0031-3998/02/5201-0019 PEDIATRIC RESEARCH Vol. 52, No. 1, 2002 Copyright © 2002 International Pediatric Research Foundation, Inc. Printed in U.S.A. Sildenafil Reverses O2 Constriction of the Rabbit Ductus Arteriosus by Inhibiting Type 5 Phosphodiesterase and Activating BKCa Channels BERNARD THÉBAUD, EVANGELOS MICHELAKIS, XI-CHEN WU, GWYNETH HARRY, KYOKO HASHIMOTO, AND STEPHEN L. ARCHER Department of Medicine (Cardiology) and the Vascular Biology Group [B.T., E.M., X.-C.W., G.H., K.H., S.L.A.], Department of Physiology [S.L.A.], University of Alberta, Edmonton, Alberta, T6G 2S2, Canada ABSTRACT Oxygen constriction causes functional closure of the ductus the DA by increasing soluble guanylyl-cyclase–derived cGMP arteriosus (DA) at birth. Although DA closure is crucial for levels and thereby activating calcium-sensitive potassium chan- postnatal adaptation, patency of the DA is critical for survival of nels, causing membrane hyperpolarization. Sildenafil, already newborns with duct-dependent cardiac malformations. In these approved for human usage, might be an alternative or a useful cases, DA patency is achieved by i.v. infusion of prostaglandin adjunct to prostaglandin E1 as a bridge to cardiac surgery. E1, which, though effective, is often associated with complica- (Pediatr Res 52: 19–24, 2002) tions. We hypothesized that sildenafil, a specific phosphodiester- ase type 5 inhibitor, is an effective DA vasodilator. In isolated Abbreviations DA rings from term (d 30) fetal rabbits, sildenafil (10Ϫ6–10Ϫ4 4-AP, 4-aminopyridine Ϫ7 Ϫ5 M) and diethylamine NONOate (10 –10 M) induced dose- BKCa, large conductance calcium-sensitive potassium channels dependent relaxation of oxygen-constricted DA (Ϫ52 Ϯ 4% and cGMP, 3',5'-cyclic GMP Ϫ51 Ϯ 6%, respectively) that was inhibited by the soluble DA, ductus arteriosus guanylyl-cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxa- DASMC, DA smooth muscle cells lin-1-one (5 ϫ 10Ϫ5 M). Sildenafil increased cyclic GMP levels. DEANO, diethylamine NONOate Iberiotoxin (200 nM), an inhibitor of calcium-sensitive potas- Em, membrane potential sium channels, decreased the vasodilatory effect of sildenafil and IBTX, iberiotoxin diethylamine NONOate (Ϫ30 Ϯ 2% and Ϫ27 Ϯ 4%, respective- IK, whole-cell potassium current ly). Oxygen inhibition of whole-cell Kϩ current and membrane NO, nitric oxide depolarization were partially restored by sildenafil, and this was O2, oxygen inhibited by iberiotoxin. Immunohistochemistry and immuno- ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one blotting confirmed the presence of phosphodiesterase type 5 and PDE-5, type 5 phosphodiesterase calcium-sensitive potassium channels in the DA smooth muscle PG, prostaglandin cells. This is the first study to demonstrate that sildenafil dilates sGC, soluble guanylate cyclase The DA is a vital fetal structure that connects the pulmonary tation, is initiated by an increase in O2. In full-term newborns, artery to the descending aorta. During fetal life, the DA shunts the DA closes 24–48 h after delivery (1). However, in new- over half of the blood flow away from the lung into the borns with duct-dependent cyanotic cardiac malformations umbilico-placental circulation, where gas exchange takes place (e.g. transposition of the great vessels) or duct-dependent aortic (1). At birth, closure of the DA, essential for postnatal adap- obstructions (including hypoplastic left heart syndrome and coarctation of the aorta), patency of the DA is critical for survival until surgery. The paramount importance of PG in the Received December 21, 2001; accepted March 1, 2002. regulation of ductal tone is well established [reviewed in (2)]. Correspondence: Stephen Archer, M.D., Cardiology, University of Alberta, 2C2.36 Walter Mackenzie Health Sciences Center, Edmonton, AB, Canada T6G 2B7; e-mail: This has resulted in successful pharmacological manipulation [email protected] of the DA (3). To date, PGE1 is the only available therapy to B.T. is supported by the Canadian Institutes for Health Research, and by the Alberta Heritage Foundation for Medical Research, a grant from the French Ministry for Foreign maintain DA patency (4). Although effective, numerous side Affairs, the Fondation pour la Recherche Médicale and the Société Française de Médecine effects are associated with PGE1 infusions (respiratory depres- Périnatale. E.M. and S.L.A. are both supported by the Alberta Heritage Foundation for sion, fever, lethargy, irritability, myoclonic jerks, flushing, Medical Research, the Canadian Institutes for Health Research, the Heart and Stroke Foundation of Canada, and the Canadian Foundation for Innovation. edema, pyloric stenosis, hyperostosis, necrotizing enterocolitis, 19 20 THÉBAUD ET AL. and structural remodeling of the DA and the pulmonary ves- potential of Ϫ70 mV and currents were evoked by steps of 200 sels) (5, 6). The reported incidence of this host of complica- ms duration from Ϫ70 to ϩ70 mV. tions ranges from 10 to 40%. To determine the effect of sildenafil on IK and Em, hypoxic Ϫ5 Although PGs are major endogenous regulators of DA tone, DASMC were successively exposed to O2 and sildenafil (10 endothelium-derived NO also modulates DA tone (7, 8). NO M). To characterize the type of current activated by sildenafil, activates smooth muscle sGC, which increases intracellular the DASMC were exposed to 4-AP (5 mM) and then IBTX cGMP levels, causing vasodilatation. cGMP in turn, is rapidly (100 nM). In additional experiments, the effects of sildenafilon degraded by the cGMP-specific PDE-5. Basal stimulation of Em in normoxic DASMC were recorded after exposure to sGC and increased smooth muscle cGMP levels contribute to IBTX or 4-AP. DA relaxation (9, 10). Consequently, we hypothesized that Immunoblotting. Immunoblots were performed on fresh inhibition of cGMP breakdown might be efficacious in achiev- hypoxic DA using a panel of protease inhibitors, as previously ing dilatation of the DA. Sildenafil, readily available orally and described (15). Antibodies were obtained from Calbiochem widely used in the treatment of erectile dysfunction (11), is a (San Diego, CA, U.S.A.; PDE-5) and Alomone Lab (Jerusa- potent vasodilator that enhances and prolongs the action of lem, Israel; BKCa). cGMP by selectively inhibiting PDE-5. Furthermore, cGMP Immunohistochemistry. Immunohistochemistry was per- induces vasodilatation, in large part, by activating protein formed on paraffin-embedded, formaldehyde-fixed DA, as pre- kinase G and thereby opening BKCa, causing membrane hy- viously described (15). After overnight incubation with pri- perpolarization (12, 13). Therefore, we assessed the efficacy mary antibody (4°C), slides were incubated for 20 min with the and mechanism of action of sildenafilinO2-constricted, term secondary antibody, either biotinylated anti-Rabbit IgG (for rabbit DA. PDE-5 and BKCa protein) or anti-Mouse IgG [for endothelial nitric-oxide synthase (eNOS) protein] (Vector Laboratories, METHODS Burlingame, CA, U.S.A.) each at 1/200 dilution. The signal was revealed using a Chromagen (DAB) kit (Biogenex, San All procedures were approved by the Animal Health Care Ramon, CA, U.S.A.). In all cases, the secondary antibody Committee of the University of Alberta. Fetal New Zealand created reddish-brown staining. Antibodies for PDE-5 (1/1000) White rabbits were delivered by cesarean section at 30 d of and BKCa (1/100) were the same as for the immunoblotting. gestation (term ϭ 31 d) and killed with an i.p. overdose of Antibodies for eNOS were obtained from BD Transduction pentobarbital. The DA was excised under a dissecting micro- Lab (Oakville, ON, Canada). scope and used within 5 min in the following studies. Statistics and drugs. Values are expressed as mean Ϯ SEM. Isolated DA rings. The isolated DA was placed in an organ Intergroup comparisons were performed with a t test or a bath and studied in Krebs solution containing meclofenamate factorial, repeated-measures ANOVA, as appropriate. Fisher’s (10Ϫ5 M), as previously described (14). After 1 h equilibration probable least significant differences test was used for post hoc ϭ Ϯ Ͻ in hypoxia (PO2 31 1 mm Hg, a level similar to that in comparisons. A p value of 0.05 was considered to be ϭ utero), the DA was exposed to a normoxic solution (PO2 127 statistically significant. All drugs, except sildenafil, a generous Ϯ 1 mm Hg) and the vasodilatory effect of sildenafil (10Ϫ6– gift from Pfizer Pharmaceuticals (Sandwich, England), were 10Ϫ4 M), the NO donor DEANO (10Ϫ7–10Ϫ5 M), and PGE1 purchased from Sigma Chemical (St. Louis, MO, U.S.A.). (10Ϫ12–10Ϫ9 M) were compared by administering each agent Sildenafil, ODQ, and IBTX were dissolved in DMSO. PGE1 at the peak of O2 constriction. To further assess the mechanism was dissolved in ethanol. DEANO was dissolved in water of sildenafil-induced DA dilatation, we compared the vasodi- immediately before use and kept refrigerated. latory effect of these drugs after preincubation of the DA with ϫ Ϫ5 the sGC inhibitor, ODQ (5 10 M), or the specificBKCa RESULTS channel blocker IBTX (200 nM). In additional experiments, we studied the effects of preincubation with vehicle, sildenafil Sildenafil relaxes O2-constricted DA. In O2-constricted DA, Ϫ4 ϫ Ϫ5 ϩ (10 M), ODQ (5 10 M), or sildenafil ODQ on O2 sildenafil, DEANO, and PGE1 induce cumulative, dose- constriction. Relaxation was expressed as percentage relax- dependent, relaxations (Ϫ52 Ϯ 4%, Ϫ51 Ϯ 6%, and Ϫ73 Ϯ ation of the active tone. 4%, respectively, Fig. 1, A–C). Treatment of DA rings with Ϫ4 cGMP levels. To assess whether sildenafil causes relaxation sildenafil (10 M) before exposure to O2 also attenuates by increasing cGMP levels in the DA, we determined cGMP O2-induced constriction (Fig. 1D). levels in the Krebs solution taken from the ring bath under sGC inhibition reduces sildenafil-induced relaxation and hypoxia and normoxia in the following conditions: after pre- cGMP accumulation. ODQ (5 ϫ 10Ϫ5 M) significantly re- incubation of the DA with vehicle, sildenafil (10Ϫ4 M), or duces sildenafil- and DEANO-induced relaxation but not PGE1 sildenafil ϩ ODQ (5 ϫ 10Ϫ5 M).