Free [Ca2+] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process
1998; Springer Nature; Volume: 17; Issue: 7 Linguagem: Inglês
10.1093/emboj/17.7.1986
ISSN1460-2075
AutoresAldebaran M. Hofer, Barbara Landolfi, Lucantonio Debellis, Tullio Pozzan, Silvana Curci,
Tópico(s)Pancreatic function and diabetes
ResumoArticle1 April 1998free access Free [Ca2+] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process Aldebaran M. Hofer Aldebaran M. Hofer University of Padova, CNR Center for Biomembranes, Viale G.Colombo 3, I-35121 Padova, Italy Search for more papers by this author Barbara Landolfi Barbara Landolfi Istituto di Fisiologia Generale, Università degli Studi di Bari, Via Amendola 165/A, I-70126 Bari, Italy Search for more papers by this author Lucantonio Debellis Lucantonio Debellis Istituto di Fisiologia Generale, Università degli Studi di Bari, Via Amendola 165/A, I-70126 Bari, Italy Search for more papers by this author Tullio Pozzan Corresponding Author Tullio Pozzan University of Padova, CNR Center for Biomembranes, Viale G.Colombo 3, I-35121 Padova, Italy Search for more papers by this author Silvana Curci Silvana Curci Istituto di Fisiologia Generale, Università degli Studi di Bari, Via Amendola 165/A, I-70126 Bari, Italy Search for more papers by this author Aldebaran M. Hofer Aldebaran M. Hofer University of Padova, CNR Center for Biomembranes, Viale G.Colombo 3, I-35121 Padova, Italy Search for more papers by this author Barbara Landolfi Barbara Landolfi Istituto di Fisiologia Generale, Università degli Studi di Bari, Via Amendola 165/A, I-70126 Bari, Italy Search for more papers by this author Lucantonio Debellis Lucantonio Debellis Istituto di Fisiologia Generale, Università degli Studi di Bari, Via Amendola 165/A, I-70126 Bari, Italy Search for more papers by this author Tullio Pozzan Corresponding Author Tullio Pozzan University of Padova, CNR Center for Biomembranes, Viale G.Colombo 3, I-35121 Padova, Italy Search for more papers by this author Silvana Curci Silvana Curci Istituto di Fisiologia Generale, Università degli Studi di Bari, Via Amendola 165/A, I-70126 Bari, Italy Search for more papers by this author Author Information Aldebaran M. Hofer1, Barbara Landolfi2, Lucantonio Debellis2, Tullio Pozzan 1 and Silvana Curci2 1University of Padova, CNR Center for Biomembranes, Viale G.Colombo 3, I-35121 Padova, Italy 2Istituto di Fisiologia Generale, Università degli Studi di Bari, Via Amendola 165/A, I-70126 Bari, Italy ‡A.M.Hofer and B.Landolfi contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1986-1995https://doi.org/10.1093/emboj/17.7.1986 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Free [Ca2+] in agonist-sensitive internal stores of single intact cells was measured in situ in order to examine the role of [Ca2+] in modulating the store refilling process. BHK-21 fibroblasts were loaded with the low-affinity fluorescent calcium indicator mag-fura-2-AM such that >80% of the dye was trapped in organelles, where it reported [Ca2+] changes solely in an agonist- and thapsigargin-sensitive internal store. The rates of store reloading following stimulation by 100 nM bradykinin were essentially unchanged when cytosolic [Ca2+] was clamped to resting values with BAPTA-AM. In control cells, recharging of stores totally depended on the presence of external Ca2+, but pre-loading the cells with BAPTA-AM permitted efficient refilling in Ca2+-free, EGTA-containing external medium. Our results show: (i) Ca2+ stores normally are recharged by Ca2+ which must first transit the cytoplasm; (ii) an elevation in cytoplasmic [Ca2+] is not required to replenish Ca2+ stores; (iii) the activation of the plasma membrane Ca2+ pump during the Ca2+ spike ordinarily results in complete extrusion of released Ca2+; and (iv) the buffering capacity of the cytoplasm is an essential component of the store refilling process. An interesting finding was that acute treatment of cells with BAPTA-AM activated capacitative Ca2+ entry at the plasma membrane, due to its efficient hydrolysis in the stores, and the ensuing decrease in the endoplasmic reticulum [Ca2+]. Introduction In intact cells, a multiplicity of intracellular Ca2+ transport and buffer systems serve to shape intracellular Ca2+ signals and maintain the low resting [Ca2+] typically found in the cytoplasm (Berridge, 1993; Pozzan et al., 1994; Clapham, 1995). At rest, the principal players are Ca2+ pumps and leaks residing in both the plasma membrane and the endoplasmic reticulum (ER). During agonist activation, however, there are dramatic increases in the Ca2+ permeability of both the ER, for example through the opening of inositol 1,4,5-trisphosphate (InsP3) receptors, and secondarily at the plasma membrane via store-operated or capacitative Ca2+ entry (Putney, 1990; Berridge, 1995). Endogenous Ca2+ buffers (including 'physiological' buffering by other organelles such as mitochondria; Mohr and Fewtrell, 1990; Friel and Tsien, 1994; Drummond and Fay, 1996; Babcock et al., 1997; Hoth et al., 1997) further modulate these Ca2+ signals. The fact that many of the elements of the Ca2+ signaling apparatus are themselves regulated by Ca2+ ions gives rise to additional complexity. While the integrated actions of many of these various elements on cytosolic Ca2+ dynamics are assessed readily by direct measurement of free Ca2+ in that compartment (e.g. using fluorescent indicators; Grynkiewicz et al., 1985; Tsien and Pozzan, 1989), some experimental questions (e.g. the influence of cytosolic Ca2+ on the release and reloading of intracellular stores) can be addressed directly only by monitoring free [Ca2+] changes in the ER lumen. Two main strategies have been adopted to attack the problem of measuring ER [Ca2+]: (i) targeting of Ca2+-sensitive proteins, mainly aequorins (Kendall et al., 1994; Montero et al., 1995, 1997), and very recently green fluorescent protein (GFP)-based indicators (Miyawaki et al., 1997; Persechini et al., 1997); and (ii) the use of trapped fluorescent low-affinity Ca2+ indicators, such as mag-fura-2. A major drawback of the latter approach is that since the probe accumulates in both the cytoplasm and organelles, permeabilization of the plasma membrane is generally necessary in order to eliminate the cytosolic indicator. The permeabilization process obviously destroys the opportunity to measure native interactions of ER transporters with those of the plasma membrane and with other organelles. We report here a modification of the mag-fura-2 technique which permits sensitive, non-invasive measurements of intraluminal ER Ca2+ dynamics in intact BHK-21 cells, a fibroblastic cell line. Stimulation with a Ca2+ mobilizing agonist [bradykinin (BK) or ATP] resulted in a large, rapid drop in the mag-fura-2 ratio, consistent with the indicator being trapped in, and reporting Ca2+ changes primarily from, agonist-sensitive stores. This afforded us the opportunity to follow changes in free [Ca2+] in the ER of single intact cells following physiological stimulations with Ca2+-releasing agonists. In the present study, we have examined the relationship between Ca2+ entry and cytoplasmic Ca2+ on the recharging of agonist-sensitive Ca2+ stores. Our results indicate that stores refill efficiently in the absence of elevations in cytoplasmic Ca2+, but that there is normally an absolute requirement for external Ca2+ for this process. Increasing the intracellular buffer capacity of the cell, however, allowed recycling of released Ca2+ and refilling of internal stores, even in Ca2+-free external solutions containing EGTA. These data provide an original insight into the physiological coordination of plasma membrane Ca2+ extrusion mechanisms, ER Ca2+ reuptake mechanisms and Ca2+ entry pathways during Ca2+ signaling events. Results One of the drawbacks of loading Ca2+ indicators into cells as their AM-ester derivative is that the accumulation of dyes in intracellular organelles often confounds the interpretation of cytosolic measurements (Roe et al., 1990). This feature can be turned around to yield a serendipitous bonus, however, when Ca2+ handling by organelles is to be addressed. Many groups have taken advantage of this phenomenon (see Hofer and Schulz, 1996, and references therein), although the overwhelming signal from cytoplasmic dye generally requires drastic procedures such as dilution of the cytosolic dye via the patch–clamp pipette (Tse et al., 1994; Chatton and Stucki, 1995; Hofer et al., 1998), or plasma membrane permeabilization (Hofer and Machen, 1993; Hirose and Iino, 1994; Hajnóczky and Thomas, 1997) in order to get usable information. This represents a significant obstacle to understanding organelle function in situ. We thus undertook a systematic search of cell types and dye-loading protocols that would maximize the trapping within intracellular InsP3-sensitive stores, thus allowing sensitive measurement of store [Ca2+] in single, intact, living cells. Among the many cell types examined, the BHK-21 fibroblast was that which approached our goal most closely. Golovina and Blaustein (1997) recently reported a similar approach using organelle-trapped mag-fura-2 in intact smooth muscle cells. When BHK-21 cells were incubated for extended periods (45–60 min) with mag-fura-2-AM at 37°C (or at higher temperatures, 39–41°C, for briefer periods), in the absence of any additional treatment, cells displayed a distribution of fluorophore consistent with preferential dye accumulation in organelles. We noticed that following 20 min of loading at 37°C, cells had a uniform fluorescence, but between 20 and 45 min there was a progressive loss of cytoplasmic dye, as evidenced by the reduced fluorescence in the nuclear (reflecting the cytoplasmic) region with respect to the periphery of the cell. Similar observations were made when cells were loaded in this manner with indo-1-AM and fura-2-AM (not shown). Dye accumulation in the cytoplasm, on the other hand, was encouraged by reducing the loading time (20 min) and temperature (25°C). As seen in Figure 1A, the fluorescence of mag-fura-2 after 45–60 min of loading at 37°C was clearly localized in the peripheral regions of the cell and in a non-uniform manner, often with the appearance of a delicate reticulum. The nuclear regions were, on the other hand, nearly devoid of fluorescence, with the nuclear membrane plainly delineated. Figure 1.(A) Mag-fura-2 fluorescence (excitation 351 nm) from intact BHK-21 fibroblasts loaded with dye at 37°C according to procedures described in the text. (B) Individual fluorescence intensities at 345 nm (Ca2+-insensitive) and 375 nm (Ca2+-sensitive) excitation wavelengths (top panel) and the calculated 345/375 ratio (lower panel) from mag-fura-2-AM-loaded fibroblasts. Intact cells were bathed in a standard NaCl Ringer's solution, and then stimulated with 100 nM BK, resulting in a drop in the mag-fura-2 ratio. Cells were then permeabilized with digitonin in intracellular buffer (see Materials and methods), initially in the absence of ATP to avoid stimulation of purinergic receptors. The drop in the fluorescence intensity at 345 nm is a measure of the amount of cytosolic dye released. Note that in this cell 73% of the total Triton X-100-releasable fluorescence was retained following permeabilization, which was less than the average amount of compartmentalized dye (83%) for cells used in this study. After supplementing the medium with ATP (arrow) to maintain Ca2+ uptake into stores, accumulated Ca2+ was released with 10 μM InsP3. Download figure Download PowerPoint As shown in Figure 1B, cells loaded with mag-fura-2 according to the protocol described above had high resting mag-fura-2 ratios, indicating that the signal was dominated by compartment(s) with elevated resting [Ca2+]. A rapid drop in the ratio followed the treatment with the Ca2+-mobilizing agonist BK (100 nM), as expected from a probe residing primarily in the InsP3-sensitive store. In order to assess what fraction of the probe remained in organellar versus cytosolic compartments, we then permeabilized the same cells with digitonin in an intracellular-like buffer (Hofer et al. 1995), and monitored the release of cytosolic indicator at the Ca2+-insensitive excitation wavelength of 345 nm. In the cell under study in Figure 1B, 73% of the probe was retained following permeabilization. On average, 83.7 ± 3.2% of the fluorophore was trapped in digitonin-resistant compartments (n = 16) and, in ∼3 out of 16 cells, there was no detectable loss of dye from the cytoplasm. After supplementing the permeabilization buffer with ATP to maintain the uptake of Ca2+ into stores, the cells responded to InsP3, which is membrane impermeant, demonstrating that cells were indeed permeabilized by digitonin (n = 4). The data reported above clearly demonstrate that under the selected experimental conditions, trapped mag-fura-2 was reporting [Ca2+] changes in an InsP3-sensitive internal store. Although dye was not present to a significant extent in the cytoplasm, we could not, however, exclude that the probe was not also contained within structures other than the ER (e.g. mitochondria, which are notorious for compartmentalizing AM-esters). In order to test whether non-ER dye was significantly altering the kinetic behavior of the agonist-dependent ratio changes, the experiment presented in Figure 2A was carried out (n = 4). The intact cell was first challenged with BK. In this particular case, the agonist first elicited a rapid drop in the ratio, followed by a slow recovery upon which were superimposed two oscillations (see below). Addition of thapsigargin (100 nM), a selective inhibitor of the sarcoendoplasmic reticulum calcium ATPase (SERCA; Thastrup et al., 1989), reversed the recovery phase following BK stimulation, and produced a relatively slow continuous drop in the ratio. During the falling phase of the thapsigargin-induced Ca2+ loss, the mag-fura-2 ratio remained insensitive to alterations in external [Ca2+]. Thapsigargin is well known to activate capacitative Ca2+ entry in many cell types (including BHK-21 cells) by depleting internal Ca2+ stores (Putney, 1990). Therefore, under these conditions, the cytoplasmic as well as the intramitochondrial [Ca2+] is predicted to increase greatly upon Ca2+ readdition. However, this maneuver had no consequence on the mag-fura-2 ratio. Addition of the Ca2+ ionophore ionomycin (10 μM) after the ratio had reached its minimum also had no effect, further confirming that, for all practical purposes, the dye reports [Ca2+] movements only in the thapsigargin-sensitive internal store. The simplest explanation for this finding is that, because of the relatively low Kd of mag-fura-2 for Ca2+ (53 μM), changes in [Ca2+] in the range of 1–10 μM (such as those occurring in the cytoplasm and in mitochondria) hardly affect the fluorescence of the dye which might be sequestered in those compartments. Figure 2.(A) Mag-fura-2 measures [Ca2+] changes only in thapsigargin- and agonist-releasable internal stores of intact BHK-21 cells. BK at 100 nM caused a rapid drop in the mag-fura-2 ratio, and evoked oscillations in store [Ca2+] in this particular cell. Treatment with 100 nM thapsigargin caused additional release of Ca2+, but removal and readdition of extracellular Ca2+ had no effect on the mag-fura-2 ratio. No further release of Ca2+ was detected upon addition of 10 μM ionomycin. (B) Agonist-induced oscillations of internal store [Ca2+]. A subpopulation of BHK-21 cells exhibited oscillations in internal store [Ca2+] upon treatment with 100 nM BK. (C) Receptor desensitization: stores can rapidly recover released Ca2+ in the continued presence of 100 nM BK. This is an example of a cell where the recovery was particularly rapid. Download figure Download PowerPoint There are two noteworthy observations regarding the profile of Ca2+ release and reloading in BHK-21 fibroblasts. The first is that following stimulation with 100 nM BK, the [Ca2+] in internal stores of ∼20% of cells was seen to oscillate with an average frequency of about one complete cycle per minute (Figure 2B; see also Figure 3A). Oscillations in intraluminal [Ca2+] have been reported previously by a number of investigators (Tse et al., 1994; Chatton et al., 1995; Golovina and Blaustein, 1997; Hajnóczky and Thomas, 1997). Oscillations occurred upon a background of store refilling, but generally ceased before recovery was complete. These fluctuations were abolished by removal of external Ca2+ (illustrated in Figure 3A), indicating that Ca2+ entry and/or the replenishment of stores is important for sustaining oscillations. Figure 3.(A) Intact mag-fura-2-loaded cells stimulated with 100 nM BK in the presence of external Ca2+. Removal of external Ca2+ blocked the recovery. The luminal [Ca2+] of this cell was oscillating initially. (B) Cells stimulated with BK in nominally Ca2+-free solution. No recharging of Ca2+ stores occurred until Ca2+ (250 μM) was reintroduced into the bath. Following a second challenge with BK, the rate of recovery in 1 mM Ca2+ was enhanced. Download figure Download PowerPoint The second point is that internal stores of BHK-21 cells were able to re-sequester Ca2+ effectively in the continued presence of agonists. The rate at which this reuptake occurred was highly variable from cell to cell. Shown in Figure 2C is a striking example of a cell in which stored Ca2+ was completely recovered in <2 min in spite of continuous exposure to 100 nM BK. This observation is explained most readily by the phenomenon of homologous receptor desensitization (see Böhm et al., 1997); cells were generally refractory to further stimulation with the same agonist for several minutes, but subsequent stimulation of purinergic receptors using 100 μM ATP allowed this resequestered Ca2+ to be released again (not shown). Very often the rate of Ca2+ reaccumulation following stimulation was indistinguishable in the absence and presence of the agonist, suggesting that cessation of InsP3 production had occurred within a very short (1–2 min) time period. One implication of these findings is that the stimulus for capacitative Ca2+ entry at the plasma membrane will be terminated rapidly simply because Ca2+ within the ER is restored, and may account in part for the relatively small plateau phase of the cytoplasmic Ca2+ signal in BHK-21 cells. Having established experimental conditions for measuring [Ca2+] changes selectively in agonist-sensitive stores in intact cells, we could then start asking specific questions about the Ca2+ handling properties of cells, the first of which regards the effects of external Ca2+ on ER [Ca2+] under resting and stimulated conditions. Removing Ca2+ from the bathing solution (nominally Ca2+-free solution) for up to 10 min had no effect on the resting mag-fura-2 ratio (not shown; n = 10), nor did increasing the bath [Ca2+] from 1 to 5 mM (n = 3). However, as illustrated in Figure 3A and B, external Ca2+ was necessary in order to realize an efficient refilling of stores, either in the presence or absence of agonist. The luminal [Ca2+] of the cell shown in Figure 3A was oscillating upon a general background of store refilling (probably as a result of receptor desensitization as described above). Removal of external Ca2+ resulted in cessation of the oscillations and an arrest of refilling that was reversed when Ca2+ was reintroduced into the perfusate. A similar effect of Ca2+-free solution on store refilling was observed in cells which were not oscillating (n = 3) and for cells recovering in the absence of agonist (n = 2; not shown). In Figure 3B, the cell was stimulated briefly with BK in a nominally Ca2+-free solution, the agonist washed off, and the cell maintained in zero Ca2+ for 10 min. No recovery was observed until 0.25 mM Ca2+ was readmitted to the bath (n = 12). A second stimulation in zero Ca2+ was performed, and this time 1 mM Ca2+ was readded, resulting in an enhanced rate of recovery compared with that seen in 0.25 mM Ca2+. The notion that extracellular Ca2+ is necessary for store refilling is well documented in the literature, although admittedly based on indirect measurements, i.e. of cytoplasmic signals. Though never questioned, this finding is in apparent contradiction to another well known property of the store, namely, that in the absence of InsP3, stores are able to refill effectively at or below resting cytoplasmic [Ca2+]. The question arises therefore as to why, after removal of the stimulus, no ER filling is observed until Ca2+ is added back to the medium. A possible clue to understanding this paradox is offered by the experiment presented in Figure 4A. In this case, cells were loaded at room temperature with fura-2 in order to monitor cytoplasmic [Ca2+]. As observed previously by a number of investigators (see, for example, Muallem et al., 1988, 1990), cells stimulated in the absence of external Ca2+ display a small (apparently ∼20 nM) undershoot in Ca2+ below the basal level following the peak of Ca2+ release (n = 3; data from 19 cells). It is conceivable, however, that the magnitude of this decrease below the basal level might be underestimated because of the low sensitivity of fura-2 below 100 nM and because of problems of calibration (Roe et al., 1990). We therefore hypothesized that the lack of refilling in Ca2+-free medium could be due to this drop in cytoplasmic [Ca2+] below resting, to a level which was below the Kd of the SERCA. The experiments in Figure 4B–E were thus carried out in order to test this possibility. Cells were loaded with 1,2-bis(2-amino-phenoxy)ethand-N, N, N′, N′-tetraacetic acid (BAPTA)-AM, thereby providing a massive increase in cytosolic buffering capacity which prevented [Ca2+] changes in the cytoplasm. Figure 4B depicts the signal from cells co-loaded with BAPTA-AM and fura-2 following stimulation with BK in Ca2+-free medium. Both the cytosolic peak and the undershoot seen in Figure 4A were eliminated completely (n = 3). When the luminal ER [Ca2+] was measured with mag-fura-2, loading with BAPTA-AM (40 μM for 20 min at 37°C) had a dramatic effect on the refilling, i.e. it abolished the requirement for extracellular Ca2+ in the refilling of stores, as seen in Figure 4C (n = 14; 25 cells). An almost complete recharging of stores occurred, even though the Ca2+-free external solution contained 100 μM EGTA. The refilling was mediated by the SERCAs of the internal store. As seen in Figure 4D, following a control stimulation with BK in zero Ca2+, BAPTA-AM-pre-treated cells were stimulated a second time with BK. The SERCA inhibitor 2, 5-di(tert-butyl)hydroquinone (tBHQ), reversibly blocked the refilling phase (always in 0 Ca2+/EGTA; n = 7). Thus, not only does recharging of the store occur directly from the cytosol, but extended recycling of Ca2+ released into the cytoplasm is possible when the intracellular buffer capacity is augmented by BAPTA. Figure 4.(A) 'Undershoot' in cytoplasmic [Ca2+] as measured by fura-2 was observed following stimulation with 100 nM BK in nominally Ca2+-free solution. Readmission of 250 μM Ca2+ resulted in a small increase in cytoplasmic [Ca2+]. (B) BAPTA-AM (co-loaded with fura-2) abolished both the peak and the undershoot following stimulation in Ca2+-free solutions containing 100 μM EGTA. (C) Intact mag-fura-2-loaded cells pre-loaded with 40 μM BAPTA-AM for 20 min. Complete recovery in store [Ca2+] following BK challenge in Ca2+-free external solution containing 100 μM EGTA. (D) Intact mag-fura-2-loaded cells pre-loaded with 40 μM BAPTA-AM for 20 min. Store refilling in zero Ca2+ is mediated by SERCA pumps. Cells were maintained in Ca2+-free solutions plus 100 μM EGTA throughout the experiment, and 100 nM BK given where indicated. During the recovery phase following the second BK stimulation, application of 20 μM tBHQ reversibly blocked the refilling. (E) Control mag-fura-2-loaded cells (no BAPTA) stimulated with BK in zero Ca2+ (100 μM EGTA). tBHQ (20 μM) was added at the time indicated, resulting in a slight drop in the mag-fura-2 ratio, but no recovery when tBHQ was washed out. Readdition of extracellular Ca2+ resulted in complete refilling. Control response to BK in the presence of external Ca2+. Download figure Download PowerPoint The question then arises as to whether the undershoot of cytoplasmic Ca2+ results in a complete blockade of the SERCA or only a major reduction in its activity. Since agonists normally release only a portion of the thapsigargin-sensitive store (see Figure 3A; Hofer et al., 1995), we could test whether the SERCA pump was still active at sub-basal cytosolic Ca2+ by treating the cells acutely with SERCA inhibitors during the 'undershoot' phase, as shown in Figure 4E. Control cells loaded with mag-fura-2 (but not BAPTA) were stimulated briefly with BK in 0 Ca2+/EGTA external solution. Five minutes later, at a time when cytosolic [Ca2+] was predicted to be well below the resting value (Figure 4A), tBHQ was administered, resulting in a small decrease in the mag-fura-2 ratio (n = 7). There was, however, no recovery of released Ca2+ when tBHQ was washed out. These data indicate that the SERCA was still somewhat active, but only at a level sufficient to maintain a modest recycling of the cation, and not enough to refill the store efficiently. The effect of cytoplasmic buffering on Ca2+ handling by the stores was next investigated in the presence of extracellular Ca2+. We measured store [Ca2+] changes from a number of cells which had been pre-loaded with BAPTA-AM (20 μM; co-loaded with mag-fura-2 for the last 20 min of dye loading at 37°C). We observed that release and reloading of stores as measured by mag-fura-2 following agonist activation appeared essentially to be similar to that in control cells (not shown; n = 14). However, given the heterogeneity of responses to BK as measured at the single cell level, we decided to compare directly, in the same cell, the agonist response before and after BAPTA-AM loading. Shown in Figure 5A are typical control recordings of luminal [Ca2+] (top panel) and cytoplasmic [Ca2+] (bottom panel) from different cells, each stimulated twice with BK. The rates of release and reloading, as well as the response measured in the cytoplasm following two sequential stimulations, were quite reproducible in a given cell (n = 8). The brief application of agonist (1 min) and the long interval between stimulations (at least 15 min) ensured that receptor desensitization was minimal. Figure 5.The upper panels of both parts of the figure depict the responses from intact mag-fura-2-loaded cells, while the corresponding measurement of cytosolic [Ca2+] from a different coverslip of fura-2 loaded cells is illustrated in the bottom panels. (A) Control responses to two sequential stimulations with 100 nM BK with a 15 min interval between agonist applications. (B) Brief control stimulation with 100 nM BK followed by superfusion with 20 μM BAPTA-AM. The response to the second BK challenge following BAPTA-AM loading was largely unchanged in the ER lumen compared with the control response, but highly attenuated in the cytoplasm. Download figure Download PowerPoint Figure 5B depicts the responses to a brief control stimulation with agonist, followed by a loading period with 40 μM BAPTA-AM for 10–15 min at 37°C on the microscope stage. Cells were then washed for 3–5 min in normal Ringer's solution, and then restimulated with agonist. The two applications of BK yielded responses which were quite similar in spite of the BAPTA-induced increase in cytoplasmic buffering. The rate of release during the control stimulation with a supramaximal dose of BK (100 nM) was 97 ± 12% (SEM) of that following BAPTA treatment, while the rate of the control recovery was 147 ± 17% (SEM) of that observed after BAPTA loading (n = 14); neither were significantly different. Subtle kinetic differences in the release phase similar to those reported by Montero et al. (1997) (i.e. a more abrupt termination of the release) were sometimes observed. This aspect was not investigated in any detail. As a control that the BAPTA-AM loading protocol had indeed reduced the cytosolic [Ca2+] changes, the same protocol was repeated in fura-2-loaded cells (Figure 5B, bottom panel). The response to a second challenge with BK was highly attenuated (on average 16 ± 3% of control, with 58% of cells giving no measurable response). The tendency of BHK-21 cells to sequester AM-ester derivatives efficiently suggested that it should be possible to manipulate the [Ca2+] within the stores during acute loading with BAPTA-AM. In fact, when mag-fura-2-loaded cells were exposed to BAPTA-AM, a clear decrease in the store [Ca2+] was observed (Figure 6A; n = 14). This decrease was more profound when BAPTA-AM was applied in Ca2+-free medium (Figure 6B; n = 3). This lowering of store [Ca2+] should activate capacitative influx, and therefore a paradoxical effect of treating cells with the Ca2+ chelator is expected in the cytosol, i.e. a transient increase in intracellular [Ca2+] during BAPTA-AM loading. This was indeed the case, as seen in Figure 6C and D. When BAPTA-AM was added to fura-2-loaded cells maintained in 1 mM external Ca2+ (Figure 6C), there was an increase in cytosolic [Ca2+] that was quite variable (n = 50; see figure legend for details). In contrast, when the loading was allowed to proceed for a few minutes in Ca2+-free external medium, and the cells then reperfused with 1 mM Ca2+, a large and reproducible increase in cytoplasmic [Ca2+] was observed (n = 7). Figure 6.Effects of acute treatment of BAPTA-AM on luminal ER and cytoplasmic [Ca2+]. (A) In the presence of external Ca2+, addition of BAPTA-AM caused a small decrease in the mag-fura-2 ratio. (B) BAPTA-AM applied in Ca2+-fre
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