Low [Mg(2+)](o) induces contraction of cerebral arteries: roles of tyrosine and mitogen-activated protein kinases

The present study was designed to investigate the mechanism of action of low extracellular magnesium ion concentration ([Mg(2+)](o)) on isolated canine basilar arteries and single cerebral vascular smooth muscle cells from these arteries. Low-[Mg(2+)](o) medium (0-0.6 mM) produces endothelium-independent contractions in isolated canine basilar arteries in a concentration-dependent manner; the lower the concentration of [Mg(2+)](o), the stronger the contractions. The low-[Mg(2+)](o) medium-induced contractions are significantly attenuated by pretreatment of the arteries with low concentrations of either SB-203580, U-0126, PD-98059, genistein, or an Src homology 2 (SH2) domain inhibitor peptide. IC(50) levels obtained for these five antagonists are consistent with reported inhibitor constant (K(i)) values for these tyrosine kinase and mitogen-activated protein kinase (MAPK) antagonists. Low-[Mg(2+)](o) medium (0-0.6 mM) produces transient intracellular calcium ion concentration ([Ca(2+)](i)) peaks followed by a slow, sustained, and elevated plateau of [Ca(2+)](i) in primary single smooth muscle cells from canine basilar arteries. Low-[Mg(2+)](o) medium induces rapid and stable increases in [Ca(2+)](i); these increases are inhibited markedly in the presence of either SB-203580, U-0126, PD-98059, genistein or a SH2 domain inhibitor peptide. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the low-[Mg(2+)](o)-induced contractions or the elevation of [Ca(2+)](i). The present study suggests that activation of several cellular signaling pathways, such as protein tyrosine kinases (including the Src family) and MAPK, appears to play important roles in low-[Mg(2+)](o)-induced contractions and the elevation of [Ca(2+)](i) in smooth muscle cells from canine basilar arteries.     

Lipoteichoic acid-stimulated p42/p44 MAPK activation via Toll-like receptor 2 in tracheal smooth muscle cells

Lipoteichoic acid (LTA), the principal component of the cell wall of gram-positive bacteria, triggers several inflammatory responses. However, the mechanisms underlying its action on human tracheal smooth muscle cells (HTSMCs) were largely unknown. This study was to investigate the mechanisms underlying LTA-stimulated p42/p44 mitogen-activated protein kinase (MAPK) using Western blotting assay. LTA stimulated phosphorylation of p42/p44 MAPK via a Toll-like receptor 2 (TLR2). Pretreatment with pertussis toxin attenuated the LTA-induced responses. LTA-stimulated phosphorylation of p42/p44 MAPK was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (PLC; D609), phosphatidylinositol (PI)-PLC (U-73122), PKC (staurosporine, G?-6976, rottlerin, or Ro-318220), MEK1/2 (U-0126), PI 3-kinase (LY-294002 and wortmannin), and an intracellular Ca(2+) chelator (BAPTA-AM). LTA directly evoked initial transient peak of [Ca(2+)](i), supporting the involvement of Ca(2+) mobilization in LTA-induced responses. These results suggest that in HTSMCs, LTA-stimulated p42/p44 MAPK phosphorylation is mediated through a TLR2 receptor and involves tyrosine kinase, PLC, PKC, Ca(2+), MEK, and PI 3-kinase.     

Sphingosine 1-phosphate induces cyclooxygenase-2 via Ca2+-dependent, but MAPK-independent mechanism in rat vascular smooth muscle cells

The effects of sphingosine 1-phosphate (S1P) on prostaglandin I(2) (PGI(2)) production and cyclooxygenase (COX) expression in cultured rat vascular smooth muscle cells (VSMCs) were investigated. S1P stimulated PGI(2) production in a concentration-dependent manner, which was completely suppressed by NS-398, a selective COX-2 inhibitor, as determined by radioimmunoassay. S1P stimulated COX-2 protein and mRNA expressions in a concentration- and time-dependent manner, while it had no effect on COX-1 expression. S1P(2) and S1P(3) receptors mRNA were abundantly expressed in rat VSMCs. Suramin, an antagonist of S1P(3) receptor, almost completely inhibited S1P-induced COX-2 expression. Pretreatment of VSMCs with pertussis toxin (PTX) partially, but significantly inhibited S1P-induced PGI(2) production and COX-2 expression. S1P also activated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). However, neither PD 98059, a selective inhibitor of ERK activation, nor SB 203580, a selective inhibitor of p38 MAPK, had a significant inhibitory effect on S1P-induced COX-2 expression, suggesting that the MAPK activation does not play main roles in S1P-induced COX-2 induction. S1P-induced COX-2 expression was inhibited by PP2, an inhibitor of Src-family tyrosine kinase, Ca(2+) depletion, and GF 109203X, an inhibitor of protein kinase C (PKC). These results suggest that S1P stimulates COX-2 induction in rat VSMCs through mechanisms involving Ca(2+)-dependent PKC and Src-family tyrosine kinase activation via S1P(3) receptor coupled to PTX-sensitive and -insensitive G proteins.     

Calcium-sensing receptor modulates extracellular Ca(2+) entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells

Ca-sensing receptor (CaSR), a member of the G protein-coupled receptor family, regulates the synthesis of parathyroid hormone in response to changes in serum Ca(2+) concentrations. The functions of CaSR in human vascular smooth muscle cells are largely unknown. Here we sought to study CaSR activation and the underlying molecular mechanisms in human aortic smooth muscle cells (HASMC). Extracellular Ca(2+) ([Ca(2+)](o)) dose-dependently increased free cytosolic Ca(2+) ([Ca(2+)](cyt)) in HASMC, with a half-maximal response (EC(50)) of 0.52 mM and a Hill coefficient of 5.50. CaSR was expressed in HASMC, and the [Ca(2+)](o)-induced [Ca(2+)](cyt) rise was abolished by dominant negative mutants of CaSR. The CaSR-mediated increase in [Ca(2+)](cyt) was also significantly inhibited by pertussis toxin, the phospholipase C inhibitor U-73122, or the general protein kinase C (PKC) inhibitor chelerythrine, but not by the conventional PKC inhibitor, G?6976. Depletion of membrane cholesterol by pretreatment with methyl-β-cyclodextrin markedly decreased CaSR-induced increase in [Ca(2+)](cyt). Blockade of TRPC channels with 2-aminoethoxydiphenyl borate, SKF-96365, or La(3) significantly inhibited [Ca(2+)](o) entry, whereas activation of TRPC6 channels with flufenamic acid potentiated [Ca(2+)](o) entry. Neither cyclopiazonic acid nor caffeine or ionomycin had any effect on [Ca(2+)](cyt) in [Ca(2+)](o)-free solutions. TRPC6 and PKCε mRNA and proteins were detected in HASMC, and [Ca(2+)](o) induced PKCε phosphorylation, which could be prevented by chelerythrine. Our data suggest that CaSR activation mediates [Ca(2+)](o) entry, likely through TRPC6-encoded receptor-operated channels that are regulated by a PLC/PKCε cascade. Our study therefore provides evidence not only for functional expression of CaSR, but also for a novel pathway whereby it regulates [Ca(2+)](o) entry in HASMC.     

Involvement of intracellular Ca2+ elevation but not cyclic AMP in PACAP-induced p38 MAP kinase activation in PC12 cells

We have recently shown that in PC12 cells, pituitary adenylate cyclase-activating polypeptide (PACAP) and NGF synergistically stimulate PACAP mRNA expression primarily via a mechanism involving a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Here we have analyzed p38 MAPK activation by PACAP and the mechanism underlying this action of PACAP in PC12 cells. PACAP increased phosphorylation of p38 MAPK with a bell-shaped dose-response relationship and a maximal effect was obtained at 10(-8) M. PACAP (10(-8) M)-induced p38 MAPK phosphorylation was already evident at 2.5 min, maximal at 5 min, and rapidly declined thereafter. PACAP-induced p38 MAPK phosphorylation was potently inhibited by depletion of Ca(2+) stores with thapsigargin and partially inhibited by the phospholipase C inhibitor U-73122, L-type voltage-dependent calcium channel inhibitors nifedipine and nimodipine, and the Ca(2+) chelator EGTA, whereas the protein kinase C inhibitor calphostin C, the protein kinase A inhibitor H-89, the cAMP antagonist Rp-cAMP, and the nonselective cation channel blocker SKF96365 had no effect. These results indicate that PACAP activates p38 MAPK in PC12 cells through activation of a phospholipase C, mobilization of intracellular Ca(2+) stores, and Ca(2+) influx through voltage-dependent Ca(2+) channels, but not cyclic AMP-dependent mechanisms.     

Activation of the neurokinin-1 receptor in rat spinal astrocytes induces Ca2+ release from IP3-sensitive Ca2+ stores and extracellular Ca2+ influx through TRPC3

Substance P (SP) plays an important role in pain transmission through the stimulation of the neurokinin (NK) receptors expressed in neurons of the spinal cord, and the subsequent increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) as a result of this stimulation. Recent studies suggest that spinal astrocytes also contribute to SP-related pain transmission through the activation of NK receptors. However, the mechanisms involved in the SP-stimulated [Ca(2+)](i) increase by spinal astrocytes are unclear. We therefore examined whether (and how) the activation of NK receptors evoked increase in [Ca(2+)](i) in rat cultured spinal astrocytes using a Ca(2+) imaging assay. Both SP and GR73632 (a selective agonist of the NK1 receptor) induced both transient and sustained increases in [Ca(2+)](i) in a dose-dependent manner. The SP-induced increase in [Ca(2+)](i) was significantly attenuated by CP-96345 (an NK1 receptor antagonist). The GR73632-induced increase in [Ca(2+)](i) was completely inhibited by pretreatment with U73122 (a phospholipase C inhibitor) or xestospongin C (an inositol 1,4,5-triphosphate (IP(3)) receptor inhibitor). In the absence of extracellular Ca(2+), GR73632 induced only a transient increase in [Ca(2+)](i). In addition, H89, an inhibitor of protein kinase A (PKA), decreased the GR73632-mediated Ca(2+) release from intracellular Ca(2+) stores, while bisindolylmaleimide I, an inhibitor of protein kinase C (PKC), enhanced the GR73632-induced influx of extracellular Ca(2+). RT-PCR assays revealed that canonical transient receptor potential (TRPC) 1, 2, 3, 4 and 6 mRNA were expressed in spinal astrocytes. Moreover, BTP2 (a general TRPC channel inhibitor) or Pyr3 (a TRPC3 inhibitor) markedly blocked the GR73632-induced sustained increase in [Ca(2+)](i). These findings suggest that the stimulation of the NK-1 receptor in spinal astrocytes induces Ca(2+) release from IP(3-)sensitive intracellular Ca(2+) stores, which is positively modulated by PKA, and subsequent Ca(2+) influx through TRPC3, which is negatively regulated by PKC.     

Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK

Sphingosylphosphorylcholine (SPC) has been implicated in a variety of cellular responses, including proliferation and differentiation. In this study, we demonstrate that d-erythro-SPC, but not l-threo-SPC, stereoselectively stimulated the proliferation of human adipose tissue-derived mesenchymal stem cells (hADSCs), with a maximal increase at 5 microM, and increased the intracellular concentration of Ca(2+) ([Ca(2+)](i)) in hADSCs, which do not express known SPC receptors (i.e., OGR1, GPR4, G2A, and GPR12). The SPC-induced proliferation and increase in [Ca(2+)](i) were sensitive to pertussis toxin (PTX) and the phospholipase C (PLC) inhibitor U73122, suggesting that PTX-sensitive G proteins, Gi or Go, and PLC are involved in SPC-induced proliferation. In addition, SPC treatment induced the phosphorylation of c-Jun and extracellular signal-regulated kinase, and SPC-induced proliferation was completely prevented by pretreatment with the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 but not with the MEK-specific inhibitor U0126. Furthermore, the SPC-induced proliferation and JNK activation were completely attenuated by overexpression of a dominant negative mutant of JNK2, and the SPC-induced activation of JNK was inhibited by pretreatment with PTX or U73122. Treatment of hADSCs with lysophosphatidic acid (LPA) receptor antagonist, Ki16425, had no impact on the SPC-induced increase in [Ca(2+)](i). However, SPC-induced proliferation was partially, but significantly, attenuated by pretreatment of the cells with Ki16425.These results indicate that SPC stimulates the proliferation of hADSCs through the Gi/Go-PLC-JNK pathway and that LPA receptors may be responsible in part for the SPC-induced proliferation.     

Endothelins regulate arachidonic acid release and mitogen-activated protein kinase activity in Schwann cells

Immortalized rat Schwann cells (iSC) express endothelin (ET) receptors coupled to inhibition of adenylyl cyclase and stimulation of phospholipase C (PLC). These effects precede phenotypic changes and increased DNA synthesis. We have investigated the role of ETs in the regulation of arachidonic acid (AA) release and mitogen-activated protein kinases (MAPKs). Both ET-1 and ET-3 increased AA release in iSC. This effect was sensitive to the phospholipase A(2) (PLA(2)) inhibitors E:-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H:-pyran-2-one and arachidonyl-trifluoromethyl ketone but was insensitive to inhibitors of PLC or phospholipase D-dependent diacylglycerol generation. ET-1-dependent AA release was also unaffected by removal of extracellular Ca(2+) and blocking the concomitant elevation in [Ca(2+)](i), consistent with participation of a Ca(2+)-independent PLA(2). Treatment of iSC with ETs also resulted in activation of extracellular signal-regulated kinase, c-Jun-NH(2)-terminal kinase (JNK), and p38 MAPK. A cause-effect relationship between agonist-dependent AA release and stimulation of MAPKs, but not the opposite, was suggested by activation of JNK by exogenous AA and by the observation that inhibition of MAPK kinase or p38 MAPK was inconsequential to ET-1-induced AA release. Similar effects of ETs on AA release and MAPK activity were observed in cultures expanded from primary SC and in iSC. Regulation of these effectors may mediate the control of proliferation and differentiation of SC by ETs during peripheral nerve development and regeneration.     

Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] suppresses the expression of cyclooxygenase-2 in endotoxin-treated monocytic cells

The anti-inflammatory activity of the predominant procyanidin dimer in cocoa, dimer B2, was investigated in this study. Pretreatment of the procyanidin dimer B2 reduced COX-2 expression induced by the endotoxin lipopolysaccharide (LPS) in differentiated human monocytic cells (THP-1) in culture. To further elucidate the underlying mechanism of COX-2 inhibition by procyanidin, we examined their effects on the activation of extracellular signal-regulated protein kinase (ERK), Jun-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), which are upstream enzymes known to regulate COX-2 expression in many cell types. Pretreatment with procyanidin dimer B2 decreased the activation of ERK, JNK, and p38 MAPK. In addition, procyanidin dimer B2 suppressed the NF-kappaB activation through stabilization of IkappaB proteins, suggesting that these signal-transducing enzymes could be potential targets for procyanidin dimer B2. By affecting the expression rather than the activity of COX-2, these in vitro data reported herein give further evidence on the anti-inflammatory protection by procyanidins.     

CaV1.3 channels and intracellular calcium mediate osmotic stress-induced N-terminal c-Jun kinase activation and disruption of tight junctions in Caco-2 CELL MONOLAYERS

We investigated the role of a Ca(2+) channel and intracellular calcium concentration ([Ca(2+)](i)) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-mediated intracellular Ca(2+) depletion. Depletion of extracellular Ca(2+) at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca(2+) depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca(2+) depletion partially reduced osmotic stress-induced rise in [Ca(2+)](i), whereas thapsigargin and extracellular Ca(2+) depletion together resulted in almost complete loss of rise in [Ca(2+)](i). L-type Ca(2+) channel blockers (isradipine and diltiazem) or knockdown of the Ca(V)1.3 channel abrogated [Ca(2+)](i) rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca(2+) depletion, thapsigargin, or Ca(V)1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca(2+) depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin, which was attenuated by BAPTA, SP600125 (JNK inhibitor), or PP2. These results demonstrate that Ca(V)1.3 and rise in [Ca(2+)](i) play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca(2+)](i) mediate osmotic stress-induced JNK activation and subsequent c-Src activation and tyrosine phosphorylation of tight junction proteins. Additionally, inositol 1,4,5-trisphosphate receptor-mediated release of ER Ca(2+) also contributes to osmotic stress-induced tight junction disruption.     

Green tea proanthocyanidins inhibit cyclooxygenase-2 expression in LPS-activated mouse macrophages: molecular mechanisms and structure-activity relationship

The inhibitory effects of green tea proanthocyanidins on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) release were investigated in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. Prodelphinidin B2 3,3' di-O-gallate (PDGG) caused a dose-dependent inhibition of COX-2 at both mRNA and protein levels with the attendant release of PGE(2). Molecular evidence revealed that PDGG inhibited the degradation of Ikappa-B, nuclear translocation of p65 and CCAAT/enhancer-binding protein (C/EBP)delta, and phosphorylation of c-Jun, but not CRE-binding protein (CREB), which regulate COX-2 expression. Moreover, PDGG suppressed the activations of mitogen-activated protein kinase (MAPK) including c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 kinase. The results demonstrated that PDGG suppressed COX-2 expression via blocking MAPK-mediated activation of nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and C/EBPdelta. Furthermore, studies on structure-activity relationship using five kinds of proanthocyanidins revealed that the galloyl moiety of proanthocyanidins appeared important to their inhibitory actions. Thus, our findings provide the first molecular basis that green tea proanthocyanidins with the galloyl moiety might have anti-inflammatory properties through blocking MAPK-mediated COX-2 expression.     

Production of beta-defensin-2 by human colonic epithelial cells induced by Salmonella enteritidis flagella filament structural protein.

We recently showed that FliC of Salmonella enteritidis increased human beta-defensin-2 (hBD-2) expression, and now describe the signaling responsible pathway. FliC increased the intracellular Ca(2+) concentration ([Ca(2+)](in)) in Caco-2 cells. The [Ca(2+)](in) increase induced by FliC was prevented by U73122 and heparin, but not by chelating extracellular Ca(2+) or pertussis toxin. The FliC-induced increase in hBD-2 promoter activity via nuclear factor kappaB (NF-kappaB) was also inhibited by chelation of intracellular Ca(2+) or by U73122. We conclude that FliC increased [Ca(2+)](in) via inositol 1,4,5-trisphosphate, which was followed by up-regulating hBD-2 mRNA expression via an NF-kappaB-dependent pathway.     

Platelet-derived growth factor-induced arachidonic acid release for enhancement of prostaglandin E(2) synthesis in human gingival fibroblasts pretreated with interleukin-1beta

Platelet-derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E(2) (PGE(2)) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time- and dose-dependent manner, and simultaneously induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), but less provoked PGE(2) release and cyclooxygenase-2 (COX-2) mRNA expression. When [Ca(2+)](i) was increased by Ca(2+)-mobilizing reagents, arachidonic acid release was increased. The PDGF-induced arachidonic acid release and increase in [Ca(2+)](i) were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre-stimulated with interleukin-1beta (IL-1beta), PDGF clearly increased PGE(2) release. The PDGF-induced PGE(2) release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL-1beta, arachidonic acid strongly enhanced PGE(2) release and COX-2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca(2+)](i) via tyrosine kinase activation, and which contributes to PGE(2) production via COX-2 expression in HGF primed with IL-1beta.     

Zn2+-induced ERK activation mediated by reactive oxygen species causes cell death in differentiated PC12 cells

Recent studies have provided evidence that Zn2+ plays a crucial role in ischemia- and seizure-induced neuronal death. However, the intracellular signaling pathways involved in Zn2+-induced cell death are largely unknown. In the present study, we investigated the roles of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), and of reactive oxygen species (ROS) in Zn2+-induced cell death using differentiated PC12 cells. Intracellular accumulation of Zn2+ induced by the combined application of pyrithione (5 microM), a Zn2+ ionophore, and Zn2+ (10 microM) caused cell death and activated JNK and ERK, but not p38 MAPK. Preventing JNK activation by the expression of dominant negative SEK1 (SEKAL) did not attenuate Zn2+-induced cell death, whereas the inhibition of ERK with PD98059 and the expression of dominant negative Ras mutant (RasN17) significantly prevented cell death. Inhibition of protein kinase C (PKC) and phosphatidylinositol-3 kinase had little effect on Zn2+-induced ERK activation. Intracellular Zn2+ accumulation resulted in the generation of ROS, and antioxidants prevented both the ERK activation and the cell death induced by Zn2+. Therefore, we conclude that although Zn2+ activates JNK and ERK, only ERK contributes to Zn2+-induced cell death, and that ERK activation is mediated by ROS via the Ras/Raf/MEK/ERK signaling pathway.     

Functional characterization of P2Y1 versus P2X receptors in RBA-2 astrocytes: elucidate the roles of ATP release and protein kinase C

A physiological concentration of extracellular ATP stimulated biphasic Ca(2+) signal, and the Ca(2+) transient was decreased and the Ca(2+) sustain was eliminated immediately after removal of ATP and Ca(2+) in RBA-2 astrocytes. Reintroduction of Ca(2+) induced Ca(2+) sustain. Stimulation of P2Y(1) receptors with 2-methylthioadenosine 5'-diphosphate (2MeSADP) also induced a biphasic Ca(2+) signaling and the Ca(2+) sustains were eliminated using Ca(2+)-free buffer. The 2MeSADP-mediated biphasic Ca(2+) signals were inhibited by phospholipase C (PLC) inhibitor U73122, and completely blocked by P2Y(1) selective antagonist MRS2179 and protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) whereas enhanced by PKC inhibitors GF109203X and Go6979. Inhibition of capacitative Ca(2+) entry (CCE) decreased the Ca(2+)-induced Ca(2+) entry; nevertheless, ATP further enhanced the Ca(2+)-induced Ca(2+) entry in the intracellular Ca(2+) store-emptied and CCE-inhibited cells indicating that ATP stimulated Ca(2+) entry via CCE and ionotropic P2X receptors. Furthermore, the 2MeSADP-induced Ca(2+) sustain was eliminated by apyrase but potentiated by P2X(4) allosteric effector ivermectin (IVM). The agonist ADPbetaS stimulated a lesser P2Y(1)-mediated Ca(2+) signal and caused a two-fold increase in ATP release but that were not affected by IVM whereas inhibited by PMA, PLC inhibitor ET-18-OCH(3) and phospholipase D (PLD) inhibitor D609, and enhanced by removal of intra- or extracellular Ca(2+). Taken together, the P2Y(1)-mediated Ca(2+) sustain was at least in part via P2X receptors activated by the P2Y(1)-induced ATP release, and PKC played a pivotal role in desensitization of P2Y(1) receptors in RBA-2 astrocytes.     

Low [Mg(2+)](o) induces contraction and [Ca(2+)](i) rises in cerebral arteries: roles of ca(2+), PKC, and PI3

Removal of extracellular Ca(2+) concentration ([Ca(2+)](o)) and pretreatment of canine basilar arterial rings with either an antagonist of voltage-gated Ca(2+) channels (verapamil), a selective antagonist of the sarcoplasmic reticulum Ca(2+) pump [thapsigargin (TSG)], caffeine plus a specific antagonist of ryanodine-sensitive Ca(2+) release (ryanodine), or a D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]- mediated Ca(2+) release antagonist (heparin) markedly attenuates low extracellular Mg(2+) concentration ([Mg(2+)](o))-induced contractions. Low [Mg(2+)](o)-induced contractions are significantly inhibited by pretreatment of the vessels with G?-6976 [a protein kinase C-alpha (PKC-alpha)- and PKC-betaI-selective antagonist], bisindolylmaleimide I (Bis, a specific antagonist of PKC), and wortmannin or LY-294002 [selective antagonists of phosphatidylinositol-3 kinases (PI3Ks)]. These antagonists were also found to relax arterial contractions induced by low [Mg(2+)](o) in a concentration-dependent manner. The absence of [Ca(2+)](o) and preincubation of the cells with verapamil, TSG, heparin, or caffeine plus ryanodine markedly attenuates the transient and sustained elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) induced by low-[Mg(2+)](o) medium. Low [Mg(2+)](o)-produced increases in [Ca(2+)](i) are also suppressed markedly in the presence of G?-6976, Bis, wortmannin, or LY-294002. The present study suggests that both Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from intracellular stores [both Ins(1,4,5)P(3) sensitive and ryanodine sensitive] play important roles in low-[Mg(2+)](o) medium-induced contractions of isolated canine basilar arteries. Such contractions are clearly associated with activation of PKC isoforms and PI3Ks.     

Involvement of Na+/H+ exchangers in induction of cyclooxygenase-2 by vacuolar-type (H+)-ATPase inhibitors in RAW 264 cells

In the mouse macrophage-like cell line RAW 264, vacuolar-type (H(+))-ATPase (V-ATPase) inhibitors, bafilomycin A(1) and concanamycin A, increased the level of cyclooxygenase (COX)-2 protein and its mRNA. The V-ATPase inhibitor-induced expression of COX-2 was suppressed by inhibitors of c-jun N-terminal kinase (JNK) and nuclear factor-kappaB, and by inhibitors of Na(+)/H(+) exchangers (NHEs). The bafilomycin A(1)-induced activation of JNK but not degradation of IkappaB-alpha was suppressed by NHE inhibitors and by an inhibitor of Na(+)/Ca(2+) exchanger SN-6. These results suggested that V-ATPase inhibitors induce the expression of COX-2 via NHE-dependent and -independent pathways.