Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca2+ and PI3-kinase

Primary cortical neurones exposed to an oxidative insult in the form of hydrogen peroxide (H(2)O(2)) for 30 min showed a concentration-dependent increase in oxidative stress followed by a delayed NMDA receptor-dependent cell death measured 24 h later. Extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK) and the kinase Akt/PKB may regulate neuronal viability in response to oxidative insults. Using phospho-specific antibodies, a 15-min stimulation of neurones with H(2)O(2) (100 microm - 1 mm) produced a concentration-dependent phosphorylation of ERK1/2 and Akt/PKB that was partly dependent on extracellular Ca(2+) and phosphatidylinositol 3-kinase (PI3-K). Higher concentrations of H(2)O(2) (1 mm) also stimulated a phosphorylation of JNK which was totally dependent on extracellular Ca(2+) but not PI3-K. H(2)O(2)-induced phosphorylation of ERK1/2, Akt/PKB or JNK were unaffected by the NMDA channel blocker MK801. Blocking ERK1/2 activation with the upstream inhibitor U0126 (10 microm) enhanced H(2)O(2)-induced (100-300 microm range) neurotoxicity and inhibited H(2)O(2)-mediated phosphorylation of the cyclic AMP regulatory binding protein (CREB), suggesting that ERK1/2 signals to survival under these conditions. At higher concentrations (mm), H(2)O(2)-stimulated a phosphorylation of c-jun. It is likely, therefore, that subjecting neurones to moderate oxidative-stress recruits pro-survival signals to CREB but during severe oxidative stress pro-death signals through JNK and c-jun are dominant.     

Mechanical stretch stimulates growth of vascular smooth muscle cells via epidermal growth factor receptor

We have studied whether activation of epidermal growth factor receptor (EGFR) is involved in stretch-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation and protein synthesis in cultured rat vascular smooth muscle cells (VSMC). Cyclic stretch (1 Hz) induced a rapid (within 5 min) phosphorylation of ERK1/2, an effect that was time and strength dependent and inhibited by an EGFR kinase inhibitor (AG-1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG-1296). The stretch rapidly (within 2 min) induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR as revealed by blockade with AG-1478 as well as immunoprecipitation with anti-EGFR antibody coupled with immunoblotting with anti-phosphotyrosine antibody. The stretch rapidly (within 2 min) induced association of tyrosine-phosphorylated EGFR with adaptor proteins (Shc/Grb2) as revealed by coprecipitation with glutathione-S-transferase-Grb2 fusion protein coupled with immunoblotting with anti-phosphotyrosine, anti-EGFR, and anti-Shc antibodies. Transfection of a dominant-negative mutant of H-Ras also inhibited stretch-induced ERK1/2 activation. Treatment with a stretch-activated ion channel blocker (Gd(3+)) and an intracellular Ca(2+) antagonist (TMB-8) inhibited stretch-induced phosphorylation of EGFR and ERK1/2. Treatment with AG-1478 and a mitogen-activated protein kinase kinase inhibitor (PD-98059), but not AG-1296, blocked [(3)H]leucine uptake stimulated by a high level of stretch. These data suggest that ERK1/2 activation by mechanical stretch requires Ca(2+)-sensitive EGFR activation mainly via stretch-activated ion channels, thereby leading to VSMC growth.     

Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid

This study was conducted on human Jurkat T-cells to investigate the role of depletion of intracellular Ca(2+) stores in the phosphorylation of two mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK) 1 and ERK2, and their modulation by a polyunsaturated fatty acid, docosahexaenoic acid (DHA). We observed that thapsigargin (TG) stimulated MAPK activation by store-operated calcium (SOC) influx via opening of calcium release-activated calcium (CRAC) channels as tyrphostin-A9, a CRAC channel blocker, and two SOC influx inhibitors, econazole and SKF-96365, diminished the action of the former. TG-stimulated ERK1/ERK2 phosphorylation was also diminished in buffer containing EGTA, a calcium chelator, further suggesting the implication of calcium influx in MAPK activation in these cells. Moreover, TG stimulated the production of diacylglycerol (DAG) by activating phospholipase D (PLD) as propranolol (PROP) (a PLD inhibitor), but not U73122 (a phospholipase C inhibitor), inhibited TG-evoked DAG production in these cells. DAG production and protein kinase C (PKC) activation were involved upstream of MAPK activation as PROP and GF109203X, a PKC inhibitor, abolished the action of TG on ERK1/ERK2 phosphorylation. Furthermore, DHA seems to act by inhibiting PKC activation as this fatty acid diminished TG- and phorbol 12-myristate 13-acetate-induced ERK1/ERK2 phosphorylation in these cells. Together these results suggest that Ca(2+) influx via CRAC channels is implicated in PLD/PKC/MAPK activation which may be a target of physiological agents such as DHA.     

Role of ca(2+) in the control of h(2)o(2)-modulated phosphorylation pathways leading to eNOS activation in cardiac myocytes

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H(2)O(2)-modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H(2)O(2) treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca(2+) ([Ca(2+)](i)), and document that activity of the L-type Ca(2+) channel is necessary for the H(2)O(2)-promoted increase in sarcomere shortening and of [Ca(2+)](i). Using the chemical NO sensor Cu(2)(FL2E), we discovered that the H(2)O(2)-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca(2+) channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase 1/2 (MEK1/2). Moreover, H(2)O(2)-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca(2+)](i) as well as the activation of protein kinase C (PKC). We also found that H(2)O(2)-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca(2+) channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H(2)O(2)-promoted eNOS phosphorylation. Here we present evidence documenting that H(2)O(2)-promoted Akt phosphorylation is dependent on activation of the L-type Ca(2+) channel, but is independent of PKC. These studies establish key roles for Ca(2+)- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H(2)O(2).     

A serpentine receptor-dependent, Gbeta- and Ca(2+) influx-independent pathway regulates mitogen-activated protein kinase ERK2 in Dictyostelium.

Ca(2+) influx and mitogen-activated protein (MAP) kinase activation are important phenomena in signal transduction, which are often interconnected. We investigated whether serpentine receptor-dependent, Gbeta-independent activation of MAP kinase ERK2 by chemoattractant cyclic AMP (cAMP) is mediated by Ca(2+) influx in the social amoeba Dictyostelium discoideum. We generated a D. discoideum double mutant, which harbours a temperature-sensitive Gbeta subunit and expresses the apoaequorin protein. Utilizing this mutant, we demonstrate that cAMP induced Ca(2+) influx into intact D. discoideum cells can be blocked completely at both the permissive and the restrictive temperature, by using either gadolinium ions or Ruthenium Red. Under the same experimental conditions, these substances do not abolish cAMP stimulation of ERK2 at either temperature. We conclude that there is a Gbeta- and Ca(2+) influx-independent pathway for the receptor-dependent activation of MAP kinase ERK2 in D. discoideum.     

Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells

Nicotine treatment triggers calcium influx into neuronal cells, which promotes cell survival in a number of neuronal cells. Phosphoinositide (PI) 3-kinase and downstream PI3-kinase target Akt have been reported to be important in the calcium-mediated promotion of survival in a wide variety of cells. We investigated the mechanisms of nicotine-induced phosphorylation of Akt in PC12h cells, in comparison with nicotine-induced ERK phosphorylation. Nicotine induced Akt phosphorylation in a dose-dependent manner. A nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitor had no significant effect on nicotine-induced Akt phosphorylation, while a non-selective nAChR antagonist inhibited the phosphorylation. L-type voltage-sensitive calcium channel (VSCC) antagonists, calmodulin antagonist, and Ca2+/calmudulin-dependent protein kinase (CaM kinase) inhibitor prevented the nicotine-induced Akt phosphorylation. Three epidermal growth factor receptor (EGFR) inhibitors prevented the nicotine-induced phosphorylation of both extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and Akt. In contrast, an inhibitor of the Src family tyrosine kinase prevented the nicotine-induced Akt phosphorylation but not ERK phosphorylation. These results suggested that nicotine induces the activation of both PI3-kinase/Akt and ERK pathways via common pathways including non-alpha7-nAChRs, L-type VSCC, CaM kinase II and EGFR in PC12h cells, but Src family tyrosine kinases only participate in the pathway to activate Akt.     

The neuropeptide pituitary adenylate cyclase activating polypeptide modulates Ca2+ and pro-inflammatory functions in human monocytes through the G protein-coupled receptors VPAC-1 and formyl peptide receptor-like 1

In human neutrophils, the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) acting via the G protein-coupled receptors vasoactive intestinal peptide/PACAP receptor 1 (VPAC-1) and formyl peptide receptor-like 1 (FPRL1) modulates Ca2+ and pro-inflammatory activities. We evaluated in human monocytes the importance of the Ca2+ signal and the participation of FPRL1 in PACAP-associated signaling pathways and pro-inflammatory activities. PACAP-evoked Ca2+ transient involved both Ca2+ influx and intracytoplasmic Ca2+ mobilisation. This was pertussis toxin, protein kinase A and adenylate cyclase dependent indicating the participation of Galphai and Galphas with mobilisation of both InsP3 sensitive and insensitive stores. Intra- or extracellular Ca2+ depletion resulted in the inhibition of PACAP-induced, Akt, ERK, p38 and NF-kappaB activations as well as a decrease in PACAP-associated reactive oxygen species (ROS) production and integrin CD11b membrane upregulation. The FPRL1 antagonist, Trp-Arg-Trp-Trp-Trp (WRW4), decreased PACAP-evoked Ca2+ signal, Akt, ERK phosphorylation, ROS and CD11b upregulation without affecting p38 phosphorylation. NF-kappaB inhibitors prevented PACAP-induced Ca2+ mobilisation. Monocytes pre-treatment with fMLP but not with LPS desensitised cells to the pro-inflammatory effects of PACAP. Thus, both intra- and extracellular Ca2+ play a role in controlling pro-inflammatory functions stimulated by PACAP which acts through a VPAC-1, FPRL1/Galphai/PI3K/ERK pathway and a VPAC-1/Galphas/PKA/p38 pathway to fully activate monocytes.     

Cyclic ADP ribose is a novel regulator of intracellular Ca2+ oscillations in human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine. However, the cellular biology of these cells is not fully understood. The present study characterizes the cyclic ADP-ribose (cADPR)-mediated Ca(2+) signals in human MSCs and finds that externally applied cADPR can increase the frequency of spontaneous intracellular Ca(2+) (Ca(2+) (i) ) oscillations. The increase was abrogated by a specific cADPR antagonist or an inositol trisphosphate receptor (IP3R) inhibitor, but not by ryanodine. In addition, the cADPR-induced increase of Ca(2+) (i) oscillation frequency was prevented by inhibitors of nucleoside transporter or by inhibitors of the transient receptor potential cation melastatin-2 (TRPM2) channel. RT-PCR revealed mRNAs for the nucleoside transporters, concentrative nucleoside transporters 1/2 and equilibrative nucleoside transporters 1/3, IP3R1/2/3 and the TRPM2 channel, but not those for ryanodine receptors and CD38 in human MSCs. Knockdown of the TRPM2 channel by specific short interference RNA abolished the effect of cADPR on the Ca(2+) (i) oscillation frequency, and prevented the stimulation of proliferation by cADPR. Moreover, cADPR remarkably increased phosphorylated extracellular-signal-regulated kinases 1/2 (ERK1/2), but not Akt or p38 mitogen-activated protein kinase (MAPK). However, cADPR had no effect on adipogenesis or osteogenesis in human MSCs. Our results indicate that cADPR is a novel regulator of Ca(2+) (i) oscillations in human MSCs. It permeates the cell membrane through the nucleoside transporters and increases Ca(2+) oscillation via activation of the TRPM2 channel, resulting in enhanced phosphorylation of ERK1/2 and, thereby, stimulation of human MSC proliferation. This study delineates an alternate signalling pathway of cADPR that is distinct from its well-established role of serving as a Ca(2+) messenger for mobilizing the internal Ca(2+) stores. Whether cADPR can be used clinically for stimulating marrow function in patients with marrow disorders remains to be further studied.     

RISK pathway is involved in oxytocin postconditioning in isolated rat heart

The reperfusion injury salvage kinase (RISK) pathway is a fundamental signal transduction cascade in the cardioprotective mechanism of ischemic postconditioning. In the present study, we examined the cardioprotective role of oxytocin as a postconditioning agent via activation of the RISK pathway (PI3K/Akt and ERK1/2).Animals were randomly divided into 6 groups. The hearts were subjected under 30 minutes (min) ischemia and 100 min reperfusion. OT was perfused 15 min at the early phase of reperfusion. RISK pathway inhibitors (Wortmannin; an Akt inhibitor, PD98059; an ERK1/2 inhibitor) and Atosiban (an OT receptor antagonist) were applied either alone 10 min before the onset of the ischemia or in the combination with OT during early reperfusion phase. Myocardial infarct size, hemodynamic factors, ventricular arrhythmia, coronary flow and cardiac biochemical marker were measured at the end of reperfusion.OT postconditioning (OTpost), significantly decreased the infarct size, arrhythmia score, incidence of ventricular fibrillation, Lactate dehydrogenase and it increased coronary flow. The cardioprotective effect of OTpos was abrogated by PI3K/Akt, ERK1/2 inhibitors and Atosiban.Our data have shown that OTpost can activate RISK pathway mostly via the PI3K/Akt and ERK1/2 signaling cascades during the early phase of reperfusion.     

Involvement of extracellular signal-regulated protein kinase in gliosis induced during recovery from metabolic inhibition

Brain reperfusion may be of particular importance in the etiology of periventricular leukomalacia, of which the common findings are gliosis and ventricular dilatation. To investigate the mechanism of this pathogenesis, we used a metabolic inhibition (MI) model using cyanide plus deoxyglucose treatment of cultured glia isolated from fetal rat brain and examined the activity of extracellular signal-regulated protein kinase (ERK) during MI and also during the recovery from MI of 30 min. ERK activation was stimulated during MI and the recovery from MI. The time course and extent of activation of ERK during MI and the recovery from MI, however, were distinctly different. Activation of ERK was stimulated within 5 min of MI and declined thereafter. Activation of ERK was sustained during the recovery phase from MI and the extent of the activation was much greater than that during MI. Pretreatment with EGTA to eliminate extracellular Ca(2+), or with APV, an NMDA receptor antagonist, to inhibit Ca(2+) influx through the NMDA receptor, attenuated the activation of ERK. Moreover, pretreatment with PMA to downregulate PKC abolished the activation of ERK. PD98059, an inhibitor of ERK kinase, attenuated the cell proliferation induced by MI followed by recovery from MI. These results suggest that ERK is involved in gliosis during the recovery phase from MI and may play a role in the etiology of periventricular leukomalacia.     

Cellular mechanism of sodium oleate-stimulated secretion of cholecystokinin and secretin

Long-chain fatty acids are potent stimulants of secretin and CCK release. The cellular mechanisms of fatty acid-stimulated secretion of these two hormones are not clear. We studied the stimulatory effect and mechanism of sodium oleate (SO) on secretin- and CCK-producing cells. SO stimulated the release of secretin or CCK from isolated rat mucosal cell preparations enriched in either secretin- or CCK-producing cells, respectively. SO also time- and dose-dependently stimulated secretin and CCK release from STC-1 cells. In STC-1 cells, SO-stimulated secretin and CCK release was potentiated by IBMX and inhibited by a protein kinase A-selective inhibitor and a cAMP-specific antagonist. SO-stimulated releases of the two hormones were also inhibited by downregulation or inhibitors of protein kinase C, a calmodulin antagonist and an inhibitor of calmodulin-dependent protein kinase II. Chelating of extracellular Ca(2+) or addition of an L-type calcium channel blocker diminished SO-stimulated hormone releases. SO caused an increase in intracellular Ca(2+) concentration that was partially reversed by diltiazem but had no effect on production of cAMP, cGMP, or inositol-1,4,5-triphosphate. These results indicate that SO acts on secretin- and CCK-producing cells. Its stimulatory effect is potentiated by endogenous protein kinase A and mediated by activation of Ca(2+) influx through the L-type channels and of protein kinase C and Ca(2+)/calmodulin-dependent protein kinase II.     

Follicle-stimulating hormone activates extracellular signal-regulated kinase but not extracellular signal-regulated kinase kinase through a 100-kDa phosphotyrosine phosphatase

In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca(2+) channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca(2+) channel blocker, and chelation of extracellular Ca(2+). These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase.     

Endoplasmic reticulum Ca2+ release modulates endothelial nitric-oxide synthase via extracellular signal-regulated kinase (ERK) 1/2-mediated serine 635 phosphorylation

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca(2+) and protein phosphorylation, but the interrelationship between intracellular Ca(2+) mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca(2+) release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca(2+) release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca(2+) release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca(2+) and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca(2+) release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca(2+)/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca(2+) release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca(2+) release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca(2+) is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca(2+)-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.     

Glutamatergic regulation of the p70S6 kinase in primary mouse neurons

Brief glutamatergic stimulation of neurons from fetal mice, cultured in vitro for 6 days, activates the mTOR-S6 kinase, ERK1/2 and Akt pathways, to an extent approaching that elicited by brain-derived neurotrophic factor. In contrast, sustained glutamatergic stimulation inhibits ERK, Akt, and S6K. Glutamatergic activation of S6K is calcium/calmodulin-dependent and is prevented by inhibitors of calcium/calmodulin-dependent protein kinase 2, phosphatidylinositol 3-OH-kinase and by rapamycin. 2-Amino-5-phosphonovaleric acid, an inhibitor of N'-methyl-D-aspartate receptors, abolishes glutamatergic activation of ERK1/2 but not the activation of mTOR-S6K; the latter is completely abolished by inhibitors of voltage-dependent calcium channels. Added singly, dopamine gives slight, and norepinephrine a more significant, activation of ERK and S6K; both catecholeamines, however, enhance glutamatergic activation of S6K but not ERK. After 12 days in culture, the response to direct glutamatergic activation is attenuated but can be uncovered by suppression of gamma-aminobutyric acid interneurons with bicuculline in the presence of the weak K(+) channel blocker 4-aminopyridine (4-AP). This selective synaptic activation of mTOR-S6K is also resistant to APV and inhibited by Ca(2+) channel blockers and higher concentrations of glutamate. Elongation factor 2 (EF2) is phosphorylated and inhibited by the eEF2 kinase (CaM kinase III); the latter is inhibited by the S6K or Rsk. Bicuculline/4-AP or KCl-induced depolarization reduces, whereas higher concentrations of glutamate increases, EF2 phosphorylation. Thus the mTOR-S6K pathway in neurons, a critical component of the late phase of LTP, is activated by glutamatergic stimulation in a calcium/calmodulin-dependent fashion through a calcium pool controlled by postsynaptic voltage-dependent calcium channels, whereas sustained stimulation of extrasynaptic glutamate receptors is inhibitory.     

Targeted deletion of A2A adenosine receptors attenuates the protective effects of myocardial postconditioning

Endogenous adenosine is an important ligand trigger for the cardioprotective effects of postconditioning (POC), yet it is unclear which adenosine receptor subtype is primarily responsible. To evaluate the role of A(2A) adenosine receptors in POC-induced protection, global ischemia-reperfusion was performed with and without POC in isolated wild-type (WT) and A(2A) adenosine receptor knockout (A(2A)KO) mouse hearts. Injury was measured in terms of postischemic functional recovery and release of cardiac troponin I (cTnI). Activation of protective signaling with POC was assessed by Akt and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. In WT hearts, POC improved recovery of postischemic developed pressure in early (81.6 +/- 6.4% of preischemic baseline vs. 37.5 +/- 5.6% for non-POC WT at 1 min) and late (62.2 +/- 4.2% of baseline vs. 45.5 +/- 5.3% for non-POC WT at 30 min) reperfusion, reduced cTnI release by 37%, and doubled the phosphorylation of both Akt and ERK1/2. These beneficial effects of POC were blocked by treatment with the selective A(2A) adenosine receptor antagonist ZM-241385 during reperfusion. Postischemic functional recovery, cTnI release, and phosphorylation of Akt and ERK1/2 were not different between non-POC WT and A(2A)KO hearts. In A(2A)KO hearts, POC did not improve functional recovery, reduce cTnI release, nor increase phosphorylation of Akt or ERK1/2. Thus the protective effects of POC are attenuated by both selective A(2A) receptor antagonism and targeted deletion of the gene encoding A(2A) adenosine receptors. These observations support the conclusion that endogenous activation of A(2A) adenosine receptors is an essential trigger leading to the protective effects of POC in isolated murine hearts.     

Clozapine, a neuroleptic agent, inhibits Akt by counteracting Ca2+/calmodulin in PTEN-negative U-87MG human glioblastoma cells

Clozapine (CZP), a dibenzodiazepine derivative with a piperazinyl side chain, is in clinical use as an antipsychotic drug. This study investigated the effect of CZP on the modulation of the PI3K/Akt/GSK-3beta pathway in PTEN-negative U-87MG glioblastoma cells. Treatment with CZP rapidly inhibited the basal and EGF-induced phosphorylation of Akt. The inhibition of Akt resulted in the dephosphorylation of GSK-3beta and increased GSK-3beta kinase activity. A voltage-sensitive Ca(2+) channel blocker and calmodulin (CaM) antagonists inhibited Akt phosphorylation, whereas elevation of the intracellular Ca(2+) concentration prevented CZP-induced dephosphorylation of Akt and GSK-3beta, suggesting that Ca(2+)/CaM participates in the inhibition of Akt by CZP in U-87MG cells. In addition, similar to LY294002, CZP arrested cell cycle progression at G0/G1 phase, which was accompanied by decreased expression of cyclin D1. The reduction in the cyclin D1 level induced by CZP was abrogated by the inhibition of GSK-3beta, the inhibition of proteasome-dependent proteolysis, or an increase in the intracellular Ca(2+) concentration. These results suggest that the antipsychotic drug CZP modulates the PI3K/Akt/GSK-3beta pathway by counteracting Ca(2+)/CaM in PTEN-negative U-87MG glioblastoma cells.     

Requirements of calcium fluxes and ERK kinase activation for glucose- and interleukin-1β-induced β-cell apoptosis

Increasing evidence indicates that beta-cell apoptosis and impaired secretory function were partly mediated by interleukin (IL)-1beta and/or high-glucose-induced beta-cell production of IL-1beta. However, the specific signal transduction pathways and molecular events involved in beta-cell dysfunction remain largely unresolved. In this study, we investigated whether Ca(2+) and extracellular signal-regulated kinase (ERK) activation plays a role for IL-1beta action in rat islets. Exposure of rat islets for 4 days to 33.3 mM glucose and 140 ng/ml IL-1beta- induced beta-cell apoptosis and impaired glucose-stimulated insulin secretion. By Western blotting with phosphospecific antibodies, glucose and IL-1beta were shown to activate ERK. Ca(2+) channel blocker nimodipine or ERK inhibitor PD98059 prevented glucose- and IL-1beta-induced ERK activation, beta-cell apoptosis, and impaired function. Furthermore, treatment with Ca(2+) ionophore ionomycin, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, all caused an amplification of IL-1beta-induced ERK activation in rat islet. On the other hand, a chelator of intracellular free Ca(2+) [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl] (BAPTA/AM) and an inhibitor of calmodulin (W7) diminished IL-1beta-induced phosphorylation of ERK. Finally, islet release of IL-1beta in response to high glucose could be abrogated by nimodipine, mibefradil, or PD98059. Together, these data suggest that glucose- and IL-1beta-induced beta-cell secretory dysfunction and apoptosis are Ca(2+) influx and ERK dependent in rat islets.     

Calcium mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) in rat astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to release intracellular Ca(2+) in several types of cells. We have used Ca(2+)-sensitive fluorescent dyes (Fura-2, Fluo-4) to measure intracellular Ca(2+) in astrocytes in culture and in situ. Bath-applied NAADP elicited a reversible and concentration-dependent Ca(2+) rise in up to 90% of astrocytes in culture (EC(50)=7 microM). The NAADP-evoked Ca(2+) rise was maintained in the absence of extracellular Ca(2+), but was suppressed after depleting the Ca(2+) stores of the ER with ATP (20 microM), with cyclopiazonic acid (10 microM) or with ionomycin (5 microM). P(2) receptor antagonist pyridoxalphosphate-6-azophenyl-2'4'-disulfonic acid (PPADS, 100 microM), IP(3) receptor blocker 2-aminoethoxydiphenyl borate (2-APB, 100 microM) and PLC inhibitor U73122 (10 microM) also reduced or suppressed the NAADP-evoked Ca(2+) rise. NAADP still evoked a Ca(2+) response after application of glycyl-l-phenylalanine-beta-naphthylamide (GPN, 200 microM), which permeabilizes lysosomes, or preincubation with H(+)-ATPase inhibitor bafilomycin A1 (4 microM) and of p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, 2 microM), that impairs mitochondrial Ca(2+) handling. In acute brain slices, NAADP (10 microM) evoked Ca(2+) transients in cerebellar Bergmann glial cells and in hippocampal astrocytes. Our results suggest that NAADP recruits Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores in mammalian astrocytes, at least partly by activating metabotropic P(2)Y receptors.