Density Of Muscarinic Cholinergic Receptors Research Articles

Biochemical characterization of the rabbit urinary bladder base and body

AbstractFunctionally, the urinary bladder can be divided into two sections: the bladder body and bladder base. Pharmacologically, the bladder body responds to a greater extent than the bladder base to cholinergic agonists (contraction), beta‐adrenergic agonists (relaxation), and purinergic agonists (contraction), whereas the bladder base responds to a greater extent than the bladder body to alpha‐adrenergic agonists (contraction). The autonomic receptor density parallels the pharmacological response; that is, the bladder body contains higher densities of muscarinic cholinergic and beta‐adrenergic receptors than the bladder base, whereas the base contains a higher density of alpha‐adrenergic receptors. The purpose of this present study was to compare a variety of biochemical factors between the rabbit urinary bladder base and body.Sections of rabbit bladder body and base were rapidly dissected and frozen in liquid nitrogen. Tissue concentrations of DNA, RNA, hydroxyproline, lipids, glycogen, cyclic AMP, CP, ATP, ADP, and AMP were determined. The results demonstrated that the bladder base contained a 34% greater concentration of DNA, whereas the bladder body contained a 127% greater concentration of RNA. The bladder base contained 39.6% and 36% greater concentrations of hydroxyproline and glycogen, respectively. The concentration of lipids, cyclic AMP, and adenine nucleotides was equal in the two areas. The observed differences between bladder body and base may be related to the significantly greater work (energy utilized to actively expel urine) performed by the bladder body.

Effect of Estrogens on the Weight and Muscarinic Cholinergic Receptor Density of the Rabbit Bladder and Urethra

We have determined the muscarinic cholinergic receptor (MChR) density in the adult female rabbit bladder body and bladder base/urethra following three weeks of estrogen treatment. Fifteen female rabbits were separated into three treatment groups. Group I remained intact, Group II underwent bilateral ovariectomy, and Group III underwent simultaneous bilateral ovariectomy followed by subcutaneous placement of a 250mg. estradiol pellet. The weight, total DNA content, and MChR density were determined for each of the tissues in the three treatment groups. The mean weight of the bladder body following ovariectomy and estrogen treatment (Group III) was threefold greater than in the intact rabbits (Group I) (p <0.001) and two-fold greater than in the ovariectomized rabbits (Group II) (p <0.05). Similar increases were observed in the mean weights of the bladder base/urethral segments in the three treatment groups. The mean total DNA content in Group III was also significantly greater than Group I (p <0.01) and Group II (p <0.05) for both the bladder body and bladder base/urethral segment.The MChR density in the three treatment groups was determined using radioligand receptor binding using [3H]N-methylscopolamine ([3H]NMS). The MChR density in the bladder body (1.72 fmol./gm. DNA) for Group III was significantly decreased compared to the receptor density in both Group I (3.17 fmol./gm. DNA) and Group II (3.14 fmol./gm. DNA) (p <0.05). The binding of [3H] NMS to the MChR in bladder body was of high affinity (Kd = 0.07-0.15nM) and was unaltered by hormonal treatment. No difference was found in the MChR density in the bladder base/urethral segment in any of the three treatment groups. In this study, we have shown that estrogens increase the weight and modulate the MChR density of the female rabbit bladder body. The ability to modulate neurotransmitter receptor density by sex steroid hormones may have important clinical implications.

Neurotransmitter receptors in olivopontocerebellar atrophy

We used in vitro receptor autoradiography to study four cases of olivopontocerebellar atrophy (OPCA) and three age- and postmortem delay-matched controls. In OPCA, benzodiazepine receptors were unchanged in cerebellar cortex but increased in the dentate nucleus, perhaps related to loss of Purkinje cell or brainstem afferents. Muscimol binding was reduced primarily in the granule cell layer. The density of muscarinic cholinergic receptors was reduced in molecular and granule cell layers, but appeared increased in the dentate.

Laminar organization of cholinergic circuits in human frontal cortex in Alzheimer's disease and aging.

Cholinergic enzyme activity (choline acetyltransferase, CAT; acetylcholinesterase, AChE) and muscarinic cholinergic receptor density were measured in frontal cortex (Brodmann's area 9) of normal patients over the life span and in brains of patients with Alzheimer's disease (AD). CAT, but not AChE activity, declined with normal aging. Significant loss of CAT and AChE activity occurred in the AD brains, but later onset AD was associated with less severe loss of frontal cortex CAT activity. The majority of normal CAT activity resided in lamina I, II, and upper lamina III; CAT loss in AD resulted in large losses from all depths, most notably the upper cortical layers. AChE did not precisely correspond to the localization of CAT; loss of AChE in AD was consistent across all six laminae. No differences were seen in muscarinic cholinergic receptor binding between AD and age-matched controls; the distribution of binding was equal in all layers of normal frontal cortex, and no laminar differences were detected in distribution of cholinergic receptors between normal and AD samples.

Muscarinic Cholinergic Receptors in Bladder Exstrophy: Insights Into Surgical Management

The surgical management of classical bladder exstrophy (functional bladder closure or urinary diversion) should be influenced by the inherent detrusor function of the exstrophied bladder. Cystometrograms performed previously on individuals with successful exstrophy closures demonstrate normal bladder function. The biochemical and neurophysiological properties of the exstrophied bladder have otherwise not been investigated. In this study radioligand receptor binding techniques were used to compare the density and equilibrium dissociation constant of muscarinic cholinergic receptors in control and exstrophy bladders. The density of muscarinic cholinergic receptors in the control and exstrophy groups was 1.97 plus or minus 0.29 and 1.44 plus or minus 0.21fmol. per μg. deoxyribonucleic acid (mean plus or minus standard error of mean), respectively. The dissociation constant of the control and exstrophy groups was 0.15 plus or minus 0.02 and 0.14 plus or minus 0.02nM. (mean plus or minus standard error of mean), respectively. These data show that the muscarinic receptor density and binding affinity in control and exstrophy bladders are similar. Therefore, the neurophysiological composition of the exstrophied bladder is not grossly altered during the anomalous development.

Down Regulation of the Muscarinic Cholinergic Receptor of the Rat Prostate Following Castration

Prostatic secretion is dependent upon the integrity of the endocrine and autonomic nervous systems and is dramatically influenced by muscarinic cholinergic analogs. In this study, we have used radioligand receptor binding methods on whole tissue homogenates and slide mounted tissue sections of rat prostate to determine whether androgens regulate the density of muscarinic cholinergic receptors in the prostate. The muscarinic cholinergic receptor binding affinities (Kd) of [3H] N-methylscopolamine in prostatic homogenates obtained from intact, castrate, and castrate rats receiving testosterone replacement (castrate + T) were similar (0.07 to 0.10nM). The muscarinic cholinergic receptor binding capacity decreased 73 per cent following castration. Testosterone administration restored the density of muscarinic cholinergic receptors in castrate rats to intact levels. In order to ensure that the loss of receptor density was not due to a decrease in the epithelial: stromal cell ratio, the number of muscarinic cholinergic receptors per unit area of epithelium was determined in the 3 treatment groups using autoradiography on slide mounted tissue sections. The density of muscarinic cholinergic receptors in a unit area of epithelium was decreased 91 per cent following castration. Testosterone administration restored the density of muscarinic cholinergic receptors in the castrate rats to intact levels. The modulation of neurotransmitter receptors by steroid hormones may be a mechanism by which sex steroids regulate biological responsiveness of target tissues.

Mechanisms of supersensitivity to sympathomimetic amines in the chronically denervated heart of the conscious dog.

Mechanisms of denervation supersensitivity to sympathomimetic amines were studied in conscious animals. Norepinephrine, 0.1 micrograms/kg, increased left ventricular dP/dt significantly more (4208 +/- 828 mm Hg/sec) in dogs with cardiac denervation than in intact dogs (1029 +/- 280 mm Hg/sec), P less than 0.01, whereas responses to isoproterenol were similar in both groups. Denervation supersensitivity to isoproterenol could be demonstrated only after opposing reflex effects were blocked. After ganglionic blockade, norepinephrine still induced 2- to 3-fold greater increases in left ventricular dP/dt and 3- to 7-fold greater increases in heart rate in cardiac-denervated dogs, whereas isoproterenol and prenalterol, not taken up by presynaptic nerve endings, elicited only 40%-50% greater increases in left ventricular dP/dt and heart rate in dogs with cardiac denervation. The density of beta-adrenergic receptors [( 3H]dihydroalprenolol) was elevated (P less than 0.01) in denervated left ventricles (105 +/- 6.9 fmol/mg protein, n = 8) compared to normal left ventricles (70 +/- 6.3 fmol/mg protein, n = 12). This was accompanied by enhanced isoproterenol-mediated adenylate cyclase activity. However, muscarinic cholinergic receptor density, [( 3H]quinuclidinyl benzilate), decreased from control levels of 251 +/- 11 fmol/mg protein (n = 7) to 193 +/- 14 fmol/mg protein (n = 6). Thus, chronic cardiac denervation results in upregulation of the beta-adrenergic receptor and down-regulation of the muscarinic receptor. The increased beta-adrenergic receptor density and adenylate cyclase stimulation correlated well with the amount of denervation supersensitivity to isoproterenol and prenalterol, but accounted for only a minor fraction of denervation supersensitivity to norepinephrine. The major mechanism of denervation supersensitivity to norepinephrine appears to involve lack of the norepinephrine reuptake.

Quantitative in vivo receptor binding. I. Theory and application to the muscarinic cholinergic receptor

A novel approach to in vivo receptor binding experiments is presented which allows direct quantitation of binding site densities. The method is based on an equilibrium model of tracer uptake and is designed to produce a static distribution proportional to receptor density and to minimize possible confounding influences of regional blood flow, blood-brain barrier permeability, and nonspecific binding. This technique was applied to the measurement of regional muscarinic cholinergic receptor densities in rat brain using [3H]scopolamine. Specific in vivo binding of scopolamine demonstrated saturability, a pharmacologic profile, and regional densities which are consistent with interaction of the tracer with the muscarinic receptor. Estimates of receptor density obtained with the in vivo method and in vitro measurements in homogenates were highly correlated. Furthermore, reduction in striatal muscarinic receptors following ibotenic acid lesions resulted in a significant decrease in tracer uptake in vivo, indicating that the correlation between scopolamine distribution and receptor density may be used to demonstrate pathologic conditions. We propose that the general method presented here is directly applicable to investigation of high affinity binding sites for a variety of radioligands.

Autonomic nerve receptors in the allergic nasal mucosa in human subjects and sensitized guinea pigs

The purpose of this study is firstly to evaluate the abnormality of autonomic nerve receptors in human nasal mucosa in nasal allergy, and secondly to define whether this abnormality of these receptors are congenital or acquired. Autonomic nerve receptors in the nasal mucosa were measured using receptor binding assay in 14 subjects with perenial nasal allergy (ages 13-41, average age 35) and 13 subjects with chronic sinusitis (ages 15-44, average age 37).As radioligands we used [3H]-quinuclidinyl benzilate (QNB) for muscarinic cholinergic receptor, [3H]-prazosin for α1 adrenergic receptor and [3H]-dihydroalprenolol (DHA) for β adrenergic receptor. Compared with the nasal mucosa with chronic sinusitis an increased density (Bmax) of muscarinic cholinergic receptors (44% increased) and decreased densities of α1 adrenergic (37% decreased) and β adrenergic (41% decreased) receptors were observed in the nasal mucosa with nasal allergy. No differences were observed between both groupes in the dissociation constant (Kd) of each ligand to adrenergic and cholinergic receptors. The organ parasympathicotonia may explain the mucosal hyperreactivity to acetylcholine observed in subjects with nasal allergy. Decreased density of α1 adrenergic receptors will result in less constriction of the nasal vasculature in response to sympathetic stimulation. Decreased density of β adrenergic receptors may promote the release of chemical mediators from mast cells and basophils. We have already suggested that hypersensitivity of nasal mucosa observed in nasal allergy is nonspecific and that it is largely a result of antigen-antibody reaction. The organ parasympathicotonia in the nasal mucosa could be reproduced with TDI (toluene-2, 4-diisocyanate) sensitized guinea pigs which showed typical symptoms both of nasal allergy and bronchial asthma. But in the guinea pigs sensitized with bacterial crystalline α amylase, which appeared typical symptoms of bronchial asthma and no apparent symptoms of nasal allergy, the increased density of muscarinic cholinergic receptors could not be observed. These results suggest that the organ parasympathicotonia in the allergic nasal mucosa is not congenital, but is acquired secondarily to antigen-antibody reaction in the nasal mucosa.

Alterations in muscarinic cholinergic receptor densities induced by thiamine deficiency: autoradiographic detection of changes in high- and low-affinity agonist binding.

Animals fed a diet deficient in thiamine or treated with a drug preventing the utilization of thiamine (thiamine antagonist) exhibited alterations in ligand binding to muscarinic receptors in several brain regions. Using quantitative techniques of receptor autoradiography, an increase in muscarinic receptor binding was demonstrated in such regions as the corpus callosum, lamina VI of the parietal cortex, caudate-putamen, ventral nucleus of the thalamus, stratum lacunosum moleculare and stratum oriens of the hippocampus, and the hilus of the area dentata. As a result of thiamine deficiency, this increase in muscarinic receptor populations was primarily due to an increase in the binding of the low-affinity agonist site. In the same experiment, a decrease in muscarinic receptor binding was found in the ventromedial region of the hypothalamus. Thiamine deficiency thus causes an up-regulation of muscarinic receptor binding in several regions of rat brain while causing a down-regulation of these same receptors in other brain areas.

Limbic muscarinic cholinergic and benzodiazepine receptor changes with chronic intravenous morphine and self-administration

Muscarinic cholinergic and benzodiazepine receptor affinities and densities were evaluated in membranes from seven brain regions of rats intravenously self-administering morphine and in littermates receiving yoked-morphine or yoked vehicle infusions to identify neuronal systems potentially involved in mediating opiate reinforcement processes. Passive morphine infusion resulted in increases in muscarinic cholinergic receptor densities in the pyriform cortex and in decreases in the cingulate cortex while benzodiazepine receptor densities were decreased in both the hippocampal formation and entorhinal-subicular cortex compared to littermates receiving passive infusions of vehicle. Morphine self-administration resulted in decreased muscarinic cholinergic receptor densities in the frontal and entorhinal-subicular cortices and increases in the amygdaloid complex compared to littermates receiving yoked passive drug. These data are in agreement with acetylcholine turnover rate measurements in these animals and support the proposed role of cholinergic innervations of the frontal and entorhinal-subicular cortices and amygdaloid complex in opiate reinforcement processes.

Lack of effects of thyroid hormones on muscarine cholinergic receptors in rat brain and heart.

The muscarinic cholinergic antagonist 3H-quinuclidinyl benzilate was used to study muscarinic cholinergic receptors in the brain and the heart from triiodothyronine, thyroxine and 6-propyl-2-thiouracil treated rats. Scatchard analysis of the saturation binding studies revealed for each rat in both brain and heart tissue a single group of muscarinic cholinergic receptor binding sites of high affinity. The density and the affinity of muscarinic cholinergic receptors in brain and heart homogenates from treated rats were not different from values obtained in control rats with the exception of triiodothyronine treated rats which showed a slightly but significantly increased equilibrium dissociation constant in the brain homogenates. Furthermore, we observed that the density of the muscarinic cholinergic receptors in the brain was significantly lower in the month of August as compared to March-April, which suggests a seasonal variation. No similar change was observed in heart homogenates. Our results suggest that cardiovascular and central nervous symptoms in patients with thyroid diseases cannot be ascribed to changes in muscarinic cholinergic receptors.

A subcortico-cortical cholinergic system is affected in Parkinson's disease

CAT activity was decreased in the frontal cortex and the substantia innominata of parkinsonian subjects, post-mortem. The decrease was greater in the frontal cortex of parkinsonians with dementia. The density of muscarinic cholinergic receptors increased in the cortex. This increase was inversely correlated with tremor. The effects on these parameters of both neuronal degeneration and anticholinergic therapy are discussed.

Effects of hypoxia on atrial muscarinic cholinergic receptors and cardiac parasympathetic responsiveness

Chronic exposure of rats to hypoxia resulted in a lower resting heart rate and a supranormal increase in heart rate in response to parasympathetic blockade by atropine. The density of muscarinic cholinergic receptors labeled by the antagonist [ 3H]quinuclidinyl benzilate was elevated significantly in the atria of animals kept hypoxic for 2–4 weeks. Chronic hypoxia did not change the affinity of the receptor for [ 3H]quinuclidinyl benzilate, the weight of the atria, or the amount of protein per atrial pair. Thus, the decrease in resting heart rate may be explained by the increase in the density of atrial muscarinic cholinergic receptors.

The chronically reserpinized rat as a model for cystic fibrosis: alterations in the mucus-secreting sublingual gland.

Summary: The chronic administration of reserpine (0.5 mg/kg body weight, daily for 7 days) increases the density of adrenergic receptors in the sublingual gland of the rat. The Bmax values (in pmole/g tissue) were 5.8 ± 0.9 for [3H]-prazosin, 22 ± 4 for [3H]-clonidine and 11.9 ± 1.7 for [3H]-dihydroalpranolol. These radioligands are used, respectively, for the study of α1, α2 and β-adrenergic receptors and the Bmax values indicated are 181, 226, and 331%, respectively, of the corresponding values in control sublingual glands. The increase in the density of α1-adrenregic receptors was accompanied by the development of a clear secretory response to norepinephrine and phenylephrine, as judged by the ability to release K+ in vitro. This response, which was not observed in control sublingual glands, amounted to 21.9 ± 2.5 and 16.8 ± 3.1% of the tissue content of K+, respectively, for the two agonists. Neither the density of muscarinic cholinergic receptors (Bmax = 106% of control value) nor the extent of K+ release elicited by carbamylcholine was modified by the drug treatment, when compared to those observed in control glands. In vivo, the volume of sublingual saliva secreted in response to i.v. infusions of acetylcholine was significantly reduced in the treated animals to 45% of that secreted by control rats. This was accompanied by increases in salivary concentrations of protein-bound carbohydrates, with no change in the individual carbohydrate ratios. No significant changes in salivary electrolyte concentrations (Na+, K+, Ca++) were observed in sublingual saliva of reserpine-treated rats. In summary, reserpine administration alters adrenergic receptor density and sensitivity in the rat sublingual gland and modifies its secretory responses both in vitro and in vivo. The chronic administration of reserpine to rats causes widespread exocrine gland disturbances resembling those of cystic fibrosis. The results of this study show that the drug treatment also affects a mucus-secreting salivary gland and support the view that the reserpine-treated rat is a useful experimental model of the human disease.

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 effect of entorhinal cortical ablation on the distribution of muscarinic cholinergic receptors in the rat hippocampus

Removal of the entorhinal cortical projection to the hippocampus in adult rats decreased the density of muscarinic cholinergic receptors in the denervated dentate gyrus outer molecular layer at two days postlesion. Thirty days following the lesion (in adults and neonates) there is a small receptor density increase in the outer molecular layer (may be due to tissue shrinkage), and a larger increase in the lacunosum-moleculare. The receptor density decrease seen two days postlesion suggests the presence of presynaptic muscarinic receptors on the lost entorhinal cortical fibers. The distribution and extent of the receptor changes seen at 30 days postlesion are inconsistent with the cholinergic fiber reorganization which follows an entorhinal cortical lesion, but are consistent with a proposed model of non-cholinergic afferent mediated control of muscarinic receptor density in the rat hippocampus.

Hypoxia-induced changes in parasympathetic neurochemical markers in guinea pig heart.

Exposure of animals to hypoxia produces cardiovascular changes including right ventricular hypertrophy and alterations in heart rate. The activity of choline acetyltransferase, a neurochemical marker of parasympathetic innervation, and the density of muscarinic cholinergic receptors, measured by the binding of [3H]quinuclidinyl benzilate, were analyzed in the hearts of guinea pigs exposed to hypobaric hypoxia. We found a significant increase in the activity of choline acetyltransferase in the sinoatrial node in response to hypoxia after 7 or 14 days. In addition, we found significant decreases in the muscarinic receptor density in several contractile and conducting regions. The decrease in receptor density may reflect regulation by the level of occupancy; an increased occupancy may produce a decrease in muscarinic receptor number.

Effects of thyroid state on brain development: Muscarinic acetylcholine and GABA receptors

A study was made of the effects of age, neonatal hypothyroidism and hyperthyroidism on the development in rat brain of muscarinic cholinergic and GABA receptors. The former receptors were estimated by the binding of [ 3H]quinuclidinyl-benzilate and the latter by binding of [ 3H]muscimol to crude membrane preparations from the forebrain and the cerebellum. In the normal forebrain, the density of muscarinic cholinergic receptors (in terms of unit membrane proteins) doubled during the period 6–35 days after birth. Thyroid state had relatively little effect on this development. In contrast, in the normal cerebellum the peak of the density of the receptors was attained in the early neonatal period followed by a progressive decline reaching about half of the maximum at day 35. Furthermore, in the cerebellum this development was significantly influenced by thyroid disorders: the rate of decrease in receptor density was accelerated by hyperthyroidism and retarded in thyroid deficiency. In comparison with euthyroid rats, the density of muscarinic receptors in the cerebellum was 30% lower in the hyperthyroidism (at day 21) and 40% higher in thyroid deficiency (at day 35). The increase in the density of GABA receptors with age was very small in the normal forebrain relative to the marked rise in the cerebellum. In the forebrain, thyroid state had no significant effect on this development. In contrast, in the cerebellum the ontogenesis of GABA receptors was advanced by thyroid hormone treatment and retarded in thyroid deficiency. However, by day 35 receptor density was normal in both conditions. Thyroid state had no significant influence on the affinity of either [ 3H]muscimol or the [ 3H]quinuclidinylbenzilate binding. The results suggest that thyroid hormone disorders during early life may lead to distortions rather than synchronized shifts in the relative development of several central transmitter systems.

Muscarinic receptor binding and behavioral effects of atropine following chronic catecholamine depletion or acetylcholinesterase inhibition in rats

Rats were subjected to one of two experimental treatments: (1) intraventricular infusion of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA), known to permanently reduce brain dopamine and norepinephrine levels, or (2) chronic administration of the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). Both treatments are believed to produce relative overactivity of cholinergic systems and to suppress forward locomotion. The anticholinergic agent atropine sulfate yielded excessive forward walking in otherwise chronically akinetic 6-OHDA-treated rats, whereas atropine slightly decreased locomotion in controls. The hypothesis that such supersensitivity to atropine may be related to a reduction in the density of muscarinic cholinergic receptors was not supported: First, 3H-quinuclidinyl benzilate (QNB) binding to membrane preparations was not decreased in the 6-OHDA-treated rats; secondly, atropine did not induce excessive forward locomotion in the DFP-treated rats in which 3H-QNB binding was decreased. There were other changes in the DFP-treated rats consistent with muscarinic receptor alteration, including tolerance to the locomotor suppressive effects of DFP, cross tolerance to the cholinergic agonist pilocarpine, and exaggerated atropine-induced increases in core temperature and stereotypy. It is concluded that 6-OHDA and DFP produce different long-term changes in cholinergic brain systems and atropine-sensitive behaviors.