PKC-independent Pathway Research Articles

Protein kinase inhibitor responses in uveal melanoma reflects a diminished dependency on PKC-MAPK signaling

Uveal melanoma (UM) is a rare cancer arising from melanocytes in the uveal tract of the eye. Despite effective primary treatment, there is no approved therapy for metastatic UM and prognosis and survival remain poor. Over 90% of UM are driven by mutations affecting the Gα subunits encoded by the GNAQ and GNA11 genes. These mutations activate downstream and targetable signaling pathways, including the protein kinase C (PKC) cascade. PKC inhibitors have been used in clinical trials for metastatic UM but have shown limited efficacy. In this study, we examined the signaling and functional effects of two PKC inhibitors (AEB071 and IDE196) in a panel of UM cell models. In response to PKC inhibition, all UM cell lines showed potent suppression of PKC activity, but this was not sufficient to predict PKC inhibitor sensitivity and only two UM cell lines showed substantial PKC inhibitor-induced cell death. The differences in UM cell responses to PKC inhibition were not attributable to the degree or timing of PKC suppression or inhibition of the downstream mitogen-activated protein kinase (MAPK) or phosphatidylinositol-3-kinase (PI3K) pathways. Instead, UM cell show complex, PKC-independent signaling pathways that contribute to their survival and resistance to targeted therapies.

Handling has an anxiolytic effect that is not affected by the inhibition of the protein kinase C pathway in adult prenatal undernourished male rat offspring.

Environmental enrichment (EE) after birth has been reported as an intervention improving the anxiety-like behavior and cognitive deficit due to maternal restraint, foot-shock, or social stress during pregnancy. However, it remains unclear whether EE after birth could benefit the early prenatal undernourished offspring. In this study, we examined the effect of daily handling as a simple EE intervention on the aberrant behavior of prenatally undernourished rats. The male rat offspring exhibited anxiety-like behavior at 9 weeks of age due to maternal food restriction in early pregnancy. Our study shows that the daily handling after weaning has an anxiolytic effect in the prenatally undernourished offspring without affecting the behavior of prenatally well-nourished offspring. Conversely, the concentrations of dopamine, serotonin, norepinephrine, and their metabolites were not altered in the prefrontal cortex by prenatal undernutrition or daily handling after weaning. We investigated whether the anxiolytic effect of daily handling was mediated by the protein kinase C (PKC) pathway using the PKC inhibitor, chelerythrine. The anxiolytic effect of the handling was not canceled by chelerythrine injection in prenatally undernourished offspring, whereas chelerythrine induced an anxiety-like behavior in control rats. Our results suggest that maternal undernutrition in early pregnancy induces an anxiety-like behavior accompanied with a PKC pathway-hyporesponsiveness; however, daily handling ameliorates the anxiety-like behavior through a PKC-independent pathway.

Muscarinic suppression of ATP-sensitive K+ channels mediated by the M3/Gq/11/phospholipase C pathway contributes to mouse ileal smooth muscle contractions.

ATP-sensitive K+ (KATP) channels are expressed in gastrointestinal smooth muscles, and their activity is regulated by muscarinic receptor stimulation. However, the physiological significance and mechanisms of muscarinic regulation of KATP channels are not fully understood. We examined the effects of the KATP channel opener cromakalim and the KATP channel blocker glibenclamide on electrical activity of single mouse ileal myocytes and on mechanical activity in ileal segment preparations. To explore muscarinic regulation of KATP channel activity and its underlying mechanisms, the effect of carbachol (CCh) on cromakalim-induced KATP channel currents ( IKATP) was studied in myocytes of M2 or M3 muscarinic receptor-knockout (KO) and wild-type (WT) mice. Cromakalim (10 µM) induced membrane hyperpolarization in single myocytes and relaxation in segment preparations from WT mice, whereas glibenclamide (10 µM) caused membrane depolarization and contraction. CCh (100 µM) induced sustained suppression of IKATP in cells from both WT and M2KO mice. However, CCh had a minimal effect on IKATP in M3KO and M2/M3 double-KO cells. The Gq/11 inhibitor YM-254890 (10 μM) and PLC inhibitor U73122 (1 μM), but not the PKC inhibitor calphostin C (1 μM), markedly decreased CCh-induced suppression of IKATP in WT cells. These results indicated that KATP channels are constitutively active and contribute to the setting of resting membrane potential in mouse ileal smooth muscles. M3 receptors inhibit the activity of these channels via a Gq/11/PLC-dependent but PKC-independent pathways, thereby contributing to membrane depolarization and contraction of smooth muscles. NEW & NOTEWORTHY We systematically investigated the regulation of ATP-sensitive K+ channels by muscarinic receptors expressed on mouse ileal smooth muscles. We found that M3 receptors inhibit the activity of ATP-sensitive K+ channels via a Gq/11/PLC-dependent, but PKC-independent, pathway. This muscarinic suppression of ATP-sensitive K+ channels contributes to membrane depolarization and contraction of smooth muscles.

α-Ketoglutarate stimulates pendrin-dependent Cl- absorption in the mouse CCD through protein kinase C.

α-Ketoglutarate (α-KG) is a citric acid cycle intermediate and a glutamine catabolism product. It is also the natural ligand of 2-oxoglutarate receptor 1 (OXGR1), a Gq protein-coupled receptor expressed on the apical membrane of intercalated cells. In the cortical collecting duct (CCD), Cl-/[Formula: see text] exchange increases upon α-KG binding to the OXGR1. To determine the signaling pathway(s) by which α-KG stimulates Cl- absorption, we examined α-KG-stimulated Cl- absorption in isolated perfused mouse CCDs. α-KG increased electroneutral Cl- absorption in CCDs from wild-type mice but had no effect on Cl- absorption in pendrin knockout mice. Because Gq protein-coupled receptors activate PKC, we hypothesized that α-KG stimulates Cl- absorption through PKC. If so, PKC agonists should mimic, whereas PKC inhibitors should abolish, α-KG-stimulated Cl- absorption. Like α-KG, PKC agonist (phorbol-12,13-dibutyrate, 500 nM) application increased Cl- absorption in wild-type but not in pendrin null CCDs. Moreover, PKC inhibitors (2.5 mM GF109203X and 20 nM calphostin C), Ca2+ chelators (BAPTA, 10-20 μM), or PKC-α or -δ gene ablation eliminated α-KG-stimulated Cl- absorption. We have shown that STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) gene ablation increases urinary α-KG excretion, renal pendrin abundance, and CCD Cl- absorption. However, in SPAK null CCDs, Cl- absorption was not activated further by luminal α-KG application nor was Cl- absorption reduced with the PKC inhibitor GF109203 . Thus SPAK gene ablation likely acts through a PKC-independent pathway to produce a chronic adaptive increase in pendrin function. In conclusion, α-KG stimulates pendrin-dependent Cl-/[Formula: see text] exchange through a mechanism dependent on PKC and Ca2+ that involves PKC-α and PKC-δ.

Intracellular Ca2+ is an essential factor for cell damage induced by unsaturated carbonyl compounds

The unsaturated carbonyl compounds are known as the environmental pollutants. Acrolein (ACR) and methyl vinyl ketone (MVK) are representative unsaturated carbonyl compounds. ACR is contained in smoke, automobile exhaust, industrial waste, and several foods. MVK is widely used as the industrial chemical. Although ACR and MVK are highly toxic, the molecular mechanism for their cytotoxicity has been unclear. We have previously reported that ACR and MVK are major cytotoxic compounds in the gas phase of cigarette smoke, and protein kinase C (PKC) inhibitor and NADPH oxidases inhibitor partially rescued cells from ACR- or MVK-induced cell death (Noya etal., Toxicology, 314, 1-10, 2013). PKC translocation, which is hallmark for PKC activation, and cell damage were induced by treatment of cultured cells with ACR or MVK. Intracellular Ca2+ chelator completely suppressed ACR- or MVK-induced PKC translocation to the cell membrane and cell damage, while extracellular Ca2+ chelator had no effects on ACR- and MVK-induced cytotoxicity. These results suggest that intracellular Ca2+ is an essential factor for cell damage caused by both PKC-dependent and PKC-independent pathways, and mobilization of Ca2+ from intracellular Ca2+ stores is induced by ACR or MVK.

Inhibition of Chikungunya Virus-Induced Cell Death by Salicylate-Derived Bryostatin Analogues Provides Additional Evidence for a PKC-Independent Pathway.

Chikungunya virus (CHIKV) has been spreading rapidly, with over one million confirmed or suspected cases in the Americas since late 2013. Infection with CHIKV causes devastating arthritic and arthralgic symptoms. Currently, there is no therapy to treat this disease, and the only medications focus on relief of symptoms. Recently, protein kinase C (PKC) modulators have been reported to inhibit CHIKV-induced cell death in cell assays. The salicylate-derived bryostatin analogues described here are structurally simplified PKC modulators that are more synthetically accessible than the natural product bryostatin 1, a PKC modulator and clinical lead for the treatment of cancer, Alzheimer's disease, and HIV eradication. Evaluation of the anti-CHIKV activity of these salicylate-derived bryostatin analogues in cell culture indicates that they are among the most potent cell-protective agents reported to date. Given that they are more accessible and significantly more active than the parent natural product, they represent new therapeutic leads for controlling CHIKV infection. Significantly, these analogues also provide evidence for the involvement of a PKC-independent pathway. This adds a fundamentally distinct aspect to the importance or involvement of PKC modulation in inhibition of chikungunya virus replication, a topic of recent and growing interest.

Calcium-Dependent PKC Isoforms Have Specialized Roles in Short-Term Synaptic Plasticity

Posttetanic potentiation (PTP) is a widely observed form of short-term plasticity lasting for tens of seconds after high-frequency stimulation. Here we show that although protein kinase C (PKC) mediates PTP at the calyx of Held synapse in the auditory brainstem before and after hearing onset, PTP is produced primarily by an increased probability of release (p) before hearing onset, and by an increased readily releasable pool of vesicles (RRP) thereafter. We find that these mechanistic differences, which have distinct functional consequences, reflect unexpected differential actions of closely related calcium-dependent PKC isoforms. Prior to hearing onset, when PKCγ and PKCβ are both present, PKCγ mediates PTP by increasing p and partially suppressing PKCβ actions. After hearing onset, PKCγ is absent and PKCβ produces PTP by increasing RRP. In hearing animals, virally expressed PKCγ overrides PKCβ to produce PTP by increasing p. Thus, two similar PKC isoforms mediate PTP in distinctly different ways.

P22 High content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC independent pathway in myotonic dystrophy cell lines

P22 High content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC independent pathway in myotonic dystrophy cell lines

GATA-dependent regulation of TPO-induced c-mpl gene expression during megakaryopoiesis.

Thrombopoietin (TPO) and its receptor, c-Mpl, play the crucial role during megakaryocytopoiesis. Previously, we have shown that the promoter activity of c-mpl induced by TPO is modulated by transcription through a PKC-dependent pathway and that GATA(-77) is involved as a positive regulatory element in TPO-induced c-mpl gene expression in the megakaryoblastic CMK cells. In this research, to examine participating possibility of GATA promoter element in TPO- induced c-mpl gene expression through a PKC-independent pathway, the promoter activity of site-directed mutagenesis and the effect of potein kinase C modulator were measured by a transient transfection assay system. Together with our previous results on the TPO-induced c-mpl promoter, this study indicates destruction of -77GATA in c-mpl promoter decreased the activity by 47.3% under existence of GF109203. These results suggest that GATA promoter element plays significant role in TPO-induced c-mpl gene expression through a PKC-independent pathway.

Shengmaisan regulates pacemaker potentials in interstitial cells of cajal in mice.

Objectives:Shengmaisan (SMS) is a traditional Chinese medicine prescription widely used for the treatment of diverse organs in Korea. The interstitial cells of Cajal (ICCs) are pacemaker cells that play an important role in the generation of coordinated gastrointestinal (GI) motility. We have aimed to investigate the effects of SMS in the ICCs in the mouse small intestine.Methods:To dissociate the ICCs, we used enzymatic digestions from the small intestine in a mouse. After that, the ICCs were identified immunologically by using the anti-c-kit antibody. In the ICCs, the electrophysiological whole-cell patch-clamp configuration was used to record pacemaker potentials in the cultured ICCs.Results:The ICCs generated pacemaker potentials in the mouse small intestine. SMS produced membrane depolarization with concentration-dependent manners in the current clamp mode. Pretreatment with a Ca2+ free solution and thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum, stopped the generation of the pacemaker potentials. In the case of Ca2+-free solutions, SMS induced membrane depolarizations. However, when thapsigargin in a bath solution was applied, the membrane depolarization was not produced by SMS. The membrane depolarizations produced by SMS were inhibited by U-73122, an active phospholipase C (PLC) inhibitors. Furthermore, chelerythrine and calphostin C, a protein kinase C (PKC) inhibitors had no effects on SMS-induced membrane depolarizations.Conclusions:These results suggest that SMS might affect GI motility by modulating the pacemaker activity through an internal Ca2+- and PLC-dependent and PKC-independent pathway in the ICCs.

High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.

Abstract 160: Contribution of IP3 Receptors in the Electromechanical Response of LV Myocytes to Gq-protein Coupled Receptor Agonists

Gq-protein coupled receptor (GPCR) stimulation promotes PLC function, generating diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). The latter may promote Ca2+ translocation from intracellular stores altering Ca2+ homeostasis in cardiomyocytes. The aim of this study was to establish whether GPCR agonists enhance IP3 receptor (IP3R) activity, affecting the electromechanical properties of LV myocytes. For this purpose, the functional responses of myocytes to GPCR agonists ATP or ET-1 were established. In field-stimulated cells, GPCR activators increased diastolic Ca2+, transient amplitude, and contractility; extra-systolic Ca2+ releases and aftercontractions were promoted. These responses were prevented by inhibition of PLC, or blockade of IP3Rs. Since DAG promotes PKC activity, the effects of GPCR stimulation were tested in the presence of the PKC inhibitor chelerythrine. This compound failed to abrogate the effects of ATP and ET-1, indicating that PKC-independent pathways play a critical role in mediating the observed cellular responses to GPCR stimulation. Additionally, an AAV9 vector carrying EGFP and sh-RNA targeting IP3R type-2 was employed in vivo to downregulate IP3Rs. GPCRs activation failed to increase Ca2+ transients and to induce extra-systolic Ca2+ elevations in EGFP-positive myocytes. Conversely, these responses were preserved in EGFP-negative cells. In patch-clamped myocytes, changes in Ca2+ transient properties following GPCR activation were accompanied by a decrease in resting potential, action potential (AP) prolongation, and emergence of arrhythmic events. Similar electrical disturbances were detected by direct activation of IP3R with IP3 dialysis, or by enhancing the affinity of the receptors to its ligand, with thimerosal. To establish whether Ca2+ mobilized from the sarcoplasmic reticulum (SR) to the cytoplasm via IP3Rs was responsible for the electrical alterations caused by GPCR agonists, experiments were performed in which SR Ca2+ was depleted, or cytosolic Ca2+ was buffered. Under these conditions, ATP and ET-1 failed to prolong the AP and to induce arrhythmias. In conclusion, the GPCR/IP3R axis regulates Ca2+ homeostasis, contractile performance and the electrical stability of LV myocytes.

Differential PKC-dependent and -independent PKD activation by G protein α subunits of the Gq family: Selective stimulation of PKD Ser748 autophosphorylation by Gαq

Protein kinase D (PKD) is activated within cells by stimulation of multiple G protein coupled receptors (GPCR). Earlier studies demonstrated a role for PKC to mediate rapid activation loop phosphorylation-dependent PKD activation. Subsequently, a novel PKC-independent pathway in response to Gαq-coupled GPCR stimulation was identified. Here, we examined further the specificity and PKC-dependence of PKD activation using COS-7 cells cotransfected with different Gq-family Gα and stimulated with aluminum fluoride (AlF4−). PKD activation was measured by kinase assays, and Western blot analysis of activation loop sites Ser744, a prominent and rapid PKC transphosphorylation site, and Ser748, a site autophosphorylated in the absence of PKC signaling. Treatment with AlF4− potently induced PKD activation and Ser744 and Ser748 phosphorylation, in the presence of cotransfected Gαq, Gα11, Gα14 or Gα15. These treatments achieved PKD activation loop phosphorylation similar to the maximal levels obtained by stimulation with the phorbol ester, PDBu. Preincubation with the PKC inhibitor GF1 potently blocked Gα11-, Gα14-, and Gα15-mediated enhancement of Ser748 phosphorylation induced by AlF4−, and largely abolished Ser744 phosphorylation. In contrast, Ser748 phosphorylation was almost completely intact, and Ser744 phosphorylation was significantly activated in cells cotransfected with Gαq. Importantly, the differential Ser748 phosphorylation was also promoted by treatment of Swiss 3T3 cells with Pasteurella multocida toxin, a selective activator of Gαq but not Gα11. Taken together, our results suggest that Gαq, but not the closely related Gα11, promotes PKD activation in response to GPCR ligands in a unique manner leading to PKD autophosphorylation at Ser748.

ANG II inhibits insulin-mediated production of PI 3,4,5-trisphosphates via a Ca2+-dependent but PKC-independent pathway in the cardiomyocytes

Insulin resistance (IR) is a condition where different organs are refractory to insulin stimulation of glucose uptake. ANG II has been suggested to be involved in the development of IR in the heart. The precise mechanism by which this occurs is still unknown. Here we have used dynamic fluorescent imaging techniques to show that ANG II inhibits insulin production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] in cardiac myocytes. Fluorophore (Venus)-conjugated cAMP-dependent protein kinase-pleckstrin homology domain, which specifically binds to PI(3,4,5)P(3), was transfected in neonatal rat cardiac myocytes. Insulin induced a robust increase in the fluorescence intensity at the cell surface, which was diminished by application of ANG II. The inhibitory action of ANG II was antagonized by RNH-6270 (an angiotensin type 1 receptor antagonist) but not by PD-122370 (an angiotensin type 2 receptor antagonist). BAPTA-AM (Ca(2+) chelator) largely attenuated the ANG II effect, whereas K-252b (PKC inhibitor) did not. Furthermore, an elevation of intracellular Ca(2+) induced by ionomycin mimicked the ANG II effect. Therefore, it is suggested that ANG II antagonizes insulin-mediated production of PI(3,4,5)P(3) via a Ca(2+)-dependent but PKC-independent pathway in cardiac myocytes.

S1708 PKD Selectively Prolongs ERK Signaling and Potentiates DNA Synthesis in Response to Gq-Coupled Receptor Agonists Through a PKC-Independent but EGFR-Dependent Pathway

S1708 PKD Selectively Prolongs ERK Signaling and Potentiates DNA Synthesis in Response to Gq-Coupled Receptor Agonists Through a PKC-Independent but EGFR-Dependent Pathway

Signaling and Dynamics of Activation of LFA-1 and Mac-1 by Immobilized IL-8

The dynamic response of neutrophils to interleukin-8 (IL-8) is of central interest in inflammation. Chemokine -induced β(2) integrin dependent adhesion can take several minutes after initial contact with IL-8 as evidenced by increased cell adhesion to intracellular adhesion molecule 1 (ICAM-1). The goal of this study is to identify signaling events that are critical for this response. We demonstrate that neither the PI3K inhibitor wortmannin, nor the PKC inhibitor bisindolymaleimide had any effect on IL-8 induced adhesion to ICAM-1. However, inhibition of PLC with U73122 or stopping the release of intracellular calcium by its downstream effector IP3 with caffeine or 2-aminoethoxydiphenyl borate completely blocked the adhesive response. Chelation of intracellular calcium with BAPTA or extracellular calcium with EGTA completely abrogated neutrophil adhesion to ICAM-1. This adhesion is mediated by LFA-1 (α(L)β(2)) within first 300 seconds after chemokine stimulation, followed by Mac-1 (α(M)β(2)) mediated adhesion, beginning 350 seconds after stimulus. Inhibition of p38MAP kinase results in a time course similar to Mac-1 inhibition, consistent with published evidence that Mac-1 mediated adhesion is p38MAP kinase dependent. These findings confirm a PLC dependent, PKC independent pathway from chemokine stimulus to integrin activation previously identified in other cell types, and demonstrate distinct dynamics and different requirements for LFA-1 vs. Mac-1 activation in primary human neutrophils.

Protein Kinase D Mediates Mitogenic Signaling by Gq-coupled Receptors through Protein Kinase C-independent Regulation of Activation Loop Ser744 and Ser748 Phosphorylation

Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to G(q)-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3-10 nm) or with vasopressin, a different G(q)-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser(744) and Ser(748). Transphosphorylation targeted Ser(744), whereas autophosphorylation was the predominant mechanism for Ser(748) in cells stimulated with G(q)-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to G(q)-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of G(q)-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.

Protective mechanisms of NO preconditioning against NO-induced apoptosis in H9c2 cells: role of PKC and COX-2

Cardiomyocyte apoptosis is involved in several cardiovascular diseases, including ischemia, hypertrophy and heart failure. Nitric oxide (NO) signalling is crucial in the regulation of cardiomyocyte apoptosis, capable of both inducing and preventing apoptosis depending upon the level of NO production. Growing evidence suggests that NO preconditioning has cardioprotective effects, but the mechanism remains unclear. The purpose of this study was to elucidate how NO preconditioning inhibits subsequent NO-induced apoptosis in H9c2 cells. According to the data, preconditioning with a low concentration of sodium nitroprusside (SNP, 0.3 mM) inhibited subsequent high-concentration-SNP (1.5 mM)-induced apoptosis and this effect was reversed by the protein kinase C (PKC) inhibitor chelerythrine and the cyclooxygenase-2 (COX-2) inhibitor rofecoxib. Low-concentration-SNP-mediated protection involved extracellular signal regulated kinase 1/2 (ERK1/2), a signal transducers and activators of transcription 1/3 (STAT1/3) activation and increased COX-2 expression. Activation of ERK1/2 and STAT1/3 was abolished by chelerythrine. However, COX-2 expression was not inhibited, implying that the COX-2-mediated protective effect occurred via a PKC-independent pathway. The results showed that low-concentration-SNP preconditioning suppresses subsequent high-concentration-SNP-induced apoptosis by ERK1/2-STAT 1/3 activation via PKC-dependent mechanisms in H9c2 cells. COX-2 also plays a role in NO-induced preconditioning, but is independent of PKC.

Panax ginseng induces anterograde transport of pigment organelles in Xenopus melanophores

Melanophores from Xenopus laevis are pigmented cells, capable of quick colour changes through cyclic adenosine 3′:5′-monophosphate (cAMP) coordinated transport of their intracellular pigment granules, melanosomes. In this study we use the melanophore cell line to evaluate the effects of Panax ginseng extract G115 on organelle transport. Absorbance readings of melanophore-coated microplates, Correlate-EIA direct cAMP enzyme immunoassay kit, and western blot were used to measure the melanosome movement and changes in intracellular signalling. We show that Panax ginseng induces a fast concentration-dependent anterograde transport of the melanosomes. No significant increase in the cAMP level was seen and pre-incubation of melanophores with the protein kinase C (PKC) inhibitor EGF-R Fragment 651–658 (M-EGF) only partly decreased the ginseng-induced dispersion. We also demonstrate that Panax ginseng, endothelin-3 (ET-3) and α-melanocyte stimulating hormone (MSH) stimulate an activation of mitogen activated protein kinase (MAPK). Pre-incubation with M-EGF decreased the MAPK activity induced by ET-3 and MSH, but again only marginally affected the response of Panax ginseng. Thus, in melanophores we suggest that Panax ginseng stimulates an anterograde transport of pigment organelles via a non-cAMP and mainly PKC-independent pathway.

Involvement of Thromboxane A2in the Modulation of Pacemaker Activity of Interstitial Cells of Cajal of Mouse Intestine

Although many studies show that thromboxane A(2) (TXA(2)) has the action of gastrointestinal (GI) motility using GI muscle cells and tissue, there are no reports on the effects of TXA(2) on interstitial cells of Cajal (ICC) that function as pacemaker cells in GI tract. So, we studied the modulation of pacemaker activities by TXA(2) in ICC with whole cell patch-clamp technique. Externally applied TXA(2) (5microM) produced membrane depolarization in current-clamp mode and increased tonic inward pacemaker currents in voltage-clamp mode. The tonic inward currents by TXA(2) were inhibited by intracellular application of GDP-beta-S. The pretreatment of ICC with Ca(2+) free solution and thapsigargin, a Ca(2+)-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the TXA(2)-induced tonic inward currents. However, chelerythrine or calphostin C, protein kinase C inhibitors, did not block the TXA(2)-induced effects on pacemaker currents. These results suggest that TXA(2) can regulate intestinal motility through the modulation of ICC pacemaker activities. This modulation of pacemaker activities by TXA(2) may occur by the activation of G protein and PKC independent pathway via extra and intracellular Ca(2+) modulation.