Involvement of cyclic nucleotide-gated channels in spontaneous activity generated in isolated interstitial cells of Cajal from the rabbit urethra.

Cyclic nucleotide-gated (CNG) channels are non-selective cation channels that mediate influx of extracellular Na+ and Ca2+ in various cell types. L-cis-Diltiazem, a CNG channel blocker, inhibits contraction of urethral smooth muscle (USM), however the mechanisms underlying this effect are still unclear. We investigated the possibility that CNG channels contribute to spontaneous pacemaker activity in freshly isolated interstitial cells of Cajal (ICC) isolated from the rabbit urethra (RUICC). Using immunocytochemistry, we found intense CNG1-immunoreactivity in vimentin-immunoreactive RUICC, mainly within patches of the cellular body and processes. In contrast, α-actin immunoreactive smooth muscle cells (SMC) did not show significant reactivity to a specific CNGA1 antibody. Freshly isolated RUICC, voltage clamped at -60mV, developed spontaneous transient inward currents (STICs) that were inhibited by L-cis-Diltiazem (50µM). Similarly, L-cis-Diltiazem (50µM) also inhibited Ca2+ waves in isolated RUICC, recorded using a Nipkow spinning disk confocal microscope. L-cis-Diltiazem (50µM) did not affect caffeine (10mM)-induced Ca2+ transients, but significantly reduced phenylephrine-evoked Ca2+ oscillations and inward currents in in RUICC. L-type Ca2+ current amplitude in isolated SMC was reduced by ~18% in the presence of L-cis-Diltiazem (50µM), however D-cis-Diltiazem, a recognised L-type Ca2+ channel blocker, abolished L-type Ca2+ current but did not affect Ca2+ waves or STICs in RUICC. These results indicate that the effects of L-cis-diltiazem on rabbit USM could be mediated by inhibition of CNG1 channels that are present in urethral ICC and therefore CNG channels contribute to spontaneous activity in these cells.

Proliferation of Interstitial Cells in the Cyclophosphamide-Induced Cystitis and the Preventive Effect of Imatinib.

Cyclophosphamide- (CYP-) induced cystitis in the rat is a well-known model of bladder inflammation that leads to an overactive bladder, a process that appears to involve enhanced nitric oxide (NO) production. We investigated the changes in the number and distribution of interstitial cells (ICs) and in the expression of endothelial NO synthase (eNOS) in the bladder and urethra of rats subjected to either intermediate or chronic CYP treatment. Pronounced hyperplasia and hypertrophy of ICs were evident within the lamina propria and in the muscle layer. IC immunolabeling with CD34, PDGFRα, and vimentin was enhanced, as reflected by higher colocalization indexes of the distinct pairs of markers. Moreover, de novo expression of eNOS was evident in vimentin and CD34 positive ICs. Pretreatment with the receptor tyrosine kinase inhibitor Imatinib prevented eNOS expression and ICs proliferation, as well as the increased voiding frequency and urinary tract weight provoked by CYP. As similar results were obtained in the urethra, urethritis may contribute to the uropathology of CYP-induced cystitis.

Altered neuronal and endothelial nitric oxide synthase expression in the bladder and urethra of cyclophosphamide-treated rats.

Increased nitric oxide (NO) production seems to play a key role in cyclophosphamide (CYP)-induced cystitis, although the underlying mechanisms and the relative involvement of the different NO synthase (NOS) isoforms remain to be elucidated. Moreover, the role of the urethra in this process is also unclear. In this study, we have analyzed the changes in the expression and distribution of the inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) isoforms of NOS, and the alterations in nerve-mediated contractility in the bladder and urethra of CYP-treated rats. Accordingly, Wistar rats were treated with 150 mg kg(-1) CYP for 4 (acute treatment) or 48 h (intermediate treatment), or with 70 mg kg(-1) CYP every 3 days for 10 days (chronic treatment), and the changes in protein expression were assessed by immunohistofluorescence and in Western blots, while mRNA expression was assessed by conventional and quantitative PCR. Similarly, nerve-mediated contractility was analyzed in vitro. Unexpectedly, no iNOS expression was detected in CYP-treated animals, while a transient downregulation of nNOS expression and a progressive upregulation of eNOS was observed, although the eNOS accumulated was not in the active phosphorylated form. Qualitative changes in mRNA expression were also observed in the bladder and urethra, although contractility only diminished in the bladder and this change was not dependent on NOS activity. These findings suggest that spatiotemporal alterations in NO production by constitutive NOS may be involved in the pathogenicity of CYP. Further studies will be necessary to understand the contribution of eNOS to the increases in NO associated with bladder inflammation, or that of free radicals.

Presence of the Ca2+-activated chloride channel anoctamin 1 in the urethra and its role in excitatory neurotransmission.

We investigated the cellular distribution of the calcium-activated chloride channel (CaCC), anoctamin 1, in the urethra of mice, rats, and sheep by both immunofluorescence and PCR. We studied its role in urethral contractility by examining the effects of chloride-free medium and of several CaCC inhibitors on noradrenergic and cholinergic excitatory responses, and on nitrergic relaxations in urethral preparations. In all species analyzed, CaCC played a key role in urethral contractions, influencing smooth muscle cells activated by increases in intracellular calcium, probably due to calcium influx but with a minor contribution by IP(3)-mediated calcium release. The participation of CaCC in relaxant responses was negligible. Strong anoctamin 1 immunoreactivity was detected in the smooth muscle cells and urothelia of sheep, rat, and mouse urethra, but not in the interstitial cells of Cajal (ICC) in any of these species. RT-PCR confirmed the expression of anoctamin 1 mRNA in the rat urethra. This anoctamin 1 in urethral smooth muscle probably mediates the activity of chloride in contractile responses in different species, However, the lack of anoctamin 1 in ICCs challenges its proposed role in regulating urethral contractility in a manner similar to that observed in the gut.

Direct coupling through gap junctions is not involved in urethral neurotransmission.

Interstitial cells of Cajal (ICC) are believed to participate in urethral neurotransmission and it was proposed that direct coupling of ICC and smooth muscle cells (SMC) through gap junctions (GJ) is involved, although this still remains unclear. Hence, we investigated the distribution of different connexins (Cx 43, Cx40, and Cx37) in the sheep and rat urethra, as well as their possible role in neurotransmission. Conventional PCR confirmed that three Cxs are expressed in the urethra. Moreover, both Cx43 and Cx37-immunoreactivity (-ir) were present in SMC, ICC, and the urothelium, although Cx37-ir was significantly weaker and Cx40-ir was limited to the endothelium. While these results indicate that GJ intercellular communication could occur between SMC and ICC, neither the contractile (noradrenergic) nor the relaxant (nitrergic) responses of the rat and sheep urethra to electrical field stimulation were significantly modified by two different GJ inhibitors: 18α-glycyrrhetinic acid and a cocktail of Cx mimetic peptides ((Cx43)Gap 26, (Cx37, Cx43)Gap 27, and (Cx40)Gap 27). By contrast, contractions induced by high K(+) were effectively reduced by both blockers, evidence that they effectively inhibit intercellular communication. These results indicate that GJ are not implicated in urethral neurotransmission, although the question of whether ICC modulate neurotransmission through some other mechanism remains to be determined.

Refractoriness of urethral striated muscle contractility to nitric oxide-dependent cyclic GMP production.

The purpose of this study was to investigate the role of cyclic GMP (cGMP) in the effects of nitric oxide (NO) on urethral striated muscle and its involvement in contractile function. The localization of cGMP, neuronal NO synthase (nNOS), vimentin, and neuronal markers was assessed by immunofluorescence in the sheep and rat urethra and the expression of nNOS was determined in Western blots. Nerve-mediated contractile responses to electrical field stimulation (EFS) were recorded in the sheep urethra. The scant nitrergic innervation of the striated muscle layer suggests that autonomic control of its activity is unlikely. The striated fiber itself may be the source of high levels NO produced by sarcolemmal and/or cytosolic mu or alpha variant of nNOS. This endogenous NO may provoke high basal production of soluble guanylate cyclase (GC) dependent cGMP, mainly in non-NO producing muscle fibers, which is not further enhanced by NO donors. cGMP co-localizes with neurofilament and PGP 9.5 at muscle endplates. Modulators of the cGMP pathway did not affect nerve-mediated contractile activity induced by EFS, suggesting that cGMP is not a significant mediator of neuromuscular transmission. In addition, NO donors did increase the accumulation of cGMP in dense networks of vimentin immunoreactive interstitial cells of Cajal (ICC), whose function is not yet known. These data suggest that there is a strong but non-regulated production of cGMP under resting conditions, which does not seem to affect contractile function. Modulation of cholinergic neurotransmission by NO through cGMP-independent mechanisms cannot be discarded.

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

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.

Interstitial cells of Cajal in the urethra are cGMP-mediated targets of nitrergic neurotransmission.

While interstitial cells of Cajal (ICC) in the urethra respond to nitric oxide (NO) donors by increasing cGMP, it remains unclear whether urethral ICC are functionally innervated by nitrergic nerves. We have addressed this issue in the rat and sheep urethra, where cGMP production and relaxation were compared in preparations subjected to electrical field stimulation (EFS; 2 Hz, 4 min) of nitrergic nerves or to exogenous S-nitroso-L-cysteine (SNC; 0.1 mM, 4 min). Upon EFS, cGMP immunoreactivity (cGMP-ir) was observed in both smooth muscle cells (SMC) and in spindle-shaped cells that contained c-kit and vimentin, features of ICC. Similarly, cGMP-ir was preferentially, but inconsistently, found in ICC of the outer muscle layer on exposure to SNC. We found separate functional groups of ICC within the urethra. Thus only ICC present in the muscle layers (ICC-M) but not those in the serosa (ICC-SR) and lamina propia (ICC-LP) seem to be specifically influenced by activation of neuronal NO synthase (nNOS). Thus the increase in cGMP-ir in the ICC-M induced by EFS was prevented by Nomega-nitro-L-arginine and ODQ. Urethral ICC did not express nNOS, although they were closely associated with nitrergic nerves. cGMP-ir was also present in the urothelium (in the rat but not in the sheep) and the vascular endothelium but not in neural structures, such as the nerve trunks and nerve terminals. Together, these results suggest a model of parallel innervation in which both SMC and ICC-M are effectors of nerve-released NO in the urethra.

Atypical relaxation by scorpion venom in the lamb urethral smooth muscle involves both NO-dependent and -independent responses.

The sustained depolarisation induced by alpha-toxins from scorpion venom (20 microg/ml(-1)) was used to test the hypothesis that an endogenous, photo-sensitive, nitrocompound could act as a stable nitrergic transmitter in the sheep (lamb) urethra. Scorpion venom-treatment effectively abolished neurogenic responses to electrical field stimulation, but it did not modify the spontaneous urethral photorelaxation. On the other hand, scorpion venom induced an atypical relaxation in noradrenaline-contracted preparations, which could be reverted, but not prevented, by tetrodotoxin (TTX, 1 microM). However, after TTX-pretreatment, relaxations elicited by scorpion venom were significantly delayed and slowed down, and similar responses were obtained in the presence of ouabain (10 microM), low sodium medium, or after the inhibition of the NO-cGMP pathway. Although the involvement of K(+) and Cl(-) channels can be ruled out since both charybdotoxin (300 nM) and chlorotoxin (50 nM) did not elicit any urethral relaxation nor modified the scorpion venom-induced one. However, a slow Ca(2+) channel seems to be involved. GVIA omega-conotoxin (1 microM), but not MVIIC omega-conotoxin (1 microM), significantly inhibited both EFS- and scorpion venom-induced relaxations and almost abolished the partial relaxation that was resistant to NO synthase inhibition. On the other hand, the presence of L-cis-diltiazem (0.3 mM), a selective inhibitor of cyclic nucleotide gated channels (CNGCs), also delayed and slowed down relaxation induced by scorpion venom, as well as abolish its reversal by TTX. L-cis-diltiazem pre-treatment induced a progressive decay in urethral relaxation brought about by electrical field stimulation only when repetitive, long duration stimulation protocols were used. Taken together, our results do not support the hypothesis of the endogenous, photo-sensitive, urethral nitrocompound as reflecting a stable nitrergic transmitter instead of NO. However, they suggest the involvement of both a NO-cGMP-dependent and TTX-sensitive component and a NO-independent response, mediated by GVIA omega-conotoxin-sensitive Ca(2+) channels, in the neurogenic relaxation of the urethral muscle. In addition, the likely involvement of CNGCs as an additional component of the cGMP signalling mechanism is suggested.

Nitric oxide synthase in the external urethral sphincter of the sheep: immunohistochemical and functional study.

PURPOSE: We studied the distribution of neuronal nitric oxide synthase (nNOS) and the effects of nitric oxide (NO) modulating drugs on contractile function of the external urethral sphincter of lambs. Gender differences were evaluated. MATERIALS AND METHODS: Longitudinal and transverse sections of the external urethral sphincter from 10 female and 10 male lambs were studied using reduced nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry and nNOS immunocytochemistry. Isometric contractile responses to electrical field stimulation were recorded from external urethral sphincter preparations from 47 female and 45 male lambs and the effects of NO modulating drugs were evaluated. RESULTS: We detected nNOS in the sarcolemma of some but not all striated fibers, where nNOS seems to be concentrated at the neuromuscular junction. In addition, nNOS was present in nerve fibers and intramural ganglia. The density of innervation decreased toward the distal part of the external urethral sphincter and was higher in male preparations. No significant functional effects of the NOS inhibitor NG-nitro-L-arginine (10 mM.) or the NO donors diethylamine and spermine NONOate (Sigma Chemical Co., St. Louis, Missouri) (5 mM. each) on external urethral sphincter isometric contractility were found in either gender. CONCLUSIONS: Despite the evidence for nNOS at the sarcolemma and nerve fibers of the external urethral sphincter the physiological relevance of these immunohistochemical findings remains to be determined.