N-n-Butyl haloperidol iodide ameliorates liver fibrosis and hepatic stellate cell activation in mice.

N-n-Butyl haloperidol iodide (F2) is a novel compound that has antiproliferative and antifibrogenic activities. In this study we investigated the therapeutic potential of F2 against liver fibrosis in mice and the underlying mechanisms. Two widely used mouse models of fibrosis was established in mice by injection of either carbon tetrachloride (CCl4) or thioacetamide (TAA). The mice received F2 (0.75, 1.5 or 3 mg·kg-1·d-1, ip) for 4 weeks of fibrosis induction. We showed that F2 administration dose-dependently ameliorated CCl4- or TAA-induced liver fibrosis, evidenced by significant decreases in collagen deposition and c-Jun, TGF-ß receptor II (TGFBR2), α-smooth muscle actin (α-SMA), and collagen I expression in the liver. In transforming growth factor beta 1 (TGF-ß1)-stimulated LX-2 cells (a human hepatic stellate cell line) and primary mouse hepatic stellate cells, treatment with F2 (0.1, 1, 10 µM) concentration-dependently inhibited the expression of α-SMA, and collagen I. In LX-2 cells, F2 inhibited TGF-ß/Smad signaling through reducing the levels of TGFBR2; pretreatment with LY2109761 (TGF-ß signaling inhibitor) or SP600125 (c-Jun signaling inhibitor) markedly inhibited TGF-ß1-induced induction of α-SMA and collagen I. Knockdown of c-Jun decreased TGF-ß signaling genes, including TGFBR2 levels. We revealed that c-Jun was bound to the TGFBR2 promoter, whereas F2 suppressed the binding of c-Jun to the TGFBR2 promoter to restrain TGF-ß signaling and inhibit α-SMA and collagen I upregulation. In conclusion, the therapeutic benefit of F2 against liver fibrosis results from inhibition of c-Jun expression to reduce TGFBR2 and concomitant reduction of the responsiveness of hepatic stellate cells to TGF-ß1. F2 may thus be a potentially new effective pharmacotherapy for human liver fibrosis.

Pharmacological and functional comparisons of α6/α3ß2ß3-nAChRs and α4ß2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line.

Neuronal nicotinic acetylcholine receptors containing α6 subunits (α6*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of α6*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of α6*-nAChRs remain poorly understood because of the lack of selective agonists for α6*-nAChRs and the challenging heterologous expression of functional α6*-nAChRs in mammalian cell lines. In particular, the α6 subunit is commonly co-expressed with α4*-nAChRs in the midbrain, which masks α6*-nAChR (without α4) function and pharmacology. In this study, we systematically profiled the pharmacology and function of α6*-nAChRs and compared these properties with those of α4ß2 nAChRs expressed in the same cell line. Heterologously expressed human α6/α3 chimeric subunits (α6 N-terminal domain joined with α3 trans-membrane domains and intracellular loops) with ß2 and ß3 subunits in the human SH-EP1 cell line (α6*-nAChRs) were used. Patch-clamp whole-cell recordings were performed to measure these receptor-mediated currents. Functionally, the heterologously expressed α6*-nAChRs exhibited excellent function and showed distinct nicotine-induced current responses, such as kinetics, inward rectification and recovery from desensitization, compared with α4ß2-nAChRs. Pharmacologically, α6*-nAChR was highly sensitive to the α6 subunit-selective antagonist α-conotoxin MII but had lower sensitivity to mecamylamine and dihydro-ß-erythroidine. Nicotine and acetylcholine were found to be full agonists for α6*-nAChRs, whereas epibatidine and cytisine were determined to be partial agonists. Heterologously expressed α6*-nAChRs exhibited pharmacology and function distinct from those of α4ß2-nAChRs, suggesting that α6*-nAChRs may mediate different cholinergic signals. Our α6*-nAChR expression system can be used as an excellent cell model for future investigations of α6*-nAChR function and pharmacology.

Brain cannabinoid receptor 2: expression, function and modulation.

Cannabis sativa (marijuana) is a fibrous flowering plant that produces an abundant variety of molecules, some with psychoactive effects. At least 4% of the world's adult population uses cannabis annually, making it one of the most frequently used illicit drugs in the world. The psychoactive effects of cannabis are mediated primarily through cannabinoid receptor (CBR) subtypes. The prevailing view is that CB1Rs are mainly expressed in the central neurons, whereas CB2Rs are predominantly expressed in peripheral immune cells. However, this traditional view has been challenged by emerging strong evidence that shows CB2Rs are moderately expressed and function in specific brain areas. New evidence has demonstrated that brain CB2Rs modulate animal drug-seeking behaviors, suggesting that these receptors may exist in brain regions that regulate drug addiction. Recently, we further confirmed that functional CB2Rs are expressed in mouse ventral tegmental area (VTA) dopamine (DA) neurons and that the activation of VTA CB2Rs reduces neuronal excitability and cocaine-seeking behavior. In addition, CB2R-mediated modulation of hippocampal CA3 neuronal excitability and network synchronization has been reported. Here, we briefly summarize recent lines of evidence showing how CB2Rs modulate function and pathophysiology in the CNS.

Effects of ghrelin on the intracellular calcium concentration in rat aorta vascular smooth muscle cells.

BACKGROUND/AIMS: Ghrelin has been regarded as a cardioprotective factor with complicated mechanisms. Whether ghrelin is vasodilative or vasoconstrictive in nature is controversial, and the effects of ghrelin on intracellular calcium concentration are still unclear. To explore the mechanisms involved in the vasoactive regulation of ghrelin at the cellular level, we investigated the effects of ghrelin on calcium concentrations in rat aorta vascular smooth muscle cells (VSMCs). METHODS: We obtained VSMCs via cell culture and stained the cells with Furo-2 AM. Western blotting was used to verify growth hormone secretagogue receptor (GHS-R1a) expression in VSMCs. The intracellular calcium variations affected by ghrelin and the interactions of ghrelin with angiotensin II (AngII), Sq22536, and potassium chloride (KCl) were observed using a calcium imaging and analysis system. RESULTS: Western blotting revealed good GHS-R1a expression in VSMCs. The most prominent finding in the present study was that ghrelin inhibited the AngII-induced increase in the calcium concentration. This inhibition was reversed by the adenylate cyclase inhibitor Sq22536 and the GHS-R1a antagonist (D-Lys(3))- GHRP-6. This finding revealed the potential vasodilative effects of ghrelin at the cellular level. We did not observe any effects of ghrelin on intracellular calcium concentrations in resting VSMCs or the increase of calcium concentration induced by KCl. CONCLUSION: Ghrelin inhibited the increase in the intracellular calcium concentration of rat aorta VSMCs induced by AngII, which may depend on the activation of the cAMP/PKA pathway.

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.

Reformative cannula electrodes for His bundle electrogram recording in isolated rat hearts.

His bundle electrogram (HBE) recording is an important method for the study of the atrioventricular conduction system. However, the current HBE recording methods in isolated animal hearts have some disadvantages, such as unstable recording due to the difficulty in fixing electrodes as a result of intense heart beat, the small amplitude of the His signal or the possibility to destroy the integrity of heart structure. To overcome these disadvantages, we designed and manufactured reliable, inexpensive and easy-made bipolar cannula electrodes, which combine the functions of Langendorff-perfusion aortic cannula and recording electrodes. With the cannula electrodes, the operation of HBE recording becomes easier and clearer; hence, more stable recordings can be obtained in isolated rat hearts.

Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injury.

Neuronal apoptosis is mediated by intrinsic and extrinsic signaling pathways such as the membrane-mediated, mitochondrial, and endoplasmic reticulum stress pathways. Few studies have examined the endoplasmic reticulum-mediated apoptosis pathway in the penumbra after traumatic brain injury, and it remains unclear whether endoplasmic reticulum stress can activate the caspase-12-dependent apoptotic pathway in the traumatic penumbra. Here, we established rat models of fluid percussion-induced traumatic brain injury and found that protein expression of caspase-12, caspase-3 and the endoplasmic reticulum stress marker 78 kDa glucose-regulated protein increased in the traumatic penumbra 6 hours after injury and peaked at 24 hours. Furthermore, numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells in the traumatic penumbra also reached peak levels 24 hours after injury. These findings suggest that caspase-12-mediated endoplasmic reticulum-related apoptosis is activated in the traumatic penumbra, and may play an important role in the pathophysiology of secondary brain injury.

Characterization and bioactivity of novel calcium antagonists - N-methoxy-benzyl haloperidol quaternary ammonium salt.

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.

Protective effects of N-n-butyl haloperidol iodide on myocardial ischemia-reperfusion injury in rabbits.

N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, was synthesized by our drugs research lab. The present study aims to evaluate the protective effects of F2 on myocardial ischemia-reperfusion injury in vivo, and to try to find the protective mechanism of F2. The animal model of myocardial ischemia-reperfusion injury was established by ligaturing rabbit's left ventricular branch of coronary artery for 40 min and removing the ligation later to reperfuse for 40 min. Different doses of F2 were intravenously injected before the onset of ischemia. The changes of hemodynamics were recorded during the experiment, and the activities of superoxide dismutase (SOD), creatine kinase (CK), Ca2+-ATPase, Na+,K+-ATPase and the level of malondialdehyde (MDA) of myocardial tissue were detected after reperfusion. Administration of F2 could dose-dependently ameliorate the hemodynamics of ischemia-reperfusion injured myocardium. During the course of reperfusion, MAP, LVSP, +/-dP/dt(max) in all F2 groups were obviously higher than those in the ischemia-reperfusion control group, and LVEDP were lower. F2 could also reduce the production of MDA, and maintain the activities of SOD, Ca2+-ATPase, Na+,K+-ATPase, and minimize the leakage of CK out of myocardial cells in a dose-dependent manner. These results suggested that F2 had apparent protective effects against myocardial ischemia-reperfusion injury.

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.