Functional and biochemical characteristics of urinary bladder muscarinic receptors in long-term alloxan diabetic rats / Características funcionais e bioquímicas dos receptores muscarínicos da bexiga de ratos com diabetes crônico induzido por aloxana

Objective To re-examine the function of the urinary bladder in vivoas well as to determine the functional and biochemical characteristics of bladder muscarinic receptors in long-term alloxan-induced diabetes rats.Methods Two-month-old male Wistar rats were injected with alloxan and the animals showing blood glucose levels >300mg/dL together with age-paired untreated animals were kept for 11 months. Body weight, bladder weight, blood glucose, and urinary volume over a period of 24 hours were determined in both groups of animals. A voiding cystometry in conscious control and diabetic rats was performed to determine maximal micturition pressure, micturition contraction interval and duration as well as voided and post-voiding residual volume. In addition, concentration-response curves for bethanechol in isolated bladder strips, as well as [3H]-N methyl-scopolamine binding site characteristics in bladder homogenates were determined.Results Mean bladder weight was 162.5±21.2mg versus 290±37.9mg in control and treated animals, respectively (p<0.05). Micturition contraction amplitude (34.6±4.7mmHg versus 49.6±2.5mmHg), duration (14.5±1.7 seconds versus 23.33±4.6 seconds) and interval (87.5±17.02 seconds versus 281.11±20.24 seconds) were significantly greater in alloxan diabetic rats. Voided urine volume per micturition contraction was also significantly higher in diabetic animals. However the post-voiding residual volume was not statistically different. Bethanechol potency (EC50 3µM versus 5µM) and maximal effect (31.2±5.9g/g versus 36.1±6.8g/g) in isolated bladder strips as well as number (169±4fmol/mg versus 176±3fmol/mg protein) and affinity (0.69±0.1nM versus 0.57±0.1nM) of bladder muscarinic receptors were also not statistically different.Conclusion Bladder function in vivo is altered in chronic alloxan-induced diabetes rats without changes in functional and biochemical characteristics of bladder muscarinic receptors.

Objetivo Reestudar o funcionamento da bexiga in vivo e determinar as características funcionais e bioquímicas dos receptores muscarínicos vesicais de ratos com diabetes crônico induzido por aloxana.Métodos Ratos Wistar de dois meses de idade receberam injeção de aloxana, e os animais que apresentaram glicemia >300mg/dL foram mantidos por 11 meses junto de outros não tratados e pareados por idade. Nos dois grupos de animais, peso corpóreo, peso da bexiga, glicemia e volume urinário de 24 horas foram medidos. Em ambos os grupos, realizou-se a cistometria miccional em animais não anestesiados. Foram determinados os seguintes parâmetros: pressão máxima de micção, intervalo e contração de micção, bem como o volume de esvaziamento e o volume residual pós-miccional. Além disso, foram determinadas as curvas de concentração-resposta a betanecol em preparações isoladas de bexiga e também as características dos sítios de ligação da [3H]-N-metil-escopolamina em homogenatos de bexiga.Resultados O peso médio da bexiga foi de 162,5±21,2mg versus290±37,9mg nos animais controles e tratados, respectivamente (p<0,05). A amplitude de contração (34,6±4,7mmHg versus 49,6±2,5mmHg), a duração (14,5±1,7 segundos versus 23,33±4,6 segundos) e o intervalo (87,5±17,02 segundos versus 281,11±20,24 segundos) de micção foram significantemente maiores nos ratos tratados com aloxana. O volume de urina eliminada durante a contração miccional também foi maior nos animais diabéticos. Contudo, o volume residual pós-miccional não foi estatisticamente diferente. Não foram observadas diferenças na resposta ao betanecol (EC50 3µM versus 5µM) e no seu efeito máximo (31,2±5,9g/g versus 36,1±6,8g/g) em preparações isoladas de bexiga, bem como no número total (169±43fmol/mgversus 176±3fmol/mg) e na afinidade (0,69±0,1nMversus 0,57±0,1nM) dos receptores muscarínicos da bexiga.Conclusão O funcionamento da bexiga in vivo está alterado no diabetes crônico induzido por aloxana, porém sem alterações funcionais e bioquímicas nos receptores muscarínicos da bexiga.

Pharmacodynamic study on acute hypotensive activities of Carissa carandas extract in normal rats

This study aims to evaluate the effect of Carissa carandas extract on cardiovascular function of normal rats. Intravenous bolus injection of this extract in the doses of 5 mg kg[-1]-45 mg kg[-1], produced dose dependent reduction in arterial blood pressure [p<0.001]. The 45mg/kg dose caused a 50.75% +/- 2.71 decrease in MABP which was highly significant with P value< 0.0005 when compared with its controls. Significant reduction in heart rate frequency was observed after CC injection at a dose of 45 mg kg[-1] [p<0.001]. The results were comparable with Acetylcholine 10[-4] M. The receptor activity performed for which Atropine 10[-4]M was administered I.V. and then the extract [45mg/kg] was administered. A highly Non Significant fall in Mean Arterial Blood pressure was observed 1.51% +/- 0.22 [P>0.05].It was concluded that the Carissa carandas Ethanol extract possess potent acute hypotensive effect in normal rats. It stimulates the muscarinic receptors located on the endothelial cells of the vasculature. This stimulation results in the release of endothelial-derived relaxing factors [EDRFs] or nitric oxide that diffuses to vasculature smooth muscles and causes their relaxation

Zataria multiflora Boiss inhibits muscarinic receptors of incubated tracheal smooth muscle with propranolol

In the present study, the effect of tissue incubation with propranolol on functional antagonism of Zataria multiflora Boiss [Z. multiflora] at muscarinic receptors of tracheal smooth muscle was examined. The effects of three concentrations of aqueous-ethanolic extract, 10 nM atropine, and saline on muscarinic receptors were tested on incubated tracheal smooth muscle with propranolol [n=5]. The EC[50] of all concentration of the extract and atropine was significantly higher than that of saline. There was parallel right ward shift in concentration response curves obtained in the presence of all concentrations of the extract. There was not any significant difference in the maximum response and slope obtained in the presence of different concentrations of extract compared to saline. There was significant positive correlation between the concentrations and the values of EC[50] [p<0.001]. The value of [CR-1] obtained in the presence of highest concentration of the extract was significantly higher than that of atropine [p<0.05]. These results indicated that functional antagonism of Z. multiflora at muscarinic receptors of tracheal smooth muscle was mainly due to beta-adrenoceptor stimulatory effect of plant

Ptychodiscus brevis toxin decreases the spontaneous activity of rat right atria involving muscarinic receptors and potassium channels.

Marine dinoflagellate Ptychodiscus brevis toxin (PbTx), is known to produce toxic effects on cardiovascular system. The present experiments were conducted to evaluate the effect of synthetic phosphorus containing Ptychodiscus brevis toxin on spontaneously beating right atrium in vitro. The PbTx (0.84-84 microM) decreased the rate and force of right atrial contractions in a concentration-dependent manner. Ethanol, a vehicle present in highest concentration of PbTx, had no effect on atrial rate or force of contraction. Pretreatment with atropine blocked the PbTx-induced decrease in atrial rate and force of contraction. The tetraethylammonium, a potassium channel blocker, blocked the PbTx-induced decrease in atrial rate and force, where as, L-type of calcium channel blocker, nifedipine blocked the PbTx-induced force of contraction but not the rate changes. The results indicate that the PbTx decreased the atrial rate and force of contraction via cholinergic receptors involving K+ channel.

Ginger facilitates cholinergic activity possibly due to blockade of muscarinic autoreceptors in rat stomach fundus

Ginger [Zingiber officinale] is universally known food plant reputed for its medicinal use in gastrointestinal disorders as a prokinetic and laxative. We recently showed that 70% aqueous-methanolic extract of ginger [Zo.Cr] exhibits prokinetic activity in rats via activation of post-synaptic muscarinic M[3] receptor in rat stomach fundus. In view of the physiological significance of pre-synaptic muscarinic M[1] and M[2] autoreceptors, this study was undertaken to further look into the possible mode of action of the prokinetic effect of ginger through inhibition of pre-synaptic muscarinic receptors. Isolated tissue bath experiments were performed with Sprague-Dawley rat stomach fundus strip preparations immersed in Kreb's solution at 37 degree C. Carbachol [CCh] maximum responses [1 microM] were obtained in rat stomach fundus. Zo.Cr, given in multiple increasing bolus concentrations [0.01-0.1 mg/ml] 10 min prior to administration of CCh, potentiated the CCh peak responses showing that it is possibly inhibiting the pre-synaptic muscarinic receptors. Like wise, increasing bolus concentrations of pirenzepine [0.03-0.3 micro M] and himbacine [0.01-0.03 micro M], standard muscarinic M[1] and M[2] antagonists respectively, also potentiated the CCh responses. These results show that ginger, in addition to having a direct cholinergic effect on the post-synaptic M[3] receptors, also has a possible inhibitory effect on pre-synaptic muscarinic autoreceptors, similar to standard muscarinic antagonists, thus reiterating the gastric stimulant effect of this age-old plant

The challenge of overactive bladder therapy: alternative to antimuscarinic agents

Contemporary, the management of overactive bladder (OAB), a medical condition characterized by urgency, with or without urge urinary incontinence, frequency and nocturia, in absence of genitourinary pathologies or metabolic factors that could explain these symptoms, is complex, and a wide range of conservative treatments has been offered, including bladder training, biofeedback, behavioral changes, oral or intravesical anticholinergic agents, S3 sacral neuromodulation and peripheral electrical stimulation. Clinical efficacy of these treatments remains an open issue and several experimental and clinical studies were carried out in the last years improving the results of medical treatment.Here we review the pathophysiology of micturition reflex, the current therapies for OAB and the rationale for alternative treatments. Furthermore we critically address the potential use of medications targeting the central nervous system (CNS) and the primary sensory nerves of the bladder wall, we review the use of agonists of nociceptin/orphanin protein (NOP) receptor and finally we report the results obtained by intradetrusor injection of botulinum toxin.

Supersensitivity of the cholinergic muscarinic system in the rat's brain is induced by high concentrations of Cu+2

Transition metals have been described as regulators of receptor's function. here, we studied the effects of chronic administration of Cu2+ or the Cu2+ chelator penicillamine (PA) on the functional and binding properties of the muscarinic receptors (MR) on selected areas of rat's brain. Groups of 10 Sprague-Dawley rats were treated daily, for 45 days with either 1) 1 mg/Kg CuSO4 (Cu2+), 2) 100 mg/Kg PA, or 3) saline solution. Double T-maze and motility cages were used for behavioral testing and the binding assays were performed using [3H]-QNB or [3H]-N-MSCP as MR's ligands. Cu2+ brain levels were measured in the cerebral cortex by atomic absorption spectrophotometer. Results showed that PA treated rats displayed a significant decrease of locomotor's activity (LA) and rearing behavior (RB), but a significant increases in memory efficiency (ME). Cu2+ treated rats displayed diminished RB with no significant changes in LA. Cu2+ treated rats displayed higher MR's density (Bmax) in cortex (C), striatum (S), and hippocampus (H). An increase in Bmax was also observed in PA treated rats, but only in C and S. Finally, Cu2+ tissue concentration was significantly higher in C of both Cu2+ and with PA treated animals. In conclusion, 45 days of Cu2+ or PA treatment induced brain hypercuprosis, which was associated with MR binding supersensitivity; however, change in ME was only observed in PA treated rats suggesting that might be still another factor in these experiments besides Cu2+ (i.e., Zn2+ or PA itself) involved in memory modulation.

Inhibition of carbachol-induced inositol phosphate accumulation in the embryonic retina promoted by kainate and veratridine

In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 muM carbachol was used, the estimated IC50 value for kainate was 0.2 muM and the maximal inhibition of ~50 percent was obtained with 1 muM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 muM veratridine, but not 50 mM KCl, inhibited ~65 percent of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 + 38.0 to 2044.5 + 299.9 cpm/mg protein, retinal response decreased to 861.6 + 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.

Multireceptor interactions of haloperidol on rat cerebral frontal cortex "in vitro"

As severall side effects of neuroleptics would be related to their interactions with several neurotransmitter receptors (R) haloperidol action on muscarinic cholinergic (mACh) R on frontal cerebral cortex preparations was analyzed. Here we shown that haloperidol was able to inhibit in a concentration dependent manner the binding of specific mAChR radiollabeled antagonist on cerebral cortex membranes. This effect would be related to its interaction on mAChR of the M1 subtype as haloperidol blocked the stimulation of phosphoiinositides (Pis) turnover induced by low concentrations of carbachol similarly as the M1 antagonist pirenzepine. However at high carbachol concentrations haloperidol triggered a potentiating stimulation of Pis hydrolysis that was only blocked by the alpha1 adrenergic antagonist prazosin indicating and alpha1 agonistic action of haloperidol on these Rs. These multireceptor actions of haloperidol found "in vitro"would strengthen its assocation with "in vivo"neuroleptic-induced side effects.

Lack of interaction of angiotensin converting enzyme inhibitors with muscarinic and neuropeptide Y receptors

In studies conducted in patients undergoing cardiac catheterizations, some hemodynamic changes were observed after the acute sublingual administration of the angiotensin converting enzyme inhibitors (ACEI) captopril, enalapril, and lisinopril. These changes consisted of an increase in pulmonary artery pressure, pulmonary vascular resistance (PVR) and induction of hypoxia. The pressure changes were transitory and disappeared after 25 min. The possible mechanisms involved in these changes may relate to interactions of the ACEI with peripheral receptor systems for hormones and neurotransmitters. We have thus undertaken the task of evaluating the potential effect of ACEI on biological receptor molecules. We have begun with studies on muscarinic receptors, and the recently characterized neuropeptide Y (NPY) receptors of endothelial cells. Equilibrium binding assays with 3H-QNB have been conducted for muscarinic receptors using rat brain synaptosomes, due to its expression of multiple muscarinic receptors subtypes. In addition 125BH-NPY binding assays were conducted on intact adrenal medullary endothelial cells. Enalapril and captopril, 10(-7) to 10(-3) M, were not able to produce significant inhibition of either muscarinic or NPY receptor probes. The paradoxical changes elicited by sublingual ACEI seems not to involve interaction with muscarinic or NPY receptors

Intoxicación crónica con manganeso: cuantificación autorradiográfica de los receptores colinérgicos muscarínicos en el cerebro de ratón / Chronic manganese poisoning: autoradiograohic cuantitation of muscarinic cholinergic receptors in mouse brain

El tratamiento crónico con cloruro de manganeso (5 mg Mn/kg/día), durante 9 semanas, no afectó la unión del radioligando [3H]-quinuclidinil benzilato a los receptores colinérgicos muscarínicos en el cerebro de ratón. Mediante técnica autorradiográfica se determinó la localización anatómica precisa de los receptores y se procedió a la cuantificació de los mismos en los cortes coronales del bulbo olfatorio y del cerebro medio. A la luz de los resultados obtenidos podemos concluir que, en nuestras condiciones experimentales, no se producen alteraciones en la densidad de los receptores colinérgicos muscarínicos en el cerebro de ratones intoxicados con manganeso

Effects of muscarinic receptor agonists and antagonists on response of non-extensor rats to maximal electroshock.

Rats which do not respond consistently to maximal electroshock by exhibiting the classical hindlimb extensor response, are designated as 'flexors', and can serve as a useful experimental model for investigating seizure mechanisms. 20-25% Charles Foster rats exhibit the flexor status and were used in this study. The flexor rats were converted to extensors by acetylcholine (icv), physostigmine (ip) and the selective muscarinic M1 receptor agonists, arecholine (ip) and McN-A-343 (icv). This conversion of flexors to extensors was significantly attenuated by M1 receptor antagonists scopolamine (ip) and pirenzepine (icv). The M2 receptor agonist, carbachol (icv), had no effect in lower doses but induced conversion of flexor rats to the extensor status only in very high doses which may be due to loss of receptor specificity on dose increment. The M2 receptor antagonists, gallamine (icv) and AF-DX 116 (ip), also induced significant conversion of flexors to extensors, which was dependent upon the availability of neuronal acetylcholine since the effects were attenuated following pretreatment with hemicholinium, an inhibitor of acetylcholine synthesis. The results suggest that the central cholinergic system has a facilitatory pro-convulsant effect, mediated through the muscarinic M1 receptors, an action modulated by the M2 receptors.

Thermic response of selective muscarinic agonists and antagonists in rat.

Effect of some selective agonists and antagonists of cholinergic M receptor subtypes on rectal temperature was investigated in rats at an ambient temperature of 25 degrees +/- 2 degrees C. Centrally administered acetylcholine (ACh) induced transient hypothermia, whereas the muscarinic M1 receptor agonists, arecholine (ip) and McN-A-343 (McN) (icv), induced sustained and dose-related hypothermia. However, the nonspecific muscarinic receptor agonist, oxotremorine, and physostigmine, induced hypothermia at a lower dose and hyperthermia, accompanied by tremors, at higher doses. The muscarinic M2 receptor agonist, carbachol (icv) also produced a dose-related dual effect, hyperthermia and hypothermia being induced by the lower and higher doses, respectively. The M1 receptor antagonists, scopolamine (ip) and pirenzepine (icv), induced hyperthermia, whereas the M2 receptor antagonists, gallamine (icv) and AF-DX 116 (AFDX) (ip), produced hypothermia. The hypothermic effects of ACh. arecholine, McN, physostigmine, oxotremorine and carbachol were attenuated by scopolamine and pirenzepine. However, although scopolamine also inhibited the hyperthermic and tremorogenic effects of the higher dose of oxotremorine, it had a synergistic effect with the hyperthermia-inducing higher dose of physostigmine. AFDX attenuated the hyperthermic effect of the lower dose of carbachol, indicating that it was M2 receptor-mediated. Hemicholinium, an ACh synthesis inhibitor, had a transient hypothermic effect followed by slight hyperthermia. However, it markedly antagonized the hypothermic effects of gallamine and AFDX, indicating that their effects were dependent upon the availability of neuronal ACh. The results indicate that cholinergic hypothermia is a function of central muscarinic M1 receptors, with the M2 receptors serving as automodulators.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of selective muscarinic receptor agonists and antagonists on active-avoidance learning acquisition in rats.

Effect of some selective muscarinic receptor agonists and antagonists was investigated on learning acquisition in an active-avoidance paradigm in rats which records an anticipatory conditioned avoidance apart from the classical conditioned avoidance response. The muscarinic M1 agonists, arecholine, pilocarpine and McN-A-343, facilitated learning acquisition, which was attenuated by the selective M1 antagonist, pirenzepine. On the other hand, M2 receptor agonist, carbachol, and physostigmine, induced a dose-related dual response, with lower doses retarding and higher doses facilitating the learning acquisition. The former effect was attenuated by gallamine, a muscarinic M2 antagonist, while the latter response was inhibited by pirenzepine, indicating that these putative M2 receptor agonist lose their receptor specificity on dose increment. The selective M2 receptor antagonists, gallamine and AF-DX 116, facilitated learning acquisition, which was inhibited by pirenzepine and the acetylcholine synthesis inhibitor hemicholinium. The results support the cholinergic hypothesis of learning and memory and indicate that M1 receptor agonists and M2 receptor antagonists are likely to prove beneficial in memory deficits. The data also indicates that the clinical dose of some drugs, like physostigmine, needs to be carefully established for optimum therapeutic benefit.

Prevention of oculocardiac reflex (O.C.R) during extraocular muscle surgery.

In the present study the effectiveness of intravenous atropine sulphate which blocks the peripheral muscarinic receptors at the heart and retrobulbar xylocaine hydrochloride which blocks the conduction at ciliary ganglion on the afferent limb of OCR was studied during strabismus surgery. The study was conducted on fifty three patients of either sex having squint of more than ten years duration. The patients were randomly allocated into four groups. No preanaesthetic medication with atropine or retrobulbar block with xylocaine was given in control group of patients. In the second group, only preanaesthetic medication with atropine was given; while in the third group only retrobulbar injection of xylocaine was given five minutes before operation. In the last group both atropine as preanaesthetic medication and xylocaine as retrobulbar block were given. The electrocardiographic recordings were taken before and throughout the operative procedure. It was interesting to note that in the group where atropine and xylocaine were used none of the patients exhibited activation of OCR. Results have been discussed.

The role of adrenergic mechanism in tremorine-induced tremors in rats: antitremor effect of beta-adrenoceptor antagonists.

Tranylcypromine (TCP) pretreatment was found to accelerate the tremorogenic activity of tremorine in rats. Conversely, reserpinization delayed the onset of induction of tremors, and a significant diminution in their intensity was observed in these rats. A comparative study of the antitremor activity of beta-adrenoceptor antagonists against this tremor-model showed that butoxamine (beta 2-antagonist) and propranolol (nonselective antagonist) were able to afford a rapid and powerful protection, whereas a weaker and delayed effect was observed in rats treated with the beta 1-antagonist, acebutolol. Furthermore, the antitremor activity of butoxamine and propranolol but not that of acebutolol was found to be potentiated and diminished in rats pretreated with reserpine and TCP, respectively. It was inferred that beta 2-receptor modulated the tremorogenic activity of tremorine, and that inhibition by propranolol or butoxamine of this subtype beta-adrenoceptor resulted in rapid and powerful suppression of tremors, and that the antiadrenergic activity of acebutolol was unlikely to have a role in its antitremor effect.