RESUMO
BACKGROUND: Despite its clinical importance, a dearth of information exists on the cellular and molecular mechanisms that underpin brain stem death. A suitable neural substrate for mechanistic delineation on brain stem death resides in the rostral ventrolateral medulla (RVLM) because it is the origin of a life-and-death signal that sequentially increases (pro-life) and decreases (pro-death) to reflect the advancing central cardiovascular regulatory dysfunction during the progression towards brain stem death in critically ill patients. The present study evaluated the hypothesis that heme oxygnase-1 (HO-1) may play a pro-life role as an interposing signal between hypoxia-inducible factor-1 (HIF-1) and nitric oxide synthase I (NOS I)/protein kinase G (PKG) cascade in RVLM, which sustains central cardiovascular regulatory functions during brain stem death. METHODS: We performed cardiovascular, pharmacological, biochemical and confocal microscopy experiments in conjunction with an experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of adult male Sprague-Dawley rats. RESULTS: Western blot analysis coupled with laser scanning confocal microscopy revealed that augmented HO-1 expression that was confined to the cytoplasm of RVLM neurons occurred preferentially during the pro-life phase of experimental brain stem death and was antagonized by immunoneutralization of HIF-1α or HIF-1ß in RVLM. On the other hand, the cytoplasmic presence of HO-2 in RVLM neurons manifested insignificant changes during both phases. Furthermore, immunoneutralization of HO-1 or knockdown of ho-1 gene in RVLM blunted the augmented life-and-death signals exhibited during the pro-life phase. Those pretreatments also blocked the upregulated pro-life NOS I/PKG signaling without affecting the pro-death NOS II/peroxynitrite cascade in RVLM. CONCLUSIONS: We conclude that transcriptional upregulation of HO-1 on activation by HIF-1 in RVLM plays a preferential pro-life role by sustaining central cardiovascular regulatory functions during brain stem death via upregulation of NOS I/PKG signaling pathway. Our results further showed that the pro-dead NOS II/peroxynitrite cascade in RVLM is not included in this repertoire of cellular events.
Assuntos
Morte Encefálica/fisiopatologia , Heme Oxigenase-1/metabolismo , Inseticidas/toxicidade , Bulbo/efeitos dos fármacos , Mevinfós/toxicidade , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Western Blotting , Morte Encefálica/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Imunofluorescência , Técnicas de Silenciamento de Genes , Heme Oxigenase-1/genética , Inseticidas/administração & dosagem , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Bulbo/citologia , Mevinfós/administração & dosagem , Microscopia Confocal , Proteínas Mitocondriais , Proteínas de Neoplasias/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Oligonucleotídeos/genética , Ratos , Ratos Sprague-DawleyRESUMO
As inhibitors of acetylcholinesterase, clinical presentations of poisoning from organophosphate compounds are generally believed to entail overstimulation by the accumulated acetylcholine on muscarinic receptors at peripheral and central synapses. That some patients still yielded to acute organophosphate poisoning despite repeated dosing of atropine suggests that cellular mechanisms that are independent of muscarinic receptor activation may also be engaged in organophosphate poisoning. The present study was undertaken to test the hypothesis that muscarinic receptor-independent activation of cyclic adenosine monophosphate-dependent protein kinase A (PKA) in rostral ventrolateral medulla (RVLM), a medullary site where sympathetic vasomotor tone originates and where the organophosphate poison mevinphos (Mev) acts, is involved in the cardiovascular responses exhibited during organophosphate intoxication. In Sprague-Dawley rats, microinjection bilaterally of Mev (10 nmol) into the RVLM significantly augmented PKA activity in ventrolateral medulla that was not antagonized by coadministration of an equimolar concentration (1 nmol) of atropine or selective muscarinic receptor type M1 (pirenzepine), M2 (methoctramine), M3 (4-diphenyl-acetoxy-N-dimethylpiperidinium), or M4 (tropicamide) inhibitor. Comicroinjection of two selective PKA antagonists (100 pmol), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide and (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolol[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-1][1,6]benzodiazocine-10-carboxylic acid, significantly blunted the initial sympathoexcitatory cardiovascular response and the accompanying augmentation of nitric oxide synthase (NOS I) expression in the ventrolateral medulla exhibited during Mev intoxication; the secondary sympathoinhibitory phase and associated elevation in NOS II expression were unaffected. We conclude that whereas a muscarinic receptor-independent augmentation of PKA activity in the ventrolateral medulla was manifested throughout acute Mev intoxication, this activation was preferentially involved in the sympathoexcitatory phase by an upregulation of NOS I expression.
Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Bulbo/efeitos dos fármacos , Mevinfós/toxicidade , Receptores Muscarínicos/metabolismo , Animais , Atropina/farmacologia , Western Blotting , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patologia , Inibidores da Colinesterase/administração & dosagem , Inibidores da Colinesterase/toxicidade , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Masculino , Bulbo/metabolismo , Bulbo/patologia , Mevinfós/administração & dosagem , Antagonistas Muscarínicos/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Piperidinas/farmacologia , Pirenzepina/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Tropicamida/farmacologiaRESUMO
Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) is a deubiquitinating enzyme that is responsible for making ubiquitin, which is required to target proteins for degradation by the ubiquitin-proteasome pathway in neurons, available. We investigated whether UCH-L1 plays a neuroprotective role at the rostral ventrolateral medulla (RVLM), the origin of sympathetic neurogenic vasomotor tone in the medulla oblongata where the organophosphate insecticide mevinphos (Mev) acts to elicit cardiovascular toxicity. In Sprague-Dawley rats maintained under propofol anesthesia, Mev (960 microg/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 bilateral microinjection into the RVLM of a transcription inhibitor, actinomycin D (5 nmol), or a translation inhibitor, cycloheximide (20 nmol). Compared with aCSF or sense 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 "pro-life" phase, blunted the increase in ubiquitin expression in ventrolateral medulla, and augmented the induced hypotension in rats that received Mev. These findings suggest that de novo synthesis of UCH-L1, leading to an enhanced disassembly of ubiquitin-protein conjugates in the RVLM, is essential to maintenance of the "pro-life" phase of Mev intoxication via prevention of cardiovascular depression, leading to neuroprotection.
Assuntos
Hidrolases/biossíntese , Bulbo/enzimologia , Mevinfós/toxicidade , Ubiquitina/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Hidrolases/metabolismo , Injeções Intravenosas , Isoenzimas/biossíntese , Isoenzimas/metabolismo , Masculino , Bulbo/efeitos dos fármacos , Mevinfós/administração & dosagem , Mevinfós/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Análise de Sobrevida , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Ubiquitina/genéticaRESUMO
We investigated possible changes in bioenergetics at the rostral ventrolateral medulla (RVLM), a medullary site where sympathetic vasomotor tone originates and where the organophosphate poison mevinphos (Mev) acts to elicit cardiovascular intoxication. In Sprague-Dawley rats maintained under propofol anesthesia, microinjection bilaterally of Mev (10 nmol) into the RVLM induced progressive hypotension that was accompanied by an early augmentation (80-100 min post-Mev; Phase I), followed by a decrease (>100 min post-Mev; Phase II) in the power density of the vasomotor components (0-0.8 Hz) in systemic arterial pressure (SAP) signals. Enzyme assay revealed that local application of Mev into the RVLM also significantly and progressively depressed the activity of NADH cytochrome c reductase (marker for Complexes I and III) and cytochrome c oxidase (marker for Complex IV) in the mitochondrial respiratory chain of the RVLM, but not the heart. On the other hand, the activity of succinate cytochrome c reductase (marker for Complexes II and III) remained unaltered. Both the cardiovascular consequences and depression of mitochondrial respiratory chain enzymes elicited by Mev were significantly antagonized on comicroinjection of atropine (3.5 or 7 nmol) bilaterally into the RVLM. We conclude that Mev adversely effects cardiovascular control by acting as a cholinesterase inhibitor in the RVLM, whose neuronal activity is intimately related to the death process. The resulting accumulation of acetylcholine and prolonged activation of muscarinic receptors in the RVLM is manifested by a selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain that underlies cardiovascular toxicity associated with organophosphate poisons such as Mev.
Assuntos
Bulbo/efeitos dos fármacos , Mevinfós/administração & dosagem , Mevinfós/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Animais , Atropina/farmacologia , Respiração Celular/efeitos dos fármacos , Modelos Animais de Doenças , Frequência Cardíaca/efeitos dos fármacos , Masculino , Bulbo/citologia , Bulbo/fisiologia , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
We evaluated the relationship between the toxicity induced by the organophosphate mevinphos (Mev) and inducible nitric oxide synthase (iNOS) in the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone. Adult Sprague-Dawley rats that were anesthetized and maintained with propofol were used. Laser scanning confocal microscopic analysis revealed colocalization of the M2 subtype of muscarinic receptors (M(2)R) and iNOS immunoreactivity in RVLM neurons. Comicroinjection bilaterally of Mev (10 nmol) and artificial cerebrospinal fluid (aCSF) into the RVLM elicited a progressive decline in systemic arterial pressure (SAP) and heart rate. This was accompanied during phase 1 Mev intoxication by an increase in the power density of the very high-frequency (VHF; 5-9 Hz), high-frequency (HF; 0.8-2.4 Hz), low-frequency (LF; 0.25- 0.8 Hz) and very low-frequency (VLF; 0-0.25 Hz) components of SAP signals. Phase 2 exhibited a reversal of the VHF and VLF power to control levels and a further reduction in the power density of both HF and LF components to below baseline. Hypotension and bradycardia promoted by Mev were significantly blunted on coadministration into the RVLM of the selective iNOS inhibitors S-methylisothiourea (250 pmol) or aminoguanidine (250 pmol). Not only was the augmented power density of HF and LF components during phase 1 Mev intoxication further enhanced, the reduced power of these two spectral components during phase 2 was appreciably antagonized. On the other hand, the temporal changes in VHF and VLF power were essentially the same as with coadministration of Mev and aCSF. We conclude that, as a cholinesterase inhibitor, Mev may induce toxicity via nitric oxide produced by iNOS on activation of the M(2)R by the accumulated acetylcholine in the RVLM.
Assuntos
Inseticidas/farmacologia , Bulbo/enzimologia , Mevinfós/toxicidade , Óxido Nítrico Sintase/efeitos dos fármacos , Acetilcolina/metabolismo , Animais , Inibidores da Colinesterase/administração & dosagem , Inibidores da Colinesterase/farmacologia , Eletrofisiologia , Hemodinâmica/efeitos dos fármacos , Imuno-Histoquímica , Inseticidas/administração & dosagem , Masculino , Bulbo/química , Bulbo/citologia , Mevinfós/administração & dosagem , Microinjeções , Microscopia Eletrônica de Varredura , Neurônios/química , Óxido Nítrico/metabolismo , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M2 , Receptores Muscarínicos/metabolismo , Sistema VasomotorRESUMO
We investigated the cardiovascular consequences of acute intoxication by the organophosphate poison, mevinphos (Mev), and delineated the underlying mechanism. Based on on-line power spectral analysis of systemic arterial pressure (SAP) signals in rats anesthetized and maintained by propofol, we identified two distinct phases after intravenous administration of Mev (160 or 320 microg/kg). Phase I was characterized by transient hypertension and mild tachycardia, concurrent with an increase in the very high-frequency (BVHF; 5-9 Hz), high-frequency (BHF; 0.8-2.4 Hz), low-frequency (BLF; 0.25-0.8 Hz),and very low-frequency (BVLF; 0-0.25 Hz) components of SAP signals. Phase II exhibited significant hypotension, a reversal of the BVHF and BVLF power to control levels, and further reduction in the power density of both BHF and BLF components to below baseline. Microinjection of Mev (2 microg) into the bilateral nucleus reticularis ventrolateralis (NRVL), the medullary origin of sympathetic neurogenic vasomotor tone, essentially duplicated those phasic cardiovascular changes. Similarly, sympathoexcitatory NRVL neurons exhibited respectively an elevation and a decline in their spontaneous activities during Phase I and Phase II Mev intoxication. We conclude that the progressive accumulation of acetylcholine over time induced by a direct inhibition of Mev on cholinesterase in the NRVL may be responsible for the phasic changes in cardiovascular events over the course of acute Mev intoxication. Whereas the initial amount of acetylcholine is excitatory to NRVL neurons, overstimulation by the amassed acetylcholine results instead of an inhibitory action.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Inseticidas/intoxicação , Mevinfós/intoxicação , Intoxicação/fisiopatologia , Animais , Artérias , Determinação da Pressão Arterial/métodos , Frequência Cardíaca/efeitos dos fármacos , Injeções Intravenosas , Masculino , Mevinfós/administração & dosagem , Microinjeções , Músculo Liso Vascular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos , Formação Reticular/efeitos dos fármacos , Formação Reticular/fisiologiaRESUMO
The efficacy of two new monopyridinium oximes (2-PAAM, 2-PAEM) and two bispyridinium oximes (obidoxime. HI-6) was tested in combination with atropine sulphate against acute poisoning with the organophosphorus insecticide mevinphos in mice. When mice were treated two min. after mevinphos poisoning, no significant differences in the therapeutic effect of tested oximes were observed. The oximes increased the 24 hr LD50 values of mevinphos about two times in comparison with the 24 hr LD50 values of mevinphos in mice protected with atropine sulphate alone and more than three times in comparison with non-treated intoxicated animals. On the other hand, both monopyridinium oximes were significantly more efficacious than HI-6 and as efficacious as obidoxime when they were administered 30 sec. after mevinphos poisoning. Both monopyridinium oximes and obidoxime increased the 24 hr values of mevinphos almost three times in comparison with the 24 hr values of mevinphos in mice protected with atropine sulphate alone and about twenty-five times in comparison with non-treated intoxicated animals, while the oxime HI-6 less than two times in comparison with the 24 hr values of mevinphos in mice protected with atropine sulphate alone and about fifteen times in comparison with non-treated intoxicated animals. Use of new monopyridinium oximes seems to be the improvement in the antidotal treatment of poisoning with organophosphorous insecticide mevinphos in comparison with HI-6 but not in comparison with obidoxime when oximes are used in equimolar doses.
Assuntos
Reativadores da Colinesterase/farmacologia , Inseticidas/toxicidade , Mevinfós/toxicidade , Cloreto de Obidoxima/farmacologia , Compostos de Piridínio/farmacologia , Animais , Atropina/administração & dosagem , Atropina/farmacologia , Reativadores da Colinesterase/administração & dosagem , Dose Letal Mediana , Masculino , Mevinfós/administração & dosagem , Camundongos , Antagonistas Muscarínicos/administração & dosagem , Antagonistas Muscarínicos/farmacologia , Cloreto de Obidoxima/administração & dosagem , Oximas , Compostos de Piridínio/administração & dosagem , Relação Estrutura-AtividadeRESUMO
A case report of fatal ingestion of mevinphos with several points of interest: There was a known ingested dosage of 28 g. Several observers witnessed events from ingestion to death. Death was rapid--apparently within 1 min and certainly within 14 min.
Assuntos
Inseticidas/intoxicação , Mevinfós/intoxicação , Suicídio , Idoso , Humanos , Masculino , Mevinfós/administração & dosagem , Fatores de TempoRESUMO
The authors carried out studies on 10 cats, anesthetized by urethane, under atropine protection and under the conditions of artificial respiration and examined the influence of therapeutic antidote of phosphorganic pesticides-Doline on the induced eletromyographic activity during total neuro-muscular block, induced by superlethal doses of phosphadrine. It was established that superlethal doses of phosphadrine induced total block of electropotentials in neuro-muscular conduction of the peripheral musculature, which was removed immediately after venous and muscular administration of the antidote Doline.
Assuntos
Antídotos/uso terapêutico , Eletromiografia , Inseticidas/intoxicação , Meperidina/uso terapêutico , Mevinfós/intoxicação , Animais , Gatos , Potenciais Evocados/efeitos dos fármacos , Mevinfós/administração & dosagem , Condução Nervosa/efeitos dos fármacos , Bloqueadores Neuromusculares , Junção Neuromuscular/efeitos dos fármacos , Fatores de TempoRESUMO
The interaction of the organophosphate mevinphos and atropine was examined in two pigeons performing in a variable interval schedule of reinforcement. When administered separately, both atropine and mevinphos produced a dose-related decrement in responding. The combined exposure to these drugs produced a performance decrement greater than that caused by exposure to each component drug alone. These findings suggest that prophylactic use of atropine may increase the detrimental behavioral effects of organophosphate exposure and that the atropine exposure alone may produce serious behavioral deficits.