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Overproduction of nitric oxide (NO) by gene transduction of endothelial NO synthase (eNOS) in rostral ventrolateral medulla (RVLM), which is responsible for maintenance of vasomotor tone, reduces arterial pressure in spontaneously hypertensive rats (SHR). This NO-induced vasodepression, however, is not sustained and is followed by rebound hypertension. Because superoxide anion (O(2)(*-)) level is increased and synthesis or activity of mitochondrial manganese superoxide dismutase (SOD2) is reduced in RVLM during hypertension, we hypothesized that an interaction between NO and O(2)(*-) in RVLM, using mitochondrial respiratory enzyme complexes (MRC) as the cellular target, contributes to those cardiovascular outcomes after eNOS gene transduction in SHR. The present study assessed this hypothesis using adenoviral vectors to overexpress eNOS (AdeNOS) and/or SOD2 (AdSOD2) in RVLM of SHR or normotensive Wistar-Kyoto (WKY) rats. Microinjection of AdeNOS bilaterally into RVLM elicited 35% depression of MRC-I enzyme activity and evoked 60% and 50% increase in O(2)(*-) and peroxynitrite level in RVLM of SHR, but not WKY rats, which was reversed by cotransduced AdSOD2 or treatment with peroxynitrite decomposition catalyst. Cotransduction of AdeNOS and AdSOD2 in RVLM of SHR elicited significantly greater decreases in arterial pressure and heart rate than those promoted by the individual transgene and prevented the AdeNOS-induced rebound hypertension. We conclude that an interactive action between NO and O(2)(*-) on MRC-I in RVLM via formation of peroxynitrite contributes to the unsustained hypotensive effects of NO after overexpression of eNOS in SHR. The mitochondria-derived O(2)(*-) also mediates the rebound hypertension induced by eNOS transgene in RVLM of SHR.

Sympathetic premotor neurons for the maintenance of vasomotor tone are located in rostral ventrolateral medulla (RVLM). We demonstrated previously that overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in RVLM, leading to caspase 3-dependent apoptotic cell death, plays a pivotal role in cardiovascular depression during endotoxemia induced by intravenous administration of Escherichia coli lipopolysaccharide. The interposing intracellular events remain unknown. We evaluated the hypothesis that these events encompass protein kinase C (PKC) activation, which triggers activation and translocation of Bax that opens mitochondrial permeability transition pore by interacting with adenine nucleotide translocase (ANT) or voltage-dependent anion protein (VDAC), followed by cytosolic release of cytochrome c. In Sprague-Dawley rats, coimmunoprecipitation and Western blot analyses revealed sequential manifestations during endotoxemia of membrane-bound translocation of PKC, dissociation of cytosolic PKC/Bax complex, mitochondrial translocation of activated Bax, augmented Bax/ANT or Bax/VDAC association, elevated cytosolic cytochrome c and caspase 3, and DNA fragmentation in ventrolateral medulla. Microinjection of iNOS inhibitor into bilateral RVLM significantly retarded PKC and Bax activation. The induced association of translocated Bax with ANT or VDAC and the triggered mitochondrial apoptotic signaling cascade were blunted by blockade in RVLM of PKC, mitochondrial translocation of Bax, Bax channels, ANT, or caspase 3, alongside significant amelioration of cardiovascular depression. We conclude that formation of mitochondrial Bax/ANT or Bax/VDAC complex that initiates caspase 3-dependent apoptosis in the RVLM as a result of PKC-dependent mitochondrial translocation of activated Bax activated by iNOS-derived NO plays a pivotal role in the manifestation of endotoxin-induced cardiovascular depression.

Whereas glucocorticoids are important blood pressure regulators via an action on peripheral circulation, their roles in central cardiovascular regulation are less known. This study evaluated the short-term cardiovascular effect of glucocorticoid in the nucleus tractus solitarii (NTS) and delineated the underlying molecular mechanisms. In Sprague-Dawley rats maintained under propofol anesthesia, microinjection bilaterally into the NTS of a synthetic glucocorticoid, dexamethasone (Dex; 12.5, 25, 50, or 100 pmol), elicited hypertensive and tachycardiac responses. The initial cardiovascular responses, which lasted 15 to 30 min, were blunted by coadministration of a selective GABA(A) or GABA(B) receptor antagonist, bicuculline (15 pmol) or 2-hydroxy saclofen (150 pmol). The delayed responses, which endured at least 90 min and entailed maintained hypertension and tachycardia, were reversed by selective glucocorticoid type II receptor (GR) antagonist mifepristone (100 or 200 pmol), phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] (20 nmol), or nitric-oxide synthase inhibitor N(G)-monomethyl-l-arginine acetate (5 nmol), but not by the RNA synthesis inhibitor actinomycin D (20 nmol). Moreover, Dex induced an association of GR with the regulatory subunit of PI3K, p85alpha, in a ligand-dependent manner and promoted serine/threonine kinase Akt phosphorylation that was blocked by coadministration of mifepristone or LY294002. These cardiovascular and molecular responses occurred when translocation of activated GR into the nucleus was minimal. Our results indicate that Dex acts on the NTS to elicit hypertension and tachycardia via both a GR-independent interaction with GABA(A) and GABA(B) receptors and a GR-dependent but nontranscriptional mechanism that involves activation of PI3K/Akt pathway.

Activation of the arterial baroreceptors induces expression of the proto-oncogene c-fos in the nucleus tractus solitarii (NTS), the terminal site of baroreceptor afferents in the medulla oblongata. This induced expression is an intracellular event that is crucial to long-term maintenance of stable blood pressure. Using Sprague-Dawley rats maintained under propofol anesthesia, we evaluated the role and delineated the underlying molecular mechanisms of nitric oxide (NO) in this process. Baroreceptor activation induced by 30 min of sustained hypertension significantly and sequentially increased the level of cyclic GMP-dependent protein kinase I (PKG-I), phosphorylated cyclic AMP response element-binding protein (pCREB), c-fos mRNA, and Fos protein in the NTS. All of these up-regulated expressions were significantly attenuated in animals that were pretreated immediately before baroreceptor activation with bilateral microinjection into the NTS of a selective neuronal nitric-oxide synthase (nNOS) inhibitor, 7-nitroindazole (2.5 pmol), or a soluble guanylyl cyclase (sGC) inhibitor, 1-H-[1,2,4]oxadiaolo[4,3-a]quinoxalin-1-one (1 nmol). Bilateral NTS microinjection of a cell-permeable cGMP analog, 8-bromoguanosine-3',5'-cyclic monophosphate (10 nmol) significantly elevated the level of pCREB or c-fos mRNA in the NTS. On the other hand, the up-regulated CREB phosphorylation or c-fos induction evoked in the dorsomedial medulla by baroreceptor activation was significantly antagonized by NTS application of a cell-permeable cGMP antagonist, (R)p-8-bromoguanosine-3',5'-cyclic monophosphorothioate (5 nmol), or a PKG inhibitor, (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H,-2,7b,11a-trizadizo-benzo(a,g)cycloocta(c,d,e)-trinden-1-one (1 nmol). We conclude that NO derived from nNOS in the NTS on baroreceptor activation may participate in c-fos expression via phosphorylation of CREB in a process that engages the sGC/cGMP/PKG-I signaling cascade.

Whereas induction of the 70-kDa heat shock protein (HSP70) in the nucleus tractus solitarii (NTS), the terminal site in the brain stem for primary baroreceptor afferents, augments baroreceptor reflex (BRR) response, the underlying cellular and molecular mechanism is essentially unexplored. In Sprague-Dawley rats, we evaluated the hypothesis that HSP70 may potentiate BRR response by up-regulating the molecular synthesis and functional expression of glutamate receptors in the NTS. Animals subjected to brief hyperthermic heat shock (HS; 42 degrees C for 15 min) exhibited augmented expression of NR1 or NR2A subunit of N-methyl-D-aspartate (NMDA) receptors, GluR1 or GluR4 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors and KA1 subunit of kainate receptors in the NTS. Intriguingly, this up-regulation of glutamate receptors was preceded by an increase in HSP70 expression at the NTS. The HS-induced augmentation in responsiveness of barosensitive NTS neurons to transient hypertension or potentiation of BRR response was discernibly blunted by MK-801 or 6-cyano-7-nitroquinoxaline-2,3-dione. Bilateral microinjection into the NTS of an antisense hsp70 oligonucleotide (50 pmol) before HS significantly suppressed the induced expression of HSP70 or the increase in glutamate receptor subunits in the dorsal medulla and discernibly attenuated the potentiation of BRR response. Control microinjection into the NTS of sense or scrambled hsp70 oligonucleotide (50 pmol) was ineffective. These findings suggest that HSP70 induced by HS may enhance BRR response by up-regulating the molecular synthesis and functional expression of NR1 or NR2A subunit of NMDA receptors and GluR1, GluR4, or KA1 subunit of non-NMDA receptors in the NTS.