There are some controversies over the contribution of Na⁺/Ca²⁺ exchanger (NCX) to the regulation of cytosolic Ca²⁺ concentration ([Ca²⁺] _c) in smooth muscle. To prove the functional role of Na⁺/Ca²⁺ exchanger, we examined whether the removal of extracelluar Na⁺ could affect [Ca²⁺] _c of rabbit cerebral arterial smooth muscle. The fluorescence ratio of fura-2 (R_340/380) was measured in the single myocyte of rabbit middle cerebral artery and Na⁺ was substituted with the same concentration of NMDG⁺ or Li⁺. In 21 out of 230 cells tested, Na⁺-removal increased R_340/380 (ΔR_340/380) by 115±16.5% of the ΔR_340/380 induced by 10mM caffeine in the same cell. The Na⁺ removal-induced ΔR_340/380 was blocked by a selective inhibitor of cardiac type NCX exchanger (KB-R7943, (2- [2- [4-(4-nitrobenzyloxy) phenyl] ethyl] isothiourea, 10μM). In those cells where the Na⁺-removal by itself did not increase R_340/380, the caffeine-induced ΔR_340/380 was increased by Na⁺-removal (130±9.8% of control response, n=30). Under the whole-cell patch clamp condition, short application of caffeine induced transient increase of outward current (I_{k, Ca}-transient) which reflect the change of subsarcolemmal [Ca²⁺]. The application of KB-R7943 increased the amplitude of Ik, Ca-transient (n=4). These results suggest the functional existence of NCX in rabbit cerebral artery smooth muscle.

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The effects of neuropeptide Y (NPY) and agonists selective for NPY Y1 and Y2 receptors were studied on contraction and Ca²⁺ currents in arterial smooth muscle. In isolated arterioles from the guinea-pig small intestine, small brief constrictions were evoked by depolarising the arteriolar smooth muscle using high K⁺ solution applied from a micropipette. The constrictions were reduced in amplitude by the Y2-selective agonists PYY (13-36) and N-acetyl [Leu²⁸, Leu³¹] NPY-(24-36) in concentrations from 20-100 nM. NPY or the Y1-selective agonist [Leu31 Pro34] NPY in concentrations from 50 pM to 100 nM increased the amplitude of the constrictions, with a maximum effect at 10nM. Smooth muscle cells were isolated from rat small mesenteric arteries, and voltage-activated Ca²⁺ currents measured by whole cell patch clamping. The peak amplitude of the Ca²⁺ currents was decreased by N-acetyl [Leu²⁸, Leu³¹] NPY-(24-36), and by NPY (100 nM). [Leu³¹, Pro³⁴] NPY either had no effect or slightly increased the Ca²⁺ currents. We conclude that Y2 receptors on vascular smooth muscle can reduce Ca²⁺ currents induced by depolarisation, and thus oppose constriction caused by smooth muscle depolarisation.

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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 recovered 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 reduction 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.

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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 supersensitivities 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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In the present study, we tried to determine whichα₁-adrenoceptor subtypes are involvedin the guinea pig thoracic aorta by using in vitro functional analysis. In first, we tried toestimate the ρA₂ values of some key α₁-adrenoceptor antagonists (prazosin, 5-methylurapidil, WB4101, BMY7378 and tamsulosin) against responses to norepinephrine in thethoracic aorta of guinea pigs. The concentration-response curves of norepinephrine wererightward shifted by the presence of prazosin, 5-methylurapidil, WB4101, BMY7378 and tamsulosin. The ρA₂ values for these antagonists against norepinephrine were 7.83, 7.78, 8.20, 5.73 and 9.57, respectively. In second, we tried to compare the estimated ρA₂ values obtained in the present study with reported ρKi and ρpA₂ values for cloned and native α₁-adrenoceptor subtypes. In rabbit mesenteric artery, trigone, urethra, prostate and human lower urinary tract which were proposed to contain the putative α_1L-adenoceptor, weobtained the good correlation for the ρA₂ values reported in these tissues with ρA₂ values estimated in guinea pig thoracic aorta. Moreover, regression lines were close to the line of identity. These results suggest that the α₁-adenoceptors mediating contraction of guineapig thoracic aorta are similar pharmacologically to the putative α_1L-adenoceptor subtype inrabbit mesenteric artery, trigone, urethra, prostate and human lower urinary tract. As afinal point, guinea pig thoracic aorta may be able to use as a tool to develop the new α₁-adrenoceptor antagonist which is therapeutically advantageous in the treatment of urinarytract obstruction (e.g., in benign prostatic hyperplasia).

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