Effects of pentobarbitone and decerebration on the clonidine-induced circulatory changes.

Clonidine 5 and 15 mug/kg i.v. was given to conscious rats, to rats under pentobarbitone anaesthesia and to decerebrated rats. Clonidine 100 mug/kg was given to conscious and to decerebrated rats. Blood pressure and heart rate were recorded via indwelling catheters. The low dose of clonidine gave virtually no response in the conscious rats but produced hypotension and bradycardia in decerebrated and in anaesthetized rats. After clonidine 15 mug/kg the blood pressure of decerebrated rats decreased further, while conscious and anaesthetized rats showed a hypertensive response. The basal blood pressure and heart rate were significantly higher in the decerebrated rats than in the conscious rats. After the highest dose of clonidine, however, the blood pressure of the decerebrated rats decreased below the basal level of the conscious rats and the blood pressure of the conscious rats increased above the basal level of the decerebrated rats. The results suggest that the hypotensive response is caused by a mechanism located in the medulla oblongata. At higher concentrations clonidine may elicit a hypertensive effect by activating suprabulbar centers. The importance of this suprabulbar effect is attenuated by pentobarbitone anaesthesia.

Brain ATP:L-methionine S-adenosyltransferase (MAT), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH): regional distribution and age-related changes.

The distribution of the activity of the enzyme methionine adenosyltransferase (ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6, MAT) was investigated in human postmortem brains of individuals without a known history of neuropsychiatric disorders. The brain regions were the frontal, temporal, parietal and occipital cortices, nucleus caudatus, putamen, globus pallidus, thalamus and white matter. The activities in the nucleus caudatus and putamen were approximately 25% higher than the activities in the seven other brain regions, however, not on a statistically significant level. The apparent values of MAT Km and Vmax in the parietal cortex were 11.41 +/- 3.51 microM methionine and 25.72 +/- 3.90 nmol/mg protein/h, respectively. In the frontal cortex, a significant positive correlation between age and the activity of MAT was found (r = 0.997, P < 0.01). Concerning MAT stability in the rat brain, there was a steady decrease in the activity with postmortem time in the brains kept for 0-72 h at room temperature (23 degrees C), which reached the level of significance at 24 h. The activity did not change significantly when the brains were kept for 120 h at 4 degrees C, or by freezing and thawing the tissue before analysis. In a parallel study in rats of different ages (2-22 months), a homogeneous distribution of SAM and SAH was observed in the cortex, striatum, midbrain, hypothalamus, brainstem and cerebellum. The lowest levels of SAM and the highest levels of SAH observed in the striatum gave the lowest SAM/SAH ratio. The SAH content of rat cerebral cortex was highest in the oldest group.(ABSTRACT TRUNCATED AT 250 WORDS)

On the importance of GABA-ergic neurons for the AOAA induced accumulation of GABA in the rat brain.

The accumulation of GABA induced by an intravenous injection of aminooxyacetic acid (AOAA) was followed for 1 h in five parts of the rat brain, i.e. cerebellum, medulla oblongata-pons, striatum, ventral mesencephalon and hypothalamus. The accumulation was similar in all brain parts studied when expressed as percentual increase from the basal value; an initial rapid accumulation indicating a mean turnovertime of 12--16 min during the first 5 min was gradually decreased to turnovertime of about 1--3 h during the last 30 min of observation. In order experiments brains were transected unilaterally between the substantia nigra and the striatum. Six days after the hemisection the GABA concentration of the substantia nigra of the transected side had decreased to less than 30% of the control side. The AOAA induced accumulation of GABA in the substantia nigra of the transected side was less pronounced than that of the control side. The initial rapid accumulation seen in all brain parts studied was completely lacking in the substantia nigra of the transected side. In the striatum, the transection did neither alter the GABA concentration nor the AOAA induced accumulation of GABA. As the initial rapid accumulation of GABA disappears after degeneration of GABA-ergic neurons, it is suggested that this initial phase of GABA accumulation produced by an intravenous injection of AOAA probably is the result of GABA accumulation in neurons.

Effect of aminooxyacetic acid (AOAA) on GABA levels in some parts of the rat brain.

The accumulation of GABA in the cerebellum and medulla oblongata-pons of rats has been studied after inhibition of GABA-T (EC 2.6.1.19) by different doses of AOAA. It was found that intraperitoneal (i.p.) injections of AOAA were, at least during the first hour after injection, much less effective than intravenous (i.v.) injections probably due to poor absorption i.p. After i.v. injection, AOAA caused a maximal accumulation of GABA in the cerebellum at a dose of 50 mg/kg. This maximal effect was virtually unchanged up to a dose of 150 mg/kg (the highest dose tested i.v.). If GAD (EC 4.1.1.15) was inhibited by 3-mercaptopropionic acid 30 min after AOAA (90 mg/kg i.v.) the GABA level was stable for at least another 30 min. The rate of GABA accumulation in the cerebellum during the first 15 min after AOAA (50-150 mg/kg i.v.) was 0.086 mumol/g/min and thereafter 0.034 mumol/g/min. It is concluded that AOAA in vivo in a wide dose range inhibits GABA-T almost 100% without affecting GAD to any great extent, and that the onset of action is rapid after i.v. but not after i.p. injection.

Effects of morphine on central catecholamine turnover, blood pressure and heart rate in the rat.

In the unanaesthetized rat morphine caused increased dopamine (DA) turnover, unchanged or possibly increased central noradrenaline (NA) turnover (utilization), hypertension and tachycardia. In the anaesthetized rat, brain DA turnover was not affected, whereas the NA-turnover was decelerated, particularly in some brain regions, e.g. cerebral cortex and medulla oblongata, and hypotension and bradycardia was obtained. Both biochemical and cardiovascular effects of morphine were antagonized by naloxone. A very small dose of morphine (1 mg/kg) caused tachycardia also in the anaesthetized rat. Decerebration just inferior to the inferior colliculus abolished the conscious rat, but left the circulatory, depressant actions of the drug unchanged. The morphine-induced cardiovascular effects, particularly the hypotension and bradycardia in the anaesthetized animal, are suggested to be related to, or mediated by, the effects of the drug on brain NA-mechanisms, especially in view of several similarities between morphine and the antihypertensive alpha-adrenergic agonist clonidine. Whereas higher brain structures appear important in the excitatory, circulatory effects of morphine, structures below the decerebration level, e.g. medulla oblongata, appear primarily involved in the hypotension and bradycardia obtained in the anaesthetized animal. Possibly, morphine has a diphasic dose-response curve with respect to cardiovascular function and, by inference, on brain noradrenergic mechanisms.

Aminooxyacetic acid induced accumulation of GABA in the rat brain. Interaction with GABA receptors and distribution in compartments.

The effect of aminooxyacetic acid (AOAA, 90 mg/kg i.v.) on bicuculline, picrotoxin and 3-mercaptopropionic acid (3-MPA) induced convulsions and on GABA concentrations in cerebellum, whole brain and a synaptosomal fraction of whole brain was investigated. At various intervals after AOAA the rats were either injected with one of the convulsive drugs or sacrificed for analysis of the GABA concentration. AOAA caused a rapid initial (0-30 min) and a later slower increase of GABA in cerebellum and whole brain. In the synaptosomal fraction the GABA accumulation was delayed and less pronounced when compared to the whole brain. The bicuculline induced convulsions were markedly potentiated during the first hour but completely blocked from 2-6 h after AOAA. Picrotoxin showed a somewhat different pattern to bicuculline in the interactions with AOAA. The initial strong potentiation was not observed but the later phase of protection was present. In the interactions with 3-MPA, the effect of AOAA was always protective. The time to onset of convulsions was gradually increased during the first 30 min after AOAA. This protective effect remained practically unchanged up to 6 h after AOAA. However, once started, the convulsions were generally of the same duration and intensity. The results can be interpreted as GABA accumulating after AOAA stimulates GABA receptors to a degree more or less proportional to the whole brain GABA concentration and further that GABA synthetized in neurons is liberated, stimulates inhibitory bicuculline sensitive (predominant) and excitatory bicuculline insensitive receptors and is captured to a large extent by non-neuronal cells.

Screening in mice of some medicinal plants used for analgesic purposes in the state of São Paulo.

Twelve medicinal plants used popularly for their reputed analgesic properties were tested in mice by the writhing and tail-flick methods. All extractions were made using 50% aqueous ethanol at low temperatures. The oral dose administered was always 1 g solids/kg. While several extracts showed a positive effect in one of the tests, significant effects in both tests were produced by Serjania communis only. Morphine and acetylsalicylic acid were used as reference drugs.

Screening in mice of some medicinal plants used for analgesic purposes in the state of São Paulo. Part II.

Seventeen medicinal plants used popularly in Brazil for their reputed analgesic properties were tested in mice by the writhing and tail flick methods. All extractions were made in 50% aqueous ethanol at low temperatures. The oral dose administered was always 1 g extract/kg. Significant effects in both tests were produced by Lippia alba, Piper abutiloides, Piper cincinnatoris, Piper lindbergii and Tillandsia usneoides.

Acute antihypertensive effect in conscious rats produced by some medicinal plants used in the state of São Paulo.

Thirty two medicinal plants used popularly for their proposed diuretic and/or antihypertensive properties have been tested for antihypertensive effects in conscious unrestrained rats. All extractions were made in aqueous ethanol (50:50 by vol.) at low temperature. Before administration of the extracts the alcohol was evaporated. The extracts (40 ml/kg) were always administered per os. Antihypertensive effects in SHR rats were observed after the administration of Allium sativum Linn. (bulb), Olea europaea Linn. (leaf) and Hedychium coronarium Koen. (leaf-blade).

Acute diuretic effects in conscious rats produced by some medicinal plants used in the state of São Paulo, Brasil.

Extracts of 32 medicinal plants used popularly for their presumed diuretic and/or antihypertensive properties were tested for diuretic effects in conscious unrestrained rats. Extracts were made using aqueous ethanol (50:50, v/v) at 4-10 degrees C. When given orally at a dose of 40 ml/kg, a majority of the ethanol-free extracts produced a more pronounced diuresis than would be expected from the potassium concentration of the extracts. The most significant diuretic effect was observed with Hedychium coronarium sheath and leaf-blade extracts.

Convulsive action of (25S)-isosolafloridine isolated from Solanum pseudo-quina bark.

The crude ethanolic extract prepared from the stem bark of Solanum pseudo-quina produced excitatory effects dominated by convulsions in rats and mice. Solvent extraction followed by alumina column chromatography resulted in the isolation of a pharmacologically active material (AP) which was identified to be (25S)-isosolafloridine. The convulsions produced by AP were predominantly clonic and invariably preceded by generalized fine and coarse tremors. This convulsive behaviour did not entirely resemble the convulsions produced by strychnine, pentylenetetrazol, bicuculline, picrotoxin or 3-mercaptopropionic acid. The tremor and convulsions were only slightly affected by drugs interfering with cholinergic, catecholaminergic, serotoninergic or encephalinergic neurotransmission. Only diazepam and particularly gamma-vinyl-GABA blocked AP-induced effects. After section of the spinal cord at a mid-theoretic level, AP produced convulsions only in the anterior part of the body. After intracerebroventricular administration, AP produced only sedation. A depressive effect was also observed on the blood pressure of conscious rats before and after the convulsions. In subconvulsive doses AP enhanced spontaneous motor activity in mice.

GABA turnover in mouse brain: agreement between the rate of GABA accumulation after aminooxyacetic acid and the rate of disappearance after 3-mercaptopropionic acid.

GABA levels of the whole mouse brain were studied after in vivo inhibition of GABA synthesis by 3-mercaptopropionic acid (3-MPA, 100 mg/kg i.p.) and of GABA degradation by aminooxyacetic acid (AOAA, 3.8-60 mg/kg i.v.). The influence of 3-MPA on GABA levels was investigated in brains where postmortal GABA accumulation was allowed to occur and in brains where this phenomenon was avoided by very rapid dissection and homogenization of the brain in acid (within 50 sec after decapitation). The post-mortal GABA increase was blocked by 86% after injection of 3-MPA and 3 min before decapitation. In the group where the postmortal accumulation was avoided by very rapid homogenization of the brain in acid, GABA levels decreased by 15% within 2 min after 3-MPA (mean turnover time = 14 min). From 2 to 4 min the GABA concentration remained stable at this decreased level. GABA accumulation after AOAA was maximal after a dose of 7.5 to 15 mg/kg. i.v. Doses higher than 60 mg/kg always produced convulsions. The phase of most rapid accumulation of GABA after AOAA indicates a mean turnover time of about 10 min. The first rapid phase of accumulation was followed by a slower phase. It is probable that the turnover time of whole mouse brain GABA is approximately 10-14 min. It is also concluded that AOAA in a dose of around 15 mg/kg i.v. hardly can inhibit GAD in vivo in the mouse brain and that this dose, by this route of administration, could be used for studies of GABA synthesis in vivo in the mouse.

Nitric oxide (NO) in expired air at rest and during exercise.

Nitric oxide (NO) was analysed in expired air from 27 healthy human subjects. At rest the NO concentration was 10.5 +/- 0.9 ng 1-1 (mean +/- SEM) corresponding to 8.6 +/- 0.7 parts per billion (ppb). The expired NO concentration did not change when the subjects were switched from breathing NO-free tank gas to room air which contained 7.7 ng l-1 NO. Repeated measurements of expired NO with an interval of 1 day showed a mean variation of 2.2 +/- 0.7 ng l-1 NO. The NO concentration in the first portion in the expired tidal volume (44%) was insignificantly higher than in the latter expired portion, 6.9 +/- 1.9 vs. 5.1 +/- 1.0 ng l-1 (n = 5). During moderately heavy exercise on an ergometer bicycle (90 W for women, n = 4, 150 W for men, n = 4) the expired concentration of NO decreased, however because of increased minute ventilation, the expired amount of NO almost doubled (from 111 +/- 12 to 209 +/- 30 ng min-1). The source of the expired NO is not clear and both the airways and the pulmonary circulation may contribute.

Purification of GABA on small columns of Dowex 50W; Combination with a method for separation of biogenic amines.

Elution of GABA has been included in the method for separation of biogenic amines on small Dowex 50W columns described by Atack & Magnusson (1978). After extraction with perchloric acid and neutralization to pH 3.0, the sample is passed through the column. 5-HIAA might thereafter be eluted as described by Lindqvist (1971). After washes with water and 8 ml of a 0.025 M sodium citrate buffer pH 4.5, GABA is eluted in 3 ml of a 0.05 M sodium citrate buffer pH 5.3-5.4. The elution of tryptophane and the above mentioned amines can thereafter be performed. The recovery of GABA from the column is 100%. Silica thin-layer chromatography of the GABA eluate gives only one ninhydrine positive spot with a Rf value identical to GABA standard. When the GABA eluate from a whole brain and spinal cord is run on an automatic amino acid analyser, only one peak is detectable, eluted in the same position as GABA standard. Fluorescence analysis of the GABA eluate gives similar values if a( ninhydrine, b) acetylacetone plus formaldehyde or c) fluorescamine is used to form the flurophore. The GABA values are identical whether the samples reacted with ninhydrine are diluted with water or copper tartrate. The GABA concentrations found in some parts of the rat brain are of the same order of magnitude as reported by other authors.

Pre- and postsynaptic alpha-adrenoceptor antagonists: differentiated cardiovascular effects in the rat.

Intravenous (i.v.) injections of yohimbine, phentolamine, prazosine and phenoxybenzamine lowered blood pressure and increased heart rate in conscious rats. Intracerebroventricular (i.c.v.) injections of yohimbine and phentolamine increased blood pressure and heart rate; this was antogonized by pretreatment with clonidine. Phenoxybenzamine and prozosine had no effect or gave hypotension and tachycardia on i.c.v. injection. Pentobarbitone anaesthesia partly antagonized the cardiovascular effects of all alpha-adrenoceptor antagonist. Synthesis and utilization of central noradrenaline was increased by i.v. or i.c.v. yohimbine; anaesthesia partly antagonized this effect. In peripheral tissues others have found that yohimbine, tolazoline, piperoxan and phentolamine are potent blockers of the presynaptic alpha-adrenoceptors while phenoxybenzamine and prazosine act preferentially on postsynaptic alpha-adrenoceptors. The differentiated cardiovascular response to i.c.v. injection of these blockers may reflect their different affinity to central pre- and postsynaptic alpha-adrenoceptors.

Evidence for involvement of central noradrenergic neurons in the cardiovascular depression induced by morphine in the rat.

Morphine caused in the anaesthetized rat reduction in brain noradrenaline (NA) turnover, hypotension and bradycardia, similarly to the antihypertensive, alpha-adrenergic agonist, clonidine. All effects of morphine were antagonized by naloxone, as well as the alpha-receptor antagonist, yohimbine. In contrast, naloxone did not affect the circulatory depression and reduction in brain NA utilization by clonidine which both previously have been found to be antagonized by yohimbine. In contrast to clonidine, morphine even in high doses did not facilitate the flexor reflex activity of acutely spinalized rats. Pretreatment with protriptylin largely attenuated the circulatory depressive effects of morphine, as it has previously been found to block the corresponding effects of clonidine. Thus, the morphine-induced cardiovascular depressive effects are primarily elicited by activation of opiate receptors. However, the inhibition of brain NA neurotransmission by morphine appears critically involved in the mediation of the circulatory depression.