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Prostaglandins and cyclic nucleotides in the urinary bladder of a rabbit model of partial bladder outlet obstruction
Prostaglandins, Leukotrienes and Essential Fatty Acids (1999) 61(5), 307–314 © 1999 Harcourt Publishers Ltd Article no. plef.1999.0105
Prostaglandins and cyclic nucleotides in the urinary bladder of a rabbit model of partial bladder outlet obstruction M. A. Khan,1 C. S. Thompson,2 G. D. Angelini,3 R. J. Morgan,1 D. P. Mikhailidis,2 J. Y. Jeremy3 1
Department of Urology, Royal Free Hospital, London NW3 2QG, UK
Summary Bladder outlet obstruction (BOO) is a common disorder that is associated with altered bladder structure and function. For example, it is well established that BOO results in hypertrophy and hyperplasia of the bladder smooth muscle as well as detrusor instability. Since prostaglandins (PGs) and cyclic nucleotides (cyclic AMP [cAMP] and cyclic GMP [cGMP]) mediate both smooth muscle tone and proliferation, it is reasonable to suggest that changes in their levels may be involved in the pathophysiology of BOO-associated bladder disorders. Hence, the objective of this study was to investigate cyclic AMP, cyclic GMP and prostaglandins in the bladder of a rabbit model of BOO. BOO was induced in adult male New Zealand White rabbits. After 3 weeks, urinary bladders were excised, weighed and cut into segments. They were then incubated with stimulators of PGs, cAMP and cGMP and the formation of PGs, cAMP and cGMP were measured using radioimmunoassays. There was a significant increase in the obstructed bladder weights (P=0.002). The formation of PGE2, PGI2, cAMP and cGMP was significantly diminished in the detrusor (P<0.05) and bladder neck (P<0.05) in the BOO bladders compared to age-matched controls. Since PGE2, PGI2, cAMP and cGMP are known to inhibit the proliferation of smooth muscle cells (SMCs), the decreased synthesis of these factors, in BOO, may play a role in bladder SMC hypertrophy/hyperplasia. Our study points to the possible use of drugs that modulate the NO-cGMP and/or PG-cAMP axes in BOO-associated bladder pathology. © 1999 Harcourt Publishers Ltd.
INTRODUCTION Bladder outlet obstruction (BOO) is a common disorder that is principally a consequence of benign prostatic hyperplasia,1 urethral stricture or congenital anomaly.2 Structural changes associated with BOO include smooth muscle cell (SMC) hyperplasia and hypertrophy.3 Functional changes associated with BOO include detrusor instability, elevated voiding pressures and the presence of residual urine.4 The mechanisms underlying the complications associated with BOO remain largely unknown. Prostaglandins (PGs) are found in virtually all tissues5–7 Received 1 June 1999 Accepted 19 July 1999 Correspondence to: Dr M. A. Khan, Research Registrar, Department of Urology, Royal Free Hospital, Pond Street, London NW3 2QG, UK. Tel: + 44(0) 171 794 0500; Fax: + 44(0) 171 794 9537
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where they exert a wide variety of functions including modulating smooth muscle activity, haemostasis and cytoprotection. The urinary bladder is no exception. Although PGs do not play a major role in controlling contraction of the urinary tract smooth muscle,8 they may be involved in the enhancement of micturition elicited by cholinergic/adrenergic innervation9 and hyperplastic changes in the diabetic bladder.10 The effects of PGs are mediated via cyclic AMP (cAMP), which in turn has been shown to elicit relaxation of urinary tract tissues.11 The PG-cAMP axis also plays a role in preventing SMC proiferation.12 Urinary bladder tissue contains the enzyme nitric oxide synthase (NOS),13 which through the generation of nitric oxide (NO) and cyclic GMP (cGMP) elicits relaxation of urinary tract tissues.13,14 NO also inhibits SMC proliferation.15 It follows that any impairment of the PG-cAMP and
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NO-cGMP axes may contribute to the pathophysiology of BOO. In support of this proposition, impaired relaxation in response to NO and the diminished formation of PGs, cAMP and cGMP in the bladder of a rabbit model of diabetes, in which detrusor hypertrophy is present, has been reported.16,17 Numerous animal models have been developed to study BOO.18–20 For example, it has been reported that the rabbit model of BOO causes hypertrophy and hyperplasia of bladder smooth muscle, an increase in voiding pressure and bladder wall compliance, a decrease in residual urine volume and hypersensitivity to contractile agents.3,18 In order to further investigate the possible roles for PGs, cAMP and cGMP in obstructive bladder disease, these variables were measured in the detrusor and bladder neck of a rabbit model of BOO in the present study. MATERIALS AND METHODS Animals BOO was induced in six adult male New Zealand White rabbits (3kg). Six age-matched, sham operated rabbits acted as controls. All animals were fed ad libitum with SDS standard plain diet (SDS, Witham, UK) and allowed free access to water.
Induction of partial bladder outlet obstruction Under general anaesthesia (1–2% halothane in O2), a urinary catheter (Foley, C.R. Bard international Ltd, Crawley, UK) size 8 Fr gauge was inserted. After performing a lower midline laparotomy, a silk ligature was applied around the proximal urethra. The urinary catheter was then removed. The laparotomy incision was then closed and the animal allowed to recover. Sham operated controls underwent the same surgical procedure without inserting a proximal urethral ligature.
Blood sampling Blood was sampled at weekly intervals, via the ear vein, for plasma urea and electrolytes to detect early signs of renal failure.
Preparation of bladder tissue After 3 weeks, the BOO and age-matched control rabbits were killed by cervical dislocation and the bladders rapidly excised. The bladders were weighed and divided into detrusor and bladder neck sections. The tissues were then immediately placed in Dulbecco’s Minimum Essential Medium (DMEM) pregassed with 95% O2 / 5% CO2. The urothelium was carefully excised from the detrusor and
bladder neck smooth muscle. The detrusor and bladder neck segments were then cut longitudinally into two equal lengths and then transversely to give segments of approximately 2mm.2 Tissue segments from animals in each study group were pooled and incubated in DMEM at 37°C with regular changes of medium to allow the tissues to recover from preparative handling. PGI2 and PGE2 formation Detrusor and bladder neck discs were pre-incubated for 4 h with frequent changes of medium. One disc, in duplicate for each drug dose, was placed in pregassed DMEM containing the following drugs which are known to stimulate PGI2 and PGE2 synthesis in the rabbit vascular tissue: acetylcholine (receptor agonist), phorbol ester dibutyrate (activator of protein kinase C, PKC), calcium ionophore A23187 (increases intracellular calcium) and arachidonate (substrate).21 Tissues were then incubated for 1 h at 37°C. Supernatants were then removed and 6-oxo-PGF1α concentrations (the stable spontaneous hydrolysate of PGI2) and PGE2 measured by radioimmunoassay.22 Briefly, aliquots were diluted with Tris HCl – 1% gelatine buffer, pH 7.4. To these and 6-oxo- PGF1α or PGE2 standards (0–10ng) was added 200 µL of diluted 6-oxoPGF1α or PGE2 antisera containing 3.7 3 104 Bq [3H]-6-oxoPGF1α or [3H]-PGE2. Tubes were incubated overnight at 4°C. Activated charcoal (1% w/v) in Tris HCl – gelatine buffer was added to each tube, centrifuged and incubated on melting ice for 15 min. Tubes were then centrifuged at 1000 g for 10 min. Supernatants were decanted into vials, scintillation fluid added and counted in a β-particle counter (LKB; Copenhagen, Sweden). Standard curves were compiled and unknown values calculated. Assessment of cyclic nucleotide formation Following pre-incubation, detrusor and bladder neck discs were placed in DMEM in polypropylene tubes containing 250 µM isobutylmethylxanthine (a phosphodiesterase inhibitor) and various concentrations of cyclic nucleotide formation stimulators: forskolin (cAMP) and sodium nitroprusside (cGMP) and A23187 (activates NOS through the elevation of cytosolic Ca2+). Tubes were incubated for a further 20 min at 37°C. Reactions were stopped by the addition of 1M perchloric acid and the tissues sonicated (3 3 30 s; Soniprep, MSE, Bucks, UK), which extracts the cyclic nucleotides. Following centrifugation at 1000 g for 15 min, supernatants were taken and neutralized with 1M K3PO4. Aliquots were then taken and acetylated with triethylamine/acetic anhydride (1:2, v/v) and diluted with phosphate buffer, pH 7.4. To these and cAMP and cGMP standards (0–256 fmol) 200 µL diluted antisera against cAMP or cGMP antisera containing [125I]-
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cAMP or [125I]-cGMP was added. After overnight incubation at 4°C, antisera against rabbit globulins in phosphate buffer was added to each tube and incubated on melting ice for 15 min. Tubes were then centrifuged at 1000 g for 10 min. Supernatants were decanted into vials, scintillation fluid added and counted in a gamma particle counter (LKB). Standard curves were compiled and unknown values calculated. Data analysis and statistics Bladder weights, plasma urea and electrolytes between the 3-week BOO and age-matched sham operated control groups were compared using the Mann-Whitney U-test (paired values). For the cAMP, cGMP, 6-oxo- PGF1α and PGE2 measurements, data are expressed as the mean (SEM) values per milligram of tissue per minute (wet weight) from six samples. Data are analysed using ANOVA for multiple comparisons, with paired comparisons between groups assessed using paired student’s ttest where the ANOVA indicated significance for the multiple comparison. Statistical significance was accepted with P < 0.05. RESULTS Plasma urea and electrolytes There was no significant difference in the plasma urea and electrolytes between the 3-week partial BOO and agematched sham operated controls (results not shown). Bladder weights The weights of the control and BOO rabbits did not differ significantly. However, there was a significant increase (P = 0.002) in the bladder weights of the 3-week partial BOO rabbits compared to age-matched sham operated controls (Table 1). Table 1 Animal and bladder weights of age-matched sham operated controls and 3-week partial bladder outlet obstruction (BOO) rabbits. Results are given as median and range.
Rabbit weights Bladder weights
Control rabbits (n=6)
Three-week BOO rabits (n=6)
3.1 Kg (2.9–3.3) 2.1 g (1.9–2.3)
3.0 Kg (2.8–3.4) *12 g (10–16)
Control bladder weight vs 3-week BOO bladder weight *P = 0.002.
PGI2 and PGE2 formation PGI2 release in response to acetylcholine, phorbol ester and calcium ionophore A23187 was significantly diminished in both the detrusor (Fig. 1a,b,c) and bladder neck © 1999 Harcourt Publishers Ltd
Fig. 1 Prostacyclin formation (as assessed by 6-oxo-PGF1α) in the detrusor from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by (a), acetylcholine; (b), phorbol ester; and (c), calcium ionophore A23187. Each point is the mean (± SEM) of six rabbits. #, P<0.05.
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(Fig. 2a,b,c) tissue from the 3-week BOO rabbits compared to controls. In response to arachidonic acid, there was no significant difference in PGI2 formation by the detrusor and bladder neck in either group (data not shown). PGE2 release in response to acetylcholine, phorbol ester and calcium ionophore A23187 was significantly diminished in both the detrusor (Fig. 3a,b,c) and bladder neck (Fig. 4a,b,c) tissue from the 3-week BOO rabbits compared to controls. In response to arachidonic acid, there was no significant difference in PGI2 formation by the detrusor and bladder neck in either group (data not shown). Cyclic nucleotide formation In response to forskolin and PGE1, cAMP formation by the detrusor (Fig. 5a,b) and bladder neck (Fig. 5c,d) from the 3-week BOO rabbits was significantly reduced compared with controls. In response to calcium ionophore A23187, cGMP formation by the detrusor (Fig. 6a) and bladder neck (Fig 6b) from the 3-week BOO rabbits was significantly reduced compared to controls. In response to sodium nitroprusside, there were no significant differences in cGMP formation by the detrusor and bladder neck from either group (data not shown). DISCUSSION The present study demonstrates that there is a significant reduction in the formation of PGI2, PGE2, cAMP and cGMP and a concomitant significant increase in bladder weights in the 3-week BOO bladders compared to controls. Since the biological effect of these PGs are mediated by cAMP formation,8,10 it is reasonable to suggest that the reduction of the PG-cAMP axes may relate to the hypertrophic/ hyperplastic changes of the bladder seen in this model. It is also notable that in animal models of diabetes mellitus where there is marked polyuria, chronic distension and hypertrophy of detrusor smooth muscle there is also a decrease in PGs, cAMP and cGMP formation.17,33 It is reasonable to suggest, therefore, that down-regulation of the PG-cAMP and NO-cGMP axes may relate to the profound hyperplastic alterations in the BOO model investigated here. Guanylyl cyclase activity was decreased in urinary tract tissue from rabbits subjected to BOO. PGI2 and PGE2 and analogues of cAMP and cGMP are associated with the inhibition of SMC proliferation.23 Little is known about the impact of PGs or cAMP on urinary tract SMCs. However, in porcine saphenous vein grafts, where there is a 10-fold increase in tissue weight over 4 weeks, there is also a significant depression of PGI2, PGE2, cAMP and cGMP formation.12,24 As in the BOO model the saphenous vein graft is subjected to a marked increase in intra-luminal pressure to which the graft adapts by rapid remodel-
Fig. 2 Prostacyclin formation (as assessed by 6-oxo-PGF1α) in the bladder neck from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by (a), acetylcholine; (b), phorbol ester; and (c), calcium ionophore A23187. Each point is the mean (± SEM) of six rabbits. #, P<0.05.
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Prostaglandins and cyclic nucleotides in the urinary bladder of a rabbit model
Fig. 3 Prostaglandin E2 formation in the detrusor from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by (a), acetylcholine; (b), phorbol ester; and (c), calcium ionophore A23187. Each point is the mean (± SEM) of six rabbits. # P<0.05.
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Fig. 4 Prostaglandin E2 formation in the bladder neck from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by (a), acetylcholine; (b), phorbol ester; and (c), calcium ionophore A23187. Each point is the mean (± SEM) of six rabbits. #, P<0.05.
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Fig. 5 Cyclic AMP formation in the detrusor (a,b) and bladder neck (c,d) from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by forskolin or prostaglandin E1. Each point is the mean (± SEM) of six rabbits. #, P<0.05.
ling which involves SMC proliferation and deposition of matrix proteins.12 Another agent that may also play a role in BOO-associated bladder pathology is endothelin-1 (ET-1). ET-1 is a potent vasoconstrictor peptide.25 Two major ET receptors have been identified and cloned: ETA and ETB.26 In humans and rabbits, ET-1 is synthesized by vascular and nonvascular SMCs and by fibroblasts in the urinary bladder.27 The extensive distribution of ET-1 synthesis in the urinary bladder, occurring in almost all cell types, suggests that this peptide could play a role in bladder wall modeling, the control of bladder smooth muscle tone and the regulation of local blood flow.27 The role of ET-1 is modulated by NO released by the endothelium.28 Interestingly, we have recently demonstrated an increase in ET-1 receptors and ET-1 content in both the BOO
model used here29,30 and in porcine vein grafts,31 as well as a concomitant decrease in NOS and guanylyl cyclase activity in both models.12,24,29,30 ET-1, as well as having vasoconstrictor properties, is also mitogenic for SMC.32 The present data may have relevance to detrusor instability due to BPH, which is a common clinical problem associated with bladder obstruction.34 The decreased synthesis of PGs in the obstructed bladders may be beneficial, at least initially. This is because the decreased synthesis may play a role in the bladder SMC hypertrophy and hyperplasia that occur in both humans with BPH and animal models of BOO. The increase in the bladder weights may be a compensatory response to the increased resistance to urinary flow produced by BOO. This change may enable the bladder to generate increased intravesical pressure to allow micturition to
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Fig. 6 Cyclic GMP formation in the detrusor (a) and bladder neck (b) from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by calcium ionophore A232187. Each point is the mean (± SEM) of six rabbits. #, P<0.05.
occur and may reflect the symptomatic improvement experienced by many patients with BPH that are managed by ‘watchful waiting’.35 However, detrusor instability occurs in the majority of men with BOO due to BPH,34 and similar results have also been demonstrated in animal models of BOO.36 Hence, in the long term the decreased PG synthesis may play a role in the development of detrusor instability. The finding that in BOO there is a reduction of both NOS and guanylyl cyclase activity in the urinary bladder may further compound this. In summary, there is a significant reduction in the activity of the PG-cAMP and NO-cGMP axes in the urinary bladder of a rabbit model of BOO. These changes are consistent with the pathophysiological alterations associated with clinical BOO; that is, bladder hypertrophy/ hyperplasia and detrusor instability. Hence, our study provides a possible rationale for the investigation of drugs that modulate the NO-cGMP and/or PG-cAMP axes in the clinical management of BOO-associated bladder pathology.
REFERENCES 1. Napalkov P., Maisonneuve P., Boyle P. Worldwide patterns of prevalence and mortality from benign prostatic hyperplasia. Urology 1995; 46: 41–46. 2. O’Connor T. L., Vaughan D. E., Felsen D. In vivo cystometric evaluation of progressive bladder outlet obstruction in rats. J Urol 1997; 158: 631–635. 3. Ghoniem G. M., Regnier C., Biancani P., Johnson L., Susset J. G. Effect of vesical outlet obstruction on detrusor contractility and passive properties in rabbits. J Urol 1986; 135:1284–1289. 4. Turner–Warwick R. Bladder outflow obstruction in the male. In: Mundy A. R., Stephenson T. P., Wein A. J., eds. Urodynamics: Principles, Practice and Applications. Edinburgh: Churchill–Livingstone, 1984: 183.
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5. Moncada S., Flower R. J., Vane J. R. Prostaglandins, prostacyclin, thromboxane A2, and leukotrienes. In: Goodman L. S., Gilman A.G. eds. The Pharmacological Basis of Therapeutics. New York: MacMillan, 1985: 660. 6. Anderson N. H., Hartzell C. J. De B. Chemistry and structure of cyclooxygenase-derived eicosanoids: a historical perspective. In: Pike J. E., Morton J. R. eds. Advances in Prostaglandin, Thromboxane and Leukotriene Research. New York: Raven Press, 1985: 1. 7. Johnson R. A. Synthesis of thromboxanes, prostacyclin and the endoperoxides. In: Pike J. E., Morton D. R. eds. Advances in Prostaglandin, Thromboxane and Leukotriene Research. New York: Raven Press, 1985: 131. 8. Mikhailidis D. P., Jeremy J. Y., Dandona P. Eicosanoids and the urinary bladder. J Urol 1987; 137: 577–582. 9. Jeremy J. Y., Mikhailidis D. P., Dandona P. Prostanoid synthesis by the rat urinary bladder: evidence for stimulation through muscarinic receptor linked calcium channels. Naunyn Schmiedeberg’s Arch Pharmacol 1986; 334: 463–467. 10. Khan M. A., Thompson C. S., Mumtaz F. H., Jeremy J. Y, Morgan R. J., Mikhailidis D. P. Role of prostaglandins in the urinary bladder: an update. Prostagl Leukotr Essentl Fatty Acids 1998; 59: 415–422. 11. Morita T., Wheeler M. A., Weiss R. M. Relaxant effect of forskolin in rabbit detrusor smooth muscle: role of cyclic AMP. J Urol 1986; 135: 1293–1295. 12. Jeremy J. Y., Dashwood M., Timm M. et al. Nitric oxide synthase and cyclic nucleotide synthesis by porcine venous–arterial grafts. Ann Thorac Surg 1997; 63: 470–476 13. Chung B. H., Seung K. C., Ki C. C. Effects of nitric oxide on detrusor relaxation. J Urol 1996; 155: 2090–2096. 14. Andersson K-E. Pharmacology of lower urinary tract smooth muscles and penile erectile tissues. Pharmacol Revs 1993; 45: 253–308. 15. Jeremy J. Y., Rowe D., Emsley A. M., Newby A. C. Nitric oxide and the proliferation of vascular smooth muscle cells. Cardiovasc Res 1999; in press. 16. Mumtaz F. H., Sullivan M. E., Thompson C. S. et al. Alterations in the nitric oxide synthase sites and non-adrenergic mediated smooth muscle relaxation in the diabetic rabbit bladder:
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possible relevance to the pathogenesis of diabetic cystopathy. J Urol 1999; in press. Mumtaz F. H., Thompson C. S., Khan M. A. et al. Prostaglandins and cyclic nucleotides synthesis in the lower urinary tract of the diabetic rabbit. Br J Urol 1998; 81: 32. Kato K., Wein A. J., Kitada S., Haugaard N., Levin R. M. The functional effects of mild outlet obstruction on the rabbit urinary bladder. J Urol 1988; 140: 880–884. Sibley G. N. A. The physiological response of the detrusor muscle to experimental bladder outflow obstruction in the pig. Br J Urol 1987; 60: 332–336. Radzinski C., Mcguire E. J., Smith D., et al. Creation of a feline model of obstructive uropathy. J Urol 1991; 154: 859–863. Jeremy J. Y., Mikhailidis D. P., Dandona P. Excitatory receptor linked prostanoid synthesis in mammalian smooth muscle: the role of calcium, protein kinase C and G proteins. Prostagl Leukotr Essential Fatty Acids 1988; 34: 215–221. Jeremy J. Y., Mikhailidis D. P., Thompson C. S., Dandona P. Experimental diabetes mellitus inhibits prostacyclin synthesis by the rat penis: pathological implications. Diabetalogia 1985; 28: 365–370. Jeremy J. Y., Jackson C. L., Bryan A. J., Angelini G. D. Eicosanoids, fatty acids and restenosis following balloon angioplasty and vein graft surgery. Prostagl Leukotr Essential Fatty Acids 1996; 54: 385–402. Jeremy J. Y., Dashwood M., Mehta D., Izzat M. B., Bryan A. J., Angelini G. D. Nitric oxide synthase, prostacyclin and cyclic nucleotide production in externally stented porcine vein grafts. Atherosclerosis 1999; in press. Yanagisawa M., Kurihara H., Kimura S. et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332: 411–415. Arai H., Hori S., Aramori L., Ohkubo H., Nakanishi S. Cloning and expression of cDNA encoding an endothelin receptor. Nature 1990; 348: 730–732.
27. Saenz de Tejada I., Mueller J. D. et al. Endothelin in the urinary bladder. Synthesis of endothelin–1 by epithelia, smooth muscle and fibroblasts suggests autocrine and paracrine cellular regulation. J Urol 1992; 148: 1290–1298. 28. Levin R. E. Endothelins. N Engl J Med 1995; 333: 356–363. 29. Khan M. A., Thompson C. S., Dashwood M. R., Mumtaz F. H., Mikhailidis D. P., Morgan R. J. Upregulation of endothelin (ETA and ETB) receptors and down regulation of nitric oxide synthase in the detrusor of a rabbit model of bladder outlet obstruction. J Urol 1999; 161: 46. 30. Khan M. A., Dashwood M. R., Thompson C. S., Mumtaz F. H., Mikhailidis D. P., Morgan R. J. Up-regulation of ETB receptors and ETB receptor-mediated rabbit detrusor contraction in partial bladder outlet obstruction. Br J Urol 1999; in press. 31. Dashwood M. R., Mehta D., Izzat M. B. et al. Distribution of endothelin-1 (ETA /ETB) receptor binding to different regions of porcine interposition venous-arterial grafts. Atherosclerosis 1998; 137: 233–242. 32. Bobik A., Grooms A., Miller J. A., Mitchell A., Grinpukel S. Growth factor activity of endothelin on vascular smooth muscle. Am J Physiol 1990; 258: C408-C415. 33. Sullivan M. E., Thompson C. S., Mikhailidis D. P., Morgan R. J., Angelini G. D., Jeremy J. Y. Differential alterations of prostacyclin, cyclic AMP and cyclic GMP formation in the corpus cavernosum of the diabetic rabbit. Br J Urol 1998; 82: 578–584. 34. Ameda K., Koyanagi T., Nantani M., Taniguchi K., Matsuno T. The relevance of preoperative cystometrography in patients with benign prostatic hyperplasia: correlating the findings with clinical features and outcome after prostatectomy. J Urol 1994; 152: 443–447. 35. Gray M. Progressive changes in detrusor function with bladder outlet obstruction. J Urol 1997; 158: 318. 36. Speakman M. J., Brading A. F., Gilpin C. J., Dixon J. S., Gilpin S. A., Gosling J. A. Bladder outflow obstruction – a cause of denervation supersensititivty. J Urol 1987; 138: 1461–1466.
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Prostaglandins and cyclic nucleotides in the urinary bladder of a rabbit model of partial bladder outlet obstruction M. A. Khan,1 C. S. Thompson,2 G. D. Angelini,3 R. J. Morgan,1 D. P. Mikhailidis,2 J. Y. Jeremy3 1
Department of Urology, Royal Free Hospital, London NW3 2QG, UK
Summary Bladder outlet obstruction (BOO) is a common disorder that is associated with altered bladder structure and function. For example, it is well established that BOO results in hypertrophy and hyperplasia of the bladder smooth muscle as well as detrusor instability. Since prostaglandins (PGs) and cyclic nucleotides (cyclic AMP [cAMP] and cyclic GMP [cGMP]) mediate both smooth muscle tone and proliferation, it is reasonable to suggest that changes in their levels may be involved in the pathophysiology of BOO-associated bladder disorders. Hence, the objective of this study was to investigate cyclic AMP, cyclic GMP and prostaglandins in the bladder of a rabbit model of BOO. BOO was induced in adult male New Zealand White rabbits. After 3 weeks, urinary bladders were excised, weighed and cut into segments. They were then incubated with stimulators of PGs, cAMP and cGMP and the formation of PGs, cAMP and cGMP were measured using radioimmunoassays. There was a significant increase in the obstructed bladder weights (P=0.002). The formation of PGE2, PGI2, cAMP and cGMP was significantly diminished in the detrusor (P<0.05) and bladder neck (P<0.05) in the BOO bladders compared to age-matched controls. Since PGE2, PGI2, cAMP and cGMP are known to inhibit the proliferation of smooth muscle cells (SMCs), the decreased synthesis of these factors, in BOO, may play a role in bladder SMC hypertrophy/hyperplasia. Our study points to the possible use of drugs that modulate the NO-cGMP and/or PG-cAMP axes in BOO-associated bladder pathology. © 1999 Harcourt Publishers Ltd.
INTRODUCTION Bladder outlet obstruction (BOO) is a common disorder that is principally a consequence of benign prostatic hyperplasia,1 urethral stricture or congenital anomaly.2 Structural changes associated with BOO include smooth muscle cell (SMC) hyperplasia and hypertrophy.3 Functional changes associated with BOO include detrusor instability, elevated voiding pressures and the presence of residual urine.4 The mechanisms underlying the complications associated with BOO remain largely unknown. Prostaglandins (PGs) are found in virtually all tissues5–7 Received 1 June 1999 Accepted 19 July 1999 Correspondence to: Dr M. A. Khan, Research Registrar, Department of Urology, Royal Free Hospital, Pond Street, London NW3 2QG, UK. Tel: + 44(0) 171 794 0500; Fax: + 44(0) 171 794 9537
© 1999 Harcourt Publishers Ltd
where they exert a wide variety of functions including modulating smooth muscle activity, haemostasis and cytoprotection. The urinary bladder is no exception. Although PGs do not play a major role in controlling contraction of the urinary tract smooth muscle,8 they may be involved in the enhancement of micturition elicited by cholinergic/adrenergic innervation9 and hyperplastic changes in the diabetic bladder.10 The effects of PGs are mediated via cyclic AMP (cAMP), which in turn has been shown to elicit relaxation of urinary tract tissues.11 The PG-cAMP axis also plays a role in preventing SMC proiferation.12 Urinary bladder tissue contains the enzyme nitric oxide synthase (NOS),13 which through the generation of nitric oxide (NO) and cyclic GMP (cGMP) elicits relaxation of urinary tract tissues.13,14 NO also inhibits SMC proliferation.15 It follows that any impairment of the PG-cAMP and
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NO-cGMP axes may contribute to the pathophysiology of BOO. In support of this proposition, impaired relaxation in response to NO and the diminished formation of PGs, cAMP and cGMP in the bladder of a rabbit model of diabetes, in which detrusor hypertrophy is present, has been reported.16,17 Numerous animal models have been developed to study BOO.18–20 For example, it has been reported that the rabbit model of BOO causes hypertrophy and hyperplasia of bladder smooth muscle, an increase in voiding pressure and bladder wall compliance, a decrease in residual urine volume and hypersensitivity to contractile agents.3,18 In order to further investigate the possible roles for PGs, cAMP and cGMP in obstructive bladder disease, these variables were measured in the detrusor and bladder neck of a rabbit model of BOO in the present study. MATERIALS AND METHODS Animals BOO was induced in six adult male New Zealand White rabbits (3kg). Six age-matched, sham operated rabbits acted as controls. All animals were fed ad libitum with SDS standard plain diet (SDS, Witham, UK) and allowed free access to water.
Induction of partial bladder outlet obstruction Under general anaesthesia (1–2% halothane in O2), a urinary catheter (Foley, C.R. Bard international Ltd, Crawley, UK) size 8 Fr gauge was inserted. After performing a lower midline laparotomy, a silk ligature was applied around the proximal urethra. The urinary catheter was then removed. The laparotomy incision was then closed and the animal allowed to recover. Sham operated controls underwent the same surgical procedure without inserting a proximal urethral ligature.
Blood sampling Blood was sampled at weekly intervals, via the ear vein, for plasma urea and electrolytes to detect early signs of renal failure.
Preparation of bladder tissue After 3 weeks, the BOO and age-matched control rabbits were killed by cervical dislocation and the bladders rapidly excised. The bladders were weighed and divided into detrusor and bladder neck sections. The tissues were then immediately placed in Dulbecco’s Minimum Essential Medium (DMEM) pregassed with 95% O2 / 5% CO2. The urothelium was carefully excised from the detrusor and
bladder neck smooth muscle. The detrusor and bladder neck segments were then cut longitudinally into two equal lengths and then transversely to give segments of approximately 2mm.2 Tissue segments from animals in each study group were pooled and incubated in DMEM at 37°C with regular changes of medium to allow the tissues to recover from preparative handling. PGI2 and PGE2 formation Detrusor and bladder neck discs were pre-incubated for 4 h with frequent changes of medium. One disc, in duplicate for each drug dose, was placed in pregassed DMEM containing the following drugs which are known to stimulate PGI2 and PGE2 synthesis in the rabbit vascular tissue: acetylcholine (receptor agonist), phorbol ester dibutyrate (activator of protein kinase C, PKC), calcium ionophore A23187 (increases intracellular calcium) and arachidonate (substrate).21 Tissues were then incubated for 1 h at 37°C. Supernatants were then removed and 6-oxo-PGF1α concentrations (the stable spontaneous hydrolysate of PGI2) and PGE2 measured by radioimmunoassay.22 Briefly, aliquots were diluted with Tris HCl – 1% gelatine buffer, pH 7.4. To these and 6-oxo- PGF1α or PGE2 standards (0–10ng) was added 200 µL of diluted 6-oxoPGF1α or PGE2 antisera containing 3.7 3 104 Bq [3H]-6-oxoPGF1α or [3H]-PGE2. Tubes were incubated overnight at 4°C. Activated charcoal (1% w/v) in Tris HCl – gelatine buffer was added to each tube, centrifuged and incubated on melting ice for 15 min. Tubes were then centrifuged at 1000 g for 10 min. Supernatants were decanted into vials, scintillation fluid added and counted in a β-particle counter (LKB; Copenhagen, Sweden). Standard curves were compiled and unknown values calculated. Assessment of cyclic nucleotide formation Following pre-incubation, detrusor and bladder neck discs were placed in DMEM in polypropylene tubes containing 250 µM isobutylmethylxanthine (a phosphodiesterase inhibitor) and various concentrations of cyclic nucleotide formation stimulators: forskolin (cAMP) and sodium nitroprusside (cGMP) and A23187 (activates NOS through the elevation of cytosolic Ca2+). Tubes were incubated for a further 20 min at 37°C. Reactions were stopped by the addition of 1M perchloric acid and the tissues sonicated (3 3 30 s; Soniprep, MSE, Bucks, UK), which extracts the cyclic nucleotides. Following centrifugation at 1000 g for 15 min, supernatants were taken and neutralized with 1M K3PO4. Aliquots were then taken and acetylated with triethylamine/acetic anhydride (1:2, v/v) and diluted with phosphate buffer, pH 7.4. To these and cAMP and cGMP standards (0–256 fmol) 200 µL diluted antisera against cAMP or cGMP antisera containing [125I]-
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cAMP or [125I]-cGMP was added. After overnight incubation at 4°C, antisera against rabbit globulins in phosphate buffer was added to each tube and incubated on melting ice for 15 min. Tubes were then centrifuged at 1000 g for 10 min. Supernatants were decanted into vials, scintillation fluid added and counted in a gamma particle counter (LKB). Standard curves were compiled and unknown values calculated. Data analysis and statistics Bladder weights, plasma urea and electrolytes between the 3-week BOO and age-matched sham operated control groups were compared using the Mann-Whitney U-test (paired values). For the cAMP, cGMP, 6-oxo- PGF1α and PGE2 measurements, data are expressed as the mean (SEM) values per milligram of tissue per minute (wet weight) from six samples. Data are analysed using ANOVA for multiple comparisons, with paired comparisons between groups assessed using paired student’s ttest where the ANOVA indicated significance for the multiple comparison. Statistical significance was accepted with P < 0.05. RESULTS Plasma urea and electrolytes There was no significant difference in the plasma urea and electrolytes between the 3-week partial BOO and agematched sham operated controls (results not shown). Bladder weights The weights of the control and BOO rabbits did not differ significantly. However, there was a significant increase (P = 0.002) in the bladder weights of the 3-week partial BOO rabbits compared to age-matched sham operated controls (Table 1). Table 1 Animal and bladder weights of age-matched sham operated controls and 3-week partial bladder outlet obstruction (BOO) rabbits. Results are given as median and range.
Rabbit weights Bladder weights
Control rabbits (n=6)
Three-week BOO rabits (n=6)
3.1 Kg (2.9–3.3) 2.1 g (1.9–2.3)
3.0 Kg (2.8–3.4) *12 g (10–16)
Control bladder weight vs 3-week BOO bladder weight *P = 0.002.
PGI2 and PGE2 formation PGI2 release in response to acetylcholine, phorbol ester and calcium ionophore A23187 was significantly diminished in both the detrusor (Fig. 1a,b,c) and bladder neck © 1999 Harcourt Publishers Ltd
Fig. 1 Prostacyclin formation (as assessed by 6-oxo-PGF1α) in the detrusor from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by (a), acetylcholine; (b), phorbol ester; and (c), calcium ionophore A23187. Each point is the mean (± SEM) of six rabbits. #, P<0.05.
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(Fig. 2a,b,c) tissue from the 3-week BOO rabbits compared to controls. In response to arachidonic acid, there was no significant difference in PGI2 formation by the detrusor and bladder neck in either group (data not shown). PGE2 release in response to acetylcholine, phorbol ester and calcium ionophore A23187 was significantly diminished in both the detrusor (Fig. 3a,b,c) and bladder neck (Fig. 4a,b,c) tissue from the 3-week BOO rabbits compared to controls. In response to arachidonic acid, there was no significant difference in PGI2 formation by the detrusor and bladder neck in either group (data not shown). Cyclic nucleotide formation In response to forskolin and PGE1, cAMP formation by the detrusor (Fig. 5a,b) and bladder neck (Fig. 5c,d) from the 3-week BOO rabbits was significantly reduced compared with controls. In response to calcium ionophore A23187, cGMP formation by the detrusor (Fig. 6a) and bladder neck (Fig 6b) from the 3-week BOO rabbits was significantly reduced compared to controls. In response to sodium nitroprusside, there were no significant differences in cGMP formation by the detrusor and bladder neck from either group (data not shown). DISCUSSION The present study demonstrates that there is a significant reduction in the formation of PGI2, PGE2, cAMP and cGMP and a concomitant significant increase in bladder weights in the 3-week BOO bladders compared to controls. Since the biological effect of these PGs are mediated by cAMP formation,8,10 it is reasonable to suggest that the reduction of the PG-cAMP axes may relate to the hypertrophic/ hyperplastic changes of the bladder seen in this model. It is also notable that in animal models of diabetes mellitus where there is marked polyuria, chronic distension and hypertrophy of detrusor smooth muscle there is also a decrease in PGs, cAMP and cGMP formation.17,33 It is reasonable to suggest, therefore, that down-regulation of the PG-cAMP and NO-cGMP axes may relate to the profound hyperplastic alterations in the BOO model investigated here. Guanylyl cyclase activity was decreased in urinary tract tissue from rabbits subjected to BOO. PGI2 and PGE2 and analogues of cAMP and cGMP are associated with the inhibition of SMC proliferation.23 Little is known about the impact of PGs or cAMP on urinary tract SMCs. However, in porcine saphenous vein grafts, where there is a 10-fold increase in tissue weight over 4 weeks, there is also a significant depression of PGI2, PGE2, cAMP and cGMP formation.12,24 As in the BOO model the saphenous vein graft is subjected to a marked increase in intra-luminal pressure to which the graft adapts by rapid remodel-
Fig. 2 Prostacyclin formation (as assessed by 6-oxo-PGF1α) in the bladder neck from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by (a), acetylcholine; (b), phorbol ester; and (c), calcium ionophore A23187. Each point is the mean (± SEM) of six rabbits. #, P<0.05.
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Fig. 3 Prostaglandin E2 formation in the detrusor from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by (a), acetylcholine; (b), phorbol ester; and (c), calcium ionophore A23187. Each point is the mean (± SEM) of six rabbits. # P<0.05.
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Fig. 4 Prostaglandin E2 formation in the bladder neck from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by (a), acetylcholine; (b), phorbol ester; and (c), calcium ionophore A23187. Each point is the mean (± SEM) of six rabbits. #, P<0.05.
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Fig. 5 Cyclic AMP formation in the detrusor (a,b) and bladder neck (c,d) from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by forskolin or prostaglandin E1. Each point is the mean (± SEM) of six rabbits. #, P<0.05.
ling which involves SMC proliferation and deposition of matrix proteins.12 Another agent that may also play a role in BOO-associated bladder pathology is endothelin-1 (ET-1). ET-1 is a potent vasoconstrictor peptide.25 Two major ET receptors have been identified and cloned: ETA and ETB.26 In humans and rabbits, ET-1 is synthesized by vascular and nonvascular SMCs and by fibroblasts in the urinary bladder.27 The extensive distribution of ET-1 synthesis in the urinary bladder, occurring in almost all cell types, suggests that this peptide could play a role in bladder wall modeling, the control of bladder smooth muscle tone and the regulation of local blood flow.27 The role of ET-1 is modulated by NO released by the endothelium.28 Interestingly, we have recently demonstrated an increase in ET-1 receptors and ET-1 content in both the BOO
model used here29,30 and in porcine vein grafts,31 as well as a concomitant decrease in NOS and guanylyl cyclase activity in both models.12,24,29,30 ET-1, as well as having vasoconstrictor properties, is also mitogenic for SMC.32 The present data may have relevance to detrusor instability due to BPH, which is a common clinical problem associated with bladder obstruction.34 The decreased synthesis of PGs in the obstructed bladders may be beneficial, at least initially. This is because the decreased synthesis may play a role in the bladder SMC hypertrophy and hyperplasia that occur in both humans with BPH and animal models of BOO. The increase in the bladder weights may be a compensatory response to the increased resistance to urinary flow produced by BOO. This change may enable the bladder to generate increased intravesical pressure to allow micturition to
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Fig. 6 Cyclic GMP formation in the detrusor (a) and bladder neck (b) from control (■) and 3-week obstructed (■ ■ ) rabbits, stimulated by calcium ionophore A232187. Each point is the mean (± SEM) of six rabbits. #, P<0.05.
occur and may reflect the symptomatic improvement experienced by many patients with BPH that are managed by ‘watchful waiting’.35 However, detrusor instability occurs in the majority of men with BOO due to BPH,34 and similar results have also been demonstrated in animal models of BOO.36 Hence, in the long term the decreased PG synthesis may play a role in the development of detrusor instability. The finding that in BOO there is a reduction of both NOS and guanylyl cyclase activity in the urinary bladder may further compound this. In summary, there is a significant reduction in the activity of the PG-cAMP and NO-cGMP axes in the urinary bladder of a rabbit model of BOO. These changes are consistent with the pathophysiological alterations associated with clinical BOO; that is, bladder hypertrophy/ hyperplasia and detrusor instability. Hence, our study provides a possible rationale for the investigation of drugs that modulate the NO-cGMP and/or PG-cAMP axes in the clinical management of BOO-associated bladder pathology.
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