Molecular Analysis of Non-specific Supersensitivity Induced by AF64A in Rat Iris Smooth Muscle

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Characteristics of supersensitivity induced by the pretreatment with AF64A, an inhibitor of choline uptake at parasympathetic nerve endings, were examined in rat iris sphincter. In preparations isolated and skinned by β-escin after the micro-injection of AF64A to eyes in vivo, the amplitude of maximum contraction in pCa 4.5 solution was increased by 180% of the control from the contralateral eyes. the Ca^<2+> sensitivity of the contractile system was slightly but significantly increased by AF64A injection;the half maximum contraction was obtained at pCa 5.87 and 6.05 in the control and AF64A-injected eyes, respectively. The increase in maximum contraction in AF64A-injected ones was neither affected by the addition of calmodulin, GTPγS nor H-7. The increase in Ca^<2+> sensitivity by AF64A injection was not affected by calmodulin, enhanced by GTPγS and abolished by H-7. AF64A injection increased the total protein content only by 30% of the control. The contents of contractile proteins per iris were quantified using Western blotting with mono-clonal antibodies. The contents of actin and calponin were increased by AF64A, whereas those of myosin, calmodulin and caldesmon were not affected. The results indicate that AF64A-induced enhancement of the maximum contraction is not mainly due to the increase in the contents of major contractile proteins and that the increase in Ca^<2+> sensitivity could be due to the mechanism in which changes in protein kinase C and/or GTP binding protein activity are involved.
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Molecular Analysis of Non-specific Supersensitivity Induced by AF64A in Rat Iris Smooth Muscle

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