RESUMO
This study determined the role of the reverse mode Na(+)/Ca(2+) exchanger (NCX) in cardioprotection of metabolic inhibition preconditioning in isolated ventricular myocyctes. Activity of the reverse mode NCX was assessed by changes of [Ca(2+)](i) upon withdrawal of extracellular Na(+). [Ca(2+)](i) was measured by spectrofluorometry, using Fura-2 as Ca(2+) indicator. The amplitude of contraction and exclusion of trypan blue by myocytes served as indices of contractile function and viability, respectively. Firstly, NCX activity significantly decreased during simulated reperfusion after severe metabolic inhibition (index ischaemia) in myocytes subjected to metabolic inhibition preconditioning. This inhibitory effect on NCX activity correlated with the enhancing effect of metabolic inhibition preconditioning on cell viability following ischaemic insult. Treatment myocytes with E4031, an activator of reverse mode NCX, during index ischaemia and reperfusion attenuated the enhancing effects of metabolic inhibition preconditioning on cell contraction and viability. Secondly, NCX activity was significantly higher at the end of metabolic inhibition preconditioning. More importantly, E4031 pretreatment mimicked the beneficial effects of metabolic inhibition preconditioning in myocytes and ischaemic preconditioning in the isolated perfused heart, respectively, and these effects were abolished by KB-R7943, an inhibitor of reverse mode NCX. The results indicate that increased reverse mode NCX activity during preconditioning triggered cardioprotection, and reduced reverse mode NCX activity during reperfusion after index ischaemia conferred cardioprotection.
Assuntos
Cálcio/metabolismo , Precondicionamento Isquêmico Miocárdico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/fisiologia , Animais , Antiarrítmicos/farmacologia , Sobrevivência Celular , Fura-2/análise , Masculino , Contração Miocárdica/efeitos dos fármacos , Isquemia Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Miócitos Cardíacos/efeitos dos fármacos , Piperidinas/farmacologia , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Trocador de Sódio e Cálcio/antagonistas & inibidores , Espectrometria de Fluorescência , Tioureia/análogos & derivados , Tioureia/farmacologia , Função Ventricular/efeitos dos fármacos , Função Ventricular/fisiologiaRESUMO
AIM: To investigate the relaxation effect and underlying mechanism of U50,488H (a selective kappa-opioid receptor agonist) in pulmonary artery in the rat. METHODS: Isolated pulmonary artery ring was perfused and the tension of the vessel was measured. RESULTS: U50,488H relaxed the pulmonary artery ring in a dose-dependent manner and the effect was abolished by nor-binaltorphimine, a selective kappa-opioid receptor antagonist. The relaxation effect of U50,488H in pulmonary artery was partially endothelium-dependent and was significantly attenuated in the presence of L-NAME. The relaxation effect of U50,488H was significantly attenuated by K(V) channel blocker 4-AP (4-aminopyridine), but not by glibenclamide (ATP-sensitive K+ channel blocker) nor TEA (tetraethylamonium, Ca2+-activated K+ channel blocker). Further study also showed that endothelium denudation and 4-AP have an additive inhibitory effect on pulmonary artery relaxation caused by U50,488H. CONCLUSION: Kappa-opioid receptor activation by U50,488H relaxes pulmonary artery via two separate pathways: one is endothelium-derived nitric oxide, the other is K(V) channel in pulmonary artery smooth muscle.
Assuntos
(trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Anti-Hipertensivos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Atropina/farmacologia , Dinorfinas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Indometacina/farmacologia , Masculino , Relaxamento Muscular/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/fisiologia , Propranolol/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides kappa/agonistasRESUMO
The purpose of this study was to investigate the different effects of ACh on the action potential and force contraction in guinea pig atrial and ventricular myocardium by using standard microelectrodes and force transducer. The results showed that the duration of the action potential (APD) of atrial myocardium was shortened from 208.57+/-36.05 to 101.78+/-14.41 ms (n=6, P<0.01), and the APD of the ventricular myocardium was shortened from 286.73+/-36.11 to 265.16+/-30.06 ms (n=6, P<0.01).The amplitude of the action potential (APA) of the atrial myocardium was decreased from 88.00+/-9.35 to 62.62+/-20.50 mV (n=6, P<0.01), while the APA of the ventricular myocardium did not change significantly.The force contraction of atrial myocardium was inhibited completely (n=6, P<0.01), while the force contraction of ventricular myocardium was inhibited by 37.57+/-2.58% (n=6, P<0.01). The ACh effects correlated with its concentration. The K(D) of the APD shortening effects in the atrial and ventricular myocardium were 0.275 and 0.575 micromol/L. The K(D) of the negative inotropic in the atrial and ventricular myocardium were 0.135 and 0.676 micromol/L, respectively. The corresponding data points were compared using t test between the atrial and ventricular myocardium, and the differences were significant when the ACh concentration was above 10 nmol/L. Furthermore, atropine (10 micromol/L) and CsCl (20 mmol/L) blocked the effects of 10 micromol/L ACh on the APD of ventricular myocardium, while CdCl2 (0.1 mmol/L) had no influence on these effects. In conclusion, ACh could shorten the action potential duration and inhibit the force contraction of atrial and ventricular myocardium in a concentration-dependent manner. There are differences in the effects of ACh on the atrial and ventricular myocardium. The atrial myocardium is more sensitive to ACh than the ventricular myocardium. It is probable that the muscarinic receptor and the potassium channel, but not the calcium channel, are involved in the ACh-induced shortening of the ventricular APD.
Assuntos
Acetilcolina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Átrios do Coração/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Cobaias , Microeletrodos , Canais de Potássio/metabolismo , Receptores Muscarínicos/metabolismoRESUMO
Using whole-cell patch clamp technique this study investigated the effects of adenosine (Ado) on action potential, L-type calcium current (I(Ca.L)), delayed afterdepolarizations (DADs), and transient inward current (I(ti)) induced by isoproterenol (Iso) in guinea pig isolated single ventricular myocytes. The results showed: (1) Ado alone had no significant direct effects on action potential and I(Ca.L) in guinea pig ventricular myocytes at 20-100 micromol/L. However, Ado significantly attenuated the prolongation of action potential duration (APD) and the increase of the peak amplitude of I(Ca.L) induced by Iso. Iso (10 nmol/L) markedly increased APD(50) and APD(90) from 340+/-21 ms to 486+/-28 ms and from 361+/-17 ms to 501+/-29 ms, respectively (P<0.01), and increased the amplitude of I(Ca.L) from 6.53+/-1.4 pA/pF to 18.28+/-2.4 pA/pF (P<0.01). The peak potential of current-potential relationship shifted to the left. Ado (50 micromol/L) abbreviated APD(50), APD(90) to 403+/-19 ms and 419+/-26 ms (P<0.01), and decreased the peak amplitude of I(Ca.L) to 10.2+/-1.5 pA/pF (P<0.01 vs Iso), but did not change resting membrane potential (RMP), action potential amplitude (APA), and overshoot (OS). (2) Iso (30 nmol/L) reproducibly elicited DADs with 100% incidence of DADs under this condition. Ado (50 micromol/L) completely inhibited Iso from inducing DADs. Iso (30 nmol/L) elicited I(ti) with 2-second depolarizing voltage-clamp pulses rising to +20 mV from a holding potential of -40 mV, the incidence of I(ti) being 100%, and the I(ti) was suppressed in the presence of Ado (50 micromol/L) with the incidence of I(ti) decreased to 14.3% (P<0.05). These data indicate that Ado antagonizes the stimulatory effect of Iso, and that the antiarrhythmic mechanism of Ado preventing Iso-induced DADs is due to the inhibition of intracellular Ca(2+) overload through attenuating the prolongation of APD, the enhance of I(Ca.L) and I(ti).
Assuntos
Potenciais de Ação/efeitos dos fármacos , Adenosina/farmacologia , Antiarrítmicos/farmacologia , Isoproterenol/antagonistas & inibidores , Miócitos Cardíacos/fisiologia , Animais , Arritmias Cardíacas/fisiopatologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Feminino , Cobaias , Ventrículos do Coração/citologia , Masculino , Técnicas de Patch-ClampRESUMO
The gating mechanism of ClC-1 chloride channel was studied in this paper by heteroexpression of rat wild type ClC-1 gene in Xenopus oocytes and by two-electrode voltage clamping technique. The deactivation gating kinetic parameters were obtained by applying two exponential fitting of the deactivating currents at various extracellular chloride concentrations. It was found that decrease in extracellular chloride concentration increased the fractional amplitude of fast deactivating component, and depressed the fractional amplitude of slow deactivating component accompanied by a decrease in fast and slow deactivating time constants. These results demonstrate that the deactivation kinetic parameters of ClC-1 are largely dependent on the extracellular chloride concentration, which induces changes in channel gating.
Assuntos
Canais de Cloreto/efeitos dos fármacos , Cloretos/farmacologia , Animais , Canais de Cloreto/fisiologia , Eletrofisiologia , Feminino , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Oócitos/fisiologia , Ratos , XenopusRESUMO
BACKGROUND: HER-2 overexpression is an independent predictor for poor prognosis of breast cancer patients. Recently, extracellular domain of HER-2 (ECD) was found detectable in the serum of breast cancer patients. In this prospective study, we wonder whether ECD levels predict the clinical outcome of metastatic breast cancer patients. METHODS: ECD were measured in 190 women with metastatic breast cancer. Chi-square test was performed to determine the relationship between ECD status and clinical outcomes. Kaplan-Meier curves were applied for survival analysis. RESULTS: Elevated ECD levels were significantly associated with short-term response to Herceptin treatment. The median PFS was significantly longer in ECD-Low patients. The patients who remained low ECD levels or achieved low ECD levels after treatments have significantly longer PFS than those whose levels remained high or converted from low to high. CONCLUSIONS: Overall, our results support the clinical utility of measuring serum HER2 ECD levels in patients with advanced breast cancer. Baseline and serial measurements of serum ECD levels are reliably predictive of clinical outcome of breast cancer patients.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Receptor ErbB-2/sangue , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Prognóstico , Estrutura Terciária de Proteína , TrastuzumabRESUMO
AIM: To observe the regulatory volume decrease (RVD) process of human intestine cells and investigate its ion channel mechanism. METHODS: Cultured human intestine cells were exposed to hypotonic solution and the cell volume was measured using Coulter Counter System. RT-PCR was explored to detect the mRNA expression of Ca(2+) -activated K+ channel. RESULTS: Human intestine cells showed a RVD process and this process could be blocked by Cl- channel blocker NPPB and K+ channel blocker TEA. Further results demonstrated the subtype of K+ channel involved in RVD was an intermediate-conductance, Ca(2+) -activated K+ channel (IK) because of its high sensitivity to clotrimazole. RT-PCR results also showed the expression of IK in this cell line. CONCLUSION: The RVD process of intestine cell was dependent on the parallel activation of Cl- channel and K+ channel. The subtype of K+ channel in volved in the RVD process was IK channel.
Assuntos
Tamanho Celular/efeitos dos fármacos , Canais de Cloreto/metabolismo , Células Epiteliais/citologia , Intestino Delgado/citologia , Canais de Potássio/metabolismo , Linhagem Celular , Canais de Cloreto/antagonistas & inibidores , Humanos , Soluções Hipotônicas , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Cálcio-Ativados/metabolismoRESUMO
In this study, we determined the effect of U50,488H (a selective kappa-opioid receptor agonist) on pulmonary artery in rats and investigated its prevention and treatment effects on hypoxic pulmonary hypertension (HPH). Isolated pulmonary arterial rings were superfused and the tension of the vessel was measured. The model of HPH was developed and indexes for hemodynamics and right ventricular hypertrophy were measured. We found that U50,488H relaxed the pulmonary artery rings in a dose-dependent manner and the effect was abolished by nor-binaltorphimine, a selective kappa-opioid receptor antagonist. Intravenous administration of U50,488H significantly lowered mean pulmonary artery pressure (mPAP) in normal rats and this effect was also abolished by nor-binaltorphimine. Hypoxia induced severe HPH in rats and intraperitoneal administration of U50,488H (every other day) during chronic hypoxia reduced mPAP and attenuated right ventricular hypertrophy compared with the control group. Moreover, acute intravenous administration of U50,488H after the rats subjected to chronic hypoxia for 4 weeks significantly lowered mPAP. Thus, U50,488H has significant vasorelaxant effect in rat pulmonary artery and has certain preventive and therapeutic application in HPH.
Assuntos
(trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Hipóxia/fisiopatologia , Circulação Pulmonar/efeitos dos fármacos , Animais , Doença Crônica , Relação Dose-Resposta a Droga , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/prevenção & controle , Hipertrofia Ventricular Direita/fisiopatologia , Hipertrofia Ventricular Direita/prevenção & controle , Técnicas In Vitro , Injeções Intravenosas , Masculino , Perfusão , Artéria Pulmonar/fisiopatologia , Ratos , Ratos Sprague-DawleyRESUMO
This study was designed to examine whether physiologically tolerable insulin, which maintains lower blood glucose, can protect the myocardium against ischemia/reperfusion (I/R) injury in a preclinical large animal model. Adult dogs were subjected to 50 minutes of myocardial ischemia (80% reduction in coronary blood flow) followed by 4 hours of reperfusion and treated with vehicle, glucose-insulin-potassium (GIK; glucose, 250 g/L; insulin, 60 U/L; potassium, 80 mmol/L), GK, or low-dose insulin (30 U/L) 10 minutes before reperfusion. Treatment with GIK exerted significant cardioprotective effects as evidenced by improved cardiac function, improved coronary blood flow, reduced infarct size, and myocardial apoptosis. In contrast, treatment with GK increased blood glucose level and aggravated myocardial I/R injury. It is interesting that treatment with insulin alone at the dose that reduced blood glucose to a clinically tolerable level exerted significant cardioprotective effects that were comparable to that seen in the GIK-treated group. This low-dose insulin had no effect on coronary blood flow after reperfusion but markedly reduced coronary reactive hyperemia and switched myocardial substrate uptake from fat to carbohydrate. Our results suggest that lower glucose supply to the ischemic myocardium at early reperfusion may create a "metabolic postconditioning" and thus reduce myocardial ischemia/reperfusion injury after prolonged reperfusion.
Assuntos
Insulina/uso terapêutico , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Glicemia/fisiologia , Circulação Coronária/efeitos dos fármacos , Circulação Coronária/fisiologia , Creatina Quinase/sangue , Cães , Relação Dose-Resposta a Droga , Feminino , Glucose/administração & dosagem , Glucose/uso terapêutico , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/uso terapêutico , Marcação In Situ das Extremidades Cortadas , Infusões Intravenosas , Insulina/administração & dosagem , L-Lactato Desidrogenase/sangue , Masculino , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Potássio/administração & dosagem , Potássio/uso terapêutico , Fatores de Tempo , Função Ventricular Esquerda/efeitos dos fármacos , Função Ventricular Esquerda/fisiologiaRESUMO
AIM: To investigate the fundamental nature of calcium release events (Ca2+ "sparks") evoked in rat ventricular myocytes during excitation-contraction (E-C) coupling. METHODS: High-resolution line-scan confocal imaging with the fluorescent calcium indicator and patch-clamp techniques were used to study the spontaneous Ca2+ sparks and sparks evoked by depolarization. RESULTS: 1) Line scans oriented along the length of the cell showed that both spontaneous sparks and sparks evoked by depolarization to -35 mV appeared to arise at single sites spacing about 1.8 microm apart (ie, the sarcomere length), and measurements of their longitudinal spread (full-width at half-maximal amplitude: FWHM) followed single Gaussian distributions with means of 2.6 microm. 2) Different to this, transverse line scans often revealed spontaneous and evoked sparks that appeared to arise near-synchronously from paired sites. Measurements of transverse FWHM of both spontaneous and evoked sparks showed bimodal distributions, which were fit well by the sums of two Gaussian curves with means of 1.8 and 2.9 microm for spontaneous sparks and with means of 1.9 and 3.1 microm for evoked sparks. Relative areas under the two Gaussian curves were 1.73:1 and 1.85:1, respectively, for spontaneous and evoked sparks. CONCLUSIONS: Ca2+ sparks evoked by depolarization are not "unitary" events, but often involve multiple sites of origin along Z-lines, as previously shown for spontaneous sparks. Thus, Ca2+ released during sparks directly triggered by influx through L-type Ca2+ channels may, in turn, trigger neighboring sites. The restricted involvement of only a few transverse release sites preserves the essential feature of the "local control" theory of E-C coupling.