Grandordy_1986_J.Pharmacol.Exp.Ther_238_273

Hydrolysis of membrane phosphatidylinositol (PI) and polyphosphonoinositides (PPI) may be the coupling mechanism between receptor stimulation and the rise in intracellular calcium concentration that leads to smooth muscle contraction. In bovine tracheal smooth muscle, we correlated PI/PPI turnover, contraction and muscarinic receptor occupancy by carbamoylcholine (10(-9) to 10(-2) M). Inositol monophosphate formation after agonist stimulation, in the presence of lithium, provided a direct measurement of PI/PPI breakdown, and receptor occupancy was determined by [3H]quinuclidinyl benzylate binding. Carbamoylcholine caused a concentration-dependent contraction (EC50 = 7.4 X 10(-8) M), PI/PPI response (EC50 = 3.8 X 10(-5) M) and [3H]quinuclidinyl benzylate displacement (with high and low affinity binding sites have dissociation constants (Kd) of 3 X 10(-7) and 6 X 10(-4) M, respectively). This indicates the presence of spare receptors as maximal contraction is obtained when less than 20% of receptors are occupied. The concentration of carbamoylcholine inhibiting 50% of the PI/PPI response and 50% of maximal receptor occupancy (IC50) were similar for atropine (IC50 = 1 X 10(-9) and 5.3 X 10(-9) M, respectively), and for pirenzepine (IC50 = 3 X 10(-6) and 2.3 X 10(-6) M, respectively); the pA2 of the contraction was 8.3 +/- 0.12 for atropine and 7.2 +/- 0.08 for pirenzepine, indicating that M2 receptors may be largely predominant among bovine tracheal smooth muscle muscarinic receptors. Bovine tracheal smooth muscle may be a useful model to study the effects of other spasmogens, as it allows comparison of functional effects, PI breakdown and receptor occupancy in the same preparation.