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¹ School of Biomedical Sciences, Worsley Building, University of Leeds, Leeds, LS2 9NQ, UK

	Abstract

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A global and transient rise of intracellular Ca²⁺ (Ca²⁺_i) is central to the operation of pump–leak coupling in the frog early distal tubule (EDT). The endoplasmic reticulum (ER) is the site of this Ca²⁺ release and reuptake; however, it is likely that other intracellular pools, such as mitochondria, also contribute to cellular Ca²⁺ homeostasis. The present study was performed to seek evidence of mitochondrial Ca²⁺ transport in the frog EDT. Experiments were performed on isolated and permeabilized EDT segments from the frog kidney loaded with the low-affinity, Ca²⁺-sensitive fluorescent indicator, mag-fura-2. Ca²⁺ uptake in the absence of SarcoEndoplasmic Reticulum Calcium ATPase (SERCA) activity (inhibition by 2,5-di-t-butyl hydroquinone, TBQ) was evident at a bath [Ca²⁺] of 1 µM, but not at 200 nM, in the presence of ATP. This uptake was sensitive to the protonophore FCCP and the ATP-synthase inhibitor oligomycin. Ca²⁺ uptake was also stimulated by respiratory substrates; this uptake was enhanced by oligomycin and reversed by the application of FCCP. These findings provide the first evidence of mitochondrial Ca²⁺ transport in renal tubules, which appears to occur via a low-affinity pathway and which will act as a physiological Ca²⁺ buffer, protecting the cell from large increases in Ca²⁺_i.

(Received 1 September 2004; accepted after revision 8 November 2004; first published online 30 November 2004)
Corresponding author M. Hunter: School of Biomedical Sciences, Worsley Building, University of Leeds, Leeds, LS2 9NQ, UK. Email: m.hunter{at}leeds.ac.uk

	Introduction

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Calcium (Ca²⁺) is a ubiquitous second messenger within cells. In epithelial cells from the early distal tubule (EDT) of frog kidney, the movement of NaCl across the epithelium is regulated by the activity of apical K⁺ channels (Cooper & Hunter, 1997). Inhibition of NaCl transport, which is the dominant physiological role of this segment (and can be achieved experimentally with frusemide) causes the release of Ca²⁺ from the endoplasmic reticulum (ER) and a transient rise in intracellular Ca²⁺ (Ca²⁺_i) that precedes upregulation of the apical K⁺ channels (Cooper & Hunter, 1997; Cooper et al. 2001). Subsequently, Ca²⁺ is sequestered back into the ER via an ATP-driven pump of the SERCA family (Cooper et al. 2001; Fowler et al. 2004). The ER, however, is often not alone in its ability to sequester Ca²⁺_i following elevations in its concentration; in particular, mitochondria have long been reported to have this ability (Vasington & Murphy, 1962; Barritt & Lamont, 1982; Coll et al. 1982). Hitherto, the relatively low affinity of the mitochondrial Ca²⁺ uptake pathway was thought to preclude a role for mitochondria in cellular Ca²⁺ homeostasis (e.g. Heaton & Nicholls, 1976). This notion has now been challenged by the concept that Ca²⁺ signalling relies on increases in Ca²⁺_i that may bring its concentration into the range in which it may be susceptible to transport by mitochondria. Mitochondrial Ca²⁺ transport has been demonstrated in a number of cell types: for example, in permeabilized gastric epithelial cells, Ruthenium Red (an inhibitor of the mitochondrial Ca²⁺ uniporter) and FCCP (a protonophore that collapses the highly negative mitochondrial membrane potential) induced the release of Ca²⁺ as reported by a low-affinity Ca²⁺ indicator trapped within the organelle (Hofer & Machen, 1994).

In cells, Ca²⁺_i signals occur in a spatially and temporally coordinated manner and such elevations in Ca²⁺_i have been shown to be modulated by mitochondrial transport in cerebellar granule cells (Budd & Nicholls, 1996), adrenal chromaffin cells (Herrington et al. 1996; Babcock et al. 1997), dorsal root ganglion cells (Werth & Thayer, 1994), rat gonadotrophs (Hehl et al. 1996), pancreatic ß cells (Kindmark et al. 2001) and cardiac myocytes (Miyata et al. 1991; Chacon et al. 1996; Duchen et al. 1998; Trollinger et al. 2000; Robert et al. 2001). Thus it appears that rises in Ca²⁺_i may be modulated by mitochondrial Ca²⁺ transport and this implicates these organelles in mechanisms of cellular Ca²⁺ handling.

The present experiments sought evidence for mitochondrial Ca²⁺ transport in permeabilized EDTs of the frog. The findings show the capacity of mitochondria within the frog EDT to accumulate Ca²⁺ when exposed to relatively high cytosolic Ca²⁺ concentrations, and stimulation of Ca²⁺ uptake upon exposure to substrates that augment mitochondrial respiration.

	Methods

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Early distal tubule preparation

The procedure for isolation and loading of EDT segments with mag-fura-2 AM has been described in detail previously (Fowler et al. 2004). Briefly, frogs (Rana temporaria, Blades Biological, Edenbridge, UK) of either sex were kept in tap water at 4°C. Following killing by a Schedule 1 procedure in accordance with the Animals (Scientific Procedures) Act 1986 (concussion followed by pithing), the kidneys were removed, cut into sections and stored in ice-cold Leibovitz incubation medium (Sigma, Poole, UK). Individual EDT segments were manually dissected in ice-cold amphibian Ringer solution (mM): NaCl, 97; KCl, 3; CaCl₂, 2; MgCl₂, 1; and HEPES, 10; titrated to pH 7.4 with NaOH.

Loading of EDT segments with mag-fura-2 AM and recording of fluorescence

Acutely isolated EDT segments were incubated in 14 µM mag-fura-2 AM (Molecular Probes, Leiden, Netherlands) for 1 h in amphibian Ringer solution. Tubules were held at each end with a pair of microperfusion pipettes but were not perfused. The fluorescence system (Newcastle Photometrics, Newcastle, UK) was based around a Nikon Diaphot inverted microscope (Nikon, Japan). Mag-fura-2 was excited (350 and 380 nm) at 1 Hz; fluorescence emission at 520 nm was collected using a 40x objective and a photomultiplier tube, digitized, displayed and stored on the hard disk of a PC. The resulting changes in the 350:380 ratios were used as an indication of changes in [Ca²⁺]. The K_D of mag-fura-2 is 25 µM, and the effective lower measurement limit is about 5 µM Ca²⁺, so the Ca²⁺ ratio measurements in this paper reflect changes within the micromolar range (Hofer & Machen, 1993). All experiments were performed at room temperature.

Permeabilization

Initially, probe accumulates in both the cytosol and intracellular organelles. Probe is washed out of the cytosol following permeabilization of the basolateral membrane with the detergent saponin. The rationale for this approach has been described and validated previously (Fowler et al. 2004). Permeabilization and subsequent experiments were carried out in an intracellular solution of the following composition (mM): K-gluconate, 88; NaCl, 12; MgSO₄, 2 (free [Mg²⁺], 1); Ca(NO₃)₂, 4 (free [Ca²⁺], 200 nM); EGTA, 10; and HEPES, 10; titrated to pH 7 with KOH/glucuronic acid lactone as appropriate. In some experiments, intracellular [Na⁺] and [Cl^–] were reduced from the normal intracellular value of 12 mM to 4 mM; this was achieved by replacement of 8 mM NaCl with either KCl or with Na-gluconate. In this way, independent manipulation of [Na⁺] and [Cl^–] was achieved. Ca²⁺ activities were calculated using React 2 software (G. Smith, University of Glasgow, Glasgow, UK). Tubules were exposed to saponin (25 ng ml^–1) and the loss of fluorescent probe into the bath solution monitored on-line until the 350 nm signal was approximately 30% of its maximum, when saponin was washed from the preparation. This left a 350 nm signal that was approximately 15–20% of the unpermeabilized value and was about 50–100 times background. Mn²⁺ (0.5 mM) was present during the permeabilization to quench any cytosolic probe not washed away by permeabilization. Additions to the intracellular bathing solution were made from the following stock solutions (unless otherwise stated all chemicals were obtained from Sigma and dissolved in distilled water): K-ATP, 1 M; K-ADP, 0.5 M (Fluka, Buchs, Switzerland); KH₂PO₄, 1 M; succinic acid, 0.5 M; 2,5-di-t-butyl hydroquinone (TBQ), 10 mM in dimethyl sulphoxide (DMSO); thapsigargin, 1 mM in DMSO; Ca(NO₃)₂, 1 M; saponin, 50 mg ml^–1; MnCl₂, 100 mM; oligomycin (mix of oligomycin A, B and C), 5 mg ml^–1 in DMSO; and carbonyl cyanide p-(trifluoromethoxy)phenyl hydrazone (FCCP), 10 mM in DMSO.

Data analysis and statistics

Data are presented as continuous experimental recordings with time on the x-axis and the 350:380 fluorescence ratio on the y-axis or as mean ratio changes taken at the steady state. No attempt has been made to calibrate the mitochondrial signals because the presence of multiple Ca²⁺ stores biases such an approach, as previously described (Hofer & Schulz, 1996). Statistical analysis was carried out in Excel (Microsoft, USA) using Student's paired or unpaired t tests. Where appropriate, ANOVA was performed with Minitab statistical software (Minitab Inc., State College, PA, USA). Significance was assumed at the 5% level.

	Results

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Ca²⁺ uptake

Elevated Ca²⁺ and ATP hydrolysis.  The movement of Ca²⁺ into mitochondria is mediated by a Ca²⁺ uniporter (Lehninger et al. 1967; Scarpa & Azzone, 1970; for review see Gunter & Pfeiffer, 1990), which has an affinity in the micromolar range (Heaton & Nicholls, 1976; see also Gunter & Pfeiffer, 1990 for review). Energy for the translocation of Ca²⁺ is stored in the highly negative potential difference (_m) that exists across the inner mitochondrial membrane (Mitchell, 1961) and which occurs by the extrusion of protons via the enzyme complexes of the respiratory chain. This gradient can be maintained in the presence of ATP by reverse activity of the F₁-F_o ATPase or ATP-synthase enzyme, which results in ATP hydrolysis (Ulrich, 1965), extrusion of protons and the maintenance of _m. Figure 1 summarizes data describing store loading under the conditions described above with respect to changes in the 350:380 ratio. Following the addition of ATP, with a bath [Ca²⁺] of 200 nM, the 350:380 ratio increased by 0.05 ± 0.009 (n = 6); this store filling was completely abolished by the addition of TBQ (10 µM) such that there was no change in the 350:380 ratio (350:380 ratio: control, 0.28 ± 0.005; TBQ, 0.28 ± 0.005, n = 6, P < 0.05), identifying these stores as the ER. Increasing bath Ca²⁺ to 1 µM resulted in a mean increase in the 350:380 ratio of 0.18 ± 0.03 (350:380 ratio: control, 0.27 ± 0.006; 1 µM Ca²⁺, 0.45 ± 0.03, n = 5, P < 0.05). Inhibiting SERCA activity in the presence of 1 µM bath Ca²⁺ resulted in a large increase in the 350:380 ratio, indicative of Ca²⁺ movement into a non-ER pool (since SERCA pumping is inhibited; Foskett & Wong, 1992), which, on average, was 0.13 ± 0.02 (350:380 ratio: control, 0.28 ± 0.01; 1 µM Ca²⁺, 0.41 ± 0.04, n = 8, P < 0.05). Similar results were also seen when thapsigargin was used to inhibit SERCA activity, in which case the 350:380 ratio increased from 0.27 ± 0.01 to 0.36 ± 0.02 when bath Ca²⁺ was elevated to 1 µM (n = 4, P < 0.05). The use of thapsigargin, which is non-reversible, essentially rules out the possibility that the elevation in bath Ca²⁺ leads to an increase in Ca²⁺ uptake as a result of competition with TBQ for sites on the SERCA pump.

View larger version (10K): [in this window] [in a new window]   Figure 1.  Uptake of store Ca2+ at low and high Ca2+ activities With a bath Ca2+ of 200 nM, Ca2+ uptake is completely abolished by the SERCA pump inhibitor TBQ. Raising bath Ca2+ to 1 µM caused a substantial increase in store Ca2+ that was only partly sensitive to TBQ. * Significant difference with respect to uptake at 200 nM Ca2+ in the absence of TBQ. Bathing [Ca2+] is indicated below the bars. Data are given as mean ratio change ± S.E.M.

View larger version (11K): [in this window] [in a new window]   Figure 2.  Release of store Ca2+ by inhibitors of mitochondrial transport Comparison of the effects of FCCP (n = 8) and oligomycin (n = 6) on the percentage release of Ca2+ from stores following loading at either 200 nM Ca2+ or at 1 µM Ca2+ in the presence of TBQ. Data are given as mean percentage release + S.E.M. * Significance compared to steady-state filled store conditions.

View larger version (16K): [in this window] [in a new window]   Figure 3.  Operation of the respiratory chain promotes Ca2+ uptake Addition of respiratory substrates (Subs; ADP, phosphate and succinate) at 200 nM Ca2+ in the presence of TBQ has no effect on store Ca2+. Elevation of bath Ca2+ to 1 µM promoted a rapid, transient increase in store Ca2+ that was enhanced by the F1-Fo ATPase inhibitor oligomycin. These effects were rapidly reversed by the protonophore FCCP. Trace is representative of 7 experiments.

In a previous study in permeabilized EDT segments (Cooper et al. 2001), lowering bath [Cl^–] to 4 mM from 12 mM promoted a rapid release of Ca²⁺ from the internal store; a decrease in [Cl^–]_i was suggested as a putative regulator of Ca²⁺ release from the ER during pump–leak coupling. However, lowering [Na⁺]_i by the same amount was without effect. Figures 4 and 5 illustrate the effect of identical reductions in both intracellular Cl^– and Na⁺, respectively, following the accumulation of Ca²⁺ into the mitochondrial store. In contrast to the ER, where a rapid loss of store Ca²⁺ occurs, lowering [Cl^–]_i results in a rapid increase in Ca²⁺ whereby the 350:380 ratio increased from 0.39 ± 0.01 to 0.45 ± 0.02 (n = 7, P < 0.05). Similarly, a reduction in intracellular [Na⁺] also promoted rapid store filling (350:380 ratio: 12 mM Na⁺, 0.32 ± 0.01; 4 mM Na⁺, 0.34 ± 0.01, n = 6, P < 0.05).

View larger version (14K): [in this window] [in a new window]   Figure 4.  TBQ-insensitive store Ca2+ uptake is stimulated by lowering intracellular Cl– Reducing intracellular [Cl–] from 12 to 4 mM in the presence of TBQ, to inhibit SERCA activity, promotes mitochondrial Ca2+ accumulation. Original recording representative of 7 experiments.

View larger version (18K): [in this window] [in a new window]   Figure 5.  Reduction of intracellular [Na+] stimulates filling of the TBQ-insensitive pool Decreasing intracellular [Na+] from 12 to 4 mM in the presence of TBQ, to inhibit SERCA activity, increases store Ca2+. Original recording representative of 6 experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Permeabilized cell preparations have been used extensively for many cell types to examine intracellular Ca²⁺ storage. Permeabilization, following loading of kidney tubules with low-affinity Ca²⁺ indicator, has been used previously to examine aspects of Ca²⁺ storage in the frog EDT (Cooper et al. 2001; Fowler et al. 2004). We have therefore used this technique to identify and examine mitochondrial Ca²⁺ uptake. The activity of mitochondria has been exposed following inhibition of the SERCA pump using TBQ and by elevating bath [Ca²⁺]. The application of TBQ prior to elevating bath [Ca²⁺] never resulted in a decline in the 350:380 ratio (which would be indicative of Ca²⁺ loss via the leak pathway), which is consistent with the idea that there is negligible ER loading during the ATP-free period during probe incubation. We therefore suggest that the increase in Ca²⁺ observed is probably due to movement into a non-ER pool. Furthermore, the sensitivity of this uptake to the protonophore FCCP and mitochondrial inhibitor oligomycin strongly suggests that the uptake is a result of mitochondrial activity. The low affinity of this pathway (see Fig. 1) is also consistent with operation of the mitochondrial Ca²⁺ uniporter (Heaton & Nicholls, 1976). Additionally, under conditions that abolish SERCA activity (absence of ATP and presence of TBQ), there is a significant movement of Ca²⁺ into an intracellular pool during exposure to respiratory substrates and elevated Ca²⁺. The proton efflux that accompanies operation of the respiratory chain re-enters the mitochondrial matrix via the ATP-synthase. Inhibiting this route for proton entry whilst maintaining respiratory chain turnover will hyperpolarize _m; the resulting increase in the driving force for Ca²⁺ uptake potentiates transport and a large, additional rise in accumulation is observed (Fig. 3). Once again, dissipation of _m with the protonophore FCCP abolished Ca²⁺ accumulation, strongly implicating mitochondria as the source of this Ca²⁺ transporting activity.

The present data do not directly address the role of mitochondria in the clearance of Ca²⁺ in the intact cell following NaCl transport inhibition with frusemide. In addition, the data do not describe a titration of the uptake response with respect to the bathing [Ca²⁺], but it is conceivable that a rise of intracellular Ca²⁺ from 0.2 to 0.5 µM (Cooper & Hunter, 1997) is sufficient to induce a significant component of mitochondrial uptake. Indeed, uptake by mitochondria from physiological-like pulses of Ca²⁺ has been described by Gunter et al. (1996) in liver mitochondria and is ascribed to a mechanism called ‘rapid uptake mode’ or ‘RaM’. It is also suggested that domains of high Ca²⁺ are created by the close apposition of ER release sites with mitochondria; Ca²⁺ is then accumulated from this spatially restricted area (Robb-Gaspers & Rutter, 1998).

Ca²⁺ accumulation by mitochondria provides a coupling between cytosolic effectors and cellular energy ([ATP]) balance. Evidence exists for alterations in mitochondrial [Ca²⁺] modulating the activity of three key enzymes in mitochondrial oxidative phosphorylation; pyruvate dehydrogenase (Denton et al. 1972), -ketoglutarate dehydrogenase (McCormack & Denton, 1979) and isocitrate dehydrogenase (Denton et al. 1978), thereby upregulating the synthesis of ATP. A global Ca²⁺ transient would thus not only upregulate the activity of the apical K⁺ channels responsible for K⁺ recycling but also increase the ATP:ADP ratio. Thus, the active transport of Na⁺ and Ca²⁺ would not be limited by the supply of cellular ATP.

In a previous study we proposed that the ER was the source of Ca²⁺ in pump–leak coupling (Fowler et al. 2004). In this study we describe a second pool, attributed to a functionally distinct organelle such as mitochondria. The existence of two distinct pools is supported by the responses towards changes in intracellular Na⁺ and Cl^–. While decreasing the concentration of these ions with a functionally intact ER promotes either release (promoted by decreasing Cl^–) or no change (Na⁺) in ER Ca²⁺ (Cooper et al. 2001), identical changes to the concentrations of these ions promote Ca²⁺ uptake when the ER Ca²⁺ pump is inhibited. Mitochondria possess a number of pathways for the movement of ions across the inner membrane. In particular, sodium–calcium exchange has been reported in a variety of tissues, although its activity in mitochondria of epithelial tissue is debated (see Bernardi, 1999 for review). If present, lowering the [Na⁺] may slow or reverse the activity of the exchanger and elevate the Ca²⁺ content of the mitochondria. The observations following a decrease in intracellular [Cl^–] are a little harder to resolve. Several reports document the activity of Cl^– channels to various intracellular membranes (Glickman et al. 1983; Morier & Sauve, 1994); indeed, the presence of Cl^– channels in the outer mitochondrial membrane has long been known (Colombini, 1980), but to our knowledge there is no evidence for anion channels in the inner mitochondrial membrane. Should a Cl^– conductance be present, however, it is conceivable that the loss of mitochondrial Cl^– as the intracellular concentration is lowered would promote a depolarization of _m; in turn this depolarization may slow or reverse the sodium calcium exchanger and increase mitochondrial Ca²⁺ content. Whatever the mechanism that couples intracellular Na⁺ and Cl^– to mitochondrial Ca²⁺ function, it is clear that these responses delineate a functionally distinct intracellular compartment and that these mechanisms may act in concert with the mitochondrial Ca²⁺ uniporter to clear Ca²⁺ loads from the cytosol and modulate the energy balance of the cell.

In conclusion, these data demonstrate that mitochondria from the frog EDT can accumulate and release Ca²⁺. Such ‘physiological’ Ca²⁺ buffering may attenuate increases in intracellular [Ca²⁺] to levels consistent with cell survival.

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	Acknowledgements

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

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This Article Abstract Full Text (PDF) All Versions of this Article: 90/2/195    most recent expphysiol.2004.028886v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal 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 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. Related Collections GI & Epithelial