Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells

Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca²⁺]_c) and nuclear calcium ([Ca²⁺]_n) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca²⁺]_n detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca²⁺]_n and [Ca²⁺]_c in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.     

Fast loading ester fluorescent Ca<Superscript>2+</Superscript> and pH indicators into pollen of <Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>

Loading of Ca²⁺-sensitive fluorescent probes into plant cells is an essential step to measure activities of free Ca²⁺ ions in cytoplasm with a fluorescent imaging technique. Fluo-3 is one of the most suitable Ca²⁺ indicators for CLSM. We loaded pollen with fluo-3/AM at three different temperatures. Fluo-3/AM was successfully loaded into pollen at both low (4°C) and high (37°C) temperatures. However, high loading temperature was best suited for pollen, because germination rate of pollen and growth of pollen tubes were relatively little impaired and loading time was shortened. Moreover, Ca²⁺ distribution increased in the three apertures of pollen after hydration and showed a Ca²⁺ gradient, similar to the tip of growing pollen tubes. The same protocol can be used with the AM-forms of other fluorescent dyes for effective labeling. When loading BCECF-AM into pollen at high temperature, the pollen did not show a pH gradient after hydration. Ca²⁺ activities and fluxes had the same periodicity as pollen germination, but pH did not show the same phase and mostly lagged behind. However, the clear zone was alkaline when pollen tube growth was slowed or stopped and turned acidic when growth recovered. It is likely that apical pH_i regulated pollen tube growth.     

Chelation of intracellular Ca2+ inhibits murine keratinocyte differentiation in vitro

The role of intracellular Ca²⁺ in the regulation of Ca²⁺-induced terminal differentiation of mouse keratinocytes was investigated using the intracellular Ca²⁺ chelator 1,2-bis(o-aminophenoxy)-ethane-N, N, N′, N′-tetraacetic acid (BAPTA). A cell permeable acetoxymethyl (AM) ester derivative BAPTA (BAPTA/AM) was loaded into primary mouse keratinocytes in 0.05 mM Ca²⁺ medium, and then the cells were induced to differentiate by medium containing 0.12 or 0.5 mM Ca²⁺. Intracellular BAPTA loaded by BAPTA/AM (15–30 μM) inhibited the expression of epidermal differentiation-specific proteins keratin 1 (K1), keratin 10 (K10), filaggrin and loricrin as detected by immunoblotting. The differentiation-associated redistribution of E-cadherin on the cell membrane was delayed but not inhibited as determined by immunofluorescence. BAPTA also inhibited the expression of K1, K10 and Ioricrin mRNA. Furthermore, BAPTA prevented the decrease in DNA synthesis induced by 0.12 and 0.5 mM Ca²⁺, indicating the drug was inhibiting differentiation but was not toxic to keratinocytes. To evaluate the influence of BAPTA on intracellular Ca²⁺, the concentration of intracellular free Ca²⁺ (Ca_i) in BAPTA-loaded keratinocytes was examined by digital image analysis using the Ca²⁺-sensitive fluorescent probe fura-2, and Ca²⁺ influx was measured by ⁴⁵Ca²⁺ uptake studies. Increase in extracellular Ca²⁺ (Ca_o) in the culture medium of keratinocytes caused a sustained increase in both Ca_i and Ca²⁺ localized to ionomycin-sensitive intracellular stores in keratinocytes. BAPTA lowered basal Ca_i concentration and prevented the Ca_i increase. After 12 hours of BAPTA treatment, the basal level of Ca_i returned to the control value, but the Ca²⁺ localized in intracellular stores was substantially decreased. ⁴⁵Ca²⁺ uptake was initially (within 30 min) increased in BAPTA-loaded cells. However, the total ⁴⁵Ca²⁺ accumulation over 24 hours in BAPTA-loaded cells remained unchanged from control values. These results indicate that keratinocytes can maintain Ca_i and total cellular Ca²⁺ content in the presence of increased amount of intracellular Ca²⁺ buffer (e.g., BAPTA) by depleting intracellular Ca²⁺ stores over a long period. The inhibition by BAPTA of keratinocyte differentiation marker expression may result from depletion of the Ca²⁺-stores since this is the major change in intracellular Ca²⁺ detected at the time keratinocytes express the differentiation markers. In contrast, the redistribution of E-cadherin on the cell membrane may be more directly associated with Ca_i change. © 1995 Wiley-Liss, Inc.     

Calcium is essential for fructan synthesis induction mediated by sucrose in wheat

The role of Ca²⁺ in the induction of enzymes involved in fructan synthesis (FSS) mediated by sucrose was studied in wheat (Triticum aestivum). Increase of FSS enzyme activity and induction of the expression of their coding genes by sucrose were inhibited in leaf blades treated with chelating agents (EDTA, EGTA and BAPTA). Ca²⁺ channel blockers (lanthanum chloride and ruthenium red) also inhibited the FSS response to sucrose, suggesting the participation of Ca²⁺ from both extra- and intra- cellular stores. Sucrose induced a rapid Ca²⁺ influx into the cytosol in wheat leaf and root tissues, shown with the Ca²⁺ sensitive fluorescent probe Fluo-3/AM ester. Our results support the hypothesis that calcium is a component of the sucrose signaling pathway that leads to the induction of fructan synthesis.     

Signal transduction in red bloodcells of the lizards Ameiva ameiva and Tupinambis merianae (Squamata, Teiidae)

The fluorescent calcium probe, Fluo-3, AM was used to measure the intracellular calcium concentration in red blood cells (RBCs) of the teiid lizards Ameiva ameiva and Tupinambis merianae. The cytosolic [Ca²⁺] is maintained around 20nM and the cells contain membrane-bound Ca²⁺pools. One pool appears to be identifiable with the endoplasmic reticulum (ER) inasmuch as addition of the sarco-endoplasmic reticulum Ca²⁺ATPase, SERCA, inhibitor thapsigargin induces an increase in cytosolic [Ca²⁺both in the presence and in the absence of extracellular Ca²⁺. In addition to the ER, an acidic compartment appears to be involved in Ca²⁺storage, as collapse of intracellular pHgradients by monensin, a Na⁺–H⁺exchanger, and nigericin, a K⁺–H⁺exchanger, induce the release of Ca²⁺from internal pools. A vacuolar H⁺pump, sensitive to NBD-Cl and bafilomycin appears to be necessary to load the acidic Ca²⁺pools. Finally, the purinergic agonist ATP triggers a rapid and transient increase of [Ca²⁺]_cin the cells from both lizard species, mostly by mobilization of the cation from internal stores.     

Native and recombinant ASIC1a receptors conduct negligible Ca2+ entry

Acid Sensing Ion Channels (ASICs) are a family of proton-gated cation channels that play a role in the sensation of noxious stimuli. Of these, ASIC1a is the only family member that is reported to be permeable to Ca²⁺, although the absolute magnitude of the Ca²⁺ current is unclear. Here, we used patch-clamp photometry to determine the contribution of Ca²⁺ to total current through native and recombinant ASIC1a receptors. We found that acidification of the extracellular medium evoked amiloride and psalmotoxin 1-sensitive currents in isolated chick dorsal root ganglion neurons and human embryonic kidney cells, but did not alter fura-2 fluorescence when the bath concentration of Ca²⁺ was close to that found in normal physiological conditions. Further, activation of recombinant ASIC1a receptors also failed to produce measurable changes in fluorescence despite of the fact that the total cation current through the over-expressed receptor was ten-fold larger than that of the native channels. Finally, we imaged a field of intact DRG neurons loaded with the Ca²⁺-sensing dye Fluo-4, and found that acidification increased [Ca²⁺]_i in a small population of cells. Thus, although our whole-field imaging data agree with previous studies that activation of ASIC1a receptors can potentially cause elevations in intracellular free Ca²⁺, our single cell data strongly challenges the view that Ca²⁺ entry through the ASIC1a receptor itself contributes to this response.     

Intracellular Free Calcium Concentration and Cisplatin Resistance in Human Lung Adenocarcinoma A549 Cells

Human lung adenocarcinoma A₅₄₉ cells sensitive and A₅₄₉/DDP cells resistantto Cis-dichlorodiammine platinum[II] (cisplatin) exhibit differentintracellular free calcium and calcium fluorescence images labeled withFura-2/AM and Fluo-3/AM as judged by dual-excitation fluorescence assay,Miracal Imaging and Laser Scanning Confocal Microscopy (LSCM) of singlecells. The concentration of intracellular free calcium of the resistantA₅₄₉/DDP cells is one third that of the sensitive A₅₄₉ cells. The effluxof Rhodamine 123 in resistant A₅₄₉/DDP cells is faster than that insensitive A₅₄₉ cells. In addition, A₅₄₉/DDP cells have an increase ofPhosphatidylinositol 4-kinase (PtdIns 4-kinase) activity in the plasmamembrane. So it is tentatively suggested that the increase in PtIns4-kinase activity resulting from lower intracellular Ca²⁺concentration leads to an increase of its enzymaticproducts——PIP and PIP₂, which may stimulate the activity ofP-glycoprotein.     

Effect of Celecoxib on Ca2+ Fluxes and Proliferation in MDCK Renal Tubular Cells

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca^{2 +} concentration ([Ca^{2 +}]_i) and proliferation was examined by using the Ca^{2 +}-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca^{2 +}]_i in a concentration-dependent manner. Celecoxib-induced [Ca^{2 +}]_i increase was partly reduced by removal of extracellular Ca^{2 +}. Celecoxib-induced Ca^{2 +} influx was independently suggested by Mn^{2 +} influx-induced fura-2 fluorescence quench. In Ca^{2 +}-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca^{2 +}-ATPase, caused a monophasic [Ca^{2 +}]_i increase, after which celecoxib only induced a tiny [Ca^{2 +}]_iincrease; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca^{2 +}]_i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca^{2 +}]_i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca^{2 +}]_i increase in renal tubular cells by stimulating both extracellular Ca^{2 +} influx and intracellular Ca^{2 +} release and is highly toxic to renal tubular cells in vitro.     

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca²⁺-sensitive dyes is used for measurement of spatial and temporal aspects of Ca²⁺ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca²⁺-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca²⁺-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca²⁺ response as % change in fluorescence is obtained.     

Quenching of a proton gradient and concomitant increase of intragranular calcium in interstitial cells of Mytilus retractor muscle

Summary The content of specific glio-interstitial granules in situ was studied in Mytilus retractor muscle using fluorescent probes and X-ray microanalysis. The granules readily take up the fluorescent monoamine dye acridine orange added to sea water (2.7×10^-6 M) and appear red in fluorescence microscopy. The addition of ammonium chloride (10 mM) or various proton ionophores results in extinction of the granule fluorescence. In addition, a step-wise decrease in granule fluorescence is observed when the tissue is perfused with artificial sea water of decreasing pH. These granules thus appear to be acidic inside. The animals were maintained in artificial sea water containing 8.36 mM Ca²⁺ and 528.90 mM Na⁺, the ratio R=[Ca²⁺]₀/[Na⁺]² ₀ being thus equal to 3x10^-5. Perfusions of the tissue with artificial sea water containing a higher calcium concentration (12.2 mM) and/or a higher [Ca²⁺]₀/[Na⁺]² ₀ ratio (R=4.5×10^-5) result in a drastic reduction of the proton gradient, evidenced by a quenching of the acridine orange fluorescence. Under the same conditions, a significant increase of the total intragranular calcium concentration was demonstrated by quantitative X-ray micro-analysis of the tissue processed by quick freezing and freeze-substitution in the presence of oxalic acid. The fluorescence of the probe Fluo-3/AM, indicative of ionized calcium, is higher in the granules than in the surrounding cytoplasm; this suggests that calcium is accumulated in the granule against its concentration gradient. The acidic gradient of specific glio-interstitial cell granules could provide the energy needed for this calcium accumulation through a Ca²⁺/H⁺ exchange. These results are discussed with regard to the hypothesis that the glio-interstitial tissue can regulate pericellular calcium and/or hydrogen ion ioncentration in the vicinity of nerve and muscle cells.     

Intracellular calcium chelator BAPTA protects cells against toxic calcium overload but also alters physiological calcium responses

The effect of the membrane-permeant calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N−'tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) on ionomycin-induced cellular calcium overload was studied in single differentiated NH15-CA2 neuroblastoma x glioma hybrid cells. To monitor [Ca²⁺]_i, we used the fluorescent indicator Fura-2. Preincubation of the cells with 3 μM BAPTA/AM reduced the number of cells showing deregulation of [Ca²⁺]_i during ionomycin-induced calcium influx. The calcium transients elicited by application of KCI were also severely affected by the chelator. These transients, although varying from cell to cell in shape, amplitude and duration, are well reproducible in individual cells. After incubation of cells for 1 h with 0.3–30 μM BAPTA/AM the time course of these cellular transients was markedly slowed. At 1 μM BAPTA/AM, the time constant of decline of [Ca²⁺]_i was increased by a factor of 4.1 ± 2.4 (n = 14) and the amplitude was reduced to about 50%. With 30 μM BAPTA/AM, the K⁺-induced calcium transients were almost completely inhibited. We conclude that intracellularly loaded calcium chelators may be used for the prevention of [Ca²⁺]_i-induced cell damage, however, at the expense of a disturbed calcium signalling.     

Investigation of carvedilol-evoked Ca2+ movement and death in human oral cancer cells

The effect of carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in OC2 human oral cancer cells is unknown. This study examined if carvedilol altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Carvedilol at concentrations between 10 and 40 µM increased [Ca²⁺]_i in a concentration-dependent fashion. The Ca²⁺ signal was decreased by 50% by removing extracellular Ca²⁺. Carvedilol-induced Ca²⁺ entry was not affected by the store-operated Ca²⁺ channel blockers nifedipine, econazole, and SK&F96365, but was enhanced by activation or inhibition of protein kinase C. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin did not change carvedilol-induced [Ca²⁺]_i rise; conversely, incubation with carvedilol did not reduce thapsigargin-induced Ca²⁺ release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibited carvedilol-induced [Ca²⁺]_i release. Inhibition of phospholipase C with U73122 did not alter carvedilol-induced [Ca²⁺]_i rise. Carvedilol at 5–50 µM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca²⁺ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA/AM). Annexin V/propidium iodide staining assay suggests that apoptosis played a role in the death. Collectively, in OC2 cells, carvedilol induced [Ca²⁺]_i rise by causing phospholipase C-independent Ca²⁺ release from mitochondria and non-endoplasmic reticulum stores, and Ca²⁺ influx via protein kinase C-regulated channels. Carvedilol (up to 50 μM) induced cell death in a Ca²⁺-independent manner that involved apoptosis.     

Ca2+ Release from Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Store by Antidepressant Drugs in Cultured Neurons of Rat Frontal Cortex

Abstract: The ability of antidepressant drugs (ADs) to increase the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) was examined in primary cultured neurons from rat frontal cortices using the Ca²⁺-sensitive fluorescent indicator fura-2. Amitriptyline, imipramine, desipramine, and mianserin elicited transient increases in [Ca²⁺]_i in a concentration-dependent manner (100 μM to 1 mM). These four AD-induced [Ca²⁺]_i increases were not altered by the absence of external Ca²⁺ or by the presence of La³⁺ (30 μM), suggesting that these ADs provoked intracellular Ca²⁺ mobilization rather than Ca²⁺ influx. All four ADs increased inositol 1,4,5-trisphosphate (IP₃) contents by 20–60% in the cultured cells. The potency of the IP₃ production by these ADs closely correlated with the AD-induced [Ca²⁺]_i responses. Pretreatment with neomycin, an inhibitor of IP₃ generation, significantly inhibited amitriptyline- and imipramine-induced [Ca²⁺]_i increases. In addition, by initially perfusing with bradykinin (10 μM) or acetylcholine (10 μM), which can stimulate the IP₃ generation and mobilize the intracellular Ca²⁺, the amitriptyline responses were decreased by 76% and 69%, respectively. The amitriptyline-induced [Ca²⁺]_i increases were unaffected by treatment with pertussis toxin. We conclude that high concentrations of amitriptyline and three other ADs mobilize Ca²⁺ from IP₃-sensitive Ca²⁺ stores and that the responses are pertussis toxin-insensitive. However, it seems unlikely that the effects requiring high concentrations of ADs are related to the therapeutic action.     

Zinc release from thapsigargin/IP3-sensitive stores in cultured cortical neurons

Background

Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP₃-gated thapsigargin sensitive intracellular calcium (Ca²⁺) storage provides a source of the ion for many cellular signaling events. Less is known about the dynamics of other intracellular ions. The present study investigated the intracellular source of zinc (Zn²⁺) that has been reported to play a role in cell signaling.

Results

In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn²⁺ indicators, we showed that intracellular regions of Zn²⁺ staining co-localized with the endoplasmic reticulum (ER). The latter was identified with ER-tracker Red, a marker for ER. The colocalization was abolished upon exposure to the Zn²⁺ chelator TPEN, indicating that the local Zn²⁺ fluorescence represented free Zn²⁺ localized to the ER in the basal condition. Blockade of the ER Ca²⁺ pump by thapsigargin produced a steady increase of intracellular Zn²⁺. Furthermore, we determined that the thapsigargin-induced Zn²⁺ increase was not dependent on extracellular Ca²⁺ or extracellular Zn²⁺, suggesting that it was of intracellular origin. The applications of caged IP₃ or IP₃-3Kinase inhibitor (to increase available IP₃) produced a significant increase in intracellular Zn²⁺.

Conclusions

Taken together, these results suggest that Zn²⁺ is sequestered into thapsigargin/IP₃-sensitive stores and is released upon agonist stimulation.     

The exploration of effect of terfenadine on Ca2+ signaling in renal tubular cells

Terfenadine, an antihistamine used for the treatment of allergic conditions, affected Ca²⁺-related physiological responses in various models. However, the effect of terfenadine on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) and its related physiology in renal tubular cells is unknown. This study examined whether terfenadine altered Ca²⁺ signaling and caused cytotoxicity in Madin–Darby canine kidney (MDCK) renal tubular cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i. Cell viability was measured by the fluorescent reagent 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1) assay. Terfenadine at concentrations of 100–1000?μM induced [Ca²⁺]_i rises concentration dependently. The response was reduced by approximately 35% by removing extracellular Ca²⁺. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) partly inhibited terfenadine-evoked [Ca²⁺]_i rises. Conversely, treatment with terfenadine abolished BHQ-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 inhibited 95% of terfenadine-induced Ca²⁺ release. Terfenadine-induced Ca²⁺ entry was supported by Mn²⁺-caused quenching of fura-2 fluorescence. Terfenadine-induced Ca²⁺ entry was partly inhibited by an activator of protein kinase C (PKC), phorbol 12-myristate 13 acetate (PMA) and by three modulators of store-operated Ca²⁺ channels (nifedipine, econazole, and SKF96365). Terfenadine at 200–300?μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in MDCK cells, terfenadine induced [Ca²⁺]_i rises by evoking PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ entry. Furthermore, terfenadine caused cell death that was not triggered by preceding [Ca²⁺]_i rises.     

Bending the MDCK Cell Primary Cilium Increases Intracellular Calcium

We tested the hypothesis that the primary cilium of renal epithelia is mechanically sensitive and serves as a flow sensor in MDCK cells using differential interference contrast and fluorescence microscopy. Bending the cilium, either by suction with a micropipette or by increasing the flow rate of perfusate, causes intracellular calcium to substantially increase as indicated by the fluorescent indicator, Fluo-4. This calcium signal is initiated by Ca²⁺-influx through mechanically sensitive channels that probably reside in the cilium or its base. The influx is followed by calcium release from IP₃-sensitive stores. The calcium signal then spreads as a wave from the perturbed cell to its neighbors by diffusion of a second messenger through gap junctions. This spreading of the calcium wave points to flow sensing as a coordinated event within the tissue, rather than an isolated phenomenon in a single cell. Measurement of the membrane potential difference by microelectrode during perfusate flow reveals a profound hyperpolarization during the period of elevated intracellular calcium. We conclude that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by greatly increasing intracellular calcium. Received: 4 April 2001/Revised: 28 June 2001     

In situ Ca2+ titration in the fluorometric study of intracellular Ca2+ binding

Imaging with Ca²⁺-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca²⁺ signaling. The spatiotemporal evolution of intracellular free Ca²⁺ observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca²⁺ buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca²⁺ imaging data, and can be exploited to investigate endogenous Ca²⁺ buffer properties. Here we extended the use of Ca²⁺ fluorometry in the characterization of Ca²⁺ binding molecules within cells, building on a method of titration of intracellular Ca²⁺ binding sites in situ with measured amounts of Ca²⁺ entering through voltage-gated Ca²⁺ channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca²⁺ binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca²⁺ indicator fluo-8. Focusing on data sets well described by a single endogenous Ca²⁺ buffer and dye, this method yielded estimates of the endogenous buffer concentration and K_d, the dye K_d, and the fraction of Ca²⁺ inaccessible cellular volume. The in situ K_d of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca²⁺-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca²⁺ titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca²⁺ binding species. This method offers a versatile approach to the study of endogenous Ca²⁺ binding molecules in their physiological milieu.     

Asante Calcium Green and Asante Calcium Red—Novel Calcium Indicators for Two-Photon Fluorescence Lifetime Imaging

For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca²⁺-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720–900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca²⁺-free and Ca²⁺-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca²⁺-dependent way, unraveling in vitro dissociation constants K _D of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca²⁺-free and Ca²⁺-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K _D of 180 nM was determined. Thus, quantitative [Ca²⁺]_i recordings were realized, unraveling a reversible dopamine-induced [Ca²⁺]_i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca²⁺ indicator dye for 2P-FLIM recordings applicable in diverse biological systems.     

USE OF FLUORESCENT DYES FOR MEASUREMENT AND LOCALIZATION OF ORGANELLES ASSOCIATED WITH CA2+ STORE RELEASE IN HUMAN NEUTROPHILS

Fura-2 and its lipid analogue, FFP-18, were used to measure changes in cytosolic free Ca²⁺concentration within human neutrophils. Whereas fura-2 was employed to monitor cytosolic Ca²⁺increases throughout the cytosol, FFP-18 was used to monitor Ca²⁺changes only near the membrane. This latter probe was incorporated into the plasma membrane as its acetoxymethyl ester (FFP-18-AM) but as de-esterification was catalysed by cytosolic esterases, the Ca²⁺-sensing probe (FFP-18 acid) accumulated on the inner face of membrane. The fluorescence of esterified probe on the extracellularly facing membrane leaflet was quenched by the membrane-impermeant ion Ni²⁺. Under these conditions, near membrane Ca²⁺changes which resulted from the release of Ca²⁺from intracellular stores was possible by conventional ratio fluorescence measurement of FFP-18. From the timing of arrival of Ca²⁺at the plasma membrane, it was proposed that there were two Ca²⁺storage sites, liberated by different stimuli, one close to the plasma membrane and the other more distant. In order to discover whether organelles within the neutrophil had distributions which correlate with the Ca²⁺release sites, fluorescent dyes for structures within the cytosol were employed. We have previously shown that the location of the intracellular membrane stain, DiOC₆(3) corresponds to the distant Ca²⁺release site. Here a second stain, BODIPY-C₅ceramide, has also been used and is shown to stain a peripheral region of the neutrophil, in a similar pattern to the near membrane Ca²⁺storage site. These data therefore raise the question of whether these stains mark the organelles in neutrophils which are the two Ca²⁺storage and release sites.