RESUMEN
BACKGROUND AND PURPOSE: Calcium antagonists play an important role in clinical practice. However, most of them have serious side effects. We have synthesized a series of novel calcium antagonists, quaternary ammonium salt derivatives of haloperidol with N-p-methoxybenzyl (X1), N-m-methoxybenzyl (X2) and N-o-methoxybenzyl (X3) groups. The objective of this study was to investigate the bioactivity of these novel calcium antagonists, especially the vasodilation activity and cardiac side-effects. The possible working mechanisms of these haloperidol derivatives were also explored. EXPERIMENTAL APPROACH: Novel calcium antagonists were synthesized by amination. Compounds were screened for their activity of vasodilation on isolated thoracic aortic ring of rats. Their cardiac side effects were explored. The patch-clamp, confocal laser microscopy and the computer-fitting molecular docking experiments were employed to investigate the possible working mechanisms of these calcium antagonists. RESULTS: The novel calcium antagonists, X1, X2 and X3 showed stronger vasodilation effect and less cardiac side effect than that of classical calcium antagonists. They blocked L-type calcium channels with an potent effect order of X1 > X2 > X3. Consistently, X1, X2 and X3 interacted with different regions of Ca2+-CaM-CaV1.2 with an affinity order of X1 > X2 > X3. CONCLUSIONS: The new halopedidol derivatives X1, X2 and X3 are novel calcium antagonists with stronger vasodilation effect and less cardiac side effect. They could have wide clinical application.
Asunto(s)
Aorta/efectos de los fármacos , Bloqueadores de los Canales de Calcio/síntesis química , Bloqueadores de los Canales de Calcio/farmacología , Haloperidol/análogos & derivados , Corazón/efectos de los fármacos , Animales , Femenino , Hemodinámica/efectos de los fármacos , Técnicas In Vitro , Masculino , Microscopía Confocal , Simulación del Acoplamiento Molecular , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacos , Difracción de Rayos XRESUMEN
N-n-butyl haloperidol iodide (F2), a novel compound, has shown palliative effects in myocardial ischemia/reperfusion (I/R) injury. In this study, we investigated the effects of F2 on the extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/Na(+)/H(+) exchanger (NHE)/Na(+)/Ca(2+) exchanger (NCX) signal-transduction pathway involved in H2O2-induced Ca(2+) overload, in order to probe the underlying molecular mechanism by which F2 antagonizes myocardial I/R injury. Acute exposure of rat cardiac myocytes to 100 µM H2O2 increased both NHE and NCX activities, as well as levels of phosphorylated MEK and ERK. The H2O2-induced increase in NCX current (I NCX) was nearly completely inhibited by the MEK inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis[o-aminophenylmercapto] butadiene), but only partly by the NHE inhibitor 5-(N,N-dimethyl)-amiloride (DMA), indicating the I NCX increase was primarily mediated by the MEK/mitogen-activated protein kinase (MAPK) pathway, and partially through activation of NHE. F2 attenuated the H2O2-induced I NCX increase in a concentration-dependent manner. To determine whether pathway inhibition was H2O2-specific, we examined the ability of F2 to inhibit MEK/ERK activation by epidermal growth factor (EGF), and NHE activation by angiotensin II. F2 not only inhibited H2O2-induced and EGF-induced MEK/ERK activation, but also completely blocked both H2O2-induced and angiotensin II-induced increases in NHE activity, suggesting that F2 directly inhibits MEK/ERK and NHE activation. These results show that F2 exerts multiple inhibitions on the signal-transduction pathway involved in H2O2-induced I NCX increase, providing an additional mechanism for F2 alleviating intracellular Ca(2+) overload to protect against myocardial I/R injury.
Asunto(s)
Haloperidol/análogos & derivados , Peróxido de Hidrógeno/antagonistas & inhibidores , Miocitos Cardíacos/efectos de los fármacos , Intercambiador de Sodio-Calcio/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Haloperidol/farmacología , Peróxido de Hidrógeno/farmacología , Masculino , Miocitos Cardíacos/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Intercambiador de Sodio-Calcio/agonistas , Relación Estructura-ActividadRESUMEN
We have previously shown that N-n-butyl haloperidol iodide (F(2)), a newly synthesized compound, reduces ischemia/reperfusion (I/R) injury by preventing intracellular Ca(2+) overload through inhibiting L-type calcium channels and outward current of Na(+)/Ca(2+) exchanger. This study was to investigate the effects of F(2) on activity and protein expression of the rat myocardial sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) during I/R to discover other molecular mechanisms by which F(2) maintains intracellular Ca(2+) homeostasis. In an in vivo rat model of myocardial I/R achieved by occluding coronary artery for 30-60 min followed by 0-120 min reperfusion, treatment with F(2) (0.25, 0.5, 1, 2 and 4 mg/kg, respectively) dose-dependently inhibited the I/R-induced decrease in SERCA activity. However, neither different durations of I/R nor different doses of F(2) altered the expression levels of myocardial SERCA2a protein. These results indicate that F(2) exerts cardioprotective effects against I/R injury by inhibiting I/R-mediated decrease in SERCA activity by a mechanism independent of SERCA2a protein levels modulation.
Asunto(s)
Cardiotónicos/farmacología , Haloperidol/análogos & derivados , Daño por Reperfusión Miocárdica/enzimología , Miocardio/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Retículo Sarcoplasmático/efectos de los fármacos , Animales , Haloperidol/farmacología , Técnicas In Vitro , Masculino , Microscopía Electrónica de Transmisión , Daño por Reperfusión Miocárdica/patología , Miocardio/ultraestructura , Ratas , Ratas Sprague-Dawley , Retículo Sarcoplasmático/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidoresRESUMEN
AIMS: N-n-Butyl haloperidol iodide (F(2)) is a novel compound derived from haloperidol. In our previous work, F(2) was found to be an L-type calcium channel blocker which played a protective role in rat heart ischemic-reperfusion injury in a dose-dependent manner. In the current study, we aimed to investigate the effects and some possible mechanisms of F(2) on calcium transients in hypoxic/ischemic rat cardiac myocytes. METHODS AND RESULTS: Calcium transients' images of rat cardiac myocytes were recorded during simulated hypoxia, using a confocal calcium imaging system. The amplitude, rising time from 25% to 75% (RT25-75), decay time from 75% to 25% (DT75-25) of calcium transients, and resting [Ca(2+)](i) were extracted from the images by self-coding programs. In this study, hypoxia produced a substantial increase in diastolic [Ca(2+)](i) and reduced the amplitude of calcium transients. Both RT25-75 and DT75-25 of Ca(2+) transients were significantly prolonged. And F(2) could reduce the increase in resting [Ca(2+)](i)and the prolongation of RT25-75 and DT75-25 of Ca(2+) transients during hypoxia. F(2) also inhibited the reduction in amplitude of calcium transients which was caused by 30-min hypoxia. The activity of SERCA2a (sarcoplasmic reticulum Ca(2+)-ATPase, determined by test kits) decreased after 30-min ischemia, and intravenous F(2) in rats could ameliorate the decreased activity of SERCA2a. The inward and outward currents of NCX (recorded by whole-cell patch-clamp analysis) were reduced during 10-min hypoxia, and F(2) further inhibited the outward currents of NCX during 10-min hypoxia. All these data of SERCA2a and NCX might be responsible for the changes in calcium transients during hypoxia. CONCLUSION: Our data suggest that F(2) reduced changes in calcium transients that caused by hypoxia/ischemia, which was regarded to be a protective role in calcium homeostasis of ventricular myocytes, probably via changing the function of SERCA2a.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Haloperidol/análogos & derivados , Hipoxia/metabolismo , Isquemia/metabolismo , Miocitos Cardíacos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Retículo Sarcoplasmático/metabolismo , Compuestos de Anilina/análisis , Animales , Bloqueadores de los Canales de Calcio/farmacología , Colorantes Fluorescentes/análisis , Haloperidol/farmacología , Corazón/efectos de los fármacos , Corazón/fisiopatología , Homeostasis , Hipoxia/fisiopatología , Isquemia/fisiopatología , Masculino , Microscopía Confocal , Imagen Molecular , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Retículo Sarcoplasmático/efectos de los fármacos , Xantenos/análisisRESUMEN
OBJECTIVE: To investigate the actions of protease-activated receptor 1 (PAR1) agonists and thrombin on the secretion of interleukin-8 (IL-8) from human lung epithelial cells. METHODS: A549 cells were cultured in a 12-well culture plate. The challenge was performed by addition of various concentrations of PAR1 agonist peptides SFLLR and its reverse peptides RLLFS, thrombin or hirudin, a thrombin inhibitor, into each well, respectively. After 2 or 16 h, the reactions were terminated by removal of the supernatant from each well. A sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of IL-8 in the supernatants. RESULTS: Following a 16-hour incubation, SFLLR was able to induce concentration-dependent secretion of IL-8. The maximum release of IL-8 was increased nearly 16 fold more than the baseline release. The reverse PAR1 agonists had little effects on IL-8 release. Thrombin was also able to induce concentration- dependent secretion of IL-8. As low as 1 kU/L thrombin was able to induce IL-8 release from the epithelial cells, and the maximum accumulated release of IL-8 was observed with 10 kU/L thrombin, which was 7.5 fold the baseline release. Thrombin inhibitor hirudin could inhibit thrombin-induced secretion of IL-8. The time course showed that the actions of PAR1 agonist peptides SFLLR and thrombin initiated at 2 h and reached the peak at 16 h. CONCLUSION: PAR1 agonist peptides and thrombin are potent secretogogues of IL-8 release from cultured human lung epithelial cells, and PAR1 antagonists and thrombin inhibitor may possess the ability to inhibit airway inflammation.