Nephrol Dial Transplant (1996) 11: 1532-1537
Nephrology
Dialysis
Transplantation
Original Article
Actions of endothelin-1 on calcium homeostasis in Madin-Darby
canine kidney tubule cells
N. A. Parkinson, A. F. James and B. M. Hendry
Renal Group, Department of Medicine, Kings College School of Medicine & Dentistry, London, UK
Abstract
Background. In both ischaemic and nephrotoxic
models, renal failure is associated with increased
endothelin-1 (ET-1) and cell calcium overload, and
ET receptor antagonists are protective. Vascular and
tubular actions of endothelins appear to be involved.
This study examines the actions of ET-1 on intracellular Ca ([Ca2+]s) in the tubule model cell line MDCK
(Madin-Darby canine kidney).
Methods. Single-cell [Ca2+]; was measured using fura-2
and actions of ET-1 were compared with thapsigargin,
which empties IP3-sensitive intracellular Ca stores.
Results. Mean resting [Ca2+]; was 84 nM (s.e.m. 6, n =
87). 1 uM thapsigargin and 100 nM ET-1 each caused
a transient increase in [Ca2+]j by 696 nM (s.e.m. 160,
n = 9) and 727 nM (s.e.m. 121, « = 5) respectively. After
1 uM thapsigargin, 100 nM ET-1 had no effect on
[Ca2+]j. Oscillations in [Ca2+]; were frequently observed
following 100 nM ET-1. In Ca2+-free extracellular
solution, mean resting [Ca2+]j was reduced by 37 nM
(s.e.m. 5, «=11) and the mean transient increase in
[Ca2+]j in response to ET-1 was 419 nM (s.e.m. 97,
n = 5). Inhibition of the plasma membrane Ca-ATPase
with La 3+ halved the rate of [Ca2+]j removal from the
cytoplasm following ET-1. The PKC inhibitor, chelerythrine (1 uM), reduced the ET-1 induced increase in
[Ca2+]; to 349 nM (s.e.m. 97, n = 5) and also reduced
the rate of removal of [Ca2+];. Ligand binding studies
demonstrated ETA receptor expression in MDCK cells
sensitive to ET-1.
Conclusions. ET-1 releases Ca 2+ from IP3-sensitive
stores in MDCK cells as well as stimulating extracellular Ca 2+ entry leading to oscillations of [Ca2+]j. Ca2+
responses to ET-1 are potentiated by PKC; the plasma
membrane Ca-ATPase contributes to removal of Ca2+
from the cytoplasm.
Introduction
The endothelins are a family of potent vasoconstrictor
peptides which are also capable of stimulating cell
proliferation and modulating epithelial transport and
appear to play an important role in renal microcirculation and tubular function [1], Endothelin-1 (ET-1)
has been found to be distributed throughout the renal
cortex, medulla, and papilla, in vascular endothelial
cells and non-vascular cells including epithelial cells
[2,3]. At least two different ET receptors have been
identified (ETAR and ETBR) with different intrarenal
distributions [4, 5].The nominally distal tubule cell line
from Madin-Darby canine kidney (MDCK) can synthesize ET-1, secrete ET-1, and express ET-1 receptors [6-8].
There is an increasing body of evidence that the
endothelins play a role both in the pathogenesis of
acute renal failure and in the progression of chronic
renal damage [9, 10]. Renal failure is associated with
an increase in plasma ET-1 concentration. Animal
models of acute and chronic renal failure also show
increased renal ET-1 production [11,12]. ET-1 is
known to have a variety of effects on kidney function.
Following systemic infusion of ET-1 in rats and dogs,
an increased renal vascular resistance is seen, resulting
in a reduced blood flow through the renal microcirculation and subsequent decreased glomerular nitration
rate [1]. ET is also associated with the inhibition of
renin secretion, inhibition of vasopressin-stimulated
water permeability in inner medullary collecting duct
cells, and inhibition of the Na+/K+-ATPase in rabbit
medullary collecting ducts [13-15]. Many of these
effects have been proposed to be dependent on altered
intracellular free calcium concentration ([Ca2+];).
Cellular calcium overload is important in the pathoof both acute and chronic renal damage in
genesis
Key words: calcium; endothelin-1; epithelial cells;
animal
models.
Stimulation of rat isolated medullary
fura-2; MDCK; protein kinase C
interstitial cells and porcine isolated inner medullary
collecting duct cells with ET-1 causes an increase in
[Ca2+];, dependent on calcium influx and intracellular
Correspondence and offprint requests to: BM Hendry, Renal Group, calcium release [3,16]. ET receptor antagonists are
Department of Medicine, Kings College School of Medicine & protective in various animal models of renal failure.
Dentistry, Bessemer Road, London SE5 9PJ, UK.
For example, in the rat model of ischaemic acute renal
B 1996 European Dialysis and Transplant Association-European Renal Association
N. A. Parkinson et al.
failure, the ET-receptor antagonist BQ-123, prevented
tubular cell mitochondrial Ca2+ accumulation and
reduced postischaemic tubular degeneration [17].
This paper presents a study of the physiological
balance of Ca2 + within MDCK cells, and of the effects
of ET-1 on intracellular Ca 2+ homeostasis.
Thapsigargin has also been used in this work to define
the importance of intracellular Ca2 + stores sensitive to
inositol 1,4,5-trisphosphate (IP3) [18].
Subjects and methods
Materials and solutions
MDCK cells, obtained from the European Collection of
Animal Cell Cultures (ECACC), were grown to near confluence in DMEM-Hams F12 (1:1), with 10% fetal calf serum,
100 U/ml streptomycin and 100 ug/ml penicillin on glass
coverslips. Fura-2AM (the acetylmethoxy ester of fura-2)
was supplied by Molecular probes (Cambridge Bioscience,
Cambridge, UK). Thapsigargin, ET-1, ET-3 and BQ-123
were supplied by Sigma (Poole, Dorset, UK). [125I]ET-1
was obtained from Amersham International, Amersham,
UK. Chelerythrine, ionomycin and pluronic F12 was supplied
by Calbiochem-Novabiochem (UK) Ltd. Other chemicals
were of AnalaR grade.
The physiological saline solution (PSS) used consisted of
127 mM NaCl, 5.9 mM KC1, 1.2 mM MgCl2, 14 mM glucose, 10.6 mM N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid] (HEPES), buffered to pH 7.4 with 1M
NaOH. PSS was nominally Ca-free and an identical solution
containing 1.0 mM Ca was created by addition of CaCl2 and
designated PSS^ A Ca2+-free solution (designated PSS0) was
made by addition of 2mM ethylene glycol-bis(P-aminoethyl
ether) N,N,N',N',-tetraacetic acid (EGTA) to PSS.
1533
ET receptor studies
Competitive inhibition binding studies were performed with
a clone of MDCK cells (known to exhibit Ca2+ responses
to ET-1) grown to confluence in 24-well culture plates.
Culture medium was aspirated and the cells washed three
times with serum-free DMEM: Hams F12 and then incubated
for 2 h at 37°C with 25 pM [U5I]ET-1 (925 Bq/well) in the
presence or absence of various amounts of unlabelled ET-1,
ET-3, or the ETA receptor antagonist BQ-123 in a total
volume of 0.5 ml of DMEM:Hams F12 containing 0.1%
fetal calf serum. Cells were then washed three times and
harvested in IN NaOH. Bound radioactivity was counted
using a gamma camera. Non-specific binding was measured
in the presence of a saturating amount of ET-1 (1 uM) and
was subtracted from the total binding to obtain specific
ET-1 binding.
Results
In the presence of 1 mM extracellular Ca 2 + , the mean
stable resting [Ca 2+ ], in single MDCK cells was 84 nM
(s.e.m. 6, n = 87). The addition of 10-100 nM ET-1
caused a transient rise in [Ca 2+ ];. This was similar in
form to the transient increase caused by 1.0 uM thapsigargin. Figure 1 shows examples of these responses
and illustrates that the response to ET-1 was abolished
by prior treatment with thapsigargin. This was a
reproducible finding. In contrast, prior treatment with
ET-1 reduced but did not abolish the thapsigargin
response. After a single application of thapsigargin
and washout, further application of thapsigargin elicited no response.
Incubation of MDCK cells in PSS 0 for 1 min reduced
mean resting [Ca 2+ ]; by a factor of 2; on continued
incubation in PSS 0 [Ca 2+ ] ; then remained approxiIntracellular Ca2+ Measurements.
mately stable for 3 min. After 4 min incubation in
2+
(s.e.m. 27,
MDCK cells were grown on coverslips and remained in situ PSS0, the mean basal [Ca ]j was 47 nM
2+
n
=
l).
The
mean
peak
responses
of
[Ca
]j
to 100 nM
for fura-2AM loading and measurements of intracellular Ca.
After growth to subconfluence the cells were washed twice ET-1 and 1.0 uM thapsigargin are summarized in
in PSS. Cells were then incubated for 30 min with 5 uM Figure 2 where data obtained in PSS! and after 1 min
Fura-2AM in 0.1% DMSO, 0.01 mg/ml pluronic, 0.1 mg/ml in PSS0 are compared. In PSS, the mean increase
albumin, in PSS at 37°C. The cells were then washed three caused by 100 nM ET-1 was 727 nM (s.e.m. 121, « =
times in PSS! and the coverslip mounted in a temperature- 5) compared with 696 nM (s.e.m. 160, n = 9) for thapsicontrolled cell perfusion chamber (Intracel Ltd) on an gargin. In PSSi 10 nM ET-1 produced a mean increase
inverted Nikon Diaphot-TMD microscope and maintained in [Ca2+]i of 149 nM (s.e.m. 85, n = 5).
at 37°C. Epifluorescence measurements were made using a
After 1 min in PSS 0 the mean Ca responses were
Deltascan W1009 (Photon Technology International Inc.,
2+
USA) high-intensity dual-wavelength light source using reduced. In PSS0, 100 nM ET-1 increased [Ca ] ; by
excitation wavelengths at 345 and 375 nm. Emitted light at 419 nM (s.e.m. 97, « = 5) and the thapsigargin response
510 nm from a single cell was quantified by photomultiplier was 146 nM (s.e.m. 37, n = 6, P<0.05 compared to
tube followed by A/D conversion at a sampling rate of 1 Hz. PSS^. After a further 3 min incubation in PSS 0 , the
The data were used to give an on-line measurement of R, transient increase in [Ca 2+ ]; produced by luM thapsithe ratio of emission amplitudes at the two excitation wave- gargin was reduced to 39 nM (s.e.m. 20, n = 4 , data
lengths (345/375).
not shown). Figure 2 also shows mean data obtained
Calibration of [Ca2+]j was achieved according to the in the presence of 500 uM La 3 + in PSS,; the mean
method of Grynkiewicz and colleagues [19]. To estimate the response to ET-1 was slightly reduced while the
removal rate of [Ca2+]i from the cytosol after an ET-1 response to thapsigargin was increased but became
induced transient peak, linear regression analysis was perof cells with La 3 + for 1 min
formed over the initial part of the declining phase. Statistical very variable. Incubation
2+
comparisons were made using two-tailed unpaired Student t did not alter basal [Ca ] ; .
tests and regarded as significantly different when /"<0.05.
The response to 100 nM ET-1 was often not confined
Results are expressed as means ±s.e.m.
to a single spike of Ca 2 + , in 30-50% of experiments
1534
OUU
2000 -
600
(nM)
2+
[Ca ],
(nM)
400
ET-1
I
200
0
1 ill
w.o.
1000 -
w
100
200
Time (seconds)
B
4
r
0
PSSn
(MM)
Tg
w.o.
ET-1
w.o.
I \ \
100
200
300
400
Time (seconds)
500
Fig. 1. Intracellular Ca responses to 100 nM ET-1 and 1.0 |iM
thapsigargin (Tg) in single MDCK cells. A, actions of ET-1 followed
by thapsigargin; B, effects of application in the reverse order, w.o.,
washout. The extracellular solution was PSSj.
PSSX
+
La 3+
Fig. 2.Summary of the effects of 1 uM thapsigargin (Tg) and 100 nM
ET-1 on cytosolic free calcium concentration ([Ca2+],). The first
solid bar of each group indicates the mean basal [Ca2+], immediately
prior to the addition of either thapsigargin or ET-1. The middle
(stippled) bar of each group indicates the mean peak [Ca2+],. after
application of 1.0 uM Thap. The last (shaded) bar of each group
indicates the mean peak [Ca2+],. after application of 100 nM ET-1.
Data obtained in PSSi are compared with data obtained after 1 min
in PSS0, and results obtained in PSS, 1 min after addition of 500 uM
La3 + . Error bars are s.e.m. *Significant difference from the result
obtained in PSS^ (P<0.05).
the basal [Ca2+], increased to >700nM (data not
shown).
The factors underlying the removal of Ca from the
cytoplasm were investigated by estimating the initial
rate of fall of [Ca2+], after the peak stimulated by ET-1
2+
oscillations in [Ca ] ; were elicited as illustrated in or thapsigargin. These results are summarized in
Figure 3. These oscillations occurred about once each Figure 6. In PSSx the mean rate of fall after either
—10 nM/s. In the presence
90 s and slowly reduced in amplitude over several stimulus was approximately
3+
minutes. Oscillations were not seen in the absence of of 500 uM La the mean rate of fall after ET-1 was
extracellular Ca 2+ in PSS0. However, immediately after markedly reduced to —4.3 nM/s (s.e.m. 1.2, « = 5).
application of ET-1 in PSS0, oscillations could be Prior incubation for 1 min in 1.0 uM Chel had a
elicited by replacing the medium with PSSj as shown similar effect, reducing the rate to —4.973+nM/s (s.e.m.
1.3, « = 5). In the presence of 500 uM La the [Ca2+];
in Figure 3(B).
The role of protein kinase C (PKC) activation in response to thapsigargin was sustained and did not
severe disthe Ca 2+ responses to ET-1 was investigated by meas- show a significant falling phase, indicating
2+
urements in the presence of the PKC inhibitor cheler- ruption of the mechanisms of Ca removal from the
ythrine (Chel). The effects of 1 min incubation with cytoplasm.
1.0 uM Chel are illustrated in Figure 4. Basal [Ca2+]j
The competitive inhibition binding data were wellwas unaltered but the peak transient increase in fitted with a two-ligand, single receptor model which
response to 100 nM ET-1 was reduced (by a factor of gave estimates for dissociation constants (K d ) and Hill
2) to 349 nM (« = 5, P<0.05). Figure 5 illustrates that coefficients for ET-1, ET-3 and BQ-123 (data not
oscillations in [Ca 2+ ] ; could still be observed when shown). The fitted Kd values were ET-1, 175 pM; ET-3,
ET-1 was added in the presence of Chel. In two 40 nM; BQ-123, 12 nM. Hill coefficients were ET-1,
experiments after 10 min incubation in 1.0 uM Chel 1.8; ET-3, 0.9; BQ-123, 1.0. The selectivity for ET-1
1535
N. A. Parkinson et al.
A
1000
1.0
(nM)
800
(MM)
600
0.5
400
ET-1
200
0.0
200
400
600
800
1000
Basal
Time (seconds)
B
3.0
Fig. 4. The effects of incubating MDCK cells with 1 uM of PKC
inhibitor chelerythrine (chel) for 1 min. The left hand pair of bars
indicate that resting or basal [Ca 2+ ]j was not significantly altered.
The right hand pair indicate the mean peak [Ca 2+ ], after addition
of 100 nM ET-1. * Chelerythrine significantly reduced the response
to ET-1 (/ > <0.05). Error bars are s.e.m.
Calciumfree
2.5
2.0
Ratio
345nm/375nm
1.5
1.0
0.5
ET-|
W.O.
600 r
4
200
ET-1
400
400
600
800
1000
(nM)
Time (seconds)
Fig. 3. Examples of oscillatory Ca 2 + responses following 100 nM
ET-1. A, application of ET-1 in standard conditions (PSS,). B,
Application of ET-1 shortly after replacement of PSS, with Ca-free
solution (PSS 0 ), the solution is changed back to PSS, after the
initial spike in [Ca 2+ ], and oscillations are seen about 250 s later. Ca
calibration was not achieved in this latter experiment, the fluorescence ratio (345/375) is an estimate of changes in [Ca 2+ ]j.
200
Chel
200
400
600
Time (seconds)
over ET-3 and the activity of BQ-123 are consistent
with the expression of ET A receptors in these cells.
Discussion
Fig. 5. An example of [Ca 2+ ], in a single MDCK cell recorded
during addition of 1.0 uM PKC inhibitor chelerythrine (chel) and
(1 min later) 100 nM ET-1. Chelerythrine did not immediately alter
basal [Ca 2+ ],; the subsequent response to ET-1 was an initial spike
followed by oscillations.
The mean resting level of [Ca2 + ] ; of 84 nM is consistent
with other reports from MDCK cells [20]. Basal [Ca2+];
was strikingly sensitive to removal of extracellular
Ca2+ for 1 min indicating that cellular Ca 2+ is in
dynamic equilibrium with the extracellular Ca 2+ pool.
Intracellular Ca 2+ stores also appear to be rapidly
depleted by removal of extracellular Ca2 + . The thapsigargin response was markedly reduced by removal of
extracellular Ca 2+ for 1 min (Figure 2) and almost
completely abolished after 4 min; consistent with significant depletion of thapsigargin-sensitive intracellular
Ca2+ stores. The possibility that a component of the
thapsigargin response may involve Ca2+ entry stimulated by release of Ca 2+ stores cannot be excluded.
Preliminary observations of Mn 2+ quenching of the
intracellular fura-2 signal on application of thapsigargin in the presence of extracellular Mn 2+ suggested
the activation of a divalent cation pathway in the cell
membrane (data not shown). This is consistent with
Ca2+-induced Ca2+ entry in MDCK cells but further
work is needed to characterize this.
The intracellular Ca 2+ stores released by thapsigargin have been characterized as IP3-sensitive endoplasmic reticulum [18,21]. The complete insensitivity.
of the MDCK cells to ET-1 after thapsigargin demonstrates that intact IP3-sensitive stores are essential for
the initiation of a Ca2+ response to ET-1. The data
are consistent with reports from many other cell sys-
1536
nM/sec
Tg
ET-1 Tg
ET-1 ET-1
La3+ La3+
Chel
2+
Fig. 6. The mean rates of initial Ca
removal from the cytosol
following 1 uM thapsigargin (Tg) or 100 nM ET-1 in PSS,. The first
two bars represent the mean rates under control conditions. The
third and fourth bars represent the mean rate after a preincubation
with 500 uM La 3+ for 1 min. f This rate was difficult to determine
accurately as the Ca response was sustained and the rate of fall was
near zero. The right hand bar represents the mean rate after a 1 min
incubation with 1 uM chelerythrine (chel). *Significant difference
from control data obtained with ET-1 (P<0.05). Error bars are
s.e.m.
tems in which the actions of ET-1 are mediated via
G-protein coupled receptors and activation of phospholipase C (PLC) generating IP3 leading to release
of intracellular Ca 2+ stores [1].
Oscillations of cell Ca 2+ were observed after application of ET-1 but not after thapsigargin. They depended
on the presence of external Ca 2+ , but whether they
arose from fluctuations in Ca 2+ entry or oscillatory
release of Ca 2+ from stores cannot be defined. These
oscillations may have considerable functional significance and have been associated with proliferative
responses [22].
The relative maintenance of a Ca 2+ response to
ET-1 in the absence of extracellular Ca2 + in comparison with the more marked reduction in thapsigargin
response was unexpected (Figure 2). The simplest
explanation is that the full Ca 2+ response to ET-1
involves intracellular Ca 2+ stores distinct from those
released by thapsigargin. These stores would be less
sensitive to depletion following removal of extracellular
Ca2 + . Release of this second store of Ca 2+ may depend
both on the initial release of Ca 2+ from the thapsigargin-sensitive store and on other effects of PLC activation by ET-1, namely the activation of PKC [1].
The data obtained using the inhibitor of PKC,
chelerythrine, support a dual role for PKC in this cell
model. A component of the ET-1 induced rise in Ca2 +
is PKC-dependent and this supports the idea that
activation of Ca2+ channels by IP3 does not fully
explain the ET-1-induced intracellular Ca 2+ response.
However, it also appears that the maintenance of
normal low resting free intracellular Ca 2+ depends on
basal PKC activity. This may arise from a requirement
for maintained PKC-dependent phosphorylation of the
plasma membrane Ca2+-ATPase for normal resting
rates of Ca2+ efflux. The delay between application of
the PKC inhibitor and the rise in intracellular Ca2+
may be explained by a significant half-life of the
phosphorylated protein or by the capacity of intracellular Ca 2+ buffering systems. The rate of fall of Ca2+
after the transient rise elicited by ET-1 was reduced by
PKC inhibition, consistent with a role for PKC in the
maintenance of resting plasma membrane Ca2+
pump activity.
At the concentration employed in this work, La3 +
ions have non-specific effects and block both the
plasma membrane Ca2+-ATPase and Ca 2+ entry channels. Accordingly data obtained in the presence of
La 3+ must be interpreted with caution. The reduced
mean ET-1-stimulated rise in intracellular Ca 2+ in the
presence of La3+ (Figure 2) is consistent with a contribution of the ET-1-induced initial Ca 2+ response arising from extracellular Ca 2+ entry. In support of this,
the effects of removing extracellular Ca2 + on the initial
Ca 2+ response to ET-1 were similar to those of La3 +
(Figure 2). The actions of La 3+ on the rate of fall of
Ca 2+ after an initial rise were more clear-cut
(Figure 6). These results suggest that the two major
routes for removal of cytoplasmic Ca2 + in the conditions employed here are the plasma membrane Ca2+
pump and the thapsigargin-sensitive Ca2+-ATPase of
endoplasmic reticulum.
• These experiments illustrate the dynamic nature of
Ca 2+ homeostasis in MDCK cells and emphasise the
roles of IP3-sensitive intracellular Ca2+-stores and
PKC modulation in the control of Ca 2+ movements.
The results support the view that ET-1 can directly
modulate tubule cell function and may be a significant
factor in creating intracellular calcium overload. These
may be important mechanisms in the generation of
acute interstitial renal damage and in the progression
of established renal parenchymal injury.
Acknowledgements. The assistance of Dr C. Streather is appreciated
for helping with initial cell culture work. This work was supported
by the National Kidney Research Fund and the Joint Research
Council of Kings College School of Medicine & Dentistry and Kings
College Hospital, London.
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Received for publication: 20.10.95
Accepted in revised form: 2.4.96