This study aims to investigate whether tissue oxidative stress in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside, plays an active role in age-dependent susceptibility to hypertension in response to nitric oxide (NO) deficiency induced by systemic l-NAME treatment, and to decipher the underlying molecular mechanisms. Systolic blood pressure (SBP) and heart rate (HR) in conscious rats were recorded, along with measurements of plasma and RVLM level of NO and reactive oxygen species (ROS), and expression of mRNA and protein involved in ROS production and clearance, in both young and adult rats subjected to intraperitoneal (i.p.) infusion of l-NAME. Pharmacological treatments were administered by oral gavage or intracisternal infusion. Gene silencing of target mRNA was made by bilateral microinjection into RVLM of lentivirus that encodes a short hairpin RNA (shRNA) to knock down gene expression of NADPH oxidase activator 1 (Noxa1). We found that i.p. infusion of l-NAME resulted in increases in SBP, sympathetic neurogenic vasomotor activity, and plasma norepinephrine levels in an age-dependent manner. Systemic l-NAME also evoked oxidative stress in RVLM of adult, but not young rats, accompanied by augmented enzyme activity of NADPH oxidase and reduced mitochondrial electron transport enzyme activities. Treatment with L-arginine via oral gavage or infusion into the cistern magna (i.c.), but not i.c. tempol or mitoQ10, significantly offset the l-NAME-induced hypertension in young rats. On the other hand, all treatments appreciably reduced l-NAME-induced hypertension in adult rats. The mRNA microarray analysis revealed that four genes involved in ROS production and clearance were differentially expressed in RVLM in an age-related manner. Of them, Noxa1, and GPx2 were upregulated and Duox2 and Ucp3 were downregulated. Systemic l-NAME treatment caused greater upregulation of Noxa1, but not Ucp3, mRNA expression in RVLM of adult rats. Gene silencing of Noxa1 in RVLM effectively alleviated oxidative stress and protected adult rats against l-NAME-induced hypertension. These data together suggest that hypertension induced by systemic l-NAME treatment in young rats is mediated primarily by NO deficiency that occurs both in vascular smooth muscle cells and RVLM. On the other hand, enhanced augmentation of oxidative stress in RVLM may contribute to the heightened susceptibility of adult rats to hypertension induced by systemic l-NAME treatment.

• Klíčová slova
• NADPH oxidase activator 1, aging, hypertension, l-NAME, mitochondria, nitric oxide, reactive oxygen species, rostral ventrolateral medulla,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The 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. METHODS: Young (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. RESULTS: We 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. CONCLUSION: Our 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.

• Klíčová slova
• Antioxidant response, Brain stem, NOS inhibition, Radical signaling,
• MeSH
• antioxidancia metabolismus   MeSH
• indazoly farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• krysa rodu Rattus MeSH
• metabolická inaktivace * MeSH
• mozkový kmen účinky léků   metabolismus   MeSH
• NG-nitroargininmethylester farmakologie   MeSH
• oxid dusnatý nedostatek   MeSH
• potkani Wistar MeSH
• synthasa oxidu dusnatého antagonisté a inhibitory   MeSH
• věkové faktory MeSH
• volné radikály metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 7-nitroindazole MeSH Prohlížeč
• antioxidancia MeSH
• indazoly MeSH
• inhibitory enzymů MeSH
• NG-nitroargininmethylester MeSH
• oxid dusnatý MeSH
• synthasa oxidu dusnatého MeSH
• volné radikály MeSH