Our understanding of calcium homeostasis during the crustacean moulting cycle derives from research on intermoult animals that has been extrapolated to other stages. In terms of transepithelial Ca(2+) flux, the more interesting stages are those surrounding ecdysis since crustaceans experience a sizeable negative calcium balance in immediate premoult and a significant positive calcium balance in immediate postmoult. These stages are elusive in the sense that larger species such as lobsters are rarely captured at this time, and smaller species such as blue crabs and crayfish are seldom synchronized in their moulting cycle. The reductionist approaches employed in cellular physiology, such as vesicle techniques, employ pooling of fresh tissues from many organisms. Examination of the elusive moulting stages requires more sensitive approaches that can utilize tissue from an individual crustacean to characterize Ca(2+) pumps (Sarco/Endoplasmic Reticulum Ca(2+)-ATPase, SERCA; Plasma Membrane Ca(2+)-ATPase, PMCA) and the Na(+)/Ca(2+) eXchanger (NCX). An emerging subcellular approach described in this paper is to use flow cytometry as a technique to monitor Ca(2+) uptake into Fluo-3-loaded membrane vesicles. This paper illustrates the utility of this technique for measuring ATP-dependent Ca(2+) uptake into hepatopancreatic basolateral membrane vesicles. Obstacles to progress in molecular studies have not been limited by synchronization of moulting since tissue can be snap-frozen and collected from many animals over time. Here, the problem has been the lack of specific antibodies that hybridize with the Ca(2+) transporters of interest so that they can be localized within epithelia. In this paper, we introduce polyclonal antibodies raised in rabbits against crayfish SERCA, PMCA and NCX. Immunocytochemistry of SERCA in muscle, PMCA in antennal gland and NCX in heart confirms the specificity of the antibodies.

REFERENCES

Cameron
J. N.
,
Wood
C. M.
(
1985
).
Apparent H+excretion and CO2dynamics accompanying carapace mineralization in the blue crab (Callinectes sapidus) following moulting.
J. Exp. Biol
114
,
181
–.
Flik
G.
,
Verbost
P. M.
,
Atsma
W.
,
Lucu
C.
(
1994
).
Calcium transport in gill plasma membranes of the crab Carcinusmaenas: evidence for carriers driven by ATP and a Na+gradient.
J. Exp. Biol
195
,
109
–.
Greenaway
P.
,
Dillaman
R. M.
,
Roer
R. D.
(
1995
).
Quercetin-dependent ATPase activity in the hypodermal tissue of Callinectes 966 sapidus during the moult cycle.
Comp. Biochem. Physiol
111
,
303
–.
Ishida
Y.
,
Chused
T. M.
(
1988
).
Heterogeneity of lymphocyte calcium metabolism is caused by T cell-specific calcium sensitive potassium channel and sensitivity of the calcium ATPase pump to membrane potential.
J. Exp. Med
168
,
839
–.
Kimura
C.
,
Ahearn
G. A.
,
Busquets-Turner
L.
,
Haley
S. R.
,
Nagao
C.
,
De Couet
G.
(
1994
).
Immunolocalization of an antigen associated with the invertebrate electrogenic 2Na+/1H+antiporter.
J. Exp. Biol
189
,
85
–.
Klein
M. J.
,
Ahearn
G. A.
(
1999
).
Calcium transport mechanisms of crustacean hepatopancreatic mitochondria.
J. Exp. Zool
283
,
147
–.
Nicoll
D. A.
,
Longini
S.
,
Philipson
K. D.
(
1990
).
Molecular cloning and functional expression of the cardiac sarcolemmal Na+—Ca2+exchanger.
Science
250
,
562
–.
Schoenmakers
T.
,
Visser
G. J.
,
Flik
G.
,
Theuvenet
A. P. R.
(
1992
).
Chelator: an improved method for computing metal ion concentrations in physiological solutions.
Biotechniques
12
,
870
–.
Sullivan
G. V.
,
Fryer
J. N.
,
Perry
S. F.
(
1995
).
Immunolocalization of proton pumps (H+-ATPase) in pavement cells of rainbow trout gill.
J. Exp. Biol
198
,
2619
–.
Telford
W. G.
,
Miller
R. A.
(
1996
).
Detection of plasma membrane Ca2+ATPase activity in mouse T lymphocytes by flow cytometry using fluo-3-loaded vesicles.
Cytometry
24
,
243
–.
Wheatly
M. G.
(
1999
).
Calcium homeostasis in Crustacea: the evolving role of branchial, renal, digestive and hypodermal epithelia.
J. Exp. Zool
283
,
620
–.
Wheatly
M. G.
,
Pence
R. C.
,
Weil
J. R.
(
1999
).
ATP-dependent calcium uptake into basolateral vesicles from transporting epithelia of intermoult crayfish.
Am. J. Physiol
276
,
566
–.
Wheatly
M. G.
,
Weil
J. R.
,
Douglas
P. B.
(
1998
).
Isolation, visualization, characterization and osmotic reactivity of crayfish BLMV.
Am. J. Physiol
274
,
725
–.
Zhang
Z.
,
Chen
D.
,
Wheatly
M. G.
(
2000
).
Cloning and characterization of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) from crayfish axial muscle.
J. Exp. Biol
203
,
3411
–.
Zhuang
Z.
,
Ahearn
G. A.
(
1996
).
Ca2+transport processes of lobster hepatopancreas brush border membrane vesicles.
J. Exp. Biol
199
,
1195
–.
Zhuang
Z.
,
Ahearn
G. A.
(
1998
).
Energized Ca2+transport by hepatopancreatic basolateral plasma membranes of Homarus americanus.
J. Exp. Biol
201
,
211
–.
Zhuang
Z.
,
Linser
P. J.
,
Harvey
W. R.
(
1999
).
Antibody to H+V-ATPase subunit E colocalizes with portasomes in alkaline larval midgut of a freshwater mosquito (Aedesaegypti L.).
J. Exp. Biol
202
,
2449
–.
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