NS309 restores EDHF-type relaxation in mesenteric small arteries from type 2 diabetic ZDF rats

Abstract

Background and purpose: The endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in mesenteric small arteries from 21 week old Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats was investigated using (6,7-dichloro-1H-indole-2,3-dione 3-oxime) (NS309), a potent activator of small-conductance, calcium-activated potassium channel (SK(Ca)) and intermediate-conductance, calcium-activated potassium channel (IK(Ca)).

Experimental approach: In the presence of inhibitors of cyclooxygenase and nitric oxide synthase [indomethacin and N(omega)-nitro-L-arginine methyl ester (l-NAME), respectively], acetylcholine (ACh)-induced hyperpolarization and EDHF-type relaxation were investigated under isometric conditions in the wire myograph using 0.5 and 1 microM NS309 and/or selective blockers of SK(Ca) and IK(Ca) channels. Membrane potential was recorded with glass microelectrodes, and changes in the intracellular calcium concentration of endothelial cells were visualized by confocal microscopy. SK(Ca) expression was assessed by Western blotting.

Key results: In arteries from ZDF rats, ACh-induced relaxation and membrane hyperpolarization were attenuated and, compared with arteries from ZL rats, NS309 was less potent at causing relaxation. Incubation with 0.5 microM NS309 did not increase ACh-induced relaxation in arteries from ZDF rats significantly. However, 1 microM NS309 restored it (both in the absence and in the presence of indomethacin and l-NAME) without changing endothelial intracellular calcium concentration. The restored EDHF-type relaxation was more sensitive to TRAM-34 (1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole) (1 microM) than to apamin. Expression of the SK(Ca) channel was unaltered.

Conclusions and implications: The attenuated EDHF-type relaxation in mesenteric small arteries from ZDF rats can be restored by NS309 without changes in the intracellular calcium concentration of endothelial cells. These results may have clinical implications for the treatment of endothelial dysfunction in overweight type 2 diabetic patients.

Figure 1

(A) Noradrenaline (NA)-induced contraction of small mesenteric arteries from Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats, under control conditions (n= 5). (B) Relative relaxation of small mesenteric arteries from ZL and ZDF rats to acetylcholine (ACh) under control conditions (n=5) and after 5 min incubation with 0.5 µM (6,7-dichloro-1H-indole-2,3-dione 3-oxime) (NS309) (n= 4). Arteries were precontracted with 6 µM NA. (C) Relative relaxation of small mesenteric arteries to ACh under control conditions from ZL (n= 7) and ZDF (n= 5) rats and after 5 min incubation with 1 µM NS309. Arteries were contracted with 6 µM NA. *Indicates P < 0.05, before and after NS309 treatment (one-way analysis of variance).

Figure 2

(A) Relative relaxation to acetylcholine (ACh) of small mesenteric arteries after incubation with 100 µM N^ω-nitro-L-arginine methyl ester (l-NAME) and 3 µM indomethacin in Zucker lean (ZL) (n=7) and Zucker diabetic fatty (ZDF) (n= 5) rats, before and after incubation with 1 µM (6,7-dichloro-1H-indole-2,3-dione 3-oxime) (NS309). Arteries were precontracted with 6 µM noradrenaline (NA). (B) Relative relaxation of small mesenteric arteries precontracted with 6 µM NA to ACh in the presence of 1 µM NS309, 100 µM l-NAME and 3 µM indomethacin in ZL and ZDF rats and before and after incubation with 50 nM apamin (APA; n= 7, 5; ZDF and ZL rats respectively). (C) Relative relaxation to ACh in the presence of 1 µM NS309, 100 µM l-NAME and 3 µM indomethacin in ZL and ZDF rats before and after incubation with 1 µM (1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole) (TRAM-34) (n= 7, 5; ZDF and ZL rats respectively). In (B) and (C), the relaxation to ACh without apamin and TRAM-34 is the same in the two panels and in (A). *Indicates P < 0.05, in the absence or presence of 1 µM NS309 (one-way anova). ^§Indicates P < 0.05 (one-way anova), before and after treatment with TRAM-34.

Figure 3

Relative relaxation of small mesenteric arteries precontracted with 6 µM noradrenaline to increasing concentrations of (6,7-dichloro-1H-indole-2,3-dione 3-oxime) (NS309) in Zucker lean (ZL) and in Zucker diabetic fatty (ZDF) rats (n= 3).

Figure 4

Changes in membrane potential (mV) in mesenteric small arteries from Zucker diabetic fatty (ZDF) rats and Zucker lean (ZL) rats. The arteries were contracted with 6 µM noradrenaline (NA) in the presence of indomethacin and N^ω-nitro-L-arginine methyl ester; 10 µM acetylcholine (ACh) was added to the bath. The results are mean ± standard error of the mean of arteries from at least four animals in each group. *Indicates P < 0.025 (Student's t-test).

Figure 6

A summary of the results of the intracellular calcium ([Ca²⁺]_i) measurements in endothelial cells from small mesenteric arteries from Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats. (A) Representative trace of changes in [Ca²⁺]_i before and during the incubation with 1 µM (6,7-dichloro-1H-indole-2,3-dione 3-oxime) (NS309) in ZL and ZDF rats. (B) Changes in [Ca²⁺]_i before and during the incubation with 1 µM NS309 in ZL and ZDF rats (n= 6). (C) Original trace showing changes in [Ca²⁺]_i in endothelial cells from ZL and ZDF rats during stimulation with cumulative concentrations of acetylcholine (ACh). (D) The [Ca²⁺]_i in endothelial cells during stimulation with cumulative concentrations of ACh under control conditions (n= 7) and after incubation with 1 µM NS309 (n= 6) in ZL and ZDF rats. The arrow indicates the application of NS309.

Figure 5

Image showing the selective load of the endothelial cells with Calcium Green-1 and Fura-Red. The focal plane is on the endothelial cell layer, where the individual endothelial cells are clearly visible (1). Upper (2) and right (3) panels show vertical scanning layer across and along the artery showing the selective loading of the endothelial cells.

Figure 7

Small-conductance, calcium-activated potassium channel (SK_Ca) expression in mesenteric small arteries from Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats. The y-axis shows the optical density values normalized to the panactin level. Insert shows representative blots of SK_Ca and panactin. Results are mean ± standard error of the mean (SEM), n= 4 for both ZL and ZDF.