Muscarinic Receptor mRNA Research Articles

β-Adrenergic and muscarinic receptor mRNA accumulation in the sinoatrial node area of adult and senescent rat hearts

The sinoatrial (SA) node is the cardiac pacemaker and changes in its adrenergic-muscarinic phenotype have been postulated as a determinant of age-associated modifications in heart rate variability. To address this question, right atria were microdissected, the SA node area was identified by acetylcholinesterase staining, and, using a RT-PCR method, the accumulation of mRNA molecules encoding β 1- and β 2-adrenergic ( β 1- and β 2-AR) and muscarinic (M 2-R) receptor was quantified to define the proportion between β-AR and M 2-R mRNAs within the sinoatrial area of adult (3 months) and senescent (24 months) individual rat hearts. In adult hearts, the highest M 2-R/ β-AR mRNA ratio was observed within the sinoatrial area compared with adjacent atrial myocardium, while in the senescent hearts, no difference was observed between sinoatrial and adjacent areas. This change was specific of the sinoatrial area since adult and senescent whole atrial or ventricular myocardium did not differ in their M 2-R/ β-AR mRNA ratio, and was associated with a fragmentation of acetylcholinesterase staining of the senescent SA node. Quantitative changes in the expression of genes encoding proteins involved in heart rate regulation specifically affect the sinoatrial area of the senescent heart.

Localization of the m2 muscarinic acetylcholine receptor protein and mRNA in cortical neurons of the normal and cholinergically deafferented rhesus monkey

Localization of the m2 muscarinic acetylcholine receptor protein and mRNA in cortical neurons of the normal and cholinergically deafferented rhesus monkey

Localization of the m2 muscarinic acetylcholine receptor protein and mRNA in cortical neurons of the normal and cholinergically deafferented rhesus monkey

The m2 muscarinic acetylcholine receptor in the cerebral cortex has traditionally been thought of as an autoreceptor located on cholinergic fibers that originate from neurons in the nucleus basalis of Meynert. We now provide evidence for widespread localization of the m2 receptor in noncholinergic neurons and fibers of the cerebral cortex. The cellular and subcellular distribution of the m2 receptor protein and mRNA were examined in normal monkeys and in monkeys in which the cortical cholinergic afferents were selectively lesioned by injection of the specific immunotoxin, anti-p75NTR-saporin into the nucleus basalis. Both in normal and immunolesioned monkeys, the m2 mRNA and protein were localized in pyramidal and nonpyramidal neurons. In pyramidal neurons, membrane-associated receptor immunoreactivity was found exclusively in dendritic spines receiving asymmetric synapses, indicating that the m2 receptor may modulate excitatory neurotransmission at these sites. In nonpyramidal neurons, the m2 immunoreactivity was present along the cytoplasmic surface of membranes in cell bodies, dendrites and axons. Both in pyramidal and nonpyramidal neurons of normal and lesioned monkeys, the m2 receptor was located peri- and extra-synaptically, suggesting that it may be contacted by acetylcholine via volume transmission. The localization of the m2 receptor in cortical neurons and the sparing of m2 immunoreactivity in lesioned monkeys indicates that the m2 receptor is synthesized largely within the cortex and/or is localized to noncholinergic terminals of either intrinsic or extrinsic origin. These findings open the possibility that the loss of the m2 receptor in Alzheimer's disease may in part be due to degenerative changes in m2 positive neurons of the cortex rather than entirely due to the loss of autoreceptors.

MRNA for the m4 muscarinic receptor subtype is expressed in adult rat brain cholinergic neurons

A number of pharmacological, anatomical, and immunological studies have previously addressed the subtype identity of the hippocampal muscarinic pre-synaptic autoreceptor. A preponderance of findings indicate that it is of the M2 pharmacological type. Both the m2 and m4 molecular subtypes exhibit M2 pharmacology and there are few drugs that differentiate between these receptors. Pharmacological attempts at defining the hippocampal autoreceptor have yielded conflicting results. The basal forebrain is relatively enriched in m2 muscarinic receptor mRNA and protein, and lesions that denervate the hippocampus of its basal forebrain cholinergic input have shown a decrement in m2, but not m4, receptor protein in the hippocampus. Thus, the anatomical data obtained to date tend to support the view that the m2 subtype is expressed as the hippocampal autoreceptor. We have combined in situ hybridization histochemistry (ISHH) with immunocytochemistry to choline acetyltransferase to examine whether mRNA for the m4 subtype of muscarinic receptor is expressed in central cholinergic neurons. The m4 muscarinic mRNA was found at moderate levels in all subdivisions of the cholinergic basal forebrain, including the medial septum/diagonal band complex (MS/DB). The m4 mRNA was also found in striatal cholinergic interneurons, in the cholinergic reticular core of the upper brainstem, and in brainstem cholinergic motor neurons. Muscarinic m4 receptor mRNA was also found in many non-cholinergic cells in the brain. For example, the hippocampal pyramidal neurons, dentate gyrus granule cells, and entorhinal cortical pyramidal neurons express relatively high levels of m4 mRNA, while in the brainstem the dorsal raphe and pontine reticular nuclei express relatively high levels of this mRNA. The finding of m4 mRNA in the (MS/DB) cholinergic neurons suggests that this receptor protein might be expressed as an autoreceptor in hippocampal cholinergic terminals.

Increased expression of M2 muscarinic receptor mRNA and binding sites in the rostral ventrolateral medulla of spontaneously hypertensive rats

A significant body of evidence suggests that the development and maintenance of elevated blood pressure in the spontaneously hypertensive rat (SHR), a genetic model for essential hypertension, is due at least partly to a central hyper-cholinergic state. For example, this strain responds with an exaggerated pressor response to pharmacological stimulation of central muscarinic receptors in certain brain regions compared to normotensive Wistar Kyoto rats (WKY). At least part of the enhanced response to central muscarinic receptor stimulation in SHR is due to the altered expression of post-synaptic receptors. In the present study, the reverse transcriptase-polymerase chain reaction and autoradiographic techniques were used to estimate the relative levels of mRNA and density of receptor binding sites for the five subtypes of muscarinic receptors within the rostral ventrolateral medulla (RVL) of SHR and WKY. Adult (12-week-old) SHR exhibited an increase in the levels of both M2 muscarinic mRNA, and M2 receptor binding sites in RVL compared to age-matched normotensive WKY. Similarly, 4-week-old pre-hypertensive SHR exhibited increased levels of M2 mRNA in whole medulla oblongata, and an increase in the number of binding sites for M2 receptors in the RVL. Since the RVL is known to integrate tonic cholinergic sympathoexcitatory input, these results suggest that the increased expression of M2 muscarinic receptors in this region represents one neurochemical correlate for the maintenance of excessive central efferent sympathetic nervous activity in the SHR. Since the neurochemical change precedes the development of hypertension, the altered medullary M2 receptor expression may play a role as an initiating or predisposing factor for the development of hypertension in SHR.

Regulation of muscarinic M2 receptors.

The molecular mechanisms involved in the regulation of muscarinic receptor gene expression are poorly understood. In an effort to gain a better understanding of the regulation of M2 receptors, we have investigated homologous and heterologous regulation of M2 muscarinic receptor protein and gene expression in human embryonic lung fibroblasts (HEL 299 cells). HEL 299 cells constitutively express m2 receptors, with no evidence of other muscarinic receptor subtypes. We have shown that M2 receptors in these cells can be down-regulated by muscarinic and beta2-adrenergic receptor agonists. Unlike the down-regulation mediated by muscarinic and beta-adrenergic stimulation, activation of PKC with PDBu was mediated through changes in m2 muscarinic receptor mRNA through reduced gene transcription. Because of the inflammatory nature of asthma, we have focused on delineating the interactions between cytokines and M2 receptors in an attempt to define potential endogenous modulators of M2 receptor expression. We have shown that the multi-functional cytokine, transforming growth factor beta1 (TGF-beta1), which is involved in several inflammatory conditions induces desensitization and down-regulation of M2 muscarinic receptor protein and gene expression that was mediated through a reduction in the rate of m2 receptor gene transcription. Other cytokines of interest are tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) which are elevated in asthma. We have demonstrated that TNF-alpha and IL-1beta synergise to induce down-regulation of M2 muscarinic receptor protein and mRNA which was associated with functional desensitization of the receptor protein. The M2 receptor mRNA down-regulation appeared to be mediated through a reduction in the rate of m2 receptor gene transcription which may be dependent on the transcription and translation of unknown protein factor(s). Moreover, a role of PKA and ceramide pathways in M2 receptor regulation is suggested. Collectively, our work provides a mechanistic explanation of previous reports indicating altered function of M2 receptors in asthma. Ours results also suggest that the expression of this receptor subtype may be under the control of a cytokine network at the airways.

TGFβ and FGF-2 down-regulate m2 muscarinic receptor mRNA expression in embryonic chick ventricular myocytes

TGFβ and FGF-2 down-regulate m2 muscarinic receptor mRNA expression in embryonic chick ventricular myocytes

Demonstration of muscarinic receptor mRNAs in parietal cells, enteric ganglia and immune cells of rat stomach by in situ hybridization histochemistry

Demonstration of muscarinic receptor mRNAs in parietal cells, enteric ganglia and immune cells of rat stomach by in situ hybridization histochemistry

Human meningiomas possess muscarinic acetylcholine receptors: stimulation of phosphatidylinositol turnover by carbachol.

The effect of carbachol, an acetylcholine receptor agonist, on rate of phosphatidylinositol (PI) turnover in cultured human meningioma cells was investigated. Exposure of meningioma cells for 2 h to carbachol (3.12-200 mumol/L) resulted in a dose-dependent stimulation of PI turnover to a maximum of 5.5-fold over basal controls. A time course study showed stimulation of IP3 formation after 30 s followed by increases in IP1 and IP2. The stimulatory effect of carbachol on PI turnover was completely abolished by the muscarinic receptor antagonist, atropine, but was unaltered by the nicotinic antagonist, hexamethonium. Reverse-transcription of meningioma-derived RNA into cDNA followed by amplification by the polymerase chain reaction using specific primers revealed presence of ml type muscarinic receptor mRNA. These results provide evidence that human meningioma cells possess muscarinic acetylcholine receptors the activation of which leads to PI hydrolysis.

Synergy between Tumor Necrosis Factor α and Interleukin 1β in Inducing Transcriptional Down-regulation of Muscarinic M2 Receptor Gene Expression: INVOLVEMENT OF PROTEIN KINASE A AND CERAMIDE PATHWAYS

Stimulation of HEL 299 cells with tumor necrosis factor alpha (TNF-alpha) or interleukin 1beta (IL-1beta) had no effect on M2 muscarinic receptor expression. However, the combination of these two cytokines markedly down-regulated muscarinic M2 receptor protein and mRNA expression and uncoupled M2 receptors from adenylyl cyclase. There was no effect of TNF-alpha and IL-1beta on the m2 muscarinic receptor mRNA stability, and nuclear run-on assays showed reduced m2 receptor gene transcription. Sequential cytokine addition suggests that the synergy involves postreceptor events. Although the cAMP-dependent protein kinase inhibitor H8 provided a significant protection against receptor down-regulation, the protein kinase C inhibitor GF109203X had no effect. The ceramide analog C2-ceramide (N-acetylsphingosine) was without effect on m2 receptor expression. However, a strong synergistic effect was demonstrated when cells were treated with the combination of C2-ceramide and TNF-alpha or IL-1beta. TNF-alpha and/or IL-1beta combination also activated the 46- and 55-kDa c-Jun NH2-terminal protein kinases and to a lesser extent p42 and p44 mitogen-activated protein kinase isoforms. Cycloheximide abolished the TNF-alpha and IL-1beta effect, suggesting that de novo protein synthesis is required for receptor down-regulation. These results suggest that the TNF-alpha and IL-1beta synergize to induce transcriptional down-regulation of the M2 muscarinic receptor, which seems to be mediated through activation of both ceramide and cAMP-dependent protein kinase pathways. Furthermore, these results suggest that M2 receptor expression is under the control of a cytokine network.

Distribution of muscarinic receptor mRNAs in the stomachs of normal or immobilized rats

Distribution of muscarinic receptor mRNAs in the stomachs of normal or immobilized rats

Axotomy-induced loss of m2 muscarinic receptor mRNA in the rat facial motor nucleus precedes a decrease in concentration of muscarinic receptors.

The abundance of muscarinic receptors and m2 muscarinic receptor mRNA in the facial nuclei of rats was evaluated by autoradiographic procedures at various times up to 14 days after transection of the right facial nerve. Receptors were labelled by in vitro incubation of brain sections with L-[3H]quinuclidinyl benzilate, while in situ hybridization with a 35S-labelled oligonucleotide was used to identify m2 muscarinic receptor mRNA in neighbouring sections. The right and left facial nuclei of non-operated control rats appeared equivalent in abundance of muscarinic receptors (359 +/- 8 versus 376 +/- 9 fmol per mg tissue, n = 5) and the presence of m2 mRNA. Axotomy had no effect on the concentration of receptors in the contralateral facial nucleus but caused a gradual loss of receptors from the ipsilateral side. No change was detected at 1 day after nerve transection, but a 23% decrease relative to the contralateral facial nucleus had occurred by 3 days. A maximum decrease of 51% was achieved by 1 week after nerve transection. By comparison, m2 mRNA was nearly eliminated from the ipsilateral facial nucleus at 1 day post-taxonomy and remained depleted for the duration of study. Previous work has established that no significant loss of motoneurons occurs within this period. Accordingly, it is postulated that axonal injury inhibits transcription of the m2 muscarinic receptor gene, resulting in a later decrease in muscarinic receptor protein expression.

Expression of m1 and m4 muscarinic receptor mRNA in the striatum following a selective lesion of striatonigral neurons

Expression of m1 and m4 muscarinic receptor mRNA in the striatum following a selective lesion of striatonigral neurons

Expression of m1 and m4 muscarinic receptor rnRNA in the striatum following a selective lesion of striatonigral neurons

Cholinergic stimulation has opposing effects on striatopallidal and striatonigral neurons. Most striatal projection neurons express m1 muscarinic receptor mRNA with m4 mRNA found in 40–50%. Expression of m4 mRNA is found in most preprotachykinin neurons but only a subset of preproenkephalin neurons, suggesting preferential localization of m4 receptors to striatonigral neurons. A volkensin lesion of striatonigral neurons reduced striatal m4 mRNA by 63% and m1 mRNA by only 18%, suggesting that preferential expression of m4 receptors by striatonigral neurons may contribute to their differential response.

Pre-and postsynaptic M3 muscarinic receptor mRNAs in the rodent peripheral auditory system

Pre-and postsynaptic M3 muscarinic receptor mRNAs in the rodent peripheral auditory system

Pre- and postsynaptic M3 muscarinic receptor mRNAs in the rodent peripheral auditory system.

The medial and lateral efferent innervations originate from distinct parts of the superior olivary complex. Both use acetylcholine, respectively, to modulate the activity of outer hair cells (OHC), and spiral ganglion neurons (SGN) which are postsynaptic to the inner hair cells (IHC). Besides predominantly activating nicotinic receptors, acetylcholine recognizes muscarinic M3 receptors, whose the role(s) and cellular localization(s) are not yet firmly established. We used reverse transcription and polymerase chain reaction to amplify the M3 receptor cDNA in the rat and guinea pig organ of Corti and spiral ganglion. Then, we localized the M3 receptor mRNAs in cochleas and superior olivary complex of both species. The M3 receptor cDNA was amplified from samples of brain, organ of Corti and spiral ganglion. Indeed, its corresponding mRNA was localized in SGNs, OHCs and IHCs. However, in the apical turns, OHCs were often found unlabeled. In the superior olivary complex, M3 mRNAs were colocalized with choline acetyltransferase mRNAs in neurons of the lateral superior olive and ventral nucleus of the trapezoid body. These results suggest that the M3 receptor-induced inositol phosphate formation described in previous studies [21] takes place in both postsynaptic (SGNs, OHCs) and presynaptic components of efferent cochlear synapses, and in cells that are not contacted by efferents in the adult cochlea (IHCs).

Temporal and spatial expression patterns of two G-protein coupled receptors inDrosophila melanogaster

Temporal and spatial expression patterns of a muscarinic acetylcholine receptor (Acr60C) and an octopamine/tyramine receptor (Octyr) were determined in Drosophila melanogaster using quantitative Northern analysis and in situ hybridization to tissue sections. Expression of mRNA encoding both of these G-protein coupled receptors peaks initially in 18 to 21 hour embryos following the formation of the mature larval nervous system. Levels of mRNA then decline during larval stages, rising to a second peak in 3 to 4-day-old pupae after a period of major nervous system reorganization. The muscarinic acetylcholine receptor mRNA is expressed throughout the cortical regions of the central nervous system in adults and embryos. Particularly high levels of expression of Acr60C are observed in cell bodies adjacent to the antennal lobes, suggesting a major role for this muscarinic receptor in the processing of olfactory information. In contrast, the octopamine/tyramine receptor mRNA is distributed diffusely throughout the adult brain, with patches of signal concentrated in the cortex of the dorsal protocerebrum near the mushroom bodies. These patches may represent individual cells expressing Octyr receptors.

Muscarinic and beta-adrenergic receptor expression in peripheral lung from normal and asthmatic patients.

We used human peripheral lung from 8 mildly asthmatic patients and 11 normal donors to study the expression of muscarinic and beta-adrenergic receptors in asthma. There was no significant difference in the affinity or the density of muscarinic (labeled with [N-methyl-3H]scopolamine) and beta 1- and beta 2-adrenergic receptors (labeled with [125I]iodocyanopindolol) in peripheral lung from asthmatics compared with nonasthmatics. Only the muscarinic m1 receptor mRNA was detected in human lung using Northern blot analysis. Additionally, peripheral lung cellular mRNA hybridized to human beta 1 and beta 2 cDNA probes, giving 3.2- and 2.2-kb hands corresponding to beta 1 and beta 2-adrenergic receptors mRNA, respectively. Densitometric scanning of the autoradiograms suggests that there was no significant difference in the relative abundance of muscarinic m1 and beta 1- and beta 2-adrenergic receptor mRNA in asthmatic compared with nonasthmatic lungs. Functional experiments obtained in trachea suggest that there was an increase in the cholinergic neural response evoked by electrical field stimulation in asthmatic compared with nonasthmatic tissues which was not due to a reduction in inhibitory noncholinergic nonadrenergic relaxations.

Preventive effects of bifemelane hydrochloride on decreased levels of muscarinic acetylcholine receptor and its mRNA in a rat model of chronic cerebral hypoperfusion

Changes in muscarinic acetylcholine receptor (mACh-R) binding and muscarinic cholinergic m1 receptor (m1-R) mRNA levels were determined in a rat model of cerebral hypoperfusion in which hypoperfusion was induced by permanent bilateral occlusion of the common carotid arteris. After 6 weeks of hypoperfusion, mACh-R binding activity was significantly reduced in the frontal cortex (79.0%, P < 0.01), striatum (74.2%, P < 0.01) and hippocampus (78.6%, P < 0.01), and the m1-R mRNA levels in the frontal cortex (86.6%, P < 0.05) and striatum (89.4%, P < 0.0) compared with sham-operated control. Repeated administration of bifemelane hydrochloide (15 mg/kg/day, p.o., once a day from the day of operation for 6 weeks) prevented the hypoperfusion-induced loss of mACh-R binding and m1-R mRNA levels above described. Since the central cholinergic systems play an important role in learning and memory, these findings suggest that bifemelane hydrochloride is useful to treat and/or prevent vascular dementia which is closely related to cerebral hypoperfusion.

Induction of muscarinic receptor subtypes in monocytic/macrophagic cells differentiated from EoL-1 cells

We demonstrated that eosinophilic leukemia cell line-1 (EoL-1 cells) differentiated into monocytic/macrophagic cells by a treatment with interferon-γ. Muscarinic receptor mRNA was not detected in untreated EoL-1 cells, and the cytosolic concentration of Ca 2+ ([Ca 2+] i) did not rise either in these cells. Interestingly, when EoL-1 cells were treated with interferon-γ, mRNAs for muscarinic M 3 and M 5 receptors could be detected in these cells, along with an increase in [Ca 2+] i and chemotaxis induced by carbachol that could be blocked with 4-diphenylacetoxy- N-methylpiperidine methiodide (4-DAMP) and pirenzepine. These data support the functional importance of muscarinic M 3 and M 5 receptors in monocytic/macrophagic cells differentiated from EoL-1 cells. This model also provides evidence of a significant functional interaction between muscarinic M 3 and M 5 receptors.