Possible involvement of GABA mechanisms in central cardiovascular and respiratory control. Studies on the interaction between diazepam, picrotoxin and clonidine

Various doses (0.1-1 mg/kg) of diazepam were given to chloralose anesthetized rats, with both systemic (i.p.) and central injections being tested. Arterial pressure, heart rate, respiration depth and frequency were recorded. Diazepam caused a dose-dependent decrease in the arterial pressure after systemic administration and also decreased it after central administration. However, only intraventricular but not intracisternal injections of diazepam were effective. The hypotensive effect of systemic diazepam was competitively counteracted by pretreatment with picrotoxin, a putative gamma-aminobutyric acid receptor blocking agent. The hypotensive effect of the centrally acting alpha-adrenoreceptor agonist clonidine was not influenced by picrotoxin pretreatment. The effect of diazepam on heart rate was inconsistent. Diazepam caused a reduction of respiratory frequency, which was not counteracted by picrotoxin pretreatment. It is concluded that central gabergic mechanisms are to some extent involved in the hypotensive effect of diazepam, probably at a supramedullary level. The hypotensive effect of a threshold dose of diazepam was blocked by a small dose of clonidine. Likewise, diazepam pretreatment could counteract the hypotension, bradycardia and respiratory frequency reduction caused by a threshold dose of clonidine. These results suggest that gabergic and/or other benzodiazepine-sensitive receptors may interact with alpha-adrenoreceptors in the control of central cardiovascular and respiratory mechanisms.     

Convulsions induced by submaximal dose of pentylenetetrazol in mice are antagonized by the benzodiazepine antagonist Ro 15-1788

The effects of benzodiazepine antagonist Ro 15–1788, alone or with diazepam, were studied in mice on convulsions induced by pentylenetetrazol (PTZ). We found that Ro 15–1788 (1 mg/kg) was able to antagonize the anticonvulsive effects of diazepam (1 mg/kg), but also had, with submaximal doses of PTZ (65 mg/kg), its own anti-convulsive action. At very low doses (0.1 mg/kg), it even potentiated the anticonvulsive effects of diazepam (0.05 mg/kg). This dual action provides evidence for partial agonist properties of the antagonist Ro 15–1788.     

Conductance of recombinant GABA (A) channels is increased in cells co-expressing GABA(A) receptor-associated protein

High conductance gamma-aminobutyric acid type A (GABA(A)) channels (>40 picosiemens (pS)) have been reported in some studies on GABA(A) channels in situ but not in others, whereas recombinant GABA(A) channels do not appear to display conductances above 40 pS. Furthermore, the conductance of some native GABA(A) channels can be increased by diazepam or pentobarbital, which are effects not reported for expressed GABA(A) channels. GABARAP, a protein associated with native GABA(A) channels, has been reported to cause clustering of GABA(A) receptors and changes in channel kinetics. We have recorded single channel currents activated by GABA in L929 cells expressing alpha(1), beta(1), and gamma(2S) subunits of human GABA(A) receptors. Channel conductance was never higher than 40 pS and was not significantly increased by diazepam or pentobarbital, although open probability was increased. In contrast, in cells expressing the same three subunits together with GABARAP, channel conductance could be significantly higher than 40 pS, and channel conductance was increased by diazepam and pentobarbital. GABARAP caused clustering of receptors in L929 cells, and we suggest that there may be interactions between subunits of clustered GABA(A) receptors that make them open co-operatively to give high conductance "channels." Recombinant channels may require the influence of GABARAP and perhaps other intracellular proteins to adopt a fuller repertoire of properties of native channels.     

Diazepam effects on Ehrlich tumor growth and macrophage activity in mice

Besides the central gabaergic receptors described for benzodiazepines, peripheral type binding sites (PBR) were also identified for these molecules in endocrine steroidogenic tissues, immune organs and cells, such as macrophages and lymphocytes. PBR activation was reported to decrease innate immunity and host defense. The present experiment was designed to analyze the effects of diazepam on Ehrlich tumor growth, and on macrophage activity of Ehrlich tumor bearing mice. Results showed that diazepam (3.0 mg/kg/day, for 7 days) increased the number of Ehrlich tumor cells and the volume of tumor-induced ascitic fluid. These effects were not observed after smaller doses of diazepam, suggesting a dose-dependant effect. Furthermore, our results show that 3.0 mg/kg of diazepam, administered daily, for 2 days, decreased (1) the number of peritoneal leukocytes retrieved after injection of the Ehrlich tumor, (2) the percents of macrophage spreading, and (3) the levels of macrophage NO production. Diazepam (3.0 mg/kg/day for 2 days) had no effect on macrophage phagocytosis or on H2O2 production. The present data is discussed based on a direct and/or indirect action of diazepam. Particularly, our findings might be due to a direct effect of diazepam on PBRs present on macrophages and tumor cells, or could still be mediated by PBR stimulation within the hypothalamus-pituitary-adrenal (HPA) axis.     

Modification of the anticonvulsant efficacy of diazepam by Ro-15-1788 in the kindled amygdaloid seizure model

The effects of various doses of diazepam and the new central benzodiazepine antagonist Ro-15-1788 were investigated in fully amygdaloid kindled rats. Diazepam had a pronounced dose-dependent anticonvulsant effect in this model. Ro-15-1788 dose-dependently reduced the behavioral ranks of the elicited kindled seizures to a maximum of 60% of control without consistently modifying the afterdischarge duration. No prestimulation convulsant effects were seen with Ro-15-1788. When 2 mg/kg i.p. of Ro-15-1788 was given after various doses of diazepam, the prestimulation sedation and ataxia anticonvulsant effects of diazepam (0.5-2.0 mg/kg) were attenuated by treatment with 2 mg/kg dose of Ro-15-1788. At the low dose of diazepam (0.25 mg/kg), increased reduction of behavioral rank and after discharge duration was seen after the 2 mg/kg dose of Ro-15-1788. Thus, Ro-15-1788 appears not to have proconvulsant properties in the kindled amygdaloid seizure model. Further, Ro-15-1788 appears to have some anticonvulsant properties of its own. Mixed agonist and antagonist effects were seen with Ro-15-1788 when given after various doses of diazepam in this model.     

Differential mediation of the anticonvulsant effects of carbamazepine and diazepam

Possible mechanisms of action of carbamazepine and diazepam on amygdala-kindled seizures were studied using compounds acting at the central and "peripheral-type" benzodiazepine binding sites. Ro-15-1788, a selective antagonist at the central benzodiazepine site, blocked the anticonvulsant effect of diazepam, but not of carbamazepine. In contrast, Ro5-4864, which acts at the "peripheral-type" benzodiazepine site, blocked the anticonvulsant effect of carbamazepine, but not of diazepam. The effect of Ro5-4864 was itself reversed by PK-11195, a compound that displaces Ro5-4864 binding in vitro and in vivo. These data indicate that the anticonvulsant effects of carbamazepine and diazepam on amygdala-kindled seizures are differentially mediated and suggest that the "peripheral-type" benzodiazepine binding site is functionally involved in the anticonvulsant effect of carbamazepine.     

Morphine impairs Acetylcholine Release but facilitates Acetylcholine Action at a Skeletal Neuromuscular Junction

THERE is considerable evidence that morphine impairs the release of acetylcholine (ACh) at cholinergic synapses in the brain^1–5, although there are considerable problems in determining the exact site and mechanism of this action. A simple synaptic model would be useful for pursuing this problem and the question arises whether this action of morphine is universal for cholinergic synapses or is restricted to particular sites. Morphine impairs the release of ACh at peripheral muscarinic sites^6–8 but there are no reports about the effects of morphine on ACh release at nicotinic neuromuscular sites. We have reported that both morphine and nalorphine block neuromuscular transmission in amphibian and mammalian skeletal neuromuscular preparations^9,10, apparently as a result of impairment of ACh release. We have now determined by direct measurement that morphine impairs ACh release at a skeletal neuromuscular junction.     

Butorphanol tartrate induces feeding in rats

Peripheral administration of butorphanol tartrate markedly enhanced feeding from 0800 to 1400 hours when compared with vehicle controls. Butorphanol tartrate feeding was not antagonized by doses of naloxone as high as 10 mg/kg. These data support the concept that the kappa or sigma opiate receptors are involved in feeding behavior.It is well recognized that the endogenous opiates play a role in the central regulation of appetite (1, 2, 3, 4). Numerous studies have shown that The endogenous opioid peptides and morphine can initiate feeding under various conditions (5–12) whereas the opiate antagonist, naloxine can reduce food consumption (13–20). Recently, the endogenous opiod peptide, dynorphin, has been reported to enhance food intake (12–25).Much evidence has been accumulated indicating that a number of opiate receptors are present in the brain, each one having a high affinity for a specific endogenous opioid peptide (26, 27). Both the cyclazocine related compounds (28) and the feeding enhancer, dynorphin (29–32), have been reported to be specific kappa receptor agonists. In the present study, we report on the effect of the morphinan congener, butorphanol tartrate (33), on ingestive behaviour.     

Adenosine A2A Receptors Activation Facilitates Neuromuscular Transmission in the Pre-Symptomatic Phase of the SOD1(G93A) ALS Mice,but Not in the Symptomatic Phase

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease leading to motor neuron dysfunction resulting in impairment of neuromuscular transmission. A_2A adenosine receptors have already been considered as a potential therapeutical target for ALS but their neuromodulatory role at the neuromuscular junction in ALS remains to be clarified. In the present work, we evaluated the effects of A_2A receptors on neuromuscular transmission of an animal model of ALS: SOD1(G93A) mice either in the pre-symptomatic (4–6 weeks old) or in the symptomatic (12–14 weeks old) stage. Electrophysiological experiments were performed obtaining intracellular recordings in Mg²⁺ paralyzed phrenic nerve-hemidiaphragm preparations. Endplate potentials (EPPs), quantal content (q. c.) of EPPs, miniature endplate potentials (MEPPs) and giant miniature endplate potential (GMEPPs) were recorded. In the pre-symptomatic phase of the disease (4–6 weeks old mice), the selective A_2A receptor agonist, CGS 21680, significantly enhanced (p<0.05 Unpaired t-test) the mean amplitude and q.c. of EPPs, and the frequency of MEPPs and GMEPPs at SOD1(G93A) neuromuscular junctions, the effect being of higher magnitude (p<0.05, Unpaired t-test) than age-matched control littermates. On the contrary, in symptomatic mice (12–14 weeks old), CGS 21680 was devoid of effect on both the amplitude and q.c. of EPPs and the frequency of MEPPs and GMEPPs (p<0.05 Paired t-test). The results herein reported clearly document that at the neuromuscular junction of SOD1(G93A) mice there is an exacerbation of A_2A receptor-mediated excitatory effects at the pre-symptomatic phase, whereas in the symptomatic phase A_2A receptor activation is absent. The results thus suggest that A_2A receptors function changes with ALS progression.     

Etomidate evokes synaptic vesicle exocytosis without increasing miniature endplate potentials frequency at the mice neuromuscular junction

Etomidate is an intravenous anesthetic used during anesthesia induction. This agent induces spontaneous movements, especially myoclonus after its administration suggesting a putative primary effect at the central nervous system or the periphery. Therefore, the aim of this study was to investigate the presynaptic and postsynaptic effects of etomidate at the mouse neuromuscular junction (NMJ). Diaphragm nerve muscle preparations were isolated and stained with the styryl dye FM1-43, a fluorescent tool that tracks synaptic vesicles exo-endocytosis that are key steps for neurotransmission. We observed that etomidate induced synaptic vesicle exocytosis in a dose-dependent fashion, an effect that was independent of voltage-gated Na⁺ channels. By contrast, etomidate-evoked exocytosis was dependent on extracellular Ca²⁺ because its effect was abolished in Ca²⁺-free medium and also inhibited by omega-Agatoxin IVA (30 and 200 nM) suggesting the participation of P/Q-subtype Ca²⁺ channels. Interestingly, even though etomidate induced synaptic vesicle exocytosis, we did not observe any significant difference in the frequency and amplitude of miniature end-plate potentials (MEPPs) in the presence of the anesthetic. We therefore investigated whether etomidate could act on nicotinic acetylcholine receptors labeled with α-bungarotoxin-Alexa 594 and we observed less fluorescence in preparations exposed to the anesthetic. In conclusion, our results suggest that etomidate exerts a presynaptic effect at the NMJ inducing synaptic vesicle exocytosis, likely through the activation of P-subtype voltage gated Ca²⁺ channels without interfering with MEPPs frequency. The present data contribute to a better understanding about the effect of etomidate at the neuromuscular synapse and may help to explain some clinical effects of this agent.     

LY 294002 inhibits adenosine receptor activation by a mechanism independent of effects on PI-3 kinase or casein kinase II

Adenosine reduces both evoked and spontaneous calcium-dependent acetylcholine (ACh) release through a mechanism downstream of calcium entry at amphibian motor nerve endings (Silinsky EM. J Physiol 1984; 346: 243–56). LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), an inhibitor of both phosphoinositide-3 kinase (PI-3 kinase) and casein kinase II, has been reported to increase spontaneous ACh release reflected in miniature endplate potential (MEPP) frequencies independently of intraterminal calcium at the frog neuromuscular junction (Rizzoli SO, Betz WJ. J Neurosci 2002; 22: 10680–9). It has been suggested that the increase in MEPP frequency caused by LY 294002, is mediated through an action on synaptotagmins, vesicle associated calcium sensors believed to trigger synaptic vesicle exocytosis. We thus examined the effects of adenosine on MEPP frequencies and evoked ACh release reflected as endplate potentials (EPPs) in order to determine if the presumed calcium-independent ACh release is affected by adenosine. We also wanted to determine if PI-3 kinase or casein kinase II is involved in mediating or modulating the inhibitory effects of adenosine. To these ends, we examined the effects of adenosine in the presence of LY 294002, wortmannin (a highly selective the PI-3 kinase inhibitor), or DRB (5,6-dichlorobenzimidazole riboside, an inhibitor of casein kinase II). LY 294002 reduced the sensitivity of both MEPP frequencies and the nerve-evoked calcium dependent EPPs to adenosine. The occlusive effects of LY 294002 on the actions of adenosine on MEPPs and EPPs were overcome by increasing adenosine concentration. Neither wortmannin nor DRB had any effect on the sensitivity of the EPPs to adenosine indicating that neither PI-3 kinase nor casein kinase II inhibition mediates the reduction in motor-nerve terminal sensitivity to adenosine produced by LY 294002. The results indicate a competitive relationship between LY 294002 and adenosine at A₁ receptors at the frog neuromuscular junction. This effect is independent of the previously described effects of LY 294002 on the exocytotic process, and is also independent of PI-3 kinase or casein kinase II.     

Sensitization by chronic diazepam treatment of A2A adenosine receptor-mediated relaxation in rat pulmonary artery

The effects of a 10-day i.p. treatment of rats with diazepam on responses to subtype selective adenosine receptor agonists were studied 3 h, 2 and 8 days after termination of diazepam treatment in isolated cardiovascular tissues possessing distinct adenosine receptors. After long-lasting diazepam exposure, the relaxation elicited by the specific A2A receptor agonist CGS 21680 was enhanced in rat main pulmonary arteries (a tissue containing A2A adenosine receptors). The increased sensitivity of A2A receptors observed 3 h and 2 days after withdrawal of diazepam was completely restored by the 8th day of the wash-out period. N6-cyclopentyladenosine (CPA)-induced suppression in mechanical activity of electrically stimulated rat atrial myocardium (a tissue containing A1 adenosine receptors) was not altered following diazepam treatment. In order to reveal the possible role of inhibition of membrane adenosine transport in the effects of diazepam (a moderate inhibitor of membrane adenosine transport), the action of a 10-day treatment with dipyridamole or S-(p-nitrobenzyl)-6-thioinosine (NBTI; prototypic adenosine uptake inhibitors) was also studied. Dipyridamole or NBTI treatment, like diazepam, increased the responsiveness of rat pulmonary artery to CGS 21680, but did not influence the cardiodepressive effect of CPA in electrically driven left atrial myocardium. The CGS 21680-induced relaxations were significantly antagonized by 10 nM ZM 241385 (a selective A2A adenosine receptor antagonist) in vessels of diazepam-treated rats. The relaxation responses to verapamil were unaltered in pulmonary arteries obtained from animals chronically treated with diazepam, dipyridamole or NBTI. These results suggest that chronic diazepam treatment is able to enhance the A2A adenosine receptor-mediated vascular functions, but does not modify the responses mediated via A1 receptors of rat myocardium, where nucleoside transport inhibitory sites of membrane are of a very low density. It is possible that sensitization of A2A adenosine receptor-mediated vasorelaxation is due to a long-lasting inhibition of membrane adenosine transporter during diazepam treatment.     

Diazepam and propofol used as anesthetics during open-heart surgery do not cause chromosomal aberrations in peripheral blood lymphocytes

Diazepam is a benzodiazepine with anticonvulsant, anxiolytic, sedative and muscle-relaxing properties. Many aspects of its toxicity have been investigated, including genotoxic and carcinogenic effects in various model systems. However, it is still unclear whether diazepam is in fact a genotoxic agent. Propofol is a rapid-onset, short-acting intravenous anesthetic agent. It is used widely for the induction and maintenance of anesthesia as well as for long-term sedation in intensive care units. There is limited information in the literature on its genotoxic effects. Both drugs are commonly used as anesthetic in patients undergoing open-heart surgery. Therefore, we investigated the possible genotoxic effects of propofol and diazepam in those patients, using a chromosomal aberration (CA) assay. Peripheral blood samples were collected from 45 patients before induction of anesthesia and at the end of the anesthesia with diazepam or propofol. In Group I (n=24), anesthesia was induced with 0.2 mg kg(-1) diazepam and 10 microg kg(-1) fentanyl. In Group II (n=21), anesthesia was induced with 1 mg kg(-1) propofol and 10 microg kg(-1) fentanyl. Pancuronium bromide (0.1 mg kg(-1)) was administered for skeletal muscle relaxation in both groups. Anesthesia was maintained by diazepam administration at 5 mg kg(-1) in Group I or by continuous propofol administration at 2-4 mg (kg h)(-1) in Group II. All patients received 0.02 mg kg(-1) pancuronium and 5 microg kg(-1) fentanyl boluses at 30-40 min intervals for anesthesia maintenance. Body temperature was controlled during bypass in the two groups. We found that the mean frequency of CAs in both groups before and at the end of the anesthesia were not statistically significantly different. Our analysis also indicated that age, smoking habit and gender were not confounding factors. In conclusion, our results indicate that diazepam and propofol do not exert genotoxic effects in blood cells during open-heart surgery.     

Effects of melatonin, morphine and diazepam on formalin-induced nociception in mice

The possible analgesic effect of melatonin was investigated in young male ICR mice. The formalin test which elicits typically 2 phases of pain response, the acute (first) phase and tonic (second) phase, was used. The test was performed in the late light period when the mice have been reported to be more sensitive to pain. Compared to control mice, no significant difference in nociceptive response was observed when melatonin was injected intraperitoneally at doses of 0.1, 5, and 20, mg/kg body weight. The combined effects of melatonin with diazepam and/or morphine, were also investigated. Melatonin, injected at 20 mg/kg 15 min before formalin test, significantly increased the antinociceptive response of diazepam (1 mg/kg) or morphine (5 mg/kg) in the second phase. In addition, when melatonin was given at 20 mg/kg together with diazepam and morphine, antinociceptive responses in both the first and second phase were increased. These data indicate the synergistic analgesia effect of melatonin with morphine and diazepam and suggest the possible involvement of melatonin as an adjunct medicine for pain patients.     

Diazepam augments gender differences in cutaneous LD flux response to local cooling

Cutaneous vasoconstriction in response to local cooling is normally greater in females than in males. Cold induces amplification of alpha2-adrenoceptor affinity for norepinephrine and increases reflex sympathetic thermoregulatory output. Benzodiazepines are drugs with very well-known binding to the central and peripheral benzodiazepine receptors. Besides these effects they decrease sympathetic output and as it was shown in the last decade they act synergistically with alpha-adrenoceptors. In the present study we tested the hypothesis that the benzodiazepine diazepam interacts with an alpha-adrenoceptor mechanism at the level of microcirculation. We measured laser-Doppler blood flux changes provoked by local cooling before and after oral application of a low dose of diazepam (5 mg) in 9 healthy males and in 11 healthy females with regular menstrual cycles. The results of our experiments show that in females there is a significant reduction (ANOVA, p < 0.05) in laser-Doppler flux during the first four minutes of cooling after taking of diazepam. In males, there is no significant difference in the responses to cold before and after an application of diazepam. Our results suggest that diazepam, in addition to its well-known effect on BZ receptors may also interact with alpha2C-adrenoceptors in the vessel wall during local cooling.     

Effect of neuromuscular blocking drugs on arterial pressure and heart rate in rats

The effects of neuromuscular blocking drugs on mean arterial pressure (MAP) and heart rate (HR) were studied in rats which were anaesthetised, tracheotomized and ventilated artificially. The arterial pressure was recorded from the carotid artery. Seven neuromuscular blocking drugs were injected intravenously at doses of 1, 5, and 25 mumol/kg. d-Tubocurarine, alcuronium and vecuronium lowered MAP in a dose dependent manner (maximum 40%). Succinylcholine, 1 mumol/kg, reduced MAP and HR, whereas the two larger doses increased them. Gallamine, 25 mumol/kg, or metocurine and pancuronium, 1 or 5 mumol/kg, each, induced short-lasting rises in MAP. Pancuronium, 25 mumol/kg, decreased MAP by 25%, while the largest dose of metocurine appeared to be toxic. The cardiovascular responses to neuromuscular blocking drugs were antagonized or abolished by pretreatment with the ganglionic blocking agent pentolinium. Pentolinium itself markedly reduced MAP and HR. After ganglionic blockade and restoration of MAP by noradrenaline infusion, all the neuromuscular blocking drugs induced short-lasting increases in MAP (10-30%), except d-tubocurarine which still reduced MAP by 30%, a fall which, in contrast to the effect in the absence of the pretreatments, was transient. This response to d-tubocurarine could not be abolished by a combined pretreatment with H1 and H2 antagonists showing that the hypotensive effect of this drug was not due to the liberation of histamine. These results suggest that the cardiovascular responses to neuromuscular blocking drugs in rats might be partly due to ganglionic effects. Other mechanisms are also involved since after the restoration of blood pressure by noradrenaline during the ganglionic blockade some cardiovascular responses to these drugs still occurred.     

Differences in the central actions of arachidonic acid and prostaglandin F2α between spontaneously hypertensive and normotensive rats

Prostaglandin F_2α (PGF_2α) is one of the most common metabolites of arachidonic acid (AA) in rat brain. When administered intracerebroventricularly (i.c.v.) to rats, both AA and PGF_2α exert dose-related hypertensive, tachycardic and hyperthermic effects. Metabolic alterations in the endogenous formation of some prostaglandins in the brain-stem of spontaneously hypertensive rats (SHR) have been reported. Therefore the central effects of AA and PGF_2α on blood pressure, heart rate and body temperature were studied both in SHR and normotensive Wistar rats (NR) under urethane-anaesthesia. The hypertensive effect of AA i.c.v. (0.01–100 μg/rat) was larger in magnitude in SHR than in NR, but there was no significant difference in the AA-induced changes of heart rate and body temperature between the groups. Pretreatment of NR with sodium meclofenamate (1 mg/rat i.c.v.) antagonised the central effects of AA indicating that these effects are not due to AA itself but to its conversion to prostaglandins. Unlike the effects of AA, the central hypertensive, tachycardic and hyperthermic responses to PGF_2α (0.5–50 μg/rat i.c.v.) were significantly attenuated in SHR. The present results obtained with AA are compatible with the previous assumption that the synthesis of prostaglandins in the brain of SHR might differ from that in NR. The results also demonstrate that the central effects of PGF_2α are reduced in SHR.     

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.     

Effects of treatment with GABA(A) receptor subunit antisense oligodeoxynucleotides on GABA-stimulated 36Cl- influx in the rat cerebral cortex

GABA(A) receptor function was studied in cerebral cortical vesicles prepared from rats after intracerebroventricular microinjections of antisense oligodeoxynucleotides (aODNs) for alpha1, gamma2, beta1, beta2 subunits. GABA(A) receptor alpha1 subunit aODNs decreased alpha1 subunit mRNA by 59+/-10%. Specific [3H]GABA binding was decreased by alpha1 or beta2 subunit aODNs (to 63+/-3% and 64+/-9%, respectively) but not changed by gamma2 subunit aODNs (94+/-5%). Specific [3H]flunitrazepam binding was increased by alpha1 or beta2 subunit aODNs (122+/-8% and 126+/-11%, respectively) and decreased by gamma2 subunit aODNs (50+/-13%). The "knockdown" of specific subunits of the GABA(A )receptor significantly influenced GABA-stimulated 36Cl- influx. Injection of alpha1 subunit aODNs decreased basal 36Cl- influx and the GABA Emax; enhanced GABA modulation by diazepam; and decreased antagonism of GABA activity by bicuculline. Injection of gamma2 subunit aODNs increased the GABA Emax; reversed the modulatory efficacy of diazepam from enhancement to inhibition of GABA-stimulation; and reduced the antagonist effect of bicuculline. Injection of beta2 subunit aODNs reduced the effect of diazepam whereas treatment with beta1 subunit aODNs had no effect on the drugs studied. Conclusions from our studies are: (1) alpha1 subunits promote, beta2 subunits maintain, and gamma2 subunits suppress GABA stimulation of 36Cl- influx; (2) alpha1 subunits suppress, whereas beta2, and gamma2 subunits promote allosteric modulation by benzodiazepines; (3) diazepam can act as an agonist or inverse agonist depending on the relative composition of the receptor subunits: and (4) the mixed competitive/non-competitive effects of bicuculline result from activity at alpha1 and gamma2 subunits and the lack of activity at beta1 and beta2 subunits.