Analysis of Ca2+ uptake into the smooth endoplasmic reticulum of permeabilised sternal epithelial cells during the moulting cycle of the terrestrial isopod Porcellio scaber
Zentrale Einrichtung Elektronenmikroskopie, Universität Ulm, 89069 Ulm, Germany
* Author for correspondence (e-mail: andreas.ziegler{at}medizin.uni-ulm.de )
Accepted 18 April 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: calcium oxalate, Crustacea, epithelial Ca2+ transport, biomineralisation, smooth endoplasmic reticulum Ca2+-ATPase, SERCA, sequestration, Ca2+-ATPase, isopod, Porcellio scaber
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-1
kg-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 situ
calcium 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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
Calcium oxalate assay
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 (aCa) 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 aCa of the loading medium between 0.15 and 2.05 µmol 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 aca. 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 aCa.
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 20x/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 1.25x103 µm2 were
quantified for each image and are presented as the intensity change from that
of the first image (intensity 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 TukeyKramer multiple-comparison test was used for
statistical analysis. Calculations were performed using GraphPad Prism 3.0
software.
Electron energy-loss spectroscopy and electron energy-loss
imaging
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
2.3x108 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, L2,3 (346 eV) of
calcium, at E=340±5 eV and
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
E from 300 to
400 eV using a 60 µm objective aperture, a 100 µm spectrometer entrance
aperture and an energy resolution of 5 eV.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 CaL2,3 edge at 346 eV (Fig. 5C).
|
|
Comparison of Ca2+ uptake rate in different moulting
stages
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.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 aCa 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. scaber
revealed 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-1
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-Ca2+-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 Ca2+-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 Ca2+
transit and supports the proposal that the Ca2+ `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. scaber
demonstrated 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.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahearn, G. A. and Franco, P. (1990). Sodium and
calcium share the electrogenic 2Na+-1H+ antiporter in
crustacean antennal glands. Am. J. Physiol.
259,F758
-F767.
Ahearn, G. A. and Franco, P. (1993). Ca2+ transport pathways in brush-border membrane vesicles of crustacean antennal glands. Am. J. Physiol. 264,1206 -1213.
Ahearn, G. A. and Zhuang, Z. (1996). Cellular mechanisms of calcium transport in crustaceans. Physiol. Zool. 69,383 -402.
Baumann, O. and Walz, B. (1989). Calcium- and inositol polyphosphate-sensitivity of the calcium-sequestering endoplasmic reticulum in the photoreceptor cells of the honeybee drone. J. Comp. Physiol. A 165,627 -636.
Baumann, O., Walz, B., Somlyo, A. V. and Somlyo, A. P. (1991). Electron probe microanalysis of calcium release and magnesium uptake by endoplasmic reticulum in bee photoreceptors. Proc. Natl. Acad. Sci. USA 88,741 -744.[Abstract]
Berridge, M. J. (1993). Inositol trisphosphate and calcium signalling. Nature 361,315 -325.[Medline]
Drobne, D. and trus, J. (1996). Moult
frequency of the isopod Porcellio scaber, as a measure of
zinc-contaminated food. Env. Toxicol. Chem.
15,126
-130.
Escalante, R. and Sastre, L. (1993). Similar
alternative splicing events generate two sarcoplasmic or endoplasmic reticulum
Ca-ATPase isoforms in the crustacean Artemia franciscana and in
vertebrates. J. Biol. Chem.
268,14090
-14095.
Feher, J. J., Fullmer, C. S. and Fritzsch, G. K. (1989). Comparison of the enhanced steady-state diffusion of calcium by calbindin-D9K and calmodulin: possible importance in intestinal calcium absorption. Cell Calcium 10,189 -203.[Medline]
Flik, G., Verbost, P. M. and Atsma, W. (1994).
Calcium transport in gill plasma membranes of the crab Carcinus
maenas: evidence for carriers driven by ATP and a Na+
gradient. J. Exp. Biol.
195,109
-122.
Franklin, I., Winz, R. and Hubbard, M. (2001). Endoplasmic reticulum Ca-ATPase pump is up-regulated in calcium-transporting dental enamel cells: a non-housekeeping role for SERCA2b. Biochem. J. 358,217 -224.[Medline]
Frixione, E. and Ruiz, L. (1988). Calcium uptake by smooth endoplasmic reticulum of peeled retinal photoreceptors of the crayfish. J. Comp. Physiol. A 162,91 -100.
Glötzner, J. and Ziegler, A. (2000). Morphometric analysis of the plasma membranes in the calcium transporting sternal epithelium of the terrestrial isopods Ligia oceanica, Ligidium hypnorum and Porcellio scaber. Arthropod Struct. Dev. 29,241 -257.
Greenaway, P. (1985). Calcium balance and moulting in the Crustacea. Biol. Rev. 60,425 -454.
Greenaway, P., Dillaman, R. M. and Roer, R. D. (1995). Quercitin-dependent ATPase activity in the hypodermal tissue of Callinectes sapidus, during the moult cycle. Comp. Biochem. Physiol. 111A,303 -312.
Hubbard, M. J. (1996). Abundant calcium homeostasis machinery in rat dental enamel cells. Up-regulation of calcium store proteins during enamel mineralization implicates the endoplasmic reticulum in calcium transcytosis. Eur. J. Biochem. 239,611 -623.[Abstract]
Jorgensen, A. O., Broderick, R., Somlyo, A. P. and Somlyo, A. V. (1988). Two structurally distinct calcium storage sites in rat cardiac sarcoplasmic reticulum: an electron microprobe analysis study. Circ. Res. 63,1060 -1069.[Abstract]
Messner, B. (1965). Ein morphologisch-histologischer Beitrag zur Häutung von Porcellio scaber Latr. und Oniscus asellus I. (Isopoda Terrestria). Crustaceana 9,285 -301.
Nemere, I. (1992). Vesicular calcium transport in chick intestine. J. Nutr. 122,657 -661.[Medline]
Neufeld, D. S. and Cameron, J. N. (1993).
Transepithelial movement of calcium in crustaceans. J. Exp.
Biol. 184,1
-16.
Roer, R. D. (1980). Mechanisms of resorption and deposition of calcium in the carapace of the crab Carcinus maenas.J. Exp. Biol. 88,205 -218.
Rogers, J. V. and Wheatly, M. G. (1997). Accumulation of calcium in the antennal gland during the molting cycle of the freshwater crayfish Procambarus clarkii. Invert. Biol. 116,248 -254.
Simkiss, K. (1996). Calcium transport across calcium-regulated cells. Physiol. Zool. 69,343 -350.
Somlyo, A. P. and Walz, B. (1985). Elemental distribution in Rana pipiens retinal rods: quantitative electron probe analysis. J. Physiol., Lond. 358,183 -195.[Abstract]
Somlyo, A. V., Gonzalez-Serratos, H., Shuman, H., McClellan, G. and Somlyo, A. P. (1981). Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron-probe study. J. Cell Biol. 90,577 -594.[Abstract]
Steel, C. G. H. (1993). Storage and translocation of integumentary calcium during the moult cycle of the terrestrial isopod Oniscus asellus (L.). Can. J. Zool. 71,4 -10.
Tsien, R. Y. and Rink, T. J. (1980). Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium. Biochim. Biophys. Acta 599,623 -638.[Medline]
Ueno, M. (1980). Calcium transport in crayfish gastrolith disc: morphology of gastrolith disc and ultrahistochemical demonstration of calcium. J. Exp. Zool. 213,161 -171.
Walz, B. (1982). Ca2+-sequestering smooth endoplasmic reticulum in an invertebrate photoreceptor. II. Its properties as revealed by microphotometric measurements. J. Cell Biol. 93,849 -859.[Abstract]
Walz, B. and Baumann, O. (1989). Calcium-sequestering cell organelles: in situ localization, morphological and functional characterization. Progr. Histochem. Cytochem. 20,1 -47.
Wheatly, M. G. (1997). Crustacean models for studying calcium transport: the journey from whole organisms to molecular mechanisms. J. Mar. Biol. 77,107 -125.
Zhang, Z., Chen, D. and Wheatly, M. G. (2000). Cloning and characterization of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) from crayfish axial muscle. J. Exp. Biol. 203,1 -13.[Abstract]
Ziegler, A. (1994). Ultrastructure and electron spectroscopic diffraction analysis of the sternal calcium deposits of Porcellio scaber Latr. (Isopoda, Crustacea). J. Struct. Biol. 112,110 -116.
Ziegler, A. (1996). Ultrastructural evidence for transepithelial calcium transport in the anterior sternal epithelium of the terrestrial isopod Porcellio scaber (Crustacea) during the formation and resorption of CaCO3 deposits. Cell Tissue Res. 284,459 -466.[Medline]
Ziegler, A. (1997). Immunocytochemical
localization of Na+,K+-ATPase in the
calcium-transporting sternal epithelium of the terrestrial isopod
Porcellio scaber Latr. (Crustacea). J. Histochem.
Cytochem. 45,437
-446.
Ziegler, A. (2002). X-ray microprobe analysis of epithelial calcium transport. Cell Calcium (in press).
Ziegler, A. and Scholz, F. H. E. (1997). The ionic hemolymph composition of the terrestrial isopod Porcellio scaber Latr. during molt. J. Comp. Physiol. B 167,536 -542.
Ziegler, A., Weihrauch, D. and Towle, D. W. (2001). Increased expression of the Ca2+-ATPase and the Na+/Ca2+-exchanger in the anterior sternal tissue of Porcellio scaber (Isopoda, Crustacea) during premolt. Zoology 104, Suppl. IV,67 .