Role of the Soluble Guanylyl Cyclase A1-Subunit in Mice Corpus Cavernosum Smooth Muscle Relaxation

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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 importance 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 monophosphate (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)). In response to increasing concentrations of NOR (10 nmol lÀ1 to 10 mmol lÀ1), the penile tissue iso- À/À lated from sGCa1 mice developed an equal force þ / þ per mg tissue compared with the sGCa1 preparations (Figure 1). The ability to relax NOR-contracted CC preparations through release of endothelial NO was tested by the addition of ACh (10 mmol lÀ1) and bradykinin À1 þ / þ acetate (50 mmol l ). ACh relaxed the sGCa1 preparations, whereas it contracted the tissues of À/À sGCa1 mice (Figure 2). Inhibition of sGC by ODQ À/À resulted in a contractile effect of ACh in both sGCa1 þ / þ and sGCa1 CC tissues (Figure 2). Bradykinin acetate had a relaxant effect in the CC of both À/À þ / þ sGCa1 and sGCa1 mice, although the response À/À in the sGCa1 preparations was significantly À/À reduced (4.55%±2.80 in sGCa1 vs 26.82%±2.86 þ / þ in sGCa1 (n ¼ 6 each, Po0.05)). The effect of neuronal NO was examined by stimulating the intrinsic nerves with EFS. EFS þ / þ relaxed the tissues of sGCa1 mice in a frequency-dependent manner (Figure 3a), whereas the À/À response in the sGCa1 preparations was nearly abolished (Figures 3b and 4a). Following preincubation with ODQ, the relaxations induced by EFS in þ / þ the sGCa1 preparations were completely blocked and even resulted in a small contractile response of EFS at 8 Hz (Figure 4a). As the response to EFS in À/À the CC of the sGCa1 mice was very small, the Figure 3 Original tracing showing a response curve to EFS (Hz) influence of ODQ was negligible. The presence of À1 þ / þ (a and b) and to SNP (Àlog mol l )(c and d) in CC from sGCa1 guanethidine and atropine significantly reduced aÀ/À þ / þ (a and c) and sGC 1 (b and d) mice. EFS, electrical field relaxation by EFS on the sGCa1 CC (Figure 4b). stimulation; SNP, sodium nitroprusside. The very limited EFS-induced response in the À/À sGCa1 preparations was unaltered by guanethidine and atropine even after increasing the stimula- SNP (1 nmol lÀ1 to 10 mmol lÀ1) resulted in a con- þ / þ tion period from 20 to 40 s (data not shown). centration-dependent relaxation of the sGCa1 À/À Administration of increasing concentrations of (Figure 3c) and sGCa1 (Figure 3d) CC preparations À/À the endothelium-independent NO-donor compound that were significantly reduced in the sGCa1 mice

International Journal of Impotence Research sGC in mice corpus cavernosum S Nimmegeers et al 281

À1 þ / þ À/À Figure 4 Effect of EFS on precontracted (5 mmol l NOR) CC from sGCa1 (n ¼ 6) and sGCa1 (n ¼ 6) mice in control conditions and þ / þ À/À þ / þ À/À in the presence of ODQ (a) or guanethidine (GUA) and atropine (ATR) (b). *sGCa1 vs sGCa1 , þ sGCa1 vs sGCa1 both in the # þ / þ y presence of GUA þ ATR, ÀODQ or ÀGUA þ ATR vs þ ODQ or þ GUA þ ATR for sGCa1 mice, ÀGUA þ À/À ATR vs þ GUA þ ATR for sGCa1 mice: Po0.05. EFS, electrical field stimulation; NOR, norepinephrine; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one.

À1 þ / þ À/À Figure 5 Relaxation effect of SNP (a) and NO gas (b) on precontracted (5 mmol l NOR) CC from sGCa1 and sGCa1 mice in control þ / þ À/À þ / þ À/À # conditions (m and W) and in the presence of ODQ (K and J). *sGCa1 vs sGCa1 , þ sGCa1 ODQ vs sGCa1 ODQ, ÀODQ vs þ / þ y À/À þ ODQ in sGCa1 mice, ÀODQ vs þ ODQ in sGCa1 mice: Po0.05, (n ¼ 6–7). CC, corpus cavernosum; NO, nitric oxide; NOR, norepinephrine; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; sGCa1, soluble guanylyl cyclase; SNP, sodium nitroprusside.

þ / þ as compared to sGCa1 mice (Figure 5a). The Addition of BAY-41-2272, an NO-independent À/À 26 À1 À1 maximal relaxation to SNP in the CC from sGCa1 sGC stimulator (1 nmol l to 10 mmol l ), pro- mice was decreased by approximately 38%. Pre- duced a concentration-dependent relaxation in the À/À þ / þ incubation of the CC tissues with ODQ strongly CC preparations of both sGCa1 and sGCa1 inhibited the SNP-induced responses in both mice. However, BAY 41-2272 had a significantly þ / þ À/À À/À sGCa1 and sGCa1 mice (Figure 5a). smaller effect in the sGCa1 preparations compared À1 þ / þ Exogenous NO, delivered as gas (1–100 mmol l ) to sGCa1 penile tissue (Figure 6b). and added noncumulatively, was able to relax the There was no difference in the concentration- À/À þ / þ CC preparations of both sGCa1 and sGCa1 mice dependent response to the cell membrane perme- in a concentration-dependent way. However, able cGMP-analog, 8-pCPT-cGMP (100 nmol lÀ1 to À1 À/À þ / þ the response to NO gas was significantly reduced in 0.1 mmol l ), between the sGCa1 and sGCa1 À/À the penile tissues of the sGCa1 mice as compared preparations (Figure 6c). þ / þ to those of sGCa1 mice (Figure 5b). The maximum Also forskolin, an adenylyl cyclase stimulator, response to NO gas was significantly diminished in induced an identical concentration-dependent re- À/À À/À þ / þ sGCa1 CC by approximately 36% compared to sponse in the sGCa1 and sGCa1 preparations control. Treatment with ODQ significantly reduced (Figure 6d). þ / þ the relaxant effect of NO gas in both sGCa1 À/À and sGCa1 CC preparations (Figure 5b). The inhibition of phosphodiesterase type 5 by T-103225 (1 nmol lÀ1 to 10 mmol lÀ1) resulted in a Discussion concentration-dependent relaxant response in the À/À þ / þ penile tissue from both sGCa1 and sGCa1 mice. It is generally accepted that sGC, as the major This response was however significantly smaller in effector molecule for NO, plays a very important À/À þ / þ the sGCa1 mice than in sGCa1 mice (Figure 6a). role in penile smooth muscle cell relaxation. An

International Journal of Impotence Research sGC in mice corpus cavernosum S Nimmegeers et al 282

Figure 6 Relaxation effect of T-1032 (a), BAY 41-2272 (b), 8-pCPT-cGMP (c) and Forskolin (d) on pre-contracted (5 mmol lÀ1 NOR) CC þ / þ À/À þ / þ À/À from sGCa1 (’; n ¼ 6) and sGCa1 (m; n ¼ 7) mice. *sGCa1 vs sGCa1 : Po0.05. 8-pCPT-cGMP, 8-(4-chlorophenylthio)-guanosine 0 0 3 ,5 -cyclic monophosphate; sGCa1, soluble guanylyl cyclase.

understanding of the functional importance of the this finding does not support the putative role of the sGC isoforms in penile erection is necessary to vasoactive intestinal peptide as an inhibitory neuro- validate the sGC subunits as therapeutic targets for transmitter in penile erection.30 Vasoactive intest- the treatment of erectile dysfunction. It has been inal peptide, which is present in the nerves of shown that the main isoform of sGC expressed in the murine corpora cavernosa, as well as in other 27 corpora cavernosa is sGCa1b1. By the present species, stimulates adenylyl cyclase and subse- study, this notion is translated in its functional quently elevates the cAMP-dependent protein importance, as the response to endogenous NO from kinase.31 As it has been shown that there are cross- endothelial origin is nearly abolished in the CC modulatory functions between the sGC/cGMP- and À/À 32 preparations of sGCa1 mice compared to the adenylyl cyclase/cAMP-signaling pathways, one þ / þ À/À sGCa1 CC tissues. Cavernosum from sGCa1 could suggest that the latter has a complementary mice developed even contractions in response to role in the control of cavernous smooth muscle ACh and showed significantly less relaxation in tone. However, we show that the adenylyl response to bradykinin acetate. This indicates that cyclase-activator, forskolin, relaxes the CC prepara- À/À þ / þ endothelium-derived NO exerts its effect through tions of both sGCa1 and sGCa1 mice to a activation of the sGCa1-subunit. These observations similar extent. Therefore, there is no evidence for an are in line with our observations on aorta and upregulation and possible compensatory effect of femoral arteries.28 This is an interesting finding for the adenylyl cyclase/cAMP-transduction pathway À/À the development of new therapies for erectile in the sGCa1 mice. Furthermore, this unaltered À/À dysfunction, as the role of endothelial NO in penile forskolin-induced response in the sGCa1 mice erection is becoming more significant than origin- demonstrates that the reduced sGC-related responses ally thought.5 Moreover, our data suggest the in this study are not due to an aspecific impairment involvement of endothelium-derived NO induced of relaxation related to structural damage. by ACh released from parasympathetic nerve Our data not only illustrate the functional im- 29 fibers, as atropine inhibited the EFS-induced portance of the sGCa1b1 isoform in vasorelaxations þ / þ relaxation in the CC of sGCa1 mice. Those EFS- induced by endogenous NO but also by exogenous induced relaxations are completely mediated by NO, as the response to SNP and NO gas were À/À NO/sGC, as they were completely abolished by the significantly reduced in the CC of the sGCa1 mice. À/À sGC-inhibitor, ODQ. In the CC preparations of the However, in the sGCa1 preparations, SNP (release À/À sGCa1 mice, the response to EFS was completely of exogenous NO upon biotransformation) and NO abolished, even after prolonged stimulation (40 s), gas (represents exogenous NO as such) still elicit a indicating that sGCa1b1 also functions as the relaxing effect, indicating that sGCa1b1 is not the predominant target for neuronal NO. Furthermore, sole mechanism responsible for those relaxations.

International Journal of Impotence Research sGC in mice corpus cavernosum S Nimmegeers et al 283 ODQ, which inhibits both sGC isoforms, had construction of the adapted holders in the myo- a strong inhibitory effect on the exogenous graph. This work was supported by a grant of FWO- À/À NO-induced relaxations observed in the sGCa1 Vlaanderen and the Bijzonder Onderzoeksfonds mice. Therefore, we suggest that the minor sGCa2b1 (BOF-GOA) of Ghent University. EB was supported isoform also participates in the responses to SNP by an award from the Northeast Affiliate Research and NO gas. Additionally, we suggest that (an) sGC- Committee of the American Heart Association. independent mechanism(s) may also be involved, as À/À the relaxing effect of exogenous NO in the sGCa1 CC is not completely abolished by ODQ. It has been shown that by the stimulation of Na þ /K þ -ATPase activity, NO (derived from SNP) relaxes human References corpus cavernosum smooth muscle cells indepen- dently of its ability to increase the intracellular 1 Andersson KE, Wagner G. Physiology of penile erection. 33 Physiol Rev 1995; 75: 191–236. cGMP concentration. The ability of NO to directly 2 Kim N, Azadzoi KM, Goldstein I, Saenz de Tejada I. A nitric activate calcium-dependent potassium channels or oxide-like factor mediates nonadrenergic-noncholinergic neu- sarcoplasmic/endoplasmic reticulum Ca2 þ -ATPase, rogenic relaxation of penile corpus cavernosum smooth as described for vascular smooth muscle cells,34 muscle. J Clin Invest 1991; 88: 112–118. 3 Hurt KJ, Musicki B, Palese MA, Crone JK, Becker RE, might be involved. Moriarity JL et al. Akt-dependent phosphorylation of The administration of T-1032, which blocks the endothelial nitric-oxide synthase mediates penile erection. hydrolysis of cGMP by phosphodiesterase type 5, Proc Natl Acad Sci USA 2002; 99: 4061–4066. resulted in a relaxation that is significantly reduced in 4 Toda N, Ayajiki K, Okamura T. Nitric oxide and penile erectile À/À þ / þ function. Pharmacol Ther 2005; 106: 233–266. the sGCa1 penile tissue as compared to sGCa1 5 Musicki B, Burnett AL. eNOS function and dysfunction in the penile tissue. This observation suggests a smaller basal penis. Exp Biol Med (Maywood) 2006; 231: 154–165. À/À sGC-activity in the sGCa1 mice. Molecules such as 6 Friebe A, Koesling D. Regulation of nitric oxide-sensitive BAY 41-2272, which stimulate sGC without the need guanylyl cyclase. Circ Res 2003; 93: 96–105. for NO,26 are of particular interest for erectile 7 Lohmann SM, Vaandrager AB, Smolenski A, Walter U, de Jonge HR. Distinct and specific functions of cGMP-dependent dysfunction patients who respond less well to protein kinases. Trends Biochem Sci 1997; 22: 307–312. phosphodiesterase type 5 inhibitors because of severe 8 Hedlund P, Aszodi A, Pfeifer A, Alm P, Hofmann F, Ahmad M endothelial and/or nerve dysfunction.35 The finding et al. Erectile dysfunction in cyclic GMP-dependent kinase that the response to BAY-41-2272 was significantly I-deficient mice. Proc Natl Acad Sci USA 2000; 97: 2349–2354. diminished but not completely abolished in the 9 Garbers DL. Purification of soluble guanylate cyclase from rat À/À lung. J Biol Chem 1979; 254: 240–243. sGCa1 preparations implies that besides sGCa1b1, 10 Harteneck C, Koesling D, Soling A, Schultz G, Bohme E. sGCa2b1 and/or (an) sGC-independent mechanism(s) Expression of soluble guanylyl cyclase. Catalytic activity also participate(s) in the relaxation. BAY 41-2272 has requires two enzyme subunits. 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Func- Conclusions tional properties of a naturally occurring isoform of soluble guanylyl cyclase. Biochem J 1998; 335(Part 1): 125–130. 15 Mergia E, Russwurm M, Zoidl G, Koesling D. Major occur- The present study demonstrates the involvement of rence of the new alpha2beta1 isoform of NO-sensitive the predominantly expressed isoform, sGCa1b1,in guanylyl cyclase in brain. Cell Signal 2003; 15: 189–195. murine CC smooth muscle relaxation in response to 16 Ushiyama M, Morita T, Kuramochi T, Yagi S, Katayama S. NO and NO-independent sGC stimulator. However, as Erectile dysfunction in hypertensive rats results from impair- some responsiveness to exogenous NO (SNP and NO ment of the relaxation evoked by neurogenic carbon monoxide and nitric oxide. Hypertens Res 2004; 27: 253–261. gas) and an sGC stimulator (BAY 41-2272) remains in À/À 17 Burnett AL, Johns DG, Kriegsfeld LJ, Klein SL, Calvin DC, the sGCa1 mice, the less abundantly expressed Demas GE et al. Ejaculatory abnormalities in mice with isoform, sGCa2b1, and/or (an) sGC-independent me- targeted disruption of the gene for heme oxygenase-2. Nat chanism(s) are also suggested to participate. Med 1998; 4: 84–87. 18 Schachter M. Erectile dysfunction and lipid disorders. Curr Med Res Opin 2000; 16(Suppl 1): s9–s12. 19 Saenz de Tejada I, Goldstein I, Azadzoi K, Krane RJ, Cohen Acknowledgments RA. Impaired neurogenic and endothelium-mediated relaxation of penile smooth muscle from diabetic men with impotence. N Engl J Med 1989; 320: 1025–1030. We thank the DMBR animal caretakers for maintain- 20 Abdel-Gawad M, Huynh H, Brock GB. Experimental chronic ing the animal facility and Cyriel Mabilde for the renal failure-associated erectile dysfunction: molecular

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