Alice Y.W. Chang

Oxidative Impairment of Mitochondrial Electron Transport Chain Complexes in Rostral Ventrolateral Medulla Contributes to Neurogenic Hypertension

The role for mitochondrial electron transport chain (ETC) in neurogenic hypertension is unidentified. We evaluated the hypothesis that feedforward depression of mitochondrial ETC functions by superoxide anion (O2·−) and hydrogen peroxide (H2O2) in rostral ventrolateral medulla (RVLM), a brain stem site that maintains sympathetic vasomotor tone and contributes to oxidative stress and neural mechanism of hypertension. Compared with normotensive Wistar-Kyoto rats, spontaneously hypertensive rats exhibited mitochondrial ETC dysfunctions in RVLM in the forms of depressed complex I or III activity and reduced electron coupling capacity between complexes I and III or II and III. Microinjection of coenzyme Q10 into RVLM of spontaneously hypertensive rats reversed the depressed ETC activity and augmented O2·− production and hypertensive phenotypes. This mobile electron carrier also antagonized the elevated H2O2 in RVLM and vasopressor responses to complex I (rotenone) or III (antimycin A) inhibitor in Wistar-Kyoto or prehypertensive rats. Intracerebroventricular infusion of angiotensin II promoted mitochondrial ETC dysfunctions in Wistar-Kyoto rats, and coenzyme Q10 or gene knockdown of the p22phox subunit of NADPH oxidase antagonized the resultant elevation of H2O2 in RVLM. Overexpression of superoxide dismutase or catalase in RVLM of spontaneously hypertensive rats by gene transfer reversed mitochondrial dysfunctions and blunted the augmented O2·− and H2O2 in RVLM. We conclude that O2·−- and H2O2-dependent feedforward impairment of mitochondrial ETC complexes because of predisposed downregulation of superoxide dismutase or catalase and a cross-talk between NADPH oxidase-derived O2·− and ETC enzymes contribute to chronic oxidative stress in the RVLM of spontaneously hypertensive rats, leading to augmented sympathetic vasomotor tone and hypertension.

Mitochondrial dysfunction and ultrastructural damage in the hippocampus during kainic acid-induced status epilepticus in the rat.

Summary:  Purpose: Prolonged and continuous epileptic seizure (status epilepticus) results in cellular changes that lead to neuronal damage. We investigated whether these cellular changes entail mitochondrial dysfunction and ultrastructural damage in the hippocampus, by using a kainic acid (KA)‐induced experimental status epilepticus model.

Transcriptional Upregulation of Mitochondrial Uncoupling Protein 2 Protects Against Oxidative Stress-Associated Neurogenic Hypertension

Rationale: Mitochondrial uncoupling proteins (UCPs) belong to a superfamily of mitochondrial anion transporters that uncouple ATP synthesis from oxidative phosphorylation and mitigates mitochondrial reactive oxygen species production. Objective: We assessed the hypothesis that UCP2 participates in central cardiovascular regulation by maintaining reactive oxygen species homeostasis in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons that maintain vasomotor tone located. We also elucidated the molecular mechanisms that underlie transcriptional upregulation of UCP2 in response to oxidative stress in RVLM. Methods and Results: In Sprague–Dawley rats, transcriptional upregulation of UCP2 in RVLM by rosiglitazone, an activator of its transcription factor peroxisome proliferator-activated receptor (PPAR)&ggr;, reduced mitochondrial hydrogen peroxide level in RVLM and systemic arterial pressure. Oxidative stress induced by microinjection of angiotensin II into RVLM augmented UCP2 mRNA or protein expression in RVLM, which was antagonized by comicroinjection of NADPH oxidase inhibitor (diphenyleneiodonium chloride), superoxide dismutase mimetic (tempol), or p38 mitogen-activated protein kinase inhibitor (SB203580) but not by extracellular signal-regulated kinase 1/2 inhibitor (U0126). Angiotensin II also induced phosphorylation of the PPAR&ggr; coactivator, PPAR&ggr; coactivator (PGC)-1α, and an increase in formation of PGC-1α/PPAR&ggr; complexes in a p38 mitogen-activated protein kinase–dependent manner. Intracerebroventricular infusion of angiotensin II promoted an increase in mitochondrial hydrogen peroxide production in RVLM and chronic pressor response, which was potentiated by gene knockdown of UCP2 but blunted by rosiglitazone. Conclusions: These results suggest that transcriptional upregulation of mitochondrial UCP2 in response to an elevation in superoxide plays an active role in feedback regulation of reactive oxygen species production in RVLM and neurogenic hypertension associated with chronic oxidative stress.

Cocaine Withdrawal Impairs Metabotropic Glutamate Receptor-Dependent Long-Term Depression in the Nucleus Accumbens

Neuroadaptation in the nucleus accumbens (NAc), a central component of the mesolimbic dopamine (DA) system, has been implicated in the development of cocaine-induced psychomotor sensitization and relapse to cocaine seeking. However, little is known about the cellular and synaptic mechanisms underlying such adaptation. Using a mouse model of behavioral sensitization, we show that animals withdrawn from repeated cocaine exposure have a selective deficit in the ability to elicit metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) in the shell of the NAc in response to bath application of the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG). Experiments conducted in the presence of the selective mGluR1 antagonists 7-(hydroxyimino)cyclopropachromen-carboxylate ethyl ester and (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid, or the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine, demonstrated that the impaired DHPG-LTD is likely attributable to a loss of mGluR5 function. Quantitative real-time reverse transcriptase-PCR and Western blot analysis revealed significant downregulation of mGluR5, but not mGluR1, mRNA and protein levels in the NAc shell. The inhibitory effect of repeated cocaine exposure on DHPG-LTD was selectively prevented when cocaine was coadministered with the selective D1-like DA receptor antagonist (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine. Furthermore, the levels of brain-derived neurotrophic factor (BDNF) protein in the NAc shell increased progressively after cocaine withdrawal, and the impairment of DHPG-LTD in the NAc shell was not found in slices from BDNF-knock-out mice after cocaine withdrawal. These results suggest that withdrawal from repeated cocaine exposure may result in increased BDNF levels in the NAc shell, which leads to a selective downregulation of mGluR5 and thereby impairs the induction of mGluR-dependent LTD.

Heat Shock Protein 60 or 70 Activates Nitric-oxide Synthase (NOS) I- and Inhibits NOS II-associated Signaling and Depresses the Mitochondrial Apoptotic Cascade during Brain Stem Death

The cellular and molecular basis of brain stem death remains an enigma. As the origin of a “life-and-death” signal that reflects the progression toward brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate for mechanistic delineation of this phenomenon. Here, we evaluated the hypothesis that heat shock proteins (HSPs) play a neuroprotective role in the RVLM during brain stem death and delineated the underlying mechanisms, using a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult. In Sprague-Dawley rats, proteomic, Western blot, and real-time PCR analyses demonstrated that Mev induced de novo synthesis of HSP60 or HSP70 in the RVLM without affecting HSP90 level. Loss-of-function manipulations of HSP60 or HSP70 in the RVLM using anti-serum or antisense oligonucleotide potentiated Mev-elicited cardiovascular depression alongside reduced nitric-oxide synthase (NOS) I/protein kinase G signaling, enhanced NOS II/peroxynitrite cascade, intensified nucleosomal DNA fragmentation, elevated cytoplasmic histone-associated DNA fragments or activated caspase-3, and augmented the cytochrome c/caspase-3 cascade of apoptotic signaling in the RVLM. Co-immunoprecipitation experiments further revealed a progressive increase in the complex formed between HSP60 and mitochondrial or cytosolic Bax or mitochondrial Bcl-2 during Mev intoxication, alongside a dissociation of the cytosolic HSP60-Bcl-2 complex. We conclude that HSP60 and HSP70 confer neuroprotection against Mev intoxication by ameliorating cardiovascular depression via an anti-apoptotic action in the RVLM. The possible underlying intracellular processes include enhancing NOS I/protein kinase G signaling and inhibiting the NOS II/peroxynitrite cascade. In addition, HSP60 exerts its effects against apoptosis by blunting Mev-induced activation of the Bax/cytochrome c/caspase-3 cascade.

Transcriptional upregulation of brain-derived neurotrophic factor in rostral ventrolateral medulla by angiotensin II: Significance in superoxide homeostasis and neural regulation of arterial pressure

Rationale: Oxidative stress in rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of neurogenic vasomotor tone are located, contributes to neural mechanisms of hypertension. Emerging evidence suggests that brain-derived neurotrophic factor (BDNF) manifests “nontrophic” actions. Objective: We assessed the hypothesis that BDNF plays an active role in oxidative stress–associated neurogenic hypertension by maintaining superoxide anion ( ) homeostasis in RVLM. Methods and Results: In Wistar–Kyoto rats, microinjection of angiotensin II (Ang II) bilaterally into RVLM upregulated BDNF mRNA and protein and induced cAMP response element binding protein (CREB) phosphorylation. The Ang II–induced BDNF upregulation in RVLM was attenuated by coadministration of the NADPH oxidase inhibitor apocynin; the superoxide dismutase mimetic tempol; or an antisense oligonucleotide against CREB. Intracisternal infusion of Ang II elicited phosphorylation of p47phox subunit of NADPH oxidase, suppression of mitochondrial electron coupling capacity, and augmentation in mitochondrial uncoupling protein (UCP)2 expression in RVLM. The former 2 cellular events were enhanced, whereas UCP2 upregulation was attenuated by gene knockdown of BDNF or depletion of tropomyosin receptor kinase (Trk)B ligands with recombinant human TrkB-Fc fusion protein. The same treatments also significantly potentiated both Ang II–induced production in RVLM and chronic pressor response. Conclusions: Ang II induces -dependent upregulation of BDNF in RVLM via phosphorylation of CREB. The Ang II–activated BDNF/TrkB signaling, in turn, exerts negative-feedback regulation on tissue level in RVLM through inhibition of p47phox phosphorylation, preservation of mitochondrial electron transport capacity, and upregulation of mitochondrial UCP2, resulting in protection against Ang II–induced oxidative stress and long-term pressor response.Rationale: Oxidative stress in rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of neurogenic vasomotor tone are located, contributes to neural mechanisms of hypertension. Emerging evidence suggests that brain-derived neurotrophic factor (BDNF) manifests “nontrophic” actions. Objective: We assessed the hypothesis that BDNF plays an active role in oxidative stress–associated neurogenic hypertension by maintaining superoxide anion (![Graphic][1] ) homeostasis in RVLM. Methods and Results: In Wistar–Kyoto rats, microinjection of angiotensin II (Ang II) bilaterally into RVLM upregulated BDNF mRNA and protein and induced cAMP response element binding protein (CREB) phosphorylation. The Ang II–induced BDNF upregulation in RVLM was attenuated by coadministration of the NADPH oxidase inhibitor apocynin; the superoxide dismutase mimetic tempol; or an antisense oligonucleotide against CREB. Intracisternal infusion of Ang II elicited phosphorylation of p47phox subunit of NADPH oxidase, suppression of mitochondrial electron coupling capacity, and augmentation in mitochondrial uncoupling protein (UCP)2 expression in RVLM. The former 2 cellular events were enhanced, whereas UCP2 upregulation was attenuated by gene knockdown of BDNF or depletion of tropomyosin receptor kinase (Trk)B ligands with recombinant human TrkB-Fc fusion protein. The same treatments also significantly potentiated both Ang II–induced ![Graphic][2] production in RVLM and chronic pressor response. Conclusions: Ang II induces ![Graphic][3] -dependent upregulation of BDNF in RVLM via phosphorylation of CREB. The Ang II–activated BDNF/TrkB signaling, in turn, exerts negative-feedback regulation on tissue ![Graphic][4] level in RVLM through inhibition of p47phox phosphorylation, preservation of mitochondrial electron transport capacity, and upregulation of mitochondrial UCP2, resulting in protection against Ang II–induced oxidative stress and long-term pressor response. # Novelty and Significance {#article-title-48} [1]: /embed/inline-graphic-1.gif [2]: /embed/inline-graphic-2.gif [3]: /embed/inline-graphic-3.gif [4]: /embed/inline-graphic-4.gif

Differential Distribution of Nitric Oxide Synthase Isoforms in the Rostral Ventrolateral Medulla of the Rat

We evaluated the distribution of nitric oxide synthase (NOS) isoforms in the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone, and the contribution of NOS III to the cardiovascular actions of endogenous NO in the RVLM. Adult Sprague-Dawley rats were used. Reverse transcription-polymerase chain reaction or Western blot analysis revealed that NOS I, II or III was expressed in the ventrolateral medulla at the mRNA or protein level under basal conditions. However, laser scanning confocal microscopic analysis of double-immunofluorescence images showed that whereas NOS I or II immunoreactivity colocalized with cells within the confines of the RVLM that stained positively with the neuronal marker, NeuN, NOS III immunoreactivity was associated primarily with blood vessels. Furthermore, bilateral microinjection into the RVLM of the selective NOS III inhibitor, N(5)-(1-iminoethyl)-L-ornithine, elicited minimal alterations in baseline systemic arterial pressure, heart rate or sympathetic vasomotor outflow in rats anesthetized with propofol. We conclude that whereas NOS I and II are present in neurons within the confines of the RVLM, NOS III is associated primarily with blood vessels. Our results further indicate that NOS III does not appear to contribute to the maintenance of basal sympathetic vasomotor outflows and arterial pressure by the endogenous NO at the RVLM.

Upregulation of nitric oxide synthase II contributes to apoptotic cell death in the hippocampal CA3 subfield via a cytochrome c/caspase-3 signaling cascade following induction of experimental temporal lobe status epilepticus in the rat

Status epilepticus results in preferential neuronal cell loss in the hippocampus. We evaluated the hypothesis that the repertoire of intracellular events in the vulnerable hippocampal CA3 subfield after induction of experimental temporal lobe status epilepticus entails upregulation of nitric oxide synthase II (NOS II), followed by the release of mitochondrial cytochrome c that triggers the cytosolic caspase-3 cascade, leading to apoptotic cell death. In Sprague-Dawley rats, significant and temporally correlated upregulation of NOS II (3-24h), but not NOS I or II expression, enhanced cytosolic translocation of cytochrome c (days 1 and 3), augmented activated caspase-3 in cytosol (days 1, 3 and 7) and DNA fragmentation (days 1, 3 and 7) was detected bilaterally in the hippocampal CA3 subfield after elicitation of sustained seizure activity by microinjection of kainic acid into the unilateral CA3 subfield. Application bilaterally into the hippocampal CA3 subfield of a selective NOS II inhibitor, S-methylisothiourea, significantly blunted these apoptotic events; a selective NOS I inhibitor, N(omega)-propyl-l-arginine or a potent NOS III inhibitor, N(5)-(1-iminoethyl)-l-ornithine was ineffective. We conclude that upregulation of NOS II contributes to apoptotic cell death in the hippocampal CA3 subfield via a cytochrome c/caspase-3 signaling cascade following the induction of experimental temporal lobe status epilepticus.

Proteomic analysis of lipopolysaccharide-induced apoptosis in PC12 cells.

We employed rat pheochromocytoma PC12 cells as our model system to identify cellular proteins that accompany Escherichia coli lipopolysaccharide (LPS)‐induced apoptosis, based on a proteomic approach. Cell viability tests revealed that naïve PC12 cells underwent cell death in a dose‐dependent manner after treatment with LPS. Flow cytometric analysis confirmed that apoptosis was primarily responsible for the observed cell death. Two‐dimensional electrophoresis in conjunction with N‐terminal sequencing, immunoblot, matrix‐assisted laser desorption/ionization‐time of flight analysis or computer matching with protein databases further revealed that the LPS‐induced apoptosis is accompanied by an augmented level of calreticulin, calcium binding protein 50, endoplasmic reticulum protein 60 (ERP60), heat shock protein 60 (HSP60) or HSP90, and a reduced level of amphoterin, cytochrome c oxidase polypeptide VIa‐liver or ERP29. These proteins are associated with endoplasmic reticulum, mitochondria or cell membrane, and are with known or potential roles in apoptosis. Their identification therefore provides an impetus for further delineation of the cellular and molecular basis of apoptotic cell death and sepsis based on proteomic profiling of PC12 cells.

Differential contributions of NOS isoforms in the rostral ventrolateral medulla to cardiovascular responses associated with mevinphos intoxication in the rat

The organophosphate poison mevinphos (Mev) elicits cardiovascular responses via nitric oxide (NO) produced on activation of M2 muscarinic receptors (M2R) in the rostral ventrolateral medulla (RVLM), where sympathetic vasomotor tone originates. This study further evaluated the contribution of nitric oxide synthase (NOS) isoforms at the RVLM to this process, using adult Sprague-Dawley rats. Bilateral co-microinjection into the RVLM of the selective NOS I inhibitor (250 pmol), 7-nitroindazole or Nω-propyl-l-arginine antagonized the initial sympathoexcitatory cardiovascular responses to Mev (10 nmol). Co-administration of a selective NOS II inhibitor, N6-(1-iminoethyl)-l-lysine (250 or 500 pmol) further enhanced these cardiovascular responses and reversed the secondary sympathoinhibitory actions of Mev. A potent NOS III inhibitor, N5-(1-iminoethyl)-l-ornithine (46 or 92 nmol) was ineffective. We also found that M2R co-localized only with NOS I- or NOS II-immunoreactive RVLM neurons. Furthermore, only NOS I or II in the ventrolateral medulla exhibited an elevation in mRNA or protein levels during the sympathoexcitatory phase, with further up-regulated synthesis of NOS II during the sympathoinhibitory phase of Mev intoxication. We conclude that whereas NOS III is not engaged, NO produced by NOS I and II in the RVLM plays, respectively, a sympathoexcitatory and sympathoinhibitory role in the cardiovascular responses during Mev intoxication.The organophosphate poison mevinphos (Mev) elicits cardiovascular responses via nitric oxide (NO) produced on activation of M2 muscarinic receptors (M2R) in the rostral ventrolateral medulla (RVLM), where sympathetic vasomotor tone originates. This study further evaluated the contribution of nitric oxide synthase (NOS) isoforms at the RVLM to this process, using adult Sprague-Dawley rats. Bilateral co-microinjection into the RVLM of the selective NOS I inhibitor (250 pmol), 7-nitroindazole or N(omega)-propyl-L-arginine antagonized the initial sympathoexcitatory cardiovascular responses to Mev (10 nmol). Co-administration of a selective NOS II inhibitor, N6-(1-iminoethyl)-L-lysine (250 or 500 pmol) further enhanced these cardiovascular responses and reversed the secondary sympathoinhibitory actions of Mev. A potent NOS III inhibitor, N5-(1-iminoethyl)-L-ornithine (46 or 92 nmol) was ineffective. We also found that M2R co-localized only with NOS I- or NOS II-immunoreactive RVLM neurons. Furthermore, only NOS I or II in the ventrolateral medulla exhibited an elevation in mRNA or protein levels during the sympathoexcitatory phase, with further up-regulated synthesis of NOS II during the sympathoinhibitory phase of Mev intoxication. We conclude that whereas NOS III is not engaged, NO produced by NOS I and II in the RVLM plays, respectively, a sympathoexcitatory and sympathoinhibitory role in the cardiovascular responses during Mev intoxication.