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Am J Physiol Renal Physiol 295: F971-F983, 2008. First published July 16, 2008; doi:10.1152/ajprenal.90301.2008
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Interstitial cells of Cajal in the urethra are cGMP-mediated targets of nitrergic neurotransmission

Ángeles García-Pascual, María Sancho, Gonzalo Costa, and Domingo Triguero

Department of Physiology, Veterinary Faculty, Complutense University, Madrid, Spain

Submitted 9 May 2008 ; accepted in final form 11 July 2008


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 N{omega}-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.

ICC; nitric oxide; cGMP immunofluorescence; urinary tract

INTERSTITIAL CELLS OF CAJAL (ICC) were first described in the intestine by Santiago Ramón y Cajal nearly a century ago (6). They are branched, noncontractile but excitable cells that are believed to act as pacemakers of the slow waves and to be important mediators of enteric neurotransmission, or to even act as mechanoreceptors (see Ref. 27).

Similarly, smooth muscles in the urinary tract have spontaneous electrical and mechanical activities that are controlled by a rich supply of autonomic nerves. Therefore, it is not surprising that cells that are morphologically and functionally similar to gastrointestinal ICC have been described throughout the urinary tract, from the ureter to the bladder and in the urethra of different species, including humans (for a review, see Ref. 4). In urinary tissues, it is now accepted that these cells, previously named interstitial cells (IC), ICC, ICC-like cells, or myofibroblasts, should all be considered as ICC in accordance with the nomenclature used in the gut (Fifth International Symposium on Interstitial Cells of Cajal, Ireland, 2007). However, studies concerning their role in the urinary tract are scarce and somewhat controversial. In enzymatic dispersal from the rabbit urethra, a small population of noncontractile, vimentin-immunoreactive (vimentin-ir) cells that spontaneously displayed Ca2+ oscillations coupled to firing of transient inward currents was described, while smooth muscle cells (SMC) were electrically quiescent (28). These features suggested an obvious pacemaking role for the ICC in this structure. Careful studies of the ionic basis of this spontaneous excitation revealed that this activity comes from the spontaneous release of Ca2+ from intracellular stores and the subsequent activation of Ca2+-activated Cl– channels (19). However, in the guinea pig (15) and sheep (29) urethras not only ICC but also SMC were able to develop spontaneous depolarization mediated by Ca2+-activated Cl– channels. Recently, on the basis of the heterogeneous effect of cyclopiazonic acid, a known inhibitor of Ca2+ uptake into intracellular stores, it was suggested that both ICC and SMC may simultaneously be involved in urethral pacemaking in the intact rabbit urethra (16). Indeed, spontaneous Ca2+ transients in rabbit urethral ICC "in situ" were not temporally correlated with neighboring SMC, which generated Ca2+ transients by themselves (17).

The urethral smooth muscle sphincter has a high spontaneous tone during continence, which is augmented by neurally released norepinephrine (NE) and abruptly lost during micturition by neurally released nitric oxide (NO; reviewed in Ref. 2). It has been shown that NE increased the frequency of the spontaneous depolarizations in ICC isolated from the rabbit urethra through the activation of Ca2+-activated Cl– channels (30). By contrast, this electrical activity was inhibited by the NO donor DEA-NO, as well as activators of the cGMP pathway, probably by inhibiting the inositol 1,4,5-trisphosphate-mediated Ca2+ release from intracellular stores (31). Indeed, SIN-1 (another NO donor) reduced the amplitude of ICC Ca2+ transients in the intact rabbit urethra, while phenylephrine increased their frequency and induced a sustained rise in Ca2+ (17). These results suggest that, like the gut, neurotransmitters in the urethra may act through ICC, although this hypothesis has yet to be tested in experiments where the release of the endogenous neurotransmitters from intramural nerves is elicited.

One of the main features of urinary tract ICC from both the bladder and the urethra is their ability to accumulate cGMP upon exposure to NO donors (10, 13, 22, 34, 40, 43). Hence, ICC appear to express the second messengers necessary to transduce NO signals, including guanylate cyclase (GC). However, the involvement of ICC in the relaxation induced by selective stimulation of nitrergic nerves in the urethra has yet to be demonstrated. It is well known that urethral nitrergic relaxation is mediated by GC activation and cGMP accumulations (11, 25). However, urethral tissue responds quite differently to exogenous NO or NO donors and to the endogenous release of the nitrergic transmitter (12).

In the present study, we have used cGMP immunofluorescence to identify the specific cell types in the sheep and rat urethra that respond to nitrergic stimulation by elevating their intracellular cGMP. Tissues were subjected to electrical field stimulation (EFS) of nitrergic nerves or exposed to S-nitroso-L-cysteine (SNC), the NO donor that produces the fastest relaxation and highest levels of cGMP in the sheep urethra (12). Both relaxations and cGMP immunoreactivity (cGMP-ir) were compared directly. Furthermore, immunoreactivity for the protein gene product (PGP 9.5) or the neuronal isoform of NO synthase (nNOS) in nerve structures was combined with cGMP labeling to identify nerves and their relationship with the effector cells. Finally, vimentin/cGMP and c-kit/cGMP double labeling was used to confirm the mesenchymal nature of the cGMP-containing cells. Preliminary data from this study were presented at the Third International Conference on cGMP Generators, Effectors and Therapeutic Implications (Dresden, Germany, 2007).


   EXPERIMENTAL PROCEDURES
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Drugs and Solutions

NE, atropine sulfate, guanethidine monosulfate, D-tubocurarine hydrochloride, IBMX, L-cysteine, N{omega}-nitro-L-arginine (L-NNA), and sodium nitrite were obtained from Sigma-Aldrich Chemie (Steinheim, Germany). Both 1,4-dihydro-5[2-propoxy-phenyl]-7H-1,2,3 triazolo [4,5 d] pyrimidine-7-one (zaprinast) and 1H-(1,2,4)oxadiazolo (4,3) alquinoxalin-1-one (ODQ) were purchased from Alexis (Alexis Biochemicals, Lausen, Switzerland), and SQ22536 was obtained from Calbiochem (Darmstadt, Germany). Most drugs were dissolved in distilled water except IBMX and zaprinast, which were dissolved in DMSO, and ODQ in acetonitrile. Solutions were stored at –20°C, and the drugs were diluted to the working concentrations in 0.9% NaCl. Solutions of SNC were prepared by adding a sodium nitrite (100 mM) solution to the same volume of a solution containing 250 mM HCl, 1 mM EDTA, and 100 mM L-cysteine. The SNC concentration was determined spectrophotometrically (Shimadzu UV-1601 UV-visible spectrophotometer, Shimadzu, Tokyo, Japan), assuming a molar absorption coefficient of {varepsilon}544 = 16.6 M/cm. Working dilutions were prepared in deoxygenated distilled water immediately before use, and they were kept on ice and maintained in the dark.

Tissue Preparation

Lower urinary tracts from female sheep (4–6 mo old) and female Wistar rats (6–8 wk old and 200–300 g) were used in this study. Sheep urinary tracts were collected at the local slaughterhouse shortly after death and maintained at 4°C in Krebs solution (in mM): 119 NaCl, 4.6 KCl, 1.5 CaCl2, 1.2 MgCl2, 15 NaHCO3, 1.2 KH2PO4, 0.01 EDTA, and 11 glucose. From each urinary tract, the urethra was dissected out, and the fat and connective tissue were removed. Rats were killed by cervical dislocation followed by exsanguination, and after the abdomen was opened, the whole lower urinary tract was removed. All procedures were approved by the Complutense University Ethical Committee and were performed in accordance with European guidelines. Transverse strips (~3-mm wide and 5-mm long) or rings (3-mm wide) were obtained from the proximal sheep or rat urethras, respectively, immediately caudal to the bladder neck. Care was taken not to damage the mucosal and serosal layers to maintain the integrity of the tissue.

Nitrergic Stimulation of Urethral Preparations

The preparations in which cGMP immunofluorescence was assessed were previously subjected to functional experiments, facilitating the direct comparison between the relaxant responses to nitrergic stimulation and the accumulation of the cyclic nucleotide. Urethral preparations (strips or rings) were mounted between two stainless steel hooks in 5-ml organ baths containing Krebs solution at 37°C and bubbled with a mixture of 95% O2-5% CO2 (pH 7.4). The isometric tension was recorded with Grass FT03C transducers (Grass Instruments, Quincy; MA) and displayed on a MacIntosh computer with a MacLab analog-to-digital converter v5.5 (AD Instruments, Hastings, East Sussex, UK). Preparations were equilibrated at a resting tension of either 15 (sheep) or 5 mN (rat) for 60 min.

Guanethidine (50 µM) and atropine (1 µM) were present throughout the experiment to prevent the release of NE from nerves or the effects of the released acetylcholine on muscarinic receptors, respectively. D-Tubocurarine 0.1 mM was also present in the rat experiments to avoid the effect of somatic nerve stimulation that innervates the striated muscle of the urethra. Tissues were precontracted with 50 µM NE and relaxed by the addition of SNC (0.1 mM for 4 min) or by selective EFS of nitrergic nerves. EFS was achieved with a Grass S-48 stimulator (Grass Instruments) connected to platinum electrodes placed parallel to the preparation and coupled to a Med-Lab stimulus splitter (Med-Lab Instruments, Loveland, CO). Square-wave pulses of 0.8-ms duration, supramaximal voltage (current strength, 200 mA), train duration of 5 s, and a frequency of 2 Hz were delivered for 4 min, supramaximal parameters to stimulate nitrergic-inhibitory nerves in this preparation (38). Preparations that did not relax properly were excluded from the study. In all experiments, tissues were exposed to the phosphodiesterase (PDE) inhibitors IBMX (1 mM) and zaprinast (0.1 mM), 30 s before stimulation, to prevent the breakdown of cyclic nucleotides, and these inhibitors were present throughout the stimulation period (4 min). This treatment enabled the accumulation of sufficient cGMP to be visualized by immunofluorescence. Control preparations were processed identically except that the tissues were not exposed to SNC or EFS. Furthermore, some preparations were pretreated with the NO synthase inhibitor L-NNA (0.1 mM) or the GC inhibitor ODQ (0.1 mM) for 30 min before the addition of NE, and these inhibitors remained present throughout the experiment (see Fig. 1 for examples of the experimental procedure). All samples were immediately processed for immunofluorescence.


Figure 1
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  		Fig. 1. Representative tracings showing the relaxation responses induced in sheep (A) and rat (B) urethral preparations under different conditions of nitrergic stimulation. Electrical field stimulation (EFS; 2 Hz, 4 min) or S-nitroso-L-cysteine (SNC; 0.1 mM, 4 min) in the presence or the absence of N{omega}-nitro-L-arginine(L-NNA) and/or ODQ (0.1 mM, 30 min pretreatment) compared with control unstimulated preparations is shown. All preparations were precontracted with norepinephrine (NE; 50 µM), and the black arrowheads indicate the point at which phosphodiesterase (PDE) inhibitors (IBMX 1 mM and zaprinast 0.1 mM) were added, which were present throughout the experiment. The final vertical lines indicate the rapid removal of tissues to be processed for immunofluorescence.

 
Immunofluorescence

Urethral strips were fixed partially stretched to 110% of their length by pinning them to a Sylgard base, while the urethral rings were immersion-fixed following the method described by De Vente et al. (8). After fixation for 30 min in ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.0), the tissue was incubated in mixed solutions of paraformaldehyde in 0.1 M PB and increasing concentrations of sucrose (10% sucrose for 90 min followed by 20% sucrose for 120 min), and cryoprotection was terminated by incubating overnight in 30% sucrose in PB at 4°C. Tissues were snap-frozen in liquid nitrogen-cooled isopentane and stored at –80°C for up to 15 days. Cryostat sections transverse to the mucosal surface (10 µm: CM1850 UV, Leica Microsystems, Barcelona, Spain) of the urethras embedded in Tissue-Tek OCT compound were thawed onto poly-L-lysine-coated slides. From each urethra, consecutive sections were collected on 4–5 slides, and thus each contained a similar collection of 10–15 serial sections from the same animal. The slides were air-dried at room temperature for 12–24 h and then processed directly or stored at –80°C for no more than 30 days.

Urethral sections were washed three times for 5 min with PB and to avoid nonspecific antibody binding; they were then incubated for 60 min with 3% normal donkey antiserum (Chemicon International, Temecula, CA) containing 0.3% Triton X-100. The sections were incubated with the primary antibody (or antibodies for dual labeling) diluted in 2% normal donkey serum and 0.3% Triton X-100 for 24 h at 4°C in a humidified chamber. To visualize cGMP in rat urethral sections, we used a sheep anti-formaldehyde-fixed cGMP antibody (1:2,000; a generous gift from Dr. J. De Vente, Maastricht University, Maastricht, The Netherlands), the selectivity and detection limits of which were described previously (8). For the sheep urethra, we used a rabbit anti-cGMP polyclonal antiserum (1:3,000; Chemicon International). Two anti-c-kit rabbit polyclonal antisera were used: c-kit Ab-1 (1:25; Oncogene Research Products, San Diego, CA) and c-kit H-300 (1:50; sc-5535, Santa Cruz Biotechnology, Santa Cruz, CA). The other primary antibodies used were a mouse monoclonal anti-vimentin antibody (clone V9; 1:100; Chemicon International), a mouse monoclonal anti-NOS brain (clone NOS-B1; 1:3,000; Sigma-Aldrich Chemie), a rabbit nNOS polyclonal antiserum (1:300; Cayman Chemical, Ann Arbor, MI), and a mouse monoclonal antibody against the neuronal marker protein gene product 9.5 (PGP9.5; 1:50; Abcam, Cambridge, UK). Double labeling for cGMP and vimentin, nNOS, or PGP9.5 and c-kit with vimentin involved the use of the polyclonal antisera for cGMP and c-kit and the monoclonal antibodies for vimentin, nNOS, and PGP 9.5, although the polyclonal nNOS antiserum was used for the vimentin-nNOS double labeling.

The following secondary antibodies were used, appropriately matched to the species in which the primary antibody was raised: donkey anti-rabbit FITC, donkey anti-sheep FITC, and donkey anti-mouse rhodamine (all 1:100; Chemicon International). Sections were incubated with the secondary antibodies for 2 h in the dark in a humidified chamber at room temperature. After a washing (3 times, 10 min each) with PB, the nuclei were counterstained with 4',6-diamino-2-phenylindole dihydrochloride (DAPI; 10.9 mM for 20–30 min; Sigma-Aldrich Chemie), and the sections were washed again and mounted with Prolong Gold antifade reagent (Molecular Probes, Eugene, OR). In all cases, a number of controls were performed in which the specificity of the immunoreactions was established by omitting the primary antibody or antibodies.

The labeled sections were examined under an Axioplan 2 fluorescence microscope (Carl Zeiss Microimaging, Göttingen, Germany) equipped with the appropriate filter sets. They were photographed with a Spot-2 digital camera (Diagnostic Instruments, Sterling Heights, MI), and the images were stored digitally as 12-bit images using MetaMorph 6.1 software (MDS Analytical Technologies, Toronto, ON, Canada). Some sections were examined by confocal laser-scanning microscopy using a spectral confocal microscope (TCS-SP2, Leica Microsystems, Barcelona, Spain). The confocal micrographs shown are digital composites of Z-series scans of 10–12 optical sections through a depth of 10–12 µm obtained with Leica Confocal Software (LCS, Leica Microsystems). Digital images were subsequently transferred to Adobe Photoshop 8.0 (San Jose, CA) for compilation of the figures. Images of the whole-rat urethral ring were constructed using the "photomerge" tool of Adobe Photoshop software from 20–25 different microphotographs (magnification x20) taken from the different regions of the ring.

Data Analysis

Urethral relaxations were expressed as the percentage of the tension elicited by NE immediately before each stimulation. In rat preparations, the time to reach 50% of the total relaxation (half-relaxation time) was also measured since the pronounced relaxant effect of PDE inhibitors unmasks the effect of stimulation with either SNC or EFS. Each experiment was carried out using a different animal, and at least five animals were included in each experimental group.

The intensity of cGMP-ir was quantified using MetaMorph 6.1 image analysis software (MDS Analytical Technologies). For each experimental group, this measurement was performed in no more than three randomly selected tissue sections per animal from at least three animals and from at least three different staining procedures (performed on different days). Images at x20 magnification were obtained at a constant time exposure to permit direct comparisons between them. In each image, an area comprising the outer muscular [250,713 (35,829) µm2, n = 42], inner muscular [198,335 (101,298) µm2, n = 43], or mucosal [300,896 (185,011) µm2, n = 35] layer of the sheep urethra, or the complete smooth muscle coat [166,720 (110,358) µm2, n = 85] of the rat urethra, was selected, and a threshold was established to subtract the background immunofluorescence. The proportion of the selected area exceeding the threshold values was quantified. For all images, the threshold was kept constant and it was set to a level that gave a relative threshold area of <5% in the least-intense images (corresponding to the preparations treated with ODQ).

The results are given as means (SD) from n experiments. One-way ANOVA was used for multiple comparisons followed by the unpaired t-test. The level of significance was determined by applying the false discovery rate procedure (7). Data were compared using GraphPad Prism 5 software (GraphPad Software, San Diego, CA).


   RESULTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
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Functional Responses

In all sheep and rat urethra preparations used here, a direct comparison was made between the relaxation responses and cGMP-ir in response to different stimuli (EFS, addition of SNC, and pretreatment with the NOS inhibitor L-NNA or the GC inhibitor ODQ). For sufficient cGMP to accumulate in tissues for immunohistochemical assays, preparations must be pretreated with PDE inhibitors (32). However, a 30-min incubation with PDE inhibitors completely precluded the increase in contractile tension produced by NE (incubation time used in previous studies where isometric tension was not monitored: 13, 20, 34, 40, 43) and hence, the study of relaxant responses. Accordingly, the exposure to PDE inhibitors was limited to 30 s before EFS or SNC, and these compounds remained present throughout the experiment (a total time in the presence of PDE inhibitors of 4 min). Exposure to PDE inhibitors for 30 s relaxed NE-precontracted preparations by 6.9 (5.0)% (n = 20) in the sheep and 24.4 (12.7)% (n = 12) in the rat preparations. This relaxation reached 39.1 (15.0)% (n = 7) in the sheep and 125.1 (30.2)% (n = 7) in the rat, after a 4-min exposure (control preparations exposed to PDE inhibitors alone) (Figs. 1 and 2; Table 1). This prominent relaxant effect of PDE inhibitors was nearly halved upon exposure to ODQ (0.1 mM), although additional inhibition of NOS by L-NNA (0.1 mM) produced no further reduction (Table 1; Figs. 1 and 2). Furthermore, incubation with the specific adenylate cyclase inhibitor SQ22536 (0.2 mM) significantly reduced PDE inhibitor-induced relaxation in the sheep, but not in the rat preparations, although this inhibition was not further enhanced by ODQ (Table 1).


Figure 2
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  		Fig. 2. Relaxant responses induced by EFS and SNC in the presence or absence of L-NNA and/or ODQ, and compared with control unstimulated preparations from the sheep and rat urethra. The experimental protocol is as shown in Fig. 1, and the results are means (SD) from 4–13 different animals expressed as the percentage of the contractile tension induced by NE (Relaxation %) or the time to reach 50% of total relaxation (half-relaxation time). {Phi}P < 0.05, {Phi}{Phi}P < 0.01, {Phi}{Phi}{Phi}P < 0.001 differences compared with controls. **P < 0.01 and ***P < 0.001 differences with preparations stimulated with EFS or SNC but not treated with L-NNA or ODQ (ANOVA followed by t-test applying the false discovery rate procedure).

 

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  		Table 1. Effects of inhibiting guanylate cyclase with ODQ, adenylate cyclase with SQ22536, and nitric oxide synthase with L-NNNA on the relaxant response induced by PDE inhibitors in urethras from sheep and rats

 
Upon specific nitrergic stimulation (EFS in the presence of guanethidine and atropine, and with the addition of D-tubocurarine in the rat), a very rapid and prominent relaxant response was induced that peaked after 30 s at 63.7 (27.5)%, n = 13 in the sheep and 113.4 (33.1)%, n = 12 in the rat. This response was followed by a partial reversion and a sustained relaxation (Fig. 1), which was significantly higher than that produced by PDE inhibitors in control preparations in the sheep (P = 0.021), but not in the rat (P = 0.613; Fig. 2). However, a comparison of the half-relaxation times unmasked a significant (P < 0.0001) enhancement in the speed of relaxation upon EFS in the rat tissue (Fig. 2).

The addition of SNC (0.1 mM for 4 min) induced slightly slower relaxations that were as pronounced as those induced by EFS in both species (Fig. 1). As with EFS, the magnitude of relaxation was significantly higher (P = 0.005) than that induced by PDE inhibitors in the sheep tissue. Both relaxations were of a similar magnitude in the rat (P = 0.358), and only by measuring the half-relaxation time was a more rapid response to SNC observed (P = 0.0027; Fig. 2). Pretreatment with either L-NNA (0.1 mM) or ODQ (0.1 mM) induced pronounced inhibition of the relaxant responses to EFS and, similarly, ODQ reduced the response to SNC (Figs. 1 and 2).

cGMP Immunoreactivity in Sheep and Rat Urethras Under Different Experimental Conditions of Nitrergic Stimulation

In control preparations (from a total of 7 sheep and 4 rat preparations), which were immediately fixed after contraction with NE (50 µM) and incubated with PDE inhibitors for 4 min but not exposed to nitrergic stimulation, basal cGMP-ir was observed in some cells with a characteristic ICC morphology. These cells were located in the serosa (ICC-SR), especially in the rat (Fig. 3A) and in the lamina propia (ICC-LP), particularly in the sheep (see Fig. 5B). Few cGMP-ir ICC were observed in the muscle layers (ICC-M), although they were more abundant in the outer muscle layer of the sheep (Figs. 3A and 4A). cGMP-ir was also evident in some urothelial cells, especially in the most superficial cell layer in the rat (Fig. 3A), but not in the sheep urethra (Figs. 4, E and F), as well as in the vascular endothelium in both species (not shown).


Figure 3
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  		Fig. 3. Comparison of cGMP immunoreactivity (cGMP-ir) in ring sections of the rat urethra. A: under control conditions, there is a weak immunoreactivity in the epithelium and in some cells of the outer muscle layer. B: a mild reduction in basal cGMP-ir occurs in unstimulated preparations treated with L-NNA and ODQ. C: cGMP-ir is absent in a negative control (without primary antibody). D: prominent increase in cGMP-ir in a preparation subjected to EFS, showing an intense reaction in the smooth muscle cells (SMC), interstitial cells of Cajal (ICC), intramural vessels, and urothelium. E: ring stimulated with SNC displaying dense groups of highly cGMP-ir ICC in the outer circular muscle layer. F–H: pronounced inhibition of cGMP-ir in preparations stimulated by EFS in the presence of ODQ (F) or L-NNA (G) and in a preparation stimulated by SNC in the presence of ODQ (H). The experimental protocol is as shown in Fig. 1, and the images of the whole ring were constructed from 20–25 different microphotographs (x20) taken from the different regions of the ring. u, Urothelium; lp, lamina propia; lm, longitudinal muscle; cm, circular muscle; sr, serosa. Bars = 100 µm.

 

Figure 5
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  		Fig. 5. Quantification of cGMP-ir in the sheep and rat urethra under different conditions of nitrergic stimulation. Preparations were stimulated by EFS (2 Hz, 4 min) or SNC (0.1 mM, 4 min) in the presence or the absence of L-NNA (0.1 mM) and/or ODQ (0.1 mM) and compared with unstimulated tissues. All preparations were precontracted with NE (50 µ) and pretreated with PDE inhibitors (See Fig. 1 for details of the experimental procedure), and they were all fixed immediately and processed for cGMP immunofluorescence. Hand-drawn fields of urethral sections were selected, and the percent area above the intensity threshold was measured (Area %; see text for details). In the sheep urethra, measurements were made independently in the inner circular muscle layer and outer longitudinal muscle layer (A) and the lamina propia (B), whereas the whole muscle coat was selected in the rat sections (A). Values are means ± SD of 5–13 different fields from at least 3 different animals. {Phi}P < 0.05, {Phi}{Phi}P < 0.01 differences with respect to the controls. **P < 0.01 and ***P < 0.001 differences with respect to preparations stimulated with EFS or SNC but not treated with L-NNA or ODQ (ANOVA followed by t-test applying the false discovery rate procedure).

 

Figure 4
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  		Fig. 4. Comparison of cGMP-ir in the sheep urethra. A: faint reaction in muscle layers under control conditions. B and C: prominent cGMP-ir in ICC scattered in the outer muscle layer of SNC- stimulated preparations. D: pronounced inhibition of the cGMP-ir induced by SNC due to the presence of ODQ. E and F: mucosal layer showing the absence of urothelial cGMP-ir and the presence of strong cGMP-ir in the ICC of the lamina propia, which did not change after stimulation with SNC (E) and was not inhibited by the presence of ODQ in a EFS-stimulated preparation (F). G and H: intense cGMP-ir in muscle layers of preparations stimulated by EFS where some ICC (ICC-M) can be clearly visualized. I: higher magnification of H showing 2 bipolar ICC-M with long interconnected prolongations running parallel to the SMC of the inner circular muscle layer. J: reduction in cGMP-ir in the inner circular muscle layer in a preparation stimulated by EFS in the presence of L-NNA. The experimental protocol is as shown in Fig. 1. olm, Outer longitudinal muscle; icm, inner circular muscle; u, urothelium; lp, lamina propia. Bars = 100 µm except in I (bar = 20 µm).

 
There seemed to be less cGMP-ir in preparations pretreated with ODQ and L-NNA (both at 0.1 mM; 5 from sheep and 3 from rats) than in control preparations (Fig. 3B), although the difference in the % area above threshold was not statistically significant in any of the urethral layers examined from sheep (outer muscle layer, P = 0.333 and inner muscle layer, P = 0.324), or the rat muscle wall (P = 0.133; Fig. 5A). Moreover, the fluorescence intensity did not seem to change in either the epithelium (Fig. 3B) or in the serosa and lamina propia (Fig. 5B).

Selective stimulation of nitrergic nerves by EFS (2 Hz, 4 min) induced a pronounced increase in cGMP-ir in the urethral muscle wall in both the rat (10 urethras; Figs. 3D and 5A) and the sheep (13 urethras; Figs. 4, G–I, and 5A). We observed cGMP-ir in cells with a characteristic ICC morphology as well as in SMC. This muscle staining makes the differentiation of cGMP-ir ICC-M more difficult as ICC-M were scattered in between the SMC or surrounding the muscle bundles. Other ICC-M were observed within the septa that separated muscle bundles. For instance, two strong cGMP-ir spindle-shaped intramuscular ICC-M with long interconnected prolongations running parallel to the inner circular SMC in the sheep urethra are shown in Fig. 4, H and I. The presence of cGMP-ir in ICC-LP in the sheep urethra was not affected by EFS (Fig. 5B), nor were ICC-SR affected in the rat urethra (Fig. 3D). After EFS, cGMP-ir was also conspicuous in the rat epithelium and vascular endothelium (Fig. 3D).

Exposure to SNC (0.1 mM for 4 min; 8 sheep and 6 rat preparations) induced very conspicuous cGMP-ir in ICC found in compact groups throughout the serosa and outer smooth muscle layers of the rat (Fig. 3E) and sheep (Fig. 4, B and C) urethra. Paradoxically, cGMP-ir in the SMC seemed to be weaker than in EFS-stimulated preparations, making the ICC stand out against a less intense background. Furthermore, the cGMP-ir ICC were distributed heterogeneously, with dense groups of cells appearing at certain sites in the urethral wall, while other areas were devoid of them (Fig. 3E). Furthermore, despite showing similar relaxation responses, significant cGMP-ir ICC populations were not identified in some preparations exposed to SNC (2 of 6 in the rat and 3 of 8 in the sheep). The less intense staining of SMC together with the variability in ICC labeling may explain the lack of significant differences in the % area above the threshold when SNC-stimulated preparations of sheep urethra were compared with control unstimulated ones (P = 0.066 in the inner muscle layer and P = 0.093 in the outer muscle layer; Fig. 5A). Intense cGMP-ir was also detected in the ICC-LP of the sheep urethra (Figs. 4E and 5B), the urothelium of the rat urethra (Fig. 3E), and the vascular endothelium in both species (Figs. 3E and 4C).

Inhibiting NOS with L-NNA (0.1 mM) impaired the increase in cGMP-ir induced by EFS (Figs. 3G, 4J, and 5A), showing that cGMP accumulation was directly related to NOS activation in nitrergic nerves (4 preparations from sheep and rats). Selective inhibition of soluble GC with ODQ (0.1 mM) also produced pronounced inhibition of cGMP-ir in preparations stimulated by EFS (4 sheep and 6 rats, Figs. 3F and 5A) or SNC (5 sheep and 6 rats, Figs. 3H, 4D, and 5A). As stated above, the high variability in ICC labeling in SNC-stimulated preparations could underlie the failure to detect significant differences in the % area above the threshold for SNC and SNC plus ODQ treatment in the sheep urethra (P = 0.852 in the inner and P = 0.088 in the outer muscle layers, Fig. 5A). The epithelium (Fig. 3, F–H) and vascular endothelium (not shown) of the rat urethra seemed to be less intensely stained following exposure to L-NNA, and especially ODQ than the EFS- or SNC-stimulated tissue. Surprisingly, most of the cGMP-ir ICC-LP in the sheep urethra (Figs. 4F and 5B) as well as the ICC-SR in the rat urethra (not shown) were not affected by exposure to either L-NNA or ODQ.

Pyriform or spindle cell bodies containing a big ovoid nucleus surrounded by scant cytoplasm was the most common cGMP-ir ICC morphology (Fig. 4I and see Figs. 7J and 8, A, E, F, and G). In these cells, cGMP-ir was mainly observed in the cell body, extending into the cellular processes in some cases (Figs. 4, G–I and see Figs. 7J and 8, A and D–H), while in other cases cGMP-ir was distributed in irregular patches that did not clearly define the cell contour (not shown). cGMP-ir ICC were distributed irregularly throughout the urethral wall, and in the serosa, cGMP-ir ICC-SR formed groups of interconnected cells around the urethral surface (Fig. 3, A, D, and E). In both circular and longitudinal muscle layers, some cGMP-ir ICC-M had long cytoplasmic extensions that typically ran parallel to muscle fibers within the smooth muscle bundles (Figs. 4, G–I and see Fig. 8A). There were also cGMP-ir ICC that seemed to run between muscular bundles with long processes that formed interconnected "networks" (Figs. 4, B and C and see Fig. 8H). The population of cGMP-ir ICC-LP in the sheep urethra consisted of bipolar cells parallel to the urethral surface, particularly evident beneath the epithelium, and in which cGMP-ir was condensed in the soma (Figs. 4, E and F). Some cGMP-ir ICC in the lamina propia of the rat were orientated perpendicular to the epithelial surface (not shown).


Figure 7
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  		Fig. 7. Colocalization of vimentin-ir (red) with c-kit-ir (green) and cGMP-ir (green) in ICC from the sheep and rat urethra. Examples of c-kit-ir (A and D) and vimentin-ir (B and E) are shown along with their respective merged images (C and F) in the outer longitudinal muscle coat (A–C) and lamina propia (D–F) of the sheep urethra. Examples of cGMP-ir (G and J) and vimentin-ir (H and K) are shown, along with their respective merged images (I and L), in the muscle layer of a rat urethra subjected to EFS (G–I) or to SNC (J–L). u: urothelium; lp: lamina propia. Bars = 100 µm (A–I) and = 20 µm (J–L).

 

Figure 8
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  		Fig. 8. There is no colocalization but rather a close relationship between cGMP-ir ICC and neuronal nitric oxide synthase (nNOS)-ir or PGP9.5-ir nerve structures. A–C: confocal images showing z-stacks of a 12-µm section of the circular muscle layer from a sheep urethra stimulated by SNC. cGMP-ir (green) in ICC together with nuclear counterstaining with 4',6-diamino-2-phenylindole dihydrochloride (DAPI; blue, A) and nNOS-ir (red) in intramural nerves (B) are shown, along with the merged image (C). D–F: higher magnification of double cGMP-ir(green)/nNOS-ir (red) and nuclear counterstaining with DAPI (blue), showing the close relationship between cGMP-ir ICC and nNOS containing nerve terminals in the rat lamina propia treated with ODQ (D), in the muscular (E) and serosal (G) layers of rat urethra upon exposure to SNC, and in the muscular layer of sheep urethra following EFS (F). Double labeling for PGP9.5-ir (red)/cGMP-ir (green) in the muscular layers of sheep urethra exposed to SNC (H) shows no colocalization. Similarly, vimentin-ir (red) never colocalized with nNOS-ir (green) in double-labeled sections (I and J) of the muscular layer of the sheep urethra, although some vimentin-ir ICC were observed in close apposition to nitrergic nerves (J). Arrows indicate the points of close contact between ICC and nerve structures. cm, Circular muscle. Bars = 20 µm except in H (100 µm).

 
Vimentin, cGMP/Vimentin, and c-kit/Vimentin Immunolabeling: Are ICC-Like Cells in the Urethra Real ICC?

In paraformaldehyde-fixed preparations, urethral ICC are at best only weakly stained by antibodies against c-kit, the tyrosine kinase receptor that specifically marks ICC in the gastrointestinal tract, although they are stained for vimentin (26, 28). Since we must use prefixed tissues (the cGMP antibodies were developed against cGMP in fixed tissues), we assessed whether vimentin is a suitable marker for ICC in the urethra and whether vimentin-ir cells are true ICC.

A total of 23 sheep and 14 rat urethras were immunostained for vimentin, and in both species vimentin-ir prominently stained cell processes (Fig. 6). Vimentin-ir cells were distributed throughout the urethral wall, and their morphology and distribution were similar but denser than for cGMP-ir cells. Thus spindle-shaped cells with one or more processes were found in the serosa (ICC-SR), muscular (ICC-M), and lamina propia (ICC-LP), forming interconnected networks (Fig. 6, A–C and F). In some cases, multiple interconnected cells formed "tracks" running across the entire urethral surface (Fig. 6A). In the muscle layers, both interfascicular and intramuscular vimentin-ir cells were observed (Fig. 6, C–E). Finally, in the sheep lamina propia bipolar cells accumulated densely parallel to the urothelial surface (Fig. 6F). In the rat, some cells lay perpendicular to the urothelium (Fig. 6B), and a dense ring of labeled cells was located between the inner longitudinal muscle layer and the lamina propia (Fig. 6A). Smooth muscle cells or nerve structures were not stained for vimentin.


Figure 6
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  		Fig. 6. Vimentin-ir in sheep and rat urethra. Low-power magnification of a complete ring (A), or a higher magnification of a section (B) of 2 different rat preparations, shows dense networks of vimentin-ir ICC in the lamina propia and muscle layers of the rat urethra. Note the presence of long interconnected processes surrounding the whole ring, which were especially dense at the junction of the lamina propia with the muscle layer, and at the outer circular muscle coat (A). In B, note the presence of vimentin-ir ICC distributed perpendicularly to the urothelial surface. C–F: vimentin-ir in sheep urethral sections. C: dense vimentin-ir ICC-M in between or inside smooth muscle bundles in both muscle layers of the urethral wall. D and E: high-magnification images showing parallel distribution of ICC-M along the smooth muscle fibers (D) and interconnected interfascicular ICC-M in between smooth muscle bundles (E). F: dense vimentin-ir in bipolar ICC in the lamina propia running parallel to the urothelium. u, Urothelium; lp, lamina propia; lm, longitudinal muscle; cm, circular muscle; olm, outer longitudinal muscle; icm, inner circular muscle. Bars = 100 µm (A–C and F) and = 20 µm (D and E).

 
The c-kit-ir was determined in four sheep and six rat urethras with two different antibodies (c-kit Ab-1 from Oncogene and c-kit H-300 from Santa Cruz). While neither antibody produced considerable c-kit-ir in rat preparations (not shown), the c-kit H-300 antibody produced weak c-kit-ir in the sheep urethra ICC. The cells recognized by this antibody had a morphology and distribution similar to those described for vimentin-ir and cGMP-ir, although there was a mild nonspecific reaction in SMC and epithelial cells (Fig. 7, A and D).

Double-labeling confirmed that both vimentin and c-kit colocalized in the sheep tissue, as did cGMP and vimentin in EFS or SNC stimulated tissues (Fig. 7). However, while all ICC from the sheep urethra that were labeled for c-kit were also vimentin-ir (Fig. 7, A–F), only a fraction of the vimentin-ir ICC were cGMP-ir in urethral preparations stimulated by SNC or EFS from both species (Fig. 7, G–L). It is noteworthy that vimentin-ir was concentrated at the periphery of the cell and particularly in the cell processes, which were much more clearly labeled by vimentin than by cGMP antibodies (Figs. 6 and 7).

Double Labeling for cGMP/nNOS and cGMP/PGP9.5: Relationship of ICC to Neuronal Structures

A total of 12 sheep preparations (6 stimulated by EFS and 6 by SNC) and 11 rat preparations (6 stimulated by EFS and 5 by SNC) were used to study the relationship between the ICC and neurons. Both combinations of double labeling showed that cGMP never colocalized with nNOS or PGP 9.5, although they were closely related (Figs. 8, 9, and 10). Nerve varicosities with PGP 9.5-ir or nNOS-ir were frequently found in close contact with cGMP-ir ICC throughout the urethral wall, in the muscle layer (Fig. 8, A–C, E, F, and H), lamina propia (Figs. 8D and 9, A–C), and serosa (Fig. 8G). Perivascular nNOS-ir nerves were found in intramural vessels with cGMP-ir in the endothelium (Fig. 9, D–F). By contrast, there was no nNOS-ir in ICC in sheep or rat urethras when they were double labeled for cGMP/nNOS (Fig. 8, A–G) or vimentin/nNOS (Fig. 8, I and J), indicating that these cells did not contain the enzymatic machinery for NO synthesis. Finally, no cGMP-ir was detected in nNOS-ir or PGP 9.5-ir nerve terminals in the muscle layer (Fig. 8, A–C, E, F, and H) or the lamina propia (Fig. 8D). Furthermore, when either nNOS-ir or PGP 9.5-ir nerve trunks were observed, cGMP-ir was present in adjacent and long nonneural cells that could be glia (Fig. 10).


Figure 9
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  		Fig. 9. A–C: double cGMP-ir (green)/nNOS-ir (red) in the rat urethra showing the presence of cGMP-ir in urothelial cells and the relationship between ICC and nitrergic nerves in the lamina propia. D–F: cGMP-ir (green) in the endothelium of an intramural vessel in the muscle layer of a sheep urethra and the perivascular distribution of nNOS-ir (red) nerves. cGMP-ir (A and D) and nNOS-ir (B and E) are shown along with their merged images (C and F). Bars = 20 µm.

 

Figure 10
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  		Fig. 10. Double cGMP-ir (green)/nNOS-ir (red; A–I) or cGMP-ir (green)/PGP9.5-ir (red; J–L) showing no colocalization of either pair of markers in intramural nerve trunks in the sheep (A–F) and rat (G–L) urethra. cGMP-ir (A, D, G, and J) and either nNOS-ir (B, E, and H) or PGP9.5-ir (K) are shown along with their merged images (C, F, I, and L). In A, I, and L, the nuclei are counterstained with DAPI (blue). Bars = 20 µm.

 

   DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study presents the first evidence of cGMP production in both SMC and ICC in response to selective stimulation of urethral nitrergic nerves, supporting the functional innervation of ICC in the urethra. The specificity of this reaction was demonstrated, as NOS and GC inhibition impaired both the relaxation response and cGMP accumulation. In addition, the close association between EFS-stimulated ICC and nitrergic intramural nerve terminals provides further morphological evidence of this relationship.

Only one previous study has examined the accumulation of cGMP-ir in ICC by EFS of intrinsic nitrergic nerves, carried out in the canine proximal colon (32). In this tissue, cGMP-ir was present in ICC located at the internal surface of the circular muscle layer but not in the ICC-M. By contrast, strong cGMP-ir was observed in networks of urethral ICC-M upon exposure to NO donors (SNP or DEA-NO) in rabbits, pigs, guinea pigs, and humans (34, 40, 43). Here, SNC induced a similar pattern of strong cGMP-ir in groups of interconnected ICC in the outer muscle layers of the urethra in both sheep and rats. Moreover, we found weaker cGMP-ir in SMC similar to that described in the rabbit (40) and pig (43) when challenged with SNP or DEA-NO, respectively. These differences may reflect the higher capacity of ICC to accumulate cGMP in response to the relatively high concentrations of NO released by the NO donors in the presence of PDE inhibitors. It should be noted that the cGMP levels found in sheep urethral preparations exposed to SNC or NO gas were two orders of magnitude higher than those found upon EFS for the same level of relaxation (11, 12). Thus the functional significance of this cGMP accumulation remains unclear, and it does not seem to be related to relaxation. Where nitrergic innervation is scarce in the bladder and NO does not induce muscle relaxation (37), the accumulation of cGMP-ir induced by NO donors was localized exclusively to networks of ICC similar to those found here (13, 22). It is also noteworthy that in the gastric fundus of Sl/Sld or W/Wv mutant mice that specifically lack ICC-M the hyperpolarization response to the nitrergic transmitter or to exogenously added SNP was greatly diminished, while relaxation induced by this NO donor remained unaffected (3, 5). Together, these results strongly suggest that NO donors and the nitrergic neurotransmitter have different mechanisms of action, and some caution should be exerted when these compounds are used as exogenous nitrergic stimulants.

By contrast to NO donors, a similar increase in cGMP-ir was observed in either the ICC or SMC upon EFS in both sheep and rat urethras. In the gastrointestinal tract, it was suggested that the ICC may be interposed between nerves and SMC, acting as mediators in a serial process of neurotransmission. Indeed, while the gastric fundus of Sl/Sld or W/Wv mutant mice did not develop inhibitory or excitatory junction potentials in response to enteric nerves stimulation, the responses to exogenous acetylcholine were maintained (3, 5), suggesting that the loss of ICC led to the loss of neurotransmission. Alternatively, parallel ICC and SMC innervation has been proposed in some regions of the gut. In the colon of W/Wv rats, both rapid ATP-derived and the slow NO-mediated inhibitory neurotransmission is preserved, suggesting that NO released by nerves may directly affect SMC (1). Similarly, nitrergic relaxation of both the internal anal sphincter (36) and the lower esophageal sphincter in vivo (33) remains in W/Wv mice. This model of parallel innervation could explain our results, whereby ICC and SMC make sufficiently close contact with nerve terminals to allow both cells to respond to the transmitter released. These results agree with the recent proposal that both ICC and SMC may be simultaneously involved in urethral pacemaking (16, 17), and they suggest that both cell types may share several functions in the urethra. Whether ICC are regulatory or modulatory and how they affect neurotransmission and/or other SMC functions remain to be determined.

Our results show that only the population of cGMP-ir ICC found within the muscle layers of the urethras from sheep and rats (ICC-M) would be functionally coupled to nitrergic neurotransmission. Thus they accumulate more cGMP following EFS, which can be prevented by both L-NNA and ODQ. In addition, the cGMP-ir induced by SNC was blocked in the presence of ODQ. These features correlate with the pronounced relaxations induced by EFS and SNC in the same urethral preparations, which were also inhibited by L-NNA and/or ODQ. In contrast, the dense networks of ICC-SR and ICC-LP showed permanent and conspicuous cGMP-ir under all experimental conditions. Indeed, the presence of cGMP-ir is not sufficient to consider a cell responsive to NO, or even as a mediator of nitrergic neurotransmission. More recently, particulate GC was shown to be present in ICC of both the lamina propia and the serosa of the guinea pig bladder, but not in the ICC within the muscle coat (9). Moreover, these ICC responded to the natriuretic peptides ANP and BNP by augmenting their cGMP-ir, a phenomenon that was not inhibited by ODQ. Whether activation of particulate GC is behind the nonspecific accumulation of cGMP in ICC-SR and ICC-LP remains unclear. Together, these data suggest separate roles for ICC throughout the urethral wall. Only ICC-M seem to act as NO-dependent modulators of contractile activity, while ICC in the lamina propia and serosa could be involved in other functional aspects like sensorial perception or act as pacemaker cells.

It should be noted that the preparations here were exposed to PDE inhibitors for 4 min. While considerably shorter than that used when the effect of NO donors on cGMP-ir was studied previously in urinary tissues (~30 min) (13, 20, 34, 40, 43), a prominent relaxant effect was observed in basal conditions (without EFS or SNC stimulation), especially high in the rat urethra. In the human urethra, the PDE-5 inhibitors sildenafil, vardenafil, and tadalafil also induced complete relaxation of NE-precontracted strips, and they increased cGMP levels but not those of cAMP (43). Thus strong basal NO or GC activity may induce the basal cGMP-ir detected in some cells, including ICC, urothelium, and vascular endothelium. However, since both basal cGMP-ir and PDE inhibitor-induced relaxation were only partially reduced by ODQ and L-NNA, nonspecific mechanisms unrelated to GC or NOS activation may also be involved. There is significant activation of adenylate cyclase and the ensuing accumulation of cAMP that was inhibited by SQ22536 in the sheep but not the rat urethra. This activation seems likely to be secondary to the increase in cGMP because it was not further inhibited by ODQ treatment, and it could be related to cGMP-dependent modulation of PDE for cAMP. In addition, we cannot rule out the involvement of other ODQ-independent mechanisms such as the activation of particulate GC (9) or the direct effects of PDE inhibitors on K+ channels (24).

ICC in the urethra appear to be very weakly labeled for c-kit (a tyrosine kinase receptor encoded by the c-kit proto-oncogene), which has become the standard means of recognizing ICC in the gastrointestinal tract (18). Similarly, a small population of dispersed noncontractile cells in the rabbit urethra contained vimentin but were not c-kit-ir (28). Indeed, no c-kit-ir was detected in paraformaldehyde-fixed mouse urinary bladder and urethra, while networks of c-kit-ir ICC were described in the ureter (26). Similarly, in fixed gut tissue, no c-kit-ir were detected in ICC from the deep muscular plexus, a type of gastrointestinal ICC clearly involved in neurotransmission (41), bringing into question the use of c-kit as an exclusive marker for ICC. Despite the relatively weak c-kit-ir in our preparations, we considered that cells present in the rat and sheep urethras that showed vimentin- and cGMP-ir can be considered as true ICC for the following reasons: 1) their characteristic spindle-shaped morphology with long processes, a big ovoid nucleus, and scant cytoplasm; 2) vimentin-ir and c-kit-ir colocalized in the sheep urethra; and 3) these cells were around and within muscle bundles, following the long the axis of the bundle and maintaining intimate contacts with intramural nerves, including nitrergic nerves. It cannot be ruled out that other cell types besides ICC were present, such as fibroblasts that are also immunoreactive for vimentin or c-kit. In fact, cells with morphological characteristics very similar to ICC were described as myofibroblasts in the bladder lamina propia (35), although, these ICC-LP play no role in nitrergic neurotransmission. It should also be noted that only a fraction of the total vimentin-ir ICC population in the urethra seems to respond to NO by accumulating cGMP, as found in the guinea pig and human bladder (34). Consequently, ICC might have different functions other than being the target for the NO action and they might develop distinctly into different types of ICC.

It has been suggested that ICC in the gut are themselves able to produce NO and, indeed, both nNOS and endothelial NOS have been identified in the ICC of the colon (39, 44). As in the human intestinal ICC (42), nNOS-ir was never found in the ICC from either the rat or sheep urethra. While the expression of an nNOS isoform not recognized by our nNOS antibodies cannot be ruled out, this possibility is not supported by the fact that NADPH-diaphorase histochemistry did not identify ICC in the sheep urethra (11, 14). Moreover, our results agree with those from the guinea pig urethra (34) and bladder (13) where no nNOS-ir was observed in ICC. In fact no cGMP-ir structures detected here colocalized with nNOS-ir. This concords with the control mechanism proposed in the central nervous system (21) by which GC would be inactivated by the same rise in Ca2+ needed to activate NOS in the NO-synthesizing cell. Indeed, our results do not confirm the presence of cGMP-ir in nerve structures, as described in the nerve trunks of the rabbit urethra (10) and in the varicose nerve terminals of the bladder from guinea pigs, humans (13, 34), or mice (22). It should also be noticed that in both the rat and sheep urethra, neither nNOS nor PGP 9.5 colocalized with cGMP in intramural nerve trunks, although cGMP-ir was present in nucleated structures parallel to nerve fibers that could be glia. Similar, parallel staining of thin nerve terminals was observed (not shown) that may be due to the close apposition of Schwann cells. In visceral smooth muscle, it is known that Schwann cells of axon varicosities respond to endogenous transmitters such as ATP, producing Ca2+ transients (23). Whether Schwann cells in the urethra also respond to the neurally derived NO via cGMP requires further study. Nevertheless, it is clear that there is a close relationship between cGMP-ir ICC and nerve terminals, especially those containing nNOS, which is in accordance with previous studies (10, 13, 22, 34).

Finally, besides ICC and SMC, cGMP-ir was also observed in urothelial cells (although only in the rat urethra) and in the vascular endothelium. The presence of cGMP-ir in epithelial cells in response to NO donors is thought to be associated with sensory functions (13), and it seems to be species dependent. Indeed, this feature was described in the guinea pig urethra (34) and bladder (13) but not in the mouse bladder (10, 22) or in the human bladder and urethra (34). In the endothelium, cGMP-ir was observed under basal conditions, and it appears to increase upon stimulation, suggesting a role for cGMP in regulating blood flow to the urethra. Similar endothelial cGMP-ir in the wall of the human and pig urethra has also been described, where it colocalizes with endothelial NOS (43). Nevertheless, cGMP-ir has only rarely been observed in the vascular smooth muscle (not shown), even though perivascular nitrergic nerves are present.

Conclusions

In conclusion, for the first time we demonstrated that cGMP-ir accumulates in ICC following stimulation of intramural nitrergic nerves in the rat and sheep urethra, suggesting that they may act as cells responsive to nitrergic neurotransmission. Different functional types of cGMP-ir ICC form interconnecting networks at different locations in the urethral wall: serosa, mucosa, and muscle layer. Only muscular ICC (ICC-M) seem to be clearly involved in nitrergic transmission, in which nerve stimulation-induced cGMP responses were specifically prevented by NOS and GC inhibitors. No ICC were nNOS-ir, although there was a close relationship between cGMP-ir ICC and nNOS-containing nerve terminals, suggesting a functional relationship between them. Since, cGMP-ir was induced in both ICC-M and SMC upon EFS, these results agree with a parallel innervation model, in which both type of cells are effectors of the NO released by nerves. Whether ICC act as mediators and/or as regulators of neurotransmission is still to be elucidated.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Spanish "Ministerio de Educación y Ciencia" (BFU2006-15135-C02-01) and the "Comunidad de Madrid-Universidad Complutense de Madrid" (UCMGR85/06-920307).


   ACKNOWLEDGMENTS
 
The authors are grateful to Jean de Vente (Department of Psychiatry and Neuropsychology, European Graduate School of Neuroscience, University of Maastricht, Maastricht, The Netherlands) for kindly providing the sheep antiserum against cGMP. The microphotographs were acquired and analyzed at the Microscopy and Cytometry Center (Complutense University, Madrid, Spain). Also, we thank Alfonso Cortés and Luis M. Alonso for technical assistance with the fluorescence and confocal microscopy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. García-Pascual, Dept. of Physiology, Veterinary Faculty, Complutense Univ., Avda. Puerta de Hierro s/n, 28040 Madrid, Spain (e-mail: angarcia{at}vet.ucm.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
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D. Triguero, M. Sancho, M. Garcia-Flores, and A. Garcia-Pascual
Presence of cyclic nucleotide-gated channels in the rat urethra and their involvement in nerve-mediated nitrergic relaxation
Am J Physiol Renal Physiol, November 1, 2009; 297(5): F1353 - F1360.
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