Further study of the correlation between na-pump activity and membrane chemosensitivity

Using internally dialyzed neurons of Helix, we have examined the effects of sodium-pump activity and intracellular ATP concentration on transmembrane currents induced by acetylcholine (ACh) and gamma-aminobutyric acid (GABA). We also report on the effects of pump activity and levels of intracellular ATP on binding by Helix ganglia of 3H-alpha-bungarotoxin (3H-alpha-BT) and 3H-GABA. Both ouabain-containing and potassium-free solutions depressed the neurotransmitter-induced transmembrane current of one type of dialyzed neurons. An increase in the intracellular ATP concentration led to a depression of ACh-induced currents and to the disappearance of the blocking effect of ouabain on these currents. Intracellular ADP had a similar but smaller effect on transmitter-induced currents, and intracellular AMP was ineffective. The depressing effect of internal ATP on ACh-induced currents was absent in the presence of an inhibitor of membrane phosphorylation (dinitrophenol). The binding of tritium-labeled alpha-BT and GABA to the membranes was depressed by both ouabain-containing and K-free solutions and also by compounds (theophylline and NaF) which increase the levels of intracellular ATP. The results suggest that the Na pump modulates the affinity of ACh and GABA membrane receptors by the regulation of the phosphorylated state of membrane receptors.     

Electrophysiological effects of GABA on cat pancreatic neurons

In mammalian peripheral sympathetic ganglia GABA acts presynaptically to facilitate cholinergic transmission and postsynaptically to depolarize membrane potential. The GABA effect on parasympathetic pancreatic ganglia is unknown. We aimed to determine the effect of locally applied GABA on cat pancreatic ganglion neurons. Ganglia with attached nerve trunks were isolated from cat pancreata. Conventional intracellular recording techniques were used to record electrical responses from ganglion neurons. GABA pressure microejection depolarized membrane potential with an amplitude of 17.4 +/- 0.7 mV. Electrically evoked fast excitatory postsynaptic potentials were significantly inhibited (5.4 +/- 0.3 to 2.9 +/- 0.2 mV) after GABA application. GABA-evoked depolarizations were mimicked by the GABA(A) receptor agonist muscimol and abolished by the GABA(A) receptor antagonist bicuculline and the Cl(-) channel blocker picrotoxin. GABA was taken up and stored in ganglia during preincubation with 1 mM GABA; beta-aminobutyric acid application after GABA loading significantly (P < 0.05) increased depolarizing response to GABA (15.6 +/- 1.0 vs. 7.8 +/- 0.8 mV without GABA preincubation). Immunolabeling with antibodies to GABA, glial cell fibrillary acidic protein, protein gene product 9.5, and glutamic acid decarboxylase (GAD) immunoreactivity showed that GABA was present in glial cells, but not in neurons, and that glial cells did not contain GAD, whereas islet cells did. The data suggest that endogenous GABA released from ganglionic glial cells acts on pancreatic ganglion neurons through GABA(A) receptors.     

1-Methyl-4-phenylpridinium (MPP+)-induced functional run-down of GABA(A) receptor-mediated currents in acutely dissociated dopaminergic neurons

We have evaluated GABA(A)receptor function during treatment of 1-methyl-4-phenylpridinium (MPP+) using patch-clamp perforated whole-cell recording techniques in acutely dissociated dopaminergic (DAergic) neurons from rat substantia nigra compacta (SNc). Gamma-aminobutyric acid (GABA), glutamate or glycine induced inward currents (I(GABA), I(Glu), I(Gly)) at a holding potential (VH) of -45 mV. The I(GABA) was reversibly blocked by the GABA(A) receptor antagonist, bicuculline, suggesting that I(GABA) is mediated through the activation of GABA(A) receptors. During extracellular perfusion of MPP+ (1-10 microm), I(GABA) , but neither I(Glu) nor I(Gly), declined (termed run-down) with repetitive agonist applications, indicating that the MPP+-induced I(GABA) run-down occurred earlier than I(Gly) or I(Glu) under our experimental conditions. The MPP+-induced I(GABA) run-down can be prevented by a DA transporter inhibitor, mazindol, and can be mimicked by a metabolic inhibitor, rotenone. Using conventional whole-cell recording with different concentrations of ATP in the pipette solution, I(GABA) run-down can be induced by decreasing intracellular ATP concentrations, or prevented by supplying intracellular ATP, indicating that I(GABA) run-down is dependent on intracellular ATP concentrations. A GABA(A) receptor positive modulator, pentobarbital (PB), potentiated the declined I(GABA) and eliminated I(GABA) run-down. Corresponding to these patch-clamp data, tyrosine hydroxylase (TH) immunohistochemical staining showed that TH-positive cell loss was protected by PB during MPP+ perfusion. It is concluded that extracellular perfusion of MPP+ induces a functional run-down of GABA(A) receptors, which may cause an imbalance of excitation and inhibition of DAergic neurons.     

Interaction of penicillin and pentobarbital with inhibitory synaptic mechanisms in neocortex

In this study we characterized the responses of neocortical neurons to iontophoretically applied gamma-aminobutyric acid (GABA) and examined how these GABA responses as well as the inhibitory postsynaptic potentials (IPSPs) were affected by the presence of penicillin or pentobarbital. Intracellular recordings were obtained from slices of rat neocortex maintained in vitro; injection of the dye Lucifer yellow indicated that recordings were primarily from pyramidal neurons. Orthodromically evoked responses were always depolarizing at the cell's resting membrane potential. Such depolarizing responses could easily be reversed in polarity by depolarizing the cell 10-15 mV, suggesting that the response consisted partly of an IPSP. In some cases, depolarization unmasked a small, short-latency excitatory postsynaptic potential (EPSP). Responses to iontophoretically applied GABA were also depolarizing at rest. Biphasic hyperpolarizing-depolarizing responses were occasionally observed upon depolarization of the neuron. Bath application of penicillin (1.7-3.4 mM) decreased the amplitude of the IPSPs and increased their time to peak, an effect associated with the development of epileptiform activity. Penicillin also reduced the maximum response to iontophoretically applied GABA without affecting the dose required to obtain a half-maximal response, suggesting a noncompetitive antagonism. Pentobarbital (100-200 microM) prolonged the time course and increased the amplitude of the IPSPs while producing a leftward shift in the GABA charge-response relation. These results suggest that the convulsant penicillin and the anticonvulsant pentobarbital have opposing actions on GABAergic inhibition in the neocortex.     

P物质抑制培养大鼠海马大锥体细胞GABA—激活电流

研究主要探讨P物质（SP）对GABA－激活电流的调制。实验在培养的新生大鼠海马大锥体细胞上进行。应用全细胞膜片箝技术记录GABA激活的内向电流。在被检的大锥体细胞中,有72％（66／92）的神经元对GABA和SP同时敏感,预后SP后,GABA激活电流明显地被抑制,此抑制作用是呈剂量依赖性的。在预加10^-8,10^-7,10^-6,10^-5mol/LSP后,GABA的激活电流分别降低18％,24．8％,25．9％和28％,用SP的拮抗剂 spantide能阻断此种抑制作用,在电极中灌注H7（PKC抑制剂）能取消此抑制作用,上述结果提示：SP对GABA激活电流的抑制作用是SP作用于SP受体,通过胞内第二信使,使GABAA受体通道复合体胞内磷酸化所致。     

Functional assay for GABA receptor subtypes of a cockroach giant interneuron.

gamma-Aminobutyric acid (GABA) receptors were examined in the cockroach central nervous system (CNS) using the single fiber-oil gap method applied to an identified giant interneuron. Short-lasting pressure application of 10 mM GABA developed a multiphasic response composed of a fast hyperpolarization followed by a transient depolarizing component and a stable hyperpolarization. This triphasic characteristic shape of the response was modified according to the dose of GABA injected or bath-applied and to the precise localization of the injection within the dendritic area. The transient depolarizing phase showed a negative reversal potential of -70 mV. Both hyperpolarizing phases reversed at a more negative level ranging to -80 mV. A positive shift of these values was caused by a decrease in external chloride concentration. Bath-application of 0.1 mM picrotoxin (Ptx) decreased the depolarizing phase which was progressively replaced by a stable hyperpolarization. The transient depolarizing component desensitized quickly and was the most sensitive phase to Ptx action. The Ptx-resistant response reversed at a mean value of -100 mV close to the equilibrium potential for potassium ions (EK+), suggesting that it was generated by a K(+)-channel coupled receptor. Although baclofen was unable to mimic the Ptx-resistant GABA response, the compound CGA 147823, known to bind with a high specificity to vertebrate GABAB receptors, has been successfully used to reproduce the Ptx-resistant GABA response. It is suggested that, in addition to GABA receptors linked to chloride channels, the insect CNS possesses GABA receptors sharing ionic characteristics of GABAB receptors especially those located in the vertebrate CNS, although they are insensitive to baclofen.     

P2X7 nucleotide receptor activation enhances IFN gamma-induced type II nitric oxide synthase activity in BV-2 microglial cells

Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFN gamma-induced NO release in murine BV-2 microglial cells was investigated. BV-2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), and 2-methylthio-ATP (2-MeSATP), but not UTP, enhanced IFN gamma-induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y2 and P2Y6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal-regulated kinases ERK1/2, decreased IFN gamma-induced NO production. BzATP, a potent P2X7 receptor agonist, was more effective than ATP, ADP, or 2-MeSATP at enhancing IFN gamma-induced ERK1/2 phosphorylation. Consistent with activation of the P2X7 receptor, periodate-oxidized ATP, a P2X7 receptor antagonist, and suramin, a non-specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFN gamma-induced NO production, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.     

Distinct signal transduction pathways for GABA-induced GABA(A) receptor down-regulation and uncoupling in neuronal culture: a role for voltage-gated calcium channels

Changes in GABA receptor (GABA(A)R) gene expression are detected in animal models of epilepsy, anxiety and in post-mortem schizophrenic brain, suggesting a role for GABA(A)R regulation in neurological disorders. Persistent (48 h) exposure of brain neurons in culture to GABA results in down-regulation of GABA(A)R number and uncoupling of GABA and benzodiazepine (BZD) binding sites. Given the central role of GABA(A)Rs in fast inhibitory synaptic transmission, GABA(A)R down-regulation and uncoupling are potentially important mechanisms of regulating neuronal excitability, yet the molecular mechanisms remain unknown. In this report we show that treatment of brain neurons in culture with tetrodotoxin, glutamate receptor antagonists, or depolarization with 25 mM K(+) fails to alter GABA(A)R number or coupling. Changes in neuronal activity or membrane potential are therefore not sufficient to induce either GABA(A)R down-regulation or uncoupling. Nifedipine, a voltage-gated Ca(2+) channel (VGCC) blocker, inhibits both GABA-induced increases in [Ca(2+)](i) and GABA(A)R down-regulation, suggesting that VGCC activation is required for GABA(A)R down-regulation. Depolarization with 25 mM K(+) produces a sustained increase in intracellular [Ca(2+)] without causing GABA(A)R down-regulation, suggesting that activation of VGCCs is not sufficient to produce GABA(A)R down-regulation. In contrast to GABA(A)R down-regulation, nifedipine and 25 mM K(+) fail to inhibit GABA-induced uncoupling, demonstrating that GABA-induced GABA(A)R down-regulation and uncoupling are mediated by independent molecular events. Therefore, GABA(A)R activation initiates at least two distinct signal transduction pathways, one of which involves elevation of intracellular [Ca(2+)] through VGCCs.     

The ATP-sensitivity of K+ channels in rat pancreatic B-cells is modulated by ADP

ATP-sensitive K+ channels in inside-out membrane patches from dispersed rat pancreatic B-cells were studied using patch-clamp methods. The dose-response curve for ATP-induced channel inhibition was shifted to higher concentrations in the presence of ADP (2 mM). In glucose-free solution, the total intracellular concentration of ATP was 3.8 mM and of ADP 1.5 mM; glucose (20 mM) increased ATP and decreased ADP by approx. 40%. These results suggest that both ADP and ATP may be involved in regulating the activity of the glucose-sensitive K+ channel in intact B-cells.     

ATP Dependence of Na+-Driven Cl–HCO3 Exchange in Squid Axons

Squid giant axons recover from acid loads by activating a Na(+)-driven Cl-HCO(3) exchanger. We internally dialyzed axons to an intracellular pH (pH( i )) of 6.7, halted dialysis and monitored the pH(i) recovery (increase) in the presence of ATP or other nucleotides, using cyanide to block oxidative phosphorylation. We computed the equivalent acid-extrusion rate (J(H)) from the rate of pH(i) increase and intracellular buffering power. In experimental series 1, we used dialysis to vary [ATP](i), finding that Michaelis-Menten kinetics describes J (H) vs. [ATP](i), with an apparent V(max) of 15.6 pmole cm(-2 )s(-1) and K (m) of 124 microM. In series 2, we examined ATP gamma S, AMP-PNP, AMP-PCP, AMP-CPP, GMP-PNP, ADP, ADP beta S and GDP beta S to determine if any, by themselves, could support transport. Only ATP gamma S (8 mM) supported acid extrusion; ATP gamma S also supported the HCO (3)(-) -dependent (36)Cl efflux expected of a Na(+)-driven Cl-HCO(3) exchanger. Finally, in series 3, we asked whether any nucleotide could alter J (H) in the presence of a background [ATP](i) of approximately 230 microM (control J (H) = 11.7 pmol cm(-2 )s(-1)). We found J (H) was decreased modestly by 8 mM AMP-PNP (J (H) = 8.0 pmol cm(-2 )s(-1)) but increased modestly by 1 mM ADP beta S (J (H) = 16.0 pmol cm(-2 )s(-1)). We suggest that ATP gamma S leads to stable phosphorylation of the transporter or an essential activator.     

Tonic GABAergic inhibition of taste-responsive neurons in the nucleus of the solitary tract

The effects of gamma-aminobutyric acid (GABA) and the GABAA receptor antagonist bicuculline methiodide (BICM) on the activity of taste- responsive neurons in the nucleus of the solitary tract (NST) were examined electrophysiologically in urethane-anesthetized hamsters. Single neurons in the NST were recorded extracellularly and drugs (21 nl) were microinjected into the vicinity of the cell via a multibarrel pipette. The response of each cell was recorded to lingual stimulation with 0.032 M NaCl, 0.032 M sucrose, 0.0032 M citric acid and 0.032 M quinine hydrochloride (QHCl). Forty-six neurons were tested for the effects of GABA; the activity of 29 cells (63%) was inhibited by 5 mM GABA. Whether activity was elicited in these cells by repetitive anodal current stimulation (25 microA, 0.5 s, 0.1 Hz) of the tongue (n = 13 cells) or the cells were spontaneously active (n = 13 cells), GABA produced a dose-dependent (1, 2 and 5 mM) decrement in activity. Forty- seven NST neurons were tested for the effects of BICM on their responses to chemical stimulation of the tongue; the responses of 28 cells (60%) were enhanced by 10 mM BICM. The gustatory responses of 26 of these cells were tested with three concentrations (0.2, 2 and 10 mM) of BICM, which produced a dose-dependent increase in both spontaneous activity and taste-evoked responses. Nine of these neurons were sucrose- best, seven were NaCl-best, eight were acid-best and two responded best to QHCl. The responses to all four tastants were enhanced, with no difference among neuron types. For 18 cells that were tested with two or more gustatory stimuli, BICM increased their breadth of responsiveness to their two most effective stimuli. These data show that approximately 60% of the taste-responsive neurons in the rostral NST are inhibited by GABA and/or subject to a tonic inhibitory influence, which is mediated by GABAA receptors. The modulation of these cells by GABA provides a mechanism by which the breadth of tuning of the cell can be sharpened. Modulation of gustatory activity following a number of physiological changes could be mediated by such a GABAergic circuit.      

Exogenous ATP induces electrical membrane responses in fibroblasts

Mouse fibroblastic L cells responded to exogenous ATP (0.2 mM) with a transient hyperpolarization due to increased membrane permeability to K⁺. By contrast, intracellular injection of ATP (up to about 3 mM) produced no noticeable effects on the membrane potential. The effects of a non-hydrolysable analogue of ATP (AMP-PNP) were similar to those of ATP. After successive applications of ATP, the cell membrane became virtually unresponsive (desensitized). Extracellular ADP was also effective, but AMP or adenosine was not. Antazoline suppressed the ATP response. Thus, exogenous ATP and ADP appear to stimulate P₂-purinoceptors. Similar responses to ATP (or ADP) were also observed in human normal diploid fibroblasts (Flow 1000 line).     

Block of an insect central nervous system GABA receptor by cyclodiene and cyclohexane insecticides

The effects of a cyclodiene (endrin) and a cyclohexane (lindane) insecticide have been tested on gamma-aminobutyric acid (GABA) receptors in the central nervous system of the cockroach (Periplaneta americana), by using electrophysiological methods and an in vitro functional receptor assay. In electrophysiological experiments on an identified motor neuron (Df), endrin blocked the GABA response with a 50% inhibition concentration of 5.0 x 10(-7) M in a non-competitive manner. The actions of endrin were irreversible under the experimental conditions adopted. Increasing the intracellular chloride concentration reduced the effectiveness of endrin, whereas a change in the potassium concentration failed to influence the block by endrin of GABA responses. Lindane exhibited similar actions to endrin on insect GABA receptors, but was approximately an order of magnitude less effective. In a microsac preparation from cockroach nerve cords, endrin, at a concentration of 1.0 x 10(-5) M, completely blocked GABA-stimulated 36Cl- uptake, whereas the same concentration of lindane was less potent, only blocking about 40% of uptake under similar conditions. Neither insecticide had any effect on L-glutamate-activated chloride channels. The results demonstrate that endrin and lindane block functional insect neuronal GABA receptors.     

ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.     

Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and phosphatidylinositol bisphosphate (PIP(2)): a study of channel rundown and reactivation

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6. 2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of K(ATP) channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP(2)), and phosphorylation. Upon excision of inside-out patches into a Ca(2+)- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6. 2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca(2+) accelerated this process, suggesting a role for PIP(2) hydrolysis mediated by a Ca(2+)-dependent phospholipase C. PIP(2) could reactivate channel activity after a brief exposure to Ca(2+), but not after prolonged exposure. However, in both cases, PIP(2) reversed the increase in ATP sensitivity, indicating that PIP(2) lowers the ATP sensitivity by increasing P(o) as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca(2+) facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5-15 min, increased ATP sensitivity and loss of reactivation by PIP(2) and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP(2) responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP(2) and phosphorylation.     

Glutamate receptors on the somata of dorsal unpaired median neurons in cockroach,Periplaneta americana,thoracic ganglia

Effects of application of glutamate and glutamatergic ligands were studied to characterize the receptors for glutamate present on the soma membrane of the dorsal unpaired median (DUM) neurons in the thoracic ganglia of the cockroach, Periplaneta americana, using the intracellular recording technique. Application of L-glutamate did not block the GABA-response, and application of beta-guanidino-propionic acid, a competitive antagonist for GABA, failed to block the response to L-glutamate. These results indicate that most of L-glutamate action may not be mediated by a GABA-activated channel. To examine glutamate receptor types on the DUM neurons, glutamate receptor agonists were applied. The ionotropic glutamate receptor (iGluR) agonists evoked depolarizations with the following relative rank of order of potency: kainate > AMPA > quisqualate. Metabotropic glutamate receptor (mGluR) agonists also elicited membrane depolarizations or hyperpolarizations associated with an increase in membrane conductance. The mGluR agonists evoked depolarizations or hyperpolarizations with the following relative rank of order: L-CCG-1 > 1S, 3R-ACPD > L-AP4. Depolarization of the same DUM neuron was detected following exposure of kainate and L-CCG-I, suggesting the coexistence of distinct iGluR and mGluR types. A membrane permeable cAMP analog, CPT-cAMP, could not mimic the effect of mGluR agonists. The mGluR selective antagonists, MCCG and MCPG, failed to antagonize the response to mGluR agonists. The involvement of cAMP in the mGluR response was not confirmed in DUM neurons. Although the functional roles of these receptors are unknown, it might be possible then that these extrasynaptic receptors have a modulatory effect on the excitability of the DUM neurons.     

Differential sensitivity of two insect GABA-gated chloride channels to dieldrin,fipronil and picrotoxinin

In the central nervous system of both vertebrates and invertebrates inhibitory neurotransmission is mainly achieved through activation of γ-aminobutyric acid (GABA) receptors. Extensive studies have established the structural and pharmacological properties of vertebrate GABA receptors. Although the vast majority of insect GABA-sensitive responses share some properties with vertebrate GABAA receptors, peculiar pharmacological properties of these receptors led us to think that several GABA-gated chloride channels are present in insects. We describe here the pharmacological properties of two GABA receptor subtypes coupled to a chloride channel on dorsal unpaired median (DUM) neurones of the adult male cockroach. Long applications of GABA induce a large biphasic hyperpolarization, consisting of an initial transient hyperpolarization followed by a slow phase of hyperpolarization that is not quickly desensitized. With GABA, the transient hyperpolarization is sensitive to picrotoxinin, fipronil and dieldrin whereas the slow response is insensitive to these insecticides.When GABA is replaced by muscimol and cis-4-aminocrotonic acid (CACA) a biphasic hyperpolarization consisting of an initial transient hyperpolarization followed by a sustained phase is evoked which is blocked by picrotoxinin and fipronil. Exposure to dieldrin decreases only the early phase of the muscimol and CACA-induced biphasic response, suggesting that two GABA-gated chloride channel receptor subtypes are present in DUM neurones. This study describes, for the first time, a dieldrin resistant component different to the dieldrin- and picrotoxinin-resistant receptor found in several insect species.     

Functional regulation of gamma-aminobutyric acid transporters by direct tyrosine phosphorylation

Tyrosine phosphorylation regulates multiple cell signaling pathways and functionally modulates a number of ion channels and receptors. Neurotransmitter transporters, which act to clear transmitter from the synaptic cleft, are regulated by multiple second messenger pathways that exert their effects, at least in part, by causing a redistribution of the transporter protein to or from the cell surface. To test the hypothesis that tyrosine phosphorylation affects transporter function and to determine its mechanism of action, we examined the regulation of the rat brain gamma-aminobutyric acid (GABA) transporter GAT1 expressed endogenously in hippocampal neurons and expressed heterologously in Chinese hamster ovary cells. Inhibitors of tyrosine kinases decreased GABA uptake; inhibitors of tyrosine phosphatases increased GABA uptake. The decrease in uptake seen with tyrosine kinase inhibitors was correlated with a decrease in tyrosine phosphorylation of GAT1 and resulted in a redistribution of the transporter from the cell surface to intracellular locations. A mutant GAT1 construct that was refractory to tyrosine phosphorylation could not be regulated by tyrosine kinase inhibitors. Activators of protein kinase C, which are known to cause a redistribution of GAT1 from the cell surface, were additive to the effects of tyrosine kinase inhibitors suggesting that multiple signaling pathways control transporter redistribution. Application of brain-derived neurotrophic factor, which activates receptor tyrosine kinases, up-regulated GAT1 function suggesting one potential trigger for the cellular regulation of GAT1 signaling by tyrosine phosphorylation. These data support the hypothesis that transporter expression and function is controlled by the interplay of multiple cell signaling cascades.     

Enhancement of acid-sensing ion channel activity by metabotropic P2Y UTP receptors in primary sensory neurons

Peripheral purinergic signaling plays an important role in nociception. Increasing evidence suggests that metabotropic P2Y receptors are also involved, but little is known about the underlying mechanism. Herein, we report that selective P2Y receptor agonist uridine 5′-triphosphate (UTP) can exert an enhancing effect on the functional activity of acid-sensing ion channels (ASICs), key sensors for extracellular protons, in rat dorsal root ganglia (DRG) neurons. First, UTP dose-dependently increased the amplitude of ASIC currents. UTP also shifted the concentration–response curve for proton upwards, with a 56.6?±?6.4 % increase of the maximal current response to proton. Second, UTP potentiation of proton-gated currents can be mimicked by adenosine 5′-triphosphate (ATP), but not by P2Y1 receptor agonist ADP. Potentiation of UTP was blocked by P2Y receptor antagonist suramin and by inhibition of intracellular G protein, phospholipase C (PLC), protein kinase C (PKC), or protein interacting with C-kinase 1 (PICK1) signaling. Third, UTP altered acidosis-evoked membrane excitability of DRG neurons and caused a significant increase in the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, UTP dose-dependently exacerbated nociceptive responses to injection of acetic acid in rats. These results suggest that UTP enhanced ASIC-mediated currents and nociceptive responses, which reveal a novel peripheral mechanism underlying UTP-sensitive P2Y₂ receptor involvement in hyperalgesia by sensitizing ASICs in primary sensory neurons.     

Mechanisms of ammonia-induced cell death in rat cortical neurons: roles of NMDA receptors and glutathione

The occurrence, nature and prevention of ammonia-induced cell death were assayed in cultured primary cortical neurons from newborn rats. Treatment with 1-10 mM ammonium chloride for 24 or 48 h, dose-dependently decreased neuronal survival (MTT assay) and GSH/GSSG ratio in the cultures, whereas total GSH content was significantly reduced only with 10mM ammonia. Treatment with a glutathione synthesis inhibitor, buthionyl sulfoximine (BSO) (10 microM), decreased the GSH content and GSH/GSSG ratio to a degree similar to that of 10 mM ammonia, but it did not decrease cell survival in control cells. This indicates that glutathione depletion per se is not a cause of ammonia-induced neuronal death. However, ammonia-induced decrease of cell viability was attenuated by incubation with glutathione diethyl ester (GEE), which transiently increased the intracellular GSH level in both control and ammonia-treated cells. Neuronal survival in the presence of ammonia was partly improved by the NMDA receptor antagonists MK-801 and APV. Morphological analysis revealed that ammonia treatment causes both apoptotic and non-apoptotic neuronal death, the former not being inhibited by MK-801. Apoptosis was the dominant type of cell death at 10mM ammonia, as concluded both from morphologic examination and the absence of survival improvement in the presence of GABA+nipecotic acid or taurine, model anti-excitotoxic treatments of cortical neurons. The mechanism underlying apoptosis may include inhibition of a survival kinase, Akt, whose activatory phosphorylation at Ser473 is reduced in neurons treated with 10 mM, but not 1 mM ammonia.