R56865 and flunarizine as Na(+)-channel blockers in isolated Purkinje neurons of rat cerebellum.

Dose-related blocking effects of R56865, flunarizine and nimodipine on voltage-activated Na+ currents recorded in the whole-cell voltage clamp mode were studied in acutely isolated Purkinje neurons of rat cerebellum. The dose-dependences of blocking action were obtained for all drugs at a holding potential of -110 mV and rare stimulation. At stimulation frequencies 5 and 15 Hz the block produced by R56865 was increased showing a shift of dose-dependence to lower concentrations of antagonist. This shift was less pronounced for flunarizine, practically absent for nimodipine, and increased for all drugs with an increase in the amplitude of stimulating voltage pulse. With the change in holding potential to -80 mV the block produced by R56865 and flunarizine increased showing a dose-dependence shift to lower concentrations of antagonists. All the drugs tested induced parallel shifts of the steady-state voltage-dependence of inactivation of Na+ channels to more negative membrane potentials. R56865, and to a lesser extent flunarizine, slowed down the recovery of Na+ channels from steady-state inactivation increasing the relative number of channels which showed slow recovery. In the absence of Na+ current inactivation (treatment by intracellular pronase) R56865 at a concentration of 1 microM blocked modified channels preferentially in the open state, while the block produced by flunarizine showed no dependence on voltage pulse protocol. R56865 was shown to decrease the cell leakage while other drugs produced little or no effect. It is concluded that R56865 and flunarizine block Na+ currents predominantly by interacting with inactivated Na+ channels. The higher ability of R56865 to block open channels and to increase slow inactivation underlies its higher frequency-dependence. These characteristics suggest the use of R56865 and flunarizine in the treatment of cerebral ischemia.

A highly potent and selective N-methyl-D-aspartate receptor antagonist from the venom of the Agelenopsis aperta spider.

Agatoxin-489, extracted from the venom of the Agelenopsis aperta spider, was studied on acutely isolated perfused hippocampal neurons of rat using the concentration clamp technique. Agatoxin-489 proved to be a selective N-methyl-D-aspartate antagonist; responses to applications of N-methyl-D-aspartate or L-aspartate were blocked by concentrations of agatoxin-489 ranging between 0.1 nM and 1 microM, while responses to kainate were not affected by agatoxin-489 at concentrations up to 10 microM. The actions of agatoxin-489 against responses to N-methyl-D-aspartate or L-aspartate were use- and voltage-dependent, being less pronounced with an increase in the holding potential from -100 to -30 mV. The action of agatoxin-489 could be completely or partially reversed only after washout in the presence of an N-methyl-D-aspartate agonist. The washout was more effective at positive membrane potentials ranging from 0 to +20 mV. These results imply that the spider toxin agatoxin-489, like dizocilpine, is a potent and selective N-methyl-D-aspartate antagonist which preferentially interacts with activated N-methyl-D-aspartate receptors and/or open N-methyl-D-aspartate-activated ionic channels.

Two types of steady-state desensitization of N-methyl-D-aspartate receptor in isolated hippocampal neurones of rat.

1. Whole-cell voltage-clamp recordings were made from rat isolated hippocampal neurones. Aspartate (Asp) and/or glycine (Gly) were applied by a method in which the external solution could be changed within 30 ms and thereafter held constant. 2. Asp and Gly applied together at maximal concentrations (5 mM and 10 microM, respectively) evoked an inward current due to activation of N-methyl-D-aspartate (NMDA) receptors. The current peaked and then declined to a steady state during the application. The time constant of desensitization (tau) was about 1 s when the agonists were applied soon after the onset of whole-cell recording. The desensitization became more rapid (tau = 0.3 s) and more complete during the first 15 min of recording, and thereafter remained stable; the amplitude of the peak response did not change throughout. In solutions containing 10 microM-Gly, Asp had an apparent Kd of 51 microM at the peak of response and 20 microM measured at the steady state. The steady-state current was 14% of the peak current. 3. Asp was applied after a conditioning exposure of the cell of Gly (from 1 to 50 microM), together with the same Gly concentration. The maximum current evoked by the application of Asp was increased while increasing Gly in the conditioning solution, with no change in the apparent Kd for Asp at the peak of Asp-activated response. 4. Various concentrations of Asp (plus 10 microM-Gly) were applied after a conditioning exposure to Asp (which alone was without effect). The maximum current induced by Asp applications was only 28% of that observed without conditioning Asp application, but the apparent Kd was unchanged (about 57 microM). 5. Test solution containing maximal concentrations of Asp and Gly was applied after conditioning exposure to both Asp (varying concentrations) and Gly (10 microM). Complete desensitization was caused by 200 microM-Asp. The apparent Kd for Asp to induce desensitization (8.7 microM) was less than the Kd as an agonist (51 microM). 6. Test solution containing maximal concentrations of Asp and Gly was applied after conditioning exposure to both Gly (varying concentrations) and Asp (5 mM). Complete desensitization was caused by 1 microM-Gly. The apparent Kd for Gly to induce desensitization (120 nM) was less than the Kd as a co-agonist (about 1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

A novel selective NMDA agonist, N-phthalamoyl-L-glutamic acid (PhGA).

Ionic currents elicited by N-phthalamoyl-L-glutamic acid (PhGA) were investigated on freshly isolated hippocampal neurons with the whole-cell voltage clamp and concentration clamp techniques. PhGA elicited desensitizing inward currents in Mg(2+)-free salines only in the presence of glycine. The dose-response relationship for PhGA was close to a Langmuir isotherm with Kd = 3.7 mM and saturating level 0.75 of that for L-aspartate (L-Asp). PhGA-activated currents were blocked by Mg2+, D-2-amino-5-phosphonovalerate and kynurenate, and had the same reversal potential as L-Asp-activated currents. Complete cross-desensitization was obtained between the responses to PhGA and L-Asp. We conclude that PhGA is a new selective 'superacidic' agonist of the N-methyl-D-aspartate type of glutamate receptor.

Cross-desensitization Reveals Pharmacological Specificity of Excitatory Amino Acid Receptors in Isolated Hippocampal Neurons.

Ionic currents elicited by excitatory amino acids were studied, using the concentration clamp method, in enzymatically isolated rat hippocampal neurons. Cross-desensitization between the responses to various agonists was applied to separate the activity of two types of receptors, N-methyl-d-aspartate (NMDA) and non-NMDA. NMDA receptors were selectively activated by NMDA, l- and d-aspartate, d-glutamate and quinolinate. Kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate appeared to be selective, and quisqualate relatively less selective non-NMDA agonists, acting on the same receptor type. l-Glutamate, l- and d-homocysteate activated both receptor types. It is supposed that two receptor sites, activation site and desensitization site, control the action of agonists at the non-NMDA receptor. When examined in the cross-desensitization experiments, NMDA and non-NMDA receptors appear to be represented by the two homogeneous and independent receptor populations operating different ionic channels.

Desensitization of NMDA receptors does not proceed in the presence of kynurenate.

The agonist-induced steady-state desensitization of the N-methyl-D-aspartate (NMDA) receptor was investigated by means of whole cell patch and concentration clamp in isolated pyramidal neurons from rat hippocampus. When administered against a background of previously applied agonist, glycine (Gly) produced a response that was smaller than the response elicited by simultaneous application of agonist and Gly. This feature could be explained by an agonist-induced suppression of NMDA receptor sensitivity to the facilitatory action of Gly. This type of 'steady-state desensitization' did not develop when the preincubating solutions contained kynurenate (250 microM). It is proposed that NMDA receptors at all concentrations of agonist may acquire two distinct and interconverting conformations that are sensitive or insensitive to the facilitatory action of Gly. Kynurenate shifts the equilibrium between these states keeping the receptor in a Gly-sensitive conformation.

Glycine action on N-methyl-D-aspartate receptors in rat hippocampal neurons.

The modulatory effect of glycine and other amino acids on the activity of N-methyl-D-aspartate (NMDA) receptors has been investigated using a concentration clamp method in the acutely isolated pyramidal neurons from rat hippocampus. The dose-response curves for L-aspartate, measured on the background of several glycine concentrations, were close to Langmuir isotherms with Kd values practically independent of the concentration of glycine. The facilitatory action of glycine appeared at concentrations below 0.1 microM and saturated between 10 and 100 microM. This effect demonstrated marked densensitization, at least at concentrations higher than 5 microM. Facilitation of the responses was shared by amino acids with the potencies diminishing as follows: glycine, D-serine, D-alanine greater than L-proline, D-proline, hydroxy-L-proline, taurine greater than L-alanine, L-serine. This sequence did not correlate with the ability of these amino acids to activate strychnine-sensitive glycine receptors.

[Argiopine, argiopinines and pseudoargiopinines--blockers of the glutamate receptors in hippocampal neurons]. / Argiopin, argiopininy i psevdoargiopininy--blokatory glutamatnykh retseptorov v neironakh gippokampa.

Several homologous low-molecular weight compounds were purified from venom of spider Argiope lobata. They selectively blocked ionic currents elicited by glutamate and its agonist kainate in the membrane of isolated hippocampal neurons of rat. Three groups of these compounds--argiopine, argiopinines and pseudoargiopinines, blocked glutamate- and kainate-activated ionic currents in a voltage-dependent manner, acting preferentially on agonist-activated ionic channels. These compounds did not change the Kd values for both agonists. The blocking action was partially reversible for argiopine and poorly reversible for other compounds. Rate constants for the interaction of toxins with membrane receptors were estimated from the dependences of two-component kinetics of the blocking action and its recovery on toxin concentrations. Argiopine, argiopinines and pseudoargiopinines may be useful tools for further electrophysiological and biochemical investigation of the mammalian CNS glutamate receptors.

[Blocking action of Nephila clavata spider toxin on ionic currents activated by glutamate and its agonists in isolated hippocampal neurons]. / Blokiruiushchee deistvie iada pauka Nephila clavata na ionnye toki, aktiviruemye glutamatom i ego agonistami v izolirovannykh neironakh gippokampa.

The blocking action of the Nephila clavata spider neurotoxin was studied using the concentration clamp method in isolated neurons of the rat hippocampus. Crude venom JSTX blocked L-glutamate-, quisqualate- and kainate-activated ionic currents mediated by activation of the non-N-methyl-D-aspartate (non-NMDA) membrane receptors. Ionic currents elicited by all agonists were depressed by crude JSTX venom to 34-35% of its initial amplitude with no recovery during prolonged washing. An active fraction of JSTX venom blocked ionic currents almost completely, but its action was partially reversible. The concentration dependences of blocking kinetics allowed determining the rate constants of JSTX interaction with glutamate receptors. It is supposed that JSTX blocks the non-NMDA ionic channels in some of their open states and may be one of useful tools in further biochemical and electrophysiological characterization of the glutamate-mediated synaptic transmission.

Spider toxin blocks excitatory amino acid responses in isolated hippocampal pyramidal neurons.

Using the 'concentration-clamp' technique we have investigated the action of Joro spider toxin (JSTX), as a specific blocker of glutamate receptor, on freshly isolated rat hippocampal pyramidal neurons. The neurons showed prominent responses to L-glutamate (L-Glu), quisqualate (QA) and kainate (KA) and JSTX blocked completely the both responses. The blocking action of toxin was dose-dependent at the concentrations of toxin between 4.8 X 10(-12) and 4.8 X 10(-8) M, and was remarkably similar in cell to cell trials. The kinetics of blockade was revealed by applying the toxin to the non-desensitizing KA response. We suggest that the JSTX spider toxin may be a valuable tool in ligand binding studies of QA/KA receptors in the central nervous system.

Are sulfhydryl groups essential for function of the glutamate-operated receptor-ionophore complex?

We monitored glutamate- or kainic acid-induced inward currents in pyramidal neurons of the rat hippocampus to determine a possible chemical modification of sulfhydryl residues on glutamate-receptor-ionophore complexes. Organic and inorganic mercury compounds such as HgCl2 and n-chloromercuribenzoate, 5,5'-dithiobis (2-nitrobenzoic acid) and N-ethylmaleimide depressed glutamate- or kainate-induced responses while the mercury compounds inhibited the kainate response, in a non-competitive manner. The responses inhibited by these agents were dose-dependently recovered by adding cysteine or glutathione. These observations suggest that the excitatory glutamate-receptor-ionophore complex includes sulfhydryl residues, components which may play a role in related functions.

Excitatory amino acid receptors in hippocampal neurons: kainate fails to desensitize them.

The excitatory responses to L-glutamate (L-Glu), quisqualate (QA) and kainate (KA) have been investigated in isolated pyramidal cells from rat hippocampus using intracellular perfusion and concentration clamp techniques. The responses to L-Glu and QA demonstrated rapid desensitization and complete cross-desensitization, while KA produced a non-desensitizing response. The activation of KA response was determined by the level of desensitization induced by L-Glu or QA pretreatment. It is concluded that QA, KA and L-Glu activate the same excitatory receptors with apparent Kd values of 9.3 X 10(-5) M, 5.0 X 10(-4) M and 1.1 X 10(-3) M, respectively.