Analysis of Ca²⁺ uptake into the smooth endoplasmic reticulum of permeabilised sternal epithelial cells during the moulting cycle of the terrestrial isopod Porcellio scaber

The rapid bidirectional Ca²⁺ dynamics and high flux rates of Ca²⁺ during the moulting cycle(Neufeld and Cameron, 1993)make crustacean epithelia excellent model systems for the analysis of epithelial Ca²⁺ transport(Wheatly, 1997). The crustacean exoskeleton is mineralised mainly by CaCO₃(Greenaway, 1985) and, in order to grow, crustaceans have to moult their cuticle periodically. The terrestrial isopod Porcellio scaber moults every 6 weeks and develops large sternal reservoirs containing amorphous, possibly hydrated,CaCO₃ (Drobne and Štrus,1996; Ziegler,1994) before every moult. The deposition and resorption of these reservoirs are linked to the unique biphasic moulting cycle of isopods in which first the posterior and then the anterior cuticle are shed. During premoult, calcium is resorbed from the posterior cuticle into the haemolymph and transported across the anterior sternal epithelium (ASE) to form CaCO₃ deposits between the epithelium of the integument and the old cuticle (Messner, 1965; Steel, 1993). Between the posterior and anterior halfbody moults (intramoult), these CaCO₃deposits are resorbed and used to mineralise the new posterior cuticle. Ultrastructural studies have shown that the ASE differentiates for epithelial ion transport during the formation and resorption of these CaCO₃deposits (Glötzner and Ziegler,2000; Ziegler,1996).

Ca²⁺ transport is generally either paracellular, between the epithelial cells, or transcellular. Transcellular Ca²⁺ transport can be divided into three phases: the passive influx of Ca²⁺ at one side of the epithelial cell, Ca²⁺ transport from one side of the cell to the other, and the energy-consuming extrusion of Ca²⁺. In crustaceans, entry probably occurs through Ca²⁺ channels or by a Ca²⁺/H⁺ exchanger(Ahearn and Franco, 1990; Ahearn and Zhuang, 1996). Active extrusion of Ca²⁺ probably involves a Ca²⁺-ATPase(Flik et al., 1994; Greenaway et al., 1995; Roer, 1980) or a Na⁺/Ca²⁺ exchanger(Ahearn and Franco, 1993). In the ASE of P. scaber, electronprobe microanalysis(Ziegler, 2002), expression analysis of the plasma membrane Ca²⁺-ATPase and the Na⁺/Ca²⁺ exchanger(Ziegler et al., 2001) and the abundance of Na⁺/K⁺-ATPase in the basolateral membrane(Ziegler, 1997) suggest that the transcellular pathway dominates.

How Ca²⁺ is transported within the epithelial cells is still unknown. Because of the multiple physiological functions of Ca²⁺,the mean free cytosolic Ca²⁺ concentration in cells is maintained at approximately 0.1 μmol l^-1. Transient rises in Ca²⁺ concentration are tolerated, but high sustained Ca²⁺ concentrations in the cytoplasm are toxic and can lead to cell death (Berridge, 1993). A cytosolic route with Ca²⁺ bound to proteins(Feher et al., 1989) and an organellar route (Nemere,1992; Simkiss,1996) have therefore been proposed for Ca²⁺ transit. Simkiss (1996) proposed a model in which the smooth endoplasmic reticulum (SER) could function as a transient Ca²⁺ store, leading only to micromolar gradients in the cytoplasm. Electron-probe microanalysis on sternal epithelial cells of P. scaber showed a significant increase in the total (free plus bound)cytoplasmic Ca²⁺ concentration during the Ca²⁺-transporting stages(Ziegler, 2002). It is of particular interest that this increase is due to an increase in the number of areas with Ca²⁺ concentrations of up to 50 mmol l^-1kg^-1 dry mass because this is similar to the concentrations measured in the SER of bee photoreceptors(Baumann et al., 1991) and vertebrate skeletal muscle (Somlyo et al.,1981).

In an attempt to test the hypothesis that the SER contributes to epithelial Ca²⁺ transport, we investigated the smooth endoplasmic reticulum Ca²⁺-ATPase (SERCA)-dependent uptake of Ca²⁺ during four different moulting stages in P. scaber employing an in situcalcium oxalate assay (Walz and Baumann,1989). The results indicate an increase in SERCA-dependent Ca²⁺ uptake into the SER between the nontransporting and the Ca²⁺-transporting moulting stages.

Porcellio scaber Latreille were maintained in plastic containers filled with soil and bark and fed on pieces of fresh potatoes and carrots. Adult non-breeding animals (body length 8.5-12 mm) at four different stages of the moulting cycle (early premoult, mid premoult, late premoult and intramoult) were used for the experiments. The animals in early and mid premoult were used 6 days and 2 weeks after the moult of the anterior part of the body, respectively. Well-developed sternal CaCO₃ deposits defined the late premoult stage. For intramoult (the period between posterior and anterior moult) animals, specimens with partly degraded CaCO₃deposits were selected.

Animals were dissected in nominally Ca²⁺-free (no calcium added)physiological saline containing 248 mmol l^-1 NaCl, 8 mmol l^-1 KCl, 10 mmol l^-1 MgCl₂, 5 mmol l^-1 glucose and 10 mmol l^-1 Tris, pH 7.4. Anterior and posterior sternal epithelia were dissected, and fatty tissue was carefully removed if necessary. Epithelia of animals in late premoult or intramoult already carried the first layers of unmineralised cuticle. Clean sternal epithelial cell layers were transferred onto nickel grids (200 mesh, thin bar,Plano Corporation) with the apical side facing away from the grid. The specimen and grid were then mounted in a perfusion chamber with the apical side facing up and with ready access of the solution to the basal side of the epithelium. A coverslip was placed on top of the perfusion chamber leaving only a narrow gap above the mounted specimen(Fig. 1A,B). The chamber was then placed on the stage of a light microscope (Zeiss, Axiophot) equipped with polarisation filters. Whenever possible, anterior and posterior sternal epithelia were analysed simultaneously.

The principle of the calcium oxalate assay for measurement of relative Ca²⁺ uptake rates into the Ca²⁺-sequestering SER was described by Walz and Baumann(1989). In permeabilised cells, oxalate moves from a loading medium into the SER by an unknown mechanism. In the presence of ATP, the SERCA pumps Ca²⁺ into the lumen of the SER. When the oxalate and Ca²⁺ concentrations exceed the solubility product, birefringent calcium oxalate precipitates form within the SER lumen, and crystal growth can be monitored in a polarisation microscope as long as Ca²⁺ is transported into the lumen of the endoplasmic reticulum. After a calcium oxalate loading experiment, the birefringent calcium oxalate crystals appear bright against a dark background(Fig. 2).

For calcium oxalate loading experiments, the sternal epithelial cells were first permeabilised with 20 μg ml^-1 saponin in 2 mmol l^-1 K₂EGTA, 125 mmol l^-1 KCl, 5 mmol l^-1 MgCl₂, 5 mmol l^-1 Na₂ATP, 20 mmol l^-1 Hepes, pH 7.0 (adjusted with KOH), for 20 min. After permeabilisation, the tissue was incubated under constant stirring in standard loading medium containing 1 mmol l^-1 K₂EGTA, 125 mmol l^-1 KCl, 5 mmol l^-1 MgCl₂, 5 mmol l^-1 Na₂ATP, 25 mmol l^-1 potassium oxalate, 4 mmol l^-1 CaEGTA, 2.5 μg ml^-1 oligomycin, 5 μmol l^-1 carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 20 mmol l^-1 Hepes, pH 7.0 (adjusted with KOH). Oligomycin and CCCP were added to the loading medium to prevent calcium oxalate forming in the mitochondria. The Ca²⁺ activity (a_Ca) of the loading medium (0.68 μmol l^-1) was measured using Ca²⁺-sensitive mini-electrodes (ETH129) prepared as described previously (Ziegler and Scholz,1997) and calibrated using the solutions of Tsien and Rink(1980).

To measure the Ca²⁺-dependency of calcium oxalate formation, we varied the a_Ca of the loading medium between 0.15 and 2.05μmol l^-1 by changing the ratio of CaEGTA to K₂EGTA,keeping the total EGTA concentration constant at 5 mmol l^-1. ATP-dependency was demonstrated by omitting Na₂ATP from the loading medium. The effects of cyclopiazonic acid (CPA) (1 μmol l^-1),ryanodine (10 μmol l^-1 and 0.5 mmol l^-1) and caffeine(25 and 50 mmol l^-1) were investigated by adding the reagents to the loading medium at various values of a_ca. For experiments with inositol trisphosphate (InsP₃) (3 and 5 μmol l^-1), we reduced the MgCl₂ concentration of the loading medium to 2 mmol l^-1 and repeated the experiments at various values of a_Ca.

All chemicals were purchased from Merck Corporation except Na₂ATP, saponin, CPA, CCCP and oligomycin, which were obtained from Sigma Corporation, and EGTA, which was obtained from Fluka Chemika Corporation.

A CCD camera (Visitron, Spot) was used to take sequences of grey-scale images with a 20×/0.50 objective (Zeiss) at 1 or 2 min intervals. We used TINA software (Raytest) for the digital analysis of transmitted light through the polariser and analyser (aligned in the crossed position). Mean grey-scale values in areas of 1.25×10³ μm² were quantified for each image and are presented as the intensity change from that of the first image (Δintensity units). Linear regression was used to calculate the relative amount of calcium oxalate formed from each series of images. Rates are given as intensity units min^-1, and values are presented as means ± S.E.M. One-way analysis of variance (ANOVA)followed by the Tukey—Kramer multiple-comparison test was used for statistical analysis. Calculations were performed using GraphPad Prism 3.0 software.

Sternal epithelia were prepared, permeabilised and incubated in loading medium as described above. After loading the epithelial cells with calcium oxalate for 50 min, single sternites were high-pressure frozen at 2.3×10⁸ N m^-2 (Leica, EMHPF) and freeze-substituted in acetone containing 1 % H₂O and 1 %OsO₄ (P. Walther and A. Ziegler, unpublished data). Freeze-substitution was performed over a 22 h period using a custom-built computer-controlled device with the temperature rising exponentially from -90 to 0 °C. After washing the specimens with pure acetone at room temperature(22 °C), the samples were embedded in Epon resin. Ultrathin sections (20 nm) were cut on a Leica Ultracut microtome with the sections floating on glycerol to prevent loss of water-soluble calcium oxalate precipitates. The sections were viewed unstained in an energy-filtering transmission electron microscope (Zeiss CEM 902) at 80 kV using a 30 μm diameter objective aperture. Electron energy-loss imaging (ESI) micrographs were taken below and above the specific element energy loss edge, L_2,3 (346 eV) of calcium, at ΔE=340±5 eV andΔ E=360±5 eV, where E is the energy of the electrons. Electron energy-loss (EEL) spectra were recorded in serial mode with a scintillator-PMT detector over a range of ΔE from 300 to 400 eV using a 60 μm objective aperture, a 100 μm spectrometer entrance aperture and an energy resolution of 5 eV.

Calcium oxalate formation in sternal epithelial cells shows a biphasic time course (Fig. 3A). After a lag phase of approximately 15 min, the signal rises first slowly and then linearly for up to 1 h. After perfusion with ATP-free loading medium, the increase in optical signal was blocked immediately(Fig. 3B); it resumed after exposure to standard loading medium containing 5 mmol l^-1 ATP(N=7). Exposure to 1 μmol l^-1 CPA, a specific blocker of the SERCA, caused an immediate inhibition of calcium oxalate formation(Fig. 3C). The block by CPA was not affected by changing a_Ca to 0.38 μmol l^-1 (N=2), 0.68 μmol l^-1 (N=7) and 1.3 μmol l^-1 (N=2). InsP₃ (N=7),ryanodine (N=5) and caffeine (N=4) had no effect on the rate of calcium oxalate formation. The rate of calcium oxalate formation depended on a_Ca within the range 0.15-2.05 μmol l^-1(Fig. 3D), which shows the relative Ca²⁺ uptake rate to have a half-maximum at 0.4 μmol l^-1, indicating that active Ca²⁺ uptake into the SER was stimulated at physiological Ca²⁺ concentrations. The Hill coefficient was 1.8.

Transmission electron microscopy confirmed that the precipitates were formed in the sternal epithelial cells(Fig. 4). Although the cells were permeabilised and incubated in loading medium for a relatively long time,most specimens showed good structural preservation. The oxalate crystals appeared either as large elongated structures(Fig. 4A) or as small needle-like crystals (Fig. 4B). Electron-dense, calcium-containing precipitates were confined to the cytoplasm and were surrounded by smooth membranes(Fig. 4C,D). It is possible that the smaller crystals resulted from vesiculation of the membranous structures in some cells as a result of the permeabilisation and calcium oxalate loading procedures. Calcium oxalate appeared only occasionally in mitochondria (Fig. 4A,B). Electron energy-loss spectroscopic images of precipitates produced bright signals when the energy loss was switched from 320 to 360 eV, indicating the presence of calcium (Fig. 5A,B). Electron energy-loss spectroscopy of precipitates confirmed this result because of the large signal at the Ca_L2,3 edge at 346 eV (Fig. 5C).

Ca²⁺ uptake rates in sternal epithelial cells changed significantly during the moulting cycle(Fig. 6). Calcium oxalate formation increased from undetectable rates in early premoult to considerable rates in mid premoult. A significant increase in calcium oxalate formation occurred between mid premoult (0.045±0.027 intensity units min^-1) and late premoult (0.23±0.016 intensity units min^-1, P<0.001) and between mid premoult and intramoult(0.19±0.03 intensity units min^-1, P<0.01) in the anterior sternal epithelium. No significant differences were observed between uptake rates in the anterior and posterior sternal epithelia during the late premoult and intramoult stages. In the posterior sternal epithelium of the early premoult and mid premoult stages, we obtained no conclusive results since we could not separate the cuticle from the epithelium. In these stages,birefingent structures formed within the mineralised cuticle, probably as a result of crystallisation of amorphous CaCO₃. Generally, a slight and insignificant (P>0.05) decrease in uptake rate was measured between late premoult and intramoult. During intramoult, large birefringent crystals appeared in the new cuticle of most of the posterior sternal epithelium, again probably as a result of crystallisation of amorphous CaCO₃ within the partly calcified cuticle. These specimens were omitted from the analysis.

The calcium oxalate method is a valuable tool for identifying and characterising Ca²⁺-sequestering organelles in situ. It meets all the requirements, such as specificity and linearity, to provide a good approach for monitoring the kinetics of Ca²⁺ uptake into the SER (Walz, 1982). A comprehensive discussion of this and related methods is given in the review by Walz and Baumann (1989). Here,we use this method to compare SERCA activity in the sternal integument of the terrestrial isopod Porcellio scaber among four different moulting stages. For the first time, we report a correlation between the increase in SERCA-dependent Ca²⁺ uptake into the SER and the moult-related increase in epithelial Ca²⁺ transport in Crustacea.

The dependency of calcium oxalate formation on ATP in the sternal epithelial cells of P. scaber and its almost total block by cyclopiazonic acid (CPA) indicate a SERCA-mediated sequestration of cytoplasmic Ca²⁺ into membranous compartments. This is supported by the dependency on the Ca²⁺ concentration in the loading medium,with the half-maximal uptake rate at a_Ca being 0.4 μmol l^-1. This value is similar to the submicromolar values of calcium oxalate formation found in SER cisternae of photoreceptors in insects(Baumann and Walz, 1989; Walz, 1982) and crayfish(Frixione and Ruiz, 1988). Electron microscopy, electron energy-loss imaging and electron energy-loss spectroscopy verified that the calcium-containing precipitates formed within the epithelial cells of P. scaber during a calcium oxalate loading experiment are formed in smooth membranous cisternae, most probably the smooth endoplasmic reticulum.

Within cells, Ca²⁺ functions as a ubiquitous second messenger regulating a vast variety of physiological processes. Ca²⁺ signals are mediated by an influx of extracellular Ca²⁺ across Ca²⁺ channels and/or by release of Ca²⁺ from the SER(Berridge, 1993). The SERCA replenishes Ca²⁺ stores by re-uptake of Ca²⁺ into the SER and restores low cytosolic free Ca²⁺ concentrations. In Ca²⁺-transporting epithelia, in which cytoplasmic Ca²⁺loads are high, regulation of cytosolic Ca²⁺ concentrations by the SERCA may be of particular importance. Recently, Simkiss(1996) reviewed the conflict between bulk cytosolic transport of Ca²⁺ in epithelial Ca²⁺ transport, its function as a second messenger and the toxicity of sustained high Ca²⁺ concentrations. Simple diffusion of free Ca²⁺ through the cytosol is impeded by its relatively slow diffusion rate, and increasing the Ca²⁺ gradient from one side of the epithelial cell to the other would result in high and toxic Ca²⁺ concentrations. Therefore, mechanisms including facilitated diffusion by Ca²⁺-binding proteins(Feher et al., 1989) and compartmentalisation (Simkiss,1996) have been proposed for transcellular Ca²⁺transit.

A recent electron-probe X-ray-microanalysis (EPMA) of freeze-dried cryosections of the sternal integument of shock-frozen P. scaberrevealed high concentrations of in situ total (bound plus free)calcium, [Ca]_t, of between 4.5 and 5.7 mmol kg^-1 dry mass, suggesting the presence of high concentrations of Ca²⁺-binding proteins (Ziegler,2002). However, comparison of [Ca]_t in the sternal epithelium of P. scaber between the early premoult, late premoult and intramoult stages indicates that the concentration of Ca²⁺-binding proteins does not change throughout the moulting cycle, arguing against a direct role of cytosolic Ca²⁺-binding proteins in epithelial Ca²⁺ transit. In contrast, the EPMA study demonstrated an in situ increase in the number of areas with high [Ca]_t (15-50 mmoll^-1 kg^-1 dry mass) between early premoult and intramoult resulting from the contribution of Ca²⁺ `hot spots' to the analysed area (Ziegler,2002). The highest values of approximately 50 mmol kg<sup>-1</sup>dry mass are similar to those measured in the SER of vertebrate(Jorgensen et al., 1988; Somlyo and Walz, 1985) and invertebrate (Baumann et al.,1991) cells. Comparison of the rates of calcium oxalate formation reported here indicates that the SERCA activity increases from undetectable values in the non-Ca<sup>2+</sup>-transporting early premoult stage to measurable values in the mid premoult stage, and increases further by a factor of up to five between mid premoult and the Ca<sup>2+</sup>-transporting late premoult and intramoult stages. This suggests that, in the ASE and the PSE of P. scaber, the SER plays a direct role in epithelial Ca<sup>2+</sup>transit and supports the proposal that the Ca<sup>2+</sup> `hot spots'revealed by EPMA represent SER cisternae. A role for the SER in epithelial Ca²⁺ transit was recently suggested in rat dental ameloblasts, in which SERCA activity and SER Ca²⁺-binding proteins are upregulated during the calcification process (Franklin et al., 2001; Hubbard,1996).

It is of interest that, in rat dental enameloblasts, the cytoplasmic 28 kDa Ca²⁺-binding protein calbindin is expressed in high concentrations,although the temporal expression pattern is not consistent with a primary role in Ca²⁺ transport (Hubbard,1996). This situation seems to be similar to that in the sternal epithelium of P. scaber, in which EPMA also suggests a high, but invariable, concentration of Ca²⁺-binding proteins in the cytosol. Ameloblasts, like the sternal epithelium of P. scaber, are involved in mineralisation processes that require massive transport of Ca²⁺in a very short time. Ca²⁺ flux rates through those epithelia are expected to be much higher than in the kidney and intestine. It seems possible that organellar routes evolved in mineralising tissues since high Ca²⁺ transit rates generally exceed the capacity of a cytosolic route.

Simkiss (1996) suggested that the loading and discharge of membranous compartments would lead to a vectorial translocation of Ca²⁺. Another possibility would be that Ca²⁺ diffuses through the lumen of the SER, possibly facilitated by low-affinity Ca²⁺-binding proteins. Alternatively, the SER could function as a Ca²⁺ buffer to prevent the formation of high cytosolic Ca²⁺ concentrations during SER-independent Ca²⁺ transit. It is important to note that a route through the SER would be in conflict with the SER's role in Ca²⁺ signalling. This conflict could be avoided if functions related to Ca²⁺ signalling and epithelial Ca²⁺ transport were regulated via different SERCA isoforms, possibly in different SER subcompartments. Recent investigations have revealed at least four different SERCA isoforms in crayfish tissues (Zhang et al.,2000) and two isoforms in whole brine shrimps(Escalante and Sastre,1993).

At this stage of the investigation, other routes for Ca²⁺transit, such as vesicular transport or co-secretion of Ca²⁺,cannot be ruled out for the sternal epithelium of P. scaber. In fact,electron microscopy, electron energy-loss spectroscopy and electron-probe X-ray-microanalysis demonstrated secretion of calcium-, phosphorus- and nitrogen-containing granules at the lateral plasma membranes of the ASE during resorption of the sternal CaCO₃ deposits during intramoult(Glötzner and Ziegler,2000; Ziegler, 1996, 2002), suggesting co-secretion of protein and Ca²⁺. A contribution of mitochondria to Ca²⁺ storage and/or transport during epithelial Ca²⁺transit has also been discussed for several crustacean Ca²⁺-transporting epithelia(Rogers and Wheatly, 1997; Ueno, 1980). However,electron-probe X-ray-microanalysis of ASE cells of P. scaberdemonstrated a decrease in the total mitochondrial calcium concentration rather than an increase between early premoult and late premoult and between early premoult and intramoult (Ziegler,2002), excluding the possibility that mitochondria could store or transport Ca²⁺ during Ca²⁺ transit.

As a working hypothesis, we propose that in Porcellio scaber the SER actively contributes to Ca²⁺ transit through the ASE and PSE cells during the formation and resorption of the CaCO₃ deposits. Future investigations should attempt to develop methods for the direct monitoring of epithelial Ca²⁺ transport and the use of pharmacological tools to analyse the role of the SER in epithelial Ca²⁺ transport.

We thank Dr Tom Carefoot for critically reading the manuscript and Oliver Schmid for his help in optimising the calcium oxalate assay. This work was supported by the Deutsche Forschungsgemeinschaft Zi 368/3-3.