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Results for “Chloride Channel Agonists”

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PubMed2006

Inhibitory effect of taurine on veratridine-evoked D-[3H]aspartate release from murine corticostriatal slices: involvement of chloride channels and mitochondria.

We have previously shown that the inhibitory neuromodulator taurine attenuates the release of preloaded D-[3H]aspartate from murine corticostriatal slices evoked by ischemic conditions or by application of the sodium channel agonist veratridine. The release of D-[3H]aspartate (a non-metabolized analog of glutamate) was used as an index of glutamate release. The aim of the present study was to reveal the molecular mechanisms responsible for this inhibitory effect of taurine. It was shown that 10 mM taurine suppresses D-[3H]aspartate release evoked by 0.1 mM veratridine, but does not affect the high-K+ -(50 mM) or ouabain- (0.1 mM) evoked release. Taurine had no effect in Ca2+ -free medium when the synaptic exocytosis of D-[3H]aspartate was inhibited. Nor did it suppress the release from slices preloaded with the competitive glutamate uptake blocker DL-threo-beta-hydroxyaspartate (THBA), which inhibits D-[3H]aspartate release mediated by the reverse action of glutamate transporters. Omission of Cl- from the incubation medium reduced the effect of taurine, signifying the involvement of a Cl- channel. The glycine receptor antagonist strychnine and the GABA(A) receptor antagonist bicuculline did not block the taurine effect, although picrotoxin, a less specific blocker of agonist-gated chloride channels, completely prevented the effect of taurine on veratridine-induced D-[3H]aspartate release. The respiratory chain blocker rotenone or mitochondrial protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in combination with the mitochondrial ATPase inhibitor oligomycin, which inhibits the mitochondrial Ca2+ uniporter, also reduced the effect of taurine. The results obtained in the present study show that taurine acts specifically on the release of preloaded D-[3H]aspartate evoked by veratridine, but not on that evoked by other depolarizing agents, and affects the release mediated both by synaptic exocytosis and the reverse action of glutamate transporter. Taurine may attenuate D-[3H]aspartate release by regulation of mitochondrial Ca2+ sequestration and by activation of a chloride channel, but not that governed by GABA(A) or strychnine-sensitive glycine receptors.

Amino Acid Transport System X-AG↗

PubMed1996

Contractile agonists preferentially activate CL- over K+ currents in arterial myocytes.

Simultaneous patch-clamp and Ca2+ fluorescence measurements have revealed depolarising oscillations in the membrane potential of arterial (pulmonary) myocytes in response to adenosine 5'-triphosphate (ATP) and endothelin-1 (ET-1). These oscillations (i) are due to the preferential activation of Ca(2+)-activated Cl-, over K+ currents, (ii) occur through a mechanism involving Ca2+ release from intracellular Ca2+ stores and (iii) are likely to promote constriction. These results provide a novel perspective into the relative contribution and importance of Ca2+ activated Cl- and K+ channels in controlling membrane potential of arterial smooth muscle in response to contractile agonists.

Adenosine Triphosphate↗

PubMed1998

Importance of basolateral K+ conductance in maintaining Cl- secretion in murine nasal and colonic epithelia.

1. Epithelia lining the nasal passages and descending colon of wild-type and cystic fibrosis (CF) mice were examined by the short-circuit current technique. Additionally, intracellular Ca2+ ion determinations were made in nasal epithelial cells. Forskolin produced anion secretory currents in wild-type and CF nasal epithelia. It produced similar effects in wild-type colonic epithelia, but not in colonic epithelia from CF mice. 2. After electrogenic Na+ transport was blocked with amiloride and electrogenic Cl- secretion was stimulated with forskolin, the ability of K+ channel blockers to inhibit the forskolin-induced Cl- current was determined. The order of efficiency for nasal epithelium was: Ba2+ > clofilium >>> TEA = azimilide >>> trans-6-cyano-4-(N-ethylsulphonyl-N-methylamino)-3-hydroxy-2, 2-dimethyl-chromane (293B) = charybdotoxin, whereas for the colonic epithelium the order was: Ba2+ = 293B >>> azimilide = TEA >>> clofilium = charybdotoxin. 3. 1-Ethyl-2-benzimdazolinone (1-EBIO) was able to generate large Cl--secretory currents in colonic epithelia which were partially sensitive to charybdotoxin, with the remaining current being inhibited by 293B. In nasal epithelia 1-EBIO produced only a small transient effect on current. 4. Forskolin released intracellular Ca2+ in nasal epithelial cells; this activity was attenuated when more powerful Ca2+-releasing agents were applied first. 5. It is concluded that an action on basolateral cAMP-sensitive K+ channels is an important determinant of the maintained responses to forskolin in nasal and colonic epithelia, in addition to the effects on the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. In CF nasal epithelia the activation of calcium-activated chloride channels (CACs) substitutes for the effect on CFTR. On the basis of the different orders of potency of the blocking agents and the differential response to 1-EBIO it is concluded that the cAMP-sensitive K+ channels are different in the airways and the gut.

Amiloride↗

PubMed2005

DCEBIO stimulates Cl- secretion in the mouse jejunum.

We investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one(DCEBIO) on the Cl- secretory response of the mouse jejunum using the Ussing short-circuit current (Isc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in Isc (EC50 41 +/- 1 microM). Pretreating tissues with 0.25 microM forskolin reduced the concentration-dependent increase in Isc by DCEBIO and increased the EC50 (53 +/- 5 microM). Bumetanide blocked (82 +/- 5%) the DCEBIO-stimulated Isc consistent with Cl- secretion. DCEBIO was a more potent stimulator of Cl- secretion than its parent molecule, 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated Isc by >80% indicating the participation of CFTR in the DCEBIO-stimulated Isc response. Clotrimazole reduced DCEBIO-stimulated Isc by 67 +/- 15%, suggesting the participation of the intermediate conductance Ca2+-activated K+ channel (IKCa) in the DCEBIO-activated Isc response. In the presence of maximum forskolin (10 microM), the DCEBIO response was reduced and biphasic, reaching a peak response of the change in Isc of 43 +/- 5 microA/cm2 and then falling to a steady-state response of 17 +/- 10 microA/cm2 compared with DCEBIO control tissues (61 +/- 6 microA/cm2). The forskolin-stimulated Isc in the presence of DCEBIO was reduced compared with forskolin control tissues. Similar results were observed with DCEBIO and 8-BrcAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated Isc, providing evidence that DCEBIO increased Cl- secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl- secretion of the mouse jejunum and that DCEBIO targets components of the Cl- secretory mechanism.

Animals↗

PubMed1990

Benzodiazepine agonist and inverse agonist actions on GABAA receptor-operated chloride channels. II. Chronic effects of ethanol.

Mice were made tolerant to and dependent on ethanol by administration of a liquid diet. Gamma-aminobutyric acid (GABA) receptor-dependent uptake of 36Cl- by mouse cortical microsacs was used to study the actions of benzodiazepine (BZ) agonists and inverse agonists. Chronic exposure to ethanol attenuated the ability of a BZ agonist, flunitrazepam, to augment muscimol-stimulated uptake of 36Cl- and enhanced the actions of BZ inverse agonists, Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,4]-benzodiazepine - 3-carboxylate) and DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate), to inhibit GABAA receptor-operated chloride channels. Augmentation of chloride flux by pentobarbital was not reduced by chronic ethanol exposure. Attenuation of flunitrazepam efficacy was transient and returned to control levels within 6 to 24 hr after withdrawal from ethanol, but increased sensitivity to Ro15-4513 was observed as long as 8 days after withdrawal. Chronic exposure to ethanol did not alter [3H]SR 95531 ([2-(3'-carbethoxy-2'propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide] binding to low-affinity GABAA receptors or muscimol stimulation of chloride flux; and did not alter [3H]Ro15-4513 or [3H]flunitrazepam binding to central BZ receptors or allosteric modulation of this binding by muscimol (i.e., muscimol-shift). These results suggest that chronic exposure to ethanol reduces coupling between BZ agonist sites and the chloride channel, and may be responsible for the development of cross-tolerance between ethanol and BZ agonists. In contrast, coupling between BZ inverse agonist sites and the chloride channel is increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1997

Nasal polyposis: immunohistochemistry and bioelectrical findings (a hypothesis for the development of nasal polyps).

Nasal polyps and turbinates were obtained from individuals undergoing surgery for symptomatic nasal obstruction caused by nonatopic rhinosinusitis or allergic rhinosinusitis. One part of the tissue from each patient was fixed in neutral buffered formalin and prepared for study by histochemical and immunohistochemical methods. Monoclonal antibodies were used to identify macrophages, lymphocytes, and plasma cells. In most cases (12 of 16, 75%) the remainder of the polyp and turbinate samples was treated with protease to achieve disaggregation of the epithelial cells. Those cells were cultured on permeable collagen matrix supports. Transepithelial potential difference and resistance were measured daily. At the time of maximal transepithelial potential difference, the epithelial cells were mounted in modified. Ussing chambers and exposed to a sodium-positive channel blocker (amiloride hydrochloride) and to selected chloride-negative channel agonists (isoproterenol bitartrate and adenosine triphosphate). Middle turbinates and polyps were found to have more macrophages, lymphocytes, plasma cells, HLA-DR-positive cells, and eosinophils than the inferior turbinates. Epithelial cells obtained from polyps exhibited higher transepithelial potential differences and equivalent short-circuit currents than turbinate cell cultures. The responses to amiloride, isoproterenol, and adenosine triphosphate were also greater for polyp than for turbinate cultures. A theory for the pathogenesis of nasal polyps is proposed. Local release of inflammatory mediators could cause sodium absorption and chloride permeability to be higher in polyps than in turbinate epithelia. Increased sodium absorption is consistent with the hypothesis that epithelial fluid absorption contributes to the development of nasal polyps and is a result of the increased recruitment of inflammatory cells, which are present in nasal polyps.

Adenosine Triphosphate↗

PubMed2001

Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists.

Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl(-) permeability, the goal of screening is to identify compounds that restore cell Cl(-) transport. We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor. CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I(-) followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I(-) entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity. In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl(-) channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl(-) channels and transporters.

Animals↗

PubMed1994

Increased ion transport in cultured nasal polyp epithelial cells.

OBJECTIVE: To measure transepithelial bioelectric properties of cultured human nasal polyp and turbinate epithelial cells to test the hypothesis that nasal polyps have increased rates of ion transport. DESIGN: Cohort study. SETTING: Private referral center. PATIENTS: Individuals undergoing surgery for symptomatic nasal obstruction due to polyps caused by cystic fibrosis, nonatopic rhinosinusitis, or allergic rhinosinusitis. METHODS: Epithelial cells were removed from separated polyp and turbinate samples by protease disaggregation and cultured on permeable collagen matrix supports. Transepithelial potential difference and resistance were measured daily. At the time of maximal transepithelial potential difference, the cultures were mounted in modified Ussing chambers and exposed to a sodium-positive channel blocker (amiloride hydrochloride) and to selected chloride-negative channel agonists (isoproterenol bitartrate, adenosine triphosphate). OUTCOME MEASURES: Maximal transepithelial potential difference, resistance, and equivalent short-circuit current. Bioelectric responses to amiloride, isoproterenol, and adenosine triphosphate. RESULTS: Polyp cultures had higher transepithelial potential difference and equivalent short-circuit current than turbinate cultures. The mediator responses were greater for polyp than for turbinate cultures. CONCLUSIONS: Polyp epithelia have increased Na+ absorption and Cl- permeability relative to turbinate epithelia. These results are consistent with the hypothesis that increased epithelial fluid absorption contributes to the development of nasal polyps.

Absorption↗

PubMed1995

Potentiation by neurosteroids of muscimol/adenosine interactions in rat hippocampus.

Extracellular recordings were made from the CA1 pyramidal cell layer of hippocampal slices in response to stimulation of Schaffer collateral fibres in stratum radiatum. Alphaxalone and 5 alpha-pregnan-3 alpha-ol-20-one potentiated the inhibitory effect of muscimol on the population spike size at low concentrations (0.5 and 1 microM) that had no significant effect on the spike size by themselves. This profile is in agreement with other reports which have described the effect of these neurosteroids as barbiturate-like. Alphaxalone and 5 alpha-pregnan-3 alpha-ol-20-one also at low concentrations potentiated the inhibitory effect of adenosine alone and in the presence of 1 mM barium which blocked adenosine activated potassium channels. Alphaxalone failed to potentiate the inhibitory effect of adenosine in the presence of 1 microM bicuculline. It is concluded that these neurosteroids enhanced the potentiative interaction between adenosine and muscimol in the presence of barium. The results indicate that adenosine's effects are normally enhanced by virtue of the potentiative interaction occurring with endogenous GABA.

Adenosine↗

PubMed2024

Longitudinal effects of elexacaftor/tezacaftor/ivacaftor on the oropharyngeal metagenome in adolescents with cystic fibrosis.

BACKGROUND: Triple modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) improves lung function and impacts upon the respiratory microbiome in people with Cystic fibrosis (pwCF) with advanced lung disease. However, adolescents with cystic fibrosis (CF) are less colonized with bacterial pathogens than adult pwCF but their microbiota already differs from healthy individuals. The aim of this study was to longitudinally analyze the impact of ETI on the respiratory metagenome in adolescents with predominantly mild CF lung disease. METHODS: In this prospective observational study, we included pwCF aged 12-20 years with at least one F508del mutation, who collected oropharyngeal swabs before and after initiation of ETI therapy twice per week to biweekly over three months. We performed whole metagenome shotgun sequencing, followed by host DNA filtering and taxonomic profiling. We used linear and additive mixed effects models adjusted for known confounders and corrected for multiple testing to study longitudinal development of the microbiome. We analyzed bacterial diversity, abundance, and strain-level phylogeny. RESULTS: We analyzed the metagenomic data of 297 swabs of 20 pwCF. Microbiome composition changed after initiation of ETI therapy. We observed a slight diversification of the microbiome over time (Inv Simpson, Coef 0.085, 95 %CI 0.003, 0.17, p = 0.04). Strain-level analysis and clustering showed that strain retention of the most frequent bacterial species is predominant even during ETI therapy. CONCLUSIONS: During three months of ETI therapy, commensal bacteria increased, which may help to prevent overgrowth of bacterial pathogens.

Humans↗

PubMed1998

Propofol analogues. Synthesis, relationships between structure and affinity at GABAA receptor in rat brain, and differential electrophysiological profile at recombinant human GABAA receptors.

A number of propofol (2,6-diisopropylphenol) congeners and derivatives were synthesized and their in vitro capability to affect GABAA receptors determined by the inhibition of the specific [35S]-tert-butylbicyclophosphorothionate ([35S]TBPS) binding to rat whole brain membranes. Introduction of halogen (Cl, Br, and I) and benzoyl substituents in the para position of the phenyl group resulted in ligands with higher potency at inhibiting [35S]TBPS binding. A quantitative structure-affinity relationship (QSAR) study demonstrated that affinity is enhanced by increases in lipophilicity of the ligand whereas affinity is adversely affected by increases in size of the substituent para to the phenolic hydroxyl group. Consistent with the displacement of [35S]TBPS and with the activation of GABAA receptors, we demonstrate that ligands displaying high affinity (i.e., 2-4, and 8) are able to increase GABA-stimulated chloride currents in oocytes expressing human GABAA receptors and to directly activate chloride currents in an electrophysiological assay. Among them, compound 4 showed a rather peculiar profile in the electrophysiological examination with cloned alpha1beta2gamma2 GABAA receptors. Indeed, compared to propofol, it displayed a much greater efficacy at potentiating GABA-elicited chloride currents, but a much lower efficacy at producing a direct activation of the chloride channel in the absence of GABA. This behavior may give to compound 4 pharmacological properties that are more similar to anxiolytic and anticonvulsant drugs than to those of general anesthetics.

Anesthetics, Intravenous↗

PubMed1997

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta.

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.

Adenoviridae↗

PubMed1999

Adenophostin A and inositol 1,4,5-trisphosphate differentially activate Cl- currents in Xenopus oocytes because of disparate Ca2+ release kinetics.

Depletion of endoplasmic reticulum Ca2+ stores induces Ca2+ entry from the extracellular space by a process termed "store-operated Ca2+ entry" (SOCE). It has been suggested that the novel fungal metabolite adenophostin-A may be able to stimulate Ca2+ entry without stimulating Ca2+ release from stores. To test this idea further, we compared Ca2+ release, SOCE, and the stimulation of Ca2+-activated Cl- currents in Xenopus oocytes in response to inositol 1,4,5-trisphosphate (IP3) and adenophostin-A injection. IP3 stimulated an outward Cl- current, ICl1-S, in response to Ca2+ release from stores followed by an inward current, ICl2, in response to SOCE. In contrast, low concentrations of adenophostins (AdAs) activated ICl2 without activating ICl1-S, consistent with the suggestion that AdA can activate Ca2+ entry without stimulating Ca2+ release. However, when Ca2+ entry has been stimulated by AdA, Ca2+ stores are largely depleted of Ca2+, as assessed by the inability of ionomycin to release additional Ca2+. The Ca2+ release stimulated by AdA, however, was 7 times slower than the release stimulated by IP3, which could explain the minimal activation of ICl1-S; when Ca2+ is released slowly, the threshold level required for ICl1-S activation is not attained.

Adenosine↗

PubMed1996

A cholecystokinin-B receptor antagonist potentiates GABAergic and glycinergic inhibition in the nucleus of the solitary tract of the rat.

In both rodent and primate in vivo models, cholecystokininB (CCKB) antagonists such as PD134,308 have anxiolytic effects that may involve the potentiation of GABAergic transmission. We have investigated this interaction using exogenous application of GABA and whole cell patch recording techniques in neurons of the nucleus of the solitary tract (NTS) in brainstem slice preparations. In the presence of PD143,308 the magnitude of the GABA-evoked decrease in membrane input resistance was enhanced by 41.2 +/- 3.1% and the duration of the response was prolonged by 34.8 +/- 2.2%. Also, PD134, 308 potentiated glycine-evoked decreases in membrane input resistance, increasing the amplitude of the response by 62.8 +/- 4. 85 and prolonging the duration of the response by 23.5 +/- 3.6%. The effect of PD134,308 persisted in the presence of tetrodotoxin, after reversal of the transmembrane gradient of chloride ions and under conditions of exaggerated GABAA receptor desensitization. Our results demonstrate that at least part of the functional link between PD134,308 and the GABAA response occurs postsynaptically.

Animals↗

PubMed2004

Genistein stimulates electrogenic Cl(-) secretion in mouse jejunum.

We used the short-circuit current (I(sc)) technique to investigate the effects of the isoflavone genistein on the electrogenic Cl(-) secretion of the mouse jejunum. Genistein stimulated a sustained increase in I(sc) that was dose dependent. Bumetanide inhibited 76 +/- 5% of the genistein-stimulated I(sc) consistent with activation of Cl(-) secretion. Genistein failed to stimulate I(sc) following maximal activation of the cAMP pathway by forskolin. In addition, forskolin had a reduced effect on I(sc) of the mouse jejunum in the presence of genistein. Glibenclamide, a blocker of CFTR, eliminated the genistein-stimulated increase of I(sc) and reduced the forskolin-activated I(sc). Clotrimazole, a Ca(2+)-activated K(+) channel blocker, failed to reduce the genistein-stimulated I(sc). Vanadate, a blocker of tyrosine-dependent phosphatases, reduced the genistein-activated I(sc). Tyrphostin A23, a tyrosine kinase inhibitor, reduced basal I(sc), after which genistein failed to stimulate I(sc). These data suggest that genistein activated a sustained Cl(-) secretory response of the mouse jejunum and that the effect of genistein was via a tyrosine-dependent phosphorylation pathway.

Animals↗

PubMed1996

On bipolar cell responses in the teleost retina are generated by two distinct mechanisms.

1. ON Bipolar cells were recorded in slices obtained from hybrid bass retinas. Cells were identified as bipolar cells by position in the slice, by characteristic voltage- and ligand-gated currents, and by filling with the fluorescent dye Lucifer yellow. Cells were recorded with the use of either whole cell or perforated-patch techniques. Standard electrophysiological protocols were used. Drugs were applied by puffing and by local superfusion. 2. Application of exogenous glutamate to ON bipolar cells generated two characteristic responses. One effect of glutamate was to open a conductance with a reversal potential close to the chloride equilibrium potential. The other effect of glutamate was to close a conductance with a reversal potential near 0 mV. These two effects of glutamate on ON bipolar cells match the effects of light described previously with the use of intracellular recordings. Thus the effects of glutamate that we report here appear to underlie the rod and cone inputs to these cells. 3. Many of the ON bipolar cells recorded demonstrated both classes of responses to glutamate. To isolate the two responses, 500 microM glutamate was first applied, and then glutamate in the presence of 5 microM 2-amino-4-phosphonobutyric acid (APB). APB specifically blocks the effects of glutamate on the putative roddriven glutamate receptor (the glutamate-elicited conductance decrease), allowing us to study in isolation the effects of glutamate on the cone component, the glutamate-activated chloride current (IGlu). 4. By isolating IGlu as described above, and taking advantage of the fact that amphotericin-perforated-patch recordings limit the diffusion of chloride ions between the patch pipette and the cell body, we found the physiological reversal potential of IGlu to be -58.9 +/- 7.7 (SD) mV. 5. Both the putative rod- and cone-mediated glutamatergic inputs to these bipolar cells could be activated by driving the photoreceptors with puffs of potassium. The currents recorded with this technique were very similar to those seen with direct application of glutamate.

Aminobutyrates↗

PubMed2000

Glycine inhibits growth of T lymphocytes by an IL-2-independent mechanism.

Previously, it was shown that glycine prevented increases in intracellular calcium ([Ca2+]i) in Kupffer cells. Since Kupffer cells and T lymphocytes are derived from the same pluripotent stem cell, it was hypothesized that glycine would prevent increases in [Ca2+]i in lymphocytes and inhibit cell proliferation. Lymphocyte proliferation was measured in one-way MLC with spleen cells from DA and Lewis rats and in enriched T lymphocyte preparations stimulated by immobilized anti-CD3 Ab. Glycine caused a dose-dependent decrease in cell proliferation to about 40% of control. Con A caused a dose-dependent increase in [Ca2+]i in Jurkat cells which was blunted maximally with 0.6 mM glycine. The effect of glycine was dependent on extracellular chloride and reversed by strychnine, an antagonist of the glycine-gated chloride channel. Similar results were obtained with rat T lymphocytes stimulated by anti-CD3 Ab. Surprisingly, glycine had no effect on IL-2 production in the mixed lymphocyte culture; therefore, the effect of glycine on IL-2-dependent proliferation was tested. Glycine and rapamycin caused dose-dependent decreases in IL-2-stimulated growth of Ctll-2 cells to about 60% and 40%, respectively, of control. Moreover, glycine also inhibited the IL-2-stimulated growth of rat splenic lymphocytes. It is concluded that glycine blunts proliferation in an IL-2-independent manner. This is consistent with the hypothesis that glycine activates a glycine-gated chloride channel and hyperpolarizes the cell membrane-blunting increases in [Ca2+]i that are required for transcription of factors necessary for cell proliferation.

Animals↗

PubMed1990

Identification and functional expression of a novel ligand binding subunit of the inhibitory glycine receptor.

By homology screening of a rat brain cDNA library we have identified a novel alpha subunit variant of the inhibitory glycine receptor, alpha 3. The deduced protein exhibits 82-83% amino acid identity to the previously characterized rat and human alpha 1 and alpha 2 subunit sequences. Upon heterologous expression in Xenopus laevis oocytes, the alpha 3 subunit formed functional agonist-gated chloride channels which displayed a low affinity for glycine and only small responses to taurine. Amplification of alpha 3 transcripts by polymerase chain reaction revealed high levels in spinal cord at later postnatal stages of development. These data indicate alpha subunit heterogeneity of the inhibitory glycine receptor in adult rats that may result in pharmacologically distinct receptor subtypes.

Amino Acid Sequence↗