International Journal of Impotence Research (2012) 24, 69–76 & 2012 Macmillan Publishers Limited All rights reserved 0955-9930/12 www.nature.com/ijir ORIGINAL ARTICLE Increased cavernosal relaxation by Phoneutria nigriventer toxin, PnTx2-6, via activation at NO/cGMP signaling KP Nunes1,2, BM Wynne2, MN Cordeiro3, MH Borges3, M Richardson3, R Leite4, ME DeLima1 and RC Webb2 1Department of Biochemistry, Biological Science Institute Federal University of Minas Gerais Gerais, Belo Horizonte, Brazil; 2School of Medicine—Department of Physiology, Medical College of Georgia, Augusta, GA, USA; 3Ezequiel Dias Foundation, Belo Horizonte, Brazil and 4School of Pharmacy—Federal University of Ouro Preto, Ouro Preto, Brazil Erectile dysfunction (ED) mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cyclic guanosine monophosphate (cGMP). This effect is neuronal nitric À5 oxide synthase (nNOS) dependent. Cavernosal strips were contracted with phenylephrine (10 M) and relaxed by electrical field stimulation (20 V, 1–32 Hz) in the presence or absence of PnTx2-6 À8 (10 M). Cavernosal strips from nNOS- and endothelial nitric oxide synthase (eNOS)-knockout À5 (KO) mice, besides nNOS inhibitor (10 M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with À4 PnTx2-6 in presence or absence of N-nitro-L-arginine methyl ester (L-NAME) (10 M)andx- À6 conotoxin GVIA (10 M), an N-type calcium channel inhibitor. Results showed that PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however, L-NAME abolished this enhancement as well as x-conotoxin GVIA. We conclude that PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing nitric oxide/cGMP production. International Journal of Impotence Research (2012) 24, 69–76; doi:10.1038/ijir.2011.47; published online 6 October 2011 Keywords: Phoneutria nigriventer; erectile dysfunction; PnTx2-6 toxin; NO; cGMP Introduction channels.5,11–13 The PhTx2 fraction as well as some of its isolated peptides has been shown to alter Venoms from arthropods are rich source of bioactive the kinetics of neuronal sodium (Na2 þ ) channel molecules, which can be useful as pharmacological inactivation.14–16 tools.1 It has been described that patients bitten by PnTx2-6, a polypeptide isolated from this fraction the Phoneutria nigriventer spider present various has shown a high affinity for Na2 þ channels, as symptoms including priapism.2–6 The venom from demonstrated in its affect on the voltage-dependent this arthropod, referred to as the ‘armed spider’,6 is gating of the Na2 þ channel. Previously published composed of a variety of distinct polypeptides papers from our group have demonstrated that which have been shown to elicit various biological incubation of PnTx2-6 in cavernosal strips increases activities.7–10 The majority of isolated toxins from relaxation, and others have shown that direct this venom have been described as neurotoxins, injection of this toxin into the penis leads to an which primarily induce effects on various ion erectile response.7,8 Additionally, this fraction preferentially localizes in penile tissue,17 which makes PnTx2-6 a direct target for research into the mechanisms of erectile function and reversal of Correspondence: Dr KP Nunes, Medical College of erectile dysfunction (ED). Georgia, Department of Physiology, 1120 15th Street, CA3135, Augusta, GA 30912, USA. It is widely accepted that regulation of erection is E-mail: [email protected] a complicated system under neuro-regulatory Received 7 January 2011; revised 28 April 2011; accepted control and consists of cholinergic, adrenergic and 26 August 2011; published online 6 October 2011 non-adrenergic non-cholinergic (NANC) effector PnTx2-6 toxin increased cavernosal relaxation via NO/cGMP KP Nunes et al 70 18,19 systems. Most investigators agree that nitric 1.17 mM MgSO4.7H2O, 1.6 mM CaCl2.2H2O and oxide (NO) is the primary mediator of the erectile 0.026 mM EDTA. Bretylium tosylate [(o-bromoben- response.18,20–26 Given that deregulated events in zyl) ethyldimethylammonium p-toluenesulfonate], the NO/cGMP pathway can cause impaired caver- o-conotoxin GVIA and 7-nitroindozole were nosal relaxation leading to ED, which is defined as purchased from Sigma Chemical (St Louis, MO, an inability to attain or to maintain a penile erection USA). N-nitro-L-arginine methyl ester (L-NAME) (NIH Consensus Conference, 1993). This condition was purchased from Cayman Chemical (Ann Arbor, has become increasingly prevalent throughout MI, USA). All reagents used were of analytical the United States and the world, and is said to grade. Stock solutions were prepared in deionized affect 19–64% of men.27,28 water and stored in aliquots at À20 1C dilutions ED risk factors include cardiovascular disease, were freshly prepared before use. The toxin lifestyle choices, such as obesity, smoking and PnTx2-6 was kindly provided by the Ezequiel Dias a sedentary lifestyle in addition to chronic Foundation (Brazil) and purified as given in reference illness.28–32 According to the Massachusetts Male Cordeiro.37 Aging Study, one such chronic illness is diabetes mellitus; these men were shown to have a 28% prevalence rate of ED as compared with 9.6% in the general population.33 Diabetes mellitus is a meta- Functional studies in cavernosal strips bolic disorder, where patients have hyperglycemia The mice were anesthetized (ketamine/xylazine, and insulin resistance and it frequently co-exists (100:10 mg kgÀ1, intraperitoneal injection) and ex- with a sedentary lifestyle and increased body mass sanguinated by aorta abdominal. After euthanasia, index. In diabetic patients and animal models, penes were excised and dissected in ice-cold buffer. vascular and endothelial dysfunction are a common The tunica albuginea was removed and one crural theme, with current literature suggesting that there strip preparation (1 Â 1 Â 10 mm) was obtained from is an impairment of vasodilatory signaling, de- each corpus cavernosum (two crural strips from creased NO in addition to NANC dysfunction.34,35 each penis). Cavernosal strips were mounted in 4-ml PnTx2-6 has been demonstrated to mediate penile myograph chambers (Danish Myo Technology, erection through an increase in NO, making it a Aarhus, Denmark) containing buffer at 37 1C con- possible therapeutic approach for diabetes-induced tinuously aerated with a mixture of 95% O2 and 5% ED. In addition, toxins have been used as a pharma CO2. The tissue was stretched to a passive force cological tool to study ED1 and recently it has been of 3.0 mN and allowed to equilibrate for 60 min; demonstrated that PnTx2-6 recombinant toxin the solutions were replaced every 10 to 15 min. (rPnTx2-6) was active in rat erectile function;36 as Changes in isomeric force were recorded using well as PnTx2-6. In this manuscript, we propose that a PowerLab/8SP data acquisition system (Chart PnTx2-6 can increase NO via neuronal nitric software, version 5.0; ADInstruments, Colorado oxide synthase (nNOS), as well as cGMP, which Springs, CO, USA). To verify the contractile ability can reverse the cavernosal hyperreactivity in strep- of the preparations, a high potassium chloride (KCl) tozocin (STZ)-diabetic mice. solution (120 mM) was added to the organ bath at the end of the equilibration period. All preparations were incubated for 35 min with bretylium tosylate Materials and methods (30 mM) to block sympathetic nerve discharge. Cavernosal strips were contracted with phenylephr- Animals ine (PE; 10 mM) and relaxation was evoked by Male C57Bl6 mice (10–12 weeks old, 22–28 g) and electrical field stimulation (EFS). Electrical stimuli genetically altered mice, which lack genes for nNOS were applied to strips placed between platinum and eNOS were used in these studies (Jackson pin electrodes, which were attached to a stimulus Laboratories, Bar Harbor ME, USA). All procedures splitter unit (Grass Technologies, an Astro-Med were carried out in accordance with the Guiding subsidiary, West Warwick, RI, USA) connected to a Principles in the Care and Use of Animals, approved Grass S88 stimulator (Grass Technologies, an Astro- by the Medical College of Georgia Committee and by Med subsidiary). EFS was conducted at 20 V, 1-ms the Federal University of Minas Gerais on the use of pulse width and trains of stimuli lasting 10 s at animals in research and education. The animals varying frequencies (1–32 Hz) before and after À8 were housed four per cage on a 12-h light/dark cycle incubation with PnTx2-6 (10 M, 4 min) and 7- À5 and fed a standard rat chow diet and water. nitroindozole (nNOS inhibitor, 10 M). The calcium channel blocker o-conotoxin GVIA À6 (10 M) was used to evaluate the participation of Drugs and solutions N-type Ca þ 2 channels in cGMP production in the Physiological salt solution of the following compo- presence of PnTx2-6. This specific Ca þ 2 channel À4 sition was used: 130 mM NaCl, 14.9 mM NaHCO3, inhibitor and/or the NOS inhibitor, L-NAME (10 M), 5.5 mM dextrose, 4.7 mM KCl, 1.18 mM KH2PO4, were added 35 min before cGMP measure levels. International Journal of Impotence Research PnTx2-6 toxin increased cavernosal relaxation via NO/cGMP KP Nunes et al 71 STZ-induced diabetes a dose-dependent increase in cavernosal relaxation, Diabetes is chiefly characterized by hyperglycemia, reaching significance at 4 Hz and 8 Hz (n ¼ 9, which was induced by using STZ.