As the origin of a ¡§life-and-death¡¨ signal that reflects central cardiovascular regulatory failure during brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate to evaluate the cellular mechanism of this fateful phenomenon. Based on a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult, this study evaluated two hypotheses. First, transcriptional upregulation of nitric oxide synthase I or II (NOS I or II) gene expression by nuclear factor-£eB (NF-£eB) on activation of phosphoinositide 3-kinases (PI3K)/Akt/phosphatase and tensin homologue deleted on chromosome ten (PTEN) cascade in the RVLM underlies brain stem death. Second, muscarinic receptor-independent activation of cyclic adenosine monophosphate-dependent protein kinase A (PKA) in the RVLM is involved in the cardiovascular responses exhibited during Mev intoxication. In Sprague-Dawley rats, our results showed that microinjection bilaterally of Mev (10 nmol) into RVLM induced a progressive augmentation in NF-£eB, PI3K, Akt or PTEN activity that paralleled the increase in NOS II or peroxynitrite level in RVLM. Loss-of-function manipulations that included pharmacological blockade, gene knockdown, or immunoneutralization of NF-£eB, PI3K or Akt in RVLM significantly potentiated and prolonged the initial increase in ¡§life-and-death¡¨ signal, reversed the cardiovascular depression, and blunted the augmented expression of NOS II or nitrotyrosine on induced by Mev. Blockade of PI3K or Akt in RVLM also significantly blunted the Mev-induced activation of NF-£eB in the RVLM. However, immunoneutralization of PTEN in RVLM significantly diminished the increase in ¡§life-and-death¡¨ signal and potentiated the increase in Akt activity. We conclude that the PI3K/Akt cascade plays a ¡§pro-death¡¨ role in our Mev intoxication model of brain stem death by upregulating NF-£eB/NOS II/peroxynitrite in the RVLM, subject to antagonism by PTEN in this process. Microinjection bilaterally of Mev (10 nmol) into the RVLM induced a significantly augmentation in PKA activity in ventrolateral medulla that was not antagonized by coadministration of a nonselevtive or selective muscarinic receptor inhibitor. However, pharmacological blockade PKA in RVLM significantly blunted the initial increase in ¡§life-and-death¡¨ signal and the accompanying augmentation of NOS I expression in the ventrolateral medulla exhibited during Mev intoxication. We conclude that a muscarinic receptor-independent activation of PKA plays a ¡§pro-life¡¨ role in our Mev intoxication model of brain stem death by up regulating NOS I/PKG in the RVLM. According to this study, we proved that Mev stimulates different mechanism, muscarinic receptor-independent/PKA and PI3K/Akt/NF-£eB, to regulate NOS I and NOS II expression respectively, and induces cardiovascular responses during ¡§pro-life¡¨ and ¡§pro-death¡¨ phases. This information should provide further insights on the cellular mechanism of central cardiovascular regulation during the progression towards brain stem death, and offer news vistas in our search for therapeutic remedies or management strategies against fatal organophosphate poisoning and brain stem death.

Mevinphos (Mev) is an orgnophosphate insectide used for suicidal purposes in Taiwan; seizure and cardiovascular depression are commom syptoms observed in organophosphate-poisoned patients. The amygdala (AMG) is part of the limbic system and the basolateral nucleus of AMG (BLA) is one of the most seizure-prone brain structures. The central neucleus of AMG (CeA) is thought to play a central role in behavioral, physiological response and cardiovascular regulation. However, detailed mechanisms in Mev-induced seizure and cardiovascular depression by an action on AMG are lacking. Based on electroencephalographic (EEG) activity to indicate neuronal electrical activity and arterial blood pressure (AP) and heart rate (HR) to indicate cardiovascular responses, the present study investigated whether Mev acts on AMG to elicit seizure or cardiovascular depression. Microinjection of Mev into BLA of adult male Sprague-Dawley (SD) rats maintained under propofol anesthesia increased EEG activity in AMG, cortex and CA3 of hippocampus leading to seizure initiation; however AP, HR, respiration rate (RR) and the power density of low-frequency (LF) component of AP was not significantly changed. Microinjection of Mev into BLA also time-dependently increased protein level and mRNA of cytokines interleukin (IL)-12, IL-13, tumor suppressor factor alpha (TNF£\) and interferon gamma (IFN£^) and cyclooxygenase (COX) activity in AMG. Microinjection of Mev into CA3 induced less seizure activity in cortex and CA3 than that induced by microinjection of Mev into BLA. In addition, microinjection Mev into CA3 did not induce seizure in AMG. These results suggest that Mev acted on BLA to initiate limbic seizures. Intraperitoneal injection of muscarinic receptor antagonist atropine (ATR), which can pass the blood-brain barrier (BBB), activator of GABAergic neurotransmission midazolam (MDZ) or antiinflaamatory agent pentoxifylline (PTX) and Lisofylline (LSF), but not muscarinic receptor antagonist atropine methyl nitrate (AMN), which can not pass BBB, inhibited Mev-induced seizure and increase of cytokines in AMG by an action on BLA. Microinjection of ATR, COX-1 inhibitor naproxen (NPX) or COX-2 inhibitor NS-398, antiserum against receptor of IL-12, IL-13, TNF£\ or IFN£^, but not nicotinic receptor antagonist mecamylamine (MEL), into BLA inhibited Mev-induced seizure and increase of cytokines and COX activity in AMG by an action on BLA. However, caspase 3 activity and DNA fragmentation at AMG were not changed by microinjection of Mev into BLA. Microinjection of Mev into CeA induced a decrease in AP and RR leading to cardiovascular depression and an increase of power desity of LF, accompanied with insignificant HR and EEG activity change. Microinjection of Mev into CeA induced the time-dependently increase of caspase 3 activity and DNA fragmentation leading to apoptosis in AMG. Microinjection of ATR or caspase 3-dependent apoptosome inhibitor NS-3694, but not MEL, into CeA inhibited cardiovascular depression and the increase of caspase 3 activity and DNA fragmentation induced by Mev action on CeA. However, the levels of cytokines were not changed by Mev treatment. Intravenous injection of Mev did not induce changes of partial pressure of oxygen, blood flow and the level of superoxide anion in AMG. In addition, microinjection of Mev into BLA or CeA did not affect the level of superoxide anion in AMG. These results suggest that AMG mediates the initiation of seizure and cardiovascular depression induced by Mev. Furthermore, inflammation in BLA and apoptosis in CeA individually play an important role in Mev-induced seizure and cardiovascular depression.

We investigated the role of Shc¡BPYK2¡BGrb2-sos binding complex¡BRas and Raf proteins at the rostral ventrolateral medulla (RVLM), the origin of sympathetic neurogenic vasomotor tone, in mevinphos (Mev) intoxication. Adult Sprague-Dawley rats anesthetized by sodium pentobarbital (45 mg/kg) and maintained by propofol (20-25 mg/kg/hr) were used. Bilateral microinjection of Mev (10 nmol) into the RVLM elicited two distinct phases of cardiovascular responses, designated Phase I (sympathoexcitatory) and Phase II (sympathoinhibitory) Mev intoxication. Pretreatment with microinjection of a phospho-Shc-tyrosine 317, phospho-PYK2-tyrosine 402, phospho-PYK2-tyrosine 579/580 antibody (1:20), Grb2-sos complex inhibitor (SH3b-p), Ras specific inhibitors (manumycin A or FTA) or Raf specific inhibitor (GW5074) into the bilateral RVLM blunted the magnitude of the Mev-elicited sympathoexcitatory cardiovascular effect without affecting the duration. The Mev-elicited sympathoinhibitory cardiovascular effect was not influenced. Our results suggest that signaling pathways that involve Shc, PYK2, Grb2-sos complex, Ras or Raf protein in the RVLM participate in the sympathoexcitatory phase of Mev intoxication.

We investigated the role of muscarinic receptor subtypes at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone, in mevinphos (Mev) intoxication. Adult Sprague-Dawley rats anesthetized by pentobarbital (45 mg/kg) and maintained by propofol (30 mg/kg/h) were used. Co-microinjection bilaterally of Mev (10 nmol) and artificial cerebrospinal fluid (aCSF) into the RVLM resulted in an increase (Phase I) followed by a decrease (Phase II) in the power density of the vasomotor components of systemic arterial pressure spectrum, our experimental index for sympathetic vasomotor tone. These changes in sympathetic vasomotor outflow in both phases of Mev intoxication were significantly and dose-dependently reduced on co-microinjection of Mev and the M2 subtype of muscarinic receptor (M2R) antagonist methoctramine (0.5 or 1 nmol) or M4R antagonist tropicamide (0.5 or 1 nmol). On the other hand, the M1R antagonist pirenzepine (0.5 or 1 nmol) or M3R antagonist 4-DAMP (0.5 or 1 nmol) was ineffective. Western blot analysis further revealed that the increase in NOS I protein levels at the RVLM during Phase I Mev intoxication or the augmented level of NOS II during both phases were significantly blunted on co-microinjection bilaterally of Mev and methoctramine (1 nmol) or tropicamide (1 nmol) into the RVLM. Pirenzepine (1 nmol) or 4-DMAP (1 nmol) was again ineffective. We conclude that both M2R and M4R subtypes in the RVLM may be involved in Mev intoxication. Whereas the prevalence of NOS I over NOS II at the RVLM during Phase I results in sympathoexcitation, sympathoinhibition induced by NO from NOS II in the RVLM is primarily involved in Phase II Mev intoxication.

In eukaryotic cells, most proteins in the cytosol and nucleus are degraded via the ubiquitin-proteasome pathway. Ubiquitin is best known for its role in targeting proteins for degradation by the proteasome. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is found specifically in central and peripheral neurons, and is responsible for the removal of small peptide fragments from the ubiquitin chain and for co-translational processing of ubiquitin gene products to generate free monomeric ubiquitin. In response to extreme conditions, cells exhibit an up-regulation of heat shock protein (HSP) expression, which contributes to repair and protective mechanisms. Within the HSP family, HSP70 is the major inducible member that protects against cell death. Based on the pharmacologic property of organophosphates as an inhibitor of cholinesterase, it is generally contended that manifestations of organophosphate poisoning, including secretion and muscle fasciculation, stupor, cardiopulmonary collapse, respiratory failure, coma or death, result from accumulation of, and over-stimulation by acetylcholine at peripheral of central synapses. One approach in furthering our understanding on organophosphate poisoning is delineation of its potential protective mechanisms. In this regard, the information on the cellular and molecular mechanisms that underlie organophosphate poisoning has received attention. Our laboratory demonstrated previously that a crucial brain site via which mevinphos (Mev), an organophosphate insecticide of the P=O type, acts is the rostral ventrolateral medulla (RVLM), the medullary origin of premotor sympathetic neurons that are responsible for the maintenance of vasomotor tone. The phasic changes in cardiovascular events over the course of acute Mev intoxication also parallel fluctuations of the ¡§life-and-death¡¨ signals that emanate form the RVLM. Based on a rat model of organophosphate poisoning that provides continuous information on cellular and molecular mechanisms in the RVLM, the present study was undertaken to evaluate whether changes in protein level of UCH-L1 or HSP70 are associated with death arising from Mev intoxication. We also evaluated the efficacy of both of them in the neuroprotection against fatality during Mev intoxication. The first part of this study investigated whether UCH-L1 plays a neuroprotective role at the RVLM, where Mev acts to elicit cardiovascular toxicity. In Sprague-Dawley rats maintained under propofol anesthesia, Mev (960 µg/kg, i.v.) induced a parallel and progressive augmentation in UCH-L1 or ubiquitin expression at the ventrolateral medulla during the course of Mev intoxication. The increase in UCH-L1 level was significantly blunted on pretreatment with microinjection bilaterally into the RVLM of a transcription inhibitor, actinomycin D (5 nmol) or a translation inhibitor, cycloheximide (20 nmol). Compared to artificial cerebrospinal fluid (aCSF) or sense uch-L1 oligonucleotide (100 pmol) pretreatment, microinjection of an antisense uch-L1 oligonucleotide (100 pmol) bilaterally into the RVLM significantly increased mortality, reduced the duration of the phase I (¡§pro- life¡¨ phase), blunted the increase in ubiquitin expression in ventrolateral medulla, and augmented the induced hypotension in rats that received Mev. The second part of this study investigated whether HSP70 plays a neuroprotective role at the RVLM. Intravenous administration of Mev (960

The rostral ventrolateral medulla (RVLM) regulates vasomotor activity via sympathoexcitation and sympathoinhibition to maintain blood pressure. Nitric oxide synthesized by nitric oxide synthase (NOS) I and NOS II within RVLM is responsible for sympathoexcitation and sympathoinhibition respectively. In our previously study, under physiological condition RVLM neurons contain both NOS I and NOS II protein, and NOS III protein is expressed mainly on blood vessels. Under Mevinphos (Mev) intoxication, our previously study demonstrates that the expression of RVLM NOS I and II mRNA or protein are both increased under Mev intoxication phase I, and NOSII mRNA or protein are further increased under Mev intoxication phase II. On the other hand, in rat central nervous system, about 65% of total cells are glial cells, including astrocytes, microglia and oligodendrocytes. However, the expressions of NOS isoforms in RVLM glial cells still need to be determined. We used double immunofluorescence staining and confocal microscopy to investigate the distributions of NOS isoforms protein in RVLM neurons and glial cells under physiological condition and under pathological condition using Mev intoxication as our model. We further compared the distributions of NOS isoforms in RVLM neurons and glial cells under physiological or pathological conditions. The confocal images indicate that NOS I protein reactivity co-localized with neurons and microglia in the RVLM. NOS II protein reactivity co-localized with neurons, astrocytes and microglia. NOS III protein reactivity co-localized with blood vessels and microglia. The distributions of NOS isoforms protein reactivity in RVLM neurons and glial cells under Mev intoxication are the same as under physiological condition. Furthermore, the expressions of NOS I protein within neurons or microglia and NOS II in neurons, astrocytes or microglia are progressively increased under Mev intoxication. On the other hand, the expression of NOS III within microglia under Mev intoxication was similar to physiological condition. The population of NOS I-positive neurons or microglia, and NOS II-positive neurons, astrocytes or microglia increased under Mev intoxication. However the population of NOS III-positive microglia decreased under Mev intoxication. These results indicate that within RVLM, the distributions of NOS I are in neurons and microglia; NOS II are in neurons, astrocytes and microglia; NOS III are in blood vessels and microglia. We suggest that under Mev intoxication, the source of up-regulated NOS I protein includes neurons and microglia; and the up-regulated NOS II protein comes from neurons, astrocytes and microglia.

Mevinphos (Mev) is an organophosphate insecticide that acts on the rostral ventrolateral medulla (RVLM), the origin of sympathetic vasomotor tone, to induce cardiovascular responses. This study investigated the role of Trk (tropomyosin-related kinase) (receptor form) or Src (non-receptor form) tyrosine kinase at the RVLM in Mev-induced cardiovascular responses. Bilateral microinjection of Mev (10 nmol) into the RVLM elicited two distinct phases of cardiovascular responses, designated Phase I (sympathoexcitatory) and Phase II (sympathoinhibitory) Mev intoxication. Western blot assay showed that whereas p-Trk490 was increased during Phase I, p-Src416 was increased only during Phase II Mev intoxication. Interestingly, application of a Trk specific inhibitor (K252a; 1 pmol) or Src specific inhibitor (SU6656; 100 pmol) into the bilateral RVLM blunted the Mev-elicited sympathoexcitatory or sympathoinhibitory effect, respectively. Besides, K252a was limited to block NOS I protein expression in the RVLM during Mev intoxication, SU6656 only inhibited NOS II protein expression in the RVLM during Mev intoxication. We conclude that Trk tyrosine kinase (p-Trk490) in the RVLM participates in the Phase I cardiovascular responses during Mev intoxication, Src tyrosine kinase (p-Src416) in the RVLM participates in the Phase II cardiovascular responses associated with Mev intoxication.