GF 109203X Research Articles

Dendrobium nobile Lindl. alkaloid decreases Tau hyperphosphorylation via regulating PI3K/Akt/GSK-3β pathway in vitro and in vivo

Ethnopharmacological relevantDendrobium is a traditional and precious Chinese medicinal herb. The Compendium of Materia Medica describes its effects as “benefiting intelligence and dispelling shock, lightning the body and extending life”. Dendrobium nobile Lindl. is a precious variety of Dendrobium. Our previous data showed Dendrobium nobile Lindl. alkaloid (DNLA) has significant neuroprotective effects and can improve cognitive dysfunction. However, the specific effects and mechanisms of action of its main active component, DNLA, on cognitive dysfunction caused by Tau hyperphosphorylation, are still unclear. Aim of the researchThis study aimed to determine the effects of DNLA on phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) pathway, thus to explore the mechanisms of DNLA to inhibit Tau hyperphosphorylation. Materials and methodsWe used wortmannin (WM) and GF-109203X (GFX)-induced hyperphosphorylation of Tau in N2a cells and rats to detect the protective mechanism of DNLA in vivo and in vitro. In vitro, the effect of modeling method on Tau hyperphosphorylation was screened and verified by Western Blotting (WB), and the regulation of Tau hyperphosphorylation and PI3K/Akt/GSK-3β pathway by different concentrations of DNLA was detected by WB. In vivo, MWM was used to detect the effect of DNLA on model rats, and then Nissl staining was used to detect the loss of neurons. Finally, WB was used to detect the regulation of Tau hyperphosphorylation and PI3K/Akt/GSK-3β pathway by different concentrations of DNLA. ResultsDNLA could rescue the abnormal PI3K/Akt/GSK-3β pathway and reverse the hyperphosphorylation of Tau induced by WM and GFX in N2a cells. Furthermore, DNLA improved the learning and memory of WM and GFX-induced model rats. Moreover, DNLA regulated PI3K/Akt/GSK-3β pathway and reduced the p-Tau and neuronal damage in the hippocampus of model rats. ConclusionDNLA may be a promising candidate for reducing hyperphosphorylation of Tau.

The role of rhoA/rho-kinase and PKC in the inhibitory effect of L-cysteine/H2S pathway on the carbachol-mediated contraction of mouse bladder smooth muscle.

We investigated the role of RhoA/Rho-kinase (ROCK) and PKC in the inhibitory effect of L-cysteine/hydrogen sulfide (H2S) pathway on the carbachol-mediated contraction of mouse bladder smooth muscle. Carbachol (10-8-10-4M) induced a concentration-dependent contraction in bladder tissues. L-cysteine (H2S precursor; 10-2M) and exogenous H2S (NaHS; 10-3M) reduced the contractions evoked by carbachol by ~ 49 and ~ 53%, respectively, relative to control. The inhibitory effect of L-cysteine on contractions to carbachol was reversed by 10-2M PAG (~ 40%) and 10-3M AOAA (~ 55%), cystathionine-gamma-lyase (CSE) and cystathionine-β-synthase (CBS) inhibitor, respectively. Y-27632 (10-6M) and GF 109203X (10-6M), a specific ROCK and PKC inhibitor, respectively, reduced contractions evoked by carbachol (~ 18 and ~ 24% respectively), and the inhibitory effect of Y-27632 and GF 109203X on contractions was reversed by PAG (~ 29 and ~ 19%, respectively) but not by AOAA. Also, Y-27632 and GF 109203X reduced the inhibitory responses of L-cysteine on the carbachol-induced contractions (~ 38 and ~ 52% respectively), and PAG abolished the inhibitory effect of L-cysteine on the contractions in the presence of Y-27632 (~ 38%). Also, the protein expressions of CSE, CBS, and 3-MST enzymes responsible for endogenous H2S synthesis were detected by Western blot method. H2S level was increased by L-cysteine, Y-27632, and GF 109203X (from 0.12 ± 0.02 to 0.47 ± 0.13, 0.26 ± 0.03, and 0.23 ± 0.06nmol/mg respectively), and this augmentation in H2S level decreased with PAG (0.17 ± 0.02, 0.15 ± 0.03, and 0.07 ± 0.04nmol/mg respectively). Furthermore, L-cysteine and NaHS reduced carbachol-induced ROCK-1, pMYPT1, and pMLC20 levels. Inhibitory effects of L-cysteine on ROCK-1, pMYPT1, and pMLC20 levels, but not of NaHS, were reversed by PAG. These results suggest that there is an interaction between L-cysteine/H2S and RhoA/ROCK pathway via inhibition of ROCK-1, pMYPT1, and pMLC20, and the inhibition of RhoA/ROCK and/or PKC signal pathway may be mediated by the CSE-generated H2S in mouse bladder.

Effects of Aloe vera on the Regulation of Thyroxine Release in FRTL-5 Thyroid Cells

To determine whether Aloe vera (AV), a traditional herbal medicine, could maintain homeostasis, we investigated its ability to regulate the production of hormones, particularly thyroid hormones. T4 ELISA assay with (6H media) and without thyrotropin (TSH) (5H media) showed that AV functions to maintain endocrine homeostasis. To understand the mechanisms by which AV regulates thyroxine release, we confirmed the protein expression of key upstream factors of thyroid hormone synthesis, such as phosphorylated extracellular signal-regulated kinase (ERK), phosphorylated cAMP response element binding protein (CREB), and phosphorylated protein kinase B (p-Akt). Although the protein expression of phosphorylated ERK and CREB were decreased in 6H media, they were increased and unchanged, respectively, in 5H media. However, protein expression of phosphorylated Akt was not changed by AV in both 6H and 5H media. Furthermore, inhibition of protein kinase A (PKA) by H89 eliminated AV-induced phosphorylation of ERK in 5H media. However, the inhibition of protein kinase C (PKC) by GF 109203X did not eliminate AV-induced ERK phosphorylation in 5H media. These results suggested that AV has TSH-like activity and could regulate thyroxine release through the TPO and PKA pathways. AV may regulate thyroid function to maintain a constant level of thyroid hormones in the body, and could be a candidate for thyroid disease therapies.

Tolfenamic acid inhibits GSK-3\u03b2 and PP2A mediated tau hyperphosphorylation in Alzheimer\u2019s disease models

Tolfenamic acid, a nonsteroidal anti-inflammatory drug, alleviated learning and memory deficits and decreased the expression of specificity protein 1 (SP1)-mediated cyclin-dependent kinase-5 (CDK5), a major protein kinase that regulates hyperphosphorylated tau, in Alzheimer’s disease (AD) transgenic mice. However, whether tolfenamic acid can regulate the major tau protein kinase, glycogen synthase kinase-3β (GSK-3β), or tau protein phosphatase, protein phosphatase 2A (PP2A), further inhibiting hyperphosphorylation of tau, remains unknown. To this end, tolfenamic acid was administered i.p. in a GSK-3β overactivation postnatal rat model and orally in mice after intracerebroventricular (ICV) injection of okadaic acid (OA) to develop a PP2A inhibition model. We used four behavioural experiments to evaluate memory function in ICV-OA mice. In this study, tolfenamic acid attenuated memory dysfunction. Tolfenamic acid decreased the expression of hyperphosphorylated tau in the brain by inhibiting GSK-3β activity, decreasing phosphorylated PP2A (Tyr307), and enhancing PP2A activity. Tolfenamic acid also increased wortmannin (WT) and GF-109203X (GFX) induced phosphorylation of GSK-3β (Ser9) and prevented OA-induced downregulation of PP2A activity in PC12 cells. Altogether, these results show that tolfenamic acid not only decreased SP1/CDK5-mediated tau phosphorylation, but also inhibited GSK-3β and PP2A-mediated tau hyperphosphorylation in AD models.

The important role of connexin 43 in subarachnoid hemorrhage-induced cerebral vasospasm

BackgroundGap junctions are involved in the development of cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH). However, the specific roles and regulatory functions of related connexin isoforms remain unknown. The aim of this study was to investigate the importance of connexin 43 (Cx43) in CVS and determine whether Cx43 alterations are modulated via the protein kinase C (PKC) signaling transduction pathway.MethodsOxyhemoglobin (OxyHb)-induced smooth muscle cells of basilar arterial and second-injection model in rat were used as CVS models in vitro and in vivo. In addition, dye transfer assays were used for gap junction-mediated intercellular communication (GJIC) observation in vitro and delayed cerebral ischemia (DCI) was observed in vivo by perfusion-weighted imaging (PWI) and intravital fluorescence microscopy.ResultsIncrease in Cx43 mediated the development of SAH-induced CVS was found in both in vitro and in vivo CVS models. Enhanced GJIC was observed in vitro CVS model, this effect and increased Cx43 were reversed by preincubation with specific PKC inhibitors (chelerythrine or GF 109203X). DCI was observed in vivo on day 7 after SAH. However, DCI was attenuated by pretreatment with Cx43 siRNA or PKC inhibitors, and the increased Cx43 expression in vivo was also reversed by Cx43 siRNA or PKC inhibitors.ConclusionsThese data provide strong evidence that Cx43 plays an important role in CVS and indicate that changes in Cx43 expression may be mediated by the PKC pathway. The current findings suggest that Cx43 and the PKC pathway are novel targets for developing treatments for SAH-induced CVS.

Role of Extracellular Calcium and Calcium Sensitization in the Anti-Contractile Effect of Perivascular Adipose Tissue in Pregnant Rat Aorta

Previous studies have shown that the anti-contractile effect of the perivascular adipose tissue (PVAT) is attenuated in pregnancy. In the present investigation, we have examined the possibility that this loss of anti-contractile effect could be due to changes in calcium mobilization. PVAT exerted anti-contractile effect against 5-hydroxytryptamine (5-HT)-induced contractions of aorta segments from pregnant and non-pregnant rats and this anti-contractile effect was attenuated in segments from pregnant rats. Nifedipine (10–6 mol/L), an inhibitor of L-type dihydropyridine calcium channels, significantly reduced 5-HT-induced contraction of aorta segments from non-pregnant and pregnant rats with and without PVAT. The inhibitory effect of nifedipine against 5-HT-induced contractions was attenuated in PVAT-free aorta segments from pregnant rats. However, while PVAT reduced the effectiveness of nifedipine in aorta segments from non-pregnant rats, it partially restored the inhibitory effect of nifedipine in aorta segments from pregnant rats. Inhibitors of calcium sensitization, Y-27632 (10–6 mol/L) and GF 109203X (10–6 mol/L), significantly reduced 5-HT-induced contractions of PVAT-free aorta segments from non-pregnant and pregnant rats. Both inhibitors, however, were less effective in aorta segments from pregnant rats. The presence of PVAT reduced the effectiveness of Y-27632 and GF 109203X in aorta segments from pregnant and non-pregnant rats. Protein expression of Rho-associated protein kinase (ROCK) I and II was detected in aorta segments and PVAT from pregnant and non-pregnant rats. There was a reduction in the expression of both isoforms in aorta segments but not PVAT from pregnant rats. In addition, there was no significant difference in the expression of ROCK-I and ROCK-II in PVAT from pregnant and non-pregnant rats. We concluded that the loss of anti-contractile effect of PVAT in aorta segments from pregnant rats could be due to increased influx of extracellular calcium through nifedipine-sensitive dihydropyridine channels.

Evidence for metabotropic function of epithelial nicotinic cholinergic receptors in rat colon.

ACh exerts its actions via nicotinic (nAChR) and muscarinic receptors. In the peripheral nervous system, ionotropic nAChR mediate responses in excitable cells. However, recent studies demonstrate the expression of nAChR in the colonic epithelium, which are coupled to an induction of Cl- secretion via activation of the Na+ -K+ -pump. In order to find out whether these epithelial nAChR function as ionotropic receptors, intracellular microelectrode and imaging experiments were performed in isolated crypts from rat colon. Apically permeabilized epithelia were used to measure pump current across the basolateral membrane. Imaging experiments with the Na+ -sensitive dye SBFI revealed that nicotine induced a decrease in the cytosolic Na+ concentration concomitant with a fall in the cytosolic Ca2+ concentration in about 50% of the cells. as shown in fura-2 experiments. Nicotine hyperpolarized the membrane by 6.4±2.1mV. These observations contradict the assumption that epithelial nAChR function as ligand-gated non-selective cation channels. The decrease in the cytosolic Na+ concentration was strongly delayed, when the Na+ -K+ -pump was inhibited by scilliroside. Ussing chamber experiments revealed a strong dependence of the nicotine-induced pump current on the presence of Ca2+ , and chelation of cytosolic Ca2+ with BAPTA prevented the fall in the cytosolic Na+ concentration in SBFI-loaded crypts. Inhibition of PKC with GF 109203X or Goe 6983 significantly reduced the nicotine-induced pump current. These results suggest that epithelial nAChR activate the Na+ -K+ -pump via a PKC dependent on a sufficient cytosolic Ca2+ concentration.

Role of Calcium Sensitization in 5‐Hudroxytryptamine‐Induced Contraction of Rat Aorta with or without Perivascular Adipose Tissue

We have previously shown that the perivascular adipose tissue (PVAT) exerted an anti‐contractile effect against 5‐hydroxytryptamine (5‐HT)‐induced contraction of aorta segments from pregnant and non‐pregnant rats. This anti‐contractile effect was significantly reduced in aorta segments from pregnant rats. In the present investigation, we have examined the possible role of calcium sensitization involving ROCK and PKC in this loss of anti‐contractile effect of PVAT. Both Y‐27632 (10−6M) and GF 109203X (10−6M), inhibitors of calcium sensitization, significantly reduced 5‐HT‐induced contractions of PVAT‐free aorta segments from non‐pregnant and pregnant rats. However, they were less effective in aorta segments from pregnant rats. PVAT reduced the effectiveness of Y‐27632 and GF 109203X in aorta segments from pregnant and non‐pregnant rats. ROCK‐I and ROCK‐II protein were expressed in aorta segments and PVAT from pregnant and non‐pregnant rats. There was a reduction in the expression of both isoforms in aorta segments but not PVAT from pregnant rats. In addition, there was no significant difference in the expression of ROCK‐I and ROCK‐II in PVAT from pregnant and non‐pregnant rats. We concluded that the loss of anti‐contractile effect of PVAT in aorta segments from pregnant rats could not be explained by altered calcium‐sensitization mechanisms.Support or Funding InformationResearch Sector, Kuwait University, KuwaitThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic low dose of AM404 ameliorates the cognitive impairment and pathological features in hyperglycemic 3xTg-AD mice

RationaleHyperglycemia accelerates the progression of Alzheimer’s disease (AD), and GSK3β plays a potential link between AD and hyperglycemia. Therefore, a direct or indirect GSK3β inhibition may have potential to delay the progression of AD. Our previous biochemical assay identified AM404 as a GSK3β inhibitor at high dose (IC50 = 5.353 μM); however, other study suggests that AM404 impaired synaptic plasticity of hippocampus at high dose (10 mg/kg; i.p.). Therefore, the dose and duration of treatment are crucial for the effects of AM404.ObjectiveThe effects and molecular mechanisms of AM404 at low dose were evaluated from in vitro to in vivo models.MethodsAM404 (0.1–0.5 μM) was tested on tau hyperphosphorylated mouse hippocampal primary cultures treated with Wortmannin (WT) and GF109203X (GFX). Hyperglycemic triple transgenic AD (3×Tg-AD) mice at 6 months old were intraperitoneally injected with AM404 (0.25 mg/kg) for 4 weeks. The spatial learning and memory of mice were measured using the Morris water maze. Mouse brain and serum samples were collected for pathological analyses.ResultsAM404 (0.5 μM) exhibited significantly augmented neuroprotection toward tau hyperphosphorylation in primary cultures. The chronic systemic administration of AM404 (0.25 mg/kg) attenuated cognitive deficits in hyperglycemic 3×Tg-AD mice. Moreover, chronic low dose of AM404 significantly attenuated Aβ production, tau protein phosphorylation, and inflammation associated with an increase of pS473Akt and pS9-GSK3β. Therefore, AM404 at low dose, not only increased neuroprotection, but also ameliorated cognitive deficit, could be partly by regulating the Akt/GSK3β signaling, which may contribute to downregulation of Aβ, tau hyperphosphorylation, and inflammation in hyperglycemic 3×Tg-AD mice.ConclusionsThese results highlight that chronic administration of AM404 at low dose may be through the Akt/GSK3β pathway to ameliorate the impairment in hyperglycemic 3×Tg-AD mice.

Intracellular emetic signaling evoked by the L-type Ca2+ channel agonist FPL64176 in the least shrew (Cryptotis parva)

Ca2+ plays a major role in maintaining cellular homeostasis and regulates processes including apoptotic cell death and side-effects of cancer chemotherapy including vomiting. Currently we explored the emetic mechanisms of FPL64176, an L-type Ca2+ channel (LTCC) agonist with maximal emetogenic effect at its 10 mg/kg dose. FPL64176 evoked c-Fos immunoreactivity in shrew brainstem sections containing the vomit-associated nuclei, nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus. FPL64176 also increased phosphorylation of proteins ERK1/2, PKCα/βII and Akt in the brainstem. Moreover, their corresponding inhibitors (PD98059, GF 109203X and LY294002, respectively) reduced FPL64176-evoked vomiting. A 30 min subcutaneous (s.c.) pretreatment with the LTCC antagonist nifedipine (10 mg/kg) abolished FPL64176-elicited vomiting, c-Fos expression, and emetic effector phosphorylation. Ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs) mediate intracellular Ca2+ release from the sarcoplasmic/endoplasmic reticulum. The RyR antagonist dantrolene (i.p.), or a combination of low doses of nifedipine and dantrolene, but not the IP3R antagonist 2-APB, significantly attenuated FPL64176-induced vomiting. The serotonin type 3 receptor (5-HT3R) antagonist palonosetron (s.c.), the neurokinin 1 receptor (NK1R) antagonist netupitant (i.p.) or a combination of non-effective doses of netupitant and palonosetron showed antiemetic potential against FPL64176-evoked vomiting. Serotonin (5-HT) and substance P immunostaining revealed FPL64176-induced emesis was accompanied by an increase in 5-HT but not SP-immunoreactivity in the dorsomedial subdivision of the NTS. These findings demonstrate that Ca2+ mobilization through LTCCs and RyRs, and subsequent emetic effector phosphorylation and 5-HT release play important roles in FPL64176-induced emesis which can be prevented by 5-HT3R and NK1R antagonists.

Cornel Iridoid Glycoside Inhibits Tau Hyperphosphorylation via Regulating Cross-Talk Between GSK-3β and PP2A Signaling.

Neurofibrillary pathology contributes to neuronal dysfunction and correlates with the clinical progression of Alzheimer’s disease (AD). Tau phosphorylation is mainly regulated by a balance of glycogen synthase kinase-3β (GSK-3β) and protein phosphatase 2A (PP2A) activities. Cornel iridoid glycoside (CIG) is a main component extracted from Cornus officinalis. The purpose of this study was to investigate the effects of CIG on GSK-3β and PP2A, thus to explore the mechanisms of CIG to inhibit tau hyperphosphorylation. The rat model of tau hyperphosphorylation was established by intraventricular injection of wortmannin and GF-109203X (GFX) to activate GSK-3β. The results showed that intragastrical administration of CIG inhibited tau hyperphosphorylation in the brain of rats induced by wortmannin/GFX. The results in vivo and in vitro exhibited that CIG inhibited tau hyperphosphorylation and GSK-3β over-activation. In the mechanism of action, CIG’s attenuating GSK-3β activity was found to be dependent on PI3K/AKT signaling pathway. PP2A catalytic C subunit (PP2Ac) siRNA abrogated the effect of CIG on PI3K/AKT/GSK-3β. Additionally and crucially, we also found that CIG inhibited the demethylation of PP2Ac at Leu309 in vivo and in vitro. It enhanced PP2A activity, decreased tau hyperphosphorylation, and protected cell morphology in okadaic acid (OA)-induced cell model in vitro. PP2Ac siRNA abated the inhibitory effect of CIG on tau hyperphosphorylation. Moreover, CIG inhibited protein phosphatase methylesterase-1 (PME-1) and demethylation of PP2Ac, enhanced PP2A activity, and decreased tau hyperphosphorylation in PME-1-transfectd cells. Taken together, CIG inhibited GSK-3β activity via promoting P13K/AKT and PP2A signaling pathways. In addition, CIG also elevated PP2A activity via inhibiting PME-1-induced PP2Ac demethylation to inhibit GSK-3β activity, thus regulated the cross-talk between GSK-3β and PP2A signaling and consequently inhibited tau hyperphosphorylation. These results suggest that CIG may be a promising agent for AD therapy.

PKC Modulates sFlt1 Production by Human Placental Trophoblasts in Response to Oxidized LDL

Preeclampsia (PE) is a leading cause of maternal and perinatal mortality and mortality, and its prevalence is 4 fold higher in women with diabetes than those without: the underlying mechanism is unclear. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt1) plays a key role in PE pathogenesis. Evidence suggests that PKC activation, which is associated with the development of diabetic vascular complications, may mediate the enhanced sFlt1 release in non-trophoblast cells. We aimed to determine the role of PKC in the regulation of sFlt1 expression in placental trophoblasts, and whether diabetic conditions upregulate sFlt1 expression via the PKC pathway. Human trophoblasts HTR8/SVneo were treated in three separate experiments with PKC activator phorbol-12-myristate-13-acetate (PMA), or diabetes stimuli ’heavily oxidized, glycated’ low-density lipoproteins (HOG-LDL) vs. native LDL ± high glucose (30 mM), over 24h, with or without PKC inhibitor GF109203X (GX). Both sFlt1 mRNA expression (RT-PCR) and protein release (ELISA) were measured. Compared to the control, PMA (5 nM) increased sFlt1 mRNA expression (2.5 fold, p<0.001) and protein release (383.8 ± 27.4 vs. 118.3 ± 21.3 pg/ml, p<0.001) in trophoblasts; these effects were entirely abrogated by the pre-treatment with 5 uM GX for mRNA expression (by 54%, p<0.001) and protein release (115.5 ± 20.9 pg/ml, p<0.001) vs. PMA-treated cells. Similarly, HOG-LDL (50 µg/ml) increased sFlt1 mRNA expression (3.7 fold, p<0.001) and protein release (126.4 ± 16.3 vs. 55.1 ± 2.4 pg/ml, p<0.001), and the both effects were partially abolished by GX (by 33-37%, p<0.05). High glucose did not significantly affect sFlt1 expression, or alter the effect of HOG-LDL. The data suggest that modified lipoproteins may promote PE development in women with diabetes, at least in part via PKC-mediated upregulation and release of sFlt1. These findings provide new insights into disease mechanisms, and may lead to new targets for prevention and treatment of PE. Disclosure R.P. Chow: None. J. Zhao: None. J. Yu: None.

Adreno-muscarinic synergy in the male human urinary outflow tract.

To examine putative interaction between adrenergic and muscarinic contractile activation in the human urinary outflow tract. Tissue from the trigone and prostatic urethra was obtained from 12 cystectomy and 16 prostatectomy specimen. Contractions were elicited by exposure to exogenous agonists before and after inhibition of Rho kinase and protein kinase c (PKC). Immunofluorescence and Western-blot studies were performed using antibodies to muscarinic M3-receptors (M3-R) and alpha1A-adrenoreceptors (alpha1A-AR). The study is registered with ClinicalTrials.gov, number NCT01227447. There was strong co-localization of M3-R and alpha1A-AR on trigonal and urethral myocytes. Western blot analysis revealed a significantly higher expression of alpha1A-AR in the superficial than in the deep trigone. Phenylephrine (PE, 1 μm) augmented contractions induced by carbachol (CA, 3 μm) to more than threefold control in the male superficial trigone, and to about sevenfold control in the proximal urethra. No such potentiation could be detected in female bladder outlet. Both PKC inhibitor GF 109203X and Rho kinase inhibitor Y-27632 reduced responses to 1 μM PE as well as to 3 μM CA significantly. However, the synergistic effect of the combined intervention remained proportionally unaffected. Muscarinic and adrenergic receptor activation exerts a strong synergistic effect in the male human bladder trigone and proximal urethra.

The Effects of Immunomodulatory Molecule-Lipoxin A4- on the Physiology of Cystic Fibrosis Human Bronchial Epithelium

ABSTRACT Cystic fibrosis (CF) is a disease characterised by the depletion of Airway Surface Liquid (ASL) height. Lipoxin A4 was found to be effective in restoring ASL back to normal by acting on ENaC. The signalling mechanisms involved in lipoxin effect were investigated by testing the action of a calcium chelator (Bapta-AM) and Protein kinase C inhibitor (GF 109203X) on NuLi-1 and CuFi-1 cells. Drug’s efficacy was monitored by conducting Ussing chamber experiments and confocal microscopy. Results showed that calcium has a major contribution to the lipoxin A4 effects while PKC has a limited role. However, our data might lack accuracy due to shortage of time to conduct more experiments.

O-GlcNAc on PKCζ Inhibits the FGF4-PKCζ-MEK-ERK1/2 Pathway via Inhibition of PKCζ Phosphorylation in Mouse Embryonic Stem Cells.

SummaryMouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ.

Calcium-dependent and calcium-mimicking pathways regulate lead-induced myometrial contraction in water buffaloes

Lead produced dose-dependent contractile effect on buffalo myometrium and it was more marked on tonicity and amplitude while almost negligible on phasic contractions and frequency. In the presence of Ca2+-free Ringer Locke solution (RLS) and nifedipine, lead-induced uterotonic effect was significantly attenuated with decrease in Emax thus, suggesting major role of extracellular Ca2+ in uterotonic effect of lead and involvement of L-type Ca2+-channels in mediating this response. Direct excitatory effect of cyclopiazonic acid (CPA) on myometrium indicates possible Ca2+-influx via store operated calcium channels (SOCC). Lead failed to evoke any excitatory effect in the concurrent presence of nifedipine (1μM) and CPA (10μM), thus intracellular calcium seems to have negligible role in lead-induced myometrial contraction. In Ca2+-free high K+ (80 mM) depolarizing solution, lead-induced excitatory effect was attenuated by GF 109203X; which suggests permeation of lead through L-type Ca2+ channels to replace Ca2+ which directly activates protein kinase C (PKC) to produce excitatory effect. Thus lead seems to interact with calcium in buffalo myometrium in a dynamic fashion and exerts calcium-depending and calcium-mimicking effects; and therefore, lead is likely to have adverse effect on reproductive functions in buffaloes.

Activation of GSK-3 disrupts cholinergic homoeostasis in nucleus basalis of Meynert and frontal cortex of rats.

The cholinergic impairment is an early marker in Alzheimer's disease (AD), while the mechanisms are not fully understood. We investigated here the effects of glycogen synthase kinse‐3 (GSK‐3) activation on the cholinergic homoeostasis in nucleus basalis of Meynert (NBM) and frontal cortex, the cholinergic enriched regions. We activated GSK‐3 by lateral ventricular infusion of wortmannin (WT) and GF‐109203X (GFX), the inhibitors of phosphoinositol‐3 kinase (PI3‐K) and protein kinase C (PKC), respectively, and significantly decreased the acetylcholine (ACh) level via inhibiting choline acetyl transferase (ChAT) rather than regulating acetylcholinesterase (AChE). Neuronal axonal transport was disrupted and ChAT accumulation occurred in NBM and frontal cortex accompanied with hyperphosphorylation of tau and neurofilaments. Moreover, ChAT expression decreased in NBM attributing to cleavage of nuclear factor‐κB/p100 into p52 for translocation into nucleus to lower ChAT mRNA level. The cholinergic dysfunction could be mimicked by overexpression of GSK‐3 and rescued by simultaneous administration of LiCl or SB216763, inhibitors of GSK‐3. Our data reveal the molecular mechanism that may underlie the cholinergic impairments in AD patients.

The effect of sphingosine-1-phosphate on colonic smooth muscle contractility: Modulation by TNBS-induced colitis.

AimIncreased levels of circulating sphingosine-1-phosphate (S1P) have been reported in ulcerative colitis. The objective of this study was to examine the effect of S1P on colonic smooth muscle contractility and how is it affected by colitis.MethodsColonic inflammation was induced by intrarectal administration of trinitrobenzene sulfonic acid. Five days later colon segments were isolated and used for contractility experiments and immunoblotting.ResultsS1P contracted control and inflamed colon segments and the contraction was significantly greater in inflamed colon segments. S1P-induced contraction was mediated by S1PR1 and S1PR2 in control and S1PR2 in inflamed colon segments. S1PR3 did not play a significant role in S1P-induced contractions in control or inflamed colon. S1PR1, S1PR2 and S1PR3 proteins were expressed in colon segments from both groups. The expression of S1PR1 and S1PR2 was significantly enhanced in control and inflamed colon segments, respectively. S1PR3 levels however were not significantly different between the two groups. Nifedipine significantly reduced S1P-induced contraction in control but not inflamed colon segments. Thapsigargin significantly reduced S1P-induced contraction of the inflamed colon. GF 109203X and Y-27632, alone abolished S1P-induced contraction of the control but not inflamed colon segments. Combination of GF 109203X, Y-27632 and thapsigargin abolished S1P-induced contraction of inflamed colon segments.ConclusionS1P contracted control colon via S1PR1 and S1PR2 and inflamed colon exclusively via S1PR2. Calcium influx (control) or release (inflamed) and calcium sensitization are involved in S1P-induced contraction. Exacerbated response to S1P in colitic colon segments may explain altered colonic motility reported in patients and experimental models of inflammatory bowel disease.

Biphasic augmentation of alpha-adrenergic contraction by plumbagin in rat systemic arteries.

Plumbagin, a hydroxy 1,4-naphthoquinone compound from plant metabolites, exhibits anticancer, antibacterial, and antifungal activities via modulating various signaling molecules. However, its effects on vascular functions are rarely studied except in pulmonary and coronary arteries where NADPH oxidase (NOX) inhibition was suggested as a mechanism. Here we investigate the effects of plumbagin on the contractility of skeletal artery (deep femoral artery, DFA), mesenteric artery (MA) and renal artery (RA) in rats. Although plumbagin alone had no effect on the isometric tone of DFA, 1 µM phenylephrine (PhE)-induced partial contraction was largely augmented by plumbagin (ΔTPlum, 125% of 80 mM KCl-induced contraction at 1 µM). With relatively higher concentrations (>5 µM), plumbagin induced a transient contraction followed by tonic relaxation of DFA. Similar biphasic augmentation of the PhE-induced contraction was observed in MA and RA. VAS2870 and GKT137831, specific NOX4 inhibitors, neither mimicked nor inhibited ΔTPlum in DFA. Also, pretreatment with tiron or catalase did not affect ΔTPlum of DFA. Under the inhibition of PhE-contraction with L-type Ca2+ channel blocker (nifedipine, 1 µM), plumbagin still induced tonic contraction, suggesting Ca2+-sensitization mechanism of smooth muscle. Although ΔTPlum was consistently observed under pretreatment with Rho A-kinase inhibitor (Y27632, 1 µM), a PKC inhibitor (GF 109203X, 10 µM) largely suppressed ΔTPlum. Taken together, it is suggested that plumbagin facilitates the PKC activation in the presence of vasoactive agonists in skeletal arteries. The biphasic contractile effects on the systemic arteries should be considered in the pharmacological studies of plumbagin and 1,4-naphthoquinones.

Mechanical activation of angiotensin II type 1 receptors causes actin remodelling and myogenic responsiveness in skeletal muscle arterioles.

Candesartan, an inverse agonist of the type 1 angiotensin II receptor (AT1 R), causes a concentration-dependent inhibition of pressure-dependent myogenic tone consistent with previous reports of mechanosensitivity of this G protein-coupled receptor. Mechanoactivation of the AT1 R occurs independently of local angiotensin II production and the type 2 angiotensin receptor. Mechanoactivation of the AT1 R stimulates actin polymerization by a protein kinase C-dependent mechanism, but independently of a change in intracellular Ca2+ . Using atomic force microscopy, changes in single vascular smooth muscle cell cortical actin are observed to remodel following mechanoactivation of the AT1 R. The Gq/11 protein-coupled angiotensin II type 1 receptor (AT1 R) has been shown to be activated by mechanical stimuli. In the vascular system, evidence supports the AT1 R being a mechanosensor that contributes to arteriolar myogenic constriction. The aim of this study was to determine if AT1 R mechanoactivation affects myogenic constriction in skeletal muscle arterioles and to determine underlying cellular mechanisms. Using pressure myography to study rat isolated first-order cremaster muscle arterioles the AT1 R inhibitor candesartan (10-7 -10-5 m) showed partial but concentration-dependent inhibition of myogenic reactivity. Inhibition was demonstrated by a rightward shift in the pressure-diameter relationship over the intraluminal pressure range, 30-110mmHg. Pressure-induced changes in global vascular smooth muscle intracellular Ca2+ (using Fura-2) were similar in the absence or presence of candesartan, indicating that AT1 R-mediated myogenic constriction relies on Ca2+ -independent downstream signalling. The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) reversed the inhibitory effect of candesartan, while this rescue effect was prevented by the protein kinase C (PKC) inhibitor GF 109203X. Both candesartan and PKC inhibition caused increased G-actin levels, as determined by Western blotting of vessel lysates, supporting involvement of cytoskeletal remodelling. At the single vascular smooth muscle cell level, atomic force microscopy showed that cell swelling (stretch) with hypotonic buffer also caused thickening of cortical actin fibres and this was blocked by candesartan. Collectively, the present studies support growing evidence for novel modes of activation of the AT1 R in arterioles and suggest that mechanically activated AT1 R generates diacylglycerol, which in turn activates PKC which induces the actin cytoskeleton reorganization that is required for pressure-induced vasoconstriction.