Biomedical subjects

M A Desai

Publications and source records attributed to M A Desai.

At least 19 recordsLinked to original sources

PubMed1996

Pharmacological characterization of desensitization in a human mGlu1 alpha-expressing non-neuronal cell line co-transfected with a glutamate transporter.

1. Stimulation of phosphoinositide hydrolysis by human mGlu1 alpha (HmGlu1 alpha) was examined in a non-neuronal cell line (AV12-664) co-expressing both HmGlu1 alpha and a rat glutamate/aspartate transporter (GLAST). 2. Desensitization of HmGlu1 alpha could be elicited by inhibition of the GLAST transporter with the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). Maximal inhibition of HmGlu1 alpha-mediated phosphoinositide hydrolysis was induced upon 24 h pretreatment with trans-PDC. The concentration of glutamate in the extracellular medium also rose significantly in cells pretreated with trans-PDC. Glutamate levels increased upon incubation with trans-PDC in a time-dependent manner, with maximal glutamate levels attained after 24 h incubation with trans-PDC. 3. The time required for desensitization of HmGlu1 alpha by trans-PDC was compared to the time course for desensitization elicited by the direct-acting mGlu receptor agonists, 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) and (R,S)-3,5-dihydroxyphenylglycine (3,5-DHPG). Both direct-acting mGlu receptor agonists elicited desensitization of HmGlu1 alpha more rapidly than did trans-PDC, with maximal inhibition of agonist-induced phosphoinositide hydrolysis upon 12 h pretreatment. Agonist-induced desensitization could be fully reversed upon washout of agonist for 12 h. 4. Both mGlu receptor agonist- and trans-PDC-induced desensitization of HmGlu1 alpha could be blocked by inclusion of (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), an mGlu receptor antagonist, in the pretreatment medium. 5. Agonist-stimulated phosphoinositide hydrolysis by HmGlu1 alpha was found to parallel closely agonist-induced desensitization of HmGlu1 alpha. Thus, the EC50 values for 1S,3R-ACPD- and 3,5-DHPG-stimulated phosphoinositide hydrolysis were similar to the EC50 values for eliciting desensitization of HmGlu1 alpha. 6. These studies demonstrate desensitization of recombinant human mGlu1 alpha receptor in a non-neuronal cell line in which the receptor can be regulated by direct activation or by manipulation of glutamate transporter activity. Desensitization of HmGlu1 alpha was found to be mediated by activation of the receptor since the mGlu receptor antagonist, MCPG, blocked both mGlu receptor agonist- and trans-PDC-induced desensitization of HmGlu1 alpha. Furthermore, agonist-induced desensitization of HmGlu1 alpha was found to parallel receptor-mediated stimulation of phosphoinositide hydrolysis.

Amino Acid Transport System X-AG↗

PubMed1995

1-BCP, a memory-enhancing agent, selectively potentiates AMPA-induced [3H]norepinephrine release in rat hippocampal slices.

It is now clear that the AMPA subtype of ionotropic glutamate receptors (iGluRs) undergoes a rapid desensitization in response to activation by AMPA receptor agonists. This desensitization is inhibited by compounds such as aniracetam and cyclothiazide, which act at a distinct site on the AMPA receptor complex. In particular, cyclothiazide greatly potentiates AMPA receptor-mediated depolarizing responses in the hippocampus. We have recently shown cyclothiazide also increases AMPA-induced release of [3H]norepinephrine ([3H]NE). More, recently, a benzamide compound, 1-(1,3-benzodioxol-5-ylcarbonyl)-piperidine (1-BCP), has been reported to enhance AMPA-induced currents and to facilitate memory retention in rats in a number of memory tasks. In this study, the effects of 1-BCP on excitatory amino acid agonist-induced [3H]NE release in rat hippocampal slices were determined. We report that 1-BCP, like cyclothiazide, selectively potentiates AMPA-induced [3H]NE release. However, cyclothiazide was more potent and efficacious than 1-BCP. Nevertheless, these data suggest a role for AMPA receptor-mediated enhancement of norepinephrine release as a mechanism of action for nootropic compounds such as 1-BCP.

Animals↗

PubMed1995

Selective inhibition of forskolin-stimulated cyclic AMP formation in rat hippocampus by a novel mGluR agonist, 2R,4R-4-aminopyrrolidine-2,4- dicarboxylate.

Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G-protein coupled receptors that are linked to multiple second messengers in the rat hippocampus. The compound 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) has been widely used to activate this class of receptors and study their functions in situ. However, 1S,3R-ACPD acts on multiple mGluR subtypes to produce multiple alterations in second messengers. We report here that the aza-substituted analog of 1S,3R-ACPD, 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), is a highly selective agonist for negatively-coupled cAMP-linked mGluRs in the rat hippocampus, with similar potency in mGluR2 expressing cells. 1S,3R-ACPD decreases forskolin-stimulated cAMP formation, increases basal cAMP formation, and increases phosphoinositide hydrolysis in the rat hippocampus. However, 2R,4R-APDC inhibited forskolin-stimulated cAMP, but had none of the other activities of 1S,3R-ACPD. Furthermore, 2R,4R-APDC had no measurable ionotropic glutamate receptor affinity in rat hippocampus, as indicated by lack of effects on basal and glutamate agonist-evoked [3H]norepinephrine release. 2R,4R-APDC also inhibited forskolin-stimulated cAMP formation in human mGluR2 expressing cells with about three-fold greater potency than 1S,3R-ACPD, but unlike 1S,3R-ACPD, showed no appreciable activation of phosphoinostide hydrolysis in human mGluR1 alpha expressing cells. Thus, 2R,4R-APDC should be a useful pharmacological agent to explore the functions of mGluRs coupled to inhibition of adenylate cyclase.

Animals↗

PubMed1995

Cloning and expression of a human metabotropic glutamate receptor 1 alpha: enhanced coupling on co-transfection with a glutamate transporter.

We cloned and expressed a human metabotropic glutamate receptor 1 alpha (HmGluR1 alpha) in a novel cell line. The human mGluR1 alpha cDNA was found to be 86% identical to rat mGluR1 alpha, and the predicted protein sequence was found to be 93% identical to rat mGluR1 alpha. We expressed HmGluR1 alpha in AV12-664, an adenovirus-transformed Syrian hamster cell line. To prevent tonic activation of HmGluR1 alpha by glutamate that may be released by these cells into the extracellular medium, HmGluR1 alpha was co-expressed in AV12-664 cells with a rat glutamate/aspartate transporter (GLAST). This allowed investigation of the effect that clearance of glutamate from the extracellular space would have on HmGluR1 alpha function. A comparison of mRNA levels revealed that HmGluR1 alpha was similarly expressed in cells with or without co-expression of GLAST. However, HmGluR1 alpha-mediated phosphoinositide hydrolysis was efficiently elicited only in cells co-expressing rat GLAST. Blockade of glutamate transport by L-trans-pyrrolidine-2,4-dicarboxylic acid resulted in an increase in glutamate levels in the media and an increase in basal HmGluR1 alpha-mediated phosphoinositide hydrolysis. Long-term pretreatment of cells with L-trans-pyrrolidine-2,4-dicarboxylic acid resulted in media glutamate levels similar to those in cells not expressing GLAST. However, this resulted in a dramatic decrease in 1-aminocyclopentane-1S,3R-dicarboxylic acid- and glutamate-stimulated phosphoinositide hydrolysis. These studies suggest that co-expression of mGluR1 alpha with a glutamate transporter prevents desensitization of the receptor, thus achieving optimal coupling of the receptor with its effector system.

Amino Acid Sequence↗

PubMed1995

Cyclothiazide acts at a site on the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor complex that does not recognize competitive or noncompetitive AMPA receptor antagonists.

Activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of ionotropic glutamate receptors by certain agonists, including AMPA and glutamate, has been shown to result in a rapid desensitization of the receptor. This desensitization is profoundly inhibited by the benzothiadiazide diuretic, cyclothiazide. We previously reported that cyclothiazide potentiates AMPA-induced [3H]norepinephrine ([3H]NE) release from rat hippocampal slices. We used this system to investigate the possible interaction of cyclothiazide with various AMPA receptor antagonists, including the competitive antagonist LY293558 and the 2,3-benzodiazepine noncompetitive antagonist GYKI 53655. Cyclothiazide significantly potentiated both AMPA- and KA-induced [3H]NE release from slices of the rat hippocampus. LY293558 and GYKI 53655 inhibited the potentiated and nonpotentiated AMPA- and KA-induced [3H]NE release in a concentration-dependent manner. The IC50 values for inhibition of AMPA- or KA-induced [3H]NE release by either antagonist were not affected by the presence of cyclothiazide. Thus, cyclothiazide seems to interact at a site on the AMPA receptor complex which differs from either the glutamate recognition site or the 2,3-benzodiazepine allosteric site.

Animals↗

PubMed1994

Metabotropic glutamate receptor-induced disinhibition is mediated by reduced transmission at excitatory synapses onto interneurons and inhibitory synapses onto pyramidal cells.

Previous studies suggest that activation of metabotropic glutamate receptors (mGluRs) reduces GABA-mediated synaptic inhibition in area CA1 of the hippocampus. However, the mechanisms involved in this disinhibition are not known. We report that mGluR activation reduces both the GABAA and GABAB receptor-mediated components of evoked inhibitory postsynaptic potentials (IPSPs) by reducing transmission at excitatory synapses onto inhibitory interneurons and inhibitory synapses onto CA1 pyramidal cells.

Animals↗

PubMed1994

Cyclothiazide selectively potentiates AMPA- and kainate-induced [3H]norepinephrine release from rat hippocampal slices.

Activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of ionotropic glutamate receptors has been shown to result in a rapid desensitization of the receptor in the presence of certain agonists. One effect of AMPA receptor desensitization in the hippocampus may be to decrease the efficacy of AMPA receptor agonists at stimulating the release of norepinephrine from noradrenergic terminals. Recently, cyclothiazide was reported to inhibit AMPA receptor desensitization by acting at a distinct site on AMPA receptors. We have examined the effect of cyclothiazide on AMPA- and kainate (KA)-induced norepinephrine release from rat hippocampal slices to determine whether cyclothiazide would increase the efficacy of AMPA-induced [3H]norepinephrine release by inhibiting AMPA receptor desensitization. Cyclothiazide was observed to potentiate markedly both AMPA- and KA-induced [3H]-norepinephrine release. This potentiation is selective for AMPA/KA receptors as cyclothiazide did not potentiate N-methyl-D-aspartate-induced [3H]norepinephrine release or release induced by the nonspecific depolarizing agents veratridine and 4-aminopyridine. These results demonstrate that AMPA receptor-mediated modulation of [3H]norepinephrine release from rat brain slices is a useful approach to studying the cyclothiazide modulatory site on the AMPA receptor complex.

4-Aminopyridine↗

PubMed1993

Enhanced H+ diffusion by NH4+/HCO3-: implications for Helicobacter-pylori-associated peptic ulceration.

The in vitro effect of ammonium bicarbonate buffer on mucus H+ permeability is reported here. The diffusional resistance of mucus and water was demonstrated to be dependent on buffer concentration, and the contrast between the two types of layers was most pronounced for low buffer concentration near neutrality. Moreover, the pKa values of HCO3- and NH3 had a profound effect on measured DHCl. These in vitro studies suggest that a potentially damaging high local concentration of NH3 and HCO3- within the mucus layer generated by the action of Helicobacter pylori urease on endogenous intragastric urea could greatly accelerate proton flux to the surface epithelium by operation of a buffer shuttle. This results in enhanced H+ permeability, particularly at pKa values of HCO3- and NH3, and that in extreme circumstances this may result in gastric ulcer formation.

Animals↗

PubMed1993

Internal membranes and laminates for adaptation of amperometric enzyme electrodes to direct biofluid analysis.

This study describes the use of discrete, highly selective covering inner membranes in amperometric enzyme electrodes for direct biofluid analysis. Such inner membranes when interposed between the enzyme layer and the electrode working surface has been found to be both selective, as well as biocompatible. Cellulose acetate or detergent (Triton X-100 and Tween 80) modified cellulose acetate and polyethersulphone polymeric membranes, were both found to be highly selective against electroactive solutes normally present in biofluids. The inclusion of such inner perm-selective membranes greatly enhanced the biocompatibility of the enzyme electrode and reduced the passivation of the working electrode on exposure to whole blood or serum. The study indicates that electrode drift in biological solutions may also be due to passivation of the working electrode by small diffusible surface active species.

Biocompatible Materials↗

PubMed1993

An in vitro study of enhanced H+ diffusion by urease action on urea. Implications for Helicobacter pylori-associated peptic ulceration.

The in vitro effect of urea and hydrolysis of urea by urease on mucus H+ permeability is reported here. The effective DHCl values indicate a strong pH dependence for H+ diffusion in both water and mucus layers, with no apparent trend at concentrations between 1 and 50 mM urea. However, the estimated DHCl at near-neutral and alkaline pH are 4- to 10-fold lower through mucus than through aqueous films. Moreover, the pKa values of HCO3- and NH3 (generated by urease action on urea) had a profound effect on measured DHCl. These in vitro studies suggest that a high local concentration of NH3 and HCO3- within the mucus layer, generated by the action of Helicobacter pylori urease on endogenous intragastric urea, could greatly accelerate proton flux to the surface epithelium by operation of a buffer shuttle. This results in enhanced H+ permeability, particularly at pKa values of HCO3- and NH3, and in extreme circumstances it may result in gastric ulcer formation.

Animals↗

PubMed1992

Trans-ACPD-induced phosphoinositide hydrolysis and modulation of hippocampal pyramidal cell excitability do not undergo parallel developmental regulation.

The selective metabotropic glutamate receptor agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), stimulates phosphoinositide hydrolysis and elicits a number of electrophysiological responses in the hippocampus. If these effects are mediated by the same receptor subtype, they should undergo parallel developmental regulation. Therefore, we compared the phosphoinositide hydrolysis response and the electrophysiological responses to trans-ACPD at two different developmental stages. Trans-ACPD-stimulated phosphoinositide hydrolysis was significantly greater in hippocampal slices from immature (6-11-day-old) rats than from adults. In contrast, trans-ACPD elicited decreases in spike frequency adaptation and in the amplitude of the slow afterhyperpolarization in roughly equal percentages of immature and adult CA1 pyramidal cells. Similar results were obtained using the putative endogenous agonist, glutamate. These data support the hypothesis that certain electrophysiological effects of trans-ACPD are mediated by a metabotropic glutamate receptor that is distinct from the phosphoinositide hydrolysis-linked glutamate receptor.

Action Potentials↗

PubMed1992

Bicarbonate and other buffer systems can enhance the rate of H+ diffusion through mucus in vitro.

The effect of various diffusible buffers on mucus H+ permeability, and in particular the potency of the HCO3-/CO2 buffer system relative to other selected buffers is reported here. The diffusional resistance of mucus and water was demonstrated to be dependent on buffer concentration, and the contrast between the two types of layer was most pronounced for low DH+ values near neutrality. This concentration dependence was most marked with mucus layers in the buffer systems investigated. Furthermore, the nature and pKa values of the diffusible buffer systems used in this study had a profound effect on measured DH+. The effect was particularly striking in the case of HCO3- buffer with mucus. Possible implications of these in vitro findings in mucosal protection from acid are discussed.

Bicarbonates↗

PubMed1992

A study of macromolecular diffusion through native porcine mucus.

A diffusion chamber technique based on time-lag analysis for the estimation of effective diffusion coefficients of radiolabelled macromolecules of varying molecular weights through native mucus gel is reported. For all solutes studied, a reduction in effective diffusion coefficients was observed with a retardation of solute flux in both aqueous and mucus layers. Over the molecular weight range of solutes investigated (126-186,000 Daltons), a consistent effect of molecular weight was evident with regard to the retarding effect of mucus. No apparent or absolute molecular weight cut-off for macromolecular transfer was exhibited. However, at high molecular weights (greater than 30,000 Daltons) the retardation was greatly enhanced. The results confirm that mucus can be regarded as a gel with finite pores, but that it does not constitute an absolute barrier to even high molecular weight solutes.

Animals↗

PubMed1992

Multiple metabotropic glutamate receptors regulate hippocampal function.

Selective activation of metabotropic glutamate receptors with trans-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) stimulates phosphoinositide hydrolysis and elicits three major physiological responses in area CA1 of the hippocampus. These include direct excitation of pyramidal cells, blockade of synaptic inhibition, and decreased transmission at the Schaffer collateral-CA1 pyramidal cell synapse. Physiological effects of trans-ACPD are thought to be mediated by activation of phosphoinositide hydrolysis. However, it is now clear that multiple metabotropic glutamate receptor subtypes exist, some of which are not coupled to phosphoinositide hydrolysis. Thus, we performed a series of studies aimed at determining whether the physiological effects of trans-ACPD in the hippocampus are mediated by activation of the predominant phosphoinositide hydrolysis-linked glutamate-receptor. We report that L-2-amino-3-phosphonopropionic acid (L-AP3), an antagonist of trans-ACPD-stimulated phosphoinositide hydrolysis, does not inhibit the physiological effects of trans-ACPD in area CA1 at concentrations that maximally inhibit trans-ACPD-stimulated phosphoinositide hydrolysis in this region. Furthermore, 1S,3S-ACPD activates the phosphoinositide hydrolysis-linked glutamate receptor but does not reduce evoked field excitatory postsynaptic potentials (EPSPs) in area CA1. However, we report that the physiological effects of 1R,3S- and 1S,3R-ACPD are consistent with the hypothesis that these effects are mediated by activation of a metabotropic glutamate receptor. Thus, our data are consistent with the hypothesis that the physiological effects of trans-ACPD in area CA1 of the hippocampus are mediated by metabotropic glutamate receptors that are distinct from the AP3-sensitive phosphoinositide hydrolysis-linked glutamate receptor.

Alanine↗

PubMed1992

Amygdala kindling alters protein kinase C activity in dentate gyrus.

Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.

Amygdala↗

PubMed1991

Estimation of effective diffusion coefficients of model solutes through gastric mucus: assessment of a diffusion chamber technique based on spectrophotometric analysis.

A diffusion chamber technique based on spectrophotometric analysis to determine effective diffusion coefficients for solutes with various relative molecular mass (Mr) values and properties, passing through native mucus gel, is reported. For all solutes studied, a reduction in effective diffusion coefficients is observed with a retardation of solute flux by a factor of at least two. For the solutes investigated (with Mr values ranging from 126-14,400 u), no consistent effect of solutes of low Mr was evident with regard to the retarding effect of mucus; however, at high Mr values (greater than 4000 u) the retardation was greatly enhanced. A possible relationship between charged solutes of low Mr and the degree of retardation was observed, which possibly suggests the presence of ionic interactions of the solutes with the largely negatively charged mucus gel. The results provide further evidence for the suggestion that mucus is acting more than simply as a gel support for an unstirred water layer and is perhaps a more potent diffusion barrier to specific solutes.

5-Hydroxytryptophan↗

PubMed1991

Electrochemical determination of the permeability of porcine mucus to model solute compounds.

An electrochemical approach to the determination of permeability through native mucus gel of simple electrochemically active solutes is reported. For all the solutes studied, a reduction in effective diffusion coefficients was observed, with retardation of solute flux by a factor of at least two. However, NADH and the dicarboxylic acid derivative of ferrocene demonstrated a substantial, almost ten-fold, reduction in permeability through mucus. Results for the controls were in reasonable agreement with literature values where available. No consistent effect of molecular weight was evident with regard to the barrier properties of mucus over the molecular weight range of solutes investigated (34-660 daltons). The results suggest that mucus is acting more than as a gel support for an unstirred water layer.

Animals↗

PubMed1991

Excitatory effects of ACPD receptor activation in the hippocampus are mediated by direct effects on pyramidal cells and blockade of synaptic inhibition.

1. Phosphoinositide hydrolysis-linked excitatory amino acid (EAA) receptors (ACPD receptors) are selectively activated by the glutamate analogue trans-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD). Regional analysis of trans-ACPD-induced phosphoinositide hydrolysis indicates that this response is greater in the hippocampus than in other brain regions. Therefore we designed a series of studies aimed at testing the hypothesis that activation of this receptor modulates synaptic function in the hippocampal region. 2. We report that trans-ACPD dramatically altered field population spikes at each of the three major synapses in the hippocampal trisynaptic circuit at concentrations that are effective in activating phosphoinositide hydrolysis. At the perforant path-dentate gyrus synapse, bath application of trans-ACPD resulted in a decrease in the amplitude of field population spikes. In contrast, trans-ACPD markedly enhanced field population spike amplitude at the mossy fiber-CA3 synapse and the Schaffer collateral-CA1 synapse. In area CA1, but not area CA3, trans-ACPD also induced generation of multiple population spikes. 3. Simultaneous field potential recordings from the s. pyramidale and s. radiatum in area CA1 revealed that the effect of trans-ACPD on population spikes in this region was not accompanied by an increase in the initial slope of the field EPSP. This suggests that the effect of trans-ACPD was not mediated by a presynaptic action but must be mediated by direct effects on CA1 pyramidal cells or by a decrease in synaptic inhibition. 4. trans-ACPD had a number of direct excitatory effects on CA1 pyramidal cells. These included 1) cell depolarization (with an increase in input resistance), 2) inhibition of the slow afterhyperpolarization, and 3) blockade of spike frequency adaptation. trans-ACPD also had effects on CA1 pyramidal cells that were not excitatory in nature. These included an increase in the threshold for initiation of calcium spikes and an increase in interspike interval during prolonged current injection. None of these effects were mimicked by an ACPD analogue that does not activate the ACPD receptor (trans-methanoglutamate), nor were they blocked by kynurenate, a nonselective EAA receptor antagonist that does not block the ACPD receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗