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.     

Hg2+ signaling in trout hepatoma (RTH-149) cells: involvement of Ca2+-induced Ca2+ release

Mercury is a non-essential heavy metal affecting intracellular Ca2+ dynamics. We studied the effects of Hg2+ on [Ca2+]i in trout hepatoma cells (RTH-149). Confocal imaging of fluo-3-loaded cells showed that Hg2+ induced dose-dependent, sustained [Ca2+]i transient, triggered intracellular Ca2+ waves, stimulated Ca2+-ATPase activity, and promoted InsP3 production. The effect of Hg2+ was reduced by the Ca2+ channel blocker verapamil and totally abolished by extracellular GSH, but was almost unaffected by cell loading with the heavy metal chelator TPEN or esterified GSH. In a Ca2+-free medium, Hg2+ induced a smaller [Ca2+]i transient, that was unaffected by TPEN, but was abolished by U73122, a PLC inhibitor, and by cell loading with GDP-betaS, a G protein inhibitor, or heparin, a blocker of intracellular Ca2+ release. Data indicate that Hg2+ induces Ca2+ entry through verapamil-sensitive channels, and intracellular Ca2+ release via a G protein-PLC-InsP3 mechanism. However, in cells loaded with heparin and exposed to Hg2+ in the presence of external Ca2+, the [Ca2+]i rise was maximally reduced, indicating that the global effect of Hg2+ is not a mere sum of Ca2+ entry plus Ca2+ release, but involves an amplification of Ca2+ release operated by Ca2+ entry through a CICR mechanism.     

Effect of anti-Ig on cytosolic Ca2+ in Daudi lymphoblastoid cells

We examined the response in the free intracellular calcium concentration ([Ca2+]i) of Daudi (human lymphoblastoid) cells to antibodies against human immunoglobulins (anti-Ig), and the relationship of [Ca2+]i to anti-Ig-induced capping. At 80 microM intracellular quin-2 (a fluorescent probe for [Ca2+]i), anti-Ig (10 micrograms/ml) caused a rapid increase in [Ca2+]i from 100 to 600 nM; the signal returned to baseline with approximately 1 min. At 450 microM intracellular quin-2, [Ca2+]i rose to only approximately 250 microM, and the signal declined gradually, returning to base line after greater than 7 min. In subsequent experiments, the lower concentrations of quin-2 were employed. Plots of the amplitude of the [Ca2+]i transients and of the binding of 125I-anti-Ig to Daudi cells versus the concentrations of anti-Ig showed similar saturation kinetics, with half-saturation occurring at 2-3 micrograms/ml. Part of the calcium in the transient is derived from the extracellular medium, and part from the nonmitochondrial intracellular stores. Caffeine (4 mM) and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate HCl (0.5 mM) suppressed the release of calcium from internal stores and the entry of calcium from outside the cells, but permitted capping in more than half of the cells. Phorbol esters (1-2 nM) inhibited both capping and the anti-Ig-induced decrease in [Ca2+]i. None of these agents blocked the binding of anti-Ig to the cells. It appears that receptor capping is not dependent on the anti-Ig-induced transient increase in calcium concentration.     

Interference of heavy metal cations with fluorescent Ca2+ probes does not affect Ca2+ measurements in living cells

In studies about the effects of heavy metals on intracellular Ca2+, the use of fluorescent probes is debated, as metal cations are known to affect the probe signal. In this study, spectrofluorimetric experiments in free solution, using Fluo-3 and Fura-2, showed that Zn2+ and Cd2+ enhanced the probe signal, Cu2+ quenched it, and Hg2+ had no effect. Addition of GSH prevented most of these effects, suggesting the occurrence of a similar protective role in living cells. Digital imaging of living mussel haemocytes loaded with Fura-2/AM or Fluo-3/AM showed that Hg2+, Cu2+ and Cd2+ induced a rise in probe fluorescence, whereas up to 200 microM Zn2+ had no effect. In particular, Cd2+ produced the strongest probe signal rise in free solution, but the lowest fluorescence increase in cells. Probe calibration yielded [Ca2+]i values characteristic of resting levels in control and Zn2+-exposed cells, and, as expected, indicated Ca2+ homeostasis impairment in cells exposed to Cd2+, Cu2+ and Hg2+. Our results show that Ca2+ probe responses to heavy metals in living cells are completely different from those obtained in free solution, indicating that fluorescent probes can be a suitable tool to record the effects of heavy metals on [Ca2+]i.     

Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in dissociated bovine parathyroid cells using the fluorescent indicator quin-2 or fura-2. Small increases in the concentration of extracellular Ca2+ produced relatively slow, monophasic increases in [Ca2+]i in quin-2-loaded cells, but rapid and transient increases followed by lower, yet sustained (steady-state), [Ca2+]i increases in fura-2-loaded cells. The different patterns of change in [Ca2+]i reported by quin-2 and fura-2 appear to result from the greater intracellular Ca2+-buffering capacity present within quin-2-loaded cells, which tends to damp rapid and transient changes in [Ca2+]i. In fura-2-loaded parathyroid cells, other divalent cations (Mg2+, Sr2+, Ba2+) also evoked transient increases in [Ca2+]i, and their competitive interactions suggest that they all affect Ca2+ transients by acting on a common site. In contrast, divalent cations failed to cause increases in steady-state levels of cytosolic Ca2+. Low concentrations of La3+ (0.5-10 microM) depressed steady-state levels of cytosolic Ca2+ elicited by extracellular Ca2+ but were without effect on transient increases in [Ca2+]i elicited by extracellular Ca2+, Mg2+ or Sr2+, suggesting that increases in the steady-state [Ca2+]i arise from the influx of extracellular Ca2+. Mg2+- and Sr2+-induced cytosolic Ca2+ transients persisted in the absence of extracellular Ca2+ but were abolished by pretreatment with ionomycin. These results show that cytosolic Ca2+ transients arise from the mobilization of cellular Ca2+ from a nonmitochondrial pool. Extracellular divalent cations thus appear to act at some site on the surface of the cell, and this site can be considered a "Ca2+ receptor" which enables the parathyroid cell to detect small changes in the concentration of extracellular Ca2+.     

Uptake of Ca2+ and refilling of intracellular Ca2+ stores in Ehrlich-ascites-tumour cells and in rat thymocytes.

We have studied the uptake of Ca2+ and its redistribution between the cytoplasm and the intracellular stores in Ehrlich-ascites-tumour cells and rat thymocytes previously depleted of Ca2+ by incubation in Ca2(+)-free medium. Measurements included changes of the cytoplasmic Ca2+ concentration ([Ca2+]i), uptake of 45Ca2+ and uptake of Mn2+, a Ca2+ surrogate for Ca2+ channels. Refilling of the Ca2+ stores in thymocytes was very fast (half-filling time: 4 s at 37 degrees C) and very sensitive to temperature (10 times slower at 20 degrees C). It was always preceded by increase of [Ca2+]i. In the Ehrlich cell, both refilling and increase of [Ca2+]i were about one order of magnitude slower. The increase of [Ca2+]i and the refilling of the intracellular stores were both almost completely blocked by Ni2+ in thymocytes, but only partially in the Ehrlich cell. The rates of 45Ca2+ and Mn2+ uptake varied consistently with temperature and the kind of cell. These results suggest that the intracellular stores are refilled by Ca2+ taken up from the cytoplasm. We also find that filling of the Ca2+ stores decreases by about 90% the rate of Mn2+ uptake in thymocytes. This is direct evidence of modulation of the plasma-membrane Ca2+ entry by the degree of filling of the intracellular stores. This modulation occurs in the absence of agonists, suggesting some kind of signalling between the intracellular stores and the Ca2+ entry pathways of the plasma membrane.     

Evidence for TRH-induced influx of extracellular Ca2+ in pituitary GH4C1 cells

The aim of the study was to investigate the relationship between thyrotropin-releasing hormone (TRH)-induced changes in intracellular free Ca2+ ([Ca2+]i), and influx of extracellular Ca2+ in Fura 2 loaded pituitary GH4C1 cells. Stimulating the cells with TRH in a Ca(2+)-containing buffer induced a biphasic change in [Ca2+]i. First, a transient increase in [Ca2+]i, followed by a sustained phase. In cells stimulated with TRH in a Ca(2+)-free buffer, the transient increase in [Ca(2+)]i was decreased (p less than 0.05), and the sustained phase was totally abolished. Addition of Ni2+ prior to TRH blunted the component of the TRH-induced transient increase in [Ca2+]i dependent on influx of Ca2+. In the presence of extracellular Mn2+, TRH stimulated quenching of Fura 2 fluorescence. This quenching was blocked by Ni2+. The results indicate that both the TRH-induced transient increase in [Ca2+]i as well as the sustained phase in [Ca2+]i in GH4C1 cells is dependent on influx of extracellular Ca2+.     

Properties of different Ca2+ pools in permeabilized rat thymocytes

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen-induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin-permeabilized and intact rat thymocytes using a Ca2+-selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin-2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non-mitochondrial. Ca2+ uptake by the non-mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 microM. The non-mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 microM. At free Ca2+ concentration close to the cytoplasmic (0.1 microM) the main calcium pool in permeabilized thymocytes is localized in the non-mitochondrial compartment. Ca2+ accumulated in the non-mitochondrial pool can be released by inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP-dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non-mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3-induced Ca2+ mobilization. Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin-2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non-mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+-containing an Ca2+-free medium. This indicates a mitogen-induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.     

Ca2+ dependence of Ca2+ release from intracellular stores of intact and permeabilized Ehrlich ascites tumour cells

Effects of Ca2+ ions on the mobilization of Ca2+ from intracellular stores of intact and permeabilized (15 microM digitonin) Ehrlich ascites tumour cells (EATC) have been compared. For permeabilized cells, the dependences of the initial rate and amplitude of Ca2+ mobilization evoked by the addition of 100 nM inositol 1,4,5-trisphosphate (IP3) on preexisting [Ca2+] were bell-shaped within a [Ca2+] range 10(-7)-10(-6) M with the maxima at [Ca2+] = 166 nM. In intact cells, different concentrations of free cytosolic Ca2+ ([Ca2+]i) were produced using low (up to 0.005%) concentrations of digitonin which selectively increased the permeability of the plasma membrane. Stimulation of cells by exogenous ATP at [Ca2+]i = 10(-8)-10(-6) M resulted in Ca2+ mobilization the rate and amplitude of which were maximal at 102-115 nM Ca2+. The experimental Ca2+ dependences were fit by a model which includes channel opening upon Ca2+ binding and transition to the inactive states upon Ca2+ binding to the closed and open channel forms. Three inactivation types (including two particular cases) demonstrate a slight priority of inhibitory binding of Ca2+ only to the open channel, but predict markedly different parameter values. We conclude that an increase in [Ca2+] can stimulate IP3-induced mobilization, but in intact EATC, deviations of [Ca2+]i from the resting level (about 100 nM) attenuate responses to the agonist stimulation.     

Free cytosolic Ca2+ measurements with fluorine labelled indicators using 19FNMR

Characterisation by 19F NMR of fluorine-labelled indicators of cytosolic free Ca2+ concentration (by 5FBAPTA) and pH (by Fquene) is described, together with the techniques used to load the cell suspensions with the indicators for NMR spectroscopy. Useful features of the 19F NMR indicators include direct identification of the intracellular cation bound to the indicators, internal calibration of [Ca]i and pHi from the spectra, and simultaneous measurements of two or more indicators in the same cell suspension. Perturbations of cellular functions by 5FBAPTA and quin 2 are very similar, but vary widely in different cell systems. The [Ca]i and pHi responses of normal and transformed cells to mitogens and growth factors in serum are compared with data from similar experiments using fluorescence indicators. The only major discrepancy in [Ca]i measurements using the two independent assays was observed in Ehrlich ascites tumour cells. These cells have a high intracellular Zn2+ content which substantially quenches the quin 2 fluorescence, but does not affect [Ca]i measurements by 5FBAPTA. The Zn2+ present in the cells is detected as a separate response in the 5FBAPTA spectrum. The time course of the Ca signal in 2H3 cells stimulated by antigen to release histamine by exocytosis has been defined using 5FBAPTA and quin 2. Extension of the 19F NMR technique to [Ca] i and pHi measurements in perfused organs is illustrated in rat heart and responses to pharmacological agents are demonstrated. Developments in prospect to improve sensitivity and to measure [Na]i with a new family of indicators are outlined.     

A reassessment of the effects of luminal [Ca2+] on inositol 1,4,5-trisphosphate-induced Ca2+ release from internal stores

Inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from intracellular stores displays complex kinetic behavior. While it well established that cytosolic [Ca2+] can modulate release by acting on the InsP3 receptor directly, the role of the filling state of internal Ca2+stores in modulating Ca2+ release remains unclear. Here we have reevaluated this topic using a technique that permits rapid and reversible changes in free [Ca2+] in internal stores of living intact cells without altering cytoplasmic [Ca2+], InsP3 receptors, or sarcoendoplasmic reticulum Ca2+ ATPases (SERCAs). N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a membrane-permeant, low affinity Ca2+ chelator was used to manipulate [Ca2+] in intracellular stores, while [Ca2+] changes within the store were monitored directly with the low-affinity Ca2+ indicator, mag-fura-2, in intact BHK-21 cells. 200 microM TPEN caused a rapid drop in luminal free [Ca2+] and significantly reduced the extent of the response to stimulation with 100 nm bradykinin, a calcium-mobilizing agonist. The same effect was observed when intact cells were pretreated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(acetoxymethyl ester) (BAPTA-AM) to buffer cytoplasmic [Ca2+] changes. Although inhibition of Ca2+ uptake using the SERCA inhibitor tBHQ permitted significantly larger release of Ca2+ from stores, TPEN still attenuated the release in the presence of tBHQ in BAPTA-AM-loaded cells. These results demonstrate that the filling state of stores modulates the magnitude of InsP3-induced Ca2+release by additional mechanism(s) that are independent of regulation by cytoplasmic [Ca2+] or effects on SERCA pumps.     

Upregulation of endothelial cell Ca2+ signaling contributes to pregnancy-enhanced vasodilation of rat uteroplacental arteries

Normal pregnancy is characterized by an increased uterine blood flow due to growth and remodeling of the maternal uterine vasculature and enhanced vasodilation of the uterine arteries. The objective of the present study was to examine the role of endothelial cell Ca2+ signaling in augmented endothelium-mediated vasodilation of uteroplacental arteries in late pregnancy. We performed fura-2-based measurements of the intracellular Ca2+ concentration ([Ca2+]i) in the cytoplasm of endothelial cells simultaneously with diameter in pressurized uterine arteries from nonpregnant (NP) and late-pregnant (LP) rats. Basal levels of endothelial cell [Ca2+]i were higher in arteries from LP rats compared with NP controls. Withdrawal of extracellular Ca2+ resulted in a decrease in the level of basal [Ca2+]i that was significantly larger in arteries of LP than NP rats. The rate of Mn2+ -induced quenching of fura-2 fluorescence was significantly elevated in late pregnancy, implicating augmented Ca2+ influx as a cause of increased basal levels of [Ca2+]i in endothelial cells. Elevation of intraluminal pressure resulted in a transient increase in endothelial [Ca2+]i that was markedly potentiated in late gestation. ACh-induced [Ca2+]i and vasodilator responses were significantly augmented in arteries of LP compared with NP rats and were abolished by BAPTA treatment, demonstrating a critical role of [Ca2+]i elevation in the production of endothelium-derived vasodilators. Together, these results indicate that late pregnancy is a state of enhanced basal and stimulated Ca2+ signaling in endothelial cells of uterine vessels, which may represent an important underlying mechanism for augmented vasodilation in the maternal uterine circulation.     

Activation of protein kinase C assists insulin producing cells in recovery from raised cytoplasmic Ca2+ by stimulating Ca2+ efflux

The effects of protein kinase C (PKC) activation on the cytoplasmic free Ca2+ concentration ([Ca2+]i) were studied in clonal insulin-producing RINm5F cells, using the fluorescent Ca2+ indicators quin-2 and fura-2. Both under basal and stimulatory conditions PKC activation lowered [Ca2+]i in these cells by promoting an active extrusion of Ca2+ to the extracellular space. PKC activation therefore assists insulin-producing cells in recovery from raised [Ca2+]i. Such an effect might be part of the signal regulating the insulin secretory process.     

Cytokinin increases intracellular Ca2+ in Funaria: detection with Indo-1.

Changes in intracellular [Ca2+] ([Ca2+]i) after cytokinin-treatment in protonema cells of the moss Funaria hygrometrica have been measured using the pentapotassium salt of Indo-1. The extent of dye loading strongly depended on lowering the pH of the incubation medium to 5.0. Exposing dye-loaded cells briefly with Mn2+ did not quench fluorescence suggesting that the source of fluorescence is from the cytoplasm and not from the cell wall. Indo-1 remains responsive to changes in [Ca2+]i in Funaria cells. The [Ca2+]i in quiescent cells (with and without extracellular Ca2+) is 250 nM, which is within the range of reported [Ca2+]i of other plant cells. Treatment of cells with extracellular cytokinin in 4 mM Ca2+ induced a three-fold increase in [Ca2+]i to 750 nM in target caulonema cells. This increase was not observed in Ca(2+)-free medium. These target cells respond to cytokinin treatment by an asymmetrical division, while non-target chloronema cells do not divide. Cytokinin appears to increase [Ca2+]i by extracellular Ca2+ uptake. However, non-target chloronema cells and tip cells also respond to cytokinin treatment by increasing [Ca2+]i. The differential physiological response of these cell types to hormonal stimulation must lie further down the signal transduction chain.     

Internalization of metallochromic Ca2+ indicators in mammalian cells

Two new techniques for internalizing metallochromic indicators into the cytosol of mammalian cells are described. One method consists of hypertonically treating the cells in the presence of the indicator, followed by a hypoosmotic treatment. The second method consists of incubating the cells at high density in a concentrated indicator solution in physiological saline. Using either method, arsenazo III or antipyrylazo III was internalized into Ehrlich Ascites tumor (EAT) cells at concentrations yielding measurable differential absorbance changes which correspond to changes in the intracellular Ca2+ concentration. In the case of antipyrylazo III, the amount of indicator internalized ranged between 140 and 350 microM, and was dependent on the metabolic state of the cell during loading. Control and loaded cells possessed virtually identical ATP/ADP ratios, as measured by high performance liquid chromatography (HPLC) in cell extracts. Antipyrylazo III was also internalized by rat hepatocytes without detectable cell damage. Treatment of metabolically active EAT cells with the calcium ionophore A23187 results in only a slight increase in the intracellular free Ca2+ concentration, [Ca2+]i, whereas treatment with the calcium ionophore ionomycin induces a substantial but transient increase in the [Ca2+]i. In contrast, metabolically inhibited EAT cells show a large rise in the [Ca2+]i upon addition of A23187. Thus, these techniques offer another way of measuring intracellular free Ca2+ changes in mammalian cells and may prove useful, especially where concentrations of free cytosolic Ca2+ larger than 1 microM are expected.     

Effects of Helicobacter pylori on intracellular Ca2+ signaling in normal human gastric mucous epithelial cells

In stomach, Helicobacter pylori (Hp) adheres to gastric mucous epithelial cells (GMEC) and initiates several different signal transduction events. Alteration of intracellular Ca2+ concentration ([Ca2+]i) is an important signaling mechanism in numerous bacteria-host model systems. Changes in [Ca2+]i induced by Hp in normal human GMEC have not yet been described; therefore, we examined effects of Hp on [Ca2+]i in normal human GMEC and a nontransformed GMEC line (HFE-145). Cultured cells were grown on glass slides, porous filters, or 96-well plates and loaded with fura 2 or fluo 4. Hp wild-type strain 60190 and vacA-, cagA-, and picB-/cagE- isogenic mutants were incubated with cells. Changes in [Ca2+]i were recorded with a fluorimeter or fluorescence plate reader. Wild-type Hp produced dose-dependent biphasic transient [Ca2+]i peak and plateau changes in both cell lines. Hp vacA- isogenic mutant produced changes in [Ca2+]i similar to those produced by wild type. Compared with wild type, cagA- and picB-/cagE- isogenic mutants produced lower peak changes and did not generate a plateau change. Preloading cultures with intracellular Ca2+ chelator BAPTA blocked all Hp-induced [Ca2+]i changes. Thapsigargin pretreatment of cultures to release Ca2+ from internal stores reduced peak change. Extracellular Ca2+ removal reduced plateau response. Hp-induced peak response was sensitive to G proteins and PLC inhibitors. Hp-induced plateau change was sensitive to G protein inhibitors, src kinases, and PLA2. These findings are the first to show that H. pylori alters [Ca2+]i in normal GMEC through a Ca2+ release/influx mechanism that depends on expression of cagA and picB/cagE genes.     

Effect of diethylstilbestrol on Ca2+ handling and cell viability in human breast cancer cells

In human breast cancer cells, the effect of the widely prescribed estrogen diethylstilbestrol (DES) on intracellular Ca2+ concentrations ([Ca2+]i) and cell viability was explored by using fura-2 and trypan blue exclusion, respectively. DES caused a rise in [Ca2+]i in a concentration-dependent manner (EC50 = 15 microM). DES-induced [Ca2+]i rise was reduced by 80 % by removal of extracellular Ca2+. DES-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that DES induced extracellular Ca2+ influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of DES on [Ca2+]i was greatly inhibited. Conversely, pretreatment with DES to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+, whereas ionomycin added afterward still released some Ca2+. These findings suggest that in human breast cancer cells, DES increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum. Acute trypan blue exclusion studies suggest that 10-20 NM DES killed cells in a time-dependent manner.     

Simultaneous analysis of cell Ca2+ and Ca2(+)-stimulated chloride conductance in colonic epithelial cells (HT-29).

We used perforated patch, whole-cell current recordings and video-based fluorescence ratio imaging to monitor the relation of plasma membrane ionic conductances to intracellular free Ca2+ within individual colonic epithelial cells (HT-29). The Ca2(+)-mediated agonist, neurotensin, activated K+ and Cl- conductances that showed different sensitivities to [Ca2+]i. The Cl- conductance was sensitive to increases or decreases in [Ca2+]i around the resting value of 76 +/- 32 (mean +/- SD) nM (n = 46), whereas activation of the K+ conductance required at least a 10-fold rise in [Ca2+]i. Neurotensin increased [Ca2+]i by stimulating a transient intracellular Ca2+ release, which was followed by a sustained rise in [Ca2+]i due to Ca2+ influx from the bath. The onset of the initial [Ca2+]i transient, monitored at a measurement window over the cell interior, lagged behind the rise in Cl- current during agonist stimulation. This lag was not present when the [Ca2+]i rise was due to Ca2+ entry from the bath, induced either by the agonist or by the Ca2+ ionophore ionomycin. The temporal differences in [Ca2+]i and Cl- current during the agonist-induced [Ca2+]i transient can be explained by a localized Ca2+ release from intracellular stores in the vicinity of the plasma membrane Cl- channel. Chloride currents recover toward basal values more rapidly than [Ca2+]i after the agonist-induced [Ca2+]i transient, and, during a sustained neurotensin-induced [Ca2+]i rise, Cl- currents inactivate. These findings suggest that an inhibitory pathway limits the increase in Cl- conductance that can be evoked by agonist. Because this Cl- current inhibition is not observed during a sustained [Ca2+]i rise induced by ionomycin, the inhibitory pathway may be mediated by another agonist-induced messenger, such as diacylglycerol.     

The caffeine-sensitive Ca2+ store in bovine adrenal chromaffin cells; an examination of its role in triggering secretion and Ca2+ homeostasis

The effect of caffeine on catecholamine secretion and intracellular free Ca2+ concentration [( Ca2+]i) in bovine adrenal chromaffin cells was examined using single fura-2-loaded cells and cell populations. In cell populations caffeine elicited a large (approximately 200 nM) transient rise in [Ca2+]i that was independent of external Ca2+. This rise in [Ca2+]i triggered little secretion. Single cell measurements of [Ca2+]i showed that most cells responded with a large (greater than 200 nM) rise in [Ca2+]i, whereas a minority failed to respond. The latter, whose caffeine-sensitive store was empty, buffered a Ca2+ load induced by a depolarizing stimulus more effectively than those whose store was full. The caffeine-sensitive store in bovine chromaffin cells may be involved in Ca2+ homeostasis rather than in triggering exocytosis.     

Nordihydroguaiaretic acid-induced Ca2+ handling and cytotoxicity in human prostate cancer cells

The effect of nordihydroguaiaretic acid (NDGA), a compound commonly used as a lipoxygenases inhibitor, on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells was investigated. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. NDGA increased [Ca2+]i in a concentration-dependent manner with an EC50 of 30 microM. The Ca2+ signal comprised a gradual and sustained increase. Removal of extracellular Ca2+ partly decreased the NDGA-induced [Ca2+]i increase, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and intracellular Ca2+ release. NDGA-induced Ca2+ influx was independently confirmed by measuring NDGA-induced Mn2+ -coupled quench of fura-2 fluorescence. The NDGA-induced Ca2+ influx was not affected by L-type Ca2+ channel blockers. In Ca2+ -free medium, the NDGA-induced [Ca2+]i increase was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with NDGA abolished thapsigargin-induced [Ca2+]i increase. NDGA-induced intracellular Ca2+ release was not altered by inhibition of phospholipase C. Overnight treatment with 20-50 microM NDGA inhibited cell proliferation rate in a concentration-dependent manner. Several other lipoxygenases inhibitors did not alter [Ca2+]i. Collectively, this study shows that in prostate cells, NDGA induced a [Ca2+]i increase via releasing stored Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca2+ influx. NDGA also caused cytotoxicity at higher concentrations.