0022-3565/97/2833-1177$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 283:1177–1184, 1997
Vol. 283, No. 3
Printed in U.S.A.
Calpains Mediate Calcium and Chloride Influx During the Late
Phase of Cell Injury2
SHAYLA L. WATERS,1,3 SATINDER S. SARANG,3 KEVIN K. W. WANG4 and RICK G. SCHNELLMANN
Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas
Accepted for publication August 22, 1997
Cell death is generally thought to occur through one of two
pathways: necrosis (oncosis) or apoptosis (Majno and Joris,
1995). Necrosis, oncosis or necrotic cell death is the form of
cell death normally associated with inflammation and organ
failure. In necrotic cell death, the organelles swell, cell volume increases and the cell ruptures/lyses, releasing its contents and triggering inflammation. In general, most toxicants that produce organ dysfunction are thought to produce
cell death through necrosis.
The role of Ca11 in oncosis has received much attention
over the past three decades and remains controversial (Choi,
1995; Harman and Maxwell, 1995; Trump and Berezesky,
Received for publication April 29, 1997.
1
S. L. W. was supported by an American Heart Association, Arkansas
Affiliate, Predoctoral Fellowship.
2
Portions of this work were presented at the XIIth International Congress
of Pharmacology, Montreal, Canada, on July 24 –26, 1994; 6th Congress on
Nephrotoxicity and Nephrocarcinogenicity in Noordwijkhout, Netherlands, on
September 22–24, 1994; 28th Annual Meeting of the American Society of
Nephrology, San Diego, CA, on November 5– 8, 1995; and 36th Annual Meeting
of the Society of Toxicology, Cincinnati, OH, on March 9 –13, 1997.
3
S. L. Waters and S. S. Sarang contributed equally.
4
Present address: Department of Neuroscience Therapeutics, Parke-Davis
Pharmaceutical Research Division, Division of Warner-Lambert Company,
2800 Plymouth Rd., Ann Arbor, MI 48105.
inhibitor 1, PD150606 and nifedipine. Finally, nifedipine, calpain
inhibitor 1, PD150606 and the Cl2 channel inhibitors [5-nitro2-(3-phenylpropylamino)-benzoate, niflumic acid, diphenylamine-2-carboxylate, and indanyloxyacetic acid] blocked
the increase in Cl2 influx that occurs during the late phase of
cell injury and triggers terminal cell swelling and death. These
data suggest that Ca11 and calpains play a common and
critical role in renal proximal tubule cell death produced by
diverse agents. In addition, calpain activation appears to play a
dual role during the late phase of cell injury. Initial calpain
activation elicits extracellular Ca11 influx through a nifedipinesensitive pathway, resulting in calpain translocation to the
membrane and in turn Cl2 influx.
1995). In a 1979 landmark report, Schanne et al. suggested
that an increase in cytosolic free Ca11 (Ca11f) represented
the final common pathway in cell death/lysis. However, a
number of investigators have questioned this hypothesis. For
example, Weinberg et al. (1991) and Jacobs et al. (1991) used
rabbit RPT subjected to anoxia or exposed to mitochondrial
inhibitors and the Ca11-sensitive fluorescent dye fura 2 and
observed an increase in Ca11f immediately before cell death/
lysis. Similar results were reported by Lemasters et al. (1987)
using hepatocytes and chemical hypoxia. These authors concluded that an increase in Ca11f occurred in concert with the
loss of cell viability and thus was not an obligatory step in cell
death. In contrast, Kribben et al. (1994), using fura 2 and rat
RPT subjected to hypoxia, demonstrated that intracellular
Ca11f levels increased significantly before cell death. In addition, Takano et al. (1985), Wetzels et al. (1993) and Rose et
al. (1994) showed that decreasing the extracellular Ca11
concentration reduced the release of LDH from rabbit RPT
subjected to anoxia and rat RPT subjected to hypoxia. Therefore, the exact role that Ca11 plays during cell injury and
death is still not clear.
In support of the hypothesis that Ca11 plays an important
role in cell injury, calcium channel blockers have been re-
ABBREVIATIONS: RPT, renal proximal tubules; LDH, lactate dehydrogenase; PD150606, 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid;
SLLVY-AMC, N-succinyl-Leu-Leu-Val-Tyr-AMC; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoate; DPC, diphenylamine-2-carboxylate; IAA-94,
indanyloxyacetic acid.
1177
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ABSTRACT
The role of Ca11 in cell death is controversial. Extracellular
Ca11 influx and calpain activation occurred during the late
phase of renal proximal tubule cell injury produced by the
mitochondrial inhibitor antimycin A. Chelation of intracellular
Ca11, extracellular Ca11, the calcium channel blocker nifedipine, calpain inhibitor 1 and the dissimilar calpain inhibitor
PD150606 blocked antimycin A-induced influx of extracellular
Ca11 and cell death. The calcium channel blocker verapamil
was ineffective. Calpain inhibitor 1 and PD150606 were cytoprotective also against tetrafluoroethyl-L-cysteine-, bromohydroquinone-, oxidant (t-butylhydroperoxide)- and calcium ionophore (ionomycin)-induced cell death. Extracellular Ca11 influx
was associated with the translocation of calpain activity from
the cytosol to the membrane and was prevented by calpain
1178
Waters et al.
Fig. 1. The effect of calpain inhibitor 2 (CI2) (top; Schnellmann, 1997)
and PD150606 (PD) (bottom) on TFEC-, BHQ- and TBHP-induced LDH
release. CI2 (1 mM) and PD (100 mM) were added 30 min before the
toxicants, and LDH release was determined 3 hr subsequent to toxicant
addition. CON, control. Bars, mean 6 S.E.M. (n 5 3–5). Bars with
different letters, significantly different (P , .05).
after antimycin A exposure and that Cl2 influx occurs after a
lag period during the late phase of cell injury through a
niflumic acid-, 5-nitro-2-(3-phenylpropylamino)benzoic acid-,
diphenyl-2-carboxylate- and indanyloxyacetic acid-sensitive
channel (Miller and Schnellmann, 1993, 1995; Waters and
Schnellmann, 1996). The goal of this study was to obtain a
more complete understanding of the events that occur during
the late phase of cell death by exploring the roles and interactions of calpains, Ca11 influx and Cl2 influx using rabbit
RPT suspensions as a model. Specifically, we addressed (1)
whether Ca11 influx occurs and plays a role in cell death, (2)
the mechanism and pathway by which Ca11 influx occurs,
(3) whether calpains are activated and play a role in cell
death, (4) the subcellular localization of calpain activity during cell injury and (5) whether calpains play a role in the
extracellular Cl2 influx that occurs during the late phase of
cell injury.
Materials and Methods
Reagents. Tetrafluoroethyl-L-cysteine was a gift from Dr. Edward A. Lock (Zeneca, Cheshire, UK). SLLVY-AMC was purchased
from Bachem Bioscience (Philadelphia, PA). Ionomycin and
EGTA-AM were obtained from Calbiochem (San Diego, CA). Calpain
inhibitors 1 and 2 were obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN). Bromohydroquinone was purchased
from ICN Pharmaceuticals (Plainview, NY). 36Cl2(Na1),
45
Ca11(2Cl2) and [14C]dextran were obtained from Dupont NEN
(Boston, MA). NPPB and IAA-94 were obtained from Research Biochemicals (Natick, MA). Antimycin A, 3,4-dichloroisocoumarin, dim-
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ported to be protective against various forms of cell injury.
For example, verapamil or nifedipine decreased cell death in
rat RPT subjected to hypoxia and anoxia and rabbit RPT
subjected to anoxia (Almeida et al., 1992; Rose et al., 1993;
Wetzels et al., 1992). Furthermore, McCarty and O’Neil
(1991) reported that rabbit RPT contain both a “base-line”
verapamil-sensitive Ca11 entry pathway and a nifedipinesensitive Ca11 entry pathway that is activated during regulatory volume decreases. These studies provide additional
evidence that extracellular Ca11 influx may play a role in
renal cell injury.
It is generally hypothesized that if Ca11f plays a role in
cell death, it is the consequence of a supraphysiological
and/or prolonged increase in Ca11f that activates degradative enzymes, including proteases and phospholipases (Choi,
1995; Harman and Maxwell, 1995; Trump and Berezesky,
1995). Investigators have suggested that nonlysosomal calcium-activated cysteine proteases, calpains (E.C., 3.4.22.17),
are activated and contribute to anoxia- or toxicant-induced
cell death (Bronk and Gores, 1993; Croall and Demartino,
1991; Edelstein et al., 1995; Nicotera et al., 1986; Saido et al.,
1994; Wang and Yuen, 1994). These results are primarily
based on the inhibition of cell death by calpain inhibitors and
to a limited extent on the measurement of calpain activity or
calpain-mediated protein degradation. For example, Edelstein et al. (1996) reported an increase in calpain activity in
rat RPT subjected to hypoxia, and Bronk and Gores (1993)
demonstrated an increase in calpain-like protease activity in
rat hepatocytes subjected to anoxia. Both studies showed
that inhibition of calpains resulted in cytoprotection. We
demonstrated that calpain inhibitor 2 was cytoprotective to
RPT exposed to anoxia and a diverse group of toxicants that
included an alkylating quinone (bromohydroquinone), an oxidant (t-butylhydroperoxide) and a toxicant that forms a reactive electrophile (tetrafluoroethyl-L-cysteine) (fig. 1;
Schnellmann et al., 1994). These results suggest that calpains play a critical role in diverse forms of cell injury;
however, progress in this area has been limited due to difficulties in measuring calpain activity, the lack of specific
calpain substrates and inhibitors and the identification of
endogenous intracellular substrates (Saito et al., 1993;
Sasaki et al., 1984; Wang and Yuen, 1994).
The majority of calpain inhibitors, including calpain inhibitor 1 and calpain inhibitor 2, are modified peptides that bind
to the active site of calpain (Wang and Yuen, 1994). The
disadvantage of these compounds is diminished selectivity, a
direct result of the similarity of the active site among the
different classes of cysteine proteases (Wang and Yuen,
1994). Recently, Wang et al. (1996a) identified a novel class of
calpain inhibitors, including the compound PD150606. As
opposed to binding to the active site of the protease,
PD150606 inhibits calpains by binding to the calcium-binding domain of the enzyme. Because calcium-binding domains
are not located in other proteases, PD150606 selectively inhibits the calpain enzyme.
The early events in anoxia- and toxicant-induced cell death
have been well characterized in numerous models and include inhibition of cellular respiration followed by the loss of
intracellular ATP, K1 efflux and Na1 influx. Those events
that occur during the late phase of cell injury have not been
completely elucidated; however, we have shown that Cl2
influx does not occur passively with the initial Na1 influx
Vol. 283
1997
1179
Cl2 and Ca11 influxes. Cl2 and Ca11 influxes were determined
by adding a tracer amount of 36Cl2(Na1) or 45Ca11(2Cl2) to RPT
suspensions 0 or 15 min after antimycin A addition (Miller and
Schnellmann, 1993, 1995; Waters and Schnellmann, 1996). At 15
min later, aliquots were removed, and RPT was separated from the
surrounding buffer by rapid centrifugation through a layer of dibutylphthalate:dioctylphthalate (2:1). RPT 36Cl2 and 45Ca11 contents
were determined by resuspending the pellets in Triton X-100 solubilization buffer (100 mM Tris, 150 mM NaCl and 0.05% Triton
X-100 at pH 7.5), and aliquots were taken for liquid scintillation
spectrometry and protein determination. Extracellular 36Cl2 and
45
Ca11 were corrected for using the extracellular water marker
[14C]dextran. RPT protein concentration was determined using the
biuret method (Gornall et al., 1949).
Cell death. Cell death/lysis was assessed by measuring the release of LDH activity as described previously (Moran and Schnellmann, 1996).
Statistics. The data are presented as mean 6 S.E.M. RPT suspensions isolated from one rabbit represented a single experiment
(n 5 1). Data were analyzed by analysis of variance, and multiple
mean values were compared using Fisher’s protected LSD test with
a level of significance of P , .05.
Results
Previous studies have demonstrated that exposure of rabbit RPT suspensions to anoxia or mitochondrial inhibitors
increases Ca11f just before cell death/lysis (Jacobs et al.,
1991; Weinberg et al., 1991). To document that Ca11 influx
occurs during the late phase of cell injury in our model, a
tracer amount of 45Ca11 was added simultaneously with
antimycin A, and RPT 45Ca11 content determined. Ca11
influx did not increase above control values during the first
15 min of antimycin A exposure (fig. 2, top), a time frame
during which ATP levels are depleted and Na1 influx and K1
efflux occur. In contrast, when a tracer concentration of
45
Ca11 was added to RPT suspensions 15 min after antimycin A and RPT 45Ca11 content determined 15 min later, RPT
45
Ca11 content increased 3.5-fold compared with controls
(fig. 2, top). The calcium channel blocker nifedipine, but not
verapamil, and chelation of intracellular Ca11 with
EGTA-AM blocked the antimycin A-induced increase in RPT
45
Ca11 content (fig. 2, bottom).
To confirm that the influx of extracellular Ca11 is necessary for cell death in our model, the extracellular Ca11
chelator EGTA, nifedipine, verapamil or EGTA-AM was
added to RPT treated with antimycin A. Chelation of extracellular Ca11 or intracellular Ca11 with EGTA or EGTAAM, respectively, decreased LDH release (table 1). Likewise,
nifedipine inhibited antimycin A-induced LDH release in a
concentration-dependent manner and inhibited LDH release
when added 15 min after antimycin A (table 1). In contrast,
verapamil was not cytoprotective and potentiated LDH release at the highest concentration tested. These results show
that influx of extracellular Ca11 occurs in the late phase of
cell injury and plays a key role in RPT cell death and that the
Ca11 influx occurs through a nifedipine-sensitive pathway.
To determine whether calpains play a role in cell death, the
effects of two calpain inhibitors on antimycin A-induced cell
death were examined. A 30-min pretreatment with calpain
inhibitor 1 or 2 was equally effective in blocking LDH release
from RPT exposed to antimycin A (fig. 3, top). No differences
were noted between calpain inhibitors 1 and 2. The calpain
inhibitor PD150606 also inhibited antimycin A-induced LDH
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ethylsulfoxide, t-butyl hydroperoxide, niflumic acid, DPC, N-[N(L-3trans-ethoxycarbonyl-oxirane-2-carbonyl)-L-leucyl]-3-methylbutylamine and N-p-tosyl-L-lysine chloromethyl ketone were
purchased from Sigma Chemical (St. Louis, MO). The sources of the
remaining chemicals have been reported previously (Rodeheaver et
al., 1990). Stock solutions of all inhibitors were prepared daily in
dimethylsulfoxide. All glassware was silanized and autoclaved before use. All media and buffers were sterilized by filtering before use.
Preparation and incubation of RPT. Rabbit RPT were isolated
and purified as described by Rodeheaver et al. (1990) and suspended
in an incubation buffer containing 1 mM alanine, 4 mM dextrose, 2
mM heptanoate, 4 mM lactate, 5 mM malate, 115 mM NaCl, 15 mM
NaHCO3, 5 mM KCl, 2 mM NaH2PO4, 1 mM MgSO4, 1 mM CaCl2
and 10 mM HEPES (pH 7.4, 295 mOsm/kg). RPT suspensions (1 mg
of cellular protein/ml) were incubated at 37°C in an orbital shaking
water bath (180 rpm) under 95% air/5% CO2 (40 ml/min flow rate).
All experiments contained a 15-min preincubation period with no
experimental manipulations. EGTA (2 mM, pH 7.4), EGTA-AM (100
mM), nifedipine (10–100 mM), verapamil (10–100 mM), NPPB (100
mM), IAA-94 (1 mM), DPC (100 mM), niflumic acid (100 mM) or
diluent (#1% dimethylsulfoxide) was added immediately before antimycin A (1 mM), and the incubation continued for an additional 30
min. Aliquots of RPT suspensions were removed, and LDH release
determined. RPT suspensions were incubated for 30 min with the
protease inhibitors before calpain activity was determined. Calpain
inhibitor 1 or 2 (1 mM) or PD150606 (3–100 mM) was added 30 min
before the addition of antimycin A or the calcium ionophore ionomycin (5 mM).
In situ calpain assay. Calpain activity was determined in RPT
suspensions by measuring the release of the fluorescent product
7-amido-4-methyl coumarin (AMC) from the membrane permeant
calpain substrate SLLVY-AMC (Sasaki et al., 1984; Wang et al.,
1996b). Briefly, a 1-ml aliquot of RPT suspension was diluted with 3
ml of 37°C incubation buffer, and a 1.5-ml aliquot was placed in a
thermostatically controlled 37°C stirred cuvette in a Hitachi F-2000
spectrofluorometer. Calpain substrate (50 mM) was added, and fluorescence (360 nm excitation, 430 nm emission) was monitored every
minute. The increase in fluorescence was linear between 7 and 30
min, with calpain activity determined between 7 and 11 min.
Calpain activity in cytosolic and membrane-associated
fractions. This assay is based on the method of Edelstein et al.
(1995) with the following modifications. Briefly, an aliquot of RPT
was removed and centrifuged at 1000 3 g for 1 min, and the supernatant was aspirated. The pellet was resuspended in imidazole
buffer (63 mM imidazole, 10 mM 2-mercaptoethanol, 1 mM EDTA
and 10 mM EGTA, pH 7.3) and incubated in the presence of digitonin
(100 mM) for 10 min at 37°C. Digitonin permeabilizes the plasma
membrane releasing the cytosolic contents. Under these conditions,
LDH release is .94%. An aliquot is removed, the tubule pellet and
supernatant are separated by centrifugation for 1 min at 1000 3 g
and the pellet is resuspended in imidazole buffer. Total calpain
activity present in the supernatant and membrane-associated fraction was determined as follows: in Costar 24-well plates, 0.25 ml of
supernatant or pellet was preincubated in imidazole buffer in the
presence and absence of 3 mM CaCl2 for 5 min on an orbital shaker
placed in a 37°C incubator. The samples incubated in the presence of
CaCl2 were incubated in an imidazole-HCl buffer without EDTA and
EGTA. Total volume in each well was 1 ml. After the preincubation,
50 mM SLLVY-AMC was added, and fluorescence was determined at
10, 20 and 30 min after substrate addition in a CytoFluor 2350
Fluorescence Measurement System (Perseptive Biosystems, Bedford, MA; 380 nm excitation; 460 nm emission). An AMC standard
curve was included in each experiment, and calpain activity was
determined as the time-dependent difference between the calciumdependent fluorescence and the calcium-independent fluorescence.
Activity was normalized to cytosolic or membrane-associated protein
according to the method of Lowry et al. (1951).
Calpains Mediate Cell Injury
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Waters et al.
Vol. 283
TABLE 1
Effects of EGTA, EGTA-AM, nifedipine and verapamil on
antimycin A-induced LDH release
Nifedipine, verapamil and EGTA were added immediately before antimycin A or
15 min after antimycin A. EGTA-AM was added 10 min before antimycin A. LDH
release was determined 30 min after antimycin A addition. Nifedipine (100 mM),
verapamil (100 mM), EGTA (2 mM) or EGTA-AM (100 mM) alone had no effect on
LDH release.
Treatment
LDH release
%
8 6 2a
34 6 2b
16 6 2d
10 6 1a
24 6 1c
15 6 2d
16 6 4d
19 6 3d
34 6 3b
35 6 1b
43 6 4e
Values are mean 6 S.E.M. (n 5 3).
Values with different superscripts are significantly different from each other
(P , .05).
Fig. 2. Temporal aspects of extracellular Ca11 influx (top) and the effect
of nifedipine (NIFED), verapamil (VERAP), EGTA-AM, calpain inhibitor 1
(CI1) and PD150606 (PD) (bottom) on antimycin A (AA)-induced extracellular Ca11 influx. NIFED (100 mM) and VERAP (100 mM) were added
immediately before antimycin A. EGTA-AM (100 mM) was added 10 min
before antimycin A. CI1 (1 mM) and PD150606 (100 mM) were added 30
min before antimycin A. For the 15-min time points, 45Ca11 was added
simultaneously with antimycin A, and RPT 45Ca11 content was determined 15 min later. For the 30-min time points, 45Ca11 was added 15
min after antimycin A, and RPT 45Ca11 content was determined 15 min
later. Control (CON) RPT 45Ca11 content was 781 6 95 dpm/mg of
protein (bottom). Bars, mean 6 S.E.M. (n 5 3 or 4). Bars with different
letters, significantly different (P , .05).
release in a concentration-dependent manner (fig. 3, bottom).
In addition, PD150606 (100 mM) was cytoprotective against a
variety of toxicant-induced injuries, including t-butyl hydroperoxide, bromohydroquinone and tetrafluoroethyl-L-cysteine (fig. 1, bottom). These results, as well as those reported
previously (fig. 1, Schnellmann et al., 1994), demonstrate
that calpain inhibitors are cytoprotective against diverse toxicant insults, strongly suggesting that calpains play a common and critical role in RPT cell death.
To document that calpain inhibitor 1 and PD150606 block
calpain activity, calpain activity was measured in situ by
adding the cell permeant calpain substrate SLLVY-AMC to
RPT suspensions and monitoring the formation of the fluorescent product AMC over time. To test whether SLLVYAMC was a substrate for other proteases under these conditions, a series of cysteine, serine and acid protease inhibitors
were added to RPT, and calpain activity determined 30 min
later. Protease inhibitors were added at their maximal nontoxic concentration (data not shown). E64d, leupeptin and
pepstatin A had no effect on calpain activity, whereas 3,4dichloroisocourmarin and N-p-tosyl-L-lysine chloromethyl
Fig. 3. Effect of calpain inhibitor 1 (CI1) and 2 (CI2) (top) and PD150606
(PD) (bottom) on antimycin A (AA)-induced LDH release. CI1 (1 mM),
CI2 (1 mM) and PD150606 (3–100 mM) were added 30 min before
antimycin A. LDH release was determined 30 min after antimycin A
addition. CON, control. Bars, mean 6 S.E.M. (n 5 3 or 4). Bars with
different letters, significantly different (P , .05).
ketone decreased calpain activity by ;10% (table 2). In contrast, calpain inhibitor 1 and PD150606 decreased calpain
activity by ;62% and ;34%, respectively. These results suggest that SLLVY-AMC is hydrolyzed by calpains and other
proteases in this assay, that calpain inhibitor 1 may inhibit
calpains and other proteases and that PD150606 inhibits
calpain activity. It is unlikely that lysosomal cysteine pro-
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Control
Antimycin A (1 mM)
1EGTA 2 mM
1EGTA-AM 100 mM
1Nifedipine 10 mM
1Nifedipine 30 mM
1Nifedipine 100 mM
1Nifedipine 100 mM (added 15
min after antimycin A)
1Verapamil 10 mM
1Verapamil 30 mM
1Verapamil 100 mM
1997
Calpains Mediate Cell Injury
TABLE 2
Effects of serine, cysteine and aspartic acid protease inhibitors
and calpain inhibitor 1 on calpain activity measured in situ in
RPT suspensions
The protease inhibitors, calpain inhibitor 1 or PD150606 were added after a
15-min preincubation period, and calpain activity was determined 30 min later.
See the text for a detailed description of the calpain assay.
Treatment
Calpain activity
% of control
Control
E64d (0.05 mM)
Leupeptin (0.5 mM)
Pepstatin A (0.2 mM)
3,4-Dichloroisocoumarin (0.2 mM)
N-p-tosyl-L-lysine chloromethyl ketone (0.1 mM)
Calpain inhibitor 1 (1 mM)
PD150606 (100 mM)
100 6 0a
106 6 6a
98 6 4a
97 6 3a
89 6 5b
82 6 4b
38 6 4d
66 6 6c
teases are responsible for SLLVY-AMC hydrolysis because
the concentrations of E64d and leupeptin used inhibit lysosomal cysteine proteases by 98% and 76%, respectively, in
this model (Yang and Schnellmann, 1996).
To determine whether calpains play a role in extracellular
Ca11 influx, the effects of calpain inhibitor 1 and PD150606
on antimycin A-induced Ca11 influx were examined. Both
calpain inhibitor 1 and PD150606 completely inhibited the
Ca11 influx (fig. 2, bottom). To determine whether calpains
play a role after extracellular Ca11 influx, the effect of calpain inhibitor 1 and PD150606 on calcium ionophore (ionomycin)-induced cell death was examined. A 30-min pretreatment with calpain inhibitor 1 or PD150606 decreased
ionomycin-induced cell death (fig. 4). These results provide
evidence that calpains may play a dual role in RPT cell
injury, calpains may mediate Ca11 influx and also act subsequent to Ca11 influx.
The effect of antimycin A on calpain activity in cytosolic
and membrane-associated cell fractions of RPT was exam-
Fig. 4. Effect of calpain inhibitor 1 (CI1), PD150606 (PD) and the Cl2
channel inhibitors NPPB, niflumic acid, IAA-94 and DPC on ionomycin
(IONO)-induced LDH release. CI1 (1 mM) and PD (100 mM) were added
30 min before IONO (5 mM). NPPB (100 mM), niflumic acid, (100 mM),
IAA-94 (1 mM) and DPC (100 mM) were added 5 min before IONO. LDH
release was determined 30 min after IONO addition. CON, control.
Bars, mean 6 S.E.M. (n 5 3). Bars with different letters, significantly
different (P , .05).
ined. At both 0- and 15-min time points, cytosolic and membrane-associated calpain activities in control and antimycin
A-treated RPT were equivalent. However, at 30 min, antimycin A caused a 2-fold increase in membrane-associated calpain activity that was associated with a corresponding decrease in cytosolic activity (fig. 5). Total calpain activity was
equivalent in all samples (data not shown). Figure 6 illustrates that the addition of calpain inhibitor 1 results in an
87% and 84% decrease in both cytosolic and membraneassociated activity, respectively. Preincubation of RPT with
calpain inhibitor 1 ameliorated calpain translocation in the
presence of antimycin A. Similarly, PD150606 or inhibition of
extracellular Ca11 influx with nifedipine decreased antimycin A-induced calpain translocation (fig. 6). These studies
suggest that extracellular Ca11 influx mediates calpain
translocation in the late phase of RPT cell injury.
Cl2 influx also occurs in the late phase of cell injury and
triggers the terminal cell swelling and lysis (Miller and
Schnellmann, 1993, 1995; Waters and Schnellmann, 1996).
To determine the temporal relationships among Ca11 influx,
calpain translocation and Cl2 influx, the effect of nifedipine,
verapamil, calpain inhibitor 1 and PD150606 on antimycin
A-induced Cl2 influx was examined. Antimycin A increased
RPT 36Cl2 content by ;2.4-fold (fig. 7, top). Nifedipine, calpain inhibitor 1 and PD150606 blocked antimycin A-induced
Fig. 5. Time-dependent effects of antimycin A (AA) on membraneassociated (top) and cytosolic (bottom) calpain activity. RPT cell samples were taken at times 0, 15 and 30 min after AA addition. Calpain
activity was determined for each time point in both cytosolic and
membrane associated fractions. CON, control. Bars, mean 6 S.E.M.
(n 5 6). Bars with different letters, significantly different (P , .05).
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Values are mean 6 S.E.M. (n 5 3).
Values with different superscripts are significantly different from one another
(P , .05).
1181
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Waters et al.
Vol. 283
Cl2 influx, whereas verapamil did not. Because nifedipine,
calpain inhibitor 1 and PD150606 all inhibited the translocation of calpains to the membrane, these data suggest that
Ca11 influx results in calpain translocation to the membrane
and in turn Cl2 influx.
To determine whether the calcium ionophore-induced extracellular Cl2 influx was calpain mediated, the effect of
calpain inhibitor 1 and PD150606 on ionomycin-induced Cl2
influx was examined. Ionomycin increased RPT 36Cl2 content by ;4.1-fold (fig. 7, bottom). Calpain inhibition completely blocked ionomycin-induced Cl2 influx. We have previously shown that the Cl2 channel inhibitors NPPB,
niflumic acid, IAA-94 and DPC inhibit antimycin A-induced
cell death and Cl2 influx (Waters and Schnellmann, 1996).
To determine whether Cl2 channel inhibitors also block calcium ionophore-induced Cl2 influx and cell death, the effects
of NPPB, niflumic acid, IAA-94 and DPC on ionomycin-induced LDH release and Cl2 influx were examined. All four
Cl2 channel inhibitors ameliorated ionomycin-induced LDH
release (fig. 4) and Cl2 influx (fig. 7, bottom).
Discussion
11
The role of Ca
in cell death is controversial. Some studies have demonstrated increases in Ca11f levels before cell
death and/or that decreasing medium Ca11 levels ameliorates anoxia- or toxicant-induced extracellular Ca11 influx
and cell death (Almeida et al., 1992; Choi, 1995; Harman and
Maxwell, 1995; Rose et al., 1994; Takano et al., 1985; Trump
and Berezesky, 1995). Other studies have shown that Ca11f
does not increase early but late in the cell injury process just
before the loss of calcium-sensitive dyes or cell death, sug-
gesting that Ca11 does not play a key role in cell death
(Jacobs et al., 1991; Lemasters et al., 1987; Weinberg et al.,
1991). With rabbit RPT subjected to mitochondrial inhibition, a model in which Ca11f does not increase until just
before cell death (Jacobs et al., 1991; Weinberg et al., 1991),
we show that (1) extracellular Ca11 influx occurs during the
late phase of cell injury, (2) inhibition of extracellular Ca11
influx blocks cell death, (3) chelation of intracellular Ca11
blocks cell death and (4) inhibition of calpain activity blocks
cell death produced by diverse toxicants. Thus, influx of
extracellular Ca11 in the late phase of RPT cell injury does
indeed play a key role in cell death.
Because the influx of extracellular Ca11 is an important
event during RPT cell injury, additional studies were conducted to determine the pathway of Ca11 entry. Nifedipine
and verapamil are two dissimilar calcium channel blockers
(Vanhoutte, 1987). Nifedipine inhibited antimycin A-induced
Ca11 influx and LDH release in a concentration-dependent
manner, whereas verapamil was ineffective. Furthermore,
nifedipine added 15 min after antimycin A, a time point after
ATP depletion, Na1 influx and K1 efflux, was completely
cytoprotective. These data are consistent with our observation that Ca11 influx did not increase above control values
during the first 15 min after antimycin A addition. Collectively, these results show that extracellular Ca11 influx occurs in the late phase of cell injury through a nifedipinesensitive pathway. Although a complete calcium channel has
not been demonstrated in RPT (Yu, 1995), a nifedipine-sensitive Ca11 entry pathway has been reported previously in
rabbit RPT (McCarty and O’Neil, 1991). Further studies are
required to identify the Ca11 entry pathway observed during
cell injury.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017
Fig. 6. Effect of calpain inhibitor 1 (CI1), PD150606 (PD) and nifedipine
(NIF) on membrane-associated (top) and cytosolic (bottom) calpain
activity. CI1 (1 mM) and PD (100 mM) were added 30 min before
antimycin A (AA). NIF (100 mM) was added immediately before AA. RPT
cell samples were taken 30 min subsequent to antimycin A addition,
and calpain activity was determined in both cytosolic and membraneassociated fractions. CON, control. Bars, mean 6 S.E.M. (n 5 3). Bars
with different letters, significantly different (P , .05).
Fig. 7. Effect of nifedipine (NIFED), verapamil (VERAP), calpain inhibitor
1 (CI1) and PD150606 (PD) on antimycin A (AA)- (top) and ionomycin
(IONO) (bottom)-induced RPT 36Cl2 influx. In addition, the effects of
NPPB, IAA-94, niflumic acid and DPC on IONO-induced 36Cl2 influx are
illustrated. NIFED (100 mM) and VERAP (100 mM) were added immediately before antimycin A. CI1 (1 mM) and PD (100 mM) were added 30
min before antimycin A. 36Cl2 was added 15 min after antimycin A, and
RPT 36Cl2 content was determined 15 min later. See figure 4 for details
of Cl2 channel inhibitor addition and concentrations. Control RPT 36Cl2
content was 1203 6 257 dpm/mg of protein. Bars, mean 6 S.E.M. (n 5
5). Bars with different letters, significantly different (P , .05).
1997
1183
current study also provides evidence that calpains play a
dual role in cell death. Calpain inhibition with both calpain
inhibitor 1 and PD150606 not only blocked antimycin Ainduced extracellular Ca11 influx but also inhibited calciumionophore (ionomycin)-induced cell death. Furthermore, calpain inhibition with calpain inhibitor 1 or PD150606 and
inhibition of extracellular Ca11 influx with nifedipine
blocked calpain translocation to the membrane. Collectively,
these data suggest that calpains play a role both before and
subsequent to extracellular Ca11 influx. Thus, the mechanism of cytoprotection provided by calpain inhibitors probably involves both the inhibition of calpain-mediated extracellular Ca11 influx and extracellular Ca11 influx-mediated
calpain translocation.
We have shown previously that the Cl2 influx that occurs
during the late phase of RPT cell death/lysis is sensitive to
Cl2 channel inhibitors (Waters and Schnellmann, 1996). The
current study shows that nifedipine, calpain inhibitor 1 and
PD150606 also inhibit this Cl2 influx. Furthermore, calpain
inhibitor 1 and PD150606 blocked calcium ionophore (ionomycin)-induced Cl2 influx and cell death. The inhibition of
Cl2 influx with the Cl2 channel inhibitors DPC, NPPB,
IAA-94 and niflumic acid is consistent with previous results
observed with antimycin A (Waters and Schnellmann, 1996).
In conjunction with the translocation observations, these
studies suggest that during the late phase of cell injury,
calpains are involved in substrate proteolysis at or near the
plasma membrane that is associated with extracellular Cl2
influx.
Previous results combined with the current data have led
us to propose the following sequence of events that lead to
RPT cell death/lysis after mitochondrial inhibition (fig. 8).
First, an increase in intracellular Ca11f levels triggers calpain-mediated extracellular Ca11 influx through a nifedip-
Fig. 8. Proposed sequence of events that occur after mitochondrial
inhibition (antimycin A) in the late phase of cell injury that lead to rabbit
RPT cell death. An initial increase in cytosolic free Ca11 activates
calpains (1), calpain activation results in extracellular Ca11 influx (2, 3)
and the further increase in cysotolic free Ca11 causes additional calpain activation (4) and translocation (5, 6) to the plasma membrane and
Cl2 influx (7, 8). Note that the events illustrated are subsequent to ATP
depletion, K1 efflux, Na1 influx and plasma membrane depolarization.
The initial calpain activation may represent one calpain isozyme (1), and
the subsequent calpain activation and translocation represent a second
calpain isozyme (4 – 6). Alternatively, initial and subsequent calpain
activations may represent the same isozyme activated to different
degrees depending on the cytosolic free Ca11 levels.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017
Cytoprotection with Ca11 channel blockers has been reported in other renal cell models subjected to anoxia or hypoxia. For example, Almeida et al. (1992) reported that verapamil transiently inhibited Ca11 uptake and LDH release in
rat RPT subjected to hypoxia and subsequently reported that
verapamil may act intracellularly on the mitochondrion. In
contrast, we did not observe cytoprotection with verapamil in
rabbit RPT suspensions subjected to mitochondrial inhibition. The difference between our findings and those of
Almeida et al. (1992) may reflect the different species used
and/or the non-plasma membrane effects of verapamil in the
rat. Rose et al. (1994) reported that methoxyverapamil decreased anoxia-induced Ca11 influx and LDH release in rabbit RPT cells subjected to anoxia, whereas felodipine was
protective by attenuating potassium loss during hypoxia.
Possible explanations for the discrepancy between verapamil
and methoxyverapamil include differences in potency, selectivity or actions at non-plasma membrane sites (Fleckenstein-Grun, 1992). It is unlikely that nifedipine attenuated
potassium loss after mitochondrial inhibition because nifedipine was protective when added 15 min after antimycin A, a
time point after potassium loss has occurred. These varying
results with different Ca11 channel blockers may also explain the conflicting actions of Ca11 channel blockers seen in
in vivo renal protection studies (Almeida et al., 1992; Rose et
al., 1994).
Investigators have postulated that one potential mechanism of Ca11-induced cellular injury involves the activation
of calpains (Bronk and Gores, 1993; Croall and Demartino,
1991; Edelstein et al., 1995; Nicotera et al., 1986; Saido et al.,
1994; Wang and Yuen, 1994). However, the role of calpains in
cell injury has been difficult to determine due to problems
with calpain assays and the lack of specific calpain substrates and selective calpain inhibitors (Sasaki et al., 1984).
We have shown that calpain inhibitor 2 and PD150606 are
cytoprotective to RPT exposed to a group of diverse toxicants
with different mechanisms of action (current results;
Schnellmann, 1997; Schnellmann et al., 1994). Furthermore,
the two inhibitors inhibited calpain activity as measured by
an in situ calpain assay. Therefore, these results strongly
suggest that calpains play a critical and common role in most
types of necrotic renal cell death. The specific calpain isoform
that is activated during the late phase of cell injury remains
to be determined.
Because calpains are known to interact with a variety of
intracellular substrates at both cytosolic and membrane sites
(Saido et al., 1994), examination of the subcellular distribution of calpains during injury may indicate their site of action. In control samples, the subcellular distribution of calpain activity was ;33% and ;66% in the membraneassociated and cytosolic fractions, respectively. In RPT
exposed to antimycin A, calpain activity translocated from
cytosolic to membrane-associated fractions. Studies by Ostwald et al. (1993, 1994) reported similar distributions of
calpain activity in normal rabbit hippocampal cells as well as
calpain translocation after hypoxia. The observation that
calpain translocation was inhibited by calpain inhibitor 1,
PD150606 and nifedipine provides evidence that one calpain
substrate involved in cell death is at or near the RPT plasma
membrane.
Although the above data demonstrate that extracellular
Ca11 influx and calpains mediate necrotic cell death, the
Calpains Mediate Cell Injury
1184
Waters et al.
Acknowledgments
The authors would like to thank Dr. Charles Edelstein for his
helpful comments and suggestions on the calpain assay, Dr. Philip R.
Mayeux for the use of his spectrofluorometer, Dr. Grazyna Nowak for
her review of the manuscript and Mr. Jeffrey H. Moran, Ms. Mary
Elizabeth Maris and Mr. Jay Harriman for their technical assistance.
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Send reprint requests to: Rick G. Schnellmann, Ph.D., Department of
Pharmacology and Toxicology, University of Arkansas for Medical Sciences,
4301 W. Markham St., Slot 638, Little Rock, AR 72205-7199. E-mail:
[email protected].
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ine-sensitive pathway. This results in a large influx of extracellular Ca11 that in turn mediates calpain activation and
translocation that either directly or indirectly results in Cl2
channel opening. The resulting Cl2 influx triggers H2O influx, causing cell swelling and death/lysis. The mechanisms
and identification of the calpain isozyme or isozymes responsible for these effects remain to be elucidated.
In summary, we demonstrated that (1) chelation of extracellular or intracellular Ca11 prevents cell death from mitochondrial inhibition, (2) the Ca11 channel blocker nifedipine but not
verapamil is cytoprotective and inhibits Ca11 and Cl2 influxes,
(3) antimycin A causes calpain translocation from cytosolic to
membrane-associated cell fractions, (4) calpain inhibitor 1,
PD150606 and nifedipine block antimycin A-induced calpain
translocation, (5) calpain inhibitor 1 and PD150606 provide
cytoprotection both before and subsequent to extracellular
Ca11 influx and (6) Cl2 influx during the late phase of cell
injury is inhibited by nifedipine, calpain inhibitor 1 and
PD150606.
Vol. 283