This study was designed to characterize the muscarinic acetylcholine receptor (mAChR) subtype present in rat exorbital lacrimal gland as well as its biochemical coupling. The nonselective muscarinic antagonist [N-methyl-3H]scopolamine ([3H]NMS) binds with high affinity to a homogeneous population of binding sites in both membranes [dissociation constant (Kd) = 82.3 +/- 3.2 pM] and acinar cell (Kd = 170.3 +/- 20 pM) preparations. Muscarinic antagonist inhibition of [3H]NMS binding is homogeneous with the following order of potency: atropine > or = 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) > pirenzepine > 11-([2-(diethylamino)-ethyl]-1-piperidinyl)-acetyl- 5,11-dihydro-6H-pirido[2,3-b]1,4,benzo diazepine-6-one (AFDX 116). Both the affinity of the selective antagonists 4-DAMP, pirenzepine, and AFDX 116 and Northern blot analysis of lacrimal gland mRNAs show a single mAChR population of the M3 subtype. Muscarinic agonist inhibition of [3H]NMS binding displays both high (approximately 20%)- and low-affinity sites (approximately 80%). Both the receptor occupancy and the stimulation by agonists or the inhibition by antagonists of the accumulation of [3H]inositol phosphate were examined under identical conditions with respect to tissue preparations (acinar cells) and buffer (Krebs-Ringer). Results demonstrate 1) the efficient coupling of the M3 mAChR subtype with the phosphatidylinositol (4,5))bisphosphate-specific phospholipase C activity and 2) that the efficacy of a muscarinic agonist is dependent on its structure. Lastly, comparison of the agonists affinity and potency to trigger the [3H]inositol phosphate accumulation suggests that the occupation of the high-affinity agonist binding state of the M3 mAChR was involved in the cellular response.
The secretion of proteins and fluids from the exorbital lacrymal gland of rat is mainly controlled by muscarinic receptors. In a recent pharmacological study. Mauduit et al (Am J Physiol (1993) 264, C1550-C1560) identified a homogeneous population of M3 muscarinic receptors in preparations of acini from these tissues. In order to define the cellular composition of these acini and localize the muscarinic receptors, we have performed an immunofluorescent labelling study combined with confocal scanning microscopy. Antibodies raised against components of the different cytoskeletal networks (alpha-smooth muscle actin, cytokeratin peptide 14 and alpha-tubulin) revealed the presence of two different cell types. Cells with a stellate form are identified as myoepithelial cells, whereas rounded cells are secretory acinar cells. Both cell types are reactive with an antibody specifically directed against the muscarinic receptor. However, myoepithelial cells appear more intensely labelled than acinar cells. The roles of myoepithelial cells and secretory cells in the physiological function of the gland are discussed in terms of the distribution of muscarinic receptors.
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