Alterations In Muscarinic Receptors Research Articles

Muscarinic receptor alterations with ageing: The effectiveness of clinical antimuscarincs on the porcine urinary bladder

Muscarinic receptor alterations with ageing: The effectiveness of clinical antimuscarincs on the porcine urinary bladder

Cadmium induced ROS alters M1 and M3 receptors, leading to SN56 cholinergic neuronal loss, through AChE variants disruption

Cadmium, an environmental neurotoxic compound, produces cognitive disorders, although the mechanism remains unknown. Previously, we described that cadmium induces a more pronounced cell death on cholinergic neurons from basal forebrain (BF). This effect, partially mediated by M1 receptor blockade, triggering it through AChE splices variants alteration, may explain cadmium effects on learning and memory processes. Cadmium has been also reported to induce oxidative stress generation leading to M2 and M4 muscarinic receptors alteration, in hippocampus and frontal cortex, which are necessary to maintain cell viability and cognitive regulation, so their alteration in BF could also mediate this effect. Moreover, it has been reported that antioxidant treatment could reverse cognitive disorders, muscarinic receptor and AChE variants alterations induced by cadmium. Thus, we hypothesized that cadmium induced cell death of BF cholinergic neurons is mediated by oxidative stress generation and this mechanism could produce this effect, in part, through AChE variants altered by muscarinic receptors disruption. To prove this, we evaluated in BF SN56 cholinergic neurons, whether cadmium induces oxidative stress and alters muscarinic receptors, and their involvement in the induction of cell death through alteration of AChE variants. Our results show that cadmium induces oxidative stress, which mediates partially the alteration of AChE variants and M2 to M4 muscarinic receptors expression and blockage of M1 receptor. In addition, cadmium induced oxidative stress generation by M1 and M3 receptors alteration through AChE variants disruption, leading to cell death. These results provide new understanding of the mechanisms contributing to cadmium harmful effects on cholinergic neurons.

Spinal cord regeneration by modulating bone marrow with neurotransmitters and Citicholine: Analysis at micromolecular level

BackgroundSpinal cord injury results in disruption of brain-spinal cord fibre connectivity, leading to progressive tissue damage at the site of injury and resultant paralysis of varying degrees. The current study investigated the role of autologous bone marrow modulated with neurotransmitters and neurotransmitter stimulating agent, Citicholine, in spinal cord of spinal cord injured rats. MethodsRadioreceptor assay using [3H] ligand was carried out to quantify muscarinic receptor. Gene expression studies were done using Real Time PCR analysis. ResultsScatchard analysis of muscarinic M1 receptor showed significantly decreased Bmax (p < 0.001) and Kd (p < 0.01) compared to control and significant reversal (p < 0.001) in both the treatment groups (spinal cord injury treated with 5HT and GABA, and spinal cord injury treated with Citicholine). Muscarinic M1 receptor gene expression in spinal cord injured group showed significant down regulation (p < 0.001) compared to control, and both the treatment groups significantly reversed (p < 0.001) these changes to near control when compared to spinal cord injured group. The confocal microscopic study using specific antibody of muscarinic M1 confirmed the gene expression studies. ConclusionThus our results suggest that the neurotransmitters combination along with bone marrow or Citicholine with bone marrow can reverse the muscarinic receptor alterations in the spinal cord of spinal cord injured rats, which is a promising step towards a better therapeutic intervention for spinal cord injury because of the positive role of cholinergic system in regulation of both locomotor activity and synaptic plasticity.

558 Alterations in Muscarinic Receptors in Human Heart Failure and Effects of LVAD Support

It is well established that the sympathetic nervous system (SNS) is over stimulated in the failing human heart, resulting in down regulation of beta-adrenergic receptors on cardiac myocytes. The goal of the current study was to test the hypothesis that the parasympathetic nervous system (PNS) may be concomitantly dysregulated in human heart failure (HF).

Blood pressure and vascular reactivity to endothelin-1, phenylephrine, serotonin, KCl and acetylcholine following chronic alcohol consumption in vitro.

Ethanol has been reported to cause hypertension, the mechanism of which is unknown. Therefore, the effect of chronic ethanol consumption on vascular responsiveness and blood pressure was investigated. Systolic blood pressure was recorded weekly by tail-cuff method. Aortic rings from rats fed chow ad libitum or pair-fed liquid diets containing either ethanol (7.2% v/v) or isocaloric carbohydrate for 4 weeks were placed in organ chambers for isometric tension measurement. There was a mild but significant elevation of the systolic blood pressure in the alcohol-fed rats by week 1 compared to baseline measurements and this remained higher. No significant changes in reactivity of rat isolated aortas to phenylephrine, serotonin, endothelin-1 (ET-1) and KCl were seen in chronic ethanol consumption. In addition, the sensitivity (i.e. pD2) of alcohol-fed aortic rings to the vasoconstrictors was also unchanged compared to controls. Chronic ethanol consumption, however, increased relaxation to acetylcholine with increased pD2 values, but did not alter relaxation to sodium nitroprusside, a cyclic guanosine monophosphate (cGMP)-dependent direct smooth muscle dilator. The results indicate that chronic ethanol consumption significantly potentiates endothelium-dependent relaxations in aortic rings, probably through interference with the production and/or the release of nitric oxide (NO) or adaptive alterations in muscarinic receptors on the endothelial cells, and that increased vascular responsiveness to several vasoconstrictors is not a mechanism responsible for the blood pressure elevation in the chronic alcohol consumption in rats.

Reversibility of Diabetes- and Diuresis-Induced Alterations in Rat Bladder Dome Muscarinic Receptors

Previous studies from our laboratory demonstrated that 8 weeks of streptozocin (STZ)-induced diabetes and sucrose-fed diuresis resulted in increases in the density of muscarinic receptors in rat bladder dome and that early insulin treatment (started 3 days after the onset of diabetes) prevented the diabetes-induced upregulation (J Pharmacol Exp Ther 248:81-88, 1989; Diabetes 40: 1150-1156, 1991; J Urol 147:760-763, 1992). To determine whether diabetes- and diuresis-induced alterations in muscarinic receptors in rat bladder dome are reversible, we administered insulin (beginning 8 weeks after the onset of diabetes) or removed sucrose from drinking water of diuretic rats (beginning 8 weeks after the onset of diuresis). Five groups of rats were maintained for 16 weeks: 1) STZ-induced diabetic rats (65 mg/kg intravenously); 2) insulin-treated diabetic rats (5-8 U/day insulin subcutaneously beginning 8 weeks after the onset of diabetes); 3) sucrose-fed diuretic rats (5% sucrose in drinking water throughout 16 weeks); 4) sucrose-removed rats (sucrose withdrawn from drinking water after 8 weeks of the sucrose-induced diuretic state); and 5) age-matched control rats. Radioligand receptor binding experiments with [3H]quinuclidinyl benzilate showed an increase in the density of muscarinic receptors in bladder dome of diabetic and sucrose-fed rats compared with age-matched control rats. Removing the 5% sucrose from the drinking water of diuretic rats reversed the increased water intake and urine output, decreased the bladder hypertrophy that accompanied the diuretic state, and corrected the upregulation of the muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of anticholinergic drugs and brain muscarinic receptor alterations in streptozotocin diabetic rats.

We studied the effects of experimental diabetes on the pharmacokinetics of biperiden (BP) and scopolamine (SP) and brain muscarinic receptor alterations in rats after the injection of streptozotocin (STZ) (60 mg kg-1 i.v.). The serum levels of BP and SP differed significantly between the rats 14 weeks after the STZ treatment and age-matched control rats. The values of total body clearance (CLtot) of BP and SP were significantly increased by STZ treatment. The values of volume of distribution (Vdss) of SP were slightly increased in the STZ-treated rats, although Vdss of BP was decreased. Because of the high lipophilicity of BP, Vdss of BP may be decreased due to the reduced fat tissue volume caused by STZ treatment. The density of the muscarinic receptors in whole brain was measured by a radioligand receptor binding assay using [3H]-quinuclidinyl-benzylate ([3H]-QNB). The density in the diabetic rats two weeks after the STZ treatment was significantly decreased compared to age-matched control rats. However in the diabetic rats 14 weeks after the STZ treatment, there was no difference in the density of muscarinic receptors. The IC50 of muscarinic antagonist for the binding of [3H]-QNB to the receptor did not change on STZ treatment. Modulation of the receptor following repeated anticholinergic drug exposure was studied. In control rats, the number of muscarinic receptors in the brain increased by 6.9% on chronic treatment with BP for two weeks. When diabetic rats were treated with BP and SP, the number of muscarinic receptors in the brain increased by 9.6% and 33.8%, respectively.

Effect of Insulin and Dietary Myoinositol on Muscarinic Receptor Alterations in Diabetic Rat Bladder

Previous studies from our laboratory demonstrated that there is an up-regulation of muscarinic receptor density in the bladder dome of the 8-wks diabetic rat compared to control. To determine whether the changes observed in receptor density can be corrected by insulin or dietary myoinositol, five groups of rats were maintained for eight weeks: control (C), diabetic (D), diabetic insulin-treated (DI), diabetic myoinositol-treated (DMI), and control myoinositol-treated (CMI). Diabetes was induced by i.v. injection of 65mg./kg. of streptozotocin. D and DMI animals were smaller, had higher serum glucose and lower serum insulin levels, higher water intakes and urine outputs, and larger bladder domes than the other groups. The density of the muscarinic receptors measured by radioligand receptor binding assays using [3H]quinuclidinyl benzilate were (in fmol./mg. protein): C, 88 ± 13; D, 176 ± 19; DI, 94 ± 5; DMI, 158 ± 8; CMI, 112 ± 10. These data indicate that insulin, but not myoinositol treatment normalized diabetes induced abnormalities observed in the general features of streptozotocin-injected rats and prevented the diabetic-induced upregulation of bladder dome muscarinic receptors.

Downregulation of muscarinic receptors in the rat caudate-putamen after lesioning of the ipsilateral nigrostriatal dopamine pathway with 6-hydroxydopamine (6-OHDA): normalization by fetal mesencephalic transplants

The autoradiographic distribution and density of muscarinic receptors was studied in the neostriatum of rats with long-term unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopaminergic pathway and in lesioned rats who had additionally received embryonic substantia nigra grafts in the dopamine denervated striatum. Muscarinic receptors were labeled with [ 3H]quinuclidinyl benzilate (QNB), M 1 receptors were directly labeled with [ 3H]pirenzepine (PZ) and non-M 1 receptors were labeled by the competition of 100 nM PZ with [ 3H]QNB. The density and distribution of muscarinic receptors were directly compared to the sodium-dependent, high-affinity, choline uptake sites as labeled with [ 3H]hemicholinium-3 (HC-3). In the 6-OHDA-lesioned animals, there was a 25% reduction in muscarinic receptors labeled with [ 3H]QNB. Subtype analysis showed that there was a reduction of both M 1 (−26%) and non-M 1 (−33%) receptors. A normal density of both muscarinic receptor populations was found in animals with successful transplants. Saturation analysis demonstrated that the changes, in muscarinic receptor density, were due to a change in receptor number ( B max) and not affinity ( K d). There was no significant change in [ 3H]HC-3 binding in the 6-OHDA-lesioned or transplanted animals, indicating that alterations in muscarinic receptors were not due to transynaptic degeneration of striatal cholinergic interneurons. The findings of downregulation of muscarinic receptors following long-term dopamine denervation and the subsequent normalization of muscarinic receptor density after fetal mesencephalic transplantation suggests that transplanted substantia nigra cells are able to restore inhibitory control on striatal cholinergic interneurons.

Hippocampal muscarinic supersensitivity after AF64A medial septal lesion excludes M 1 receptors

Stereotaxic injection of AF64A, into the medial septum of the rat, resulted in significant loss of presynaptic cholinergic markers in this structure. No significant change was observed for the presynaptic neuronal markers for dopamine- and serotonin-containing neurons in either the medial septum or hippocampus. The AF64A lesion also resulted in a significant reduction of muscarinic receptors as demonstrated by a loss of [ 3H]QNB binding in the medial septum. Subtype analysis showed the decrease of receptor binding in the medial septum to be due to a loss of M 1 receptors as well as other muscarinic receptor subtypes. In the hippocampal formation, [ 3H]hemicholinium-3 binding was significantly reduced in the molecular layer of the dentate gyrus, and in the stratum oriens and stratum radiatum of the hippocampus. AF64A lesion resulted in a significant increase (B max) in non-M 1 muscarinic receptors in hippocampal stratum oriens, in areas CA2, CA3, and CA4. AF64A lesion of the medial septum did not result in muscarinic receptor alterations in any other region of the hippocampal formation examined. These results indicate that postsynaptic muscarinic receptors in the stratum oriens of the CA2 to CA4 region of the hippocampus mediate primarily the function of the cholinergic cell bodies of the medial septum. These receptors are not of the M 1 subtype.

Alterations in Muscarinic Receptors of Ventricular Muscle in Carbachol-Induced Short-Term Desensitization

The inhibitory action of a muscarinic agonist on the contractile response of cardiac muscle is transient due to short-term desensitization of muscarinic cholinergic receptors. Studies were made on the binding of the muscarinic antagonist L-[3H]-qutnuclidinyl benzilate ([3H]QNB) to the muscarinic receptor in the membrane fraction of ventricular muscle of guinea pigs desensitized by perfusion with carbachol for 10 min. Desensitization did not change the maximum binding or equibilium dissociation constant (Kd) of [3H]QNB, but shifted the inhibition curves of [3H]ONB binding by carbachol to the right both in the presence and absence of 5 -guanylyl imidodiphosphate (GppNHp). Analysis of these inhibition curves with a multiple site model suggested that superhigh and high affinity agonist binding sites were convened to low affinity sites in the desensitized state. GppNHp has additive effects to the prior exposure to carbachol, suggesting a different site of action from shon-term exposure of agonist. We conclude that agonist-induced shon-term desensitization of the muscarinic receptor of ventricular muscle is caused by reduction in the affinity of the receptor for agonist without reduction in its amount or affinity for antagonist.

Muscarinic receptor alterations as a mechanism of anticholinesterase tolerance

Tolerance to the toxic signs of the organophosphorus ester acetylcholinesterase inhibitor, O,O-diethyl S-[2-(ethylthio)ethyl] phosphorodithioate (disulfoton), was induced in rats by giving 10 doses of 2.0 mg/kg/day. Concurrent with the induction of tolerance, decreased sensitivity to the cholinergic agonists carbachol and oxotremorine could be demonstrated in studies of heart rate in vivo and in isolated preparations of ileum and atria. A significant decrease in the binding of the muscarinic antagonist [ 3H]quinuclidinyl benzilate could be demonstrated in ileum from disulfoton-tolerant animals. However, no alterations in the binding of [ 3H]quinuclidinyl benzilate, [ 3H]oxotremorine-M, or oxotremorine were evident in atria from tolerant animals. The results suggest that, in addition to receptor loss, other mechanisms distal to ligand recognition sites or removed from the receptor complex may contribute to the subsensitivity of tissues to muscarinic cholinergic agonists.

Muscarinic receptor alterations following neostigmine treatment

Male mice administered neostigmine in the drinking water at daily increasing concentrations (20–1000 mg/l) for four days became tolerant to its toxicity and presented a reduced binding of [ 3H]quinuclidinyl benzilate ([ 3H]QNB) in the small intestine. An increased binding of [ 3H]QNB was found in the forebrains of neostigmine-treated animals. This was due to an increase in muscarinic cholinergic receptor density. Neostigmine-treated animals were also more sensitive than control to the hypothermic effect induced by oxotremorine. Dopaminergic and α- and β-adrenoceptors were not affected. Administration of methylatropine together with neostigmine prevented the decrease of [ 3H]QNB binding in the small intestine as well as the increase in the forebrain.

The muscarinic receptors of airway smooth muscle: their characterization in vitro.

We have used radioligand binding and in vitro muscle contraction techniques to characterize the muscarinic cholinergic receptors of canine tracheal smooth muscle. The tritiated muscarinic antagonist, quinuclidinyl benzilate ([3H]QNB), bound no particulate preparations of tracheal smooth muscle with high affinity, saturability, pharmacologic specificity, and stereoselectivity. The equilibrium dissociation constant for the binding reaction was 33 +/- 3 pM (mean +/- SE). The concentration of binding sites was 410 +/- 34 pmol/g protein. The ability of muscarinic agents to inhibit [3H]QNB binding paralleled their relative effects on tracheal smooth muscle contraction in vitro. We found the anesthetic agents, lidocaine and tetracaine, and the nicotonic cholinergic antagonist d-tubocurarine, were competitive inhibitors of [3H]QNB binding to the tracheal smooth muscle preparations. Histamine, aminophylline, and adrenergic agonists did not act as competitive inhibitors. The guanine nucleotide, guanyl-5'-imidodiphosphate, reduced the ability of the muscarinic agonist, acetylcholine, to compete with high affinity for [3H]QNB binding site(s) in tracheal smooth muscle but had minimal effects on the binding of the muscarinic antagonist, atropine. THe radioligand binding and in vitro contraction techniques described are being used to study the possible alteration of muscarinic receptors of airway smooth muscle in disease or from treatment.

Properties of muscarinic acetylcholine receptors in heart cell cultures

The binding of acetylcholine to receptors in the intact heart causes a decrease in the frequency (chronotropic effect) and force (ionotropic effect) of contraction. The studies reported here demonstrate a chronotropic response of cultured embryonic chicken heart cells to the muscarinic agonist carbamoylcholine. This response is markedly decreased after a 3-hr incubation with 0.1 mM carbamoylcholine. In order to determine whether agonist-induced alterations in muscarinic receptors were responsible for this decrease, we studied the effects of incubation with carbamoylcholine on the binding of the (3)H-labeled muscarinic antagonist quinuclidinyl benzilate (QNB) to homogenates of heart cell cultures. [(3)H]QNB binding to homogenates of cultures of embryonic hearts of chicks 9 days in ovo was characterized and shown to have properties similar to those of muscarinic receptors in intact hearts. Binding was both specific and saturable. [(3)H]QNB was displaced by muscarinic agonists and antagonists in concentrations consistent with their known potency. Binding was poorly inhibited by the nicotinic antagonist D-tubocurarine. Kinetic analysis of the binding of QNB by muscarinic receptors showed that initially the reaction proceeds by formation of a rapidly reversible complex with a K(d) of 1.8 nM, which is converted to a slowly reversible form. These properties of muscarinic receptors in heart cell cultures are strikingly similar to those observed in homogenates of intact hearts. Homogenates of heart cell cultures bound 84 +/- 6 fmol (mean +/- SD) of QNB per mg of protein. The number of receptors remained stable from day 4 to day 8 in culture. Incubation of cultures with 0.1 mM carbamoylcholine for 3 hr decreased QNB binding by 55%, to 38 +/- 5 fmol/mg protein. When cell cultures were first homogenized and then incubated with carbamoylcholine, no decrease in QNB binding sites could be detected. Thus, incubation with carbamoylcholine causes loss of muscarinic binding sites as well as decreased physiologic responsiveness to muscarinic agonists.