The roles of mid-myocardial and epicardial cells in T-wave alternans development: a simulation study.

BACKGROUND: The occurrence of T-wave alternans in electrocardiographic signals was recently linked to susceptibility to ventricular arrhythmias and sudden cardiac death. Thus, by detecting and comprehending the origins of T-wave alternans, it might be possible to prevent such events. RESULTS: Here, we simulated T-wave alternans in a computer-generated human heart model by modulating the action potential duration and amplitude during the first part of the repolarization phase. We hypothesized that changes in the intracardiac alternans patterns of action potential properties would differentially influence T-wave alternans measurements at the body surface. Specifically, changes were simulated globally in the whole left and right ventricles to simulate concordant T-wave alternans, and locally in selected regions to simulate discordant and regional discordant, hereinafter referred to as "regional", T-wave alternans. Body surface potential maps and 12-lead electrocardiographic signals were then computed. In depth discrimination, the influence of epicardial layers on T-wave alternans development was significantly higher than that of mid-myocardial cells. Meanwhile, spatial discrimination revealed that discordant and regional action potential property changes had a higher influence on T-wave alternans amplitude than concordant changes. Notably, varying T-wave alternans sources yielded distinct body surface potential map patterns for T-wave alternans amplitude, which can be used for location of regions within hearts exhibiting impaired repolarization. The highest ability for T-wave alternans detection was achieved in lead V1. Ultimately, we proposed new parameters Vector Magnitude Alternans and Vector Angle Alternans, with higher ability for T-wave alternans detection when using multi-lead electrocardiographic signals processing than for single leads. Finally, QT alternans was found to be associated with the process of T-wave alternans generation. CONCLUSIONS: The distributions of the body surface T-wave alternans amplitude have been shown to have unique patterns depending on the type of alternans (concordant, discordant or regional) and the location of the disturbance in the heart. The influence of epicardial cells on T-wave alternans development is significantly higher than that of mid-myocardial cells, among which the sub-endocardial layer exerted the highest influence. QT interval alternans is identified as a phenomenon that correlate with T-wave alternans.

Distinctive transcriptome alterations of prefrontal pyramidal neurons in schizophrenia and schizoaffective disorder.

Schizophrenia is associated with alterations in working memory that reflect dysfunction of dorsolateral prefrontal cortex (DLPFC) circuitry. Working memory depends on the activity of excitatory pyramidal cells in DLPFC layer 3 and, to a lesser extent, in layer 5. Although many studies have profiled gene expression in DLPFC gray matter in schizophrenia, little is known about cell-type-specific transcript expression in these two populations of pyramidal cells. We hypothesized that interrogating gene expression, specifically in DLPFC layer 3 or 5 pyramidal cells, would reveal new and/or more robust schizophrenia-associated differences that would provide new insights into the nature of pyramidal cell dysfunction in the illness. We also sought to determine the impact of other variables, such as a diagnosis of schizoaffective disorder or medication use at the time of death, on the patterns of gene expression in pyramidal neurons. Individual pyramidal cells in DLPFC layers 3 or 5 were captured by laser microdissection from 36 subjects with schizophrenia or schizoaffective disorder and matched normal comparison subjects. The mRNA from cell collections was subjected to transcriptome profiling by microarray followed by quantitative PCR validation. Expression of genes involved in mitochondrial (MT) or ubiquitin-proteasome system (UPS) functions were markedly downregulated in the patient group (P-values for MT-related and UPS-related pathways were <10(-7) and <10(-5), respectively). MT-related gene alterations were more prominent in layer 3 pyramidal cells, whereas UPS-related gene alterations were more prominent in layer 5 pyramidal cells. Many of these alterations were not present, or found to a lesser degree, in samples of DLPFC gray matter from the same subjects, suggesting that they are pyramidal cell specific. Furthermore, these findings principally reflected alterations in the schizophrenia subjects were not present or present to a lesser degree in the schizoaffective disorder subjects (diagnosis of schizoaffective disorder was the most significant covariate, P<10(-6)) and were not attributable to factors frequently comorbid with schizophrenia. In summary, our findings reveal expression deficits in MT- and UPS-related genes specific to layer 3 and/or layer 5 pyramidal cells in the DLPFC of schizophrenia subjects. These cell type-specific transcriptome signatures are not characteristic of schizoaffective disorder, providing a potential molecular-cellular basis of differences in clinical phenotypes.

Local and distant neuronal degeneration following intrastriatal injection of kainic acid.

After intrastriatal injection, the neurotoxin, kainic acid, was cleared from the rat forebrain in a biphasic manner with 70% eliminated within 2 hours; by 24 hours after infusion, less than 1% of the kainic acid remained in the forebrain. The kainic acid diffused into adjacent brain structures, achieving mu molar concentrations in several regions ipsilateral to the injected striatum. At various times after intrastriatal injection of 9.3 nmoles of kainic acid, the brain was serially sectioned; the sections were stained for Nissl substance with cresyl violet or for degenerating neurons with the ammoniacal silver method. Neuronal degeneration spread unevenly into contiguous structures from the central sphere in the injected striatum and affected the ipsilateral pyriform cortex and amygdala, the deep layers of the overlying cerebral cortex, and the medial aspects of the bed nucleus of the stria terminalis and of the nucleus accumbens. In half of the rats, the pyriform cortex contralateral to the side of injection also underwent degeneration. A subpopulation of pyramidal cells in layer IV of the lateral neocortex and the CA3-CA4 pyramidal cells in the ipsilateral hippocampus were selectively affected, whereas adjacent neurons remained intact. The distribution of agyrophilic fibers and terminals in subcortical structures was consistent with the degeneration of neurons of origin in the affected striatal and extrastriatal regions. Brain sections stained by the gold sublimate technique from rats perfused 20 days after injection revealed an intense astrocytic response in all areas affected by acute neuronal degeneration. Extrastriatal damage could be markedly reduced by injection of lower doses of kainic acid (2.3 nmoles) with brief anesthesia; under these conditions, however, the subpopulation of large striatal neurons were relatively resistant, as compared to the Golgi II neurons. These studies demonstrate significant and variable neuronal degeneration beyond the primary site of the lesion after intracerebral injection of kainic acid; several factors affect the pattern of degeneration, including the amount of kainic acid injected, its biological activity, its diffusion, duration of anesthesia, and variable sensitivity of neurons. Consequently, care must be exercised in the use of this neurotoxin to determine the extent and selectivity of neuronal damage, particularly with reference to neuronal vulnerability beyond the central sphere of intrinsic neuronal degeneration.

Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: characterization of an active digitonin-solubilized receptor complex.

The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of [125I]Tyro-ovine CRF ([125I]oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for [125I] oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, [125I]oCRF labeled the same size receptor complex. These data suggest that either the guanine nucleotide-binding protein (Ns) is tightly associated with the CRF receptor after solubilization and is insensitive to guanine nucleotides, or that high affinity binding for soluble CRF receptors is not dependent on the coupling of a guanine nucleotide-binding protein. The solubilization of CRF receptors from membranes in digitonin should allow for the more complete molecular and functional characterization of CRF-mediated events and purification of the receptor.

Role for brain corticotropin-releasing factor in the weight-reducing effects of chronic fenfluramine treatment in rats.

Fenfluramine is an amphetamine derivative which is used as a weight-reducing agent in the treatment of obesity. It has been postulated that fenfluramine affects brain serotonin (5HT) neurons resulting in decreased food intake and altered autonomic outflow which, in turn, increases metabolism. CRF decreases food intake and, in addition, has been demonstrated to reduce body weight in genetically obese rats through selective activation of sympathetic and inhibition of parasympathetic outflows. Because 5HT is a potent CRF secretagogue, we tested the hypothesis that the weight-reducing effects of fenfluramine administration may be mediated, in part, through altered CRF secretion. Chronic fenfluramine treatment (1-24 mg/kg sc, twice daily, 4 days) resulted in a dose-dependent decrease in hypothalamic CRF concentration at 30 min after the final drug injection and was accompanied by a significant reciprocal increase in plasma corticosterone concentration. These data suggest that the decrease in hypothalamic CRF was a consequence of increased CRF secretion. These changes in hypothalamic CRF and plasma corticosterone correlated with brain fenfluramine levels. In contrast, high dose fenfluramine treatment significantly increased hippocampus, midbrain, and spinal cord CRF concentrations whereas levels in cerebral cortex, caudate putamen, thalamus, pons/medulla, and cerebellum were unaffected. There was no effect of this fenfluramine treatment protocol on regional brain TRH or neurotensin concentrations. In keeping with the well known development of tolerance to the weight-reducing effects of fenfluramine, chronic fenfluramine treatment resulted in lesser increases in corticosterone secretion than after acute treatment. Whereas weight loss observed after chronic fenfluramine treatment was associated with stimulation of hypothalamic-pituitary-adrenocortical hormone secretion, the weight-recovery phase after cessation of drug treatment was associated with decreased levels of plasma corticosterone. These data, demonstrating fenfluramine-induced alterations in brain CRF and plasma corticosterone, suggest that CRF may represent an important endogenous transmitter which mediates the weight-reducing effects of the drug.

Cerebrospinal fluid correlates of depression in Huntington's disease.

Patients with Huntington's disease (HD) commonly have concomitant depressive disorders. Prompted by reports of elevated corticotropin releasing factor (CRF) and reduced 5-hydroxyindoleacetic acid (5-HIAA) concentrations in lumbar cerebrospinal fluid (CSF) of patients with major depression, these CSF constituents were examined in 56 nonmedicated patients who were in the early stages of HD. Elevated CRF concentrations were found in patients with HD in comparison with a control group of 21 subjects without neurologic illness. The CSF 5-HIAA concentrations in patients with HD did not differ from that in four normal volunteers. Patients with HD who had depressive disorders (major depression or dysthymia) did not differ from those without depression with respect to CSF 5-HIAA or CRF concentration. However, a positive correlation was observed between severity of major depression and CRF concentration. These findings suggest that the depression associated with HD may differ neurochemically from that seen in other major depressive disorders, and support the notion that clinically significant depressive symptoms reflect heterogeneous pathophysiologic conditions with different neurochemical correlates.

Excitatory amino acid analogues: neurotoxicity and seizures.

The neurotoxic and convulsant properties of conformationally restricted and synthetic analogues of excitatory acidic amino acids were examined after stereotaxic injection into the striatum and the dentate gyrus of the hippocampal formation. In the striatum, neurotoxicity was quantified by the reduction in the activity of choline acetyltransferase and glutamate decarboxylase, markers for striatal intrinsic neurons. The following sequence of neurotoxic potencies was defined; kainic acid approximately equal to domoic acid much greater than alpha-keto kainic acid approximately equal to alpha-allo kainic acid greater than ibotenic acid approximately equal to cis-cyclopentyl glutamic acid greater than quisqualic acid approximately equal to N-methyl-D-aspartic acid. When normalized for neurotoxic potencies, a wide variation in the convulsant effects of the agents was observed after hippocampal injection. N-Methyl-D-aspartate produced nearly continuous electroencephalographic seizures for 2 hr after injection, where alpha-keto-kainate and kainate and quisqualate caused seizure activity for 64 and 45% respectively of this period; kainate, alpha-allo kainate and domoate caused intermittent seizure activity during approximately 30% of the recording period; ibotenate and cyclopentylglutamate had minimal convulsant effects. Seizures were associated with a significant reduction in the levels of norepinephrine and with increases in the levels of 5-hydroxyindoleacetic acid in the cortex and hippocampal formation and increases in the levels of gamma-aminobutyric acid in the hippocampal formation. Kainate, domoate, keto-kainate and alpha-allo-kainate caused extensive lesions of the hippocampal formation that also involved the pyriform cortex; ibotenate and cyclopentylglutamate caused uniform but substantial lesions limited to the dentate gyrus, whereas quisqualate and N-methyl-D-aspartate produced small and restricted lesions. The results demonstrate a poor correlation between the neurotoxic and convulsant potencies of these excitatory amino acid analogues and suggest that receptor-specific interactions may account for these disparities.

Characteristics of chloride-dependent incorporation of glutamate into brain membranes argue against a receptor binding site.

Although membrane sites from brain, labelled with [3H]glutamate (Glu) under sodium-free conditions, are thought to represent excitatory receptors, certain anomalous characteristics of the kinetics of apparent binding raised the question of whether transport might contribute to this process, prompting a closer examination of it. Hyperosmolar media and low incubation temperatures (4 degrees C) both led to decreases in the apparent specific binding of [3H]glutamate to membranes from the brain of the rat in the presence of chloride. Furthermore, only 15% of the [3H]glutamate, bound at 37 degrees C, was dissociable when the membranes were then cooled to 4 degrees C. The binding of [3H]glutamate was increased in the presence of certain dipeptides such as L-phenylalanyl-L-glutamate (Phe-Glu); and the binding augmented by the presence of Phe-Glu, was also sensitive to temperature and osmolarity of the incubation buffer. Sonication of membranes in 5 mM glutamate increased the apparent binding of [3H]glutamate and abolished the stimulatory effect of Phe-Glu. These findings are consistent with the hypothesis that chloride-dependent association of [3H]glutamate with membranes from brain reflects, in part, a sequestration process, which may be driven by glutamate exchange.

4-(1,3-Dimethoxyprop-2-ylamino)-2,7-dimethyl-8-(2, 4-dichlorophenyl)pyrazolo[1,5-a]-1,3,5-triazine: a potent, orally bioavailable CRF(1) receptor antagonist.

Structure-activity studies in the pyrazolo[1,5-a]-1,3,5-triazine series led to the discovery that compound 11i (DMP696) is a potent hCRF(1) receptor antagonist (K(i) = 1.7 nM vs 7.5 nM for alpha-hel-CRF(9-41), hCRF(1) adenylate cyclase IC(50) = 82 nM vs 286 nM for alpha-hel-CRF(9-41)). Compound 11i has excellent oral pharmacokinetic profiles in rats and dogs (37% and 50% oral bioavailabilities, respectively). This compound displays good activity in the rat situational anxiety model (MED = 3 mg/kg (po)), whereas a literature standard 1 (CP154526-1) was inactive (MED > 30 mg/kg (po)). Analogue 11i reduced stereotypical mouth movements in rhesus monkeys by 50% at 21 mg/kg (po) using the human intruder paradigm. Overall, the profile of pyrazolotriazine 11i indicates that hCRF(1) receptor antagonists may be anxiolytic agents, which have reduced motor side effect profiles.

2-Fluoro-4-pyridinylmethyl analogues of linopirdine as orally active acetylcholine release-enhancing agents with good efficacy and duration of action.

In an effort to improve the pharmacokinetic and pharmacodynamic properties of the cognition-enhancer linopirdine (DuP 996), a number of core structure analogues were prepared in which the 4-pyridyl pendant group was systematically replaced with 2-fluoro-4-pyridyl. This strategy resulted in the discovery of several compounds with improved activity in acetylcholine (ACh) release-enhancing assays, in vitro and in vivo. The most effective compound resulting from these studies, 10, 10-bis[(2-fluoro-4-pyridinyl)methyl]-9(10H)-anthracenone (9), is between 10 and 20 times more potent than linopirdine in increasing extracellular hippocampal ACh levels in the rat with a minimum effective dose of 1 mg/kg. In addition to superior potency, 9 possesses an improved pharmacokinetic profile compared to that of linopirdine. The half-life of 9 (2 h) in rats is 4-fold greater than that of linopirdine (0.5 h), and it showed a 6-fold improvement in brain-plasma distribution over linopirdine. On the basis of its pharmacologic, pharmacokinetic, absorption, and distribution properties, 9 (DMP543) has been advanced for clinical evaluation as a potential palliative therapeutic for treatment of Alzheimer's disease.

Synthesis, corticotropin-releasing factor receptor binding affinity, and pharmacokinetic properties of triazolo-, imidazo-, and pyrrolopyrimidines and -pyridines.

The synthesis and CRF receptor binding affinities of several new series of N-aryltriazolo- and -imidazopyrimidines and -pyridines are described. These cyclized systems were prepared from appropriately substituted diaminopyrimidines or -pyridines by nitrous acid, orthoester, or acyl halide treatment. Variations of amino (ether) pendants and aromatic substituents have defined the structure-activity relationships of these series and resulted in the identification of a variety of high-affinity agents (Ki's < 10 nM). On the basis of this property and lipophilicity differences, six of these compounds (4d,i,n,x, 8k, 9a) were initially chosen for rat pharmacokinetic (PK) studies. Good oral bioavailability, high plasma levels, and duration of four of these compounds (4d,i,n,x) prompted further PK studies in the dog following both iv and oral routes of administration. Results from this work indicated 4i,x had properties we believe necessary for a potential therapeutic agent, and 4i1 has been selected for further pharmacological studies that will be reported in due course.

Dopamine transporter: expression in Xenopus oocytes.

Xenopus oocytes can express biologically relevant transport activity after injection of mRNAs encoding several carrier molecules. mRNA from PC12 cells, as well as transcripts from a rat ventral midbrain library, can be expressed in these oocytes and allow them to display pharmacologically specific dopamine uptake. mRNA-injected oocytes incubated with tritiated dopamine contain tritiated dopamine and metabolites; lower amounts of radiolabeled dopamine and more radiolabeled metabolites are found in oocytes co-incubated with cocaine or in water-injected oocytes. Tritiated dopamine uptake into mRNA-injected oocytes is time, sodium, and temperature dependent. It is blocked by cocaine and mazindol, but not by haloperidol. It is not found after injection of mRNA from other brain regions. A size-selected rat midbrain library constructed in the plasma vector pCDM8 yields mRNA transcripts whose injection into oocytes causes cocaine-blockable [3H]dopamine uptake. These findings provide an assay for purification of the dopamine transporter cDNA by sib selection techniques.

A simple method for quantitative electroencephalographic assessment of drugs with convulsant and anticonvulsant properties.

A simple, inexpensive and reliable microcomputer-assisted method to detect and quantify abnormal EEG spiking is described. High frequency wave forms (20-40 Hz) with high amplitude are discriminated using a beta-2 bandpass filter and a threshold comparater. The spikes are then compiled and reported by an Apple II+ microcomputer. The method was validated by measuring seizures generated by intraperitoneal injection of pentylenetetrazol (PTZ), by microinjection of excitatory amino acids into the dorsal hippocampus, and by the antagonism of these seizures by diazepam and 2-amino-7-phosphono heptanoic acid, respectively.

Differential effects of DSP-4 on noradrenaline axons in cerebral cortex and hypothalamus may reflect heterogeneity of noradrenaline uptake sites.

The effect of the noradrenergic neurotoxin DSP-4 on high affinity transport of noradrenaline (NAT) was studied using rat brain synaptosomes. DSP-4 decreased NAT with the characteristics of a competitive inhibitor. The neurotoxin was more potent in inhibiting NAT into cortical synaptosomes (Ki = 179 +/- 39 nM) than into hypothalamic synaptosomes (Ki = 460 +/- 35 nM). Differences in NAT into cortical and hypothalamic synaptosomes were also observed with noradrenaline itself (Km = 39.5 +/- 7.5 nM and 100 +/- 12.1 nM, respectively) and with the catecholamine uptake blocker mazindol (Ki = 0.55 +/- 0.05 nM and 0.30 +/- 0.08 nM, respectively). The differences in the pharmacological properties of the noradrenaline uptake carrier in cerebral cortex and hypothalamus may account for the differential effects of DSP-4 on noradrenergic axons in these two brain regions.

Effects of anaesthetics and anticonvulsants on the action of kainic acid in the rat hippocampus.

The effects of anaesthetics and anticonvulsants on the acute behavioral response and neurochemical alterations following intrahippocampal injection of kainic acid were examined. When compared to the effects in animals anaesthetized with a combination of chloral hydrate and pentobarbital (Equithesin), brief anaesthesia with ether or with hexobarbital potentiated whereas prolonged anaesthetia attenuated the action of 0.5 microgram of kainic acid. Of the constituents of Equithesin, chloral hydrate offered less protection than pentobarbital. The anticonvulsants, phenobarbital, diazepam, diphenylhydantoin, and carbamazepine offered partial protection when administered in conjunction with ether anaesthetia.

Microinjection of kainic acid into the rat hippocampus.

The effects of unilateral injection of kainic acid into the rate hippocampus have been examined in terms of morphologic, neurochemical and behavioral sequelae. Infusion of 10 nmoles if kainate causes a rapid and complete degeneration of neuronal perikarya in the entire hippocampal formation followed by gliosis and atrophy of the region. This unilateral neuronal loss is accompanied by a 50% decrease in the specific activity of the biochemical markers for GABAergic neurons including glutamic acid decarboxylase, endogenous GABA and synaptosomal uptake of [3H]GABA. The extrinsic hippocampal cholinergic and noradrenergic afferents also exhibit significant alteration. Although the specific activity of choline acetyltransferase is unaffected and the specific activity of tyrosine hydroxylase is significantly increased in the injected hippocampus, the synaptosomal high affinity uptake process for [3H]choline and [3H]norepinephrine are significantly reduced at 10 days after injection. Whereas the level of endogenous acetylcholine is elevated in the lesioned hippocampus at 2 days after injection, the level of endogenous norepinephrine is reduced. For several hours after intrahippocampal injections of 5 nmoles or more of kainate, rats exhibit epileptiform behavior. Intrahippocampal injection of kainate may be a useful rodent model for temporal lobe seizure disorders.

Unique properties of norepinephrine release from terminals arising from the locus coeruleus: high potassium sensitivity and lack of linopirdine (DuP 996) enhancement.

The dose-response of K+ to elicit the release of norepinephrine (NE), acetylcholine (ACh), dopamine (DA) and serotonin (5-HT) from rat brain slices was examined. Cerebral cortical and hippocampal [3H]NE release had steeper K+ dose-response curves than those observed for apparent hippocampal [3H]ACh, striatal [3H]DA and striatal [3H]5-HT release. In contrast, the apparent release of [3H]NE from the hypothalamus had a K+ dose-response curve similar to those observed for the release of [3H]ACh, [3H]DA and [3H]5-HT. Linopirdine, a drug which enhances K(+)-stimulated release of [3H]Ach, [3H]DA and [3H]5-HT, had no effect on cerebral cortical [3H]NE release even at submaximal K+ stimulation. Hippocampal [3H]NE release was also not affected by linopirdine, however the compound significantly enhanced K(+)-evoked [3H]NE release from hypothalamic slices. These data point to unique properties of [3H]NE release from terminals arising from the locus coeruleus (i.e. those found in the cerebral cortex and hippocampus) when compared to [3H]NE release from terminals derived from the lateral tegmentum (i.e. those found in the hypothalamus) and the release properties of other neurotransmitters. The relative high K+ sensitivity of NE release from coerulear terminals may be related to the lack of linopirdine effects on cerebral cortical and hippocampal [3H]NE release.

N-Methyl-D-aspartate acid: a convulsant with weak neurotoxic properties.

N-Methyl-D-aspartic acid (NMDA) is 100-fold less potent as a neurotoxin than kainic acid when injected into the rat striatum. However, NMDA, when injected into the hippocampus, causes a more severe seizure disorder than kainic acid and doses of NMDA than produce much smaller lesions than those caused by kainate. These results indicate a poor correlation between convulsant and neurotoxic properties of acidic excitatory amino acids.

Glutamate-containing dipeptides enhance specific binding at glutamate receptors and inhibit specific binding at kainate receptors in rat brain.

The dipeptide, L-phenylalanyl-L-glutamate (PG), augments the specific binding of the excitatory amino acid receptor antagonist, [3H]2-amino-7-phosphonoheptanoic acid (APH), to rat forebrain membranes by 5-fold at 100 microM with an EC50 of 4.9 microM. The increase in the specific binding of [3H]AHP induced by PG results exclusively from an increase in Bmax. In contrast, PG inhibits the specific binding of [3H]kainic acid to forebrain membranes with a Ki of 6.8 microM. Of several related peptides examined, active ones affected the two receptor sites in a reciprocal fashion. The results suggest an allosteric interaction between [3H]APH and kainate receptors modulated by glutamate-containing peptides.

Blockade of N- and Q-type Ca2+ channels inhibit K(+)-evoked [3H]acetylcholine release in rat hippocampal slices.

In the present study, we examined the contribution of specific Ca2+ channels to K(+)-evoked hippocampal acetylcholine (ACh) release using [3H]choline loaded hippocampal slices. [3H]ACh release was Ca(2+)-dependent, blocked by the nonspecific Ca2+ channel blocker verapamil, but not by blockade of L-type Ca2+ channels. The N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CgTx GVIA; 250 nM) inhibited [3H]ACh release by 44% and the P/Q-type Ca2+ channel blocker omega-agatoxin IVA (omega-Aga IVA; 400 nM) inhibited [3H]ACh release by 27%, with the combination resulting in a nearly additive 79% inhibition. Four hundred or one thousand nM omega-Aga IVA was necessary to inhibit [3H]ACh release. omega-Conotoxin MVIIC (omega-CTx-MVIIC) was used after first blocking N-type Ca2+ channels with omega-CgTx GVIA (1 microM). Under these conditions, 500 nM omega-CTx-MVIIC led to a nearly maximal inhibition of the omega-CgTx GVIA-insensitive [3H]ACh release. Based on earlier reports about the relative sensitivity of cloned and native Ca2+ channels to these toxins, this study indicates that N- and Q-type Ca2+ channels primarily mediate K(+)-evoked hippocampal [3H]ACh release.