Tranilast increases vasodilator response to acetylcholine in rat mesenteric resistance arteries through increased EDHF participation

Abstract

Background and purpose: Tranilast, in addition to its capacity to inhibit mast cell degranulation, has other biological effects, including inhibition of reactive oxygen species, cytokines, leukotrienes and prostaglandin release. In the current study, we analyzed whether tranilast could alter endothelial function in rat mesenteric resistance arteries (MRA).

Experimental approach: Acetylcholine-induced relaxation was analyzed in MRA (untreated and 1-hour tranilast treatment) from 6 month-old Wistar rats. To assess the possible participation of endothelial nitric oxide or prostanoids, acetylcholine-induced relaxation was analyzed in the presence of L-NAME or indomethacin. The participation of endothelium-derived hyperpolarizing factor (EDHF) in acetylcholine-induced response was analyzed by preincubation with TRAM-34 plus apamin or by precontraction with a high K+ solution. Nitric oxide (NO) and superoxide anion levels were measured, as well as vasomotor responses to NO donor DEA-NO and to large conductance calcium-activated potassium channel opener NS1619.

Key results: Acetylcholine-induced relaxation was greater in tranilast-incubated MRA. Acetylcholine-induced vasodilation was decreased by L-NAME in a similar manner in both experimental groups. Indomethacin did not modify vasodilation. Preincubation with a high K+ solution or TRAM-34 plus apamin reduced the vasodilation to ACh more markedly in tranilast-incubated segments. NO and superoxide anion production, and vasodilator responses to DEA-NO or NS1619 remained unmodified in the presence of tranilast.

Conclusions and implications: Tranilast increased the endothelium-dependent relaxation to acetylcholine in rat MRA. This effect is independent of the nitric oxide and cyclooxygenase pathways but involves EDHF, and is mediated by an increased role of small conductance calcium-activated K+ channels.

Figure 1. Mast cell localization by toluidine blue staining.

Figure is representative of preparations from four rats. Magnification: 400X (general vision) and 600X (inset).

Figure 2. Effect of tranilast on endothelial function.

NA-induced vasoconstriction in control and tranilast-treated mesenteric resistance arteries (A).Endothelium-dependent relaxation induced by ACh in NA-precontracted control and tranilast-treated rat resistance arteries (B) Results are expressed as mean ± S.E.M. *P<0.05 control vs. tranilast. N = 6–7 animals each group.

Figure 3. Participation of NO on the vasodilator response to acetylcholine.

Effect of L-NAME (100 µM) on the concentration-dependent relaxation to ACh in control (A) and tranilast-treated (B) mesenteric resistance arteries. Insert graph shows the differences of area under the curve (dAUC) in control and tranilast-treated arteries pre-treated with L-NAME. Results are expressed as mean ± SEM. N = 6–7 animals in each group. (C) Vasodilator response to DEA-NO in control and tranilast-incubated mesenteric resistance arteries, precontracted with either noradrenaline or KCl. Results are expressed as mean ± S.E.M. N = 5–6 animals each group. (D) Effect of tranilast on basal and acetylcholine-induced NO release in rat mesenteric resistance arteries. Results (mean ± S.E.M.) are expressed as arbitrary fluorescence units (A.U.)/mg tissue. N = 4 animals each group. *P<0.05 vs. basal.

Figure 4. Participation of EDHF in the vasodilator response to acetylcholine.

Relaxation to acetylcholine in control (A) and tranilast-treated arteries (B) pre-contracted with KCl. Effect of preincubation with 1 µM apamin plus 0.1 µM TRAM-34 on endothelium-dependent relaxation to acetylcholine in noradrenaline-pre-contracted control (C) and tranilast-treated arteries (D). Insert graph shows the differences of area under the curve (dAUC) in control and tranilast-treated arteries either preconstricted with KCl or pre-treated with TRAM-34 plus Apamin. Results are expressed as mean ± SEM. *P<0.05 control vs. tranilast. N = 5–7 animals in each group.

Figure 5. Participation of potassium channels in the vasodilator response to acetylcholine.

Effect of preincubation with 100 µmol/L L-NAME plus 1 µM apamin or plus 0.1 µM TRAM-34 on endothelium-dependent relaxation to acetylcholine in noradrenaline-pre-contracted control (A) and tranilast-treated arteries (B). Results are expressed as mean ± SEM. *P<0.05 control vs. tranilast N = 5–7 animals in each group. (C) Differences of area under curve (dAUC) in the absence or presence of 100 µmol/L L-NAME plus 1 µM apamin or plus 0.1 µM TRAM-34. Results are expressed as mean ± SEM. dAUC values are expressed as percentage. *P<0.05 control vs. tranilast. N = 5–7 animals each group. (D) Representation of remnant acetylcholine-induced vasodilation after preincubation with 100 µmol/L L-NAME plus 1 µmol/L apamin or plus 0.1 µM TRAM-34, expressed as mean ± SEM of percentage of AUC. *P<0.05 control vs. tranilast. N = 5–7 animals each group.

Figure 6. Vasodilator response to K⁺-channel openers.

Effect of tranilast on the relaxation to the large conductance calcium-activated K⁺-channel opener NS1619 in de-endothelized rat mesenteric arteries. Results are expressed as mean ± SEM. N = 5–7 animals in each group.

Figure 7. Participation of prostanoids in the vasodilator response to acetylcholine.

Effect of preincubation with 10 µM indomethacin or with 100 mmol/L L-NAME plus 1 µM apamin plus 0.1 µM TRAM-34 on the concentration-dependent relaxation to ACh in control (A) and tranilast-treated (B) rat mesenteric resistance arteries. Results are expressed as mean ± S.E.M. *P<0.05 control vs. tranilast. N = 6–7 animals in each group.