We have developed an atypical adrenergic β₃-agonist, GS-332: Sodium (2R)- [3- [3- [2-(3-Chlorophenyl)-2-hydroxyethylamino] cyclohexyl] phenoxy] acetate, which has an unique structure compared to other β₃-agonists. In vitro study, we compared effects of GS-332 on rat urinary bladder muscle strip contractility with those of clenbuterol hydrochloride (clenbuterol), an adrenergic β₂-agonist. GS-332 relaxed isolated rat urinary bladder strips in a concentration dependent manner with 50% effective concentration (EC₅₀) of 15.7 nM, and the relaxant activity of GS-332 was as potent as that of clenbuterol (EC₅₀ ; 30.8 nM). The concentration-response curve of GS-332 on isolated rat urinary bladder was competed by a specific β₃-antagonist, SR59230A in a concentration-dependent manner, in which Schild slope was 1.1 and pA₂ value of SR59230A was 7.1. In vivo study, cystometory investigated in anesthetized rats demonstrated that GS-332 was more potent in increasing the urinary storage volume than clenbuterol and less potent in inhibiting the contractile force of urinary bladder at micturition reflex than clenbuterol. These data demonstrate that GS-332, a new adrenergic β₃-agonist, may be more useful to maintain continence than clenbuterol, an adrenergic β₃-adrenergic agonist.

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Based on the affinity of α_1D-adrenoceptor subtype for a selective antagonist BMY 7378, we studied its functional role in rabbit thoracic aorta and iliac artery, and evaluated the subtypes of the α₁-adrenoceptors that are activated by phenylephrine (a full agonist) and tizanidine (a partial agonist). In thoracic aorta, the concentration-response curves of phenylephrine and tizanidine were antagonized by BMY 7378 with low potency (pA₂ values 6.68±0.06 and 6.67±0.06, slopes of Schild plot 1.06±0.04. and 1.01±0.04, respectively). On the other hand, in iliac artery concentration-response curves for phenylephrine were potently antagonized by a low concentration of BMY 7378, and the slope (0.75±0.02) of the Schild plot was significantly different from unity. In iliac artery, a concentration-response curve of tizanidine was antagonized by BMY 7378 with low potency (pA₂ value 6.64±0.08, slope of Schild plot 1.01±0.05). These results suggest that an α_1D-adrenoceptor subtype contributes to α₁-adrenoceptor mediating muscle contraction in iliac artery, but not in thoracic aorta of rabbit, and that it is activated by a full agonist phenylephrine but not by a partial agonist tizanidine.

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In smooth muscle cells, the sarcoplasmic reticulum (SR) has been identified as the primary storage site for intracellular Ca²⁺. The peripheral SR is in close proximity with plasma membrane to make a narrow subsarcolemmal space. In this study, we investigated the regulation of subsarcolemmal [Ca²⁺] ([Ca²⁺] _s1) and global cytosolic [Ca²⁺] ([Ca²⁺] _c) of rabbit arterial smooth muscle using whole-cell patch clamp technique and microspectrofluorimetry. The Ca²⁺-activated K⁺ current (I_{K (Ca)} ) and the ratio of fura-2 fluorescence (R_340/380) were considered to reflect the [Ca²⁺] _s1 and [Ca²⁺] _c, respectively. At a holding potential of 0mV, extracellular application of 10mM caffeine, a well-known Ca²⁺-releasing agent, induced transient increase of I_{K (Ca)} and R_340/380 (I_{K (Ca)} -transient and R_340/380-transient, respectively). The increase and decay of I_{K (Ca)} -transient was faster than R_340/380-transient. By repetitive application of caffeine, when the refilling state of SR was supposed to be lower than the control condition, I_{K (Ca)} -transient and R_340/380-transient were suppressed to different levels; e.g. the second application 20sec after the first could induce smaller I_{k (Ca)} -transient than R_340/380-transient. Dissociation of I_{k (Ca)} -transient and R_340/380-transient was removed by sufficient (>3min) washout of caffeine. Recovery from the dissociation was also dependent upon the membrane potential; faster recovery was observed at negative (-40mV) holding potential than at depolarized (0mV) condition. Dissociation of I_{K (Ca)} from [Ca²⁺] _c was also partially prevented by perfusion with Na⁺-free (replaced by NMDG⁺) extracellular solution. These results suggest that, 1) there is prominent spatial inhomogeneity of [Ca²⁺] in cerebral arterial myocyte, 2) [Ca²⁺] _s1 is preferentially affected by the interference from nearby plasmalemmal Ca²⁺ regulation mechanism which is partly dependent upon extracellular Na⁺.

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The most distal part of the circular muscle layer functions as the internal anal sphincter, which constitutes a high pressure zone at rest, but maintains a relaxed state during defecation. To elucidate such sphincter mechanisms of the smooth muscle cells, the circular muscle layer in the canine anal canal was examined within 2cm from the anal verge. Both the mechanical and intracellular electrical activities were recorded simultaneously. The examined region could be divided into three different regions according to the pattern of spontaneous activity and innervation and consisted of an upper region (20-15mm from the anal verge), a transitional region (15-5mm from the anal verge) and a lower region (within 5mm from the anal verge), respectively. The spontaneous membrane activity was characterized by ongoing slow potential changes and each potential change was associated with a phasic contraction in the three regions. The mean frequencies of spontaneous electrical activity were 6.8, 15.9, and 24.1 c/min in the upper, transitional and lower regions, respectively. In the transitional and lower region, muscle tone generation was observed. Transmural field stimulation (0.4 msec in pulse duration) evoked membrane depolarization and contractions in the lower region. The application of an alfa-adrenergic blocking agent completely suppressed the generation of excitatory responses, leaving a long-lasting hyperpolarization associated with relaxation. In the transitional and upper region, stimulation consistently evoked membrane hyperpolarization with relaxation. The characteristics of this hyperpolarization response varied among the three regions. The total duration of hyperpolarization increased distally, while the time to peak-hyperpolarization became decreases in a reverse direction. These regional differences in the characteristics of spontaneous membrane activity and innervation indicate that the transitional and lower region might therefore function as the internal anal sphincter.

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