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

International Journal of Impotence Research (2002) 14, 121–127 ß 2002 Nature Publishing Group All rights reserved 0955-9930/02 $25.00 www.nature.com/ijir 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.

Load more