M2 muscarinic receptor subtype is associated with inositol trisphosphate accumulation, myosin light chain phosphorylation and contraction in sphincter smooth muscle of rabbit iris

Abstract

The relationships between occupancy of muscarinic acetylcholine receptors on iris sphincter muscle, measured by [3H]quinuclidinylbenzylate (QNB) binding, carbachol (CCh)-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, measured as myo-inositol trisphosphate (IP3) accumulation, myosin light chain (MLC) phosphorylation and contraction were analyzed by examination of the dose-response relationships and the effects of the muscarinic antagonists, atropine and pirenzepine (PZ). CCh caused a concentration-dependent accumulation of IP3 (EC50 = 2.3 X 10(-6) M), MLC phosphorylation (EC50 = 3.8 X 10(-6) M), contraction (EC50 = 0.55 X 10(-6) M) and [3H]QNB displacement [KH (high affinity dissociation constant) = 2.9 X 10(-6) M]. The time course of atropine reversal of CCh-induced IP3 accumulation and muscle contraction revealed that the continued presence of activated muscarinic acetylcholine receptors was required to maintain IP3 production and contraction. Atropine was about 2 orders of magnitude more potent than PZ in inhibiting the CCh-induced biochemical and pharmacological responses and [3H] QNB binding, indicating the preponderance of M2 receptors in this smooth muscle. Thus, the PA2 values for atropine antagonism of CCh-stimulated IP3 accumulation, MLC phosphorylation and contraction were 9.1, 9.05 and 9.39, respectively, and for PZ antagonism were 7.12, 7.10 and 7.29, respectively. Furthermore, the KD values for atropine and PZ antagonism of [3H]QNB binding were 6.9 X 10(-10) and 1.5 X 10(-7) M, respectively. In addition, AF-DX116 (11-[(2-[(diethylamino)methyl]-1-piperidinyl) acetyl]-5,11-dihydro-6 H-pyrido[2,3-b][1,4]benzodiazepine-6-one), a M2 cardioselective antagonist, significantly inhibited the CCh-induced IP3 accumulation and muscle contraction.(ABSTRACT TRUNCATED AT 250 WORDS)