Evidence that the substance P‐induced enhancement of pacemaking in lymphatics of the guinea‐pig mesentery occurs through endothelial release of thromboxane A 2

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 n M (115±3% of control, n =11) with 1 μ M , the highest concentration tested, increasing the rate to 153±4% of control ( n =9). 3 Repetitive 5 min applications of substance P (1 μ M ) 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 μ M ) and the specific NK 1 receptor antagonist, WIN51708 (10 μ M ) both prevented the enhancement of constriction rate induced by 1 μ M substance P. 5 Endothelial cells loaded with the Ca 2+ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca 2+ ] i upon application of 1 μ M substance P. 6 Inhibition of nitric oxide synthase by N G nitro‐ L ‐arginine ( L ‐NOARG; 100 μ M ) had no significant effect on the response induced by 1 μ M substance P. 7 The enhancement of constriction rate induced by 1 μ M substance P was prevented by the cyclo‐oxygenase inhibitor, indomethacin (3 μ M ), the thromboxane A 2 synthase inhibitor, imidazole (50 μ M ), and the thromboxane A 2 receptor antagonist, SQ29548 (0.3 μ M ). 8 The stable analogue of thromboxane A 2 , U46619 (0.1 μ M ) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μ M ). 9 Treatment with pertussis toxin (PTx; 100 ng ml −1 ) completely abolished the response to 1 μ M substance P without inhibiting either the perfusion‐induced constriction or the U46619‐induced enhancement of constriction rate. 10 Application of the phospholipase A 2 inhibitor, antiflammin‐1 (1 n M ) prevented the enhancement of lymphatic pumping induced by substance P (1 μ M ), without inhibiting the response to either U46619 (0.1 μ M ) or acetylcholine (10 μ M ). 11 The data support the hypothesis that the substance P‐induced increase in pumping rate is mediated via the endothelium through NK 1 receptors coupled by a PTx sensitive G‐protein to phospholipase A 2 and resulting in generation of the arachidonic acid metabolite, thromboxane A 2 , this serving as the diffusible activator.British Journal of Pharmacology (1997) 121 , 1589–1596; doi: 10.1038/sj.bjp.0701306