Carbachol-induced desensitization of PLC-β pathway in rat myometrium: downregulation of Gqα/G11α

In the estrogen-treated rat myometrium, carbachol increased the generation of inositol phosphates by stimulating the muscarinic receptor-Gq/G11-phospholipase C-β3 (PLC-β3) cascade. Exposure to carbachol resulted in a rapid and specific (homologous) attenuation of the subsequent muscarinic responses in terms of inositol phosphate production, PLC-β3 translocation to membrane, and contraction. Refractoriness was accompanied by a reduction of membrane muscarinic binding sites and an uncoupled state of residual receptors. Protein kinase C (PKC) altered the functionality of muscarinic receptors and contributed to the initial period of desensitization. A delayed phase of the muscarinic refractoriness was PKC independent and was associated with a downregulation of Gqα/G11α. Atropine failed to induce desensitization as well as Gqα/G11α downregulation, indicating that both events involve active occupancy of the receptor. Prolonged exposure to A[Formula: see text] reduced subsequent A[Formula: see text] as well as carbachol-mediated inositol phosphate responses and similarly induced downregulation of Gqα/G11α. Data suggest that a decrease in the level of Gqα/G11α is subsequent to its activation and may account for heterologous desensitization.

M2 muscarinic ([3H]N-methyl scopolamine) binding in micropunches of rat ventricular myocardium: characterization and modification by progesterone

A new technique is outlined for the characterization and quantification of M2 muscarinic binding sites (receptors) in micro-punches (1 mm diam.), cut from slices (350 μm), of fresh cardiac tissue using the hydrophilic antagonist [3H]N-methyl scopolamine. The use of this water-soluble ligand allows us to label, and quantify, M2 receptors on the cell surface of intact cells contained within the micropunch. We believe that cardiac micropunches offer a simple but powerful approach to the investigation of membrane receptor regulation in tissue that largely retains the in vivo cytoarchitecture. Specific binding is reversible, stereospecific, saturable, of high affinity, and has the drug specificity typical of an M2 muscarinic receptor. In rat left ventricle, Bmax was 151.2 ± 10.3 fmol/mg protein while KD was 1.0 ± 0.1 nM. Nonspecific binding of the ligand was very low, varying from 2.8% (at 0.27 nM) to 7.7% (at 3.58 nM). This micropunch assay was used to determine that progesterone can compete with the muscarinic ligand for the M2 receptor in vitro (IC50 = 50 × 10−6 M). The steroids estradiol and testosterone, as well as ouabain, were without effect. Progesterone inhibited [3H]N-methyl scopolamine binding competitively (KD reduced from 1.9 to 4.3 nM) without affecting the rate of association of the ligand. However, progesterone induced a rapid dissociation of the ligand from its receptor. We conclude that the micropunch assay described here is suitable for the continued study of sex hormone effects on cardiac function.Key words: cardiac micropunches, muscarinic receptor, progesterone, [3H]N-methyl scopolamine.