Effects of ifenprodil on the N-methyl-D-aspartate receptor ionophore complex in rat brain.

The effects of a cerebral anti-ischemic drug ifenprodil on the receptor ionophore complex of an N-methyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors were examined using [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine (MK-801) binding in rat brain synaptic membrane preparations as a biochemical measure. The binding in membrane preparations not extensively washed was markedly inhibited not only by competitive NMDA antagonists such as (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic, D-2-amino-5-phosphonovaleric and D-2-amino-7-phosphonoheptanoic acids, but also by competitive antagonists at the strychnine-insensitive glycine (Gly) site including 7-chlorokynurenic acid and 6,7-dichloroquinoxaline-2,3-dione. Among several proposed ligands for alpha-adrenergic receptors tested, ifenprodil most potently inhibited the binding in these membrane preparations due to a decrease in the density of the binding sites without significantly affecting the affinity. Ifenprodil also inhibited the binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine as well as of [3H]MK-801 to open NMDA channels in a concentration-dependent manner at concentrations above 10 nM in membrane preparations extensively washed but not treated by a detergent, with a Hill coefficient of less than unity. Further treatment of extensively washed membrane preparations with a low concentration of Triton X-100 resulted in an almost complete abolition of [3H]MK-801 binding, and the binding was restored to the level found in membrane preparations not extensively washed following the addition of both L-glutamic acid (Glu) and Gly. Ifenprodil was effective in inhibiting [3H]MK-801 binding via reducing both initial association and dissociation rates in Triton-treated membrane preparations, irrespective of the presence of Glu and Gly added. The binding in Triton-treated membrane preparations was additionally potentiated by the polyamine spermidine in a concentration-dependent manner at concentrations above 10 microM in the presence of both Glu and Gly at maximally effective concentrations. Ifenprodil invariably diminished the abilities of these three stimulants to potentiate [3H]MK-801 binding at concentrations over 1 microM in a manner that the maximal responses each were reduced. These results suggest that ifenprodil does not interfere with the NMDA receptor complex as a specific isosteric antagonist at the polyamine domain in contrast to the prevailing view.

Profiles of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine binding in brain synaptic membranes treated with Triton X-100.

Binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) was examined using rat brain synaptic membranes treated with a low concentration of Triton X-100. This compound is assumed to be a non-competitive antagonist for the N-methyl-D-aspartate(NMDA)-sensitive subclass of central excitatory amino acid receptors. Binding was quite low but detectable in Triton-treated membranes irrespective of the incubation temperature, and the temperature-dependent portion of the binding was greatly reduced in these Triton-treated membranes. However, binding was drastically potentiated by the inclusion of L-glutamate and its analogous amino acids in a concentration-dependent manner at a concentration range of 10 nM to 0.1 mM. Agonists for the NMDA-sensitive subclass also potentiated binding, with agonists for the other subclasses being ineffective. Glycine at a concentration above 10 nM was not only effective as a stimulant of potentiated binding by glutamate, but was also active in enhancing binding in the absence of added glutamate. Glycine increased both the association and dissociation rates without significantly affecting the dissociation constant. Pharmacological profiles of binding in Triton-treated membranes were not significantly different from those in untreated membranes, except for that of haloperidol. Haloperidol is proposed to be highly selective for brain sigma-receptors on the basis of a potent inhibition of sigma-receptor binding. The inhibitory potency of this sigma-ligand was markedly attenuated in the presence of both glutamate and glycine in Triton-treated membranes, as compared with that in untreated membranes. These results suggest that [3H]TCP binding in Triton-treated membranes is a useful biochemical tool to evaluate predominantly the activated state of ion channels associated with the NMDA-sensitive receptors in terms of freedom from the confounding effects of endogenous amino acids.

N-methyl-D-aspartate-sensitive [3H]glutamate binding sites in brain synaptic membranes treated with Triton X-100.

Specific binding activity of radiolabeled L-glutamic acid, a putative central excitatory neutrotransmitter, was drastically increased with increasing concentrations of Triton X-100 used for pretreatment of rat brain synaptic membranes. The binding in these Triton-treated membranes was a protein dependent, inversely temperature-dependent, stereospecific, structure-selective and saturable process with a high affinity for the amino acid. The binding activity was invariably inhibited by agonists and antagonists for the N-methyl-D-aspartic acid (NMDA)-sensitive subclass, but not by agonists for the other subclasses of excitatory amino acid neurotransmitter receptors in the brain. Scatchard analysis revealed that the binding sites consisted of a single component with a Kd of 24.4 +/- 2.5 nM and a Bmax of 0.94 +/- 0.09 pmol/mg protein. Some endogenous tryptophan metabolites such as kynurenic acid and quinolinic acid also inhibited the binding. These results suggest that synaptic membranes may indeed contain the NMDA-sensitive receptors which are disclosed by Triton X-100 treatment.

[Effects of grayanotoxin III on liver function and renal function in rats].

The grayanotoxin III (GTX III) was given intraperitoneally to rats at a dose of 0.8 or 2.8 mg/kg. To study the effects of GTX III on rats, biological tests in serum for functions of liver and kidney and their pathological observation were performed 1 h after the administration. Using analysis of variance, multiple comparison and correlation on biological parameters, activities of glutamic-pyruvic transaminase (GPT), guanase and leucine aminopeptidase and concentrations of total protein, albumin, creatinine, uric acid and K increased significantly. These parameters showed dose-effect relations with GTX III. Though GPT and free fatty acid increased significantly, dose-effect relations were not shown. The activity of choline esterase and the concentrations of bilirubin, urea-N, lipoperoxide, cholesterol, triglycerides, Na and Cl were not significantly different. Pathological changes were not observed in the liver and kidney of rats. These results show that GTX III may affect the functions of liver and kidney in rats.

High concentrations of calmodulin antagonists inhibit accumulation and binding activities of [(3)H]glutamate in rat brain.

Sodium- and energy-dependent accumulation of [(3)H]l-glutamic acid (Glu) into rat cerebral cortical slices was inhibited by relatively high concentrations (40-100 ?M) of calmodulin antagonists, such as N-(6-aminohexyl)-1-naphthalenesulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, in concentration-dependent and noncompetitive manners. The latter antagonist not only diminished the basal binding activity of [(3)H]Glu in synaptic membranes of the rat brain, but also eliminated the activities found in the presence of Cl(?) and Cl(?)/Ca(2+) ions. However, N- methyl- d -aspartate-sensitive [(3)H]Glu binding was not affected by these antagonists. These results suggest the possible involvement of the calmodulin system in glutamatergic neurotransmission in the brain.

The repair of thymine and thymidine bromohydrins, models of damaged nucleic acids, by a copper(II) and ascorbic acid system.

Bromohydrins (1), (3), (4), and (5), damaged products of thymine derivatives, were repaired smoothly to regenerate the original thymine derivatives when exposed to ascorbic acid in the presence of a catalytic amount of Cu2+ in water at room temperature.

Preventive action of quisqualic acid against grayanotoxin-induced suppression of locomotor activity in mice.

Grayanotoxin-III (GTX-III) is a constituent in leaves of Pieris japonica D. Don which exhibits, in vitro, the ability to open voltage-sensitive sodium channels in various excitable tissues. Effects of systemic administration of GTX-III were studied in vivo using Std-ddy mice. Salivation, vomiting and paralysis of the hind paws invariably occurred in mice injected intraperitoneally with 0.1 or 0.25 mg/kg of GTX-III. The writhing response to an intraperitoneal injection of acetic acid was considerably diminished by pretreatment of animals with the toxin. The grayanoid also caused a profound attenuation of the response to caudal compression, while inducing no significant alteration of that to thermal injury. Pretreatment with GTX-III resulted in a significant decrement of the time required for loss of the righting reflex induced by pentobarbital, with a concomitant delay in recovery. Mice injected with the toxin exhibited a significant and restorable suppression of coordination, and a long-lasting suppression of spontaneous locomotor activity in both horizontal and vertical directions. Neither tetrodotoxin (1-5 micrograms/kg, i.p.) nor Ro15-1788 (1-5 mg/kg, i.p.) prevented the GTX-III-induced suppression of locomotion. Atropine (5-10 mg/kg, i.p.) failed to antagonize the GTX-III-induced suppression but protected against salivation induced by the toxin without affecting other symptoms. Intracerebroventricular injection of quisqualic acid (0.5 microgram), one of the agonists for central glutamate receptors, but not that of tetrodotoxin (5 ng) prevented the GTX-III-induced suppression of horizontal movement. These results suggest that GTX-III may elicit its depressant action on horizontal locomotion possibly through interacting with central glutamatergic neurons rather than activating voltage-sensitive sodium channels in the brain. Possible involvement of muscarinic cholinergic neurons in the GTX-III-induced salivation is also suggested.

Tetrodotoxin-insensitive central depression by grayanotoxin-III in mice.

The effect of systemic administration of grayanotoxin (GTX)-III, a constituent in leaves of Pieris japonica D. Don with an ability to activate the voltage-sensitive sodium channels in excitable tissues, on general behaviors of animals was studied using Std-ddy mice. Intraperitoneal administration of the toxin (0.1-0.25 mg/kg b. wt.) resulted in a dose-dependent manner in a significant and reversible muscle relaxation, and a profound and long lasting (greater than or equal to 60 min) depression of locomotor activity. Pretreatment with GTX-III caused a profound potentiation of the duration of loss of righting reflex by pentobarbital with a concomitant delay of the onset of convulsive seizures by various convulsants such as strychnine, picrotoxin and pentetrazol. Neither tetrodotoxin (1-5 micrograms/kg, i.p.) nor Ro15-1788 (1-5 mg/kg, i.p.) prevented the GTX-III-induced suppression of locomotor activity. These results suggest that GTX-III may elicit a central depressant action in mice through a molecular mechanism other than activation of the voltage-sensitive sodium channels in the brain.

Possible interaction of [(3)H]glutamate binding sites with anion channels in rat neural tissues.

The effect of various ions on [(3)H]l-glutamic acid (Glu) binding was examined using crude synaptic membrane preparations from the rat brain. In vitro addition of sodium acetate (1-100 mM) exhibited a significant enhancement of the binding in a concentration dependent manner. Ammonium chloride (20 mM) prevented the potentiation by sodium acetate at 2 degrees C, whereas sodium acetate exerted an inhibitory action on the ammonium chloride-induced augmentation of the binding at 30 degrees C. Ammonium chloride (1-100 mM) itself elicited a temperature dependent stimulation of the binding, which was invariably attenuated by an antagonist for the anion channels such as picrotoxinin (10(?3) M) as well as by inhibitors of anion transport including ethacrynic acid (10(?3) M) and 4,4?-diisothiocyanatostilbene-2,2?-disulfonic acid (10(?4)?10(?3) M), respectively. The later two inhibitors also caused a significant additional raise of the sodium acetate-induced enhancement of the binding. A significant augmentation of the binding resulted from the addition (20 mM) of various anions known to penetrate the anion channels such as bromide, iodide, nitrate, bicarbonate and thiocyanate in a permeability related manner, while that of non-permeable anions including fluoride, sulfate, acetate, formate, phosphate, oxalate, lactate, succinate and tartarate had no such a profound effect on the binding. Addition of d-aspartic acid resulted in the complete abolition of the Na(+)-dependent binding while sparing the Cl(?)-dependent binding. Scatchard analysis revealed that Cl(?) ions induced a two-fold increase in the number of the binding sites without affecting their affinity, whereas Na(+) ions reduced the affinity with a concomitant increase of the number of the binding sites. Addition of quisqualic acid (10(?5)?10(?3) M) inhibited the Cl(?)-dependent binding of [(3)H]Glu to a significantly greater extent than the inhibition on Na(+)-dependent binding. N- Methyl- d -aspartic acid and kainic acid exerted no preventive action on the basal, Cl(?)-dependent and Na(+)-dependent binding. respectively. The highest basal binding activity was found in the retina among various central structures examined. A significant basal binding activity of [(3)H]Glu was also detected in the pituitary and adrenal but not in the kidney. Chloride ions exhibited a significant facilitation of [(3)H]Glu binding to central regions without altering that to peripheral tissues such as pituitary and adrenal. In contrast, Na(+) ions induced significant attenuation of the binding to the pituitary, adrenal and retina despite the occurrence of augmentation of the binding to other central structures. These results suggest the Glu binding sites may be linked to the anion channels in the rat central nervous system and that this linkage may be absent from the pituitary, adrenal and retina.