Supersensitivity by AF64A, a Novel Inhibitor of Neuronal Choline-Uptake, of the Rat Iris Sphincter Smooth Muscle.

Abstract

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.

Effects of AF64A on the mRNA Levels of Muscarinic Receptor Subtypes in the Rat Iris Sphincter.

“…In the preliminary experiments, the treatment with 50 nmol AF64A twice resulted in both specific increase in sensitivity to muscarinic agonist, acetylcholine, by the shift of pD2 from 5.0 to 5.6 and the increase in the maximum response by 53% at the forth day after the second injection (n =10). These changes were comparable to and slightly smaller than those in the preceding study, respectively (Tanaka et al, 1999). As shown in the preced ing study, the injection of 100 nmol AF64A occasionally gave irreversible damages to eyes, we preferred to 50 nmol injection, which did not induced apparent damages in over 20 eyes used in the preliminary experiments.…”

“…In addition to specific supersensitivity to muscarinic agonists, our previous work showed the increase in maximum contractile response to 80 mM K+ in the iris sphincter treated with AF64A (Tanaka et al, 1999). In the present study, the total protein contents were increased in AF64A-treated iris sphincter by 25%.…”

“…The administra tion of AF64A to cerebral ventricle elicits the following phenomena : 1) several biochemical markers such as choline acetyltansferase activity and high affinity choline uptake are reduced (Walsh et al, 1984), 2) the density of muscarinic recognition sites in brain remains virtually unchanged (Vickroy et al, 1985), and 3) the activation of phosphoinositide turnover or the inhibition of cyclic AMP accumulation elicited by muscarinic receptor agonists is facilitated (Eva et al, 1987). Our preceding study (Tanaka et al, 1999) showed that the intraocular injection of AF64A induces both specific and non-specific supersensitivities which are the same as the results of parasympathetic denervation by ciliary ganglionectomy in the rat iris sphincter (Hasegawa et al, 1987). In the rat urinary bladder, denervation induces both hypertrophy and a supersensitivity to muscarinic agonists (Gunasena et al, 1995) and an increase in M2 subtype density with no change in M3 subtype density (Braverman et al, 1998).…”

“…The PCR fragments amplified with brain cDNA and specific primers (m2-1, m3-1, and m4-1 ; see Table) were ligated into pBluescript II SK(+) (Stratagene For application of AF64A (RBI) in vivo, 8-9 week-old male Wistar rats were anesthetized with an i. p. injection of thiopental sodium (25 mg/kg) and amobarbital sodium (50 mg/kg). As described by Tanaka et al (1999), AF64A was applied into the anterior chamber of the eye by 26 gage injection needle. On the third day after the first injection, the another addition of 50 nmol of AF64A was performed to same eyes.…”

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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.

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