The mechanisms underlying nonspecific supersensitivity induced by parasympathetic denervation in rat iris sphincter

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The mechanisms underlying nonspecific supersensitivity induced by parasympathetic denervation were examined using RT-PCR, semi-quantitative PCR and skinned fiber techniques. It was found that microinjection of AF64A, a novel choline uptake inhibitor, into anterior eye chamber of the rat resulted in changes in iris sphincter smooth muscle similar to those elicited by surgical parasympathetic denervation by removal of ciliary gangrionectomy. The changes were characterized in vitro by reduced contractile response to nerve stimulation presumably via the decrease in acetylcholine (ACh) release from parasympathetic nerve endings and by enhanced contractile responses both specific to exogenously applied ACh and nonspecific to serotonin and high KィイD1+ィエD1. Semi-quantitative PCR analyses of muscarinic receptors in rat iris indicate that mRNAs of m2, m3 and m4 subtypes are expressed in iris and that, unexpectedly, the expression level of m4 is much higher than that of m2 and m3. The sequence homology of m2, m3 and m4 in rat iris to those reported in genome or cDNA in other tissues of the rat was over 99%, while several differences in amino acid sequence in intracellular loops were found. Following the injection of AF64A, mRNA levels of m2 and m3 increased and that of m4 decreased, respectively. The CaィイD12+ィエD1 sensitivity of the contractile machinery measured in skinned fiber was slightly but significantly increased by AF64A injection. The increase in CaィイD12+ィエD1 sensitivity was abolished by addition of H7, protein kinase C inhibitor. AF64A injection resulted in marked increase in the maximum contractile response of iris sphincter muscle. A slight increase in amount of actin and no change in those of myosin and calmodulin in iris can not explain the increase in the maximum contractile response. A novel mechanism distinctive from the changes in major contractile and related regulatory proteins may underlie the nonspecific supersensitivity induced by AF64A.