Effects of ethylcholine mustard aziridinium ion (AF64A) on contractile responses to agonists or transmural nerve stimulation (TNS) were examined in rat iris sphincter muscle.The responses to TNS of isolated sphincter muscle were abolished within 1 hr after the addition of 0.1 mM AF64A to the bathing solution, while responses to acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) were not changed significantly. In sphincter muscles which were isolated 3 days after the micro-injection of 100 nmol AF64A into the anterior chamber of eyes in vivo, the response to TNS were decreased to about 30% of the control.The injection of AF64A at higher concentrations often resulted in serious chronic damage of the eye. When 100 nmol AF64A was injected twice with an interval of 3 days, the response to TNS was decreased to about 20% of the control. Maximum responses and sensitivities to ACh and 5-HT were markedly enhanced in sphincters from eyes which had been treated with AF64A twice. The sensitivity of depolarized sphincters to external Ca2+ was also increased significantly. Seven weeks after the second injection, responses to TNS recover ed to more than 50% of the control and the effects of AF64A mostly disappeared. In conclusion, the acute inhibition of parasympathetic transmission without postsynaptic changes can be achieved within 1 hr after bath-application of 0.1 mM AF64A. The reduc tion of parasympathetic nerve activity by treatment with AF64A in vivo induces nonspecific supersensitivity in iris sphincter, whereas effects of AF64A were mostly reversible under the present experimental conditions.

Muscarinic receptor subtypes that involved in cholinergic responses in rat iris were identified by reverse transcription-polymerase chain reaction (RT-PCR) analysis . mRNAs encoding m2, m3, and m4 subtypes were abundantly expressed in iris, whereas ml and m5 subtypes were not detected. Selective amplification of the coding regions of m2 , m3, and m4 subtypes in iris was carried out using specific primers based on the sequence of each subtype previously cloned from rat brain and heart by RT-PCR. The amino acid sequence for iris m2 was different from published heart and genomic m2 by nine and one residue(s) , respective ly. It was also found that the sequence for m2 that in brain , heart, and several smooth muscles determined in the present study is completely identical to that in iris but not to that reported previously in heart. The sequence for iris m4 was completely identical to that for m4 in brain. The sequence identity between m3 subtype in iris and that in brain is 99.3%, with four amino acid substitutions at the sites of the position 165 and 184 in the edge of second intracellular loop and the sites of the position 337 and 406 in the central of i3 loop.It was found that iris m3 is slightly but substantially different in amino acid sequence from that in brain of the rat.

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 beta-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 Ca2+ 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, GTPgammaS nor H-7. The increase in Ca2+ sensitivity by AF64A injection was not affected by calmodulin, enhanced by GTPgammaS 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 monoclonal 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 Ca2+ sensitivity could be due to the mechanism in which changes in protein kinase C and/or GTP binding protein activity are involved.

The reduction of parasympathetic nerve activity by the treatment with ethylcholine mustard aziridinium ion (AF64A) in vivo induced both specific and non-specific supersen sitivities in the rat iris sphincter (Tanaka et al., 1999). Changes in the expression of muscarinic receptor subtypes, which could be a cause of specific supersensitivity induced by the treatment with AF64A, were examined using competitive PCR techniques. Muscarinic receptor population is composed of m2, m3, and m4 subtypes in the rat iris (Furuta et al., 1998). Interestingly, m4 mRNA was much more abundantly expressed than m2 and m3 in the rat iris sphincter. The treatment with AF64A significantly increased the mRNA levels of m2 and m3 subtypes to 370 and 330% of the control but not that of m4 (approximately 90% of the control). In addition, the total protein contents were increased to approximately 125% of the control. The up-regulation of the mRNA levels of m2 and m3 subtypes by the treatment with AF64A was significant when they were compensated for the increase in total protein contents. The down-regulation of m4 mRNA expression was not significant even after being corrected for the protein content. These results suggest that the up-regulation of the mRNA levels of m2 and m3 subtypes may be, at least in part, responsible for the supersensitivity to muscarinic agonists after the treatment with AF64A in vivo.

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