RESUMO
Tumor suppressor p53 protein is the transcription factor responsible for various genes including DNA repair, growth arrest, apoptosis and antiangiogenesis. Recently, we showed that clathrin heavy chain (CHC), which was originally identified as a cytosolic protein regulating endocytosis, is present in nuclei and functions as a coactivator for p53. Here, we determined the detailed p53-binding site of CHC and a CHC deletion mutant containing this region (CHC833-1406) behaved as a monomer in cells. Monomeric CHC833-1406 still had a higher ability to transactivate p53 than wild-type CHC although this CHC mutant no longer had endocytic function. Moreover, similar to wild-type CHC, monomeric CHC enhances p53-mediated transcription through the recruitment of histone acetyltransferase p300. Immunofluorescent microscopic analysis exhibited that CHC833-1406 is predominantly localized in nuclei, suggesting that there may be a certain regulatory domain for nuclear export in the C-terminus of CHC. Thus, the trimerization domain of CHC is not necessary for the transactivation of p53 target genes and these data provide further evidence that nuclear CHC plays a role distinct from clathrin-mediated endocytosis.
Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Cadeias Pesadas de Clatrina/fisiologia , Transcrição Gênica , Ativação Transcricional , Proteína Supressora de Tumor p53/fisiologia , Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/farmacologia , Dimerização , Endocitose/efeitos dos fármacos , Humanos , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Análise de Sequência de Proteína , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The initiation of cardiopulmonary bypass creates significant derangements in cardiovascular volume status and both endocrine and autonomic nervous system function. To examine whether such derangements might differ in patients with different pre-operative physical status scores, we measured the plasma concentrations of calcitonin gene-related peptide, atrial natriuretic peptide and brain natriuretic peptide, catecholamines and antidiuretic hormone, as well as haemodynamic variables, during and after cardiopulmonary bypass in 27 consecutive patients undergoing coronary artery bypass grafting. The pre-operative levels of atrial natriuretic peptide and brain natriuretic peptide differed significantly between ASA II patients and III and IV patients [mean (SD) brain natriuretic peptide levels = 14 (8.2) vs. 129 (51) pg.ml-1]. Plasma calcitonin gene-related peptide increased significantly in both groups after the initiation of cardiopulmonary bypass, and remained increased throughout cardiopulmonary bypass. The changes in plasma epinephrine, norepinephrine and antidiuretic hormone were similar to those reported previously. The changes in plasma calcitonin gene-related peptide, atrial natriuretic peptide and brain natriuretic peptide did not correlate with any changes in haemodynamic variables before or after cardiopulmonary bypass. Measurement of plasma brain natriuretic peptide might usefully be included in the pre-operative evaluation of patients with cardiac disease.
Assuntos
Fator Natriurético Atrial/sangue , Peptídeo Relacionado com Gene de Calcitonina/sangue , Ponte de Artéria Coronária , Peptídeo Natriurético Encefálico/sangue , Gasometria , Temperatura Corporal , Eletrólitos/sangue , Epinefrina/sangue , Feminino , Hematócrito , Hemodinâmica , Humanos , Masculino , Pessoa de Meia-Idade , Norepinefrina/sangue , Vasopressinas/sangueRESUMO
Several kinds of stress such as psychological stress, restraint, and foot shock inhibit feeding behavior through corticotropin-releasing factor (CRF). In contrast, a mild tail pinch increases food intake in rats. Although dopamine and opioid are thought to be involved in tail-pinch-induced food intake, it is unknown whether CRF participates in this phenomenon. Therefore, we attempted to clarify this issue using rats. A 30-s tail pinch increased food intake in 30 min after the tail pinch, and this increase was blocked by intraperitoneal injection of CRF receptor type 1 selective antagonist. CRF increased food intake in 30 min after intracerebroventricular injection at a dose of 2 or 10 ng, and this increase was also blocked by CRF receptor type 1 antagonist. Tail-pinch- or CRF-induced food intake was blocked by naloxone, pimozide, and spiperone. These results suggest that CRF, through CRF receptor type 1 as well as opioid and dopaminergic systems, are involved in the mechanism of tail-pinch-induced food intake. The results also suggest that brain CRF has dual effects on food intake, hyperphagia and anorexia, in a stress-dependent manner.
Assuntos
Hormônio Liberador da Corticotropina/farmacologia , Dopamina/fisiologia , Ingestão de Alimentos/fisiologia , Receptores Opioides/fisiologia , Estresse Fisiológico/fisiopatologia , Animais , Antagonistas de Dopamina/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Estimulação Física , Pimozida/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Wistar , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/fisiologia , Espiperona/farmacologia , CaudaRESUMO
The biological basis for the therapeutic mechanisms of depression is still unknown. We have previously performed expressed-sequence tag (EST) analysis to identify some molecular machinery responsible for antidepressant effect. Then, we developed our original cDNA microarray, on which cDNA fragments identified as antidepressant-related genes/ESTs were spotted. In this study, with this microarray followed by Western blot analysis, we have demonstrated the induction of vesicle-associated membrane protein 2(VAMP2/synaptobrevin-2) in rat frontal cortex not only after chronic antidepressant treatment, but also after repeated electroconvulsive treatment. On the other hand, expression of SNAP-25 and syntaxin-1 was not changed by these treatments. These components make a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex with VAMP2 and mediate the synaptic vesicle docking/fusion machinery. In conclusion, it is suggested that VAMP2/synaptobrevin-2 plays important roles in the antidepressant effects. Our results may contribute to a novel model for the therapeutic mechanism of depression and new molecular targets for the development of therapeutic agents.
Assuntos
Antidepressivos/farmacologia , Eletroconvulsoterapia/métodos , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Proteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Masculino , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Proteínas R-SNARE , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
UNLABELLED: We investigated whether the stereoisomers of ropivacaine and bupivacaine exert differential effects on the cerebral microcirculation. Pentobarbital-anesthetized dogs (n = 16) were prepared for measurement of cerebral pial vessel diameters by using a closed cranial window preparation. We administered three different concentrations (10(-7), 10(-5), and 10(-3) M) of each of three drug solutions [R(+), racemic, and S(-) forms of ropivacaine (n = 8) or bupivacaine (n = 8)] under the window in a randomized manner and measured cerebral pial arteriolar diameters. Various physiologic data were obtained before and after topical application of each test solution. All three forms of ropivacaine constricted cerebral pial arterioles, each in a concentration-dependent manner. The rank order for degree of vasoconstriction was S(-) ropivacaine > racemic ropivacaine > R(+) ropivacaine. In contrast, R(+) and racemic bupivacaine dilated, but S(-) bupivacaine constricted, cerebral pial arterioles, each in a concentration-dependent manner. We could find no difference in vascular reactivity to these drugs between large (> or = microm) and small (<100 microm) arterioles. Topical application of these drugs induced no changes in mean blood pressure or heart rate. The observed differences in the microvascular alterations induced by the stereoisomers of ropivacaine and bupivacaine suggest that the vasoactive effects of these drugs on cerebral arterioles could, at least in part, depend on their chirality. IMPLICATIONS: The differential effects of the stereoisomers of ropivacaine and bupivacaine on cerebral pial vessels could, at least in part, depend on their chirality.
Assuntos
Amidas/farmacologia , Anestésicos Locais/farmacologia , Bupivacaína/farmacologia , Pia-Máter/irrigação sanguínea , Animais , Arteríolas/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Cães , Frequência Cardíaca , Ropivacaina , Estereoisomerismo , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
We previously observed Ca2+ release from intracellular Ca2+ stores caused by reduction in extracellular Na+ concentration ([Na+]o). The purpose of this study was to determine whether lowering [Na+]o can elicit Ca2+ release from Ca2+ stores via the Na+/Ca2+ exchanger and to elucidate the mechanisms related to the Ca2+ release pathway in cultured longitudinal smooth muscle cells obtained from guinea pig ileum. Low [Na+]o-induced Ca2+ release was inhibited by antisense oligodeoxynucleotides for Na+/Ca2+ exchanger type 1 (anti-NCX). Application of anti-NCX to cells attenuated both the number of Ca2+ responding cells and the expression of the exchanger. Moreover, microinjection of heparin, a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, into the cells inhibited low [Na+]o-induced Ca2+ release. These findings suggest that low [Na+]o-induced Ca2+ release occurs through an IP3-induced Ca2+ release mechanism due to changes in the Ca2+ flux regulated by the Na+/Ca2+ exchanger.
Assuntos
Cálcio/metabolismo , Músculo Liso/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Atropina/farmacologia , Carbacol/farmacologia , Células Cultivadas , Colina/farmacologia , Relação Dose-Resposta a Droga , Fura-2/farmacologia , Glutamatos/farmacologia , Cobaias , Heparina/farmacologia , Histamina/farmacologia , Íleo , Immunoblotting , Masculino , Microinjeções , Oligonucleotídeos Antissenso/farmacologia , Trocador de Sódio e Cálcio/efeitos dos fármacos , Trocador de Sódio e Cálcio/genéticaAssuntos
Aminoácidos/urina , Colágeno/urina , Osteoporose/terapia , Osteoporose/urina , Peptídeos/urina , Fosfatase Alcalina/urina , Biomarcadores/urina , Reabsorção Óssea , Colecalciferol/uso terapêutico , Colágeno/química , Colágeno Tipo I/química , Difosfonatos/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Terapia de Reposição Hormonal , Humanos , Osteoporose/diagnóstico , Peptídeos/químicaRESUMO
Single smooth muscle cells (SMCs) isolated from guinea pig ileum using collagenase and papain were cultured on laminin-coated dishes in MEM containing fetal calf serum. Temporal changes in intracellular calcium ion concentration in response to carbachol and to ATP were investigated using fluo-3/AM and fluorescence microscopy. It was observed that carbachol caused an increased intracellular calcium ion in freshly isolated single SMCs but a reduced or negative response of cultured SMCs before confluence. On the other hand, ATP was observed to cause an increase in the calcium ion content of SMCs throughout the culture. SDS-PAGE and Western blot analyses revealed changes in the expression of contractile proteins as follows. l-Caldesmon and non-muscle type myosin heavy chain (NMHC) (considered to be marker molecules for dedifferentiation in smooth muscle cells) and non-muscle type tropomyosin were not observed in freshly isolated single SMCs. l-Caldesmon and NMHC appeared in the cultured SMCs within 2 days and the tropomyosin isoform was observed 6 days following seeding. Simultaneously, smooth muscle type myosin heavy chain (SMHC) decreased strikingly and the 41 kDa tropomyosin monomer was lost. The content of alpha-actin decreased gradually to a minimum on day 6 when non-muscle type tropomyosin appeared, and the cells began to proliferate rapidly. These results suggest that the loss of contractility in cultured smooth muscle cells is more closely related to changes in contractile protein profiles than to receptor-mediated signal transduction and that in addition to NMHC and l-caldesmon, non-muscle type tropomyosin may be useful as a marker molecule for de-differentiation of smooth muscle cells.
Assuntos
Sinalização do Cálcio , Proteínas Musculares/metabolismo , Músculo Liso/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Carbacol/farmacologia , Divisão Celular , Células Cultivadas , Cobaias , Íleo , Líquido Intracelular/metabolismo , Masculino , Músculo Liso/citologia , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas/metabolismo , Tropomiosina/metabolismoRESUMO
The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on the release of Ca(2+) from intracellular stores were investigated in isolated rat hepatocytes. Isolated hepatocytes permeabilized with digitonin were suspended in solution, and the concentration of extracellular Ca(2+) was measured, using a fluorescent Ca(2+) dye, fura-2. In the solution containing permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min, the extracellular Ca(2+) concentration was high, but the inositol 1,4,5-trisphosphate (InsP(3))-induced increase in Ca(2+) concentration was suppressed, suggesting that the extracellular release of Ca(2+) in response to TBT treatment was from intracellular stores. Images of the Ca(2+) concentration in the intracellular stores of primary cultured hepatocytes loaded with fura-2 were obtained after digitonin-permeabilization, using digitalized fluorescence microscopy. The permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min had a very low fura-2 fluorescence ratio (340/380 nm), suggesting that stored Ca(2+) was released. When the hepatocytes were treated with 4.0 microM TBT after digitonin-permeabilization, the decrease in the fura-2 fluorescence ratio was very small. However, when the permeabilized hepatocytes were incubated with 4.0 microM TBT and 2.0 microM NADPH, the decrease was enhanced, raising the possibility that TBT might be metabolized to the active form(s), thus releasing Ca(2+) from intracellular stores. When the hepatocytes were preincubated with 0.1 microM TBT for 30 min and then were permeabilized, the fura-2 fluorescence ratio was almost the same as that in the control permeabilized hepatocytes. However, the InsP(3)-induced decrease in the fluorescence ratio was suppressed significantly in the permeabilized hepatocytes. These results suggest that TBT released Ca(2+) from the intracellular stores at high concentrations, and suppressed the InsP(3)-induced Ca(2+) release at non-toxic low concentrations. It is probable that the latter effect was responsible for the previously reported suppression of Ca(2+) response induced by hormonal stimulations (Kawanish et al., Toxicol Appl Pharmacol 1999;155:54-61).
Assuntos
Cálcio/metabolismo , Hepatócitos/efeitos dos fármacos , Compostos de Trialquitina/farmacologia , Animais , Compartimento Celular , Permeabilidade da Membrana Celular , Células Cultivadas , Interações Medicamentosas , Fluorescência , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Hepatócitos/metabolismo , Masculino , Fosfotransferases (Aceptor do Grupo Álcool)/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
To evaluate the effects of ketamine on cerebral arterioles, we used a closed cranial window technique in mechanically ventilated, anesthetized dogs. Fourteen dogs were assigned to one of the following two basal-anesthesia groups: pentobarbital 2 mg. kg(-1). h(-1) or isoflurane 0.5 MAC (n = 7 each). We administered three different concentrations of ketamine (10(-7), 10(-5), and 10(-3) M) under the window and measured arteriolar diameters. For comparison, in another 14 dogs we examined the effect of systemic (IV) ketamine (1 mg/kg and 5 mg/kg) using the same two basal anesthetics. We measured diameters before and after ketamine administration, and we evaluated the effect of ketamine on CO(2) reactivity of the cerebral arterioles. Neither topical nor systemic ketamine dilated pial arterioles in either basal-anesthesia group. CO(2) reactivity of pial arterioles was reduced under systemic ketamine in both basal-anesthesia groups. The results indicate that although ketamine does not dilate pial arteriolar diameters when topically or IV administered, IV ketamine does attenuate hypercapnic vasodilation in dogs under basal pentobarbital or isoflurane anesthesia. These results provide some insight that ketamine is suitable for supplementary neurosurgical anesthesia.
Assuntos
Adjuvantes Anestésicos/farmacologia , Anestésicos Dissociativos/farmacologia , Anestésicos Inalatórios/farmacologia , Artérias Cerebrais/efeitos dos fármacos , Isoflurano/farmacologia , Ketamina/farmacologia , Pentobarbital/farmacologia , Administração Tópica , Anestésicos Dissociativos/administração & dosagem , Animais , Arteríolas/efeitos dos fármacos , Gasometria , Cães , Hemodinâmica/efeitos dos fármacos , Ketamina/administração & dosagemRESUMO
Using real-time confocal microscopy, we have demonstrated that lysophosphatidic acid (LPA), a bioactive phospholipid existing in plasma, positively regulates fluid flow-induced [Ca(2+)](i) response in fluo 4-loaded, cultured, bovine aortic endothelial cells. The initial increase in [Ca(2+)](i) was localized to a circular area with a diameter of <4 microm and spread concentrically, resulting in a mean global increase in [Ca(2+)](i). The local increase often occurred in a stepwise manner or repetitively during constant flow. The percentage of cells that responded and the averaged level of increase in [Ca(2+)](i) were dependent on both the concentration of LPA (0.1 to 10 micromol/L) and the flow rate (25 to 250 mm/s). The response was inhibited by removing extracellular Ca(2+) or by the application of Gd(3+), an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticular Ca(2+)-ATPASE: It was also inhibited by 8-bromo-cGMP, and the inhibition was completely reversed by KT5823, an inhibitor of protein kinase G (PKG). These results suggest that the [Ca(2+)](i) response arises from Ca(2+) influx through Gd(3+)-sensitive MS channels, which are negatively regulated by the activation of PKG. The spatiotemporal properties of the [Ca(2+)](i) response were completely different from those of a Ca(2+) wave induced by ATP, a Ca(2+)-mobilizing agonist. Therefore, we called the phenomenon Ca(2+) spots. We conclude that LPA positively regulates fluid flow-induced local and oscillatory [Ca(2+)](i) increase, ie, the Ca(2+) spots, in endothelial cells via the activation of elementary Ca(2+) influx through PKG-regulating MS channels. This indicates an important role for LPA as an endogenous factor in fluid flow-induced endothelial function.
Assuntos
Cálcio/metabolismo , Endotélio Vascular/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Trifosfato de Adenosina/farmacologia , Compostos de Anilina/farmacologia , Animais , Cálcio/farmacologia , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Corantes Fluorescentes/farmacologia , Gadolínio/farmacologia , Microscopia Confocal , Estresse Mecânico , Tapsigargina/farmacologia , Fatores de Tempo , Xantenos/farmacologiaRESUMO
We investigated the prevalence and characteristics of ischemic heart disease especially silent myocardial ischemia (SMI) and arrhythmia in need of careful observation in the exercise stress tests in the Total Health Promotion Plan (THP), which was conducted between 1994-96 for the purpose of measuring cardiopulmonary function. All workers (n = 4,918, 4,426 males) aged 18-60 yr old in an occupational field were studied. Exercise tests with an ergometer were performed by the LOPS protocol, in which the maximal workload was set up as a presumed 70-80% maximal oxygen intake, or STEP (original multistage protocol). ECG changes were evaluated with a CC5 lead. Two hundred and fifteen people refused the study because of a common cold, lumbago and so on. Of 4,703 subjects, 17 with abnormal rest ECG and 19 with probable anginal pain were excluded from the exercise tests. Of 4,667 who underwent the exercise test, 37 (0.79%) had ischemic ECG change, and 155 (3.32%) had striking arrhythmia. These 228 subjects then did a treadmill exercise test with Bruce protocol. Twenty-two (0.47% of 4,703) showed positive ECG change, 9 (0.19%) of 22 had abnormal findings on a 201Tl scan. 8 (0.17%) were diagnosed as SMI (Cohn I), in which the prevalence of hypertension, hyperlipidemia, diabetes mellitus, smoker and positive familial history of ischemic heart disease was greater than that of all subjects. In a 15-30 month follow up, none has developed cardiac accidents. Exercise-induced arrhythmia was detected in 11 (0.23%) subjects. Four were non-sustained ventricular tachycardia without any organic disease, 4 were ventricular arrhythmia based on cardiomyopathy detected by echocardiography, 2 were atrial fibrillation and another was WPW syndrome. It is therefore likely that the ergometer exercise test in THP was effective in preventing sudden death caused by ischemic heart disease or striking arrhythmia.
Assuntos
Arritmias Cardíacas/diagnóstico , Teste de Esforço , Isquemia Miocárdica/diagnóstico , Serviços de Saúde do Trabalhador , Adolescente , Adulto , Arritmias Cardíacas/complicações , Arritmias Cardíacas/etiologia , Morte Súbita Cardíaca/prevenção & controle , Eletrocardiografia , Teste de Esforço/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/complicações , Consumo de Oxigênio , PrevalênciaRESUMO
Local increases in the intracellular Ca2+ concentration ([Ca2+]i) in several regions within the bovine lens epithelial cell during application of mechanical stress were clearly visualized in the presence of lysophosphatidic acid (LPA), a bioactive lysophospholipid, using real-time confocal microscopy. We called the phenomenon 'Ca2+ spots'. Ca2+ spots started in a circular area with a radius of about 1.5 m. These Ca2+ spots spread concentrically, resulting in a mean global increase in [Ca2+]i. The local increase often occurred in a stepwise manner or repetitively at the same region. The spatiotemporal properties of the Ca2+ spots were completely different from those of the Ca2+ wave induced by ATP, a Ca2+-mobilizing agonist. Ca2+ spots were inhibited by decreasing the extracellular Ca2+ concentration or by the presence of Gd3+, an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, suggesting that Ca2+ spots arise from Ca2+ influx through Gd3+-sensitive MS channels. On the assumption that, in lens epithelial cells, the open probability of the MS channel is 0.4, the membrane potential is 56 mV and the channel conductance is 50 pS, the estimated maximum flux of Ca2+ in a Ca2+ spot (0.4 x 10-17 to 4.7 x 10-17 mol x s(-1)) was comparable to currents of one or a few MS channels. On real-time three-dimensional confocal imaging analysis, which permitted simultaneous imaging of basal and apical planes of cells at 37.6 ms intervals, Ca2+ spots on the apical plane were more clearly visualized than those on the basal plane. From these results, we propose that the Ca2+ spot is an elementary Ca2+-influx event through MS channels directly coupled with the first step in mechanoreception In addition, our results strongly suggest that LPA functions as an endogenous factor affecting mechanotransduction systems.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Cápsula do Cristalino/metabolismo , Lisofosfolipídeos/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Gadolínio/farmacologia , Cinética , Cápsula do Cristalino/citologia , Cápsula do Cristalino/efeitos dos fármacos , Microscopia Confocal , Estresse Mecânico , Tapsigargina/farmacologia , Fatores de TempoRESUMO
Changes in intracellular calcium concentration ([Ca2+]i) and focal adhesion sites of cultured bovine aortic endothelial cells (BAECs) were simultaneously visualized in real time. Local [Ca2+]i transients were observed at the rear edges of spontaneously migrating BAECs. Furthermore, the majority of starting regions of [Ca2+]i transients retracted continuously. Frequency of [Ca2+]i transients increased with the application of fluid flow. The majority of starting regions of flow-induced [Ca2+]i transients retracted following the occurrence of [Ca2+]i transients. In addition, retracted areas were distributed in the upstream regions of the cell. Application of GdCl3, a mechanosensitive cation channel blocker, resulted in a clear reduction of [Ca2+]i transients and rear retractions in cases of spontaneous and flow-induced BAEC migration. Flow-induced directional rear retractions were also inhibited. Consequently, we conclude that local [Ca2+]i transients play an important role in the migration of BAECs with respect to rear retraction. Furthermore, flow-induced [Ca2+]i transients regulate directional rear retraction under flow conditions.
Assuntos
Cálcio/metabolismo , Endotélio Vascular/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Cátions , Bovinos , Adesão Celular , Movimento Celular , Células Cultivadas , Endotélio Vascular/citologia , Guanidina/farmacologia , Microscopia Confocal , Fatores de TempoRESUMO
STUDY OBJECTIVES: To investigate the effects of ketamine and propofol on the cerebrovascular response to carbon dioxide (CO(2)) in humans during isoflurane anesthesia. DESIGN: Randomized clinical investigation. SETTINGS: University hospital of a medical school. PATIENTS: 30 ASA physical status I and II adult, elective surgical patients. INTERVENTIONS AND MEASUREMENTS: With each patient given air/oxygen/isoflurane anesthesia, the flow velocity in the middle cerebral artery (Vmca) and pulsatility index were measured using the transcranial Doppler method under hypocapnic [arterial CO(2)tension (PaCO(2)) 28-32 mmHg], normocapnic (PaCO(2) 38-42 mmHg), and hypercapnic conditions (PaCO(2) 48-52 mmHg). PaCO(2) was altered by supplementing the inspired gas with CO(2) without changing the respiratory conditions. Patients were then randomly assigned to receive either ketamine 1 mg. kg(-1) or propofol (2 mg. kg(-1)followed by an infusion of 6-10 mg. kg(-1). hr(-1)) (n = 15 for each drug), and the measurements were repeated. MAIN RESULTS: Ketamine reduced both absolute and relative cerebrovascular reactivity to CO(2) significantly [2.9 +/- 0.8 (control) vs. 2.6 +/- 1.0 (ketamine) cm. sec(-1). mmHg(-1): p < 0.05; and 3.5 +/- 0.7 (control) vs. 2.8 +/- 0.9 (ketamine) %. mmHg(-1): p < 0.01, respectively]. However, ketamine did not reduce Vmca during hypercapnic conditions (117 +/- 29 cm. sec(-1)) compared with controls (120 +/- 28 cm. sec(-1)). Although propofol decreased Vmca during all conditions, it did not cause any change in either absolute or relative CO(2) reactivity [2.5 +/- 0.8 (control) vs. 2.5 +/- 1.0 (propofol) cm. sec(-1). mmHg(-1), and 3.3 +/- 1.3 (control) vs. 4.1 +/- 1.0 (propofol) %. mmHg(-1), respectively]. CONCLUSIONS: In humans given isoflurane anesthesia, a) ketamine reduced cerebrovascular response to CO(2), but cerebral blood flow (CBF) during hypercapnic conditions was comparable with controls, and b) although propofol decreases CBF, it maintains the cerebrovascular response to CO(2).
Assuntos
Anestesia por Inalação , Anestésicos Inalatórios , Anestésicos Intravenosos/farmacologia , Dióxido de Carbono/sangue , Circulação Cerebrovascular/efeitos dos fármacos , Isoflurano , Ketamina/farmacologia , Propofol/farmacologia , Adulto , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Masculino , Artéria Cerebral Média/fisiologia , N-Metilaspartato/antagonistas & inibidoresRESUMO
The mechanotransduction mechanism is believed to play an important role in maintenance of cellular homeostasis in a wide variety of cell types. In particular, the mechanotransduction system in vascular endothelial cells may be an essential mechanism for local hemodynamic control. Elevations in intracellular free Ca2+ concentration ([Ca2]i) are an important signal in the initial step of mechanotransduction and mechanosensitive (MS) cation channels are thought to be a putative pathway; however, the molecular mechanisms remain unclear. We found that lysophosphatidic acid (LPA), a bioactive phospholipid, sensitizes the response of [Ca2+]i to mechanical stress in several cell types. Employing real-time confocal microscopy, local increases in [Ca2+]i in several regions within the cell during application of mechanical stress were clearly visualized in bovine lens epithelial and endothelial cells in the presence of LPA. The phenomenon was termed "Ca2+ spots". Pharmacological studies revealed that Ca2+ spots arise due to influx through MS channels. In this report, our data indicating the possible significance of LPA as an endogenous factor involved in regulation of mechanotransduction is reviewed. Furthermore, our findings suggest that the Ca2+ spot is a novel phenomenon occurring as an elementary Ca2+-influx event through MS channels directly coupled with the initial step in mechanotransduction.
Assuntos
Cálcio/metabolismo , Lisofosfolipídeos/fisiologia , Estresse Mecânico , Animais , Endotélio Vascular/citologia , Humanos , Lisofosfolipídeos/farmacologiaRESUMO
Inhalation of lysophosphatidic acid (LPA, 1-100 microg/ml) for 2 min enhanced the airway response induced by intravenous injection of ACh in guinea pigs. At 30 min after inhalation of LPA, the airway response to ACh was two fold higher than that before inhalation. This enhancement of airway response to ACh was partially inhibited by capsaicin desensitization or bilateral vagotomy. These results suggested that the enhancement of airway response to ACh induced by LPA may be due to the activation of capsaicin-sensitive fibers. It can be also contribute to bronchial asthma or other types of pulmonary disease such as cough variant asthma and atopic cough.
Assuntos
Acetilcolina/farmacologia , Alérgenos/farmacologia , Hiper-Reatividade Brônquica/induzido quimicamente , Lisofosfolipídeos/farmacologia , Acetilcolina/administração & dosagem , Administração por Inalação , Obstrução das Vias Respiratórias/induzido quimicamente , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Testes de Provocação Brônquica , Capsaicina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Cobaias , Injeções Intravenosas , Lisofosfolipídeos/administração & dosagem , Masculino , VagotomiaAssuntos
Cistos/patologia , Hepatopatias/patologia , Omento/patologia , Idoso , Cistos/microbiologia , Humanos , Infecções por Klebsiella/patologia , Klebsiella pneumoniae/isolamento & purificação , Hepatopatias/microbiologia , Masculino , Infecções Estreptocócicas/patologia , Streptococcus/isolamento & purificaçãoRESUMO
Urocortin (UCN), a member of the corticotropin-releasing factor (CRF) family, inhibits food intake when it is injected intracerebroventricularly in rats. To explore the site of action of UCN in feeding behavior, we examined the effects of injection of UCN into various hypothalamic nuclei on food and water intake in 24-h fasted rats. Injection of UCN into the ventromedial hypothalamic nucleus (VMH) significantly inhibited food and water intake over 3 h without sedative effect, but no significant effect was observed following injection either into the lateral hypothalamic area, or the paraventricular nucleus of the hypothalamus. To further explore the physiological significance of endogenous UCN of the VMH in feeding behavior, the effect of immunoneutralization of hypothalamic UCN on food intake was examined. Injection of anti-rat UCN rabbit gamma-globulin into the bilateral VMH in freely fed rats significantly potentiated food and water intake compared with rats that received normal rabbit gamma-globulin. These results suggest that endogenous UCN in the VMH exert inhibitory control on ingestive behavior.
Assuntos
Hormônio Liberador da Corticotropina/farmacologia , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Núcleo Hipotalâmico Ventromedial/efeitos dos fármacos , Animais , Hormônio Liberador da Corticotropina/fisiologia , Ingestão de Líquidos/fisiologia , Ingestão de Alimentos/fisiologia , Comportamento Alimentar/fisiologia , Masculino , Coelhos , Ratos , Ratos Wistar , Urocortinas , Núcleo Hipotalâmico Ventromedial/fisiologia , gama-Globulinas/farmacologiaRESUMO
In the epileptic hippocampus, newly sprouted mossy fibers are considered to form recurrent excitatory connections to granule cells in the dentate gyrus and thereby increase seizure susceptibility. To study the effects of mossy fiber sprouting on neural activity in individual lamellae of the dentate gyrus, we used high-speed optical recording to record signals from voltage-sensitive dye in hippocampal slices prepared from kainate-treated epileptic rats (KA rats). In 14 of 24 slices from KA rats, hilar stimulation evoked a large depolarization in almost the entire molecular layer in which granule cell apical dendrites are located. The signals were identified as postsynaptic responses because of their dependence on extracellular Ca(2+). The depolarization amplitude was largest in the inner molecular layer (the target area of sprouted mossy fibers) and declined with increasing distance from the granule cell layer. In the inner molecular layer, a good correlation was obtained between depolarization size and the density of mossy fiber terminals detected by Timm staining methods. Blockade of GABAergic inhibition by bicuculline enlarged the depolarization in granule cell dendrites. Our data indicate that mossy fiber sprouting results in a large and prolonged synaptic depolarization in an extensive dendritic area and that the enhanced GABAergic inhibition partly masks the synaptic depolarization. However, despite the large dendritic excitation induced by the sprouted mossy fibers, seizure-like activity of granule cells was never observed, even when GABAergic inhibition was blocked. Therefore, mossy fiber sprouting may not play a critical role in epileptogenesis.