Role of NADPH oxidase and iNOS in vasoconstrictor responses of vessels from hypertensive and normotensive rats

Abstract

Background and purpose: To analyse the influence of hypertension in the modulation induced by inducible NOS (iNOS)-derived NO and superoxide anion (O(2) (*-)) of vasoconstrictor responses and the sources of O(2) (*-) implicated.

Experimental approach: Vascular reactivity experiments were performed in segments of aorta from normotensive, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR); protein and mRNA expressions were respectively measured by western blot and quantitative reverse transcription-polymerase chain reaction and O(2) (*-) production was evaluated by ethidium fluorescence.

Key results: The contractile responses to phenylephrine (1 nM-30 microM) and 5-hydroxytryptamine (0.1-100 microM) were greater in aortic segments from SHR than WKY. The selective iNOS inhibitor, 1400W (10 microM), increased the phenylephrine contraction only in WKY segments; however, iNOS protein and mRNA expressions were greater in aorta from SHR than WKY. Superoxide dismutase (SOD, 150 U ml(-1)) reduced phenylephrine and 5-hydroxytryptamine responses only in aorta from SHR; the NAD(P)H oxidase inhibitor apocynin (0.3 mM) decreased phenylephrine and 5-hydroxytryptamine responses more in vessels from SHR than WKY. Co-incubation with SOD plus 1400W potentiated the phenylephrine and 5-hydroxytryptamine responses more in segments from SHR than WKY. O(2) (*-) production was greater in aorta from SHR than WKY; apocynin abolished this difference.

Conclusions and implications: Increased O(2) (*-) formation from NADP(H) oxidase in vessels from hypertensive rats contributes to the vasoconstrictor responses and counteract the increase of NO from iNOS and the consequent modulation of these responses.

Figure 1

Concentration–response curve to phenylephrine in intact aortic segments from normotensive (WKY) and hypertensive (SHR). Results are expressed as a percentage of the response to 75 mM KCl for the number of animals indicated in parentheses. ANOVA (two-way): ^*P<0.05 vs control. WKY, Wistar Kyoto rat; SHR, spontaneously hypertensive rat.

Figure 2

(a) Representative western blot and densitometric analysis for the inducible isoform of nitric oxide synthase (iNOS) in aortic segments from WKY and SHR; mouse macrophages were used for iNOS positive controls (C+). (b) Quantitative RT-PCR assessment of iNOS mRNA expression in the same segments. Results are expressed as the relative expression of protein or mRNA in SHR compared to WKY. ANOVA (one-way): ^*P<0.05. The number of animals are indicated in parentheses. WKY, Wistar Kyoto rat; SHR, spontaneously hypertensive rat.

Figure 3

Effect of 1400W (10 μM) on the concentration–response curve to phenylephrine in: (a) intact aortic segments from WKY and SHR, (b) segments incubated with dexamethasone (1 μM) and (c) endothelium-denuded segments (E−). Results are expressed as a percentage of the response to 75 mM KCl for the number of animals indicated in parentheses. ANOVA (two-way): ^*P<0.05 vs control. WKY, Wistar Kyoto rat; SHR, spontaneously hypertensive rat.

Figure 4

(a) Representative fluorescent photomicrographs and (b) quantitative analysis of confocal microscopic sections of aortic segments from WKY and SHR incubated without (control) or with apocynin (0.3 mM). Vessels were labelled with the oxidative dye dihydroethidium, which produces a red fluorescence when oxidized to ethidium bromide by superoxide anion (O₂^•−). Natural autofluorescence of elastin was used to delimit the different layers of the vascular wall (appears green). Image size 375 × 375 μm. Images were captured with a fluorescence confocal microscope (× 40 oil immersion objective, zoom 1). E=endothelial layer; M=media layer; A=adventitial layer. ^*P<0.05 vs WKY, ^#P<0.05 vs control by Student's t-test. WKY, Wistar Kyoto rat; SHR, spontaneously hypertensive rat.

Figure 5

Effect of (a) superoxide dismutase (SOD; 150 U ml⁻¹) and (b) catalase (1000 U ml⁻¹) on the concentration–response curve to phenylephrine in aortic segments from SHR and WKY. (c) Effect of 1400W (10 μM) on the concentration–response curve to phenylephrine in aortic segments from WKY and SHR treated with SOD. Results are expressed as a percentage of the response to 75 mM KCl for the number of animals indicated in parentheses. ANOVA (two-way): ^*P<0.05 vs control; ^#P<0.05 vs SOD. SHR, spontaneously hypertensive rat; WKY, Wistar Kyoto rat.

Figure 6

Effect of (a) apocynin (0.3 mM) and (b) allopurinol (0.3 mM) on the concentration–response curve to phenylephrine in aortic segments from WKY and SHR. Results are expressed as a percentage of the response to 75 mM KCl for the number of animals indicated in parentheses. ANOVA (two-way): ^*P<0.05 vs control. WKY, Wistar Kyoto rat; SHR, spontaneously hypertensive rat.

Figure 7

Effect of (a) apocynin (0.3 mM) and (b) superoxide dismutase (SOD; 150 U ml⁻¹) on the concentration–response curve to 5-hydroxytryptamine in aortic segments from SHR and WKY. (c) Effect of 1400W (10 μM) on the concentration–response curve to 5-hydroxytryptamine in aortic segments from WKY and SHR treated with SOD. Results are expressed as a percentage of the response to 75 mM KCl for the number of animals indicated in parentheses. ANOVA (two-way): ^*P<0.05 vs control; ^#P<0.05 vs SOD. SHR, spontaneously hypertensive rat; WKY, Wistar Kyoto rat.