thapsigargin and nickel-chloride

βig-h3 is a TGF-β (transforming growth factor β)-induced ECM (extracellular matrix) protein that induces the secretion of MMPs (matrix metalloproteinases). However, the mechanism of induction is yet to be established. In this study, siRNAs (small interfering RNAs) targeted against βig-h3 were transfected into SMMC-7721 cells [a HCC (human hepatocellular carcinoma) cell line] to knockdown the expression of βig-h3. We found that NiCl2, a potent blocker of extracellular Ca2+ entry, reduced βig-h3-induced secretion of MMP-2 and -9. Further investigation suggested that reduction in the levels of βig-h3 decreased the secretion of MMP-2 and -9 that was enhanced by an increase in the concentration of extracellular Ca2+. SNAP (S-nitroso-N-acetylpenicillamine), a NO (nitric oxide) donor, and 8-Br-cGMP (8-bromo-cGMP) inhibited thapsigargin-induced Ca2+ entry and MMP secretion in the invasive potential of human SMMC-7721 cells. Further, the inhibitory effects of 8-Br-cGMP and SNAP could be significantly enhanced by down-regulating βig-h3. βig-h3 attenuates the negative regulation of NO/cGMP-sensitive store-operated Ca2+ entry. Our findings suggest that the expression of βig-h3 might play an important role in the regulation of store-operated Ca2+ entry to increase the invasive potential of HCC cells.

Topics: Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclic GMP; Extracellular Matrix Proteins; Fluorescent Antibody Technique; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Nickel; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; S-Nitroso-N-Acetylpenicillamine; Thapsigargin; Transfection; Transforming Growth Factor beta

Lysophosphatidic acid (LPA) is a membrane-derived lysophospholipid that can induce pleomorphic effects in neural progenitor cells (NPCs) from the cerebral cortex, including alterations in ionic conductance. LPA-induced, calcium-mediated conductance changes have been reported; however, the underlying molecular mechanisms have not been determined. We show here that activation of specific cognate receptors accounts for nearly all intracellular calcium responses evoked by LPA in acutely cultured nestin-positive NPCs from the developing mouse cerebral cortex. Fast-onset changes in intracellular calcium levels required release from thapsigargin-sensitive stores by a pertussis toxin-insensitive mechanism. The influx of extracellular calcium through Cd(2+)/Ni(2+)-insensitive influx pathways, approximately one-half of which were Gd(3+) sensitive, contributed to the temporal diversity of responses. Quantitative reverse transcription-PCR revealed the presence of all five known LPA receptors in primary NPCs, with prominent expression of LPA(1), LPA(2), and LPA(4). Combined genetic and pharmacological studies indicated that NPC responses were mediated by LPA(1) (approximately 30% of the cells), LPA(2) (approximately 30%), a combination of receptors on single cells (approximately 30%), and non-LPA(1,2,3) pathways (approximately 10%). LPA responsivity was significantly reduced in more differentiated TuJ1(+) cells within cultures. Calcium transients in a large proportion of LPA-responsive NPCs were also initiated by the closely related signaling lipid S1P (sphingosine-1-phosphate). These data demonstrate for the first time the involvement of LPA receptors in mediating surprisingly diverse NPC calcium responses involving multiple receptor subtypes that function within a single cell. Compared with other known factors, lysophospholipids represent the major activator of calcium signaling identified within NPCs at this early stage in corticogenesis.

Topics: Animals; Cadmium Chloride; Calcium; Calcium Signaling; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Embryo, Mammalian; Extracellular Fluid; Female; Gene Expression Regulation, Developmental; Glutamic Acid; Intracellular Signaling Peptides and Proteins; Isoxazoles; Lysophospholipids; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Neurogenesis; Neurons; Nickel; Organophosphates; Pertussis Toxin; Pituitary Adenylate Cyclase-Activating Polypeptide; Pregnancy; Propionates; Pyridines; RNA, Messenger; Sphingosine; Thapsigargin

We studied centrifugation-mediated mechanical stress-induced tumor necrosis factor-alpha (TNF-alpha) production in the monocyte-like cell line THP-1. The induction of TNF-alpha by mechanical stress was dependent on the centrifugation speed and produced the highest level of TNF-alpha after 1 h of stimulation. TNF-alpha production returned to normal levels after 24 h of stimulation. Mechanical stress also induced Toll-like receptor-2 (TLR2) mRNA in proportion to the expression of TNF-alpha. The inhibition of TLR2 signaling by dominant negative myeloid differentiation factor 88 (MyD88) blocked TNF-alpha expression response to mechanical stress. After transient overexpression of TLR2 in HEK-293 cells, mechanical stress induced TNF-alpha mRNA production. Interestingly, mechanical stress activated the c-Src-dependent TLR2 phosphorylation, which is necessary to induce Ca(2+) fluxes. When THP-1 cells were pretreated with BAPTA-AM, thapsigargin, and NiCl(2).6H(2)O, followed by mechanical stimulation, both TLR2 and TNF-alpha production were inhibited, indicating that centrifugation-mediated mechanical stress induces both TLR2 and TNF-alpha production through Ca(2+) releases from intracellular Ca(2+) stores following TLR2 phosphorylation. In addition, TNF-alpha treatment in THP-1 cells induced TLR2 production in response to mechanical stress, whereas the preincubation of anti-TNF-alpha antibody scarcely induced the mechanical stress-mediated production of TLR2, indicating that TNF-alpha produced by mechanically stimulated THP-1 cells affected TLR2 production. We concluded that TNF-alpha production induced by centrifugation-mediated mechanical stress is dependent on MyD88-dependent TLR2 signaling that is associated with Ca(2+) release and that TNF-alpha production induced by mechanical stress affects TLR2 production.

Topics: Antibodies; Calcium; Calcium Signaling; Cell Line; Cell Line, Tumor; Egtazic Acid; Endotoxins; Enzyme Inhibitors; Epithelial Cells; Gene Expression; Humans; Inositol 1,4,5-Trisphosphate; Monocytes; Myeloid Differentiation Factor 88; NF-kappa B; Nickel; Phosphorylation; Polymyxin B; Pyrazoles; Pyrimidines; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Stress, Mechanical; Thapsigargin; Toll-Like Receptor 2; Transfection; Tumor Necrosis Factor-alpha

Previously, we reported that Ca(2+) influx through nicotinic acetylcholine (ACh) receptors (nAChRs) activates a fulfenamic acid (FFA)-sensitive inward current, presumably a Ca(2+)-activated nonselective cation current (I(CAN)), in mesencephalic dopamine (DA) neurons. This current exhibited a negative slope conductance in the voltage range between -80 and -40mV and its activation led to a dramatic change in the responses to a transient application of glutamate, from single spikes to burst discharges. In this study, to examine the effect of activation of the FFA-sensitive current on EPSPs, we applied ACh (1mM) by transient air pressure shortly before electrical stimulation to evoke EPSPs in DA neurons. Application of ACh enhanced the amplitude of EPSPs when it preceded the electrical stimulation by less than 2 s, but not when the interval was longer than 3 s. In addition, this enhancement was critically dependent on intracellular Ca(2+) and the membrane potentials of the postsynaptic cell. Furthermore, the enhancing effect of ACh on EPSPs was sensitive to FFA and phenytoin. These results suggest that Ca(2+) influx caused by cholinergic inputs enhances EPSPs via activation of the FFA- and phenytoin-sensitive current.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Acetylcholine; Animals; Animals, Newborn; Atropine; Chelating Agents; Dopamine; Drug Interactions; Egtazic Acid; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Mesencephalon; Muscarinic Antagonists; Neurons; Nickel; ortho-Aminobenzoates; Patch-Clamp Techniques; Pertussis Toxin; Phenytoin; Rats; Thapsigargin; Time Factors; Valine

The detection of focal Ca(2+) transients (called neuroeffector Ca(2+) transients, or NCTs) in smooth muscle of the mouse isolated vas deferens has been used to detect the packeted release of ATP from nerve terminal varicosities acting at postjunctional P2X receptors. The present study investigates the sources and sequestration of Ca(2+) in NCTs. Smooth muscle cells in whole mouse deferens were loaded with the Ca(2+) indicator Oregon Green 488 BAPTA-1 AM and viewed with a confocal microscope. Ryanodine (10 microM) decreased the amplitude of NCTs by 45 +/- 6 %. Cyclopiazonic acid slowed the recovery of NCTs (from a time course of 200 +/- 10 ms to 800 +/- 100 ms). Caffeine (3 mM) induced spontaneous focal smooth muscle Ca(2+) transients (sparks). Neither of the T-type Ca(2+) channel blockers NiCl2 (50 microM) or mibefradil dihydrochloride (10 microM) affected the amplitude of excitatory junction potentials (2 +/- 5 % and -3 +/- 10 %) or NCTs (-20 +/- 36 % and 3 +/- 13 %). In about 20 % of cells, NCTs were associated with a local, subcellular twitch that remained in the presence of the alpha1-adrenoceptor antagonist prazosin (100 nM), showing that NCTs can initiate local contractions. Slow (5.8 +/- 0.4 microm s(-1)), spontaneous smooth muscle Ca(2+) waves were occasionally observed. Thus, Ca(2+) stores initially amplify and then sequester the Ca(2+) that enters through P2X receptors and there is no amplification by local voltage-gated Ca(2+) channels.

Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Calcium Channels, T-Type; Calcium Signaling; Chelating Agents; Electric Stimulation; Electrophysiology; In Vitro Techniques; Indoles; Male; Mibefradil; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Models, Biological; Muscle Contraction; Muscle, Smooth; Nickel; Prazosin; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2X; Ryanodine; Thapsigargin; Vas Deferens

Nickel compounds induce cell transformation in cell culture models and tumor formation in experimental animals. However, the molecular mechanisms by which nickel compounds induce tumors are not yet well understood. The present study found that exposure of cells to either Ni(3)S(2) or NiCl(2) could result in specific transactivation of nuclear factor of activated T cells (NFAT), although it did not show any activation of p53 or AP-1. Furthermore, nickel compounds were also able to cause generation of reactive oxygen species (ROS). The scavenging of nickel-induced H(2)O(2) with N-acety-L-cyteine (a general antioxidant) or catalase, or the chelation of nickel with deferoxamine, resulted in inhibition of NFAT activation. In contrast, pretreatment of cells with sodium formate (an .OH radical scavenger) or superoxide dismutase (an O(-.)(2) radical scavenger) did not show any inhibitory effects. These results demonstrate that nickel compounds are able to induce NFAT activation, and that the mechanism of NFAT activation seems to be mediated by the generation of H(2)O(2) by these metal compounds. This study should help us understand the signal transduction pathways involved in carcinogenic effects of these nickel compounds.

Topics: Acetylcysteine; Animals; Calcineurin; Calcium; Carcinogens; Cells, Cultured; Chelating Agents; Cyclosporine; Deferoxamine; DNA-Binding Proteins; Drug Synergism; Fibroblasts; Free Radical Scavengers; Hydrogen Peroxide; Mice; NFATC Transcription Factors; Nickel; Nuclear Proteins; Reactive Oxygen Species; Thapsigargin; Transcription Factors; Transcriptional Activation; Transfection

Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (L(p)) by stimulating Ca(2+) influx into endothelial cells. To determine whether VEGF-mediated Ca(2+) influx is stimulated by release of Ca(2+) from intracellular stores, we measured the effect of Ca(2+) store depletion on VEGF-mediated increased L(p) and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) of frog mesenteric microvessels. Inhibition of Ca(2+) influx by perfusion with NiCl(2) significantly attenuated VEGF-mediated increased [Ca(2+)](i). Depletion of Ca(2+) stores by perfusion of vessels with thapsigargin did not affect the VEGF-mediated increased [Ca(2+)](i) or the increase in L(p). In contrast, ATP-mediated increases in both [Ca(2+)](i) and L(p) were inhibited by thapsigargin perfusion, demonstrating that ATP stimulated store-mediated Ca(2+) influx. VEGF also increased Mn(2+) influx after perfusion with thapsigargin, whereas ATP did not. These data showed that VEGF increased [Ca(2+)](i) and L(p) even when Ca(2+) stores were depleted and under conditions that prevented ATP-mediated increases in [Ca(2+)](i) and L(p). This suggests that VEGF acts through a Ca(2+) store-independent mechanism, whereas ATP acts through Ca(2+) store-mediated Ca(2+) influx.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Channel Blockers; Capillary Permeability; Endothelial Growth Factors; Endothelium, Vascular; Enzyme Inhibitors; Intracellular Membranes; Lymphokines; Male; Manganese; Microcirculation; Nickel; Osmolar Concentration; Rana pipiens; Rana temporaria; Splanchnic Circulation; Thapsigargin; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The aim of our study was to investigate the physiologic mechanisms involved in eosinophil activation as an essential prerequisite to disrupting the biochemical cascade that triggers inflammation, thereby attenuating the effect of this activation or, ideally, preventing it from occurring. We have, therefore, examined the nature of the fMLP- and PAF-induced [Ca2+]i rise and the relationship between the [Ca2+]i rise and O2- production in human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5. These cells responded to fMLP or PAF (1 microM each) with an increase in [Ca2+]i (217.3 +/- 22.1 and 197.8 +/- 22.1 nM respectively) which was associated with production of O2- (40.2 +/- 8.2 and 35.2 +/- 7.6 pmol/min/10(6) cells respectively). The role of Ca2+ in the induced respiratory burst was studied by changing the availability of Ca2+ in the intra- and extracellular compartments. Removal or chelation of extracellular Ca2+ induced a reduction of both the fMLP and PAF-induced [Ca2+]i rise and O2- production. Chelation of intracellular Ca2+ induced a concentration-dependent inhibition of fMLP- and PAF-induced [Ca2+]i rise and caused a decrease in O2- production. SK&F 96365 had a stimulatory effect on PAF-induced [Ca2+]i rise and on fMLP-induced O2- production, this phenomenon was not observed with extracellular Ca2+ removal or chelation. Furthermore, Ni2+ exhibited an inhibition of both fMLP and PAF-induced [Ca2+]i rise and O2- production. Finally, both fMLP and PAF induced an increase in divalent cation influx that was further augmented by thapsigargin. Our results indicate that fMLP and PAF dependent O2- production in human eosinophils require intra- and extracellular Ca2+ and that Ca2+ influx is necessary for optimal activation.

Topics: Calcium; Calcium Channel Blockers; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Eosinophils; Fetal Blood; Humans; Imidazoles; Ionomycin; Magnesium; N-Formylmethionine Leucyl-Phenylalanine; Nickel; Oxygen; Platelet Activating Factor; Respiratory Burst; Spectrometry, Fluorescence; Thapsigargin

The mitogen-activated protein kinases (MAPK), including stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and extracellular signal-related kinase (ERK), are believed to be important biomolecules in cell proliferation, survival, and apoptosis induced by extracellular stimuli. In Chinese hamster V79 cells exposed to hydrogen peroxide (H2O2), we recently demonstrated that SAPK/JNK was activated by tyrosine kinase and intracellular Ca2+ ([Ca2+]i). In this study, we report that [Ca2+]i release from intracellular stores is important in the activation of SAPK/JNK but not p38 and ERK. H2O2-induced elevation of [Ca2+]i was observed in Ca2+-free medium. Pretreatment with thapsigargin, a Ca2+-ATPase inhibition of endoplasmic reticulum (ER), did not influence H2O2-induced elevation of [Ca2+]i in the absence of external Ca2+. An intracellular Ca2+ chelator (BAPTA-AM) inhibited H2O2-induced phosphorylation of SAPK/JNK, but an extracellular Ca2+ chelator (EDTA) or a Ca2+ entry blocker (NiCl2) did not. Activation of p38 and ERK in V79 cells exposed to H2O2 was observed in the presence of these inhibitors. These results suggest that [Ca2+]i release from intracellular stores such as mitochondria or nuclei but not ER, occurred after H2O2 treatment and Ca2+-dependent tyrosine kinase-induced activation of SAPK/JNK, although [Ca2+]i was unnecessary for the H2O2-induced activation of p38 and ERK.

Topics: Animals; Calcium; Cell Line; Chelating Agents; Cricetinae; Edetic Acid; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Humans; Hydrogen Peroxide; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Nickel; p38 Mitogen-Activated Protein Kinases; Protein-Tyrosine Kinases; Thapsigargin

An in vitro technique for the measurement of calcium uptake into the maternal-facing fetal chorionic membrane (apical trophoblast) was used to study the relationship between calcium uptake and stage of pregnancy in the sheep. The effects on calcium uptake of varying calcium concentration and temperature of the incubation medium, of adding calcium channel blockers or heavy metals (lanthanum and nickel) or calcium ionophore/agonist were also studied. The data indicate a saturable calcium uptake process, plateauing after 15 min incubation. This uptake remained constant throughout the last third of gestation until a significant fall in uptake was noted during the final week prior to parturition. This uptake was not due to extracellular cellular diffusion since there was no significant uptake of tritiated inulin over the same period in each case. Calcium uptake in this system was also shown to be a temperature dependent process which was abolished at temperatures of 0-4 degrees C. A decrease in calcium concentration to 0.12 mM in the incubation medium also caused a corresponding decrease in calcium uptake to 21 per cent of control (1.2 mM). The addition of the heavy metals lanthanum and nickel also significantly reduced calcium uptake as did the calcium channel blockers verapamil, metoprolol and diltiazem. The calcium channel ionophore A23187 increased calcium uptake into the material facing chorion. Although the interplacentomal chorion may not be representative of the whole of the placental unit, it clearly contains a specific calcium uptake process under local physiological control. The blocking of calcium uptake by the specific I-type calcium channel blocker verapamil may indicate the presence of I-type channels of unusually low sensitivity since the concentration needed to block them was much higher than would be required for excitable I-type channels in isolated cells.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Biological Transport; Calcimycin; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Chorion; Female; Gestational Age; Ionophores; Kinetics; Lanthanum; Nickel; Placenta; Pregnancy; Sheep; Temperature; Thapsigargin

Staurosporine (Stp) is an inhibitor of protein kinase C (PKC) that has been used to address the role of this enzyme in a variety of cells. However, Stp can also inhibit protein tyrosine kinases (PTK). We have investigated the effects of Stp on the InsP3-(using mAb C305 directed against the beta chain of the T cell receptor (TcR/CD3 complex) and the thapsigargin (Tg)-dependent release and influx of Ca2+ in human (Jurkat) T cells. The addition of Stp (200 nM) during the sustained phase of the TcR-dependent Ca2+ response resulted in a rapid inhibition of the influx of Ca2+ that was not seen when Ca2+ mobilization was triggered by Tg (1 microM). When the cells were preincubated with Stp (200 nM), there was an inhibition of the mAb C305- but not the Tg-dependent Ca2+ response. The effect of Stp was not the result of the inhibition of PKC as shown by down-regulation of PKC and with the use of the specific PKC inhibitor bis-indolyl maleimide GF 109203X. The effect of Stp on the entry of Ca2+ in activated (mAb C305) Jurkat lymphocytes was dose-related and was not the result of a direct inhibition of plasma membrane Ca2+ channels based on an absence of effect on the Tg-dependent entry of Ca2+ and the use of Ca2+ channel blockers (econazole and Ni2+). These blockers terminated the influx of Ca2+ but the Tg-sensitive Ca2+ reserves were not refilled in marked contrast to the effect of Stp. Quantification of InsP3 revealed that the addition of Stp resulted in an approximate 40% reduction in mAb C305-activated Jurkat cells. The effects of Stp can be explained as follows. Stp decreases the mAb C305-induced production of InsP3 by inhibiting the TcR/CD3-dependent activation of PTK associated with the stimulation of phospholipase C-gamma 1. A decrease in [InsP3] without a return to baseline is sufficient to close the InsP3 Ca2+ channel, endoplasmic Ca2+ ATPases use the incoming Ca2+ to refill the Ca2+ pools and that terminates the capacitative entry of Ca2+. A simple kinetic model reproduced the experimental data.

Topics: Antibodies, Monoclonal; Biological Transport; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Membrane Permeability; Chelating Agents; Computer Simulation; Econazole; Egtazic Acid; Enzyme Inhibitors; Fura-2; Humans; Jurkat Cells; Kinetics; Lymphocyte Activation; Models, Immunological; Nickel; Protein Kinase C; Receptors, Antigen, T-Cell, alpha-beta; Staurosporine; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thapsigargin

Recent data suggest an important role for calcium (Ca2+) in human placental endocrinology. Thus, the regulation of Ca2+ influx seems to be implicated in the modulation of human placental lactogen and hCG release. A possible mechanism of influx regulation is through receptor-operated channels. One of the most characterized receptor gating Ca2+ channels, the ATP receptor, stimulates the intracellular calcium concentration ([Ca2+]i) in various tissues. The aim of this study was to determine whether ATP receptors gating Ca2+ channels are also present in placental cells. We thus determined the effect of ATP on [Ca2+]i in human term trophoblastic cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. ATP stimulated a 4.3 +/- 0.4 (+/- SE)-fold increase in [Ca2+]i, with a half-maximal effective concentration (EC50) of 1.5 mumol/L. The pharmacological activation profile suggests the presence of purinergic P2u receptors (nucleotide receptors), because uridine 5'-triphosphate (UTP) also stimulated [Ca2+]i (4.0-fold increase, with an EC50 of 10 mumol/L). The ATP-stimulated [Ca2+]i was partly sensitive to pertussis toxin; we observed a 58% inhibition of ATP-induced [Ca2+]i with the toxin without effect on basal [Ca2+]i. The ATP- and UTP-stimulated [Ca2+]i declined with time in the presence of ATP (or UTP). The rate of deactivation was rapid (t1/2, < 60 s with 10(-5) mol/L ATP) and concentration dependent. The deactivation occurring during one application of ATP or UTP resulted in a diminution of subsequent responses. The recovery was incomplete even with long waiting times (up to 30 min). ATP and UTP also stimulated inositol phosphate production with EC50 values of 11 and 15 mumol/L, respectively, but not human placental lactogen or hCG release in experiments in which known secretagogues were effective. The results suggest the presence in human term placental cells of P2u receptors pharmacologically similar to those observed in other tissues, especially in the pituitary and amnion. The physiological significance of this stimulation of [Ca2+]i by ATP and UTP in the human placenta remains to be investigated.

Topics: Adenosine Triphosphate; Calcium; Calcium Channels; Cells, Cultured; Cobalt; Female; Humans; Ion Channel Gating; Kinetics; Nickel; Nifedipine; Pertussis Toxin; Placenta; Pregnancy; Receptors, Purinergic P2; Terpenes; Thapsigargin; Trophoblasts; Uridine Triphosphate; Virulence Factors, Bordetella

The membrane potential of the endothelium of isolated rat aorta was recorded using the patch-clamp technique. The inhibitor of phospholipase C, U-73122, reduced or abolished the electrical response of the endothelium evoked by acetylcholine, but its inactive analogue, U-73343, was without effect. Depletion of intracellular Ca2+ stores with thapsigargin, cyclopiazonic acid, 2,5-di-(tertbutyl)-1,4-benzohydroquinone, or ionomycin evoked an acetylcholine-like response, which in some aortas included oscillations in endothelial membrane potential and abolished electrical responses evoked by acetylcholine. All stages of acetylcholine- and ionomycin-evoked responses could be evoked in endothelium isolated from underlying smooth muscle. Buffering of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid reduced or abolished acetylcholine-evoked responses. Both buffering of intracellular Ca2+ and inhibition of phospholipase C abolished the inhibitory effect of acetylcholine on phenylephrine-evoked oscillations in endothelial membrane potential. These results suggest that acetylcholine exerts its effect on the membrane potential of intact rat aorta through activation of phospholipase C, the release of Ca2+ from intracellular stores, and so the elevation of intracellular Ca2+ by a mechanism typical for inositol(1,4,5)-trisphosphate-mediated responses.

Topics: Acetylcholine; Animals; Aorta; Calcium; Calcium-Transporting ATPases; Egtazic Acid; Endothelium, Vascular; In Vitro Techniques; Intracellular Membranes; Ionomycin; Membrane Potentials; Nickel; Osmolar Concentration; Rats; Terpenes; Thapsigargin; Type C Phospholipases

Using the fluorescent dyes sodium-binding-benzofuran-isophthalate and fura-2 cytosolic free sodium concentration ([Na+]i) and cytosolic free calcium concentration ([Ca2+]i) were investigated in intact human platelets in order to characterize the effect of elevated [Ca2+]i on [Na+]i. Spectrofluorometric studies of [Ca2+]i and [Na+]i in intact platelets were done after specific inhibition of endoplasmic Ca-ATPase by thapsigargin. Thapsigargin increased [Ca2+]i and [Na+]i in platelets. Addition of thapsigargin increased [Na+]i from 23.5 +/- 2.9 mM to 51.6 +/- 11.1 mM (mean +/- S.E., P < 0.05). The thapsigargin induced [Na+]i increase was also seen in the absence of extracellular calcium. In the absence of external sodium the thapsigargin induced [Na+]i increase was abolished, indicating that thapsigargin induced [Na+]i increase was due to sodium influx. Thapsigargin induced sodium influx was blocked after administration of NiCl2. The present results support the idea that the filling state of intracellular calcium stores regulate plasma permeability for sodium.

Topics: Blood Platelets; Calcium; Calcium-Transporting ATPases; Cytosol; Fura-2; Humans; In Vitro Techniques; Kinetics; Nickel; Sodium; Terpenes; Thapsigargin; Time Factors

The effects of cytochrome P-450 inhibitors of different chemical structures, including several imidazole antimycotics, SKF525A, 5,8,11,14-eicosatetraynoic acid (ETYA), gossypol and nordihydroguaiaretic acid (NDGA), were tested on the entry of Ca2+ and Mn2+ induced either by emptying the intracellular Ca2+ stores with thapsigargin or by stimulation with platelet activating factor (PAF). Most of the drugs inhibited thapsigargin-induced Ca2+ and Mn2+ entry with the same affinity, with the striking exceptions of econazole and miconazole, which were 5- and 2-fold more potent to inhibit the thapsigargin-induced Mn2+ entry than to inhibit Ca2+ entry, respectively. Additionally, high doses of every drug (3-10-times the Ki) activated a pathway permeable to Mn2+ and Ni2+ but not to Ca2+. These findings indicate that Mn2+ entry data should be interpreted with caution and always be cross-checked with Ca2+ uptake measurements. Most of the drugs inhibited PAF-induced Mn2+ uptake with an affinity similar to that found for thapsigargin-induced Mn2+ uptake. PAF- and thapsigargin-induced Ca2+ uptake were also inhibited similarly by NDGA, SKF525A and gossypol, but PAF-induced Ca(2+)-uptake was inhibited about 5-fold more strongly by econazole and ETYA and two-fold more strongly by miconazole and clotrimazole. These findings suggest that the Ca2+/Mn2+ entry pathway opened by agonists in human neutrophils is the same that activates on emptying the Ca2+ stores and that cytochrome P-450 activity may be involved en the activation of the channels.

Topics: Calcium; Cell Membrane Permeability; Cytochrome P-450 Enzyme Inhibitors; Econazole; Fura-2; Humans; Ion Channels; Ionomycin; Manganese; Neutrophils; Nickel; Platelet Activating Factor; Terpenes; Thapsigargin