HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 286/6/F1219    most recent 00255.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Fowler, M. R. Articles by Hunter, M. Search for Related Content PubMed PubMed Citation Articles by Fowler, M. R. Articles by Hunter, M.

Regulation and identity of intracellular calcium stores involved in membrane cross talk in the early distal tubule of the frog kidney

¹School of Biomedical Sciences, Worsley Building, University of Leeds, Leeds, West Yorkshire LS2 9NQ; and ²Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom

Submitted 18 July 2003 ; accepted in final form 9 February 2004

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The early distal tubule (EDT) of the frog nephron, similar to the thick ascending limb in mammals, mediates the transepithelial absorption of NaCl. The continued absorption of NaCl in the face of varying Na⁺ load is maintained by coordination of the activity of ion-transporting proteins in the apical and basolateral membranes, so-called pump-leak coupling. Previous studies identified intracellular Ca²⁺, originating from an intracellular Ca²⁺ store, as playing a key role in pump-leak coupling in the EDT (Cooper GJ, Fowler MR, and Hunter M. Pflügers Arch 442: 243–247, 2001). The purpose of the experiments described in this paper was to identify the intracellular Ca²⁺ storage pools in the renal diluting segment. Store Ca²⁺ movements were monitored by the fluorescence of mag-fura 2 in permeabilized segments of frog EDTs. The presence of both ATP and Ca²⁺ was required to maintain store Ca²⁺ content. Removal of either of these substrates resulted in a passive leak of Ca²⁺ from the stores. The uptake of Ca²⁺ into the store was sensitive to the SERCA inhibitor 2,5-di(tert-butyl) hydroquinone, whereas Ca²⁺ release from the store was stimulated by IP₃ but not cADPR. Store Ca²⁺ was insensitive to the mitochondrial ATP synthase inhibitor oligomycin, and, under conditions that energized _m, the complex 1 inhibitor rotenone and the protonophore FCCP. Ionomycin was able to mobilize store Ca²⁺ following exposure to IP₃. These results suggest that the endoplasmic reticulum is a dominant Ca²⁺ store in the frog EDT. A second pool, sensitive to ionomycin but not IP₃, may overlap with the IP₃-sensitve pool. The data also rule out any contribution by mitochondria to EDT Ca²⁺ cycling.

pump-leak coupling; permeability

NaCl reabsorption is energized by the basolateral Na⁺-K⁺-ATPase, which maintains a low intracellular Na⁺ concentration ([Na⁺]) and establishes a favorable chemical gradient for Na⁺ uptake from the luminal fluid. The movement of Na⁺ across the apical membrane is coupled to that of Cl^– and K⁺ via an electroneutral cotransporter. Working in concert, these two mechanisms complete the transcellular transport of Na⁺. The basolateral passage of Cl^– is coupled to that of K⁺ via either cotransport or parallel movement through channels. The K⁺ that exits the cell via the basolateral membrane is immediately available for reuptake via the Na⁺-K⁺-ATPase. In this manner, K⁺ recycles across the basolateral membrane and acts as a substrate supporting Na⁺ efflux from the cell. Peculiar to this nephron segment, K⁺ also recycles across the apical membrane. After its absorption from the lumen, K⁺ reenters the tubule fluid via an apical channel, where it is once again taken up by the electroneutral NKCC1 cotransporter (17). This apical recycling is mandatory for the continued reabsorption of NaCl, because the luminal delivery via the glomerular filtrate of K⁺ to the diluting segment is not of itself sufficient to sustain NaCl reabsorption; mutations of the apical K⁺ channels in humans lead to the salt-wasting symptoms of Bartter's syndrome (29). Thus the uptake of NaCl is determined by the availability of luminal K⁺ (16) and is obligatorily coupled to its movement on the cotransporter. Furthermore, the secretory flux of K⁺ via the apical channels is the rate-limiting step in NaCl absorption and is the principal regulator of salt absorption in this segment (16, 17).

Previous work demonstrated an indirect link between the activity of the apical K⁺ channels and cytosolic Ca²⁺ concentration ([Ca²⁺]); the activity of the apical K⁺ channels is directly regulated by the intracellular pH, where raising pH increases channel activity (23). Intracellular pH is itself principally determined by the activity of the Na⁺/H⁺ exchangers located on the basolateral membrane (8). Inhibition of the apical cotransporters by the loop-acting diuretic furosemide results in a rise in cytosolic [Ca²⁺] and is associated with increased activity of the apical K⁺ channels. Furosemide inhibits Cl^– uptake on the apical cotransporter and results in a rapid fall in intracellular [Cl^–]. This fall in intracellular [Cl^–] results in Ca²⁺ release from intracellular stores, thereby elevating cytosolic [Ca²⁺] (7). The increase in intracellular [Ca²⁺] activates the basolateral Na⁺/H⁺ exchangers, via calmodulin, and the consequent intracellular alkalinization directly upregulates apical K⁺ channel activity, increasing the availability of luminal K⁺ (9). This complex series of regulatory steps can be seen as a mechanism to maintain Na⁺ absorption in the face of reduced Na⁺ delivery to the diluting segment. Therefore, a rise in cytosolic Ca²⁺ is central to membrane cross talk, pump-leak coupling, and, therefore, the regulation of salt absorption in this nephron region.

The principal question remaining from the experiments described above is the source of intracellular Ca²⁺ involved in this feedback response. Previous work suggested that this was the endoplasmic reticulum (ER), because depletion of the intracellular Ca²⁺ stores with the SERCA-specific inhibitor 2,5-di(tert-butyl) hydroquinone (TBQ) ablated the furosemide-induced rise in intracellular Ca²⁺ (9). The purpose of the experiments described in this paper was to seek functional evidence for the identity of intracellular Ca²⁺ storage pools in the renal diluting segment, which may be involved in pump-leak coupling. In this manuscript, we describe experiments designed to elucidate functional calcium storage pools within the amphibian diluting segment. To our knowledge, this is the first time that such experiments have been conducted in native renal tissue. The results show that the predominant calcium pool is the ER and further show modulation of ER calcium content with physiological regulators. These studies provide strong corroborative evidence in support of our earlier findings concerning the ER as the source of calcium in pump-leak coupling in the amphibian EDT.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Preparation of EDT Segments for Monitoring Compartmentalized Probe

Frogs (Rana temporaria) of either sex were kept in tap water at 4°C. Animals were stunned by concussion, and the brain and spinal cord were destroyed by pithing in accordance with the UK legislature. The kidneys were removed, cut into 1-mm sections, and stored in ice-cold Leiboitz incubation medium (pH 7.4, 204 mosmol/kgH₂O). Individual EDT segments were then manually dissected in ice-cold amphibian Ringer solution (for composition see below) and incubated for an hour in amphibian Ringer solution containing 14 µM mag-fura 2-AM at room temperature. Tubules were transferred to a perfusion chamber mounted on the stage of a Nikon Diaphot inverted microscope and held at each end with a pair of microperfusion pipettes (9). Tubule segments were unperfused. Figure 1A illustrates a mag-fura 2-loaded EDT segment before permeabilization. Permeabilization of the basolateral membrane with the detergent saponin (15) (25 ng/ml) selectively disrupts the basolateral cell membrane and permits washout of the probe in the cytosol. Other detergents used in similar studies are also accepted to preferentially permeabilize the cell membrane, which means that internal membranes are left intact (5, 15, 19, 33). This permeabilization procedure was monitored by following the fluorescence signal online and terminated by washing off the saponin when the 350-nm fluorescence signal had fallen to 30% of its maximum, indicating loss of cytosolic probe into the bath solution. By the end of the permeabilization period, the signal was 15–20% of the unpermeabilized value and was 50–100 times background (Fig. 1B). In this way, the mag-fura present in the cytosol was removed and access to the intracellular Ca²⁺ stores could be gained via the bath solution; 0.5 mM Mn²⁺ was added to the bath solution during permeabilization to quench any cytosolic probe that was not washed out by the permeabilization process, such that changes in fluorescence from dye trapped within intracellular compartments could be followed.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Frog early distal tubule (EDT) segments loaded with mag-fura 2-AM. Images captured using a 35-mm Nikon SLR camera of unperfused frog EDT segments following incubation in mag-fura 2-AM (A) and after the addition of saponin (B). After permeabilization, the remaining fluorescence is clearly confined to the cytosol, with some punctuate juxta-nuclear located regions that may represent compartments with high esterase activity or very low Ca2+ content (see DISCUSSION). Scale bar: 30 µm.

Amphibian Ringer solution was used as dissection medium and for incubation of tubule segments in fluorescent probe and had the following composition (in mM): 97 NaCl, 3 KCl, 2 CaCl₂, 1 MgCl₂, and 10 HEPES, titrated to pH 7.4 with NaOH. The "intracellular" solution had the following composition (in mM): 88 K-gluconate, 12 NaCl, 2 MgSO₄, 1 free [Mg²⁺], 4 Ca(NO₃)₂, 200 nM free [Ca²⁺], 10 EGTA, and 10 HEPES, titrated to pH 7 with KOH/glucuronic acid lactone as appropriate.

Where required, additions to the intracellular solution were made from the following stock solutions (unless otherwise stated, all chemicals were obtained from Sigma, Poole, UK, and dissolved in water): 1 mM or 1 M K-ATP, 0.5 M K-ADP (FLUKA), 1 M KH₂PO₄, 0.5 M succinic acid, 1 M Ca(NO₃)₂, 50 mg/ml saponin, 100 mM MnCl₂, 10 mM rotenone in DMSO, 10 mM FCCP in DMSO, 10 mM TBQ in DMSO, 10 mM IP₃, 100 mg/ml heparin, 10 mM ionomycin in DMSO, and 5 mg/ml oligomycin in DMSO (mix of A, B, and C).

Fluorescence Microscopy

The compartmentalization within intracellular organelles of the low-affinity calcium-sensitive probe mag-fura 2 (Molecular Probes, Leiden, The Netherlands) has been exploited here to monitor changes in intrastore Ca²⁺ following permeabilization of the basolateral membrane. Although the fluorescent probe chosen is also sensitive to Mg²⁺, it is of much lower affinity than that for Ca²⁺ and has been shown to be insensitive to Mg²⁺ in physiological type intracellular buffers (20, 33). The basic recording system (Newcastle Photometrics) has been described in detail previously (9). Briefly, mag-fura 2 was alternately excited at 350 and 380 nm for 500 ms, and emitted light was collected at 520 nm by a photomultiplier tube and digitized before being displayed and stored on an IBM-compatible computer.

Data Analysis and Statistics

Data are presented as continuous experimental recordings with time on the x-axis and 350/380 ratio on the y-axis, or as mean steady-state ratio values ± SE. No attempt has been made to calibrate the intrastore signals because a mixed population of Ca²⁺ stores invalidates such an approach (21). On the other hand, it was necessary to ascertain that the probe was sensitive to changes in Ca²⁺ following permeabilization, which was achieved by exposure to a Ca²⁺ ionophore and different levels of Ca²⁺: after incubation with 5 µM ionomycin, the 350/380 fluorescence ratio was 0.26 ± 0.003 in Ca²⁺-free and 1.07 ± 0.01 (n = 3) in a 5 mM Ca²⁺ solution, respectively. All starting ratios were above this minimum and all peak fluorescence ratios fell below the maximum. Additionally, changes in fluorescence while adding intracellular regulators occurred rapidly, suggesting that the probe was effectively reporting changes in Ca²⁺, even with very low levels of substrates (e.g., see ATP and TBQ dose reponses in RESULTS), without any appreciable delay other than that caused by the lag in solution exchange.

Statistical analysis was carried out in Excel (Microsoft) using paired or unpaired t-tests as appropriate. Where appropriate, ANOVA analysis was peformed with Minitab (Mintab, State College). Significance was assumed at the 5% level. Sigma Stat (Jandel Scientific) was used for nonlinear curve fitting in the determination of K_d values.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Factors Modulating Ca²⁺ Uptake into the Internal Store

ATP and Ca²⁺. Addition of ATP to the bath solution in the presence of 200 nM Ca²⁺ promoted a dose-dependent increase in the 350/380 ratio, consistent with the movement of Ca²⁺ into the internal store (Fig. 2). The mean data from these experiments are summarized in the inset and are described well by saturation kinetics with a K_d of 2.6 ± 0.6 µM (n = 5, P < 0.05). The degree of store filling also depended on the Ca²⁺ concentration of the bath fluid: in the presence of a saturating concentration of ATP (0.1 mM), incremental increases in the 350/380 ratio were seen on the addition of Ca²⁺ to the bathing solution over the range of 10 nM to 10 µM (data not shown) and reflect the ability of the store to accumulate Ca²⁺ over a wide concentration range. These results suggest that Ca²⁺ is moved against its concentration gradient at the expense of ATP and that the transport mechanisms operate over a wide range of intracellular Ca²⁺ concentrations.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Store loading in response to low levels of ATP. Filling of the internal stores was dose dependent (as shown by the recording under the horizontal bars) and appeared to have saturated in resposnse to 0.1 mM ATP. Inset: mean data from 5 tubules. Solid line is best fit to an equation representing saturation kinetics, yielding a Kd of 2.6 ± 0.66 µM. R.U., ratio units.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Attenuation of store loading by the SERCA-specific inhibitor 2,5-di(tert-butyl) hydroquinone (TBQ). TBQ was added to the bath (before the addition of ATP), and its concentration was decreased sequentially as indicated by the numbers over the horizontal bars. Inset: mean data from 6 tubules. Solid line connects points and has no theoretical significance.

Passive Ca²⁺ leak. In the steady state, the store Ca²⁺ content reflects equal rates of influx and efflux, a change in either of these processes will alter the store Ca²⁺ equilibrium. In an attempt to determine the pathway of the passive leak, the effect of heparin, an IP₃ receptor antagonist (14), on unstimulated store Ca²⁺ release was examined. Permeabilized tubules were exposed to 0.1 mM ATP to fill the internal store, and ATP was subsequently removed from the bath solution to reveal the passive loss of store Ca²⁺ (Fig. 4A). Calcium leak from the store followed an exponential time course that was unaffected by the addition of the IP₃ receptor blocker heparin (100 µg/ml, a concentration that has been previously determined to cause complete reversal of IP₃-mediated release, n = 6; Fig. 4B); time constant for 350/380 ratio decline following removal of ATP: control 1,750 ± 690 s, heparin 1,633 ± 529 s, n = 5, P > 0.05 (Fig. 4A). Loss of Ca²⁺ from the store was also apparent when Ca²⁺ was removed from the bath (data not shown). It appears therefore that the presence of both ATP and Ca²⁺ is required to maintain store Ca²⁺ load.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Passive leak of store Ca2+. A: passive leak of store Ca2+ as revealed by heparin, an IP3 receptor antagonist. A typical experiment in which ATP (0.1 mM) was present throughout the period is indicated by the bar. On removal of ATP (but with a bath [Ca] of 200 nM), the passive leak is exposed. Heparin (100 µg/ml) was added during the period of passive store leak for the duration of the horizontal bar. Trace representative of 5 separate experiments. B: heparin (100 µg/ml) is an effective antagonist of the effects of IP3.

IP₃-sensitive Ca²⁺ efflux. Store filling was promoted by the addition of 2 mM ATP to the bath, a concentration known to maximize the open probability of the IP₃ receptor (3), before the addition of IP₃ to the bath. The efflux of Ca²⁺ was clearly dependent on the concentration of IP₃ (Fig. 5), with an apparent K_d of 1 µM (see inset) and showing saturation at 3 µM. Because virtually all Ca²⁺ was released with 3 µM IP₃, this concentration was used in all subsequent experiments. Furthermore, the IP₃ receptor antagonist heparin (100 µg/ml) abolished the effect of IP₃ on store calcium (see Fig. 4B as previously discussed, n = 6). IP₃ therefore appears to be an important regulator of store Ca²⁺ content.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Release of store Ca2+ by IP3. Store filling was induced with 2 mM ATP, which was present throughout the remainder of the experiment. IP3 was added for the periods indicated by the horizontal bars and at the indicated concentrations. Inset: mean data from 6 tubules. Solid line is best fit to Michaelis-Menten equation with an apparent Kd of 1 µM.

Permeabilized tubules were exposed to ionomycin in the absence of ATP, i.e., without store loading. There was no effect on the 350/380 ratio following this intervention (350/380 ratio: control 0.27 ± 0.003; ionomycin 0.27 ± 0.003, n = 7, P > 0.05). As previously shown, after Ca²⁺ loading with ATP, IP₃ (3 µM) promoted a rapid fall in store Ca²⁺ that represented 58 ± 5% of the total release. However, addition of ionomycin promoted a further reduction in the 350/380 ratio, consistent with a release from an IP₃-insensitive store that was 42 ± 5% of the total release (n = 6; Fig. 6A). It is possible that these data represent Ca²⁺ release from a single compartment that occurs with two different time courses. However, we excluded this time-dependent effect on Ca²⁺ within the store because Fig. 6, A and B, shows that store Ca²⁺ decreases only after the addition of ionomycin, despite differences in the time course of initial application of the compound. Furthermore, Fig. 5 demonstrates that steady-state levels of store Ca²⁺ are only altered by additions of IP₃ and not by any effect of time (total exposure time to IP₃ >5 min).

View larger version (10K): [in this window] [in a new window]   Fig. 6. Ca2+ store mobilization by ionomycin and IP3. A and B: stores were loaded with 2 mM ATP, which was present throughout the experiment. A: IP3 (3 µM) was added during the period indicated by the horizontal bar and promoted a release of store Ca2+. Tubules were also exposed to ionomycin (5 µM) for the time indicated, which also promoted a release of store Ca2+. B: after addition of IP3, the tubule was exposed to oligomycin (10 µg/ml) for the period indicated by the horizontal bar. Oligomicin failed to elicit Ca2+ release, although there was a further release of Ca2+ in response to ionomycin.

View larger version (9K): [in this window] [in a new window]   Fig. 7. Identification of the IP3-insensitive store. A: in vivo mitochondria are likely to be exposed to respiratory substrates in addition to ATP. Exposure to this combination of compounds induced Ca2+ uptake that was insensitive to inhibition of the respiratory chain by rotenone but could be released by IP3. B: exposure to respiratory substrates after ATP-induced uptake does not promote further Ca2+ uptake; FCCP was without effect on store Ca2+, whereas IP3 was able to induce Ca2+ release.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

The ER appears to be a major Ca²⁺ store within the cells of the diluting segment because Ca²⁺ accumulation is sensitive to the SERCA-specific blocker TBQ; inhibition of SERCA activity with this compound prevents the global, furosemide-induced increase in intracellular Ca²⁺ (9). Furthermore, the application of TBQ alone, before store loading, never resulted in a decline in the 350/380 ratio (an effect indicative of Ca²⁺ loss via the leak pathway), which is consistent with the idea that there is little SERCA activity and thus ER loading following time spent in an ATP-free environment during dye loading. The data display an apparent K_i of 2 µM toward TBQ. This is in good agreement with inhibitory assays on rat liver microsomes (26) that demonstrated a K_i of 1 µM and a maximal inhibitory effect at 10 µM. The depletion of accumulated Ca²⁺ in the absence of ATP (or Ca²⁺) is also consistent with the notion that the continued presence of both these substrates is required to maintain a constant luminal Ca²⁺ concentration and thus the integrity of the pump-leak coupling mechanism. The affinity of Ca²⁺ uptake toward ATP strongly suggests that ATP is unlikely to be rate limiting in maintaining store Ca²⁺ content, assuming an intracellular ATP concentration in excess of 0.1 µM (1). Furthermore, our calculated K_d for ATP with respect to the uptake process of 2.6 µM is several orders of magnitude lower than other estimates using a similar technique in gastric epithelial cells (1.5 mM, see Ref. 19). However, both these estimates are considerably higher than values obtained using COS cell microsomes whereby the K_1/2 for the formation of a phosphorylated intemediate for SERCA 2b and SERCA3 (the most likely nonmuscle isoforms) are 20 and 50 nM, respectively (24). These differences are probably due to the fact that the fluorescence assay represents a global average of all ATP-dependent uptake processes. Similarly, data where Ca²⁺ transport varied with bath [Ca²⁺] could be fitted using a K_d of 230 nm. This value is similar to Ca²⁺ transport values published for SERCA2b, which elicited a K_d of 270 nM (24).

Release of Ca²⁺ from the ER appears to be dominated by the IP₃ receptor; there was no effect of the endogenous ryanodine receptor regulator cADPR. This is in agreement with our earlier work, in which addition of ryanodine directly to the bathing medium was without effect on store Ca²⁺ (7). Therefore, it is unlikely that there is a mixed expression of intracellular Ca²⁺ release channels in the EDT region. In vivo, it is therefore conceivable that release from the ER may well be mediated by IP₃. In human fibroblasts, it has been shown that a reduction in extracellular Na⁺ promotes the formation of inositol polyphosphates and a release of Ca²⁺ (30). Similarly furosemide, which causes an abrupt fall in intracellular Na⁺ in the EDT (27), also results in the release of store Ca²⁺, raising the possibility that this mechanism is mediated by IP₃.

Additional Ca²⁺ release following store loading with ATP was induced by the addition of ionomycin to the bath following store depletion with IP₃. There are several possibilities to explain this observation. 1) This represents anatomic homogeneity but functional heterogeneity (i.e., not all the Ca²⁺ within the store is released) within the IP₃-sensitive pool. This may explain the ATP-dependent uptake but the lack of effect of ionomycin in the absence of store loading. An example of such a store would be the nuclear envelope, which is not only continuous with the ER (13) but has been shown to have functional Ca²⁺ uptake (13) and release pathways (12, 22, 31). 2) It is also conceivable that this result represents a differential expression of IP₃ receptors to the ER membrane and as such Ca²⁺ release in the presence of IP₃ occurs from the part of the store with the highest level of receptor expression. We have been unable to image the patterns of release and are therefore unable to resolve this possibility, but it is tempting to speculate that focal Ca²⁺ release could target the Ca²⁺ signal to discrete calmodulin-rich areas to accomplish activation of the sodium hydrogen exchanger. 3) This observation may represent the activity of a spatially inhomogenous store such as mitochondria (19) or another endomembranous compartment, e.g., lysosomes (18). Under the current conditions, the fluorescence signal is unaffected by addition of the protonphore FCCP, the actions of which are not confined to mitochondria, but to all compartments across which a proton gradient is maintained (see Fig. 6B and also Ref. 25). This has two implications. It suggests 1) that lysosomes do not make a significant contribution to Ca²⁺ cycling in the frog EDT and rules out any artefactual, pH-induced, fluorescence changes that result from the activity of proton-transporting ATPases on the lysosomal membrane and 2) that mitochondria do not participate directly in the uptake and release of Ca²⁺ under these conditions. This may not be too surprising given that the affinity of the Ca²⁺ uniporter is in excess of 1 µM (2), which is an order of magnitude higher than the Ca²⁺ levels used in the present study.

The data presented in this investigation rely on the compartmentalization into cellular pools of a low-affinity Ca²⁺-sensitive probe. In our system, mag-fura 2 appears to respond to large changes in Ca²⁺ (from 0 to 5 mM), equilibrated across the organelle membranes using ionomycin, which produces concomitantly large changes in the fluorescence ratio that would be well in excess of the levels of Ca²⁺ anticipated to reside within the stores (4). However, signal responses from this preparation may be limited because of 1) a low pumping activity with low levels of "resting" Ca²⁺, 2) washout of cellular regulators during permeabilization that modulate the activity of store-filling mechanisms, or 3) that "silent" compartments bias the fluorescence signal in favor of probe trapped in pools where Ca²⁺ resides below the detection limit of the probe (5 µM) (21). This occurs because fluorescence ratio increases rely on quenching of fluorescent probe by Ca²⁺. Therefore, a probe trapped equally in two compartments will return an average fluorescence signal of the two pools; the compartment containing high Ca²⁺ will have a quenched signal compared with the compartment containing low Ca²⁺. Thus a proportionately larger part of the signal is composed of fluorescence from the low-Ca²⁺ compartment. If Ca²⁺ is not moved into this pool at all during uptake, then changes in the fluorescence signal will arise from movements into and out of just one store, which will suppress the magnitude of the observed changes (21). Due to these complications, the current data remain uncalibrated.

In summary, frog EDT cells maintain a constant internal store Ca²⁺ content at the expense of ATP. The availability of ATP is unlikely to be rate limiting to Ca²⁺ accumulation, given the high affinity of the Ca²⁺ uptake mechanism relative to the expected intracellular concentration of ATP. Ca²⁺ is released via the IP₃ receptor, and not the ryanodine receptor, whereas the sensitivity toward the SERCA-specific inhibitor TBQ and the endogenous releasing agent IP₃ strongly implicates the ER as the major site of Ca²⁺ storage within the EDT.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The support of the National Kidney Research Fund is gratefully acknowledged.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Hunter, School of Biomedical Sciences, Worsley Bldg., Univ. of Leeds, Leeds, West Yorkshire LS2 9NQ, UK (E-mail: m.hunter{at}leeds.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Beck JB, Breton S, Mairbaurl H, Laprade R, and Giebisch G. Relationship between sodium transport and intracellular ATP in isolated perfused rabbit proximal convoluted tubule. Am J Physiol Renal Fluid Electrolyte Physiol 261: F634–F639, 1991.[Abstract/Free Full Text]
2. Bernardi P. Mitochondrial transport of cations: channels, exchangers and permeability transition. Physiol Rev 79: 1127–1155, 1999.[Abstract/Free Full Text]
3. Bezprozvanny I and Ehrlich BE. ATP modulates the function of inositol 1,4,5-trisphosphate-gated channels at two sites. Neuron 10: 1175–1184, 1993.[CrossRef][Web of Science][Medline]
4. Bygrave FL and Benedetti A. What is the concentration of calcium ions in the endoplasmic reticulum? Cell Calcium 19: 547–551, 1996.[CrossRef][Web of Science][Medline]
5. Churchill GC and Louis CF. Imaging of intracellular calcium stores in single permeabilized lens cells. Am J Physiol Cell Physiol 276: C426–C434, 1999.[Abstract/Free Full Text]
6. Clapper DL and Lee HC. Inositol trisphosphate induces calcium release from nonmitochondrial stores in sea urchin egg homogenates. J Biol Chem 260: 13947–13954, 1985.[Abstract/Free Full Text]
7. Cooper GJ, Fowler MR, and Hunter M. Membrane cross-talk in the early distal tubule sgment of frog kidney: role of calcium stores and chloride. Pflügers Arch 442: 243–247, 2001.[CrossRef][Web of Science][Medline]
8. Cooper GJ and Hunter M. Na⁺-H⁺ exchange in frog early distal tubule: effect of aldosterone on the set-point. J Physiol 479: 423–432, 1994.[Abstract/Free Full Text]
9. Cooper GJ and Hunter M. Intracellular pH and calcium in frog early distal tubule: effects of transport inhibitors. J Physiol 498.1: 49–59, 1997.
10. Foskett JK and Wong D. Calcium oscillations in parotid acinar cells induced by microsomal Ca²⁺-ATPase inhibition. Am J Physiol Cell Physiol 262: C656–C663, 1992.[Abstract/Free Full Text]
11. Galione A, Lee HC, and Busa WB. Ca²⁺-induced Ca²⁺ release in sea urchin egg homogenates: modulation by cyclic ADP-ribose. Science 253: 1143–1146, 1991.[Abstract/Free Full Text]
12. Gerasimenko OV, Gerasimenko JV, Tepikin AV, and Petersen OH. ATP-dependent accumulation and inositol trisphosphate- or cyclic ADP-ribose-mediated release of Ca²⁺ from the nuclear envelope. Cell 80: 439–444, 1995.[CrossRef][Web of Science][Medline]
13. Gerasimenko OV, Gerasimenko JV, Tepikin AV, and Petersen OH. Calcium transport pathways in the nucleus. Pflügers Arch 432: 1–6, 1996.[CrossRef][Web of Science][Medline]
14. Ghosh TK, Eis PS, Mullaney JM, Ebert CL, and Gill DL. Competitive, reversible and potent antagonism of inositol 1,4,5-trisphosphate-activated calcium release by heparin. J Biol Chem 263: 11075–11079, 1988.[Abstract/Free Full Text]
15. Goldenthal KL, Hedman JW, Chen JW, August JT, and Willingham MC. Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins. J Histochem Cytochem 33: 813–820, 1985.[Abstract]
16. Greger R and Schlatter E. Presence of luminal K⁺, a prerequisite for active NaCl transport in the cortical thick ascending limb of Henle's loop of rabbit kidney. Pflügers Arch 392: 92–94, 1981.[CrossRef][Web of Science][Medline]
17. Guggino WB, Oberleithner H, and Giebisch G. The amphibian diluting segment. Am J Physiol Renal Fluid Electrolyte Physiol 254: F615–F627, 1988.[Abstract/Free Full Text]
18. Haller T, Dietl P, Deetjen P, and Volkl H. The lysosomal compartment as intracellular calcium store in MDCK cells: a possible involvement in Insp₃-mediated Ca²⁺ release. Cell Calcium 19: 157–165, 1996.[CrossRef][Web of Science][Medline]
19. Hofer AM and Machen TE. Direct measurement of free Ca in organelles of gastric epithelial cells. Am J Physiol Gastrointest Liver Physiol 267: G442–G451, 1994.[Abstract/Free Full Text]
20. Hofer AM, Schule WR, Curci S, and Machen TE. Spatial distribution and quantitation of free luminal [Ca] within the InsP₃-sensitive internal store of individual BHK-21 cells: ion dependence of InsP₃-induced Ca release and reloading. FASEB J 9: 788–798, 1995.[Abstract]
21. Hofer AM and Schulz I. Quantification of intralumina free [Ca] in the agonist-sensitive internal calcium store using compartmentalized fluorescent indicators: some considerations. Cell Calcium 20: 235–242, 1996.[CrossRef][Web of Science][Medline]
22. Humber JP, Matter N, Artault JC, Koppler P, and Malviya AN. Inositol 1,4,5-trisphosphate receptor is located to the inner nuclear membrane vindicating regulation of nuclear calcium signalling by inositol 1,4,5-trisphosphate. J Biol Chem 271: 478–485, 1996.[Abstract/Free Full Text]
23. Hurst A and Hunter M. Apical K⁺ channels of frog diluting segment: inhibtion by acidification. Pflügers Arch 415: 115–117, 1989.[CrossRef][Web of Science][Medline]
24. Lytton J, Westlin M, Burk SE, Shull GE, and MacLennan DH. Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps. J Biol Chem 267: 14483–14489, 1992.[Abstract/Free Full Text]
25. Mojet MH, Jacobson DJ, Keelan J, Vergun, and Duchen MR. Monitoring mitochondrial function in single cells. In: Calcium Signalling (2nd edition), edited by Tepikin AV. Oxford, UK: Oxford Univ. Press, 2001.
26. Moore GA, McConkey DJ, Kass GEN, O'Brien PJ, and Orreniun S. 2,5-Di(tert-butyl)-1,4-benzohydroquinone-a novel inhibitor of liver microsomal Ca²⁺ sequestration. FEBS Lett 224: 331–336, 1987.[CrossRef][Web of Science][Medline]
27. Oberleithner H, Lang F, Wang W, and Giebisch G. Effects of inhibition of chloride transport on intracellular sodium activity in distal amphibian nephron. Pflügers Arch 402: 55–60, 1982.
28. Prentki M, Biden TJ, Janjic D, Irvine RF, Berridge MJ, and Wollheim CB. Rapid mobilization of Ca²⁺ from rat insulinoma microsomes by inositol-1,4,5-trisphosphate. Nature 309: 562–564, 1984.[CrossRef][Medline]
29. Simon DB, Karet FE, Rodriguez-Soriana J, Hamdan JH, DiPietro A, Trachtman H, Sanjard SA, and Lifton RP. Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K⁺ channel, ROMK. Nat Genet 14: 152–156, 1996.[CrossRef][Web of Science][Medline]
30. Smith JB, Dwyer SC, and Smith L. Decreasing extracellular Na concentration triggers inositiol polyphosphate production and Ca²⁺ mobilization. J Biol Chem 264: 831–837, 1989.[Abstract/Free Full Text]
31. Stehno-Bittel L, Luckhoff A, and Clapham DE. Calcium release from the nucleus by InsP₃ receptor channels. Neuron 14: 163–167, 1995.[CrossRef][Web of Science][Medline]
32. Streb H, Bayerdorfer E, Haase W, Irvine RF, and Schulz I. Effect of inositol-1,4,5-trisphospahte on isolated subcellular fractions of rat pancreas. J Membr Biol 81: 241–253, 1984.[CrossRef][Web of Science][Medline]
33. Van de Put FHMM and Elliot AC. Imaging of intracellular calcium stores in individual permeabilized pancreatic acinar cells. J Biol Chem 271: 4999–5006, 1996.[Abstract/Free Full Text]

This article has been cited by other articles:

				B. M. Walsh, H. B. Naik, J. M. Dubach, M. Beshire, A. M. Wieland, and D. I. Soybel Thiol-oxidant monochloramine mobilizes intracellular Ca2+ in parietal cells of rabbit gastric glands Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1687 - C1697. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 286/6/F1219    most recent 00255.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Fowler, M. R. Articles by Hunter, M. Search for Related Content PubMed PubMed Citation Articles by Fowler, M. R. Articles by Hunter, M.