A characterization of the spontaneous activity of the mouse bladder and the contribution of nitric oxide to activity

In order to investigate causes of detrusor instability, spontaneous electrical and Ca2+ activity in the mouse bladder were explored. The mouse bladder is a useful model organ for investigating bladder dysfunction as it represents a diseased state of the human bladder. In addition, the ability to manipulate the functioning of various genes in this species allows an in-depth study of the roles of neuronal- and urothelium-derived nitric oxide (NO), whose functions in the modulation of spontaneous activity of detrusor remain to be established. In the present study spontaneous electrical activity of mouse detrusor smooth muscle cells (SMCs) was characterized using conventional intracellular microelectrodes. The activity of SMCs was characterized by occasional nifedipine-sensitive spontaneous action potentials (sAPs) and in most cells, many ATP-mediated spontaneous depolarizations (SDs). The SDs are likely to be spontaneous excitatory junction potentials. Additionally, cells in which sAPs were not observed and hyperactive cell with frequent (>15 min-1 ) sAPs were also observed. Two types of sAPs, termed 'spike-type' and 'pacemaker-type', were identified according to their shape. In SMCs with very frequent sAPs, pacemaker-type sAPs represent a greater proportion of sAPs than in cells in which they were less frequent. Simultaneous microelectrode intracellular recording and Ca2+ imaging demonstrated that spontaneous whole cell Ca2+flashes are correlated with electrical activity. The frequency of sAPs and whole cell Ca2+ flashes was lower in bladder preparations from neuronal nitric oxide synthase gene-disrupted (nNOS'') mice compared to that from their wild type siblings (nNOS+1+). An NO donor, diethylamine NONOate sodium salt, enhanced the frequency of sAPs in nNOS-1 - detrusor even when the cGMP pathway had been blocked. In in vivo urodynamic studies, an nNOS inhibitor, S-methyl-L-thiocitrulline acetate, increased the voiding interval. These data suggest that NO may be continuously released from nerves and may directly facilitate the generation of spontaneous activity in mouse detrusor without employing a cGMP-dependent pathway. In addition, the falling phase of sAPs more prolonged in nNOS·I-than in nNOS+1+detrusor SMCs, indicating that neuronal NO is also crucial for the function of the channels which contribute to the repolarizing phase. The role of the urothelium in the spontaneous activity of SMCs was investigated by comparing preparations in which the urothelium was carefully dissected away, with preparations in which the urothelium remained intact. Detrusor strips without the urothelium showed greater fre~uency of sAPs and whole cell Ca2+flashes compared to the urothelium-intact strips. A NOS inhibitor, N -mono-methylL- arginine, augmented the frequency of sAPs in nNOS·1 • detrusor. Furthermore, a c-Kit inhibitor, imatinib mesylate (GleeveC®), decreased the frequency of sAPs only when the urothelium was absent. These results imply that the urothelium may generate an inhibitory agent, likely to be NO, which suppresses the spontaneous activity possibly through suburothelial ICs. 'Immunohistochemistry confirmed the existence of nNOS-containing neurons and nerve fibres in nNOS+/+ bladders, which were absent in nNOS·1 • bladders. eNOS-containing basal urothelial cells were observed in both nNOS+1+ and nNOS-1 - bladders. c-Kit positive ICs were demonstrated in both suburothelial and detrusor layers of the mouse bladder. The present study characterizes the spontaneous electrical activity of the mouse detrusor and demonstrates that the mouse detrusor shares properties of detrusor muscle with other species, but also shows novel characteristics, specific to this species. Immunohistochemical characterization has for the first time demonstrated c-Kit immunopositive ICs in the mouse bladder. The study suggests that neuronal-derived NO facilitates the generation of spontaneous activity via a cGMP-independent pathway, whereas urothelium-derived NO inhibits spontaneous activity of the mouse detrusor probably by suppressing the function of suburothelial ICs. The overall degree of excitation of the bladder will thus be a balance between these two effects of NO. Upsetting the balance may result in bladder dysfunction.EThOS - Electronic Theses Online ServiceGBUnited Kingdo