N-n-butyl haloperidol iodide inhibits H2O2-induced Na+/Ca2+-exchanger activation via the Na+/H+ exchanger in rat ventricular myocytes.

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.

Effects of N-n-butyl haloperidol iodide on the rat myocardial sarcoplasmic reticulum Ca(2+)-ATPase during ischemia/reperfusion.

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.

N-n-butyl haloperidol iodide preserves cardiomyocyte calcium homeostasis during hypoxia/ischemia.

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.

Cardiac electrophysiological and antiarrhythmic effects of N-n-butyl haloperidol iodide.

AIMS: N-n-butyl haloperidol (F(2)), a novel compound of quaternary ammonium salt derivatives of haloperidol, was reported to antagonize myocardial ischemia/reperfusion injuries. The antiarrhythmic potential and electrophysiological effects of F(2) on rat cardiac tissues were investigated. METHODS AND RESULTS: In Langendorff-perfused rat hearts, the ventricular arrhythmias were induced by left anterior descending coronary artery of rat heart ligated for 20 min before the release of the ligature. F(2) provided some inhibitive effects against ischemia- and reperfusion-induced ventricular arrhythmias. In His bundle electrogram and epicardial ECG recordings, the drug produced bradycardia, delayed the conduction through the atrioventricular node and prolonged the Wenckebach cycle length and atrioventricular nodal effective refractory period. In whole-cell patch-clamp study, F(2) primarily inhibited the L-type Ca2+ current (I(Ca,L)) (IC(50) = 0.17 microM) with tonic blocking properties and little use-dependence. And the drug also decreased the Na+ current (IC(50) = 77.5 microM), the transient outward K+ current (IC(50) = 20.4 microM), the steady-state outward K+ current (IC(50) = 56.2 microM) and the inward rectifier K+ current (IC(50) = 127.3 microM). CONCLUSION: F(2) may be a promising drug for the treatment of ischemic heart disease with cardiac arrhythmia.

Egr-1, a central and unifying role in cardioprotection from ischemia-reperfusion injury?

AIMS: Our previous studies have shown that N-n-butyl haloperidol iodide (F(2)) can antagonize myocardial ischemia/reperfusion (I/R) injury by blocking intracellular Ca(2+) overload and suppressing Egr-1 overexpression. The present study is to investigate the relation between the reduction of Ca(2+) overload and the inhibition of Egr-1 overexpression. METHODS: The Sprague-Dawley rat myocardial I/R model and cultured cardiomyocyte hypoxia-reoxygenation (H/R) model were established. Administration of Egr-1 antisense oligodeoxyribonucleotide (AS-ODN) only or combining with F(2), Egr-1 protein expression was examined by Western-blot analyses. Hemodynamic parameters, creatine kinase (CK) and lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA), myeloperoxidase (MPO), cardiac troponin I (cTnI), and tumor necrosis factor-alpha (TNF-alpha) were measured to assess the degree of injury and inflammation of myocardial tissues and cells. RESULTS: Treatment with Egr-1 AS-ODN significantly reduced Egr-1 protein expression and attenuated injury and inflammation of myocardium caused by I/R or H/R evidenced by the amelioration of hemodynamics, the decrease in leakage of CK, LDH, cTnI, the increase in MDA generation, the decrease in SOD activity, the reduction of MPO activity in myocardial tissues and release of TNF-alpha from cultured cardiomyocytes. Treatment with F(2) combined with Egr-1 AS-ODN, the inhibition of Egr-1 protein expression and inflammation (MPO activity and TNF-alpha level) were not enhanced, but the protection from myocardial I/R (or H/R) injury was significantly increased in hemodynamics and cytomembrane permeability relative to the using of Egr-1 AS-ODN only. CONCLUSION: These data suggest that the inhibition of Egr-1 overexpression cannot involve all mechanisms of cardioprotection from I/R injury.

Effects of N-n-butyl haloperidol iodide on rat myocardial ischemia and reperfusion injury and L-type calcium current.

AIM: To study the effects of N-n-butyl haloperidol iodide (F2) on rat heart ischemia/reperfusion (I/R) injury and L-type calcium current (ICa) in rat ventricular myocytes. METHODS: Rat heart I/R injury was induced by occluding the left anterior descending coronary artery for 30 min and restoring perfusion for 30 min. F2 (1, 2, and 4 mg/kg) were i.v. injected before ischemia. Plasma creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB), lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBDH), glutamic-oxaloacetic transaminase (GOT), malondialdehyde (MDA) concentrations, and superoxide dismutase (SOD) activity were measured. The pathologic changes of I/R myocardium were assessed by the transmission electron microscopy. Single rat ventricular myocyte was obtained by enzymatic dissociation method. The currents were recorded with the whole-cell configuration of the patch-clamp technique. RESULTS: F2 reduced the release of CK, CK-MB, LDH, HBDH and GOT, preserved the activity of SOD, and decreased the MDA contents dose-dependently. For morphology, F2 mollified the pathologic changes of myocardium induced by I/R injury. F2 1 micromol/L decreased ICa from (1775+/-360) pA to (464+/-129) pA (n=8, P<0.01) and shifted the current-voltage of ICa upward, without affecting the voltage-depend-ent properties of ICa. CONCLUSION: F2 played a protective role against rat heart I/R injury in a dose-dependent manner, and inhibited ICa in rat ventricular myocytes. The cardioprotective and vasodilatory mechanisms of F2 may be related to its inhibitory effect on L-type calcium channel.