A thromboxane A 2 system in the Atlantic stingray, Dasyatis sabina

Abstract

Thromboxane B 2 ( TXB 2) is the stable metabolite of thromboxane A 2 ( TXA 2) and thromboxane B 2-like immunoreactivity (iTXB 2) has been identified in the plasma of the Atlantic stingray, Dasyatis sabina (0.57±0.03 ng/ml). Plasma levels of iTXB 2 increase if the blood is allowed to clot (3.0±0.27 ng/ml). When clotting occurs in the presence of indomethacin, this increase is partially inhibited (1.5±0.17 ng/ml), indicating the presence of a cyclooxygenase activity. Radioligand binding analysis using the TXA 2 analog [ 125 I ]BOP in isolated kidney membranes revealed a receptor of K d=2.88±0.51 nM and B max=25.6±5.9 fmol/mg protein. [ 125 I ]BOP binding was displaced by the TXA 2 receptor (TP receptor) agonists U46619 (IC 50=106.4±15.7 nM) and U44069 (IC 50=88.7±13.0 nM), and the antagonist SQ29548 (IC 50=51.0±12.9 nM). Binding was also displaced stereoselectively by the antagonists (−)L657925 (IC 50=18.9±3.8 nM) and (+)L657926 (IC 50=2025±280 nM). Tissue bath studies revealed that U46619, a stable TXA 2 mimetic, elicited concentration-dependent contractions in the ventral aorta which were inhibited in a concentration-dependent manner by the TP receptor antagonist SQ29548. Using a human TP receptor riboprobe, Northern blotting of mRNA isolated from the stingray kidney identified transcripts of 2.8 and 6 kb. The 2.8 kb transcript is similar to a 2.8 kb transcript found in human cells or tissues, but the 6 kb transcript may be unique. These data indicate the presence of a TXB 2-like substance in the blood, a TP receptor in the kidney, TXA 2 biological activity in the ventral aorta, and expression of a TP receptor-like gene.