RESUMO
In the changing legal environment of obstetric care in the USA, with laws in many states banning termination at all stages of pregnancy with narrow exemptions, healthcare providers are encountering cases in which risk to maternal safety is increased. This report presents a case of a 28-year-old primigravida with an anencephalic fetus who was legally unable to pursue termination in her home state. She traveled to another state in order to pursue safe and legal abortion of a non-viable fetus. Due to an unrecognized cornual ectopic gestation, the delivery resulted in uterine rupture, the need for hysterectomy, and significant morbidity in a patient with a strong desire for future fertility.
Assuntos
Gravidez Ectópica , Gravidez , Feminino , Humanos , Adulto , Gravidez Ectópica/cirurgia , Aborto Legal , HisterectomiaRESUMO
A 37-year-old man presented with symptoms of intermittent claudication. Investigations revealed atypical calf vessel disease but no obvious aetiology. Ten years later he re-presented with worsening symptoms. CT angiography confirmed the atypical pattern of lower limb arterial disease but also noted calcification of the renal parenchyma, myocardium and scrotum. A diagnosis of pseudo-xanthoma elasticum was confirmed by skin biopsy. Pseudo-xanthoma elasticum is a rare condition that presents infrequently to vascular surgeons. Early recognition should prompt aggressive risk factor management to slow accelerated atherosclerosis. Clinicians should be aware of the clinical features of this condition to allow early diagnosis.
Assuntos
Claudicação Intermitente/etiologia , Pseudoxantoma Elástico/complicações , Pseudoxantoma Elástico/diagnóstico , Adulto , Humanos , MasculinoRESUMO
Src family protein kinases (SFKs) -mediated tyrosine-phosphorylation regulates N-methyl-D-aspartate (NMDA) receptor synaptic function. Some members of the membrane-associated guanylate kinase (MAGUK) family of proteins bind to both SFKs and NMDA receptors, but it is unclear whether the MAGUK family of proteins is required for SFKs-mediated tyrosine-phosphorylation of the NMDA receptors. Here, we showed by co-immunoprecipitation that post-synaptic density (PSD) -93, a member of the MAGUK family of proteins, interacts with the NMDA receptor subunits NR2A and NR2B as well as with Fyn, a member of the SFKs, in mouse cerebral cortex. Using a biochemical fractionation approach to isolate subcellular compartments revealed that the expression of Fyn, but not of other members of the SFKs (Lyn, Src, and Yes), was significantly decreased in synaptosomal membrane fractions derived from the cerebral cortex of PSD-93 knockout mice. Interestingly, we found that PSD-93 disruption causes reduction of tyrosine-phosphorylated NR2A and NR2B in the same fraction. Moreover, PSD-93 deletion markedly blocked the SFKs-mediated increase in tyrosine-phosphorylated NR2A and NR2B through the protein kinase C pathway after induction with 4-phorbol 12-myristate 13-acetate in cultured cortical neurons. Our findings indicate that PSD-93 appears to mediate tyrosine-phosphorylation of the NMDA receptors and synaptic localization of Fyn.
Assuntos
Córtex Cerebral/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Guanilato Quinases , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Sinaptossomos/metabolismo , Quinases da Família src/metabolismoRESUMO
We conducted a randomised double-blinded study of 960 women undergoing day-case surgery to determine whether combination anti-emetic therapy of granisetron and cyclizine was more effective at decreasing the incidence of postoperative nausea and vomiting than these agents used alone. The women were randomly allocated to three groups to receive intravenous granisetron 1 mg, cyclizine 50 mg or both before induction of general anaesthesia. The incidence of postoperative nausea and vomiting was 77/322 (24%) in the granisetron group, 73/316 (23%) in the cyclizine group and 53/322 (17%) in those women given both drugs (p = 0.04). There was no difference in the requirement for rescue anti-emetic drugs. There were no differences in the anaesthetic techniques used in the three groups. We conclude that the risk of postoperative nausea and vomiting is less with cyclizine and granisetron given together than with either given alone.
Assuntos
Antieméticos/uso terapêutico , Ciclizina/uso terapêutico , Granisetron/uso terapêutico , Náusea e Vômito Pós-Operatórios/prevenção & controle , Adulto , Procedimentos Cirúrgicos Ambulatórios , Antieméticos/efeitos adversos , Ciclizina/efeitos adversos , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Granisetron/efeitos adversos , Humanos , Estudos Prospectivos , Fatores de RiscoRESUMO
The present study investigated the role of neuronal nitric oxide synthase (nNOS) in carrageenan-induced inflammatory pain by combining genomic and pharmacological strategies. Intrathecal injection of the nNOS inhibitor 7-nitroindazole dose-dependently inhibited carrageenan-induced thermal hyperalgesia in both early and late phases in wild-type mice. However in nNOS knockout mice, carrageenan-induced thermal hyperalgesia remained intact in the early phase but was reduced in the late phase. Spinal Ca2+ -dependent nitric oxide synthase (NOS) activity in nNOS knockout mice was significantly lower than that in wild-type mice. Following carrageenan injection, although the spinal Ca2+ -dependent NOS activity in both wild-type and knockout mice increased, the enzyme activity in nNOS knockout mice reached a level similar to that in wild-type mice. On the other hand, no significant difference in spinal Ca2+ -independent NOS activity was noted between wild-type and nNOS knockout mice before and after carrageenan injection. Furthermore, intrathecal administration of the endothelial NOS (eNOS) inhibitor L-N5-(1-iminoethyl)-ornithinein nNOS knockout mice inhibited the thermal hyperalgesia in both early and late phases, though this inhibitor had no effect in wild-type mice. Meanwhile, Western blot showed that eNOS expression in the spinal cord of nNOS knockout mice was up-regulated compared with wild-type mice; immunohistochemical staining showed that the spinal eNOS was mainly distributed in superficial laminae of the dorsal horn. Finally, double staining with confocal analysis showed that the enhanced spinal eNOS was expressed in astrocytes, but not in neurons. Our current results indicate that nNOS plays different roles in the two phases of carrageenan-induced inflammatory pain. In this model, enhanced spinal eNOS appears to compensate for the role of nNOS in nNOS knockout mice.
Assuntos
Carragenina , Hiperalgesia/fisiopatologia , Inflamação/fisiopatologia , Óxido Nítrico Sintase/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Hiperalgesia/induzido quimicamente , Imuno-Histoquímica , Indazóis/administração & dosagem , Indazóis/farmacologia , Inflamação/induzido quimicamente , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Medula Espinal/metabolismo , Distribuição TecidualRESUMO
The expression and distribution of the neuronal glutamate transporter, excitatory amino acid carrier-1 (EAAC1), are demonstrated in the dorsal root ganglion neurons and their central terminals. Reverse transcriptase-polymerase chain reaction shows expression of EAAC1 mRNA in the dorsal root ganglion. Immunoblotting analysis further confirms existence of EAAC1 protein in this region. Immunocytochemistry reveals that approximately 46.6% of the dorsal root ganglion neurons are EAAC1-positive. Most EAAC1-positive neurons are small and around 250-750 microm2 in surface area, and some co-label with calcitonin gene-related peptide (CGRP) or isolectin IB4. In the spinal cord, EAAC-1 immunoreactive small dot- or patch-like structures are mainly localized in the superficial dorsal horn, and some are positive for CGRP or labeled by isolectin IB4. Unilateral dorsal rhizotomy experiments further show that EAAC1 immunoreactivity is less intense in superficial dorsal horn on the side ipsilateral to the dorsal rhizotomy than on the contralateral side. The results indicate the presence of EAAC1 in the dorsal root ganglion neurons and their central terminals. Our findings suggest that EAAC1 might play an important role in transmission and modulation of nociceptive information via the regulation of pre-synaptically released glutamate.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Gânglios Espinais/citologia , Neurônios/metabolismo , Simportadores/metabolismo , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Western Blotting/métodos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Contagem de Células , Transportador 3 de Aminoácido Excitatório , Lateralidade Funcional , Proteínas de Transporte de Glutamato da Membrana Plasmática , Imuno-Histoquímica/métodos , Lectinas/metabolismo , Masculino , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rizotomia/métodos , Medula Espinal/metabolismo , Simportadores/genéticaRESUMO
The activation of spinal cord N-methyl-D-aspartate (NMDA) receptors and subsequent intracellular cascades play a pivotal role in the development of opioid tolerance. Postsynaptic density protein-95 (PSD-95), a molecular scaffolding protein, assembles a specific set of signaling proteins around NMDA receptors at neuronal synapses. The current study investigated the possible involvement of PSD-95 in the development of opioid tolerance. Opioid tolerance was induced by intrathecal injection of morphine sulfate (20 microg/10 microl) twice a day for 4 consecutive days. Co-administration of morphine twice daily and PSD-95 antisense oligodeoxynucleotide (50 microg/10 microl) once daily for 4 days not only markedly reduced the PSD-95 expression and its binding to NMDA receptors in spinal cord but also significantly prevented the development of morphine tolerance. In contrast, co-administration of morphine twice daily and PSD-95 missense oligodeoxynucleotide (50 microg/10 microl) once daily for 4 days did not produce these effects. The PSD-95 antisense oligodeoxynucleotide at the doses we used did not affect baseline response to noxious thermal stimulation or locomotor function. The present study indicates that the deficiency of spinal cord PSD-95 attenuates the development of opioid tolerance. These results suggest that PSD-95 might be involved in the central mechanisms of opioid tolerance and provide a possible new target for prevention of development of opioid tolerance.
Assuntos
Tolerância a Medicamentos/fisiologia , Morfina/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/fisiologia , Medula Espinal/metabolismo , Animais , Proteína 4 Homóloga a Disks-Large , Injeções Espinhais , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana , Proteínas do Tecido Nervoso/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/farmacologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacosRESUMO
To date, the exact role of inducible nitric oxide synthase (iNOS) in inflammatory pain remains controversial. In the present study, we combined a pharmacological strategy (using a selective iNOS inhibitor) with a genomic strategy (using mice lacking the iNOS gene) to address the function of iNOS in the central mechanism of carrageenan-induced persistent inflammatory pain. In the wild type mice, intrathecal administration of L-N(6)-(1-iminoethyl)-lysine, a selective iNOS inhibitor, significantly inhibited thermal hyperalgesia in the late phase but not in the early phase of carrageenan inflammation. Moreover, iNOS mRNA expression in the lumbar enlargement segments of the spinal cord was dramatically induced at 24 h (late phase) after injection of carrageenan into a hind paw. Interestingly, targeted disruption of iNOS gene did not affect carrageenan-induced thermal hyperalgesia in either the early (2-6 h) or late phase. In the lumbar enlargement segments of iNOS knockout mice, nitric oxide synthase (NOS) enzyme activity remained at a similar level to that of the wild type mice at 24 h after carrageenan injection. We found that intrathecal administration of 7-nitroindazole (a selective neuronal NOS inhibitor), but not L-N(5)-(1-iminoethyl)-ornithine (a selective endothelial NOS inhibitor), significantly reduced carrageenan-induced thermal hyperalgesia in both the early phase and the late phase in iNOS knockout mice. We also found that expression of neuronal NOS but not endothelial NOS in the lumbar enlargement segments was significantly increased in iNOS knockout mice compared with wild type mice at 24 h after carrageenan injection. Our results indicate that neuronal NOS might compensate for the function of iNOS in the late phase of carrageenan-induced inflammatory pain in iNOS knockout mice. This suggests that iNOS may be sufficient, but not essential, for the late phase of the carrageenan-induced thermal hyperalgesia.
Assuntos
Carragenina , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Óxido Nítrico Sintase/metabolismo , Medula Espinal/metabolismo , Animais , Comportamento Animal , Western Blotting , Inibidores Enzimáticos/farmacologia , Temperatura Alta , Hiperalgesia/tratamento farmacológico , Inflamação/induzido quimicamente , Injeções Espinhais , Região Lombossacral , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Dor/induzido quimicamente , Dor/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Our previous work has demonstrated that postsynaptic density protein-95, a molecular scaffolding protein that binds and clusters N-methyl-D-aspartate receptors at neuronal synapses, plays an important role in the development of peripheral nerve injury-induced neuropathic pain. The current study further investigated the possible involvement of postsynaptic density protein-95 in the maintenance of neuropathic pain. Mechanical and thermal hyperalgesia were induced within 3 days and maintained for 15 days or longer after unilateral injury to the fifth lumbar spinal nerve. The rats injected intrathecally with postsynaptic density protein-95 antisense oligodeoxynucleotide every 24 h for 4 days from day 7 to day 10 post-surgery exhibited not only a marked decrease in spinal cord postsynaptic density protein-95 protein expression but also a significant reduction in mechanical and thermal hyperalgesia on day 11 post-surgery. The rats injected with sense oligodeoxynucleotide did not display these changes. However, in the rats without nerve injury, postsynaptic density protein-95 antisense oligodeoxynucleotide given intrathecally every 24 h for 4 days did not affect responses to mechanical and thermal stimulation. In addition, postsynaptic density protein-95 antisense oligodeoxynucleotide did not change locomotor activity of experimental animals. Our results indicate that the deficiency of postsynaptic density protein-95 protein in the spinal cord significantly attenuates nerve injury-induced mechanical and thermal hyperalgesia during both the development and maintenance of chronic neuropathic pain. These results suggest that postsynaptic density protein-95 might be involved in the central mechanisms of chronic neuropathic pain and provide a novel target for development of new pain therapies.
Assuntos
Proteínas do Tecido Nervoso/fisiologia , Dor/fisiopatologia , Traumatismos dos Nervos Periféricos , Nervos Periféricos/fisiopatologia , Animais , Western Blotting , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Transtornos de Estresse por Calor , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Região Lombossacral , Masculino , Atividade Motora/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Dor/etiologia , Dor/metabolismo , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Nervos Periféricos/metabolismo , Estimulação Física , Ratos , Ratos Sprague-Dawley , Tempo de Reação , Medula Espinal/metabolismo , Medula Espinal/fisiopatologiaRESUMO
Nitric oxide synthase is expressed abundantly in the spinal cord, and nitric oxide (NO) has been shown to play important roles in the central mechanism of inflammatory hyperalgesia. However, the expression and function of the NO receptor, soluble guanylate cyclase, is not fully understood in this processing at the spinal cord level. In the present study, we report that the soluble guanylate cyclase alpha(1) subunit but not the beta(1) subunit was expressed in rat spinal cord, particularly in the dorsal horn. We showed that intrathecal administration of a selective inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, produced a significant anti-nociception demonstrated by the decrease in the number of flinches and shakes in the formalin-induced inflammatory pain model. This was accompanied by a marked reduction in formalin-induced c-fos expression in the spinal cord. During formalin-induced long-lasting inflammation, we found that the expression of the alpha(1) subunit of soluble guanylate cyclase was dramatically increased in the lumbar spinal cord on the second and fourth days after formalin injection into the dorsal side of a hind paw. Intraperitoneal pretreatment with an N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine maleate (MK-801), and a neuronal NO synthase inhibitor, 7-nitroindazole, not only significantly blocked formalin-induced secondary thermal hyperalgesia but also suppressed formalin-produced increase in the alpha(1) subunit of soluble guanylate cyclase in the spinal cord. The present results indicate that peripheral inflammation not only initially activates but also later up-regulates soluble guanylate cyclase expression via the NMDA receptor-NO signaling pathway, suggesting that soluble guanylate cyclase might be involved in the central mechanism of formalin-induced inflammatory hyperalgesia in the spinal cord.
Assuntos
Guanilato Ciclase/metabolismo , Hiperalgesia/fisiopatologia , Inflamação/fisiopatologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Medula Espinal/fisiopatologia , Animais , Comportamento Animal/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Formaldeído , Guanilato Ciclase/antagonistas & inibidores , Membro Posterior/efeitos dos fármacos , Hiperalgesia/induzido quimicamente , Hiperalgesia/complicações , Hiperalgesia/tratamento farmacológico , Inflamação/induzido quimicamente , Inflamação/complicações , Injeções Intraperitoneais , Injeções Espinhais , Masculino , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I , Oxidiazóis/administração & dosagem , Medição da Dor/efeitos dos fármacos , Subunidades Proteicas , Quinoxalinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Guanilil Ciclase Solúvel , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Distribuição Tecidual , Regulação para Cima/efeitos dos fármacosRESUMO
Our previous work has shown that PSD-95/SAP90 is required for NMDA receptor-mediated thermal hyperalgesia. To address the role of PSD-95/SAP90 in chronic pain, the present study investigated the effect of the deficiency of PSD-95/SAP90 on nerve injury-induced neuropathic pain. Following unilateral L5 spinal nerve injury, mechanical and thermal hyperalgesia developed within 3 days and persisted for 9 days or longer on the injured side. The intrathecal administration of antisense oligodeoxynucleotide specifically against PSD-95/SAP90, but not sense or missense oligodeoxynucleotide, dose-dependently delayed the onset of tactile allodynia and thermal hyperalgesia. These results suggest that PSD-95/SAP90 might be involved in the central mechanisms of the development of chronic neuropathic pain.
Assuntos
Proteínas do Tecido Nervoso/deficiência , Neuralgia/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Nervos Periféricos/metabolismo , Doenças do Sistema Nervoso Periférico/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Proteína 4 Homóloga a Disks-Large , Relação Dose-Resposta a Droga , Pé/inervação , Pé/fisiopatologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neuralgia/tratamento farmacológico , Neuralgia/fisiopatologia , Medição da Dor/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Traumatismos dos Nervos Periféricos , Nervos Periféricos/fisiopatologia , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/fisiopatologia , Estimulação Física , Ratos , Ratos Sprague-Dawley , Raízes Nervosas Espinhais/lesões , Raízes Nervosas Espinhais/fisiopatologia , Raízes Nervosas Espinhais/cirurgiaRESUMO
The effect of prolonged hypoxia as well as the molecular mechanisms on cardiac cell death is not well established. A possible role of Bcl-2 and Bax in hypoxia-induced apoptosis in different cell types has been proposed. Here we demonstrate the effect of hypoxia on the induction of apoptosis and the expression of Bcl-2-like proteins in vivo and in vitro. Hearts from rats exposed to chronic hypoxia (n = 4) showed an increased rate of apoptosis compared to normoxic hearts (n = 4). The induction of apoptosis in hypoxic hearts correlated with a significant decrease of Bcl-2 protein level, whereas Bax protein expression was increased. Exposure of isolated neonatal rat cardiac myocytes to hypoxia also resulted in a significant increase in apoptosis. However, Bcl-2 and Bax protein levels essentially remained unchanged. Our results may suggest a different molecular mechanism of hypoxia-induced apoptosis in vivo and in vitro.
Assuntos
Apoptose , Cardiomiopatias/etiologia , Hipóxia/patologia , Miocárdio/patologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Animais Recém-Nascidos , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Hipóxia Celular , Células Cultivadas , Hipóxia/metabolismo , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína X Associada a bcl-2RESUMO
BACKGROUND: Spinal N-methyl-D-aspartate (NMDA) receptor activation has been demonstrated to play an important role in the processing of spinal nociceptive information and in the determination of the minimum alveolar anesthetic concentration (MAC) of inhalational anesthetics. Postsynaptic density-95 (PSD-95)/synapse-associated protein-90 (SAP90), a molecular scaffolding protein that binds and clusters the NMDA receptor perferentially at synapses, was implicated in NMDA-induced thermal hyperalgesia. The current study investigated the possible involvement of PSD-95/SAP9O in determining MAC for isoflurane anesthesia. METHODS: Sprague-Dawley rats were pretreated intrathecally with PSD-95/SAP90 antisense oligodeoxyribonucleotide (ODN), sense ODN, missense ODN, or saline every 24 h for 4 days. After initial baseline determination of the MAC, NMDA or saline was injected intrathecally. Ten minutes later, MAC measurement was repeated. The rats also were evaluated for the presence of locomotor dysfunction by intrathecal administration of NMDA or saline in the saline- and ODN-treated rats. RESULTS: In the groups treated with antisense ODNs, but not in those treated with sense or missense ODNs, there was a significant decrease in isoflurane MAC that was not accompanied by marked changes in either blood pressure or heart rate. In the saline-treated group, intrathecal NMDA caused an increase in isoflurane MAC. In contrast, in the antisense ODN-treated group, intrathecal NMDA did not produce a significant change in isoflurane MAC. An NMDA-induced increase in blood pressure but not heart rate was found in both saline- and antisense ODN-treated groups. Locomotor activity was not changed in any of the treated animals. CONCLUSION: The results indicate not only a significant decrease in MAC for isoflurane but also an attenuation in the NMDA-induced increase in isoflurane MAC in the PSD-95/SAP90 antisense-treated animals, which suggests that PSD-95/SAP90 may mediate the role of the NMDA receptor in determining the MAC of inhalational anesthetics.
Assuntos
Anestésicos Inalatórios/farmacocinética , Isoflurano/farmacocinética , Proteínas do Tecido Nervoso/deficiência , Alvéolos Pulmonares/metabolismo , Medula Espinal/metabolismo , Anestésicos Inalatórios/farmacologia , Animais , Proteína 4 Homóloga a Disks-Large , Agonistas de Aminoácidos Excitatórios/administração & dosagem , Agonistas de Aminoácidos Excitatórios/farmacologia , Injeções Espinhais , Peptídeos e Proteínas de Sinalização Intracelular , Isoflurano/farmacologia , Masculino , Proteínas de Membrana , Atividade Motora/efeitos dos fármacos , N-Metilaspartato/administração & dosagem , N-Metilaspartato/farmacologia , Oligorribonucleotídeos Antissenso/farmacologia , Ratos , Ratos Sprague-Dawley , Reflexo/efeitos dos fármacosRESUMO
Hypoxia upregulates endothelial (e) nitric oxide synthase (NOS), but how eNOS affects soluble guanylate cyclase (sGC) protein expression in hypoxia-induced pulmonary hypertension is unknown. Wild-type (WT), eNOS-deficient [eNOS(-/-)], and inducible NOS (iNOS)-deficient [iNOS(-/-)] mice were used to investigate the effects of lack of NO from different NOS isoforms on sGC activity and protein expression and its relationship to the muscularization of the pulmonary vasculature. After 6 days of hypoxic exposure (10% O2), the ratios of the right ventricle to left ventricle + septum weight (RV/LV+S) and right ventricle weight to body weight, the lung sGC activity, and vascular muscularization were determined, and protein analysis for eNOS, iNOS, and sGC was performed. Results demonstrated that there were significant increases of RV/LV+S in all animals treated with hypoxia. In hypoxic WT and iNOS(-/-) mice, eNOS and sGC alpha1- and beta1-protein increased twofold; cGMP levels and the number of muscularized vessels also increased compared with hypoxic eNOS(-/-) mice. There was a twofold increase of iNOS protein in WT and eNOS(-/-) mice, and the basal iNOS protein concentration was higher in eNOS(-/-) mice than in WT mice. In contrast, the eNOS(-/-) mouse lung showed no eNOS protein expression, lower cGMP concentrations, and no change of sGC protein levels after hypoxic exposure compared with its normoxic controls (P > 0.34). These results suggest that eNOS, but not iNOS, is a major regulator of sGC activity and protein expression in the pulmonary vasculature.
Assuntos
Deleção de Genes , Guanilato Ciclase/metabolismo , Hipóxia/enzimologia , Hipóxia/genética , Pulmão/enzimologia , Óxido Nítrico Sintase/genética , Animais , Vasos Sanguíneos/patologia , Peso Corporal , Doença Crônica , Septos Cardíacos/patologia , Ventrículos do Coração , Hipóxia/patologia , Pulmão/irrigação sanguínea , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Miocárdio/enzimologia , Miocárdio/patologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Tamanho do Órgão , Regulação para CimaRESUMO
Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.
Assuntos
Óxido Nítrico Sintase/metabolismo , Células Parietais Gástricas/enzimologia , Animais , Western Blotting , Células Cultivadas , Histocitoquímica , Masculino , Óxido Nítrico Sintase Tipo I , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hypoxia-inducible factor-1 (HIF-1) is an essential transcription factor involved in the oxygen-dependent regulation of gene expression. Thiol groups in HIF-1 or in proteins that modify HIF-1 are conventional targets for regulation by nitric oxide (NO). Moreover, NO delivery to tissue by hemoglobin appears to be oxygen dependent. Therefore, the role NO plays in regulating HIF-1 activity and expression was examined. The 1-substituted diazen-1-ium-1, 2-diolate NOC-18 induced HIF-1 DNA-binding activity in normoxic bovine pulmonary artery endothelial cells and rat aortic smooth muscle cells in a time- and dose-dependent manner. Induction of HIF-1-binding activity was consistent with an increased expression of HIF-1 subunit proteins HIF-1alpha and HIF-1beta. The effect of NOC-18 on HIF-1 activity was blocked by cycloheximide, consistent with a post-transcriptional effect. NOC-18 induction of HIF-1 DNA-binding activity was not blocked with oxyhemoglobin, nor was it related to the rate of NO evolution, arguing against NO-mediation of the effect. Additionally, the effect of NOC-18 could not be mimicked by Angeli's salt, arguing against nitroxyl mediation. However, the NOC-18 effect could be reproduced by S-nitrosoglutathione (GSNO), an endogenous nitrosonium donor formed in the presence of deoxyhemoglobin. Furthermore, the GSNO effect could be reversed by dithiothreitol as well as acivicin, an inhibitor of GSNO bioactivation. Taken together, these results suggest that an S-nitrosylation reaction stabilizes HIF-1 protein expression and activity. We speculate that one signaling mechanism by which deoxyhemoglobin may activate HIF-1 involves NO.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Doadores de Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição , Animais , Bovinos , Células Cultivadas , Cisteína Endopeptidases , DNA/efeitos dos fármacos , DNA/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Ditiotreitol/farmacologia , Interações Medicamentosas , Expressão Gênica/efeitos dos fármacos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Complexos Multienzimáticos/antagonistas & inibidores , Óxido Nítrico/química , Óxidos de Nitrogênio/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Oxirredução , Complexo de Endopeptidases do Proteassoma , Transcrição GênicaRESUMO
Pulmonary hypertension is characterized by structural and morphological changes to the lung vasculature. To determine the potential role of nitric oxide in the vascular remodeling induced by hypoxia, we exposed wild-type [WT(+/+)] and endothelial nitric oxide synthase (eNOS)-deficient [(-/-)] mice to normoxia or hypoxia (10% O(2)) for 2, 4, and 6 days or for 3 wk. Smooth muscle alpha-actin and von Willebrand factor immunohistochemistry revealed significantly less muscularization of small vessels in hypoxic eNOS(-/-) mouse lungs than in WT(+/+) mouse lungs at early time points, a finding that correlated with decreases in proliferating vascular cells (5-bromo-2'-deoxyuridine positive) at 4 and 6 days of hypoxia in the eNOS(-/-) mice. After 3 wk of hypoxia, both mouse types exhibited similar percentages of muscularized small vessels; however, only the WT(+/+) mice exhibited an increase in the percentage of fully muscularized vessels and increased vessel wall thickness. eNOS protein expression was increased in hypoxic WT(+/+) mouse lung homogenates at all time points examined, with significantly increased percentages of small vessels expressing eNOS protein after 3 wk. These results indicate that eNOS deficiency causes decreased muscularization of small pulmonary vessels in hypoxia, likely attributable to the decrease in vascular cell proliferation observed in these mice.
Assuntos
Hipóxia/fisiopatologia , Pulmão/fisiologia , Microcirculação/citologia , Músculo Liso Vascular/citologia , Óxido Nítrico Sintase/metabolismo , Circulação Pulmonar/fisiologia , Actinas/análise , Animais , Doença Crônica , Cruzamentos Genéticos , Hipóxia/genética , Pulmão/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcirculação/patologia , Músculo Liso Vascular/patologia , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Circulação Pulmonar/genética , Fator de von Willebrand/análiseRESUMO
Recent pharmacological evidence showed that metabotropic glutamate receptors (mGluRs), particularly mGluRs1/5, had a potential role in spinal nociceptive processing. However, previous morphological studies on mGluRs have been limited mainly to their distribution in the spinal cord. In the present study, electron microscopic immunocytochemistry was employed to identify the synaptic relationship of the neurons containing mGluR5, with nociceptive primary afferent and gamma-aminobutyric acid-ergic (GABAergic) terminals in the superficial dorsal horn of the spinal cord. Nociceptive C- and A(delta)-primary afferent terminals selectively labeled with horseradish peroxidase conjugated to wheat-germ agglutinin were in asymmetric synaptic contacts with or in direct apposition to mGluR5 positive dendritic profiles. The double-labeling studies revealed that mGluR5 immunoreactive dendrites also received symmetric synaptic contacts from axon terminals labeled with immunogold particles indicating GABA. The present demonstration of mGluR5 neurons receiving inputs from both nociceptive primary afferents and GABAergic terminals of presumed interneurons further supports the involvement of mGluR5 in the transmission and modulation of nociceptive information in the spinal cord.
Assuntos
Terminações Nervosas/fisiologia , Neurônios/fisiologia , Nociceptores/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Medula Espinal/fisiologia , Sinapses/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Terminações Nervosas/ultraestrutura , Neurônios/ultraestrutura , Ratos , Ratos Sprague-Dawley , Receptor de Glutamato Metabotrópico 5 , Medula Espinal/ultraestrutura , Sinapses/ultraestrutura , Conjugado Aglutinina do Germe de Trigo-Peroxidase do Rábano SilvestreRESUMO
Nitric oxide (NO) upregulates ciliary beat frequency (CBF). The present study evaluates mechanisms of the NO-cyclic guanosine monophosphate (cGMP) pathway regulation of CBF. Rat tracheal explants were loaded with 4,5-diaminofluorescein diacetate for the demonstration of NO production by ciliated epithelial cells after L-arginine (L-Arg) stimulation. CBF was measured using phase contrast microscopy and videotape analysis. The roles of NO, soluble guanylate cyclase (sGC), cGMP-dependent protein kinase (PK) G, and phosphodiesterase (PDE) V in regulation of CBF were evaluated. NO synthase (NOS) was activated with L-Arg or inhibited with N(G)-monomethyl-L-Arg. sGC was stimulated with NO donors 1-hydroxy-2-oxo-3- (N-ethyl-2-aminoethyl)-3-ethyl-1-triazene and S-nitroso-L-glutathione or mimicked by 8-bromo-guanosine 3', 5'-cyclic monophosphate (8-Br-cGMP) and inhibited with 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one. The effects of the PKG inhibition with KT5823 and PDE V inhibition with Zaprinast were also examined. The studies demonstrate that ciliated epithelial cells produce NO, which is correlated with CBF stimulation. L-Arg dose- and time-dependently increases CBF, and NO donors, 8-Br-cGMP, and Zaprinast also enhance CBF. Inhibitors of NOS, sGC, and PKG can block the stimulant effect of L-Arg on CBF. Thus, NO is a regulator of CBF acting via sGC and PKG. The NO-cGMP signaling pathway regulates CBF in an autocrine manner in cultured rat ciliated airway epithelium.