sq-23377 and nickel-chloride

Most mechanoreceptor neurons encode mechanical signals into action potential trains within the same cell. Evidence suggests that intracellular calcium ion concentration, [Ca2+], increases during mechanotransduction, either by direct entry through mechanically activated channels or indirectly through voltage-activated calcium channels. However, little is known about the amounts of calcium involved or its roles in mechanotransduction. We estimated [Ca2+] in mechanoreceptor neurons of the spider, Cupiennius salei, during mechanical stimulation using Oregon Green BAPTA-1, and a single-compartment model of [Ca2+] as a function of action potential firing rate. Resting [Ca2+] was approximately 400 nM and increased to up to 2 microM at 30 action potentials/s. Similar levels of resting and stimulated [Ca2+] were obtained in the cell soma, axon and two parts of the sensory dendrite, including the region immediately adjacent to the site of sensory transduction. The time constant of rise and fall of [Ca2+] was 1-5 s in the dendrite and axon, but up to 15 s in the soma. Calcium elevation was dependent on action potentials and could not be induced by the receptor potential alone. Blockade of voltage-activated calcium channels by nickel ions prevented calcium increase, but thapsigargin, which empties intracellular calcium stores, had no effect. Estimates of calcium entry per action potential from fluorescence changes agreed approximately with estimates based on action potential voltage-time profile and previous reports of calcium channel properties. This first report of calcium levels during transduction in spiking mechanoreceptors suggests that calcium signaling plays important roles in primary somatosensory neurons.

Topics: Action Potentials; Animals; Calcium; Egtazic Acid; Electric Stimulation; In Vitro Techniques; Ionomycin; Ionophores; Mechanoreceptors; Neurons; Nickel; Signal Transduction; Spiders; Tetrodotoxin; Time 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

Interactions between human platelets and human umbilical vein endothelial cells (HUVEC) were studied by monitoring changes in cytosolic [Ca2+]i in both cell types. Confluent monolayers of Fura-2-loaded HUVEC, grown on gelatin-coated coverslips, responded to repeated addition of a suspension of unstimulated platelets by transient increases in cytosolic [Ca2+]i. These platelet-evoked Ca2+ responses were not caused by products secreted from the platelets and were insensitive to inhibitors of platelet activation and/or platelet aggregation. The platelet-evoked rises in [Ca2+]i in endothelial cells, similarly as the thrombin-evoked rises, were blocked by preincubation of HUVEC with the phospholipase C inhibitor U73122 or the Ca2+ influx blocker Ni2+. In contrast, treatment with the protein tyrosine kinase inhibitor genistein was without effect. Video imaging experiments, in which the fluorescence signal was analysed from the individual cells of an endothelial monolayer, showed that only 2-20% of the cells, scattered over the monolayer, responded to the addition of platelets by a transient increase in [Ca2+]i, whereas most of the cells responded to thrombin. This leads to the conclusion that unstimulated platelets can activate HUVEC putatively by mechanical interaction with individual endothelial cells in the monolayer.

Topics: Blood Platelets; Calcium; Cell Communication; Cells, Cultured; Chelating Agents; Cytosol; Endothelium, Vascular; Enzyme Inhibitors; Estrenes; Fura-2; Humans; Ionomycin; Ionophores; Nickel; Pyrrolidinones; Thrombin; Type C Phospholipases; Umbilical Veins

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

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

Human umbilical-vein endothelial cells stimulated with thrombin or histamine show an increase in [Ca2+]i (cytoplasmic free calcium concn.) that is maintained well above the basal pre-stimulated value as long as agonist and a source of extracellular Ca2+ are present. These results provide circumstantial evidence that agonists stimulate influx of Ca2+ across the plasma membrane and into the cytoplasm. Here, we have used Mn2+ as the extracellular bivalent cation which can bind to the fluorescent Ca2+ indicator fura-2 to quench its fluorescence completely. Human umbilical-vein endothelial cells were loaded with fura-2 and, in the presence of extracellular Mn2+, thrombin and histamine were shown to cause quenching of the intracellular dye. This result demonstrates conclusively that agonists can stimulate the influx of bivalent cations. Stimulated discharge of Ca2+ from intracellular stores and influx of Mn2+ were temporally resolved in the same cells to show that release of Ca2+ from intracellular stores clearly precedes influx. Influx of Mn2+ was also demonstrated when extracellular Mn2+ was added after agonist at a time when [Ca2+]i had fallen back to the basal value, showing that influx is not dependent on elevated [Ca2+]i.

Topics: Calcium; Chlorides; Endothelium, Vascular; Ethers; Female; Histamine; Humans; In Vitro Techniques; Ionomycin; Manganese; Manganese Compounds; Nickel; Spectrometry, Fluorescence; Thrombin