Supersensitivity of muscarinic receptors in rat isolated detrusor smooth muscle (DSM) after chronic nitric oxide inhibition

Background

It has been suggested that disturbance of the NO-cGMP pathway lead to impaired relaxation of the urethral outflow region, increased bladder afferent activity and overactive bladder, but the precise role of NO in regulating the detrusor smooth muscle (DSM) functions remains to be determined.

Purpose

Our present work aimed to examine the functional and biochemical alterations of rat DSM after chronic NO blockade.

Methods

Male Wistar rats were treated orally with L-NAME (20 mg/rat/day) for 30 days. Age-matched control animals received tap water alone. Concentration-response curves to full agonist carbachol (CCh, 1 nM-30 μM) in the DSM were obtained. The values of potency (pEC₅₀) and maximal responses (E_max) were calculated. The IP₃ accumulation and the nitric oxide synthase (NOS) activity, as well as morphometric analyses were evaluated in the urinary bladder of control and L-NAME-treated rats.

Results

L-NAME-treated rats presented a marked arterial hypertension (ctrl:124 ± 2 vs treated: 198 ± 1 mmHg) and a reduction of 86% in the total NOS activity in the isolated rat bladder. Four-weeks treatment with L-NAME increased by 5-fold the CCh potency (6.09 ± 0.02 vs 6.82 ± 0.06), without modifying the E_max (ctl: 3.50 ± 0.10 vs treated: 3.40 ± 0.07) (Figure 1A). Incubation of urinary bladder with CCh (10^-9–10^-3 M) concentration-dependently increased the total [³H]-inositol phosphates in rat urinary bladder that was markedly higher in L-NAME-treated rats compared with control animals (Figure 1B). The measurement of the thickness of rat isolated bladder revealed that L-NAME treatment for 30 days caused no alterations in the thickness of submucosa and muscular layers of the DSM when compared with control animals. However, in the trigone smooth muscle (TSM), L-NAME treatment significantly increased the thickness of the muscular layer without changing the thickness of the sub-mucosa layer (Table 1).

Figure 1

(A) Concentration-response curve to carbachol in rat DSM from control and L-NAME-treated rats in 30 days (n = 6–8, p < 0.05). (B) Effects of carbachol on total [³H]-inositol phosphate accumulation in both control and L-NAME-treated group in 30 days (n = 3–6, P < 0.05).

Table 1 Morphometric analyses in isolated rat detrusor and trigone smooth muscle in control and L-NAME-treated groups in 30 days. *p < 0.05.

Conclusion

Our findings show that long-term NO inhibition significantly increases the sensitivity of DSM for the muscarinic agonist carbachol via accumulation of IP₃, suggesting that NO exerts a modulatory effect on the contractility mediated by muscarinic receptors.