Proc. West. Pharmacol. Soc. 47: 6-17 (2004) 6 INVITED REVIEW Nucleotides in the Blood Stream Robert A. Kaiser 1 and Iain L. O. Buxton 2 1 Department of Pediatrics, Cincinnati Children’s Hospital, Cincinnati, Ohio and 2 Department of Pharmacology, University of Nevada School of Medicine, Reno, NV 89509 Receptors for Nucleotides: The role of nucleotides as effectors in the blood stream is continually being defined and broadened. It is understood that nucleotides such as ATP and UTP are capable of modulating smooth muscle contraction, platelet aggregation, pain, and cardiac function [1]. These signaling roles of nucleotides occur through a series of ionotropic P2X receptors and metabotropic P2Y receptors. There are at least 7 cloned P2X receptor ion channels, P2X1-7. The P2Y receptors are a family of 7 TMD G-protein coupled receptors. To date there are at least 7 cloned P2Ys expressed in mammalian tissues, P2Y1,2,4,6,11,12 [2] and P2Y13 [3]. Both P2X and P2Y receptors are classified by their affinities for various nucleotide ligands [2]. The two distinct receptor classes are often co- expressed in tissues each in multiple subtypes, leading to a heterogeneous nucleotide response that is complex and difficult to characterize [4]. This complex pattern of expression is tissue specific and of great consequence to the overall responses to nucleotides exhibited by target cells. This has presented researchers with the daunting task of characterizing receptors individually, ultimately interpreting the interactions of multiple receptors as is necessary to understand tissue specific responses to nucleotides. For example, in rat intrapulmonary arteries UTP, UDP, and to a lesser extent ATP cause vasoconstriction in a suramin-insensitive manner while ATP, but not UDP or UTP, is capable of vasodilatory activity in methoxamine (α1 agonist) stimulated vessels [5]. This is in contrast to another study in rabbit pulmonary artery where both ATP and UTP are capable of inducing vasodilation under the same conditions [6]. This dual regulation of vessel tone by nucleotides is theoretically explained by the presence of P2 receptors on the endothelium as well as the vascular smooth muscle. The vasoconstrictor activity of these nucleotides is believed to be mediated by the P2X ligand-gated ion channels on the vascular smooth muscle cells, and the vasodilatory functions are mediated through the P2Y G-protein coupled receptors of the endothelium as has been demonstrated in rat aorta [7]. This may yet prove an oversimplification as P2X receptors are being found on some endothelial cells as well [8]. In 1990 it was demonstrated that inositol phosphates measured from ATP treated bovine aorta demonstrated a disparate pattern of induction and signaling than did comparable analysis of inositol phosphates derived from bovine adrenal medulla microvasculature [9]. This was an early indication that P2 receptor expression was dynamic and prone to vary based on the source of the tissue observed. This study was also a warning that any model of the P2 response that did not include the possibility of different receptors coupling to different second messengers would be an over-simplification. Most P2Y receptors are coupled to phospholipase C through a G q/11 protein [10] while still others like P2Y 4 may also be coupled to inhibition of adenylyl cyclase through G i proteins, as evidenced by pertussis toxin sensitivity [11]. There is also evidence that not only are different G-proteins involved for adenylyl cyclase (AC) and phospholipase C (PLC) interaction [12], but there may also be coupling of these receptors to other messenger systems such as prostaglandin formation and nitric-oxide production [13], possibly via chronic signaling resulting in activation of mitogen activated protein kinases (MAPKs) p42 and p44 [14]. Attempts to identify the type and number of receptors on a given cell type have come to rely heavily on known antagonists of these receptors including pyridoxal phosphate 6-azophenyl 2’,4’-disulphonic acid (PPADS) [15,16], reactive blue 2 (cibachron Blue) [17], suramin [18,19], MRS2179 [20,21], and the more recent MRS2279 [22]. These studies and others have provided many insights to both the receptors and the feasibility of using these antagonists to characterize the P2 receptor subtypes expressed in various tissues and cells. Attempts have been made to test these antagonists on cloned receptors expressed in recombinant systems and apply the results of these studies to the endogenously expressed proteins in their multiple-receptor environments. The human P2Y 4 receptor is reported to be sensitive to inhibition by PPADS with only mild sensitivity to reactive blue-2 and no effect of suramin. In contrast, the rat P2Y 4 receptor is reported to be most sensitive to reactive blue-2 with far less effect of either PPADS or suramin. It has also been proposed that this effect of PPADS on the inhibition of inositol phosphate production in response to UTP and ATP is