ENSAYOS PRELIMINARES

This study demonstrates that in both control and diabetic animals, the density of

E Ta receptor binding sites was significantly greater in the detrusor compared to the

bladder neck. Also the contractile responses to ET-1 were significantly greater in the detrusor compared to the bladder neck o f both control and diabetic animals. The contractile responses to ET- 1 are largely E Ta receptor-mediated since the E Ta selective

receptor antagonist (BQ123) caused a competitive inhibition (Eguchi et al 1992) o f contractile responses to ET-1 while the E Tb antagonist (BQ788) failed to inhibit these

responses. Furthermore, the E Tb selective receptor agonist (IRL 1620) did not elicit

contractions, suggesting that E Tbreceptor activation does not initiate contraction.

The findings o f a significant increase in E Tb receptor binding sites in the diabetic

urinary bladder are not clear, but are certainly provocative. These E Tb changes were not

due to a toxic effect o f alloxan since no differences in receptor expression were identified (at six months) in animals given alloxan who did not develop DM (table-6). The possible

mechanisms contributing to a significant decrease in the contractile responses to ET-1 in the diabetic bladder neck associated with an increase in the E Tbreceptor density certainly

merits further investigation. Since E Tb receptors have been implicated in the release of

endogenous NO (Clozel et al 1992), the activation o f E Tb receptors may oppose ET-1

contractile responses in the bladder neck as NO is thought to play a prominent role in bladder neck smooth muscle relaxation (Persson et al 1994). Whether the increased intracellular Ca^^ levels observed in DM (Levy J et al 1993) contribute to NO production (via activation o f Ca^^-dependent NOS) remains unclear (Razmjouei et al 1997).

In contrast to the bladder neck, the contractile responses to ET-1 were not decreased in the diabetic detrusor compared to controls, despite a similar increase in E Tb

receptor binding sites. This difference in ET-1-induced contractile responses between diabetic detrusor and bladder neck may be associated with the lack o f responsiveness o f detrusor smooth muscle to NO (Persson et al 1994).

Experimental DM may also precipitate a nonspecific decrease in the responsiveness o f bladder neck smooth muscle to vasoactive agents. Alterations in responses to ET-1 and NOS inhibitors have been reported in the cutaneous microcirculation o f diabetic rats (Kiff et al 1991). We have also shown that despite an increase in NOS in the diabetic bladder neck/urethra, NO-mediated smooth muscle relaxation was impaired (Chapter 4 ). It is therefore possible that an increase in E Tb

receptors and NOS in the diabetic urinary bladder neck could represent an attempted compensatory response to altered NO bioactivity. Overproduction o f NO, though beneficial in many systems, could also be potentially toxic due to the increased production o f free radicals (Ceriello et al 1991, Nussler et al 1993). It is conceivable, therefore, that a similar increase in free radicals in the diabetic urinary bladder can cause cytotoxic damage and thus alter bladder function. Alterations in the synthesis of another potent vasodilator, PGI2, have been well documented in several diabetic tissues including

the urinary bladder (Weisbrod et al 1993, Jeremy et al 1986 & 1987, Mikhailidis et al 1987). Hence, the interactions between NO, PGI2 and ET, which are thought to be

important in maintaining vascular tone (Luscher et al 1993), may also contribute to the regulation o f urinary tract smooth muscle tone. The precise relationship between NO,

PGI: and ET-1 in bladder function, however, has not been elucidated. We are currently investigating this relationship in the pathogenesis o f diabetic cystopathy.

ET may also influence other aspects o f bladder function. For example, ET is synthesised by SMC and it stimulates mitogenesis (Bobik et al 1990, Clozel et al 1992). Therefore, the DM-associated detrusor smooth muscle hyperplasia may be related to the significant increase in E Tb receptor density. The proliferation o f vascular SMC in

response to ET-1 has been shown to correlate with ET-receptor density (Kanse et al 1995). Both E Taand E Tb receptors have been implicated in this response. For example,

E Tb receptors participate in the development o f intimai hyperplasia after endothelial

injury (Azuma et al 1995) while human airway smooth muscle cell proliferation is E Ta

receptor-dependent (Panettieri et al 1996). ET-1 is also reported to enhance detrusor cholinergic and NANC responses (Saenz de Tejada et al 1992, Donso et al 1994), presumably by autocrine (release o f ET-1 by SMC) or by paracrine mechanisms. Thus, alterations in circulating ET levels in DM (Takahashi et al 1990) and expression of ET receptors in the diabetic urinary bladder may contribute to changes in bladder smooth muscle contractility. Furthermore, the dense E Ta binding to the blood vessels within the

bladder may also be involved in the control o f the blood supply to the detrusor and hence its function. In this context, it is o f interest to note that, bladder vascular insufficiency has recently been shown to alter bladder smooth muscle contractile responses to ET-1 (Azadzoi et al 1997).

In conclusion, this study has demonstrated that alloxan-induced DM in the rabbit is associated with a significant increase in the density o f E Tb receptors in the urinary

neck. These findings may contribute to the pathophysiological changes in the bladder, which are associated with DM. The manipulation o f E Tb receptor-mediated responses

CHAPTER 4

LOCALISATION OF NITRIC OXIDE SYNTHASE AND IN VITRO FUNCTIONAL