.

Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.

Sevoflurane Preconditioning Reduces Intestinal Ischemia-Reperfusion Injury: Role of Protein Kinase C and Mitochondrial ATP-Sensitive Potassium Channel

Ischemic preconditioning (IPC) has been considered to be a potential therapy to reduce ischemia-reperfusion injury (IRI) since the 1980s. Our previous study indicated that sevoflurane preconditioning (SPC) also reduced intestinal IRI in rats. However, whether the protective effect of SPC is similar to IPC and the mechanisms of SPC are unclear. Thus, we compared the efficacy of SPC and IPC against intestinal IRI and the role of protein kinase C (PKC) and mitochondrial ATP-sensitive potassium channel (mKATP) in SPC. A rat model of intestinal IRI was used in this study. The superior mesenteric artery (SMA) was clamped for 60 min followed by 120 min of reperfusion. Rats with IPC underwent three cycles of SMA occlusion for 5 min and reperfusion for 5 min before intestinal ischemia. Rats with SPC inhaled sevoflurane at 0.5 minimum alveolar concentration (MAC) for 30 min before the intestinal ischemic insult. Additionally, the PKC inhibitor Chelerythrine (CHE) or mKATP inhibitor 5-Hydroxydecanoic (5-HD) was injected intraperitoneally before sevoflurane inhalation. Both SPC and IPC ameliorated intestinal IRI-induced histopathological changes, decreased Chiu’s scores, reduced terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) positive cells in the epithelium, and inhibited the expression of malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α). These protective effects of SPC were similar to those of IPC. Pretreatment with PKC or mKATP inhibitor abolished SPC—induced protective effects by increasing Chiu’s scores, down-regulated the expression of Bcl-2 and activated caspase-3. Our results suggest that pretreatment with 0.5 MAC sevoflurane is as effective as IPC against intestinal IRI. The activation of PKC and mKATP may be involved in the protective mechanisms of SPC.

Editorial: Cell and molecular signaling, and transport pathways involved in growth factor control of synaptic development and function.

Since the discovery of nerve growth factor (NGF) more than a half century ago (Levi-Montalcini and Cohen, 1960), the prototypic neurotrophin family has included brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Neurotrophins bind to the Trk family of receptors, as well as the p75 receptor, to activate multiple intracellular signaling cascades (reviewed by Reichardt, 2006). BDNF receptor tropomyosin receptor kinase B (TrkB) signaling has been extensively studied for its roles in the central nervous system (CNS) ranging from cell survival, axonal and dendritic growth and synapse formation. The pathway mediates long-lasting activity-modulated synaptic changes on excitatory and inhibitory neurons and plays critical roles in circuit development and maintenance. In addition to BDNF, many studies have identified other “growth” or signaling factors in the CNS that play important roles in the development, maintenance, and control of synaptic and circuit function. However, details of the intracellular signaling systems downstream of these events are frequently unexplored. In this Research Topic, we have collected original studies and review articles that present cellular and molecular mechanisms concerning activity-dependent synapse formation and their implications for behavior and brain disorders.

Gabapentin inhibits the activity of the rat excitatory glutamate transporter 3 expressed in Xenopus oocytes

Gabapentin, a derivative of γ-aminobutyric acid (GABA), is used to treat epilepsy and neuropathic pain. The pharmacological mechanisms for gabapentin effects are not completely elucidated. We investigated the effect of gabapentin on the activity of excitatory amino acid transporter 3 (EAAT3) that can regulate extracellular glutamate concentrations. EAAT3 was expressed in Xenopus oocytes. Membrane currents were recorded after application of l-glutamate in the presence or absence of different concentrations of gabapentin (1–300μM) by using a two-electrode voltage clamp. To determine the effect of gabapentin on Vmax and Km of EAAT3 for l-glutamate, l-glutamate at 3–300μM was used. To study the effects of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) on gabapentin-induced changes in EAAT3 activity, oocytes were incubated with the PKC activator (Phorbol 12-myristate 13-acetate, PMA), the PKC inhibitors (chelerythrine or staurosporine), and the PI3K inhibitor wortmannin. Gabapentin decreased EAAT3 activity in a concentration-dependent manner and EAAT3 activity was significantly inhibited by 10–300μM gabapentin. Gabapentin significantly decreased Vmax without affecting Km. PMA increased EAAT3 activity; however, gabapentin attenuated the PMA-induced increase in EAAT3 activity. Pre-incubation of oocytes with chelerythrine, staurosporine, or wortmannin decreased basal EAAT3 activity, which was further reduced by gabapentin. We conclude that gabapentin decreases EAAT3 activity at clinically relevant and higher concentrations, in which PKC and PI3K may not be involved. The results suggest that EAAT3 might not be a target for the anticonvulsant action of gabapentin.

Downregulation of natriuretic peptide clearance receptor mRNA in vascular smooth muscle cells by angiotensin II.

Angiotensin II can downregulate atrial natriuretic peptide binding to rat vascular smooth muscle cells (VSMCs), but the mechanism is not known. Because protein kinase C (PKC) mimetic phorbol myristate acetate (PMA) can destabilize natriuretic peptide clearance receptor (NPR-C) mRNA and angiotensin II activates several PKC isoforms in VSMCs, we hypothesized that angiotensin II treatment decreases NPR-C mRNA stability and exerts this effect through PKC. This study demonstrated that angiotensin II induced time- and concentration-dependent downregulation of NPR-C, which was completely inhibited by an angiotensin II type I receptor blocker losartan. NPR-C mRNA disappearance rate over 6h was nearly doubled by exposure of VSMCs to 100nm angiotensin II, compared with that observed after inhibition of RNA synthesis alone. However, this response to angiotensin II was undiminished by the PKC inhibitor chelerythrine, or by depletion of PKC by prior exposure of cells to PMA for 48h. Inhibitors of tyrosine kinases, phospholipase C, or mitogen-activated protein kinase kinase also failed to reverse the angiotensin II effect. We conclude that at least two distinct proximal signaling pathways, one involved and one independent of phorbol ester-sensitive protein kinase C, lead to downregulation of NPR-C gene expression by destabilizing its mRNA.

Protein kinase C inhibition rescues manic-like behaviors and hippocampal cell proliferation deficits in the sleep deprivation model of mania.

Background:Recent studies revealed that bipolar disorder may be associated with deficits of neuroplasticity. Additionally, accumulating evidence has implicated alterations of the intracellular signaling molecule protein kinase C (PKC) in mania.Methods:Using sleep deprivation (SD) as an animal model of mania, this study aimed to examine the possible relationship between PKC and neuroplasticity in mania. Rats were subjected to SD for 72h and tested behaviorally. In parallel, SD-induced changes in hippocampal cell proliferation were evaluated with bromodeoxyuridine (BrdU) labeling. We then examined the effects of the mood stabilizer lithium, the antipsychotic agent aripiprazole, and the PKC inhibitors chelerythrine and tamoxifen on both behavioral and cell proliferation impairments induced by SD. The antidepressant fluoxetine was used as a negative control.Results:We found that SD triggered the manic-like behaviors such as hyperlocomotion and increased sleep latency, and reduced hippocampal cell proliferation. These alterations were counteracted by an acute administration of lithium and aripiprazole but not of fluoxetine, and only a single administration of aripiprazole increased cell proliferation on its own. Importantly, SD rats exhibited increased levels of phosphorylated synaptosomal-associated protein 25 (SNAP-25) in the hippocampus and prefrontal cortex, suggesting PKC overactivity. Moreover, PKC inhibitors attenuated manic-like behaviors and rescued cell proliferation deficits induced by SD.Conclusions:Our findings confirm the relevance of SD as a model of mania, and provide evidence that antimanic agents are also able to prevent SD-induced decrease of hippocampal cell proliferation. Furthermore, they emphasize the therapeutic potential of PKC inhibitors, as revealed by their antimanic-like and pro-proliferative properties.

Exercise preconditioning-induced late phase of cardioprotection against exhaustive exercise: possible role of protein kinase C delta.

The objective of this study was to investigate the late cardiac effect of exercise preconditioning (EP) on the exhaustive exercise-induced myocardial injury in rats and the role of protein kinase C (PKC) in EP. Rats were subjected to a run on the treadmill for four periods of 10 min each at 30 m/min with intervening periods of rest of 10 min as an EP protocol. The exhaustive exercise was performed 24 h after EP. PKC inhibitor chelerythrine (CHE) was injected before EP. The results showed that EP increased the running ability of rats, and alleviated the exhaustive exercise-induced injury in cardiomyocytes, but pretreatment with PKC inhibitor CHE did not abolish the late phase cardioprotection of EP. A significant increase of PKCδ, both at the protein level and the mRNA level in the left ventricular myocardium of rats, accompanied by its activated form (phosphorylated on Thr507, p-PKCδThr507) translocated to intercalated disks and was found in the late phase of EP. This circumstance was not attenuated by CHE. These results suggested that a high level of PKCδ might be involved in cardioprotection against myocardial damage, but if activated PKCδ at reperfusion took on a key role in cardioprotection was still an outstanding question.

Dexmedetomidine increases the activity of excitatory amino acid transporter type 3 expressed in Xenopus oocytes: The involvement of protein kinase C and phosphatidylinositol 3-kinase

Dexmedetomidine, an α2 adrenergic agonist, has neuroprotective and anticonvulsant properties in addition to its sedative and anxiolytic effects. We hypothesized that dexmedetomidine would increase the activity of excitatory amino acid transporter type 3 (EAAT3) and that this effect would involve protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), two protein kinases known to regulate EAAT3 activity. EAAT3 was expressed in Xenopus oocytes by injecting its mRNA. Two-electrode voltage clamping was used to record membrane currents before, during, and after application of 30μM l-glutamate in the presence of 0.1–30nM dexmedetomidine. Dexmedetomidine-treated oocytes were also exposed to a PKC activator (phorbol-12-myristate-13-acetate [PMA]), PKC inhibitors (chelerythrine, staurosporine, and calphostin C), and PI3K inhibitors (wortmannin and LY294002) before current measurement. Dexmedetomidine application resulted in a concentration-dependent increase in the EAAT3 activity in response to l-glutamate. The kinetic study showed that dexmedetomidine significantly increased the Vmax without changing Km. Treatment of oocytes with PMA significantly increased transporter currents compared with controls, but treatment with dexmedetomidine plus PMA did not further increase the response compared with PMA or dexmedetomidine alone. In addition, pre-treatment of oocytes with PKC inhibitors and PI3K inhibitors significantly abolished the dexmedetomidine-enhanced EAAT3 activity. These results suggest that dexmedetomidine increases the activity of EAAT3 expressed in Xenopus oocytes. PKC and PI3K seem to mediate this effect. These findings may explain the neuroprotective and anticonvulsant effects of dexmedetomidine.

Ondansetron attenuates the activity of excitatory amino acid transporter type 3 expressed in Xenopus oocytes

The purpose of this study was to evaluate the effect of ondansetron on excitatory amino acid transporter type 3 (EAAT3) and to elucidate the roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in the effect. EAAT3 was expressed in Xenopus oocytes following the injection of rat EAAT3 mRNAs. Using the two-electrode voltage clamping method, the inward currents induced by l-glutamate were measured for 1min in the presence and absence of ondansetron (1–1000μM). Different concentrations of l-glutamate (3–300μM) were used to determine the kinetic characteristics of EAAT3. To identify the involvement of PKC and PI3K in the effect, oocytes were exposed to a PKC activator and to PKC inhibitors and PI3K inhibitors, and l-glutamate-induced currents were recorded. Ondansetron decreased EAAT3 activity in a dose-dependent manner. In a kinetic study, ondansetron (10μM for 3min) reduced Vmax, but not Km compared with the control group. The PKC activator abolished the ondansetron-induced decrease in EAAT3 activity. The PKC inhibitors (staurosporine and chelerythrine) and ondansetron had not additive or synergistic effects on EAAT3 activity. The PI3K inhibitors (wortmannin and LY294002) decreased the EAAT3 response, although there were no differences among the groups comprising ondansetron, PI3K inhibitors, and PI3K inhibitors plus ondansetron. Our results demonstrate that ondansetron attenuates EAAT3 activity and this effect seems to be mediated by PKC and PI3K.

Role of protein kinase C in reduction of mitochondrial injury during myocardial ischemia-reperfusion by ischemic preconditioning in rats

Objective To evaluate the role of protein kinase C （PKC ） in reduction of mitochondrial injury during myocardial ischemia-reperfusion （I/R） by ischemic preconditioning in rats .Methods Forty male Sprague-Dawley rats ,aged 12-13 weeks ,weighing 280-320 g ,were randomly divided into 4 groups （ n=10 each） using a random number table：sham operation group （S group） ,I/R group ,ischemic preconditioning group （IP group） and PKC inhibitor chelerythrine group （C group） .Myocardial I/R was produced by 35 min occlusion of left anterior descending branch of coronary artery followed by 120 min reperfusion .Ischemic preconditioning was induced by 3 episodes of 5 min occlusion of left anterior descending branch at 5 min intervals before myocardial ischemia . Chelerythrine 1 mg/kg was injected intravenously via the caudal vein before ischemic preconditioning in group C . At 120 min of reperfusion ,the animals were sacrificed and the hearts were immediately removed .Mitochondrial suspension was prepared for determination of activities of succinate dehydrogenase （SDH ） , xanthine oxidase （XOD ） , glutathione peroxidase （GSH-Px ） and Ca2＋-ATPase , content of Ca2＋ , myocardial mitochonerial permeability transition pore （mPTP） opening and membrane potential （Δψm ） .Results Compared with S group , the activities of XOD and Ca2＋-ATPase ,content of Ca2＋ and mPTP opening were significantly increased ,and the＆nbsp;activities of SDH and GSH-Px and Δψm were decreased in I/R group （ P〈0.05） .Compared with I/R group ,the activities of XOD and Ca2＋-ATPase , content of Ca2＋ and mPTP opening were significantly decreased , and the activities of SDH and GSH-Px and Δψm were increased in IP group （ P〈0.05） .Compared with IP group ,the activities of XOD and Ca2＋-ATPase , content of Ca2＋ and mPTP opening were significantly increased , and the activities of SDH and GSH-Px and Δψm were decreased in C group （ P〈0.05） .Conclusion PKC is involved in reduction of mitochondrial injury during myocardial I/R by ischemic preconditioning in rats . Key words: Protein kinase C; Ischemic preconditioning; Myocardial reperfusion injury; Mitochondria

Dexmedetomidine-induced contraction involves phosphorylation of caldesmon by JNK in endothelium-denuded rat aortas.

Caldesmon, an inhibitory actin binding protein, binds to actin and inhibits actin-myosin interactions, whereas caldesmon phosphorylation reverses the inhibitory effect of caldesmon on actin-myosin interactions, potentially leading to enhanced contraction. The goal of this study was to investigate the cellular signaling pathway responsible for caldesmon phosphorylation, which is involved in the regulation of the contraction induced by dexmedetomidine (DMT), an alpha-2 adrenoceptor agonist, in endothelium-denuded rat aortas. SP600125 (a c-Jun NH2-terminal kinase [JNK] inhibitor) dose-response curves were generated in aortas that were pre-contracted with DMT or phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Dose-response curves to the PKC inhibitor chelerythrine were generated in rat aortas pre-contracted with DMT. The effects of SP600125 and rauwolscine (an alpha-2 adrenoceptor inhibitor) on DMT-induced caldesmon phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) were investigated by western blot analysis. PDBu-induced caldesmon and DMT-induced PKC phosphorylation in rat aortic VSMCs was investigated by western blot analysis. The effects of GF109203X (a PKC inhibitor) on DMT- or PDBu-induced JNK phosphorylation in VSMCs were assessed. SP600125 resulted in the relaxation of aortas that were pre-contracted with DMT or PDBu, whereas rauwolscine attenuated DMT-induced contraction. Chelerythrine resulted in the vasodilation of aortas pre-contracted with DMT. SP600125 and rauwolscine inhibited DMT-induced caldesmon phosphorylation. Additionally, PDBu induced caldesmon phosphorylation, and GF109203X attenuated the JNK phosphorylation induced by DMT or PDBu. DMT induced PKC phosphorylation in rat aortic VSMCs. These results suggest that alpha-2 adrenoceptor-mediated, DMT-induced contraction involves caldesmon phosphorylation that is mediated by JNK phosphorylation by PKC.

Nicotine decreases the activity of glutamate transporter type 3

Nicotine, the main ingredient of tobacco, elicits seizures in animal models and cigarette smoking is regarded as a behavioral risk factor associated with epilepsy or seizures. In the hippocampus, the origin of nicotine-induced seizures, most glutamate uptake could be performed primarily by excitatory amino acid transporter type 3 (EAAT3). An association between temporal lobe epilepsy and EAAT3 downregulation has been reported. Therefore, we hypothesized that nicotine may elicit seizures through the attenuation of EAAT3 activity. We investigated chronic nicotine exposure (72h) cause reduction of the activity of EAAT3 in a Xenopus oocyte expression system using a two-electrode voltage clamp. The roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) were also determined. Nicotine (0.001–1μM) resulted in a time- and dose-dependent decrease in EAAT3 activity with maximal inhibition at nicotine concentrations of 0.03μM or higher and at an exposure time of 72h. Vmax on the glutamate response was significantly reduced in the nicotine group (0.03μM for 72h), but the Km value of EAAT3 for glutamate was not altered. When nicotine-exposed oocytes (0.03μM for 72h) were pretreated with phorbol-12-myristate-13-acetate (PMA, a PKC activator), the nicotine-induced reduction in EAAT3 activity was abolished. PKC inhibitors (staurosporine, chelerythrine, and calphostin C) significantly reduced basal EAAT3 activity, but there were no significant differences among the PKC inhibitors, nicotine, and PKC inhibitors+nicotine groups. Similar response patterns were observed among PI3K inhibitors (wortmannin and LY294002), nicotine, and PI3K inhibitors+nicotine. In conclusion, this study suggests that nicotine decreases EAAT3 activity, and that this inhibition seems to be dependent on PKC and PI3K. Our results may provide an additional mechanism for nicotine-induced seizure.

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.