Loss Of Ryanodine Recepter Channel Associated With Overactive Urinary Bladder Smooth Muscle Contractions In Detrusor Instability Model

Objective: A variety of changes occur during detrusor instability ( DI ) and many studies suggest that there is a myogenic basis of the increased excitability and contractile activity. Ryanodine receptors ( RyR ) in the sarcoplasmic reticulum ( SR ) act as an important modulator of excitation-contraction coupling in detrusor smooth muscle, contributing transient, localized Ca²⁺ release events ( Ca²⁺ sparks ) associated with activation of Ca²⁺-activated K⁺ channels ( Kca ) and negative feedback regulation of voltage-dependent Ca²⁺ channels ( VDCC ), and thus regulation corresponding in spontaneous contractility. In this study, we investigated the changes that occur to spontaneous bladder smooth muscle contractions during DI and the possibility that alterations in RyR mRNA & protein expression and function could account for the increased bladder contractions which was observed during DI. In addition, to provide evidence in application of DI model, a comparison was taken between proximal and distal urethra ligation way in producing post-obstruction DI model.Materials and Methods: Producing partial lower urethra obstruction by these two ways in Wistar rats and confirming DI rats 8 weeks later by filling cystometry, we took statistics the bladder wet weight, the number of DI rats, the number of excluded rats (including death, infection, calculus, leakage ), the number of non-DI rats, bladder volume, max vesica pressure in two ways groups as well as in control. Muscle strips were dissected from freshly isolated bladders and isometric tension was recorded in detruosr small strips from DI and normal bladders. The contractions were recorded during electric stimulation or exposure to various agents. RT-PCR test investigated the sub-type of RyR in DI bladdermusle. Semiquantitative RT-PCR test and western blot analysis determined RyR expression respectively in the level of mRNA & protein in DI and normal bladder muscle preparations.Results: ? The ratio of DI in proximal way is higher than that of in distal way but it failed to have significant difference. The similar results happened in death, infection, and calculus. The prevalence of leakage in proximal way had a significant low ratio than that of in distal way. ? There are significant differences between in two ways DI models and in control on the results of bladder wet weight, volume, and max vescia pressure. ? In DI muscle, spontaneous contractile activity persisted in the presence of a cocktail containing blockers for known neurotransmitter receptors in bladder wall. ? RyR2 sub-type was confirmed in rat DI detrusor tissue by RT-PCR test, accordingly the same sub-type result in control. ? Semiquantitative RT-PCR test and western blot analysis showed that RyR expression of mRNA as well as protein significantly decreased in DI muscle compared to normal preparations. ? With the RyR blocker ryanodine, spontaneous contractile frequency significantly increased in normal bladder strips, while ryanodine failed to affect spontaneous contractions in DI muscle. ? Caffeine applications inhibited spontaneous contractile activity in both the DI and normal preparations. ? After the administration of L-type Ca2+ channel antagonist nimodipine, the myogenic contractile activity was almost abolished except for the remained rhythm under accountableness in DI muscle.Conclusion: ? A relative high ratio of leakage happened in distal way. Both of ways are effective approach to post-obstruction DI model. ?In the presence of the cocktail, spontaneous contractions persisted in DI strips. This persistence of contractile activity in the presence of neurotransmitter antagonists suggests that the spontaneous contractions in DI preparations have a myogenic basis. ? Our results provide the first characterization of a loss of regulation of spontaneous contractile activity by RyR in DI muscle associated with a significant decrease in RyR expression. RyR in normal detrusor act as negative feedback regulators of spontaneous contractile activity, presumably by releasing Ca + that activates Kca channels to decrease contractility. ? This cross talk mechanism may be weakened in DI muscle, which results in spontaneous contractile overactivity.