The "antidementia drug" pantoyl-gamma-aminobutyric acid increases high affinity uptake of choline by slices of rat brain.

Studies were made on the effects of an "antidementia drug", pantoyl-gamma-aminobutyric acid (pantoyl-GABA), on high affinity uptake of choline by slices of rat brain. Depolarization caused by increasing the K+ concentration to 25 mM for 30 min before incubation with [3H]choline enhanced the uptake of radioactivity by slices of cerebral cortex, hippocampus and striatum during a 5 min incubation in the presence of 1 microM [3H]choline. Pantoyl-GABA (1 mM) increased the depolarization-induced uptake of radioactivity by the slices of cortex and hippocampus, but not by the slices of striatum; it had little effect on the uptake when the slices were not depolarized. It also had no effect when the slices were depolarized in the incubation medium without Ca2+. Since the high affinity uptake of choline is considered to be a regulatory step in the synthesis of acetylcholine (ACh), these results suggest that pantoyl-GABA increases the synthesis of ACh in cholinergic terminals in the cerebral cortex and hippocampus. This action may be involved in its effect as an antidementia drug, because cholinergic deficits are assumed to occur in Alzheimer's disease.

Effect of the 'antidementia drug' pantoyl-GABA on high affinity transport of choline and on the contents of choline and acetylcholine in rat brain.

1. Effect of pantoyl-gamma-aminobutyric acid (pantoyl-GABA) on high affinity transport of choline into synaptosomes and on the choline (Ch) and acetylcholine (ACh) concentrations of rat brain were studied. 2. Pantoyl-GABA was injected intraperitoneally four times at a dose of 500 mg kg-1 at intervals of 30 min. One hour after the last injection, rats were killed by decapitation for measurement of high affinity transport of Ch into synaptosomes or by microwave irradiation for the measurement of Ch and ACh concentrations. 3. Transport of Ch was increased into synaptosomes prepared from the cerebral cortex and hippocampus, but not into those from the striatum. 4. In the cerebral cortex and hippocampus, Ch concentration was increased and ACh concentration decreased. 5. Since treatments that enhance the activity of cholinergic neurones in vivo are reported to increase high affinity transport of Ch measured in vitro, the present results suggest that pantoyl-GABA may increase cholinergic activity in vivo. This action of the drug may be related to changes in the Ch and ACh concentrations.

Endogenous inhibitor of GABA(B) and GABA(A) receptors.

An endogenous inhibitor of ?-aminobutyric acid (GABA) receptors was partially purified from bovine brain striatum. It was obtained as a low molecular weight fraction by gel filtration on Biogel P-2 and was adsorbed to Dowex AG 50W-X8, but not to Dowex AG 1-X8. It was ninhydrin-negative, basic, heat-stable substance. It caused dose-dependent inhibition of Na(+)-independent [(3)H]GABA bindings. Scatchard plot analysis of the [(3)H]GABA binding to GABA "B" receptor recognition site showed this inhibitor increased the K(d) value (24.1 nM to 3.6 nM) without changing the B(max). On the other hand, Scatchard plot analysis of the [(3)H]GABA binding to GABA "A" receptor recognition site showed that the inhibitor decreased number of binding sites (706 fmol/mg protein to 494 fmol/mg protein) without affecting the K(d) value. These results suggest that the endogenous inhibitor functions as a modulator for GABA(B) and GABA(A) receptors.

Selective enhancement in striatal [(3)H]nitrendipine binding following chronic treatment with morphine.

Two parameters of Ca(2+) dynamics in brain preparations ((45)Ca-uptake to slices and [(3)H]nitrendipine binding to membrane fractions) were measured in naive and chronic morphine-administered rats. While morphine did not have any effect on (45)Ca-uptake to striatal slices in normal Krebs-Ringer solution, it inhibited K(+)-stimulated (45)Ca-uptake to slices. Furthermore, the effect of morphine was antagonized by naloxone. Inhibition of K(+)-stimulated (45)Ca-uptake to striatal slices by morphine was not observed in preparations obtained from chronic morphine-administered rats (6 mg/kg/b.i.d./7 days). In membrane fractions, [(3)H]nitrendipine binding increased by 34% in striatum following chronic morphine treatment, whereas no change was observed in the cortex and hippocampus. The results will be discussed in relation to the phenomena underlying chronic morphine administration.

Endogenous modulator of dopamine receptor(s).

An endogenous modulator(s) of the dopamine receptor(s) in bovine and rat brain striatum was detected by demonstrating that water extracts of the striatum inhibited [(3)H]apomorphine binding. This modulator(s) was partially purified by methanol extraction and then successive ion exchange chromatographies on SP-Sephadex C-25 and QAE-Sephadex A-25, and gel chromatography on Sephadex G-25. The partially purified (about 1,500-fold) modulator was a fluorescamine-positive substance, Mr = 500 1000, which was heat-stable (95 degrees C, 10 min), and was destroyed by acid- and alkali-treatment, but not by treatments with various peptidases. The modulator inhibited binding of the dopamine agonist, [(3)H]apomorphine non-competitively, but did not inhibit binding of the dopamine antagonist, [(3)H]spiroperidol. Direct injection of the modulator into rat brain striatum depressed apomorphine-induced locomotor activity. Moreover the modulator inhibited dopamine-sensitive adenylate cyclase activity. These findings indicate that the modulator acts at a site(s) other than the ligand binding site of the dopamine receptor(s) and modulates the activities of dopamine agonists.

Effects of morphine and (D-Ala, Met)-enkephalinamide on 45Ca-uptake to rat brain striatal slices.

Effects of morphine and (D-Ala, Met)-enkephalinamide on 45Ca-uptake to rat striatal slices were examined. While morphine didn't give any effect on 45Ca-uptake to striatal slices in normal Krebs-Ringer solution, it inhibited K+ (40 mM)-stimulated 45Ca-uptake to slices. In high K+ medium, 45Ca-uptake was enhanced to 170% and this was reduced to 147 and 137% by 10(-5) and 10(-4) M morphine, respectively. Furthermore, the effect of morphine was antagonized by 10(-5) M naloxone. (D-Ala, Met)-enkephalinamide (10(-4) M) also inhibited K+-stimulated 45Ca-uptake to the slices to the same extent as was observed by morphine. K+-stimulated 45Ca-uptake to striatal slices obtained from chronically morphine administered rat (6 mg/kg, twice/day, 7 days) was not inhibited by morphine.

Mercury modulation of GABA-activated chloride channels and non-specific cation channels in rat dorsal root ganglion neurons.

The effects of mercuric chloride and methylmercury chloride on the rat dorsal root ganglion neurons in primary culture were studied by the whole-cell patch clamp technique. gamma-Aminobutyric acid-induced chloride currents were augmented by mercuric chloride in a potent and efficacious manner; at concentrations of 1 and 10 microM, the current amplitude was increased to 130% and 200% of the control. Methylmercury even at 100 microM did not augment but rather decreased the GABA-induced chloride current. Both mercuric chloride and methylmercury generated slow inward currents by themselves. These currents are not mediated by the GABA-activated chloride channels or by voltage-activated sodium, potassium or calcium channels, and are likely to be due to non-specific cation channels.

Chloride current induced by alcohols in rat dorsal root ganglion neurons.

We have recently demonstrated that ethanol and longer-chain alcohols (n-alcohols) enhance gamma-aminobutyric acid (GABA)-induced chloride currents before desensitization takes place. The potencies of n-alcohols increase with lengthening of the carbon chain. We now report that n-alcohols induce chloride currents by themselves in rat dorsal root ganglion neurons in primary culture. The whole cell variation of the patch clamp techniques was used to record currents as induced by external application of alcohols and other test compounds. Ethanol, n-butanol, n-hexanol and n-octanol induced inward currents with their potencies increasing in that order. The potencies were approximately one order of magnitude less than those to augment GABA-induced currents. The maximum amplitudes of currents induced by the alcohols were less than those produced by GABA. The n-octanol-induced currents were carried largely by chloride ions because the reversal potentials were changed according to the Nernst chloride potential as the internal chloride concentration was changed. Bicuculline and picrotoxin suppressed the n-octanol-induced current, and chlordiazepoxide and pentobarbital augmented the n-octanol-induced current. Therefore, the alcohol-induced chloride currents flow through the chloride channels associated with the GABAA receptors. When applied after the GABA-induced current was desensitized to a lower level, n-octanol suppressed rather than augmented the current. Thus, n-alcohols mimic barbiturates in augmenting the GABA-induced currents and in generating chloride currents by themselves. These actions of both agents may play a role in causing anxiolytic, sedative and/or anesthetic effects.

Effects of pertussis toxin on D2-dopamine receptor in rat striatum: evidence for coupling of Ni regulatory protein with D2-receptor.

It is well established that a pertussis toxin, islet activating protein (IAP), interacts directly with the Ni regulatory protein involved in the receptor-adenylate cyclase system. In this study we investigated the effect of the toxin on the dopaminergic function of the central nervous system in conjunction with the adenylate cyclase system. Direct bilateral microinjection of the toxin into rat striatum reduced the stereotyped behavior induced by apomorphine. This inhibitory effect was observed even 40 h after administration of the toxin, whereas the inhibition by haloperidol disappeared within 15 h. Toxin administration did not influence either the Bmax or affinity of specific binding of [3H]spiroperidol to the striatal membrane. However, it increased the IC50 value of apomorphine for the specific binding of [3H]spiroperidol. GTP (10(-4) M) had little effect on the apparent affinity of apomorphine to the [3H]spiroperidol binding sites in IAP-treated membrane, though an effect was clearly observed in untreated membrane. [3H]Spiroperidol binding sites were increased 34 +/- 8% (n = 4) on chronic treatment of haloperidol for 2 weeks. On the contrary, on long-term administration of IAP the ligand binding sites were decreased by 25 +/- 10% (n = 4). These results indicate that pertussis toxin can interact with the Ni protein coupled with striatal D2-dopamine receptor. Inhibition of the coupling between Ni protein and the D2-dopamine receptor attenuated the manifestation of rat stereotyped behavior. Furthermore, chronic administration of dopamine antagonist resulted in the up-regulation of the D2-dopamine receptor, while long-term inhibition of coupling between the D2-dopamine receptor and Ni regulatory protein reduced the amount of receptors.

Blockade of cholinergic receptors by an irreversible antagonist, propylbenzilylcholine mustard (PrBCM), in the rat cerebral cortex causes deficits in passive avoidance learning.

Studies were made on the effects of blockade of muscarinic acetylcholine (mACh) receptors in the rat cerebral cortex on learning and memory assessed by performance of a step-through passive avoidance task. Bilateral injection of propylbenzilylcholine mustard (PrBCM) into both the frontal and parietal cortex at doses of 2.25 X 4 to 22.5 X 4 micrograms decreased mACh receptors dose-dependently, as assessed by [3H]quinuclidinyl benzilate binding studies. When the training trial of a step-through passive avoidance task was performed 24 h after injection of 7.5 X 4 to 22.5 X 4 micrograms PrBCM into the frontal and parietal cortex, and then a retention test was made 24 h after the training trial, the treated rats showed shorter latencies than controls. In contrast, injection of PrBCM into the occipital cortex had no significant effect on performance in the test. These results confirm the notion that cholinergic neurotransmission in the cerebral cortex, especially the frontoparietal cortex, is important in learning and memory. The effects of injection of PrBCM (22.5 X 4 micrograms) into the frontoparietal cortex on 3 postulated phases of the learning and memory process (i.e. registration, retention and recall) were also examined. When PrBCM was injected 24 h before the training trial, no retention of the task was observed 14 days after the training trial. However, when PrBCM was injected 24 h after the training trial, retention of the task 14 days after the training trial was not affected. When PrBCM was injected 3-24 h after the initial training trial, the latencies in the retention test examined 24 h later were shorter than those of control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

D-2 dopamine receptor synthesis and turnover in rat striatum.

Direct injection of phenoxybenzamine into rat striatum inhibited apomorphine-induced stereotyped behavior. This inhibition corresponded well with the inhibition of D-2 dopamine receptor labelling with [3H]spiroperidol. Both the behavioral response and the receptor level were completely restored within 5 days after the injection. The recoveries of both were blocked by cycloheximide. The rate of synthesis and half-life of the D-2 receptor associated with the stereotyped behavior were calculated to be 6.9 fmol/mg protein per h and 28 h, respectively.

Potentiation of GABA-induced Cl- current by a series of n-alcohols disappears at a cutoff point of a longer-chain n-alcohol in rat dorsal root ganglion neurons.

We studied the effects of n-alcohols on gamma-aminobutyric acid (GABA)-induced Cl- current of rat dorsal root ganglion neurons in primary culture by a whole-cell, patch-clamp technique. n-Alcohols (C1-C11) at the concentrations inducing anesthesia in whole animals enhanced the current evoked by GABA application to the neurons. Their potencies for current enhancement increased with their carbon chain length, leveled off for higher alcohols and completely disappeared at C12. The potency of the alcohols for current enhancement correlated well with their anesthetic potencies.

Antagonistic effect of delta-aminovaleric acid on bicuculline-insensitive gamma-aminobutyric acid B (GABA B) sites in the rat's brain.

The effect of delta-aminovaleric acid (delta-AV) on bicuculline-insensitive gamma-aminobutyric acid B (GABA B) sites in the central nervous system (CNS) was investigated by binding studies and experiments on slices in vitro. delta-AV inhibited [3H]GABA (10 nM) binding to GABA B sites in a rat brain membrane preparation with an IC50 value of 10(-4) M. It also inhibited [3H]baclofen (20 nM) binding with an IC50 value of 10(-4) M. In preparations of hippocampal slices, (-)-baclofen (5 microM) reduced the population spikes evoked by stimulating the Schaffer collaterals in CA1 pyramidal cells in the presence of 100 microM bicuculline. delta-AV (1 mM) antagonized this inhibitory action of baclofen. Since baclofen is an agonist of GABAB sites, our results indicate that delta-AV has an antagonistic effect on GABAB sites in the CNS.

Dimethyl sulfoxide (DMSO) blocks GABA-induced current in rat dorsal root ganglion neurons.

The effects of dimethyl sulfoxide (DMSO) on gamma-aminobutyric acid (GABA)-induced Cl- currents of rat dorsal root ganglion neurons in primary culture were studied by the whole-cell patch-clamp technique. Bath application of 5 microM GABA evoked sustained inward and outward currents at membrane potentials of -60 mV and +30 mV, respectively, when the external and internal solutions both contained 142 mM Cl-. DMSO at concentrations of 0.3-3% (v/v) caused a dose-dependent inhibition of the inward current induced by 5 microM GABA at -60 mV. DMSO at 3% inhibited the GABA-induced inward and outward currents at -60 mV and +30 mV to similar extents (54% and 53% of the control, respectively), but the time-course of inhibition of the outward current at +30 mV was much faster than that of the inward current at -60 mV. These results suggest that DMSO suppressed the currents by interacting with GABA receptor-Cl- channel complex protein rather than by affecting the lipid-bilayer of the cell membrane.

Blockade of ACh receptors by PrBCM causes deficits in shuttle avoidance performance.

Studies were made examining the effect of blockade of muscarinic acetylcholine (mACh) receptors in the cerebral cortex of rats on their shuttle avoidance after training. Rats were given a session of shuttle avoidance tests once a day for 12 days. Then the irreversible antagonist of mACh receptors, propylbenzilylcholine mustard (PrBCM), was injected bilaterally into the cerebral cortex of rats showing avoidance rates of more than 75% in the last session, and avoidance rates were examined 24 hr later. The avoidance rates of the rats treated with 100 micrograms PrBCM were lower than those in the last session before treatment. The amount of mACh receptors in the cerebral cortex was decreased by PrBCM treatment, as shown by [3H]quinuclidinyl benzilate (QNB) binding studies performed just after measurement of the avoidance response. The present study indicates that cholinergic neurotransmission in rat cerebral cortex is involved in performing a learned shuttle avoidance.

Modulation of gamma-aminobutyric acid receptor-channel complex by alcohols.

The electrophysiological effects of ethanol on the gamma-aminobutyric acid (GABA) system have been a matter of controversies; some observed an enhancement of GABA response whereas others failed to see the effect. Acute effects of n-alcohols (ethanol, n-butanol, n-hexanol and n-octanol) on chloride current activated by bath application of GABA were studied with the rat dorsal root ganglion neurons maintained in primary culture. The whole-cell patch-clamp technique was used to record the current. Ethanol (30-300 mM), n-butanol (1-30 mM), n-hexanol (30-1000 microM) and n-octanol (3-100 microM) enhanced the initial peak current evoked by 30 microM GABA in a dose-dependent manner. The potency of alcohols increased with the chain length and was well correlated with the membrane/buffer partition coefficient. The observed low potency of ethanol is also predicted from this correlation. The dose-response curve for the GABA-induced nondesensitized current was sigmoidal with an apparent dissociation constant of 55 microM and a Hill coefficient of 1.5 to 1.9. Ethanol (300 mM) and n-octanol (100 microM) shifted the dose-response curve in the direction of low concentration without greatly changing the Hill coefficient. Ethanol (300 mM) and n-octanol (30 and 100 microM) shortened the decay time course of the current induced by 30 microM GABA to 66, 74 and 52% of control, respectively. Unlike the peak nondesensitized current, the desensitized steady-state current induced by high concentration (300 microM) of GABA was suppressed by long-chain alcohols.(ABSTRACT TRUNCATED AT 250 WORDS)

General anesthetics modulate GABA receptor channel complex in rat dorsal root ganglion neurons.

The effects of halothane, isoflurane, and enflurane on ionic currents induced by bath application of gamma-amino-butyric acid (GABA) were studied with the rat dorsal root ganglion neurons maintained in primary culture. The whole-cell patch clamp technique was used to record the current. In normal neurons before exposure to anesthetics, GABA at low concentrations (1-3 x 10(-6) M) induced a small sustained inward current. At higher concentrations (3 x 10(-5) M-1 x 10(-3) M), GABA induced a large inward current, which decayed to a steady-state level (desensitization). Halothane (0.86 mM), isoflurane (0.96 mM), and enflurane (1.89 mM), each equivalent to the respective 2 minimum alveolar concentration (MAC) units, augmented the sustained current evoked by 3 x 10(-6) M GABA to 330-350% of control and the peak current evoked by 3 x 10(-5) M of GABA to 136-145% of control. The decay phase of the current was accelerated by the anesthetics, the time for the current to decline to 70% of the peak being reduced to 23-39% of control. In contrast, the densitized steady-state current evoked by high concentrations of GABA was decreased by anesthetics. In conclusion, general anesthetics exert a dual effect on the GABA receptor channel complex: to potentiate the nondesensitized (both peak and sustained) current and to suppress the desensitized steady-state current. The potentiation of the GABA receptor channel response may be a primary action of anesthetics leading to surgical anesthesia.

The "antidementia drug", pantoyl-GABA, increases choline acetyltransferase activity in mouse brain.

Studies were made on the effects of in vivo administration of an "anti-dementia drug", pantoyl-GABA, on choline acetyltransferase (ChAT) activity in mouse brain. Male ICR mice were treated intraperitoneally with 500 mg/kg of pantoyl-GABA, once a day, for various periods. Then they were killed, and the ChAT activities of their cerebral cortex, hippocampus and striatum were measured. Results showed that ChAT activity increased significantly in the cerebral cortex and hippocampus from day 5 and 7, respectively, of treatment, but did not increase in the striatum even after treatment for 14 days. The kinetics of ChAT activity were investigated in tissues from the cerebral cortex and hippocampus of mice treated with the drug for 7 days. Drug-treatment increased the Vmax value, but did not affect the Km values for choline and acetyl-CoA. These results indicate that long-term treatment with pantoyl-GABA enhanced ChAT activity, without altering the affinity of the enzyme for either substrate.