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Soluble Guanylate Cyclase and Cgmp-Dependent Protein Kinase I Expression in the Human Corpus Cavernosum

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Soluble Guanylate Cyclase and Cgmp-Dependent Protein Kinase I Expression in the Human Corpus Cavernosum 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 1Department 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 regulated phosphodiesterases, cGMP-gated ion channels and cGMP dependent protein kinases.4 cGMP-dependent protein kinases have emerged as 0 Cyclic guanosine 5 -monophosphate (cGMP) medi- important signal transduction mediators and may ates as a second messenger the physiological action regulate the function of numerous proteins.5±7 of nitric oxide (NO), which is known to be one of the Recently it has been shown that activation of main mediators of smooth muscle relaxation in the cGMP-dependent kinase I relaxes smooth muscle corpus cavernosum.1,2 NO, which can diffuse freely cells by decreasing the cytosolic calcium concentra- across cell membranes, acts on the soluble isoform tion.6,7 Because of the fact that smooth muscle of guanylate cyclase (sGC) binding to the heme relaxation is essential for an erection, it is necessary moiety of the protein and thereby activating this to investigate the cGMP-dependent protein kinases enzyme.3 The resultant increase of cGMP is thought in cavernosal tissue. Two distinct types of cGMP- to underlie the physiological action of NO eg in dependent protein kinases have been identi®ed. The smooth muscle cells. There appear to be three main type I kinase (cGK I) occurs in two isoforms, arising classes of intracellular targets for cGMP, cGMP- 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 *Correspondence: T Klotz, Department of Urology, University of Cologne, Joseph-Stelzmannstr. 9, D-50924 KoÈln, Germany. intestine and is expressed in widespread areas of the 9±12 Received 8 September 1999; revised 22 October 1999; brain. Recently we have shown that the endo- accepted 14 January 2000 thelial NO-synthase is one of the main sources of sGC and cGMP-dependent protein kinase I expression TKlotzet al 158 13 nitric oxide in the cavernosal tissue. Nitrinergic 3% H2O2 and 60% methanol PBS for 30 min, then innervation and eNOS expression have shown a permeabilized with 0.2% Triton-X 100 in 0.1 M PBS. broad heterogenicity and no correlation between The sections were then treated with 5% normal goat NOS expression and erectile function was observed. serum (NGS) and 5% bovine serum albumin (BSA) The cGMP kinase I lies `downstream' of the NO solution on PBS. Before each step, the sections were mediated effects, thus it may be possible that there rinsed three times in PBS buffer. Incubation with the are differences between potent and impotent pati- primary antibody occured in a PBS-based solution ents. A loss of cGK I abolishes nitric oxide=cGMP of 0.8% BSA and 20 mM NaN3 for 12 h at 4 C. The dependent relaxation of smooth muscle, at least in anti-sGC antibody was applied in a dilution of the vascular and intestinal system.14 Up to now, 1:1000 and the anti-cGMP-kinase I antibody at cavernosal tissue has not been examined for the 1:800. After rinsing with PBS the sections were cGMP dependent kinase I enzyme. The aim of this incubated with the corresponding secondary bio- study was to investigate the distribution of the tinylated antibody for 1 h at room temperature. A guanylate cylcase and the cGMP dependent protein streptavidin-horseradish peroxidase complex was kinase I in human corpus cavernosum. In this then applied as a detection system (1:200 dilution) context, we examined cavernosal tissues from for 1 h. Finally the staining was developed for 3 ± potent and impotent patients in order to elucidate 5 min with 3,3-diaminobenzidine tetrahydrochlor- possible differences between these groups. ide (DAB) in 0.05 M Tris-HCI buffer and 0.1% H2O2. Counterstaining was performed using methyl green. Negative control sections were incubated without Patients and methods the primary antibody. Patients and collection of tissue Immun¯uorescence Twenty-three human corpus cavernosum tissue specimens were obtained with informed consent Sections of 20 mm, pretreated with BSA and NGS from patients subjected to penile surgery. The were incubated with primary antibody (PECAM specimens were immediately ®xed in 4% parafor- 1:300 or undiluted a-actin smooth muscle solution maldehyde for 4 h and then rinsed in 0.1 M supplied by the manufacturer) and sGC (1:1000) or phosphate-buffered saline (PBS) for 24 h. The tissue cGMP-kinase I (1:800) antibody over night at 4C. was stored for 12 consecutive hours in a PBS After washing three times in PBS the slices were solution containing 18% sucrose for cryoprotection incubated at room temperature for 1 h with anti and then frozen to 780C. mouse Cy2 (1:100) and biotinylated anti rabbit Eleven of the patients (age range 16 ± 63 y; mean (1:400) antibody. Following washing with PBS the age 42 y) with normal erectile functions suffered slices were treated with extravidin Cy3. After from penile deviations. All patients were treated via dehydration in a series of graded ethanol the Nesbit's surgical method. Normal erectile function specimens were embedded in entelan. Analysis was ascertained by anamnestic evaluation. was performed using a Zeiss Axiophot ¯uorescence Nine patients (age range 27 ± 66 y; mean age 46 y) microscope. had shown complete erectile dysfunction for more than 2 y. This group underwent implantation of ¯exible hydraulic penile protheses. One of the Material patients suffered from Klinefelter's syndrome, four patients from severe venous leakage, one patient from impotence due to radical surgery of a bladder The primary anti-GC antibody were from Alexis carcinoma. Two patients had diabetes and a com- (San Diego, CA, USA). The monoclonal primary bined arterial-venous insuf®ciency of the corpus mouse antibodies PECAM-1 and a-actin smooth carvernosum. In one patient, the impotence was of muscle were from Biogenex (San Ramon, CA, unknown origin. USA). The polyclonal cGMP-kinase I antibody was Three patients underwent trans-sexual operations obtained from Professor Peter Ruth from the In- (male ± female). These patients had a long-term stitute of Pharmocology in Munich. The antibody hormonal pretreatment, but reported normal production method was as described previously.4 erections. The secondary biotinylated goat anti-rabbit and goat anti-mouse antibodies were from Vector Labora- Immunohistochemistry tories (Burlingame, USA), mouse Cy2 (Dianova, Hamburg, Germany), biotinylated rabbit anti-mouse (Dako, Denmark), extravidin Cy3 (Sigma, Deisenho- Prior to immunohistochemical examination tissue fen, Germany). BSA was purchased from Sigma sections (20 mm) were placed in a bathing solution of (Deisenhofen, Germany), which also provided the International Journal of Impotence Research sGC and cGMP-dependent protein kinase I expression T Klotz et al 159 normal goat serum as well as the necessary chemi- and tunica albuginea vessels, without a speci®c cals required for staining with the streptavidin- subcellular localization (Figure 2a ± f). The endo- biotin-peroxidase complex. thelium of the cavernosal sinus and of cavernosal arteries and veins also showed an immunoreactivity against sGC (Figure 2c). The cavernosal tissue Results revealed a known dense nerve network in the ®bromuscular stroma surrounding cavernosal ves- In all specimens of cavernosal tissue, a distinct sels.10 These nerve ®bers were partially immuno- immunoreactivity was observed in different parts positive against sGC (Figure 2b). This observation and structures. We found a high expression of demonstrates the localization of sGC not only in soluble guanylate cyclase and cGMP-kinase I in smooth muscle cells, but also in the endothelium smooth muscle cells of the corpus carvernosum and nerve ®bers (Figure 2a ± d).
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