Soluble guanylate cyclase and cGMP-dependent protein ... - Nature

International Journal of Impotence Research (2000) 12, 157±164 ß 2000 Macmillan Publishers Ltd All rights reserved 0955-9930/00 $15.00 www.nature.com/ijir

Soluble guanylate cyclase and cGMP-dependent protein kinase I expression in the human corpus cavernosum T Klotz1*, W Bloch2, J Zimmermann1, P Ruth3, U Engelmann1 and K Addicks2 1

Department of Urology, University of Cologne; 2Institute I of Anatomy, University of Cologne; and 3Institute of Pharmacology, TU University of Munich, Germany Nitric oxide (NO) as a mediator in smooth muscle cells causes rapid and robust increases in cGMP levels. The cGMP-dependent protein kinase I has emerged as an important signal transduction mediator for smooth muscle relaxation. The purpose of this study was to examine the existence and distribution of two key enzymes of the NO=cGMP pathway, the cGMP-dependent kinase I (cGK I) and the soluble guanylate cyclase (sGC) in human cavernosal tissue. The expression of the enzymes were examined in corpus cavernosum specimens of 23 patients. Eleven potent patients suffered from penile deviations and were treated via Nesbit's surgical method. Nine long-term impotent patients underwent implantation of ¯exible hydraulic prothesis. Three potent patients underwent trans-sexual operations. Expression of the sGC and cGK I were examined immunohistochemically using speci®c antibodies. In all specimens of cavernosal tissue a distinct immunoreactivity was observed in different parts and structures. We found a high expression of sGC and cGK I in smooth muscle cells of vessels and in the ®bromuscular stroma. The endothelium of the cavernosal sinus, of the cavernosal arteries, and the cavernosal nerve ®bers showed an immunoreactivity against sGC. The distribution analysis of cGK I revealed a predominately vesicular localization in smooth muscle cells. The examination of the endothelium showed no clear immunoreactivity against cGK I. There was no distinct difference in immunoreactivity and cellular distribution between potent and impotent patients. cGMP-dependent kinase I is predominately expressed in cavernosal smooth muscle cells. sGC is expressed in smooth muscle cells, endothelium and nerve ®bers of the corpus cavernosum. In this study, there is no correlation between the distribution of soluble guanylate cyclase and cGMP dependent kinase I in potent or impotent patients. International Journal of Impotence Research (2000) 12, 157±164. Keywords: corpus cavernosum; cGMP-dependent kinase I; impotence; soluble guanylate cyclase

Introduction Cyclic guanosine 50 -monophosphate (cGMP) mediates as a second messenger the physiological action of nitric oxide (NO), which is known to be one of the main mediators of smooth muscle relaxation in the corpus cavernosum.1,2 NO, which can diffuse freely across cell membranes, acts on the soluble isoform of guanylate cyclase (sGC) binding to the heme moiety of the protein and thereby activating this enzyme.3 The resultant increase of cGMP is thought to underlie the physiological action of NO eg in smooth muscle cells. There appear to be three main classes of intracellular targets for cGMP, cGMP-

*Correspondence: T Klotz, Department of Urology, University of Cologne, Joseph-Stelzmannstr. 9, D-50924 KoÈln, Germany. Received 8 September 1999; revised 22 October 1999; accepted 14 January 2000

regulated phosphodiesterases, cGMP-gated ion channels and cGMP dependent protein kinases.4 cGMP-dependent protein kinases have emerged as important signal transduction mediators and may regulate the function of numerous proteins.5 ± 7 Recently it has been shown that activation of cGMP-dependent kinase I relaxes smooth muscle cells by decreasing the cytosolic calcium concentration.6,7 Because of the fact that smooth muscle relaxation is essential for an erection, it is necessary to investigate the cGMP-dependent protein kinases in cavernosal tissue. Two distinct types of cGMPdependent protein kinases have been identi®ed. The type I kinase (cGK I) occurs in two isoforms, arising from alternative mRNA splicing.4,7,8 cGK I is present at high levels in smooth muscle, lung and platelets.4 A second form, type II cGMP-dependent kinase (cGK II) was cloned from mouse brain and rat intestine and is expressed in widespread areas of the brain.9 ± 12 Recently we have shown that the endothelial NO-synthase is one of the main sources of

sGC and cGMP-dependent protein kinase I expression T Klotz et al 158

nitric oxide in the cavernosal tissue.13 Nitrinergic innervation and eNOS expression have shown a broad heterogenicity and no correlation between NOS expression and erectile function was observed. The cGMP kinase I lies `downstream' of the NO mediated effects, thus it may be possible that there are differences between potent and impotent patients. A loss of cGK I abolishes nitric oxide=cGMP dependent relaxation of smooth muscle, at least in the vascular and intestinal system.14 Up to now, cavernosal tissue has not been examined for the cGMP dependent kinase I enzyme. The aim of this study was to investigate the distribution of the guanylate cylcase and the cGMP dependent protein kinase I in human corpus cavernosum. In this context, we examined cavernosal tissues from potent and impotent patients in order to elucidate possible differences between these groups.

Patients and methods Patients and collection of tissue Twenty-three human corpus cavernosum tissue specimens were obtained with informed consent from patients subjected to penile surgery. The specimens were immediately ®xed in 4% paraformaldehyde for 4 h and then rinsed in 0.1 M phosphate-buffered saline (PBS) for 24 h. The tissue was stored for 12 consecutive hours in a PBS solution containing 18% sucrose for cryoprotection and then frozen to 780 C. Eleven of the patients (age range 16 ± 63 y; mean age 42 y) with normal erectile functions suffered from penile deviations. All patients were treated via Nesbit's surgical method. Normal erectile function was ascertained by anamnestic evaluation. Nine patients (age range 27 ± 66 y; mean age 46 y) had shown complete erectile dysfunction for more than 2 y. This group underwent implantation of ¯exible hydraulic penile protheses. One of the patients suffered from Klinefelter's syndrome, four patients from severe venous leakage, one patient from impotence due to radical surgery of a bladder carcinoma. Two patients had diabetes and a combined arterial-venous insuf®ciency of the corpus carvernosum. In one patient, the impotence was of unknown origin. Three patients underwent trans-sexual operations (male ± female). These patients had a long-term hormonal pretreatment, but reported normal erections. Immunohistochemistry Prior to immunohistochemical examination tissue sections (20 mm) were placed in a bathing solution of International Journal of Impotence Research

3% H2O2 and 60% methanol PBS for 30 min, then permeabilized with 0.2% Triton-X 100 in 0.1 M PBS. The sections were then treated with 5% normal goat serum (NGS) and 5% bovine serum albumin (BSA) solution on PBS. Before each step, the sections were rinsed three times in PBS buffer. Incubation with the primary antibody occured in a PBS-based solution of 0.8% BSA and 20 mM NaN3 for 12 h at 4 C. The anti-sGC antibody was applied in a dilution of 1:1000 and the anti-cGMP-kinase I antibody at 1:800. After rinsing with PBS the sections were incubated with the corresponding secondary biotinylated antibody for 1 h at room temperature. A streptavidin-horseradish peroxidase complex was then applied as a detection system (1:200 dilution) for 1 h. Finally the staining was developed for 3 ± 5 min with 3,3-diaminobenzidine tetrahydrochloride (DAB) in 0.05 M Tris-HCI buffer and 0.1% H2O2. Counterstaining was performed using methyl green. Negative control sections were incubated without the primary antibody. Immun¯uorescence Sections of 20 mm, pretreated with BSA and NGS were incubated with primary antibody (PECAM 1:300 or undiluted a-actin smooth muscle solution supplied by the manufacturer) and sGC (1:1000) or cGMP-kinase I (1:800) antibody over night at 4 C. After washing three times in PBS the slices were incubated at room temperature for 1 h with anti mouse Cy2 (1:100) and biotinylated anti rabbit (1:400) antibody. Following washing with PBS the slices were treated with extravidin Cy3. After dehydration in a series of graded ethanol the specimens were embedded in entelan. Analysis was performed using a Zeiss Axiophot ¯uorescence microscope. Material The primary anti-GC antibody were from Alexis (San Diego, CA, USA). The monoclonal primary mouse antibodies PECAM-1 and a-actin smooth muscle were from Biogenex (San Ramon, CA, USA). The polyclonal cGMP-kinase I antibody was obtained from Professor Peter Ruth from the Institute of Pharmocology in Munich. The antibody production method was as described previously.4 The secondary biotinylated goat anti-rabbit and goat anti-mouse antibodies were from Vector Laboratories (Burlingame, USA), mouse Cy2 (Dianova, Hamburg, Germany), biotinylated rabbit anti-mouse (Dako, Denmark), extravidin Cy3 (Sigma, Deisenhofen, Germany). BSA was purchased from Sigma (Deisenhofen, Germany), which also provided the

sGC and cGMP-dependent protein kinase I expression T Klotz et al

normal goat serum as well as the necessary chemicals required for staining with the streptavidinbiotin-peroxidase complex.

Results In all specimens of cavernosal tissue, a distinct immunoreactivity was observed in different parts and structures. We found a high expression of soluble guanylate cyclase and cGMP-kinase I in smooth muscle cells of the corpus carvernosum (Figure 1a ± d). All specimens from impotent patients with different primary diseases and transsexual patients showed an expression of sGC and cGMP-kinase I. There was no distinct difference in immunoreactivity and distribution between potent and impotent patients (Figure 1a ± d). Concerning the soluble guanylate cylase (sGC) we could describe a distinct pattern of distribution in the cavernosal tissue. sGC was predominately found in smooth muscle cells of the ®bromuscular stroma and the vascular smooth muscle layer of cavernosal

and tunica albuginea vessels, without a speci®c subcellular localization (Figure 2a ± f). The endothelium of the cavernosal sinus and of cavernosal arteries and veins also showed an immunoreactivity against sGC (Figure 2c). The cavernosal tissue revealed a known dense nerve network in the ®bromuscular stroma surrounding cavernosal vessels.10 These nerve ®bers were partially immunopositive against sGC (Figure 2b). This observation demonstrates the localization of sGC not only in smooth muscle cells, but also in the endothelium and nerve ®bers (Figure 2a ± d). The immunohistochemical detection of cGK I in smooth muscle cells shows a distinct pattern (Figure 3a ± d). A vesicular-like distribution of cGK I in the smooth muscle cells was found. No immunostaining against cGK I was found in the endothelium of corpus cavernosum veins and in the endothelium of tunica albuginea vessels. The smooth muscle layer of vessels is cGK I positive (Figure 3c, d). Immunostaining using a polyclonal cGMP-dependent protein kinase I antibody revealed that cavernosal smooth muscle cells and vascular smooth

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Figure 1 A representative comparison of the soluble guanylate-cyclase (sGC) (a, b) and cGMP-kinase I (c, d) expression in corpus cavernosum of patients with (b, d) and without (a, c) erectile dysfunction. At this magni®cation smooth muscle (SM) and venous sinus (S) are recognizable. A distinct staining of sGC and cGK I are observable in the smooth muscle of potent (a,c) and impotent (b, d) patients without recognizable differences. (Cause of impotence in this sections was venous leakage.) Bar a, b 150 mm, c, d 80 mm.

International Journal of Impotence Research


Figure 2 Immunohistochemical detection of soluble guanylate cyclase (sGC) of human corpus cavernosum tissue (a ± f) from potent patients: The smooth muscles are distinct immunostained (a). Nerve ®bers (arrow heads) innervating the ®bromuscular stroma are positive for sGC (b). The endothelium (arrows) of corpus cavernosum veins also contains sGC (c). At higher magni®cation a sGC expression in the endothelial cells (arrows) of the sinus cavernosum is demonstrated (e). A sGC immunoreactivity is also found in endothelial cells and smooth muscle of tunica albuginea vessels (V) (d). Without the ®rst antibody no immunoreaction is found (f) Bar a ± d 50 mm, e 15 mm, f 75 mm.

muscle layer contained this enzyme (Figures 1 and 4). We found a double labeling of sGC and PECAM (Figure 4b, d). In the double immunostaining of the human corpus cavernosum with antibodies for sGC or cGK I and PECAM or a-smooth muscle actin. Furthermore we can also reveal a double labeling with cGK I and a-smooth muscle actin (Figure 4e, f). International Journal of Impotence Research

Discussion Recently we have shown that the endothelial NOsynthase is one of the important sources of NO in cavernosal smooth muscle cells.13,15 Furthermore we have demonstrated that erectile dysfunction cannot solely be reduced to pathological ®ndings


Figure 3 Distribution of cGMP-kinase I in human corpus cavernosum: the immunohistochemical detection of cGMP-kinase I in smooth muscle cells shows a distinct pattern. In cross section of the smooth muscle cells, a vesicular like distribution (arrow heads) of cGMPkinase I is found (a). In longitudinal sectioned smooth muscle, a cross striation of the immunoreaction products of cGMP-kinase I staining is seen (b). No immunostaining is found in the endothelium (arrows) of tunica albuginea vessels (c), while the smooth muscle layer (SM) of the vessel (V) is cGMP-kinase I positive (c, d). Bar a ± d 25 mm.

in penile NO-synthases, because there has been no correlation between the expression of the NOsynthases and the erectile function in cavernosal tissue of potent and impotent patients. NO has the capacity to activate guanylate cyclase. Submicromolar concentrations of NO cause rapid and robust increases in cGMP levels in target cells.16 Changes of the cellular cGMP level as a second messenger lead to physiological responses in these target cells. There are some targets of cGMP action such as cGMP-gated channels, cGMP-regulated posphodiesterases and cGMP-dependent protein kinases. Many of these responses are mediated by the activation of the cGMP-dependent kinase I.5,14,17 The cGMPdependent protein kinase I has emerged as an important signal transduction mediator.4,18,19 Subsequent cGMP-mediated phosphorylation of proteins appears to be involved in intracellular Ca2 sequestration or removal. The removal of cytosolic Ca2 in response to cGMP mediated protein phos-

phorylation is responsible for smooth relaxation and reduction in vascular tone (Figure 4).17 Smooth muscle relaxation is one of the most important effects of the NO=cGMP pathway concerning erectile function. In intact smooth muscle cells, cGMP dependent kinase I phosphorylates the inositol 1,4,5-trisphosphate receptor (IP3R).17 This phosphorylation may result either in inhibition of Ca2 release or in increased compartmentalization. Thus cGMP-dependent kinase I is one of most relevant trigger enzymes for smooth muscle relaxation. For the understanding of the complete signal transduction in the corpus cavernosum it is necessary to elucidate the whole NO=cGMP pathway with its `up-and downstream' target proteins. To our knowledge this is the ®rst study that deals with the expression of the guanlyate cyclase (sGC) and GMP-dependent kinase I (cGK I) in human corpus cavernosum. sGC was found in smooth muscle cells without speci®c subcellular localization. International Journal of Impotence Research


Figure 4 Double immunostaining of human corpus cavernosum with antibodies for sGC or cGMP-kinase I and PECAM or a-smooth muscle actin: (a, b) The sGC-immun¯ourescence (b) is more expanded as the immuno¯uorescence for a-smooth muscle actin (a). The comparison of immuno¯uorescence labeling with the endothelial speci®c marker PECAM and the sGC labeling shows a double labeling of sGC and PECAM (arrows). The ¯uorescence staining for cGMP-kinase I (f) is restricted to the a-smooth muscle actin positive areas (e) Bar 25 mm.

The endothelium of cavernosal sinus, of cavernosal arteries and cavernosal nerves also showed an immunoreactivity. This observation demonstrates the localization of sGC not only in smooth muscle cells, but also in the endothelium. The results were con®rmed by double immunostaining with endotheInternational Journal of Impotence Research

lium speci®c PECAM. Concerning the cGMPdependent kinase I we found that cavernosal smooth muscle cells contain this enzyme. This results were also con®rmed by double immunostaining with speci®c a-smooth muscle actin antibody.

sGC and cGMP-dependent protein kinase I expression T Klotz et al

The distribution analysis of cGK I showed a vesicular localization in smooth muscle cells. In our study, the examination of the endothelium and nerve ®bers revealed no clear expression of cGK I, although cGK I expression was found in the nerve ®bers and endothelium of other organs.20,21 Furthermore we have not found in our study any distinct differences between potent and impotent patients in regards to the distribution of sGC and cGMP dependent kinase I. We have to stress the limitations of our study regarding the small number of patients which did not allow a statistical analysis. For example, immunohistochemistry is not a quantitative technique and another assay such as immuno-blotting with more patients might reveal quantitiative changes in the enzymes between potent and impotent patients. Thus our results have a premature character regarding differences between potent and impotent patients. As for the NO-synthases erectile dysfunction cannot be reduced to pathological ®ndings in sGC and cGK I.13,15 It should be stressed that our study shows only the expression or distribution of the enzymes but not the functional status. In the case of the cGMP-dependent kinases, this problem is alleviated by recent developments for the analysis of the function of cGMP dependent kinases.23 Because of the selective expression of cGK I in smooth muscle cells of the corpus cavernosum, it seems possible that cGMP kinase could be therapeutically activated or inhibited by speci®c substances.23,24 Gene knockout approaches may promise new insights into major functions controlled by cGK I and justify a more detailed investigation into the underlying mechanisms. The challenge remains to de®ne the speci®c effects of agonists and cGMP that are dependent on cGK I as a key enzyme for smooth muscle relaxation.

Conclusions This study indicates that cGK I is present in the corpus cavernosum. It remains to be determined whether the enzyme is important for erectile dysfunction. The distribution of the cGK I correlates with the distribution of the soluble guanylate cylcase (sGC) in smooth muscle cells. cGK I is a key enzyme for smooth muscle relaxation and the development of speci®c agonists may have therapeutical consequences.

Acknowledgements This study was supported by a ®nancial grant of P®zer GmbH Karlsruhe, Germany.

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