Baclofen modifies via the release of monoamines the synaptic depression, frequency facilitation, and posttetanic potentiation observed at an identified cholinergic synapse of Aplysia californica

1. The mechanism of action of baclofen was studied at a cholinergic synapse of Aplysia. This synapse, called RC1-R15, can be activated by a minimal stimulation of the right pleurovisceral connective and is recorded in cell R15 of Aplysia californica. Repeated stimulation of synapse RC1-R15 produces depression, followed by frequency facilitation and by posttetanic potentiation (PTP). 2. Perfusion with baclofen (3 x 10(-5) or 5 x 10(-5) M) reduces the size of all excitatory postsynaptic potentials (EPSPs) of synapse RC1-R15 produced by a train of 100 stimuli at 1.5 Hz. It also reduces the synaptic depression and PTP but increases the frequency facilitation. These effects are similar to those produced by gamma-aminobutyric acid (GABA), serotonin, and dopamine on this synapse. 3. When the preparation is perfused with bicuculline or picrotoxin, two antagonists of GABA, the effects of baclofen are not antagonized. However, when baclofen is perfused in the presence of SQ10,631 (an antagonist of serotonin) or butaclamol (an antagonist of dopamine), its effects are partially blocked. 4. To determine if baclofen produces its action by direct interaction with aminergic receptors or by liberating the amines from some nerve endings, a few animals were treated with reserpine to deplete the aminergic pool. Following this treatment no effects were obtained with baclofen suggesting that it acts by liberating dopamine and serotonin or some other amines. 5. In animals treated with reserpine, GABA still produces its normal effects (reduction of EPSP size, synaptic depression, posttetanic potentiation, and increase of facilitation), indicating that baclofen does not act directly on the GABA receptor located on the presynaptic terminal.     

Reserpine-induced central effects: pharmacological evidence for the lack of central effects of reserpine methiodide

Reserpine, an alkaloid from Rauwolfia serpentina, was widely used for its antihypertensive action. However, its use has been reduced because of its sedative and extra pyramidal symptoms. In the present investigation, reserpine methiodide (RMI), a quaternary analogue of reserpine, was synthesized and pharmacologically evaluated in rats and mice for its central (barbiturate hypnosis, spontaneous motor activity, body temperature, and avoidance of conditioned response) and peripheral actions (blood pressure) in comparison with reserpine. The results indicate that reserpine produced a dose-dependent depression of the central nervous system. RMI at doses equal to and double the equimolar doses of reserpine did not produce any behavioural changes compared with control animals. Nevertheless, both reserpine and RMI were found to produce dose-dependent reduction in the blood pressure of anaesthetized rats, although only at higher doses of RMI, indicating that quaternization of reserpine not only attenuated the entry of RMI into the central nervous system, but also reduced its access to the target tissue in the periphery. It is speculated that the hypotensive actions of RMI may also be due to peripheral depletion of catecholamines.     

Increased acoplasmic transport of H3-dopamine in nigro-neostriatal neurons after reserpine

Recent evidence suggests that dopamine can undergo axoplasmic transport in nigro-neostriatal neurons by binding to amine storage granules. In the present experiments it was demonstrated that reserpine pretreatment (10 mg/kg) 24 hours before stereotaxic injections of ³H-DOPA or ³H-dopamine into the substantia nigra increases the amount of ³H-dopamine transported to the neostriatum by about 300 percent. The activity recovered from the substantia nigra was significantly reduced by reserpine pretreatment however. Stereotaxic injection of ¹⁴C-leucine into the substantia nigra indicated that neither fast nor slow axoplasmic transport of protein was influenced by reserpine pretreatment in these same neurons. The increased transport of dopamine appears therefore to be due to a relatively selective action of reserpine. The results suggest that reserpine either (i) increases the binding of dopamine to newly synthesized amine storage granules, (ii) increases the number of newly synthesized amine storage granules, or (iii) accelerates the rate of transport of amine storage granules. In addition, the results support the view that reserpine can increase the membrane permeability of adrenergic neurons to the outward movement of catecholamines.     

Reserpine-Induced Processing of Chromogranin A in Cultured Bovine Adrenal Chromaffin Cells

The effect of reserpine on the processing of the secretory granule protein chromogranin A (CgA) in isolated bovine adrenal chromaffin cells was investigated using two radioimmunoassays employing site-specific antisera. The two antisera were directed against closely associated regions of the CgA molecule which would be exposed by specific processing: antiserum L331 was raised against the C-terminus of the regulatory peptide pancreastatin, and the second antiserum, L300, was raised against the synthetic peptide [Tyr0]CgA306-313 (YLSKEWEDA), a sequence that lies immediately C-terminal to pancreastatin and adjacent to a dibasic amino acid cleavage site. Chronic reserpine treatment of chromaffin cells produced a time- and dose-dependent increase in processing, as demonstrated by an increase in pancreastatin- and YLSKEWEDA-immunoreactivity (ir). The reserpine-induced rise in pancreastatin-ir was due predominantly to an increase in pancreastatin 1-47, whereas the rise in YLSKEWEDA-ir was due to increases in three polypeptides: a 51-kDa YLSKEWEDA-ir polypeptide, CgA297-313, and CgA248-313. The latter predominated. The action of reserpine on both pancreastatin- and YLSKEWEDA-ir was found to be largely inhibited by the protein synthesis inhibitor cycloheximide. The results show that treatment of isolated chromaffin cells with reserpine induces both the selective proteolytic processing and peptidyl-glycine amidation of CgA and its derived fragments. As reserpine has a similar effect on proenkephalin in chromaffin cells, the results suggest that reserpine induces a general increase in the activity of the processing enzymes, partially by an increase in protein synthesis.     

Modifying action of drug preparations on the effect of promutagen compounds

The Ames test has shown that the action of nitrosomorpholine and cyclophosphane promutagens on bacteria of Salmonella typhimurium TA 1950 increases while using S-9 liver fraction of rats treated with pharmaceuticals of amidopyrine, reserpine and pyrazidol and decreases while using those treated with phenazepam.     

Behavior of plasma cortisol during testing with insulin and with LVP in obese subjects compared to normal subjects after resperine administration]

The behaviour of plasma cortisol levels was studied in fifteen overweight subjects with comparison to normal subjects. In eight subjects the insulin hypoglycemic test was performed before and after medication with reserpine (4mg/24HR); the other seven subjects were tested with LVP before and after treatment with reserpine. In both cases, premedication with reserpine significantly reduce the cortisolemic response. This behaviour is similar to the response observed in normal subjects.     

Characterization of the interference of T cell activation by reserpine

It has been suggested that reserpine blocks expression of delayed hypersensitivity (DH) reactions by depleting tissue mast cells of serotonin, thereby preventing a T cell-dependent release of mast cell serotonin necessary to localize and to amplify the DH response. However, reserpine blocks expression of DH in mast cell-deficient mice. Recently, we showed that the ability of reserpine to interfere with the expression of contact sensitivity was independent of an effect on mast cells, but reflected an effort of the drug on effector T cell function. In the present study we evaluated the mechanisms by which reserpine abrogates the expression of T cell functions. By using human peripheral blood mononuclear cells or enriched T cell populations we found that the drug inhibited, in a dose-dependent fashion, the proliferation of T cells after mitogen stimulation. Reserpine also interfered with the mitogen-induced IL-2 production by these cells, but the IL-2 receptor expression, as measured by immunofluorescence, was unaffected. Despite this, in the continuous presence of reserpine, exogenous IL-2 did not bypass reserpine inhibition of PHA-induced proliferation. By using the fluorescent indicator quin-2 we have demonstrated that preincubation with reserpine prevented the increase of cytosolic free calcium, which accompanies PHA-induced proliferative responses of human T lymphocytes. These results identify the sites of action of reserpine in human T lymphocytes and are sufficient to explain its ability to block cell-mediated immune responses in vitro and in vivo.     

Effects of the sulfhydryl reagentN-ethylmaleimide on reserpine binding to the catecholamine transporter in chromaffin granule membranes

1. Catecholamines are transported into chromaffin granules via a carrier-mediated, active-transport process which is inhibited by micromolar concentrations of the sulfhydryl reagent, N-ethylmaleimide (NEM). Reserpine is a very potent, competitive inhibitor of the catecholamine transporter and can be used to investigate the characteristics of the catecholamine transporter. 2. The purpose of this study was to determine whether [3H]reserpine binding to the catecholamine transporter present in chromaffin granule membranes isolated from bovine adrenal glands was also inhibited by NEM and, if so, whether this was a direct or an indirect effect of NEM on the catecholamine transporter. 3. Both [3H]norepinephrine transport into and [3H]reserpine binding to the chromaffin granule ghosts isolated from bovine adrenal glands are inhibited by NEM, with IC50 values of 0.63 +/- 0.02 and 2.8 +/- 0.66 microM, respectively. 4. Mg and ATP protected both the [3H]norepinephrine transport into the ghosts and the [3H]reserpine binding to the transporter from inhibition by NEM, shifting the IC50 values to 260 +/- 43 and 120 +/- 29 microM, respectively. 5. NEM inhibition of the catecholamine transport and reserpine binding appears to be due to an action on the proton translocator associated with the Mg ATPase enzyme rather than a direct action on the catecholamine transporter since (a) the concentration of NEM required to inhibit formation of a membrane potential is similar to that required to inhibit [3H]norepinephrine transport into and [3H]reserpine binding to the ghosts and (b) Mg and ATP protected the proton translocation and [3H]norepinephrine transport into the ghosts, and [3H]reserpine binding to the ghosts, from inhibition by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reproduction of passive avoidance reactions after neurotensin microinjection into nucleus accumbens of rat brain against the background of reserpine action

The purpose of the study was to reveal the features of the influence of neurotensin injected into the nucleus accumbens on behaviour of rats after systemic administration of reserpine in the dose of 2 mg/kg. Reprodution of passive avoidance conditioned reactions, painful stimulation aftereffects on locomotor activity in the "open field", and behavior in the elevated plus-maze were studied. It was shown that reserpine administration impaired the reproduction of passive avoidance reactions and weakened the oppressing aftereffect of painful stimulation, which can be due to a decrease in anxiety in rats. Neurotensin prevented disorders in the defensive behavior evoked by reserpine and intensified the state of anxiety in the elevated plus-maze. The positive influence ofneurotensin on the reproduction of passive avoidance can be associated with the recovery of the anxiogenic effect of painful stimulation destroyed by reserpine. Thus, neurotensin injected into the nucleus accumbens could normalize the balance of brain monoaminergic systems.     

Comparative effects of 6-hydroxy-dopamine and of reserpine on quabain toxicity in the rat

The toxic effects of ouabain were compared in normal, and in 6-OH-DA and reserpine pretreated rats. Reserpine significantly reduced the lethality of intravenous ouabain. This effect does not seem related to depletion of peripheral catecholamines, since 6-OH-DA did not reduce mortality significantly. Since reserpine, but not 6-OH-DA, does cross the blood brain barrier it is suggested that the protective effect of reserpine might be related to its action on the central nervous system.     

Increase of the peroxidase activity of mouse submaxillary gland by reserpine

The peroxidase activity of mouse submaxillary gland was found to be elevated by about 128% at 22 hr. after the administration of reserpine (0.5 mg/kg). This effect of reserpine was observed in rat also. Neither pretreatment with 6-hydroxy dopamine (6-OH dopamine) nor surgical sympathetic denervation could abolish the increase of the peroxidase activity elicited by reserpine. Also, treatment with propranolol, dibenamine or atropine sulfate failed to reverse the effect of reserpine. These results suggest that neither catecholamine nor acetyl choline is involved in this reserpine action.     

Animal model for cystic fibrosis: pulmonary clearance of Staphylococcus aureus in mice treated with reserpine.

Since it has been demonstrated that chronic administration of reserpine to rats produces CF-like alterations, we have studied the lung resistance to bacterial infections in animals treated with reserpine. Mice of the BALB/c inbred strain were injected subcutaneously with different doses of reserpine for 7 days; after, they were submitted to an infective aerosol containing $\text{Staphylococcus aureus}$ in a nebulization chamber. With each pair of exposed animals, an individual value of the uncleared bacteria ratio (UBR) at 4 hours was obtained. Reserpinized animals showed a significant increase in UBR values when compared to control mice (p < 0.05 and p < 0.01 depending on the doses), suggesting an impairment of the lung antibacterial defenses. Although pharmacological doses of reserpine produce catecholamine depletion, we can not conclude that this action upon the nervous system is the only cause of the CF-like response we detected. In conclusion, the observed UBR decrease lends support to the concept of a reserpine-induced CF animal model.     

Differential Roles of Raphe Nuclei in the Regulation of Dopamine β-Hydroxylase in the Adrenal Gland of the Rat

The effect of reserpine on the activity of dopamine beta-hydroxylase (DBH) in the adrenal gland of the rat was determined following electrolytic lesion of the dorsal raphe nucleus (DRN) or medial raphe nucleus (MRN). In sham-operated rats, as well as in those with a lesion of the DRN, there was no significant modification of the action of reserpine on this enzyme. However, a lesion of MRN potentiated the inducing action of the drug. A specific role of MRN in the serotonergic regulation of adrenal DBH is suggested by this work.     

Apomorphine induced biting and fighting behaviour in reserpinized rats and an approach to the mechanism of action

Various patterns of aggressive behaviour have been induced by drugs. In the present study, the biting and the fighting responses were induced in rats by apomorphine alone, and reserpine plus apomorphine combination respectively, and these could be blocked completely by a dopamine receptor blocking agent. Dopamine, norepinephrine and clonidine given intraperitoneally or intraventricularly failed to induce these responses. Chemical agents known to increase the concentration of dopamine in the brain, induced the biting, but not the fighting response, whereas these behavioural patterns were more intense due to apomorphine in the rats pretreated with reserpine and dopamine or α-methyltyrosine and reserpine combinations. In amphetamine pretreated rats, apomorphine induced intense biting after 10 min and a few bouts of fighting after 30 min. It is suggested that (i) the receptors on which apomorphine acts may be called ‘Apomorphine Receptor’ rather than ‘Dopamine Receptor’, (ii) dopamine incompletely activates these receptors which are sensitised in the absence of catecholamines and induce a higher degree of stereotyped behaviour i.e. fighting, due to apomorphine.     

Monoamine oxidase inhibitors and the depletion of 5-hydroxytryptamine from enterochromaffin cells by reserpine

Summary The influence of Nialamide (a monoamine oxidase inhibitor) on the depletion of 5-hydroxytryptamine from enterochromaffin cells, has been studied in rabbits. Experimental animals were given varying doses of Nialamide in N/10 HCl by single or repeated injections, followed by injections of reserpine. Control animals were given N/10 HCl followed by reserpine or by reserpine solvent. Nialamide did not prevent the depletion of enterochromaffin cell granules by reserpine in any of the experimental animals.     

Effect of reserpine administered during infancy on brain catecholamines and adult behaviour in the rat

Reserpine (0.1 mg/kg/day) was administered to rats from 11 through 30 days of age. During and after administration of reserpine, concentrations of catecholamines, epinephrine and norepinephrine, in the brain were estimated. Levels of catecholamines were about 30 per cent of normal during the period of reserpine administration. Approximately 3 weeks were required for these levels to return to normal. When animals were 95-100 days of age, they were deprived of food and were trained to press a bar for food. When the rate of responding became stable, the animals were subjected to three successive extinctions at daily intervals and the increase in response rate after the onset of each extinction was determined. This extinction-induced increase in response rate was greater for previously reserpinized animals than controls during the second and third extinctions, but not the first. These findings are interpreted as a decreased ability of the animals, reserpinized during infancy, to learn to respond discriminatively during non-reinforcement (extinction). Thus, an effect of reserpine administration during infancy on a type of behaviour in the adult has been demonstrated. This occurs after the catecholamine-depleting effect of the reserpine has been fully dissipated.     

Effects of verapamil on behavioral and microcirculatory disorders in pain syndrome of spinal etiology

In the experiments on Wistar rats with pain syndrome of spinal origin (PSSO) caused by the generator of pathologically enhanced excitation (GPEE) in dorsal horns of the spinal cord lumbosacral segments, it was shown that the intravenous verapamil injection (1.25 mg/kg) undoubtedly decreased behaviour response and improves the state of microcirculation. The compound of 10-fold decreased dose does not affect the behaviour response and microcirculation. When PSSO exists, the intravenous injection of analgin (150 mg/kg) produced an effect on the behaviour response and does not produce any action on microcirculation. When verapamil reaches the dorsal surface of the spinal cord (GPEE area) it decreases the behaviour response and microcirculation disorders created in PSSO. The obtained data make it clear that the GPEE depression caused by the verapamil calcium channel blocker weakens PSSO and normalizes the microcirculation.     

Metorphamide Levels in Chromaffin Cells Increase After Treatment with Reserpine

Exposure of bovine chromaffin cells in primary culture to 0.01-1 microM reserpine caused a dose- and time-dependent increase in intracellular levels of the amidated enkephalin peptide metorphamide. Maximal levels (approximately 800% of control) were obtained at 0.1 microM reserpine and increased levels were apparent by 16 h of treatment. Metorphamide increases were at least fivefold more than that of either Met- or Leu-enkephalin, suggesting that reserpine stimulates both enkephalin processing and amidation in the secretory vesicle. Treatment with elevated potassium, which increases enkephalin levels by stimulating production of preproenkephalin messenger RNA, elicited an increase in metorphamide levels equivalent to, but not greater than, the increase in Met-enkephalin pentapeptide. The ratio of Met-enkephalin to metorphamide in untreated chromaffin cells is approximately 140:1, whereas the final Met-enkephalin: metorphamide ratio in reserpinized chromaffin cells is approximately 30:1, similar to the Met-enkephalin:metorphamide ratio in enkephalinergic neurons of the CNS.     

Neuromodulation of central inhibition on peripheral mechanosensory afferents

100 g reserpine, suspended in sunflower oil, was injected into the crop of six leeches (Hirudo medicinalis). Five leeches were injected with oil alone. The efficacy of delivering reserpine in this manner was assessed by measuring swimming bout duration. Four days after injection, swimming bouts were significantly longer in reserpine-treated leeches than in oil-treated or normal leeches. The amplitude of excitatory potentials elicited in velocity sensitive skin mechanoreceptors (T cells) by action potentials in mechanosensory cells which respond to sustained deformation of the skin (P cells) was no different in reserpine-treated leeches than in untreated leeches. The amplitude of inhibitory potentials in these T cells, produced by interneurons excited by P cells, was significantly larger in reserpine-treated leeches. Depletion of monoamines enhances inhibition in T cells. This is consistent with the established roles played by monoamines in promoting leech swimming and feeding behaviour.     

Anti-depressant-like activity of a novel serotonin type-3 (5-HT3) receptor antagonist in rodent models of depression

N-Cyclohexyl-3-methoxyquinoxalin-2-carboxamide (QCM-13), a novel 5-HT3 antagonist identified from a series of compounds with higher pA2 (7.6) and good log P (2.91) value was screened in rodent models of depression such as forced swim test (FST), tail suspension test (TST), interaction studies with standard anti-depressants and confirmatory studies such as reversal of parthenolide induced depression and reserpine induced hypothermia. In FST (2 and 4 mg/kg) and TST (2 and 4 mg/kg), QCM-13 significantly reduced the duration of immobility in mice without affecting the base line locomotion. QCM-13 (2 and 4 mg/kg) was also found to have significant interaction with standard anti-depressants (fluoxetine and bupropion in FST and TST respectively). Further, reversal of parthenolide induced depression in mice and reserpine induced hypothermia in rat models indicate the serotonergic influence of QCM-13 for anti-depressant potential.