RESUMO
Laccase (LAC) is a blue multicopper oxidase that contains four copper ions, which is involved in lignin polymerization and flavonoid biosynthesis in plants. Although dozens of LAC genes have been identified in Salvia miltiorrhiza Bunge (a model medicinal plant), most have not been functionally characterized. Here, we explored the expression patterns and the functionality of SmLAC25 in S. miltiorrhiza. SmLAC25 has a higher expression level in roots and responds to methyl jasmonate, auxin, abscisic acid, and gibberellin stimuli. The SmLAC25 protein is localized in the cytoplasm and chloroplasts. Recombinant SmLAC25 protein could oxidize coniferyl alcohol and sinapyl alcohol, two monomers of G-lignin and S-lignin. To investigate its function, we generated SmLAC25-overexpressed S. miltiorrhiza plantlets and hairy roots. The lignin content increased significantly in all SmLAC25-overexpressed plantlets and hairy roots, compared with the controls. However, the concentrations of rosmarinic acid and salvianolic acid B decreased significantly in all the SmLAC25-overexpressed lines. Further studies revealed that the transcription levels of some key enzyme genes in the lignin synthesis pathway (e.g., SmCCR and SmCOMT) were significantly improved in the SmLAC25-overexpressed lines, while the expression levels of multiple enzyme genes in the salvianolic acid biosynthesis pathway were inhibited. We speculated that the overexpression of SmLAC25 promoted the metabolic flux of lignin synthesis, which resulted in a decreased metabolic flux to the salvianolic acid biosynthesis pathway.
Assuntos
Salvia miltiorrhiza , Salvia miltiorrhiza/genética , Salvia miltiorrhiza/metabolismo , Lignina/metabolismo , Alcenos/metabolismo , Polifenóis/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
This study aimed to explore the positive inotropic effect of phosphodiesterase type 9 (PDE9) inhibitor PF-04449613 in ratsand its cellular and molecular mechanisms. The heart pressure-volume loop (P-V loop) analysis was used to detect the effects of PF-04449613 on rat left ventricular pressure-volume relationship, aortic pressures and peripheral vessel resistance in healthy rats. The Langendorff perfusion of isolated rat heart was used to explore the effects of PF-04449613 on heart contractility. The cardiomyocyte sarcoplasmic reticulum (SR) Ca2+ transients induced by field stimulation and caffeine were used to analyze the mechanism underlying the effect of PF-04449613 using Fluo-4 AM as a Ca2+ indicator. The results indicated as follows: (1) PF-04449613 (5.5 mg/kg, ip) significantly increased the stroke work, cardiac output, stroke volume, end-systolic pressure and ejection fraction (P < 0.05), and decreased the end-systolic volume, end-diastolic volume and end-diastolic pressure (P < 0.05). Meanwhile, the systolic blood pressure was increased and diastolic blood pressure and arterial elastance were decreased after PF-04449613 treatment (P < 0.05). (2) PF-04449613 (0.001, 0.01, 0.1, 1 µmol/L) significantly increased the left ventricular developed pressure (LVDP) in a concentration-dependent manner in vitro (P < 0.05). (3) PF-04449613 (5 µmol/L) significantly increased the amplitude of SR Ca2+ transients mediated by facilitating sarcoplasmic reticulum Ca2+-ATPase-2a (SERCA2a) (P < 0.05). (4) PF-04449613 (5 µmol/L) decreased the SR Ca2+ leak rate via ryanodine receptor 2 (RyR2) (P < 0.05). In conclusion, PF-04449613 exerted positive inotropic effect both in vivo and in vitro by enhancing SERCA2a activity.
Assuntos
Cálcio , Inibidores de Fosfodiesterase , Animais , Cálcio/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Diester Fosfórico Hidrolases , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina , Retículo SarcoplasmáticoRESUMO
This study investigated the hemodynamic effect of Bay 60-7550, a phosphodiesterase type 2 (PDE2) inhibitor, in healthy rat hearts both in vivo and ex vivo and its underlying mechanisms. In vivo rat left ventricular pressure-volume loop, Langendorff isolated rat heart, Ca2+ transient of left ventricular myocyte and Western blot experiments were used in this study. The results demonstrated that Bay 60-7550 (1.5 mg/kg, i. p.) increased the in vivo rat heart contractility by enhancing stroke work, cardiac output, stroke volume, end-diastolic volume, heart rate, and ejection fraction. The simultaneous aortic pressure recording indicated that the systolic blood pressure was increased and diastolic blood pressure was decreased by Bay 60-7550. Also, the arterial elastance which is proportional to the peripheral vessel resistance was significantly decreased. Bay 60-7550 (0.001, 0.01, 0.1, 1 µmol/l) also enhanced the left ventricular development pressure in non-paced and paced modes with a decrease of heart rate in non-paced model. Bay 60-7550 (1 µmol/l) increased SERCA2a activity and SR Ca2+ content and reduced SR Ca2+ leak rate. Furthermore, Bay 60-7550 (0.1 µmol/l) increased the phosphorylation of phospholamban at 16-serine without significantly changing the phosphorylation levels of phospholamban at 17-threonine and RyR2. Bay 60-7550 increased the rat heart contractility and reduced peripheral arterial resistance may be mediated by increasing the phosphorylation of phospholamban and dilating peripheral vessels. PDE2 inhibitors which result in a positive inotropic effect and a decrease in peripheral resistance might serve as a target for developing agents for the treatment of heart failure in clinical settings.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cardiotônicos/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/antagonistas & inibidores , Imidazóis/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Triazinas/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Hemodinâmica/efeitos dos fármacos , Técnicas In Vitro , Masculino , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fosforilação , Ratos , Ratos Sprague-Dawley , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Resistência Vascular/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
NEW FINDINGS: What is the central question of this study? The therapeutic effect of ivabradine on patients with chronic heart failure and chronic stable angina pectoris is mediated through a reduction in heart rate: what are the haemodynamic characteristics and the mechanism of the inotropic effect? What is the main finding and its importance? Ivabradine has a positive inotropic effect and lowers the heart rate both in vivo and in vitro. These effects are likely mediated by ivabradine's significant increase of the fast component rate constant mediated by sarcoplasmic/endoplasmic reticulum calcium ATPase 2a and decrease of the slow component rate constant that is mediated by the Na+ /Ca2+ exchanger and sarcolemmal Ca2+ -ATPase during the Ca2+ transient decay phase. ABSTRACT: Ivabradine's therapeutic effect is mediated by a reduction of the heart rate; however, its haemodynamic characteristics and the mechanism of its inotropic effect are poorly understood. We aimed to investigate the positive inotropic effect of ivabradine and its underlying mechanism. The results demonstrated that ivabradine increased the positive inotropy of the rat heart in vivo by increasing the stroke work, cardiac output, stroke volume, end-diastolic volume, end-systolic pressure, ejection fraction, ±dP/dtmax , left ventricular end-systolic elastance and systolic blood pressure without altering the diastolic blood pressure and arterial elastance. This inotropic effect was observed in both non-paced and paced rat isolated heart. Ivabradine increased the Ca2+ transient amplitude and the reuptake rates of sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a), lowered the diastolic Ca2+ level and suppressed the combined extrusion rate of the Na+ /Ca2+ exchanger and the sarcolemmal Ca2+ -ATPase. In addition, ivabradine widened the action potential duration, hyperpolarized the resting membrane potential, increased sarcoplasmic reticulum Ca2+ content and reduced Ca2+ leak. Overall, ivabradine had a positive inotropic effect brought about by enhanced SERCA2a activity, which might be mediated by increased phospholamban phosphorylation. The positive inotropic effect along with the lowered heart rate underlies ivabradine's therapeutic effect in heart failure.
Assuntos
Cálcio/metabolismo , Ivabradina/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Volume Sistólico/efeitos dos fármacosRESUMO
Objective: To explore the positive inotropic effect of atractylodin which is major active component of Rhzoma Atractylodis Lanceae and its underlying mechanism. Methods: For in vivo study, six male SD rats were randomly selected for the heart pressure-volume loop (P-V loop) experiment. The effects of atractylodin (3 mg/kg, intraperitoneal injection) on hemodynamic parameters such as LVDP (left ventricular developed pressure), SW (stroke work), HR (heart rate), CO (cardiac output), SBP (systolic blood pressure) and DBP (diastolic blood pressure) were analyzed. For in vitro study, left ventricular developed pressure (LVDP) from the Langendroff-perfused isolated rat heart was analyzed before as the control and after atractylodin perfusion. For in vitro study, the effects of atractylodin and atractylodin with H89 (PKA inhibitor) or KN-93 (CaMKII inhibitor or Calyculin A (PP1, PP2A inhibitor) on LVDP were analyzed. The experiments were separated into four parts with six isolated hearts for each as follows: (1) Controlâ0.1â1â10 µmol/L atractylodin; (2) Controlâ200 nmol/L H89 (PKA inhibitor)â200 nmol/L H89+10 µmol/L atractylodin; (3) Controlâ500 nmol/L KN-93 (CaMKII inhibitor)â500 nmol/L KN-93+10 µmol/L atractylodin; (4) Controlâ10 nmol/L Calyculin A (PP1, PP2A inhibitor)â10 nmol/L Calyculin A+10 µmol/L atractylodin. For the study of rat left ventricular myocyte Ca2+ transient induced by field stimulation, the experiment design was the same as in vitro study. The six cells from the different rats were used for each part experiment. Results: â Atractylodin (3 mg/kg) significantly increased the heart rate, cardiac output and stroke work (Pï¼0.05) and decreased the diastolic blood pressure (Pï¼0.05). â¡ Atractylodin (0.1, 1, 10 µmol/L) significantly increased the LVDP in a concentration dependent manner (Pï¼0.05). The positive inotropic effect of atractylodin could be blocked by PKA inhibitor H89. ⢠Atractylodin (10 µmol/L) significantly increased the amplitude of SR Ca2+ transient amplitude mediated by facilitating sarcoplasmic reticulum SERCA2a. The enhanced amplitude of SR Ca2+ transient could be blocked by PKA inhibitor H89. Conclusion: Atractylodin had positive inotropic effect in rat heart both in vivo and in vitro with decreased diastolic blood pressure and its underlying mechanism was mediated by PKA. Based on the fact that the atractylodin exerted its positive inotropic effect was mediated by PKA, the PKA-SERCA2a signaling pathway might be the mechanism of the atractylodin's positive inotropy.