Central benzodiazepine involvement in clonidine cardiovascular actions

It is well known that the GABAergic and noradrenergic systems play an important role in blood pressure and heart rate regulation. Benzodiazepines and beta-carbolines, respectively, increase or decrease the probability of chloride-channel opening induced by GABA. The aim of this study was to determine, in conscious rats, the interaction existing between the central alpha2-adrenoceptor stimulation induced by clonidine and the facilitation or impairment of benzodiazepine receptor activity through the administration of either diazepam, a benzodiazepine receptor agonist, or methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), an inverse benzodiazepine agonist. Clonidine (5-10 microg, intracerebroventricularly) reduced heart rate and increased mean blood pressure by activation of central alpha2-adrenoceptors. Diazepam (2 mg/kg, intravenously (i.v.)) induced an increase in heart rate, while DMCM (0.3 mg/kg, i.v.) elicited a bradycardic effect. The bradycardic effects induced by both clonidine and DMCM were antagonized by the prior administration of methylatropine (1.5 mg/kg, i.v.). DMCM (0.3 mg/kg, i.v.) prevented the clonidine effects on heart rate and mean blood pressure, while diazepam (2 mg/kg, i.v.) failed to modify these effects. Our results suggest that the bradycardic effects of clonidine are mediated by a vagal stimulation and are related to the activation of a GABAergic pathway.     

The effects of ouabain in the medullary site of the hypotensive action of clonidine

Blood pressure was studied in pentobarbital anesthetized rats and cats after central administration of ouabain. Intracerebroventricular (i.c.v.) injections caused a classical biphasic effect, a short lasting hypotension followed by a hypertensive phase. When injected directly into the nucleus reticularis lateralis region (NRL), ouabain (0.01–2 μg/kg) caused a dose-dependent pressor effect. In the same region, kryptofix 221, a sodium complexing agent, produced a fall in blood pressure. Moreover, central administration of ouabain prevented the hypotensive effect of i.v. clonidence whereas the central hypotensive effect of muscimol was not affected. It is concluded that sodium movements play an important role in the blood pressure regulation within the NRL region. We also report here that ouabain antagonizes the hypotensive effect of clonidine suggesting that sodium movement might be the essential link of this action.     

Influence of analgesic antipyretics on the central cardiovascular effects of clonidine in rats

The centrally acting antihypertensive drug clonidine has been found to stimulate the synthesis of PGF_2α in the brain. Centrally administered PGF_2α, in turn, induces rises of blood pressure and heart rate. We therefore studied the influence of inhibitors of prostaglandin (PG) synthesis on the cardiovascular effects of clonidine in urethane-anaesthetised rats. Pretreatment with indomethacin or paracetamol (100 μg/rat into the fourth cerebral ventricle) antagonised the central hypotensive effect of clonidine (0.125–16.0 μg/rat into the fourth cerebral ventricle). The bradycardic effect of centrally administered clonidine was, however, enhanced by pretreatment with paracetamol but not influenced by indomethacin pretreatment. Sodium meclofenamate (100 μg/rat into the fourth cerebral ventricle) did not significantly affect the clonidine-induced changes in blood pressure and heart rate.These results suggest that the clonidine-induced hypotension on one hand and bradycardia on the other hand may be mediated by partly different mechanisms. An interference of the formation of PGF_2α with the cardiovascular effects of clonidine cannot be completely excluded since paracetamol pretreatment potentiated the bradycardic effect of clonidine. However, inhibitors of PG synthesis did not enhance but antagonised the hypotensive effect of clonidine. Therefore it is likely that the synthesis of PGF_2α does not interfere with the hypotensive effect of clonidine. Moreover, the antagonism of the hypotensive effect by inhibitors of PG synthesis suggests that some hypotensive metabolite of arachidonic acid in the brain could be involved in the central hypotensive effect of clonidine.     

Evidence for antagonistic activity of endothelin for clonidine induced hypotension and bradycardia.

Effect of endothelin (ET) on clonidine induced cardiovascular effects was studied in male Sprague-Dawley rats. Clonidine (75 micrograms/kg, iv) produced significant decrease in blood pressure and heart rate. ET-1 (50 ng/kg, iv) pretreatment completely antagonized the hypotension and bradycardia induced by clonidine. ET-2 (50 ng/kg, iv) and ET-3 (50 ng/kg, iv) had similar antagonistic effect on clonidine induced hypotension and bradycardia. The antagonistic effect of ET lasted for several hours, however, 4 hours after ET pretreatment only partial blockade of clonidine induced hypotension and bradycardia was observed. This indicated that the antagonistic effect of ET was reversible. Initial hypertensive response induced by high dose of clonidine (750 micrograms/kg, iv) could not be antagonized by ET-1, ET-2 or ET-3, while phenoxybenzamine, an alpha adrenoceptor antagonist, blocked the hypertensive response of clonidine. Thus, ET has no antagonistic effect on the initial hypertensive response but antagonizes the hypotensive and bradycardic effect induced by clonidine. Clonidine induced hypotension and bradycardia are mediated through central alpha 2 adrenoceptors while hypertension is mediated through peripheral alpha 2 adrenoceptors. It is concluded that central alpha 2 adrenoceptors are different from peripheral alpha 2 adrenoceptors and ET antagonizes the effect of clonidine only on central alpha 2 adrenoceptors but has no antagonistic activity on peripheral alpha 2 adrenoceptors.     

安定减低家兔膈神经放电幅度的机制分析

本实验室曾经报道静脉注射安定对于清醒、麻痹、人工呼吸的家兔具有减低膈神经放电幅度、加快呼吸频率,缩短吸气时程(T_I)和呼气时程(T_E),降低动脉血压等作用。本工作在35只家兔中进一步分析了某些药物对安定的这些作用的影响。GABA 降低膈神经放电幅度和动脉血压,这与安定的作用相同,但 GABA 延长T_I、T_E和减慢呼吸频率,与安定的作用相反。事先用氨基酸脱羧酶抑制剂异烟肼处理,或用 GABA 受体拮抗剂印防己毒素处理,可阻遏安定减低膈神经放电幅度的作用。在事先用印防己毒素处理的家兔中,可见安定缩短 T_IT_E的作用不受影响。异烟肼或印防己毒素还能部分对抗安定的降压效应。阿片受体拮抗剂纳洛酮和5-HT 受体拮抗剂赛庚啶都不能阻遏安定降低膈神经放电幅度和动脉血压的作用。上述结果提示安定降低膈神经放电幅度的作用可能通过 GABA 这一中间环节,而内啡肽和5-HT可能不起重要作用。安定的降压作用需要有内源性 GABA 参与才得以持续较长时间,在减少GABA 或阻断 GABA 受体后,安定只有短暂的降压作用。     

Clonidine and morphine increase atrial natriuretic peptide secretion in anesthetized rats

In order to determine whether the activity of central alpha 2-adrenergic and opioid receptors influence plasma atrial natriuretic peptide (ANP) levels, clonidine and morphine were infused into the lateral cerebral ventricle for 45 min in anesthetized Sprague-Dawley rats. The central administration of a low dose of clonidine (10 ng/min) caused a significant increase in plasma ANP without changing arterial blood pressure or central venous pressure. Pretreatment with yohimbine (5 micrograms/min) completely blocked the effect of clonidine. Central infusion of morphine (100 ng/min) also elevated plasma ANP levels and naloxone (5 micrograms/min) blunted this effect. Intravenous infusion of the same dose of clonidine or morphine did not affect plasma ANP levels. Moreover, the effect of clonidine on plasma ANP was partially blocked by pretreatment with naloxone (5 micrograms/min). These results suggest that central alpha 2-adrenergic and opioid receptors may be involved in ANP secretion.     

An inhibitory role of beta-endorphin in central cardiovascular regulation

The cardiovascular effects of beta-endorphin after administration directly into the nucleus tractus solitarii (NTS) of urethane anaesthetised rats were investigated. Unilateral injection resulted in a dose related fall in mean arterial pressure and heart rate. No change in respiratory frequency was prevented and the bradycardia reduced by pretreatment with locally applied naloxone (10 ng). This dose of the opiate antagonist had no effect on mean arterial pressure or heart rate when administered alone. Antiserum to beta-endorphin (1:50 dilution) caused a rise in pressure and a tendency towards tachycardia on injection into the NTS, while it completely blocked the depressor response and bradycardia induced by beta-endorphin. These results are consistent with the view that a beta-endorphin-like peptide has a depressor role in the central nervous system. The hypotension may result from an effect within the central connections of the baroreceptor reflex arc, probably at the level of the NTS.     

Gabamimetic properties of anxiolytic drugs

Diazepam (5 mg/kg, ip) and tracazolate (40 mg/kg, ip), a nonbenzodiazepine anxiolytic, blocked electrically-induced head-turning without producing sedation. Bicuculline and picrotoxin, GABA antagonists, at doses not affecting head-turning (2 mg/kg, ip) antagonized the effects of diazepam and tracazolate on head-turning. However, at the same dose, bicuculline was more effective as an antagonist of diazepam whereas picrotoxin was more effective as an antagonist of tracazolate. These results suggest that benzodiazepine as well as nonbenzodiazepine anxiolytics possess GABAmimetic activity. The difference in potency between bicuculline and picrotoxin as antagonists of diazepam and tracazolate may be related to their reported differences as GABA antagonists (e.g., site of receptor interaction).     

安定对家兔呼吸的作用

在切断迷走神经和局部麻醉的家兔中观察了静脉注射安定(2mg/kg)对呼吸和循环的影响。在动物进行自然呼吸的条件下,安定使每分通气量减少,心率变慢和血压降低。在将动物肌肉麻痹并进行正压人工呼吸的条件下,安定使呼吸频率加快,吸气时程和呼气时程缩短,即呼吸周期缩短。此外,膈神经放电的高频振荡频率增高而电位的幅度变小,动物的血压也降低。在桥脑头端去大脑而表现长吸式呼吸,以及在延體髓纹水平横断脑干而表观喘息式呼吸的动物中,安定亦使膈神经放电的幅度减小,呼吸周期延长和血压降低。以上结果表明安定可使呼吸抑制、血压降低,而其作用的基本部位可能主要在延髓。     

Diazepam administered prior to coronary artery occlusion increases latency to ventricular fibrillation

Few studies have addressed the antiarrhythmic potential of pretreatment with diazepam in acute myocardial infarction. Thus, the effect of diazepam pretreatment prior to coronary artery occlusion was examined in conscious pigs. Animals were instrumented with aortic catheters to measure arterial pressure, a pulmonary artery catheter for drug administration, and a snare around the left anterior descending coronary artery for permanent occlusion one week later. Diazepam (1 mg/kg iv bolus) or vehicle was administered 10 minutes prior to occlusion. Eight of 14 animals receiving diazepam (57%) and 13 of 22 receiving vehicle animals (59%) developed ventricular fibrillation following coronary occlusion. However, the latency to ventricular fibrillation was significantly shorter (7 +/- 1 min) in animals receiving vehicle compared to animals receiving diazepam (11 +/- 1 min). Significant increases in heart rate were seen up to 5 hours after coronary occlusion only in animals receiving vehicle. The results indicate that diazepam pretreatment can increase ventricular fibrillation latency and prevent heart rate increases following acute myocardial infarction.     

A probable site of action of diazepam in rat cerebellar GABA system

Diazepam and some neurotropic drugs were examined for their effects on the cyclic guanosine 3′, 5′-monophosphate (cyclic GMP) content in rat cerebellum. Diazepam caused a marked decrease of cerebellar cyclic GMP content in a dose-dependent manner. Elevation of cerebellar gamma-aminobutyric acid (GABA) level by aminooxyacetic acid also caused a decrease in the cyclic GMP content. On the other hand, dl-amphetamine, oxotremorine, picrotoxin, and GABA-reducing agents such as isoniazid or thiosemicarbazide increased the content of this nucleotide. The increase of cyclic GMP content elicited by isoniazid was blocked completely by the premedication of diazepam in doses causing partial reduction of dl-amphetamine or oxotremorine action. Changes of cerebellar GABA level, which were caused by aminooxyacetic acid or thiosemicarbazide, did not influence the effect of diazepam. Moreover, the inhibitory action of diazepam on the picrotoxin-induced increase of cyclic GMP was a competitive type. These results suggest that diazepam facilitates the GABAergic function by acting on a picrotoxin-sensitive site of the GABA receptor complex in vivo.     

Caffeine and theophylline counteract diazepam effects in man

A total of 237 healthy subjects were studied in four placebo-controlled double-blind trials with parallel treatment groups. The subjects ingested a capsule (diazepam or placebo) and decaffeinated coffee with or without added caffeine or theophylline. Diazepam (10 and 20 mg) impaired dose dependently cognitive skills as measured by digit symbol substitution and letter cancellation, the balance of extraocular muscles, flicker fusion, and tapping speed. With diazepam 10 mg statistically significant effects were seen on digit symbols and exophoria only. Theophylline (10 mg/kg) increased tapping speed and heart rate, whereas other objective measurements were negative for the effects of theophylline or caffeine (250 and 500 mg) alone. Subjectively they reduced calmness, and caffeine also increased alertness. Caffeine 250 mg counteracted diazepam induced (10 mg) impairment of cognitive skills and relaxation of extraocular muscles whereas caffeine 500 mg counteracted the same effects of diazepam 20 mg, respectively. Theophylline antagonized diazepam-induced impairment in digit symbol substitution and tapping speed tests. Subjectively, caffeine and theophylline counteracted diazepam induced drowsiness and mental slowness. The results suggest, therefore, that the ample use of methylxanthines compensates various side-effects of benzodiazepines in man. It may also increase the need of sedation and thus the consumption of benzodiazepines.     

Involvement of GABA in palate morphogenesis and its relation to diazepam teratogenesis in two mouse strains

Previous studies have indicated that serotonin and acetylcholine stimulate palate shelf reorientation. The present studies were undertaken to determine whether gamma-aminobutyric acid (GABA) functions as an inhibitory neurotransmitter in the palate and whether diazepam mimics GABA to inhibit shelf reorientation and cause cleft palate. First, it was shown that 10(-4) M GABA inhibits palate shelf reorientation in day 14.5 AJ embryos cultured for 2 hours. Anterior palate reorientation stimulated by 10(-5) M serotonin was decreased by GABA; 10(-5) M picrotoxin (GABA antagonist) stimulated anterior shelf reorientation and reversed the effect of GABA. Diazepam (10(-4) M) partially inhibited palate shelf reorientation and that stimulated by 10(-5) M serotonin. Diazepam (400 mg/kg) was administered to AJ mice at day 13.5 of gestation and embryos were cultured at day 14.5. The inhibition produced by diazepam was significantly reduced by 10(-5) M picrotoxin. The teratogenic effect of diazepam was compared with AJ and Swiss-Webster Vancouver (SWV) inbred strains. Diazepam produced greater clefting in SWV mice (57% net) than in the AJ (18% net) when compared to their water- and food-starved controls. The greater sensitivity of the SWV strain than the AJ strain to diazepam, as well as to GABA, was also observed in embryo culture. GABA (10(-5) M) markedly inhibited posterior palate reorientation and reversed the stimulation produced by bethanechol in SWV mice. The inhibitory effects of GABA on the posterior palate were partially reversed by picrotoxin. Furthermore, diazepam inhibited palate reorientation either when administered to the pregnant dam or added in embryo culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex related differences in the response of mice, rats and cats to administration of picrotoxin

Picrotoxin, 2.5 mg/kg, which was subconvulsive in male rats was 92% convulsive in female rats. Four mg/kg of picrotoxin, a dose which did not produce death in the male rats, was 75% lethal in the female rats. Picrotoxin also produced a significantly greater increase in the frequency of the spinal motoneurons discharge in the female than in male rats (444% of control compared to 222% of control). A similar significant difference to the analogous treatment was obtained in the female and male cats (439% of control compared to 368% of control). To counteract the picrotoxin-induced increased frequency of the spinal motoneurons discharge a double dose of diazepam had to be given to females of both species. A sex related difference in the occurrence of convulsions, latency and death following picrotoxin administration was also present in mice. However, mice responded in an opposite direction to rats and cats. Three mg/kg of picrotoxin was 100% convulsive and 27% lethal in male mice, while only 40% convulsive and 0% lethal in female mice. In male mice treated with a 100% lethal dose of picrotoxin, diazepam, 3.0 mg/kg, did not diminish the occurrence of convulsions but reduced the incidence of death to 70%. In equally treated female mice the same dose of diazepam reduced the occurrence of convulsions from 100 to 70% and the incidence of death to 10%. The existence of sex related differences in the response of mice, rats and cats to administration of picrotoxin might have its origin in the dimorphisms of the GABA system in these animal species.     

Multiple treatment potentiates the anticonvulsive activity of cholecystokinin octapeptides

The dose-response curves for the anticonvulsive activity of sulfated and nonsulfated cholecystokinin octapeptide (CCK-8-SE and CCK-8-NS) against picrotoxin-induced (6 mg/kg SC) seizures were assessed either following or without pretreatment with a single high dose of CCK-8-SE or CCK-8-NS, to examine acute tolerance to the effect after IP injections in mice. As CCK-8-SE or CCK-8-NS pretreatment, a 1.6 μmole/kg dose was injected 2 hr prior to the second injection. No acute tolerance to the anticonvulsive activity was demonstrated, and CCK-8-NS pretreatment significantly potentiated its own anticonvulsive activity. Chronic (8-day) daily treatment with a 0.16 μmole/kg dose of CCK-8-SE or CCK-8-NS antagonized seizures by picrotoxin, presumably in a cumulative manner. To investigate the interactions of CCK octapeptides with other anticonvulsive agents, picrotoxin-induced seizures were antagonized with several doses of diazepam following or without acute, high-dose pretreatment with CCK-8-SE or CCK-8-NS. The two octapeptides only slightly modified the activity of diazepam: CCK-8-SE pretreatment displayed a tendency to antagonize it, while CCK-8-NS pretreatment to potentiate it. The results suggest that multiple treatment with CCK-8 induces sensitization of CCK receptors mediating anticonvulsive activity.     

Clonidine action in spontaneously hypertensive rats (SHR) depends on the GABAergic system function

Summary The effect of -aminobutyric acid (GABA)_A antagonists (bicuculline, picrotoxin) on clonidine hypotension in spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats were examined. The GABA turnover changes after clonidine injection in both strains were also studied. Administration of clonidine alone induced the stronger decrease of systolic blood pressure (SBP) in SHR. Co-dosage of clonidine with these agents reduced its hypotensive effect in dose dependent manner and the effectiveness of both antagonists was higher in SHR. We find that clonidine stimulates GABA synthesis in the hypothalamus and the pons-medulla in both strains but the GABA turnover rate is significantly slower in SHR. Therefore, the differences in inhibitory action of GABAA receptor anatgonists between WKY and SHR rats may be explained by central GABAergic system dysfunction in the hypertension. Our results indicate that the down regulation of the GABAergic system observed in hypertension may be compensated by the action of clonidine.     

The GABA-ergic system and brain edema

It has been shown in rats with experimental toxic and traumatic edemas that picrotoxin (1 mg/kg) removes the antiedematous action of diazepam, phenazepam, phenibut and amizyl and reduces the action of phentolamine. When the dose of picrotoxin is minimized to 0.5 mg/kg such an effect is not observed. Prolonged daily administration of picrotoxin in a dose of 1 mg/kg results in the development of brain edema. It is recommended that GABA-positive drugs be included into a complex of treatment measures for edema.     

Effects of diethylcarbamazine on the motility of Angiostrongylus cantonensis and Dirofilaria immitis

Effects of piperazine derivatives, especially of diethylcarbamazine (DEC) on adult Angiostrongylus cantonensis and Dirofilaria immitis were examined. Piperazine (3 X 10(-5)-10(-4) M) paralyzed A. cantonensis and the action was antagonized by picrotoxin. 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (10(-5)-10(-4) M) caused contraction but little effect was produced by strychnine. An inhibitory effect on untreated preparations was caused by lower concentrations (3 X 10(-6)-10(-5) M) of diethylcarbamazine citrate (DEC) and also on the preparations contracted by eserine. A stimulatory effect was also seen when higher concentrations (10(-4)-3 X 10(-4) M) of this drug were applied to both preparations. The inhibitory action of DEC was antagonized by gabergic antagonists such as picrotoxin and bicuculline, but not by alpha-adrenergic antagonists like dibenamine and phentolamine. When the worm preparation was paralyzed by strychnine or hexylresorcinol (inhibitors of the release of acetylcholine in this worm), the stimulatory effect of DEC was blocked, but pyrantel (a nicotinic cholinergic agonist) contracted the paralyzed preparation. However, the effect of DEC on D. immitis (10(-7)-3 X 10(-4) M) was inhibitory, and this action was also antagonized by picrotoxin. These results suggest that the DEC inhibitory and stimulatory action is through the gabergic and cholinergic mechanisms in adult A. cantonensis and D. immitis.     

Cardiovascular effects of delta 9- and delta 9(11)-tetrahydrocannabinol and their interaction with epinephrine

The administration of delta-9-tetrahydrocannabinol (delta 9-THC, 0.078-5.0 mg/kg, i.v.) to rats anesthetized with pentobarbital caused as much as a 50% decrease in mean arterial blood pressure, heart rate and respiratory rate in a dose-dependent manner. Delta-9(11)-tetrahydrocannabinol (delta 9(11)-THC) was approximately 8-fold less potent than delta 9-THC in its hypotensive effect and had smaller effects on heart and respiratory rates that were not dose-related at doses below 5 mg/kg. Alternate injections of epinephrine (2 micrograms/kg) with vehicle and increasing cannabinoid doses (1.25-5.0 mg/kg) indicated a potentiation of both the duration of the pressor effect and the magnitude of the reflex bradycardic effect of epinephrine by both delta 9- and delta 9(11)-THC. Epinephrine also produced arrhythmias in rats receiving cannabinoids, but not in rats receiving alternate injections of vehicle. It is concluded that both cannabinoids have adverse effects on the cardiovascular system and adverse interactions with epinephrine in rats anesthetized with pentobarbital.     

Pharmacological evidence for a role of gamma-aminobutyric acid A receptor mechanism in modulating nitric oxide synthase activity in rat brain

The role of gamma-aminobutyric acid (GABA) mechanism on the synthesis of nitric oxide (NO) has been investigated by measuring the activity of nitric oxide synthase (NOS) and the concentration of NO in rat brain 15 min after administration of anticonvulsant doses of diazepam (0.25 and 0.5 mg/kg) which is known to activate GABA A receptor for its anticonvulsant action. Diazepam enhanced both NOS activity and the concentration of NO in a dose-dependent manner. A reversal has been observed in animals treated with a convulsant dose of picrotoxin (5 mg/kg) which is known to produce convulsions by blocking GABA A receptor mechanism. These results suggest that a functional interaction occurs between GABA A receptor activity and NO synthesis in the brain.