Role of in vivo vascular redox in resistance arteries

Abstract

Vascular thiol redox state has been shown to modulate vasodilator functions in large conductance Ca2+ -activated K+ channels and other related channels. However, the role of vascular redox in small resistance arteries is unknown. To determine how in vivo modulation of thiol redox state affects small resistance arteries relaxation, we generated a transgenic mouse strain that overexpresses thioredoxin, a small redox protein (Trx-Tg), and another strain that is thioredoxin-deficient (dnTrx-Tg). The redox state of the mesenteric arteries (MAs) in Trx-Tg mice is found to be predominantly in reduced state; in contrast, MAs from dnTrx-Tg mice remain in oxidized state. Thus, we created an in vivo redox system of mice and isolated the second-order branches of the main superior MAs from wild-type, Trx-Tg, or dnTrx-Tg mice to assess endothelium-dependent relaxing responses in a wire myograph. In MAs isolated from Trx-Tg mice, we observed an enhanced intermediate-conductance Ca2+ -activated potassium channel contribution resulting in a larger endothelium-dependent hyperpolarizing (EDH) relaxation in response to indirect (acetylcholine) and direct (NS309) opening of endothelial calcium-activated potassium channels. MAs derived from dnTrx-Tg mice showed both blunted nitric oxide-mediated and EDH-mediated relaxation compared with Trx-Tg mice. In a control study, diamide decreased EDH relaxations in MAs of wild-type mice, whereas dithiothreitol improved EDH relaxations and was able to restore the diamide-induced impairment in EDH response. Furthermore, the basal or angiotensin II-mediated systolic blood pressure remained significantly lower in Trx-Tg mice compared with wild-type or dnTrx-Tg mice, thus directly establishing redox-mediated EDH in blood pressure control.

Keywords: endothelium; mesenteric arteries; relaxation; thioredoxins.

Figure 1

(A) Expression of Trx in mesenteric artery of wt, Trx-Tg and dnTrx-Tg mice; (B)Trx activity is increased in MAs in Trx-Tg mice, but not in dnTrx-Tg mice.(C) TrxR activity does not change in wt, Trx-Tg or dnTrx-Tg mice MAs.(D) Trx remains predominantly in the oxidized state in MAs from dnTrx mice.

Figure 2

(A) Depolarization (60 mmol/L K⁺ KRB)-induced, and (B) induced contraction in MAs derived from wt (white bars), Trx-Tg (blue bars) and dnTrx-Tg (red bars) mice. MAs derived from wild-type (circles), Trx-Tg (upward triangles) and dnTrx-Tg (downward triangles) mice were assessed for: (C) Endothelium-dependent relaxations to cumulative concentrations of ACh. (D) Endothelium-independent relaxations in MAs pretreated with L-NAME and indomethacin (INDO) contracted with a submaximal concentration of PHE, and assessed with cumulative concentrations of SNP. (E) NO-mediated endothelium-dependent relaxations in MAs pretreated for 30 min with INDO the IK1 channel blocker TRAM-34 and the SK3 channel blocker UCL-1684, to block EDH-mediated responses, contracted with PHE before assessing relaxing responses to cumulative concentrations of ACh. (F) Endothelium-dependent hyperpolarizing (EDH) responses in MAs treated with the NO synthase blocker L-NAME and INDO followed by cumulative concentrations of ACh. Values are means ± SEM (n=8–10 mice). * P< 0.05 Trx-Tg compared with wt, # P< 0.05 dnTrx-Tg compared with wtand Trx-Tg, ** P< 0.05 Trx-Tg compared with wtand dnTrx-Tg.

Figure 3

(A) EDH-mediated responses in the presence of the selective IK1 channel blocker TRAM-34 in MAs derived from wt (circles), Trx-Tg (upward triangles) and dnTrx-Tg (downward triangles) mice. Insert: Percentage inhibition of the EDH response by TRAM-34 in MAs derived from wt (white bars), Trx-Tg (blue bars) and dnTrx-Tg (red bars) mice. (B) EDH-mediated responses in the presence of the selective SK3 channel blocker UCL-1684 in MAs derived from wt (circles), Trx-Tg (upward triangles) and dnTrx-Tg (downward triangles) mice. Insert: Percentage inhibition of the EDH response by UCL-1684 in MAs derived from wt (white bars), Trx-Tg (blue bars) and dnTrx-Tg (red bars) mice. (C) EDH-mediated responses in the combined presence of TRAM-34 and UCL-1684 in MAs derived from wt (circles), Trx-Tg (upward triangles) and dnTrx-Tg (downward triangles) mice. Values are means ± SEM (n=6-8 mice). * P< 0.05 Trx-Tg compared with wt or dnTrx-Tg.

Figure 4

(A) NS309-mediated relaxing responses in endothelium-intact (+E, open symbols) and endothelium-denuded (-E, closed symbols) MAs derived from wt (circles), Trx-Tg (upward triangles) and dnTrx-Tg (downward triangles) mice. (B) NS309-mediated relaxing responses in MAs treated with L-NAME (100 μmol/L) and INDO (10 μmol/L) from wt (circles), Trx-Tg (upward triangles) and dnTrx-Tg (downward triangles) mice. (C) Effect of IK1 channel activator SKA-31 (1 μmol/L) in MAs treated with L-NAME and INDO followed by contraction with PHE and cumulative concentrations to ACh. MAs were isolated from wt (circles), Trx-Tg (upward triangles) and dnTrx-Tg (downward triangles) mice. Values are means ± SEM (n=8–10 mice). * P< 0.05 Trx-Tg compared with wt and dnTrx-Tg, # P< 0.05 dnTrx-Tg compared with wt and Trx-Tg.

Figure 5

NS309-mediated relaxing responses in MAs derived from wt (circles), Trx-Tg (upward triangles) and dnTrx-Tg (downward triangles) mice. MAs were either incubated with L-NAME (100 μmol/L), INDO (10 μmol/L) and the IK1 blocker TRAM-34 (A), or the SK3 channel blocker UCL-1684 (B) or the combined presence of TRAM-34 and UCL- 1684 (C). Inserts: shift in sensitivity (ΔpEC₅₀) for NS309 caused by IK1 inhibition by TRAM-34 for wt (white bars), Trx-Tg (blue bars), and dnTrx-Tg (red bars) mice. Values are means ± SEM (n=8–10 mice). * P< 0.05 Trx-Tg compared with wt or dnTrx-Tg.

Figure 6

(A) Cumulative concentration-response curves to diamide and(DTT) in MAs derived from wt mice. (B) DTT reverses diamide-induced inhibition of PHE-induced increase in active tension. Representative tracing showing tension changes in a MAs isolated from a wt mouse contracted with PHE before, during and after application of diamide, followed by addition of DTT. (C) EDH responses (in the presence of L-NAME and INDO in MAs derived from wt mice treated without (CON, open circles), 0.1 μmol/L diamide (open diamonds) or 1 μmol/L diamide (closed diamonds). Insert: Effect of IK1 channel inhibition by TRAM-34 on the EDH responses. (D) EDH responses in MAs derived from wt mice treated without (CON, open squares), 0.1 μmol/L DTT (open squares), or 1 μmol/L DTT (closed squares). Insert: Effect of IK1 channel inhibition by TRAM-34 on the EDH responses. (E) EDH responses in MAs derived from wt mice treated with 0.3 μmol/L diamide in the additional presence of either 0.1 μmol/L DTT (circles), 1 μmol/L DTT (squares) or 10 μmol/L DTT (diamonds). (F) MAs derived from dnTrx-Tg (downward triangles) mice were treated for 30 min with either vehicle (H₂O; CON) or = DTT and were assessed for endothelium-dependent relaxations to cumulative concentrations of ACh =. Values are means ± SEM (n=4–6 mice). * P< 0.05.

Figure 7

(A) In a subset of MAs, relaxing responses to hydrogen peroxide (H₂O₂) were analysed. Segments were contracted with PHE, followed by a CRC for H₂O₂. (B) After a 15 min washout period, the same segments were incubated with catalase for 30 min and CRCs to H₂O₂ were repeated; (C)Activity of Prx is increased in Trx-Tg MAs, but not in wt or dnTrx-Tg MAs.

Figure 8

(A) The baseline blood pressure of 12 week old mice were measured as described in the methods. The blood pressure of Trx-Tg mice is significantly (P<0.05, ANOVA) lower than the wt or dnTrx-Tg mice. (B) Angiotensin II –mediated increased blood pressure is significantly decreased in Trx-Tg mice, but not in wt mice. *Significantly higher than Trx-Tg mice (P<0.05, ANOVA).

Figure 9

A schematic diagram presenting the role of Trx system in regulation of EDHF response and consequent blood pressure control.