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Hyperglycemia is one of the main damaging factors of diabetes mellitus (DM). The severity of this disease is most clearly manifested under conditions of the inflammatory process. In this work, we have studied the activation features of rat peritoneal macrophages (MPs) under conditions of high glucose concentration in vitro. Comparison of the independent and combined effects of streptozotocin-induced DM and hyperglycemia on proliferation and accumulation of nitrites in the MPs culture medium revealed similarity of their effects. Elevated glucose levels and, to a lesser extent, DM decreased basal proliferation and NO production by MPs in vitro. The use of the protein kinase C (PKC) activator, phorbol ester (PMA), abolished the proinflammatory effect of thrombin on PMs. This suggests the involvement of PKC in the effects of the protease. At the same time, the effect of thrombin on the level of nitrites in the culture medium demonstrates a pronounced dose-dependence, which was not recognized during evaluation of proliferation. Proinflammatory activation of MPs is potentiated by hyperglycemia, one of the main pathological factors of diabetes. Despite the fact that high concentrations of glucose have a significant effect on proliferation and NO production, no statistically significant differences were found between the responses of MPs obtained from healthy animals and from animals with streptozotocin-induced DM. This ratio was observed for all parameters studied in the work, during analysis of cell proliferation and measurement of nitrites in the culture medium. Thus, the results obtained indicate the leading role of elevated glucose levels in the regulation of MPs activation, which is comparable to the effect of DM and even "masks" it.

Purpose: To observe the effects of electroacupuncture on ocular surface neuralgia and the P2X 3 R-PKC signaling pathway in guinea pigs with dry eye., Methods: A dry eye guinea pig model was established by subcutaneous injection of scopolamine hydrobromide. Guinea pigs were monitored for body weight, palpebral fissure height, number of blinks, corneal fluorescein staining score, phenol red thread test, and corneal mechanical perception threshold. Histopathological changes and mRNA expression of P2X 3 R and protein kinase C in the trigeminal ganglion and spinal trigeminal nucleus caudalis were observed. We performed a second part of the experiment, which involved the P2X 3 R-specific antagonist A317491 and the P2X 3 R agonist ATP in dry-eyed guinea pigs to further validate the involvement of the P2X 3 R-protein kinase C signaling pathway in the regulation of ocular surface neuralgia in dry eye. The number of blinks and corneal mechanical perception threshold were monitored before and 5 min after subconjunctival injection and the protein expression of P2X 3 R and protein kinase C was detected in the trigeminal ganglion and spinal trigeminal nucleus caudalis of guinea pigs., Results: Dry-eyed guinea pigs showed pain-related manifestations and the expression of P2X 3 R and protein kinase C in the trigeminal ganglion and spinal trigeminal nucleus caudalis was upregulated. Electroacupuncture reduced pain-related manifestations and inhibited the expression of P2X 3 R and protein kinase C in the trigeminal ganglion and spinal trigeminal nucleus caudalis. Subconjunctival injection of A317491 attenuated corneal mechanoreceptive nociceptive sensitization in dry-eyed guinea pigs, while ATP blocked the analgesic effect of electroacupuncture., Conclusions: Electroacupuncture reduced ocular surface sensory neuralgia in dry-eyed guinea pigs, and the mechanism of action may be associated with the inhibition of the P2X 3 R-protein kinase C signaling pathway in the trigeminal ganglion and spinal trigeminal nucleus caudalis by electroacupuncture.

Purpose: β-Adrenergic receptors (βAR) are essential targets for the treatment of heart failure (HF); however, chronic use of βAR agonists as positive inotropes to increase contractility in a G s protein-dependent manner is associated with increased mortality. Alternatively, we previously reported that allosteric modulation of β2AR with the pepducin intracellular loop (ICL)1-9 increased cardiomyocyte contractility in a β-arrestin (βarr)-dependent manner, and subsequently showed that ICL1-9 activates the Ras homolog family member A (RhoA). Here, we aimed to elucidate both the proximal and downstream signaling mediators involved in the promotion of cardiomyocyte contractility in response to ICL1-9., Methods: We measured adult mouse cardiomyocyte contractility in response to ICL1-9 or isoproterenol (ISO, as a positive control) alone or in the presence of inhibitors of various potential components of βarr- or RhoA-dependent signaling. We also assessed the contractile effects of ICL1-9 on cardiomyocytes lacking G protein-coupled receptor (GPCR) kinase 2 (GRK2) or 5 (GRK5)., Results: Consistent with RhoA activation by ICL1-9, both Rho-associated protein kinase (ROCK) and protein kinase D (PKD) inhibition were able to attenuate ICL1-9-mediated contractility, as was inhibition of myosin light chain kinase (MLCK). While neither GRK2 nor GRK5 deletion impacted ICL1-9-mediated contractility, pertussis toxin attenuated the response, suggesting that ICL1-9 promotes downstream RhoA-dependent signaling in a G i protein-dependent manner., Conclusion: Altogether, our study highlights a novel signaling modality that may offer a new approach to the promotion, or preservation, of cardiac contractility during HF via the allosteric regulation of β2AR to promote G i protein/βarr-dependent activation of RhoA/ROCK/PKD signaling., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The precise underlying mechanism of antioxidant effects of dexmedetomidine-induced neuroprotection against cerebral ischemia has not yet been fully elucidated. Activation of Nuclear factor erythroid 2-related factor (Nrf2) and Heme Oxygenase-1 (HO-1) represents a major antioxidant-defense mechanism. Therefore, we determined whether dexmedetomidine increases Nrf2/HO-1 expression after global transient cerebral ischemia and assessed the involvement of Protein Kinase C (PKC) in the dexmedetomidine-related antioxidant mechanism., Methods: Thirty-eight rats were randomly assigned to five groups: sham (n...=...6), ischemic (n...=...8), chelerythrine (a PKC inhibitor; 5...mg.kg -1 IV administered 30...min before cerebral ischemia) (n...=...8), dexmedetomidine (100.....g.kg -1 IP administered 30...min before cerebral ischemia (n...=...8), and dexmedetomidine...+...chelerythrine (n...=...8). Global transient cerebral ischemia (10...min) was applied in all groups, except the sham group; histopathologic changes and levels of nuclear Nrf2 and cytoplasmic HO-1 were examined 24...hours after ischemia insult., Results: We found fewer necrotic and apoptotic cells in the dexmedetomidine group relative to the ischemic group (p...<...0.01) and significantly higher Nrf2 and HO-1 levels in the dexmedetomidine group than in the ischemic group (p...<...0.01). Additionally, chelerythrine co-administration with dexmedetomidine attenuated the dexmedetomidine-induced increases in Nrf2 and HO-1 levels (p...<...0.05 and p...<...0.01, respectively) and diminished its beneficial neuroprotective effects., Conclusion: Preischemic dexmedetomidine administration elicited neuroprotection against global transient cerebral ischemia in rats by increasing Nrf2/HO-1 expression partly via PKC signaling, suggesting that this is the antioxidant mechanism underlying dexmedetomidine-mediated neuroprotection., (Copyright © 2021 Sociedade Brasileira de Anestesiologia. Published by Elsevier España, S.L.U. All rights reserved.)

Infection by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has posed a severe threat to global public health. The current study revealed that several inhibitors of protein kinases C (PKCs) possess protective activity against SARS-CoV-2 infection. Four pan-PKC inhibitors, Go 6983, bisindolylmaleimide I, enzastaurin, and sotrastaurin, reduced the replication of a SARS-CoV-2 replicon in both BHK-21 and Huh7 cells. A PKCδ-specific inhibitor, rottlerin, was also effective in reducing viral infection. The PKC inhibitors acted at an early step of SARS-CoV-2 infection. Finally, PKC inhibitors blocked the replication of wild-type SARS-CoV-2 in ACE2-expressing A549 cells. Our work highlights the importance of the PKC signaling pathway in infection by SARS-CoV-2 and provides evidence that PKC-specific inhibitors are potential therapeutic agents against SARS-CoV-2. IMPORTANCE There is an urgent need for effective therapeutic drugs to control the pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We found that several inhibitors of protein kinases C (PKCs) dramatically decrease the replication of SARS-CoV-2 in cultured cells. These PKC inhibitors interfere with an early step of viral infection. Therefore, the rapid and prominent antiviral effect of PKC inhibitors underscores that they are promising antiviral agents and suggests that PKCs are important host factors involved in infection by SARS-CoV-2.

Background: Elabela is a recently discovered peptide hormone. The present study aims to investigate the vasorelaxant effect mechanisms of elabela in the rat thoracic aorta., Methods: The vascular rings obtained from the thoracic aortas of the male Wistar albino rats were placed in the isolated tissue bath system. Resting tension was set to 1 gram. After the equilibration period, the vessel rings were contracted with phenylephrine or potassium chloride. Once a stable contraction was achieved, elabela-32 was applied cumulatively (10 -9 -10 -6  molar) to the vascular rings. The experimental protocol was repeated in the presence of specific signaling pathway inhibitors or potassium channel blockers to determine the effect mechanisms of elabela., Results: Elabela showed a significant vasorelaxant effect in a concentration-dependent manner (P < 0.001). The vasorelaxant effect level of elabela was significantly reduced by the apelin receptor antagonist F13A, cyclooxygenase inhibitor indomethacin, adenosine monophosphate-activated protein kinase inhibitor dorsomorphin, protein kinase C inhibitor bisindolmaleimide, large-conductance calcium-activated potassium channel blocker iberiotoxin, and intermediate-conductance calcium-activated potassium channel blocker TRAM-34 (P < 0.001). However, the vasorelaxant effect level of elabela was not significantly affected by the endothelial nitric oxide synthase inhibitor nitro-L-arginine methyl ester and mitogen-activated protein kinase inhibitor U0126., Conclusions: Elabela exhibits a prominent vasodilator effect in rat thoracic aorta. Apelin receptor, prostanoids, adenosine monophosphate-activated protein kinase, protein kinase C, and calcium-activated potassium channels are involved in the vasorelaxant effect mechanisms of elabela., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Irisin, a newly identified exercise-derived myokine, has been found involved in a peripheral vasodilator effect. However, little is known regarding the potential vascular activity of irisin, and the mechanisms underlying its effects on vascular smooth muscle have not been fully elucidated. This study was aimed to investigate the effects of irisin on vascular smooth muscle contractility in rat thoracic aorta, and the hypothesis that protein kinase C (PKC) may have a role in these effects., Methods: Isometric contraction-relaxation responses of thoracic aorta rings were measured with an isolated organ bath model. The steady contraction was induced with 10 µM phenylephrine (PHE), and then the concentration-dependent responses of irisin (0.001-1 µM) were examined. The time-matched vehicle control (double distilled water) group was also formed. To evaluate the role of PKC, endothelium-intact thoracic aorta rings were incubated with 150 nM bisindolylmaleimide I (BIM I) for 20 min before the addition of 10 µM PHE and irisin. Also, a vehicle control group was formed for dimethyl sulfoxide (DMSO)., Results: Irisin exerted the vasorelaxant effects at concentrations of 0.01, 0.1, and 1 µM compared to the control group (p < 0.001). Besides, PKC inhibitor BIM I incubation significantly inhibited the relaxation responses induced by varying concentrations of irisin (p: 0.000 for 0.01 µM; p: 0.000 for 0.1 µM; p: 0.000 for 1 µM). However, DMSO, a solvent of BIM I, did not modulate the relaxant effects of irisin (p > 0.05)., Discussion: In conclusion, physiological findings were obtained regarding the functional relaxing effects of irisin in rat thoracic aorta. The findings demonstrated that irisin induces relaxation responses in endothelium-intact thoracic aorta rings in a concentration-dependent manner. Furthermore, this study is the first to report that irisin-induced relaxation responses are regulated probably via activating signaling pathways implicating PKC.

Introduction: The long-term inhibition of bone resorption suppresses new bone formation because these processes are coupled during physiological bone remodeling. The development of anti-bone-resorbing agents that do not suppress bone formation is urgently needed. We previously demonstrated that Wnt5a-Ror2 signaling in mature osteoclasts promoted bone-resorbing activity through protein kinase N3 (Pkn3). The p38 MAPK inhibitor SB202190 reportedly inhibited Pkn3 with a low Ki value (0.004 μM). We herein examined the effects of SB202190 on osteoclast differentiation and function in vitro and in vivo., Materials and Methods: Bone marrow cells were cultured in the presence of M-csf and GST-Rankl to differentiate into multinucleated osteoclasts. Osteoclasts were treated with increasing concentrations of SB202190. For in vivo study, 10-week-old female mice were subjected to ovariectomy (OVX). OVX mice were intraperitoneally administered with a Pkn3 inhibitor at 2 mg/kg or vehicle for 4 weeks, and bone mass was analyzed by micro-CT., Results: SB202190 suppressed the auto-phosphorylation of Pkn3 in osteoclast cultures. SB202190 significantly inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation. SB202190 reduced c-Src activity in osteoclast cultures without affecting the interaction between Pkn3 and c-Src. A treatment with SB202190 attenuated OVX-induced bone loss without affecting the number of osteoclasts or bone formation by osteoblasts., Conclusions: Our results showed that Pkn3 has potential as a therapeutic target for bone loss due to increased bone resorption. SB202190 is promising as a lead compound for the development of novel anti-bone-resorbing agents., (© 2021. The Japanese Society Bone and Mineral Research.)

HIV latency prevents cure of infection with antiretroviral therapy (ART) alone. One strategy for eliminating latently infected cells involves the induction of viral protein expression via latency-reversing agents (LRAs), allowing killing of host cells by viral cytopathic effects or immune effector mechanisms. Here, we combine a barcoded HIV approach and a humanized mouse model to study the effects of a designed, synthetic protein kinase C modulating LRA on HIV rebound. We show that administration of this compound during ART results in a delay in rebound once ART is stopped. Furthermore, the rebounding virus appears composed of a smaller number of unique barcoded viruses than occurs in control-treated animals, suggesting that some reservoir cells that would have contributed virus to the rebound process are eliminated by LRA administration. These data support the use of barcoded virus to study rebound and suggest that LRAs may be useful in HIV cure efforts., Competing Interests: Stanford University has filed patent applications on SUW133 and related technology, which has been licensed by Neurotrope BioScience for the treatment of neurological disorders and by BryoLogx for use in HIV/AIDS eradication and cancer immunotherapy. P.A.W. is an adviser to both companies and a cofounder of the latter. J.A.Z. is on the scientific advisory board for BryoLogx and is a co-founder of CDR3 Therapeutics., (© 2020 The Author(s).)

This study investigated the acute in vitro effect of low-concentration bisphenol A (BPA) on calcium ( 45 Ca 2+ ) influx in zebrafish (Danio rerio) testis and examined whether intracellular Ca 2+ was involved in the effects of BPA on testicular toxicity. In vitro studies on 45 Ca 2+ influx were performed in the testes after incubation with BPA for 30 min. Inhibitors were added 15 min before the addition of 45 Ca 2+ and BPA to testes to study the mechanism of action of BPA. The involvement of intracellular calcium from stores on lactate dehydrogenase (LDH) release and on triacylglycerol (TAG) content were carried out after in vitro incubation of testes with BPA for 1 h. Furthermore, gamma-glutamyl transpeptidase (GGT) and aspartate aminotransferase (AST) activities were analyzed in the liver at 1 h after in vitro BPA incubation of D. rerio. Our data show that the acute in vitro treatment of D. rerio testes with BPA at very low concentration activates plasma membrane ionic channels, such as voltage-dependent calcium channels and calcium-dependent chloride channels, and protein kinase C (PKC), which stimulates Ca 2+ influx. In addition, BPA increased cytosolic Ca 2+ by activating inositol triphosphate receptor (IP 3 R) and inhibiting sarco/endoplasmic reticulum calcium ATPase (SERCA) at the endoplasmic reticulum, contributing to intracellular Ca 2+ overload. The protein kinases, PKC, MEK 1/2 and PI3K, are involved in the mechanism of action of BPA, which may indicate a crosstalk between the non-genomic initiation effects mediated by PLC/PKC/IP 3 R signaling and genomic responses of BPA mediated by the estrogen receptor (ESR). In vitro exposure to a higher concentration of BPA caused cell damage and plasma membrane injury with increased LDH release and TAG content; both effects were dependent on intracellular Ca 2+ and mediated by IP 3 R. Furthermore, BPA potentially induced liver damage, as demonstrated by increased GGT activity. In conclusion, in vitro effect of BPA in a low concentration triggers cytosolic Ca 2+ overload and activates downstream protein kinases pointing to a crosstalk between its non-genomic and genomic effects of BPA mediated by ESR. Moreover, in vitro exposure to a higher concentration of BPA caused intracellular Ca 2+ -dependent testicular cell damage and plasma membrane injury. This acute toxicity was reinforced by increased testicular LDH release and GGT activity in the liver., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Traumatic brain injury (TBI) is a common cause of death and disability. Enhancing the midline-crossing of the contralateral corticospinal tract (CST) to the denervated side of spinal cord facilitates functional recovery after TBI. Activation of the gamma isoform of PKC (PKCγ) in contralateral CST implicates its roles in promoting CST remodeling after TBI. In this study, we deployed loss and gain of function strategies in N2a cells and primary cortical neurons in vitro, and demonstrated that PKCγ is not only important but necessary for neuronal differentiation, neurite outgrowth and axonal branching but not for axonal extension. Mechanically, through the phosphorylation of GSK3β, PKCγ stabilizes the expression of cytosolic β-catenin and increase GAP43 expression, thus promoting axonal outgrowth. Further, rAAV2/9-mediated delivery of constitutive PKCγ in the corticospinal tract after unilateral TBI in vivo additionally showed that specifically delivery of active PKCγ mutant to cortical neuron promotes midline crossing of corticospinal fibers from the uninjured side to the denervated cervical spinal cord. This PKCγ-mediated injury response promoted sensorimotor functional recovery. In conclusion, PKCγ mediates stability of β-catenin through the phosphorylation of GSK3β to facilitate neuronal differentiation, neurite outgrowth and axonal branching, and PKCγ maybe a novel therapeutic target for physiological and functional recovery after TBI.

Purpose: To explore the role of synapsin-Ia/b in visual cortical plasticity, the dynamic changes in total protein expression (T-) and conventional protein kinase C (cPKC)γ-modulated phosphorylation (P-) levels of synapsin-Ia/b were observed in the developing visual cortex of mice., Methods: The Western blot analysis was used to determine the levels of T- and P-synapsin-Ia/b at site of Ser9, 549, and 603; the cPKCγ gene wild-type (cPKCγ+/+) and knockout (cPKCγ-/-) mice were applied to explore the modulation of cPKCγ on synapsin-Ia/b phosphorylation status in visual cortex of mice at postnatal 7 to 60 days (P7-P60, n = 6 per group)., Results: The results showed that T-synapsin-Ia/b protein levels significantly increased at P14 to P35 and peaked at P42 to 60 (P < 0.001) in visual cortex when compared with that of P7 cPKCγ+/+ mice, and cPKCγ-/- did not affect this pattern of T-synapsin-Ia/b protein expressions. For synapsin-Ia/b phosphorylation status, the levels of P-Ser9 and 603 synapsin-Ia/b significantly elevated at P21 to P28 (P < 0.05 or 0.001), and then went down and maintained at lower levels at P35 to P60 (P < 0.05 or 0.001) compared with P7 cPKCγ+/+ mice. In addition, the cPKCγ gene knockout could significantly (P < 0.001) inhibit both the increase and decrease of P-Ser9 and 603 synapsin-Ia/b levels when compared with cPKCγ+/+ mice at P7 to P60. However, there were no significant changes of P-Ser549 synapsin-Ia/b in the developing visual cortex of both cPKCγ+/+ and cPKCγ-/- mice at P7 to P60., Conclusions: These results suggested that both protein expression levels and cPKCγ-modulated phosphorylation status at Ser9 and 603 of synapsin-Ia/b may play important role in developing visual cortex of mice.

Background: Osteoarthritis (OA) can be defined as degradation of articular cartilage of the joint, and is the most common degenerative disease. To regenerate the damaged cartilage, different experimental approaches including stem cell therapy have been tried. One of the major limitations of stem cell therapy is the poor post-transplantation survival of the stem cells. Anoikis, where insufficient matrix support and adhesion to extracellular matrix causes apoptotic cell death, is one of the main causes of the low post-transplantation survival rate of stem cells. Therefore, enhancing the initial interaction of the transplanted stem cells with chondrocytes could improve the therapeutic efficacy of stem cell therapy for OA. Previously, protein kinase C activator phorbol 12-myristate 13-acetate (PMA)-induced increase of mesenchymal stem cell adhesion via activation of focal adhesion kinase (FAK) has been reported. In the present study, we examine the effect PMA on the adipose-derived stem cells (ADSCs) adhesion and spreading to culture substrates, and further on the initial interaction between ADSC and chondrocytes., Results: PMA treatment increased the initial adhesion of ADSC to culture substrate and cellular spreading with increased expression of adhesion molecules, such as FAK, vinculin, talin, and paxillin, at both RNA and protein level. Priming of ADSC with PMA increased the number of ADSCs attached to confluent layer of cultured chondrocytes compared to that of untreated ADSCs at early time point (4 h after seeding)., Conclusion: Taken together, the results of this study suggest that priming ADSCs with PMA can increase the initial interaction with chondrocytes, and this proof of concept can be used to develop a non-invasive therapeutic approach for treating OA. It may also accelerate the regeneration process so that it can relieve the accompanied pain faster in OA patients. Further in vivo studies examining the therapeutic effect of PMA pretreatment of ADSCs for articular cartilage damage are required.

Unlabelled: Protein kinase M ζ (PKMζ), an atypical isoform of protein kinase C, has been suggested to be necessary and sufficient for the maintenance of long-term potentiation (LTP) and long-term memory (LTM). This evidence is heavily based on the use of ζ inhibitory peptide (ZIP), a supposed specific inhibitor of PKMζ that interferes with both LTP and LTM. Problematically, both LTP and LTM are unaffected in both constitutive and conditional PKMζ knock-out mice, yet both are still impaired by ZIP application, suggesting a nonspecific mechanism of action. Because translational interference can disrupt neural activity, we assessed network activity after a unilateral intrahippocampal infusion of ZIP in anesthetized rats. ZIP profoundly reduced spontaneous hippocampal local field potentials, comparable in magnitude to infusions of lidocaine, but with a slower onset and longer duration. Our results highlight a serious confound in interpreting the behavioral effects of ZIP. We suggest that future molecular approaches in neuroscience consider the intervening level of cellular and systems neurophysiology before claiming influences on behavior., Significance Statement: Long-term memory in the brain is thought to arise from a sustained molecular process that can maintain changes in synaptic plasticity. A so-called candidate for the title of "the memory molecule" is protein kinase M ζ (PKMζ), mainly because its inhibition by ζ inhibitory peptide (ZIP) interferes with previously established synaptic plasticity and memory. We show that brain applications of ZIP that can impair memory actually profoundly suppress spontaneous brain activity directly or can cause abnormal seizure activity. We suggest that normal brain activity occurring after learning may be a more primary element of memory permanence., (Copyright © 2016 the authors 0270-6474/16/366193-06$15.00/0.)

Results: The lysophosphatidic acid receptors LPA1, LPA2, and LPA3 were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA1- and LPA3-mediated effect, whereas that mediated by LPA2 was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA2 receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA1-3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA1-3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA2 subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes., Conclusion: Our data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes.

Evidence increasingly shows that astrocytes play a pivotal role in brain physiology and pathology via calcium dependent processes, thus the characterization of the calcium dynamics in astrocytes is of growing importance. We have previously reported that the epidermal growth factor and basic fibroblast growth factor up-regulate the oscillation of the calcium releases that are induced by stimuli, including glutamate in cultured astrocytes. This calcium oscillation is assumed to involve protein kinase C (PKC), which is activated together with the calcium releases as a consequence of inositol phospholipid hydrolysis. In the present study, this issue has been investigated pharmacologically by using astrocytes cultured with and without the growth factors. The pharmacological activation of PKC largely reduced the glutamate-induced oscillatory and non-oscillatory calcium increases. Meanwhile, PKC inhibitors increased the total amounts of both calcium increases without affecting the peak amplitudes and converted the calcium oscillations to non-oscillatory sustained calcium increases by abolishing the falling phases of the repetitive calcium increases. Furthermore, the pharmacological effects were consistent between both glutamate- and histamine-induced calcium oscillations. These results suggest that PKC up-regulates the removal of cytosolic calcium in astrocytes, and this up-regulation is essential for calcium oscillation in astrocytes cultured with growth factors., (Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

This study aimed to determine the role of mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channels and protein kinase C (PKC)-ε in the delayed protective effects of sevoflurane preconditioning using Langendorff isolated heart perfusion models. Fifty-four isolated perfused rat hearts were randomly divided into 6 groups (n=9). The rats were exposed for 60 min to 2.5% sevoflurane (the second window of protection group, SWOP group) or 33% oxygen inhalation (I/R group) 24 h before coronary occlusion. The control group (CON) and the sevoflurane group (SEVO) group were exposed to 33% oxygen and 2.5% sevoflurane for 60 min, respectively, without coronary occlusion. The mitoKATP channel inhibitor 5-hydroxydecanoate (5-HD) was given 30 min before sevoflurane preconditioning (5-HD+SWOP group). Cardiac function indices, infarct sizes, serum cardiac troponin I (cTnI) concentrations, and the expression levels of phosphorylated PKC-ε (p-PKC-ε) and caspase-8 were measured. Cardiac function was unchanged, p-PKC-ε expression was upregulated, caspase-8 expression was downregulated, cTnI concentrations were decreased, and the infarcts were significantly smaller (P<0.05) in the SWOP group compared with the I/R group. Cardiac function was worse, p-PKC-ε expression was downregulated, caspase-8 expression was upregulated, cTnI concentration was increased and infarcts were larger in the 5-HD+SWOP group (P<0.05) compared with the SWOP group. The results suggest that mitoKATP channels are involved in the myocardial protective effects of sevoflurane in preconditioning against I/R injury, by regulating PKC-ε phosphorylation before ischemia, and by downregulating caspase-8 during reperfusion.

Screening of a marine natural products library afforded three new analogues of the tetronic acid containing polyketide abyssomicin family and identified abyssomicin 2 as a selective reactivator of latent HIV virus. Examination of the mode of action of this new latent HIV reactivating agent demonstrated that it functions via a distinct mechanism compared to that of existing reactivating agents and is effective at reactivating latent virus in a subset of primary patient cell lines.

The human mast cell line HMC-1⁵⁶⁰,⁸¹⁶ carries activating mutations in the proto-oncogene of c-kit that cause autophosphorylation and permanent c-kit receptor activation. The compound CCT129202 is a new and selective inhibitor of Aurora kinase A and B that decreases the viability of a variety of human tumor cell lines. The effect of Aurora kinase inhibition was assessed in the HMC-1⁵⁶⁰,⁸¹⁶ line in order to find a suitable tool for mastocytosis treatment. CCT129202 treatment induces a significant decrease in cell viability in HMC-1⁵⁶⁰,⁸¹⁶ cells after 48 hours of treatment. Moreover, caspase-3 and caspase-8 activation was induced after incubation of HMC-1⁵⁶⁰,⁸¹⁶ cells in the presence of CCT129202. It has been demonstrated that Protein Kinase C (PKC) plays a crucial role in mast cell activation as well as cell migration, adhesion and apoptotic cell death. Co-treatment of Ca²⁺-independent PKCs (δ ε and θ) inhibitor GF109203X with CCT129202, reduces caspase-3 activation which controls cell levels. In contrast, Go6976, an inhibitor of Ca²⁺-dependent PKCs, increases caspase-3 activation. Oppositely, GF109203X does not modify CCT129202-induced apoptosis through the caspase-8 pathway whereas Go6976 treatment abolishes the increase on caspase-8 activity due to CCT129202. This implies that Ca²⁺-independent PKC isoforms seems to be related with CCT129202-induced apoptosis through the caspase- 3 pathway, whereas Ca²⁺-dependent PKC isoforms are related with the CCT129202 effect on the caspase-8 pathway. Interestingly, CCT129202 cytotoxic effect remains even though Ca²⁺-dependent PKCs are inhibited, which shows that the Aurora kinase inhibitor effect is acting through the caspase-3 pathway. On the other hand, Ca²⁺-independent PKCs inhibition does not affect the final apoptotic CCT129202 effect because this seems to be mediated by the caspase-8 pathway. Moreover, CCT129202 does not affect PKCδ and Ca²⁺-dependent PKC translocation, which indicates that PKC translocation pivots on its activation. This demonstrates that Aurora kinase inhibition is not related to this process. Finally, when PKC is silenced in HMC-1⁵⁶⁰,⁸¹⁶ cells, the effect of CCT129202 on the caspase-3 pathway disappears, which indicates that the CCT129202 effect is clearly PKC-dependent.

The effect of the insecticide methoxychlor on the physiology of renal tubular cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca(2+) concentrations ([Ca(2+) ](i) ) in MDCK renal tubular cells using the Ca(2+) -sensitive fluorescent dye fura-2. Methoxychlor at 5-20 μM increased [Ca(2+) ](i) in a concentration-dependent manner. The signal was reduced by 80% by removing extracellular Ca(2+) . Methoxychlor-induced Ca(2+) entry was not affected by nifedipine and SK&F96365 but was inhibited by econazole and protein kinase C modulators. In Ca(2+) -free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) partly inhibited methoxychlor-induced [Ca(2+) ](i) rise. Incubation with methoxychlor also inhibited thapsigargin- or BHQ-induced [Ca(2+) ](i) rise. Inhibition of phospholipase C with U73122 nearly abolished methoxychlor-induced [Ca(2+) ](i) rise. At 5-15 μM, methoxychlor slightly increased cell viability, whereas at 20 μM, it decreased viability. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid/AM (BAPTA/AM). Annexin V-FITC data suggest that 10 μM methoxychlor inhibited apoptosis, while 20 μM methoxychlor enhanced apoptosis. Methoxychlor (10 and 20 μM) increased the production of reactive oxygen species. Together, in renal tubular cells, methoxychlor induced [Ca(2+) ](i) rise by inducing phospholipase C-dependent Ca(2+) release from multiple stores and Ca(2+) entry via protein kinase C- and econazole-sensitive channels. Methoxychlor slightly enhanced or inhibited cell viability in a concentration-dependent, Ca(2+) -independent manner. Methoxychlor induced cell death that may involve apoptosis via mitochondrial pathways., (© 2012 The Authors Basic & Clinical Pharmacology & Toxicology © 2012 Nordic Pharmacological Society.)

Objective: To determine effects of activating protein kinase C (PKC) on ventricular action potential duration restitution (APDR) and Burst stimulus induced arrhythmia in Langendorff-perfused rabbit hearts., Methods: Male rabbits were equally divided into three groups randomly: control group (Tyrode's solution perfusion), PKC agonist phorbol-12-myristate-13-acetate (PMA, 100 nmol/L) group and PKC inhibitor bisindolylmaleimide (BIM, 500 nmol/L) group. Thirty minutes after perfusion, the monophasic action potential (MAP) and effective refractory period (ERP) were determined in right basal ventricle (RB), right apex (RA), left basal ventricle (LB) and left apex (LA) of all the animals, and APDR curve was drawn. Burst stimulus method was used to induce ventricular arrhythmia in perfused rabbit hearts; Real-time PCR was used to detect the mRNA expression of PKC in four different areas of ventricle., Results: Compared with the control group, the ERP, 90% of monophasic action potential duration (MAPD(90)) and ERP/MAPD(90) were significantly shortened (all P < 0.01), the max slopes (S(max)) of APDR curve were significantly steeper (RB: 1.22 ± 0.23 vs. 0.65 ± 0.19; RA: 2.99 ± 0.29 vs. 1.02 ± 0.18; LB: 1.84 ± 0.21 vs. 0.85 ± 0.12; LA: 4.02 ± 0.32 vs.1.12 ± 0.23, all P < 0.01) and the incidences of ventricular arrhythmia were significantly increased in the PMA group. All parameters were similar between the BIM group and the control group (all P > 0.05)., Conclusion: Activating PKC could enhance the max slopes of APDR curve at various ventricular areas and subsequently increase arrhythmia susceptibility in Langendorff-perfused rabbit hearts.

Two peptides derived from the C1B domain of protein kinase Cγ (PKCγ) were shown to associate with classical PKC isozymes and modulate their activities. These C1B peptides are designated C1B1 (amino acid residues 101-112) and C1B5 (residues 141-151). Since PKC enzyme activity is shown to be involved in colon cancer development, the effect of C1B peptides on the growth of various human colon cancer cell lines was examined in vitro and in vivo. Sub-micromolar to micromolar levels of both C1B peptides induced approximately 60-70% growth attenuation in multiple colon cancer cell lines in a soft agar tumor colony assay; however, C1B5 peptide was not cytotoxic to normal colon epithelial cells in two dimensional culture. The effect of C1B5 peptide on colony growth of COLO205 cells was reversed by treatment with the PKCα/β inhibitor, Ro-32-0432. C1B peptide treatment attenuated COLO205 cells via two mechanisms: 1) cell cycle arrest and 2) stimulation of apoptosis. This is evident in G 2 arrest and increases in levels of cleaved caspase 3 and p53 phosphorylated at serine 20. Intratumoral injection of C1B5 peptide (20 mg/kg/day, every three days) markedly attenuated the growth of subcutaneous xenografts of COLO205 cells in SCID mice by 76% compared with the control. Taken together, these results strongly suggest that C1B peptides have negligible effects on normal tissues but are potentially effective chemotherapeutic agents for colon cancer.

Ethnopharmacological Relevance: Several Asian plants are known for their anti-diabetic properties and produce alkaloids and flavonoids that may stimulate insulin secretion., Materials and Methods: Using Vietnamese plants (Nelumbo nucifera, Gynostemma pentaphyllum, Smilax glabra, and Stemona tuberosa), we extracted two alkaloids (neotuberostemonine, nuciferine) and four flavonoids (astilbin, engeletin, smitilbin, and 3,5,3'-trihydroxy-7,4'-dimethoxyflavone), and studied their insulin stimulatory effects., Results: Nuciferine, extracted from Nelumbo nucifera, stimulated both phases of insulin secretion in isolated islets, whereas the other compounds had no effect. The effect of nuciferine was totally abolished by diazoxide and nimodipine, and diminished by protein kinase A and protein kinase C inhibition. Nuciferine and potassium had additive effects on insulin secretion. Nuciferine also stimulated insulin secretion in INS-1E cells at both 3.3 and 16.7 mM glucose concentrations. Compared with glibenclamide, nuciferine had a stronger effect on insulin secretion and less beta-cell toxicity. However, nuciferine did not compete with glibenclamide for binding to the sulfonylurea receptor., Conclusions: Among several compounds extracted from anti-diabetic plants, nuciferine was found to stimulate insulin secretion by closing potassium-adenosine triphosphate channels, explaining anti-diabetic effects of Nelumbo nucifera., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The bryostatins, powerful protein kinase C (PKC) agonists, are a family of complex macrolactone natural products. They are originally isolated from the marine bryozoan Bugula neritina. So far tweenty bryostatins have been obtained naturally and exhibit a remarkable range of biological activities, including antineoplastic activity, synergistic chemotheoreputic activity, cognition and memory enhancement, etc. Of the 20 known members, the most extensively studied is bryostatin 1. The effects of bryostatin 1 are mainly linked to its ability of selectively modulating the function of various individual protein kinase C (PKC) isozymes. Moreover, bryostatin 1, or in combination with other agents, has been proposed for phase I and phase II clinical trials. The bryostatins have excellent biological properties, but are scarce in nature. Therefore, it has attracted considerable interests in structural modification over the past two decades. In this review, we will attempt to summarize the main developments that have occurred in the structure-activity relationship and biology of bryostatins over the period 1982-2011.

Paclitaxel chemotherapy is limited by a long-lasting painful neuropathy that lacks an effective therapy. In this study, we tested the hypothesis that paclitaxel may release mast cell tryptase, which activates protease-activated receptor 2 (PAR2) and, subsequently, protein kinases A and C, resulting in mechanical and thermal (both heat and cold) hypersensitivity. Correlating with the development of neuropathy after repeated administration of paclitaxel, mast cell tryptase activity was found to be increased in the spinal cord, dorsal root ganglia, and peripheral tissues in mice. FSLLRY-amide, a selective PAR2 antagonist, blocked paclitaxel-induced neuropathic pain behaviors in a dose- and time-dependent manner. In addition, blocking downstream signaling pathways of PAR2, including phospholipase C (PLC), protein kinase A (PKA), and protein kinase Cε (PKC), effectively attenuated paclitaxel-induced mechanical, heat, or cold hypersensitivity. Furthermore, sensitized pain response was selectively inhibited by antagonists of transient receptor potential (TRP) V1, TRPV4, or TRPA1. These results revealed specific cellular signaling pathways leading to paclitaxel-induced neuropathy, including the activation of PAR2 and downstream enzymes PLC, PKCε, and PKA and resultant sensitization of TRPV1, TRPV4, and TRPA1. Targeting one or more of these signaling molecules may present new opportunities for the treatment of paclitaxel-induced neuropathy., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Invasive fungal infections are common clinical complications of neonates, critically ill, and immunocompromised patients worldwide. Candida species are the leading cause of disseminated fungal infections, with Candida albicans being the most prevalent species. Candida glabrata, the second/third most common cause of candidemia, shows reduced susceptibility to a widely used antifungal drug fluconazole. Here, we present findings from a screen of 9134 C. glabrata Tn7 insertion mutants for altered survival profiles in the presence of fluconazole. We have identified two components of RNA polymerase II mediator complex, three players of Rho GTPase-mediated signaling cascade, and two proteins implicated in actin cytoskeleton biogenesis and ergosterol biosynthesis that are required to sustain viability during fluconazole stress. We show that exposure to fluconazole leads to activation of the protein kinase C (PKC)-mediated cell wall integrity pathway in C. glabrata. Our data demonstrate that disruption of a RhoGAP (GTPase activating protein) domain-containing protein, CgBem2, results in bud-emergence defects, azole susceptibility, and constitutive activation of CgRho1-regulated CgPkc1 signaling cascade and cell wall-related phenotypes. The viability loss of Cgbem2Δ mutant upon fluconazole treatment could be partially rescued by the PKC inhibitor staurosporine. Additionally, we present evidence that CgBEM2 is required for the transcriptional activation of genes encoding multidrug efflux pumps in response to fluconazole exposure. Last, we report that Hsp90 inhibitor geldanamycin renders fluconazole a fungicidal drug in C. glabrata.

The effects of a newly synthesized docosahexaenoic acid (DHA) derivative, CRBM-0244, on lung inflammation and airway hyperresponsiveness were determined in an in vitro model of TNF-α-stimulated human bronchi and in an in vivo model of allergic asthma. Mechanical tension measurements revealed that CRBM-0244 prevented bronchial hyperresponsiveness in TNF-α-pretreated human bronchi. Moreover, treatment with CRBM-0244 resulted in a decrease in NF-κB activation and cyclooxygenase-2 (COX-2) overexpression triggered by TNF-α. The inhibition of peroxisome proliferator-activated receptor-γ with GW9662 abolished the CRBM-0244-mediated anti-inflammatory effects. CRBM-0244 reduced the Ca(2+) sensitivity of bronchial smooth muscle through a decrease in the phosphorylation and expression of the PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI-17). Results also revealed an overexpression of CPI-17 protein in lung biopsies derived from patients with asthma. Furthermore, the presence of specialized enzymes such as 5-lipoxygenase and 15-lipoxygenase in the lung may convert CRBM-0244 into active mediators, leading to the resolution of inflammation. The in vivo anti-inflammatory properties of CRBM-0244 were also investigated in a guinea pig model of allergic asthma. After oral administration of CRBM-0244, airway leukocyte recruitment, airway mucus, ovalbumin-specific IgE, and proinflammatory markers such as TNF-α and COX-2 were markedly reduced. Hence, CRBM-0244 treatment prevents airway hyperresponsiveness, Ca(2+) hypersensitivity, and the overexpression of CPI-17 in lung tissue. Together, these findings provide key evidence regarding the mode of action of CRBM-0244 in the lung, and point to new therapeutic strategies for modulating inflammation in patients with asthma.

S-nitrosylation is a ubiquitous protein modification in redox-based signaling and forms S-nitrosothiol from nitric oxide (NO) on cysteine residues. Dysregulation of (S)NO signaling (nitrosative stress) leads to impairment of cellular function. Protein kinase C (PKC) is an important signaling protein that plays a role in the regulation of vascular function, and it is not known whether (S)NO affects PKC's role in vascular reactivity. We hypothesized that S-nitrosylation of PKC in vascular smooth muscle would inhibit its contractile activity. Aortic rings from male C57BL/6 mice were treated with auranofin or 1-chloro-2,4-dinitrobenzene (DNCB) as pharmacological tools, which lead to stabilize S-nitrosylation, and propylamine propylamine NONOate (PANOate) or S-nitrosocysteine (CysNO) as NO donors. Contractile responses of aorta to phorbol-12,13-dibutyrate, a PKC activator, were attenuated by auranofin, DNCB, PANOate, and CysNO. S-nitrosylation of PKCα was increased by auranofin or DNCB and CysNO as compared with control protein. Augmented S-nitrosylation inhibited PKCα activity and subsequently downstream signal transduction. These data suggest that PKC is inactivated by S-nitrosylation, and this modification inhibits PKC-dependent contractile responses. Because S-nitrosylation of PKC inhibits phosphorylation and activation of target proteins related to contraction, this posttranslational modification may be a key player in conditions of decreased vascular reactivity.

Background: The aim of this study was to investigate the protein kinase C (PKC) isoforms involved in the regulation of vascular calcium sensitivity after hemorrhagic shock, the related mechanism, and the role of integrin-linked kinase (ILK)., Methods: Using superior mesenteric artery from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells, the effects of PKC isoforms agonists and antagonists on vascular calcium sensitivity, their relationship with myosin light chain phosphatase (MLCP), myosin light chain (MLC20) phosphorylation, and ILK were observed., Results: The results indicated that PKCα and ε agonists, thymelea toxin and carbachol, restored shock-induced decrease of vascular calcium sensitivity; PKCα antagonist, Gö-6976 and PKCε pseudosubstrate inhibition peptide, aggravated shock-induced calcium desensitization, whereas the agonists and antagonists of PKCδ and ζ had no effects on shock-induced calcium desensitization. PKCα and ε agonists reversed the increased MLCP activity and the decreased MLC20 phosphorylation induced by shock or hypoxia. ILK inhibitor abolished the effects of PKCα and ε agonists on vascular calcium sensitivity, MLCP activity, and MLC20 phosphorylation., Conclusion: These findings suggested that PKCα and ε may be the main isoforms responsible for the regulation of vascular calcium sensitivity after hemorrhagic shock, which enforce their regulation through MLCP-MLC20 pathway, and ILK may be a downstream molecule of PKCα and ε.

Cholinergic neurotransmission in the medial prefrontal cortex (mPFC) is critical for normal processing of cue detection and cognitive performance. However, the mechanism by which cholinergic system modifies mPFC synaptic function remains unclear. Here we show that activation of muscarinic acetylcholine receptors (mAChRs) by carbamoylcholine (CCh) induces long-term depression (CCh-LTD) of excitatory synaptic transmission on mPFC layer V pyramidal neurons. The induction of CCh-LTD is dependent on M(1) mAChR activation but does not require N-methyl-D-aspartate receptor activation or coincident synaptic stimulation. Activation of phospholipase C (PLC), protein kinase C (PKC), and postsynaptic Ca(2+) release from inositol 1,4,5-triphosphate (IP(3)) receptor-sensitive internal stores are required for CCh-LTD induction. The expression of CCh-LTD is likely to be presynaptic because it is accompanied by a decrease in 1/(coefficient of variance)(2) and an increase in synaptic failure and paired-pulse ratio of synaptic responses. CCh-LTD is blocked by nitric oxide (NO) synthase inhibitors, soluble guanylyl cyclase (sGC) inhibitor, and protein kinase G (PKG) inhibitor. Synaptic stimulation of M(1) mAChRs with prolonged paired-pulse low-frequency stimulation also triggers LTD. These results suggest that activation of M(1) mAChRs can induce LTD on mPFC layer V pyramidal neurons through the activation of postsynaptic PLC/PKC/IP(3) receptor- and subsequently presynaptic NO/sGC/PKG-dependent signaling processes.

Background and Purpose: Sphingosine-1-phosphate and its receptors may be involved in vascular smooth muscle cell (VSMC) proliferation following vascular injury. Here, we evaluate the effect of d-erythro-N,N-dimethylsphingosine (DMS), a sphingosine kinase (SK) inhibitor, on VSMC proliferation, apoptosis and neointimal formation., Experimental Approach: Growth responses in vitro to fetal calf serum (FCS) were measured by [(3)H]-thymidine incorporation and extracellular signal-regulated kinase-1/2 (ERK-1/2) activation in quiescent primary cultures of porcine VSMC in the presence and absence of various concentrations of the SK inhibitor DMS. In vivo treatment with DMS was delivered with a local endoluminal catheter, following balloon injury of coronary arteries. The artery intimal formation was investigated by angiography, myography and histomorphometry., Key Results: In vitro experiments indicated that DMS induced a dose-dependent reduction in [(3)H]-thymidine incorporation and ERK-1/2 activation via a protein kinase C (PKC) independent mechanism with an IC(50) value of 12 +/- 6 and 15 +/- 10 microM respectively. DMS also reduced Akt signalling. Four weeks following in vivo delivery of DMS, complete functional endothelial regeneration was observed in all treatment groups, with significant reduction of intimal formation (vehicle 23.7 +/- 4.6% vs. DMS infusion 8.92 +/- 2.9%, P < 0.05)., Conclusions and Implications: Taken together, these results demonstrate that local administration of the SK inhibitor, DMS, reduced neointimal formation, and this effect could involve inhibition of ERK-1/2 and Akt signalling, and modulation of smooth muscle growth.

Background: Peroxisome proliferator activator receptor alpha (PPAR alpha), a member of the nuclear receptor superfamily, is known to increase nitric oxide (NO) production and the mechanisms by which PPAR alpha activation alleviates vascular dysfunction may predicate its activation and possible expression., Objectives: We have evaluated the effects of acute clofibrate, a PPAR alpha ligand and the role of PKC on PPAR alpha expression and NO production in cultured cerebral microvascular endothelial cell (CMVEC)., Methods: Confluent CMVEC derived from pig brain were cultured and the role of PKC in acute clofibrate-induced PPAR alpha expression and NO production was determined in the presence or absence of PKC activator phorbol myristate acetate (PMA) or inhibitor (calphostin C)., Results: Incubation of CMVEC with clofibrate or PMA increased NO production by 40% or 27%, respectively, whereas co-incubation of cells with PMA and clofibrate had no effect on NO production. Incubation of cells with Calphostin C blunted PMA but not clofibrate-induced increase in NO production. L-NAME (0.1 mM), an inhibitor of NO synthase, reduced basal (47%; p<0.01) and abolished clofibrate-induced increase in NO production. Clofibrate increased PPAR alpha expression (26%; p<0.05) while PMA with or without clofibrate reduced PPAR alpha expression (p<0.01). On the other hand, calphostin C reduced basal (69%, ap<0.01) as well as clofibrate-induced increase (59%, p<0.01) in PPAR expression, and further reduced PMA-induced down regulation of PPAR expression. eNOS expression was not significantly affected by either clofibrate or PMA, alone or in combination., Conclusion: These results show that in the brain microvascular endothelial cell, PPAR alpha activation increases NO production-independent of eNOS and PKC signaling pathways, a regulates PPAR alpha expression through a complex PKC signaling mechanism(s) as both PKC activation and inhibition reduced clofibrate-induced activation of PPAR expression (Fig. 4, Ref. 32). Full Text (Free, PDF) www.bmj.sk.

Effects of chronic hypoxia (CH) on store- and receptor-operated Ca(2+) entry (SOCE, ROCE) in pulmonary vascular smooth muscle (VSM) are controversial, although whether genetic variation explains such discrepancies in commonly studied rat strains is unclear. Since protein kinase C (PKC) can inhibit Ca(2+) permeable nonselective cation channels, we hypothesized that CH differentially alters PKC-dependent inhibition of SOCE and ROCE in pulmonary VSM from Sprague-Dawley and Wistar rats. To test this hypothesis, we examined SOCE and endothelin-1 (ET-1)-induced ROCE in endothelium-disrupted, pressurized pulmonary arteries from control and CH Sprague-Dawley and Wistar rats. Basal VSM Ca(2+) was elevated in CH Wistar, but not Sprague-Dawley, rats. Further, CH attenuated SOCE in VSM from Sprague-Dawley rats, while augmenting this response in Wistar rats. CH reduced ROCE in arteries from both strains. PKC inhibition restored SOCE in CH Sprague-Dawley arteries to control levels, while having no effect on SOCE in Wistar arteries or on ROCE in either strain. We conclude that effects of CH on pulmonary VSM SOCE are strain dependent, whereas inhibitory effects of CH on ROCE are strain independent. Further, PKC inhibits SOCE following CH in Sprague-Dawley, but not Wistar, rats but does not contribute to ET-1-induced ROCE in either strain.

Voltage-gated potassium (K(v)) channels in vascular smooth muscle cells (VSMC) are critical regulators of membrane potential and vascular tone. These channels exert a hyperpolarizing influence to counteract the depolarizing effects of intraluminal pressure and vasoconstrictors. However, the contribution of K(v) channel activity to the functional regulation of cerebral (parenchymal) arterioles within the brain is not known. Thus K(v) channel properties in parenchymal arteriolar SMCs were characterized. Isolated, pressurized parenchymal arterioles and arterioles in cortical brain slices exhibited robust constriction in the presence of the K(v) channel inhibitor 4-aminopyridine (4-AP). 4-AP also decreased the amplitude of K(v) currents recorded from SMCs. The steady-state activation and inactivation properties of K(v) currents suggested that these channels are composed of K(v)1.2 and 1.5 subunits, which was confirmed by RT-PCR. K(v) channels can be regulated by extracellular glucose, which may be involved in the functional hyperemic response in the brain. Thus the effects of glucose on K(v) channel activity and arteriolar function were investigated. Elevation of glucose from 4 to 14 mM significantly decreased the peak K(v) current amplitude and constricted arterioles. Arteriolar constriction was prevented by inhibition of protein kinase C (PKC), consistent with previous studies showing enhanced PKC activity in the presence of elevated glucose. In cortical brain slices, the dilation generated by neuronal activity induced by electrical field stimulation was decreased by 54% in 14 mM glucose when compared with the dilation in 4 mM glucose. In anesthetized mice the whisker stimulation-induced increase in local cerebral blood flow was also significantly decreased in 14 mM glucose, and this effect was similarly prevented by PKC inhibition. These findings point to a critical role for K(v) channels in the regulation of intracerebral arteriolar function and suggest that changes in perivascular glucose levels could directly alter vascular diameter resulting in a modulation of local cerebral blood flow.

Objective: The aim of the present study is to verify the ATP-induced varied responses in isolated dorsal root ganglion (DRG) neurons of the adult rat, and investigate the modulatory effects of specific P2X receptor agonist beta, gamma-me-ATP and protein kinase C (PKC) on P2X receptor-mediated inward current in DRG neurons., Methods: Whole cell patch-clamp was employed to record the currents on acutely isolated DRG neurons in the adult rats., Results: beta, gamma-me-ATP, similar as ATP, evoked 2 distinct subtypes of P2X receptor-mediated inward currents in a dose-dependent manner in DRG neurons. Activation of PKC by phorbol 12, 13-dibutyrate (PDBu) significantly inhibited both subtypes of inward currents mediated by P2X receptors in a dose-dependent manner., Conclusion: Activation of PKC negatively modulated the P2X receptor-mediated currents in rat DRG neurons, which may be of benefit to preventing the over-excitation of nociceptor under inflammatory or neuropathic conditions.

Ion channel regulation is key to controlling neuronal excitability. However, the extent that modulators and gating factors interact to regulate channels is less clear. For Aplysia, a nonselective cation channel plays an essential role in reproduction by driving an afterdischarge in the bag cell neurons to elicit egg-laying hormone secretion. We examined the regulation of cation channel voltage and Ca2+ dependence by protein kinase C (PKC) and inositol trisphosphate (IP3)-two prominent afterdischarge signals. In excised, inside-out patches, the channel remained open longer and reopened more often with depolarization from -90 to +30 mV. As previously reported, PKC could closely associate with the channel and increase activity at -60 mV. We now show that, following the effects of PKC, voltage dependence was shifted to the left (essentially enhanced), particularly at more negative voltages. Conversely, the voltage dependence of channels lacking PKC was shifted to the right (essentially suppressed). Predictably, activity was increased at all Ca2+ concentrations following the effects of PKC; nevertheless, Ca2+ dependence was actually shifted to the right. Moreover, whereas IP3 did not alter activity at -60 mV, it drastically shifted Ca2+ dependence to the right-an outcome largely reversed by PKC. With respect to the afterdischarge, these data suggest PKC initially upregulates the channel by direct gating and shifting voltage dependence to the left. Subsequently, PKC and IP3 attenuate the channel by suppressing Ca2+ dependence. This ensures hormone delivery by allowing afterdischarge initiation and maintenance but also prevents interminable bursting. Similar regulatory interactions may be used by other neurons to achieve diverse outputs.

Alterations in PKC isozyme expression and aberrant induction of cyclin D1 are early events in intestinal tumorigenesis. Previous studies have identified cyclin D1 as a major target in the antiproliferative effects of PKCalpha in non-transformed intestinal cells; however, a link between PKC signaling and cyclin D1 in colon cancer remained to be established. The current study further characterized PKC isozyme expression in intestinal neoplasms and explored the consequences of restoring PKCalpha or PKCdelta in a panel of colon carcinoma cell lines. Consistent with patterns of PKC expression in primary tumors, PKCalpha and delta levels were generally reduced in colon carcinoma cell lines, PKCbetaII was elevated and PKCepsilon showed variable expression, thus establishing the suitability of these models for analysis of PKC signaling. While colon cancer cells were insensitive to the effects of PKC agonists on cyclin D1 levels, restoration of PKCalpha downregulated cyclin D1 by two independent mechanisms. PKCalpha expression consistently (a) reduced steady-state levels of cyclin D1 by a novel transcriptional mechanism not previously seen in non-transformed cells, and (b) re-established the ability of PKC agonists to activate the translational repressor 4E-BP1 and inhibit cyclin D1 translation. In contrast, PKCdelta had modest and variable effects on cyclin D1 steady-state levels and failed to restore responsiveness to PKC agonists. Notably, PKCalpha expression blocked anchorage-independent growth in colon cancer cells via a mechanism partially dependent on cyclin D1 deficiency, while PKCdelta had only minor effects. Loss of PKCalpha and effects of its re-expression were independent of the status of the APC/beta-catenin signaling pathway or known genetic alterations, indicating that they are a general characteristic of colon tumors. Thus, PKCalpha is a potent negative regulator of cyclin D1 expression and anchorage-independent cell growth in colon tumor cells, findings that offer important perspectives on the frequent loss of this isozyme during intestinal carcinogenesis.

Ca(v)2.2 high voltage-gated calcium channels are regulated by phorbol-12-myristae, 13-acetate (PMA) via Ser/Thr protein kinase C (PKC) phosphorylation sites in the I-II linker and C-terminus of the alpha(1) 2.2 subunit. Here we show that PMA enhancement of Ca(v)2.2 currents expressed in Xenopus oocytes can be blocked by inhibitors of PKC betaII or PKC epsilon isozymes, as shown previously for Ca(v)2.3 currents, and that microinjection of PKC betaII or PKC epsilon isozymes in the oocytes expressing the WT Ca(v)2.2 channels increases the basal barium current (I(Ba)). The I-V plot shows a large increase in current amplitude with PKC betaII and PKC epsilon isozymes with only a small shift in the peak I(Ba) in the hyperpolarizing direction. The potentiation of Ca(v)2.2 currents by microinjection of PKC betaII and PKC epsilon isozymes was not altered by the inhibition of G proteins with GDPbetaS. The combination of isozyme specific inhibitors with previously generated Ser/Thr to Ala mutants of alpha(1) 2.2 subunit revealed that PKC betaII or PKC epsilon isozymes (but not PKC alpha or delta) can provide full enhancement through the stimulatory site (Thr-422) in the I-II linker but that PKC epsilon is better at decreasing channel activity through the inhibitory site Ser-425. The enhancing effect of PKC betaII or epsilon at Thr-422 is dominant over the inhibitory effect at Ser-425. Injected PKC betaII also enhances Ca(v)2.2 current when any of the potential stimulatory sites (Ser-1757, Ser-2108 and Ser-2132) are available in the C-terminus. PKC epsilon provides lesser enhancement with C-terminal sites and only with Ser-2108 and Ser-2132. Sites Ser-1757 and Ser-2132, but not Ser-2108, are dominant over the inhibitory site Ser-425. Collectively, these results reveal a hierarchy of regulatory sites in Ca(v)2.2 channels. Site-specific regulation by different PKC isozymes may allow graded levels of channel activation and susceptibility or resistance to subsequent stimulatory events.

The degree of tonic force (F) maintenance induced in vascular smooth muscle upon K(+) depolarization with 110 mM KCl can be greatly reduced by inhibition of rhoA kinase (ROCK). We explored the possibility that a protein kinase C (PKC) isotype may also play a role in causing KCl-induced Ca(2+) sensitization. In isometric rings of rabbit artery, the PKC inhibitors, Go-6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), GF-109203X (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide), and a cell-permeable (myristoylated) pseudosubstrate inhibitor of PKCzeta (PI(PKCzeta)) inhibited KCl-induced tonic F. A myristoylated pseudosubstrate inhibitor of PKCalpha/beta that inhibited phorbol dibutyrate-induced F slightly potentiated KCl-induced tonic F and attenuated 30 mM KCl-induced F. Although the ROCK inhibitor, H-1152 [(S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)-sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride], reduced basal phosphorylation of myosin light-chain phosphatase-targeting subunit at Thr853 (MYPT1-pT853), 3 and 10 muM GF-109203X inhibited only KCl-stimulated phosphorylation, not basal MYPT1-pT853. In fura-2-loaded tissues, GF-109203X and PI(PKCzeta) elevated basal [Ca(2+)](i) (calcium) and potentiated KCl-induced tonic increases in calcium while reducing KCl-induced tonic increases in F. Blockade by nifedipine of Ca(2+) entry through voltage-operated Ca(2+) channels reduced KCl-induced Ca(2+) sensitization and KCl-stimulated but not basal MYPT1-pT853. These data together support a model in which ROCK and PKCzeta are constitutively active and function in "resting" muscle to regulate the basal levels of MYPT1-pT853 and calcium, respectively. In this model, KCl-induced increases in calcium activate PKCzeta to feed forward and cause additional MYPT1-pT853 above that induced by constitutive ROCK, permitting Ca(2+) sensitization and strong F maintenance. Active PKCzeta also feeds back to attenuate the degree of KCl-induced increases in calcium.

Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia coli-expressed glutathione-S-transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho-Ser845 cGluR1 protein, but without binding to phospho-Ser831 cGluR1 protein. Moreover, pre-binding of the unphospho-cGluR1 protein with these aptamers inhibits protein kinase A-mediated phosphorylation at Ser845 residue. In contrast, the pre-binding of aptamer A2 has no effect on protein kinase C-mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1-containing AMPA receptor trafficking to the cell surface and abrogated forskolin-stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein-GluR1-transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single-residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.

Background: After ischaemia and during reperfusion, rat hearts release cardiodepressive substances that are putatively cyclooxygenase-2-dependent. The present study analyses the mechanisms by which these substances mediate their effect downstream of cyclooxygenase-2., Materials and Methods: After 10 min of global stop-flow ischaemia, isolated rat hearts were reperfused and post-ischaemic coronary effluent was collected over a period of 30 s. Non-ischaemic effluent collected before ischaemia was used as a control. We investigated the effect of the effluents on cell shortening and Ca(++)-metabolism, by application of fluorescence microscopy of field-stimulated adult rat cardiomyocytes incubated with fura-2. Cells were pre-incubated with inhibitors of protein kinase A and C and with antagonists of protein kinase A-dependent prostaglandin receptors. We examined the expression of prostaglandin receptors in cardiomyocytes by Western blotting., Results: In contrast to non-ischaemic effluent, post-ischaemic effluent induced reduction of Ca(++) transient and cell shortening in the cardiomyocytes. In contrast to protein kinase C inhibitor Myr-PKC [19-27], the protein kinase A inhibitor Rp-cAMPS completely blocked the effect of post-ischaemic effluent. Furthermore, we determined a cyclic adenosine monophosphate increase in cardiomyocytes that were pre-incubated with post-ischaemic effluent. The antagonist of prostaglandin E-receptor EP2 AH6809 and the antagonist of receptor subtype EP4 AH23848 attenuated the effect of post-ischaemic effluent in contrast to other antagonists of prostaglandin D and I receptors, which did not influence the effect. In lysates of adherend cardiomyocytes, expression of prostaglandin D, E and I receptors was detected by Western blotting., Conclusions: The effect of post-ischaemic effluent is mediated by the protein kinase A-dependent prostaglandin-receptor subtypes EP2 and EP4 downstream of cyclooxygenase-2.

Background: We showed previously that parathyroid hormone (PTH) may stimulate the endothelial expression of pro-atherosclerotic and pro-inflammatory markers. Considering the impact of PTH on vasculature, we decided to evaluate its effect on mRNA and intra-cellular protein expressions of endothelial vascular endothelial growth factor (VEGF) taking into account that VEGF may play a role in the pathogenesis of endothelial dysfunctions., Materials and Methods: Human umbilical vein cords endothelial cells (HUVEC) were stimulated for 24 h with 10(-12)-10(-10) mol L(-1) PTH. The VEGF-165 mRNA expression (critical in stimulating endothelial cell proliferation) was evaluated by RT/PCR and the intra-cellular VEGF protein expression by flow cytometry. The pathways by which PTH may have an effect on VEGF expression were also evaluated., Results: PTH (10(-10) mol L(-1)) significantly increased VEGF-165 mRNA expression (P < 0.05). The addition of 50 nmol L(-1) protein kinase C (PKC) and 10 micromol L(-1) protein kinase A (PKA) inhibitors significantly reduced the VEGF-165 mRNA expression (P = 0.01). We also examined whether nitric oxide (NO) may be involved in the PTH-induced stimulation of VEGF-165 expression. Pre-treatment of the cells with 200 micromol L-nitro arginine methyl ester (L-NAME, NO synthase inhibitor) was found to inhibit VEGF-165 mRNA expression (P = 0.006). VEGF protein could not be detected in the medium of HUVEC but it was present in the cell cytoplasm. PTH had no significant effect on cytoplasmatic VEGF protein expression., Conclusion: The stimulatory effect of PTH on endothelial VEGF-165 mRNA expression is partly through PKC and PKA pathways and is also NO dependent.

Dopamine (DA) reuptake terminates dopaminergic neurotransmission and is mediated by DA transporters (DATs). Acute protein kinase C (PKC) activation accelerates DAT internalization rates, thereby reducing DAT surface expression. Basal DAT endocytosis and PKC-stimulated DAT functional downregulation rely on residues within the 587-596 region, although whether PKC-induced DAT downregulation reflects transporter endocytosis mechanisms linked to those controlling basal endocytosis rates is unknown. Here, we define residues governing basal and PKC-stimulated DAT endocytosis. Alanine substituting DAT residues 587-590 1) abolished PKC stimulation of DAT endocytosis, and 2) markedly accelerated basal DAT internalization, comparable to that of wildtype DAT during PKC activation. Accelerated basal DAT internalization relied specifically on residues 588-590, which are highly conserved among SLC6 neurotransmitter transporters. Our results support a model whereby residues within the 587-590 stretch may serve as a locus for a PKC-sensitive braking mechanism that tempers basal DAT internalization rates.

Dexmedetomidine (Dexmd), a potent and highly specific alpha(2) adrenoreceptor agonist, is an efficient therapeutic agent for sedation. Dexmd has been recently reported to have a neuroprotective effect. Heat shock protein (HSP) 27, a low-molecular weight HSP has been shown to be expressed following cerebral ischemia in astrocytes but not in neurons. HSP27 expression is involved in ischemic tolerance of the brain. This study investigated the effect of Dexmd on HSP27 in rat C6 glioma cells. 12-O-tetradecanoylphorbol-13-actate (TPA), a direct activator of protein kinase C (PKC), stimulated the phosphorylation of HSP27 at Ser82, but not Ser15 in a time-dependent manner. Prostaglandin (PG) E(1) or PGE(2) which activates the adenylyl cyclase-cAMP system as well as forskolin and dibutyryl-cAMP, suppressed the TPA-induced phosphorylation of HSP27. Dexmd reversed the suppression of HSP27 phosphorylation by the adenylyl cyclase-cAMP system. Therefore, these results strongly suggest that Dexmd reverses the suppression of HSP27 phosphorylation by the adenylyl cyclase-cAMP system activation through the inhibition of its system in C6 cells. alpha(2) Adrenoreceptor agonists may therefore show a neuroprotective effect through the modification of HSP27 phosphorylation induced by PKC activation.

Myocardial ischaemia activates blood platelets, which in turn stimulate cardiac sympathetic afferents, leading to chest pain and sympathoexcitatory reflex cardiovascular responses. Previous studies have shown that activated platelets stimulate ischaemically sensitive cardiac sympathetic afferents, and that thromboxane A(2) (TxA(2)) is one of the mediators released from activated platelets during myocardial ischaemia. The present study tested the hypothesis that endogenous TxA(2) stimulates cardiac afferents during ischaemia through direct activation of TxA(2) (TP) receptors coupled with the phospholipase C-protein kinase C (PLC-PKC) cellular pathway. Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicantes (T(2)-T(5)) in anaesthetized cats. Single fields of 39 afferents (conduction velocity = 0.27-3.65 m s(-1)) were identified in the left or right ventricle initially with mechanical stimulation and confirmed with a stimulating electrode. Five minutes of myocardial ischaemia stimulated all 39 cardiac afferents (8 Adelta-, 31 C-fibres) and the responses of these 39 afferents to chemical stimuli were further studied in the following four protocols. In the first protocol, 2.5, 5 and 10 microg of the TxA(2) mimetic, U46619, injected into the left atrium (LA), stimulated seven ischaemically sensitive cardiac afferents in a dose-dependent manner. Second, BM13,177, a selective TxA(2) receptor antagonist, abolished the responses of six afferents to 5 microg of U46619 injected into the left atrium and attenuated the ischaemia-related increase in activity of seven other afferents by 44%. In contrast, cardiac afferents, in the absence of TP receptor blockade responded consistently to repeated administration of U46619 (n = 6) and to recurrent myocardial ischaemia (n = 7). In the fourth protocol, administration of PKC-(19-36), a selective PKC inhibitor, attenuated the responses of six other cardiac afferents to U46619 by 38%. Finally, using an immunohistochemical staining approach, we observed that TP receptors were expressed in cardiac sensory neurons in thoracic dorsal root ganglia. Taken together, these data indicate that endogenous TxA(2) contributes to the activation of cardiac afferents during myocardial ischaemia through direct stimulation of TP receptors probably located in the cardiac sensory nervous system and that the stimulating effect of TxA(2) on cardiac afferents is dependent, at least in part, upon the PLC-PKC cellular pathway.

Histone deactylase inhibitors (HDACi) are common chemotherapeutic agents that stimulate Epstein-Barr virus (EBV) reactivation; the detailed mechanism remains obscure. In this study, it is demonstrated that PKCdelta is required for induction of the EBV lytic cycle by HDACi. Inhibition of PKCdelta abrogates HDACi-mediated transcriptional activation of the Zta promoter and downstream lytic gene expression. Nuclear translocation of PKCdelta is observed following HDACi stimulation and its overexpression leads to progression of the EBV lytic cycle. Our study suggests that PKCdelta is a crucial mediator of EBV reactivation and provides a novel insight to study the regulation of the EBV lytic cycle.

Altered regulation of dopamine D(2) receptors is implicated in addiction, schizophrenia and movement disorders, as well as lactotroph growth and regulation. Dopamine D(2S) and dopamine D(2L) receptors are alternately-spliced variants that differ by 29 amino acids in the third intracellular (i3) domain and display different sensitivity to desensitization by protein kinase C (PKC). In the present studies we determined the specific phosphorylation sites on the dopamine D(2S) receptor that confer PKC-mediated desensitization. In dopamine D(2L) receptors, we identified a PKC pseudosubstrate site responsible for the relative insensitivity of the receptor to PKC-induced uncoupling. In transiently transfected Ltk(-) fibroblast cells, 2-min preactivation of PKC with 12-O-tetradecanoyl 4beta-phorbol 13alpha-acetate (TPA) completely inhibited calcium mobilization induced by the dopamine D(2S) receptor, but not the dopamine D(2L) variant. Point mutation of i3 PKC sites Ser228/229Gly rendered the dopamine D(2S) receptor resistant to PKC action, with lesser effects of other Ser and Thr mutations. Inactivation of the PKC pseudosubstrate motif in the dopamine D(2L) receptor sensitized the receptor to PKC, and this was reversed by mutation of i3 PKC sites Ser228/229. A phospho-specific antibody generated against phospho-Ser228/229 demonstrated PKC-induced phosphorylation at these sites of dopamine D(2S), but not D(2L) receptors, in Ltk(-) cells. Conversely, the pseudosubstrate dopamine D(2L) receptor mutant displayed PKC-induced phosphorylation at Ser228/229, which was abolished when these sites were mutated. Similar phosphorylation results were observed using GH4 cells stably transfected with dopamine D(2) receptors and mutants. Thus the relative location of phosphorylation and pseudosubstrate sites provides an important determinant substrate sensitivity to PKC.

Phosphorylation of various AMPA receptor subunits can alter synaptic transmission and plasticity at excitatory glutamatergic synapses in the central nervous system. Here, we identified threonine-840 (T840) on the GluR1 subunit of AMPA receptors as a novel phosphorylation site. T840 is phosphorylated by protein kinase C (PKC) in vitro and is a highly turned-over phosphorylation site in the hippocampus. Interestingly, the high basal phosphorylation of T840 in the hippocampus is maintained by a persistent activity of a protein kinase, which is counter-balanced by a basal protein phosphatase activity. To study the function of T840, we generated a line of mutant mice lacking this phosphorylation site using a gene knock-in technique. The mice generated lack T840, in addition to two previously identified phosphorylation sites S831 and S845. Using this mouse, we demonstrate that T840 may regulate synaptic plasticity in an age-dependent manner.

The repetitive activation of synaptic glutamate receptors can induce a lasting change in the number or subunit composition of synaptic AMPA receptors (AMPARs). However, NMDA receptors that are present extrasynaptically can also be activated by a burst of presynaptic activity, and thus may be involved in the induction of synaptic plasticity. Here we show that the physiological-like activation of extrasynaptic NMDARs induces a lasting change in the synaptic current, by changing the subunit composition of AMPARs at the parallel fibre-to-cerebellar stellate cell synapse. This extrasynaptic NMDAR-induced switch in synaptic AMPARs from GluR2-lacking (Ca(2+)-permeable) to GluR2-containing (Ca(2+)-impermeable) receptors requires the activation of protein kinase C (PKC). These results indicate that the activation of extrasynaptic NMDARs by glutamate spillover is an important mechanism that detects the pattern of afferent activity and subsequently exerts a remote regulation of AMPAR subtypes at the synapse via a PKC-dependent pathway.

We showed in our previous study that in hippocampal CA1 neurons the stimulation of muscarinic receptors inhibited the GIRK current (I(GIRK)) via a PLC/PKC pathway, whereas group I metabotropic glutamate receptors (mGluR) inhibited I(GIRK) via a PLA(2)/arachidonic acid pathway. In this study, we present evidence that receptor-mediated signalling pathways activated by the two G(q)-coupled receptors (G(q)PCRs) converge on the inhibition of GIRK channel-PIP(2) interaction. I(GIRK) was activated in acutely isolated hippocampal CA1 neurons by repetitive application of baclofen, a GABA(B) receptor agonist, with a 2-3 min interval. When both CCh and DHPG were pretreated before the second I(GIRK) activation, the magnitude of the second I(GIRK) was 52.2 +/- 2.5% of the first I(GIRK), which was not significantly different from the magnitude of inhibition by CCh or DHPG alone. This result shows that the effects of muscarinic receptor and group I mGluR stimulation on I(GIRK) are not additive but occlusive, suggesting that each pathway may converge to a common mechanism that finally regulates I(GIRK). To test the involvement of PIP(2) in this mechanism, the effect of CCh and DHPG on I(GIRK) was tested in cells loaded with exogenous PIP(2). The inhibition of I(GIRK) by CCh or DHPG was almost completely abolished in PIP(2)-loaded cells. We confirmed that the inhibition of I(GIRK) by direct application of phorbol ester or arachidonic acid was also completely reversed in PIP(2)-loaded cells. These results indicate that the decrease in PIP(2)-channel interactions is the final common mechanism responsible for G(q)PCR-induced inhibitions of I(GIRK) mediated by PKC and arachidonic acid.