Erythropoietin increases cytosolic free calcium concentration and thrombin induced changes in cytosolic free calcium in platelets from spontaneously hypertensive rats

Using fura-2 cytosolic free calcium concentrations were measured in intact washed platelets from 9 spontaneously hypertensive rats (SHR) and from 9 age-matched normotensive Wistar-Kyoto rats (WKY). In resting platelets cytosolic free calcium concentration was significantly higher in SHR than in WKY (171.8 +/- 64.4 nM vs 93.1 +/- 59.0 nM, p less than 0.05). After preincubation with erythropoietin cytosolic free calcium concentration was significantly higher in SHR than in WKY (197.5 +/- 83.2 vs 93.0 +/- 60.1, p less than 0.01). Using platelets from SHR erythropoietin increased mean resting cytosolic free calcium concentration by 14.9% (p less than 0.05) and mean thrombin induced changes of cytosolic free calcium by 58.3% (p less than 0.01). In contrast, erythropoietin caused no significant increase in the resting calcium concentration or in thrombin induced changes of cytosolic free calcium in platelets from WKY. It is concluded that erythropoietin is involved in the pathogenesis of hypertension by elevating cytosolic free calcium concentration.     

Fluorescence combined with excised patch: measuring calcium currents in plant cation channels

Combined application of the patch–clamp technique and fura-2 fluorescence detection enables the study of study calcium fluxes or related increases in cytosolic calcium concentration. Here we used the excised patch configuration, focusing the photomultiplier on the tip of the recording pipette where the fluorescent dye was present (FLEP, fluorescence combined with excised patch). This configuration has several advantages, i.e. a lack of delay in loading the fluorophore, of interference by internal calcium buffers and of photobleaching, due to the quasi-infinite dye reservoir inside the pipette. Upon voltage stimulation of tonoplast patches, sustained and robust fluorescence signals indicated permeation of calcium through the slow vacuolar (SV) channel. Both SV currents and fluorescence signal changes were absent in the presence of SV channel inhibitors and in vacuoles from Arabidopsis tpc1 knockout plants that lack SV channel activity. The fractional calcium currents of this non-selective cation channel were voltage-dependent, and were approximately 10% of the total SV currents at elevated positive potentials. Interestingly, calcium permeation could be recorded as the same time as oppositely directed potassium fluxes. These events would have been impossible to detect using patch–clamp measurements alone. Thus, we propose use of the FLEP technique for the study of divalent ion-selective channels or transporters that may be difficult to access using conventional electrophysiological approaches.     

The effect of oxidative stress on levels of cytosolic calcium within and uptake of calcium by synaptosomes

The ability of synaptosomes subjected to oxidative stress, to maintain homeostasis has been evaluated using various indices of cellular integrity. These include levels of cytosolic calcium and leakiness of the plasma membrane. The status of a neural characteristic; depolarization-induced calcium entry into the cytoplasm, has also been studied. The presence of 5 μM FeSO₄ and 0.1 mM ascorbic acid increased peroxidative activity as judged by the rate of thiobarbituric acid reactive material production, and depressed levels of free ionic calcium [Ca²⁺]_i as determined using the calcium-sensitive flouorescent indicator dye fura-2. Depolarization-induced influx of ⁴⁵Ca²⁺ was greatly depressed under these conditions, while basal calcium uptake was inhibited to a much lesser degree. The efflux of fura-2 from synaptosomes was enhanced in the oxidizing environment, suggesting increased permeability of the synaptosomal outer limiting membrane.

The treatment of synaptosomes with 25 μM -tocopherol succinate before and during exposure to the Fe²⁺/ascorbate mixture prevented many of the changes otherwise induced by the oxidizing system. Similar pretreatment with β-carotene or superoxide dismutase did not have any protective effect. Ganglioside GM₁ pre-exposure did not alter the Fe²⁺/ascorbate-induced changes in calcium-related parameters, but mitigated synaptosomal plasma membrane damage as judged by fura-2 leakage. Thus exogenous agents may be capable of reducing the severity of oxidative stress in nervous tissue.     

Measurement of ionized calcium in blood platelets with a new generation calcium indicator

Fura 2, a new generation calcium indicator, has a 30 fold brighter fluorescence than Quin 2, shows wavelength shifts upon calcium binding and has a relatively low buffering capacity for free calcium. Quin 2, the most widely used fluorophore, on the other hand, shows no wavelength shifts and has a very high affinity for free calcium. Therefore, we have compared the relative merits of these two fluorophores for monitoring agonist induced alterations in platelet cytosolic calcium. Platelets loaded with Fura 2 showed a significant rise in cytosolic calcium when stirred with agonists such as epinephrine, arachidonate and thrombin, whereas Quin 2 loaded platelets demonstrated a rise in cytosolic calcium only with thrombin stimulation. A rise in agonist induced calcium in Fura 2 loaded platelets was prevented when the cells were exposed first to antagonists such as aspirin or prostaglandin E1. Arachidonate refractory platelets, upon stirring with a single agonist, did not show a significant elevation in cytosolic calcium. However, when refractory platelets were first exposed to epinephrine and then challenged with arachidonate, they revealed a significant elevation in cytosolic calcium. Unlike Quin 2, Fura 2 at the highest concentration tested did not inhibit platelet function. Improved properties of Fura 2 suggest that it may be a useful agent to study agonist induced alterations in cytosolic calcium levels in blood platelets.     

Phorbol ester stimulates calcium sequestration in saponized human platelets

When platelets are activated by agonists, calcium (Ca2+) is released from an intracellular storage site. Recent studies using fura-2 show that, after thrombin stimulation, the rise in free calcium is transient and returns to base-line levels in 2-3 min, while the transient following ADP stimulation lasts only 15-20 s. We reported previously that the phorbol ester 12,13-phorbol myristate acetate (PMA), added at nanomolar levels after thrombin, immediately accelerated the rate of return of calcium to the base line severalfold (Yoshida, K., Dubyak, G., and Nachmias, V.T. (1986) FEBS Lett. 206, 273-278). In the present study, we used both intact and saponized platelets to determine whether this is due to stimulation of calcium sequestration. Using fura-2 and intact platelets, we found 1) that PMA stimulated the restoration of free Ca2+ levels after ADP as well as after thrombin, and 2) that H-7, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), slowed the return of Ca2+ to baseline levels. Using saponized platelets, we also found 3) that pretreatment of platelets with PMA before saponin treatment increased the ATP-dependent 45Ca2+ uptake 2-fold, with a half-maximal effect at 5 nm; 4) that most of the Ca2+ released by ionomycin or by myoinositol 1,4,5-trisphosphate; and 5) that a GTP-binding protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), decreased basal or PMA-stimulated 45Ca2+ uptake in saponin-treated platelets. Our data suggest that activation of protein kinase C stimulates the sequestration of Ca2+ independently of cAMP or myoinositol 1,4,5-trisphosphate.     

Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths

Free Ca2+ concentrations in the cytosol of individual small cells can be recorded with a new fluorescent Ca2+ indicator, "fura-2", and a fluorescence microscope modified to chop rapidly between two wavelengths of excitation. Both fura-2 and its Ca2+ complex fluoresce strongly, but their excitation peaks differ in wavelength. Alternation between the two preferred wavelengths allows assessment of the ratio of Ca2+-bound dye to free dye and hence cytosolic free Ca2+. This ratio measurement largely cancels out the effects of cell thickness, dye content, or instrumental efficiency, uncertainties that can jeopardize measurements at single wavelengths. We describe instrumentation that supplies rapidly alternating excitation wavelengths to either a standard cuvet or a fluorescence microscope. Its use is illustrated by experiments showing changes in cytosolic [Ca2+] accompanying activation of human platelets in suspension or single mouse thymocytes on the microscope.     

Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators.

To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds.     

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

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

Apparent cytosolic calcium gradients in T-lymphocytes due to fura-2 accumulation in mitochondria

Fura-2 is the most common dye to measure cytosolic Ca2+ concentrations ([Ca2+]i). To facilitate simultaneous imaging of many cells while preserving their cytosolic environment, fura-2 is often loaded into the cytosol in its membrane-permeant ester form. It has been reported that small amounts of fura-2 accumulate in intracellular compartments, an effect that is usually neglected. We show that either focal or non-focal stimulation methods induce large [Ca2+]i gradients in T-lymphocytes during both, Ca2+ release and Ca2+ influx across the plasma membrane. Interfering with mitochondrial Ca2+ homeostasis and by labeling mitochondria with MitoTracker, we demonstrate that [Ca2+]i gradients co-localize with mitochondria and are attributable to mitochondrial fura-2 sequestration. Gradients could not be avoided by different loading protocols, compromising measurements of "real" [Ca2+]i gradients following T-cell stimulation. They were observed in human blood and lamina propria lymphocytes, Jurkat T-cells, mast cells, but not to the same extent in HEK-293 cells. Finally, we show that T-lymphocytes can be efficiently loaded with the membrane-impermeant fura-2 salt by electroporation and by osmotic lysis of pinocytic vesicles, which result in the loss of [Ca2+]i gradients. These methods are therefore suitable to study localized Ca2+ signals in large populations of T-cells while preserving their cytosolic integrity.     

Presynaptic calcium signals during neurotransmitter release: detection with fluorescent indicators and other calcium chelators.

Synthetic calcium buffers, including fluorescent calcium indicators, were microinjected into squid 'giant' presynaptic nerve terminals to investigate the calcium signal that triggers neurotransmitter secretion. Digital imaging methods, applied in conjunction with the fluorescent calcium indicator dye fura-2, reveal that transient rises in presynaptic calcium concentration are associated with action potentials. Transmitter release terminates within 1-2 ms after a train of action potentials, even though presynaptic calcium concentration remains at micromolar levels for many seconds longer. Microinjection of the calcium buffer, EGTA, into the presynaptic terminal has no effect on transmitter release evoked by single presynaptic action potentials. EGTA injection does, however, block the change in calcium concentration measured by fura-2. Therefore, the calcium signal measured by fura-2 is not responsible for triggering release. These results suggest that the rise in presynaptic calcium concentration that triggers release must be highly localized to escape detection with fura-2 imaging. Unlike EGTA, microinjection of BAPTA--a calcium buffer with an equilibrium affinity for calcium similar to that of EGTA--produces a potent, dose-dependent, and reversible block of action-potential evoked transmitter release. The superior ability of BAPTA to block transmitter release apparently is due to the more rapid calcium-binding kinetics of BAPTA compared to EGTA. Because EGTA should bind calcium within a few tens of microseconds under the conditions of our experiments, the inability of EGTA to block release indicates that transmitter release is triggered within a few tens of microseconds after the entry of calcium into the presynaptic terminal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caffeine interaction with fluorescent calcium indicator dyes.

We report that caffeine, in millimolar concentrations, interacts strongly with four common calcium indicator dyes: mag-fura-2, magnesium green, fura-2, and fluo-3. Fluorescence intensities are either noticeably enhanced (mag-fura-2, fura-2) or diminished (magnesium green, fluo-3). The caffeine-induced changes in the fluorescence spectra are clearly distinct from those of metal ion binding at the indicator chelation sites. Binding affinities for calcium of either mag-fura-2 or magnesium green increased only slightly in the presence of caffeine. Caffeine also alters the fluorescence intensities of two other fluorescent dyes lacking a chelation site, fluorescein and sulforhodamine 101, implicating the fluorophore itself as the interaction site for caffeine. In the absence of caffeine, variation of solution hydrophobicity by means of water/dioxane mixtures yielded results similar to those for caffeine. These observations suggest that hydrophobic substances, in general, can alter dye fluorescence in a dye-specific manner. For the particular case of caffeine, and perhaps other commonly used pharmacological agents, the dye interactions can seriously distort fluorescence measurements of intracellular ion concentrations with metal indicator dyes.     

A simple method for high temporal resolution calcium imaging with dual excitation dyes.

Calcium-sensitive dual excitation dyes, such as fura-2, are now widely used to measure the free calcium concentration ([Ca2+]) in living cells. Preferentially, [Ca2+] is calculated in a ratiometric manner, but if calcium images need to be acquired at high temporal resolution, a potential drawback of ratiometry is that it requires equally fast switching of the excitation light between two wavelengths. To circumvent continuous excitation switching, some investigators have devised methods for calculating [Ca2+] from single-wavelength measurements combined with the acquisition of a single ratiometric pair of fluorescence images at the start of the recording. These methods, however, are based on the assumption that the concentration of the dye does not change during the experiment, a condition that is often not fulfilled. We describe here a method of single-wavelength calcium imaging, in which the dye concentration is estimated from ratiometric fluorescence image pairs acquired at regular intervals during the recording period, that furthermore includes a correction for the changing dye concentration in the calculation of [Ca2+].     

Concentrations of ouabain that prevent intercellular communication do not affect free calcium levels in cultured fibroblasts

To better understand inhibition of gap-junction-mediated cell communication among cultured fibroblasts treated with the sodium pump inhibitor ouabain, we tested whether such cells have higher calcium levels than normal. Using the calcium indicator dye fura-2 with fluorescence spectroscopy and digital imaging microscopy, we determined cell calcium levels during exposure of cells to ouabain. The concentration of ouabain was high enough to achieve maximum alterations of steady-state sodium and potassium content and cell communication. We found no consistent change in calcium levels in human fibroblasts as a result of this treatment. In mouse 3T3 fibroblasts, concentrations of ouabain that inhibit cell communication were associated with a significant reduction of cell calcium. It appears, therefore, that the inhibition of communication by ouabain cannot be attributed to elevated cytosolic free calcium in the treated cultures.     

Effects of cholecystokinin on cytosolic calcium in human pancreatic cancer cells.

Cholecystokinin (CCK) has been shown to increase cytosolic calcium and stimulate enzyme release from pancreatic acinar cells and a rat acinar cell line, AR42J. CCK is also trophic to normal pancreas and pancreatic cancer; however, the cellular mechanisms which regulate CCK-stimulated growth are unknown. The effect of CCK on intracellular calcium was evaluated in four human pancreatic cancer cell lines known to grow in response to CCK but not secrete enzymes (SW-1990, MIA PaCa-2, BXPC-3 and PANC-1) and a rat acinar cell line (AR42J) shown to secrete enzymes but not grow with CCK. By using single cell fluorescence microscopy in fura-2 loaded cells, intracellular calcium [Ca2+]i was measured. After obtaining baseline fluorescent cell images, synthetic CCK-octapeptide (CCK8) was added to the cells and images of cell fluorescence captured. [Ca2+]i of the rat acinar cells increased (603%) over the baseline within the first minute after the addition of CCK (4.10(-13) M to 4.10(-10) M) in 77% of cells tested. In contrast [Ca2+]i failed to significantly change in the human cancer cells treated with CCK. To further localize the defect in hormone signal transduction in cancer cells, cells were suspended in low calcium media and the plasma membranes were selectively permeabilized with digitonin. Media free calcium concentration was continuously monitored by fura-2 fluorescence. Addition of inositol 1,4,5-trisphosphate (IP3) resulted in a marked increase in medium calcium concentration indicating IP3 was capable of releasing calcium from intracellular stores in both the AR42J rat acinar cell line and in the human pancreas cancer cell lines. In conclusion, CCK does not increase cytosolic calcium in human pancreatic cancer cells in contrast to rat acinar cells although all contain IP3-sensitive intracellular Ca2+ pools. Our results suggest that growth promoting and secretory effects of CCK on pancreatic cells may occur via two independent signalling pathways.     

Influence of calcium antagonists on thrombin-induced calcium mobilization and platelet-vessel wall interactions.

Elevation of cytosolic ionized calcium plays a critical role in human platelet activation. We have evaluated three well-characterized calcium antagonists for their ability to prevent thrombin-induced calcium mobilization in Fura 2 AM-loaded platelets and also their ability to inhibit platelet-vessel wall interactions. Thrombin (0.2 U/ml) caused significant elevation of cytosolic calcium (basal 84 +/- 18, activated 546 +/- 76 nM; n = 3). Verapamil, diltiazem, and nifedipine (100 microM) did not exert any inhibitory effect on thrombin-mediated calcium elevation. Untreated platelets perfused through a Baumgartner chamber containing a rabbit aorta preparation reacted with exposed and denuded subendothelium. The percentage of the total area covered by control platelet thrombi was 39.6 +/- 3.4. Diltiazem and Nifedipine significantly reduced the percentage of area covered by platelet thrombi, but the drugs were not as effective as aspirin (8.2 +/- 1.4). Calcium antagonists studied did not inhibit thrombin-stimulated elevation of cytosolic calcium in blood platelets. Although these drugs have been shown to prevent in vitro platelet aggregation and offer some protection against risks for atherosclerosis and thrombosis, they failed to significantly inhibit platelet-vessel wall interactions leading to formation of spread platelets and aggregates.     

Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores

A new group of fluorescent indicators with visible excitation and emission wavelengths has been synthesized for measurements of cytosolic free Ca2+. The five compounds, "rhod-1," "rhod-2," "fluo-1," "fluo-2," and "fluo-3" (Figs. 2 and 3), combine the 8-coordinate tetracarboxylate chelating site of 1,2-bis(2-amino-phenoxyethane-N,N,N',N'-tetraacetic acid with a xanthene chromophore to give a rhodamine-like or fluorescein-like fluorophore. Binding of Ca2+ increases the fluorescence by up to 40-fold. The Ca2+ dissociation constants are in the range 0.37-2.3 microM, so that the new indicators should give better resolution of high [Ca2+] levels than previously obtainable with quin-2 or fura-2. The visible excitation wavelengths of the new compounds are more convenient for fluorescence microscopy and flow cytometry than the UV required by previous indicators. However, the new dyes' increase in fluorescence upon binding calcium is not accompanied by a wavelength shift, so they are unsuitable for measurements using ratios at two wavelengths. The most promising dye of this series is fluo-3, whose initial biological testing in fibroblasts is described in the following paper (Kao, J. P. Y., Harootunian, A. T., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 8171-8178).     

Fura-2 antagonises calcium-induced calcium release

Calcium-induced calcium release (CICR) from the endoplasmic reticulum (ER) takes place through ryanodine receptors (RyRs) and it is often revealed by an increase of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) induced by caffeine. Using fura-2-loaded cells, we find such an effect in bovine adrenal chromaffin cells, but not in cerebellar granule neurones or in HEK-293 cells. In contrast, a caffeine-induced [Ca(2+)](c) increase was clearly visible with either fluo-3 or cytosolic aequorin. Simultaneous loading with fura-2 prevented the [Ca(2+)](c) increase reported by the other Ca(2+) probes. Caffeine-induced Ca(2+) release was also measured by following changes of [Ca(2+)] inside the ER ([Ca(2+)](ER)) with ER-targeted aequorin in HEK-293 cells. Fura-2 loading did not modify Ca(2+) release from the ER. Thus, fura-2, but not fluo-3, antagonises the generation of the cytosolic Ca(2+) signal induced by activation of RyRs. Cytosolic Ca(2+) buffering and/or acceleration of Ca(2+) diffusion through the cytosol may contribute to these actions. Both effects may interfere with the generation of microdomains of high [Ca(2+)](c) near the ER release channels, which are essential for the propagation of the Ca(2+) wave through the cytosol. In any case, our results caution the use of fura-2 to study CICR.     

Abnormalities in the regulation of blood platelet free cytosolic calcium in malignant hyperthermia. II. Pig platelets.

Since 1966 the domestic pig has served as the animal model in Malignant Hyperthermia (MH) research [1]. The use of genetically well-defined pigs rendered it possible to test the method for diagnosing MH-susceptibility of patients presented in the preceding paper. Thus, the effect of halothane on intracellular calcium movements was studied in Quin-2- and chlorotetracycline-loaded pig platelets. In 'Ca(2+)-free' suspensions the resting level of free cytosolic Ca2+ was about 60 nM. In contrast to the results with human platelets there were no significant differences between pig genotypes either in the absence or in the presence of external calcium. After addition of halothane, a mobilization of intracellular membrane-bound calcium can be observed. However, the calcium mobilization is not accompanied by a marked increase in fluorescence intensity of Quin-2-loaded platelets. Thus, in the absence of external calcium, halothane produces only a slight increase in free cytosolic Ca2+. Nevertheless, the calcium rises measured in platelets from affected animals were statistically significantly higher than those from normal subjects. However, in the presence of 1 mM external calcium, a rapid increase in free cytosolic calcium can be detected after halothane addition. This suggests that halothane causes a marked, dose-dependent increase in Ca2+ permeability of the plasma membrane. Compared to the control group, significantly enhanced calcium permeability was found, not only in homozygous positive pigs, but also in heterozygous animals.     

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

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