M3 Distribution Research Articles

Autoradiographic distribution of M1, M2, M3, and M4 muscarinic receptor subtypes in Alzheimer's disease.

We studied the autoradiographic densities of all pharmacologically characterised muscarinic receptors (MR) in frontal, temporal, and visual cortex, hippocampal formation, and striatum in autopsied brains from 19 histopathologically verified patients of Alzheimer's disease (AD) and in matched controls. Almost all (16 of 19) of the AD cases were severe. In AD brains, total MR, M1, and M3 MR subtypes were found to be significantly decreased in entorhinal cortex and in most hippocampal strata. Total MR and M1 receptors were also significantly reduced in visual area and in frontal cortex of AD brains, respectively. M2 receptors were significantly reduced over hippocampal formation but increased significantly in striatum of AD brains as compared with controls. M3 receptors in AD were in the range of controls in neocortex and striatum, whereas the M4 receptor subtype was also preserved in all brain regions in AD brains when compared with controls. This is the first autoradiographic study analysing the distribution of all MR subtypes in AD brains. These changes in MR densities concur with the general pattern of neuronal degeneration occurring in AD brains and partly explain the poor response of AD cognitive decline to present cholinergic supplementation therapies. Although M3 and M4 MR were labelled with nonselective approaches, the preservation of M4 and to a lesser degree M3 MR subtypes in AD brains could open an alternative way for the symptomatic therapy of AD dementia.

Expression of muscarinic receptor subtypes and M2-muscarinic inhibition of adenylyl cyclase in lung.

The relative distribution and absolute quantities of muscarinic receptor subtypes m1, m2, m3, and m4 were determined in membranes of canine trachealis muscle, bronchi, and lung parenchyma by immuno-precipitation with receptor subtype-specific rabbit polyclonal antisera. Additionally, the functional coupling of muscarinic receptors to the inhibition of adenylyl cyclase was related to the presence of m2-muscarinic receptors in each region. Immunoprecipitation identified more total muscarinic receptors in trachealis muscle than in bronchi or lung. m2-Muscarinic receptor predominated in tracheal muscle (372 +/- 85 fmol/mg protein) with fewer m3 receptors (48 +/- 5 fmol/mg protein). Bronchi contained 6.6 +/- 2.0 and 9.2 +/- 1.8 fmol/mg protein of m2 and m3 receptors, respectively. Lung parenchyma contained 13.9 +/- 3.9 fmol/mg protein of m3 receptors. Adenylyl cyclase activity increased in response to guanosine triphosphate and isoproterenol in membranes from all three lung regions, but muscarinic-mediated inhibition of adenylyl cyclase occurred only in trachealis membranes. These studies provide the first quantitative assessment of muscarinic receptor subtypes in different regions of the lung and relate the ability to measure muscarinic inhibition of adenylyl cyclase to the presence of m2 receptors.

Localization of muscarinic M3 receptor protein and M3 receptor binding in rat brain

A family of receptor subtypes, defined either by molecular (ml–m5) or pharmacological (M1–M4) analysis, mediates muscarinic cholinergic neurotransmission in brain. The distribution and functions of the m3 receptor protein in brain and its relation to M3 ligand binding sites are poorly understood. To better characterize the native brain receptors, subtype-specific antibodies reactive with the putative third inner loops were used: (i) to measure the abundance of m3 protein and its regional distribution in rat brain by immunoprecipitation; (ii) to determine the cellular and subcellular distribution of m3 protein by light microscopic immunocytochemistry; and (iii) to compare the distribution of m3 immunoreactivity with the autoradiographic distribution of M3 binding sites labeled by [ 3H]4-diphenylacetoxy- N-methyl pipericline methioxide in the presence of antagonists selective for the other receptor binding sites. The m3 protein, measured by immunoprecipitation, accounted for 5–10% of total solubilized receptors in all brain regions studied. Immunocytochemistry also revealed a widespread distribution of m3-like immunoreactivity, and localized the subtype to discrete neuronal populations and distinct subcellular compartments. The distribution of m3 protein was consistent with the messenger RNA expression, and like M3 binding sites, the protein was enriched in limbic cortical regions, striatum, hippocompus, anterior thalamic nuclei, superior colliculus and pontine nuclei. However, m3 immunoreactivity and M3 binding were differentially localized in regions and lamina of cortex and hippocompus. The results confirm the presence of m3 protein in brain, its low abundance compared to other muscarinic receptor subtypes, and provide the first immunocytochemical map of its precise localization. The distribution of m3 suggests that it mediates a wide variety of cholinergic processes in brain, inclucling possible roles in learning and memory, motor function and behavioral state control. However, since the distribution of the molecularly-defined receptor protein is distinct from the pharmacologically-defined M3 binding site, investigations of the functions of m3 in brain must await development of more selective ligands or use of non-pharmacological approaches.

Laminar expression of m1-, m3- and m4-muscarinic cholinergic receptor genes in the developing rat visual cortex using in situ hybridization histochemistry. Effect of monocular visual deprivation

The postnatal development of laminar pattern of m1-, m3- and m4-mRNA-muscarinic acetylcholine receptor subtypes in the visual cortex of both normally raised and monocularly deprived rats (one eyelid sutured at the age of 11 days) was studied using in situ hybridization histochemistry and computer-assisted image analysis. From birth until day 15 the level of m1-receptor transcript in layer II/III increases markedly as compared to deeper layers. From day 15 up to day 18 a transient bimodal pattern develops with peaks in layers II/III and VI. Already on day 35 a more homogeneous distribution of m1-receptor mRNA level is detectable persisting until adulthood. In contrast, the m3-receptor mRNA shows already at birth a bimodal distribution with peaks in layers II/III and VI. Further development until adulthood results in transient changes in the ratio of the mRNA levels in these layers. In the adult visual cortex a similar laminar pattern as at birth is observed. From day 1 up to day 10 a relative increase in the mRNA level of the m4-receptor in layers II to IV is observed. From day 10 until day 15 a bimodal distribution of receptor mRNA develops with peaks in layers III and VI which is similar to the adult stage. However, between days 18 and 35 a shift in the laminar receptor mRNA distribution occurs resulting in peaks in layers IV and VI. The labeling of the m5-receptor transcript in rat visual cortex was very weak and did not show any alteration with age.Unilateral eyelid closure from postnatal day 11 resulted in transient changes in the laminar distribution of m3- and m4-receptor mRNA between postnatal days 18 and 25, whereas the development of the laminar pattern of the m1-receptor mRNA was not affected regardless of the length of visual deprivation.The distinct laminar developmental pattern of mRNA muscarinic receptor subtypes in rat visual cortex suggests specific roles of the muscarinic receptor subtypes during the first weeks of postnatal maturation of visual function.

Association of glycosphingolipids with intermediate filaments of human umbilical vein endothelial cells

Our previous studies of glycosphingolipids (GSLs) of human umbilical vein endothelial cells (HUVECs) established that globoside and ganglioside G M3 are the most abundant GSLs of HUVECs. Both compounds are located intracellularly, as well as on the cell surface. In this study, we demonstrate that the intracellular globoside and G M3 antigens are associated with the vimentin intermediate filaments of the HUVEC cytoskeleton. Immunofluorescence staining of fixed, permeabilized HUVECs showed colocalization of globoside and G M3 with vimentin but not with tubulin or actin. Both GSLs remained associated with intermediate filaments after perinuclear collapse of the filaments induced by colcemid. Indirect evidence that the globoside epitope is present on a GSL is the loss of staining by anti-globoside after methanol fixation and the absence of anti-globoside reactivity with HUVEC proteins on immunoblots. Colocalization of anti-globoside and anti-vimentin was also demonstrated in cryosections of endothelial cells, which indicates that the observed association was not an artifact induced by exposure of cells to detergent or organic solvent. Association of globoside with intermediate filaments was confirmed by immunoelectron microscopy, which demonstrated the presence of antigen along intermediate filaments, as well as on the cell surface and on lipid vesicles. Interferon-γ decreased the ratio of surface to filamentous globoside staining, but had the opposite effect on G M3 distribution. Less abundant HUVEC GSLs, including Gb 3, nLc 4, IV 2FucnLc 4, and IV 3NeuAcnLc 4, were not detected along filaments. This is the first report of the association of GSLs with intermediate filaments. We suggest that intermediate filaments may play a role in the transport of GSLs.