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Adrenoreceptors in Sheep Bladder – Relaxation Mechanisms – 1990

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Adrenoreceptors in Sheep Bladder – Relaxation Mechanisms – 1990 Research in Veterinary Science 1991, 50, 259-263 c~- and/3-adrenoceptors in the sheep urinary bladder L. RIVERA*, S. BENEDITO, D. PRIETO, M. HERNANDEZ, A. LABADIA, A. GARCIA-SACRISTAN, Departamento de Fisiolog&, Facultad de Veterinaria, Universidad Complutense, 28040-Madrid, Spain A study was undertaken to determine the presence /3-adrenoceptor subtype varies according to the and distribution of c~- and ~3-adrenoceptors in the species; the ~-adrenoceptors present in the bladder of sheep bladder body and base. In the bladder body, the cat are of the/31-subtype (Nergardh et al 1977), noradrenaline and isoproterenoi induced relaxation whereas in pigs (Larsen 1979), rabbits (Anderson and which was significantly inhibited by propranolol, Marks 1984), cattle (Garcla-Sacristgm et al 1985) and pafenolol and butoxamine. In the presence of pro- horses (Labadla et al 1988) they are of the j32-subtype. pranolol (10 -s M), noradrenaline induced a small c~-adrenoceptors predominate in the bladder base contraction, as well as phenylephrine, but B-HT 920 (Edvardsen and Setekleiv 1968) and produce its con- failed to cause any effect on the bladder body. In the traction. This contractile effect is a result of the bladder base, noradrenaline caused a contraction that stimulation of either one or both of the c~-adreno- was significantly inhibited by prazosin but not by ceptor subtypes and also varies between species. yohimbine. Phenylephrine also induced a contractile Obviously there are important differences between response in this structure which was inhibited by species relating to the presence of adrenergic receptors prazosin, lsoproterenoi caused a relaxation that was in the urinary bladder. As no information has been significantly inhibited by propranolol and pafenolol provided for the sheep, the present study was under- but not by butoxamine. Relaxation was mediated by taken to investigate the distribution and the both ~t and/~2-adrenoceptors in the detrusor muscle functional control of the different adrenergic receptor and by /~l-adrenoceptors in the bladder base. r types and subtypes in the urinary bladder body and cq-adrenoceptors contributed to maintain the base of the sheep. detrusor tone and contract the bladder base. THE autonomic nervous system plays an important Materials and methods role in regulating the activity of the smooth muscle of Twenty male lambs, three to four months old, with the urinary bladder in animals and man (Edvardsen no apparent lesions in their urinary tract, were and Setekleiv 1968, Gosling et al 1977, Larsen 1979) selected from the local slaughterhouse. Urinary and is of considerable importance during the storage bladders were removed immediately after death and phase of micturition (Edvardsen 1967), exerting an the adjacent connective and fatty tissues were inhibitory effect on the detrusor muscle and at the removed with care. same time producing contraction of the bladder base Strips of smooth muscle of the body and bladder smooth muscle (Edvardsen and Setekleiv 1968). base (8 to 10 mm long and 2 to 5 mm wide) were However, pharmacological studies have also obtained from each urinary bladder by means of demonstrated the existence of different adrenergic longitudinal incision. The strips were set up in a 30 ml receptor subtypes distributed throughout the urinary organ bath containing a modified Krebs solution bladder. These receptors belong to the c~- and (PSS) maintained at 37°C and bubbled with a mixture ~-adrenoceptor subtypes and their distribution of 95 per cent oxygen and 5 per cent carbon dioxide varies depending upon the portions of the bladder resulting in pH 7.4. The composition of the PSS was under study (Edvardsen and Setekleiv 1968, Levin et (in mM): sodium chloride 119; potassium chloride al 1980, Garcla-Sacrist~m et al 1985, Labadia et al 4-6; calcium chloride 1-5; magnesium chloride 1.2; 1988). sodium bicarbonate 24.9; glucose 11; ethylene- ~3-adrenoceptors predominate in the bladder body diamine tetraacetic acid (EDTA) 0"07. The proximal smooth muscle and produce relaxation. The portion of the strip was connected to an isotonic force transducer (Hugo Sachs Elektronik TVP B 368) when *Present address: Departarnento de Fisiologia, Facultad de recording contractile activity and to an isometric Veterinaria, Universidad Complutense, 28040-Madrid, Spain force transducer (Grass FT 03 C) when recording 259 260 L. Rivera, S. Benedito, D. Prieto, M. Herndndez, A. Labadfa, A~ Garc[a-Sacristdn TABLE 1 : pD 2 values (expressed as - log EC50) for isoproterenol pafenolol, which was dissolved in 95 per cent ethanol, in the sheep bladder body, relating to the inhibition of propranolol and prazosin, which was dissolved in distilled water at (10 -6 M), pafenolol (10 -5 M} and butoxamine (10 -5 M). Values are expressed as mean 4- SEM (n = 4) 50°C, with a pH of 4-0 and under constant agitation for 20 minutes. Agonist Antagonist pD 2 ( - log EC50) Isoproterenol 8.68 4- 0' 10 Isoproterenol Propranolol 7" 03 4- 0" 28* * * Results Isoproterenol Pafenolol 5.99 4- O- 49* ** Isoproterenol Rutoxamine 8.33 4- 0.07* Spontaneous activity * Significantly different from isoproterenol at P< 0" 05 After 60 to 90 minutes' adaptation, all muscle strips * ** Significantly different from isoproterenol at P < 0. 001 from the body and base of the sheep urinary bladder exhibited a rhythmic spontaneous activity, charac- relaxant activity. The distal end of the preparation terised by the presence of phasi c contractions. The was fixed to the organ bath base. Isometric force amplitude of the contractions was smaller at the base transducers were used for recording relaxant activity, than in the bladder body, the frequency being 2.1 which could be observed in isometric recordings but contractions rain- 1 in the detrusor muscle and 2.0 at not in isotonic ones (Karaki et al 1984). Preparations the bladder base. No significant difference between were allowed to equilibrate for at least 60 minutes the two structures was observed. under 2 g tension before the start of the experiment. Cumulative concentration response curves to Detrusor muscle agonists were obtained. The agonists were added Noradrenaline (10-8-10 -4 M) and isoproterenol directly to the bath and the concentration was ( I 0 - 9.10- 6 M) caused a dose-dependent relaxation of increased as soon as there was a stable response. detrusor smooth muscle strips. The relaxant response When studying relaxation, an increased concentra- to isoproterenol was greater in detrusor than in tion of the agonist was added to the bath at intervals bladder base, whereas the pD z values were similar in of 10 minutes. The apparent potency of an agonist both tissues (8.68 ± 0.10 and 8.12 ± 0 • 10, respec- was expressed by its pD 2 value, that is, pD 2 = log EC50 tively). Pretreatment with propranolol (10 -6 M) (- log of the molar concentration that had an effect resulted in a rightward shift of the isoproterenol dose- equal to 50 per cent of the maximal effect tested on the response curve (P<0"001) (pDz: 7.03 ± 0"28). same strip as reference). Adrenergic antagonists were Pafenolol (10 -5 M), a 31-adrenergic blocker, induced allowed to equilibrate with the preparations 20 an inhibition of the relaxant effect of isoproterenol minutes before a new concentration response curve of the agonist was repeated. The results are expressed as mean ± standard error H Isoproterenol of the mean of four experiments per dose and the Saibutamol statistical evaluation was calculated using Student's t 100- test. The following drugs were used: B-HT 920 (5-allyl- 2-amino-5,6,7,8-tetrahydro-4H-thiazolo = (4,5- d)azepin-dihydrochloride) (Dr Karl Thomae); butox- amine hydrochloride (Burroughs Wellcome) isopro- terenol hydrochloride (Boehringer Ingelheim); nor- adrenaline hydrochloride (Serva); pafenolol hydro- chloride (courtesy of Dr Nyborg, Aarhus, Denmark); 50- phenylephrine hydrochloride (Sigma); phentolamine hydrochloride (Sigma); prazosin hydrochloride (Pfizer); propranolol hydrochloride (If0; salbutamol sulphate (Glaxo); yohimbine hydrochloride (Sigma). All drugs were dissolved in PSS daily except TABLE 2: pD 2 values (expressed as - log ECS0) for salbutamol in the sheep bladder body, relating to the inhibition of butoxamine (10 -5 M). Values are expressed as mean 4- SEM (n=4) I I I 8 7 s ; Agonist Antagonist pD 2 ( - log EC50) -Log (M) Salbutamol 6.48 4- 0- 08 Salbutamol Butoxamine 4.77 4- 0.14" FIG 1 : Dose-related relaxation of the sheep bladder body induced by 3-adrenoceptor agonists: Isoproterenol and salbutamol. Each * Significantly different from salbutamol at P < 0.05 point represents the mean ± SEM of four experiments per dose Adrenoceptors in the urinary bladder 261 TABLE 4: pD 2 values (expressed as - log EC60) for phenylephrine in the sheep bladder base, relating to prazosin (10- 7 M). Values Prop'aoo,o, ; ; ; ; " are expressed as mean ± SEM (n = 4) 10~M -log (M) Agonist Antagonist DD 2 ( - log EC50) Phenylephrine 5.83 + 0.18 Phenylephrine Prazosin 3.26 ± 0- 05* * • * Significantly different from phenylephrine at P < 0-01 j~'~'-~ (b) fashion. Because of this, specific agonists of the cq p~ • • • Q • • and o/2 adrenergic receptors subtypes were used. Pro anolol 8 7 6 5 4 Phenylephrine (10-8-10 -4 M), a specific c~l-agonist, lOSM -log (M) also induced small but detectable concentration- 0.5g dependent contractions in this tissue. However, B-HT 1 920 (10-8-10 -4 M), a specific a2-agonist, failed to 4 rain cause any effect on the bladder body of the sheep (Fig 2a,b,c). (c) Bladder base Prop~anolol ; ; ; ; ; • Noradrenaline (3 x 10-7-10 -4 M) induced a dose- 10"~M -log (M) dependent contraction of the bladder base smooth FIG 2: Dose-related contraction of the sheep bladder body muscle. This contractile effect was significantly induced by c~-adrenoceptor agonists: noradrenaline (a), phenyl- ephrine (b) and B-HT 920 (c), when/~-adrenoceptors are blocked inhibited by phentolamine (10 -7 M) (P < 0-01) (pD2: with propranolol ( 10- 5 M).
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