Pflugers Arch - Eur J Physiol (2002) 445:40–50
DOI 10.1007/s00424-002-0912-z
ORIGINAL ARTICLE
Brian Faurskov · Henning F. Bjerregaard
Evidence for cadmium mobilization of intracellular calcium through
a divalent cation receptor in renal distal epithelial A6 cells
Received: 14 May 2002 /Accepted: 12 July 2002 /Published online: 6 September 2002
Springer-Verlag 2002
Abstract The effect of Cd
2+
on intracellular Ca
2+
homeostasis was examined in renal epithelial A6 cells
loaded with Fura-2. Cd
2+
(10 M to 1 mM) produced a
transient spike in cytosolic Ca
2+
in a dose-dependent
manner. The phospholipase C inhibitor U73122 and the
cation receptor agonist, neomycin, both diminish Cd
2+
-
evoked increase in intracellular Ca
2+
([DCa
2+
]
Cd
). Further,
thapsigargin, an inhibitor of intracellular Ca
2+
-ATPases,
significantly reduced [DCa
2+
]
Cd
. Extending these obser-
vations, inositol-3-phosphate (IP
3
) binding studies
showed that the resting level of intracellular IP
3
under-
went a 1.45-fold increase when exposed to Cd
2+
.
Furthermore, we found that the Cd
2+
-related heavy
metals, Zn
2+
and Ni
2+
, were even more potent inducers
of Ca
2+
mobilization and IP
3
generation than Cd
2+
. It can
be concluded that Cd
2+
, and possibly Zn
2+
and Ni
2+
, may
act as agonists of a cation-sensing receptor (CSR)
belonging to G-protein receptors capable of mediating
IP
3
release of Ca
2+
from intracellular stores. The CSR
receptor in A6 epithelia could not be stimulated with
neomycin or Gd
3+
, suggesting that the receptor is different
from the calcium-sensing receptor.
Keywords Cadmium · Calcium-sensing receptor ·
Cellular calcium · Inositol-3-phosphate · Fura-2 · Heavy
metals · Short-circuit current
Introduction
The kidney is believed to be the most critical organ
affected by cadmium toxicity. Often the mechanisms and
effects of inorganic cadmium (Cd
2+
) are focussed on
epithelial cells originating from the renal proximal tubule
since this nephron segment reabsorbs the bulk of ultra
filtered Cd
2+
(for review see [20]). However, evidence for
distal tubule toxicity suggests that cadmium may also act
at this nephron site [16].
To elucidate the effect of cadmium on epithelial cells
from the distal part of the kidney, A6 cells were used,
since this cell model exhibits the morphological and
functional properties of the mammalian distal epithelium
[30]. A6 cells respond to various hormones and intracel-
lular messengers, and exhibit Na
+
and Cl
–
transport
similar to what is observed in vivo [30, 35, 45]. In
previous studies we found that Cd
2+
administered to the
basolateral surface of renal epithelia A6 cells induced a
prompt and transient increase in short-circuit current (I
sc
),
which was independent of Na
+
absorption [13], but
exclusively involved Cl
–
secretion [14]. Furthermore,
Ca
2+
mobilization was apparently crucial for these events.
Cellular calcium homoeostasis and calcium-mediated
functions are being increasingly recognized as sensitive
and critical targets for the action of toxic metals such as
Cd
2+
[4, 33, 44]. Cd
2+
and other metals are known to bind
to and interact with receptor proteins on the cell surface,
with ion channel proteins, or with intracellular proteins
controlling Ca
2+
homoeostasis.
Recent studies have provided evidence that the distal
part of the kidney express polyvalent sensing receptors
responsive to cations such as Ca
2+
,Mg
2+
,Gd
3+
and
neomycin [1, 2, 11, 29]. These receptors belong to the
superfamily of G-protein-coupled receptors, which can be
activated by increased level of extracellular Ca
2+
, leading
to release of Ca
2+
from intracellular stores due to
activation of phospholipase C (PLC) and accumulation
of insitol-3-phosphate (IP
3
) (for review see [10]). There-
fore, the kidney is able to respond directly to alterations in
extracellular Ca
2+
with the resultant modulation of
mineral ion transport, i.e. elevations of Ca
2+
o
lead to
reduced reabsorption of Ca
2+
(and Mg
2+
) and hence
increased Ca
2+
/Mg
2+
excretion in the urine [19]. In order
to extend our previous observations we decided to
examine whether Cd
2+
was able to activate a cation-
B. Faurskov
Novozymes A/S, Kalundborg, Denmark
H.F. Bjerregaard (
)
)
Department of Life Sciences and Chemistry, Roskilde University,
P.O. Box 260, 4000 Roskilde, Denmark
e-mail: hfb@ruc.dk
Tel.: +45-46-742623
Fax: +45-46-743011