Evidence that the ATP-induced increase in vasomotion of guinea-pig mesenteric lymphatics involves an endothelium-dependent release of thromboxane A2

Abstract

1. Experiments were made to investigate mechanisms by which adenosine 5'-trisphosphate (ATP) enhanced vasomotion in mesenteric lymphatic vessels isolated from young guinea-pigs. 2. ATP (10-8 - 10-3 M) caused a concentration-dependent increase of perfusion-induced vasomotion with the endothelium mediating a fundamental role at low ATP concentrations (10-8 - 10-6 M). 3. The response to 10-6 M ATP showed tachyphylaxis when applied at intervals of 10 min but not at intervals of 20 or 30 min. 4. Suramin (10-4 M) or reactive blue 2 (3x10-5 M) but not PPADS (3x10-5 M) abolished the excitatory response to 10-6 M ATP confirming an involvement of P2 purinoceptors. 5. The excitatory response to 10-6 M ATP was abolished by treatment with either pertussis toxin (100 ng ml-1), antiflammin-1 (10-9 M), indomethacin (3x10-6 M) or SQ29548 (3x10-7 M), inhibitors of specific G proteins, phospholipase A2, cyclo-oxygenase and thromboxane A2 receptors respectively. 6. ATP simultaneously induced a suramin-sensitive inhibitory response, which was normally masked by the excitatory response. ATP-induced inhibition was mediated by endothelium-derived nitric oxide (EDNO) as the response was abolished by NG-nitro-L-arginine (L-NOARG; 10-4 M), an inhibitor of nitric oxide synthase. 7. We conclude that ATP modulates lymphatic vasomotion by endothelium-dependent and endothelium-independent mechanisms. One of these is a dominant excitation caused through endothelial P2 purinoceptors which because of an involvement of a pertussis toxin sensitive G-protein may be of the P2Y receptor subtype. Their stimulation increases synthesis of phospholipase A2 and production of thromboxane A2, an arachidonic acid metabolite which acts as an endothelium-derived excitatory factor.

Figure 1

The effects of ATP at concentrations in the range 10⁻⁹–10⁻³ M on lymphangion constriction rate in vessels with or without an endothelium. Data were normalized with respect to the corresponding control lymphangion constriction rate with n=9–12 lymphangions for all points. Vertical lines denote s.e.mean. ^*P<0.05, ^**P<0.01.

Figure 2

The effects of antagonists of P₂ purinoceptors on lymphangion constriction rate before and during application of 10⁻⁶ M ATP. The data presented are for suramin (10⁻⁴ M) (a); PPADS (3×10⁻⁵ M) (b), reactive blue 2 (RB2, 3×10⁻⁵ M) (c) and RB2 together with L-NOARG (10⁻⁴ M) an inhibitor of NO synthase (d). Data were normalized with respect to the corresponding lymphangion constriction rate just before application of either ATP or ATP plus inhibitor with each measurement based on data from n=6–12 lymphangions. Vertical lines denote s.e.mean. ^**P<0.01.

Figure 3

The effects of L-NOARG an inhibitor of NO synthase on lymphangion constriction rate before and during application of 10⁻⁶ M ATP. Data were normalized with respect to the corresponding lymphangion constriction rate just before application of either ATP or ATP plus L-NOARG with each measurement based on data from n=9 lymphangions. Vertical lines denote s.e.mean. ^*P<0.05, ^**P<0.01.

Figure 4

The effects of various inhibitors on lymphangion constriction rate before and during application of 10⁻⁶ M ATP. The data presented are for SQ29548 (3×10⁻⁷ M) an inhibitor thromboxane A₂ receptors (a), indomethacin (3×10⁻⁶ M) an inhibitor of cyclo-oxygenase (b), antiflammin-1 (10⁻⁹ M) an inhibitor of phospholipase A₂ (c) and pertussis toxin (PTx, 100 ng ml⁻¹) an inhibitor of specific G proteins (d). Data were normalized with respect to the corresponding lymphangion constriction rate just before application of either ATP or ATP plus inhibitor with each measurement based on data from n=6–12 lymphangions. All experiments were performed in the presence of 10⁻⁴ M L-NOARG. Vertical lines denote s.e.mean. ^*P<0.05, ^**P<0.01.