Cyclooxygenase involvement in thromboxane-dependent contraction in rat mesenteric resistance arteries

Abstract

The influence of cyclooxygenase pathway activation following thromboxane-endoperoxide (TP) receptor stimulation was studied in rat mesenteric resistance arteries (n=6 to 10 per group). We studied isolated, perfused, and pressurized mesenteric resistance arteries (mean internal diameter 214 microm) using an arteriograph, enabling us to study arteries in physiological conditions of flow and pressure. Changes in diameter were continuously recorded, and contractions measured as internal diameter reduction. Release of cyclooxygenase pathway metabolites was also assessed by enzyme immunoassay (EIA) analysis of mesenteric bed perfusions. The thromboxane A2 (TxA2) analog U-46619 (1 micromol/L) induced a significant contraction (108 microm maximal diameter reduction). Inhibition by 3 chemically different cyclooxygenase inhibitors (ie, flurbiprofen, indomethacin, and aspirin) potently reduced the contraction to 27%, 25%, and 6% of control, respectively. The selective cyclooxygenase-1 inhibitor SC-58560 inhibited U-46619 contraction, whereas selective cyclooxygenase-2 inhibition (SC-58236) had no effect. Thromboxane synthase inhibition (furegrelate) did not affect U-46619-induced contraction, but it was reduced by cytosolic phospholipase A2 inhibition. Measurement of cyclooxygenase derivatives produced by the isolated mesenteric bed showed that PGE2 was produced after TxA2-receptor stimulation with U-46619. Exogenous prostaglandin E2 (in the presence of the TxA2 receptor antagonist SQ 29 548) and U-46619 contracted mesenteric arteries with a similar potency (EC50: 0.30 and 0.48 micromol/L, respectively). This study provides the first evidence that TxA2-receptor-dependent contraction in a resistant artery involved cyclooxygenase stimulation and, at least in part, a PGE2 formation. This mechanism of TxA2-dependent contraction in resistant arteries might be of importance in the understanding of diseases affecting resistant arteries and involving TxA2, such as hypertension.

Figure 1

A: Concentration-response curves to U-46619 obtained in isolated perfused and pressurized rat mesenteric arteries. Concentration-response curves to U-46619 were repeated in the presence of the non-selective COX inhibitor indomethacin (indo 1 μmol/L, n = 6 per group). B: Inhibitory effect of three chemically non-related non-steroidal anti-inflammatory drugs, indomethacin (1 μmol/L, Indo), flurbiprofen (1 μmol/L, Flur) and aspirin (10 μmol/L, ASA) on U-46619 (1 μmol/L)-induced contraction. C: Inhibitory effect of indomethacin (1 μmol/L, Indo) and flurbiprofen (1 μmol/L, Flur) on phenylephrine (Phe, 1 μmol/L)- and endothelin-1 (ET-1, 10 nmol/L)-induced contraction. Contraction was obtained in perfused and pressurized rat mesenteric resistance arteries (n=10 per group). *P <0.05; two tailed Student’s paired T-test.

Figure 2

Changes in diameter in response to increasing concentrations of the TP receptor agonist U-46619, in perfused and pressurized rat mesenteric resistance arteries (n=8 per group, mean ± sem). Concentration-response curves to U-46619 were repeated in the presence of the selective COX-1 inhibitor SC-58560 (upper panel, A) or in the presence of the COX-2 selective inhibitor (SC-58236) (lower panel, B). Control experiments were conducted in the presence of the solvent (<0.1% DMSO). *P <0.05; two-factor ANOVA.

Figure 3

Changes in diameter in response to increasing concentrations (1 nmol/L to 0.1 μmol/L) of the TP receptor agonist U-46619, PGF_2α or PGE₂ in perfused and pressurized rat mesenteric resistance arteries (n=8 per group, mean ± s.e.m.). *P <0.05; two tailed Student’s T-test.

Figure 4

Inhibitory effect of the TP receptor antagonist SQ 29,548 (SQ, 10 μmol/L) on U-46619 (U46, 1 μmol/L)-, PGF_2α (10 μmol/L)- or PGE₂ (1 μmol/L)-induced contraction. Contraction was obtained in perfused and pressurized rat mesenteric resistance arteries (n=8 per group). *P <0.05; two tailed Student’s T-test.

Figure 5

Immunometric analysis of PGE₂ were done using specific antibodies and tracers in the presence or in the absence (control) of U-46619, in the PSS collected from rat perfused mesenteric beds. *P <0.05; two tailed Student’s T-test.