We first carried out microscopic observation of the intramural nerves of a bullfrog gallbladder which were fixed and stained with a solution of OsO₄ and ZnI₂. We then investigated if the responses of isolated frog gallbladder evoked by electrical stimulation are mediated through the intramural nerves. The following results were obtained: 1. The nerve plexus and the perivascular nerves were observed in the subserosal layer of the wall of the gallbladder. These nerves do not have a ganglia. That is to say, no ganglionated plexus or ganglia were observed in the subserosal layer of the wall of the gallbladder. 2. Electrical stimulation caused the gallbladders to contraction with rectangular pulses (50 volt, 40 Hz) of durations of 0.5, 1, 2, 3, 4 and 5 msec for a period of 10 sec. Three blockers of nerve-mediated responses, atropine (1×10^-6 M), guanethidine (1×10^-6 M) and tetrodotoxin (3×10^-7M), had no effect on the gallbladder contractions induced by stimulation with pulses as short as 0.5 msec or as long as 5 msec. These results suggest that the bullfrog gallbladder may not contain nerves related to movement. Thus, the contraction of the bullfrog gallbladder induced by electrical stimulation does not seem to be modulated by extrinsic nerve terminals distributed in the gallbladder wall.

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Single smooth muscle cells isolated from guinea pig ileum using collagenase and papainproduce contractile response to muscarinic agents, while the cultured cells do not. UsingHuo-3/AM and a confocal laser scanning Huorescence microscope, it was observed thatcarbachol, a muscarinic agent, caused an increase in the intracellular Ca²⁺ of both single andcultured cells. SDS-PAGE and Western Blot analyses revealed the expression of myosinheavy chain isoforms of SM1 (204 kDa) and SM2 (200 kDa) in single smooth muscle cells, andnon-muscle isoform (196 kDa) of myosin heavy chain only in the cultured cells. With respect to actin isoforms, α-actin was predominant in single cells and β-actin was major in the cultured cells. Two types of tropomyosin monomer, 39 kDa and 41 kDa, were detected in single cells, while the 41 kDa monomer was lost in cultured cells. These differences in contractile protein profiles between single and cultured cells were collaborated with the observation of cells using immunofluorescence microscope with responsible antibodies to isoforms of myosin heavy chain, actin and tropomyosin. These results suggest that the loss of contractility in cultured smooth muscle cells is profoundly related to changes in contractile protein profiles from smooth muscle type to non-muscle type.

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Influences of endothelium on contraction of aortic smooth muscle by various agents were studied and those in the preparations from Wistar Kyoto rat (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) were compared. Endothelium depressed the contractions induced by noradrenaline and 5-hydroxytryptamine (5-HT). The time course of the contraction was bi- or tri-phasic in the former and slow rising monophasic in the latter. On the other hand, the depression was weaker in the contraction by prostaglandin F_2α-and high K⁺. The depression was blocked by the removal of endothelium or in thepresence of N^ω-nitro-L-arginine (L-NNA), indicating that nitric oxide (NO) released from endothelium was responsible for the inhibition. The inhibition was weaker in the preparation from SHRSP when compared to that in the preparation from WKY. Relaxation by acetylcholine (ACh) of the preparation precontracted in the presence of respective contractile agent was impaired in the preparation from SHRSP. It is concluded that mode of inhibition of the contraction varies depending on the agents used to initiate the contraction, i.e. depending on the mode of the release of NO.In the preparation from SHRSP, the influence of endothelium is impaired due to the reduced release of NO.

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Control of the active tone by endothelium in aortae from various strains of spontaneously hypertensive rats was studied. The active tone was negligibly observed in endotheliumintact preparation. The application of N^G-nitro-L-arginine (L-NNA, 10^-4 M) induced slowly developed active tone in the preparations from hypertensive rats but no active tone was induced in the preparation from normotensive Wistar Kyoto rats (WKY). The developed tension was stronger in preparations from rats with higher blood pressure as observed in endothelium-denuded preparations. The developed active tone in the presence of L-NNA was greater than that observed in endothelium-denuded preparations. The active tone was abolished by the removal of extracellular Ca²⁺ or by the application of Caantagonists. L-arginine counteracted the effects of L-NNA and depressed the developed active tone in the presence of the latter drug. The application of indomethacin (10^-5 M) depressed the active tone of the preparations from SHRSP by 25.5±5.2%.Increasing extracellular K⁺ concentration or application of tetraethylammonium (TEA) could not be used to observe the effect of endothelium-derived factors on the active tone, because of their strong contractile effect. Simultaneous application of apamin and charybdotoxin induced an elevation of tension which was often associated with spontaneous tension oscillation.
It is concluded that the active tone, which is smooth muscle origin, is depressed by endothelium-derived nitric oxide (NO) strongly and potentiated by a product of arachidonic acid cascade through cyclooxygenase pathway. The involvement of endothelium-derived hyperpolarizing factor (EDHF) in the depressing effect of endothelium is thought to be small.

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In the present study, we tried to determine which β-adrenoceptor subtypes are involved in the inhibitory regulation of bladder motility in the guinea pig by using in vitro functional analysis. Isoprenaline, norepinephrine and epinephrine decreased spontaneous contractile activity of the bladder concentration-dependently, the rank order of their potency being isoprenaline (pD₂=7.38) > norepinephrine (6.71) > epinephrine (6.31). Dobutamine was also effective in inhibiting the spontaneous contraction (5.81). However, CGP-12177, BRL37344, salbutamol, and clenbuterol were not effective at the concentration of 30μM. The concentration-response curves of catecholamines (isoprenaline, norepinephrine and epinephrine) were rightward shifted by the presence of atenolol. Schild regression analyses carried out for atenolol against isoprenaline, norepinephrine and epinephrine gave pA₂ values of 6.93, 6.78 and 6.69, respectively. The concentration-response curve for isoprenaline was unaffected by 10μM butoxamine. In the presence of 1μM propranolol, bupranolol produced shifts of the concentration-response curve for isoprenaline, and the apparent pA₂ value for bupranolol against isoprenaline was 5.50. These results suggest that the inhibition of spontaneous contraction of the guinea pig bladder by β-adrenoceptor agonists is mediatedmainly via β₁-adrenoceptor and the effect of isoprenaline is mediated partly via atypical β-adrenoceptor subtype.

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