Protein kinase A-dependent Neuronal Nitric Oxide Synthase Activation Mediates the Enhancement of Baroreflex Response by Adrenomedullin in the Nucleus Tractus Solitarii of Rats

<p>Abstract</p> <p>Background</p> <p>Adrenomedullin (ADM) exerts its biological functions through the receptor-mediated enzymatic mechanisms that involve protein kinase A (PKA), or neuronal nitric oxide synthase (nNOS). We previously demonstrated that the receptor-mediated cAMP/PKA pathway involves in ADM-enhanced baroreceptor reflex (BRR) response. It remains unclear whether ADM may enhance BRR response via activation of nNOS-dependent mechanism in the nucleus tractus solitarii (NTS).</p> <p>Methods</p> <p>Intravenous injection of phenylephrine was administered to evoke the BRR before and at 10, 30, and 60 min after microinjection of the test agents into NTS of Sprague-Dawley rats. Western blotting analysis was used to measure the level and phosphorylation of proteins that involved in BRR-enhancing effects of ADM (0.2 pmol) in NTS. The colocalization of PKA and nNOS was examined by immunohistochemical staining and observed with a laser confocal microscope.</p> <p>Results</p> <p>We found that ADM-induced enhancement of BRR response was blunted by microinjection of NPLA or Rp-8-Br-cGMP, a selective inhibitor of nNOS or protein kinase G (PKG) respectively, into NTS. Western blot analysis further revealed that ADM induced an increase in the protein level of PKG-I which could be attenuated by co-microinjection with the ADM receptor antagonist ADM_22-52or NPLA. Moreover, we observed an increase in phosphorylation at Ser1416 of nNOS at 10, 30, and 60 min after intra-NTS administration of ADM. As such, nNOS/PKG signaling may also account for the enhancing effect of ADM on BRR response. Interestingly, biochemical evidence further showed that ADM-induced increase of nNOS phosphorylation was prevented by co-microinjection with Rp-8-Br-cAMP, a PKA inhibitor. The possibility of PKA-dependent nNOS activation was substantiated by immunohistochemical demonstration of co-localization of PKA and nNOS in putative NTS neurons.</p> <p>Conclusions</p> <p>The novel finding of this study is that the signal transduction cascade that underlies the enhancement of BRR response by ADM in NTS is composed sequentially of cAMP/PKA and nNOS/PKG pathways.</p

Bioenergetics Failure and Oxidative Stress in Brain Stem Mediates Cardiovascular Collapse Associated with Fatal Methamphetamine Intoxication

Background: Whereas sudden death, most often associated with cardiovascular collapse, occurs in abusers of the psychostimulant methamphetamine (METH), the underlying mechanism is much less understood. The demonstration that successful resuscitation of an arrested heart depends on maintained functionality of the rostral ventrolateral medulla (RVLM), which is responsible for the maintenance of stable blood pressure, suggests that failure of brain stem cardiovascular regulation, rather than the heart, holds the key to cardiovascular collapse. We tested the hypothesis that cessation of brain stem cardiovascular regulation because of a loss of functionality in RVLM mediated by bioenergetics failure and oxidative stress underlies the cardiovascular collapse elicited by lethal doses of METH. Methodology/Principal Findings: Survival rate, cardiovascular responses and biochemical or morphological changes in RVLM induced by intravenous administration of METH in Sprague-Dawley rats were investigated. High doses of METH induced significant mortality within 20 min that paralleled concomitant the collapse of arterial pressure or heart rate and loss of functionality in RVLM. There were concurrent increases in the concentration of METH in serum and ventrolateral medulla, along with tissue anoxia, cessation of microvascular perfusion and necrotic cell death in RVLM. Furthermore, mitochondrial respiratory chain enzyme activity or electron transport capacity and ATP production in RVLM were reduced, and mitochondria-derived superoxide anion level was augmented. All those detrimental physiological and biochemica

sj-pdf-1-cep-10.1177_03331024241230466 - Supplemental material for Vagus nerve stimulation inhibits cortical spreading depression via glutamate-dependent TrkB activation mechanism in the nucleus tractus solitarius

Supplemental material, sj-pdf-1-cep-10.1177_03331024241230466 for Vagus nerve stimulation inhibits cortical spreading depression via glutamate-dependent TrkB activation mechanism in the nucleus tractus solitarius by Tzu-Ting Liu, Shih-Pin Chen, Shuu-Jiun Wang and Jiin-Cherng Yen in Cephalalgia</p

Midostaurin Modulates Tumor Microenvironment and Enhances Efficacy of Anti-PD-1 against Colon Cancer

Immunotherapy modulating the tumor microenvironment (TME) immune function has a promising effect on various types of cancers, but it remains as a limited efficacy in colon cancer. Midostaurin (PKC412) has been used in the clinical treatment of fms-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia and has demonstrated immunomodulatory activity. We aimed to evaluate the effect of midostaurin on the modulation of TME and the efficacy of anti-programmed cell death protein 1 (PD-1) against colon cancer. Midostaurin inhibited the growth of murine CT26 and human HCT116 and SW480 cells with multinucleation and micronuclei formation in morphology examination. The cell cycle arrested in the G2/M phase and the formation of the polyploid phase was noted. The formation of cytosolic DNA, including double-strand and single-strand DNA, was increased. Midostaurin increased mRNA expressions of cGAS, IRF3, and IFNAR1 in colorectal adenocarcinoma cells and mouse spleen macrophages. The protein expressions of Trex-1, c-KIT, and Flt3, but not PKC&alpha;/&beta;/&gamma; and VEGFR1, were down-regulated in midostaurin-treated colorectal adenocarcinoma cells and macrophages. Trex-1 protein expression was abrogated after FLT3L activation. In vivo, the combination of midostaurin and anti-PD-1 exhibited the greatest growth inhibition on a CT26-implanted tumor without major toxicity. TME analysis demonstrated that midostaurin alone decreased Treg cells and increased neutrophils and inflammatory monocytes. NKG2D+ and PD-1 were suppressed and M1 macrophage was increased after combination therapy. When combined with anti-PD-1, STING and INF&beta; protein expression was elevated in the tumor. The oral administration of midostaurin may have the potential to enhance anti-PD-1 efficacy, accompanied by the modulation of cytosolic DNA-sensing signaling and tumor microenvironment

Proposed mechanisms in RVLM that underlies METH-induced cardiovascular collapse.

<p>Intravenous administration of METH rapidly reaches RVLM to induce anoxia and cessation of tissue perfusion, followed by bioenergetics failure and oxidative stress because of mitochondrial dysfunction that lead to necrotic cell death. The loss of functionality in RVLM results in cessation of central cardiovascular regulation and the eventual cardiovascular collapse.</p

Methamphetamine did not induce apoptotic cell death in RVLM.

<p>Changes of activated caspase-3 or histone-associated DNA fragments in fold relative to sham-control group in samples collected from RVLM of rats that received saline or died of intravenous administration of METH. Values are mean ± SEM, n = 5–7 animals per experimental group. <i>P></i>0.05 among all groups in one-way ANOVA.</p

Methamphetamine induced mitochondrial dysfunction in RVLM.

<p>Fold changes relative to sham-control group in the activity of Complex I (A), II (B), III (C), IV (D) or V (E) and electron transfer capacity between Complexes I and III (NCCR; F)or II and III (SCCR; G) in the mitochondrial respiratory chain in samples collected from RVLM of rats died of METH (24 mg/kg, i.v.) or survived with pretreatment by microinjection of Mito-TEMPO (500 pmol) or coenzyme Q10 (CoQ10; 7 nmol) into bilateral RVLM prior to METH administration. Values are mean ± SEM, n = 4–5 animals per experimental group. *<i>P</i><0.05 versus Vehicle+Saline group, and ^#<i>P</i><0.05 versus Vehicle+METH group in the post hoc Scheffé multiple-range test.</p

Methamphetamine dose-dependently and time-dependently elicited mortality and cardiovascular collapse.

<p>Temporal changes in survival rate (A), mean arterial pressure (MAP; B), heart rate (HR; C) or power density of the low-frequency (LF) component of systolic blood pressure signals (D) in rats that received (at arrow) intravenous (i.v.) METH or saline (vehicle control). Values are mean ± SEM, n = 5–62 animals per experimental group. *<i>P</i><0.05 versus saline group in the Fisher Exact Test (A), or at corresponding time-points in the post hoc Scheffé multiple-range test (C–D). B, baseline.</p

Methamphetamine induced bioenergetics failure in RVLM.

<p>(A). Fold changes relative to sham-control group in the level of ATP, ADP or ADP/ATP ratio in samples collected from RVLM of rats that died of METH (24 mg/kg, i.v.) or survived with pretreatment by microinjection of Mito-TEMPO (500 pmol) or coenzyme Q10 (CoQ10; 7 nmol) into RVLM prior to METH administration. Values are mean ± SEM, n = 3–5 animals per experimental group. *<i>P</i><0.05 versus Vehicle+Saline group, and^#<i>P</i><0.05 versus Vehicle+METH group in the post hoc Scheffé multiple-range test. (B). Representative laser scanning confocal microscopic images superimposed on phase contrast images showing neurons in RVLM that were immunoreactive to propidium iodide in rats that received saline (B1), died of METH (24 mg/kg, i.v.; B2), or survived with pretreatment by microinjection of CoQ10 (7 nmol; B3) or IM-54 (3 pmol; B4) into bilateral RVLM prior to METH administration. These results are typical of 4 animals from each experimental group. Scale bar, 100 µm. NA, nucleus ambiguus.</p