Ling-Lin Wang

NADPH Oxidase–Derived Superoxide Anion Mediates Angiotensin II–Induced Pressor Effect via Activation of p38 Mitogen–Activated Protein Kinase in the Rostral Ventrolateral Medulla

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 (O2·−) 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 (AT1) 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 AT1 receptor-dependent increase in O2·− 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 AT1 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 O2·− 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.

Differential cardiovascular responses to blockade of nNOS or iNOS in rostral ventrolateral medulla of the rat

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. 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 significant hypotension and bradycardia. 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), N6‐(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. Both hypotension and bradycardia induced by the NO precursor, L‐arginine (100 nmoles), were significantly blunted when aminoguanidine (250 pmoles) was co‐microinjected bilaterally into the RVLM. On the other hand, co‐administered 7‐nitroindazole (2.5 pmoles) was ineffective. 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. Reverse transcription – polymerase chain reaction analysis revealed that both iNOS and nNOS mRNA were expressed in the ventrolateral medulla. 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 outflow and stable systemic arterial pressure by the endogenous NO.

Heat Shock Protein 70 Confers Cardiovascular Protection During Endotoxemia via Inhibition of Nuclear Factor-κB Activation and Inducible Nitric Oxide Synthase Expression in the Rostral Ventrolateral Medulla

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-&kgr;B (NF-&kgr;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-&kgr;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-&kgr;B were detected during endotoxemia. Heat shock preconditioning significantly decreased DNA binding activity of NF-&kgr;B, which was reversed by microinjection of an hsp70 antisense oligonucleotide bilaterally into the RVLM. Heat shock preconditioning also blocked inhibitory &kgr;B (I&kgr;B) kinase activity or degradation of I&kgr;B in the RVLM during endotoxemia. Conclusions—We conclude that HSP70 confers protection against sepsis-related circulatory fatality via inhibition of iNOS gene expression in the RVLM through prevention of NF-&kgr;B activation in cellular processes that include prevention of I&kgr;B kinase activation and inhibition of I&kgr;B&agr; degradation.

Upregulation of AT1 receptor gene on activation of protein kinase C??/nicotinamide adenine dinucleotide diphosphate oxidase/ERK1/2/c-fos signaling cascade mediates long-term pressor effect of angiotensin II in rostral ventrolateral medulla

Objective Angiotensin II induces the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2 via the activation of nicotinamide adenine dinucleotide diphosphate (NADPH) oxidase on stimulation of the angiotensin subtype 1 receptor (AT1R) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone and blood pressure are located. Angiotensin II-activated p38 MAPK in RVLM promotes a short-term pressor effect via augmented glutamatergic neurotransmission. We tested the hypothesis that the NADPH oxidase-dependent phosphorylation of ERK1/2 after the activation of conventional protein kinase C (PKC) mediates the AT1R-dependent long-term pressor effects of angiotensin II via transcriptional induction of the proto-oncogene c-fos gene in RVLM. Methods and results In Sprague–Dawley rats, a microinjection of angiotensin II bilaterally into the RVLM induced membrane-bound translocation of the conventional PKCα, PKCβ or PKCγ isoform, phosphorylation of the p47phox subunit of NADPH oxidase and ERK1/2, followed by phosphorylation of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB), and c-fos induction. The PKC inhibitor antagonized angiotensin II-induced p47phox phosphorylation, and an antisense oligonucleotide (ASON) complementary to PKCβ messenger RNA suppressed angiotensin II-induced ERK1/2 activation, phosphorylation or DNA binding activity of CREB, and upregulation of c-fos mRNA expression in the ventrolateral medulla. Furthermore, a microinjection of ERK1/2, CREB or c-fos ASON into the RVLM significantly reduced the long-term pressor effect and augmented AT1R expression in the ventrolateral medulla induced by intracerebroventricular infusion of angiotensin II. Conclusion We concluded that the PKCβ/NADPH oxidase/ERK1/2/CREB/c-fos cascade represents a novel signaling cascade that mediates the long-term pressor effect induced by angiotensin II in the RVLM.

Differential engagements of glutamate and GABA receptors in cardiovascular actions of endogenous nNOS or iNOS at rostral ventrolateral medulla of rats

We evaluated in Sprague–Dawley rats anaesthetized with propofol the engagement of soluble guanylyl cyclase (sGC)/cGMP cascade, glutamatergic and GABAergic neurotransmission in the cardiovascular actions of endogenous nitric oxide (NO) at the rostral ventrolateral medulla (RVLM). Microinjection bilaterally into the RVLM of a selective iNOS inhibitor, S‐methylisothiourea (SMT, 250 pmoles), or a selective nNOS inhibitor, 7‐nitroindazole (7‐NI, 5 pmoles), induced respectively an enhancement or a reduction in systemic arterial pressure, heart rate and power density of the vasomotor components in the spectrum of arterial blood pressure signals, our experimental index for sympathetic neurogenic vasomotor tone. The cardiovascular actions of SMT or 7‐NI in the RVLM were significantly antagonized by co‐administration into the RVLM of the sGC inhibitor, 1H‐[1,2,4]Oxadiazole[4,3‐α]quinoxalin‐1‐one (ODQ, 250 or 500 pmoles). The cardiovascular excitatory effects after blockade of endogenous iNOS activity were significantly attenuated when N‐methyl‐D‐aspartate (NMDA) receptor antagonist, dizocilpine (20 or 50 pmoles), or non‐NMDA receptor antagonist, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (250 or 500 pmoles), was co‐microinjected bilaterally into the RVLM. On the other hand, the cardiovascular depressive responses to blockade of endogenous nNOS activity were significantly antagonized on co‐administration of GABAA receptor antagonist, bicuculline methiodine (5 or 10 pmoles), but not GABAB receptor antagonist, 2‐hydroxy saclofen (50 or 100 pmoles). We conclude that the cardiovascular actions of endogenous NO in the RVLM engage the sGC/cGMP pathway. In addition, whereas NO derived from nNOS induced sympathoexcitation via both NMDA and non‐NMDA receptors in the RVLM, NO generated by iNOS elicited sympathoinhibition via GABAA receptors.

Augmented Upregulation by c-fos of Angiotensin Subtype 1 Receptor in Nucleus Tractus Solitarii of Spontaneously Hypertensive Rats

Our laboratory demonstrated previously that spontaneously hypertensive rats (SHR) exhibited an elevated basal Fos expression in the nucleus tractus solitarii (NTS), the terminal site for primary baroreceptor afferents, and that Fos protein is required for the re-expression of angiotensin subtype 1 receptor (AT1R) mRNA in the NTS after baroreceptor activation. The present study evaluated the hypothesis that this re-expression of AT1R is augmented in SHR and is promoted by the heightened Fos expression. Reverse transcription–polymerase chain reaction analysis revealed that baroreceptor activation via sustained increase in systemic arterial pressure resulted in a discernible reduction in the expression of AT1R mRNA at the dorsomedial medulla of SHR and normotensive Wistar-Kyoto rats. However, SHR manifested an appreciably larger magnitude of decline, followed by a faster time course of re-expression in AT1R mRNA. Parallel findings were obtained from the pressor response induced by microinjection unilaterally of angiotensin II (40 pmol) into the NTS. Whereas the re-expression of AT1R at both transcriptional and functional expression levels after baroreceptor activation was discernibly blunted by prior bilateral application into the NTS of an antisense c-fos oligonucleotide (50 pmol), the suppression in SHR was again significantly more intense. Control pretreatment with the corresponding sense or scrambled c-fos oligonucleotide was ineffective. We conclude that the heightened Fos expression in SHR is causatively related to the augmented re-expression of AT1R in the NTS at both transcriptional and functional levels.

Downregulation of Basal iNOS at the Rostral Ventrolateral Medulla Is Innate in SHR

Abstract—We demonstrated recently that a significant reduction in both the molecular synthesis and functional expression of inducible nitric oxide synthase (iNOS) in the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor outflow, underlies the augmented sympathetic vasomotor tone during hypertension. This study further evaluated the hypothesis that this downregulation of basal iNOS at the RVLM during hypertension is innate. In adult spontaneously hypertensive rats (SHR) treated for 4 weeks with the antihypertensive captopril to normalize elevated blood pressure or in young prehypertensive SHR, the significantly lower iNOS mRNA and protein levels at the ventrolateral medulla under basal conditions or on activation by microinjection bilaterally into the RVLM of lipopolysaccharide (10 ng) remained unaltered. The retarded efficacy of lipopolysaccharide (10 ng) to elicit cardiovascular depression (hypotension, bradycardia, and reduction in sympathetic vasomotor tone) also persevered in captopril-treated adult or young normotensive SHR. On the other hand, compared with Wistar-Kyoto normotensive rats, the magnitude of cardiovascular depression induced in adult SHR by local administration into the RVLM of the NO precursor l-arginine (40 nmol) was significantly smaller. In addition, microinjection bilaterally into the RVLM of a selective iNOS inhibitor, aminoguanidine (125 or 250 pmol), was discernibly less efficacious in unmasking hypertension, tachycardia, and the increase in sympathetic vasomotor tone in adult SHR. We conclude that a predisposed reduction in molecular synthesis and functional expression of basal iNOS in the RVLM is associated with the sympathetic vasomotor overactivity during hypertension.

Altered Temporal Profile of Heat Shock Factor 1 Phosphorylation and Heat Shock Protein 70 Expression Induced by Heat Shock in Nucleus Tractus Solitarii of Spontaneously Hypertensive Rats

Background—We demonstrated recently that heat shock (HS)–induced heat shock protein 70 (HSP70) expression in bilateral nucleus tractus solitarii (NTS), the terminal site in the brain stem for primary baroreceptor afferents, confers cardiovascular protection against heatstroke by potentiating baroreceptor reflex (BRR) response. This study evaluated the hypothesis that altered regulation of HSP70 expression may be associated with the heightened susceptibility to heatstroke during hypertension. Methods and Results—Spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats anesthetized with propofol were used. Compared with WKY rats, significant induction in HSP70 or phosphorylation of heat shock factor 1 (HSF1), but not HSF2, in the NTS and potentiation of BRR response in SHR occurred earlier (4 versus 8 hours), reaching peak magnitude sooner (16 versus 24 hours), and declined more rapidly after a brief hyperthermic HS (42±0.5°C for 15 minutes). The protection conferred by HS against hypotension and bradycardia during the onset of heatstroke (45°C for 60 minutes), although effective, was less effective in SHR. Microinjection bilaterally into the NTS of the selective protein kinase A (PKA) inhibitor H-89 (100 pmol) or the selective PKC inhibitor calphostin C (100 pmol) significantly attenuated all of the above events induced in SHR by HS. However, only H-89 was effective in WKY rats. Conclusions—An altered temporal profile of HS-induced HSP70 expression or potentiation of BRR response by concurrent activation via both PKA and PKC pathways of phosphorylation of HSF1 in the NTS may be associated with greater susceptibility to heatstroke during hypertension.

Downregulation of Angiotensin Subtype 1 Receptor in Rostral Ventrolateral Medulla During Endotoxemia

Abstract—We reported recently that an upregulation of the inducible nitric oxide synthase (iNOS) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, is a crucial determinant for the elicitation of cardiovascular depression during experimental endotoxemia. The current study evaluated the hypothesis that a downregulation of the molecular synthesis and functional expression of angiotensin subtype 1 receptor (AT1R) in the RVLM is consequential to this upregulated iNOS. In adult Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of Escherichia coli lipopolysaccharide (15 mg/kg) elicited a reduction, followed by an augmentation and a secondary decrease in sympathetic vasomotor outflow, together with progressive hypotension and bradycardia. There was also a progressive increase in iNOS mRNA and protein level in the ventrolateral medulla. This was followed by a significant downregulation of both mRNA and protein levels of AT1R in the ventrolateral medulla, alongside reduced efficacy of angiotensin II (50 pmol) to induce an increase in systemic arterial pressure, heart rate, or sympathetic vasomotor outflow on unilateral microinjection into the RVLM. Pretreatment with microinjection of a selective iNOS inhibitor, S-methylisothiourea (250 pmol) bilaterally into the RVLM significantly reversed the reduction in both synthesis and activity of AT1R. We conclude that a downregulation of molecular synthesis and functional expression of AT1R in the ventrolateral medulla is consequential to the overproduction of NO through upregulation of iNOS in the RVLM and may underlie the cardiovascular depression that takes place during experimental endotoxemia.