Activation of Soluble Guanylate Cyclase Causes Relaxation of Corpus Cavernosum Tissue: Synergism of Nitric Oxide and YC-1

Home , Nitric oxide synthase, Soluble guanylyl cyclase

Activation of soluble guanylate cyclase causes relaxation of corpus cavernosum tissue: synergism of nitric oxide and YC-1

M Nakane1*, G Hsieh1, LN Miller1, R Chang1, MA Terranova1, RB Moreland1, T Kolasa1 and JD Brioni1

1Neuroscience, Global Pharmaceutical Research, Abbott Laboratories, Abbott Park, Illinois, USA

Nitric oxide (NO) activates corpus cavernosum smooth muscle soluble guanylate cyclase (sGC) and increases the synthesis of cGMP that results in smooth muscle relaxation and ultimately, penile erection. To characterize sGC and define the potential synergy between NO and the allosteric activator YC-1 in corpus cavernosum, rat sGC was activated by either sodium nitroprusside (SNP) or YC-1, and YC-1 potentiated the effects of SNP with a 200-fold activation of sGC. Both SNP and YC-1 decreased the Km and increased the Vmax. ODQ significantly inhibited sGC activated by SNP with IC50 of 0.5 nM, but did not affect the sGC activated by YC-1 as well as basal sGC activity. SNP and YC-1 synergistically increased intracellular cGMP levels in rabbit corpus cavernosum smooth muscle cell cultures. YC-1 significantly relaxed rabbit cavernosum tissue strips in organ baths with an EC50 of 8.4 mM. In the presence of L-nitroarginine methyl ester to block endogenous NO production, co-administration of SNP shifted the dose response of YC-1 to the left, showing the synergism of SNP and YC-1 in tissue strips. In view of the clinical efficacy of phosphodiesterase-5 inhibitors, activation of sGC may provide an alternative means for enhancing the activity of neurally derived NO during sexual stimulation in the corpus cavernosum, representing a novel approach for the treatment of erectile dysfunction. International Journal of Impotence Research (2002) 14, 121–127. DOI: 10.1038=sj=ijir=3900843

Keywords: guanylate cyclase; activator; nitric oxide; YC-1; sodium nitroprusside; corpus cavernosum

Introduction cGMP. On the other hand, NO or NO donors including nitroglycerine and sodium nitroprusside (SNP) are well-known sGC activators. Nitric oxide (NO), a key neurotransmitter mediating A novel type of sGC activators is represented by penile erection, exerts its effects by activating YC-1.3 This agent is not an NO donor, but causes corpus cavernosum smooth muscle soluble guany- activation of sGC especially in the presence of 1,2 late cyclase (sGC). sGC is responsible for the NO.4,5 The finding that YC-1 activates sGC by enzymatic conversion of GTP to cyclic GMP (cGMP), binding to an allosteric site on the enzyme opened and the increase of cGMP mediates relaxation the possibility to discover a new class of compounds of cavernosal smooth muscle leading to penile with a different pharmacological profile in compar- erection. ison to the NO donors. The goal of this study was to The discovery that NO-sGC-cGMP system is one characterize sGC and define the potential synergy of the major effectors in penile smooth muscle between NO and the allosteric activator YC-1 in relaxation has led to the development of two classes corpus cavernosum, and to discuss the possibility of agents that increase cGMP levels in cavernosal that the regulation of sGC by activators may smooth muscle: (a) agents that inhibit cGMP represent a new approach for the treatment of degradation (phosphodiesterase inhibitors); and (b) erectile dysfunction. agents that elevate cGMP synthesis (sGC activators). The latter class can be divided into NO-dependent and NO-independent sGC activators. Sildenafil increases cGMP levels by inhibiting Materials and methods phosphodiesterase-5, the enzyme that degrades Chemicals *Correspondence: M Nakane, D4ND, AP9, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064- 0 0 6119, USA. YC-1 (3-(5 -hydroxymethyl-2 -furyl)-1-benzyl inda- E-mail: [email protected] zole), ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin- Received 31 August 2001; revised 20 December 2001; 1-one), L-NAME (No-nitro-L-arginine methyl ester) accepted 11 January 2002 and SNP (sodium nitroprusside) were purchased Activation of guanylate cyclase causes relaxation of cavernosal smooth muscle M Nakane et al 122 from Sigma RBI (St Louis, MO, USA), and GTP sGC expression obtained from Roche Molecular Biochemicals (In- dianapolis, IN, USA). Oligonucleotides were synthe- The cells were grown to 26106 cells=ml and then sized and purified in-house. Other reagents were infected with a mixture of the virus stocks for the a1- obtained from Sigma, otherwise indicated. and b1-subunits for protein production. We found that the best expression of sGC activity was achieved by a multiplicity of infection of 0.1 pfu each sGC expression in human corpus cavernosum subunit=cell and the three days incubation at 28C. Cells were harvested and stored at 7 80C. The cell To identify the sGC isoforms expressed in human pellets were thawed on ice and were homogenized penile corpus cavernosum, reverse transcriptase- with Tekmer Sonic Disruptor (Cincinnati, OH, USA) polymerase chain reaction (RT-PCR) analysis was in 5 vol of 20 mM Tris – HCl (pH 7.6) containing performed. Human penile corpus cavernosum tis- 1 mM EDTA, 5 mM dithiothreitol, 90 mM NaCl, 10% sues were obtained from Zoion Diagnostics (Shrews- glycerol, and 1 Protease Inhibitor Cocktail Tablet per bury, MA, USA). Total RNA was isolated using every 50 ml (Roche Molecular Biochemicals, India- TRIzol reagent (Life Technologies). Five micrograms napolis, IN, USA). The homogenate was centrifuged of total RNA was reverse-transcribed in a 20 ml at 50000 g for 30 min, and the supernatant was used reaction mixture containing 500 ng of oligo(dT)12 – 18, for GC purification. Purification was performed from 0.5 mM deoxynucleoside triphosphates, 10 mM 3 l culture of High5 cells as described previously,9 dithiothreitol, 5 mM MgCl2, 40 units RNase inhibitor and the purified preparation was aliquoted and in RT reaction buffer and 50 units of SuperScript II stored at 7 80C until use. RT (Life Technologies) at 42C for 50 min. PCR reactions were carried out in a 50 ml reaction mixture containing 0.2 mM deoxynucleoside triphosphates, GC enzyme assay 1.5 mM MgCl2, 20 nM of specific primers, cDNA, and two units of Taq DNA polymerase in PCR GC enzyme activity assay was performed as de- reaction buffer (Life Technologies). Amplified pro- scribed previously with modifications to allow us to ducts were resolved on a 1% agarose gel containing assay large number of samples at the same time.7 ethidium bromide. The following PCR primer pairs Assay mixtures contained 50 mM Tris – HCl (pH and conditions were used: sGC a1 subunit (488 bp 7.6), 4 mM MgCl2, 5 mM creatin phosphate, 0.5 mM product), 50-TGC CTC CCT GCT TCC ATA AT-30 IBMX, 4 units=ml creatin kinase and 1 mM GTP in a (sense) and 50-GTA GAG CCC TCG TCC TGT AAA volume of 100 ml in 96-well plate (Thermowell) 0 ATC-3 (antisense); sGC a2 subunit (392 bp product), (Costar, Corning, NY, USA). The incubation was 50-TGT ACA CCA GAT TTG ACC ACC AGT-30 carried out for 10 min at 37C followed by 95C for (sense) and 50-ACG AGA CCG CGG AAT GAA TG- 2 min to terminate the reaction using GeneAmp PCR 0 0 3 (antisense); sGC b1 subunit (846 bp product), 5 - System 9600 (PE Biosystems, Foster City, CA, USA). TAA GAG CCC TGG AAG ATG AAA AGA-30 (sense) Produced cGMP from GTP was quantified by and 50-TGG GGT AAT GGA CAA GGA CAA A-30 enzyme immunoassay (Amersham Pharmacia Bio- 0 (antisense); sGC b2 subunit (460 bp product), 5 -AGG tech, Piscataway, NJ, USA). GAA GAA GGA GCA TGT TGT GTT-30 (sense) and 50-TCT GCG GAT GCT GAA AAT GTT GA-30 (antisense).1 Samples were denatured at 94C for cGMP measurement in smooth muscle cell culture 30 s, followed by 30 cycles at 94C for 30 s, 55C for 30 s, and 72C for 60 s. Corpus cavernosal smooth muscle tissues were prepared from adult male New Zealand white rabbits (Covance Research Production, Kalamazoo, Recombinant baculovirus MI, USA). Explant cavernosal tissues were placed in small petri dishes in Dulbecco’s Modified Eagle 6,7 Rat sGC a1-andb1-subunits were subcloned into Medium containing 10% fetal bovine serum, anti- pVL1392 (Invitrogen, Carlsbad, CA, USA) and pre- biotics, and amphotercin B. The explants were kept pared recombinant baculoviruses encoding the a1 undisturbed for 3 – 4 days in 37 C5%CO2 incuba- 8 and the b1 subunits of sGC by the standard protocol. tor. Smooth muscle cells migrated out from the tissues underwent proliferation within 7 days. The explants were removed and the medium was Insect cell culture changed every 3 days. Once confluent, cells were detached with trypsin – EDTA and sub-cultured at a Sf9 or High5 cells were grown in suspension culture density of 0.56104 cells=well in 24-well culture in flasks or roller bottles at 28C. SF900II serum-free plates. All experiments were performed in caverno- medium (Life Technologies) was used for culture sum cells that had been grown for 48 h ( > 90% without any supplements. confluent).

International Journal of Impotence Research Activation of guanylate cyclase causes relaxation of cavernosal smooth muscle M Nakane et al 123 The culture medium was changed and washed Results with Hank’s BSS solution three times immediately before each experiment. The cells were incubated with pharmacological agents with or without To characterize sGC expressed in human corpus 100 mM IBMX prepared in Hank’s BSS for 10 min. cavernosum, RNA was extracted from tissue sam- Cells were lysed with 0.5% (w=v) dodecyl triammo- ples obtained from eight patients and RT-PCR was nium bromide in 50 mM sodium acetate buffer (pH performed. As shown in Figure 1, specific amplifi- 5.8). Total cellular cGMP levels were determined cation products encoding the a1 and b1 subunits using enzyme immunoassay (Amersham Pharmacia were detected after 30 cycles of PCR. Very weak Biotech) after acetlylation of cGMP. products encoding a2 subunits were detected from four patients, but none of the b2 subunit products were detected after 30 cycles. Although a2 and b2 subunits were detected after 45 cycles of PCR (data not shown), we conclude that the main isoform of sGC expressed in human corpus cavernosum is Organ bath studies a1=b1. Based on the results above, we expressed a1=b1 Rabbit corpus cavernosum strips were obtained from isoform of rat sGC in baculovirus system, and euthanized New Zealand White rabbits (Covance purified it to homogeneity. The purified rat sGC Research Production, Denver, PA, USA), weighing was highly activated by either SNP or YC-1 (Figure 3.5 – 4.0 kg. The two corpus cavernosum strips were 2). SNP showed a concentration-dependent activa- dissected from the tunica albugenea and each was tion of sGC with a maximal effect of 50-fold longitudinally cut into two to three strip prepara- activation at 10 – 100 mM and an EC50 value of 2 mM tions. The strips were mounted in an organ bath (Figure 2A). YC-1 also activated sGC with an effect (10 ml) containing Kreb-Henseleit buffer (Sigma, St of 50-fold activation at 100 mM (Figure 2B). The activation by YC-1 did not reach maximum at Louis, MO, USA) with 2.5 mM CaCl2,25mM 100 mM, which is the highest concentration tested NaHCO3 and 4 mM DL-propranolol, pH 7.4 at 37 C, 10 because of the solubility of YC-1. Interestingly, YC-1 which were aerated with 95% O2 and 5% CO2. The strips were equilibrated for 90 min with a resting and SNP showed synergistic effects over the range of tension of 2 g. Isometric tension of muscle tissue was the dose tested with a maximal activation of sGC by measured using a force-displacement transducer 10 mM SNP and 100 mM YC-1 of around 200-fold (FT-202, Kent Scientific, Litchfield, CT, USA) and activation and one magnitude shift of EC50 to lower recorded on a Powerlab 800 (AD Instruments, Castle concentrations (Figure 2A and B). Hill, Australia) and calculated by Prism (GraphPad Effect of SNP and=or YC-1 on the kinetic proper- ties of sGC was examined. Ten micromoles of SNP Software, San Diego, CA, USA). EC50 of phenylephrine-induced contraction was determined by cumula- (maximum effect of activation, see Figure 2) in- tively adding phenylephrine (161078 to 361074 M) creased Vmax from 1.19 mmol=min=mg to 13.3 mmol= to the tissue bath. The strips were washed, re- min=mg (11-fold increase), and lowered Km from equilibrated and stretched with tension of 2 g for 446 mM to 130 mM (3.4-fold decrease). Also, 100 mM YC-1 (maximum effect of activation, see Figure 2) 90 min. The strips were pre-contracted by EC50 concentration of phenylephrine for 15 min. Drugs increased Vmax from 1.19 mmol=min=mg to were cumulatively added from low to high to the 16.2 mmol=min=mg (14-fold increase), and lowered chamber with 7 min intervals. Relaxation response Km from 446 mM to 239 mM (1.9-fold decrease, Figure was expressed as percentage of total tone induced by 3). When 10 mM SNP and 100 mM YC-1 were combined, a 30-fold increase of the V and a 14- the addition of EC50 of phenylephrine. All data were max expressed as means Æ s.e.m. fold decrease of the Km were observed, indicating

Figure 1 sGC expression in human corpus cavernosum. Total RNA from eight (number 1 to number 8) human penile corpus cavernosum tissues was isolated and ran RT-PCR as described in Materials and methods using the primer pairs of a1 subunit (488 bp product), a2 subunit (392 bp product), b1 subunit (846 bp product) and b2 subunit (460 bp product).

International Journal of Impotence Research Activation of guanylate cyclase causes relaxation of cavernosal smooth muscle M Nakane et al 124 that the conformational changes brought about by strips in vitro. YC-1 significantly relaxed caverno- NO and by YC-1 are synergistic. This result also sum tissue strips in a dose-dependent manner with supports the proposal that YC-1 binding site would an EC50 of 8.4 mM (Figure 6A). When the strips were be different from NO binding site (the heme domain pre-treated with 100 mM of nitric oxide synthase of sGC,5 and YC-1 is an allosteric activator of sGC. inhibitor, L-NAME to avoid the endogenous NO ODQ has been reported to be a potent inhibitor of effect, YC-1 induced a concentration-dependent 11,12 NO-activated sGC. ODQ binds to sGC in an NO- relaxation, but less potently (EC50 ¼ 50 mM) (Figure competitive manner and inhibits NO-stimulated 6B). This indicates that endogenous NO signifi- activity by oxidizing the ferrous heme in the first cantly contributes to the relaxation caused by YC-1 complex to a five liganded ferric intermediate. This prevents both binding and activation by NO, leaving basal activity unchanged.12,13 We tested the effect of ODQ on sGC activated by SNP and YC-1 (Figure 4). ODQ significantly inhibited sGC activated by 10 mM SNP with IC50 of 0.5 nM, but did not affect the sGC activated by 100 mM YC-1 as well as basal sGC activity, indicating that YC-1 binds to sGC at a different site from the heme (NO binding site). Having established in biochemical characteristics of sGC activation, we wanted to characterize this activity in smooth muscle cells in vitro.We prepared a primary culture of rabbit corpus cavernosum smooth muscle cells and observed the effect of SNP and=or YC-1 on the intracellular concentration of cGMP. YC-1 increased cGMP level dose- dependently (Figure 5). In the presence of 1 mM (sub-optimal concentration, data not shown) SNP, YC-1 drastically increased cGMP, much more than YC-1 alone, indicating that SNP and YC-1 synergistically increased intracellular cGMP levels in rabbit Figure 3 SNP and YC-1 change both Vmax and Km. sGC activity was measured with the increasing concentration of substrate GTP corpus cavernosum smooth muscle cells. in the absence or presence of 10 mM SNP, 100 mM YC-1, or 10 mM We examined the ability of sGC activators to relax SNP and 100 mM YC-1. Results are the means Æ s.e.m. from three phenylephrine pre-contracted cavernosum tissue independent experiments performed in duplicate.

Figure 2 SNP and YC-1 synergistically activate soluble guanylate cyclase. (A) sGC activity was measured in the presence of increasing concentration of SNP in the absence or presence of 1, 10, or 100 mM YC-1. (B) The same data shown as a function of the YC-1 concentration in the absence or presence of 0.1, 1, 10, or 100 mM SNP. Results are the means Æ s.e.m. from four independent experiments performed in duplicate.

International Journal of Impotence Research Activation of guanylate cyclase causes relaxation of cavernosal smooth muscle M Nakane et al 125 alone. Co-administration of SNP dose-dependently YC-1 has been identiﬁed as an inhibitor of shifted the dose response of YC-1 to leftward, platelet aggregation that led to an increase of showing the synergism of SNP and YC-1 in tissue intracellular cGMP.3,14 Soon after the ﬁnding the strips (Figure 6B). EC50 shifted from 50 mMto3mM pharmacological effect of YC-1 on platelets, YC-1 with 1 mM SNP. was found to be a modulator of sGC activity.15 Interestingly, YC-1 highly potentiates the stimula- tory effect of submaximally activating NO (Figure 1). In the presence of YC-1, the NO concentration – Discussion response curve is shifted to the left, indicating that YC-1 sensitized sGC towards NO. YC-1 binds to sGC at a different site from the In this study, YC-1 synergistically activates sGC in heme.5,16 This is evidenced by studies in which YC- the presence of sub-maximal concentration of NO, 1 can act as an allosteric activator in the absence of increases intracellular cGMP levels in cavernosal NO and lead to even more activation in the presence smooth muscle cells, and causes relaxation of corpus cavernosum.

Figure 5 SNP and YC-1 synergistically increase intracellular cyclic GMP in cultured smooth muscle cells from corpus Figure 4 ODQ inhibits NO-stimulated sGC activity, but does not cavernosum. Rabbit corpus cavernosum smooth muscle tissues inhibit YC-1-stimulated and basal sGC activity. sGC activity was were prepared as described in Materials and methods. The cells measured in the presence of increasing concentration of ODQ in were incubated with the increasing concentration of YC-1 with or the absence or presence of 10 mM SNP or 100 mM YC-1. Results are without 1 mM SNP in the presence of 100 mM IBMX for 10 min. the means Æ s.e.m. from three independent experiments per- Total cellular cGMP levels were determined using enzyme formed in duplicate and normalized with the activity without immunoassay. Results are the means Æ s.e.m.: **P < 0.05, ODQ as 100%. **P < 0.05, ***P < 0.01 vs basal. ***P < 0.01 vs vehicle.

Figure 6 SNP potentiates relaxation by YC-1 with pretreatment of L-NAME. Rabbit corpus cavernosum strips were dissected and prepared as described in Materials and methods. (A) The strips were pre-contracted by EC50 concentration of phenylephrine for 15 min and YC-1 was cumulatively added from low to high to the chamber with 7 min intervals. (B) The strips were pre-contracted by EC50 concentration of phenylephrine in the presence of 100 mM L-NAME for 15 min and YC-1 in the absence or presence of 0.1 mMor1mM SNP were cumulatively added from low to high to the chamber with 7 min intervals. Relaxation response was expressed as percentage of total tone induced by the addition of EC50 of phenylephrine and the results were expressed as means Æ s.e.m. **P < 0.05, ***P < 0.01 vs control.

International Journal of Impotence Research Activation of guanylate cyclase causes relaxation of cavernosal smooth muscle M Nakane et al 126 of NO, decreasing the Km for GTP and increasing the disorders, YC-1 as well as activators of sGC can also Vmax of cGMP formation (Figure 2). This site has be beneficial for the treatment of male erectile recently been mapped using a photoaffinity labeling dysfunction. technique to the a-subunit cysteines 238 and 243 of human sGC.17 This report does not agree with earlier work that suggested that cysteine 541 of the b- subunit was key for YC-1 interaction,18 and the Acknowledgements binding of YC-1 to the sGC heme-binding domain 19 leading to conformational changes. This may We thank Annemarie Rueter and Robert Simmer for reflect the nature of the conformational change as helping the expression of sGC in Baculovirus well as the initial binding of YC-1. system. For sGC to possess two different allosteric activators is fortuitous therapeutically. sGC activator binding to the allosteric non-heme site that in it has little effect on increasing the basal activity, but in References the presence of NO, can greatly amplify the effects of NO, would be a good candidate for male erectile 1 Behrends S, Steenpass A, Porst H, Scholz H. Expression of dysfunction. YC-1 activates sGC through a pathway nitric oxide-sensitive guanylyl cyclase subunits in human that does not involve the interaction with the heme corpus cavernosum. Biochem Pharmacol 2000; 59: 713 – 717. moiety and cause a pronounced sensitization of the 2 Klotz T et al. Soluble guanylate cyclase and cGMP-dependent enzyme for stimulation by NO.5 protein kinase I expression in the human corpus cavernosum. Int J Impot Res 2000; 12: 157 – 164. YC-1 induced a concentration-dependent relaxa- 3 Wu CC, Ko FN, Kuo SC, Lee FY, Teng CM. YC-1 inhibited tion in rabbit aortic rings pre-contracted with human platelet aggregation through NO-independent activa- phenylephrine by increasing intracellular cGMP tion of soluble guanylate cyclase. Br J Pharmacol 1995; 116: levels.20 We also observed that YC-1 synergistically 1973 – 1978. increased intracellular cGMP levels in rabbit caver- 4 Stone JR, Marletta MA. Synergistic activation of soluble guanylate cyclase by YC-1 and carbon monoxide: implications nosal smooth muscle cells (Figure 4) and induced a for the role of cleavage of the iron-histidine bond during relaxation in rabbit corpus cavernosum strips and activation by nitric oxide. Chem Biol 1998; 5: 255 – 261. the potencies shifted to the left in the presence of 5 Friebe A, Koesling D. Mechanism of YC-1-induced activation NO donor, SNP (Figure 5). The synergistic responses of soluble guanylyl cyclase. Mol Pharmacol 1998; 53: 123 – 127. to NO-donor and YC-1 in cultured cavernosal 6 Nakane M, Saheki S, Kuno T, Ishii K, Murad F. Molecular smooth muscle cells and isolated corpus caverno- cloning of a cDNA coding for 70 kilodalton subunit of soluble sum strips apparently reflect the direct synergistic guanylate cyclase from rat lung. Biochem Biophys Res action of YC-1 and NO on the sGC. Commun 1988; 157: 1139 – 1147. In sGC enzyme assay, we observed that ODQ did 7 Nakane M, Arai K, Saheki S, Kuno T, Buechler W, Murad F. Molecular cloning and expression of cDNAs coding for soluble not block the activation of sGC by YC-1 (Figure 3). guanylate cyclase from rat lung. J Biol Chem 1990; 265: However, YC-1 induced a concentration-dependent 16841 – 16845. relaxation in rat aortic rings pre-contracted with 8O’Reilly DR. Use of baculovirus expression vectors. Methods phenylephrine, and the relaxant effects of YC-1 were Mol Biol 1997; 62: 235 – 246. 21 9 Hoenicka M et al. Purified soluble guanylyl cyclase expressed reversed by ODQ and potentiated by NO donor. in a baculovirus=Sf9 system: stimulation by YC-1, nitric oxide, The endogenous NO in tissue preparations pro- and carbon monoxide. J Mol Med 1999; 77:14– 23. duced by nitric oxide synthase can explain this 10 Gupta S et al. The expression of functional postsynaptic difference of ODQ effect. In pre-constricted rat aortic alpha2-adrenoceptors in the corpus cavernosum smooth rings, YC-1 produced a concentration-dependent muscle. Br J Pharmacol 1998; 123: 1237 – 1245. 11 Hussain AS, Marks GS, Brien JF, Nakatsu K. The soluble relaxation, and the concentration – response curve guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-alpha] was shifted towards the right, to higher concentra- quinoxalin-1-one (ODQ) inhibits relaxation of rabbit aortic tions of YC-1 when the aortas were pre-treated with rings induced by carbon monoxide, nitric oxide, and glyceryl nitric oxide synthase inhibitor, L-NAME.22 trinitrate. Can J Physiol Pharmacol 1997; 75: 1034 – 1037. 12 Zhao Y et al. Inhibition of soluble guanylate cyclase by ODQ. In view of the clinical efficacy of phosphodiester- Biochemistry 2000; 39: 10848 – 10854. ase-5 inhibitors, agents that activate sGC may 13 Wegener JW, Closs EI, Forstermann U, Nawrath H. Failure of provide a means for enhancing the activity of NO 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) to in the corpus cavernosum, representing a novel inhibit soluble guanylyl cyclase in rat ventricular cardiomyo- approach for the treatment of erectile dysfunction. cytes. Br J Pharmacol 1999; 127: 693 – 700. 14 Ko FN et al. YC-1, a novel activator of platelet guanylate Pharmacological studies in vivo support this con- cyclase. Blood 1994; 84: 4226 – 4233. cept. Systemic injections of YC-1 facilitated penile 15 Teng CM et al. YC-1, a nitric oxide-independent activator of erections in rats, indicating that the activation of soluble guanylate cyclase, inhibits platelet-rich thrombosis in sGC via this allosteric site in the enzyme can lead to mice. Eur J Pharmacol 1997; 320: 161 – 166. 16 Brandish PE, Buechler W, Marletta MA. Regeneration of the penile erection in mammals (manuscript in prepara- ferrous heme of soluble guanylate cyclase from the nitric oxide tion). Although YC-1 has already been proposed for complex: acceleration by thiols and oxyhemoglobin. Biochem- the treatment of cardiovascular and thrombotic istry 1998; 37: 16898 – 16907.

International Journal of Impotence Research Activation of guanylate cyclase causes relaxation of cavernosal smooth muscle M Nakane et al 127 17 Stasch JP et al. NO-independent regulatory site on soluble 21 Wegener JW, Gath I, Forstermann U, Nawrath H. Activation of guanylate cyclase. Nature 2001; 410: 212 – 215. soluble guanylyl cyclase by YC-1 in aortic smooth muscle but 18 Friebe A, Russwurm M, Mergia E, Koesling D. A point- not in ventricular myocardium from rat. Br J Pharmacol 1997; mutated guanylyl cyclase with features of the YC-1-stimulated 122: 1523 – 1529. enzyme: implications for the YC-1 binding site? Biochemistry 22 Wohlfart P et al. Release of nitric oxide from endothelial cells 1999; 38: 15253 – 15257. stimulated by YC-1, an activator of soluble guanylyl cyclase. 19 Denninger JW et al. Interaction of soluble guanylate cyclase Br J Pharmacol 1999; 128: 1316 – 1322. with YC-1: kinetic and resonance Raman studies. Biochemistry 2000; 39: 4191 – 4198. 20 Mulsch A et al. Effect of YC-1, an NO-independent, super- oxide-sensitive stimulator of soluble guanylyl cyclase, on smooth muscle responsiveness to nitrovasodilators. Br J Pharmacol 1997; 120: 681 – 689.

International Journal of Impotence Research