Protein Kinase C Inhibitor Ro31-8220 Research Articles

Progesterone attenuates aquaporin-4 expression in an astrocyte model of ischemia/reperfusion.

Previous studies have suggested that progesterone may be involved in neuroprotection by preventing brain edema. In this study, we assessed the effects of progesterone on aquaporin-4 (AQP4) expression in an ischemia/reperfusion model of cultured rat astrocytes, and further explored the possible role of the protein kinase C (PKC) pathway in this course. We evaluate primary culture astrocytes exposed to 4 h oxygen-glucose deprivation (OGD) followed by 24 h reperfusion (OGD4h/R24h) as a means of simulating cortex ischemia and reperfusion, and test the effect of progesterone on AQP4 expression in response to OGD4h/R24h. Besides, the cell viability was assessed by MTT reduction and lactate dehydrogenase release assay, accompanied by cell morphology survey. At a concentration of 1 and 2 μM, progesterone significantly attenuated AQP4 at the level of both protein and mRNA and ameliorated the cell viability of astrocytes from OGD/reperfusion injury. Moreover, this effect was blocked by the PKC inhibitor Ro31-8220, which was employed before the OGD. These results indicate that progesterone exerts the protective effects and attenuates AQP4 expression in an astrocyte model of ischemia/reperfusion depending on the PKC signal pathway.

Role of a Janus kinase 2-dependent signaling pathway in platelet activation

IntroductionJanus kinases (JAKs) are intracellular non-receptor tyrosine kinases that transduce cytokine-mediated signals through a pathway mediated by JAK and the signal transducer and activator of transcription (STAT) proteins. The JAK-STAT pathway is involved in immune response, inflammation, and tumorigenesis. Platelets are anuclear blood cells that play a central role in hemostasis. MethodsThe aggregometry, immunoblotting, and platelet functional analysis used in this study. ResultsWe found that the JAK2 inhibitor AG490 (25 and 50μM) attenuated collagen-induced platelet aggregation and calcium mobilization in a concentration-dependent manner. In the presence of AG490, the phosphorylation of PLCγ2, protein kinase C (PKC), Akt or JNK in collagen-activated aggregation of human platelets was also inhibited. In addition, we found that various inhibitors, such as the PLCγ2 inhibitor U73122, the PKC inhibitor Ro318220, the phospoinositide 3-kinase inhibitor LY294002, the p38 mitogen-activated protein kinase inhibitor SB203580, the ERK inhibitor PD98059, and the JNK inhibitor SP600125, had no effects on collagen-induced JAK2 activity. However, U73122, Ro318220 and SP600125 significantly diminished collagen-induced STAT3 phosphorylation. These findings suggest that PLCγ2-PKC and JNK are involved in JAK2-STAT3 signaling in collagen-activated platelets. ConclusionOur results demonstrate that the JAK2-STAT3 pathway is involved in collagen-induced platelet activation through the activation of JAK2-JNK/PKC-STAT3 signaling. The inhibition of JAK2 may represent a potential therapeutic strategy for the preventing or treating thromboembolic disorders.

Inhibition of Protein Kinase C/Twist1 Signaling Augments Anticancer Effects of Androgen Deprivation and Enzalutamide in Prostate Cancer

The progression of prostate cancer to metastatic and castration-resistant disease represents a critical step. We previously showed that the transcription factor Twist1, which promotes epithelial-mesenchymal transition, was involved in castration-resistant progression. Similarly, protein kinase C (PKC) has been implicated in both metastatic progression and castration resistance in prostate cancer. In this study, we aimed to elucidate the role of PKC/Twist1 signaling in castration resistance, and to apply this information to the development of a novel therapeutic concept using PKC inhibitor Ro31-8220 against prostate cancer using various prostate cancer cell lines. Androgen deprivation and the next-generation antiandrogen enzalutamide induced PKC activation and Twist1 expression, which were reversed by the PKC inhibitor Ro31-8220. Ro31-8220 suppressed cell proliferation in androgen-dependent prostate cancer LNCaP cells, which was augmented by its combination with androgen deprivation or enzalutamide. The favorable anticancer effects of the combination of Ro31-8220 and enzalutamide were also observed in castration-resistant C4-2 and 22Rv1 cells. Furthermore, PKC phosphorylation was elevated in castration-resistant and enzalutamide-resistant cells compared with their parental cells, leading to persistent sensitivity to Ro-31-8220 in castration- and enzalutamide-resistant cells. Taken together, these findings indicate that PKC/Twist1 signaling contributes to castration resistance as well as enzalutamide resistance in prostate cancer, and suggest that therapeutics targeting PKC/Twist1 signaling, such as PKC inhibitors, represent a promising novel therapeutic strategy for prostate cancer, especially castration-resistant prostate cancer, when combined with enzalutamide.

A novel role of andrographolide, an NF-kappa B inhibitor, on inhibition of platelet activation: the pivotal mechanisms of endothelial nitric oxide synthase/cyclic GMP

Andrographolide is a novel NF-κB inhibitor from the leaves of Andrographis paniculata. Platelet activation is relevant to a variety of thrombotic diseases. However, no data are available concerning the effects of andrographolide in platelet activation. The aim of this study was to examine the mechanisms of andrographolide in preventing platelet activation. Andrographolide (25-75μΜ) exhibited a more potent activity of inhibiting platelet aggregation stimulated by collagen. Andrographolide inhibited collagen-stimulated platelet activation accompanied by relative Ca(2+) mobilization; thromboxane A(2) formation; and phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Andrographolide markedly increased cyclic GMP, but not cyclic AMP levels. Andrographolide also stimulated endothelial nitric oxide synthase (eNOS) expression, NO release, and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. ODQ, an inhibitor of guanylate cyclase, markedly reversed the andrographolide-mediated inhibitory effects on platelet aggregation, p38 MAPK and Akt phosphorylation, and the andrographolide-mediated stimulatory effect on VASP phosphorylation. Furthermore, a PI3 kinase inhibitor (LY294002) but not a PKC inhibitor (Ro318220) significantly diminished p38 MAPK phosphorylation; nevertheless, a p38 MAPK inhibitor (SB203580) and LY294002 diminished PKC activity stimulated by collagen. Andrographolide also reduced collagen-triggered hydroxyl radical (OH([Symbol: see text])) formation. In vivo studies revealed that andrographolide (22 and 55μg/kg) is effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism and significantly prolonged platelet plug formation in mice. This study demonstrates for the first time that andrographolide possesses a novel role of antiplatelet activity, which may involve the activation of the eNOS-NO/cyclic GMP pathway, resulting in the inhibition of the PI3 kinase/Akt-p38 MAPK and PLCγ2-PKC cascades, thereby leading to inhibition of platelet aggregation.

Adhesion of renal carcinoma cells to endothelial cells depends on PKCμ

BackgroundThe formation of metastases includes the separation of tumor cells from the primary tumor, cell migration into subendothelial tissue and cell proliferation in secondary organ. In this process, cell adhesion of tumor cells to the endothelium is an essential requirement for formation of metastases. Protein kinase C (PKC) regulates adhesion and proliferation. To identify a relation between PKC isoforms and tumor progression in renal cell carcinoma (RCC), the influence of PKC isoforms on cell adhesion and proliferation, and possible influences of integrins were analyzed in RCC cells.MethodsThe experiments were performed in the RCC cell lines CCF-RC1 and CCF-RC2 after pre-incubation (16 h) with the PKC inhibitors GF109203X (inhibits PKCα, βI, βII, γ, δ and ε), GÖ6976 (inhibits PKCα, βI and μ), RO31-8220 (inhibits PKCα, βI, βII, γ and ε) and rottlerin (inhibits PKCδ). Cell adhesion was assessed through adherence of RCC cells to an endothelial monolayer. Cell proliferation was analyzed by a BrdU incorporation assay. The expression of β1 integrins was analyzed by flow cytometry.ResultsIn CCF-RC1 cells, cell adhesion was significantly reduced by GÖ6976 to 55% and by RO31-8220 to 45% of control. In CCF-RC2 cells, only GÖ6976 induced a significant reduction of cell adhesion to 50% of control levels. Proliferation of both cell lines was reduced by rottlerin to 39% and 45% of control, respectively. The β1 integrin expression on the cell surface of CCF-RC1 and CCR-RC2 cells was decreased by RO31-8220 to 8% and 7% of control, respectively. β2 and β3 integrins were undetectable in both cell lines.ConclusionsThe combination of the PKC inhibitors leads to the assumption that PKCμ influences cell adhesion in CCF-RC1 and CCF-RC2 cells, whereas in CCF-RC1 cells PKCε also seems to be involved in this process. The expression of β1 integrins appears to be regulated in particular by PKCε. Cell proliferation was inhibited by rottlerin, so that PKCδ might be involved in cell proliferation in these cells.

Reactive Oxygen Species-generating Mitochondrial DNA Mutation Up-regulates Hypoxia-inducible Factor-1α Gene Transcription via Phosphatidylinositol 3-Kinase-Akt/Protein Kinase C/Histone Deacetylase Pathway

Lewis lung carcinoma-derived high metastatic A11 cells constitutively overexpress hypoxia-inducible factor (HIF)-1alpha mRNA compared with low metastatic P29 cells. Because A11 cells exclusively possess a G13997A mutation in the mitochondrial NADH dehydrogenase subunit 6 (ND6) gene, we addressed here a causal relationship between the ND6 mutation and the activation of HIF-1alpha transcription, and we investigated the potential mechanism. Using trans-mitochondrial cybrids between A11 and P29 cells, we found that the ND6 mutation was directly involved in HIF-1alpha mRNA overexpression. Stimulation of HIF-1alpha transcription by the ND6 mutation was mediated by overproduction of reactive oxygen species (ROS) and subsequent activation of phosphatidylinositol 3-kinase (PI3K)-Akt and protein kinase C (PKC) signaling pathways. The up-regulation of HIF-1alpha transcription was abolished by mithramycin A, an Sp1 inhibitor, but luciferase reporter and chromatin immunoprecipitation assays indicated that Sp1 was necessary but not sufficient for HIF-1alpha mRNA overexpression in A11 cells. On the other hand, trichostatin A, a histone deacetylase (HDAC) inhibitor, markedly suppressed HIF-1alpha transcription in A11 cells. In accordance with this, HDAC activity was high in A11 cells but low in P29 cells and in A11 cells treated with the ROS scavenger ebselene, the PI3K inhibitor LY294002, and the PKC inhibitor Ro31-8220. These results suggest that the ROS-generating ND6 mutation increases HIF-1alpha transcription via the PI3K-Akt/PKC/HDAC pathway, leading to HIF-1alpha protein accumulation in hypoxic tumor cells.

The PKC inhibitor Ro31-8220 blocks acute amphetamine-induced dopamine overflow in the nucleus accumbens

Acute administration of the psychostimulant amphetamine increases extracellular levels of dopamine (DA) by reversing the DA transporter on ascending midbrain DA neurons. In vitro studies using striatal synaptosomal, slice and nucleus accumbens (NAcc) tissue preparations have implicated protein kinase C (PKC) in this effect. The present study further examined this effect in vivo by assessing the ability of the PKC inhibitor, Ro31-8220 (10 μM), to inhibit acute amphetamine-induced DA overflow when applied with this drug to the NAcc via reverse dialysis. Amphetamine was applied at a concentration of 30 μM, and the core and shell subregions of the NAcc were assayed separately in freely moving rats. These brain regions play a role in the acute locomotor-activating and motivational effects of amphetamine. Consistent with the findings of previous in vitro experiments, reverse dialysis of Ro31-8220 with amphetamine robustly attenuated the ability of this drug to increase extracellular levels of dopamine in both the core and shell subregions of the NAcc. These results confirm that amphetamine stimulates dopamine overflow via a PKC-dependent mechanism.

Inhibitory effect of glutamate release from rat cerebrocortical synaptosomes by dextromethorphan and its metabolite 3-hydroxymorphinan

Dextromethorphan (DM), a widely used antitussive, has demonstrated an effective neuroprotective effect. Excessive release of glutamate is considered to be an underlying cause of neuronal damage in several neurological diseases. In the present study, we investigated whether DM or its metabolite 3-hydroxymorphinan (3-HM) could affect glutamate release in rat cerebral cortex nerve terminals (synaptosomes). DM or 3-HM inhibited the Ca 2+-dependent release of glutamate that was evoked by exposing synaptosomes to the K + channel blocker 4-aminopyridine (4-AP), and this presynaptic inhibition was concentration-dependent. Inhibition of glutamate release by DM or 3-HM was resulted from a reduction of vesicular exocytosis, because the vesicular transporter inhibitor bafilomycin A1 completely blocked DM or 3-HM-mediated inhibition of 4-AP-evoked glutamate release. DM or 3-HM did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization, but significantly reduced depolarization-induced increase in [Ca 2+] C. DM or 3-HM-mediated inhibition of 4-AP-evoked glutamate release was blocked by ω-conotoxin MVIIC, an antagonist of N- and P/Q-type Ca 2+ channel, not by dantrolene, an intracellular Ca 2+ release inhibitor. DM or 3-HM modulation of 4-AP-evoked glutamate release appeared to involve a protein kinase C (PKC) signaling cascade, insofar as pretreatment of synaptosomes with the PKC inhibitors GF109203X or Ro318220 all effectively occluded the inhibitory effect of DM or 3-HM. Furthermore, 4-AP-induced phosphorylation of PKC was reduced by DM or 3-HM. These results suggest that DM or 3-HM inhibits glutamate release from rat cortical synaptosomes through the suppression of presynaptic voltage-dependent Ca 2+ entry and PKC activity. This may explain the neuroprotective effects of DM against neurotoxicity.

A ROLE FOR RHO‐KINASE IN Ca2+‐INDEPENDENT CONTRACTIONS INDUCED BY PHORBOL‐12,13‐DIBUTYRATE

1. Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca(2+)-depleted solutions. In the present study, we tested the hypothesis that Rho-kinase plays a role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. 2. In Ca(2+)-free solution, 0.1 and 1 micromol/L PDBu induced contraction and myosin light chain (MLC(20)) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 micromol/L), an inhibitor of Rho-kinase, but not ML7 (10 micromol/L), an inhibitor of myosin light chain kinase, inhibited Ca(2+)-independent contractions induced by PDBu. 3. In Ca(2+)-free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI-17 almost completely abolished by 1 micromol/L Ro31-8220, an inhibitor of PKC. 4. In Ca(2+)-free solution, PDBu increased the amount of GTP-RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31-8220, but not by the Rho kinase inhibitor H1152. 5. In conclusion, RhoA/Rho-kinase plays an important role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca(2+)-independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP-RhoA and subsequent phosphorylation of MYPT1 and CPI-17.

Mechanisms underlying the honokiol inhibition of evoked glutamate release from glutamatergic nerve terminals of the rat cerebral cortex

The effect of honokiol, an active component of Magnolia officinalis, on glutamate release from isolated nerve terminals (synaptosomes) was examined. Honokiol potently inhibited 4-aminopyridine (4-AP)-evoked glutamate release in a concentration-dependent manner, and this effect resulted from a reduction of vesicular exocytosis and not from an inhibition of Ca(2+)-independent efflux via glutamate transporter. The inhibitory action of honokiol was not due to decreasing synaptosomal excitability or directly interfering with the release process at some point subsequent to Ca(2+) influx, because honokiol did not alter the 4-AP-evoked depolarization of the synaptosomal plasma membrane potential or Ca(2+) ionophore ionomycin-induced glutamate release. Rather, examination of the effect of honokiol on cytosolic [Ca(2+)] revealed that the diminution of glutamate release could be attributed to a reduction in voltage-dependent Ca(2+) influx. Consistent with this, the honokiol-mediated inhibition of 4-AP-evoked glutamate release was completely prevented in synaptosomes pretreated with a wide-spectrum blocker of N-, P-, and Q-type Ca(2+) channels, omega-conotoxin MVIIC. In addition, honokiol modulation of 4-AP-evoked glutamate release appeared to involve a protein kinase C (PKC) signaling cascade, in so far as pretreatment of synaptosomes with the PKC inhibitors Ro318220 or GF109203X all effectively occluded the inhibitory effect of honokiol. Furthermore, honokiol attenuated 4-AP-induced phosphorylation of PKC. Together, these results suggest that honokiol effects a decrease in PKC activation, which subsequently attenuates the Ca(2+) entry through voltage-dependent N- and P/Q-type Ca(2+) channels to cause a decrease in evoked glutamate exocytosis. These actions of honokiol may contribute to its neuroprotective effect in excitotoxic injury.

CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin αIIbβ3 in platelets

AbstractSecond messenger-mediated inside-out activation of integrin αIIbβ3 is a key step in platelet aggregation. We recently showed strongly impaired but not absent αIIbβ3-mediated aggregation of CalDAG-GEFI–deficient platelets activated with various agonists. Here we further evaluated the roles of CalDAG-GEFI and protein kinase C (PKC) for αIIbβ3 activation in platelets activated with a PAR4 receptor–specific agonist, GYPGKF (PAR4p). Compared with wild-type controls, platelets treated with the PKC inhibitor Ro31-8220 or CalDAG-GEFI–deficient platelets showed a marked defect in aggregation at low (< 1mM PAR4p) but not high PAR4p concentrations. Blocking of PKC function in CalDAG-GEFI–deficient platelets, how-ever, strongly decreased aggregation at all PAR4p concentrations, demonstrating that CalDAG-GEFI and PKC represent separate, but synergizing, pathways important for αIIbβ3 activation. PAR4p-induced aggregation in the absence of CalDAG-GEFI required cosignaling through the Gαi-coupled receptor for ADP, P2Y12. Independent roles for CalDAG-GEFI and PKC/Gαi signaling were also observed for PAR4p-induced activation of the small GTPase Rap1, with CalDAG-GEFI mediating the rapid but reversible activation of this small GTPase. In summary, our study identifies CalDAG-GEFI and PKC as independent pathways leading to Rap1 and αIIbβ3 activation in mouse platelets activated through the PAR4 receptor.

The ATP-gated P2X1 Receptor Plays a Pivotal Role in Activation of Aspirin-treated Platelets by Thrombin and Epinephrine

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.

Migration of renal carcinoma cells is dependent on protein kinase Cδ via β1 integrin and focal adhesion kinase

Migration and adhesion of tumor cells are essential prerequisites for the formation of metastases in malignant diseases. Protein kinase C (PKC) has been shown to regulate cell migration, adhesion and proliferation. In order to identify a connection between PKC isoforms and tumor progression in renal cell carcinoma (RCC), the influence of PKC isoforms on cell migration, adhesion and proliferation and possible influences of the activity of integrins and focal adhesion kinase (FAK) were analyzed in RCC cells. The experiments were performed in the RCC cell line CCF-RC1 after pre-incubation of the cells with the PKC inhibitors GF109203X, GO6976, RO31-8220 and rottlerin. Cell migration and adhesion were assessed through chemotaxis analysis and adhesion to an endothelial monolayer, respectively. Cell proliferation was analysed by a BrdU incorporation assay. The expression and activity of beta1 integrins and FAK were analysed by Western blot analysis. GF109203X reduced cell migration to 69%, the activity of beta1 integrins to 63% and FAK expression to 82% compared to untreated cells. Rottlerin reduced cell migration in a concentration-dependent manner to 36%, cell proliferation to 81%, expression and activity of beta1 integrins to 72 and 79%, and expression and activity of FAK to 56 and 76% of untreated cells, respectively. RO31-8220 also reduced the expression and activity of beta1 integrins as well as the expression of FAK to 84, 66 and 66% of untreated cells, respectively. GO6976 reduced the expression of FAK to 60% of untreated cells. Cell migration was only slightly reduced by GO6976 to 84% of untreated cells, and cell adhesion remained uninfluenced. These findings show a critical role of PKCdelta in the regulation of tumor cell migration, which seems to be caused by affecting the expression and activity of beta1 integrins and FAK. These results can provide a basis for new strategies in preventing metastases of renal cell carcinoma.

Protein Kinase C Synergistically Stimulates Tumor Necrosis Factor-α–Induced Secretion of Urokinase-Type Plasminogen Activator in Human Dental Pulp Cells

Plasminogen activator (PA) is the enzyme converting plasminogen to its active form, plasmin, involved in various physiological and pathological phenomena. The conversion is catalyzed by two types of PA, urokinase-type PA (uPA) and tissue-type PA (tPA). When human dental pulp cells were stimulated by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), PA activity in the conditioned medium was increased, indicating that TNF-alpha provoked PA secretion. The TNF-alpha-induced PA release was significantly enhanced in the presence of phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator. The PKC inhibitor Ro31-8220 abolished the effect of PMA on the PA release. The activity of PA secreted from the cells stimulated by TNF-alpha and PMA was reduced by immunoprecipitation using anti-uPA antibody. PMA failed to enhance the TNF-alpha-induced expression of uPA mRNA. These results suggest that protein kinase C synergistically enhances the secretion of uPA in TNF-alpha-stimulated human dental pulp cells.

Regulation of protein kinase D activity in adult myocardium: Novel counter-regulatory roles for protein kinase Cε and protein kinase A

Protein kinase D (PKD) is activated downstream of protein kinase C (PKC) in many cell types, although little is known about the mechanisms that regulate PKD in adult myocardium. Exposure of cultured adult rat ventricular myocytes (ARVM) to phorbol 12-myristate 13-acetate (PMA; 100 nM for 5 min) activated PKD, as evidenced by significantly increased phosphorylation at Ser744/8 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). PKD activation occurred concomitantly with translocation of the enzyme from the cytosolic to the particulate fraction. The role of PKC was confirmed by pretreatment (15 min) of ARVM with the PKC inhibitors GF109203X (1 μM) and Ro31-8220 (1 μM), both of which prevented PKD phosphorylation on subsequent exposure to PMA. Exposure of ARVM to endothelin-1 (ET1; 100 nM for 10 min) also activated PKD by a PKC-dependent mechanism. To determine the PKC isoform(s) involved in the ET1-induced PKD activation, ARVM were infected with adenoviral vectors encoding dominant-negative (DN) mutants of PKCα, PKCδ and PKCε. Expression of DN-PKCα and DN-PKCδ had little effect on ET1-induced PKD activation, whilst this was significantly attenuated by expression of DN-PKCε, indicating that PKCε plays a predominant role in the pertinent ET1 signaling pathway. Intriguingly, prior exposure to the adenylyl cyclase activator forskolin (1 μM for 5 min) or the β-adrenergic agonist isoprenaline (100 nM for 5 min) markedly attenuated ET1-induced PKD activation, but not PMA-induced PKD activation. The ET1-induced response was rescued when protein kinase A (PKA) was inhibited (H89, 10 μM) before exposure to isoprenaline. These results show that ET1-induced PKD activation in ARVM is mediated by PKC, primarily the PKCε isoform, and is suppressed by PKA activation.

The effects of protein kinase C (PKC) on the tension of normal and passively sensitized human airway smooth muscle and the activity of voltage-dependent delayed rectifier potassium channel (Kv)

The effects of protein kinase C (PKC) on the tension and the activity of voltage-dependent delayed rectifier potassium channel (K(y)) were examined in normal and passively sensitized human airway smooth muscle (HASM), by measuring tones and whole-cell patch clamp techniques, and the K(v) activities and membrane potential (E (m)) were also detected. The results showed that phorbol 12-myristate 13-acetate (PMA), a PKC activator, caused a concentration-dependent constriction in normal HASM rings. The constriction of the passively sensitized muscle in asthma serum group was significantly higher than that of the normal group (P<0.05), and the constrictions of both groups were completely abolished by PKC inhibitor Ro31-8220 and calcium channel inhibitor nifedipine. K(v) activities of HASM cells were significantly inhibited by PMA, and the E (m) became more positive, as compared with the DMSO (a PMA menstruum)-treated group (P<0.01). This effect could be blocked by Ro31-8220 (P<0.01). It was concluded that activation of PKC could increase the tones of HASM, which might be related to the reduced K(v) activity. In passively sensitized HASM rings, this effect was more notable.

Injury‐induced Janus kinase/protein kinase C‐dependent phosphorylation of growth‐associated protein 43 and signal transducer and activator of transcription 3 for neurite growth in dorsal root ganglion

Elevation of corticosteroids and excessive glutamate release are the two major stress responses that occur sequentially during traumatic CNS injury. We have previously reported that sequential application of corticosterone and kainic acid (CORT + KA) mimicking the nerve injury condition results in synergistic enhancement of neurite outgrowth and expression of growth-associated protein 43 (GAP-43) in cultured dorsal root ganglion (DRG). GAP-43 is known to promote neurite extension when phosphorylated by protein kinase C (PKC). In addition, PKC can phosphorylate the signal transducer and activator of transcription 3 (STAT3) at Ser727, which is phosphorylated primarily by Janus kinase (JAK) at Tyr705. In this study, we further examine the role of PKC in this stress-induced growth-promoting effect. In the cultured DRG neurons, the JAK inhibitor AG-490 and the PKC inhibitor Ro-318220 reduced the CORT + KA-enhanced neurite growth effect when applied prior to CORT and KA treatment, respectively. Both AG-490 and Ro-318220 diminished the CORT + KA-enhanced GAP-43 expression, phosphorylation, and axonal localization. Furthermore, CORT + KA treatment synergistically phosphorylated STAT3 at Ser727 but not at Tyr705. Similar phenomena were observed in an animal model of acute spinal cord injury (SCI), in which phosphorylation of GAP-43 and phospho-Ser727-STAT3 was elevated in the injured DRG 4 hr after the impact injury. Further treatment with the therapeutic glucocorticoid methylprednisolone enhanced the phosphorylation of GAP-43 in both the DRG and the spinal cord of SCI rats. These results suggest that elevated glucocorticoids and overexcitation following CNS injury contribute to nerve regeneration via induction of JAK/PKC-mediated GAP-43 and STAT3 activities.

Salviae miltiorrhizae radix increases dopamine release of rat and pheochromocytoma PC12 cells

The Radix of Salvia miltiorrhiza Bunge (Labiatae) (SMR), an eminent herb, is often included as an ingredient in various herbal remedies recommended for vascular circulation therapies. The present study investigated the effect of SMR on dopaminergic neurotransmission. Various extracts prepared from the stems of SMR were tested for cytotoxic activity on pheochromocytoma PC12 cells using the XTT assay method. The ethanol extract (IC50 > 100 microg/mL), water extract (IC50 > 100 microg/mL) and chloroform (IC50 = 90 microg/mL) fraction exhibited weak cytotoxic activity. However, the butanol (IC50 = 80 microg/mL) and ethyl acetate (EtOAc; IC50 = 70 microg/mL) fractions exhibited strong cytotoxic activity. Also, the extracts and fractions were investigated for dopamine release effects. The EtOAc fraction showed a stronger stimulatory effect on dopamine release activity than the other fractions. The effect of the crude EtOAc fraction (50 microg/mL) of SMR on K+ (20 mm)-stimulated dopamine (DA) release from rat striatal slices was compared with amphetamine (10(-4) m) using high-performance liquid chromatography with electrochemical detection to measure endogenous DA. The EtOAc fraction significantly increased K+ -stimulated DA release (p < 0.001) from rat striatal slices when compared with K+ -stimulated alone. The EtOAc fraction potentiated the effect of amphetamine on K+ -stimulated DA release (p < 0.001) when compared with amphetamine alone. To examine whether in vitro the EtOAc fraction treatment induces DA release in PC12 cells, the role of protein kinases was investigated in the induction of the EtOAc fraction-mediated events by using inhibitors of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) or protein kinase A (PKA). The PKC inhibitors chelerythrine (50 nm and 100 nm) and Ro31-8220 (100 nm) and the MAP kinase kinase inhibitor, PD98059 (20 microm), inhibited the ability of the EtOAc fraction of SMR to elicit the EtOAc fraction-stimulated DA release. The PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 100 nm) mimicked the ability of the EtOAc fraction of SMR to elicit DA release. In contrast, a selective PKA inhibitor, 50 microm Rp-8-Br-cAMP, blocked the development of EtOAc fraction-stimulated DA release. It was demonstrated that the EtOAc fraction of SMR stimulated DA release. Therefore the mechanism by which the EtOAc fraction of SMR induced the enhancement in EtOAc fraction-stimulated DA release is apparent.

Differential participation of protein kinase C and Rho kinase in α1-adrenoceptor mediated contraction in rat arteries

The major functional alpha1-adrenoceptor in the rat aorta is of the alpha1Dsubtype and that in the caudal artery is of the alpha1A subtype. In the present study, the participation of protein kinase C (PKC) and Rho kinase (RhoK) in contractile responses to stimulation of the alpha1-adrenoceptors in these two arteries was investigated. Both the PKC inhibitor Ro-318220 and the RhoK inhibitor Y-27632 significantly blocked contractile responses of the aorta to phenylephrine (PE) and the selective alpha1A-adrenoceptor agonist A61603. When used in combination, the inhibitors had an additive blocking effect. In the caudal artery, Y-27632 but not Ro-318220 inhibited contractile responses to PE and A61603, and, in combination, the antagonism produced was no greater than that by Y-27632 alone. Contractile responses to direct activation of PKC with phorbol 12,13-dibutyrate were much smaller and levels of CPI-17 (PKC-activated protein phosphatase inhibitor of 17 kDa) were much lower in the caudal artery than the aorta. The results suggest that both PKC and RhoK contribute independently to contractile responses to stimulation of alpha1D-adrenoceptors in the aorta. However, RhoK, but not PKC, participates in contractile responses to stimulation of alpha1A-adrenoceptors in the caudal artery. This difference may largely be due to differences between the two arteries in the extent to which PKC participates in contraction.

Identifying pathways involved in leptin-dependent aggregation of human platelets.

To investigate the role of phospholipase C (PLC), phospholipase A(2) (PLA(2)), calcium, and protein kinase C (PKC) in mediating leptin-enhanced aggregation of human platelets. In vitro, ex vivo study. Outpatient's Service for Prevention and Treatment of Obesity at the University Hospital of Messina, Italy. In total, 14 healthy normal-weight male (age 31.4+/-1.9 y; body mass index 22.7+/-0.6 kg/m2) subjects. Adenosine diphosphate-(ADP-) induced platelet aggregation and platelet free calcium were measured after incubation of platelets with leptin alone (5-500 ng/ml), or leptin (50 and 100 ng/ml) in combination with anti-human leptin receptor long form antibody (anti-ObRb-Ab, 1:800-1:100 dilutions), PLC inhibitor U73122 (3.125-25 microM), PLA(2) inhibitor AACOCF3 (1.25-10 microM), or PKC inhibitor Ro31-8220 (1.25-10 microM). Platelet stimulation with leptin leads to a significant and dose-dependent increase in ADP-induced platelet aggregation and platelet free calcium concentrations. Leptin effects on both platelet aggregation and calcium mobilization were completely abated by the co-incubation with leptin and anti-ObRb-Ab. Leptin-induced platelet aggregation was dose-dependently inhibited by U73122, AACOCF3, or Ro31-8220. The effect of leptin on intracellular calcium was inhibited in a dose-dependent manner by incubation with U73122 and AACOCF3, but not with Ro31-8220. Our study confirms that leptin is able to enhance ADP-induced aggregation of human platelets, and raise the possibility that PLC, PKC, PLA(2), and calcium could play a relevant role in mediating the proaggregating action of leptin.