Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists.

The N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors has been well described as a result of the early appearance of NMDA antagonists, but no potent antagonist for the "non-NMDA" glutamate receptors has been available. Quinoxalinediones have now been found to be potent and competitive antagonists at non-NMDA glutamate receptors. These compounds will be useful in the determination of the structure-activity relations of quisqualate and kainate receptors and the role of such receptors in synaptic transmission in the mammalian brain.

New developments in the molecular pharmacology of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and kainate receptors.

Separation of non-N-methyl-D-aspartate subtypes of glutamate receptors, known as alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate receptors, is traced through conventional pharmacology to molecular biology. The physiology and pharmacology of recombinant receptor subtypes (GluR1-7 and KA1-2) are described. Competitive antagonists, e.g., the quinoxalinedione, 2,3-dihyroxy-6-nitro-7-sulphamoyl-benz(F)quinoxaline, and the decahydroisoquinoline, 3S,4aR,6R, 8aR-6-[2-(1(2)H-tetrazol-5-yl)ethyl]-decahydroisoquinolin e-3-carboxylate, have a broad antagonist spectrum, except that the latter is inactive on GluR6. The 2,3-benzodiazepines noncompetitively antagonise the AMPA receptor GluR1-4. Desensitisation of AMPA (GluR1-4) and kainate (GluR5-7 and KA1-2) receptors is blocked by cyclothiazide and concanavalin A, respectively. Polyamine toxins block AMPA receptors not containing GluR2 and unedited kainate receptors (GluR5-6). These data correlate well with results on native neurons characterised by techniques such as in situ hybridisation.

Efflux of rubidium in rat cortical synaptosomes is blocked by sigma and dextromethorphan binding site ligands.

Large concentrations of potassium were used to stimulate the release of rubidium-86 from preloaded cortical synaptosomes, so that the pharmacological sensitivity of this efflux could be examined. Potassium channel blockers, 4-aminopyridine and tetraethylammonium, inhibited the evoked release of rubidium. Sigma ligands, e.g. pentazocine, cyclazocine, rimcazole, 1,3-di(2-tolyl)guanidine (DTG) and haloperidol, as well as the antitussives, carbetapentane, caramiphen and dextromethorphan, significantly reduced potassium-stimulated efflux of rubidium. By contrast, 3-hydroxyphenyl-propylpiperidine (3-PPP), 5-methyl-10,11-dihydro-5H-dibenzo(a,d)-cyclohepten-5,10-imine (MK-801), phencyclidine (PCP), ketamine and D-2-amino-5-phosphonovalerate (D-AP5) were all inactive. This suggests that inhibition of potassium-stimulated efflux of rubidium is correlated with activity at the sigma and/or dextromethorphan binding sites rather than at the N-methyl-D-aspartate (NMDA)/PCP receptor-channel complex.

Blockade by sigma site ligands of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones.

1. The effects of a series of structurally-dissimilar sigma site ligands were examined on high voltage-activated Ca2+ channel activity in two preparations of cultured hippocampal pyramidal neurones. 2. In mouse hippocampal neurones under whole-cell voltage-clamp, voltage-activated Ca2+ channel currents carried by barium ions (IBa) were reduced with the rank order (IC50 values in microM): 1S,2R-(-)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]- 2-(1-pyrrolidinyl)cyclohexylamine (7.8) > rimcazole (13) > haloperidol (16) > ifenprodil (18) > opipramol (32) > carbetapentane (40) = 1-benzylspiro[1,2,3,4-tetrahydronaphthalene-1,4-piperidine] (42) > caramiphen (47) > dextromethorphan (73). At the highest concentrations tested, the compounds almost abolished IBa in the absence of any other pharmacological agent. 3. The current-voltage characteristics of the whole-cell IBa were unaffected by the test compounds. The drug-induced block was rapid in onset and offset, with the exceptions of carbetapentane and caramiphen where full block was achieved only after two to three voltage-activated currents and was associated with an apparent increase in the rate of inactivation of IBa. 4. In rat hippocampal neurones loaded with the Ca(2+)-sensitive dye Fura-2, rises in intracellular free Ca2+ concentration evoked by transient exposure to 50 mM K(+)-containing medium, either in the absence or in the presence of 10 microM nifedipine (to block L-type high voltage-activated Ca2+ channels), were also reversibly attenuated by the sigma ligands. The rank order potencies for the compounds in these experimental paradigms were similar to that observed for blockade of IBa in the electrophysiological studies. 5. These results indicate that, at micromolar concentrations, the compounds tested block multiple subtypes of high voltage-activated Ca2+ channels. These actions, which do not appear to be mediated by high-affinity sigma binding sites, may play a role in some of the functional effects previously described for the compounds.

Selective reduction of N-methyl-D-aspartate-evoked responses by 1,3-di(2-tolyl)guanidine in mouse and rat cultured hippocampal pyramidal neurones.

1. The effects of 1,3-di(2-tolyl)guanidine (DTG) were examined on the responses of cultured hippocampal neurones to the excitatory amino acid analogues N-methyl-D-aspartate (NMDA), kainate, quisqualate and (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). 2. In rat hippocampal neurones loaded with the Ca(2+)-sensitive dye Fura-2, DTG (10-100 microM) produced a concentration-dependent depression of the NMDA-evoked rises in intracellular free calcium ([Ca2+]i), an effect that was not modified by changes in the extracellular glycine concentration. DTG (at 50 and 100 microM) also attenuated, although to a lesser extent, the rises in [Ca2+]i evoked by naturally-derived quisqualate. In contrast, 50 and 100 microM DTG did not depress responses evoked by kainate, AMPA and synthetic, glutamate-free (+)-quisqualate although on occasions DTG enhanced kainate- and AMPA-evoked rises in [Ca2+]i. 3. DTG attenuated NMDA-evoked currents recorded from mouse hippocampal neurones under whole-cell voltage-clamp with an IC50 (mean +/- s.e. mean) of 37 +/- 5 microM at a holding potential of -60 mV. The DTG block of NMDA-evoked responses was not competitive in nature and was not dependent on the extracellular glycine or spermine concentration. The block did, however, exhibit both voltage-, and use-, dependency. The steady-state current evoked by naturally-derived quisqualate was also attenuated by DTG whereas those evoked by kainate and AMPA were not. 4. We conclude that DTG, applied at micromolar concentrations, is a selective NMDA antagonist in cultured hippocampal neurones, the block exhibiting both Mg(2+)- and phencyclidine-like characteristics. Given the nanomolar affinity of DTG for sigma binding sites it is unlikely that the antagonism observed here is mediated by sigma-receptors, but the data emphasize the potential danger of ascribing the functional consequences of DTG administration solely to sigma receptor-mediated events.

Blockade by sigma site ligands of N-methyl-D-aspartate-evoked responses in rat and mouse cultured hippocampal pyramidal neurones.

1. The effects of a range of structurally-dissimilar compounds which possess affinity for sigma binding sites were examined on the responses of cultured hippocampal pyramidal neurones to the excitatory amino acid analogues N-methyl-D-aspartate (NMDA), kainate and (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). 2. In mouse hippocampal neurones under whole-cell voltage-clamp, the compounds tested reversibly attenuated NMDA-, but not kainate- or AMPA-, evoked currents with a rank order potency (IC50 values in microM): ifenprodil (0.8) > (+)-N-allylnormetazocine (1.1) > dextromethorphan (1.8) = haloperidol (1.9) > (+)-pentazocine (7.2) > 1S,2R-(-)-cis-N-methyl-N-[2-(3, 4-dichlorophenyl) ethyl]-2-(1-pyrrolidinyl)cyclohexylamine (17) = rimcazole (18) > 1,3-di(2-tolyl)guanidine (37) > opipramol (96) > caramiphen (110) = carbetapentane (112) > > (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (485). 3. The attenuation of NMDA-evoked responses was not mediated through interactions with the agonist, glycine (except haloperidol) or polyamine (except ifenprodil) binding sites on the NMDA receptor-channel complex but, in the light of the voltage- and, in some cases, use-dependent nature of their antagonism, an interaction with the ion channel appears to be a likely mechanism of action for many of the compounds. 4. Micromolar concentrations of selected sigma site ligands also reduced NMDA-evoked rises in intracellular free calcium concentration in Fura-2-loaded cultured hippocampal neurones of the rat with the same rank order potency as observed in the electrophysiological studies. 5. The data indicate that, at micromolar concentrations, the sigma site ligands tested act as NMDA receptor antagonists, an action which does not appear to be mediated by high-affinity sigma binding site(s). The functional effects of micromolar concentrations of sigma site ligands cannot, therefore, be attributed exclusively to interactions with high-affinity sigma binding sites.

Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions.

1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o. The antagonist effects of ifenprodil 20 micro M on high-[K+]0-evoked rises in [Ca2+]. were attenuated by spermine 0.25 mM but not by putrescine 1 or 5 mM. In contrast,spermine 0.1 mM increased rises in [Ca2+]i evoked by NMDA and enhanced the ifenprodil (5 micro M) block of NMDA-evoked rises in [Ca2+]i.4. Similar results were obtained in mouse cultured hippocampal pyramidal neurones under whole-cell voltage-clamp. Ifenprodil attenuated both the peak and delayed whole-cell IB. with an IC% value of 18 +/- 2 micro M, whilst it attenuated steady-state NMDA-evoked currents with an IC50 of 0.8 +/- 0.2 micro M. Block of IBa by ifenprodil 10 JaM was rapid in onset, fully reversible and occurred without change in thecurrent-voltage characteristics of Ba. The ifenprodil block of IBa was enhanced on membrane depolarization and was weakly dependent on the frequency of current activation. Spermine 0.1 mM potentiated control NMDA-evoked currents but attenuated IB,. In agreement with the microspectrofluorimetric studies, co-application of spermine produced a small enhancement of the inhibitory effect of ifenprodil 10 micro M on NMDA-evoked responses whereas the reduction of I4 by ifenprodil 10 micro M in the presence of spermine was less than expected if the inhibitory effects of ifenprodil and spermine on IBa were simply additive.5. The results indicate that ifenprodil blocks high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones. Although the Ca2+ channel blocking actions of ifenprodil are observed at higher concentrations than those associated with NMDA antagonist activity, Ca2+ channel blockade may contribute, at least in part, to the established neuroprotective and anticonvulsant properties of the compound.

Quinoxalinediones selectively block quisqualate and kainate receptors and synaptic events in rat neocortex and hippocampus and frog spinal cord in vitro.

1. Two quinozalinediones, FG9041 and FG9065, which had previously been shown to displace binding to the quisqualate receptor, were tested on rat neocortex and frog spinal cord in vitro against depolarizations induced by quisqualate, kainate and N-methyl-D-aspartate (NMDA). In both preparations effects of quisqualate were reduced the most and those of NMDA the least. 2. The near unitary slopes of the Schild plots were consistent with a competitive type of interaction. pA2 values for FG9041 were estimated to be 6.6, 6.1 and 5.1 in frog cord and 5.9, 5.3 and and about 4 in the rat neocortex for quisqualate, kainate and NMDA antagonism, respectively. FG9065 gave equivalent pA2 values of 6.2, 5.6 and 4.5. 3. At concentrations, which were without effect on depolarizations induced by NMDA, FG9041 and FG9065 reduced or blocked synaptically-evoked field potentials in hippocampal and neocortical slices superfused with normal magnesium-containing medium. Since these synaptic components are also insensitive to NMDA antagonists, these results are consistent with their mediation by postsynaptic receptors of the quisqualate (or kainate) type. 4. By contrast, quinoxalinediones had only limited effects on spontaneous epileptiform activity seen in both neocortical and hippocampal preparations when superfused with magnesium-free medium. These burst discharges were, however, abolished by NMDA antagonists. 5. In the frog spinal cord the early component of the dorsal root to ventral root reflexes was selectively reduced by FG9041 whereas NMDA antagonists reduced the longer latency components. 6. Our results suggest that the quinoxalinediones are likely to be useful pharmacological probes for elucidating the role of non-NMDA receptors in the vertebrate central nervous system.

Expression of human glutamate receptors (GluR) in neuroblastoma cell lines.

Neuroblastoma cell lines are useful for the investigation of neuronal receptor regulation since these cells display various neuronal features. Here we report the analysis of human AMPA and kainate receptor expression in four neuroblastoma cell lines (SK-N-MC, IMR-32, CHP 126 and NMB/N7). The reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated the presence of hGluR1, hGluR4 and EAA4 transcripts in all four cell lines whereas hGluR3 mRNA was undetectable. The pattern of expression of hGluR2, EAA1, EAA2, EAA3 and EAA5 was more complex and differed among the cell lines tested. Immunoblot analysis and electrophysiological recordings failed to demonstrate expression of hGluR1-hGluR4, EAA1/EAA2 proteins and the formation of functional AMPA/kainate receptor channels. These cell lines might provide a valuable model to study the transcriptional and post-transcriptional regulation of AMPA and kainate receptor expression.

Haloperidol interacts with the strychnine-insensitive glycine site at the NMDA receptor in cultured mouse hippocampal neurones.

N-Methyl-D-aspartate (NMDA)-evoked responses in voltage-clamped hippocampal neurones in culture were reversibly, but not completely, attenuated on superfusion with micromolar concentrations (0.1-100 microM) of haloperidol with an IC50 (+/- S.E.M.) value of 1.9 +/- 0.2 microM (n = 7). In contrast, kainate- and (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-evoked responses were relatively unaffected on application of 50 microM haloperidol. The NMDA receptor antagonist action of haloperidol was neither competitive in nature nor voltage-dependent but was reduced upon elevation of the extracellular concentration of glycine. Furthermore, in the absence of added glycine haloperidol (at 0.1 microM) frequently potentiated NMDA-evoked responses. Haloperidol thus appears to be a partial agonist for the strychnine-insensitive glycine site associated with the NMDA receptor-channel complex.

Haloperidol blocks voltage-activated Ca2+ channels in hippocampal neurones.

The Ca2+ channel antagonist action of the antipsychotic haloperidol was investigated using two functional assays of Ca2+ channel activity. Haloperidol dose dependently attenuated the rise in intracellular free Ca2+ ([Ca2+]i) evoked by 50 mM extracellular K+ in Fura-2 loaded cultured rat hippocampal neurones with an IC50 (+/- S.E.M.) of 7.8 +/- 0.5 microM and similarly reduced whole-cell Ba2+ currents (IBa) in voltage-clamped mouse hippocampal neurones with an IC50 value of 15.6 +/- 1.1 microM. Block of whole-cell IBa by haloperidol was rapid, fully reversible, and was greater at more depolarized membrane potentials. Our data indicate that haloperidol non-selectively blocks neuronal voltage-gated Ca2+ channels at micromolar concentrations.

Inhibition of high affinity L-glutamic acid uptake into rat cortical synaptosomes by the conformationally restricted analogue of glutamic acid, cis-1-aminocyclobutane-1,3-dicarboxylic acid.

The action of two cyclobutane derivatives of L-glutamic acid on the high affinity uptake of L-glutamic acid was investigated using a preparation of synaptosomes from rat cerebral cortex. cis-1-Aminocyclobutane-1,3-dicarboxylic acid (also known as trans-2,4-methanoglutamic acid) potently inhibited L-glutamic acid uptake (IC50 30 microM), whereas trans-1-aminocyclobutane-1,3-dicarboxylic acid (also known as cis-2,4-methanoglutamic acid), a potent N-methyl-D-aspartate (NMDA) agonist, was inactive. Analysis of the kinetics of L-glutamic acid uptake in the presence and absence of cis-1-aminocyclobutane-1,3-dicarboxylic acid (CACB) suggests that it may act as a competitive inhibitor (Ki 8 microM). CACB may be substrate for the L-glutamic acid high-affinity uptake carrier since preincubation of CACB with the synaptosomal preparation increased its potency in inhibiting L-glutamic acid uptake. The conformationally restricted structure of CACB may be indicative of the conformations of L-glutamic acid that interact with the high affinity uptake carrier.

Effect of contamination of pharmaceutical equipment on powder triboelectrification.

Triboelectrification of pharmaceutical powders may cause problems during processing and manufacture due to adhesion/cohesion effects. The aim of this work was to investigate the role of adhered particles and moisture as contact surface contaminants on the electrostatic charging of size fractionated lactose, following contact with a surface, i.e. stainless steel, typically used in pharmaceutical process and manufacturing operations. Replicated experimental runs without cleaning the contact surface showed a successive decrease in the net electronegative charge due to adhered lactose particles. Removal of these contaminating particles by different cleaning methods had a considerable effect on the charge after triboelectrification. The charge on the lactose samples was found to decrease when humidity in the cyclone apparatus was increased from 2 to 100% relative humidity. These results clearly demonstrate that moisture, particulate contamination and method of cleaning of processing equipment during pharmaceutical manufacturing operations may influence the electrostatic behaviour of powders.

Regional heterogeneity of L-glutamate and L-aspartate high-affinity uptake systems in the rat CNS.

The high-affinity uptake of L-[3H]glutamate and L-[3H]aspartate into synaptosomes prepared from rat cerebral cortical, hippocampal, and cerebellar tissue was reduced by a number of structural analogues of L-glutamate and L-aspartate. threo-3-Hydroxy-L-aspartic acid was a more potent inhibitor of L-glutamate uptake than of L-aspartate uptake in the cerebral cortex, but not in the hippocampus or cerebellum. A similar pattern of selectivity was observed for cis-1-aminocyclobutane-1,3-dicarboxylic acid. Dihydrokainate was also more potent against L-glutamate than against L-aspartate in the cerebral cortex, but in the hippocampus, it was more potent against L-aspartate than against L-glutamate. By contrast, L-alpha-aminoadipate was significantly more potent in the cerebellum than in the cerebral cortex and hippocampus as an antagonist of both L-glutamate and L-aspartate. These results support other evidence that there is regional heterogeneity in acidic amino acid uptake sites and that the amino acids L-glutamate and L-aspartate may be taken up by a number of transport systems with overlapping substrate specificity but different inhibitor profiles.

Non-competitive antagonism of N-methyl-d-aspartate by displacement of an endogenous glycine-like substance.

N-methyl-D-aspartate (NMDA; 40 microM) induced depolarizations of cortical wedges that were reduced by 30 - 60% in the presence of D-2-amino-5-phosphonovalerate (D-AP5; 5 microM), ketamine (5 microM), dextrorphan (5 microM), magnesium (500 microM), kynurenate (200 microM), and 1-hydroxy-3-aminopyrrolidone-2 (HA-966; 200 microM). Superfusion with glycine (1 microM - 1 mM) did not enhance the action of NMDA in control medium and in media containing D-AP5, ketamine, dextrorphan, or magnesium. In the presence of kynurenate and HA-966, however, NMDA-induced depolarizations were enhanced in a dose-dependent manner by glycine (10 microM - 3.16 mM). NMDA antagonism produced by HA-966 appeared to be more completely reversed than that produced by kynurenate. This action of glycine was mimicked by D-serine but not by GABA or L-serine, and was resistant to strychnine (10 - 50 microM). Reduction of responses to quisqualate by kynurenate was not reversed by glycine. In these cortical wedges, spontaneous synaptic activity was observed in nominally magnesium-free medium and this epileptiform activity could be blocked by the above NMDA antagonists. Glycine and D-serine reversed only the effect of kynurenate and HA-966 on such synaptic activity. These results suggest there is an endogenous glycine-like compound acting on NMDA receptor-ionophore complexes and that displacement of this compound by HA-966 or kynurenate produces antagonism of NMDA.

Measurement of electrostatic charge decay in pharmaceutical powders and polymer materials used in dry powder inhaler devices.

The electrostatic charge generated on drug/excipient particles during the formulation, manufacture, and use of pharmaceutical dry powder inhaler (DPI) devices may significantly affect the performance of such devices. An experimental investigation has been undertaken of charge accumulation and decay on compacts of selected powders (lactose and salbutamol sulfate) and a device material (polyvinyl chloride, PVC) used during the formulation, manufacture, and use of DPIs. Significant differences in charge acquisition and decay for the three materials have been demonstrated after charging using a corona electrode. PVC acquired the highest charge, which decayed rapidly in 30 min toward the value prior to exposure to corona. Lactose and salbutamol acquired similar charge values, which decayed to zero after 30 min for lactose, whereas salbutamol retained a significant charge after 120 min. The significant differences in charging propensity among drug, excipient, and device materials may have relevance in DPI formulation, manufacture, and use.

Loperamide blocks high-voltage-activated calcium channels and N-methyl-D-aspartate-evoked responses in rat and mouse cultured hippocampal pyramidal neurons.

The effects of the antidiarrheal agent loperamide on high-voltage-activated (HVA) calcium channel activity and excitatory amino acid-evoked responses in two preparations of cultured hippocampal pyramidal neurons were examined. In rat hippocampal neurons loaded with the calcium-sensitive dye fura-2, rises in intracellular free calcium concentration ([Ca2+]i) evoked by transient exposure to 50 mM K(+)-containing medium [high extracellular potassium concentration ([K+]o)] were mediated by Ca2+ flux largely through nifedipine-sensitive Ca2+ channels, with smaller contributions from omega-conotoxin GVIA (omega-CgTx)-sensitive Ca2+ channels and channels insensitive to both nifedipine and omega-CgTx. Loperamide reversibly blocked rises in [Ca2+]i evoked by high [K+]o in a concentration-dependent manner, with an IC50 of 0.9 +/- 0.2 microM. At the highest concentration tested (50 microM), loperamide eliminated rises in [Ca2+]i evoked by high [K+]o, a result otherwise achieved only in Ca(2+)-free medium or by the combined application of nifedipine, omega-CgTx, and funnel web spider venom to Ca(2+)-containing medium. The action of loperamide was neither naloxone sensitive nor mimicked by morphine and was seen at concentrations substantially less than those required to block influx of Ca2+ through the N-methyl-D-aspartate (NMDA) receptor-operated ionophore. Similar results were obtained in cultured mouse hippocampal pyramidal neurons under whole-cell voltage clamp. Voltage-activated Ca2+ channel currents carried by barium ions (IBa) could be discriminated pharmacologically into nifedipine-sensitive (L-type) and nifedipine-resistant, omega-CgTx-sensitive (N-type) components. Loperamide (0.1-50 microM) produced a concentration-dependent reduction of the peak IBa with an IC50 value of 2.5 +/- 0.4 microM and, at the highest concentration tested, could fully block IBa in the absence of any other pharmacological agent. The loperamide-induced block was rapid in onset and offset, was fully reversible, and did not appear to be related to the known calmodulin antagonist actions of loperamide. The current-voltage characteristics of the whole-cell IBa were unaffected by loperamide and the block was not voltage dependent. Loperamide also attenuated NMDA-evoked currents recorded at a membrane potential of -60 mV, with an IC50 of 73 +/- 7 microM. The block of NMDA-evoked currents was not competitive in nature, was not reversed by elevation of the extracellular glycine or spermine concentration, and was not affected by changes in the membrane holding potential. Steady state currents evoked by kainate and DL-alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid were, in contrast, relatively unaffected by 100 microM loperamide.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of two alternatively spliced forms of a metabotropic glutamate receptor coupled to phosphoinositide turnover.

A comparison of the pharmacological and physiological properties of the metabotropic glutamate 1 alpha and 1 beta receptors (mGluR1 alpha and mGluR1 beta) expressed in baby hamster kidney (BHK 570) cells was performed. The mGluR1 beta receptor is an alternatively spliced form of mGluR1 alpha with a modified carboxy terminus. Immunoblots of membranes from the two cell lines probed with receptor-specific antipeptide antibodies showed that mGluR1 alpha migrated with an M(r) = 154,000, whereas mGluR1 beta migrated with an M(r) = 96,000. Immunofluorescence imaging of receptors expressed in BHK 570 cells revealed that the mGluR1 alpha receptor was localized to patches along the plasmalemma and on intracellular membranes surrounding the nucleus, whereas mGluR1 beta was distributed diffusely throughout the cell. Agonist activation of the mGluR1 alpha and the mGluR1 beta receptors stimulated phosphoinositide hydrolysis. At both receptors, glutamate, quisqualate, and ibotenate were full agonists, whereas trans-(+)-1-aminocyclopentane-1,3-dicarboxylate appeared to act as a partial agonist. The stimulation of phosphoinositide hydrolysis by mGluR1 alpha showed pertussis toxin-sensitive and insensitive components, whereas the mGluR1 beta response displayed only the toxin-insensitive component. The mGluR1 alpha and mGluR1 beta receptors also increased intracellular calcium levels by inducing release from intracellular stores. These results indicate that the different carboxy terminal sequences of the two receptors directly influences G protein coupling and subcellular deposition of the receptor polypeptides and suggest that the two receptors may subserve different roles in the nervous system.

Functional expression and pharmacological characterization of the human EAA4 (GluR6) glutamate receptor: a kainate selective channel subunit.

A cDNA encoding an ionotropic glutamate receptor subunit protein humEAA4 (GluR6), has been cloned from a human fetal brain library. This cDNA when expressed in COS or HEK-293 cells is associated with high-affinity kainate receptor binding and ion channel formation. We have successfully established cell lines stably expressing humEAA4 in HEK-293 cells This is the first report of the establishment of stable cell lines expressing a glutamate receptor channel. The relative potency of compounds for displacing [3H]-kainate binding to humEAA4 receptors expressed in COS or HEK-293 cells is domoate > kainate > quisqualate > 6-cyano-7-nitroquinoxaline-2,3-dione > L-glutamate = 6,7- dinitroquinoxaline-2,3-dione > dihydrokainate. Applications of kainate, glutamate, and domoate but not AMPA evoked rapidly desensitizing currents in cells expressing homo-oligomeric humEAA4 in a concentration dependent manner. The order of potency was: domoate > kainate > L-glutamate. Although AMPA did not itself activate humEAA4 receptors it did reduce, to a limited extent, kainate-evoked responses. AMPA may therefore be a weak partial agonist for this receptor. To date this effect has not been demonstrated with rat GluR6. It is possible that subtle species differences may exist in the nature of agonist receptor interaction. Kainate evoked currents were attenuated by the quinoxalinediones CNQX and DNQX but not by DAP5. The receptor desensitization was attenuated on application of concanavalin A. Ion-permeability studies indicated that the receptor-linked ion channel is permeable to both Na+ and Ca2+ ions.