Metabotropic glutamate receptor 5 activation enhances tyrosine phosphorylation of the N-methyl-D-aspartate (NMDA) receptor and NMDA-induced cell death in hippocampal cultured neurons.

The activation of group I metabotropic glutamate receptors (mGluRs), which are coupled with Gq-protein, initiates a variety physiological responses in different types of cells. While Gq-protein-coupled receptors can upregulate N-methyl-D-aspartate (NMDA) receptor function, group I mGluR-mediated regulations of NMDA receptor function are not fully understood. To determine biochemical roles of group I mGluRs in the regulation of the NMDA receptor, we have investigated changes in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B induced by a selective mGluR5 agonist, (RS)-chloro-5-hydroxyphenylglycine (CHPG) in hippocampal neuronal cultures. Activation of mGluR5 by CHPG increased active-forms of Src. CHPG also enhanced tyrosine phosphorylation of NR2A and NR2B in hippocampal neuronal cultures. In addition, NMDA-induced cell death was enhanced by CHPG-induced mGluR5 stimulation at the concentration, which increased tyrosine phosphorylation of Src and NR2A/2B but did not induce cell death. This effect was inhibited by selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP). The results suggest that in hippocampal neurons, mGluR5 may regulate NMDA receptor activity, involving tyrosine phosphorylation of NR2A and NR2B and may be involved in NMDA receptor-mediated cell injury.

Effects of metabotropic glutamate mGlu5 receptor antagonist on tyrosine phosphorylation of NMDA receptor subunits and cell death in the hippocampus after brain ischemia in rats.

Tyrosine phosphorylation of the N-methyl-D-aspartate (NMDA) receptor appears to be associated with the regulation of the receptor's ion channel. This study focused on the effect of a metabotropic glutamate mGlu5 receptor antagonist on tyrosine phosphorylation of NMDA receptor subunits and cell death in the hippocampal CA1 region after transient global ischemia and sought to explore their mechanisms. Pretreatment with the mGlu5 receptor antagonist reduced cell death in the hippocampal CA1 region on day 3 after the transient ischemia. Transient ischemia increased the tyrosine phosphorylation of NMDA receptor subunits, which are a major target of Src family tyrosine kinases. Therefore, we investigated the effect of the antagonist on tyrosine phosphorylation of the NMDA receptor subunits after transient ischemia. Tyrosine phosphorylation of the NR2A subunit, but not that of the NR2B one, was inhibited by the mGlu5 receptor antagonist. The administration of the antagonist also attenuated the increase in the amount of active form of Src after the reperfusion. We further demonstrated that the administration of a Src-family kinase inhibitor prevented cell death in the hippocampal CA1 region and attenuated the increase in the tyrosine phosphorylation of the NMDA receptor subunits after the reperfusion. These findings suggest that mGlu5 receptor in the hippocampal CA1 region after transient ischemia is involved in the activation of Src and subsequent tyrosine phosphorylation of NMDA receptor subunits, which actions may contribute to alterations of properties of the NMDA receptor and may be related to pathogenic events leading to neuronal cell death.

Possible pathway of Na(+) flux into mitochondria in ischemic heart.

Previous studies showed that myocardial Na(+) overload during ischemia directly induced mitochondrial damage. The pathway for Na(+) flux into mitochondria remains unclear. We examined possible routes for Na(+) flux into mitochondria in the ischemic heart. Isolated perfused rat hearts were subjected to 15- to 35-min ischemia followed by 60-min reperfusion and then Na(+) content and respiratory function in mitochondria of the ischemic heart were determined. The mitochondrial Na(+) content of the ischemic heart was ischemic duration-dependently increased, associated with a reduction in mitochondrial respiratory function. To mimic induction of mitochondrial Na(+) overload in vitro, isolated mitochondria were incubated with 6.25 to 50 mM NaCl or sodium lactate, a metabolite of anaerobic glycolysis, in the presence and absence of a mitochondrial Na(+)/Ca(2+) exchanger inhibitor CGP37157 and a monocarboxylate transporter (MCT) inhibitor α-cyano-4-hydroxy cinnamic acid (CHCA). Incubation of mitochondria with NaCl or sodium lactate increased the mitochondrial Na(+) concentration. This increase in mitochondrial Na(+) was partially attenuated by the presence of either inhibitor. Combined treatment of mitochondria with both inhibitors attenuated sodium lactate-induced increase in Na(+) content to a greater degree than that treated with either agent. These results suggest that mitochondrial Na(+)/Ca(2+) exchanger and MCT inhibitor-sensitive Na(+) transporter are possible pathways for the mitochondrial Na(+) overload in the ischemic myocardium.

Transient focal cerebral ischemia differentially decreases Homer1a and 1b/c contents in the postsynaptic density.

Homer is a scaffold protein in the postsynaptic density (PSD) and binds to the intracellular tail of group I metabotropic glutamate receptors (mGluRs). Although Homer contributes to the regulation of physiological function in synapses, the role of Homer proteins under pathophysiological conditions, such as cerebral ischemia, is still not fully clear. In the present study, we sought to determine whether transient focal cerebral ischemia would affect the level of Homer1 in the isolated-PSD fraction from rats. We showed that Homer1a (short form) and Homer1b/c (long form) as well as group I mGluR were localized in the cortical PSD. Cerebral ischemia decreased the content of Homer1a, which is a dominant-negative inhibitor of the long form of Homer proteins, in the PSD at 4 h of reperfusion without changing the level of Homer1a in cortical homogenates. On the other hand, the levels of Homer1b/c in the both PSD and homogenates were decreased at 24 h of reperfusion. These results suggest that these decreases in the level of Homer1 proteins after cerebral ischemia may contribute to the disturbance of synaptic function and subsequent development of cerebral ischemia.

Intravenous injection of neural progenitor cells improves cerebral ischemia-induced learning dysfunction.

The ability of stem cells to enhance neurological recovery seen after cerebral ischemia has been reported. However, it remains to be clarified whether neural progenitor cells (NPCs) improve cerebral ischemia-induced learning dysfunction. We found in an earlier study that the direct injection of NPCs into the hippocampus prevents spatial learning dysfunction after cerebral ischemia. As the intravascular injection of cells represents a minimally invasive therapeutic approach, we sought to determine whether the intravenous injection of NPCs also would improve ischemia-induced spatial learning dysfunction. Cerebral ischemia was produced by the injection of 700 microspheres into the right hemisphere of rats. The injection of NPCs via a femoral vein on day 7 after the induction of ischemia improved the modified neurological severity score and reduced the prolongation of the escape latency seen in the water maze task on days 12-28 after cerebral ischemia. The intravenous injection of NPCs on day 7 did not affect the viable area of the ipsilateral hemisphere on day 28 compared with that of the non-treated ischemic rats. Furthermore, the NPCs injected via the vein were detected in the ipsilateral hemisphere; and they expressed brain-derived neurotrophic factor (BDNF) on day 28. The decrease in the BDNF level in the ipsilateral hemisphere was also inhibited by the injection of NPCs. These results suggest that the NPCs injected via the vein after cerebral ischemia improved spatial learning dysfunction, but without having any restorative effect on the damaged areas, possibly by acting as a source of neurotrophic factors.

NADPH oxidase-mediated oxidative damage to proteins in the postsynaptic density after transient cerebral ischemia and reperfusion.

NADPH oxidase is an important source of superoxide in the central nervous system. Although NADPH oxidase is localized near the postsynaptic site in neurons, little is known about the pathophysiological role of NADPH oxidase in synapses after cerebral ischemia and reperfusion. In the present study, we sought to determine the role of NADPH oxidase in oxidative damage to postsynaptic density (PSD) proteins, which were isolated from rats subjected to transient focal cerebral ischemia and reperfusion. The amounts of carbonylated PSD proteins were increased after transient focal cerebral ischemia and reperfusion. This change was accompanied by an increase in the level of NADPH oxidase subunits in the PSD. The administration of apocynin, an NADPH oxidase inhibitor, attenuated both the protein carbonylation in the PSD and cerebral infarct volume. We further demonstrated that the decreases seen in the amounts of PSD-associated proteins, such as neuroligin, N-cadherin, and SAP102, in the PSD were prevented by treatment with apocynin. These results suggest that pronounced activation of NADPH oxidase in the PSD after cerebral ischemia and reperfusion may be related to the focal oxidative damage to synaptic functions and subsequent development of ischemia and reperfusion-induced cerebral injury.

Cardioprotective effects of 2-octynyladenosine (YT-146) in ischemic/reperfused rat hearts.

The present study was aimed at investigating the cardiac receptor subtypes involved in the cardioprotective effects of 2-octynyladenosine (YT-146), a novel adenosine receptor (AR) agonist. Isolated rat hearts were perfused in the Langendorff manner, and the hearts were exposed to 30 minute of ischemia followed by 60 minutes of reperfusion. YT-146 was infused for 10 minutes just before ischemia, and selective antagonists for AR subtypes were coadministered with YT-146. YT-146 (0.03­0.3 µM) dose dependently improved postischemic recovery of the left ventricular developed pressure (LVDP) of the ischemic/reperfused rat heart (maximum 59.7% ± 2.3% of the preischemic value). Coadministration of 8-(3-chlorostyryl) caffeine (A(2A) AR antagonist), alloxazine (A(2B)AR antagonist), or MRS-1191 (A(3) AR antagonist) with YT-146 failed to alter the cardioprotective effects of YT-146, and their LVDP recoveries were 55.9% ± 5.1%, 52.1% ± 1.9%, and 47.5% ± 1.7%, respectively, at the end of the reperfusion. On the other hand, coadministration of 8-cyclopentyl-1,3-dipropylxanthine (A(1) AR antagonist) abolished the YT-146­induced enhancement of postischemic LVDP recovery (31.7% ± 4.6%). The protein kinase C inhibitor chelerythrine also abolished the YT-146­induced enhancement of postischemic LVDP recovery (22.2% ± 4.5%). YT-146 has been known as an A(2) AR agonist, but our findings suggest that the cardioprotective effects of YT-146 are exerted via cardiac A(1) AR, not A(2) AR, stimulation and the activation of protein kinase C by preischemic treatment in isolated and crystalloid-perfused rat hearts.

Stable interaction between the human proliferating cell nuclear antigen loader complex Ctf18-replication factor C (RFC) and DNA polymerase {epsilon} is mediated by the cohesion-specific subunits, Ctf18, Dcc1, and Ctf8.

One of the proliferating cell nuclear antigen loader complexes, Ctf18-replication factor C (RFC), is involved in sister chromatid cohesion. To examine its relationship with factors involved in DNA replication, we performed a proteomics analysis of Ctf18-interacting proteins. We found that Ctf18 interacts with a replicative DNA polymerase, DNA polymerase ε (pol ε). Co-immunoprecipitation with recombinant Ctf18-RFC and pol ε demonstrated that their binding is direct and mediated by two distinct interactions, one weak and one stable. Three subunits that are specifically required for cohesion in yeast, Ctf18, Dcc1, and Ctf8, formed a trimeric complex (18-1-8) and together enabled stable binding with pol ε. The C-terminal 23-amino acid stretch of Ctf18 was necessary for the trimeric association of 18-1-8 and was required for the stable interaction. The weak interaction was observed with alternative loader complexes including Ctf18-RFC(5), which lacks Dcc1 and Ctf8, suggesting that the common loader structures, including the RFC small subunits (RFC2-5), are responsible for the weak interaction. The two interaction modes, mediated through distinguishable structures of Ctf18-RFC, both occurred through the N-terminal half of pol ε, which includes the catalytic domain. The addition of Ctf18-RFC or Ctf18-RFC(5) to the DNA synthesis reaction caused partial inhibition and stimulation, respectively. Thus, Ctf18-RFC has multiple interactions with pol ε that promote polymorphic modulation of DNA synthesis. We propose that their interaction alters the DNA synthesis mode to enable the replication fork to cooperate with the establishment of cohesion.

Metabotropic glutamate mGlu5 receptor-mediated serine phosphorylation of NMDA receptor subunit NR1 in hippocampal CA1 region after transient global ischemia in rats.

Phosphorylation of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor has been implicated in the regulation of the receptor's ion channel. The contribution of metabotropic glutamate receptors to the NMDA receptor function after brain ischemia remains to be determined. Presently we investigated the effects of an antagonist of the metabotropic glutamate mGlu5 receptor on cell death and serine phosphorylation of the NR1 subunit of the NMDA receptor in the hippocampal CA1 region after transient global ischemia and sought to explore the mechanisms involved. Phosphorylation of serine residues at 890 and 896 of NR1 was increased predominantly in the deoxycholate (DOC)-insoluble fraction after transient global ischemia in rats; and the increase in the phosphorylation of S890, but not that of S896, of NR1 in this fraction was attenuated by the mGlu5 receptor antagonist. The administration of this antagonist also reduced the increase in the amount of protein kinase C (PKC)gamma, but not that of PKCalpha, in the DOC-insoluble fraction. The results suggest that the mGlu5 receptor in the hippocampal CA1 region is involved in the phosphorylation of S890 of NR1 subunit via PKCgamma following transient ischemia. As treatment with the mGlu5 receptor antagonist reduced cell death in the hippocampal CA1 region on day 3 after the start of the reperfusion, these changes in intracellular signaling through mGlu5 receptor may be linked to the pathogenesis of cerebral ischemia.

mGluR1 antagonist decreased NADPH oxidase activity and superoxide production after transient focal cerebral ischemia.

NADPH oxidase, which is activated by PKC and signaling via the NMDA receptor, is one of the crucial enzymes for superoxide production in the CNS. We showed earlier that the metabotropic glutamate receptor 1 (mGluR1) plays an important role in the activation of PKC and tyrosine phosphorylation of the NMDA receptor, which has been implicated in enhancement of the channel activity, after cerebral ischemia. In this study, we sought to determine the role of mGluR1 in the activation of NADPH oxidase and subsequent superoxide production after transient focal cerebral ischemia. The amounts of NADPH oxidase subunits in the membrane fraction were increased after the start of reperfusion. These changes were accompanied by increased NADPH oxidase activity followed by superoxide production. The administration of an mGluR1 antagonist attenuated NADPH oxidase activity, which was coincident with inhibition of superoxide production. We further showed that the increase in the amount of PKCδ, but not of PKCζ, as well as the increase in those of NADPH oxidase subunits, was attenuated by the mGluR1 antagonist. These results suggest that mGluR1 may be linked to the increase in NADPH oxidase activity that is mediated by PKCδ and subsequent superoxide production after cerebral ischemia.

Prostanoid EP1 receptor antagonist reduces blood-brain barrier leakage after cerebral ischemia.

Disruption of the blood-brain barrier (BBB) after cerebral ischemia is considered to be the initial step in the development of brain injuries, and an increase in the tyrosine phosphorylation of the tight junctional protein occludin has been shown to cause an increase in BBB permeability. Prostaglandin E2 (PGE2) appears to be associated with both toxic and protective effects on neuronal survival in vitro. However, it remains to be determined whether the prostanoid EP1 receptor is involved in the disruption of the BBB after cerebral ischemia. So we examined the effect of a prostanoid EP1 receptor antagonist, SC51089, on BBB leakage and tyrosine phosphorylation of occludin after cerebral ischemia. We demonstrated that SC51089 attenuated the increase in the tyrosine phosphorylation of occludin in isolated brain capillaries, which was coincident with a decrease in BBB leakage. These results suggest that the prostanoid EP1 receptor is involved in the tyrosine phosphorylation of occludin at tight junction, which may lead to disruption of the BBB and be linked to the development of cerebral infarctions.

Decreased susceptibility to salt-induced hypertension in subtotally nephrectomized mice lacking the vasopressin V1a receptor.

AIMS: By examining vasopressin V1a receptor (V1aR) knockout (KO) mice, we previously found that the V1aR is critically involved in the regulation of normal blood pressure. The present study was undertaken to elucidate the role of the V1aR in salt-induced hypertension. METHODS AND RESULTS: We compared haemodynamic responses induced by subtotal nephrectomy + salt loading in V1aR KO mice with those of wild-type (WT) controls. The time course of changes in the systolic blood pressure and heart rate during the salt loading was attenuated in the KO mice compared with that for the WT mice. The elevation of the plasma norepinephrine level caused by the subtotal nephrectomy + salt loading was also reduced in the V1aR KO mice. A V1aR antagonist markedly lowered the arterial blood pressure in the salt-loaded WT mice but not in the normotensive WT mice or in the salt-loaded or normotensive V1aR KO mice. Whereas arginine vasopressin (AVP) administered to the lateral ventricle of the brain induced pressor and tachycardiac responses accompanied by sympathetic activation in the WT mice, these events were completely abolished in the V1aR KO mice. Also, pressor and tachycardiac responses induced by intraventricularly administered hypertonic saline in the WT mice were diminished in the V1aR KO mice. Moreover, the pressor response induced by intraventricularly administered AVP was reduced in alpha(1d) adrenoceptor KO mice, whereas the tachycardiac response did not differ from that of the WT mice. CONCLUSION: These results suggest that the V1aR is involved in the elevation of arterial blood pressure caused by dietary salt and that a V1aR antagonist, in particular regarding its effect in the brain, could have significant therapeutic potential in the treatment of hypertension.

Alterations in dystrophin-related glycoproteins in development of right ventricular failure in rats.

Genetic depletion of the dystrophin-related glycoprotein (DRGP) complex causes cardiomyopathy in animals and humans. The present study was undertaken to explore the possible involvement of alterations in DRGP in the development of the right ventricular failure in monocrotaline-administered rats (MCT rats). At the 6th and 8th weeks after subcutaneous administration of 60 mg/kg monocrotaline, echocardiographic examination showed that cardiac output indices were decreased and that the right ventricular Tei indices were increased, suggesting that right ventricular failure occurs, at the latest, by 6 weeks after monocrotaline-administration. The levels of alpha- and beta-sarcoglycan and beta-dystroglycan in the right ventricle of the MCT rats at the 6th and 8th weeks were markedly decreased, and these decreases were inversely related to the increase in the right ventricular Tei index of the MCT-administered animals. The content and activity of the Ca(2+)-activated neutral protease m-calpain in the right ventricle of the MCT rats were increased at the 4th to 8th weeks and those of matrix metalloproteinase-2, at the 6th and 8th weeks. These results suggest that m-calpain- and/or matrix metalloproteinase-2-mediated alterations in the contents of alpha-sarcoglycan, beta-sarcoglycan, and beta-dystroglycan may be involved in the development of right ventricular failure in MCT rats.

Possible involvement of mitochondrial energy-producing ability in the development of right ventricular failure in monocrotaline-induced pulmonary hypertensive rats.

The present study was undertaken to explore the possible involvement of alterations in the mitochondrial energy-producing ability in the development of the right ventricular failure in monocrotaline-administered rats. The rats at the 6th week after subcutaneous injection of 60 mg/kg monocrotaline revealed marked myocardial hypertrophy and fibrosis, that is, severe cardiac remodeling. The time-course study on the cardiac hemodynamics of the monocrotaline-administered rat by the cannula and echocardiographic methods showed a reduction in cardiac double product, a decrease in cardiac output index, and an increase in the right ventricular Tei index, suggesting that the right ventricular failure was induced at the 6th week after monocrotaline administration in rats. The mitochondrial oxygen consumption rate of the right ventricular muscle isolated from the monocrotaline-administered animal was decreased, which was associated with a reduction in myocardial high-energy phosphates. Furthermore, the decrease in mitochondrial oxygen consumption rate was inversely related to the increase in the right ventricular Tei index of the monocrotaline-administered rats. These results suggest that impairment of the mitochondrial energy-producing ability is involved in the development of the right ventricular failure in monocrotaline-induced pulmonary hypertensive rats.

Alterations in pharmacological action of the right ventricle of monocrotaline-induced pulmonary hypertensive rats.

The present study was undertaken to elucidate pathophysiological and pharmacological alterations in the right ventricle in monocrotaline-administered (MCT) rats. Examination of tissue weights of the MCT and age-matched control (CON) rats indicated the right ventricular (RV) hypertrophy until 8 weeks after a single subcutaneous administration of 60 mg/kg MCT. Apparent fibrosis in the right ventricle of the MCT rat at the 6th week (6w-MCT) was observed. Echocardiographic measurement of the cardiac and hemodynamic parameters of the MCT rat showed decreases in cardiac output and stroke volume indices at the 6th and 8th weeks. The RV Tei index, which increase represents aggravation of RV function, was augmented at the 4th to 8th week. The results suggest the genesis of cardiac and RV failure until 6 weeks after MCT administration. Injection of dobutamine (300 ng) or colforsin daropate (1 microg) into the perfused right ventricle isolated from CON rat at the 6th week resulted in a marked increase in cardiac double product, whereas injection of either agent into the right ventricle from the 6w-MCT rat elicited a small increase in the double product, followed by a sustained decrease in the developed tension. Infusion of acetylcholine (1 microg) into the RV muscle of the 6w-MCT rat resulted in prolongation of the periods for cardiac arrest and for bradycardia of the right ventricle. The results suggest that MCT administration causes the RV hypertrophy and eventually leads to the RV failure, accompanied by abnormal inotropic and chronotropic actions of the RV muscle.

Inhibition of Src activity decreases tyrosine phosphorylation of occludin in brain capillaries and attenuates increase in permeability of the blood-brain barrier after transient focal cerebral ischemia.

Disruption of the blood-brain barrier (BBB) caused by cerebral ischemia can initiate the development and progression of brain injuries, which may lead to irreversible dysfunction of the central nervous system. It is likely that tyrosine phosphorylation of a membrane-associated tight junctional protein, occludin, is important for the interaction of occludin with intracellular proteins, ZO-1 to ZO-3, and it regulates vascular permeability. Little is known about the pathophysiological alterations of tight junctional proteins after transient focal cerebral ischemia. In this study, we examined the tyrosine phosphorylation of occludin in isolated brain capillaries after transient focal cerebral ischemia. We further examined the effects of the Src-family tyrosine kinase inhibitor, PP2, on the tyrosine phosphorylation of occludin and on vascular permeability and infarct volume. Transient focal ischemia increased the tyrosine phosphorylation of occludin in the isolated brain capillaries. The administration of PP2 attenuated this phosphorylation, which was coincident with an inhibition of BBB leakage and a decrease in infarct volume. These results suggest that the increase in the tyrosine phosphorylation of occludin in the brain capillaries may be linked to the disruption of tight junctions, whose disruption can cause dysfunction of the BBB and the consequent increase in infarct volume.

Hepatocyte growth factor suppresses ischemic cerebral edema in rats with microsphere embolism.

The present study was aimed at determining whether human recombinant hepatocyte growth factor (HGF) ameliorates cerebral edema induced by microsphere embolism (ME). Rats were injected with 700 microspheres (48 microm in diameter). Continuous administration of HGF at 13 microg/3 days/animal into the right ventricle was started from 10 min after embolism to the end of the experiment by using an osmotic pump. On day 3 after the ME, the rats were anesthetized, and their brains were perfused with an isotonic mannitol solution to eliminate constituents in the vascular and extracellular spaces. Thereafter, tissue water and cation contents were determined. A significant increase in tissue water content of the right hemisphere by ME was seen. This ME-induced increase in water content was associated with increases in tissue sodium and calcium ion contents and decreases in tissue potassium and magnesium ion contents of the right hemisphere. The treatment of the animal with HGF suppressed the increases in water and sodium and calcium ion contents, but not the decreases in potassium and magnesium ion contents. These results suggest that HGF suppresses the formation of ischemic cerebral edema provoked intracellularly in rats with ME.

Injection of neural progenitor cells improved learning and memory dysfunction after cerebral ischemia.

Accumulating evidence indicates that stem cells have the ability to improve neurological deficits seen after cerebral ischemia. However, the effects of neural progenitor cells (NPCs) on cerebral ischemia-induced learning and memory dysfunction remain to be clarified. The purpose of the present study was to determine whether the injection of exogenous NPCs could prevent learning and memory dysfunction after cerebral ischemia. Sustained cerebral ischemia was produced by the injection of 700 microspheres into the right hemisphere of each rat. We demonstrated that injection of NPCs into the hippocampus at 10 min after the induction of cerebral ischemia reduced prolongation of the escape latency seen in acquisition and retention tests of the water maze task on Days 12-28 after cerebral ischemia. Injection of NPCs partially attenuated the decrease in viable areas of the ipsilateral hemisphere on Day 28 after the cerebral ischemia. We also demonstrated that injection of NPCs prevented the decrease in the level of BDNF seen at the early period after cerebral ischemia. These results suggest that the injection of exogenous NPCs into the hippocampus can prevent cerebral ischemia-induced learning and memory dysfunction, possibly through maintenance of the BDNF level.

mGluR1 antagonist decreases tyrosine phosphorylation of NMDA receptor and attenuates infarct size after transient focal cerebral ischemia.

The contribution of metabotropic glutamate receptors to brain injury after in vivo cerebral ischemia remains to be determined. We investigated the effects of the metabotropic glutamate receptor 1 (mGluR1) antagonist LY367385 on brain injury after transient (90 min) middle cerebral artery occlusion in the rat and sought to explore their mechanisms. The intravenous administration of LY367385 (10 mg/kg) reduced the infarct volume at 24 h after the start of reperfusion. As the Gq-coupled mGluR1 receptor is known to activate the PKC/Src family kinase cascade, we focused on changes in the activation and amount of these kinases. Transient focal ischemia increased the amount of activated tyrosine kinase Src and PKC in the post-synaptic density (PSD) at 4 h of reperfusion. The administration of LY367385 attenuated the increases in the amounts of PSD-associated PKCgamma and Src after transient focal ischemia. We further investigated phosphorylation of the NMDA receptor, which is a major target of Src family kinases to modulate the function of the receptor. Transient focal ischemia increased the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B. Tyrosine phosphorylation of NR2A, but not that of NR2B, in the PSD at 4 h of reperfusion was inhibited by LY367385. These results suggest that the mGluR1 after transient focal ischemia is involved in the activation of Src, which may be linked to the modification of properties of the NMDA receptor and the development of cerebral infarction.

Alcohol preference in mice lacking the Avpr1a vasopressin receptor.

[Arg(8)]-vasopressin (Avp), a nonapeptide hormone, is known to regulate blood pressure, water balance, and a variety of behaviors such as anxiety, aggression, and bonding. Although some evidence that Avp modifies ethanol consumption and some of the effects of ethanol on behavior have been reported, the role of Avp in alcohol consumption and preference is poorly understood. The Avp1a receptor (Avpr1a) is ubiquitously expressed in the central nervous system. To determine the role of Avp signaling on the behavioral effects of alcohol, we examined voluntary ethanol consumption in mice with targeted disruptions of the Avpr1a knockout (Avpr1a KO) gene. Avpr1a KO mice displayed both increased ethanol consumption and preference compared with wild-type (WT) mice. Enhanced ethanol consumption was dramatically and reversibly reduced by treatment with N-methyl-D-aspartic acid antagonists. Basal glutamate release was elevated around the striatum in Avpr1a KO mice. Elevation of extracellular glutamate was also produced in WT mice by local application of an Avpr1a antagonist though a dialysis probe, and this elevation was quickly reversed by stopping the perfusion. These results suggest that Avp can inhibit the release of glutamate from the presynaptic terminal via the Avp1a receptor and that elevation of glutamate levels owing to loss of the inhibitory effect via Avp-Avpr1a signaling may play an important role in the preference for ethanol.