mAChR Number Research Articles

Acetylcholine receptor and behavioral deficits in mice lacking apolipoprotein E

Apolipoprotein E (apoE) is involved in the risk to develop sporadic Alzheimer's disease (AD). Since impaired central acetylcholine (ACh) function is a hallmark of AD, apoE may influence ACh function by modulating muscarinic ACh receptors (mAChRs). To test this hypothesis, mAChR binding was measured in mice lacking apoE and wild type C57BL/6J mice. Mice were also tested on the pre-pulse inhibition, delay eyeblink classical conditioning, and 5-choice serial reaction time tasks (5-SRTT), which are all modulated by ACh transmission. Mice were also given scopolamine to challenge central mAChR function. Compared to wild type mice, mice lacking apoE had reduced number of cortical and hippocampal mAChRs. Scopolamine had a small effect on delay eyeblink classical conditioning in wild type mice but a large effect in mice lacking apoE. Mice lacking apoE were also unable to acquire performance on the 5-SRTT. These results support a role for apoE in ACh function and suggest that modulation of cortical and hippocampal mAChRs might contribute to genotype differences in scopolamine sensitivity and task acquisition. Impaired apoE functioning may result in cholinergic deficits that contribute to the cognitive impairments seen in AD.

Binding and functional characterisation of allosteric agonists at M2 muscarinic acetylcholine receptors

Muscarinic acetylcholine receptors (mAChRs) contain at least one allosteric site that is topographically distinct from the acetylcholine (ACh)‐binding orthosteric site. Although numerous studies have investigated the structural basis of allosteric modulation at these receptors, far less is known about allosteric ligands that activate the receptor in their own right. We generated a number of M2 mAChRs harboring different orthosteric‐site mutations (W99A, Y403A), or allosteric‐site mutations (172EDGE175‐QNGQ, Y177A), and investigated their impact on the binding and function of the putative allosteric agonists AC‐42, 77‐LH‐28‐1, McN‐A‐343 and N‐desmethylclozapine (NDMC). As expected, orthosteric site mutations reduced the affinity and/or efficacy of the orthosteric agonists ACh and pilocarpine, whereas the allosteric site mutations had no significant effects. W99A and Y403A caused significant increases in the affinity (100–300 fold and 3–10 fold respectively) and efficacy of AC‐42 and 77‐LH‐28‐1. NDMC and McN‐A‐343 showed increased efficacy, but not affinity, at Y403A and Y177A. Homology models of the M2 mAChR have been developed around the different ligands. The experimental data and modelling together lead to a better understanding of the allosteric binding sites of mAChRs. This work is supported by National Health & Medical Research Council Project Grant No. 400134 and the Dowd Foundation.

Opposing effects on muscarinic acetylcholine receptors in the piriform cortex of odor-trained rats

We combined pharmacological studies and electrophysiological recordings to investigate modifications in muscarinic acetylcholine (ACh) receptors (mAChR) in the rat olfactory (piriform) cortex, following odor-discrimination rule learning. Rats were trained to discriminate between positive and negative cues in pairs of odors, until they reached a phase of high capability to learn unfamiliar odors, using the same paradigm ("rule learning"). It has been reported that at 1-3 d after the acquisition of odor-discrimination rule learning, pyramidal neurons in the rat piriform cortex show enhanced excitability, due to a reduction in the spike-activated potassium current I(AHP), which is modulated by ACh. Further, ACh and its analog, carbachol (CCh), lost the ability to reduce the I(AHP) in neurons from trained rats. Here we show that the reduced sensitivity to CCh in the piriform cortex results from a decrease in the number of mAChRs, as well as a reduction in the affinity of the receptors to CCh. Also, it has been reported that 3-8 d after the acquisition of odor-discrimination rule learning, synaptic transmission in the piriform cortex is enhanced, and paired-pulse facilitation (PPF) in response to twin stimulations is reduced. Here, intracellular recordings from pyramidal neurons show that CCh increases PPF in the piriform cortex from odor-trained rats more than in control rats, suggesting enhanced effect of ACh in inhibiting presynaptic glutamate release after odor training.

Effects of Estrogen on Muscarinic Acetylcholine Receptors in the Rat Hippocampus

The aim of the present study was to investigate whether different estrogen manipulations have effects on the expression of muscarinic acetylcholine receptors (mAChRs) in the adult female rat hippocampus. Hippocampus was obtained from rats in proestrus (control), ovariectomized for 2, 10 and 15 days, ovariectomized for 15 days and treated with 17β-estradiol for 7 days, and treated with 17β-estradiol immediately after ovariectomy for 21 days. Rats’ estrogen status was monitored by measuring estradiol plasma levels and uterus relative weight. [³H]quinuclidinyl benzilate ([³H]QNB) binding studies indicated that ovariectomy time-dependently increases the number of mAChRs in hippocampus when compared to those obtained from control rats. Estradiol treatments for 21 days avoid the effect of ovariectomy. However, the estradiol treatments for 7 days after 15 days of ovariectomy slightly change the number of mAChRs. In conclusion, these results showed that ovariectomy time-dependently increases mAChRs number in the rat hippocampus. In addition, these data suggest that treatment with estradiol initiated within a specific period of time after the loss of ovarian function may be effective at preventing specific effects of hormone deprivation on hippocampus.

Effects of testosterone on muscarinic acetylcholine receptors in the rat epididymis

The effect of testosterone on the expression of muscarinic acetylcholine receptor (mAChR) subtypes was studied in the rat epididymis, at mRNA and protein level. The rat androgen status was monitored by measuring plasma testosterone level and caput and cauda epididymis wet weight. Ribonuclease protection assay (RPA) and [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding assay were performed in the caput and cauda epididymis from control (50-day old), castrated, castrated and treated with testosterone and sexually immature (30-day old) rats. The expression of each mAChR transcript subtype differed depending on the epididymal region analyzed and rat testosterone and/or testicular factors status. In control rats, RPA showed the presence of mRNA for M 1, M 2 and M 3 mAChR in the caput and cauda epididymis. The abundance of m2 and m3 transcripts in the cauda was higher than that in the caput epididymis. Low amount of m1 transcript was observed in both regions. Orchidectomy increased m1 mRNA amount in the caput and cauda epididymis when compared to control rats, an effect slightly modified by testosterone replacement. Although orchidectomy down-regulated the level of m2 transcript in both epididymal regions, castration significantly increased m3 mRNA amount in the caput region. These effects on m2 and m3 transcripts were prevented by testosterone replacement to castrated rats. Similar abundance of m3 transcript, however, was detected in the cauda epididymis of all experimental group tested. [ 3H]QNB binding studies revealed that orchidectomy down-regulated the number of mAChR detected in both epididymal regions, an effect also prevented by testosterone replacement. Thus, testosterone and/or testicular factors may play a role in the regulation of mAChR expression in the rat epididymis.

Carbachol-Induced Secretion and Homologous Desensitization in Rat Basophilic Leukemia (RBL-2H3) Cells Transfected With Human m2 Muscarinic Acetylcholine Receptors

Carbachol (CCh) caused a dose-dependent release of β-hexosaminidase and an increase in the production of inositol 1,4,5-trisphosphate (IP3) in RBL-2H3 cells transfected with m2 mAChR cDNA (RBL-m2 cells). The secretion was completely inhibited by LaCl3 and pertussis toxin. The secretion was dependent on extracellular Ca2+ and mediated through the pertussis toxin-sensitive G protein. Exposing RBL-m2 cells to 100 μM CCh for 30 min in Ca2+-free medium (desensitizing treatment) inhibited the secretion induced by the subsequent addition of 10 μM CCh plus Ca2+, but not by stimulating the high affinity IgE receptor (FcεRI). Desensitizing treatment of RBL-m2 cells reduced the affinity of the lipophilic ligand [3H]quinuclidinyl benzilate to m2 mAChR without a reduction of the total m2 mAChR number. The treatment also decreased the cell surface mAChR number to 14% with a slight reduction in its affinity. Desensitizing treatment of RBL-m2 cells inhibited the CCh-induced transient increase in levels of IP3 and intracellular Ca2+ concentration. The results suggested that the CCh-induced desensitization of m2 mAChR-mediated secretion is due to the receptor sequestration followed by blocking the increase in [Ca2+]i and that this desensitizing mechanism is receptor-subtype-specific.

Role of Nitric Oxide in Ethanol‐Induced Up‐Regulation of Muscarinic Acetylcholine Receptors in SH‐SY5Y Cells

Ethanol abuse has been shown to up-regulate muscarinic acetylcholine receptors (mAChRs) in the central nervous system, both in vivo and in vitro, but the detailed mechanisms for this action are not known. Recent studies suggest that the actions of ethanol in several biological systems involve nitric oxide (NO) pathways. The aim of this work was therefore to determine whether the effects of long-term ethanol treatment on mAChRs involved actions upon NO pathways in a well defined cell culture system. Human neuroblastoma SH-SY5Y cells were used as an in vitro model system. The time- and dose-dependent effects of ethanol on endogenous NO production, as well as the effects of the NO donor 3-morpholino-sydnonimine-chloride and of the neuronal NO synthase (nNOS) inhibitor N(omega)-propyl-L-arginine on mAChR number and on ethanol effects upon these receptors, were studied. Ethanol time- and dose-dependently decreased the production of NO in the cells. Exogenous NO decreased the number of mAChRs and totally blocked the effects of ethanol upon these receptors. Inhibition of nNOS up-regulated the number of mAChRs, but this effect was not additive to the effects of ethanol. The results of this study suggest that the number of cell-surface mAChRs in SH-SY5Y cells may be correlated with changes in NO levels. The number of cell surface mAChRs decreased with NO-elevating treatment and increased with NO-lowering treatment. Because ethanol, which is known to up-regulate mAChRs in SH-SY5Y cells, also decreased NO levels and because nNOS inhibition and ethanol effects on mAChRs were not additive, it is conceivable that ethanol-induced up-regulation of mAChRs involves inhibition of nNOS.

Effects of ethanol on phosphoinositide hydrolysis and muscarinic acetylcholine receptor number in SH-SY5Y cells

Previous studies suggest that the effects of ethanol on carbachol-stimulated I(1,4,5)P 3 formation and on the number of mAChRs may be independent of each other. The aim of this work was to further study this hypothesis. Human neuroblastoma SH-SY5Y cells were used as a model system. Acute exposure of the cells to 100 mM ethanol induced a decrease in [ 3H]N-methylscopolamine ([ 3H]NMS) binding at 30 seconds which was of lower magnitude and of shorter duration than the previously described ethanol-induced inhibition of the peak of carbachol-stimulated I(1,4,5)P 3 formation. Long-term ethanol treatment of the cells induced a time- and concentration-dependent increase in [ 3H]NMS binding. Three hours of 100 mM ethanol treatment were sufficient to increase the number of mAChRs at the cell surface but these receptors were not immediately functionally active, suggesting that they may be newly synthesized. Furthermore, the ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P 3 formation, after two days, was, for all ethanol concentrations tested, of higher magnitude than the ethanol-induced increase in mAChR number. Together, these data indicate that both acute and chronic ethanol-induced changes in carbachol-stimulated I(1,4,5)P 3 formation may not only be explained by changes in mAChR density at the cell surface but may rather be the consequence of actions of ethanol down-stream of the receptor.

Effects of breeding conditions on neurochemical cholinergic and monoaminergic markers in aged rat brain

We investigated the effects of breeding conditions on neurochemical markers, muscarinic receptor (mAChR), beta-adrenoreceptor (beta-AdrR), imipramine binding sites (IMBS), choline acetyltransferase (ChAT), acetylcholinesterase (AChE) and monoamine oxidase (MAO) activity in aged rat brains. An increase of affinity (Kd) and the decrease in the number (Vmax) of mAChR were found in the individual aged rats. Concerning IMBS, Kd and Vmax values increased in the individual aged rats. However, no significant changes were observed in the beta-AdrR. The increases of ChAT and MAO activity were found in the aggregated aged rats to compare with in the individual aged rats, while AChE activity decreased in the aggregated aged rats. These changes were also particularly seen in the forebrain of aged rats. These results indicate that the functions of the central nervous system may be reduced in the individual aged rats to compare with in the aggregated aged rats under the breeding conditions.

Chronic effects of ethanol on muscarinic acetylcholine receptors are modulated by protein kinase C

We have previously demonstrated that long-term ethanol treatment increased the number and function of muscarinic acetylcholine receptors (mAChRs) in human neuroblastoma cells, but the molecular mechanisms involved in these changes are unknown. In the present study, the effect of protein kinase C (PKC) on these events was investigated in human neuroblastoma SH-SY5Y cells. Following exposure to 100 mM ethanol for 2 days, both [³H]N-methylscopolamine binding and carbachol-stimulated I(1,4,5)P₃formation were increased. When cells were cultured in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of PKC, the effects of ethanol on mAChR number were totally inhibited but ethanol still potentiated carbacholstimulated I(1,4,5)P₃ formation in TPA treated cells. TPA dose-dependently inhibited carbochol-stimulated I(1,4,5)P₃ formation and this effect appeared to be independent of PKC phosphorylating activity. On the other hand, PKC inhibitors mimicked ethanol effects on mAChR number and function. Selective inhibition of classical PKC isozymes with 1 μΜ Gö 6976 for 2 days caused an increase in mAChR number and function, suggesting a role for these isozymes in ethanol-induced upregulation of mAChRs. These data indicate that longterm ethanol treatment may upregulate the number of mAChRs by counteracting PKC-mediated phosphorylation. The effects of ethanol on receptor-coupled phosphoinositide hydrolysis appear to be independent of PKC activity.

Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells.

Binding experiments with the specific muscarinic ligand [3H]N-methylscopolamine (3H-NMS) have shown the presence of constitutive muscarinic acetylcholine receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1-M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K(D)) for 3H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B+) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B+ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells.

Chronic cerebral hypoperfusion disrupts discriminative behavior in acquired-learning rats

We describe an `acquired-learning' rat model that was used to investigate the effects of chronic cerebral hypoperfusion on the maintenance of previously acquired discriminative behavior using the discriminating learning task. Male Wistar rats, aged 11 weeks, were trained to discriminate between lamp-on and lamp-off states under an operant-type learning procedure. After 30 sessions, we selected `acquired-learning' rats with an average discrimination ratio higher than 75% recorded during the last three sessions. Chronic hypoperfusion was then induced by permanent ligation of both common carotid arteries under pentobarbital anesthesia. The rats were tested after surgery over a period of 12 weeks and brain tissue was analyzed for muscarinic acetylcholine receptor (mACh-R) binding. Cerebral hypoperfusion resulted in a significant reduction in the discrimination ratio throughout the observation period, compared with sham-operated rats. However, chronic hypoperfusion would not affect on motor function. The maximum number of mACh-R examined 12 weeks after the operation was significantly reduced in the frontal cortex and hippocampus in the hypoperfusion group. Impaired discrimination learning was associated with a reduction in mACh-R. Our findings suggest that chronic cerebral hypoperfusion in acquired-learning rats is a useful model for investigating the pathophysiology of dementia and that cortical and/or hippocampal cholinergic systems contributes to learning impairment, at least, in our learning task.

Differential cholinoceptor subtype-dependent activation of signal transduction pathways in neonatal versus adult rat atria

In this study, we investigated the expression and distribution of muscarinic cholinergic receptors (mAChRs) and the different signaling pathways associated with mAChR activation in atria isolated from adult and neonatal rats. Carbachol stimulation of mAChRs in both neonatal and adult rat atria led to a negative inotropic response with activation of phosphoinositide hydrolysis, an increase in cyclic GMP levels, and a decrease in cyclic AMP production. However, compared with adult atria, neonatal atria showed hypersensitivity in the contractile effect induced by carbachol. Pharmacological analysis with mAChR antagonists indicated that M 1 and M 2 mAChR subtypes are important mediators of the response to carbachol in neonatal atria. In contrast, in adult atria the effect of the agonist was coupled only to the M 2 mAChR subtype. Moreover, an increased number of total mAChRs was labeled in neonatal atrial membranes compared with those of adults. Although a predominant M 2 mAChR population is expressed in atria at both stages of development studied, competition binding parameters calculated for carbachol indicated the presence of high-affinity binding sites, with higher affinity in neonates than in adults. These results suggest that the differences observed between neonatal and adult atria in their response to a cholinergic agonist may be related to differential expression of mAChR subtypes and/or changes in functional coupling of mAChR subtypes during development.

Non-selectivity of the monoclonal antibody M35 for subtypes of muscarinic acetylcholine receptors.

The monoclonal antibody M35, one of the first monoclonal antibodies successfully raised against muscarinic acetylcholine receptors, has been widely used to study the distribution of this protein in a variety of tissues and cell types of different species. It is not fully known, however, to which muscarinic acetylcholine receptor subtypes M35 binds. Knowledge of subtype-selectivity of M35 is a necessary step towards a functional interpretation of the obtained immunocytochemical data. The aim of the present study was to determine the subtype-selectivity of M35 employing transfected CHO-K1 cells stably expressing human m1-m5 muscarinic acetylcholine receptors separately, and to study M35 immunoreactivity in areas of rat central and peripheral tissues known to be specifically enriched in a single muscarinic acetylcholine receptor subtype. The results show that (a) all five transfected cell lines were immunopositive for M35, (b) nontransfected control cells were immunonegative, (c) the number of mAChRs expressed per cell correlated positively with the intensity of M35 immunoreactivity, and (d) cell types in aldehyde-fixed rat tissue enriched in a single m1-m4 subtypes revealed clear M35 immunoreactivity. Taken together, the present results show that M35 does not discriminate between muscarinic acetylcholine receptor subtypes. Evidently, the epitope of M35 on the receptor-protein is preserved on all muscarinic acetylcholine receptor subtypes. The epitope for M35 must, therefore, be localized on a homologous part of each subtype.

Inhibition of Receptor Signaling to Phospholipase D by Clostridium difficile Toxin B: ROLE OF Rho PROTEINS

Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5'-O-(thio)triphosphate (GTP gamma S) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF4- and GTP gamma S, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.

Subtype-specific Regulation of Muscarinic Receptor Expression and Function by Heterologous Receptor Activation

Incubation of cultured embryonic chicken heart cells with the beta-adrenergic agonist isoproterenol resulted in a dose-dependent increase in the number of mAChR on the surface of intact cells. The isoproterenol-mediated increase in mAChR number was time dependent and reached a maximum by 48 h. Chick heart cells treated with isoproterenol exhibited a greater than 6-fold increase in the sensitivity for carbachol-mediated inhibition of adenylyl cyclase activity as compared to control. Stimulation of cultured heart cells for 24 h with isoproterenol resulted in a 25-35% increase in cm2 mRNA levels as compared to control cm2 mRNA levels. In contrast, the level of cm4 mRNA was not significantly affected by isoproterenol treatment. cm2 mRNA levels were maximally elevated by 15 h following isoproterenol stimulation and remained elevated for up to 72 h. Incubation of cells with isoproterenol in the presence of Rp-cAMP, an inhibitor of cAMP-dependent protein kinase, blocked the increase in the level of cm2 mRNA. Thus, prolonged activation of beta-adrenergic receptors results in an increase in mAChR number and muscarinic responsiveness in chick heart cells due to a cAMP-dependent protein kinase mediated increase in cm2 mRNA levels.

Sequestration of muscarinic cholinergic receptors in permeabilized neuroblastoma cells.

The feasibility of using a permeabilized preparation of human SH-SY-5Y neuroblastoma cells for studies of muscarinic acetylcholine receptor (mAChR) sequestration has been evaluated. Exposure of cells permeabilized with digitonin, streptolysin-O, or the alpha-toxin from Staphylococcus aureus to oxotremorine-M (Oxo-M) for 30 min resulted in a 25-30% reduction in the number of cell surface mAChRs, as monitored by the loss of N[3H]methylscopolamine ([3H]NMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 microM digitonin, the Oxo-M-mediated reduction in [3H]NMS binding was time (t1/2 approximately 5 min) and concentration (EC50 approximately 10 microM) dependent and was agonist specific (Oxo-M > bethanechol = arecoline = pilocarpine). In contrast, no reduction in total mAChR number, as monitored by the binding of [3H]quinuclidinyl benzilate, occurred following Oxo-M treatment. The loss of [3H]NMS sites observed in the presence of Oxo-M was unaffected by omission of either ATP or Ca2+, both of which are required for stimulated phosphoinositide hydrolysis, but could be inhibited by the inclusion of guanosine 5'-O-(2-thiodiphosphate). mAChRs sequestered in response to Oxo-M addition were unmasked when the cells were permeabilized in the presence of higher concentrations of digitonin (80 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between muscarinic receptor occupancy and response in rat parotid acinar cells

To determine whether spare muscarinic cholinergic receptors (mAChRs) exist in rat parotid acinar cells, we examined the effect of propylbenzilylcholine mustard (PBCM) on agonist (carbachol)-stimulated inositol trisphosphate (IP3) formation and on mAChR number, using l-[N-methyl-3H]scopolamine methyl chloride (NMS)-binding assays. Treatment with PBCM (1, 3, 10, 30, 50 nM) for 15 min caused a 5, 22, 60, 66, and 72% decrease, respectively, in maximal IP3 formation stimulated by carbachol as well as a large reduction in the potency of carbachol in eliciting this response. Using these data, equilibrium constants (Ka) for activation of the mAChRs by carbachol were calculated. These Ka values agreed well with Kd values of high-affinity mAChR binding sites determined from carbachol displacement of [3H]NMS binding in parotid acinar cells. Reduction in mAChR number after PBCM treatment was determined by Scatchard analysis of specific [3H]-NMS binding sites and compared with the expected reduction (q values) calculated from dose-response curves for carbachol-stimulated IP3 formation before and after PBCM treatment. PBCM (1, 3, 10, 30 nM) decreased mAChR maximal binding in cells 47.5, 68.9, 82.4, and 85.3%, respectively, which did agree with the approximately 38, 70, 90, and 92% decrease in receptor number expected from the calculated q values. Data demonstrate that PBCM irreversibly inactivates mAChRs in rat parotid cells, and the decrease in receptor number, measured directly from [3H]NMS binding or calculated from receptor theory, is greater than that observed for stimulated IP3 production. These results suggest that a modest (30-40%) population of spare receptors exists for mAChR-mediated IP3 production in rat parotid glands.

Characterization of muscarinic cholinergic receptors in intact myocardial cells in vitro

Muscarinic acetylcholine receptors (mAChR) were studied on heart cells grown in culture by the radioligand binding technique. We used [ 3H] n-methyl-scopolamine to monitor the level of receptors on intact cardiocytes. The number of mAChR was very low during the first days in culture (23 fmol/dish). It increased gradually until it reached a plateau on the 4th day (180 fmol/dish), where it remained for 1–2 weeks. To determine whether contractile activity affected the level or affinity of mAChR, the cardiocytes were exposed to agents that stimulate or arrest the heart beat. Treatment with triiodothyronine (T 3, 10–90 nM) for 48 hr caused a reduction in the level of the receptors by 20–30% without changing significantly the affinity of the receptors. Similarly, electrical stimulation caused a reduction in the level of the receptors by 30–40%, without a significant influence on creatine kinase activity. When the myocardial cells were treated with Ca-channel blocker such as metoxyverapamil (D600) (10–30 μg/mL) or diltiazem (10–25 μg/mL) the level of the receptors was also reduced by 30–40%. The reduction in the receptor binding sites was accompanied by an increase in K d from 0.8 to 3.2 nM in D600-treated cells, whereas there was no significant change in the radioligand affinity after application of diltiazem. Treatment with D600 or T 3 together with cycloheximide showed that under these experimental conditions the rate of receptor degradation was accelerated. The half-life of the receptors in the control was 27 hr, whereas the half-lives of T 3 and D600 were 15 and 18 hr, respectively. It is concluded that regulation of the amount of cholinergic receptors occurs at the level of receptor breakdown, and simple linkage does not exist between the rate of cardiac contractions and the number of mAChR.

Regulation of muscarinic acetylcholine receptor mRNA expression by activation of homologous and heterologous receptors.

Muscarinic acetylcholine receptors (mAChR) in the embryonic chicken heart undergo agonist-induced internalization and a subsequent decrease in receptor number (downregulation). Cloning studies have identified two subtypes of mAChR expressed in the embryonic chicken heart, the cm2 and cm4 receptors. We report here that persistent activation of the mAChR in cultured chicken heart cells with the cholinergic agonist carbachol causes significant decreases in the levels of both cm2 and cm4 mRNA, as measured by solution hybridization analyses. The half-lives of the cm2 and cm4 mRNAs are not altered by agonist treatment, indicating that agonist most likely regulates mRNA levels by regulating the rate of gene transcription. Activation of mAChR in chicken heart causes both inhibition of adenylate cyclase activity and stimulation of phospholipase C activity. To test whether changes in the levels of intracellular second messengers were involved in the changes in mAChR mRNAs observed following agonist exposure, we determined the effects of incubation with agonists for the A1 adenosine receptors (which inhibit adenylate cyclase in chicken heart) and angiotensin II receptors (which stimulate phospholipase C) on mAChR receptor number and mRNA levels. Activation of these pathways together through heterologous receptors resulted in decreased mAChR number and mRNA levels, although these changes were not as large as those seen with direct activation of the mAChR. These results suggest that regulation of adenylate cyclase and phospholipase C activities may be involved in the regulation of mAChR gene expression.