Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions

Fura-2, loaded into J774.2 macrophages as the acetoxymethyl ester, is sequestered into intracellular vacuoles within 90 min after the beginning of the loading at 37 degrees C. The dye is also efficiently secreted from the cells. Sequestration and secretion of fura-2 reduce the accuracy of measurements of cytosolic free Ca2+ concentration in this cell line. Fura-2 is also sequestered and secreted by J774.2 when the dye is loaded into the cytoplasm as the pentapotassium salt by reversible permeabilization of the plasma membrane. Regardless of the mechanism by which fura-2 is loaded into the cytoplasm, both sequestration and secretion are prevented by 2.5 mM probenecid, a blocker of organic anion transport. Probenecid has no effect on resting or stimulated cytosolic free Ca2+ levels or on FcR-mediated phagocytosis. These findings suggest that macrophages express a transport mechanism for the anionic form of fura-2. This transport system is responsible for the clearance of fura-2 from the cytoplasm of this cell type. Furthermore we suggest that use of probenecid to block secretion and intracellular sequestration of fura-2 may overcome problems arising in the application of this Ca2+ indicator to macrophages and perhaps to other cell types.     

Catecholamine secretion from digitonin-treated PC12 cells. Effects of Ca2+, ATP, and protein kinase C activators

PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 cells.     

Effects of a time-varying strong magnetic field on transient increase in Ca2+ release induced by cytosolic Ca2+ in cultured pheochromocytoma cells

Exposure of pheochromocytoma (PC 12) cells to a time-varying 1.51 T magnetic field inhibited an increase in the intracellular Ca2+ concentration ([Ca2+]i) induced by addition of caffeine to Ca(2+)-free medium. This inhibition occurred after a 15-min exposure and was maintained for at least 2 h. [Ca2+]i sharply increased in cells loaded with cyclic ADP-ribose, and 2-h exposure significantly suppressed the increase. Addition of ATP induced a transient increase in intracellular Ca2+ release mediated by IP3 receptor, and this increase was strongly inhibited by the exposure. Results indicated that the magnetic field exposure strongly inhibited Ca2+ release mediated by both IP3 and ryanodine receptors in PC 12 cells. However, thapsigargin-induced Ca2+ influx (capacitative Ca2+ entry) across the cell membrane was unaffected. The ATP content was maintained at the normal level during the 2-h exposure, suggesting that ATP hydrolysis was unchanged. Therefore, Mg2+ which is known to be released by ATP hydrolysis and inhibit intracellular Ca2+ release may not relate the exposure-caused inhibition. Eddy currents induced in culture medium appear to change cell membrane properties and indirectly inhibit Ca2+ release from endoplasmic reticulum and other Ca2+ stores in PC 12 cells.     

Generation of inositol phosphates, cytosolic Ca2+, and ionic fluxes in PC12 cells treated with bradykinin

Accumulation of inositol phosphates (Ins-Ps, revealed by high performance liquid chromatography), changes of the cytosolic free Ca2+ [( Ca2+]i, revealed by fura-2), membrane potential and ionic currents (revealed by bis-oxonol and patch clamping) were investigated in PC12 cells treated with bradykinin (BK). The phenomena observed were (a) due to the activation of a B2 receptor (inhibitor studies) and (b) unaffected by pertussis toxin, cAMP analogs, and inhibitors of either cyclooxygenase or voltage-gated Ca2+ channels. During the initial tens of s, three interconnected events predominated: accumulation of Ins-1,4,5-P3, Ca2+ release from intracellular stores and hyperpolarization due to the opening of Ca2+-activated K+ channels. Phorbol myristate acetate partially inhibited Ins-1,4,5-P3 accumulation at all [BK] investigated, and the [Ca2+]i increase at [BK] less than 50 nM. In PC12 cells treated with maximal [BK] in the Ca2+-containing incubation medium, Ins-1,4,5-P3 peaked at 10 s, dropped to 20% of the peak at 30 s, and returned to basal within 5 min; the peak increase of Ins-1,3,4-P3 was slower and was variable from experiment to experiment, while Ins-P4 rose for 2 min, and remained elevated for many min thereafter. Meanwhile, influx of Ca2+ from the extracellular medium, plasma membrane depolarization (visible without delay when hyperpolarization was blocked), and increased plasma membrane conductance were noticed. Evidence is presented that these last three events (which were partially inhibited by phorbol myristate acetate at all [BK]) were due to the activation of a cation influx, which was much more persistent than the elevation of the two Ins-P3 isomers. Our results appear inconsistent with the possibility that in intact PC12 cells the BK-induced activation of cation influx is accounted for entirely by the increases of either Ins-1,3,4-P3 or Ins-1,4,5-P3 (alone or in combination with Ins-1,3,4,5-P4), as previously suggested by microinjection studies in different cell types.     

Staphylococcus aureus alpha-toxin activates phospholipases and induces a Ca2+ influx in PC12 cells

Staphylococcal alpha-toxin at subcytotoxic concentrations stimulated phosphatidylinositol turnover and arachidonic acid release in undifferentiated cultures of pheochromocytoma PC12 cells. Stimulation of phospholipase A2 but not C was dependent on extracellular calcium. Addition of staphylococcal alpha-toxin to PC12 cells caused a dose-dependent, biphasic increase in intracellular calcium measured by fura-2 fluorescence technique. Elevation of intracellular Ca2+ content occurred with a time course similar to those observed for stimulation of phospholipase A2. Alteration of membrane structure and formation of staphylococcal alpha-toxin pores facilitating an influx of Ca2+, represent the probable mechanisms by which phospholipases C and A2 are activated, respectively. These results suggest a possible involvement of Ca2+, phosphoinositides and arachidonic acid metabolites in the pathogenic action of staphylococcus alpha-toxin and caution against the general usage of this toxin as a permeabilizing agent to study stimulus-secretion coupling in secretory cells.     

Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers

Fura-2 is widely used to measure the concentration of cytosolic free calcium, but in many cells the dye does not remain localized within the cytoplasmic matrix. In these cells, Fura-2 is sequestered within intracellular organelles, secreted into the extracellular medium, or both. We have found that, in mouse peritoneal macrophages, J774 cells, PC12 cells, and N2A cells, Fura-2 sequestration and secretion are mediated by organic anion transport systems and are blocked by the inhibitors probenecid and sulfinpyrazone. Under appropriate conditions these agents have little affect on calcium transients, and may facilitate the use of Fura-2 in a variety of cell types.     

Intracellular calcium during chemotaxis of Dictyostelium discoideum: a new fura-2 derivative avoids sequestration of the indicator and allows long-term calcium measurements.

During stimulation of Dictyostelium discoideum amoebae with the chemoattractant cAMP, extracellular calcium is taken up by the cells. The aim of this study was to determine the cytosolic free calcium concentration ([Ca++]i) during chemotaxis of Dictyostelium cells. In contrast to most vertebrate cells, three major drawbacks were encountered: 1) the indicator fura-2 could not be introduced into the cells by incubation with the ester form, 2) once loaded, the dye was rapidly sequestered into vesicles, 3) the organic anion transport blocker probenecid was not suitable to block sequestration. These problems were met by introducing the indicator into the cells with the scrape-loading technique adapted for use with Dictyostelium and the construction of a new fura-2 derivative, fura-2-dextran. Scrape-loading of Dictyostelium yielded up to 40% of labeled, vital cells. Fura-2-dextran fulfilled the following criteria: 1) it remained homogeneously distributed in the cytoplasm of motile Dictyostelium cells, 2) it retained the fluorescence intensity of fura-2 and the affinity for calcium binding, 3) it was very well suitable to demonstrate changes of [Ca++]i in serum-stimulated fibroblasts. [Ca++]i-measurements with fura-2-dextran in chemotactically active D. discoideum amoebae revealed that the large decrease in the extracellular calcium concentration is not accompanied by an overall change in [Ca++]i. Chemotaxis in this organism occurs in the absence of global changes in [Ca++]i. However, we cannot exclude either short-lived or local changes just beneath the plasma membrane.     

Effects of Erythropoietin on Neuronal Activity

Recently, erythropoietin (EPO) receptors and synthesis of EPO have been identified in the brain. To clarify the effects of EPO on neuronal cells, we investigated the effects of EPO on Ca2+ uptake, intracellular Ca2+ concentration, membrane potential, cell survival, release and biosynthesis of dopamine, and nitric oxide (NO) production in differentiated PC12 cells, which possess EPO receptors. EPO (10(-12)-10(-10) M) increased 45Ca2+ uptake and intracellular Ca2+ concentration in PC12 cells in a dose-related manner; these increases were inhibited by nicardipine (1 microM) or anti-EPO antibody (1:100 dilution). EPO induced membrane depolarization in PC12 cells. After a 5-day culture without serum and nerve growth factor (NGF), viable cell number decreased to 50% of that of the control cells cultured with serum and NGF. EPO (10(-13)-10(-10) M) increased the number of viable cells cultured without serum and NGF; this increase was blunted by nicardipine or anti-EPO antibody. Incubation with EPO (10(-13)-10(-10) M) stimulated mitogen-activated protein kinase activity in PC12 cells. EPO (10(-13)-10(-10) M) increased dopamine release from PC12 cells and tyrosine hydroxylase activity; these increases were sensitive to nicardipine or anti-EPO antibody. Following a 4-h incubation with EPO (10(-14)-10(-10) M), NO production was increased, which was blunted by nicardipine and anti-EPO antibody. In contrast, maximal NO synthase activity was not changed by EPO. These results suggest that EPO stimulates neuronal function and viability via activation of Ca2+ channels.     

Intracellular Ca2+ shift and signal transduction from the tubulovesicular portion of gastric parietal cells during gastrin stimulation or Ca2+ ionophore treatment: comparison between luminescent and fluorescent probes, and electron probe X-ray microanalyzer

In gastrin-stimulated, aequorin-loaded parietal cells from guinea pig gastric mucosa, a rapid but transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i), owing to Ca2+ released from the store(s), and a more prolonged Ca2+ entry from outside the cells were observed. However, there was a little increase in [Ca2+]i when similar measurements were assessed by quin 2 or fura-2 in physiological saline. However, depletion or elimination of Na+ from the incubation medium caused a significant increase in the [Ca2+]; response to gastrin as measured by quin 2. These findings suggest that aequorin and quin 2 (or fura-2) provide information about different aspects of Ca2+ homeostasis and that there is an inhomogeneity of [Ca2+]i in the cytoplasm during gastrin stimulation. By the gastrin stimulation, the intracellular Ca2+ gradients were shifted from the unidentified portion(s) to the restricted apical cytoplasm, as determined by electron probe X-ray microanalysis. Therefore, localization and identification of the source of intracellular Ca2+ as a pool were determined by an X-ray microanalyzer. In the resting state, the tubulovesicle had high Ca2+ concentration compared with the level in the apical cytoplasm. Cells treated with the Ca2+ ionophore ionomycin had a decreased tubulovesicular Ca2+ level, followed by a reciprocal increase in area of the canalicular membrane. The secretory canaliculus in stimulated cells had lower Ca2+ or higher K+ and Cl- concentrations than that of tubulovesicles or cytoplasm in the resting state, respectively. These findings suggest that the Ca2+ pool of the parietal cell is in the tubulovesicles and (or) luminal cell membrane and that the Ca2+ released from the store(s) may mediate a flow of K+ or Cl- into the secretory canaliculus.     

Agonist-induced calcium transients in cultured smooth muscle cells: measurements with fura-2 loaded monolayers

Elevation of cytosolic Ca2+ in response to depolarization and various receptor agonists was measured in several types of cultured smooth muscle cells (DDT1, A10, rabbit aorta) loaded with the either quin-2 or fura-2, and assayed either in suspension or in monolayer cultures attached to plastic cover slips. Agonists (norepinephrine, vasopressin) induced both the release of intracellular Ca2+ and the influx of extracellular Ca2+. Agonist-induced Ca2+ influx was not blocked by dihydropyridines, and depolarization did not induce Ca2+ influx. However, in fura-2 loaded monolayers of PC12 cells, depolarization did induce dihydropyridine-sensitive Ca2+ influx. Thus cultured smooth muscle cells appear to express receptor-operated Ca2+ channels, but not functional voltage-operated Ca2+ channels.     

The effect of antisense oligonucleotide treatment of plasma membrane Ca(+2)-ATPase in PC12 cells

Plasma membrane Ca(+2)-ATPase (PMCA), encoded by four separate genes, constitutes a high affinity system extruding Ca(+2) outside the cell. The nerve growth factor-treated PC12 cell line possesses all four main PMCA isoforms. To evaluate the potential role of PMCA isoforms in the differentiation process, we transiently suppressed the expression of PMCA2 and 3 using the antisense oligonucleotides. In the transfected PC12 cells, we observed morphological changes, slowed neurite extension and diminished survival of the cells. The apparent transport activity and affinity of the calcium pump to Ca(+2) were lower in the cells with suppressed PMCA2 and 3 isoforms than in the control cells. Moreover, in the transfected PC12 plasma membranes, the calcium pump was insensitive to stimulation by calmodulin. These findings suggest that PMCA2 and 3 isoforms may be involved in developmental and differentiation processes.     

Safrole-induced Ca2+ mobilization and cytotoxicity in human PC3 prostate cancer cells

The effect of the carcinogen safrole on intracellular Ca2+ mobilization and on viability of human PC3 prostate cancer cells was examined. Cytosolic free Ca2+ levels ([Ca2+]i) were measured by using fura-2 as a probe. Safrole at concentrations above 10 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 350 microM. The Ca2+ signal was reduced by more than half after removing extracellular Ca2+ but was unaffected by nifedipine, nicardipine, nimodipine, diltiazem, or verapamil. In Ca2+-free medium, after treatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release Ca2+. Neither inhibition of phospholipase C with U73122 nor modulation of protein kinase C activity affected safrole-induced Ca2+ release. Overnight incubation with 0.65-65 microM safrole did not affect cell viability, but incubation with 325-625 microM safrole decreased viability. Collectively, the data suggest that in PC3 cells, safrole induced a [Ca2+]i increase by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion, and by inducing Ca2+ influx. Safrole can decrease cell viability in a concentration-dependent manner.     

Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.

A rapid rise in the level of cytosolic free calcium ([Ca2+]i) is believed to be one of several early triggering signals in the activation of T lymphocytes by antigen. Although Ca2+ release from intracellular stores and its contribution to Ca2+ signaling in many cell types is well documented, relatively little is known regarding the role and mechanism of Ca2+ entry across the plasma membrane. We have investigated mitogen-triggered Ca2+ signaling in individual cells of the human T-leukemia-derived line, Jurkat, using fura-2 imaging and patch-clamp recording techniques. Phytohemagglutinin (PHA), a mitogenic lectin, induces repetitive [Ca2+]i oscillations in these cells peaking at micromolar levels with a period of 90-120 s. The oscillations depend critically upon Ca2+ influx across the plasma membrane, as they are rapidly terminated by removal of extracellular Ca2+, addition of Ca(2+)-channel blockers such as Ni2+ or Cd2+, or membrane depolarization. Whole-cell and perforated-patch recording methods were combined with fura-2 measurements to identify the mitogen-activated Ca2+ conductance involved in this response. A small, highly selective Ca2+ conductance becomes activated spontaneously in whole-cell recordings and in response to PHA in perforated-patch experiments. This conductance has properties consistent with a role in T-cell activation, including activation by PHA, lack of voltage-dependent gating, inhibition by Ni2+ or Cd2+, and regulation by intracellular Ca2+. Moreover, a tight temporal correlation between oscillations of Ca2+ conductance and [Ca2+]i suggests a role for the membrane Ca2+ conductance in generating [Ca2+]i oscillations in activated T cells.     

Botulinum Neurotoxin Light Chain Inhibits Norepinephrine Secretion in PC12 Cells at an Intracellular Membranous or Cytoskeletal Site

Botulinum neurotoxin (NT) is a potent inhibitor of neurotransmitter secretion, but its intracellular mechanism and site of action are unknown. In this study, the intracellular action of NT was investigated by rendering the secretory apparatus of PC12 cells accessible to macromolecules by a recently described "cell cracking" procedure. Soluble cytoplasmic factors were depleted from permeabilized cells by washing to generate cell "ghosts" which retained cellular structural components and intracellular organelles (including secretory granules). The PC12 cell ghosts exhibited Ca(2+)-activated [3H]norepinephrine release which was enhanced by cytosolic proteins and MgATP. PC12 cell ghosts provide the opportunity to distinguish the intracellular action of NT on soluble cytoplasmic components versus structural cellular components. The 150-kDa NT and the 50-kDa light chain of serotypes E and B, and to a lesser extent type A, inhibited Ca(2+)-activated [3H]norepinephrine release in PC12 ghosts, but not in intact PC12 cells. The 100-kDa heavy chain had no effect. This indicates that NT acts at an intracellular site in these cells permeabilized by "cell cracking." The inhibition of secretion by NT was rapid and irreversible under the incubation conditions used. NT inhibition of [3H]-norepinephrine release from PC12 ghosts occurred in the absence of cytosolic proteins and MgATP and was not reversed by the addition of cytosolic proteins and MgATP, indicating that NT acts at an intracellular membranous or cytoskeletal site.     

Endogenous heavy metal ions perturb fura-2 measurements of basal and hormone-evoked Ca2+ signals.

Using the membrane-permeant chelator of heavy metal ions, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), we demonstrate that in pancreatic acinar cells, hepatocytes, and a variety of mammalian cell lines, endogenous heavy metal ions bind to cytosolic fura-2 causing basal cytosolic free [Ca2+] ([Ca2+]i) to be overestimated. TPEN had most effect in cells lightly loaded with fura-2, suggesting the presence of a limited pool of heavy metal ions (> or = 12 microM in pancreatic acinar cells) that does not rapidly exchange across the plasma membrane. In fura-2-loaded hepatocytes, vasopressin failed to evoke a detectable change in fluorescence, but after preincubation of cells with TPEN, it caused fluorescence changes characteristic of an increase in [Ca2+]i. We conclude that in many mammalian cells, a slowly exchanging mixture of cytosolic heavy metal ions binds to fura-2 both to quench its fluorescence and to mimic the effects of Ca2+ binding, thereby causing basal [Ca2+]i to be overestimated. By chelating endogenous heavy metal ions, TPEN allows basal [Ca2+]i to be accurately measured and, by preventing competition between heavy metal ions and Ca2+ for binding to fura-2, unmasks the full effect of agonists in increasing [Ca2+]i.     

Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells

The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ [( Ca2+]i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37 degrees C with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15 degrees C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37 degrees C for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15 degrees C demonstrated in spotty cells a marked apparent blunting of Ca2+ transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]i was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]i measurements.     

NPC-14686 (Fmoc-l-homophenylalanine)-induced CaCa2+ increases and death in human prostate cancer cells

The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca2+ levels ([Ca2+]i) and growth in PC3 human prostate cancer cells was examined by using fura-2 as a fluorescent Ca2+ indicator and WST-1 as a fluorescent growth dye. NPC-14686 at concentrations above 10 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 100 microM. NPC-14686-induced Ca2+ influx was confirmed by Mn2+ quench of fura-2 fluorescence. The Ca2+ signal was also reduced by removing extracellular Ca2+. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ nearly abolished 200 microM NPC-14686-induced Ca2+ release; and conversely pretreatment with NPC-14686 completely inhibited thapsigargin-induced Ca2+ release. The Ca2+ release induced by 200 microM NPC-14686 was not affected by inhibiting phospholipase C with 2 microM U73122. Overnight treatment with 1-500 microM NPC-14686 decreased cell viability in a concentration-dependent manner. These findings suggest that in human PC3 prostate cancer cells, NPC-14686 increases [Ca2+]i by evoking extracellular Ca2+ influx and releasing intracellular Ca2+ from the endoplasmic reticulum via a phospholiase C-independent manner. NPC-14686 may be cytotoxic to prostate cancer cells.     

Altered calcium homeostasis in the pathogenesis of myocardial ischemic and hypoxic injury

Pathological calcification, observed in infarcted myocardium under certain conditions, is the most severe manifestation of abnormal calcium (Ca2+) homeostasis induced by ischemia and related forms of myocardial injury. Specialized techniques for measurement of intracellular electrolytes, i.e., electron probe X-ray microanalysis, and intracellular free Ca2+, i.e. carboxylate indicators including fura-2, are providing new insights into regulation of intracellular Ca2+ and the role of altered Ca2+ homeostasis in the pathogenesis of myocardial cell injury. Several lines of investigation indicate that increased intracellular Ca2+ develops in association with other electrolyte alterations, altered cell volume regulation, and altered membrane phospholipid composition during the progression of myocardial cell injury.     

Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader

Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading.     

Effect of diindolylmethane on Ca(2+) homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 20-50 μM induced [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). Diindolylmethane-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 50-100 μM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 μM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca(2+)](i) rise in PC3 cells by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive store-operated Ca(2+) channels. Diindolylmethane caused cell death in which apoptosis may participate.