Distribution Of Muscarinic Receptor Subtypes Research Articles

Distribution of muscarinic receptor subtypes and interstitial cells of Cajal in the gastrointestinal tract of healthy dairy cows

To describe the distribution of muscarinic receptor subtypes M(1) to M(5) and interstitial cells of Cajal (ICCs) in the gastrointestinal tract of healthy dairy cows. Full-thickness samples were collected from the fundus, corpus, and pyloric part of the abomasum and from the duodenum, ileum, cecum, proximal loop of the ascending colon, and both external loops of the spiral colon of 5 healthy dairy cows after slaughter. Samples were fixed in paraformaldehyde and embedded in paraffin. Muscarinic receptor subtypes and ICCs were identified by immunohistochemical analysis. Staining for M(1) receptors was found in the submucosal plexus and myenteric plexus. Antibodies against M(2) receptors stained nuclei of smooth muscle cells only. Evidence of M(3) receptors was found in the lamina propria, in intramuscular neuronal terminals, on intermuscular nerve fibers, and on myocytes of microvessels. There was no staining for M(4) receptors. Staining for M(5) receptors was evident in the myocytes of microvessels and in smooth muscle cells. The ICCs were detected in the myenteric plexus and within smooth muscle layers. Distribution among locations of the bovine gastrointestinal tract did not differ for muscarinic receptor subtypes or ICCs. The broad distribution of M(1), M(3), M(5), and ICCs in the bovine gastrointestinal tract indicated that these components are likely to play an important role in the regulation of gastrointestinal tract motility in healthy dairy cows. Muscarinic receptors and ICCs may be implicated in the pathogenesis of motility disorders, such as abomasal displacement and cecal dilatation-dislocation.

M2 mediated contractions of human bladder from organ donors is associated with an increase in urothelial muscarinic receptors

Previous studies have shown increased density of M2 receptors in hypertrophied rat bladders that possess an M2 contractile phenotype. The aim of the current study is to determine whether human bladders with an M2 contractile phenotype also have a greater density of bladder M2 receptors. Human bladders were obtained from 24 different organ transplant donors. Darifenacin and methoctramine affinity was determined by the rightward shift of cumulative carbachol concentration contractile response curves for each bladder. Radioligand binding and immunoprecipitation was used to quantify M2 and M3 subtypes in isolated detrusor muscle and urothelium. In addition, pig bladder muscle and urothelial receptors were quantified for comparison. In the human urothelium total, M2 and M3 muscarinic receptor density is significantly negatively correlated with the affinity of darifenacin for inhibition of contraction of the detrusor muscle. In the detrusor muscle there is no correlation between receptor density and darifenacin affinity for inhibition of contraction. Muscarinic receptor density is greater in the muscle than in the urothelium in human bladders whereas in the pig bladder the density is greater in the urothelium than in the muscle. The greater density of urothelial muscarinic receptors in human bladders with lower darifenacin affinity, indicative of a greater contribution of M2 receptors to the contractile response, points towards a possible role of the urothelium in controlling M2 mediated contractile phenotype. In comparison between human and pig bladders, the distribution of muscarinic receptor subtypes in the muscle and urothelium are quite different.

Quantitative analysis of the levels of expression of muscarinic receptor subtype RNA in the detrusor muscle of patients with overactive bladder.

Antimuscarinic drugs have frequently been used for the treatment for patients with an overactive bladder (OAB) and there have been many studies on the distribution of muscarinic receptor subtypes in the bladder. However, the distribution of muscarinic receptor subtypes in OAB patients has not been well investigated. In this study we investigated the distribution of muscarinic receptor subtypes with mRNA and protein expressions in patients with and without OAB, and investigated both the dome and trigone area. Samples of bladder smooth muscle were obtained from 10 individuals, five patients with OAB and a non-OAB group consisting of five patients who received radical cystectomy. The M2 receptor was predominant, but there was no significant difference in the level of M2 expression between the groups in the dome area. M5 expression in the dome area was significantly higher in the OAB group than in the non-OAB group. In the trigone area, the level of M2 mRNA expression was the highest in the non-OAB group, and was significantly lower in the OAB group. The levels of M1 and M5 mRNA expression were also observed in samples obtained from the trigone area. The multiformity of the muscarinic receptor subtypes in human bladder smooth muscle was confirmed, and our results suggest that the efficacy of a given pharmacologic therapy differs from patient to patient.

The effects of the antagonists of muscarinic acetylcholine receptor subtypes in rat brain on urinary bladder contraction

Pontine micuturition center and storage center are controlled by upper brain. Pharmacological and molecular biological data indicate the distribution of muscarinic receptor subtypes in rat brain, however, the effect of central muscarinic cholinergic mechanisms on the bladder activity has not been evaluated. We investigated the diversity of effect of each subtype in rat brain. The muscarinic agents such as muscarine (non-specific agonist), atropine (non-specific antagonist), pirenzepine (M1 receptor antagonist), AF-DX116 (M2 receptor antagonist) and 4-DAMP (M3 receptor antagonist) were administrated with intracerebroventricular injection (I.C.V. group) or intravenous injection (i.v. group). The drug effects on rhythmic bladder contraction were investigated. In I.C.V. group, the bladder contraction pressure were reduced by atropine, pirenzepine and 4-DAMP. The contraction time were shortened by pirenzepine and 4-DAMP but extended by AF-DX116. The contraction frequency was decreased by AF-DX116. Whereas AF-DX116 in i.v. group reduced the contraction pressure but did not cause any changes of the contraction time and the frequency. Other agents in i.v. group had same tendency with those of I.C.V. group except for intensity. That is to say AF-DX116 in I.C.V. group facilitate both micuturition center and storage center. In other words, these data indicate M2 receptor in rat brain inhibits both micurition and storage center. In addition, the effect of AF-DX116 in brain suggests that the development of new drug which proceed to central neuron system might provide a new treatment of neurogenic bladder.

Pharmacological strategies to selectively label and localize muscarinic receptor subtypes

Equilibrium binding assays support four (M1–M4) and molecular cloning studies have identified five (m1–m5) muscarinic receptor subtypes in the brain and peripheral tissues. However, the overlapping affinities of virtually all of the available muscarinic antagonists have permitted the unambiguous direct labeling of only two of the subclasses of muscarinic receptors, the M1 and M2 subtypes. Thus, the major obstacle to providing a detailed map of the distribution of muscarinic receptor subtypes in the brain, a tissue that expresses all five receptor proteins, has been the lack of muscarinic receptor subtype-specific ligands. Recent studies have exploited the distinct kinetic binding properties of muscarinic receptor subtypes and have demonstrated that a combination of both kinetic and equilibrium labeling approaches affords selective labeling of the five muscarinic receptors. Application of these novel labeling strategies has permitted for the first time a comparison of the distinct autoradiographic localization patterns of the M1–M5 receptor subtypes in the brain. These new labeling techniques compare well with previous pharmacological, immunological, and molecular methods for localizing and quantifying muscarinic receptor subtypes, and provide further evidence that the pharmacologically defined M1–M5 receptors correspond to the molecularly defined m1–m5 proteins. While unequivocal distribution profiles of each of the five muscarinic receptors awaits the development of more subtype-selective ligands, the labeling strategies described here provide an alternative, versatile approach for studying muscarinic receptor subtype distribution in the brain. Drug Dev. Res. 40:104–116, 1997. © 1997 Wiley-Liss, Inc.

Distribution of muscarinic receptor subtypes in rat brain from postnatal to old age

In an effort to understand the developmental changes in the distribution of muscarinic receptor subtypes (m1–m5), specific brain regions from juvenile (16-day-old), young (21-day-old) and adult (91-day-old) rats were analyzed using subtype-selective antibodies. These studies revealed significant age-dependent changes in the four brain regions examined. In cortex, and area associated with higher cognitive functions, significant increases of m2 and m4 receptors occurred between juvenile and adult rats. In the striatum, the level of m4 receptor increased with age whereas the m1, m2 and m3 receptors had reached mature levels within the first 16 days. Small but significant changes occured in the cerebellum with a decrease in m1, m3 and m4 receptor subtypes. In contrast to other brain regions, the hippocampus displayed consistent expression levels of muscarinic receptor subtypes. Suggesting that this brain region, which is involved in the foundation of numerous neural networks, requires a full complement of muscarinic receptors at a very early age. Muscarinic receptors have been shown to be important in a number of behavioral activities, including learning and memory. The changes observed in the age-dependent expression of these receptors most likely play an important role in how acetylcholine produces its effects in vivo.

Differences between rat dorsal and ventral hippocampus in muscarinic receptor agonist binding and interaction with phospholipase C

Carbachol, a full muscarinic receptor agonist, stimulated [ 3H]inositol phosphate accumulation in both the ventral and dorsal hippocampus, but its efficacy and affinity were higher in the former area. The partial agonist oxotremorine had a weak stimulatory effect in both regions. The affinity profiles of pirenzepine and AF-DX 116 in antagonizing carbachol-stimulated [ 3H]inositol phosphate accumulation indicated that M 1 and M 3 receptors contributed equally to the response in either region. On the other hand, there were no differences in the receptor density, or in the distribution of muscarinic receptor subtypes between the two regions of the hippocampus which could account for the effect as determined in binding experiments with selective antagonists. Analysis of carbachol binding curves did, instead, indicate a difference in the way the agonist interacted with the receptors within the hippocampus, i.e., carbachol recognized three agonist affinity states (superhigh, high and low) in the ventral hippocampus, and only two (high and low) in the dorsal part. The findings thus suggested that the regional diversity in the efficacy of carbachol in stimulating phosphoinositide turnover was related to the complexity with which it bound to muscarinic receptors. Transduction processes that intervene between changes in the muscarinic receptors' conformation and activation of phospholipase C might be relevant to these differences.

Visualization of muscarinic m4 mRNA and M 4 receptor subtype in rabbit lung

We have used in situ hybridization to study the localization of muscarinic receptor subtype mRNAs and compared this with the distribution of muscarinic receptor subtypes using receptor autoradiography in tissue sections of the rabbit lung. To localize muscarinic receptor subtype mRNAs, subtype-specific synthetic oligonucleotide probes or riboprobes were generated. Two muscarinic receptor mRNAs (m3 and m4) were detected. The m3 mRNA was expressed in high concentration in tracheal smooth muscle, epithelium and blood vessels and to a lesser extent in bronchiolar smooth muscle while the m4 mRNA was abundantly expressed in bronchiolar smooth muscle and alveolar walls. No expression of m1, m2 or m5 mRNAs was found in any lung structures. This was confirmed by Northern blot analysis which revealed only a single transcript of 3.6 kb for m4 mRNA in rabbit lung. With the exception of the airway epithelium and blood vessels, there was a correspondence between mRNA localization and the distribution of mucarinic receptor subtypes visualized by [ 3H](−)-quinuclidinyl benzilate ([ 3H]QNB) autoradiographically in the presence of relatively selective muscarinic subtype antagonists (M 1–M 3) and direct labelling with selective [ 3H]AF-DX 384. The significance and the reasons for the presence of m4 mRNA and M 4 receptors in the rabbit lung are uncertain and warrant further investigation.

Differential distribution of muscarinic receptor subtypes and their regulation by G-protein in rat brain

1. [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding in rat cerebral and cerebellar synaptosomes had different B max values, but similar K d values. 2. These bindings could be displaced by classic muscarinic agents: pilocarpine (partial agonist), and atropine (antagonist), which both had similar binding affinities in rat cerebral and cerebellar synaptosomes. 3. The new muscarinic M 1 selective agents: McN-A-343 (agonist), prienzepine and trihexyphenidyl (antagonists) and higher affinities for receptor sites in the cerebrum than in the cerebellum. 4. The muscarinic M 2 selective agents: carbachol, oxotremorine (agonists), and AF-DX-116 (antagonist) had higher affinities for receptor sites in the cerebellum than in the cerebrum. 5. GPP(NH)p (40 μM) decreased the binding affinities of carbachol and oxotremorine in the cerebellum, but not in the cerebrum. However, it did not decrease the binding affinities of all the antagonists studied in both brain regions. 6. These results reveal that more muscarinic M 1 sites are present in the cerebrum than in the cerebellum, while the opposite is true for M 2 sites. Furthermore, the regulatory role of G-protein on these muscarinic receptor subtypes in the brain is different.

Distribution of muscarinic receptor subtypes within architectonic subregions of the primate cerebral cortex.

The regional distributions of muscarinic receptor subtypes (M1 and M2) in the macaque brain were investigated by in vitro receptor autoradiography. Putative muscarinic receptor subtypes were distinguished by their differential affinities for pirenzepine and carbachol in competition with [3H]-quinuclidinyl benzilate. Autoradiographic visualization of muscarinic receptor subtypes demonstrated marked regional and laminar variations that respected architectonic boundaries. The M1 receptor subtype was widely distributed throughout most cortical areas and was most intense over the superficial layers. Almost all limbic and paralimbic regions including the amygdala, hippocampus, orbitofrontal, temporopolar, parahippocampal, cingulate, and parolfactory areas displayed peak densities of the M1 receptor subtype. The M2 receptor subtype was selectively elevated in the primary sensory areas of all five sensory modalities, including the visual (area 17, V1), auditory (A1), and somatosensory (3b, S1) koniocortices, the anterior olfactory nucleus, and the gustatory area. The primary motor area also displayed a relative peak of M2 receptor subtype labeling. In the hippocampal formation, M1, M2, and nicotine receptors were distributed differentially, with each subdivision having a specific complement of cholinergic receptor subtype. The M1 receptor subtype was prevalent in the dentate gyrus, the CA4-CA3 region, and the CA1 ammonic sector. The M2 receptor subtype was concentrated in the CA2 sector, the subiculum, the rhinal cortices, and the parasubiculum. Putative neural nicotinic receptors, tagged with L-[3H]-nicotine, were most concentrated within the presubiculum.

Distribution of muscarinic receptor subtypes in rat brain as determined in binding studies with AF-DX 116 and pirenzepine

In vitro competition binding experiments with the selective 3 muscarinic antagonists AF-DX 116 and pirenzepine (PZ) vs H-N-methylscopolamine as radioligand revealed a characteristic distribution of muscarinic receptor subtypes in different regions of rat brain. Based on non linear least squares analysis, the binding data were compatible with the presence of three different subtypes: the M 1 receptor (high affinity for PZ), the cardiac M 2 receptor (high affinity for AF-DX 116) and the glandular M 2 receptor (low affinity for PZ and AF-DX 116). The highest proportion of M 1 receptors was found in the hippocampus, whilst the cerebellum and the hypothalamus were the regions with the largest fraction of the cardiac M 2 and glandular M 2 receptors, respectively. In certain brain areas, depending on the relative proportions of the subtypes, flat binding curves were seen for AF-DX 116 and PZ. Based on these data, an approximate distribution pattern of the subtypes in the various brain regions is presented.