Preparation of drug-loaded chitosan microspheres repair materials.

OBJECTIVE: With the development of society and the progress of science and technology, microspheres, as a new polymer material, have been applied to all aspects of human beings. Microspheres can play a huge role in food safety, electronic technology, sewage treatment, biomedicine, etc., and are non-toxic or harmless. There are three main types of substrates for the preparation of microspheres: natural polymers, semi-synthetic polymer materials, and synthetic polymer materials. MATERIALS AND METHODS: In this study, the inorganic material kaolin was modified by the emulsification-crosslinking method with chitosan and composite microspheres with large interlayer spacing were prepared, which were characterized by Fourier Transform Ioncyclotron Resonancel (FTIR) analysis and Scanning Electron Microscope (SEM). The prepared kaolin/chitosan microspheres were then placed in different amounts of aspirin and the optimal dose was investigated by encapsulation efficiency and drug loading rate. The drug release rate of 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 6 h, and 12 h was then determined by simulating the human colon to determine the performance of the sustained-release drug. RESULTS: The experimental results showed that after the prepared composite microspheres were loaded with aspirin drug, we got the optimal dosage of 0.1 g by discussing the encapsulation efficiency and drug loading rate of the drug-loaded microspheres, and the encapsulation efficiency reached 80.80%, while the drug loading rate was 24.40%, the drug release capacity reached about 83% in about 12 hours. CONCLUSIONS: The research shows that the kaolin/chitosan drug-loaded microspheres prepared by the emulsification and cross-linking method are excellent drug-loading materials.

[Reconstruction of anal stenosis induced by scar contracture after repair of defect in perineal region with paraumbilical flap using random pattern flap].

Objective: To investigate the method and timing of reconstruction of anal stenosis induced by scar contracture after repair of defect in perineal region with paraumbilical flap using random pattern flap. Methods: Ten patients who suffered anal stenosis induced by scar contracture after the first phase repair of defect of perineal region with paraumbilical flap were hospitalized from July 2009 to September 2015. Eight patients were with central type scar contracture of perineal region after healing of burn wound, and two patients were with lesion of perineal region which had been excised. In 6 to 8 weeks after the first phase surgery, two or three random pattern flaps were designed around the narrow anus in the survived paraumbilical flap. After thorough release of the narrow anus, the random pattern flaps were transferred to enlarge the anus. The tip of the lifted triangular flap was transferred to insert into the anal canal. The skin of anal canal or rectal mucosa was pulled out to be crossly-stitched with the flap. Results: All the patients' narrow anuses were released and enlarged with one operation, and the diameters of the narrow anuses were enlarged to 2.0 to 3.0 cm. During follow-up of 6 to 36 months, the anal stenosis was totally released, and the symptom of difficult defecation was significantly improved; 7 patients were excellent and 3 patients were good with evaluation of clinical criteria of anus function; no symptom of anal stenosis or rectal mucosal prolapse was observed any more. Conclusions: In 6 to 8 weeks post repair of defect in perineal region with paraumbilical flap, designing of random pattern flap in the survived paraumbilical flap to enlarge and reconstruct the narrow anus has good therapeutic effects on anatomical narrow and difficult defecation.

Long-term exposure of K562 cells to benzene metabolites inhibited erythroid differentiation and elevated methylation in erythroid specific genes.

Benzene is a common occupational hazard and a widespread environmental pollutant. Previous studies have revealed that 72 h exposure to benzene metabolites inhibited hemin-induced erythroid differentiation of K562 cells accompanied with elevated methylation in erythroid specific genes. However, little is known about the effects of long-term and low-dose benzene metabolite exposure. In this study, to elucidate the effects of long-term benzene metabolite exposure on erythroid differentiation, K562 cells were treated with low-concentration phenol, hydroquinone and 1,2,4-benzenetriol for at least 3 weeks. After exposure of K562 cells to benzene metabolites, hemin-induced hemoglobin synthesis declined in a concentration- and time-dependent manner, and the hemin-induced expressions of α-, ß- and γ-globin genes and heme synthesis enzyme porphobilinogen deaminase were significantly suppressed. Furthermore, when K562 cells were continuously cultured without benzene metabolites for another 20 days after exposure to benzene metabolites for 4 weeks, the decreased erythroid differentiation capabilities still remained stable in hydroquinone- and 1,2,4-benzenetriol-exposed cells, but showed a slow increase in phenol-exposed K562 cells. In addition, methyltransferase inhibitor 5-aza-2'-deoxycytidine significantly blocked benzene metabolites inhibiting hemoglobin synthesis and expression of erythroid genes. Quantitative MassARRAY methylation analysis also confirmed that the exposure to benzene metabolites increased DNA methylation levels at several CpG sites in several erythroid-specific genes and their far-upstream regulatory elements. These results demonstrated that long-term and low-dose exposure to benzene metabolites inhibited the hemin-induced erythroid differentiation of K562 cells, in which DNA methylation played a role through the suppression of erythroid specific genes.

Cotransplantation of hepatic stellate cells attenuates the severity of graft-versus-host disease.

Liver allografts seem to be immunologically privileged; they are the only solid transplant that can protect cotransplanted organs from rejection. The mechanisms of this "hepatic tolerance" are poorly understood. The aim of this study was to examine the immunomodulatory effect of hepatic stellate cells (HSCs) in mixed lymphocyte reactions in vitro and in graft-versus-host disease (GVHD) in vivo. Using a carboxyfluorescein diacetate succinimidyl ester-labeling method, we observed that the percentage of proliferative T cells was reduced when HSCs were present in a mixed lymphocyte culture. In addition, the degree of reduction was the same when HSCs were co-cultured in transwell inserts. Thus, soluble factors may participate in the immunomodulatory effect of HSCs. In GVHD experiments, irradiated BALB/c (H-2d) mice simultaneously received an intravenous mixture of bone marrow and splenic T cells from C57BL/6 (H-2b) mice. The HSCs prominently reduced symptoms and pathologic severity of GVHD in target organs. HSC cotransplanted mice survived for 57.5+/-30.6 days whereas the control hosts only survived for 15.3+/-5.2 days (P<.01). We concluded that HSCs may reduce T-cell proliferation against alloantigens and suppress acute GVHD to prolong recipient survival. Our study sheds some light on the immunosuppressive nature of the liver, suggesting a biological manipulation of alloreactivity for transplantation medicine.

Determination of molecular weight profile for a bioactive beta-(1-->3) polysaccharides (Curdlan).

This paper focuses on the development of methodology based on MALDI-TOF mass spectrometry for evaluation of molecular weight profile of the water-insoluble portion of an extracellular polysaccharide, i.e. Curdlan. As previously demonstrated, MALDI analysis of water-insoluble Curdlan fraction gave number-average (M(n)) and weight-average (M(w)) molecular weights of 8000 and 8700 Da, respectively [T.W.D. Chan, K.Y. Tang, Rapid Commun. Mass Spectrom. 17 (2003) 887]. To validate the MALDI determined molecular weight information, several additional analytical schemes were used to analysis the water-insoluble Curdlan fraction. In all cases, the water-insoluble Curdlan sample was fractionated by gel permeation chromatography (GPC) using Sephadex G-75 column. The M (n) of low-mass and narrow distributed polysaccharide fractions were obtained by MALDI-MS. Good linearity was found in the calibration plot constructed from the measured M (n)-values and the corresponding elution time/volume. The relative quantity of various fractionated samples was then measured using three different approaches. These include (a) direct refractometric analysis; (b) UV-vis absorption analysis of the Aniline Blue stained sample; and (c) GC-MS analysis of the hydrolyzed and TMS-derivatized sample. Using results obtained from theses quantification methods and the correlation function between the GPC retention time and M (n), the MW and MWD of water-insoluble Curdlan were obtained. Our results demonstrated that the previous use of MALDI methods for measuring M(n), M(w) and polydispersity (PD) of water-insoluble Curdlan (with and without GPC fractionation) were unreliable. However, by standardizing the narrow distributed polysaccharides using MALDI-MS method, reliable molecular weight information for dispersed polysaccharides could be obtained. The M (n),M (w) and PD of the water-insoluble Curdlan were found to be 22,000, 31,500 Da and 1.40, respectively.

An intelligent tutoring system for trauma management (Trauma-Teach): a preliminary report.

Trauma-Teach is an interactive software for tutoring surgical trainees on medical trauma management procedures. Users of the system interact with a virtual patient suffering from trauma injuries. The task of the user is to stabilise the virtual patient, discover the underlying injuries and decide on an appropriate management plan. Artificial intelligence techniques are used to simulate the patient's pulmonary and cardiovascular systems in real time, determine the responses and results of treatments and diagnostics accordingly, model the patient deterioration if wrong actions are taken, and give a measure of reality to the system by selecting actual trauma cases from the hospital's database.

Analysis of a bioactive beta-(1 --> 3) polysaccharide (Curdlan) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

This paper focuses on the development of MALDI sample preparation protocols for the analysis of a bioactive beta-(1 --> 3) polysaccharide, i.e. Curdlan. The crude Curdlan sample was first separated into a low molecular weight water-soluble portion and a high molecular weight water-insoluble portion. The water-soluble portion was analyzed using a standard MALDI sample preparation method developed for dextran analysis. Two low-mass (<4000 Da) polysaccharide distributions differing by 16 Da were observed. For the analysis of the water-insoluble portion, several sample preparation protocols were evaluated using GPC-fractionated samples. A sample preparation method based on the deposition of the analyte solution with a mixture of 2,5-dihydroxybenzoic acid (DHB) and 3-aminoquinoline (3AQ) matrices in dimethyl sulfoxide (DMSO) at elevated temperature of 70 degrees C was found to reliably produce good MALDI spectra. MALDI analysis of the water-insoluble Curdlan portion gave number-average (Mn) and weight-average (Mw) molecular weights and polydispersity of 8000 Da, 8700 Da, and 1.10, respectively.

Influence of background exposure on detection and determination limits for a TL dosimetry system based on LiF:Mg,Cu,P(GR-200A).

Thermoluminescence dosemeters are widely used to monitor personal doses. For these low dose range applications, it is important to determine the detection limit L(D) and the determination limit L(Q) of the dosimetric system. The influence of background exposure on these limits for LiF:Mg,Cu,P(GR-200A) based TL dosimetry was investigated. Both the conventional analysis and the glow curve analysis methods were used to determinate these limits. The detection limit L(D) was compared with the recording level and the investigation level. A systematic error can occur in the occupational dose evaluation when the detection limit L(D) is more than the recording level. It was found that the L(D) of the dosimetric system-based LiF:Mg,Cu,P(GR-200A) was less than the recording level for exposure time tau > or = 10 days considering an annual dose limit of 1 mSv for the public recommended in ICRP Publication 60.

The application of computerised analysis of glow curves to personal dosimetry in LiF:Mg,Cu,P.

In personnel monitoring services, it is important to omit the high-temperature annealing process so that large numbers of TL detectors can be produced economically. There are two efficient ways of reducing the residual signal of LiF:Mg,Cu,P. One is by increasing the maximum readout temperature and the other is by improving the preparation procedure (increasing the Cu concentration and the sintering temperature) but both reduce the TL sensitivity. In personal dosimetry the real dosimetric signals are separated from the residual signals by computerised analysis of glow curves. The adverse influence of the high residual signals of LiF:Mg.Cu.P TL material has been effectively eliminated and the sensitivity remains stable. A good dosimetric result using only reader measurement without pre-irradiation oven annealing is attained in a dose range of 50-80,000 microGy.

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.

Characterization of histamine-induced increases in intracellular free Ca2+ concentrations in Chang liver cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in Chang liver cells were investigated by using fura-2 as a Ca2+ dye. Histamine (0.2-50 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 0.8 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the maximum [Ca2+]i signal and abolished the sustained phase. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase with a magnitude 7-fold greater than control. In Ca2+-free medium, after treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. Histamine (5 microM)-induced intracellular Ca2+ release was abolished

Fendiline mobilizes intracellular Ca2+ in Chang liver cells.

1. The effects of the antianginal drug fendiline (N-[3,3-diphenylpropyl]-alpha-methyl-benzylamine) on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. 2. Fendiline (1-100 micromol/L) increased [Ca2+](i) in a concentration-dependent manner, with an EC50 of 25 micromol/L. 3. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase. Removal of extracellular Ca2+ partly reduced the [Ca2+](i) signals. 4. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was reduced by 65% following pretreatment with 1 micromol/L thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete Ca2+ stored in the endoplasmic reticulum. 5. After pretreatment with 10 micromol/L fendiline in Ca2+-free medium for several minutes, addition of 3 mmol/L Ca2+ induced an increase in [Ca2+](i) of a magnitude four-fold greater than control. This increase in [Ca2+](i) was not reduced by 10 micromol/L SKF96365, econazole, nifedipine or verapamil. 6. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was not altered by inhibition of phospholipase C with 2 micromol/L 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione (U73122). 7. The results of the present study show that fendiline induces an increase in [Ca2+](i) in Chang liver cells by releasing stored Ca2+ in an inositol 1,4,5-trisphosphate-independent manner and by causing extracellular Ca2+ influx.

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.

Effect of the neuroprotective agent riluzole on intracellular Ca2+ levels in IMR32 neuroblastoma cells.

Riluzole is an effective neuroprotective drug. Its effect on intracellular free Ca2+ levels ([Ca2+]i) has not been explored. This study examined the effect of riluzole on [Ca2+]i in IMR32 neuroblastoma cells using fura-2 as a Ca2+ probe. Riluzole 0.1-1 mM increased [Ca2+]i in a concentration-dependent manner. Removal of extracellular Ca2+ inhibited the response by 52 +/- 5%. The [Ca2+]i increase induced by 0.2 mM riluzole was unaltered by 0.1 mM La3+ or 10 microM verapamil, but was inhibited by 51 +/- 4% by 10 microM nifedipine. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) reduced the 0.2 mM riluzole-induced Ca2+ release by 44 +/- 3%; this reduction was augmented to 66 +/- 5% by additionally depleting the Ca2+ stores in the Golgi complex with 50 microM brefeldin A. Inhibition of inositol 1,4,5-trisphosphate formation by 2 microM U73122, a phospholipase C inhibitor, did not affect Ca2+ release induced by 0.2 microM riluzole. It was concluded that the neuroprotective agent riluzole increased [Ca2+]i in IMR32 neuroblastoma cells concentration-dependently by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner and also by inducing nifedipine-sensitive Ca2+ influx.

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.

Effect of diethylstilbestrol (DES) on intracellular Ca(2+) levels in renal tubular cells.

The effect of the estrogen diethylstilbestrol (DES) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was investigated, using the fluorescent dye fura-2 as a Ca(2+) indicator. DES (10-50 microM) evoked [Ca(2+)](i) increases in a concentration-dependent manner. Extracellular Ca(2+) removal inhibited 45 +/- 5% of the Ca(2+) response. In Ca(2+)-free medium, pretreatment with 50 microM DES abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor); and pretreatment with CCCP and thapsigargin partly inhibited DES-induced [Ca(2+)](i) signals. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 50 microM DES in Ca(2+)-free medium, suggesting that DES may induce capacitative Ca(2+) entry. 17beta-Estradiol (2-20 microM) increased [Ca(2+)](i), but 100 microM diethylstilbestrol dipropionate had no effect. Pretreatment with the phospholipase C inhibitor U73122 (1 microM) to abolish inositol 1,4,5-trisphosphate formation inhibited 30% of DES-induced Ca(2+) release. DES (20 microM) also increased [Ca(2+)](i) in human normal hepatocytes and osteosarcoma cells. Cumulatively, this study shows that DES induced rapid and sustained [Ca(2+)](i) increases by releasing intracellular Ca(2+) and triggering extracellular Ca(2+) entry in renal tubular cells.

Fluoxetine-induced Ca2+ signals in Madin-Darby canine kidney cells.

The effect of fluoxetine on Ca2+ signaling in Madin-Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca2+]i concentration-dependently between 5 microM and 200 microM with an EC50 value of 40 microM. The response was reduced by external Ca2+ removal by 30%40%. In Ca2+-free medium pretreatment with 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, abolished 100 microM fluoxetine-induced Ca2+ release. Addition of 3 mM Ca2+ to Ca2+-free medium increased [Ca2+]i when cells were pretreated with 100 microM fluoxetine. Suppression of 1,4,5-trisphosphate (IP3) formation by 2 microM U73122 (a phospholipase C inhibitor) did not affect 100 microM fluoxetine-induced Ca2+ release. Fluoxetine (5-100 microM) also increased [Ca2+]i in neutrophils, prostate cancer cells and bladder cancer cells from human and rat glioma cells.