The excitatory and inhibitory amino acid receptors on horizontal cells isolated from the white perch retina

1993 ◽  
Vol 70 (1) ◽  
pp. 8-19 ◽  
Author(s):  
Z. J. Zhou ◽  
G. L. Fain ◽  
J. E. Dowling
Keyword(s):  
Amino Acid ◽  
White Perch ◽  
Glycine Receptor ◽  
Horizontal Cells ◽  
Hill Coefficient ◽  
Equilibrium Potential ◽  
Homogeneous Distribution ◽  
Response Analysis ◽  
Bathing Solution ◽  
Whole Cell

1. The distribution and the properties of receptors to the inhibitory amino acid glycine (GLY) and the excitatory amino acid glutamate (GLU) and its analogues kainate (KA), quisqualate (QUIS), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and N-methyl-D-aspartate (NMDA), were studied with whole-cell and outside-out patch-clamp techniques on all four types of horizontal cells isolated from the retina of white perch. 2. Glycine at concentrations above 30 microM evoked whole-cell current responses from two types of horizontal cells (H2 and H4). The other two types of horizontal cells (H1 and H3) were unresponsive to GLY (30 microM-3 mM). 3. Responses elicited by GLY from H2 and H4 cells were similar, consisting of inward currents that desensitized with a half-decay time of 0.5-2 s at glycine concentrations between 100 and 500 microM. GLY-activated currents were inhibited by the glycine receptor antagonist strychnine (STRYCH). Current responses evoked by GLY reversed at the Cl- equilibrium potential. 4. Dose-response analysis of peak currents induced by GLY revealed a Hill coefficient of 2.0 +/- 0.1 (mean +/- SD, n = 3) and an median effective concentration (EC50) of 85 +/- 2 microM (n = 3). 5. Single glycine receptor channels recorded from outside-out patches had a main-state conductance of 47 +/- 4 pS (n = 3). 6. Every type of horizontal cell from the white perch responded to GLU, KA, QUIS, and AMPA but none responded to exogenously applied NMDA (200 microM) or NMDA (200 microM) + GLY (1 microM) in a Mg+2-free bathing solution. 7. The ratio of the amplitude of responses to GLU, KA, QUIS, and AMPA remained nearly constant among all the horizontal cells tested, suggesting there might be only a single population of non-NMDA receptors on these cells. 8. QUIS and KA both elicited responses from the horizontal cells. When applied together with KA, QUIS competitively antagonized the responses of horizontal cells to KA. 9. The results demonstrated the existence of an inhomogeneous distribution of strychnine-sensitive glycine receptors and a homogeneous distribution of non-NMDA type glutamate receptors among the four types of white perch horizontal cells.

GABA-Induced Cl− Current in Cultured Embryonic Human Dorsal Root Ganglion Neurons

1999 ◽  
Vol 82 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Alexander Y. Valeyev ◽  
John C. Hackman ◽  
Alice M. Holohean ◽  
Patrick M. Wood ◽  
Jennifer L. Katz ◽  
...  
Keyword(s):  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Single Channel ◽  
Dorsal Root Ganglion Neurons ◽  
Hill Coefficient ◽  
Equilibrium Potential ◽  
Single Channels ◽  
Drg Neurons ◽  
Whole Cell ◽  
Ganglion Neurons

γ-Aminobutyric acid (GABA)-activated channels in embryonic (5–8 wk old) human dorsal root ganglion (DRG) neurons in dissociated culture were characterized by whole cell and single-channel techniques. All DRG neurons when held at negative holding membrane potentials displayed inward current to micromolar concentrations of GABA applied by pressure pulses from closely positioned micropipettes. The current was directly proportional to the concentration of GABA (EC50, 111 μM; Hill coefficient, 1.7). DRG neurons also responded to micromolar concentrations of pentobarbital and alphaxalone but not to cis-4-aminocrotonic acid (CACA), glycine, or taurine. Baclofen (100 μM) affected neither the holding currents nor K+ conductance (when patch pipettes were filled with 130 mM KCl) caused by depolarizing pulses. Whole cell GABA-currents were blocked by bicuculline, picrotoxin, and t-butylbicyclophosphorothionate (TBPS; all at 100 μM). The reversal potential of whole cell GABA-currents was close to the theoretical Cl− equilibrium potential, shifting with changes in intracellular Cl− concentration in a manner expected for Cl−-selective channels. The whole cell I-V curve for GABA-induced currents demonstrated slight outward rectification with nearly symmetrical outside and inside Cl− concentrations. Spectral analysis of GABA-induced membrane current fluctuations showed that the kinetic components were best fitted by a triple Lorentzian function. The apparent elementary conductance for GABA-activated Cl− channels determined from the power spectra was 22.6 pS. Single-channel recordings from cell-attached patches with pipettes containing 10 μM GABA indicated that GABA-activated channels have a main and a subconductance level with values of 30 and 19 pS, respectively. Mean open and closed times of the channel were characterized by two or three exponential decay functions, suggesting two or three open channel states and two closed states. Single channels showed a lack of rectification. The actions of GABA on cultured human embryonic DRG neurons are mediated through the activation of GABAA receptors with properties corresponding to those found in the CNS of human and other mammalian species but differing from those of cultured human adult DRG neurons.

Potentiation of NMDA Receptor-Mediated Responses by Dynorphin at Low Extracellular Glycine Concentrations

1997 ◽  
Vol 78 (2) ◽  
pp. 582-590 ◽  
Author(s):  
Li Zhang ◽  
Robert W. Peoples ◽  
Murat Oz ◽  
Judith Harvey-White ◽  
Forrest F. Weight ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nmda Receptor ◽  
Opioid Receptor ◽  
Hippocampal Neurons ◽  
High Performance ◽  
Hill Coefficient ◽  
Bathing Solution ◽  
Dynorphin A ◽  
Glycine Concentration ◽  
Nmda Receptor Function

Zhang, Li, Robert W. Peoples, Murat Oz, Judith Harvey-White, Forrest F. Weight, and Ulrike Brauneis. Potentiation of NMDA receptor-mediated responses by dynorphin at low extracellular glycine concentrations. J. Neurophysiol. 78: 582–590, 1997. The effect of dynorphin A(1–13) on N-methyl-d-aspartate (NMDA)-activated currents was investigated in the presence of low extracellular glycine concentrations in Xenopus oocytes expressing recombinant heteromeric NMDA receptors and in cultured hippocampal neurons with the use of voltage-clamp techniques. At an extracellular added glycine concentration of 100 nM, dynorphin A(1–13) (10 μM) greatly increased the amplitude of NMDA-activated currents for all heteromeric subunit combinations tested; on average, the potentiation was: ε1/ζ1, 3,377 ± 1,416% (mean ± SE); ε2/ζ1, 1,897 ± 893%; ε3/ζ1, 4,356 ± 846%; and ε4/ζ1, 1,783 ± 503%. Potentiation of NMDA-activated current by dynorphin A(1–13) was concentration dependent between 0.1 and 10 μM dynorphin A(1–13), with a half-maximal concentration value of 2.77 μM and an apparent Hill coefficient of 2.53, for ε2/ζ1 subunits at 100 nM added extracellular glycine. Percentage potentiation by dynorphin A(1–13) was maximal at the lowest glycine concentrations tested (0.01 and 0.1 μM), and decreased with increasing glycine concentration. No significant potentiation was observed at glycine concentrations >0.1 μM for ε1/ζ1, ε2/ζ1, and ε4/ζ1 subunits, or at >1 μM for ε3/ζ1 subunits. Potentiation of NMDA-activated currents by dynorphin A(1–13) was not inhibited by 1 μM of the κ-opioid receptor antagonist nor-binaltorphimine, and potentiation was not observed with 10 μM of the κ-opioid receptor agonist trans-3,4-dichloro- N-methyl- N-[2-(1-pyrrolidinyl)-cyclohexyl]benzene-acetamide. Potentiation of NMDA-activated current by dynorphin A(1–13) was inhibited by the glycine antagonist kynurenic acid (50 μM). NMDA-activated current was also potentiated at low glycine concentrations by 10 μM dynorphin A(2–13) or (3–13), both of which have a glycine as the first amino acid, but not by 10 μM dynorphin A(4–13), which does not have glycine as an amino acid. In hippocampal neurons, 10 μM dynorphin A(1–13) or (2–13) potentiated steady-state NMDA-activated current in the absence of added extracellular glycine. The extracellular free glycine concentration, determined by high-performance liquid chromatography, was between 26 and 36 nM for the bathing solution in presence or absence of 10 μM dynorphin A(1–13), (2–13), (3–13), or (4–13), and did not differ significantly among these solutions. The observations are consistent with the potentiation of NMDA-activated current at low extracellular glycine concentrations resulting from an interaction of the glycine amino acids in dynorphin A(1–13) with the glycine coagonist site on the NMDA receptor. Because dynorphin A is an endogenous peptide that can be coreleased with glutamate at glutamatergic synapses, the potentiation of NMDA receptor-mediated responses could be an important physiological regulator of NMDA receptor function at these synapses.

Voltage-clamp analysis of currents produced by glutamate and some glutamate analogues on horizontal cells isolated from the catfish retina

1986 ◽  
Vol 56 (1) ◽  
pp. 19-31 ◽  
Author(s):  
G. Hals ◽  
B. N. Christensen ◽  
T. O'Dell ◽  
M. Christensen ◽  
R. Shingai
Keyword(s):  
Membrane Potential ◽  
Amino Acid ◽  
Membrane Permeability ◽  
Time Course ◽  
Reversal Potential ◽  
Horizontal Cells ◽  
Resting Potential ◽  
Bathing Solution ◽  
Patch Type ◽  
Highly Nonlinear

Horizontal cells isolated from the catfish retina were exposed to radiolabeled glutamate, glycine, gamma-aminobutyric acid (GABA), and sucrose to determine if the enzymatic dissociation procedure altered the high-affinity uptake mechanism for GABA and generally reduced membrane selectivity. As in the intact retina, isolated cells could transport GABA but not the other substances. The horizontal cells were voltage clamped using a single low-resistance patch-type electrode. The acidic amino acid L-glutamate, and its analogues kainate and quisqualate, were applied to the cell by pressure ejection from a nearby pipette. All three agonists produced inward currents that reversed near O mV. Quisqualate produced a current with a similar time course as glutamate, but the time course of the response to kainate was faster. The agonists N-methyl-D-aspartate and L-aspartate had little effect on the membrane conductance. The current-to-voltage (I-V) relationship for all three agonists was nonlinear when the membrane potential was hyperpolarized. The nonlinearity was, at least in part, a result of the decreased response to the three agonists. Removal of Mg did not alter this nonlinear relationship. When the inward potassium rectifier was blocked with 100 microM Ba, the response to glutamate was increased compared with the control experiment before block by barium; however, the I-V relationship was still highly nonlinear. Thus glutamate block of the inward potassium current cannot account entirely for the nonlinear I-V. The increase in membrane permeability to specific ions in the presence of an agonist was determined by ion substitution experiments and measuring the shift in the reversal potential. The three agonists appear to increase the membrane permeability to cations but not to anions. The amino acid antagonists cis-2,3-piperidine dicarboxylic acid (PDA) and D-glutamyl glycine (DGG) were bath applied to test their ability to block the depolarizing effects of glutamate. DGG had no measureable effect at 100 microM concentration, whereas PDA reversibly reduced the glutamate response at 1 mM concentration although block was incomplete. Isolated horizontal cells responded to bath-applied glutamate in concentrations of 10-500 microM. In concentrations of glutamate greater than 50 microM, when the membrane potential was held at the resting potential, the inward current reached a maximum followed by a decrease to a steady-state level. This apparent time-dependent desensitization at high agonist concentrations was at least partially removed when Mg was removed from the bathing solution.(ABSTRACT TRUNCATED AT 400 WORDS)

Local superfusion modifies the inward rectifying potassium conductance of isolated retinal horizontal cells

1988 ◽  
Vol 60 (4) ◽  
pp. 1322-1332 ◽  
Author(s):  
I. Perlman ◽  
A. G. Knapp ◽  
J. E. Dowling
Keyword(s):  
White Perch ◽  
Reversal Potential ◽  
Ringer Solution ◽  
Inward Rectifier ◽  
Horizontal Cells ◽  
Equilibrium Potential ◽  
Steady State Current ◽  
Conductance Increase ◽  
Local Superfusion ◽  
Drug Free

1. Horizontal cells were enzymatically and mechanically dissociated from the white perch (Roccus americana) retina and voltage clamped using patch electrodes. Steady-state current-voltage (I-V) relationships of solitary horizontal cells were determined by changing the membrane potential in a rampwise fashion. 2. The I-V curve of cells bathed in normal Ringer solution exhibited a large conductance increase at negative membrane potentials. This conductance activated near the K+ equilibrium potential, had no clear reversal potential, was enhanced by raising the extracellular concentration of K+, and was suppressed by external Cs+. These properties identify the conductance as the inward (anomalous) rectifier. 3. Continuous superfusion of the cells' local environment with drug-free Ringer reduced the magnitude of the inward rectifier current and shifted its activation point to more negative potentials. This effect developed over approximately 30 s, lasted as long as superfusion continued and was reversible upon cessation of superfusion. 4. Pressure ejection of drug-free Ringer solution onto cells bathed in the identical solution also reduced the magnitude of the inward rectifier current, although the effects were more rapid and more transient than those exerted by superfusion. Pressure ejection had little effect when cells were simultaneously superfused with Ringer, suggesting a common mode of action on the inward rectifier. 5. In the absence of superfusion, pressure ejection of Ringer containing 200 microM L-glutamate had a biphasic effect on membrane conductance. At potentials above -60 mV, glutamate caused a conductance increase with a reversal potential near +10 mV. At potentials below -60 mV, glutamate caused a conductance decrease whose reversal potential could not reliably be determined. The latter effect was similar to the suppression of the inward rectifier by application of Ringer alone, suggesting that it may represent an artifact of pressure ejection rather than a direct effect of glutamate. 6. In support of this interpretation, we found that pressure ejection of glutamate in the presence of external Cs+ (which blocks the inward rectifier) or during local superfusion with Ringer (which prevents attenuation of the inward rectifier by pressure ejection) did not cause a conductance decrease at negative potentials. Under these conditions, glutamate caused primarily a conductance increase with a reversal potential near +10 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Nitric oxide mediates outward potassium currents in opossum esophageal circular smooth muscle

1996 ◽  
Vol 270 (6) ◽  
pp. G932-G938 ◽  
Author(s):  
J. Jury ◽  
K. R. Boev ◽  
E. E. Daniel
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Circular Muscle ◽  
Outward Current ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Circular Smooth Muscle ◽  
Outward Currents

Single smooth muscle cells from the opossum body circular muscle were isolated and whole cell currents were characterized by the whole cell patch-clamp technique. When the cells were held at -50 mV and depolarized to 70 mV in 20-mV increments, initial small inactivating inward currents were evoked (-30 to 30 mV) followed by larger sustained outward currents. Depolarization from a holding potential of -90 mV evoked an initial fast inactivating outward current sensitive to 4-aminopyridine but not to high levels of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The outward currents reversed near K+ equilibrium potential and were abolished when KCl was replaced by CsCl in the pipette solution. The sustained outward current was inhibited by quinine and cesium. High EGTA in the pipette solution reduced but did not abolish the sustained outward currents, suggesting that both Ca(2+)-dependent and -independent currents were evoked. The nitric oxide (NO)-releasing agents Sin-1 and sodium nitroprusside increased outward K+ currents. High levels of EGTA in the pipette solution abolished the increase in outward current induced by Sin-1. The presence of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, blocked the effects of NO-releasing agents. We conclude that NO release activates K+ outward currents in opossum esophagus circular muscle, which may depend on Ca2+ release from the SR stores.

Presence of the vesicular inhibitory amino acid transporter in GABAergic and glycinergic synaptic terminal boutons

1999 ◽  
Vol 112 (6) ◽  
pp. 811-823
Author(s):  
A. Dumoulin ◽  
P. Rostaing ◽  
C. Bedet ◽  
S. Levi ◽  
M.F. Isambert ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Amino Acid ◽  
Synaptic Vesicle ◽  
Glycine Receptor ◽  
Primary Cultures ◽  
Ultrastructural Level ◽  
Amino Acid Transporter ◽  
Vesicle Membrane ◽  
Hybridization Data ◽  
Acid Transporter

The characterization of the Caenorhabditis elegans unc-47 gene recently allowed the identification of a mammalian (gamma)-amino butyric acid (GABA) transporter, presumed to be located in the synaptic vesicle membrane. In situ hybridization data in rat brain suggested that it might also take up glycine and thus represent a general Vesicular Inhibitory Amino Acid Transporter (VIAAT). In the present study, we have investigated the localization of VIAAT in neurons by using a polyclonal antibody raised against the hydrophilic N-terminal domain of the protein. Light microscopy and immunocytochemistry in primary cultures or tissue sections of the rat spinal cord revealed that VIAAT was localized in a subset (63-65%) of synaptophysin-immunoreactive terminal boutons; among the VIAAT-positive terminals around motoneuronal somata, 32.9% of them were also immunoreactive for GAD65, a marker of GABAergic presynaptic endings. Labelling was also found apposed to clusters positive for the glycine receptor or for its associated protein gephyrin. At the ultrastructural level, VIAAT immunoreactivity was restricted to presynaptic boutons exhibiting classical inhibitory features and, within the boutons, concentrated over synaptic vesicle clusters. Pre-embedding detection of VIAAT followed by post-embedding detection of GABA or glycine on serial sections of the spinal cord or cerebellar cortex indicated that VIAAT was present in glycine-, GABA- or GABA- and glycine-containing boutons. Taken together, these data further support the view of a common vesicular transporter for these two inhibitory transmitters, which would be responsible for their costorage in the same synaptic vesicle and subsequent corelease at mixed GABA-and-glycine synapses.

Whole cell current analyses of pancreatic acinar AR42J cells. I. Voltage- and Ca(2+)-activated currents

1991 ◽  
Vol 260 (5) ◽  
pp. C934-C948 ◽  
Author(s):  
K. Kusano ◽  
H. Gainer
Keyword(s):  
Reversal Potential ◽  
Outward Current ◽  
Pancreatic Acinar Cells ◽  
Equilibrium Potential ◽  
Channel Blockers ◽  
Whole Cell ◽  
Inward Current ◽  
Tail Current ◽  
Ar42j Cells ◽  
Conductance Increase

Voltage- and Ca(2+)-activated whole cell currents were studied in AR42J cells, a clonal cell line derived from rat pancreatic acinar cells, using a patch electrode voltage-clamp technique. Four kinds of ionic currents were identified by their ionic dependencies, pharmacological properties, and kinetic parameters: 1) an outward current flow due mainly to a voltage-dependent K(+)-conductance increase, 2) an initial transient inward current due to an Na(+)-conductance increase, 3) transient and long-duration inward current due to a Ca(2+)-conductance increase, and 4) a slowly activating inward current that persists over the duration of the depolarizing pulse and deactivates slowly upon repolarization, producing a slow inward tail current. The slow inward tail current was particularly robust and was interpreted as due to a Ca(2+)-activated Cl(-)-conductance increase, since 1) the generation of this current was blocked by removing the extracellular Ca2+, applying Ca(2+)-channel blockers (Cd2+, nifedipine), or by lowering the intracellular Ca2+ concentration [( Ca2+]i) with EGTA; and 2) the reversal potential (Erev) of the slow inward tail current was close to 0 mV in the control condition (152 mM [Cl-]o/154 mM [Cl-]i), and changes of the [Cl-]o/[Cl )i ratio shifted the Erev toward the predicted Cl- equilibrium potential.

EAAT1 is involved in transport ofl-glutamate during differentiation of the Caco-2 cell line

2000 ◽  
Vol 279 (2) ◽  
pp. G366-G373 ◽  
Author(s):  
Agnès Mordrelle ◽  
Eric Jullian ◽  
Cyrille Costa ◽  
Estelle Cormet-Boyaka ◽  
Robert Benamouzig ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Excitatory Amino Acid ◽  
Kinetic Studies ◽  
Glutamate Transport ◽  
Hill Coefficient ◽  
Amino Acid Transporters ◽  
Intestinal Epithelial ◽  
Transport Capacity ◽  
Excitatory Amino Acid Transporter

Little is known concerning the expression of amino acid transporters during intestinal epithelial cell differentiation. The transport mechanism ofl-glutamate and its regulation during the differentiation process were investigated using the human intestinal Caco-2 cell line. Kinetic studies demonstrated the presence of a single, high-affinity,d-aspartate-sensitive l-glutamate transport system in both confluent and fully differentiated Caco-2 cells. This transport was clearly Na+ dependent, with a Hill coefficient of 2.9 ± 0.3, suggesting a 3 Na+-to-1 glutamate stoichiometry and corresponding to the well-characterized XA,G − system. The excitatory amino acid transporter (EAAT)1 transcript was consistently expressed in the Caco-2 cell line, whereas the epithelial and neuronal EAAT3 transporter was barely detected. In contrast with systems B0 and y+, which have previously been reported to be downregulated when Caco-2 cells stop proliferating, l-glutamate transport capacity was found to increase steadily between day 8 and day 17. This increase was correlated with the level of EAAT1 mRNA, which might reflect an increase in EAAT1 gene transcription and/or stabilization of the EAAT1 transcript.

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