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

Background Gap 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. Methods Oxyhemoglobin (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. Results Increase 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. Conclusions These 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.

Pregnancy-associated adaptations in [Ca2+]i-dependent and Ca2+ sensitization mechanisms of venous contraction: implications in pregnancy-related venous disorders

Pregnancy is associated with significant adaptations in the maternal hemodynamics and arterial circulation, but the changes in the venous mechanisms during pregnancy are less clear. We hypothesized that pregnancy is associated with alterations in venous function, intracellular free Ca2+ concentration ([Ca2+]i), and Ca2+-dependent mechanisms of venous contraction. Circular segments of inferior vena cava (IVC) from virgin and late pregnant (Preg, day 19) Sprague-Dawley rats were suspended between two hooks, labeled with fura-2, and placed in a cuvet inside a spectrofluorometer for simultaneous measurement of contraction and [Ca2+]i (fura-2 340/380 ratio). KCl (96 mM), which stimulates Ca2+ influx, caused less contraction (35.6 ± 6.3 vs. 92.6 ± 19.9 mg/mg tissue) and smaller increases in [Ca2+]i (1.67 ± 0.12 vs. 2.19 ± 0.11) in Preg vs. virgin rat IVC. The α-adrenergic receptor agonist phenylephrine (Phe; 10−5 M) caused less contraction (23.8 ± 3.4 vs. 70.9 ± 12.9 mg/mg tissue) and comparable increases in [Ca2+]i (1.76 ± 0.10 vs. 1.89 ± 0.08) in Preg vs. virgin rat IVC. At increasing extracellular Ca2+ concentrations ([Ca2+]e) (0.1, 0.3, 0.6, 1, and 2.5 mM), KCl and Phe induced [Ca2+]e-contraction and [Ca2+]e-[Ca2+]i curves that were reduced in Preg vs. virgin IVC, supporting reduced Ca2+ entry mechanisms. The [Ca2+]e-contraction and [Ca2+]e-[Ca2+]i curves were used to construct the [Ca2+]i-contraction relationship. Despite reduced contraction and [Ca2+]i in Preg IVC, the Phe-induced [Ca2+]i-contraction relationship was greater than that of KCl and was enhanced in Preg vs. virgin IVC, suggesting parallel activation of Ca2+-sensitization pathways. The Ca2+ channel blocker diltiazem, protein kinase C (PKC) inhibitor GF-109203X, and Rho-kinase (ROCK) inhibitor Y27632 inhibited KCl- and Phe-induced contraction and abolished the shift in the Phe [Ca2+]i-contraction relationship in Preg IVC, suggesting an interplay between the decrease in Ca2+ influx and possible compensatory activation of PKC- and ROCK-mediated Ca2+-sensitization pathways. The reduced [Ca2+]i and [Ca2+]i-dependent contraction in Preg rat IVC, despite the parallel rescue activation of Ca2+-sensitization pathways, suggests that the observed reduction in [Ca2+]i-dependent contraction mechanisms is likely underestimated, and that the veins without the rescue Ca2+-sensitization pathways could be even more prone to dilation during pregnancy. These pregnancy-associated reductions in Ca2+ entry-dependent mechanisms of venous contraction, if occurring in human lower extremity veins and if not adequately compensated by Ca2+-sensitization pathways, may play a role in pregnancy-related venous disorders.

Cornel Iridoid Glycoside Attenuates Tau Hyperphosphorylation by Inhibition of PP2A Demethylation

Aim. The aim of the present study was to investigate the effect of cornel iridoid glycoside (CIG) on tau hyperphosphorylation induced by wortmannin (WT) and GF-109203X (GFX) and the underlying mechanisms.Methods. Human neuroblastoma SK-N-SH cells were preincubated with CIG (50, 100, and 200 µg/ml, resp.) for 24 h and then exposed to 10 µM WT and 10 µM GFX for 3 h after washing out CIG. Immunohistochemistry was used to observe the microtubular cytoskeleton of the cultured cells. Western blotting was used to measure the phosphorylation level of tau protein, glycogen synthase kinase 3β(GSK-3β), and protein phosphatase 2A (PP2A). The activity of PP2A was detected by a biochemical assay.Results. Preincubation of CIG significantly attenuated the WT/GFX-induced tau hyperphosphorylation at the sites of Thr205, Thr212, Ser214, Thr217, Ser396, and PHF-1 and improved the damage of morphology and microtubular cytoskeleton of the cells. CIG did not prevent the decrease in p-AKT-ser473 and p-GSK-3β-ser9 induced by WT/GFX. However, CIG significantly elevated the activity of PP2A by reducing the demethylation of PP2A catalytic subunit (PP2Ac) at Leu309 and the ratio of PME-1/LCMT in the WT/GFX-treated cells. The results suggest that CIG may be beneficial to the treatment of AD.

Classical PKCs Regulate ADP-Induced Thromboxane Generation by Modulating Tyrosine Phosphorylation On Novel PKC Isoform Delta Through Shptp-1

Abstract 1064 Introduction: Adenosine Di-phosphate (ADP) is stored in dense granules of platelets and is released upon platelet activation acting as a feedback activator by binding to G-protein coupled P2Y1 and P2Y12 receptors. ADP stimulation causes platelets to change shape, aggregate, release dense and a-granule contents and synthesize thromboxane A2 that can further act as a feedback activator potentiating platelet responses by binding to thromboxane receptor (TP). Protein kinase C is a serine threonine specific kinase that regulates multiple platelet functional responses. Specific PKC isoforms regulating platelet responses downstream of ADP receptors are not completely known. Aim: The aim of the current study is to elucidate the role of PKC isoforms in regulating ADP-induced platelet functional responses in platelets. Methods: We sought to delineate the mechanism of ADP-induced platelet responses by performing platelet aggregation (aggregometry), ATP secretion (luciferin-luciferase reaction) and thromboxane generation (ELISA kit measuring TxB2) in human or murine platelets by pre-incubating the platelets with control (DMSO) or inhibitors wherever mentioned. We also evaluated the role of PKCd to ADP-induced platelet responses by using murine platelets lacking PKCd. Background and Results: Murugappan et al have shown that PKCd was not activated downstream of ADP receptors based on the inability of ADP to cause threonine 507 phosphorylation on PKCd in platelets. However, studies from other labs have shown that PKCd can be activated when it is phosphorylated on its tyrosine residues. In the current study we show that, upon stimulation with 2MeSADP, PKCd is phosphorylated on tyrosine residue 311 in a time-dependent manner in platelets (Fig A). Also, ADP-induced thromboxane generation (Fig B) and ADP-induced thromboxane-mediated dense granule secretion were significantly inhibited in PKCd knockout murine platelets compared to those of wild type platelets. Similarly, thromboxane generation downstream of ADP receptors in human platelets pre-incubated with a PKCd inhibitor is significantly inhibited compared to control indicating a role for PKCd in mediating ADP-induced responses in platelets. Bynagari et al have shown that ADP-induced thromboxane generation is potentiated in the presence of the pan-PKC inhibitor, GF 109203X and the isoform regulating this effect is PKCe. We observed that pre-incubation of PKCe knockout murine platelets with GF 109203X further potentiated ADP-induced thromboxane generation suggesting that there are other PKC isoforms negatively regulating ADP-induced thromboxane generation. We show that this potentiating effect of thromboxane generation with GF 109203X in WT or PKCe KO murine platelets correlate with an increase in the phosphorylation of Y311 on PKCd (Fig C) suggesting that ADP-induced thromboxane generation is regulated through PKCd Y311 phosphorylation. Tyrosine phosphorylation on PKCd is mediated by Src family kinases (SFKs) as the phosphorylation is abolished with PP2, a SFK inhibitor and is only partially inhibited in Fyn knockout murine platelets suggesting that other SFKs also mediate this tyrosine phosphorylation. Surprisingly, pre-incubation of platelets with LY-333531, a classical PKC isoform (a/b) inhibitor potentiated PKCd Y311 phosphorylation (Fig D) as well as thromboxane generation (Fig E) downstream of ADP receptors suggesting a role for classical PKCs. Also, platelets pre-incubated with LY-333531 showed a decrease in the phosphorylation of SHPTP-1 (Fig F), a tyrosine phosphatase, rendering it active. The active SHPTP-1 phosphatase may dephosphorylate and activate SFKs, which can now phosphorylate PKCd on Y311 in platelets. Conclusions: In the current study, we report for the first time that the novel PKC isoform d is tyrosine phosphorylated downstream of ADP receptors through which it mediates ADP-induced thromboxane generation. We also show a novel role for classical PKC isoforms a/b in regulating tyrosine phosphorylation on novel isoform, PKCd possibly through the tyrosine phosphatase SHPTP-1 and Src family kinases in platelets. Disclosures: No relevant conflicts of interest to declare.

Upregulation of ACE2-ANG-(1–7)-Mas axis in jejunal enterocytes of type 1 diabetic rats: implications for glucose transport

The inhibitory effects of the angiotensin-converting enzyme (ACE)-ANG II-angiotensin type 1 (AT1) receptor axis on jejunal glucose uptake and the reduced expression of this system in type 1 diabetes mellitus (T1DM) have been documented previously. The ACE2-ANG-(1–7)-Mas receptor axis is thought to oppose the actions of the ACE-ANG II-AT1 receptor axis in heart, liver, and kidney. However, the possible involvement of the ACE2-ANG-(1–7)-Mas receptor system on enhanced jejunal glucose transport in T1DM has yet to be determined. Rat everted jejunum and Caco-2 cells were used to determine the effects of ANG-(1–7) on glucose uptake and to study the ACE2-ANG-(1–7)-Mas receptor signaling pathway. Expression of target gene and protein in jejunal enterocytes and human Caco-2 cells were quantified using real-time PCR and Western blotting. T1DM increased jejunal protein and mRNA expression of ACE2 (by 59 and 173%, respectively) and Mas receptor (by 55 and 100%, respectively) in jejunum. One millimolar ANG-(1–7) reduced glucose uptake in jejunum and Caco-2 cells by 30.6 and 30.3%, respectively, effects that were abolished following addition of 1 μM A-779 (a Mas receptor blocker) or 1 μM GF-109203X (protein kinase C inhibitor) to incubation buffer for jejunum or Caco-2 cells, respectively. Finally, intravenous treatment of animals with ANG-(1–7) significantly improved oral glucose tolerance in T1DM but not control animals. In conclusion, enhanced activity of the ACE2-ANG-(1–7)-Mas receptor axis in jejunal enterocytes is likely to moderate the T1DM-induced increase in jejunal glucose uptake resulting from downregulation of the ACE-ANG II-AT1 receptor axis. Therefore, altered activity of both ACE and ACE2 systems during diabetes will determine the overall rate of glucose transport across the jejunal epithelium.

A late phase of LTD in cultured cerebellar Purkinje cells requires persistent dynamin-mediated endocytosis

Long-term synaptic depression (LTD) of cerebellar parallel fiber-Purkinje cell synapses is a form of use-dependent synaptic plasticity that may be studied in cell culture. One form of LTD is induced postsynaptically through an mGlu1/Ca influx/protein kinase Cα (PKCα) cascade, and its initial expression requires phosphorylation of ser-880 in the COOH-terminal PDZ-ligand region of GluA2 and consequent binding of PICK1. This triggers postsynaptic clathrin/dynamin-mediated endocytosis of GluA2-containing surface AMPA receptors. Cerebellar LTD also has a late phase beginning 45–60 min after induction that is blocked by transcription or translation inhibitors. Here, I have sought to determine the expression mechanism of this late phase of LTD by applying various drugs and peptides after the late phase has been established. Neither bath application of mGluR1 antagonists (JNJ-16259685, LY-456236) nor the PKC inhibitor GF-109203X starting 60–70 min after LTD induction attenuated the late phase. Similarly, achieving the whole cell configuration with a second pipette loaded with the peptide PKC inhibitor PKC(19–36) starting 60 min postinduction also failed to alter the late phase. Late internal perfusion with peptides designed to disrupt PICK1-GLUA2 interaction or PICK1 dimerization failed to impact late phase LTD expression. However, late internal perfusion with two different blockers of dynamin, the drug dynasore and a dynamin inhibitory peptide (QVPSRPNRAP), produced rapid and complete reversal of cerebellar LTD expression. These findings suggest that the protein synthesis-dependent late phase of LTD requires persistent dynamin-mediated endocytosis, but not persistent PICK1-GluA2 binding nor persistent activation of the upstream mGluR1/PKCα signaling cascade.

Calcium sensitization involved in dexmedetomidine-induced contraction of isolated rat aorta

Dexmedetomidine, a full agonist of the α2B-adrenoceptor that is mainly involved in vascular smooth muscle contraction, is primarily used for analgesia and sedation in intensive care units. High-dose dexmedetomidine produces hypertension in children and adults. The goal of this in vitro study was to investigate the role of the calcium (Ca2+) sensitization mechanism involving Rho-kinase, protein kinase C (PKC), and phosphoinositide 3-kinase (PI3-K) in mediating contraction of isolated rat aortic smooth muscle in response to dexmedetomidine. The effect of dexmedetomidine on the intracellular Ca2+ level ([Ca2+]i) and tension was measured simultaneously. Dexmedetomidine concentration–response curves were generated in the presence or absence of the following antagonists: rauwolscine, Y 27632, LY 294002, GF 109203X, and verapamil. Dexmedetomidine-induced phosphorylation of PKC and membrane translocation of Rho-kinase were detected with Western blotting. Rauwolscine, Y 27632, GF 109203X, LY 294002, and verapamil attenuated dexmedetomidine-induced contraction. The slope of the [Ca2+]i–tension curve for dexmedetomidine was higher than that for KCl. Dexmedetomidine induced phosphorylation of PKC and membrane translocation of Rho-kinase. These results suggest that dexmedetomidine-induced contraction involves a Ca2+ sensitization mechanism mediated by Rho-kinase, PKC, and PI3-K that is secondary to α2-adrenoceptor stimulation in rat aortic smooth muscle.

Parathyroid hormone (PTH) rapidly enhances CFTR-mediated HCO3− secretion in intestinal epithelium-like Caco-2 monolayer: a novel ion regulatory action of PTH

Besides being a Ca2+-regulating hormone, parathyroid hormone (PTH) has also been shown to regulate epithelial transport of certain ions, such as Cl−, HCO3−, and Na+, particularly in the kidney. Although the intestinal epithelium also expressed PTH receptors, little was known regarding its mechanism in the regulation of intestinal ion transport. We investigated the ion regulatory role of PTH in intestinal epithelium-like Caco-2 monolayer by Ussing chamber technique and alternating current impedance spectroscopy. It was found that Caco-2 cells rapidly responded to PTH within 1 min by increasing apical HCO3− secretion. CFTR served as the principal route for PTH-stimulated apical HCO3− efflux, which was abolished by various CFTR inhibitors, namely, NPPB, glycine hydrazide-101 (GlyH-101), and CFTRinh-172, as well as by small interfering RNA against CFTR. Concurrently, the plasma membrane resistance was decreased with no changes in the plasma membrane capacitance or paracellular permeability. HCO3− was probably supplied by basolateral uptake via the electrogenic Na+-HCO3− cotransporter and by methazolamide-sensitive carbonic anhydrase, while the resulting intracellular H+ might be extruded by both apical and basolateral Na+/H+ exchangers. Furthermore, the PTH-stimulated HCO3− secretion was markedly reduced by protein kinase A (PKA) inhibitor (PKI 14–22 amide) and phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY-294002), but not by intracellular Ca2+ chelator (BAPTA-AM) or protein kinase C inhibitor (GF-109203X). In conclusion, the present study provided evidence that PTH directly and rapidly stimulated apical HCO3− secretion through CFTR in PKA- and PI3K-dependent manner, which was a novel noncalciotropic, ion regulatory action of PTH in the intestinal epithelium.