BIOCHEMICAL CHARACTERISTICS OF MUSCARINIC CHOLINORECEPTORS IN SWINE TRACHEAL SMOOTH MUSCLE

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Abstract
1 The tritiated muscarinic cholinoreceptor antagonist quinuclidinyl benzilate, [3H]QNB, was used to characterize the muscarinic receptors associated with homogenized membrane of the smooth muscle from swine trachea. Based on receptor binding assays, the homogenate had specific, saturable, high-affinity receptors for [3H]QNB. 2 Specific binding was time- and temperature-dependent. The association of [3H]QNB with the muscarinic receptor reached equilibrium much sooner at 37.degree. C than 25.degree. C at a [3H]QNB concentration of 180 pM (30 min and 2h, respectively). Equilibrium at both temperatures was attained within 5 min at a [3H]QNB concentration of 1800 pM. All remaining experiments were performed at 37.degree. C. 3 Binding was saturable with respect to [3H]QNB and tissue concentrations. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 51 .+-. 20 pM and a maximum receptor density (Bmax) of 2.17 .+-. 0.27 pmole/mg protein. The Hill coefficient for [3H]QNB binding was 1.07 .+-. 0.16. The association (K1) and dissociation (K-1) rate constants were determined to be (5.51 .+-. 0.16) .times. 108 M-1 min-1 and (1.41 .+-. 0.18) .times. 10-2 min-1 respectively. KD calculated from the ratio of K1 and K-1 was 26.3 .+-. 3.8 pM; this value is close to the value of KD calculated from Scatchard plots of binding isotherms. 4 The density of muscarinic receptor binding sites was 10-fold in tracheal smooth muscle than in tracheal epithelium (0.20 .+-. 0.03 pmole/mg protein). There is no difference between weanling and young swine in the density of muscarinic receptors in tracheal smooth muscle. 5 The nonselective muscarinic antagonists atropine, scopolamine and quinuclidinyl benzilate (QNB) competitively inhibited [3H]QNB binding to the homogenate with Hill coefficients of 0.9-1.0 and inhibition constants (Ki) of nanomolar range. 6 Competition with selective muscarinic antagonists pirenzepine and 3-quinuclidinyl xanthene-9-carboxylate (QNX) gave Ki values, 0.26 M and 0.78 nM, respectively, and Hill coefficients of approximately 1. There was a single population of [3H]QNB binding sites of the M2 subtype for all tested muscarinic antagonists. 7 Competition with selective muscarinic agonists pilocarpine and carbachol yielded Ki values of micromolar range, Hill coefficients of less than 1, and revealed the existence of two binding sites (P < 0.01).