Sudden arrhythmia death syndrome in young victims: a five-year retrospective review and two-year prospective molecular autopsy study by next-generation sequencing and clinical evaluation of their first-degree relatives.

OBJECTIVE: Sudden arrhythmia death syndrome (SADS) accounts for about 30% of causes of sudden cardiac death (SCD) in young people. In Hong Kong, there are scarce data on SADS and a lack of experience in molecular autopsy. We aimed to investigate the value of molecular autopsy techniques for detecting SADS in an East Asian population. METHODS: This was a two-part study. First, we conducted a retrospective 5-year review of autopsies performed in public mortuaries on young SCD victims. Second, we conducted a prospective 2-year study combining conventional autopsy investigations, molecular autopsy, and cardiac evaluation of the first-degree relatives of SCD victims. A panel of 35 genes implicated in SADS was analysed by next-generation sequencing. RESULTS: There were 289 SCD victims included in the 5-year review. Coronary artery disease was the major cause of death (35%); 40% were structural heart diseases and 25% were unexplained. These unexplained cases could include SADS-related conditions. In the 2-year prospective study, 21 SCD victims were examined: 10% had arrhythmogenic right ventricular cardiomyopathy, 5% had hypertrophic cardiomyopathy, and 85% had negative autopsy. Genetic analysis showed 29% with positive heterozygous genetic variants; six variants were novel. One third of victims had history of syncope, and 14% had family history of SCD. More than half of the 11 first-degree relatives who underwent genetic testing carried related genetic variants, and 10% had SADS-related clinical features. CONCLUSION: This pilot feasibility study shows the value of incorporating cardiac evaluation of surviving relatives and next-generation sequencing molecular autopsy into conventional forensic investigations in diagnosing young SCD victims in East Asian populations. The interpretation of genetic variants in the context of SCD is complicated and we recommend its analysis and reporting by qualified pathologists.

Overexpression of Lon contributes to survival and aggressive phenotype of cancer cells through mitochondrial complex I-mediated generation of reactive oxygen species.

Lon protease is a multifunction protein and operates in protein quality control and stress response pathways in mitochondria. Human Lon is upregulated under oxidative and hypoxic stresses that represent the stress phenotypes of cancer. However, little literature undertakes comprehensive and detailed investigations on the tumorigenic role of Lon. Overexpression of Lon promotes cell proliferation, apoptotic resistance to stresses, and transformation. Furthermore, Lon overexpression induces the production of mitochondrial reactive oxygen species (ROS) that result from Lon-mediated upregulation of NDUFS8, a mitochondrial Fe-S protein in complex I of electron transport chain. Increased level of mitochondrial ROS promotes cell proliferation, cell survival, cell migration, and epithelial-mesenchymal transition through mitogen-activated protein kinase (MAPK) and Ras-ERK activation. Overall, the present report for the first time demonstrates the role of Lon overexpression in tumorigenesis. Lon overexpression gives an apoptotic resistance to stresses and induces mitochondrial ROS production through Complex I as signaling molecules to activate Ras and MAPK signaling, giving the survival advantages and adaptation to cancer cells. Finally, in silico and immunohistochemistry analysis showed that Lon is overexpressed specifically in various types of cancer tissue including oral cancer.

Initial experience of cryoballoon catheter ablation for atrial fibrillation in Hong Kong.

OBJECTIVE. To report the initial experience in using cryoballoon catheter ablation in the treatment of atrial fibrillation in Hong Kong. DESIGN. Single-centre, prospective case series. SETTING. Regional hospital, Hong Kong. PATIENTS. Sixteen patients (mean age, 55 years; standard deviation, 14 years; 11 males) with paroxysmal (n=12) or persistent (n=4) atrial fibrillation. INTERVENTIONS. Pulmonary vein isolation by ablation with a 28-mm cryoballoon catheter. MAIN OUTCOME MEASURES. Safety, effectiveness, and learning curve of this procedure. RESULTS. Of 67 pulmonary veins, 61 (91%) could be successfully isolated with the cryoballoon alone. The remaining pulmonary veins were isolated with additional ablation using an 8-mm tip cryocatheter. One phrenic nerve palsy developed during right middle pulmonary vein ablation, which resolved. Another patient endured a minor guidewire dissection of the right inferior pulmonary vein. The mean (standard deviation) procedural and fluoroscopic times were 231 (32) and 62 (18) minutes, respectively. On comparing the first nine and last seven procedures, there was a significant improvement in procedural time (mean [standard deviation], 244 [32] vs 213 [24] minutes; P=0.04) and in the fluoroscopic time (70 [21] vs 51 [7] minutes; P=0.038). With a median follow-up of 21 months, nine (75%) of the 12 patients with paroxysmal atrial fibrillation and one (25%) of those four with persistent atrial fibrillation had no recurrence, without the use of anti-arrhythmic drugs. CONCLUSIONS. Pulmonary vein isolation by cryoballoon catheter ablation is safe and effective in treating patients with paroxysmal, but not for patients with persistent atrial fibrillation. A relatively short learning curve of around 10 cases was deemed appropriate.

Is a combined enforcement and penalty strategy effective in combating red light violations? An aggregate model of violation behavior in Hong Kong.

Red light violations are a major cause of traffic crashes at signalized intersections. In Hong Kong, prosecutions for red light violations have increased in the past decade. An automated enforcement camera system has been established to combat this prohibited driver behavior. In addition, both demerit points and financial penalties were revised upwards in 2006 to strengthen the deterrent effect of the system. An observational study of driver tendency to run a red light was conducted to evaluate the effectiveness of the combined penalty and camera strategy. Both the short- and long-term effects of the strategy on red light violations were estimated. The influences of factors including temporal variation, the presence of a red light camera, geometric design, and traffic control type were also determined with an aggregate count data model. The results show that the frequency of red light violations significantly decreased after the implementation of the new penalty system, and that the reduction remained significant one year after implementation. Interestingly, no evidence was found for an association between the frequency of red light violations and the presence of a red light camera.

Gossypol, a component in cottonseed, induced increases in cytosolic Ca2+ levels in Chang liver cells.

The effect of gossypol, a compound found in cottonseed, on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. Gossypol (0.2-5microM) increased [Ca2+](i) in a concentration-dependent manner with an EC(50) value of 1.5microM. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase within 5min after drug application. Removal of extracellular Ca2+ markedly reduced the [Ca2+](i) signals by 80+/-2%. Preincubation with 0.1mM La3+ or 10microM nimodipine abolished the Ca2+ influx. Gossypol (5microM)-induced release of intracellular Ca2+ was reduced by 75% by pretreatment with 1microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+. Conversely, pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. After pretreatment with 5microM gossypol in Ca2+-free medium for several min, addition of 3mM Ca2+ induced a [Ca2+](i) increase of a magnitude nine-fold greater than control. Gossypol (5microM)-induced Ca2+ release was not affected by inhibiting phospholipase C with 2microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Together, this study shows that gossypol induced significant [Ca2+](i) increases in Chang liver cells by releasing Ca2+ from intracellular pools in a phospholipase C-dissociated fashion and by causing La3+- and nimodipine-sensitive Ca2+ influx.

Stimulation of the BK(Ca) channel in cultured smooth muscle cells of human trachea by magnolol.

BACKGROUND: Magnolol, a compound isolated from the cortex of Magnolia officinalis, has been found to possess anti-allergic and anti-asthmatic activity. METHODS: The effect of magnolol on ionic currents was studied in cultured smooth muscle cells of human trachea with the aid of the patch clamp technique. RESULTS: In whole cell current recordings magnolol reversibly increased the amplitude of K+ outward currents. The increase in outward current caused by magnolol was sensitive to inhibition by iberiotoxin (200 nM) or paxilline (1 microM) but not by glibenclamide (10 microM). In inside out patches, magnolol added to the bath did not modify single channel conductance but effectively enhanced the activity of large conductance Ca2+ activated K+ (BK(Ca)) channels. Magnolol increased the probability of these channel openings in a concentration dependent manner with an EC50 value of 1.5 microM. The magnolol stimulated increase in the probability of channels opening was independent of internal Ca2+. The application of magnolol also shifted the activation curve of BK(Ca) channels to less positive membrane potentials. The change in the kinetic behaviour of BK(Ca) channels caused by magnolol in these cells is the result of an increase in dissociation and gating constants. CONCLUSIONS: These results provide evidence that, in addition to the presence of antioxidative activity, magnolol is potent in stimulating BK(Ca) channel activity in tracheal smooth muscle cells. The direct stimulation of these BK(Ca) channels by magnolol may contribute to the underlying mechanism by which it acts as an anti-asthmatic compound.

Mechanism underlying histamine-induced intracellular Ca2+ movement in PC3 human prostate cancer cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca2+ dye. Histamine at concentrations between 0.1 and 50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1 microM. The [Ca2+]i response comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In the absence of extracellular Ca2+, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 10 microM histamine did not increase [Ca2+]i. After pretreatment with 10 microM histamine in a Ca2+-free medium for several minutes, addition of 3 mM Ca2+ induced [Ca2+]i increases. Histamine (10 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17 beta-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 microM pyrilamine but was not altered by 50 microM cimetidine. Collectively, the present study shows that histamine induced [Ca2+]i transients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca2+ release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca2+ entry.

Effect of oleamide on Ca(2+) signaling in human bladder cancer cells.

The effect of oleamide, a sleep-inducing endogenous lipid in animal models, on intracellular free levels of Ca(2+) ([Ca(2+)](i)) in non-excitable and excitable cells was examined by using fura-2 as a fluorescent dye. [Ca(2+)](i) in pheochromocytoma cells, renal tubular cells, osteoblast-like cells, and bladder cancer cells were increased on stimulation of 50 microM oleamide. The response in human bladder cancer cells (T24) was the greatest and was further explored. Oleamide (10-100 microM) increased [Ca(2+)](i) in a concentration-dependent fashion with an EC(50) of 50 microM. The [Ca(2+)](i) signal comprised an initial rise and a sustained plateau and was reduced by removing extracellular Ca(2+) by 85 +/- 5%. After pre-treatment with 10-100 microM oleamide in Ca(2+)-free medium, addition of 3 mM Ca(2+) increased [Ca(2+)](i) in a manner dependent on the concentration of oleamide. The [Ca(2+)](i) increase induced by 50 microM oleamide was reduced by 100 microM La(3+) by 40%, but was not altered by 10 microM nifedipine, 10 microM verapamil, and 50 microM Ni(2+). In Ca(2+)-free medium, pre-treatment with thapsigargin (1 microM), an endoplasmic reticulum Ca(2+) pump inhibitor, abolished 50 microM oleamide-induced [Ca(2+)](i) increases; conversely, pretreatment with 50 microM oleamide reduced 1 microM thapsigargin-induced [Ca(2+)](i) increases by 50 +/- 3%. Suppression of the activity of phospholipase C with 2 microM U73122 failed to alter 50 microM oleamide-induced Ca(2+) release. Linoleamide (10-100 microM), another sleep-inducing lipid with a structure similar to that of oleamide, also induced an increase in [Ca(2+)](i). Together, it was shown that oleamide induced significant [Ca(2+)](i) increases in cells by a phospholipase C-independent release of Ca(2+) from thapsigargin-sensitive stores and by inducing Ca(2+) entry.

Rutaecarpine-induced block of delayed rectifier K+ current in NG108-15 neuronal cells.

The effects of rutaecarpine on ionic currents of NG108-15 neuronal cells were investigated in this study. Rutaecarpine (2-100 microM) suppressed the amplitude of delayed rectifier K+ current (I(K(DR))) in a concentration-dependent manner. The IC50 value for rutaecarpine-induced inhibition of I(K(DR)) was 11 microM. I(K(DR)) present in these cells is sensitive to the inhibition by quinidine and dendrotoxin, yet not by E-4031. The presence of rutaecarpine enhanced the rate and extent of I(K(DR)) inactivation, although it had no effect on the initial activation phase of I(K(DR)). Recovery from block by rutaecarpine (5 microM) was fitted by a single exponential with a value of 2.87 s. Crossover of tail currents in the presence of rutaecarpine was also observed. Cell-attached single-channel recordings revealed that rutaecarpine decreased channel activity, but it did not alter single-channel amplitude. With the aid of the binding scheme, a quantitative description of the rutaecarpine actions on I(K(DR)) was provided. However, rutaecarpine (20 microM) had no effect on L-type Ca2+ current. Under current-clamp configuration, rutaecarpine prolonged action potential duration in NG108-15 cells. These results show that rutaecarpine is a blocker of the K(DR) channel. The increase in action potential duration induced by rutaecarpine can be explained mainly by its blocking actions on I(K(DR)).

Ceramide inhibits the inwardly rectifying potassium current in GH(3) lactotrophs.

The effects of ceramide on ion currents in rat pituitary GH(3) cells were investigated. Hyperpolarization-elicited K(+) currents present in GH(3) cells were studied to determine the effect of ceramide and other related compounds on the inwardly rectifying K(+) current (I(K(IR))). Ceramide (C(2)-ceramide) suppressed the amplitude of I(K(IR)) in a concentration-dependent manner, with an IC(50) value of 5 microM. Ceramide caused a rightward shift in the midpoint for the activation curve of I(K(IR)). Pretreatment with PD-98059 (30 microM) or U-0126 (30 microM) did not prevent ceramide-mediated inhibition of I(K(IR)). However, the magnitude of ceramide-induced inhibition of I(K(IR)) was attenuated in GH(3) cells preincubated with dithiothreitol (10 microM). TNF alpha (100 ng/g) also suppressed I(K(IR)). In the inside-out configuration, application of ceramide (30 microM) to the bath slightly suppressed the activity of large conductance Ca(2+)-activated K(+) channels. Under the current clamp mode, ceramide (10 microM) increased the firing of action potentials. Cells that exhibited an irregular firing pattern were converted to those displaying a regular firing pattern after application of ceramide (10 microM). Ceramide also suppressed I(K(IR)) in neuroblastoma IMR-32 cells. Therefore, ceramide can produce a depressant effect on I(K(IR)). The blockade of this current by ceramide may affect cell function.

Effects of the antianginal drug fendiline on Ca2+ movement in hepatoma cells.

This study investigated the effect of the anti-anginal drug, fendiline, on intracellular free Ca2+ levels ([Ca2+]i) in HA/ 22 human hepatoma cells by using fura-2 as a fluorescent Ca2+ dye. Fendiline (1-100 microM) increased [Ca2+]i with an EC50 of 25 microM. Removal of extracellular Ca2+ reduced the [Ca2+]i signals by 51 +/- 5%. Fendiline (10 microM)-induced Ca2+ release was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) did not alter 10 microM fendiline-induced Ca2+ release. Several other calmodulin antagonists, such as phenoxybenzamine (100-200 microM), trifluoperazine (5-50 microM), and fluphenazine-N-chloroethane (2-100 microM), had no effect on [Ca2+]i. Together, it was found that fendiline increased [Ca2+]i in human hepatoma cells by discharging Ca2+ from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca2+ entry. This effect of fendiline does not appear to be via antagonism of calmodulin.

Characterization of action potential waveform-evoked L-type calcium currents in pituitary GH3 cells.

The response of the L-type Ca2+ current (ICa,L) in pituitary GH3 cells to variations in the action potential (AP) waveform was examined using the whole-cell configuration of the patch-clamp technique. ICa,L evoked during an AP waveform exhibited an early and a late component. The early component occurred on the rising phase of the AP; the late component coincided with the falling phase. Prolonging the falling phase of the AP increased the Ca2+ charge carried by ICa,L, although the amplitude of the late ICa,L was reduced. Prolonging the peak voltage of the AP waveform, however, increased the amplitude of the late component. ICa,L inactivated during a train of AP waveforms. When Ba2+ was used as the charge carrier, current inactivation during a train of APs decreased. Likewise, ICa,L evoked by the AP templates with irregular bursting pattern was inactivated. When the repetitive firing of APs with depolarizing potentials was replayed to cells, Ca2+ entry was not only spread over the entire AP, but also occurred during the interspike voltage trajectory. After application of thyrotropin releasing hormone (TRH; 10 microM), ICa,L in response to rectangular pulses was increased and the current/voltage relation shifted slightly to more negative values. TRH (10 microM), thapsigargin (10 microM) or cyclopiazonic acid (30 microM) enhanced the late component of the AP-evoked ICa,L. TRH also attenuated the inactivation of ICa,L during a train of APs. These results indicate that in pituitary GH3 cells, the time course and kinetics of ICa,L during the AP waveforms is distinct from that evoked by rectangular voltage clamp. Changes in the shape and firing pattern of APs in GH3 cells can modulate Ca2+ influx through L-type Ca2+ channels. Ca2+ release from internal stores may affect the magnitude of AP-evoked ICa,L in these cells.

Fendiline, an anti-anginal drug, increases intracellular Ca2+ in PC3 human prostate cancer cells.

BACKGROUND: The effects of the anti-anginal drug fendiline on intracellular Ca2+ concentrations ([Ca2+]i) in human PC3 prostate cancer cells were examined. METHODS: [Ca2+]i was measured using the fluorescent dye fura-2. RESULTS: Fendiline (0.5-100 microM) increased [Ca2+]i in a concentration-dependent manner. Ca2+ removal partly inhibited the Ca2+ signals. In Ca2+-free medium, pretreatment with 100 microM fendiline inhibited most of the [Ca2+]i increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and pretreatment with thapsigargin abolished the fendiline-induced [Ca2+]i increases. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.5-200 microM fendiline in Ca2+-free medium. Pretreatment with 1 microM U73122 to block the formation of inositol-1.4.5-trisphosphate (IP3) did not alter fendiline-induced internal Ca2+ release. CONCLUSIONS: The anti-anginal drug fendiline induced internal Ca2+ release and external Ca2+ entry. Because prolonged increases in [Ca2+]i may lead to cell injury and death, the long-term effect of fendiline on the function of prostate cancer cells should be investigated.

Sensitivity of diffusion-weighted magnetic resonance imaging in the diagnosis of acute lacunar infarcts.

BACKGROUND AND PURPOSE: Heightened interest in the early diagnosis and treatment of acute stroke challenges neuroimaging specialists to optimize available modalities and to develop new techniques for the evaluation of cerebrovascular disease. The purpose of this study was to evaluate the sensitivity of diffusion-weighted (DW) magnetic resonance (MR) imaging in detecting early small infarcts and in differentiating acute from nonacute small infarcts when conventional MR imaging demonstrates multiple small infarcts. METHODS: Thirty-eight consecutive patients with a clinical diagnosis of lacunar infarcts (20 men and 18 women, aged 50-79 yr) who underwent DW MR imaging within 3 days of symptom onset were enrolled in this study. All patients underwent both conventional fast spin-echo (FSE) MR imaging and DW MR imaging. Apparent diffusion coefficient (ADC) maps were also acquired. All patients had at least one of the following classic lacunar syndromes: pure motor hemiparesis, ataxic hemiparesis, dysarthria-clumsy hand, pure sensory stroke, and sensorimotor stroke. RESULTS: Thirty-six patients (40 acute lesions) had focal areas of high intensity on DW MR imaging associated with their clinical symptoms. Acute lacunar infarcts were seen on DW MR imaging as bright areas of decreased ADC ratio (range 0.31-0.85, mean 0.64). Lesion conspicuity with DW MR imaging was superior to that with FSE in 33 acute lesions. In four patients with small hyperacute (within 6 hours) infarcts, DW MR imaging was particularly sensitive for infarcts that were not visible on FSE sequences. The sensitivity of DW MR imaging and ADC map for acute lacunar infarcts was 95%, specificity 94%, positive predictive value 97%, negative predictive value 90%, and accuracy 95%. In 15 patients with both acute and nonacute old small infarcts, DW MR imaging and ADC map could easily distinguish the new infarct from adjacent old ones, although this distinction was difficult to make with FSE. CONCLUSIONS: DW MR imaging accompanied by ADC map is a sensitive diagnostic modality for hyperacute and acute lacunar infarcts. It is also sensitive in distinguishing fresh small infarcts from adjacent multiple old infarcts.

Tamoxifen-induced Ca2+ mobilization in bladder female transitional carcinoma cells.

This study examined the effect of tamoxifen, an anti-breast cancer drug, on Ca2+ handling in bladder female transitional cancer cells. Changes in cytosolic free Ca2+ levels were recorded by using the Ca2+-sensitive dye fura-2. In a dose-dependent manner, tamoxifen induced intracellular free Ca2+ concentrations ([Ca2+]i) increases between 5 and 20 microM with an EC50 of 10 microM. External Ca2+ removal reduced the response by 60+/-6%. Addition of 3 mM Ca2+ caused a [Ca2+]i increase after pretreatment with 10 microM tamoxifen in Ca2+-free medium. In Ca2+-free medium, pretreatment with 10 microM tamoxifen abolished the [Ca2+]i increase induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 1 microM thapsigargin prevented tamoxifen from releasing more Ca2+. Inhibition of phospholipase C-dependent inositol 1,4,5-tris-phosphate formation with 2 microM U73122 did not alter 10 microM tamoxifen-induced Ca2+ release. The [Ca2+]i increase induced by 5 microM tamoxifen was not altered by 10 microM La3+, nifedipine, verapamil, and diltiazem. Collectively, it was found that tamoxifen increased [Ca2+]i in bladder cancer cells by releasing Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity, and by inducing Ca2+ entry from external medium.

Effect of fluoxetine on intracellular Ca2+ levels in bladder female transitional carcinoma (BFTC) cells.

The effect of the antidepressant fluoxetine on Ca2+ signaling in cultured cells was largely unknown. The effect of various concentrations of fluoxetine on [Ca 2+] i in populations of bladder female transitional cancer (BFTC) cells was evaluated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca 2+] i concentration dependently (20-100 microM) with an EC50 value of 30 microM. The response was inhibited by 50-60% on extracellular Ca2+ removal. In Ca2+ -free medium, pretreatment with 1 microM thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ pump) abolished 50 microM fluoxetine-induced Ca2+ release; whereas pretreatment with fluoxetine did not alter the thapsigargin-induced Ca2+ response. Addition of 3 mM Ca2+ increased [Ca 2+] i after pretreatment with 50 microM fluoxetine in Ca2+ -free medium, suggestive of fluoxetine-induced capacitative Ca2+ entry. Suppression of inositol 1,4,5-trisphosphate formation by 2 microM U73122 (a phospholipase C inhibitor) did not affect 50 microM fluoxetine-induced Ca2+ release. Collectively, this study shows that fluoxetine increased [Ca 2+] i in bladder cancer cells in a concentration-dependent fashion, by releasing Ca2+ from thapsigargin-sensitive Ca2+ stores in an IP3-independent manner, and by inducing Ca2+ influx from extracellular medium.

Fendiline-Induced Ca2+ movement in A10 smooth muscle cells.

The effect of fendiline, an anti-anginal drug, on cytosolic free Ca2+ levels ([Ca2+]i) in A10 smooth muscle cells was explored by using fura-2 as a Ca2+ indicator. Fendiline at concentrations between 10-50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 of 20 microM. External Ca2+ removal reduced the Ca2+ signal by 75%. Addition of 3 mM Ca2+ increased [Ca2+]i in cells pretreated with fendiline in Ca2+-free medium. The 50 microM fendiline-induced [Ca2+]i increase in Ca2+-containing medium was inhibited by 10 microM of La3+, nifedipine, or verapamil. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ store partly inhibited 50 microM fendiline-induced Ca2+ release; whereas pretreatment with 50 microM fendiline abolished 1 microM thapsigargin-induced Ca2+ release. Inhibition of phospholipase C activity with 2 microM U73122 did not alter 50 microM fendiline-induced Ca2+ release. Incubation with 50 microM fendiline for 10-30 min decreased cell viability by 10-20%. Together, the findings indicate that in smooth muscle cells fendiline induced [Ca2+]i increases. Fendiline acted by activating Ca2+ influx via L-type Ca2+ channels, and by releasing internal Ca2+ in a phospholipase C-independent manner. Prolonged exposure of cells to fendiline induced cell death.