Presence of cyclic nucleotide-gated channels in the rat urethra and their involvement in nerve-mediated nitrergic relaxation

Abstract

We have addressed the distribution of cGMP-gated channels (CNG) in the rat urethra for the first time, as well as their putative role in mediating of the relaxation elicited by electrical field stimulation of nitrergic nerves. Functional studies have shown that specifically blocking CNG with l-cis-diltiazem leads to the rapid inhibition of urethral relaxation induced either by nitric oxide (NO) released by the nerves or by soluble guanylate cyclase activated with YC-1. By contrast, nerve-mediated noradrenergic contractions were only slowly and mildly reduced by l-cis-diltiazem. This effect was mimicked by lower concentrations of the d-diltiazem isomer, probably due to the nonspecific inhibition of voltage-dependent calcium channels. However, d-diltiazem did not affect relaxation responses. The expression of heteromeric retinal-like CNGA1 channels was demonstrated by conventional PCR on mRNA from the rat urethra. These channels were located in a subpopulation of intramuscular interstitial cells of Cajal (ICC) as well as in smooth muscle cells, although they were less abundant in the latter. CNG channels could not be visualized in any nervous structure within the urethral wall, in agreement with the emerging view that a subset of ICC serves as a target for NO. These channels could provide a suitable ionic mechanism to associate the changes in cytosolic calcium with the activation of the nitric NO-cGMP pathway and relaxation although the precise mechanisms involved remain to be elucidated.

Micturition is initiated by an abrupt loss of urethral smooth muscle sphincter tone that is mediated by the release of nitric oxide (NO) from the local nitrergic fiber network (see Ref. 1 for a review). It is generally accepted that this process involves the NO-dependent activation of soluble guanylate cyclase (GC), which provokes a transient increase in intracellular cGMP levels and the subsequent activation of PKG in urethral smooth muscle cells (1, 20). Some recent reports suggest that the interstitial cells of Cajal (ICC) act as a new cellular element in the NO-cGMP pathway. These cells are of mesenchymal origin, and they exist as interconnected networks in all the layers of the urethral wall. Many authors have described the ability of vesical and urethral ICC to accumulate cGMP upon exposure to exogenous NO donors (12, 26, 30). Furthermore, we recently reported that only a muscle subpopulation of urethral ICC undergo a significant increase in cGMP upon functional nitrergic activation (11), suggesting that these cells could be also targets of the NO released by nerves.

Urethral ICC are far from electrically quiescent since they generate spontaneous Ca2+ oscillations, supporting their role as pacemakers. In a tonic organ like the urethra, pacemaker cells have been suggested to support the asynchronous recruitment of muscle units to maintain tone, very much like in skeletal muscle (22). The ionic mechanism thought to underlie this activity is initiated by inositol 1,4,5-triphosphate (IP3)-mediated Ca2+ release from intracellular stores and the subsequent opening of Ca2+-activated Cl− channels in the rabbit urethra (17). In addition, extracellular Ca2+ entry through non-voltage-dependent Ca2+ channels is also needed for the spontaneous Ca2+ oscillations (24), and a Na+/Ca2+ exchanger (NCX) working in a reverse mode has been suggested (4). The physiological relevance of the spontaneous ICC depolarization is reinforced by the fact that they can be modified by endogenous neurotransmitter release, suggesting that ICC act as mediators of neurotransmission as in the gut. Therefore, to define the involvement of ICC in the regulation of urinary motor function and their relationship with autonomic nervous control will require an analysis of the mechanisms of communication between all the cells involved in this pathway.

The hyperpolarization-activated (HCN) and the cyclic nucleotide-gated (CNG) channels are ion channels directly gated by the binding of intracellular cAMP and/or cGMP to a cytoplasmic cyclic nucleotide-binding domain. The functional roles of these ion channels are thought to be complex, and they are poorly understood (see updated reviews in Refs. 5 and 15). In clear contrast to the structurally related subfamily of HCN channels, CNG channels are weakly activated by changes in membrane potential, and their opening and gating are directly defined by the intracellular binding of cyclic nucleotides. These channels mainly carry an inward Na+ and Ca2+ current which provokes membrane depolarization or local changes in cytosolic Ca2+ concentrations. Although initially described in bovine rod photoreceptors (19), their involvement in the control of photoresponse is already found as early as in ciliate protozoa (29). Indeed, it is now generally accepted that they may be found in sensory receptors, epithelia, and blood vessels and even in spermatozoa (see Ref. 5), participating in a plethora of physiological processes from sensory transduction to the control of fluid reabsorption at the alveolar epithelia (16). However, in most of these cases the specific role of CNG channels is unknown. Although CNG channels can conduct currents carried by mono- and divalent cations, there is growing interest in this family as they provide an alternative pathway for Ca2+ entry that is virtually independent of membrane voltage and that couples the activity of Ca2+-regulated proteins to cAMP/cGMP signaling without involving protein kinases.

One of the most widely used specific inhibitors of cGMP-gated CNG channels is l-cis-diltiazem (15), while the d-isomer blocks voltage-gated Ca2+ channels. When analyzing the effect of synaptic vesicle depletion induced by the scorpion venom α-toxins in the sheep urethra, we observed that their depolarizing effect lead to pronounced NO synthase (NOS)- and GC-mediated relaxation that could be inhibited by l-cis-diltiazem (28). Similarly, l-cis-diltiazem was shown to inhibit relaxation elicited by electrical field stimulation (EFS) of intrinsic nitrergic nerves (28). These data strongly suggest that CNG channels are involved in the NO-cGMP signaling pathway active in the urethra.

In the present work, we further examine the possibility that retinal-like CNG channels (CNGA1), a functional subtype selectively gated by cGMP, are present in the rat urethra. We studied the cellular distribution of CNGA1 channels by immunofluorescence as well as the mRNA expression of the different subunits that form the functional channel. We show that CNGA1 channels are mainly present in a subpopulation of urethral ICC, and they are only weakly expressed in smooth muscle cells. In addition, the characterization of the functional effects of l-cis-diltiazem on both relaxant and contractile nerve-mediated responses induced by EFS provides new insights into the relevance of CNG channels in the regulation of urethral motility. We hypothesize that CNG could be a suitable link between the activation of the NO-cGMP pathway and the modulation of the nitrergic control of smooth muscle activity by the ICC.