Mediators and mechanisms of relaxation in rabbit urethral smooth muscle.

1. Electrophysiological and mechanical experiments were performed to investigate whether the nitric oxide (NO)-mediated relaxation of rabbit urethral smooth muscle is associated with a hyperpolarization of the membrane potential. In addition, a possible role for vasoactive intestinal peptide (VIP) and carbon monoxide (CO) as relaxant agents in rabbit urethra was investigated. 2. Immunohistochemical experiments were performed to characterize the NO-synthase (NOS) and VIP innervation. Possible target cells for NO were studied by using antisera against cyclic GMP. The cyclic GMP-immunoreactivity was investigated on tissues pretreated with 1 mM IBMX, 0.1 mM zaprinast and 1 mM sodium nitroprusside. 3. Intracellular recordings of the membrane potential in the circular smooth muscle layer revealed two types of spontaneous depolarizations, slow waves with a duration of 3-4 s and an amplitude of 30-40 mV, and faster (0.5-1 s), more irregular depolarizations with an amplitude of 5-15 mV. The resting membrane potential was 39 +/- 1 mV (n = 12). Application of NO (30 microM), CO (30 microM) or VIP (1 microM) did not change the resting membrane potential. 4. Both NO (1-100 microM) and VIP (1 nM-1 microM) produced concentration-dependent relaxations amounting to 87 +/- 4% and 97 +/- 2% (n = 6), respectively. The relaxant effect of CO (1-30 microM) amounted to 27 +/- 4% (n = 5) at the highest concentration used. 5. Immunohistochemical experiments revealed a rich supply of NOS-immunoreactive nerve fibres in the smooth muscle layers. Numerous spinous cyclic GMP-immunoreactive cells were found interspersed between the smooth muscle bundles, mainly localized in the outer layer. These cells had long processes forming a network surrounding the smooth muscle bundles. VIP-immunoreactivity was sparse in comparison to NOS-immunoreactive nerves. 6. The rich supply of NOS-immunoreactive nerve fibres supports the view that NO is an important NANC-mediator in the rabbit urethra. In contrast to several other tissues, the relaxant effect of NO in the rabbit urethra does not seem to be mediated by hyperpolarization. The network of cyclic GMP-immunoreactive cells may constitute target cells for NO, but their function remains to be established.