Dapagliflozin reduces the vulnerability of rats with pulmonary arterial hypertension-induced right heart failure to ventricular arrhythmia by restoring calcium handling.

BACKGROUND: Malignant ventricular arrhythmia (VA) is a major contributor to sudden cardiac death (SCD) in patients with pulmonary arterial hypertension (PAH)-induced right heart failure (RHF). Recently, dapagliflozin (DAPA), a sodium/glucose cotransporter-2 inhibitor (SGLT2i), has been found to exhibit cardioprotective effects in patients with left ventricular systolic dysfunction. In this study, we examined the effects of DAPA on VA vulnerability in a rat model of PAH-induced RHF. METHODS: Rats randomly received monocrotaline (MCT, 60 mg/kg) or vehicle via a single intraperitoneal injection. A day later, MCT-injected rats were randomly treated with placebo, low-dose DAPA (1 mg/kg/day), or high-dose (3 mg/kg/day) DAPA orally for 35 days. Echocardiographic analysis, haemodynamic experiments, and histological assessments were subsequently performed to confirm the presence of PAH-induced RHF. Right ventricle (RV) expression of calcium (Ca2+) handling proteins were detected via Western blotting. RV expression of connexin 43 (Cx43) was determined via immunohistochemical staining. An optical mapping study was performed to assess the electrophysiological characteristics in isolated hearts. Cellular Ca2+ imaging from RV cardiomyocytes (RVCMs) was recorded using Fura-2 AM or Fluo-4 AM. RESULTS: High-dose DAPA treatment attenuated RV structural remodelling, improved RV function, alleviated Cx43 remodelling, increased the conduction velocity, restored the expression of key Ca2+ handling proteins, increased the threshold for Ca2+ and action potential duration (APD) alternans, decreased susceptibility to spatially discordant APD alternans and spontaneous Ca2+ events, promoted cellular Ca2+ handling, and reduced VA vulnerability in PAH-induced RHF rats. Low-dose DAPA treatment also showed antiarrhythmic effects in hearts with PAH-induced RHF, although with a lower level of efficacy. CONCLUSION: DAPA administration reduced VA vulnerability in rats with PAH-induced RHF by improving RVCM Ca2+ handling.

Activation of the NFAT-Calcium Signaling Pathway in Human Lamina Cribrosa Cells in Glaucoma.

Purpose: Optic nerve cupping in glaucoma is characterized by remodeling of the extracellular matrix (ECM) and fibrosis in the lamina cribrosa (LC). We have previously shown that glaucoma LC cells express raised levels of ECM genes and have elevated intracellular calcium ([Ca2+]i). Raised [Ca2+]i is known to promote proliferation, activation, and contractility in fibroblasts via the calcineurin-NFAT (nuclear factor of activated T-cells) signaling pathway. In this study, we examine NFAT expression in normal and glaucoma LC cells, and investigate the effect of cyclosporin A (CsA, a known inhibitor of NFAT activity) on [Ca2+]i and ECM gene expression in normal and glaucoma LC cells. Methods: [Ca2+]i was measured with dual-wavelength Ca2+ imaging and confocal microscopy using Fura-2-AM and Fluo-4 under physiological isotonic and hypotonic cell stretch treatment. Human donor LC cells were cultured under normal physiological conditions or using a glaucoma-related stimulus, oxidative stress (H2O2, 100 µM), for 6 hours with or without CsA. NFATc3 protein levels were examined using Western blot analysis. Profibrotic ECM gene transcription (including transforming growth factor-ß1 [TGFß1], collagen 1A1 [Col1A1], and periostin) was analyzed using quantitative real time RT-PCR. Results: Basal and hypotonic cell membrane stretch-induced [Ca2+]i were significantly (P < 0.05) elevated in glaucoma LC cells compared to normal controls. There was a significant delay in [Ca2+]i reuptake into internal stores in the glaucoma LC cells. NFATc3 protein levels were increased in glaucoma LC cells. CsA (10 µM) significantly inhibited the H2O2-induced expression of NFATc3 in normal and glaucoma LC cells. CsA also reduced the H2O2-induced NFATc3 dephosphorylation (and nuclear translocation), and also suppressed the H2O2-induced elevation in profibrotic ECM genes (TGFß1, Col1A1, and periostin), both in normal and in glaucoma LC cells. Conclusions: Intracellular Ca2+ and NFATc3 expression were significantly increased in glaucoma LC cells. CsA reduced the H2O2-induced enhancement in NFATc3 protein expression and nuclear translocation and the profibrotic gene expression both in normal and in glaucoma LC cells. Therefore, targeting the calcineurin-NFATc3 signaling pathway may represent a potential avenue for treating glaucoma-associated LC fibrosis.

Effect of NPC-14686 (Fmoc-l-Homophenylalanine) on Ca²âº Homeostasis and Viability in OC2 Human Oral Cancer Cells.

The effect of the anti-inflammatory compound NPC-14686 on intracellular Ca²âº concentration ([Ca²âº](i)) and viability in OC2 human oral cancer cells was investigated. The Ca²âº-sensitive fluorescent probe fura-2 was used to examine [Ca²âº](i). NPC-14686 induced [Ca²âº](i) rises in a concentration-dependent fashion. The effect was reduced approximately by 10% by removing extracellular Ca²âº. NPC-14686- elicited Ca²âº signal was decreased by nifedipine, econazole, SKF96365, and GF109203X. In Ca²âº-free medium, incubation with the endoplasmic reticulum Ca²âº pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished NPC-14686-induced [Ca²âº](i) rises. Conversely, pretreatment with NPC-14686 abolished thapsigargin or BHQ-induced [Ca²âº](i) rises. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca²âº](i) rises. At 20-100 µM, NPC-14686 inhibited cell viability, which was not reversed by chelating cytosolic Ca²âº with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid-acetoxymethyl ester (BAPTA/AM). NPC-14686 between 20 µM and 40 µM also induced apoptosis. Collectively, in OC2 cells, NPC-14686 induced [Ca²âº](i) rises by evoking phospholipase C-dependent Ca²âº release from the endoplasmic reticulum and Ca²âº entry via protein kinase C-regulated store-operated Ca²âº channels. NPC-14686 also caused Ca²âº-independent apoptosis.

An integrated platform for simultaneous multi-well field potential recording and Fura-2-based calcium transient ratiometry in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes.

INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes are available from various sources and they are being evaluated for safety testing. Several platforms are available offering different assay principles and read-out parameters: patch-clamp and field potential recording, imaging or photometry, impedance measurement, and recording of contractile force. Routine use will establish which assay principle and which parameters best serve the intended purpose. METHODS: We introduce a combination of field potential recording and calcium ratiometry from spontaneously beating cardiomyocytes as a novel assay providing a complementary read-out parameter set. Field potential recording is performed using a commercial multi-well multi-electrode array platform. Calcium ratiometry is performed using a fiber optic illumination and silicon avalanche photodetectors. Data condensation and statistical analysis are designed to enable statistical inference of differences and equivalence with regard to a solvent control. RESULTS: Simultaneous recording of field potentials and calcium transients from spontaneously beating monolayers was done in a nine-well format. Calcium channel blockers (e.g. nifedipine) and a blocker of calcium store release (ryanodine) can be recognized and discriminated based on the calcium transient signal. An agonist of L-type calcium channels, FPL 64176, increased and prolonged the calcium transient, whereas BAY K 8644, another L-type calcium channel agonist, had no effect. Both FPL 64176 and various calcium channel antagonists have chronotropic effects, which can be discriminated from typical "chronotropic" compounds, like (±)isoprenaline (positive) and arecaidine propargyl ester (negative), based on their effects on the calcium transient. DISCUSSION: Despite technical limitations in temporal resolution and exact matching of composite calcium transient with the field potential of a subset of cells, the combined recording platform enables a refined interpretation of the field potential recording and a more reliable identification of drug effects on calcium handling.

Induction of TRPV5 expression by small activating RNA targeting gene promoter as a novel approach to regulate cellular calcium transportation.

AIM: Promoter-targeted small activating RNAs (saRNAs) have been shown to be able to induce target gene expression, a mechanism known as RNA activation (RNAa). The present study tested whether saRNA can induce the overexpression of TRPV5 in human cells derived from the kidney and subsequently manipulate cell calcium uptake. MAIN METHODS: Three saRNAs complementary to the TRPV5 promoter were synthesized and transfected into cells. TRPV5 expression at the RNA and protein levels was analyzed by quantitative real-time PCR and Western blotting respectively. For functional study, transcellular Ca(2+) transportation was tested by fura-2 analysis. Dihydrotestosterone (DHT), a suppressor of cellular calcium transportation, was administered to challenge the activating effect of selected saRNA. KEY FINDINGS: One of these synthesized saRNAs, ds-2939, significantly induced the expression of TRPV5 at both mRNA and protein levels. Fura-2 analysis revealed that the intracellular Ca(2+) concentration was elevated by ds-2939. DHT treatment reduced transmembrane Ca(2+) transport, which was partially antagonized by ds-2939. SIGNIFICANCE: Our results suggest that a saRNA targeting TRPV5 promoter can be utilized to manipulate the transmembrane Ca(2+) transport by upregulating the expression of TRPV5 and may serve as an alternative for the treatment of Ca(2+) balance-related diseases.

Bergamot essential oil differentially modulates intracellular Ca2+ levels in vascular endothelial and smooth muscle cells: a new finding seen with fura-2.

In this study, we compared the effect of the essential oil of Citrus bergamia Risso [bergamot, bergamot essential oil (BEO)] on the intracellular Ca levels in vascular endothelial (EA) and mouse vascular smooth muscle (MOVAS) cells, using the fura-2 fluorescence technique. BEO caused an initial transient increase in intracellular Ca concentration ([Ca]i) in EA cells, followed by a decrease, whereas it induced a sustained increase in [Ca]i in MOVAS cells. Linalyl acetate (LA) as a major component of BEO-induced [Ca]i mobilization was similar to BEO in EA cells. The increase of [Ca]i by LA was higher in EA cells than in MOVAS cells. [Ca]i rise induced by extracellular Ca application was significantly blocked by BEO or LA in EA cells but not in MOVAS cells, suggesting that BEO and LA block Ca influx in EA cells. The present results suggest that BEO and LA differentially modulate intracellular Ca levels in vascular endothelial and smooth muscle cells. In addition, blockade of Ca influx by BEO and LA in EA cells may explain the protective effects of BEO on endothelial dysfunction associated with cardiovascular disease.

Selenium modulates oxidative stress-induced TRPM2 cation channel currents in transfected Chinese hamster ovary cells.

It has been recently reported that the essential antioxidant element selenium has protective effects on cytosolic Ca(2+) levels in cell lines. However, the effects of selenium on like transient receptor potential melastatin 2 (TRPM2) in response to oxidative stress (H(2) O(2) ) are not well understood. We investigated the effects of selenium on H(2) O(2) -induced TRPM2 channel currents in the Chinese hamster ovary (CHO) cell line using patch-clamp and fura-2 fluorescence imaging techniques.

Antidepressant-like effect of hyperoside isolated from Apocynum venetum leaves: possible cellular mechanisms.

In the present work, we studied the possible cellular mechanisms of hyperoside isolated from Apocynum venetum leaves in corticosterone-induced neurotoxicity, using PC12 cells as a suitable in vitro model of depression. Cell viability was quantitated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The release amount of lactic dehydrogenase (LDH) and intracellular Ca(2+) concentration were measured using kit and transcript abundances of brain-derived neurotrophic factor (BDNF) and cAMP response element binding protein (CREB) were determined by real-time RT-PCR. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase (LDH) assays showed that 2.5, 5 and 10 µg/ml hyperoside or 10 µM fluoxetine (FLU) protected PC12 cells from the lesion induced by a 48 h treatment with 10 µM corticosterone. Fura-2/AM (acetoxymethyl ester) assays showed that 2.5, 5 and 10 µg/ml hyperoside or 10 µM FLU attenuated the intracellular Ca(2+) overloading in PC12 cells induced by corticosterone. The transcript abundance of BDNF and CREB in PC12 cells was elevated upon hyperoside treatment. These results suggest that the possible cellular mechanisms of hyperoside antidepressant-like effect is a cytoprotective action related to elevation the expression of BDNF and CREB through the signal pathway AC-cAMP-CREB.

Effects of Schisandra chinensis extract on the contractility of corpus cavernosal smooth muscle (CSM) and Ca2+ homeostasis in CSM cells.

UNLABELLED: What's known on the subject? And what does the study add? Schisandra chinensis extract (SCE) has been known to have relaxative effects on penile smooth muscle. A recent study showed that SCE could enhance slidenafil citrate-induced relaxation of penile corpus cavernosum. The current study investigated the mechanism of action of SCE and its constituents on corporal smooth muscle cells. And this study shows that SCE induced relaxation of CSM primarily through an endothelium independent pathway and the relaxation effects of SCE on corporal smooth muscle are, in part, due to the activation of K(+) channels and inhibition of TRPC6 channels, resulting in decreased [Ca(2+)]. OBJECTIVE: • To evaluate the relaxant effects of Schisandra chinensis extract (SCE) on corporal tissue in the penis and to investigate the mechanism of action of SCE and its constituents on corporal smooth muscle (CSM) cells. MATERIALS AND METHODS: • The fruit of SC was collected and extracted with ethanol. Six SC lignans (schisandrol A, schisandrol B, schisandrin A, schisandrin B, gomisin N, and schisandrin C) were isolated and purified, and the chemical structures were confirmed by (1)H-nuclear magnetic resonance (NMR) and (13)C-NMR data. • Isolated rabbit CSM strips were mounted in an organ-bath system, and the effects of SCE were evaluated. • To estimate the intracellular Ca(2+) level ([Ca(2+)](i)), we used a Fura-2 fluorescent technique, and a conventional whole-cell patch-clamp technique was used to measure the calcium-sensitive K(+) channels (K(Ca)), inward rectifier K(+) channels (K(IR)), and canonical transient receptor potential cation channel 6 (TRPC6) currents. RESULTS: • SCE induced concentration-dependent relaxation in contracted CSM tissue, and the removal of the endothelium did not significantly affect their relaxation potencies. • In CSM cells, extracellular application of SCE significantly increased whole-cell K(Ca) currents (117.4%) and K(IR) currents (110.0%). These effects were completely abolished by charybdotoxin or BaCl(2). • In contrast, carbachol-induced TRPC6 channel activity was significantly inhibited (87.3%) by SCE in green fluorescent protein-TRPC6 pcDNA transfected HEK 293 cells. [Ca(2+)](i) measurements showed that SCE effectively reduced basal [Ca(2+)](i) in both cell lines (CSM cells and A7r5 cells) and the [Arg8]-vasopressin (AVP)-induced [Ca(2+)](i) increase in A7r5 cells. • Among the six SC lignans, schisandrin A and schisandrin B most effectively attenuated the AVP-induced [Ca(2+)](i) increase. CONCLUSIONS: • SCE induced relaxation of CSM that occurred primarily via an endothelium-independent pathway. • The relaxation effects of SCE on CSM were, in part, due to the activation of K(+) channels and inhibition of TRPC6 channels, resulting in decreased [Ca(2+)](i).

Effects of erythropoietin on intracellular calcium concentration of rat primary cortical neurons.

Erythropoietin (Epo) has been shown to afford neuroprotection in many experimental models. Although the cytosolic Ca(2+) concentration ([Ca(2+)](i)) is an important factor regulating cell survival, the effects of Epo on [Ca(2+)](i) in neurons are not fully elucidated. We studied the effects of human recombinant Epo on [Ca(2+)](i) of rat primary cortical neurons in normal and excitotoxic conditions. Changes in [Ca(2+)](i) were measured using fura-2 microfluorometry in rat primary cortical cultures. In the control condition with 2mM Mg(2+) in the bath solution, Epo at 4 u/ml significantly increased the fluorescence ratio, but the Epo-induced increase in the fluorescence ratio was abolished by omission of Ca(2+) from the bath solution and by the addition of cadmium. Omission of Mg(2+) and supplementation with glycine resulted in basal and periodic increases in the fluorescence ratio, due to sustained activation of N-methyl-d-asparate (NMDA) receptors. Epo at 0.4 and 4 u/ml significantly decreased the fluorescence ratio in this condition, and this effect was attenuated by the phosphoinositide 3-kinase (PI3K) inhibitors, LY 294002 and wortmannin, and the Ca-activated K channel blocker, iberiotoxin. In the presence of Mg(2+) and exogenous glutamate, 4 but not 0.4 u/ml Epo slightly but significantly reduced the [Ca(2+)](i) elevation. These results suggest that Epo increased [Ca(2+)](i) in cortical neurons by inducing Ca(2+) entry in the control condition but decreased [Ca(2+)](i) in the Mg(2+)-free excitotoxic condition, at least in part via PI3K-dependent activation of Ca-activated K channels. Reduction of [Ca(2+)](i) by Epo in the excitotoxic condition may contribute to neuroprotection.

Novel high-throughput assay to assess cellular manganese levels in a striatal cell line model of Huntington's disease confirms a deficit in manganese accumulation.

In spite of the essentiality of manganese (Mn) as a trace element necessary for a variety of physiological processes, Mn in excess accumulates in the brain and has been associated with dysfunction and degeneration of the basal ganglia. Despite the high sensitivity, limited chemical interference, and multi-elemental advantages of traditional methods for measuring Mn levels, they lack the feasibility to assess Mn transport dynamics in a high-throughput manner. Our lab has previously reported decreased net Mn accumulation in a mutant striatal cell line model of Huntington's disease (STHdh(Q111/Q111)) relative to wild-type following Mn exposure. To evaluate Mn transport dynamics in these striatal cell lines, we have developed a high-throughput fluorescence-quenching extraction assay (Cellular Fura-2 Manganese Extraction Assay - CFMEA). CFMEA utilizes changes in fura-2 fluorescence upon excitation at 360 nm (Ca(2+) isosbestic point) and emission at 535 nm, as an indirect measurement of total cellular Mn content. Here, we report the establishment, development, and application of CFMEA. Specifically, we evaluate critical extraction and assay conditions (e.g. extraction buffer, temperature, and fura-2 concentration) required for efficient extraction and quantitative detection of cellular Mn from cultured cells. Mn concentrations can be derived from quenching of fura-2 fluorescence with standard curves based on saturation one-site specific binding kinetics. Importantly, we show that extracted calcium and magnesium concentrations below 10 µM have negligible influence on measurements of Mn by fura-2. CFMEA is able to accurately measure extracted Mn levels from cultured striatal cells over a range of at least 0.1-10 µM. We have used two independent Mn supplementation approaches to validate the quantitative accuracy of CFMEA over a 0-200 µM cellular Mn-exposure range. Finally, we have utilized CFMEA to experimentally confirm a deficit in net Mn accumulation in the mutant HD striatal cell line versus wild-type cells. To conclude, we have developed and applied a novel assay to assess Mn transport dynamics in cultured striatal cell lines. CFMEA provides a rapid means of evaluating Mn transport kinetics in cellular toxicity and disease models.

TRPV1 activation is required for hypertonicity-stimulated inflammatory cytokine release in human corneal epithelial cells.

PURPOSE: To determine whether hypertonic stress promotes increases in inflammatory cytokine release through transient receptor potential vanilloid channel type 1 (TRPV1) signaling pathway activation in human corneal epithelial cells (HCECs). METHODS: Hyperosmotic medium was prepared by supplementing isotonic Ringers solution with sucrose. Ca2+ signaling was measured in fura2-AM-loaded HCECs using a single-cell fluorescence imaging system. Western blot analysis evaluated the phosphorylation status of EGFR, ERK, p38 MAPK, and nuclear factor (NF)-κB. ELISA assessed the effect of TRPV1 activation on the release of IL-6 and IL-8. RESULTS: A 450 mOsm hypertonic stress elicited 2-fold Ca2+ transients that were suppressed by the TRPV1-selective antagonists capsazepine and JYL 1421. Such transients were enhanced by PGE2. Hypertonicity-induced EGF receptor (EGFR) transactivation was suppressed by preincubating HCECs with capsazepine, matrix metalloproteinase 1 (MMP1) inhibitor TIMP-1, broad-spectrum MMP inhibitor GM 6001, heparin-bound (HB)-EGF inhibitor CRM 197, or EGFR inhibitor AG 1478. ERK and p38 MAPK and NF-κB activation after EGFR transactivation occurred in tonicity and in a time-dependent manner. Hypertonicity-induced increases in IL-6 and IL-8 releases were suppressed by exposure to capsazepine, AG 1478, ERK inhibitor PD 98059, p38 inhibitor SB 203580, or NF-κB inhibitor PDTC. CONCLUSIONS: Hypertonic stress-elicited TRPV1 channel stimulation mediates increases in a proinflammatory cytokine IL-6 and a chemoattractant IL-8 by eliciting EGFR transactivation, MAPK, and NF-κB activation. Selective drug modulation of either TRPV1 activity or its signaling mediators may yield a novel approach to suppressing inflammatory responses occurring in dry eye syndrome.

Culturing conditions determine neuronal and glial excitability.

The cultivation of pure neuronal cultures is considered advantageous for the investigation of cell-type specific responses (such as transmitter release and also pharmacological agents), however, divergent results are a likely consequence of media modifications and culture composition. Using Fura-2 based imaging techniques, we here set out to compare calcium responses of rat hippocampal neurones and glia to excitatory stimulation with l-glutamate in different culture types and media. Neurones in neurone-enriched cultures had increased responses to 10 µM and 100 µM l-glutamate (+43 and 45%, respectively; p's< 0.001) and a slower recovery compared to mixed cultures, indicating heightened excitability. In matured (15-20 days in vitro) mixed cultures, neuronal responder rates were suppressed in a neurone-supportive medium (Neurobasal-A, NB: 65%) compared to a general-purpose medium (supplemented minimal essential medium, MEM: 96%). Glial response size in contrast did not differ greatly in isolated or mixed cultures maintained in MEM, but responder rates were suppressed in both culture types in NB (e.g. 10 µM l-glutamate responders in mixed cultures: 29% in NB, 71% in MEM). This indicates that medium composition is more important for glial excitability than the presence of neurones, whereas the presence of glia has an important impact on neuronal excitability. Therefore, careful consideration of culturing conditions is crucial for interpretation and comparison of experimental results. Especially for investigations of toxicity and neuroprotection mixed cultures may be more physiologically relevant over isolated cultures as they comprise aspects of mutual influences between glia and neurones.

Capacitative Ca2+ entry during Ca2+ undershoot in bovine airway smooth muscle.

In numerous cells, Ca2+ undershoot is commonly observed after withdrawing stimulus that release Ca2+ from intracellular stores. In airway smooth muscle (ASM), the fast intracellular Ca2+ concentration ([Ca2+]i) drop during undershoot is produced by sarcoplasmic reticulum (SR) reloading, but the mechanisms involved in the long lasting basal [Ca2+]i recovery are unknown. We investigated the post-caffeine Ca2+ undershoot recovery in ASM isolated cells from bovine trachea. [Ca2+]i determination was done by a ratiometric method by incubating cells with Fura-2/AM. After inducing a transient response, caffeine withdrawn generated a Ca2+ undershoot. SR-Ca2+ content during maximum undershoot drop was approximately 40% of SR caffeine-releasable Ca2+ (SR-Ca2+ load). Undershoot recovery rate increased in presence of cyclopiazonic acid (CPA, a SR-Ca2+ ATPase inhibitor), but SR-Ca2+ load was reduced. Genistein (a tyrosine kinase inhibitor) slowed down the Ca2+ undershoot drop and the SR-Ca2+ load but did not affect the undershoot recovery rate. Ni2+ (a capacitative Ca2+ inhibitor), but neither SKF-96365 (a passive Ca2+ entry inhibitor) nor econazole (a capacitative Ca2+ inhibitor in non-excitable cells), inhibited Ca2+ undershoot recovery and SR-Ca2+ load. Our data suggest that capacitative Ca2+ entry is involved in bovine ASM Ca2+ undershoot recovery, and that changes in Ca2+ undershoot have an impact on SR-Ca2+ loading which might affect in turn ASM excitability.

The garlic ingredient diallyl sulfide induces Ca(2+) mobilization in Madin-Darby canine kidney cells.

Diallyl sulfide (DAS), one of the major organosulfur compounds (OSCs) of garlic, is recognized as a group of potential chemoproventive compounds. In this study, we examines the early signaling effects of DAS on renal cells loaded with Ca(2+)-sensitive dye fura-2. It was found that DAS caused an immediate and sustained rise of [Ca(2+)](i) in a concentration-dependent manner (EC(50)=2.32 mM). DAS also induced a [Ca(2+)](i) elevation when extracellular Ca(2+) was removed, but the magnitude was reduced by 45%. Depletion of intracellular Ca(2+) stores with CCCP, a mitochondrial uncoupler, did not affect DAS's effect. In Ca(2+)-free medium, the DAS-induced [Ca(2+)](i) rise was abolished by depleting stored Ca(2+) with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor). DAS-caused [Ca(2+)](i) rise in Ca(2+)-containing medium was not affected by modulation of protein kinase C activity. The DAS-induced Ca(2+) influx was blocked by nicardipine. U73122, an inhibitor of phospholipase C, abolished ATP (but not DAS)-induced [Ca(2+)](i) rise. Additionally, pretreatment with DAS for 24 h decreased cell viability in a concentration-dependent manner. Furthermore, DAS-induced cell death involved apoptotic events. These findings suggest that diallyl sulfide induced a significant rise in [Ca(2+)](i) in MDCK renal tubular cells by stimulating both extracellular Ca(2+) influx and thapsigargin-sensitive intracellular Ca(2+) release via as yet unidentified mechanisms.

Inhibition of multidrug/xenobiotic resistance transporter by MK571 improves dye (Fura 2) accumulation in crustacean tissues from lobster, shrimp, and isopod.

Multidrug/xenobiotic resistance transporters are present in living organisms as a first line defence system against small, potentially harmful molecules from the environment or from internal metabolic reactions. Multidrug resistance associated proteins (MRP) are one type of ATP-Binding-Cassette (ABC) transporters, which also transport dyes such as Fura 2, a calcium chelating fluorescence indicator. The specific MRP inhibitor MK571 was used to investigate the fluorescence intensity of cells in tissues of the brain and the midgut gland of the crustaceans Homarus gammarus (lobster), Crangon crangon (brown shrimp) and Idotea emarginata (isopod) during incubation with Fura 2AM (1 microM). In the presence of the inhibitor MK571 (50 microM), the fluorescence of brain tissue significantly increased in all of the three species. The midgut gland of H. gammarus showed a significant increase of fluorescence, whereas there was no effect in the midgut glands of C. crangon and I. baltica. The half maximal concentration of MK571 was 50 microM as measured in the midgut gland of H. gammarus. In conclusion, MRP transporters are present in the three investigated crustacean nervous systems. Using the midgut glands of the three species, only in H. gammarus MK571 inhibited dye extrusion, indicating species-specific differences of transporter systems, their specificity, or tissue specific expression.

C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro.

With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.

High throughput screening assay of alpha(1G) T-type Ca2+ channels and comparison with patch-clamp studies.

Whole-cell patch-clamp-based electrophysiological techniques are powerful tools for examining the biophysical and pharmacological properties of ion channels. However, the recent validation of ion channels as novel drug targets necessitates the development of a faster screening method for ion channels. Therefore, we have developed a rapid, reliable, and sensitive cell-based high throughput screening (HTS) assay for T-type Ca2+ channels. We had previously constructed HEK293/alpha(1G)/Kir2.1 cell lines that stably expressed alpha(1G) and Kir2.1 subunits [1] and found that alpha(1G) T-type channel-sensitive Ca2+ signals were detected by the application of high concentrations of KCl under fura-2-based single cell measurements of intracellular Ca2+ concentration ([Ca2+](i)). In the present study, we applied HEK293/alpha (1G)/Kir2.1 cells to the FDSS6000 (Functional Drug Screening System) to develop a fast and reliable cell-based HTS method for alpha(1G) T-type Ca2+ channels. After detecting 70 mM KCl-induced [Ca2+](i) increases using the FDSS6000 system, we verified this new alpha(1G) channel HTS system by examining two T-type Ca2+ channel blockers, Ni2+ and mibefradil, and measuring the Z'-factor (Z' factor = 0.66) in 96-well plates. Furthermore, we assayed selected 3,4-dihydroquinazolin derivatives using this FDSS6000-based alpha(1G) channel HTS system at the level of IC(50) values and compared the results with those obtained from whole-cell patch-clamp recordings. Taken together, our results suggest that the FDSS6000-based alpha(1G) channel HTS system is a fast and feasible assay for alpha(1G) T-type Ca2+ channels. This assay can be utilized as a primary screening method for T-type Ca2+ channel-targeted chemicals and for the development of HTS systems for other types of ion channels.

Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons.

The lipid diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was used to verify the existence of DAG-sensitive channels in cortical neurons dissociated from E13 mouse embryos. Calcium imaging experiments showed that OAG increased the cytosolic concentration of Ca(2+) ([Ca(2+)]i) in nearly 35% of the KCl-responsive cells. These Ca(2+) responses disappeared in a Ca(2+)-free medium supplemented with EGTA. Mn(2+) quench experiments showed that OAG activated Ca(2+)-conducting channels that were also permeant to Ba(2+). The OAG-induced Ca(2+) responses were unaffected by nifedipine or omega-conotoxin GVIA (Sigma-Aldrich, Saint-Quentin Fallavier, France) but blocked by 1-[beta-(3-(4-Methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF)-96365 and Gd(3+). Replacing Na(+) ions with N-methyl-D-glucamine diminished the amplitude of the OAG-induced Ca(2+) responses showing that the Ca(2+) entry was mediated via Na(+)-dependent and Na(+)-independent mechanisms. Experiments carried out with the fluorescent Na(+) indicator CoroNa Green showed that OAG elevated [Na(+)]i. Like OAG, the DAG lipase inhibitor RHC80267 increased [Ca(2+)]i but not the protein kinase C activator phorbol 12-myristate 13-acetate. Moreover, the OAG-induced Ca(2+) responses were not regulated by protein kinase C activation or inhibition but they were augmented by flufenamic acid which increases currents through C-type transient receptor potential protein family (TRPC) 6 channels. In addition, application of hyperforin, a specific activator of TRPC6 channels, elevated [Ca(2+)]i. Whole-cell patch-clamp recordings showed that hyperforin activated non-selective cation channels. They were blocked by SKF-96365 but potentiated by flufenamic acid. Altogether, our data show the presence of hyperforin- and OAG-sensitive Ca(2+)-permeable channels displaying TRPC6-like properties. This is the first report revealing the existence of second messenger-operated channels in cortical neurons.

Inflammation-induced increase in evoked calcium transients in subpopulations of rat dorsal root ganglion neurons.

The concentration of intracellular Ca(2+) ([Ca(2+)](i)) influences neuronal properties ranging from excitability to neurotransmitter release. Persistent inflammation is associated with changes in the properties of primary afferent neurons ranging from excitability to transmitter release. The purpose of the present study was to determine whether previously described inflammation-induced changes in excitability and transmitter release are associated with changes in the regulation of [Ca(2+)](i). Acutely dissociated dorsal root ganglion (DRG) neurons harvested from adult rats 3 days following a hind-paw injection of complete Freund's adjuvant (CFA) or naïve controls, were stimulated with 30 mM K(+) (High K(+)). High K(+) evoked changes in [Ca(2+)](i) were assessed with fura-2 ratiometric microfluorimetry. Subpopulations of DRG neurons were defined by cell body diameter, isolectin B4 (IB4) binding, capsaicin (CAP) sensitivity and target of innervation (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbo-cyanine perchlorate labeling). Inflammation was associated with significant increases in resting [Ca(2+)](i) and increases in the magnitude and decreases in the decay, of the evoked increase in [Ca(2+)](i). The changes in evoked transients were larger in neurons innervating the site of inflammation. Furthermore, there were differences among subpopulations of DRG neurons with respect to changes in magnitude and/or decay of the evoked transient such that the increase in magnitude was larger in small- and medium-diameter neurons than in large diameter neurons while the decrease in the decay was greater in CAP responsive, IB4 positive, small- and medium-diameter neurons than in CAP unresponsive, IB4 negative and/or large-diameter neurons. These changes in the regulation of [Ca(2+)](i) were not due to inflammation-induced changes in passive or active electrophysiological properties. Importantly, an inflammation-induced increase in evoked Ca(2+) transients in putative nociceptive afferents may contribute to the pain and hyperalgesia associated with persistent inflammation via facilitation of transmitter release from these afferents.