Structure-activity relationships of diadenosine polyphosphates (Ap(n)As), adenosine polyphospho guanosines (Ap(n)Gs) and guanosine polyphospho guanosines (Gp(n)Gs) at P2 receptors in the rat mesenteric arterial bed

Abstract

1. Vascular effects of diadenosine polyphosphates (Ap(n)As), adenosine polyphospho guanosines (Ap(n)Gs) and guanosine polyphospho guanosines (Gp(n)Gs), novel families of naturally-occurring signalling molecules, were investigated in methoxamine preconstricted rat isolated perfused mesenteric arterial beds. 2. Three different types of response were elicited by Ap(n)As and Ap(n)Gs. Those with a short polyphosphate chain (n=2 - 3) elicited vasorelaxation. Ap(3)A was more potent than Ap(2)A, and both were more potent than the corresponding Ap(n)G. Relaxations to Ap(3)A and Ap(3)G, but not to Ap(2)A and Ap(2)G, were blocked by endothelium removal and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a P2 receptor antagonist. 3. Longer polyphosphate chain Ap(n)As and Ap(n)Gs (n=4 - 6) elicited dose-dependent vasoconstriction followed by prolonged vasorelaxation, with a potency order for both types of response of Ap(5)A> or =Ap(6)A>Ap(4)A. A similar order and potency was observed for Ap(n)Gs. Contractions and prolonged relaxations were blocked by PPADS and P2X(1) receptor desensitization with alpha,beta-methylene ATP (alpha,beta-meATP), and were largely endothelium-independent. 4. In the presence of alpha,beta-meATP rapid relaxations to contractile Ap(n)As and Ap(n)Gs (n=4 - 6) were revealed. 5. Gp(n)Gs were virtually inactive, except for Gp(2)G which elicited vasoconstriction via PPADS- and alpha,beta-meATP-sensitive smooth muscle P2X(1)-like receptors. 6. These data show that, as with Ap(n)As, the length of the polyphosphate chain (n) is an important determinant of the activity of Ap(n)Gs at P2 receptors in the rat mesenteric arterial bed. When the chain is short (n=2 - 3) the purines elicit rapid vasorelaxation, which for Ap(3)A and Ap(3)G is mediated via endothelial P2Y(1)-like receptors. When the chain is long (n=4 - 6) Ap(n)As and Ap(n)Gs elicit vasoconstriction via P2X(1)-like receptors, followed by prolonged endothelium-independent vasorelaxation. Rapid relaxation to contractile dinucleotides (n=4 - 6) is revealed by block of vasoconstriction. Regarding the purine moiety, one adenine is crucial and sufficient for vasoactivity as Gp(n)Gs were largely inactive, and Ap(n)As and Ap(n)Gs approximately equipotent.

Figure 1

Representative trace showing responses to Ap_nGs (n=2 – 5) in the rat isolated perfused methoxamine-preconstricted mesenteric arterial bed. Ap₂G and Ap₃G elicited only vasorelaxation, whereas Ap₄G and Ap₅G elicited vasoconstriction followed by slow and prolonged vasorelaxation. Responses to Ap₆G (not shown) were similar to those to Ap₅G. Ap_nGs were applied at the doses (nmol) indicated. The very small increases in perfusion pressure observed at low doses of all agonists are injection artefacts. Note the different vertical scale for responses to 50 nmol Ap₄G and 5 and 50 nmol Ap₅G.

Figure 2

Dose response curves to (a) Ap_nGs and (b) Ap_nAs in the rat isolated perfused methoxamine-preconstricted mesenteric arterial bed. Solid lines indicate constrictions (increase in perfusion pressure, mmHg) or rapid relaxations (% of tone) and dashed lines indicate prolonged relaxations (% of tone). n=5 – 6 for each point. Data are shown as mean+s.e.mean.

Figure 3

Dose response curves to Ap_nGs under control conditions (solid symbols; n=6) and (a) in the presence of pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10 μM) (n=4), (b) after endothelium removal (n=6), and (c) in the presence of α,β-methylene ATP (α,β-meATP; 10 μM) (n=4), in the rat isolated perfused methoxamine-preconstricted mesenteric arterial bed. Data are shown as mean+s.e.mean.

Figure 4

Representative trace showing responses to Ap_nGs (n=2 – 6) in the rat isolated perfused methoxamine-preconstricted mesenteric arterial bed without endothelium (upper trace) or in the presence of α,β-methylene ATP (α,β-meATP; 10 μM) (lower trace). Rapid vasorelaxations to Ap₃G were abolished by endothelium removal, but contraction and prolonged relaxation to Ap₄G was unaffected. α,β-meATP blocked contraction and prolonged relaxation to Ap₄G, Ap₅G and Ap₆G and revealed rapid relaxations. Ap_nGs were applied at the doses (nmol) indicated. The very small increases in perfusion pressure observed at low doses of all agonists are injection artefacts.

Figure 5

Representative trace showing responses to Ap_nAs (n=3 and 4) in the rat isolated perfused methoxamine-preconstricted mesenteric arterial bed under control conditions (top trace), in the presence of pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10 μM) (middle trace), and in the presence of α,β-methylene ATP (α,β-meATP; 10 μM) (bottom trace). Note that an increase by a single phosphate of the polyphosphate chain length of Ap₃A to produce Ap₄A leads to a loss of rapid relaxation and the response is contraction followed by slow relaxation. Rapid vasorelaxations to Ap₃A, and contractions and prolonged relaxation to Ap₄A were attenuated by PPADS. Rapid relaxation to Ap₃A was unaffected by α,β-meATP but contractions and prolonged relaxation to Ap₄A was attenuated. Ap_nAs were applied at the doses (nmol) indicated. The very small increases in perfusion pressure observed at low doses of all agonists are injection artefacts.

Figure 6

Dose response curves to Ap_nAs under control conditions (solid symbols) and (a) in the presence of pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10 μM), (b) after endothelium removal, and (c) in the presence of α,β-methylene ATP (α,β-meATP; 10 μM), in the rat isolated perfused methoxamine-preconstricted mesenteric arterial bed. n=5 – 6. Data are shown as mean+s.e.mean.

Figure 7

Effect of pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10 μM) on relaxation dose-response curves to Ap_nGs and Ap_nAs (n=2 – 6) in the rat isolated perfused methoxamine-preconstricted mesenteric arterial bed. Rapid relaxations to: (a) Ap₂G and Ap₃G (n=4 – 6); (b) Ap₂A and Ap₃A (n=4 – 6). Prolonged relaxations to: (c) Ap₄G, Ap₅G and Ap₆G (n=6); (d) Ap₄A, Ap₅A and Ap₆A (n=5 – 6). Data are shown as mean+s.e.mean.

Figure 8

Effect of α,β-methylene ATP (α,β-meATP; 10 μM) on relaxation dose-response curves to Ap_nGs and Ap_nAs (n=2 – 6) in the rat isolated perfused methoxamine-preconstricted mesenteric arterial bed. Rapid relaxations to: (a) Ap₂G and Ap₃G (n=4 – 6); (b) Ap₂A and Ap₃A (n=4 – 6). Prolonged relaxations to: (c) Ap₄G, Ap₅G and Ap₆G (n=6); (d) Ap₄A, Ap₅A and Ap₆A (n=5 – 6). Data are shown as mean+s.e.mean.

Figure 9

Effect of endothelium removal on relaxation dose-response curves to Ap_nGs and Ap_nAs (n=2 – 6) in the rat isolated perfused methoxamine-preconstricted mesenteric arterial bed. Rapid relaxations to: (a) Ap₂G and Ap₃G (n=6); (b) Ap₂A and Ap₃A (n=6). Prolonged relaxations to: (c) Ap₄G, Ap₅G and Ap₆G (n=6); (d) Ap₄A, Ap₅A and Ap₆A (n=5 – 6). Data are shown as mean+s.e.mean. In the absence of endothelium the attenuated rapid relaxation response to Ap₃A was followed by a small constriction (isolated symbol in panel b).

Figure 10

Dose-response curves to Gp_nG (n=2 – 6) in the rat isolated perfused methoxamine-preconstricted mesenteric arterial bed. (a) Control conditions; (b) responses to Gp_nG in the presence of pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10 μM) and α,β-methylene ATP (α,β-meATP; 10 μM); (c) responses to Gp_nGs with and without endothelium removal (n=4 – 6). Data are shown as mean+s.e.mean. Where error bars do not appear, these fall within the symbol.