Dinucleoside polyphosphates: strong endogenous agonists of the purinergic system

Abstract

The purinergic system is composed of mononucleosides, mononucleoside polyphosphates and dinucleoside polyphosphates as agonists, as well as the respective purinergic receptors. Interest in the role of the purinergic system in cardiovascular physiology and pathophysiology is on the rise. This review focuses on the overall impact of dinucleoside polyphosphates in the purinergic system. Platelets, adrenal glands, endothelial cells, cardiomyocytes and tubular cells release dinucleoside polyphosphates. Plasma concentrations of dinucleoside polyphosphates are sufficient to cause direct vasoregulatory effects and to induce proliferative effects on vascular smooth muscle cells and mesangial cells. In addition, increased plasma concentrations of a dinucleoside polyphosphate were recently demonstrated in juvenile hypertensive patients. In conclusion, the current literature accentuates the strong physiological and pathophysiological impact of dinucleoside polyphosphates on the cardiovascular system.

Introduction

Interest in the functional roles of nucleotides and the underlying purinergic system in cardiovascular physiology and pathophysiology continues to grow (Gabriels et al., 2002). The purinergic system consists of mononucleosides, mononucleoside polyphosphates and dinucleoside polyphosphates as agonists, as well as the respective purinergic receptors, which possess very different functions (Ralevic and Burnstock, 1998). The purinergic signalling system that controls vascular regulation displays a high degree of complexity.

Purinergic receptor system (purinoceptors)

The complexity of the purinergic signalling system is partially due to the large number of agonists that constitute this system. This is further complicated by the diversity of the purinergic receptors (purinoceptors), including the subtypes of the P1, P2X and P2Y receptors, as well as formation of heteropolymeric P2X ion channels (Nori et al., 1998; Robertson et al., 2001), and P2X splicing variants (Cheewatrakoolpong et al., 2005; Koshimizu et al., 2006). Additionally, there are numerous soluble and membrane-bound ecto-nucleotidases (Yegutkin et al., 2000; 2007; Linden, 2001; Leipziger, 2003) that transform one active purinoceptor agonist into an active agonist for another purinoceptor. In the last several years, our understanding of the role(s) the purinergic system plays in normal and pathological cardiovascular physiology has been vastly increased by the identification of new purinergic agonists and cloning of several purine receptor subtypes.

The potent actions of purine mononucleoside polyphosphates on cardiovascular system vessels were first described in 1929 (Drury and Szent-Györgyi, 1929). Since then, the impact of mononucleoside polyphosphates in various vasoregulatory processes, like immunomodulatory and prothrombotic responses in the cardiovascular system, have been described in detail elsewhere (Burnstock, 2002; Moore and MacKenzie, 2007).

Dinucleoside polyphosphates as mediators of the purinergic system

In recent years, interest in dinucleoside polyphosphates as strong purinergic agonists has been growing. Dinucleoside polyphosphates mediate vascular tone regulation (Schlüter et al., 1994; Jankowski et al., 2005), as well as vascular smooth muscle and mesangial cell proliferation (McLennan, 1992; Heidenreich et al., 1995).

Occurrence and physiological effects of dinucleoside polyphosphates

Molecular structure of dinucleoside polyphosphates

Dinucleoside polyphosphates (Xp_nX) consist of two nucleotides (ribosylated nucleic acids), which are interconnected by a polyphosphate chain of two to seven phosphates through phosphoester bonds at the 5′-position of two ribose moieties (where X = adenosine and/or guanosine, or uridine; n= number of phosphate groups). Figure 1 shows the molecular structure of P₁,P₄ uridine adenosine tetraphosphate (Up₄A) as an example of a dinucleoside polyphosphates. In comparison with mononucleoside polyphosphates, dinucleoside polyphosphates have relatively long half-lives. Furthermore, metabolites of dinucleoside polyphosphates may serve as potential sources of extracellular ATP and other purines.

Figure 1.

Molecular structure of uridine (5′)-adenosine (5′) tetraphosphate (Up₄A).

Occurrence of dinucleoside polyphosphates

P₁,P₄ diadenosine tetraphosphate (Ap₄A) was the first diadenosine polyphosphate to be identified in mammalian tissue (Rapaport and Zamecnik, 1976), was subsequently also identified in human platelets (Flodgaard and Klenow, 1982; Lüthje and Ogilvie, 1983). Following these early discoveries, numerous different nucleotides have been isolated from human tissues. For example, diadenosine polyphosphates (Ap_nA, with n= 2–7) were isolated from body fluids and cells (Pintor et al., 1992a; Schlüter et al., 1994; 1998; Hoyle et al., 1996; Jankowski et al., 1999; 2001a; 2003a;). Dinucleoside polyphosphates are released into the circulation from several cell types, including activated platelets (Flodgaard and Klenow, 1982; Lüthje and Ogilvie, 1983; Jankowski et al., 1999), chromaffin cells of the adrenal glands (Rodriguez del Castillo et al., 1988; Castillo et al., 1992; Pintor et al., 1991; 1992a;), tubular cells (Jankowski et al., 2007a; 2008;) or from synaptic vesicles (Zimmermann et al., 1993). Ap_nA are important neurotransmitter molecules in the nervous system (Delicado et al., 2006), and the importance of purines for neurotransmission in general was recently reviewed in an excellent manner by G Burnstock (Burnstock, 2007). In addition, Ap_nA stimulate different responses in the cardiovascular system, including control of vascular tone and prevention of platelet aggregation (Flores et al., 1999). Release of dinucleoside polyphosphates may result in local concentrations in the range of 10⁻⁵ mol·L⁻¹ or even higher (Ogilvie, 1992). Diadenosine polyphosphates have a direct effect on the vascular tone (Busse et al., 1988; Schlüter et al., 1994; Ralevic et al., 1995; Hoyle et al., 1996; Inscho et al., 1998; van der Giet et al., 1998; Jankowski et al., 1999; Luo et al., 1999b; Gabriels et al., 2000; Lewis and Evans, 2000). In the vasculature of isolated perfused rat kidney, Ap₅A and Ap₆A were effective at a concentration of 10⁻⁹ mol·L⁻¹, and contractions in aortic rings were elicited at 10⁻⁸ mol·L⁻¹. Intra-aortic injection in the rat caused a prolonged increase in blood pressure (Schlüter et al., 1994). The vasoconstrictive action of Ap₇A on the vasculature of the isolated perfused rat kidney Ap₇A is lower than that of Ap₆A. The threshold of the vasoconstrictive action of Ap₇A is in the range of 10⁻⁵ mol·L⁻¹ (Jankowski et al., 1999). Vasoconstriction induced by the diadenosine polyphosphates is mediated by an increase in intracellular free calcium ions, [Ca²⁺]_i (Tepel et al., 1996; 1997;).

Recently, interest in uridine (5′)-adenosine (5′) tetraphosphate (Up₄A) (Figure 1) has increased (Jankowski et al., 2005). Up₄A was isolated from the supernatant of stimulated human endothelium and was identified by mass-spectrometry. Stimulation with adenosine 5′-triphosphate (ATP), uridine 5′-triphosphate (UTP), acetylcholine, endothelin, A23187 and mechanical stress releases Up₄A from endothelium, suggesting that Up₄A contributes to vascular regulation. Up₄A plasma concentrations found in healthy subjects are high enough to cause vasoconstriction. Up₄A is the first endogenous dinucleoside polyphosphate isolated from living organisms, which contains both purine and pyrimidine moieties. Several lines of evidence strongly suggest that Up₄A has a functional role in the cardiovascular system, including its vasoactive effects (Figure 2), plasma concentrations and its release upon endothelial stimulation.

Figure 2.

Change in perfusion pressure in the isolated perfused rat kidney induced by Up₄A alone (•), with α,β-methylene ATP (×) and with L-NAME (○) [Figure adapted from Jankowski et al. (2005)].

Vasodilatory effects of dinucleoside polyphosphates

A small number of dinucleoside polyphosphates have vasodilatory effects, for example, Ap₂A on the tone of isolated mesenteric arterial bed of rats (Ralevic and Burnstock, 1996). Ap₃A and Ap₄A both induce vasodilation upon perfusion of arteries containing endothelium, whereas Ap₄A causes vasoconstriction in arteries from which the endothelium has been removed (Busse et al., 1988). Arterial infusion of Ap₄A produced a dose-dependent decrease of systemic blood pressure and coronary vascular resistance (Nakae et al., 1996). These vasodilatory effects of dinucleoside polyphosphates are mediated via endothelial A₂ receptors (Ralevic and Burnstock, 1996) and endothelial metabotropic P2Y receptors (Ralevic et al., 1995; Hilderman and Christensen, 1998; Rump et al., 1998; Gabriels et al., 2000; Malmsjo et al., 2000b). Stimulation of endothelial P2Y₁, P2Y₂ and P2Y₄ receptors causes endothelium-derived hyperpolarizing factor- and NO-mediated dilatation (Malmsjo et al., 2000a,b; Mombouli and Vanhoutte, 1993; Vanhoutte, 1991). Ohata et al. reported that in aortic strips in situ[Ca²⁺]_i waves within endothelial cells are induced by ATP via the P2Y₁ purinoceptor, but not by the P2Y₂ purinoceptor (Ohata et al., 1997).

In addition to endothelium-dependent dilation effects, Ap₄A and Ap₅A were also demonstrated to directly affect vascular smooth muscle cell (VSMC) dilation. Dilation induced by both dinucleoside polyphosphates was mediated by cAMP, which affects a decrease of [Ca²⁺]_I, possibly through activation of K⁺ channels (Sumiyoshi et al., 1997). Direct, endothelium-independent vasodilation induced by purinoceptor agonists was also observed in intrapulmonary arteries isolated from newborn piglets and found to be mediated by a P2Y receptor on the VSMCs (McMillan et al., 1999).

The different functional effects of Ap₄A (constriction in some smooth muscle and relaxation in others) may be explained by the action of a specific Ap₄A hydrolase. This Ap₄A hydrolase hydrolyses Ap₄A in an asymmetric fashion to yield AMP, which is dephosphorylated (Walker and Hilderman, 1993) to yield adenosine, and ATP, which is converted to AMP and inorganic pyrophosphate (Lüthje and Ogilvie, 1985; 1988; Ogilvie et al., 1989; Hankin et al., 1995; Thorne et al., 1995). This enzyme is presumed to be involved in the regulation of the intracellular adenosine, AMP, ADP and ATP levels (Guranowski and Sillero, 1992). Thus, it is possible that the application of Ap₄A may produce adenosine, AMP, ADP and ATP, leading to stimulation of multiple receptors such as nucleotide receptors and adenosine receptors. Conversely, there was no significant difference in the potency of Ap₄A and Ap₅A to induce relaxation of the guinea pig left atrium (Hoyle et al., 1996). Sumiyoshi et al. (1997) also observed almost equal potency for Ap₄A- and Ap₅A-induced coronary vasorelaxation. However, it is still possible that Ap₄A might stimulate receptors in its intact form (i.e. without degradation) because it has been reported that Ap_nA are substance with long half-lives (Busse et al., 1988; Pohl et al., 1991; Hoyle et al., 1996). In agreement with this speculation, the specific and saturable membrane receptors for Ap₄A have been reported to be present in brain, cardiac, liver, kidney, spleen and adipose tissue (Hilderman et al., 1991; Walker and Hilderman, 1993).

Proliferative effects of dinucleoside polyphosphates on VSMCs

Dinucleoside polyphosphates do not only directly influence the vascular physiology, but also increase the proliferation rate of VSMCs. Growth-stimulating effects of nucleoside polyphosphates have been demonstrated in numerous types of vascular beds (Erlinge, 1998; Jankowski et al., 2001b; Verspohl et al., 2004; Jankowski et al., 2007a,b;). The ATP-induced proliferation of VSMCs is coupled to a Gq-protein and triggers phosphoinositide hydrolysis with subsequent activation of protein kinase C, serine/threonine-kinase Raf-1 and mitogen-activated protein kinase (MAPK) (Yu et al., 1996; Wilden et al., 1998). Tu et al. observed that P2Y₂ receptor stimulation involves the activation of Ras/Raf/MEK/MAPK pathway, which is modulated by [Ca²⁺]_i, protein kinase C and tyrosine kinase (Tu et al., 2000). ATP-stimulated proliferation of coronary artery smooth muscle cells requires independent activation of both the extracellular signal-regulated kinase (ERK/MAPK) cascade and phosphatidylinositol 3-kinase (PI3K) signalling pathways (Wilden et al., 1998). P2Y₂ receptor stimulation results in increased c-fos mRNA expression in cultured aortic smooth muscle cells and stimulates proliferation of vascular tissue (Malam-Souley et al., 1996). Ap₄A is equipotent to ATP for induction of these effects (Erlinge et al., 1995).

Vascular smooth muscle cell proliferation and c-fos proto-oncogene expression are induced by Ap₃A, Ap₄A, Ap₅A (Jankowski et al., 2001b), Ap_nG and Gp_nG (n= 3–6) (Schlüter et al., 1998) as well as Ap₂A, Ap₂G and Gp₂G (Jankowski et al., 2001a). In vascular tissues, the proliferative effect of the diguanosine polyphosphates Gp_nG (with n= 3–6) is significantly stronger than that of ATP (Schlüter et al., 1998). Micromolar concentrations of Ap₃A, Ap₄A, Ap₅A and Ap₆A also stimulate growth in rat glomerular mesangial cells (Heidenreich et al., 1995; Schulze-Lohoff et al., 1995). Moreover, these diadenosine polyphosphates potentiate the growth response to platelet-derived growth factor, but not to insulin-like growth factor-1 (Heidenreich et al., 1995).

Effects of dinucleoside polyphosphates on platelet aggregation

The dinucleoside polyphosphates are potent antagonists of ADP-induced platelet aggregation (Jankowski et al., 1999). These inhibitory effects of dinucleoside polyphosphates on platelet aggregation are mediated by the P2Y₁, the P2Y₁₂ and the P2X₁ receptors and appears to be via competitive inhibition of ADP and ATP, with Ap₄A having a K_i of approximately 0.7 mmol·L⁻¹ (Kunapuli and Daniel, 1998). Because plasma Ap₄A concentrations are significantly lower than the K_i, a systemic impact is unlikely. However, the interaction may be caused by local effects as the concentrations close to platelets are significantly higher than plasma concentrations. A comparison of the homologous series of Ap_nA compounds with phosphate chain lengths from two to six revealed Ap₅A as the most potent inhibitor of ADP-induced platelet aggregation, followed by Ap₆A and Ap₄A, which were more potent than Ap₃A or Ap₂A. Inhibition of platelet aggregation by dinucleoside polyphosphates is proposed to be due to direct competition between the dinucleoside polyphosphates and ADP at a specific receptor site on the platelet membrane (Harrison et al., 1975; Kunapuli, 1998). Dinucleoside polyphosphates inhibit release of ADP from blood platelets with a potency that decreases with decreasing chain length. Thus, dinucleoside polyphosphates in platelets may fulfil an anti-aggregatory role. In human neutrophils, Ap₃A, Ap₄A, Ap₅A and Ap₆A produce an increase in intracellular free calcium via a G-protein-coupled receptor (Pintor et al., 1997).

Release of dinucleoside polyphosphates

Up to millimolar range concentrations of dinucleoside polyphosphates are stored in secretory granules from platelets, in adrenal chromaffin cells and in central nervous synaptosomes (Flodgaard and Klenow, 1982; Lüthje and Ogilvie, 1983; Rodriguez del Castillo et al., 1988; Pintor et al., 1991). The intracellular concentration of diadenosine (5′, 5′) tetraphosphate (Ap₄A) during normal growth (Garrison and Barne, 1992) correlates directly with the proliferative state of the cell or tissue (Rapaport and Zamecnik, 1976; Remy, 1992). Ap₄A levels are known to respond to cellular stresses, such as oxidation and heat shock. Ap₄A has been described as an alarmone that signals the onset of cellular and metabolic stress, although its precise role remains unclear (Brevet et al., 1985; Garrison et al., 1986; Remy, 1992).

Metabolism of dinucleoside polyphosphates

Subsequent inactivation of the released nucleotides is thought to be mainly regulated by vascular endothelial (Marcus et al., 2003) and lymphoid (Heptinstall et al., 2005) membrane-bound nucleoside triphosphate diphosphohydrolase (NTPDase; also known as ecto-ATPDase, CD39) and ecto-5′-nucleotidase (CD73). Ectohydrolases are present on a broad variety of cell types, including aortic endothelial cells (Mateo et al., 1997a,b;), chromaffin cells (Gasmi et al., 1998), rat mesangial cells, bovine corneal epithelial cells, the human hepatocellular liver carcinoma cell line (Hep-G2) and periodontal cells (von Drygalski and Ogilvie, 2000). The human diphosphorylated inositol phosphate phosphohydrolase was shown to be a candidate for regulating signalling of diadenosine polyphosphates by hydrolysis of Ap₅A and Ap₆A in preference to other diadenosine polyphosphates (Safrany et al., 1999). The enzymatic breakdown of dinucleoside polyphosphates leads to generation of mononucleotides and nucleotides that, in turn, are biologically active in vascular tissues. In contrast to these traditional paradigms that focus on nucleotide-inactivating mechanisms, it has now become clear that nucleotide-phosphorylating enzymes adenylate kinase and NDP kinase are also co-expressed on the cell surface and finely control the purinergic signalling cascade via two counter-balancing pathways, ATP-inactivating and ATP-regenerating respectively (Yegutkin et al., 2000; Yegutkin and Burnstock, 2000). The identification of a complex mixture of nucleotide pyrophosphatase/phosphodiesterase (NPP), NTPDase, adenylate kinase and other soluble purinergic enzymes freely circulating in the bloodstream adds another level of complexity to the understanding of the regulatory mechanisms of purine homeostasis within the vasculature (Birk et al., 2002; Yegutkin et al., 2003; 2007; 2008;). The agonistic effects of nucleotides are obviously mediated by complex mechanisms, including: (i) dinucleoside polyphosphate receptor pathway; (ii) inhibition of ecto-adenylate kinase activity; and (iii) generation of biologically active ATP and adenosine.

Table 1 gives a characteristic overview of endogenous dinucleoside polyphosphates that have been identified in human tissues and cells.

Table 1.

Isolations, identifications and characterizations of dinucleoside polyphosphates in human tissues and their receptor-mediated vascular effects

Compound	Isolation from human tissue	Known vascular effects and receptor type involved (in brackets)	References
Ap2A	Myocardium Platelets Placenta Adrenal glands	Vasodilation (A2) and vasoconstriction (A1) in coronary arteries and renal vessels Proliferation of vascular smooth muscle cells (P2Y)	Hoyle et al. (1996); van der Giet et al. (1997a); Luo et al. (1999a); Jankowski et al. (2001a,b,c; 2003b;)
Ap2G	Platelets	Proliferation of vascular smooth muscle cells (P2Y)	Jankowski et al. (2001a)
Gp2G	Platelets	Proliferation of vascular smooth muscle cells (P2Y)	Jankowski et al. (2001a)
Ap3A	Myocardium Platelets Placenta Adrenal glands Plasma Proximal tubule Epithelial cells	Vasodilation (P2Y and A2) and vasoconstriction (A1 and P2X) in coronary arteries, renal vessels and mesenteric vessels Proliferation of vascular smooth muscle cells (P2Y)	Lüthje and Ogilvie (1983); Ogilvie and Jakob (1983); Schlüter et al. (1994); van der Giet et al. (1997a); Luo et al. (1999a); Jankowski et al. (2001a,b,c; 2003a,b; 2007a;)
Ap3G	Platelets	Vasoconstriction in renal vessels (P2X) Proliferation of vascular smooth muscle cells (P2Y)	Schlüter et al. (1998)
Gp3G	Platelets	Proliferation of vascular smooth muscle cells (P2Y)	Schlüter et al. (1998)
Ap4A	Adrenal gland Plasma Platelets Brain Proximal tubule epithelial cells	Vasoconstriction in renal vessels (P2X, A1) and mesenteric vessels (P2X) Proliferation of vascular smooth muscle cells (P2Y)	Ogilvie et al. (1989); Pintor et al. (1992a); van der Giet et al. (1997a; 1998;); Jankowski et al. (2001a,b; 2003a,b;)
Ap4G	Platelets	Vasoconstriction in renal vessels (P2X) Proliferation of vascular smooth muscle cells (P2Y)	Schlüter et al. (1998)
Gp4G	Platelets	Vasoconstriction of renal vessels (P2X) Proliferation of vascular smooth muscle cells (P2Y)	Schlüter et al. (1998)
Ap5A	Adrenal glands Plasma Platelets Placenta Brain Proximal tubule epithelial cells	Proliferation in vascular smooth muscle cells (P2Y) Vasoconstriction in renal vessels (P2X) and coronary arteries (P2X) Vasodilation in coronary arteries (P2Y)	Pintor et al. (1992a); Schlüter et al. (1994); van der Giet et al. (1997a; 1999; 2001; 2002;); Jovanovic et al. (1998); Jankowski et al. (2001a,b,c; 2003a,b; 2007a;);
Ap5G	Platelets	Vasoconstriction in renal vessels (P2X) Proliferation of vascular smooth muscle cells (P2Y) Vasoconstriction in renal vessels (P2X) and coronary arteries (P2X) Vasodilation in coronary arteries (P2Y)	Schlüter et al. (1998); van der Giet et al. (2001; 2002;)
Gp5G	Platelets	Proliferation of vascular smooth muscle cells (P2Y)	Schlüter et al. (1998); van der Giet et al. (2001; 2002;)
Ap6A	Adrenals Plasma Platelets Red blood cells Placenta Proximal tubule epithelial cells	Proliferation of vascular smooth muscle cells (P2Y) Vasoconstriction in renal vessels (P2X) and coronary arteries (P2X) Vasodilation in coronary arteries (P2Y)	Pintor et al. (1992b); Schlüter et al. (1994); van der Giet et al. (1997a; 1998; 2001; 2002;); Jankowski et al. (2001a,b.c; 2003a.b; 2007a)
Ap6G	Platelets	Vasoconstriction in renal vessels (P2X) and coronary vessels (P2X) Vasodilation in coronary arteries (P2Y) Proliferation of vascular smooth muscle cells (P2Y)	Schlüter et al. (1998); van der Giet et al. (2001; 2002;)
Gp6G	Platelets	Proliferation of vascular smooth muscle cells (P2Y)	Schlüter et al. (1998); van der Giet et al. (2001; 2002;)
Ap7A	Platelets	Vasoconstriction in renal vessels (P2X)	Jankowski et al. (1999)
Up4A	Endothelial cells Tubule cells	Vasoconstriction and proliferation	Jankowski et al. (2005; 2007b; 2008;); Gui et al. (2008)

Purinoceptor system

The physiological and pathophysiological effects of mononucleosides, mononucleoside polyphosphates and dinucleoside polyphosphates are mediated via nucleotide-selective receptors. Essentially two major purine receptors subfamilies have been described based on pharmacological, functional and cloning data (Ralevic, 2000; Burnstock, 2002). P1 receptors are preferably activated by adenosine, while P2 receptors are activated by ATP, ADP, UTP and UDP and also by dinucleoside polyphosphates. Using molecular, biochemical and pharmacological criteria P1 receptors have been further subdivided into four subgroups: A₁, A_2A, A_2B and A₃ according to molecular, biochemical and pharmacological criteria. The group of P2 receptors is divided into P2X and P2Y (Figure 3) receptors according to their molecular structure and the signal transduction pathways they regulate. Selective agonists or antagonists discriminating adequately between families of P2X and P2Y receptors, or between subtypes of receptors within each of these groups have been discovered. Some selective antagonists include the P2X₇ antagonist [³H]-2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine (Donnelly-Roberts et al., 2008), the P2Y₁-selective antagonist N⁶-methyl 2′-deoxyadenosine 3′,5′-bisphosphate (Boyer et al., 1998) and the potent and relatively selective P2X₁ antagonist diinosine pentaphosphate (Ip₅I) (King et al., 1999). Purinoceptors are characterized by high plasticity, and they are dynamically regulated during development. The purinoceptor system plays a general role as a sympathetic regulator of vasomotor tone (Kennedy, 1996; Ralevic, 2000). The purinoceptor system that controls vascular homeostasis displays a high degree of complexity.

Figure 3.

Structure of P2Y membrane receptors. P2Y are G-protein-coupled receptors (Fields and Burnstock, 2006), which act via a downstream signalling cascade including G-proteins and inositol triphosphate among other factors (Barnard and Simon, 2001).

P2X receptors mediating dinucleoside polyphosphate effects

P2X receptors are ligand-gated ion channels, which are opened by purinergic messengers (Bo et al., 2000), thus mediating rapid changes in the membrane permeability of monovalent and divalent cations (Bean, 1992; Dubyak and el-Moatassim, 1993; Burnstock, 2006; Erb et al., 2006). Stimulation of ionotropic P2X receptors induces an influx of Na⁺-ions and Ca²⁺-ions into the cytosol. The increase of the concentration of these ions triggers depolarization of the membrane potential, which opens potential operated Ca²⁺-channels (Usune et al., 1996). The resulting Ca²⁺-influx increases [Ca²⁺]_i, which affects the constriction of the VSMCs (Tepel et al., 1997). There are seven P2X receptor cDNAs currently known, P2X₁–P2X₇ (Khakh et al., 2001). When expressed individually in heterologous systems, P2X₁ and P2X₃ subunits form channels activated by ATP or α,β-methylene ATP, whereas P2X₂, P2X₄ and P2X₅ form channels activated by ATP but not α,β-methylene ATP (North and Surprenant, 2000). P2X₁ and P2X₃ receptors are characterized by strong and rapid desensitization, whereas desensitization of P2X₂, P2X₄ and P2X₆ receptors is relatively modest (Ralevic and Burnstock, 1998). Therefore, P2X₁-induced vasoconstrictions are transient, whereas P2X₂- or P2X₄-mediated vasoconstrictions are permanent as long as the agonist is present.

The ion-gating pore is formed by the aggregation of three P2X monomers (Nicke et al., 1998). Co-expression of P2X subtypes in heterologous expression systems can result in the formation of heteropolymeric P2X trimers (Nori et al., 1998). Therefore, it can be assumed that heteropolymeric channel formation is also possible in vivo. Heteropolymeric P2X receptors clearly differ from their homomeric relatives (Torres et al., 1998; Bianchi et al., 1999) and may therefore constitute an important mechanism for generating functional diversity of ATP- and dinucleoside polyphosphate-mediated responses. The P2X₁ forms large, approximately elliptical clusters on the smooth muscle cells of mesenteric, renal and pulmonary arteries as well as in the aorta and in veins, which are restricted to the adventitial surface of the media. At the adventitial surface, the large clusters are immediately apposed to sympathetic varicosities. In the pulmonary artery, large receptor clusters were found throughout the media of the vessel. Smaller, spherical P2X₁ clusters occur throughout the media of arteries of all sizes. The small P2X₁ clusters are not associated with varicosities (Hansen et al., 1999). This observation may point to a paracrine role of purinergic agonists. P2X₁, P2X₂ and P2X₄ are co-expressed in smooth muscle cells of coronary vessels as well as in peripheral vessels, including aorta, pulmonary artery, renal artery, femoral artery, internal and external iliac arteries. The co-expression of P2X receptor subtypes substantiates the possibility of a heteropolymeric assembly of the P2X ion channels. In contrast, no mRNA transcripts of P2X₁, P2X₂ or P2X₄ were found in the superior mesenteric artery (Nori et al., 1998). A further aspect contributing to the complexity of the purinergic system is the existence of P2X splice variants. In the human bladder cells, Hardy et al. detected a P2X₁ receptor splice variant, which lacks part of the second transmembrane domain. It was suggested that isoforms might be potential sites for modifying or regulating putative purinergic activation (Hardy et al., 2000). This view is supported by the observation of Chen et al., who published that ATP-induced currents in cells expressing P2X_2/1 and P2X_2/2 variants were large and desensitized rapidly, whereas the current in those cells expressing the P2X_2/3 variant was much smaller and desensitized more slowly (Chen et al., 2000). Alternatively, spliced P2X₄ RNAs in human smooth muscle cells were identified (Dhulipala et al., 1998).

The P2X₄ receptor is the most prominent P2X receptor in human vascular endothelial cells from umbilical veins, aorta, pulmonary artery and skin microvessels. Vascular endothelial cells are continuously exposed to variations in blood flow, which plays an important role in vessel growth or regression and in the local development of atherosclerosis. The shear stress that occurs during changes in blood flow leads to substantial release of ATP and dinucleoside polyphosphates from endothelial cells (Burnstock, 1999; Jankowski et al., 2005), and these purines might mediate alterations in the balance between proliferation and apoptosis. This endothelium-dependent response to ATP is absent in atherosclerotic patients. Consequently, P2X₄ mRNA expression was much higher in these cells than was the expression of other subtypes, including P2X₁, P2X₃, P2X₅ and P2X₇ (Yamamoto et al., 2000). P2X₂ receptors are located on neurons and on endothelial cells in rat blood vessels (Hansen et al., 1999; Zemkova et al., 2004).

The calcium-permeable P2X₁ receptor is considered the principal mediator of vasoconstriction (Kennedy, 1996), with P2X₁ protein clusters on the adventitial surface of blood vessels immediately adjacent to sympathetic nerve varicosities (Hansen et al., 1999). However, P2X₁ transcripts co-localize with P2X₂, P2X₄ and P2X₅ mRNA in muscle cells of a number of blood vessels, which alludes the added presence of heteromeric P2X receptors (Lewis and Evans, 2000; 2001; Pulvirenti et al., 2000; Turner et al., 2003). For example, heteromeric P2X_1/5 receptors have been implicated in vasoconstriction of submucosal arterioles in the guinea pig (Surprenant et al., 2000).

P2Y receptors mediating dinucleoside polyphosphate effects

P2Y receptors are 7-membrane-spanning proteins (King et al., 2000; Abbracchio et al., 2006; Burnstock, 2006; Erb et al., 2006). Some common mechanisms of signal transduction shared by most 7-membrane-spanning receptors include activation of phospholipase C and/or regulation of adenyl cyclase activity (Burnstock, 2006; Erb et al., 2006). P2Y receptors do not act directly by inducing a cation influx, but via a downstream signalling cascade including G-proteins and inositol triphosphate among other factors (Barnard and Simon, 2001).

P2X and G-protein-coupled P2Y receptors expressed in VSMCs were reported to mediate vasoconstriction (Inscho et al., 1998; Fukumitsu et al., 1999; Hillaire-Buys et al., 1999; McMillan et al., 1999; Shen et al., 2004). The vasoconstriction inducing P2Y receptor is probably coupled to PLC-β1 via Gαq/11 and to PLC-β3 via Gβγ3 (Murthy and Makhlouf, 1998a,b;). In human coronary arteries, extracellular nucleotides elicit constriction primarily by activation of P2X and P2Y₂ receptors, whereas a role for P2Y₁ and P2Y₆ receptors was excluded (Malmsjo et al., 2000b). UTP- and ATP-induced vasoconstriction in intrapulmonary arteries is consistent with activation of the P2Y₄ receptor subtype (McMillan et al., 1999), which is sensitive to Ap₄A. Dinucleoside polyphosphate vasoconstriction is also mediated by the adenosine A₁ receptor (Vahlensieck et al., 1996; van der Giet et al., 1997b; 1998; 1999; Gabriels et al., 2000). Because the vasoconstrictive effects of Ap₂A and Ap₃A, but not the vasoconstrictive effect of Ap₅A, are mediated by A1 receptors (van der Giet et al., 1997b), vasoconstriction is likely due to a direct effect of intact dinucleoside polyphosphates on the A1 receptor rather than effects caused by dinucleoside polyphosphate metabolites.

Purinoceptor heteromerization

These principally different mechanisms led to the terminology metabotropic and ionotropic used for P2Y receptors and P2X receptors respectively. The purine receptor subtypes have been characterized according to their molecular structures and according to their pharmacological characteristics, both of which show considerable differences. Different purinoceptor subtypes may be expressed in the same cell type, and heteromeric receptors are formed among different purinoceptor subtypes (Torres et al., 1998; Le et al., 1999; Barnard and Simon, 2001). These purinoceptor heteromeres may show pharmacological properties quite different from those of purinoceptor homomeres (Torres et al., 1998). The ability to form heteromeres has been demonstrated both in P2X receptors (Le et al., 1999) and in P2Y receptors (Barnard and Simon, 2001) and contributes to the diversity of pharmacological properties manifested by the purinoceptor family.

Distribution of purinoceptor

Characterization of P2X and P2Y expression in vascular endothelium has been an especially active area of investigation in recent years. Different purine nucleotides activate endothelial cells in distinct ways, suggesting that at least two different endothelial purinoceptors exist. Pirotton et al. (1996) initially demonstrated and others have since confirmed (Bultmann et al., 1997) that both P2Y₁ and P2Y₂ receptors are co-expressed in endothelial cells from bovine aorta. P2Y receptors have also been identified in endothelial cells from rat cerebral vessels (Vigne et al., 2000; Mistry et al., 2003) and from mesenteric arteries (Malmsjo et al., 2000a,b;). These findings have been confirmed also in rat renal glomeruli (Harada et al., 2000). Currently, it remains unclear whether other purinoceptor subtypes are also expressed in endothelial cells. Undoubtedly, the endothelial P2Y₁ receptor subtype mediates vasodilation (Bultmann et al., 1997; Malmsjo et al., 2000b; Vigne et al., 2000). In addition, the P2Y₂ and P2Y₄ receptor subtypes may also play a role in vasodilation (Malmsjo et al., 2000b).

Specific dinucleoside polyphosphate receptors

Dinucleoside polyphosphates have the capacity to potentiate signalling effects via P2 receptors (primarily, via P2X₁, P2X₃ and P2Y₁ subtypes), although the existence of specific dinucleoside polyphosphate receptors has also been proposed (Flores et al., 1999; Delicado et al., 2006). However, the specificity of dinucleoside polyphosphate receptors and their implication into multiple extracellular signals are still poorly understood, primarily due to the complexity of the purinergic signalling cascade. The effects of the dinucleoside polyphosphates are blocked by desensitization of P2X receptors with α,β-methylene ATP subtype (Bo et al., 1998; Kunapuli and Daniel, 1998; Wang et al., 2002) or blockade by diinosine pentaphosphates (Ip₅I) (Hoyle et al., 1997).

Uridine adenosine tetraphosphate (Up₄A)

Physiological and pathophysiological effects of Up₄A

Mean total plasma Up₄A concentrations are significantly increased in juvenile hypertensives compared with juvenile normotensive subjects (Jankowski et al., 2007b). Accordingly, Up₄A shows a significant association with juvenile hypertension. The plasma Up₄A concentrations correlate with left ventricular mass and intima media wall thickness in hypertensive subjects. The increased intima media thickness may be related to proliferative effects of Up₄A, as Up₄A has been demonstrated to increase human VSMC proliferation. Up₄A is obviously an important risk factor of juvenile hypertension. Furthermore, Up₄A was identified in renal tissue (Jankowski et al., 2008). Stimulation of tubule cells with oleoyl-2-acetyl-sn-glycerol (OAG) increases the release rate of Up₄A from tubule cells about 10-fold. Up₄A acts as a strong vasoconstrictive mediator on afferent arterioles, but has no significant effect on the tone of efferent arterioles, suggesting a functional role of Up₄A as an autocrine hormone for glomerular perfusion. Because of the predominant effect of the Up₄A on afferent arterioles, Up₄A may decrease glomerular perfusion, intra-glomerular pressure and, hence, glomerular filtration rate. The release of Up₄A from renal tubular cells may be an additional mechanism whereby tubular cells could affect renal perfusion. Up₄A release may further contribute to renal vascular autoregulation mechanisms. Up₄A obviously plays a role in renal haemodynamics and blood pressure regulation.

Moreover, Up₄A stimulated contraction of isolated rat pulmonary arteries in a concentration-dependent manner (Gui et al., 2008). Up₄A is potent as UTP and UDP in arteries without endothelium, while much more effective than UTP and UDP in preparations with endothelium. The vasoconstrictor effect of Up₄A is inhibited by suramin, but not by P₁,P₅ diinosine pentaphosphate (Ip₅I) or desensitization of P2X receptors with α,β-methylene-ATP. Up₄A-induced contraction is inhibited by pretreatment with thapsigargin, nitrendipine or EGTA, but unaffected by the specific Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-iso-quinolinyl) sulfonyl]-homopiperazine (H-1152) (Ikenoya et al., 2002). Furthermore, unlike ATP and UTP, Up₄A does not induce relaxation of preparations with endothelium precontracted with phenylephrine. Up₄A is obviously a potent vasoconstrictor, but not a vasodilator, of the rat pulmonary artery. It is likely that Up₄A acts through a suramin-sensitive P2Y receptor. The contractile effect of Up₄A involves the entry of extracellular Ca²⁺ and release of Ca²⁺ from intracellular stores, but not Ca²⁺ sensitization via the RhoA/Rho-kinase pathway. Up₄A, therefore, may be important for regulation of pulmonary vascular tone.

Plasma concentrations of dinucleoside polyphosphates

Physiologically relevant concentrations of dinucleoside polyphosphates are present in human plasma. The mean total plasma diadenosine polyphosphate concentrations (µmol·L⁻¹; mean ± SEM) in cubital veins of normotensive subjects are 0.89 ± 0.59 for Ap₃A, 0.72 ± 0.72 for Ap₄A, 0.33 ± 0.24 for Ap₅A and 0.18 ± 0.18 for Ap₆A (Jankowski et al., 2003a). In adrenal venous plasma, significantly higher diadenosine polyphosphate concentrations are detectable than in plasma from the infrarenal and suprarenal vena cava. Mean total plasma Up₄A concentrations are in the range of 55.5 ± 15.2 nmol·L⁻¹ (Jankowski et al., 2005). Adrenal medulla (Jankowski et al., 2003a) and endothelial cells (Jankowski et al., 2005) are obviously a source of plasma dinucleoside polyphosphates in humans.

Therapeutic aspects of the purinergic system

There have been promising developments concerning purinergic anti-thrombotic drugs (Cattaneo, 2006; Gachet, 2006; Gachet et al., 2006). Platelets are known to express P2Y₁, P2Y₁₂ and P2X₁ receptors (Hollopeter et al., 2001). Clinical trials like CURE (Yusuf et al., 2001) and CREDO (Beinart et al., 2005) have provided clear evidence that the purinergic anti-thrombotic drugs clopidogrel and ticlopidine reduce the risks of recurrent strokes and heart attacks, especially when combined with aspirin (Kam and Nethery, 2003; Kunapuli et al., 2003).

Moreover, dinucleoside polyphosphates have been shown to possess beneficial properties in the treatment of various diseases, such as chronic obstructive pulmonary disease. Dinucleoside polyphosphates facilitate the clearance of mucous secretions from the lungs of mammals, including humans, being treated for diseases, such as cystic fibrosis (Picher and Boucher, 2000) and chronic bronchitis (Picher and Boucher, 2000). Furthermore, properties of diadenosine polyphosphates may serve to help in the treatment of some ocular pathologies like dry eye (Yerxa et al., 2002; Guzman-Aranguez et al., 2007) and retinal detachment (Guzman-Aranguez et al., 2007). Recent findings showing increased dinucleoside polyphosphate concentration in hypertensive patients (Jankowski et al., 2007b) may provide novel therapeutic approaches for hypertension in the future.

Patented therapeutic effects of dinucleoside polyphosphates

Some potential therapeutic effects of dinucleoside polyphosphates are protected by international patents. For example, Stutts et al. claim the rights to the therapeutic effects of dinucleoside polyphosphates in the context of asthma, bronchiectasis, post-operative mucous retention, pneumonia and primary ciliary dyskinesia (Stutts et al., 1995). Moreover, prevention and treatment of pneumonia in immobilized patients using dinucleoside polyphosphates are protected by Jacobus and Leighton (1996). Further patents claim treatment of sinusitis (Jacobus et al., 1998a,b; Jacobus et al., 1996), otitis media (Drutz et al., 1996) and nasolacrimal duct obstruction (Yerxa and Brown, 2003) with dinucleoside polyphosphates.

Glossary

Abbreviations:

ERK

extracellular signal-regulated kinase

H-1152

(S)-(+)-2-methyl-1-[(4-methyl-5-iso-quinolinyl) sulfonyl]-homopiperazine: Rho-kinase inhibitor

Hep-G2

human hepatocellular liver carcinoma cell line

MAPK

mitogen-activated protein kinase

OAG

oleoyl-2-acetyl-sn-glycerol

PI3K

phosphatidylinositol 3-kinase

Raf-1

serine/threonine-specific kinase (EC 2.7.11.1)

MAP

kinase kinase kinase (MAP3K)

VSMC

vascular smooth muscle cell

Xp_nX

dinucleoside polyphosphates [with X = adenosine, guanosine, inosine or uridine; n= number of phosphate groups (p)]

Conflicts of interest

None.