Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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ABSTRACT |
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The goals of this study were to determine 1) the expression of calpain isoforms in rabbit renal proximal tubules (RPT); 2) calpain autolysis and translocation, and calpastatin levels during RPT injury; and 3) the effect of a calpain inhibitor (PD-150606) on calpain levels, mitochondrial function, and ion transport during RPT injury. RT-PCR, immunoblot analysis, and FITC-casein zymography demonstrated the presence of only µ- and m-calpains in rabbit RPT. The mitochondrial inhibitor antimycin A decreased RPT µ- and m-calpain and calpastatin levels in conjunction with cell death and increased plasma membrane permeability. No increases in either µ- or m-calpain were observed in the membrane nor were increases observed in autolytic forms of either µ- or m-calpain in antimycin A-exposed RPT. PD-150606 blocked antimycin A-induced cell death, preserved calpain levels in antimycin A-exposed RPT, and promoted the recovery of mitochondrial function and active Na+ transport in RPT after hypoxia and reoxygenation. The present study suggests that calpains mediate RPT injury without undergoing autolysis or translocation, and ultimately they leak from cells subsequent to RPT injury/death. Furthermore, PD-150606 allows functional recovery after injury.
rabbit; mitochondrial inhibitor; hypoxia/reoxygenation; mitochondrial function; active sodium transport; calpastatin
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INTRODUCTION |
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MANY HYPOTHESES
REGARDING the mechanisms of renal proximal tubules (RPT) cell
injury and death have been proposed, including increases in
intracellular free calcium (Ca
It is generally hypothesized that a supraphysiological and/or prolonged
increase in Ca
Among the numerous members of the calpain superfamily, two ubiquitous
isoforms have been identified, µ- and m-calpain. As the names imply,
purified µ- and m-calpains are activated by micro- and millimolar
Ca2+ concentrations in vitro, respectively. In contrast to
the concept that both µ- and m-calpains are ubiquitously and
constitutively expressed in mammalian cells (7, 39),
Edelstein et al. (12) reported that rat RPT only express
µ-calpain. Both µ- and m-calpains are heterodimers and consist of
an 80-kDa large subunit, containing the active site, and a 30-kDa
regulatory subunit (7, 39). The procalpains are
predominantly localized in the cytosol, and several cellular events can
increase the activity of calpains: a rise in intracellular
Ca
The translocation of calpain from the cytosol to the plasma membrane was proposed to be a critical step in the calpain activation process in platelets and red blood cells (15, 28). A redistribution of calpain from the cytosol to the membrane fraction was detected previously after traumatic rat brain injury using casein zymography (51). However, N-methyl-D-aspartic acid-induced calpain activation was independent of calpain translocation in primary rat coritcal neurons (20). Also, Blomgren et al. (3) reported that calpain immunoreactivity decreased in the cytosolic fraction with no significant changes in the membrane fractions in cortical tissue from neonatal rats subjected to cerebral hypoxia-ischemia. Calpain activation in these models was confirmed by increased spectrin proteolysis.
With respect to calpain activation during renal cell injury, Edelstein et al. (9, 10, 12) observed an increase in calpain activity before membrane damage using a fluorescent calpain substrate and degradation of spectrin in rat RPT subjected to hypoxia. Using a calpain substrate, our laboratory (42, 43) suggested that calpain activity translocated from the cytosol to the membrane fraction during mitochondrial inhibitor-induced RPT cell injury. However, it is unknown which calpain isoform(s) are expressed in rabbit RPT and whether µ- and/or m-calpain undergoes autolysis or translocates to the membrane during RPT injury.
The aims of this study were to determine 1) the expression of calpain isoforms in rabbit RPT; 2) calpain autolysis and translocation during RPT cell injury; 3) calpastatin levels during RPT cell injury; and 4) the effect of a calpain inhibitor (PD-150606) on calpain levels, mitochondrial function, and ion transport during RPT cell injury.
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MATERIALS AND METHODS |
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Reagents. Purified µ-calpain (from porcine erythrocyte), purified m-calpain (from porcine kidney), and the calpain inhibitor PD-150606 were purchased from Calbiochem (La Jolla, CA). Antimycin A, DMSO, and casein fluorescein isothiocyanate (FITC-casein) were obtained from Sigma (St. Louis, MO). An enhanced chemiluminescence (ECL) kit and autoradiography film were obtained from Amersham Pharmacia (Arlington Heights, IL). TRIzol reagent and QIAquick gel extraction kit were purchased from GIBCO BRL (Grand Island, NY) and from Qiagen (Chatsworth, CA), respectively. Moloney murine leukemia virus reverse transcriptase (MuLV-RT) was obtained from PerkinElmer (Foster City, CA). The sources of the remaining chemicals have been reported previously (17, 33) or were from Sigma. All glassware was silanized and autoclaved before use. All media and buffers were sterilized by filtering before use.
Isolation of RPT S2 segments, RNA
extraction, and RT-PCR amplification.
Female New Zealand White rabbits (Myrtle's Rabbitry, Thompson Station,
TN) were injected with 500 units/kg heparin sulfate and euthanized with
an overdose of pentobarbital sodium (50 mg/kg), and kidneys were
removed. Rabbit RPT S2 segments were individually isolated using
microdissection as described by Zalups and Barfuss (49).
Total cellular RNA was isolated from RPT S2 segments using TRIzol
reagent following the manufacturer's instructions. The RNA was stored
in RNAse-free water at 80°C until it was used. The rabbit µ- and
m-calpain large-subunit mRNA sequences were identified in the National
Center for Biotechnology Information (NCBI) Entrez database (accession
no. M13363 and M13797 for µ- and m-calpain large subunits,
respectively). Two sets of primers were designed for use in PCR
amplification studies: µ-calpain large subunit, CU5 (sense)
5'-TGCGTACCAAGGGCTTCA-3', CU6 (antisense) 5'-AGGGGACATGAGGAAGCTGG-3';
m-calpain large subunit, CM1 (sense) 5'-ACATGCACACCATCGGCTTCG-3', and
CM2 (antisense) 5'-GGCAGCGAACGAAGTT GTCGAAG-3'. For the µ-calpain
large subunit, a specific 5' inner primer, CU3,
5'-CCATCGAGTCTGCAGGCTTCAAG-3' also was used for nested PCR
amplification. The expected product from first-round PCR was 700 base
pairs (bp) for the m-calpain large subunit and 700 bp for the
µ-calpain large subunit. The expected product from the nested PCR for
the µ-calpain large subunit was 500 bp.
Isolation and incubation of rabbit RPT.
RPT were isolated and purified by the method described by Rodeheaver et
al. (33) and Groves and Schnellmann (17) from 1.5-2.0 kg female New Zealand White rabbits. RPT were suspended at
a concentration of 2 mg/ml in an incubation buffer containing (in mM) 1 alanine, 5 dextrose, 2 heptanoate, 4 lactate, 5 malate, 115 NaCl, 15 NaHCO3, 5 KCl, 2 NaH2PO4, 1 MgSO4, 1 CaCl2, and 10 HEPES (pH 7.4, 295 mosmol/kg). RPT suspensions were incubated under air-CO2
(95-5%) at 37°C in a gyrating water bath (180 rpm). All
experiments contained a 15-min preincubation period with no experimental manipulations. After the preincubation, the mitochondrial inhibitor antimycin A (10 µM) or diluent (DMSO, 0.5% of
total volume) was added to RPT, and the incubation continued. Antimycin A has been shown to produce extensive cell death over an extended period of time in this model (35). At 5, 10, 15, 30, and
60 min after antimycin A addition, aliquots of RPT suspension were removed and processed for immunoblot analysis, zymogram assay, and LDH
release assay. In experiments with the calpain inhibitor, PD-150606 was
added 30 min before antimycin A, and the incubation continued for an
additional 30 min.
Immunoblot analysis of calpain isoforms. RPT suspensions were separated into cytosolic and membrane fractions and incubation buffer as described previously (43, 44). Briefly, RPT were centrifuged at 1,000 g for 1 min, the incubation buffer was removed, the pellet was resuspended in imidazole buffer [(in mM) 63 imidazole, 1 EDTA, 10 EGTA, 10 2-mercaptoethanol, pH 7.3], and was permeabilized with 100 µM digitonin for 10 min at 37°C. The cytosol and the membrane fraction were obtained by centrifugation (14,000 g for 2 min). The pellet was resuspended in a pellet solubilization buffer containing 1% SDS, 10 mM dithiothreitol, 10 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 5 µg/ml pepstatin and boiled for 10 min. The supernatant and pellet and incubation buffer samples were mixed with 2× loading buffer [100 mM Tris, 4% SDS, 20% (vol/vol) glycerol, 10% 2-mercaptoethanol, and 0.04% bromophenol blue] and boiled for 10 min. Matched samples were taken for protein concentrations and determined either by the method described by Lowry et al. (24), or by the BCA assay (Pierce, Rockford, IL), using BSA as standards.
To estimate the total calpain isoforms in RPT, the RPT were centrifuged at 1,000 g for 1 min, resuspended in imidazole buffer, and lysed with 1% Triton X-100 for 10 min at 37°C. The Triton X-100-soluble fraction was obtained by centrifugation (14,000 g for 2 min) and contained more than 95% of the total cellular protein (data not shown). Thirty-microgram (unless otherwise indicated) or 20-µl samples of incubation buffer were loaded onto 10% SDS-polyacrylamide gels and subjected to electrophoresis, and the proteins were transferred to a nitrocellulose membrane. The membrane was incubated overnight in blocking buffer (2.5% casein, 0.9% NaCl, 5 mM Tris, and 0.25 µM thimerosal, pH 7.6) and then incubated overnight with either the anti-µ-calpain antibody (1:2,500) (19) or the m-calpain antibody (1:2,000) (18). The membrane was washed and incubated with a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase. The membrane was washed and developed using the ECL system following the manufacturer's instructions. For µ- and m-calpains, densities of the corresponding bands were determined with National Institutes of Health (NIH) Image software. Results were expressed as percentage of controls.FITC-casein zymography. RPT suspensions were separated into cytosolic and membrane-associated fractions as described previously (42, 43) with the following modifications. RPT were centrifuged at 1,000 g for 1 min, resuspended in zymography buffer [50 mM HEPES, 150 mM NaCl, 10% (vol/vol) glycerol, 5 mM EDTA, 100 µM PMSF, 10 µg/ml leupeptin, and 10 mM 2-mercaptoethanol, pH 7.6], and permeabilized with 100 µM digitonin for 10 min at 37°C. The cytosol and the membrane fraction were obtained by centrifugation (14,000 g for 10 min at 4°C). The pellet was resuspended in zymography buffer and lysed with 1% Triton X-100 on ice for 10 min. The cytosol and the membrane-associated fractions were mixed with 2× zymography loading buffer [100 mM Tris · HCl, pH 6.8, 10 mM EDTA, 20% (vol/vol) glycerol, 10 mM 2-mercaptoethanol, and 0.02% bromophenol blue]. Matched samples were taken for protein concentration and determined using the BCA assay.
To measure total activity of µ- and m-calpain in RPT, RPT were centrifuged at 1,000 g for 1 min, resuspended in zymography buffer, and lysed with 1% Triton X-100 for 10 min at 37°C. The lysate was centrifuged (14,000 g for 10 min) and the supernatant was mixed with 2× zymography loading buffer. Ten-microgram samples (unless otherwise indicated) were loaded onto 10% polyacrylamide gels containing 0.0025% FITC-casein and subjected to electrophoresis under nondenaturing conditions with buffer containing (in mM) 25 Tris base, 125 glycine, 1 EDTA, and 10 2-mercaptoethanol, pH 8. Thirty nanograms of purified µ- and m-calpains and a mixture of the two were loaded onto the same gels as positive controls. After electrophoresis, the gels were incubated with a buffer containing (in mM) 50 Tris · HCl, 10 CaCl2, and 10 2-mercaptoethanol, pH 7.6, twice for 30 min at room temperature. The gels were then incubated with the same buffer at 4°C for 16 h. Photographs were taken under UV light and scanned, and the densities for the bands were determined with NIH Image software. Results are expressed as the percentage of control RPT.Immunoblot analysis of calpastatin. The RPT supernatant used for total calpain activity evaluation by zymography was also used for determination of calpastatin. The supernatant samples were mixed with 2× loading buffer (100 mM Tris, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.04% bromophenol blue). Immunoblot analysis of calpastatin was performed with a specific anti-calpastatin antibody (Calbiochem) as described for immunoblot analysis of calpain isoforms above. The density for the band was determined with NIH Image software, and the results are expressed as the percentage of control RPT.
Hypoxia/reoxygenation exposure and QO2 measurement. RPT were subjected to hypoxia (95% N2-5% CO2, 1 h)/reoxygenation (95% air-5% CO2, 1 h), as described previously (31). Immediately after the hypoxic period, aliquots of RPT were removed for determination of LDH release. After reoxygenation, aliquots of RPT were removed for determination of LDH release or oxygen consumption (QO2). QO2 was measured polarographically using a Clark-type electrode as described previously (34). After basal oxygen consumption was obtained, ouabain-insensitive QO2 was measured in the presence of 0.1 mM ouabain, and the ouabain-sensitive QO2 was calculated as the difference between basal and ouabain-insensitive QO2. The calpain inhibitor PD-150606 (100 µM) was added immediately before hypoxia. Protein concentration was determined by the BCA assay.
LDH analysis. The release of LDH into the incubation buffer was measured as a marker of cellular death/lysis, as described previously (30).
Statistics. The data are expressed as means ± SE. RPT suspensions isolated from one rabbit represent a single experiment (n of 1). Data were analyzed by ANOVA; multiple means were compared using Fisher's protected least significance difference test with a level of significance of P < 0.05. Two means were compared using Student's t-test with the same level of significance.
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RESULTS |
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Expression of calpain isoforms in rabbit RPT by
RT-PCR, immunoblot analysis, and
FITC-casein zymography.
Freshly isolated rabbit RPT suspensions provide a mixture of S1 and S2
segments of RPT (96-99% purity) with the majority of the
contamination being glomeruli and distal tubular segments (17). To avoid contamination of non-RPT cells in
determining the expression of calpain isoforms in RPT using RT-PCR,
individual RPT S2 segments were micro-dissected and pooled, and total
cellular RNA was extracted. RNA was subjected to RT-PCR using either
rabbit µ- or m-calpain large subunit-specific primers. First round
PCR amplified a cDNA of the expected size of 700 bp using the m-calpain large subunit primers (Fig.
1A). A product was not
observed after first-round PCR using the µ-calpain large subunit
primers. However, nested PCR using a 5' inner primer resulted in the
expected 500-bp product (Fig. 1B). No products were observed
in reactions lacking the RNA template or RT (data not shown).
Sequencing of both PCR products revealed sequences that were identical
to µ- and m-calpain large subunit mRNA sequences reported in the NCBI
Entrez database. Therefore, the RT-PCR data demonstrate that both µ-
and m-calpain large subunits are expressed in rabbit RPT S2 segments at
the mRNA level.
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Alterations of µ- and m-calpains in the cytosolic and membrane
fractions during mitochondrial inhibitor-induced RPT
cell injury.
RPT were exposed to the mitochondrial inhibitor antimycin A to induce
cell injury/death. Antimycin A treatment resulted in time-dependent
cell death, indicated by increased LDH release at 30 min of treatment
(Fig. 5A). FITC-casein
zymography was performed on the cytosolic and membrane fractions of RPT
after different times of antimycin A exposure. As demonstrated in Fig.
5, B and C, cytosolic µ- and m-calpain
activities did not change at the early stage of RPT injury produced by
antimycin A exposure (15 min). However, antimycin A treatment
resulted in decreases in cytosolic µ- and m-calpain activities after
30 min. FITC-casein zymography of the membrane fraction obtained from
RPT exposed to antimycin A for up to 15 min did not reveal any
increases in either µ- or m-calpain isoform (Fig. 5, D and
E). However, after 30 min of exposure, both µ- and
m-calpain activities in the membrane fractions were decreased, similar
to that observed in the cytosol. Therefore, the FITC-casein zymography
results demonstrate the lack of µ- or m-calpain translocation during
the early phases of cell injury and decreases in µ- and m-calpain
activities in both fractions in concert with cell death/lysis produced
by antimycin A exposure.
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Leakage of calpain isoforms from RPT during
mitochondrial inhibitor-induced cell injury.
The possibility that decreases in cytosolic µ- and m-calpains was due
to increased cell membrane permeability during RPT cell injury/death
was explored further. Total µ- or m-calpain levels were measured in
antimycin A-treated RPT and compared with that of controls. Loss of
cytosolic protein from antimycin A-treated RPT decreased the ratio of
cytosolic-to-membrane protein and would introduce an artificial
decrease in the calpain isoforms contained per milligram total protein.
Therefore, the same sample volume (20 µl) instead of the same amount
of total protein from control and antimycin A-treated RPT was used for
FITC-casein zymography or immunoblot analysis. FITC-casein zymography
revealed that antimycin A exposure resulted in no changes in both
calpain isoforms during the first 15 min of RPT injury (Fig.
7, A and B).
However, after 30 min, decreased activities for calpain
isoforms were observed (Fig. 7, A and B).
Immunoblot analysis using the anti-µ-calpain large subunit antibody
on the Triton X-100-soluble fraction revealed decreases in total 80-kDa
µ-calpain large subunit after 30 and 60 min of antimycin A exposure
(Fig. 8A), further supporting
the FITC-casein zymography results. Correlated with the decreases in
protein or activities of both calpains were similar increases in cell
death, indicated by increased LDH release and the appearance of both
µ- and m-calpain large subunits in the incubation buffer (Figs.
5A and 8). These results show that calpains leak from the RPT cells during antimycin A-induced RPT injury/death.
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Changes in calpastatin during mitochondrial inhibitor-induced
RPT cell injury.
The protein level of the endogenous calpain inhibitor calpastatin was
examined using immunoblot analysis. The antibody recognized a 120-kDa
protein in RPT, which is consistent with previous reports (25). Immunoblot analysis demonstrated that calpastatin is
present primarily in the RPT cytosol (data not shown). As shown in Fig. 9, A and B,
antimycin A exposure up to 15 min did not result in decreases in
calpastatin levels. A decrease in calpastatin protein level was found
30 min after antimycin A exposure (Fig. 9B) and corresponded
to the increased LDH release (Fig. 5A).
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Effect of PD-150606 on µ-calpain level,
mitochondrial function, and active
Na+ transport in
RPT exposed to antimycin A or subjected
to hypoxia/reoxygenation.
The present study investigated the effects of PD-150606 on total
µ-calpain levels during RPT cell injury/death induced by antimycin A
exposure. As illustrated in Fig. 10,
the presence of 100 µM PD-150606 prevented the decreases in total
80-kDa µ-calpain large subunit (A) and protected against
RPT cell injury/death produced by 30 min of antimycin A exposure
(B).
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DISCUSSION |
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Many hypotheses have been proposed for the underlying mechanisms
of oncosis. There is now a significant amount of data demonstrating that increases in intracellular Ca and cell injury/death in rabbit RPT subjected to
hypoxia or exposed to antimycin A (44).
Calpains are a family of Ca2+-activated proteases, with
µ- and m-calpains being ubiquitously and constitutively expressed in most mammalian cells (7, 39), and were proposed to be a
cell injury/death mediator after increased intracellular
Ca
Immunoblot analysis demonstrated that the majority of µ-calpain large subunits are present in the cytosol as an 80-kDa form whereas a small amount of the 78-kDa intermediate autolytic form is associated with the membrane. Localization of µ-calpain mainly in the cytosol is consistent with previous reports in the literature (15) and was supported by the FITC-casein zymography results. The presence of the 80-kDa µ-calpain large subunit and the absence of autolytic forms in the cytosol suggest that cytosolic µ-calpain is an inactive proenzyme or may have activity without undergoing autolysis. It was reported that µ-calpain has a long half-life and was suggested that it is active without autolysis (50). The presence of the autolyzed 78-kDa form only in the membrane fraction confirmed its preferential formation and association with the membrane fraction as described previously (26) and suggests that it may form and act on membrane substrates. The 80-kDa m-calpain and its autolytic 76-kDa form were present only in RPT cytosol. These results are consistent with previous observations in other tissues that m-calpain primarily localizes in the cytosol (51).
The presence of the 78-kDa µ-calpain in the membrane fraction and the 76-kDa m-calpain in the cytosol under control conditions suggests that both calpains have physiological functions in RPT. The fact that they originated from different calpain isoforms, at different autolytic stages, and localized to different cell compartments, suggests that µ- and m-calpains have different physiological functions under normal conditions. For example, selective inhibition of µ-calpain by anti-sense oligonucleotides or a specific inhibitor disrupted cell spreading and adhesion whereas inhibition of m-calpain had no effect (23). Additionally, Schoenwaelder et al. (37) reported that 78- and 76-kDa µ-calpain autolytic products have different substrates and functions during platelet activation.
Calpains have been implicated in ischemic/hypoxic cell/tissue injury. However, how calpain becomes active/activated intracellularly remains unknown. Translocation of cytosolic calpains to the membrane is thought to be a critical step before calpain activation. In previous studies from our laboratory, the mitochondrial inhibitor antimycin A decreased cytosolic calpain activity and increased calpain activity in the membrane fraction. This alteration was interpreted as translocation of calpain activity from the cytosol to the membrane fraction (42, 43). However, calpain activity translocation does not reflect the calpain isoform involved nor the form (i.e., proenzyme or autolyzed product). FITC-casein zymography and immunoblot analysis of the membrane fraction failed to detect any increases in either µ- or m-calpain after antimycin A exposure, demonstrating no translocation of either calpain isoform during RPT injury. In the previous study, an in vitro calpain assay and a fluorescent substrate were used, and the increased activity in the membrane fraction could be due to enzymes other than calpains or calpain isoforms undetectable using the anti-µ or m-calpain large subunit antibodies. However, in the present study, FITC-casein zymography did not reveal any additional calpain isoforms in RPT.
Of equal importance, no increases in autolytic forms of µ- or m-calpain large subunits were observed by immunoblot analysis in antimycin A-exposed RPT. Therefore, it is likely that µ- or m-calpain mediate RPT cell injury in their proenzyme form. The reductions in µ- and m-calpain levels observed during the late phase of RPT injury correlated with the appearance of µ- and m-calpain in the incubation buffer and the increased plasma membrane permeability to LDH release. These results demonstrate that calpains leak from RPT during the cell death process.
Calpains coexist with calpastatin, the endogenous inhibitor in the cell
(2). Under normal conditions, calpastatin is localized in
specific structures rather than distributed in a diffused form in the
cytosol (8). Calpastatin and calpain associate in the presence of Ca+ (7). Higher and prolonged
increases in intracellular Ca
The present study demonstrates that the calpain inhibitor PD-150606 decreases LDH release and calpain leakage from RPT subjected to antimycin A, consistent with previous reports (36, 44). Furthermore, the present study shows that PD-150606 promotes the recovery of mitochondrial function and active Na+ transport in RPT subjected to hypoxia/reoxygenation, suggesting that calpains mediate mitochondrial dysfunction and inhibit ion transport during RPT injury. Alternatively, the mitochondrial dysfunction and inhibition of ion transport may be due to calpain-mediated plasma membrane permeability changes. However, previous reports from our laboratory demonstrated that the Na+-K+-ATPase isolated from RPT subjected to hypoxia/reoxygenation showed no inhibition of activity when measured in vitro, indicating that calpain does not exert a direct effect on Na+-K+-ATPase (31). We hypothesize that calpain may modulate the cytoskeleton (such as spectrin and ankyrin) and interfere with Na+-K+-ATPase function or localization.
In summary, the present study demonstrates that rabbit RPT cells express µ- and m-calpain, and calpains play a critical role during RPT injury/death without undergoing autolysis or translocation. The calpain inhibitor PD-150606 is a true cytoprotecant, allowing the return of physiological functions in RPT subjected to hypoxia/reoxygenation.
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ACKNOWLEDGEMENTS |
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Present address of J. J. Rainey: The College of St. Scholastica, 1200 Kenwood Ave., Duluth, MN 55811.
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FOOTNOTES |
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This work was supported by National Institute of Environmental Health Sciences Grant ES-09129 (R. G. Schnellmann) and predoctoral fellowships (J. F. Harriman and X. Liu) from the American Heart Association, Heartland Affiliate.
Address for reprint requests and other correspondence: R. G. Schnellmann, Dept. of Pharmaceutical Sciences, Medical University of South Carolina, 280 Calhoun St., POB 250140, Charleston, SC 29425 (E-mail: schnell{at}musc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 October 2000; accepted in final form 21 May 2001.
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