l-DOPA receptor GPR143 inhibits neurite outgrowth via L-type calcium channels in PC12 cells.

The gene product of ocular albinism 1 (OA1)/G-protein-coupled receptor (GPR)143 is a receptor for L-3,4-dihydroxyphenylanine (l-DOPA), the most effective agent for Parkinson's disease. When overexpressed, human wild-type GPR143, but not its mutants, inhibits neurite outgrowth in PC12 cells. We investigated the downstream signaling pathway for GPR143-induced inhibition of neurite outgrowth. Nifedipine restored GPR143-induced neurite outgrowth inhibition to the level of control transfectant but did not affect outgrowth in GPR143-knockdown cells. Cilnidipine and flunarizine also suppressed the GPR143-induced inhibition, but their effects at higher concentrations still occurred even in GPR143-knockdown cells. These results suggest that GPR143 regulates neurite outgrowth via L-type calcium channel(s).

Understanding the Role of the SMN Complex Component GEMIN5 and Its Functional Relationship with Demethylase KDM6B in the Flunarizine-Mediated Neuroprotection of Motor Neuron Disease Spinal Muscular Atrophy.

Dysregulated RNA metabolism caused by SMN deficiency leads to motor neuron disease spinal muscular atrophy (SMA). Current therapies improve patient outcomes but achieve no definite cure, prompting renewed efforts to better understand disease mechanisms. The calcium channel blocker flunarizine improves motor function in Smn-deficient mice and can help uncover neuroprotective pathways. Murine motor neuron-like NSC34 cells were used to study the molecular cell-autonomous mechanism. Following RNA and protein extraction, RT-qPCR and immunodetection experiments were performed. The relationship between flunarizine mRNA targets and RNA-binding protein GEMIN5 was explored by RNA-immunoprecipitation. Flunarizine increases demethylase Kdm6b transcripts across cell cultures and mouse models. It causes, in NSC34 cells, a temporal expression of GEMIN5 and KDM6B. GEMIN5 binds to flunarizine-modulated mRNAs, including Kdm6b transcripts. Gemin5 depletion reduces Kdm6b mRNA and protein levels and hampers responses to flunarizine, including neurite extension in NSC34 cells. Moreover, flunarizine increases the axonal extension of motor neurons derived from SMA patient-induced pluripotent stem cells. Finally, immunofluorescence studies of spinal cord motor neurons in Smn-deficient mice reveal that flunarizine modulates the expression of KDM6B and its target, the motor neuron-specific transcription factor HB9, driving motor neuron maturation. Our study reveals GEMIN5 regulates Kdm6b expression with implications for motor neuron diseases and therapy.

Novel pharmacological modulation of dystonic phenotypes caused by a gain-of-function mutation in the Na+ leak-current channel.

The Na leak-current channel (NALCN) regulates the resting membrane potential in excitable cells, thus determining the likelihood of depolarization in response to incoming signals. Gain-of-function (gf) mutations in this channel are associated with severe dystonic movement disorders in man. Currently, there are no known pharmacological antagonists or selective modulators of this important channel. A gain-of-function mutation in NALCN of C. elegans [known as unc-77(e625)] causes uncoordinated, hyperactive locomotion. We hypothesized that this hyperactive phenotype can be rescued with pharmacological modulators. Here, we summarize the results of targeted drug screening aimed at identification of drugs that corrected locomotion deficits in unc-77(e625) animals. To assay hyperactive locomotion, animals were acutely removed from food and characteristic foraging movements were quantified. Drug screening revealed that 2-aminoethoxydiphenyl borate (2-ABP), nifedipine, nimodipine, flunarizine and ethoxzolamide significantly decreased abnormal movements in unc-77(e625) animals. 2-APB also corrected egg release and coiling deficits in this strain. In addition, serotonin and dopamine both reduced hyperactive locomotion, consistent with regulatory interactions between these systems and the NALCN. 2-APB induced movement phenotypes in wild-type animals that faithfully mimicked those observed in NALCN knockout strains, which suggested that this drug may directly block the channel. Moreover, 2-APB and flunarizine showed significant structural similarities suggestive of overlap in their mode of action. Together, these studies have revealed new insights into regulation of NALCN function and led to the discovery of a potential pharmacological antagonist of the NALCN.

Mibefradil and Flunarizine, Two T-Type Calcium Channel Inhibitors, Protect Mice against Lipopolysaccharide-Induced Acute Lung Injury.

Recent studies have illuminated that blocking Ca2+ influx into effector cells is an attractive therapeutic strategy for lung injury. We hypothesize that T-type calcium channel may be a potential therapeutic target for acute lung injury (ALI). In this study, the pharmacological activity of mibefradil (a classical T-type calcium channel inhibitor) was assessed in a mouse model of lipopolysaccharide- (LPS-) induced ALI. In LPS challenged mice, mibefradil (20 and 40 mg/kg) dramatically decreased the total cell number, as well as the productions of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF). Mibefradil also suppressed total protein concentration in BALF, attenuated Evans blue extravasation, MPO activity, and NF-κB activation in lung tissue. Furthermore, flunarizine, a widely prescripted antimigraine agent with potent inhibition on T-type channel, was also found to protect mice against lung injury. These data demonstrated that T-type calcium channel inhibitors may be beneficial for treating acute lung injury. The important role of T-type calcium channel in the acute lung injury is encouraged to be further investigated.

A central hydrophobic E1 region controls the pH range of hepatitis C virus membrane fusion and susceptibility to fusion inhibitors.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection causes chronic liver disease. Antivirals have been developed and cure infection. However, resistance can emerge and salvage therapies with alternative modes of action could be useful. Several licensed drugs have emerged as HCV entry inhibitors and are thus candidates for drug repurposing. We aimed to dissect their mode of action, identify improved derivatives and determine their viral targets. METHODS: HCV entry inhibition was tested for a panel of structurally related compounds, using chimeric viruses representing diverse genotypes, in addition to viruses containing previously determined resistance mutations. Chemical modeling and synthesis identified improved derivatives, while generation of susceptible and non-susceptible chimeric viruses pinpointed E1 determinants of compound sensitivity. RESULTS: Molecules of the diphenylpiperazine, diphenylpiperidine, phenothiazine, thioxanthene, and cycloheptenepiperidine chemotypes inhibit HCV infection by interfering with membrane fusion. These molecules and a novel p-methoxy-flunarizine derivative with improved efficacy preferentially inhibit genotype 2 viral strains. Viral residues within a central hydrophobic region of E1 (residues 290-312) control susceptibility. At the same time, viral features in this region also govern pH-dependence of viral membrane fusion. CONCLUSIONS: Small molecules from different chemotypes related to flunarizine preferentially inhibit HCV genotype 2 membrane fusion. A hydrophobic region proximal to the putative fusion loop controls sensitivity to these drugs and the pH range of membrane fusion. An algorithm considering viral features in this region predicts viral sensitivity to membrane fusion inhibitors. Resistance to flunarizine correlates with more relaxed pH requirements for fusion. LAY SUMMARY: This study describes diverse compounds that act as HCV membrane fusion inhibitors. It defines viral properties that determine sensitivity to these molecules and thus provides information to identify patients that may benefit from treatment with membrane fusion inhibitors.

Effect of flunarizine on defibrillation outcomes and early refibrillation in a canine model of prolonged ventricular fibrillation.

NEW FINDINGS: What is the central question of this study? Can successful electrical shock in combination with a delayed after-depolarization (DAD) blocker suppress early refibrillation episodes following long duration ventricular fibrillation (LDVF)? What is the main finding and its importance? Flunarizine significantly reduced the activation of LDVF and early ventricular fibrillation (VF) recurrence following LDVF, suggesting that DADs potentially contribute to refibrillation in prolonged VF. Thus, DAD inhibition can be used as an adjunctive therapy for electrical defibrillation to treat prolonged VF and suppress refibrillation following LDVF. ABSTRACT: This study attempts to detect changes in the defibrillation threshold (DFT) at different stages of ventricular fibrillation (VF) (short duration VF, SDVF; long duration VF, LDVF) and during early refibrillation following successful defibrillation of LDVF by giving flunarizine, a blocker of delayed after-depolarizations (DADs). Twelve beagles were divided into two groups (the control group, n = 6; and the flunarizine group, n = 6). Two 64-electrode basket catheters were deployed into the left and the right ventricles for global endocardium mapping. The DFTs of SDVF and LDVF were determined at 20 s and 7 min, respectively, after VF induction in each group. Any refibrillation episodes were recorded within 15 min after the first successful defibrillation of LDVF. In the flunarizine group, the SDVF-DFT values before and after the drug were not significantly different. The 7 min LDVF-DFTs were markedly reduced by 26% (P < 0.05, the control group) and 38% (P < 0.01, the flunarizine group) compared to the 20 s SDVF-DFTs within each group. The difference between SDVF-DFT and LDVF-DFT after flunarizine was larger than that in the control group (213 ± 65 vs. 120 ± 84 V, P < 0.05). The number of refibrillation episodes per animal (1.3 ± 1.0) following successful defibrillation of LDVF after flunarizine was 48% of that in controls (2.7 ± 2.0, P < 0.05). The effect of flunarizine on SDVF-DFT and LDVF-DFT indicates that the role of DADs in the defibrillation mechanism may differ as VF continues. Flunarizine significantly reduced early VF recurrence following LDVF, suggesting that DADs potentially contribute to refibrillation in a canine model of prolonged VF.

Point of View: Electrophysiological Endpoints Differ When Comparing the Mode of Action of Highly Successful Anti-arrhythmic Drugs in the CAVB Dog Model With TdP.

In the anaesthetized, chronic atrioventricular block (CAVB) dog, ventricular ectopic beats and Torsade de pointes arrhythmias (TdP) are believed to ensue from an abrupt prolongation of ventricular repolarization and increased temporal dispersion of repolarization, quantified as short-term variability (STV). These TdP stop spontaneously or, when supported by substantial spatial dispersion of repolarization (SDR), degenerate into ventricular fibrillation. However, most studies involving ventricular arrhythmias do not quantify SDR by means of an electrophysiological parameter. Therefore, we reviewed the effects of 4 highly effective anti-arrhythmic drugs (flunarizine, verapamil, SEA-0400, and GS-458967) on the repolarization duration and associated STV. All drugs were tested as anti-arrhythmic strategies against TdP in CAVB dogs, their high anti-arrhythmic efficacy was defined as suppressing drug-induced TdP in 100% of the experiments. This comparison demonstrates that even though the anti-arrhythmic outcome was similar for all drugs, distinct responses of repolarization duration and associated STV were observed. Moreover, the aforementioned and commonly adopted electrophysiological parameters were not always sufficient in predicting TdP susceptibility, and additional quantification of the SDR proved to be of added value in these studies. The variability in electrophysiological responses to the different anti-arrhythmic drugs and their inconsistent adequacy in reflecting TdP susceptibility, can be explained by distinct modes of interference with TdP development. As such, this overview establishes the separate involvement of temporal and spatial dispersion in ventricular arrhythmogenesis in the CAVB dog model and proposes SDR as an additional parameter to be included in future fundamental and/or pharmaceutical studies regarding TdP arrhythmogenesis.

Flunarizine prevents hepatitis C virus membrane fusion in a genotype-dependent manner by targeting the potential fusion peptide within E1.

UNLABELLED: To explore mechanisms of hepatitis C viral (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. CONCLUSION: These observations reveal novel details about HCV membrane fusion; moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as a cost-effective component of HCV combination therapies.

Flunarizine rescues reduced lifespan in CLN3 triple knock-out Caenorhabditis elegans model of batten disease.

CLN3 disease (Spielmeyer-Vogt-Sjogren-Batten disease, previously known as classic juvenile neuronal ceroid lipofuscinosis, NCL) is a pediatric-onset progressive neurodegenerative disease characterized by progressive vision loss, seizures, loss of cognitive and motor function, and early death. While no precise biochemical mechanism or therapies are known, the pathogenesis of CLN3 disease involves intracellular calcium accumulation that may trigger apoptosis. Our prior work in in vitro cell models of CLN3 deficiency suggested that FDA-approved calcium channel antagonists may have therapeutic value. To further evaluate the potential efficacy of this approach in an otherwise untreatable disorder, we sought to compare the therapeutic effects and underlying mechanisms in an animal model of CLN3 disease. Here, we used the well-characterized XT7 complete cln-3 knockout strain of C. elegans to evaluate the therapeutic efficacy of calcium channel antagonist therapy in a living animal model of Batten disease. Therapeutic effects of five calcium channel antagonists were evaluated on XT7 animal lifespan and in vivo mitochondrial physiology. Remarkably, maximal therapeutic efficacy in this model animal was observed with 1 µM flunarizine, the identical concentration previously identified in cell-based neuronal models of CLN3 disease. Specifically, flunarizine rescued the short lifespan of XT7 worms and prevented their pathophysiologic mitochondrial accumulation. These results confirm the treatment efficacy and dosing of flunarizine in cln-3 disease in a translational model organism. Clinical treatment trials in CLN3 human patients are now needed to test the dosing regimen and efficacy of flunarizine in individuals suffering with this otherwise untreatable and ultimately lethal neurologic disease.

AV-block and conduction slowing prevail over TdP arrhythmias in the methoxamine-sensitized pro-arrhythmic rabbit model.

INTRODUCTION: The methoxamine-sensitized rabbit model is widely used to screen drugs for proarrhythmic properties, especially repolarization-dependent TdP arrhythmias. With the change of anesthesia and/or sensitizing agent, conduction disturbances have been reported as well. Therefore, we compared currently available in-house anesthetics in order to preserve arrhythmia sensitivity and preclude conduction disturbances. METHODS AND RESULTS: Rabbits were randomly assigned to 3 groups: (1) 35 mg/kg ketamine + 5 mg/kg xylazine; (2) 0.5 mL/kg hypnorm + 3 mg/kg midazolam; (3) 35 mg/kg ketamine + 20 mg/kg propofol. Anesthesia was maintained by 1.5% isoflurane. Concomitant infusion of methoxamine (17 µg/kg/min for 40 minutes) and dofetilide (10 µg/kg/min for 30 minutes) was used to induce arrhythmias. Sole methoxamine infusion exclusively decreased HR in groups 1 and 3. Dofetilide lengthened repolarization, followed in time by PQ/QRS prolongation, second-degree AV block, and subsequently TdP arrhythmias. TdP was seen in 80%, 0%, and 33% of the rabbits in groups 1, 2, and 3, respectively. Decreasing the dose of dofetilide to 5 µg/kg/min in ketamine/xylazine anesthetized rabbits resulted in a drop in TdP incidence (25%) while conduction disturbances persisted. Flunarizine (n = 6) suppressed all TdP arrhythmias while conduction disturbances remained present. CONCLUSION: TdP incidence in the methoxamine-sensitized rabbit could be dramatically influenced by anesthesia, drug dose, and flunarizine, while conduction slowing remained present. Thus, conduction slowing seems to be the integral outcome in this model.

HOCl causes necrotic cell death in human monocyte derived macrophages through calcium dependent calpain activation.

The abundance of dead macrophages in close proximity to HOCl-modified proteins in advanced atherosclerotic plaques implicates HOCl in the killing of macrophages and the formation of the necrotic core region. The mechanism of HOCl mediated death of macrophages was unknown, so using human monocyte derived macrophages (HMDM) we here have shown that HOCl causes a rapid necrotic cell death characterized by loss of MTT reduction, cellular ATP and cell lysis without caspase-3 activation in HMDM cells. The HOCl causes a rise in cytosolic calcium level via the plasma membrane L- and T-type calcium channels and endoplasmic reticulum RyR channel. Blocking of the calcium channels or the addition of calpain inhibitors prevents the HOCl mediated loss of mitochondrial potential, lysosome failure and HMDM cell death. Blocking MPT-pore formation with cyclosporin A also prevents the loss of mitochondrial membrane potential, lysosomal destabilization and HMDM cell death. Blocking the calcium mitochondrial uniporter with ruthenium red also blocks the loss of mitochondrial potential but only at high concentrations. HOCl appears to cause HMDM cell death through destabilization of cytosolic calcium control resulting in the failure of both the mitochondria and lysosomes.

T-type Ca²âº signalling downregulates MEK1/2 phosphorylation and cross-talk with the RAAS transcriptional response in cardiac myocytes.

Cardiac dysfunction is often associated with an increase in the activity of the renin-angiotensin II-aldosterone system (RAAS). Here, we highlight the cross-talk between the Ca(2+) signalling generated by cardiac T-type current (I(CaT)) and RAAS signalling. Neonatal rat cardiomyocytes exposed to aldosterone, angiotensin II or aldosterone plus angiotensin II co-treatment (AA) show an increase in I(CaT) density, with no cumulative effect of the AA co-treatment. AA increases the amount of T-type channel Ca(v)3.1 mRNA in a time-dependent manner. Angiotensin II increases Ca(v)3.1 mRNA stability, whereas aldosterone increases the transcriptional activity of the Ca(v)3.1 gene promoter. However, in AA-treated cells, angiotensin II decreases aldosterone-induced promoter activity, and aldosterone decreases angiotensin II-induced mRNA stability. The mitogen-activated protein kinase kinase (MEK1/2), which is synergically phosphorylated in AA-treated cells, alters the translocation of glucocorticoid receptors (GR) into the nucleus and attenuates aldosterone-induced promoter activity. In contrast, MEK1/2 has no effect on the NFkB-induced increase in Ca(v)3.1 mRNA and MEK1/2 promoted CREB-target gene transcription. Aldosterone and AA-induced I(CaT) signalling result in a time-dependent activation of the phosphatase PP2A, which dephosphorylates MEK1/2 and CREB. Finally, angiotensin II alone also activates PP2A, which targets MEK1/2, but this activation is independent of I(CaT) calcium signalling and has no effect on CREB phosphorylation. In conclusion, our data demonstrate the cross-talk between a GR-mediated aldosterone response, angiotensin II and the I(CaT) signalling pathways and identify MEK1/2 as a point of connection. This cross-talk results in the fine control of GR- and/or CREB-target gene expression.

Screening for calcium channel modulators in CLN3 siRNA knock down SH-SY5Y neuroblastoma cells reveals a significant decrease of intracellular calcium levels by selected L-type calcium channel blockers.

BACKGROUND: Defects of the CLN3 gene on chromosome 16p12.1 lead to the juvenile form of neuronal ceroid-lipofuscinosis (JNCL, Batten Disease), the most common recessive inherited neurodegenerative disorder in children. Dysregulation of intracellular calcium homeostasis in the absence of a functional CLN3 protein (CLN3P, Battenin) has been linked to synaptic dysfunction and accelerated apoptosis in vulnerable neuronal cells. Prolonged increase of intracellular calcium concentration is considered to be a significant trigger for neuronal apoptosis and cellular loss in JNCL. METHODS: We examined the potential effect of 41 different calcium channel modulators on intracellular calcium concentration in CLN3 siRNA knock down SH-SY5Y neuroblastoma cells. RESULTS: Six drugs belonging to the group of voltage dependent L-type channel blockers show significant lowering of the increased intracellular calcium levels in CLN3 siRNA knock down cells. CONCLUSIONS: Our studies provide important new data suggesting possible beneficial effects of the tested drugs on calcium flux regulated pathways in neuronal cell death. Therapeutic intervention in this untreatable disease will likely require drugs that cross the blood-brain barrier as did all of the positively screened drugs in this study. GENERAL SIGNIFICANCE: Better comprehension of the mechanism of neurodegeneration in rare recessive disorders, such as neuronal ceroid-lipofuscinoses, is likely to help to better understand mechanisms involved in more complex genetic neurodegenerative conditions, such as those associated with aging.

Flunarizine suppresses Mycobacterium tuberculosis growth via calmodulin-dependent phagosome maturation.

Tuberculosis (TB), an infectious bacterial disease caused by Mycobacterium tuberculosis (Mtb), is a major cause of death worldwide. Multidrug-resistant TB remains a public health crisis and thus novel effective treatments, such as host-directed therapies (HDTs), are urgently required to overcome the challenges of TB infection. In this study, we evaluated 4 calcium modulators for their effects on Mtb growth in macrophages. Only flunarizine enhanced the bactericidal ability of macrophages against Mtb, which was induced by an increase in phosphorylated calcium/calmodulin (CaM)-dependent protein kinase II (pCaMKII) levels. We further discovered that the expression of CaM was decreased in Mtb-infected macrophages and restored following flunarizine treatment; this was associated with phagolysosome maturation and acidification. Consistent with these findings, the anti-TB ability of macrophages was reduced following the silencing of CaM or inhibition of CAMKII activity. In conclusion, our results demonstrated that flunarizine enhanced the bactericidal ability of macrophages and clarified its CaM-pCAMKII-dependent mechanism. Therefore, our findings strongly support further studies of this currently approved drug as an HDT candidate for TB therapy.

Reno-protective role of flunarizine (mitochondrial permeability transition pore inactivator) against gentamicin induced nephrotoxicity in rats.

This study was aimed to evaluate the role of flunarizine on gentamicin (GEM) induced nephrotoxicity in rat. Administration of GEM (40 mg/kg, s.c. for 10 consecutive days) significantly increased blood urea nitrogen (BUN), N-acetyl ß-d-glucosaminidase (NAG), thiobarbituric acid reactive substances (TBARS) and total calcium whereas, decreased body weight, fractional excretion of sodium (FrNa), creatinine clearance (CrCl), reduced glutathione (GSH), mitochondrial cytochrome c oxidase (Cyt-C oxidase) and ATP levels resulting in nephrotoxicity. Further, flunarizine (100, 200 and 300 µmol/kg, p.o.) was administered to evaluate its renoprotective effect against GEM induced nephrotoxicity and the results were compared with cylcosporin A (CsA, 50 µmol/kg, p.o.). Flunarizine resulted in the attenuation of renal dysfunction and oxidative marker changes in rats subjected to GEM induced nephrotoxicity in a dose dependent manner. Medium and higher doses of flunarizine produced significant renal protective effect which was comparable to cyclosporin A. The results of this study clearly revealed that flunarizine protected the kidney against the nephrotoxic effect of GEM via mitochondrial permeability transition pore (MPTP) inactivation potential.

[The pharmacological mechanism of gastrodin on calcitonin gene-related peptide of cultured rat trigeminal ganglion].

The Chinese herbal medicine Tianma (Gastrodia elata) has been used for treating and preventing primary headache over thousands of years, but the exact pharmacological mechanism of the main bioactive ingredient gastrodin remains unclear. In present study, the effects of gastrodin on calcitonin gene-related peptide (CGRP) and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) expression were observed in rat trigeminal ganglion (TG) after in vitro organ culture to explore the underlying intracellular mechanism of gastrodin on primary vascular-associated headache. CGRP-immunoreactivity (CGRP-ir) positive neurons count, positive area, mean optical density and integrated optical density by means of immunohistochemistry stain were compared at different concentrations of gastrodin, which was separately co-incubated with DMEM in SD rat TG for 24 hours. Only at 5 or 10 mmol L(-1) concentration, gastrodin demonstrated significantly concentration-dependent reduction of CGRP-ir (+) expression and its action closed to 1.2 mmol L(-1) sumatriptan succinate. While at 2.5, 20, and 40 mmol L(-1) concentration, gastrodin did not show remarkable effects on CGRP-ir (+) expression. The optimal concentration of gastrodin (5 and 10 mmol L(-1)) similarly inhibited CGRP-mRNA expression level separately compared with 1.2 mmol L(-1) sumatriptan succinate and 10 micromol L(-1) flunarizine hydrochloride, which was quantitatively analyzed by real-time PCR (RT-PCR). pERK1/2 level was examined by Western blotting after co-cultured with optimal concentration of gastrodin and effective specific ERK1/2 pathway inhibitors PD98059, U0126. The result indicated that gastrodin significantly reduced pERK1/2 protein actions similarly to ERK1/2 pathway specific blockade. It suggests ERK1/2 signaling transduction pathway may be involved in gastrodin intracellular mechanism. This study indicates gastrodin (5 and 10 mmol L(-1)) can remarkably reduce CGRP-ir (+) neuron, CGRP-mRNA and pERK1/2 expression level in cultured rat TG, with its actions similar to the effective concentration of sumatriptan succinate, flunarizine hydrochloride and specific ERK1/2 pathway blocker. The intracellular signaling transduction ERK1/2 pathway may be involved in the gastrodin reducing CGRP up-regulation in rat TG after organ culture.

Protection of flunarizine on cerebral mitochondria injury induced by cortical spreading depression under hypoxic conditions.

A rat cortical spreading depression (CSD) model was established to explore whether cerebral mitochondria injury was induced by CSD under both normoxic and hypoxic conditions and whether flunarizine had a protective effect on cerebral mitochondria. SD rats, which were divided into seven groups, received treatment as follows: no intervention (control Group I); 1 M NaCl injections (Group II); 1 M KCl injections (Group III); intraperitoneal flunarizine (3 mg/kg) 30 min before KCl injections (Group IV); 14% O(2) inhalation before NaCl injections (Group V); 14% O(2) inhalation followed by KCl injections (Group VI); 14% O(2) inhalation and intraperitoneal flunarizine followed by KCl injections (Group VII). Following treatment, brains were removed for the analysis of mitochondria transmembrane potential (MMP) and oxidative respiratory function after recording the number, amplitude and duration of CSD. The duration of CSD was significantly longer in Group VI than that in Group III. The number and duration of CSD in Group VII was significantly lower than that in Group VI. MMP in Group VI was significantly lower than that in Group III, and MMP in Group VII was significantly higher than that in Group VI. State 4 respiration in Group VI was significantly higher than that in Group III, and state 3 respiration in Group VII was significantly higher than that in Group VI. Respiration control of rate in Group VII was also significantly higher than that in Group VI. Thus, we concluded that aggravated cerebral mitochondria injury might be attributed to CSD under hypoxic conditions. Flunarizine can alleviate such cerebral mitochondria injury under both normoxic and hypoxic conditions.

Flunarizine, a drug approved for treating migraine and vertigo, exhibits cytotoxicity in GBM cells.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with a poor prognosis. The current treatment regimen, including surgical resection, radiation, and temozolomide (TMZ) chemotherapy, is still not curative. Therefore, there is an emerging need to develop a drug to treat GBM or synergistic enhance TMZ effect on GBM cells. Flunarizine (FLN), a drug approved for treating migraine and vertigo, was analyzed for its cytotoxicity and synergistic effect with TMZ on GBM cells in this study. Cell proliferation, clonogenic assay, flow cytometry, and Western blotting were used to determine the effects of FLN on three GBM cells, U-87 MG, LN-229, and U-118 MG cells. We found that FLN induced GBM cell death. FLN also interfered with U-87 MG cell cycle progression. Flow cytometric analysis showed an increase of apoptotic cells after FLN treatment. Caspase 9, caspase 3, and Poly (ADP-ribose) polymerase (PARP) activation were involved in apoptosis induction in U-87 MG and LN-229, suggesting the possible involvement of an intrinsic apoptotic pathway. We found that FLN treatment inhibited Akt pathway activation in U-87 MG cells, and synergistically increased the cytotoxicity of three GBM cells when combined with TMZ treatment. In conclusion, our current data suggested that FLN inhibited cell viability by inducing apoptosis. FLN inhibited Akt activation and enhanced the sensitivity of GBM cells to TMZ. These findings may provide important information regarding the application of FLN in GBM treatment in the future.

The Ca(2+) channel blocker flunarizine induces caspase-10-dependent apoptosis in Jurkat T-leukemia cells.

Flunarizine is a Ca(2+) channel blocker that can be either cytoprotective or cytotoxic, depending on the cell type that is being examined. We show here that flunarizine was cytotoxic for Jurkat T-leukemia cells, as well as for other hematological maligancies, but not for breast or colon carcinoma cells. Treatment of Jurkat cells with flunarizine resulted in caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and laddering of DNA fragments, all of which are hallmarks of apoptosis. Flunarizine-induced DNA fragmentation was inhibited by the caspase-3 inhibitor z-DEVD-fmk, the caspase-8/caspase-10 inhibitor z-IETD-fmk, and the caspase-10 inhibitor z-AEVD-fmk, but was not reduced in caspase-8-deficient Jurkat cells, indicating the involvement of caspase-10 upstream of caspase-3 activation. Interestingly, FADD recruitment to a death receptor was not involved since flunarizine caused DNA fragmentation in FADD-deficient Jurkat cells. Flunarizine treatment of Jurkat cells also resulted in reactive oxygen species production, dissipation of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, and caspase-9 activation, although none of these events were necessary for apoptosis induction. Collectively, these findings indicate that flunarizine triggers apoptosis in Jurkat cells via FADD-independent activation of caspase-10. Flunarizine warrants further investigation as a potential anti-cancer agent for the treatment of hematological malignancies.