Research Article 2,2 0 -Nitrophenylisatogen Potentiates P2X 1 Receptor Mediated Vascular Contraction and Blood Pressure Elevation Anna-Karin Wihlborg, 1 Johnny Sla ¨tt, 2 Xiangyang Sun, 3 Xiao-He Zhao, 3 Malin Malmsjo ¨, 1 Jan Bergman, 2 Thomas Hedner, 3 and David Erlinge 1* 1 Department of Cardiology, Lund University, Lund, Sweden 2 Organic Chemistry, Novum, Karolinska Institute, Stockholm, Sweden 3 Clinical Pharmacology, Gothenburg University, Gothenburg, Sweden Strategy, Management and Health Policy Venture Capital Enabling Technology Preclinical Research Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics Clinical Development Phases I-III Regulatory, Quality, Manufacturing Postmarketing Phase IV ABSTRACT The objective of this research was to examine the effects of chemical compounds with possible P2 receptor modulating effects and to characterize the potentiating effects of 2,2 0 -nitrophenylisatogen (NPI) on P2X 1 receptors in vitro and in vivo. Chemical compounds were tested in an in vitro pharmacological assay using vascular segments from the rat mesenteric artery stimulated by P2 receptor-specific agonists. Contractions were expressed as a percentage of 60 mM K + -induced contractions. Blood pressure was evaluated in pithed rats. NPI (30 mM) added 15 min before addition of the P2X 1 receptor-specific agonist ab-MeATP increased the maximum vasoconstriction from 23% to 49% (an increase of 113%). Furthermore, NPI prevented the desensitization of repeated ab-MeATP contractions. Related compounds were examined, and 2-(3-methoxy-phenyl)-1-oxy-indol-3-one (MPI) had similar effects as NPI, but several others lacked effect. NPI had no effect on ADPbS (P2Y 1 ) or acetylcholine-mediated vasodilatation, nor on UTP (P2Y 2/4 ), UDP (P2Y 6 ), or noradrenaline-mediated contractions. In pithed rats, the blood pressure response to 50 nmol/kg-infusion of ab-MeATP was increased from 5076 to 6375 mmHg (Po0.05), but had no effect on basal blood pressure or on the cardiovascular response to preganglionic nerve stimulation. In conclusion, NPI and MPI potentiates P2X 1 receptor vascular contractions in vitro and (NPI) blood pressure effects in vivo. It is possible that the effect is mediated by an inhibition of P2X 1 receptor desensitization. Drug Dev. Res. 59:82–87, 2003.  c Wiley-Liss, Inc. Key words: purines; pyrimidines; vasoconstriction; P2X receptor; potentiation INTRODUCTION Extracellular nucleotides have physiological ef- fects in most organ systems. Our focus has been the cardiovascular system, in which extracellular nucleo- tides mediate vasodilatation, vasoconstriction, platelet aggregation, and inotropic effects in the heart [Ralevic and Burnstock, 1998]. The effects are mediated by P2 receptors, one of the larger receptor groups identified. Recent receptor cloning has proven the existence of several different P2X and P2Y receptor subtypes, and there is evidence that at least five of these elicit DDR Contract grant sponsors: Swedish Foundation for Strategic Research (National Network for Drug Development); Swedish Heart and Lung Foundation; Franke and Margareta Bergqvist Foundation; Wiberg Foundation; Bergwall Foundation; Zoegas Foundation; Netpharma Foundation. Contract grant sponsor: Swedish Medical Research Council; Grant number: 13130. *Correspondence to: David Erlinge, M.D., Ph.D., Depart- ment of Cardiology, University Hospital, SE-221 85 Lund, Sweden. E-mail: david.erlinge@kard.lu.se Published online in Wiley InterScience (www.interscience. wiley.com) DOI: 10.1002/ddr.10205 DRUG DEVELOPMENT RESEARCH 59:82–87 (2003)  c 2003 Wiley-Liss, Inc.