P物质对大鼠骶髓后连合核神经元甘氨酸激活的氯电流的调控

采用制霉菌素穿孔膜片箍技术,研究了Ｐ物质对急性分离的大鼠骶髓后的核神经元士的宁敏感性甘氨酸反应的调控作用。在箍制电压为－４０ｍＶ时,ＳＰ时１ｍｍｏｌ／Ｌ－１μｍｏｌ／Ｌ之间呈浓度依赖性地增强３０μｍｏｌ／Ｌ甘氨酸激活的氯电流。ＳＰ既不改变ＩＧｌｙ的翻转电位,也不是影响Ｇｌｙ与其受体的亲和力。Ｓｐａｎｔｉｄｅ和选择性Ｎ中受体拮抗剂,Ｌ－６６８,１６９,可阻断ＳＰ的增强作用,而选择性ＮＫ２受体拮抗剂,     

锌离子对急性分离的大鼠骶髓后连合核神经元GABAA受体介导电流的？…

采用制霉菌素膜片箝 技术,研究了锌离子对急性分离的大鼠骶髓后连合核神经元ＧＡＢＡＡ受体介导电流的作用。     

Effect of flumazenil on GABAA receptors in isolated rat hippocampal neurons

Using whole cell patch-clamp recordings from pyramidal cells acutely dissociated from rat hippocampal slices, Ro-15 1788 (flumazenil, FLU) was shown to enhance the GABA_A-receptor mediated currents evoked by application of -aminobutyric acid (GABA) and to antagonize the enhancing effect of the benzodiazepine agonist flurazepam (FZP) on the GABA_A response. Both FLU and FZP increased the peak and the steady-state components of the responses and accelerated the current decay. This suggests that both agents act via a common mechanism on GABA transmission. It is concluded that FLU possesses high affinity for the binding site, but low efficacy on the GABA_A-benzodiazepine receptor. This suggests that FLU acts as a partial agonist on GABA_A receptors.     

Cu2+ suppresses GABA(A) receptor-mediated responses in rat sacral dorsal commissural neurons

The effect of copper ions (Cu(2+)) on gamma-aminobutyric acid (GABA)-induced responses in acutely dissociated neurons from the rat sacral dorsal commissural nucleus (SDCN) was investigated using a nystatin-perforated patch recording configuration under voltage clamp conditions. The application of Cu(2+) to SDCN neurons reversibly suppressed the GABA (10 microM)-activated Cl(-) current (I(GABA)) in a concentration-dependent manner (1-1000 microM; IC(50) = 24.5 microM). In the presence of Cu(2+) (30 microM), the concentration-response curve of GABA was shifted rightward without reducing I(GABA) recorded under the maximally effective concentration of GABA, thus indicating a dependence of Cu(2+) action on GABA concentration. Inhibition of GABA (10 microM) responses by 30 microM Cu(2+) was essentially voltage independent and was not accompanied by a shift in the reversal potential of the currents. Cu(2+) antagonized the suppressive effect of Zn(2+) in a concentration-dependent manner, suggesting competition between Cu(2+) and Zn(2+) for similar binding sites. These data demonstrate that Cu(2+) is a potent inhibitor of GABA(A) receptor-mediated responses, implying a possible modulatory effect of Cu(2+) on GABAergic synaptic transmission in the mammalian SDCN.     

Biphasic Modulation of GABAA Receptor Binding by Steroids Suggests Functional Correlates

Neuroactive steroids and other positive modulators of GABA_A receptors showed regional variation in both the efficacy and potency for modulation of [³⁵S]TBPS binding to rat brain membrane homogenates, with biphasic concentration-dependence. GABA present in the binding assays prevented the enhancement phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GABA_A receptors, expressed in insect cell line Sf9 using baculovirus, enhancement by steroids of [³⁵S]TBPS binding was sensitive to the presence of the 2 subunit and the nature of the subunit (122S > 12, 62, 622S, and 62). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are consistent with a three-state model in which the receptor mono-liganded by GABA or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for TBPS.     

Methylmercury modulates GABAA receptor complex differentially in rat cortical and cerebellar membranes in vitro

Effects of methylmercury (MetHg) on the specific [³H]flunitrazepam binding were studied in rat cortical and cerebellar P₂-fractions in vitro. MetHg did not affect significantly the specific [³H]flunitrazepam binding in unwashed P₂-fraction but increased it marginally (by 16%) at 100 M in washed P₂-fraction, in both brain regions.Muscimol (3 M), a GABA_A agonist, stimulated the [³H]flunitrazepam binding by 30% to 50% depending on the brain region. In washed cerebellar membranes the enhancing response of muscimol was 10 to 14% lower after preincubation of the tissue with MetHg but in cerebral cortex MetHg did not modulate the muscimol response at all. The results indicate that Met-Hg may have region specific effects on GABA_A receptors in vitro and the effect may depend on the occupational state of the GABA binding domain of the receptor complex.     

Characteristics of GABAA channels in rat dentate gyrus

Single channel currents were activated by GABA (0.5 to 5 m) in cell-attached and inside-out patches from cells in the dentate gyrus of rat hippocampal slices. The currents reversed at the chloride equilibrium potential and were blocked by bicuculline (100 m). Several different kinds of channel were seen: high conductance and low conductance, rectifying and nonrectifying. Channels had multiple conductance states. The open probability (P_o) of channels was greater at depolarized than at hyperpolarized potentials and the relationship between P_oand potential could be fitted with a Boltzmann equation with equivalent valency (z) of 1. The combination of outward rectification and potentialdependent open probability gave very little chloride current at hyperpolarized potentials but steeply increasing current with depolarization, useful properties for a tonic inhibitory mechanism.     

Assembly of GABAA receptors (Review)

GABA_A receptors are the major inhibitory transmitter receptors in the central nervous system. They are chloride ion channels that can be opened by γ-aminobutyric acid (GABA) and are the targets of action of a variety of pharmacologically and clinically important drugs. GABA_A receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to the formation of a large variety of distinct GABA_A receptor subtypes in the brain. The majority of GABA_A receptors seems to be composed of two α, two β and one γ subunit and the occurrence of a defined subunit stoichiometry and arrangement in αβγ receptors strongly indicates that assembly of GABA_A receptors proceeds via defined pathways. Based on the differential ability of subunits to interact with each other, a variety of studies have been performed to identify amino acid sequences or residues important for assembly. Such residues might be involved in direct protein-protein interactions, or in stabilizing direct contact sites in other regions of the subunit. Several homo-oligomeric or hetero-oligomeric assembly intermediates could be the starting point of GABA_A receptor assembly but so far no unequivocal assembly mechanism has been identified. Possible mechanisms of assembly of GABA_A receptors are discussed in the light of recent publications.     

Suppression of kainate-evoked AMPA receptor mediated responses by lanthanum in rat sacral dorsal commissural neurons

The effect of lanthanum (La) on kainate (KA) responses in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) was investigated using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. The responses to KA were mediated by activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in SDCN neurons. La(3+) reversibly inhibited KA (100 microM) activated currents (I(KA)) in a concentration-dependent manner over the range from 30 microM to 30 mM, with IC(50) values of 0.64 +/- 0.06 mM at a holding potential (V(H)) of -40 mV. Our further study indicated that the effects of La(3+) on I(KA) were voltage independent. Moreover, the inhibition was not use dependent and was not overcome by increasing the concentration of agonist. These findings indicate that La(3+) is an efficacious inhibitor of AMPA receptor mediated responses which may contribute to its cytotoxic effect.     

Differential Effects of Thiopental and Pentobarbital on Spinal GABAA Receptors

General anesthetics thiopental and pentobarbital possess very similar chemical structures whereas their clinical potency is quite different. The underlying molecular mechanism is not fully understood. This study was designed to assess the differential effects of thiopental and pentobarbital on GABA_A receptors of mechanically dissociated rat spinal dorsal horn neurons by using whole-cell patch-clamp technique. Pentobarbital, at a concentration of 30 μM, which markedly enhanced sub-saturated GABA-induced current (I _GABA), had no effect on thiopental-induced maximal current. Similarly, the pentobarbital-induced maximal current was also not affected by 30 μM thiopental. Moreover, a linear summation of thiopental-induced maximal current and pentobarbital-induced sub-maximal current was observed. In addition, pentobarbital failed to further enhance I _GABA in the presence of thiopental at a concentration with maximal modulatory effects on I _GABA, and vice versa. Our results thus suggest that thiopental and pentobarbital might exert the GABA mimetic effects independently, but share a common mechanism to produce the GABA modulatory effects. Special issue article in honor of Dr. Ji-Sheng Han.     

Effects of Bisphenol A and its Derivatives on the Response of GABAA Receptors Expressed in Xenopus Oocytes

To study the effects of bisphenol-A (BPA) known to have estrogenic actions, and its derivatives, 3,5-dimethylphenol (DMP) and p-t-butylphenol (TBP), on ionotropic γ-aminobutyric acid (GABA) receptors, GABA_A receptors were expressed in Xenopus oocytes by injecting both poly(A)⁺RNA prepared from rat whole brain and cRNAs synthesized from cloned cDNAs of α₁ and β₁ subunit of the bovine receptors, and their electrical responses were measured by the voltage clamping method. BPA caused the potentiation and inhibition of the former receptor-responses, while it caused only inhibition of the latter ones. In the presence of low concentrations of GABA, DMP and TBP potentiated the responses of both receptors. DMP and TBP also increased the rate of decay of the response, possibly by desensitization of the receptors when GABA solution was continuously bath-applied. Diethyl terephthalate (DTP), which is also known to have estrogenic actions, had little effect on both the responses and the decay of both receptors.     

GABAA Receptors in Central Nervous System Disease: Anxiety,Epilepsy, and Insomnia

Brain function is based on an exquisite balance between excitatory and inhibitory neurotransmission. GABAergic neurons provide the major inhibitory control. By controlling spike timing and sculpting neuronal rhythms they play a key role in regulating behavior. GABAergic neurons are highly diverse and operate with a corresponding diversity of GABA_A receptor subtypes. In this article, the contribution of GABA_A receptor deficits to central nervous system disorders, in particular anxiety disorders, epilepsy, schizophrenia and insomnia, is reviewed.     

Identification of inosine as an endogenous modulator for the benzodiazepine binding site of the GABAA receptors

Previously we have reported the presence of endogenous ligands that are involved in the regulation of the binding of muscimol to the GABA binding site of the GABA_A receptors. Here, we report the presence of multiple forms of endogenous ligands in the brain which modulate the binding of flunitrazepam (FNZP) to the benzodiazepine (BZ) binding site of the GABA_A receptor. Furthermore, one of the endogenous ligands for the BZ receptors, referred to as EBZ, has been identified as inosine based on the following observations: (1) standard inosine and the EBZ have identical NMR and UV spectra; (2) the elution profile of inosine and the EBZ from a HPLC column are indistinguishable, and (3) inosine and the EBZ show identical activity in inhibiting [³H]FNZP binding.     

Subunit Composition and Function of GABAA Receptors of Rat Spermatozoa

GABA triggers mammalian sperm acrosome reaction (AR). Here, evidence is presented, showing that rat spermatozoa contain GABA_A receptors, composed of ₅, ₁ and ₃ subunits. The effects of GABA_A receptor agonist and antagonist on the induction of AR in rat spermatozoa were assessed using the chlortetracycline assay. Muscimol, a GABA_A receptor agonist, triggered AR; whereas bicuculline, a GABA_A receptor antagonist and picrotoxin, a GABA_A receptor/Cl^– channel blocker, inhibited the ability of GABA or progesterone to induce AR. In conclusion, GABA_A receptors appear to mediate the action of progesterone in inducing AR in rat spermatozoa.     

Synaptic Membrane Freezing Affects Modulatory Sites in Avian Central Nervous System GABAA Receptor

Studies were carried out to determine whether barbiturates and neurosteroids share common recognition sites at the GABA_A receptor complex in avian CNS. To achieve this, differentially prepared fresh and frozen synaptic membranes were used. Both the barbiturate, pentobarbital, and the neurosteroid, 3-hydroxy-5-pregnan-20-one, were able to stimulate GABA binding in both types of membranes. Stimulation differed markedly when both drugs were added jointly to different treated tissue. In frozen membranes drugs acted synergistically and were differentially displaced by picrotoxinin, while in fresh ones, where both compounds were inhibited by the convulsant, this additivity was absent. Post-freezing wash supernatants were collected and used as a source of putative endogenous factors involved in the above mentioned membrane differences. Addition of a high molecular weight fraction from supernatants to frozen synaptic membranes led to an inhibition of barbiturate and neurosteroid potentiation, as well as a loss of their additive effect. Our results indicate that GABA_A receptor modulation by barbiturates and neurosteroids is affected by synaptic membrane treatment, with a common modulatory site in fresh membranes and separate recognition sites after a freeze-thawing procedure. There may also be endogenous factors involved in overlapping of modulatory sites, which would thus regulate GABA_A receptor functionality by direct interaction with the complex.