Abstract The expression of the seven P2X receptor sub- types and of two P2Y receptors was examined immuno- histochemically and by in situ hybridisation in thymi of adult male rats. P2X 4 , P2Y 2 and 4 receptor mRNA colo- calisation studies combining in situ hybridisation and immunohistochemistry were also carried out. P2X and P2Y receptors were found on thymocytes. P2X receptors were also abundant in cells of the thymic microenviron- ment, involved in control of T-cell maturation in vivo. We are the first to describe the expression of P2X 4 recep- tors on thymocytes and confirm the finding of P2X 1 and P2Y 2 receptors on subpopulations of lymphocytes. P2X 1,2,3,4 and 5 receptors were present in blood vessels of the thymus. P2X 1,2 and 4 receptors were detected in vascu- lar smooth muscle, while P2X 3 receptors appeared to be associated with endothelial cells; some small arteries were positive for P2X 5 , possibly labelling vascular smooth muscle or fibroblasts in the adventitia. P2X 2,3,6 and 7 receptors were found on thymic epithelial cells. P2X 2 and 3 receptors were abundant on medullary epithe- lial cells, whilst P2X 6 receptors were prominent in Hassall’s corpuscles. P2X 2 receptors were found on sub- capsular and perivascular epithelial cells. P2X 2,6 and 7 re- ceptors were detected in epithelial cells along the thymic septa. Expression of P2X receptors was also investigated by Western blotting of crude thymic tissue extracts under reducing conditions. All seven P2X receptor subtypes were found to be dimers of approximately 70 kDa and 140 kDa molecular weight. ATP-mediated apoptosis and cell proliferation of thymocytes are discussed. Key words P2 receptor · Thymus · Immunohistochemistry · In situ hybridisation · Western blot · Rat (Sprague-Dawley) Introduction ATP has long been known as an intracellular energy car- rier, but is also recognised as an extracellular signalling molecule (see Burnstock 1997). The role of ATP as a motor-neurotransmitter was first proposed by Burnstock in 1972, and many articles on its effects in the cardiovas- cular system, vascular and visceral smooth muscle, en- dothelial cells, secretory and endocrine cells and several types of blood cells are available today (Burnstock 1995). Two families of receptors to purines and pyrimidines have been defined, the G-protein-coupled P2Y receptors and the ligand-gated cation channels named P2X recep- tors (Abbracchio and Burnstock 1994). Investigations in the field have been enhanced in recent years by the clon- ing and characterisation of P2X and P2Y receptor sub- types (Burnstock and King 1996). There are now seven subtypes of P2X receptors and five subtypes of mamma- lian P2Y receptors. P2X receptors have been reported on blood and im- mune cells. The bifunctional P2X 7 subtype is a mem- brane pore for molecules up to 900 Da, in addition to its action as a fast and selective channel for small mono- and divalent cations (Surprenant et al. 1996). The P2X 7 receptor (formerly termed P2Z) has mainly been detect- ed in mast cells and macrophages and has there been re- lated to apoptosis and necrosis (Di Virgilio et al. 1989). It has also been suggested that it participates in mitoge- nic stimulation of thymocytes (which are intrathymic T- cells or T-lymphocytes), cytokine release from macro- phages, formation of macrophage polykaryons and cyto- toxicity (Collo et al. 1997). Another line of investigation of P2X receptors in the immune system has been raised since significant sequence homology of P2X 1 with the RP-2 gene, expressed in apoptotic thymocytes, was de- The support of Roche Bioscience, Palo Alto, USA, is gratefully acknowledged R. Glass · A. Townsend-Nicholson · G. Burnstock ( ✉ ) Autonomic Neuroscience Institute, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK e-mail: g.burnstock@ucl.ac.uk Tel: +44 171 830 2948, Fax: +44 171 830 2949 Cell Tissue Res (2000) 300:295–306 Digital Object Identifier (DOI) 10.1007/s004410000206 REGULAR ARTICLE Rainer Glass · Andrea Townsend-Nicholson Geoffrey Burnstock P2 receptors in the thymus: expression of P2X and P2Y receptors in adult rats, an immunohistochemical and in situ hybridisation study Received: 8 September 1999 / Accepted: 21 February 2000 / Published online: 30 March 2000 © Springer-Verlag 2000