P. Placheta

In vitro modulations by SQ 20009 and SQ 65396 of GABA receptor binding in rat CNS membranes

SQ 20009 and SQ 65396, two new pyrazolopyridines with anxiolytic properties in animals, reversibly increased Na+-independent 3H-GABA binding to rat cerabral cortex membranes in vitro. This modulation was partially chloride dependent and sensitive to Triton-X 100. Kinetic analysis of 3H-muscimol binding revealed an increase of the apparent number of binding sites. The results are compatible with a functional association between Na+-independent GABA binding sites and the GABA/benzodiazepine receptor complex.

Reduction of [3H]muscimol binding sites in rat dorsal spinal cord after neonatal capsaicin treatment

Two-day-old rats were pretreated with 50 mg/kg of capsaicin. After 3--4 months, specific binding of [3H]muscimol and [3H]strychnine was measured in membrane preparations from dorsal spinal cord. A 20-30% decrease of the number of [3H]muscimol binding sites was observed after capsaicin treatment. In contrast, [3H]strychnine binding was unchanged. The results provide indirect evidence for a presynaptic location of GABA receptors on capsaicin-sensitive primary afferent neurons.

Regional distribution of Na+-independent GABA and benzodiazepine binding sites in rat CNS.

Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the mammalian central nervous system (CNS). In various pharmacological and neurophysiological experiments benzodiazepines (BZs) have been found to facilitate GABAergic synaptic transmission s. Recent biochemical studies provide evidence for a close functional interrelationship of the GABA system and BZ receptors 3. It has been reported that BZs displace an endogenous modulator protein which may inhibit the high affinity binding site for GABA in synaptic membrane preparations 3. Furthermore, studies from several laboratories 7-9 have revealed that BZ receptor binding 2 can be stimulated by GABA and several GABA analogues. These observations suggest that GABA and BZ receptors are functionally or anatomically linked together. In order to study whether such an association between BZ and GABA receptors is mandatory we performed a kinetic analysis of the regional distribution of these two receptors in the rat brain and spinal cord. Total membrane fractions were prepared from 8 regions of the rat CNS. Tissues from adult, Sprague-Dawley rats were dissected on ice, pooled, homogenized with an Ultra-Turrax for 30 sec in 25 vol. of 100 mM K-phosphate buffer (pH 7.2) and centrifuged for 10 min at 48,000 × g. The pellets were washed twice by resuspension and recentrifugation in the same vol. of buffer and stored frozen at -20 °C for at least t 8 h. After thawing, aliquots of the membrane suspensions were pelleted and resuspended in 60 vol. of 50 mM Tris-citrate buffer (pH 7.1) for the determination of [3H]flunitrazepam binding. The remaining membrane fractions were resuspended and incubated for 30 rain at 37 °C in 25 vol. of 100 mM potassium phosphate buffer (pH 7.2) containing 0.05 ~ (v/v) Triton X-100, washed 3 times by resuspension and recentrifugation in Tris.citrate buffer, and stored at -20 °C until use for assay of Na ~independent GABA binding. Na÷-independent [3H]GABA binding was determined using a centrifugation assay 4 with minor modifications. Membrane fractions derived from 5 mg of tissue wet

Effect of azepexole (B-HT 933) on pre- and postsynaptic α-adrenoceptors at peripheral and central nervous sites☆

Abstract Azepexole (B-HT 933, 2-amino-6-ethyl-5,6,7,8-tetrahydro-4H-oxazolo-[4,5-d]-azepine dihydrochloride) is chemically different from clonidine but has been characterized pharmacologically as a typical clonidine-like drug. In this study the stimulating effect on presynaptic α-adrenoceptors was shown in pithed rats by a decrease of the tachycardia produced by electrical stimulation within the spinal canal (0.2 Hz; 50 V, 2 msec; 25 sec). The 50% inhibitory dose (D 50 ) was 236 μg/kg i.v. (clonidine: 4.9 μg/kg). Compared with the effect on postsyanptic α-adrenoceptors in spinal rats (blood pressure increase 30 mm Hg, D 30 ) this resulted in a pre/postsynaptic activity ratio D 30 /D 50 = 2.24 (clonidine 1.9; methoxamine 0.13). The question of wether this relatively high presynaptic activity was responsible for the central nervous sympathoinhibition and vagally mediated baroreceptor reflex facilitation was tested in noradrenaline-depleted cats and dogs. Cats were pretreated with reserpine (5 mg/kg i.p., 18 h) and 2 × 3 mg/kg i.p. α-methyl-p-tyrosine and 2 h); intracisternal injection of 30 μg/kg B-HT 933 decreased significantly the rate of spontaneous sympathetic discharges of splanchnic nerve fibres. Dogs were pretreated with reserpine (5 mg/kg i.p., 18 h) and α-methyl-p-tyrosine (300 mg/kg i.p., 18 h), resulting in radioenzymatically determined noradrenaline concentrations of 2.3 ± 0.6 ng/g, n = 6 as compared with controls, 396 ± 46.8 ng/g, n = 6. Antiotensin i.v. increased blood pressure and reflexly decreased heart rate. In noradrenaline-depleted dogs intracisternal injection of 30 μg/kg B-HT 933 significantly facilitated the angiotensin-induced reflex bradycardia and this facilitation was antagonized by additional intracisternal injection of 50 μg/kg piperoxan. These results clearly demonstrate that both central effects are mediated by stimulation of ‘functional’ postsynaptic α-adrenoceptors.

B-HT 920 and B-HT 958: Presynaptic effects on electrically evoked 3H-dopamine release from slices of rat nucleus accumbens

SummaryThe effects of two thiazoloazepine derivatives, B-HT 920 (6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepine) and B-HT 958 (2-amino-6-(p-chloro-benzyl)-4H-5,6,7,8-tetrahydrothiazolo[5,4-d]azepine) on electrically evoked overflow of 3H-dopamine were studied. Slices from nucleus accumbens of the rat were preincubated with 3H-dopamine and superfused at 23°C or 37°C. Electrical field stimulation was applied using frequencies of 0.5 or 5 Hz.1.At 37°C/5 Hz, B-HT 920 markedly and dose-dependently (0.01–0.1 μmol/l) reduced the stimulation evoked overflow of tritium. Its dose-response curve was shifted to the right at 23°C/0.5 Hz and 23°C/5 Hz, respectively. A similar result was obtained with the dopamine receptor agonist, apomorphine (1 μmol/l).2.B-HT 958 (0.1–10 μmol/l) also reduced electrically induced overflow of tritium at 37°C/5 Hz, had no effect at 23°C/0.5 Hz, and facilitated tritium overflow at 23°C/5 Hz.3.Sulpiride (10 μmol/l) completely prevented the effects of B-HT 920 (1 μmol/l) or B-HT 958 (1 μmol/l) at 37°C/ 5 Hz, whereas phentolamine (1 μmol/l) had no effect on the actions of the two drugs under these experimental conditions.4.From the patterns of effects obtained under the different experimental conditions it is concluded that B-HT 920 acts as full agonist at presynaptic dopamine autoreceptors whereas B-HT 958 acts as partial agonist.

Reduction of endogenous level, uptake and release of taurine after intrastriatal kainic acid injection.

Abstract Rats received single unilateral stereotaxic injections of 2 μg of kainic acid into the left caudate nucleus 10 days before sacrifice. On the lesioned side, a significant reduction of endogenous taurine levels and high affinity uptake of 3H-taurine into crude synaptosomal (P2) fractions to about 75% of control values was found. In agreement with previous reports on the neurotoxic action of kainic acid, a marked reduction of endogenous GABA levels and 14C-GABA uptake into P2-fractions was observed, while 3H-dopamine uptake was not significantly changed. In superfusion experiments with prelabeled P2-fractions prepared from lesioned striata, K+(56 mM)-induced release of 3H-taurine and 14C-GABA, but not of 3H-dopamine, was reduced. The results are compatible with the hypothesis that taurine in the striatum of the rat is partially localized in interneurones.

Properties of [3H]taurine release from crude synaptosomal fractions of rat cerebral cortex

The release of previously accumulated [3H]taurine and [14C]GABA from crude synaptosomal (P2) fractions isolated from rat cerebral cortex was studied using a superfusion system. The spontaneous efflux of [3H]taurine and [14C]GABA was stimulated by elevated concentrations of K+ (15–133 mM) in a concentration-dependent manner. This K+-stimulated release of [14C]GABA but not of [3H]taurine was enhanced in the presence of Ca2+. However, addition of 3 mM Ca2+ to the superfusion medium in the presence of the ionophore A 23187 resulted in a stimulation of the release of both [3H]taurine and [14C]GABA. These results are discussed in connection with the cellular localization of tourine in the central nervous system.

Effects of clonidine on the stimulation-evoked release of3H-noradrenaline from superfused rat brain slices as a function of the biophase concentration

SummaryThe influence of clonidine on the stimulation-evoked overflow of tritium was studied in brain slices preincubated with3H-noradrenaline. The slices were prepared from parietal cortex (Cx), nucleus anterior hypothalami (nah) and nucleus tractus solitarii (nts). After preincubation, the tissues were superfused at 23°C or 37°C with a medium containing the noradrenaline uptake inhibitor desipramine. Electrical field stimulation was applied using stimulation frequencies of 0.3–10 Hz.1.At 23°C/0.3 Hz, clonidine concentration-dependently inhibited the evoked overflow of tritium in all three brain regions. In contrast, at 23°C/3 Hz the inhibitory effect of the drug in the Cx was abolished and a facilitation was observed in the nah and nts. When tested at increasing frequencies of stimulation in the nts at 23°C, clonidine exerted a dual action, characterized by a reduction of electrically evoked responses at frequencies below 1 Hz and a facilitation at frequencies above 1 Hz.2.At 37°C, clonidine concentration-dependently decreased the evoked overflow in all brain regions studied, this effect being more pronounced at 0.3 Hz than at 3 Hz.3.The apparent lack of an effect of clonidine on the stimulation-evoked overflow of tritium in the Cx at 23°C/3 Hz was turned to a facilitation when noradrenaline (0.01 μmol/l) was included in the superfusion medium. Conversely, an inhibitory effect of clonidine was seen when the uptake blocker desipramine (as well as noradrenaline) was omitted from the superfusion medium.4.The facilitation by clonidine of the stimulus-induced release in the nah at 23°C/3 Hz was reversed to inhibition when the noradrenaline content of the slices was reduced to about 60% by exposure to a medium containing 50 mmol/l potassium before preincubation with3H-noradrenaline.5.A marked difference regarding the two superfusion temperatures was noted at the ultrastructural level: the superfusion of slices resulted in a widening of extracellular spaces, dissociation of synaptic clefts with preservation of presynaptic terminals, and intracytoplasmatic uptake of horseradish peroxidase. These changes were more pronounced after superfusion at 37°C than at 23°C. In accordance with these observations, the retention of3H-polyethyleneglycol in slices superfused at 37°C was greater than in slices superfused at 23°C.6.It is concluded that in superfusion/stimulation experiments with brain slices temperature-dependent changes at the ultrastructural level may have an influence on the concentration of noradrenaline in the synaptic cleft. The results emphasize the role of the biophase concentration of noradrenaline with regard to the magnitude and the direction of the presynaptic effect of clonidine.

Acute effects of alinidine on heart rate and blood pressure in healthy subjects and patients with hyperkinetic heart syndrome.

SummaryThe effects of a single dose of alinidine (0.5 mg/kg i.v.), the N-allyl-derivative of clonidine, on heart rate and blood pressure were investigated in healthy volunteers and in patients with hyperkinetic heart syndrome, at rest and during bicycle exercise. In healthy volunteers plasma catecholamine levels were also determined. Alinidine did not change heart rate at rest in the healthy volunteers but it did significantly reduce exercise-induced tachycardia, whereas blood pressure and plasma catecholamine levels were not significantly affected by alinidine, either at rest or during exercise. In patients with hyperkinetic heart syndrome, alinidine reduced heart rate at rest and during exercise to a similar extent as propranolol (0.1 mg/kg i.v.). The blood pressure did not change with alinidine but it was significantly reduced by propranolol. The observation that an alinidine-induced reduction of heart rate occurs without a concomitant fall in blood pressure, and without a clonidine-like symphatho-inhibitory action, is in line with experimental findings suggesting a specific bradycardic action of alinidine under short-term conditions.

Sedimentation and release properties of P2 fractions derived from rat cerebral cortex slices incubated with radiolabeled GABA for a short or long time period

On homogenization of rat cerebral cortex slices previously incubated with [3H] GABA or [14C]GABA for 5 or 30 min, respectively, particles were recovered in P2 fractions which exhibited similar buoyant density, but different sedimentation velocity on linear sucrose density gradient centrifugation. The K+-evoked release of [3H]GABA from particles isolated from slices previously incubated for 5 min with [3H]GABA was increased in the presence of exogenous Ca2+. In contrast, the K+-evoked release from particles isolated from slices previously incubated for 30 min with [3H]GABA, was not influenced by the presence of exogenous Ca2+.These results suggest that, depending on the incubation time of slices, exogenously applied GABA can be detected in differnnt pools. These pools not only seem to differ in their Ca2+ dependency of K+-evoked release but also in their subcellular localization.