Abstract-The rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, is a central site via which angiotensin II (Ang II) elicits its pressor effect. We tested the hypothesis that NADPH oxidase-derived superoxide anion (O 2 ⅐Ϫ ) in the RVLM mediates Ang II-induced pressor response via activation of mitogen-activated protein kinase (MAPK) signaling pathways. Bilateral microinjection of Ang II into the RVLM resulted in an angiotensin subtype 1 (AT 1 ) receptor-dependent phosphorylation of p38 MAPK and extracellular signal-regulated protein kinase (ERK)1/2, but not stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), in the ventrolateral medulla. The Ang II-induced p38 MAPK or ERK1/2 phosphorylation was attenuated by application into the RVLM of a NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), an antisense oligonucleotide that targets against p22phox or p47phox subunit of NADPH oxidase mRNA, or the superoxide dismutase mimetic tempol. DPI or antisense p22phox or p47phox oligonucleotide treatment also attenuated the AT 1 receptor-dependent increase in O 2 ⅐Ϫ production in the ventrolateral medulla elicited by Ang II at the RVLM. Functionally, Ang II-elicited pressor response in the RVLM was attenuated by DPI, tempol, or a p38 MAPK inhibitor, SB203580. The AT 1 receptor-mediated enhancement of the frequency of glutamate-sensitive spontaneous excitatory postsynaptic currents induced by Ang II in RVLM neurons was also abolished by SB203580. These results suggest that NADPH oxidase-derived O 2 ⅐Ϫ underlies the activation of p38 MAPK or ERK1/2 by Ang II in the ventrolateral medulla. Furthermore, the p38 MAPK signaling pathway may mediate Ang II-induced pressor response via enhancement of presynaptic release of glutamate to RVLM neurons. (Circ Res. 2005;97:772-780.)Key Words: mitogen-activated protein kinases Ⅲ angiotensin II Ⅲ superoxide anion Ⅲ NADPH oxidase Ⅲ rostral ventrolateral medulla Ⅲ blood pressure I n addition to its well-known actions on the vasculature, angiotensin II (Ang II) also plays a critical role in central regulation of circulatory functions. 1,2 One brain target where Ang II exerts its influence on cardiovascular control is rostral ventrolateral medulla (RVLM), 3,4 where premotor neurons that maintain tonic sympathetic vasomotor outflow are located. 5 The RVLM contains a high density of Ang II receptors 6 and is a major site of the sympathoexcitatory and pressor actions of the octapeptide. 7,8 It is generally accepted that activation of Ang II type 1 (AT 1 ) receptors in the RVLM contributes mainly to the cardiovascular effects of Ang II. 9,10 The reactive oxygen species, particularly superoxide anion (O 2 ⅐Ϫ ), is an important intracellular messenger for brain Ang II. The octapeptide increases the activity of NADPH oxidase, the major source of O 2 ⅐Ϫ in the vasculature, 11 and enhances O 2 ⅐Ϫ production in the central nervous system. 12,13 Intracerebroventricular infusion of NADPH oxidase inhibitor antagonizes the increase in renal sympa...

1 We investigated the contribution of neuronal or inducible nitric oxide synthase (nNOS or iNOS) at the rostral ventrolateral medulla (RVLM) to central cardiovascular regulation by endogenous nitric oxide (NO), using Sprague-Dawley rats anaesthetized and maintained with propofol. 2 Microinjection bilaterally into the RVLM of a NO trapping agent, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-l-oxy-l-3-oxide (10, 50 or 100 nmoles) resulted in signi®cant hypotension and bradycardia.3 Similar application of a selective antagonist of nNOS, 7-nitroindazole (1, 2.5 or 5 pmoles), or selective antagonists of iNOS, aminoguanidine (125, 250 or 500 pmoles), N 6 -(l-iminoethyl)-L-lysine (250 pmoles) or S-methylisothiourea (250 pmoles), induced respectively a reduction or an enhancement in systemic arterial pressure, heart rate and power density of the vasomotor components in the spectrum of arterial blood pressure signals, the experimental index for sympathetic neurogenic vasomotor tone. 4 Both hypotension and bradycardia induced by the NO precursor, L-arginine (100 nmoles), were signi®cantly blunted when aminoguanidine (250 pmoles) was co-microinjected bilaterally into the RVLM. On the other hand, co-administered 7-nitroindazole (2.5 pmoles) was ineective. 5 Whereas low doses of S-nitro-N-acetylpenicillamine (0.25 or 0.5 nmoles) elicited hypertension and tachycardia, high doses of this non-nitrate NO donor (5 nmoles) induced hypotension and bradycardia. 6 Reverse transcription ± polymerase chain reaction analysis revealed that both iNOS and nNOS mRNA were expressed in the ventrolateral medulla. 7 We conclude that the prevalence of nNOS over iNOS activity at the RVLM and the associated dominance of sympathoexcitation over sympathoinhibition may underlie the maintenance of sympathetic vasomotor out¯ow and stable systemic arterial pressure by the endogenous NO.

Background-Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, plays a pivotal role in the manifestation of fatal cardiovascular depression during endotoxemia. The iNOS gene is regulated transcriptionally by nuclear factor-B (NF-B) activation. The present study tested the hypothesis that heat shock protein 70 (HSP70) may confer protection against sepsis-induced circulatory fatality via inhibition of iNOS gene expression in the RVLM through prevention of NF-B activation. Methods and Results-Adult male Sprague-Dawley rats subjected to a brief hyperthermic heat shock (42°C for 15 minutes) exhibited significant upregulation of HSP70 in the RVLM. Brief heat shock preconditioning also significantly suppressed iNOS mRNA or protein surge and alleviated hypotension, bradycardia, and reduction in neurogenic sympathetic vasomotor activity manifested during experimental endotoxemia induced by intravenous administration of Escherichia coli lipopolysaccharide. An increase in DNA binding activity and nuclear translocation of transcription factor NF-B were detected during endotoxemia. Heat shock preconditioning significantly decreased DNA binding activity of NF-B, which was reversed by microinjection of an hsp70 antisense oligonucleotide bilaterally into the RVLM. Heat shock preconditioning also blocked inhibitory B (IB) kinase activity or degradation of IB in the RVLM during endotoxemia. Conclusions-We