Age-dependent redox status in the brain stem of NO-deficient hypertensive rats

Abstract

BackgroundThe brain stem contains important nuclei that control cardiovascular function via the sympathetic nervous system (SNS), which is strongly influenced by nitric oxide. Its biological activity is also largely determined by oxygen free radicals. Despite many experimental studies, the role of AT1R-NAD(P)H oxidase-superoxide pathway in NO-deficiency is not yet sufficiently clarified. We determined changes in free radical signaling and antioxidant and detoxification response in the brain stem of young and adult Wistar rats during chronic administration of exogenous NO inhibitors.MethodsYoung (4 weeks) and adult (10 weeks) Wistar rats were treated with 7-nitroindazole (7-NI group, 10 mg/kg/day), a specific nNOS inhibitor, with NG-nitro-L-arginine-methyl ester (L-NAME group, 50 mg/kg/day), a nonspecific NOS inhibitor, and with drinking water (Control group) during 6 weeks. Systolic blood pressure was measured by non-invasive plethysmography. Expression of genes (AT1R, AT2R, p22phox, SOD and NOS isoforms, HO-1, MDR1a, housekeeper GAPDH) was identified by real-time PCR. NOS activity was detected by conversion of [3H]-L-arginine to [3H]-L-citrulline and SOD activity was measured using UV VIS spectroscopy.ResultsWe observed a blood pressure elevation and decrease in NOS activity only after L-NAME application in both age groups. Gene expression of nNOS (youngs) and eNOS (adults) in the brain stem decreased after both inhibitors. The radical signaling pathway triggered by AT1R and p22phox was elevated in L-NAME adults, but not in young rats. Moreover, L-NAME-induced NOS inhibition increased antioxidant response, as indicated by the observed elevation of mRNA SOD3, HO-1, AT2R and MDR1a in adult rats. 7-NI did not have a significant effect on AT1R-NADPH oxidase-superoxide pathway, yet it affected antioxidant response of mRNA expression of SOD1 and stimulated total activity of SOD in young rats and mRNA expression of AT2R in adult rats.ConclusionOur results show that chronic NOS inhibition by two different NOS inhibitors has age-dependent effect on radical signaling and antioxidant/detoxificant response in Wistar rats. While 7-NI had neuroprotective effect in the brain stem of young Wistar rats, L-NAME- induced NOS inhibition evoked activation of AT1R-NAD(P)H oxidase pathway in adult Wistar rats. Triggering of the radical pathway was followed by activation of protective compensation mechanism at the gene expression level.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-017-0366-4) contains supplementary material, which is available to authorized users.

Chronic NOS Inhibition Affects Oxidative State and Antioxidant Response Differently in the Kidneys of Young Normotensive and Hypertensive Rats

“…Many studies have observed that chronic inhibition of NOS induced with L-NAME successfully decreases the activity of NOS in major systems involved in the regulation of blood pressure, such as the brain, vessels, heart, or kidney, in normotensive and hypertensive rats [21, 42, 43]. However, we did not observe this inhibitory effect in the kidneys of Wistar rats.…”

“…Administration of L-NAME to young SHRs involves kidney damage, and it is often used as a model for studying chronic kidney diseases typically observed in patients with essential hypertension [27]. Used doses of the inhibitors were adjusted based on previous experiences from our laboratory as well as observations of other experimental works [21, 28, 29]. Experimental studies have observed that higher doses of 7-NI result in nonspecific inhibition of NOS and the inhibition of eNOS has also been observed [30].…”

“…The activity of NOS was measured by conversion of radioactive [3H]-L-arginine (Amersham, UK) to 3H-L-citrulline according to a previous procedure [21]. After the incubation (6 min at 37°C) of pairs of samples and the reaction mixture (consisting of 10 mM NADPH; 0.5 M Tris, pH 7.4; 20 mM CaCl2 (MgCl2); 100 μ M L-arginine; 1 mg/ml calmodulin; FAD/FMN 1 : 1; radio-labelled L-arginine; 50 mM BH4; distilled water), the reaction was started by adding the reaction mixture to the samples.…”

“…Short- and long-term administrations of L-NAME (nonselective inhibitor of NOS) in moderate and high doses cause persistent hypertension associated with multiple renal functional and morphological damage [15–17]. Studies show that the L-NAME-induced inhibition of NOS can lead to stimulation of RAS [18–20] and may result in an increase of ROS production [21, 22]. Oxidative injury of the kidney present in L-NAME-treated normotensive rats is probably mediated by Ang II.…”

“…The neuroprotective effect of 7-NI was observed in young normotensive rats, where the antioxidant response of SOD enzymes was stimulated in the brain stem, but not in the L-NAME group. Our previous study suggests that the effect of NOS inhibition is age-dependent [21].…”