Nerve conduction block in diabetic rats using high-intensity focused ultrasound for analgesic applications.

BACKGROUND: Nerve conduction block using high-intensity focused ultrasound (HIFU) has been conducted with nerves of mixed fibres in normal animal models. This study tested the feasibility and safety of HIFU for sensory nerve conduction block in diabetic neuropathic nerves to determine its potential for pain relief. METHODS: Diabetes was induced in Sprague-Dawley rats using streptozotocin, and HIFU at 2.68 MHz was used for the block. This study consisted of two sections, in vitro and in vivo. For the in vitro experiments, the entire contiguous sciatic-sural nerves were obtained. Compound action potentials and sensory action potentials were recorded in the sciatic and sural nerves, respectively. For the in vivo experiments, compound muscle action potentials (CMAPs) were recorded from the gastrocnemius muscles. All data were expressed as median (range). RESULTS: The in vitro results showed that HIFU temporarily inhibited sensory action potentials of the control and diabetic rat nerves to 33.9 (8.2) and 14.0 (10.7)% of the baseline values, respectively, whereas the compound action potentials were suppressed to 53.6 (8.4) and 76.2 (7.5)% of baseline, respectively. The in vivo results showed that HIFU acutely blocked CMAPs to 32.9 (12.6) and 19.9 (10.9)% of baseline in control and diabetic rat nerves, respectively. Measurements of CMAPs and histological exanmination were used for indirect assessment of the safety of the HIFU technique. CONCLUSIONS: High-intensity focused ultrasound safely and reversibly suppressed nerve conduction in diabetic rat nerves when the stimulation parameters were appropriate. The results suggest that HIFU may have potential to block sensory nerves reversibly and provide peripheral pain relief.

Approaching the asymptotic regime of rapidly rotating convection: boundary layers versus interior dynamics.

Rapidly rotating Rayleigh-Bénard convection is studied by combining results from direct numerical simulations (DNS), laboratory experiments, and asymptotic modeling. The asymptotic theory is shown to provide a good description of the bulk dynamics at low, but finite Rossby number. However, large deviations from the asymptotically predicted heat transfer scaling are found, with laboratory experiments and DNS consistently yielding much larger Nusselt numbers than expected. These deviations are traced down to dynamically active Ekman boundary layers, which are shown to play an integral part in controlling heat transfer even for Ekman numbers as small as 10^{-7}. By adding an analytical parametrization of the Ekman transport to simulations using stress-free boundary conditions, we demonstrate that the heat transfer jumps from values broadly compatible with the asymptotic theory to states of strongly increased heat transfer, in good quantitative agreement with no-slip DNS and compatible with the experimental data. Finally, similarly to nonrotating convection, we find no single scaling behavior, but instead that multiple well-defined dynamical regimes exist in rapidly rotating convection systems.

A prototype axial ultrasound needle guide to reduce epidural bone contact.

We have developed an ultrasound probe through the centre of which an epidural needle can pass, intended to reduce the rate of contact between bone and needle during epidural insertion. We tested the ability of this probe to identify the lumbar interspace, using A-mode ultrasound, in a submerged plastic model, a porcine phantom and five human volunteers. In the plastic model, the minimum echo representing the interspace was only 8.8% of the maximum echo from the 'bone'. In the porcine model, the echo variations between the interspace and L3 were up to 48% and the needle was safely inserted into the interspace without bone contact under guidance. The human study also showed that the maximum bone echo was at least three times stronger than the interspace echo. Axial ultrasound guidance, with the needle passing through the probe, offers a method for reducing bone contact during epidural insertion.

Rheumatoid factor and immunoglobulin M mark hepatitis C-associated mixed cryoglobulinaemia: an 8-year prospective study.

OBJECTIVES: The prevalence and factors of hepatitis C virus (HCV) -associated mixed cryoglobulinaemia in Asia remain elusive, and we aimed to investigate these topics. METHODS: An 8-year prospective cohort study was conducted in 678 consecutive Taiwanese individuals with chronic HCV infection (438 completed an anti-HCV therapy course). RESULTS: Of 678 individuals, 437 (64.5%) had mixed cryoglobulinaemia and 20 (2.9%) had mixed cryoglobulinaemic syndrome. At baseline, IgM (cut-off >122 mg/dL), triglycerides and IgG levels, and HCV genotype 3 were independently associated with mixed cryoglobulinaemia. Rheumatoid factor (RF) levels were associated with mixed cryoglobulinaemic syndrome (cut-off >12.2 IU/mL). At 24 weeks post-therapy, the 362 individuals with a sustained virological response (SVR) had higher cured (106/362 (29.3%) versus 10/76 (13.2%), p = 0.003) and lower persistent (100/362 (27.6%) versus 33/76 (43.4%), p = 0.003) mixed cryoglobulinaemia rates than non-SVR patients. Among SVR patients, compared with baseline levels, RF, IgG and IgM levels decreased, except in individuals with new mixed cryoglobulinaemia. Pre-therapy IgM levels were associated with 24-week post-therapy new (95% CI of OR 1.002-1.023) and persistent (95% CI of OR 1.004-1.015) mixed cryoglobulinaemia in SVR patients. After up to 8 years, 24-week post-therapy IgM levels were associated with mixed cryoglobulinaemia in SVR patients (9/51; 17.64%; 95% CI of HR 1.004-1.011). Among 17 SVR patients with pre-therapy mixed cryoglobulinaemic syndrome, 5 (29.4%) had long-term mixed cryoglobulinaemia and 4 (23.5%) had mixed cryoglobulinaemic syndrome. CONCLUSIONS: Over 60% of chronic HCV-infected individuals had mixed cryoglobulinaemia, and 17.64% of SVR patients had mixed cryoglobulinaemia 8 years post-therapy. Pre-therapy RF and IgM levels marked HCV-associated mixed cryoglobulinaemic syndrome and mixed cryoglobulinaemia, respectively.

Clinical trial: percutaneous acetic acid injection vs. percutaneous ethanol injection for small hepatocellular carcinoma--a long-term follow-up study.

BACKGROUND: The long-term outcome of percutaneous acetic acid injection (PAI) and percutaneous ethanol injection (PEI) for treating small hepatocellular carcinoma (HCC) remains unclear. AIM: To compare the long-term outcome of PAI vs. PEI for treating small HCC. METHODS: From July 1998 to July 2004, 125 patients with small HCC were enrolled. Seventy patients receiving PAI and 55 patients receiving PEI were enrolled. There were no significant differences in the clinical characteristics between the two groups. Tumour recurrence and survival rates were assessed. RESULTS: Mean follow-up time was 43 months. The local recurrence rate and new tumour recurrence rate were similar between the PAI and PEI groups. The PAI group had significantly better survival than the PEI group (P = 0.027). Multivariate analysis revealed that PAI was the significant factor associated with overall survival [PAI vs. PEI, RR: 0.639, 95% CI: (0.419-1.975), P = 0.038]. The treatment sessions required to achieve complete tumour necrosis were significantly fewer in the PAI group than in the PEI group (2.4 +/- 1.0 vs. 2.9 +/- 1.3, P = 0.018). CONCLUSION: Percutaneous acetic acid injection required fewer treatment sessions than PEI and provided better survival after long-term follow-up.

Modulation of C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerosis drugs.

BACKGROUND: C-reactive protein (CRP) induces adhesion molecule expression by endothelial cells. However, the effects of CRP on chemokine expression by endothelial cells are not known. METHODS AND RESULTS: We tested the effects of CRP on the production of the chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES in cultured human umbilical vein endothelial cells. The secretion of chemokines was assessed by ELISA. Incubation with 100 microgram/mL recombinant human CRP induced a 7-fold increase in MCP-1 but no change in RANTES secretion. We showed that the effect of CRP on MCP-1 was present even at 5 microgram/mL CRP, with stepwise increases as the CRP concentration was increased to 10, 50, and 100 microgram/mL. The effect of CRP on MCP-1 induction was not influenced by aspirin (at concentrations up to 1 mmol/L), but it was significantly inhibited by 5 micromol/L simvastatin. The peroxisome proliferator-activated receptor-alpha activators fenofibrate (100 micromol/L) and Wy-14649 (100 micromol/L) almost completely abolished the induction of MCP-1, but the peroxisome proliferator-activated receptor-gamma activator ciglitazone had only a moderate effect. CONCLUSIONS: These results further strengthen the role of CRP in the pathogenesis of vascular inflammation and, likely, atherosclerosis and provide a crucial insight into a novel mechanism of action of anti-atherosclerosis drugs such as simvastatin and fenofibrate.

Naproxen-induced Ca2+ movement and death in MDCK canine renal tubular cells.

Naproxen is an anti-inflammatory drug that affects cellular calcium ion (Ca(2+)) homeostasis and viability in different cells. This study explored the effect of naproxen on [Ca(2+)](i) and viability in Madin-Darby canine kidney cells (MDCK) canine renal tubular cells. At concentrations between 50 µM and 300 µM, naproxen induced [Ca(2+)](i) rises in a concentration-dependent manner. This Ca(2+) signal was reduced partly when extracellular Ca(2+) was removed. The Ca(2+) signal was inhibited by a Ca(2+) channel blocker nifedipine but not by store-operated Ca(2+) channel inhibitors (econazole and SKF96365), a protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, and a PKC inhibitor GF109203X. In Ca(2+)-free medium, pretreatment with 2,5-di-tert-butylhydroquinone or thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+) pumps, partly inhibited naproxen-induced Ca(2+) signal. Inhibition of phospholipase C with U73122 did not alter naproxen-evoked [Ca(2+)](i) rises. At concentrations between 15 µM and 30 µM, naproxen killed cells in a concentration-dependent manner, which was not reversed by prechelating cytosolic Ca(2+) with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl. Annexin V/propidium iodide staining data suggest that naproxen induced apoptosis. Together, in MDCK renal tubular cells, naproxen induced [Ca(2+)](i) rises by inducing Ca(2+) release from multiple stores that included the endoplasmic reticulum and Ca(2+) entry via nifedipine-sensitive Ca(2+) channels. Naproxen induced cell death that involved apoptosis.

Effect of saikosaponin, a triterpene saponin, on apoptosis in lymphocytes: association with c-myc, p53, and bcl-2 mRNA.

1. The mechanisms involved in the apoptotic effect of saikosaponin-d, a triterpene saponin from Bupleurum falcatum L., were studied in human CEM lymphocytes and compared with those of dexamethasone (3 x 10(-7) M). 2. Saikosaponin-d (10(-8) to 10(-5) M) inhibited the serum-stimulated [(3)H]-thymidine incorporation in a concentration-dependent manner. Dexamethasone also inhibited serum-stimulated [(3)H]-thymidine incorporation. 3. Cell viability was unaffected by saikosaponin-d until 10(-5) - 10(-4) M. Dexamethasone significantly reduced the number of viable cells. 4. Following saikosaponin-d (10(-5) - 10(-4) M) treatment, flow cytometry analysis of propidium iodide-stained cells showed a significant increase in the percentage of cells in the apoptotic region. Dexamethasone also significantly increased the percentage of apoptotic cells. The supravital exposure to propidium iodide and annexin V labelling demonstrated that saikosaponin-d (10(-5) - 10(-4) M) induced apoptosis as well as necrosis. 5. The apoptotic effect of saikosaponin-d (3 x 10(-6) - 10(-4) M) was also demonstrated by TUNEL analysis and DNA laddering. The percentage of apoptotic cells induced by saikosaponin-d (3 x 10(-6) - 10(-5) M) was unaffected by the presence of Z-VAD-FMK, indicating that saikosaponin-d-induced apoptosis may not be mediated by caspase activity. However, the percentage of apoptotic cells induced by dexamethasone was significantly reduced by the presence of Z-VAD-FMK. 6. Levels of c-myc, p53, and bcl-2 mRNA were analysed by the reverse transcription-polymerase chain reaction. Levels of c-myc and p53 mRNA were significantly increased, while the level of bcl-2 mRNA was decreased, by saikosaponin-d (10(-5) M) treatment. Dexamethasone did not significantly change the expression of these genes. 7. It is suggested that the apoptotic effect of saikosaponin-d may be partly mediated by increases in c-myc and p53 mRNA levels accompanied by a decrease in bcl-2 mRNA level.

Mechanisms of histamine-induced intracellular Ca2+ release and extracellular Ca2+ entry in MG63 human osteosarcoma cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+](i)) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca2+ dye. Histamine increased ([Ca2+](i)) in a concentration-dependent fashion with an EC(50) value of 0.5 microM. Extracellular Ca2+ removal inhibited the ([Ca2+](i)) signals. Histamine failed to increase ([Ca2+](i)) in Ca2+-free medium after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Addition of Ca2+ induced concentration-dependent ([Ca2+](i)) increases after preincubation with histamine in Ca2+-free medium. Histamine-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The ([Ca2+](i)) increase induced by histamine in Ca2+ medium was abolished by cimetidine, but was not altered by pyrilamine, nifedipine, verapamil, and La(3+). Together, this study shows that histamine increased in ([Ca2+](i)) in osteosarcoma cells by stimulating H2 histamine receptors. The Ca2+ signal was caused by Ca2+ release from the endoplasmic reticulum in a phospholipase C-dependent manner. The Ca2+ release was accompanied by Ca(2+) influx.

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.

Eradication of Helicobacter pylori prevents ulcer development in patients with ulcer-like functional dyspepsia.

BACKGROUND: Although the eradication of Helicobacter pylori infection benefits patients with gastric or duodenal ulcers, the value of eradicating the infection in the patients with functional dyspepsia (FD) remains controversial. AIMS: To determine whether eradicating H. pylori can prevent the subsequent development of ulcers or relieve the symptoms of functional dyspepsia patients. METHODS: In a double-blind, placebo-controlled trial, 161 patients infected with H. pylori who had functional dyspepsia were randomly assigned to 7 days of treatment with a lansoprazole-based triple therapy or placebo and then followed for 1 year. The main outcome measures were the development of peptic ulcers and the resolution of symptoms. RESULTS: H. pylori was eradicated in 63 out of 81 patients (78%) in the treatment group and none of the 80 patients (0%) in the placebo group. During the follow-up period, two patients in the treatment group and six patients in the placebo group developed peptic ulcers at repeat endoscopy (2.5% vs. 7.5%; 95% CI: -12 to 2). The reduction in ulcer rates was statistically significant in the 'ulcer-like' sub-group (0% vs. 16.7%; 95% CI: -32 to -2), but not in the 'dysmotility-like' and 'unclassifiable' sub-groups. Regarding symptom response, the resolution rates of symptoms were similar between the treatment and placebo groups (58.0% vs. 55.0%, 95% CI: -12 to 18). Additionally, no significant differences existed in the symptom responses between the treatment and control arms in each of the dyspepsia sub-groups. CONCLUSIONS: Eradicating H. pylori can prevent the subsequent development of peptic ulcers in the patients with 'ulcer-like' functional dyspepsia. However, this approach does not significantly reduce the symptoms of functional dyspepsia patients.

NMDA antagonist displays anticonvulsant effect via NO synthesis inhibition in penicillin-treated rat hippocampal slices.

The present study investigated the role of nitric oxide (NO) in epileptogenesis and whether this role correlated with ionotropic glutamate receptor (IGR). Using a self-constructed NO-sensitive microelectrode (SNM), we observed the effect of nitric oxide synthase (NOS) inhibitors, NMDA and non-NMDA selective antagonists on penicillin(PEN)-treated hippocampal slices by simultaneously recording evoked field potentials and nitric oxide release from CA1 pyramidal neurons. 7-nitroindazole (7-NI),Nomega-nitro-L-arginine (L-NNA) and DL-2-amino-phospho-novaleric acid (APV), but not 6,7-dinitroquinoxaline-2,3 (1h,4h)-dione(DNQX), depressed NO release and partly reversed PEN's epileptogenetic effect, while APV + 7-NI + L-NNA did not display a further inhibitory effect. These findings suggest both NOS inhibitor and NMDA antagonist involve as anticonvulsant factors in epileptogenesis, providing direct evidence for NO release in response to NMDA receptor activation. The anticonvulsant effect of NMDA antagonist may ascribe to its action on NO release.

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 betulinic acid on intracellular-free Ca(2+) levels in Madin Darby canine kidney cells.

The effect of betulinic acid, an anti-tumor and apoptosis-inducing natural product, on intracellular-free levels of Ca(2+) ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was examined by using fura-2 as a Ca(2+) dye. Betulinic acid caused significant increases in [Ca(2+)](i) concentration dependently between 25 and 500 nM with an EC(50) of 100 nM. The [Ca(2+)](i) signal was composed of an initial gradual rise and a plateau. The response was decreased by removal of extracellular Ca(2+) by 45+/-10%. In Ca(2+)-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) abolished 250 microM betulinic acid-induced [Ca(2+)](i) increases. Conversely, pretreatment with betulinic acid only partly inhibited thapsigargin-induced [Ca(2+)](i) increases. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) increase after pretreatment with 250 nM betulinic acid in Ca(2+)-free medium for 5 min. This [Ca(2+)](i) increase was not altered by the addition of 20 microM SKF96365 and 10 microM econazole. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 microM) abolished 250 nM betulinic acid-induced Ca(2+) release. Pretreatment with 10 microM La(3+) inhibited 250 nM betulinic acid-induced [Ca(2+)](i) increases by 85+/-3%; whereas 10 microM of verapamil, nifedipine and diltiazem had no effect. In Ca(2+) medium, pretreatment with 2.5 nM betulinic aid for 260 s potentiated 10 microM ATP and 1 microM thapsigargin-induced [Ca(2+)](i) increases by 33+/-3% and 45+/-3%, respectively. Trypan blue exclusion revealed that acute exposure of 250 nM betulinic acid for 2-30 min decreased cell viability by 6+/-2%, which could be prevented by pretreatment with 2 microM U731222. Together, the results suggest that betulinic acid induced significant [Ca(2+)](i) increases in MDCK cells in a concentration-dependent manner, and also induced mild cell death. The [Ca(2+)](i) signal was contributed by an inositol 1,4, 5-trisphosphate-dependent release of intracellular Ca(2+) from thapsigargin-sensitive stores, and by inducing Ca(2+) entry from extracellular medium in a La(3+)-sensitive manner.

Effect of adenosine on adenosine triphosphate-sensitive potassium channel during hypoxia in rat hippocampal neurons.

Using the whole-cell patch clamp method, we explored the effect of adenosine on the K(ATP) current and its regulatory mechanisms in acutely dissociated rat hippocampal neurons. A chemical hypoxia model was made using 0.2 mmol/l 2,4dinitrophenol (2,4DNP). During hypoxia, the K(ATP) current was not raised significantly by adenosine alone, but was accelerated significantly by adenosine in combination with the selective A(2) receptor blocker 3, 7-dimethl-1-propargylxanth-ine. The selective A(1) receptor agonist N6-cyclopentyladenosine also accelerated the K(ATP) current. These results suggest that activation of the adenosine A(1) receptor can accelerate opening of the K(ATP) channel during hypoxia, and that the A(2) receptor may have an opposing effect to the A(1) receptor.

Taurine antagonizes calcium overload induced by glutamate or chemical hypoxia in cultured rat hippocampal neurons.

Taurine has been proposed to have antiexcitotoxic and antihypoxic activity. To explore the effect of taurine on neuronal calcium overload evoked by glutamate or hypoxia, we employed fluo-3 imaging of intracellular calcium ([Ca2+]i) in confocal laser scanning microscope to measure real-time changes of [Ca2+]i arose from glutamate/2,4-dinitrophenol (DNP, mimic hypoxia) and taurine in cultured rat hippocampal neurons. We found that 3 mM taurine could inhibit [Ca2+]i elevation ascribed to 0.5 mM glutamate or 0.2 mM DNP. Low (0.5 mM) or high (12 mM) level of taurine displayed no significantly depressant effect. However, sole application of taurine could increase [Ca2+]i transiently. The results indicate that taurine in moderate concentration may exert antiexcitotoxic and antihypoxic effect partially via its antagonism to [Ca2+]i overload.

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.

AM-404 elevates renal intracellular Ca(2+), questioning its selectivity as a pharmacological tool for investigating the anandamide transporter.

The effect of N-(4-hydroxyphenyl)-arachidonamide (AM-404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca(2+) levels ([Ca(2+)](i)) was studied in Madin Darby canine kidney (MDCK) cells. [Ca(2+)](i) was measured using fura-2 as a Ca(2+) indicator. Between 2 and 40 microM, AM-404 increased [Ca(2+)](i) in a concentration-dependent fashion with an EC(50) value of 20 microM. Removal of extracellular Ca(2+) abolished the [Ca(2+)](i) signals. The [Ca(2+)](i) increase was nearly abrogated by 10 microM La(3+), but was insensitive to 50 microM Ni(2+) and 10 microM of nifedipine, nimodipine, nicardipine, and verapamil. At a concentration that did not increase [Ca(2+)](i), AM-404 (1 microM) did not alter the [Ca(2+)](i) increases induced by 10 microM ATP and 1 microM bradykinin. AM-404 (5 microM) also increased [Ca(2+)](i) in Chang liver cells, PC3 human prostate cancer cells, BFTC human bladder cancer cells, and MG63 human osteoblast-like cells. Together, this study shows for the first time that AM-404 at concentrations commonly used to inhibit the anandamide transporter in various systems induced an increase in [Ca(2+)](i) in different cell types. The [Ca(2+)](i) increase was solely due to extracellular Ca(2+) influx. Thus caution must be exercised in using AM-404 as a selective inhibitor of the anandamide transporter.

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.