International Journal of Impotence Research (2008) 20, 278–284 & 2008 Nature Publishing Group All rights reserved 0955-9930/08 $30.00 www.nature.com/ijir ORIGINAL ARTICLE Role of the soluble guanylyl cyclase a1-subunit in mice corpus cavernosum smooth muscle relaxation S Nimmegeers1, P Sips2,3, E Buys2,3,4, K Decaluwe´1, P Brouckaert2,3 and J Van de Voorde1* 1Department of Physiology and Physiopathology, Ghent University, Ghent, Belgium; 2Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent, Belgium; 3Department of Molecular Biology, Ghent University, Ghent, Belgium and 4Department of Anesthesia and Critical Care, Anesthesia Center for Critical Care Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Soluble guanylyl cyclase (sGC) is the major effector molecule for nitric oxide (NO) and as such an interesting therapeutic target for the treatment of erectile dysfunction. To assess the functional À/À importance of the sGCa1b1 isoform in corpus cavernosum (CC) relaxation, CC from male sGCa1 and wild-type mice were mounted in organ baths for isometric tension recording. The relaxation to endogenous NO (from acetylcholine, bradykinin and electrical field stimulation) was nearly À/À À/À abolished in the sGCa1 CC. In the sGCa1 mice, the relaxing influence of exogenous NO (from sodium nitroprusside and NO gas), BAY 41-2272 (NO-independent sGC stimulator) and T-1032 (phosphodiesterase type 5 inhibitor) were also significantly decreased. The remaining exogenous À/À NO-induced relaxation seen in the sGCa1 mice was significantly decreased by the sGC-inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. The specificity of the impairment of the sGC-related responses was demonstrated by the unaltered relaxations seen with forskolin (adenylyl cyclase activator) and 8-pCPT-cGMP (cGMP analog). In conclusion, the sGCa1b1 isoform is involved in corporal smooth muscle relaxation in response to NO and NO-independent sGC stimulators. The À/À fact that there is still some effect of exogenous NO in the sGCa1 mice suggests the contribution of (an) additional pathway(s). International Journal of Impotence Research (2008) 20, 278–284; doi:10.1038/sj.ijir.3901627; published online 6 December 2007 Keywords: penile erection; nitric oxide; soluble guanylyl cyclase; vasodilatation Introduction required for penile erection. However, the impor- tance of NO produced by endothelial NOS for penile Penile erection is a complex, neurally regulated erection is becoming increasingly recognized.5 physiologic event that involves increased blood Regardless of the source, NO binds to the heme filling of the corporal tissue and restricted venous component of soluble guanylyl cyclase (sGC), lead- outflow, both resulting from corporal smooth muscle ing to a 300-fold increase in the catalytic conversion relaxation.1 Nitric oxide (NO) is widely accepted as of guanosine-50-triphosphate to cyclic guanosine- the principal mediator of the erectile response. It is 30,50-monophosphate (cGMP) and pyrophosphate.6 produced by neuronal NO synthase (nNOS) in This high amount of cGMP conveys signals through nonadrenergic noncholinergic nerves innervating activation of cGMP-dependent protein kinase I, the penis.2 Although sinusoidal and vascular en- eventually leading to smooth muscle relaxation.7,8 dothelial cells also release NO in response to sGC is a heterodimer composed of two subunits, a mechanical3 and chemical stimuli,4 neurogenic NO and b,9 both essential for catalytic activity.10 Two is generally considered as the primary source isoforms for each subunit (a1/a2 and b1/b2) have 11–13 been described, but only the a1b1 and a2b1 14 heterodimers are found active. sGCa1b1 is the *Correspondence: Professor J Van de Voorde, Department predominantly expressed isoform in most tissues of Physiology and Physiopathology, Ghent University, De Pintelaan 185, Ghent 9000, Belgium. except in the brain, in which the levels of both isoforms are comparable.15 Various diseases, includ- E-mail: [email protected] 16,17 18 Received 19 September 2007; revised 18 October 2007; ing hypertension, hypercholesterolemia, dia- 19 20 accepted 1 November 2007; published online 6 December betes mellitus and renal failure, that cause 2007 erectile dysfunction are highly associated with sGC in mice corpus cavernosum S Nimmegeers et al 279 impairments of the NO/cGMP signaling pathway. field stimulation (EFS: train duration 20 or 40 s; The central role of this pathway is demonstrated by frequency: 1, 2, 4 and 8 Hz; pulse duration: 5 ms; the phosphodiesterase type 5 inhibitor sildenafil as 80 V), delivered by a Grass stimulator via two today’s most successful therapy for the treatment of parallel platinum electrodes, was applied to the erectile dysfunction. However, as some side effects tissue or various vasodilating substances were added and limitations on use have been reported,21,22 there to the bath medium. In some experiments, increasing is increasing interest in alternative therapeutic concentrations of NOR were added at a stable resting measures. As the predominant intracellular receptor tension to analyze the contractile response. EFS was of NO, sGC is a promising therapeutic target. The repeated after incubation with atropine (1 mmol lÀ1) aim of the present study was therefore to analyze the and guanethidine (4 mmol lÀ1) for 30 min to eliminate functional importance of the sGCa1b1 isoform in responses mediated by cholinergic and noradrener- penile smooth muscle relaxation using sGCa1 gic nerves, respectively. The influence of the sGC À/À knockout (sGCa1 ) mice. inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1- one (ODQ) (1 mmol lÀ1, 20 min preincubation) was investigated on EFS and drug-induced effects. Materials and methods Between response curves, the CC were washed and allowed to recover for 20–30 min. At the end of the Animals experiments, tissues were lightly patted dry and All experiments were performed on male homo- weighed. À/À zygous sGCa1 knockout (sGCa1 ; n ¼ 6–9) mice þ / þ and sGCa1 (n ¼ 6–11) littermates (genetic back- ground: mixed Swiss-129),23 bred in the Depart- ment of Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology, Ghent, Drugs Belgium. The animals were treated in accordance The experiments were performed in a Krebs-Ringer bicarbonate solution of the following composition with the Guide for the Care and Use of Laboratory À1 Animals published by the US National Institutes of (mmol l ): NaCl, 135; KCl, 5; NaHCO3, 20; glucose, Health (NIH Publication no. 85–23, revised 1996). 10; CaCl2, 2.5; MgSO4, 1.3; KH2PO4, 1.2 and EDTA, 0.026 in H2O. ODQ, acetylcholine (ACh) chloride, On the day of experiment, the mice were sexually o bradykinin acetate, N -nitro-L-arginine, forskolin, mature (age: 10–15 weeks) and euthanized by 0 0 cervical dislocation. 8-(4-chlorophenylthio)-guanosine 3 ,5 -cyclic mono- phosphate (8-pCPT-cGMP), atropine, guanethidine and NOR bitartrate were obtained from Sigma- Aldrich (St Louis, MO, USA), BAY 41-2272 from Tissue collection Alexis (San Diego, CA, USA) and sodium nitroprus- The penile tissue was dissected free by removal of side (SNP) from Merck (Darmstadt, Germany). ODQ the connective and adventitial tissues along the and BAY 41-2272 were dissolved in dimethylsulf- shaft of the penis, the dorsal arteries, dorsal vein, oxide and ACh in 50 mmol lÀ1 potassium hydrogen corpus spongiosum, urethra and glans penis. phthalate buffer, pH 4.0. The other drugs were Corpora cavernosa were then separated by cutting dissolved in distilled water. Saturated NO solution the fibrous septum between them and were excised was prepared from gas (Air Liquide, Belgium) as at the base. They were kept in cooled Krebs-Ringer described by Kelm and Schrader.24 All concentra- bicarbonate solution until mounting. tions are expressed as final molar concentrations in the organ bath. The final concentration of dimethyl- sulfoxide in the organ bath never surpassed 0.1%. Tension measurements One corpus cavernosum from each mouse was mounted horizontally in a myograph with one end fixed to a force-displacement transducer and the other, to a micrometer. The tissue chambers con- Calculations and statistics tained 10 ml Krebs-Ringer bicarbonate solution at Data are presented as mean values±s.e.m.; 37 1C (pH 7.4) equilibrated with 95% O2 and 5% n represents the number of corpora cavernosa (each CO2. The preparations were preloaded with 0.45 g of obtained from a different mouse). Relaxations are tension and allowed to equilibrate for 60 min in bath expressed as a percentage of the tone developed by fluid that was frequently replaced with fresh Krebs- the addition of NOR. Contractions are expressed Ringer bicarbonate solution. The preparations were in mN. contracted three times with 5 mmol lÀ1 norepinephr- Statistical significance was evaluated using ine (NOR), washed and allowed to relax to resting Student’s t-test for paired and unpaired observations tension before starting the protocol. When the pre- (SPSS, version 12). Po0.05 was considered as contraction response reached a stable level, electrical significant. International Journal of Impotence Research sGC in mice corpus cavernosum S Nimmegeers et al 280 Figure 1 Cumulative concentration–contraction curve to NOR in þ / þ À/À m CC from sGCa1 (’; n ¼ 11) and sGCa1 ( ; n ¼ 9) mice. CC, Figure 2 Relaxation effect of ACh on precontracted (5 mmol lÀ1 corpus cavernosum; NOR, norepinephrine; sGCa , soluble gua- þ / þ À/À 1 NOR) CC from sGCa1 (n ¼ 7) and sGCa1 (n ¼ 7) mice in nylyl cyclase. control conditions (ÀODQ) and in the presence of ODQ ( þ ODQ). þ / þ À/À # *sGCa1 vs sGCa1 , ÀODQ vs þ ODQ: Po0.05. ACh, acetylcholine; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Results The weight of the CC preparations did not signifi- À/À þ / þ cantly differ between sGCa1 and sGCa1 mice (13.54 mg±0.80 (n ¼ 9) vs 12.77 mg±0.77 (n ¼ 11, P40.05)).