Effects of Pinacidil and Cromakalim (BRL 34915) On Bladder Function in Rats with Detrusor Instability

0022-5347/89/1424-1134$02.00/0 THE JOURNAL OF UROLOGY

Vol. 142, October

Copyright© 1989 by AMERICAN UROLOGICAL ASSOCIATION, INC.

Printed in U.S.A.

EFFECTS OF PINACIDIL AND CROMAKALIM (BRL 34915) ON BLADDER FUNCTION IN RATS WITH DETRUSOR INSTABILITY A. MALMGREN,* K.-E. ANDERSSON, C. SJOGREN

AND

P. 0. ANDERSSON

From the Departments of Clinical Pharmacology and Physiology, University of Lund and Research Laboratories, Pharmacia LEO Therapeutics AB, Helsingborg, Sweden

ABSTRACT

Normal rats as well as rats with bladder hypertrophy secondary to outflow obstruction were investigated cystometrically before and after administration of the potassium channel openers pinacidil or cromakalim one mg./kg. orally. In normal rats cromakalim decreased micturition pressure by 15 ± 6%. A diminished micturition pressure was also seen after pinacidil (by 18 ± 8%) but this did not achieve statistical significance. Further, no clear-cut effects on bladder capacity, residual volume, basal bladder pressure, threshold pressure, bladder compliance or on bladder wall tension were seen in this group of rats neither in the presence of pinacidil nor cromakalim. Rats with bladder hypertrophy exhibited a significant bladder instability during cystometrical investigations. The mean amplitude of the spontaneous bladder contractions exceeded 20 cm. H20 prior to micturition. Administration of pinacidil and cromakalim decreased the spontaneous contractions to 26 ± 12% and 22 ± 7%, respectively, of that seen in the absence of the drugs. Furthermore, pinacidil decreased micturition pressure by 61 ± 12%. Also cromakalim decreased micturition pressure (by 27 ± 13 %) but this effect did not achieve statistical significance. After both pinacidil and cromakalim these rats tended to develop residual urine. In accordance with the results in normal rats pinacidil and cromakalim showed no effects on bladder capacity, basal bladder pressure, threshold pressure, bladder compliance or on bladder wall tension in rats with bladder hypertrophy. The findings of an almost complete disappearance of spontaneous bladder contractions in rats with bladder instability and a remaining voiding ability after administration of pinacidil or cromakalim suggest that potassium channel openers may be a therapeutic alternative in the treatment of bladder instability associated with outflow obstruction. (J. Urol., 142: 1134-1138, 1989) Bladder instability is a common condition in men with benign prostatic hyperplasia (BPH) and can be demonstrated in 5075 % of the patients. 1 A main aim of drug treatment in BPH patients with bladder instability is to give symptomatic relief of frequency, urge and urinary incontinence. This should be achieved by selectively depressing the unstable bladder contractions without influencing the normal micturition contraction. The therapeutic principles used today for treating bladder instability all aim at diminishing the contractile ability of the bladder. 2 Blockade of muscarinic receptors by anticholinergic compounds as well as calcium channel blockade decrease the contractile power of the bladder muscle. However, side-effects restrict the use of presently available compounds. Pinacidil and cromakalim belong to a group of compounds which have been shown to act via a new principle, by the opening of potassium channels in the smooth muscle cell membrane. Potassium channel openers efficiently relax various types of smooth muscle. Both pinacidil and cromakalim were shown to increase potassium efflux in rat portal vein and to induce hyperpolarisation of this vessel. 3 • 4 Furthermore, cromakalim was shown to relax uterine,5 bronchial,6 colonic7 and normal detrusor smooth muscle. 8 Preliminary data have indicated that cromakalim may abolish spontaneous bladder contractions in pigs with bladder instability.8 The present investigation was performed in order to further examine the effects of pinacidil and cromakalim on bladder

function in normal rats as well as in rats with bladder instability associated with outflow obstruction. MATERIALS AND METHODS

Animals. Female Sprague-Dawley rats (weighing approximately 200 gm.) were used. Surgical procedures. To obtain a partial obstruction of the urethra, rats were anaesthetized with methohexital sodium (Brietal), 50 mg./kg. intraperitoneally. Via a lower midline incision the bladder and the proximal urethra were exposed. A ligature, Novolene D 405 (D&G Monofil Inc.), was placed around the urethra and tied in the presence of a plastic rod with a diameter of one mm. 9 The abdominal wall was closed and the animals left to recover. Six weeks after the obstruction the rats were again anaesthetized (methohexital sodium, 50 mg./kg. intraperitoneally), the abdomen opened and the urethral ligature removed. Thereafter, a polythene catheter (ClayAdams PE-50) with a cuff was inserted into the bladder top. A suture around the catheter closed the bladder incision. The catheter was tunneled subcutaneously and an orifice was made on the back of the animal. The abdominal incision was sutured and the free end of the catheter sealed. In order to prevent infections the rat received an injection of sulfadoxin (24 mg./ kg.) and trimetoprim (4.8 mg./kg.) subcutaneously. Cystometrical investigations were performed two days after removal of the ligature since this time-point was found to be the optimum one. In age-matched controls a catheter was placed in the urinary bladder as described above and cystometries performed two days thereafter. The survival rate of rats subjected to outflow obstruction and implantation of catheter were 70% and 100%, respectively. After the cystometry experiments the rat

Accepted for publication May 9, 1989. * Requests for reprints: Dept. of Clinical Pharmacology, Lund University Hospital, S-221 85 Lund, Sweden. 1134

1135

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·was killed by cervical fracture and the bladder rer.noved and weighed. Partial obstruction of the urethra induces a significant bladder hyper-trophy. 9 · 10 · 11 Removal of the ligature has been shown to lead to a successive reduction in bladder weight. 9 • 12 However, two days after removal of the ligature the bladder still exhibits a significant degree of hypertrophy. 12 For simplicity the previously obstructed rats are therefore referred to as rats with a bladder hypertrophy. Cystometrical investigations. Cystometric evaluation of the bladder function was performed using a modified method described previously. 11 The bladder catheter was connected to an infusion pump and to a pressure transducer. The rate of infusion was 10 mL/hr. in normal rats. In order to obtain approximately equal number of micturitions in the two animal groups during the cystometry period, the rate of infusion was increased to 20 ml./hr. in rats with bladder hypertrophy. The saline infusion was started and after the following first micturition the infusion was maintained during a twenty minute period. Two hours after the end of the initial cystometry period, the rats were, via a stomach tube, given solutions of vehicle, pinacidil or cromakalim one mg.jkg. orally. One hour after the drug administration another cystometry period was performed. The following urodynamic variables were investigated: Bladder capacity Micturition pressure Basal pressure

= Volume of infused saline at rnicturition = the lowest bladder pressure during cystometry

Threshold pressure = bladder pressure immediately prior to micturition Micturition volume Residual volume = volume of infused saline minus micturition volume = mean amplitude of bladder pressure fluctuaSpontaneous activity tions during two minutes prior to micturition. Bladder compliance = LI. volume/ LI. pressure, i.e., bladder capacity/ (threshold pressure minus basal pressure) = (threshold pressure X bladder radius)/2 Bladder wall tension

Drugs. The following drugs were used: Cromakalim (BRL 34915, Beecham, U.K) Pinacidil (Leo Pharmaceutical, Denmark)

Calculations and statistics. From the initial cystometry period the mean value of each investigated variable was calculated and then compared with the corresponding mean value obtained from the cystometry performed after drug treatment. Collected data are given as mean values ± SEM. Statistical analysis was performed using Student's t test. Unpaired comparisons were performed between groups and pai:red comparisons between treatments. RESULTS

Normal rats. Fig. l shows a representative original recording from a continuous cystornetry performed in a normal rat. During the cystometry period the bladder pressure was low and almost devoid of spontaneous fluctuations. However, a small increase in bladder pressure preceded the micturition contractions which occurred regularly. The mean bladder capacity was 0.65 ml. in this particular rat and the mean micturition pressure amounted to 90 cm. H 2 0. The total micturition volume during the cystometry period was similar to the total amount of infused saline. Thus, no urine accumulation during the cystometry period was found in this rat. Two hours after the initial cystometry period this rat was given pure vehicle. One hour after the administration of vehicle another cystometry period was provided. The vehicle had no effect on the cystometry. Statistical analyses of the collected data from a group of rats receiving vehicle only (n = 8) showed

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that all cystometrical parameters were similar in the absence and presence of vehicle. Fig. 2 (upper panel) shows representative recordings from continuous cystometries performed in a normal rat before and after administration of pinacidil one mg./kg. orally. Pinacidil depressed the micturition pressure form 97 to 73 cm. H 2 0. Despite this decrease, the micturition volume was similar to the infused volume during both the cystometries. Further, bladder capacity was slightly increased after pinacidil (from 0.33 to 0.39 ml.), and no spontaneous bladder activity was observed. Fig. 2 (lower panel) shows corresponding original recordings from cystometries performed in another normal rat before and after administration of cromakalim one mg.jkg. orally. This rat developed a micturition pressure of 99 cm. H 2 0 and the bladder capacity amounted to 0.53 ml. No spontaneous bladder activity was seen during filling. Cromakalim decreased the micturition pressure to 88 cm. H20 but did not alter the bladder capacity, which was 0.55 ml. The collected data on the effects of pinacidil and cromakalim on micturition pressure and bladder capacity in normal rats are given in figure 3. Pinacidil decreased the micturition pressure by 18 ± 8% in normal rats but this effect did not achieve statistical significance (n = 6, p = 0.09). Cromakalim depressed the micturition pressure, by 15 ± 6% (n = 9, p <0.05). No effect on the bladder capacity was seen in this group of rats after administration of pinacidil or cromakalim. Residual volume, basal bladder pressure, threshold pressure, bladder compliance and bladder wall tension were evaluated in the absence and presence of the drugs. However, no clear-cut effects of either drug on these parameters were observed (data not Bladder wall tension was calculated to 2.1 ± 0.2 N/cm. in these normal rats. Preliminary experiments indicated that a higher dose of the drugs (five mg./kg.) gave no further increase in effect. Rather, unwanted side-effects such as roughcoat and piloerection were seen in the presence of cromakalim five mg.jkg. Rats with bladder hypertrophy. Fig. 4 (upper panel) shows original recordings from cystometries performed in a rat with bladder hypertrophy in the absence and presence of pinacidil one mg./kg. orally. The most prominent finding in this rat was spontaneous bladder contractions developing during filling. Immediately after a micturition had ceased the bladder pressure was slow and stable. However, during the continuous saline infusion bladder pressure fluctuations appeared with a successively increasing amplitude. Prior to the micturition contractions the spontaneous activity was most pronounced with a mean amplitude amounting to 11 cm. H 2 0. Furthermore, the micturition pressure was estimated to 127 cm. H 20 and the bladder capacity to 2.2 ml. The most conspicuous effect of pinacidil was an almost complete inhibition of the spontaneous bladder contractions. This effect persisted throughout the cys-

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tometry period. The minor remaining bladder pressure fluctuations were estimated to two cm. H 2 0. Moreover, after pinacidil the bladder capacity increased (to 3.5 ml.) and the micturition pressure had decreased by approximately 50% (to 68 cm. H 2 0). Despite the increase in bladder capacity and the decreased micturition pressure, no development of residual volume was found after pinacidil. A corresponding cystometry recording of the effects of cromakalim (one mg.jkg. orally) in a rat with bladder hypertrophy is shown in fig. 4 (lower panel). This rat exhibited regular bladder pressure fluctuations amounting to 24 cm. H20. The micturition pressure was 217 cm. H 2 0 and the bladder capacity 1.3 ml. Similar to the effects observed after administration of pinacidil, cromakalim depressed the spontaneous bladder contractions significantly and induced an increase in the bladder capacity in this particular rat. Thus, the mean amplitude of the contractions was only five cm. H 20 in the presence of cromakalim and the bladder capacity was 2.2 ml. The collected data on micturition pressure, bladder capacity and spontaneous bladder activity in rats with bladder hypertrophy given pinacidil and cromakalim are shown in fig. 5. Pinacidil decreased the micturition pressure, the remaining micturition pressure being 39 ± 12% of that developed in the absence of the drug (n = 6, p <0.01, fig. 5, upper panel). Also in the presence of cromakalim the micturition pressure decreased but this did not achieve statistical significance (p = 0.16). Furthermore, pinacidil but not cromakalim decreased the micturition pressure to a significantly larger extent in rats with bladder hypertrophy than in normal rats (p <0.05). Neither cromakalim nor pinacidil showed any effect on the bladder capacity in rats with bladder hypertrophy (fig. 5, middle panel). Moreover, pinacidil as well as cromakalim significantly depressed the spontaneous bladder contractions. The mean contraction amplitude remaining in the presence of pinacidil and cromakalim was only 26 ± 12% (n = 6, p <0.01) and 22 ± 7% (n = 5, p <0.001, fig. 5, lower panel), respectively, of the amplitude seen in the absence of the drugs. When the residual volume was calculated in hypertrophied rats (volume of infused saline minus micturated volume) a tendency towards an increased residual volume was found during the second cystometry period in both controls (n = 5)

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and drug treated animals. However, this increase was larger in animals receiving pinacidil (n = 5) or cromakalim (n = 7). In the control group the residual volume was 0.03 ± 0.18 ml. during the first and 0.06 ± 0.18 ml. during the second cystometry period (p = 0.88). Residual volume was -0.02 ± 0.05 ml. and 0.44 ± 0.16 ml., respectively, in the absence and presence ofpinacidil (p = 0.06). The corresponding values obtained from rats given cromakalim were -0.16 ± 0.15 and 0.21 ± 0.17 ml., respectively, p <0.05. Further, in accordance with the results in normal rats effects were noted neither on the basal bladder pressure, the threshold pressure, the bladder compliance nor on the bladder wall tension after administration of pinacidil and cromakalim in rats with bladder hypertrophy. However, the bladder wall tension was significantly larger in this group of rats compared to normal ones (4.8 ± 0.8, n = 10, and 2.1 ± 0.2 N/cm., n = 12, respectively, p<0.01).

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DISCUSSION

Recently an experimental technique for urodynamic evaluation of the bladder function in conscious rats was developed. 11 Using this technique rats subjected to outflow obstruction were found to have many characteristics in common with patients with BPH, spontaneous bladder contractions occurring during cystometry, development of residual urine, altered micturition pattern and bladder wall hypertrophy. 9 - 13 The model seems to be of great value for the investigation of the nature of bladder instability and further, for the assessment of drug effects in this disorder. In the present investigation the model described above was subjected to some modifications. It is well-known that in rats both micturition volumes and micturition intervals differ significantly during a 24-hour period. 13 Continuous cystometries rather than a single one make it possible to investigate several voiding cycles, thereby diminishing the intraindividual variation. However, the performance of a continuous cystometry in a rat with bladder outflow obstruction is associated with an obvious risk of successive urinary accumulation and impaired bladder function in such animals, We have recently shown that the bladder instability remains two days after removal of the urethral ligature in rats subjected to obstruction for 6 weeks. 12 At this time after removal of the ligature the residual urine was virtually abolished and the micturition volumes and intervals were normalized, In the present study the cystometrical investigation was performed two days after removal of the urethral ligature and the rate of saline infusion was increased to 10 ml./hr. in normal rats and to 20 ml./hr. in rats with bladder hypertrophy in order to optimize the cystometry period. The rate of filling is known to influence the bladder pressure and the micturition reflex, 14 In a previous study the diuresis in the rat was estimated to 44 ml. (kg. x 24 hr.). 13 This will lead to an average urine flow of 0.46 ml./hr. In man a forced cystometry is usually performed with a flow rate exceeding 100 ml./min. 15 Thus, the present rate of infusion both in normal rats and in rats with bladder hypertrophy must be considered to be small. In the present investigation the micturition pressure averaged 100 cm. H 2 0 in normal rats during continuous bladder filling with a rate of 10 ml./hr. In a previous study with an infusion rate of 2.5 ml./hr. the micturition pressure amounted

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to 25 cm. H 20 during a single cystometry. Thus, the micturition pressure increased fourfold when the rate of infusion was increased four times. However, the bladder capacity in the present study was similar to the one obtained during a single cystometry and an infusion rate of 2.5 ml./hr. 16 Despite the difference in micturition pressure separate pilot experiments indicated no quantitative difference in drug response between the two cystometrical methods. Pinacidil and cromakalim are drugs reported to relax and inhibit contractions of smooth muscle by increasing the efflux of potassium thereby inducing a hyperpolarisation of the smooth muscle cells. 3 ' 4 ' 17 Foster and Brading (1987) 8 have shown that in the guinea-pig in vitro, "BRL 34915 (10- 6 and 10-5 M) had no effect on carbachol and potassium contractures or on the response to intrinsic nerve stimulation." However, in the pig "BRL 34915 (8 X 10-6 and 10-5 M) markedly inhibited

1138

MALMGREN AND ASSOCIATES

the response to carbachol and slightly but significantly inhibited potassium contractures and the frequency response relationship." Fovaeus et al. (1988) 18 showed that in the isolated human bladder pinacidil depressed the contractile response to carbachol, to low concentrations of potassium (<60 mM) and to electrical stimulation. Further, they also showed that pinacidil increased the 86Rb-efflux from preloaded detrusor cells. In the present in vivo study cromakalim as well as pinacidil in a dose of one mg./kg. orally, depressed the micturition pressure in normal rats by circa 15%. However, no effects were seen on the bladder capacity by either of the drugs (fig. 3). An increased dose (five mg./kg.) did not increase the drug effects further. This should be compared with the results of a previous study where the effects of several drugs on the bladder function in normal rats were investigated. 19 It was shown that the muscarinic receptor antagonist emepronium and the calcium antagonist nifedipine, both in a dose of 10 mg./kg. orally, depressed the micturition pressure by 34 and 33%, respectively. Furthermore, emepronium increased the bladder capacity by 31%. Thus, the effects of pinacidil as well as cromakalim on the bladder function in normal rats must be considered to be rather small in comparison with that of anticholinergic agents and calcium antagonists. Foster and Brading (1986) 8 using a model of outflow obstruction and bladder instability in the pig, reported preliminary data showing that cromakalim (0.3 mg./kg.) "abolished the abnormal spontaneous increases in detrusor pressure associated with this condition without impairing" the animals ability to void." In agreement with this the most prominent finding in the present investigation was an almost complete inhibition of spontaneous bladder contractions in rats with bladder instability after administration of pinacidil as well as cromakalim (one mg./kg.). Bladder outflow obstruction in the pig as well as in the rat has been suggested to lead to a supersensitivity of the smooth muscle of the bladder. 20 • 21 Further, the instability seen in vivo in the rat has been shown to be resistant to atropine 22 and it has therefore been suggested to be caused by myogenic activation. Since potassium channel openers have been reported to induce a hyperpolarisation of the smooth muscle cells, hyperpolarisation of the smooth muscle cells in the unstable detrusor might be the mechanism leading to the depression of the spontaneous bladder contractions. In addition to the effects on the bladder instability a clearcut decrease in micturition pressure by 60% was observed after pinacidil. A decreased micturition pressure was also seen after cromakalim. However, this effect did not achieve statistical significance. In agreement with the findings in normal rats the effect was not increased by the dose of five mg./kg. Despite the reduction in micturition pressure the rats were still able to void effectively during the cystometrical investigation period. This might be due to the fact that during cystometrical investigations normal rats and probably also rats previously subjected to outflow obstruction, micturate at a submaximal pressure whereas obstructed rats micturate at a bladder pressure nearer the maximum one. The basis for this assumption is a previous study10 showing that the maximum pressures developed under anaesthesia at volumes corresponding to the bladder capacity in the present investigation were 104 ± 11 and 92 ± 14 cm. H 2 0 in normal rats and in rats with a bladder outflow obstruction, respectively. This should be compared with the micturition pressure developed during a previous cystometrical investigation (36 ± 4 and 112 ± 2 cm. H 2 0, in control and obstructed animals, respectively). 11 In rats as well as in patients with outflow obstruction a decreased micturition pressure will probably lead to an impaired emptying of the bladder. However, it is possible that a separation between the effects on the spontaneous activity and the micturition pressure exists. The findings of an almost complete disappearance of spontaneous bladder contractions in rats with bladder instability

and a remaining voiding ability after administration of pinacidil or cromakalim suggest that potassium channel openers may be a potential therapeutic alternative in the treatment of bladder instability associated with outflow obstruction. Acknowledgment. The authors thank Mrs Gull-Britt Ask, Mrs Lillemor Halvarsson and Mrs Ingrid Persson for skilful technical assistance. REFERENCES 1. McGuire, E. J.: Detrusor response to outlet obstruction. World J. Urol., 2: 208, 1984. 2. Andersson, K-E.: Current concepts in the treatment of disorders of micturition. Drugs, 35: 477, 1988. 3. Bray, K. M., Newgreen, D. T., Small, R. C., Southerton, J. S., Taylor, S. G., Weir, S. W. and Weston, A.H.: Evidence that the mechanism of the inhibitory action ofpinacidil in rat and guineapig smooth muscle differs from that of glyceryl trinitrate. Br. J. Pharmacol., 91: 421, 1987. 4. Hamilton, T. C., Weir, S. W. and Weston, A.H.: Comparison of the effects of BRL 34915 and verapamil on electrical and mechanical activity in rat portal vein. Br. J. Pharmacol., 88: 103, 1986. 5. Hollingsworth, M., Ame'de'e, T., Edwards, D., Mironneau, J., Savineau, J.P., Small, R. C. and Weston, A.H.: The relaxant action of BRL 34915 in rat uterus. Br. J. Pharmacol., 91: 803, 1987. 6. Allen, S. L., Boyle, J.P., Cortijo, J., Foster, R. W., Morgan, G. P. and Small, R. C.: Electrical and mechanical effects ofBRL 34915 in guinea-pig isolated trachealis. Br. J. Pharmacol., 89: 395, 1986. 7. Weir, S. W. and Weston, A.H.: Effect of apamin on responses to BRL 34915, nicorandil and other relaxants in the guinea-pig taenia caeki. Br. J. Pharmacol., 88: 113, 1986. 8. Foster, C. D. and Brading, A. F.: An in vitro and in vivo study of BRL 34915 in guinea-pig and pig bladder smooth muscle. Proceedings of the Xth IUPHAR Congress, Sydney, P1002, 1987. 9. Lindn'er, P., Mattiasson, A., Persson, L. and Uvelius, B.: Reversibility of detrusor hypertrophy and hyperplasia after removal of infravesical outflow obstruction in the rat. J. Urol., 140: 642, 1988. 10. Mattiasson, A., and Uvelius, B.: Changes in contractile properties in hypertrophic rat urinary bladder. J. Urol., 128: 1340, 1982. 11. Malmgren, A., Sjogren, C., Uvelius, B., Mattiasson, A., Andersson, K.-E. and Andersson, P. 0.: Cystometrical evaluation of bladder instability in rats with infravesical outflow obstruction. J. Urol., 137: 1291, 1987. 12. Malmgren, A., Uvelius, B., Andersson, K.-E. and Andersson, P. 0.: On the reversibility of functional bladder changes induced by infravesical outflow obstruction in the rat. J. Urology. Accepted for publication, 1989. 13. Malmgren, A.: Bladder instability and abnormal micturition pattern in rats with infravesical outflow obstruction. Neurourol. Urodyn., 6: 371, 1988. 14. Klevmark, B.: Motility of the urinary bladder in cats during filling at physiological rates. Acta Physiol. Scand., 90: 565, 1974. 15. International Continence Society: First report on the standardisation of terminology of lower urinary tract function. Br. J. Urol., 48: 39, 1976. 16. Malmgren, A., Andersson, K.-E. and Andersson, P. 0.: Capsaicinpretreatment induces supersensitivity in the rat urinary bladder. Proc. XIX Nordic Congress of Physiology and Pharmacology, p. 129, 1988. 17. Cook, N. S.: The pharmacology of potassium channels and their therapeutic potential. TIPS, 9: 21, 1988. 18. Fovaeus, M., Andersson, K.-E. and Hedlund, H.: The action of pinacidil in the isolated human bladder. J. Urol., 141: 637, 1989. 19. Pietra, C., Cazzulani, P., Rossi, M. G., Comolatti, G. and Cesarini: A method for quantitative studies for urinary bladder function in conscious rats. IRCS Med. Sci., 14: 992, 1986. 20. Sibley, G. N. A.: The physiological response of the detrusor muscle to experimental bladder outflow obstruction in the pig. Br. J. Urol., 60: 332, 1987. 21. Malmgren, A., Andersson, K.-E., Andersson, P. 0., Fovaeus, M. and Sjogren, C.: Effects of nifedipine and Bay K8644 on normal andhypertrophiedratdetrusor. Neurourol. Urodyn., 7: 27, 1988. 22. Malmgren, A., Sjogren, C., Andersson, K.-E. and Andersson, P. 0.: Effects of atropine on bladder capacity and instability in rats with bladder hypertrophy. Neurourol. Urodyn., 6: 331, 1987.