14 August 1997, Volume 121, Issue 8, Pages 1589 – 1596

Article

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2 Sharyn E. Rayner1 & Dirk F. Van Helden The Neuroscience Group, Discipline of Human Physiology, Faculty of Medicine and Health Sciences, University of Newcastle, Callaghan, NSW 2308, Australia     1Author for correspondence

Keywords

Lymphatic vessels;   endothelium;   constriction rate;   pacemaking;   substance P;   thromboxane A2;   arachidonic acid;   calcium;   pertussis toxin;   NK1 receptor

Abstract

1   In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels. 2   Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 µM, the highest concentration tested, increasing the rate to 153±4% of control (n=9). 3   Repetitive 5 min applications of substance P (1 µM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively. 4   The competitive antagonist of tachykinin receptors, spantide (5 µM) and the specific NK1 receptor antagonist, WIN51708 (10 µM) both prevented the enhancement of constriction rate induced by 1 µM substance P. 5   Endothelial cells loaded with the Ca2+ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca2+]i upon application of 1 µM substance P. 6   Inhibition of nitric oxide synthase by NG nitro-L-arginine (L-NOARG; 100 µM) had no significant effect on the response induced by 1 µM substance P. 7   The enhancement of constriction rate induced by 1 µM substance P was prevented by the cyclo-oxygenase inhibitor, indomethacin (3 µM), the thromboxane A2 synthase inhibitor, imidazole (50 µM), and the thromboxane A2 receptor antagonist, SQ29548 (0.3 µM). 8   The stable analogue of thromboxane A2, U46619 (0.1 µM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 µM). 9   Treatment with pertussis toxin (PTx; 100 ng ml-1) completely abolished the response to 1 µM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate. 10   Application of the phospholipase A2 inhibitor, antiflammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 µM), without inhibiting the response to either U46619 (0.1 µM) or acetylcholine (10 µM). 11   The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK1 receptors coupled by a PTx sensitive G-protein to phospholipase A2 and resulting in generation of the arachidonic acid metabolite, thromboxane A2, this serving as the diffusible activator.

Received 20 January, 97; Revised 23 April, 97; Accepted 12 May, 97

^© Macmillan Publishers Ltd 1997