Endothelium, 15:43–51, 2008 Copyright c  Informa Healthcare USA, Inc. ISSN: 1062-3329 print/1543-5261 online DOI: 10.1080/10623320802092294 P2Y 2 Receptor Desensitization on Single Endothelial Cells Priscila Sanabria, Elizabeth Ross, Edgardo Ram´ırez, Katiria Col´ on, and Millie Hern´ andez Department of Physiology, Universidad Central del Caribe, Bayam´ on, Puerto Rico H´ ector M. Maldonado Department of Pharmacology, Universidad Central del Caribe, Bayam´ on, Puerto Rico Walter I. Silva and Carlos A. Jim´ enez-Rivera Department of Physiology, University of Puerto Rico, Medical Sciences Campus, Rio Piedras, Puerto Rico Fernando A. Gonz´ alez Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, Rio Piedras, Puerto Rico Receptor desensitization, or decreased responsiveness of a re- ceptor to agonist stimulation, represents a regulatory process with the potential to have a significant impact on cell behavior. P2Y 2 ,a G-protein–coupled receptor activated by extracellular nucleotides, undergoes desensitization at many tissues, including the vascular endothelium. Endothelial cells from a variety of vascular beds are normally exposed to extracellular nucleotides released from dam- aged cells and activated platelets. The purpose of the present study was to compare P2Y 2 receptor desensitization observed in endothe- lial cells derived from bovine retina, a model of microvascular en- dothelium, and human umbilical vein endothelial cells (HUVECs), a model of a large blood vessel endothelium. P2Y 2 receptor desensi- tization was monitored by following changes in UTP-stimulated in- tracellular free Ca 2+ in single cells using fura-2 microfluorometry. Both endothelial cell models exhibited desensitization of the P2Y 2 receptor after stimulation with UTP. However, the cells differed in the rate, dependence on agonist concentration, and percentage of maximal desensitization. These results suggest differential mecha- nisms of P2Y 2 receptor desensitization and favors heterogeneity in extracellular nucleotide activity in endothelial cells according to its vascular bed origin. Received 1 October 2007; accepted 31 March 2008. The authors thank the participation of the medical students Carlos Calisto, Arturo Arroba, and Audberto Feliciano, and the undergraduates Frankie Rodriguez and Tatitana Grajales in the processing of imaging data analysis and for the support provided to our technical staff. This publication was made possible by NIH grant number G12 RR-03035– 22 from the National Center for Research Resources and SO6GM 50695 to P.S. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Address correspondence to Priscila Sanabria, PhD, Department of Physiology, Universidad Central del Caribe, Bayam´ on, Puerto Rico 00960–6032, USA. E-mail: psanabria@uccaribe.edu Keywords Endothelial Cells, Extracellular Nucleotide Receptors, Densensitization, P2Y Receptors The role of nucleotides like ATP, UTP, and ADP as extra- cellular signaling molecules is well established (Boarder et al. 1995; Burnstock 1997, 2002). Nucleotides can reach extracellu- lar fluids following cell necrosis, exocytosis of secretory gran- ules, or efflux through membrane channels (Boeynaems et al. 2005).One of the natural targets of these compounds is the vas- cular endothelium, where extracellular nucleotides have been shown to influence endothelial cell functions associated with the action of growth factors, such as mitogenesis, cell adhesion, and migration (Kaczmarek et al. 2005; Satterwhite et al. 1999; Seye et al. 2004; Teuscher and Weidlich 1985; Van Daele et al. 1992). Extracellular nucleotides are known to activate extracellu- lar signal-regulated kinase 1/2 (ERK1/2), a critical signaling pathway that has been associated with the regulation of cell growth and division (Ralevic and Burnstock 1998). Furthermore, the phenomenon of receptor desensitization has been suggested to play a role in the transient activation of ERK1/2 in UTP- stimulated endothelial cells (Graham et al. 1996). Although there has been substantial evidence of extracellular nucleotide ability to induce early cellular responses in a variety of endothelium, not much is known about the regulatory processes of P2Y ac- tivation and how they differ in endothelia from varied sources. In view of the importance of the phenomenon of desensitization for the long-term trophic actions of extracellular nucleotides, we decided to explore the heterogeneity of P2Y receptor desensi- tization events in vascular endothelium from different sources. Specifically, we sought to test the hypothesis of differential P2Y 2 43