Multiple states of rat brain (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors as revealed by quantitative autoradiography.

The binding of (RS)-alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a selective ligand for non-N-methyl-D-aspartate excitatory amino acid receptors, was investigated in rat brain using an autoradiographic receptor binding technique. [3H]AMPA binding sites were widely distributed throughout the rat central nervous system, and the rank order of potency of displacers of [3H]AMPA binding was quisqualate greater than AMPA greater than 6,7-dinitroquinoxaline-2,3-dione = 6-cyano-7-nitroquinoxaline-2,3-dione greater than beta-N-oxalylamino-L-alanine greater than glutamate greater than kainate. Potassium thiocyanate (0-100 mM) exerted a 4-fold stimulation of [3H]AMPA binding, without changing the relative regional distribution of [3H]AMPA binding densities among rat brain regions. Scatchard analysis of equilibrium saturation binding revealed high affinity and low affinity components of [3H]AMPA binding, even in the absence of potassium thiocyanate. Addition of potassium thiocyanate increased the number of high affinity [3H]AMPA binding sites without a change in affinity. In addition, the number of low affinity [3H]AMPA binding sites was unchanged in the presence of potassium thiocyanate, but the affinity of low affinity [3H]AMPA binding was greatly increased. [3H]AMPA thus binds specifically to two affinity conformations of postsynaptic binding sites that appear to be interconverted with potassium thiocyanate. The pharmacologic profile of these sites is consistent with that of the ion channel-linked ("ionotropic") quisqualate/AMPA class of excitatory amino acid receptor in the rat central nervous system.     

YM872: a selective,potent and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist

This review focuses on the in vitro and in vivo neuropharmacology of YM872, a potential neuroprotective agent currently undergoing clinical trials in the United States (trial name: AMPA Receptor Antagonist Treatment in Ischemic Stroke - ARTIST). Its neuroprotective properties in rats and cats with induced focal cerebral ischemia are described. YM872, [2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydroquinoxalin-1-yl]-acetic acid monohydrate, is a selective, potent and highly water-soluble competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist. YM872 has a potent inhibitory effect on [(3)H]AMPA binding with a K(i) value of 0.096 microM. In contrast, YM872 has very low affinity for other ionotropic glutamate receptors. The solubility of YM872 is approximately 500 to 1000 times higher than that of the other competitive AMPA antagonists: YM90K, NBQX, or CNQX. The neuroprotective efficacy of YM872 was investigated in rats and cats subjected to permanent occlusion of the left middle cerebral artery. The animals were assessed either histologically or neurologically following ischemia. In rats with occluded middle cerebral artery (MCAO) YM872, by i.v. infusion, significantly reduced infarct volume measured at 24 h and 1 week after ischemia. Significant neuroprotection was maintained even when drug administration was delayed for up to 2 h after ischemia. In addition, YM872 significantly improved neurological deficit measured at 1 week after ischemia. In cats with MCAO YM872, by i.v. infusion, dose-dependently reduced infarct volume at 6 h after ischemia. YM872 produced no behavioral abnormalities and was not nephrotoxic. The evidence for the neuroprotective efficacy of YM872 suggests its therapeutic potential in the treatment of acute stroke in humans.     

Benzodiazepine-induced hippocampal CA1 neuron alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA) receptor plasticity linked to severity of withdrawal anxiety: differential role of voltage-gated calcium channels and N-methyl-D-aspartic acid receptors

Withdrawal from 1-week oral administration of the benzodiazepine, flurazepam (FZP) is associated with increased alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA) receptor (AMPAR) miniature excitatory postsynaptic currents (mEPSCs) but reduction of N-methyl-D-aspartic acid (NMDA) receptor (NMDAR)-evoked (e)EPSCs in hippocampal CA1 neurons. A positive correlation was observed between increased AMPAR-mediated mEPSC amplitude and anxiety-like behavior in 1-day FZP-withdrawn rats. These effects were disrupted by systemic AMPAR antagonist administration (GYKI-52466, 0.5 mg/kg, intraperitoneal) at withdrawal onset, strengthening the hypothesis that CA1 neuron AMPAR-mediated hyperexcitability is a central component of a functional anatomic circuit associated with the expression of withdrawal anxiety. Abolition of AMPAR current upregulation in 2-day FZP withdrawn rats by GYKI-52466 injection also reversed the reduction in NMDAR-mediated eEPSC amplitude in CA1 neurons from the same rats, suggesting that downregulation of NMDAR function may serve a protective, negative-feedback role to prevent AMPAR-mediated neuronal overexcitation. NMDAR antagonist administration (MK-801, 0.25 mg/kg intraperitoneally) had no effect on modifying increased glutamatergic strength or on withdrawal anxiety, whereas injection of an L-type voltage-gated calcium channel antagonist, nimodipine (10 mg/kg, intraperitoneally) averted AMPAR current enhancement and anxiety-like behavior, suggesting that these manifestations may be initiated by a voltage-gated calcium channel-dependent signal transduction pathway. An evidence-based model of likely cellular mechanisms in the hippocampus contributing to benzodiazepine withdrawal anxiety was proposed implicating regulation of multiple CA1 neuron ion channels.     

Quinazolone-alkyl-carboxylic acid derivatives inhibit transmembrane Ca(2+) ion flux to (+)-(S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid

Comparison of the kinetics of the inward Ca(2+) ion flux to (S)-alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid [(S)-AMPA] in cerebrocortical homogenates and that of the previously reported transmembrane Na(+) ion influx mediated by an AMPA receptor in hippocampal homogenates established that the agonist-induced opening of the AMPA receptor channels occurs in two kinetically distinguishable phases. Here we report that the 2-methyl-4-oxo-3H-quinazoline-3-acetic acid (Q1) inhibits the major slow-phase response specifically, whereas the acetyl piperidine derivative (Q5) is a more potent inhibitor of the fast-phase response. Both the quinazolone-3-propionic acid (Q2) and the quinazolone-3-acetic acid methyl ester (Q3) enhanced the slow-phase response to (S)-AMPA. The information provided by docking different Q1, Q2, and Q5 models at the ligand-binding core of iGluRs were used to define agonistic and antagonistic modes of interactions. Based on the effects of quinazolone-3-alkyl-carboxylic acid derivatives on specific [(3)H]Glu binding and kinetically distinguishable Ca(2+) ion permeability responses to (S)-AMPA and molecular modeling, the fast- and the slow-phase (S)-AMPA-elicited Ca(2+) ion fluxes were corresponded to different subunit compositions and degrees of S1S2 bridging interaction relative to substitution of kainate thereupon. Substitutions of agonists and antagonists into the iGluR2 S1S2 ligand binding core induced different modes of domain-domain bridging.     

Effects of thiol-reagents on [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding to rat telencephalic membranes

The binding of [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a ligand for the quisqualate subtype of excitatory amino acid receptors, was measured after chemical modifications of rat brain synaptic membranes. Treatment with oxidizing or thiol-alkylating agents did not modify [3H]AMPA binding, whereas treatment with several sulfhydryl reagents produced marked increases in binding. The involvement of free sulfhydryl groups in the regulation of the properties of [3H]AMPA binding sites was suggested by the specificity of p-chloromercuribenzoic acid (PCMB), its sulfonate analog p-chloromercuriphenyl-sulfonic acid (PCMBS), and HgCl2, plus the reversal of their effects after reduction with dithiothreitol. Pretreatment of synaptic membranes with the oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid) or the alkylating agent N-ethylmaleimide did not significantly affect [3H]AMPA binding but markedly reduced the enhancing effect of PCMBS. On the other hand, the increase in [3H]AMPA binding produced by PCMBS was not prevented by treatment with agonists such as quisqualate or L-glutamate and was produced equally well in resealed postsynaptic membranes with both lipophilic or nonlipophilic SH-reagents. Using filtration assays, two types of binding sites could be detected with high and low affinity for [3H]AMPA. Treatment with SH-reagents produced an increase in the Bmax for the high affinity component and a decrease in the Bmax for the low affinity component, accompanied by an increase in its affinity for the ligand. Using centrifugation assays, the same two types of sites could be detected under control conditions but treatment with SH-reagents produced an increase in affinity of the large component that prevented the analytical differentiation of the two sites. Treatment with SH-reagents also increased the binding of [3H] glutamate to the N-methyl-D-aspartate receptors but did not modify the binding of [3H]kainate to the kainate receptors or the strychnine-insensitive [3H]glycine binding. These results suggest that free sulfhydryl groups allosterically modulate the affinity of the quisqualate subtype of excitatory amino acid receptors and also indicate that different types of glutamate receptors might be differentially affected by chemical modification.     

Calcium directly permeates kainate/alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors in cultured cerebellar Purkinje neurons.

In cultures of rat cerebellar neurons that were enriched in Purkinje cells, the non-N-methyl-D-aspartate glutamate receptor agonist kainate (KA) stimulated Ca2+ influx into all neurons in Na(+)-containing solutions. A large Ca2+ influx was also observed in most neurons when KA was applied in Na(+)-free solutions, even when the cells were voltage-clamped at negative potentials. KA also stimulated Co2+ uptake into both Purkinje and non-Purkinje neurons. The KA-induced Ca2+ influx was insensitive to pharmacological antagonists of voltage-sensitive Ca2+ channels and antagonists of N-methyl-D-aspartate receptors. Thus, different types of cerebellar neurons possess KA-gated ionophores that are permeable to Ca2+. This Ca2+ conductance may play an important role in glutamate-mediated physiological and pathological events in the cerebellum.     

The molecular pharmacology and cell biology of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) are of fundamental importance in the brain. They are responsible for the majority of fast excitatory synaptic transmission, and their overactivation is potently excitotoxic. Recent findings have implicated AMPARs in synapse formation and stabilization, and regulation of functional AMPARs is the principal mechanism underlying synaptic plasticity. Changes in AMPAR activity have been described in the pathology of numerous diseases, such as Alzheimer's disease, stroke, and epilepsy. Unsurprisingly, the developmental and activity-dependent changes in the functional synaptic expression of these receptors are under tight cellular regulation. The molecular and cellular mechanisms that control the postsynaptic insertion, arrangement, and lifetime of surface-expressed AMPARs are the subject of intense and widespread investigation. For example, there has been an explosion of information about proteins that interact with AMPAR subunits, and these interactors are beginning to provide real insight into the molecular and cellular mechanisms underlying the cell biology of AMPARs. As a result, there has been considerable progress in this field, and the aim of this review is to provide an account of the current state of knowledge.     

Edaravone protects the vestibular periphery from free radical-induced toxicity in response to perilymphatic application of (+/-)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid

Intracochlear infusion of (+/-)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) was performed with a syringe pump in guinea pigs, and peripheral vestibular dysfunction was induced. Animals were administered edaravone systemically or topically. In the systemic application group, animals were administered edaravone once a day for 7 days after AMPA infusion. In the topical application group, edaravone-soaked gelfoam was placed on the round window membrane just after, 12 h after or 24 h after AMPA infusion. Spontaneous nystagmus was observed after AMPA infusion. Immunohistochemistry for 4-hydroxy-2-nonenal (4-HNE), a marker of free radical-induced lipid peroxidation, was performed 24 h after AMPA infusion. In addition, caloric tests were performed to evaluate vestibular function 1 week after AMPA infusion. Animals in both groups showed decreased spontaneous nystagmus, but results were not significant. Animals treated topically with edaravone within 12 h of AMPA infusion showed normal morphology of the ampullar sensory epithelia of the lateral semicircular canals and showed a good response to the caloric tests. 4-HNE immunoreactivity in the sensory epithelia was very low in these animals. In contrast, untreated animals and animals treated with edaravone systemically or topically 24 h after AMPA infusion showed morphologic hair cell damage, reduced caloric response and remarkable 4-HNE immunoreactivity in the sensory epithelia. These results indicate that topical application of edaravone within 12 h after damage protects the vestibular periphery from free radical-induced toxicity in response to intracochlear infusion of AMPA.     

Desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors facilitates use-dependent inhibition by pentobarbital

Although the mechanisms underlying the use-dependent inhibition of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) by barbiturates are not well understood, it has generally been assumed to involve open channel block. We examined the properties of the inhibition of AMPARs by the barbiturate pentobarbital (PB) in acutely isolated and cultured hippocampal neurons. PB caused a use- and concentration-dependent inhibition (IC50 = 20.7 microM) of AMPAR-mediated currents evoked by kainate. Contrary to the properties of an open channel blocker, the inhibition by PB developed with double exponential kinetics was reduced under conditions that favor the open channel state of AMPARs and was independent of membrane voltage. In addition, the inhibition was reduced at basic pH, indicating that the uncharged form of PB is active at AMPARs. Preventing AMPAR desensitization with cyclothiazide reduced the potency of inhibition by PB and prevented its trapping after the removal of agonist. PB preferentially reduced the steady-state (IC50 = 92.8 microM), rather than peak (IC50 > 1 mM) component of responses evoked by glutamate and accelerated the onset of desensitization in a concentration-dependent manner. Miniature excitatory postsynaptic currents recorded from cultured hippocampal neurons, the time course of which is minimally influenced by desensitization, are not inhibited by PB. The sensitivity of AMPAR-mediated synaptic responses to inhibition by PB therefore depends on the contribution of desensitization to these events. Our results suggest that PB does not act as an open channel blocker of AMPARs. Rather, the sensitivity, use dependence, and trapping of inhibition by PB are determined by AMPARs desensitization.     

Selectivity of amino acid transmitters acting at N-methyl-D-aspartate and amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors.

The endogenous neurotransmitter candidates L-aspartate, L-cysteine sulfinate (CSA), L-glutamate, L-homocysteate (HCA), and the endogenously occurring analogue quinolinate were compared in terms of potency, maximal activity, and selectivity for steady state activation of N-methyl-D-aspartate (NMDA) and non-NMDA [(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)] types of glutamate receptors expressed in Xenopus oocytes injected with mRNA isolated from rat brain (minus cerebellum). Selective activation of NMDA receptors was achieved by deleting Mg2+ and including 3-10 microM glycine in the perfusion medium and by applying ligands in the presence of 30 microM quisqualate, which blocks the AMPA receptor and desensitizes the oocyte's own Ca(2+)-dependent Cl- current. Oocytes were voltage clamped, and steady state inward currents were measured in response to perfusion with agonists at known concentrations. Under the NMDA receptor-preferring condition, the potency rank order was L-glutamate (EC50 = 2.2 microM, 95% confidence interval = 1.4-3.6 microM) greater than L-aspartate (13 microM) = HCA (13 microM) greater than CSA (59 microM) greater than quinolinate (greater than or equal to 7200 microM). All amino acids tested evoked similar maximal currents, which were 120-159% that of NMDA itself. The Hill coefficient was greater than 1 for all agonists except L-HCA (0.6), which might reflect heterogeneity of NMDA receptors expressed. This was supported by the finding that glycine was more potent in combination with HCA than NMDA, in activating NMDA receptors. To study the activity of agonists at AMPA receptors, glycine and quisqualate were omitted and 1 mM Mg2+ was included to block NMDA receptors. Ca(2+)-dependent Cl- currents activated by L-glutamate were prevented by inclusion of 0.4 M ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the recording electrode. All amino acids were less potent at AMPA receptors than at NMDA receptors; the potency rank order for steady state activation of AMPA receptors was L-glutamate (EC50 = 11 microM, 95% confidence interval = 7.3-18 microM) greater than HCA (430 microM) greater than CSA (3300 microM). L-Aspartate and quinolinate produced little or no inward current even up to 10 mM, i.e., were inactive at forebrain AMPA receptors. The maximal currents activated by all amino acids at steady state were 5-10% that of kainate, presumably due to severe desensitization of the AMPA receptor by the natural agonists. These results are consistent with L-glutamate acting as a mixed agonist at both AMPA and NMDA synaptic receptors and L-aspartate being involved exclusively in NMDA receptor-mediated synapses.     

Long-term alterations in benzodiazepine, muscarinic and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor density following continuous cocaine administration.

Animal models of clinical phenomena, such as stimulant-induced psychosis have focused primarily on persisting alterations that develop in brain after chronic stimulant administration. The present study utilized autoradiographic measures to examine changes in the density of benzodiazepine ([3H] flunitrazepam), muscarinic ([3H] quinuclidinyl benzilate), and non-NMDA glutamatergic (3H alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid: AMPA) receptor binding in rats 21 days following two exposures to cocaine administered continuously for 5 days via subcutaneous pellets. A marked, selective increase in [3H] flunitrazepam binding in both the lateral and medial habenula nucleus was observed. Reduced [3H] quinuclidinyl benzilate binding was observed in various brain areas, including large decreases in the anterior cingulate cortex and ventral thalamus. A reduction in [3H]AMPA binding was observed in the ventral striatum and was suggested in the nucleus accumbens. [3H] Flunitrazepam binding was also examined 12 hr following a single 5 day cocaine exposure to determine if the long-term habenular changes were evident at acute withdrawal. No alterations in [3H] flunitrazepam binding were observed in the habenula or any other structure analyzed at this time point. The relation of these results to persisting alterations in mesocorticolimbic pathways and previous findings of cocaine-induced degeneration in lateral habenula circuitry is discussed.     

Characterization of the renal tubular transport of zonampanel, a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, by human organic anion transporters.

Zonampanel monohydrate (YM872; [2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate) is a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist. The major elimination route for zonampanel has been reported to be by urine via the kidneys. The purpose of this study is to elucidate the molecular mechanism of the renal excretion of zonampanel using cells stably expressing human organic anion transporters (hOAT) 1, hOAT2, hOAT3, and hOAT4, as well as human organic cation transporters (hOCT) 1 and hOCT2. Another AMPA receptor antagonist, YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride], a decarboxymethylated form of zonampanel, was also used for comparing the substrate specificity. Zonampanel inhibited the uptake of prototypical organic anion substrates, [14C]para-aminohippurate in hOAT1 and [3H]estrone sulfate in hOAT3 and hOAT4, in a competitive manner. A time- and concentration-dependent increase in [14C]zonampanel uptake was observed in cells expressing hOAT1, hOAT3, and hOAT4. The Km values of zonampanel uptake by hOAT1, hOAT3, and hOAT4 were 1.4, 7.7, and 215 microM, respectively. Considering the localization of each transporter, results suggest that zonampanel is taken up via hOAT1 and hOAT3 from the blood into proximal tubular cells and then effluxed into the lumen via hOAT4. Probenecid and cimetidine competitively inhibited [14C]zonampanel uptake by the hOATs (hOAT1, hOAT3, and hOAT4 for probenecid; hOAT3 for cimetidine). YM90K inhibited the uptake of the prototypical substrate via hOAT3 competitively, but the uptake via hOAT1 noncompetitively. These findings suggest that the prototypical organic anion substrates (para-aminohippurate and estrone sulfate), cimetidine, probenecid, and zonampanel share binding specificity in each hOAT, whereas YM90K does not in hOAT1, possibly due to it being the decarboxymethylated form.     

The carboxyl terminus of the prolactin-releasing peptide receptor interacts with PDZ domain proteins involved in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor clustering

PDZ domain proteins use the PDZ domain binding motif to bind to the C-terminal sequence of membrane proteins to help scaffold them and spatially organize the components of the intracellular signaling machinery. We have identified a sequence at the C terminus of a G protein-coupled receptor, the PrRP receptor, that shares similarities with the C-terminal sequence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPA-R) subunits that interact with PDZ domain proteins. When coexpressed in human embryonic kidney 293 cells, PrRP receptor was able to coimmunoprecipitate the three PDZ domain proteins known to interact with AMPA receptors: glutamate receptor interacting protein (GRIP), AMPA binding protein (ABP), and protein that interacts with C-kinase (PICK1), but not the PDZ domain protein PSD-95, which does not interact with AMPA receptors. These interactions are sequence-selective as determined by mutagenesis. Furthermore, we show that PrRP receptor forms intracellular clusters when coexpressed with PICK1, and that this clustering effect is dependent on the interaction between the PICK1 PDZ domain and the last four amino acids of PrRP receptor. We found that PrRP receptor interaction with GRIP is not protein kinase C-regulated but may be regulated by other unidentified kinase because okadaic acid dramatically reduced GRIP interaction. By in situ hybridization, we show that the PrRP receptor is expressed in neurons that also express these PDZ domain proteins. We thus demonstrate that PrRP receptor interacts with the same PDZ domain proteins as the AMPA-Rs, raising the possibility that these two proteins could be scaffolded together at the synapse. These results may help to gain important insights into PrRP functions within the central nervous system.     

Galpha12/13- and rho-dependent activation of phospholipase C-epsilon by lysophosphatidic acid and thrombin receptors

Because phospholipase C epsilon (PLC-epsilon) is activated by Galpha(12/13) and Rho family GTPases, we investigated whether these G proteins contribute to the increased inositol lipid hydrolysis observed in COS-7 cells after activation of certain G protein-coupled receptors. Stimulation of inositol lipid hydrolysis by endogenous lysophosphatidic acid (LPA) or thrombin receptors was markedly enhanced by the expression of PLC-epsilon. Expression of the LPA(1) or PAR1 receptor increased inositol phosphate production in response to LPA or SFLLRN, respectively, and these agonist-stimulated responses were markedly enhanced by coexpression of PLC-epsilon. Both LPA(1) and PAR1 receptor-mediated activation of PLC-epsilon was inhibited by coexpression of the regulator of G protein signaling (RGS) domain of p115RhoGEF, a GTPase-activating protein for Galpha(12/13) but not by expression of the RGS domain of GRK2, which inhibits Galpha(q) signaling. In contrast, activation of the G(q)-coupled M1 muscarinic or P2Y(2) purinergic receptor was neither enhanced by coexpression with PLC-epsilon nor inhibited by the RGS domain of p115RhoGEF but was blocked by expression of the RGS domain of GRK2. Expression of the Rho inhibitor C3 botulinum toxin did not affect LPA- or SFLLRN-stimulated inositol lipid hydrolysis in the absence of PLC-epsilon but completely prevented the PLC-epsilon-dependent increase in inositol phosphate accumulation. Likewise, C3 toxin blocked the PLC-epsilon-dependent stimulatory effects of the LPA(1), LPA(2), LPA(3), or PAR1 receptor but had no effect on the agonist-promoted inositol phosphate response of the M1 or P2Y(2) receptor. Moreover, PLC-epsilon-dependent stimulation of inositol phosphate accumulation by activation of the epidermal growth factor receptor, which involves Ras- but not Rho-mediated activation of the phospholipase, was unaffected by C3 toxin. These studies illustrate that specific LPA and thrombin receptors promote inositol lipid signaling via activation of Galpha(12/13) and Rho.     

Long-Term Alterations in Benzodiazepine,Muscarinic and α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) Receptor Density Following Continuous Cocaine Administration

Abstract: Animal models of clinical phenomena, such as stimulant-induced psychosis have focused primarily on persisting alterations that develop in brain after chronic stimulant administration. The present study utilized autoradiographic measures to examine changes in the density of benzodiazepine ([³H] flunitrazepam), muscarinic ([³H] quinuclidinyl benzilate), and non-NMDA glutamatergic (³H α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid: AMPA) receptor binding in rats 21 days following two exposures to cocaine administered continuously for 5 days via subcutaneous pellets. A marked, selective increase in [³H] flunitrazepam binding in both the lateral and medial habenula nucleus was observed. Reduced [³H] quinuclidinyl benzilate binding was observed in various brain areas, including large decreases in the anterior cingulate cortex and ventral thalamus. A reduction in [³H]AMPA binding was observed in the ventral striatum and was suggested in the nucleus accumbens. [³H] Flunitrazepam binding was also examined 12 hr following a single 5 day cocaine exposure to determine if the long-term habenular changes were evident at acute withdrawal. No alterations in [³H] flunitrazepam binding were observed in the habenula or any other structure analyzed at this time point. The relation of these results to persisting alterations in mesocorticolimbic pathways and previous findings of cocaine-induced degeneration in lateral habenula circuitry is discussed.     

Modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor desensitization by extracellular protons

The interstitial milieu of the brain is buffered to an average pH of 7.3, but synaptic activation produces a temporal sequence of events that can affect pH in the synaptic cleft. Furthermore, pathophysiological processes such as ischemia and seizures produce global and prolonged acidification of interstitial pH. Changes in pH, in turn, can affect neuronal excitability by modulating receptors and channels. Patch-clamp recordings were made from cultured rat hippocampal neurons to determine whether physiologically relevant changes in interstitial pH (6.5-7.8) significantly affect AMPA receptor function. Acidic pH, such as that typically associated with ischemia (pH 6.5), significantly inhibited AMPA receptor-mediated responses in neurons. The effect of pH was agonist-dependent, with 2-fold greater inhibition of responses evoked by the strongly desensitizing agonists glutamate and quisqualate than the weakly desensitizing agonist kainate. Additional experiments tested the hypothesis that protons modulate AMPA receptor desensitization. In the presence of drugs that block AMPA receptor desensitization, pH 6. 5 had no effect on glutamate-evoked responses. In neuronal macropatches, protons increased equilibrium desensitization without affecting macroscopic desensitization or deactivation kinetics. The mechanisms and molecular determinants of pH-mediated effects were further investigated using human embryonic kidney 293 cells expressing recombinant AMPA receptors. Inhibition of kainate-evoked responses varied with subunit and isoform composition, ranging from 10% to >40%. Flop isoforms, which exhibit faster and more extensive desensitization, were most strongly inhibited. These findings suggest that interstitial acidification can modulate AMPA receptor-mediated synaptic transmission and that differences in receptor sensitivity to proton modulation may underlie the selective vulnerability of certain neuronal populations to ischemia.     

Spinal alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors may mediate the analgesic effects of emulsified halogenated anaesthetics

1. The present study was designed to investigate the relationship between spinal cord alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and the analgesic effects of emulsified halogenated anaesthetics. 2. After having established the mouse model of analgesia by intraperitoneal or subcutaneous injections of appropriate doses of emulsified enflurane, isoflurane or sevoflurane, we injected different doses of AMPA intrathecally and observed effects on the pain threshold using the hot-plate and acetic acid-induced writhing tests. 3. The results showed that intrathecal injection of AMPA (0.25, 0.5 and 1.0 ng) did not affect the pain threshold on the hot-plate test or the writhing times in conscious mice. In contrast, AMPA (0.25, 0.5 and 1.0 ng intrathecally) significantly and dose-dependently decreased the pain threshold on the hot-plate test and increased the writhing times in mice treated with emulsified anaesthetics. 4. These results suggest that spinal AMPA receptors may be important targets for the analgesic effects of emulsified enflurane, isoflurane and sevoflurane.     

Novel regulation of 5-HT1C receptors: down-regulation induced both by 5-HT1C/2 receptor agonists and antagonists

The 5-hydroxytryptamine_1C (5-HT_1C) receptor shares many features with the 5-HT₂ receptor. To determine if the regulation of the sites is also similar we studied the effects of chronic treatment with drugs active at 5-HT_1C/2 receptors on [³H]mesulergine-labelled 5-HT_1C binding sites in spinal cord. The 5-HT receptor agonists 1-(3-chlorophenyl)piperazine (m-CPP) (-38%), 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (-35%), quipazine (-27%) and m-trifluoromethylphenylpiperazine (TFMPP) (-27%) significantly down-regulated spinal 5-HT_1C sites with chronic injection compared to vehicle treatment. The 5-HT receptor antagonist methiothepin (-71%), mianserin (-24%), methysergide (-21%), and cyproheptadine (-27%) also induced down-regulation, and ritanserin and metergoline further reduced [³H]mesulergine specific binding to undetectable levels. There were no significant changes in K_d to implicate presence of residual drug except for mianserin, methiothepin, and TFMPP. Pindolol and spiperone had no significant effects. In acute dose-response studies, injection of a single dose of DOI did not result in a significant change in any receptor parameters. The capacity of a drug to lower B_max correlated significantly with its pK_d (r = 0.84, P < 0.0007). This drug regulation pattern for 5-HT_1C sites of down-regulation by both 5-HT_1C/2 receptor agonists and antagonists is similar to that for 5-HT₂ receptors and is consistent with the classification of 5-HT_1C and 5-HT₂ receptors in the same superfamily.     

Different modes of regulation for receptors activating phospholipase C in the rat pancreatoma cell line AR4-2J.

The inositol phosphate responses to substance P, bombesin, cholecystokinin, and the muscarinic cholinergic agonist methacholine were examined in the rat pancreatoma cell line AR4-2J. It was found that each agonist produced a distinct temporal pattern of inositol phosphate formation. Furthermore, these different response patterns resulted, at least in part, from different patterns of homologous receptor desensitization. The response to substance P desensitized rapidly and completely within 90 sec. After a 10-15-min refractory period, the response recovered with a t1/2 of approximately 1 hr. The response to methacholine also completely desensitized. However, in this case desensitization developed slowly over the course of 40 min, and no recovery of responsiveness was detected for up to 45 min after the cessation of stimulation. The inositol phosphate responses to bombesin and cholecystokinin were similar to one another and appeared to be composed of two phases. Initially, there was a robust activation of phospholipase C. This initial phase was followed within 20 sec by a second phase of lesser magnitude. For bombesin, attenuation of the initial phase was due to rapid, but only partial, desensitization of the response. Furthermore, the concentration of bombesin required to maintain the second phase of the response was about 100-fold lower than that required to maximally activate the initial phase of the response. These results may indicate multiple mechanisms for the regulation of different phospholipase C-linked receptors in this cell line.