Measurement of cytoplasmic calcium in aleurone protoplasts using indo-1 and fura-2

Previous attempts to measure cytoplasmic Ca2+ in plant cells using the new generation of fluorescent probes, indo-1 and fura-2, have been unsuccessful. We investigated the use of indo-1 and fura-2 to measure cytoplasmic Ca2+ in barley aleurone protoplasts and found that indo-1 could be successfully used when it was loaded into protoplasts in the Ca2+-sensitive form. The acetoxymethyl esters of both dyes accumulated in aleurone protoplasts, but fura-2 was sequestered in the vacuole and indo-1 was not adequately hydrolyzed. We developed a non-disruptive method for loading the Ca2+-sensitive form of indo-1 into aleurone protoplasts in mildly acidic solutions. Using this approach, protoplasts accumulate indo-1 in a pH-dependent manner. The accumulated dye is Ca2+-sensitive, it is not sequestered in vacuoles or the endomembrane system, and it is not rapidly secreted. Fluorescence from indo-1 in individual cells was quenched by Mn2+ in the presence of digitonin. We estimate the cytoplasmic Ca2+ concentration in aleurone protoplasts to be approximately 250 nM. The Ca2+ ionophore, ionomycin does not induce changes in the fluorescence of protoplasts loaded with indo-1, but fluorescence changes could be induced by changes in extracellular Ca2+ in the presence of digitonin. We conclude that the strategy of loading indo-1 at acidic pH provides a useful means of measuring cytoplasmic Ca2+ in the barley aleurone that may also be applicable to other types of plant cells.     

Determination of intracellular Ca2+ in cells of intact wheat roots: loading of acetoxymethyl ester of Fluo‐3 under low temperature

Loading of Ca²⁺-sensitive fluorescent probes into plant cells is an essential step to measuring activities of cytoplasmic free Ca²⁺ ions with a fluorescent imaging technique. A major barrier to the loading of the fluorescent probes into plant cells using the acetoxymethyl (AM) esters of the Ca²⁺-sensitive dyes is the presence of cell-wall associated esterases. These esterases hydrolyse the esterified form of the fluorescent probes, rendering the probes membrane-impermeable. A novel non-invasive loading protocol was described in this paper to load the Ca²⁺-sensitive fluorescent probe Fluo-3/AM ester into apical cells of intact wheat roots by incubating the roots in Fluo-3/AM ester solution at 4°C for 2 h followed by 2-h incubation in the dye-free solution at 20°C. The incubation at low temperature inhibited extracellular hydrolysis of Fluo-3/AM ester but had less effect on diffusion of membrane-permeable Fluo-3/AM ester across the plasma membrane, because hydrolysis of Fluo-3/AM ester by extracellular esterases is a chemical process (high Q₁₀), while diffusion of Fluo-3/AM across the plasma membrane is a physical process (low Q₁₀). The Fluo-3/AM ester, accumulated in the root cells during the low temperature incubation, was then cleaved by intracellular esterases during the incubation at 20°C, releasing the membrane-impermeable Ca²⁺-sensitive Fluo-3 in the cytoplasm. The root cells loaded with Fluo-3 showed strong intracellular fluorescence under confocal microscopy. The fluorescence from the root cells was sensitive to the Ca²⁺ ionophore and hydrogen peroxide, indicating that the intracellular fluorescence was due to intracellular Ca²⁺ ions.     

Quantitative intracellular calcium imaging with laser-scanning confocal microscopy

Laser-scanning confocal microscopy has been used to visualise the fluorescence of a visible wavelength Ca2(+)-sensitive fluorophore, Fluo-3 in isolated cardiac myocytes. A protocol for the derivation of quantitative information from this single wavelength indicator is presented. This paradigm involves co-loading cells with two Ca2(+)-sensitive fluorescent indicators, Fluo-3 and Fura-2. Wide-field ratiometric measurements of Fura-2 fluorescence provided a baseline [Ca2+] upon which changes in Fluo-3 fluorescence could be directly expressed as [Ca2+] changes. The Ca2+ changes occurring in spontaneously active cardiac cells are presented as an example of the method. Although fluorescence energy transfer between Fura-2 and Fluo-3 was detectable in some in vitro mixtures of the two fluorophores, this process was not evident in co-loaded cardiac cells under the loading conditions employed.     

Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics

The Ca(2+) dissociation constant (K(d)) of Fluo-3 was determined using confocal fluorescence microscopy in two different situations: (i) within the cytosol of a permeabilised cardiomyocyte; and (ii) in an intact cardiomyocyte after incubation with the acetoxymethyl ester form of Fluo-3 (AM). Measurements were made on isolated rabbit ventricular cardiomyocytes after permeabilisation by a brief treatment with beta-escin (0.1mg/ml) and equilibration with 10 microM Fluo-3. The K(d) of Fluo-3 within the cytosol was not significantly different from that in free solution (558 +/- 15 nM, n=6). Over a range of cytoplasmic [Ca(2+)], the minimum [Ca(2+)] values between Ca(2+) waves was relatively constant despite changes in wave frequency. After loading intact cardiomyocytes with Fluo-3 by incubation with the -AM, spontaneous Ca(2+) waves were produced by incubation with strophanthidin (10 microM). By assuming a common minimum [Ca(2+)] in permeabilised and intact cells, the intracellular K(d) of Fluo-3 in intact myocytes was estimated to be 898 +/-64 nM (n=6). Application of this K(d) to fluorescence records shows that Ca(2+) waves in intact cells have similar amplitudes to those in permeabilised cells. Stimulation of cardiac myocytes at 0.5 Hz in the absence of strophanthidin (room temperature) resulted in a Ca(2+) transient with a maximum and minimum [Ca(2+)] of 1190 +/- 200 and 158 +/- 30 nM (n=11), respectively.     

Confocal imaging of ionised calcium in living plant cells

Laser-scanning confocal microscopy has been used in conjunction with Fluo-3, a highly fluorescent visible wavelength probe for Ca2+, to visualize Ca2(+)-dynamics in the function of living plant cells. This combination has overcome many of the problems that have limited the use of fluorescence imaging techniques in the study of the role of cations (Ca2+ and H+) in plant cell physiology and enables these processes to be studied in single cells within intact plant tissue preparations. Maize coleoptiles respond to application of ionophores and plant growth hormones with elevations in cytosolic Ca2+ that can be resolved with a high degree of spatial resolution and can be interpreted quantitatively.     

Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca(2+)concentrations distribution in living plant tissue

A method for visualisation of cytosolic [Ca(2+)] distribution was applied to living plant tissue. A mixture of the fluorescent probes Fluo-3 and Fura Red was used. The emitted fluorescence was scanned simultaneously in two channels with a laser-scanning confocal microscope and rationing was performed. The homogeneity of the Fluo-3/Fura Red concentration ratio throughout the tissue after AM-ester loading was proven. In vitro calibration permitted conversion of Fluo-3/Fura Red fluorescence ratios to [Ca(2+)] values. Apparent K(D)of 286 nM, R(min)of 0.43 and R(max)of 18 were calculated. The in vivo determination of extreme ratio values was performed by permeabilizing the plasmalemma for Ca(2+)with a ionophore and manipulating the extracellular [Ca(2+)]. The resultant R(minv)of 1.33 and R(maxv)of 2.69 for vegetative apices, and R(mini)of 1.26 and R(maxi)of 3.45 for apices induced to flowering, suggested incomplete equalization of extra- and intracellular Ca(2+)levels in these experiments. In Chenopodium rubrum, the cytosolic [Ca(2+)] patterns of apical tissue obtained using Fluo-3 and Fura Red were significantly different between vegetative apices and apices after photoperiodic flower induction. This methodological approach may also be helpful for studying cytosolic [Ca(2+)] distribution in other living plant tissues.     

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.     

Characterization of Ca2+ release from heterogeneous Ca2+ stores in sarcoplasmic reticulum isolated from arterial and gastric smooth muscle

Ca2+ transport was investigated in vesicles of sarcoplasmic reticulum subfractionated from bovine main pulmonary artery and porcine gastric antrum using digitonin binding and zonal density gradient centrifugation. Gradient fractions recovered at 15-33% sucrose were studied as the sarcoplasmic reticulum component using Fluo-3 fluorescence or 45Ca2+ Millipore filtration. Thapsigargin blocked active Ca2+ uptake and induced a slow Ca2+ release from actively loaded vesicles. Unidirectional 45Ca2+ efflux from passively loaded vesicles showed multicompartmental kinetics. The time course of an initial fast component could not be quantitatively measured with the sampling method. The slow release had a half-time of several minutes. Both components were inhibited by 20 microM ruthenium red and 10 mM Mg2+. Caffeine, inositol 1,4,5-trisphosphate, ATP, and diltiazem accelerated the slow component. A Ca2+ release component activated by ryanodine or cyclic adenosine diphosphate ribose was resolved with Fluo-3. Comparison of tissue responses showed that the fast Ca2+ release was significantly smaller and more sensitive to inhibition by Mg2+ and ruthenium red in arterial vesicles. They released more Ca2+ in response to inositol 1,4,5-trisphosphate and were more sensitive to activation by cyclic adenosine diphosphate ribose. Ryanodine and caffeine, in contrast, were more effective in gastric antrum. In each tissue, the fraction of the Ca2+ store released by sequential application of caffeine and inositol 1,4,5-trisphosphate depended on the order applied and was additive. The results indicate that sarcoplasmic reticulum purified from arterial and gastric smooth muscle represents vesicle subpopulations that retain functional Ca2+ channels that reflect tissue-specific pharmacological modulation. The relationship of these differences to physiological responses has not been determined.     

维生素D3对三角帆蚌外套膜细胞内Ca2+浓度的影响

采用胰酶消化法获得三角帆蚌(Hyriopsis cumingii)外套膜细胞,用Fluo-3/AM荧光标记技术和激光共聚焦扫描显微镜(confocal laser scanning microscope,CLSM)检测外套膜内外表皮细胞在静息状态下细胞的游离ca2+浓度及不同浓度维生素D.(VD.)孵育后的外套膜细胞的...     

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.     

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.     

A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals

Quantifying the magnitude of Ca2+ signals from changes in the emission of fluorescent indicators relies on assumptions about the indicator behaviour in situ. Factors such as osmolarity, pH, ionic strength and protein environment can affect indicator properties making it advantageous to calibrate indicators within the required cellular or subcellular environment. Selecting Ca2+ indicators appropriate for a particular application depends upon several considerations including Ca2+ binding affinity, dynamic range and ease of loading. These factors are usually best determined empirically. This study describes the in-situ calibration of a number of frequently used fluorescent Ca2+ indicators (Fluo-3, Fluo-4, Calcium Green-1, Calcium Orange, Oregon Green 488 BAPTA-1 and Fura-Red) and their use in reporting low- and high-amplitude Ca2+ signals in HeLa cells. All Ca2+ indicators exhibited lower in-situ Ca2+ binding affinities than suggested by previously published in-vitro determinations. Furthermore, for some of the indicators, there were significant differences in the apparent Ca2+ binding affinities between nuclear and cytoplasmic compartments. Variation between indicators was also found in their dynamic ranges, compartmentalization, leakage and photostability. Overall, Fluo-3 proved to be the generally most applicable Ca2+ indicator, since it displayed a large dynamic range, low compartmentalization and an appropriate apparent Ca2+ binding affinity. However, it was more susceptible to photobleaching than many of the other Ca2+ indicators.     

Membrane-proximal calcium transients in stimulated neutrophils detected by total internal reflection fluorescence.

A novel fluorescence microscope/laser optical system was developed to measure fast transients of membrane-proximal versus bulk cytoplasmic intracellular calcium levels in cells labeled with a fluorescent calcium indicator. The method is based on the rapid chopping of illumination of the cells between optical configurations for epifluorescence, which excites predominantly the bulk intracellular region, and total internal reflection fluorescence, which excites only the region within approximately 100 nm of the cell-substrate contact. This method was applied to Fluo-3-loaded neutrophils that were activated by the chemoattractant N-formyl-met-leu-phe. Chemoattractant-activated cells showed 1) transient increases in both membrane-proximal and bulk cytosolic Ca2+ that peaked simultaneously; 2) a larger fractional change (20-60%) in membrane-proximal Ca2+ relative to bulk cytosolic Ca2+ that peaked at a time when the main Ca2+ transient was decreasing in both regions and that persisted well after the main transient was over. This method should be applicable to a wide variety of cell types and fluorescent ion indicators in which membrane-proximal ionic transients may be different from those deeper within the cytosol.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

Evidence that binding of Indo-1 to cardiac myocyte protein does not markedly change Kd for Ca2+

Quantitative measurement of [Ca2+]i with the fluorescent Ca(2+)-indicators Indo-1 and Fura-2 is complicated by the possibility that the value of the dissociation constant (Kd) may be influenced by binding to intracellular proteins. We investigated this question in cultured chick ventricular myocytes by use of two different Indo-1 calibration methods. First, the Indo-1 fluorescence ratio (R) (400/500 nm) was measured in beating myocytes loaded by exposure to Indo-1/AM. Then, cells were exposed to the Ca2+ ionophore Br A-23187 and fluorescence ratio was measured in the presence of 500 nM Ca2+ (EGTA-Ca2+ buffer). Subsequently cells were permeabilized to Ca2+ by a 1 min exposure to 25 microM digitonin in the presence of 'zero' Ca2+ (10 mM EGTA) and saturating 1 mM Ca2+ to obtain Rmin, Rmax and beta. We then calculated [Ca2+]i from the formula ([Ca2+]i = Kd [( R - Rmin)/(Rmax - R)]beta). With Kd = 250 nM, calculated systolic [Ca2+]i was 750 +/- 44 nM and diastolic 269 +/- 19 nM (means +/- SEM, n = 16). The R value calculated for an assumed [Ca2+]i = 500 nM using the above formula and digitonin derived constants was very similar to the value measured using Br A-23187 (digitonin, 0.67 +/- 0.03: Br A-23187, 0.66 +/- 0.03, ns). As the Br A-23187 method is independent of the value chosen for Kd, we conclude that the Kd of 250 nM for Indo-1 measured in free solutions closely approximates the Kd for intracellular Indo-1 in these cells, and that therefore the Kd of Indo-1 for Ca2+ does not appear to be markedly affected by binding to proteins or other intracellular molecules.     

Measurement of Ca2+ signaling dynamics in exocrine cells with total internal reflection microscopy

In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+ signals consist of both Ca2+ release and Ca2+ influx. We have investigated the contribution of these processes to membrane-localized Ca2+ signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+ oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+ release in the TIRF zone would be expected to saturate the Ca2+ indicator (Fluo-4), these data suggest that Ca2+ release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+ release, followed by a substantial, maintained plateau phase indicative of Ca2+ entry, was observed. The contribution of Ca2+ influx and Ca2+ release in isolation to these near-plasma membrane Ca2+ signals was investigated by using a Ca2+ readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+ release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+ influx was isolated by subsequent Ca2+ readmission, the Ca2+ signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+ readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+ influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+ influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+ signals mediated by Ca2+ influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+ influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.     

Loss and Ca2+-Dependent Retention of Scinderin in Digitonin-Permeabilized Chromaffin Cells: Correlation with Ca2+-Evoked Catecholamine Release

Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca(2+)-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca(2+)-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca(2+)-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca(2+)-dependent catecholamine release observed in permeabilized chromaffin cells.     

Calcium compartmentation in isolated renal tubules in suspension

Substantial increases of total cell Ca2+ have been observed in suspensions of isolated rabbit proximal tubules subjected to hypoxic injury or treated with exogenous ATP followed by apparent recovery with reoxygenation of the hypoxic tubules or continued incubation of ATP-treated tubules. Ca2+ compartmentation studies using digitonin and metabolic inhibitors were done to clarify the basis for these changes. Digitonin, 40-90 micrograms/mg tubule protein, rapidly permeabilized the tubule cells and did not impair mitochondrial Ca2+ sequestration. Most of the increases of tubule cell Ca2+ produced by hypoxia and ATP were accounted for by pools which could be rapidly removed by exposure of tubules to EGTA and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone without concomitant use of digitonin, suggesting that the changes of Ca2+ predominantly reflect sequestration by mitochondria in severely damaged cells or mitochondria already released to the medium from them. The time course of uptake followed by spontaneous release of mitochondrial Ca2+ from tubule cells deliberately permeabilized with digitonin, then incubated for prolonged periods, indicated that the decreases of tubule cell Ca2+ during reoxygenation of hypoxic suspensions and prolonged incubation of ATP-treated tubules were likely to be attributable to loss of Ca2+ from free mitochondria and those in damaged cells rather than to extrusion by intact cells.     

Subcellular distribution of cytosolic Ca2+ in isolated rat hepatocyte couplets: evaluation using confocal microscopy.

Ca2+ agonists induce Ca2+ waves and other non-uniform Ca2+ patterns in the cytosol of epithelial cells. To define subcellular Ca2+ transients in the cytosol of hepatocytes we examined Fluo-3-loaded isolated rat hepatocyte couplets using confocal microscopy. Optical sections of less than 1 micron in thickness were observed in couplets, and fluorescence from cytosolic Ca2+ signals was readily distinguished from nuclear, mitochondrial, and lysosomal fluorescence. The nature of the noncytosolic components of the fluorescent images was verified by double labelling with the mitochondrial dye DiOC6(3) and with the lysosomal marker acridine orange. Using the line scanning mode of confocal microscopy, measurements of cytosolic Ca2+ were made with a frequency of up to 250 Hz and without significant bleaching. It was found that phenylephrine-induced Ca2+ signals generally began at the basal pole of the hepatocytes, then spread to the canaliculus at average speeds of 80 micron/s. These findings demonstrate the utility of confocal line scanning microscopy for detecting rapid changes in the subcellular distribution of cytosolic Ca2+ in hepatocyte couplets, and suggest that phenylephrine-induced Ca2+ waves radiate in a basal-to-apical direction in this cell type.     

Signal transduction in red blood cells 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 20 nM 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+]c in the cells from both lizard species, mostly by mobilization of the cation from internal stores.