Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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ABSTRACT |
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Diverse -aminobutyric acid (GABAA) receptor
modulators exhibited novel cytoprotective effects and mechanisms of
action in rabbit renal proximal tubules subjected to mitochondrial
inhibition (antimycin A) or hypoxia. Cytoprotective potencies (50%
effective concentration,
EC50) were 0.3 nM
allopregnanolone (AP) > 0.4 nM 17
-OH-allopregnanolone
(17
-OH-AP) > 30 nM dehydroepiandrosterone sulfate (DHEAS) = 30 nM
pregnenolone sulfate (PS) > 500 nM pregnenolone (PREG) > 30 µM
muscimol > 10 mM GABA following antimycin A exposure. Maximal
protection with AP and 17
-OH-AP was 70%, whereas DHEAS, PS, PREG,
and muscimol produced 100% cytoprotection. Experiments with AP, PS,
and muscimol revealed the return of mitochondrial function and active
Na+ transport following
hypoxia/reoxygenation. Muscimol inhibited the antimycin A-induced
influx of both extracellular Ca2+
and Cl
that occurs during
the late phase of cell injury, whereas the neurosteroids only inhibited
influx of Cl
. Radioligand
binding studies with AP and PS did not reveal a specific binding site;
however, structural requirements were observed for cytoprotective
potency and efficacy. In conclusion, we suggest that the
GABAA receptor modulators muscimol
and neurosteroids are cytoprotective at different cellular sites in the
late phase of cell injury; muscimol inhibits
Ca2+ and subsequent
Cl
influx, whereas the
neurosteroids inhibit Cl
influx.
renal proximal tubules; calcium influx; chloride influx; anoxia; -aminobutyric acid receptor
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INTRODUCTION |
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EXAMINATION OF THE TEMPORAL sequence of cell
injury reveals that the onset of anoxia or the inhibition of
mitochondrial function by a toxicant results in respiratory arrest, the
loss of ATP, and decreased
Na+-K+-adenosinetriphosphatase
(Na+-K+-ATPase)
activity (6). Na+ influx and
K+ efflux occur concomitantly with
the loss of
Na+-K+-ATPase
activity, and the normally negative membrane potential is diminished.
During the late phase of cell injury in rabbit renal proximal tubules
(RPT), influx of extracellular
Ca2+ and
Cl occurs (10, 17, 20, 29,
38, 39). Leaf (11) originally demonstrated that
Cl
influx is critical for
the cell swelling produced by mitochondrial inhibition. Miller and
Schnellmann (17, 20) subsequently showed that
Cl
influx occurs
significantly after the loss of ATP and cation movement during the late
phase of cell injury produced by a variety of toxicants. Decreasing the
extracellular Cl
concentration decreased Cl
entry, cell swelling, and lysis, whereas increasing extracellular osmolality did not decrease
Cl
entry but did decrease
cell swelling and lysis. These data suggest that
Cl
influx occurs during the
late phase of cell injury and triggers the cell swelling that leads to
cell lysis.
The cytoprotective effects of the neuronal glycine receptor agonist glycine and the antagonist strychnine have been reported in a number of in vitro and in vivo models against anoxia/hypoxia and a diverse group of toxicants. For example, glycine is cytoprotective in vivo against maleate-, ifosfamide-, and cisplatin-induced nephrotoxicity in the rat (8, 12, 25), against hypoxic injury in the isolated perfused rat kidney (2, 7, 33), reperfusion injury in the rat liver (40), cold ischemic injury in the isolated perfused rabbit and dog kidney (14, 30), and in vitro in rat hepatocytes (15), pulmonary artery endothelial cells (35), and human umbilical vein endothelial cells (40). The neuronal glycine receptor antagonist strychnine exhibits cytoprotective properties similar to glycine (16, 17, 42). Therapeutic realities of these compounds are limited due to the high concentrations (mM) necessary for cytoprotection and the additional neurotoxic effects of strychnine. Investigators have screened numerous agents in the hope of identifying more potent and efficacious cytoprotective compounds, including dipeptides and tripeptides with different amino acid constituents, glycine receptor antagonists, N-methyl-D-aspartate (NMDA) receptor antagonists, and other amino acid analogs (3, 9, 18, 41). However, none of the compounds thus far tested have been shown to meet or exceed the cytoprotective characteristics of glycine.
The -aminobutyric acid (GABAA) receptor antagonist,
bicuculline, and the benzodiazepine inverse agonist, norharmane, are partially protective to rabbit RPT exposed to the mitochondrial inhibitor antimycin A (1). Recently, Venkatachalam et al. (36) demonstrated that avermectin B1a
and its analogs, agonists at both the neuronal
GABAA and glycine receptors,
prevent hypoxic cell death in Madin-Darby canine kidney cells at
concentrations as low as 100 µM. Thus avermectin
B1a is 10 times more potent than
either glycine or strychnine. They also demonstrated the cytoprotective
effects of the neuronal GABAA and
glycine receptor antagonist cyanotriphenylboron (36).
These data prompted us to investigate the cytoprotective profile of
additional GABAA receptor
modulators in rabbit RPT including potency, efficacy, structural, and
temporal aspects. We determined whether the
GABAA receptor agonists GABA,
muscimol,
4,5,6,7-tetrahydroisoxazolo[5.4-c]oyridin-3-ol hydrochloride (THIP), the GABAA
receptor antagonist -hydrastine, and the neurosteroid
GABAA receptor modulators
allopregnanolone (AP), 17
-OH-allopregnanolone (17
-OH-AP),
pregnenolone sulfate (PS), pregnenolone (PREG), and
dehydroepiandrosterone sulfate (DHEAS) (Fig.
1) 1)
prevent cell death produced by anoxia and mitochondrial inhibition,
2) provide cytoprotection through
the return of cellular functions, 3)
are cytoprotective during the late phase of cell injury,
4) modulate the influx of
extracellular Ca2+ that occurs
during the late phase of cell injury, and
5) modulate the
Cl
influx that occurs
during the late phase of cell injury. This study also examined the
structural requirements necessary for neurosteroid cytoprotective
potency or efficacy and whether the neurosteroids bind specifically to
RPT.
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MATERIALS AND METHODS |
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Materials. Antimycin A, GABA, THIP,
-hydrastine, and the neurosteroids AP (5
-pregnan-3
-ol-20-one),
17
-OH-AP (5
-pregnane-3
,17
-diol-20-one), PREG, PS, and DHEAS
were purchased from Sigma Chemical (St. Louis, MO). Muscimol was
obtained from Research Biochemicals International (Natick, MA).
Na36Cl,
[14C]dextran (85,000 mol wt), [3H]AP,
[3H]PREG, and
45CaCl2
were purchased from Dupont-NEN (Boston, MA). All other chemicals and
agents were of reagent grade and obtained from either Sigma Chemical or
Aldrich Chemical (Milwaukee, WI).
Isolation and incubation of rabbit
RPT. RPT were isolated and purified by the method of
Rodeheaver et al. (28) from female 1.5- to 2.0-kg New Zealand White
rabbits (Myrtle's Rabbitry, Thompson Station, TN). RPT were suspended
at a concentration of 1 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 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4, 295 mosmol/kgH2O).
RPT suspensions were incubated under 95% air-5%
CO2 at 37°C in a gyrating
water bath (180 revolutions/min). Following a 15-min preincubation
period, 1 µM antimycin A and/or various concentrations of AP,
17-OH-AP, PREG, PS, DHEAS, muscimol, THIP, GABA, or diluent
(dimethyl sulfoxide,
0.5% total volume) was added to the RPT
suspensions, and lactate dehydrogenase (LDH) release was determined 30 min later. In some experiments, muscimol or the neurosteroids were
added 15 min after antimycin A, and LDH release was determined 15 min
later. Tracer concentrations of
36Cl
or
45Ca2+
were added 15 min after antimycin A, and aliquots were taken for
analysis 15 min later.
Hypoxia/reoxygenation studies. Following a 15-min exposure to oxygenated conditions, RPT were subjected to hypoxia by incubating the tubules under 95% N2-5% CO2 for 1 h. Cytoprotectants were added immediately prior to the initiation of hypoxia. After the hypoxic exposure, RPT were reoxygenated with 95% air-5% CO2 for 1 h, and oxygen consumption was measured. LDH samples were taken following both hypoxia and reoxygenation.
Biochemical analysis. Aliquots of RPT
suspensions were taken at various times, and RPT were separated from
the surrounding buffer by rapid centrifugation through a layer of
dibutylphthalate-dioctylphthalate (2:1). The pellets were
resuspended in Triton solubilization buffer [100 mM
tris(hydroxymethyl)aminomethane, 150 mM NaCl, and 0.05% Triton X-100
at pH 7.5], and aliquots were taken for liquid scintillation spectrometry and protein content. Extracellular
36Cl
and
45Ca2+
were corrected for by using the extracellular water marker
[14C]dextran. The
release of LDH into the incubation buffer was used as a marker of cell
death/lysis as described previously by Moran and Schnellmann (24). RPT
ATP and protein contents were determined using high-performance liquid
chromatography analysis (31) and the biuret method (5), respectively.
Oxygen consumption analysis. Oxygen consumption in RPT was measured using a Clark-type electrode as previously described (31). Following the measurement of basal oxygen consumption, the Na+-K+-ATPase inhibitor ouabain (0.1 mM) was added to obtain ouabain-insensitive oxygen consumption. Ouabain-sensitive oxygen consumption was calculated as the difference between basal and ouabain-insensitive oxygen consumption.
Radioligand binding. Concentration-dependent binding of [3H]AP and [3H]PS was determined by incubating RPT with radiolabeled compound (10 nM to 10 µM) in the absence and presence of 1 mM unlabeled AP and PS, respectively, at 4°C for 30 min. Time-dependent neurosteroid binding was conducted by incubating RPT with 10 nM [3H]AP or 100 nM [3H]PS in the absence and presence of 1 mM unlabeled AP or PS, respectively. Samples were taken at 1, 5, 10, 15, 30, and 60 min at 4°C or at 1, 2, 5, 10, and 15 min at 37°C. RPT were separated from the surrounding media by centrifugation through a layer of dibutylphthalate-dioctylphthalate (2:1), resuspended in Triton X-100 solubilization buffer as above, and radioactivity was determined using liquid scintillation spectrophotometry. Protein concentration was determined as above.
Statistics. Data are expressed as means ± SE. Each tubule preparation represented a separate experiment (n = 1). Percent protection was calculated using the following formula
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RESULTS |
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The cytoprotective properties of GABA, THIP, muscimol, -hydrastine,
and the neurosteroids in RPT exposed to antimycin A are illustrated in
Fig. 2. The neurosteroids were the most
potent compounds examined, providing 50% cytoprotection at ~0.3 nM
AP, 0.4 nM 17
-OH-AP, 30 nM DHEAS, 30 nM PS, and 500 nM PREG. Maximal cytoprotection of ~70% was observed at 10 nM with AP and
17
-OH-AP. In contrast, complete cytoprotection was observed with
DHEAS, PS, and PREG at ~3, 3, and 30 µM, respectively. Muscimol and
-hydrastine were less potent cytoprotectants, providing 50%
protection at ~30 and 20 µM, respectively. GABA and THIP were only
partially protective at millimolar concentrations. GABA provided
~55% protection at 10 mM, whereas THIP provided ~45% protection
at 1 mM.
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To determine whether the cytoprotective properties of the neurosteroids and muscimol applied only to the prevention of cell lysis or also would allow RPT to regain mitochondrial function and active ion transport following the removal of the injurious insult, a model of RPT hypoxia/reoxygenation was utilized. The antimycin A model cannot be used for these studies, because antimycin A cannot be removed from the RPT (unpublished observations). Studies were conducted to determine the effects of AP, PS, and muscimol on LDH release, mitochondrial function, and active Na+ transport following hypoxia/reoxygenation. Following a 15-min oxygenation period, 10 nM AP, 3 µM PS, or 100 µM muscimol was added to RPT immediately prior to 1 h of hypoxia, followed by 1 h of reoxygenation. LDH release increased to 70% following the hypoxic period and did not further increase after the reoxygenation period (Fig. 3). AP, PS, and muscimol completely ameliorated hypoxia-induced LDH release both after the hypoxic exposure and following reoxygenation. Associated with hypoxia/reoxygenation was a marked inhibition of basal oxygen consumption; however, RPT incubated with muscimol, PS, or AP had basal oxygen consumption levels ~1.6 times greater than RPT subjected to hypoxia/reoxygenation (Fig. 4). Dissection of the basal respiration into its ouabain-sensitive and ouabain-insensitive components, representing respiration associated with active Na+ transport and other ATP-consuming pathways, respectively, revealed that both components were inhibited following hypoxia/reoxygenation. Ouabain-insensitive respiration decreased ~28% and was not increased by muscimol, PS, or AP (data not shown). In contrast, ouabain-sensitive respiration was decreased 90% by hypoxia/reoxygenation, and muscimol, PS, and AP increased ouabain-sensitive respiration approximately eightfold following hypoxia/reoxygenation. These data suggest that not only do muscimol, PS, and AP prevent cell lysis as indicated by the inhibition of LDH release, but that these compounds also allow RPT cells to regain mitochondrial function and active Na+ transport following hypoxia/reoxygenation.
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To determine the temporal relationship for neurosteroid and
muscimol cytoprotection, maximally cytoprotective concentrations of
muscimol or the neurosteroids were added 15 min after antimycin A
addition, during the late phase of cell injury. We have previously determined that ATP depletion occurs during the first 10 min following antimycin A addition and that ATP depletion results in
Na+ influx and
K+ efflux (unpublished
observations). Muscimol and the neurosteroids produced similar
cytoprotective effects when added 15 min after antimycin A (Fig.
5). Furthermore, 17-OH-AP and AP were
equally efficacious, and PS, PREG, DHEAS, and muscimol were equally
efficacious. Additional evidence that the neurosteroids and muscimol
act subsequent to ATP depletion was provided by the observation that
these compounds do not reverse the ATP depletion produced by antimycin
A. ATP content under control conditions was 6.8 ± 0.9 nmol/mg
protein. After a 30-min incubation in the presence of antimycin A, ATP levels decreased to 1.4 ± 0.2 nmol/mg protein and remained
decreased in the presence of 100 µM muscimol (1.3 ± 0.3 nmol/mg
protein), 10 nM AP (1.6 ± 0.3 nmol/mg protein), or 3 µM PS (1.7 ± 0.2 nmol/mg protein).
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Influx of extracellular Ca2+ occurs in the late phase of mitochondrial inhibitor-induced cell injury (10, 29, 39). To determine the effect of neurosteroids and muscimol on antimycin A-induced Ca2+ influx, a tracer amount of 45Ca2+ was added to RPT suspensions 15 min after the simultaneous addition of antimycin A and muscimol or a neurosteroid. Antimycin A increased Ca2+ influx 3.2-fold over vehicle control values (Fig. 6). Cytoprotective concentrations of muscimol, but not the neurosteroids, inhibited extracellular Ca2+ influx.
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Cl influx also occurs
during the late phase of cell injury and directly contributes to the
cell swelling and death/lysis produced by antimycin A as well as a
variety of other toxicants (17, 20, 27, 38). Therefore, we determined
whether the neurosteroids and muscimol also inhibited
Cl
influx. A tracer amount
of
36Cl
was added to RPT suspensions 15 min after the simultaneous addition of
antimycin A and muscimol or a neurosteroid. Antimycin A increased RPT
36Cl
content 2.5-fold over controls (Fig. 7).
Muscimol and the neurosteroids all blocked antimycin A-induced
Cl
influx. These results
suggest that muscimol and the neurosteroids act in the late phase of
cell injury at two distinct sites. Muscimol acts by blocking the influx
of extracellular Ca2+, whereas the
neurosteroids act subsequent to
Ca2+ influx but prior to the
influx of Cl
.
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Radioligand binding studies were conducted to determine whether a specific binding site for the neurosteroids could be identified. No difference in binding was observed with increasing concentrations of [3H]PS (10 nM to 10 µM) in the absence and presence of 1 mM unlabeled PS (Fig. 8A). No difference in binding with respect to time (from 1-60 min) was observed in RPT incubated with 100 nM [3H]PS in the absence and presence of 1 mM unlabeled PS (Fig. 8B). Incubating RPT with 3,000 nM [3H]PS in the presence of 1 µM antimycin A also did not result in specific neurosteroid binding (data not shown). Similar results were obtained using [3H]AP. These data suggest that the cytoprotective effects of the neurosteroids may not be at the extracellular surface of the plasma membrane.
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DISCUSSION |
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We have examined a variety of compounds that interact with
the neuronal GABAA receptor for
cytoprotective effects against mitochondrial inhibition and anoxia in
rabbit RPT (Table 1). The neurosteroids AP
and 17-OH-AP [50% effective concentration (EC50) of ~0.3
nM] were the most potent neurosteroids tested
followed by PS, DHEAS, and PREG
(EC50 values of ~30-500
nM). The GABAA receptor agonists
THIP and GABA were the least potent cytoprotectants (EC50 > 1 mM). Thus the
neurosteroids are ~103- to
105-fold more potent than
muscimol,
-hydrastine, and avermectin B1a and
104- to
106-fold more potent than
bicuculline, norharmane, THIP, GABA, glycine, and strychnine in
preventing antimycin A-induced RPT cell death (1, 36). The rank order
of cytoprotective potency of GABAA receptor agonists/antagonists/modulators is AP = 17
-OH-AP > DHEAS = PS > PREG >>>
-hydrastine = muscimol = avermectin
B1a >>> THIP = bicuculline = norharmane > GABA.
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Although AP and 17-OH-AP were the most potent cytoprotectants, their
efficacy was only 70%. In contrast, the neurosteroids PS, DHEAS, and
PREG were completely efficacious. These studies demonstrate that
neurosteroid structure is an important determinant of cytoprotective
efficacy and potency. Structure activity analysis revealed that the
absence of the double bond between C-5 and C-6 (AP and 17
-OH-AP)
and/or the hydroxyl group in the
-position increases
cytoprotective potency and decreases efficacy. The difference in the
degree of potency and efficacy between DHEAS and PS and between AP and
17
-OH-AP suggests that substitution at the C-17 position is not as
critical. Although Majewska (13) has suggested that PREG is not likely
to be an active GABAA receptor
modulator without a sulfate group, it is not clear whether the
decreased cytoprotective potency of PREG is due to the absence of the
sulfate group per se or decreased ability of RPT to sulfate PREG.
Considering the cytoprotective potency of the neurosteroids and the
structural requirements for cytoprotection, it is likely that the
neurosteroids are acting in a specific manner.
To test whether the neurosteroids were acting at a receptor on the RPT plasma membrane, ligand binding studies were conducted. Radioligand binding assays did not reveal any specific binding of cytoprotective concentrations of the neurosteroids to the extracellular surface of the RPT plasma membrane. In contrast, this assay method did identify a specific strychnine binding site on the basolateral membrane of RPT (19), indicating the validity of the binding assay. These results suggest that, although the neurosteroids are very potent and require strict structural requirements for cytoprotection, they do not bind specifically to the extracellular site of a plasma membrane receptor in a classic ligand/receptor fashion. Since the neurosteroids are very lipophilic, it is possible that they act within the lipid environment of the membrane or intracellularly.
Investigations have revealed temporal markers of initial and late
phases in rabbit RPT cell injury. Gullans et al. (6) have shown that
respiration is inhibited in RPT within 1 min of addition of antimycin
A. During the next 10 min, ATP levels decrease, Na+-K+-ATPase
activity is inhibited, intracellular
K+ levels decrease, and
intracellular Na+ levels increase.
During the late phase of cell injury in rabbit RPT,
Cl influx occurs followed
by cell swelling and lysis (17, 20). The observations that the
neurosteroids and muscimol prevented cell death/lysis when added 15 min
after antimycin A, did not preserve ATP levels, and blocked antimycin
A-induced Cl
influx
indicate that these compounds are acting subsequent to mitochondrial
dysfunction, ATP depletion, and
Na+/K+
imbalances and prior to Cl
influx in the late phase of cell injury. The inability of the neurosteroids and muscimol to preserve ATP concentrations is consistent with studies showing that cytoprotective concentrations of glycine do
not reverse the ATP depletion caused by antimycin A (36).
Ca2+ influx also occurs in the
late phase of cell injury in rabbit RPT (10, 29, 39). Furthermore, we
have recently shown that 1)
Ca2+ influx occurs in the late
phase of cell injury produced by antimycin A,
2) the
Ca2+ channel blocker nifedipine
blocks antimycin A-induced
Ca2+ influx,
3) nifedipine blocks cell
death/lysis, and 4) nifedipine blocks antimycin A-induced
Cl influx (37). These
results are consistent with the previous observation that
Cl
channel inhibitors
reported to inhibit Ca2+-activated
Cl
channels (34),
5-nitro-2-(3-phenylpropylamino)benzoate, indanyloxyacetic acid
(i.e., IAA-94), and niflumic acid, inhibit
Cl
influx and are
cytoprotective against antimycin A-induced RPT cell death/lysis (27,
38). Experiments designed to determine whether the neurosteroids and
muscimol inhibit antimycin A-induced Ca2+ influx revealed that muscimol
inhibited Ca2+ influx, whereas the
neurosteroids did not. Therefore, the cytoprotective actions of
muscimol and the neurosteroids are at two different sites; muscimol
acts prior to and blocks Ca2+
influx, whereas the neurosteroids act subsequent to
Ca2+ influx and prior to
Cl
influx. Figure
9 illustrates our current hypothesis
for the mechanism of cytoprotection produced by the neurosteroids and
muscimol in the late phase of cell injury. Further research is needed
to thoroughly dissect the cytoprotective mechanisms of action of the
neurosteroids and muscimol and correlate cytosolic free
Ca2+ levels with
cell injury and cytoprotection.
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Our laboratory has observed that a variety of compounds including
glycine receptor agonists and antagonists,
GABAA receptor agonists,
antagonists, and modulators (1, 16-18; also, above data), and
Cl channel inhibitors (38)
inhibit toxicant- and anoxia-induced extracellular
Cl
influx and cell
death/lysis in rabbit RPT suspensions. These results support Leaf's
(11) original demonstration that
Cl
influx is critical for
the cell swelling produced by mitochondrial inhibition. In contrast,
Venkatachalam et al. (36) have suggested that the cytoprotective
effects of glycine, strychnine, cyanotriphenylboron, and avermectin
B1a and its analogs
are not the result of the inhibition of
Cl
influx. However, in
their experiments, Cl
influx was not examined. In addition, their
Cl
substitution experiments
were conducted under conditions of complete Cl
substitution with either
gluconate or sucrose, a condition which is inherently damaging to the
cells (Miller and Schnellmann, unpublished observations). Although it
is not clear whether glycine and
GABAA receptor modulators act
directly on a Cl
channel to
inhibit Cl
influx or
indirectly immediately prior to
Cl
influx, the data
strongly suggest that the inhibition of
Cl
influx is involved in
the cytoprotection.
Characterization of a large variety of
GABAA receptor modulators
demonstrated that agonists, antagonists, and modulators
were cytoprotective in RPT (Table 1). The original
characterizations of the actions of these
GABAA receptor compounds and their
subsequent categorization into pharmacological groups were conducted in
neuronal tissue. Their action as cytoprotectants in nonneuronal tissue does not appear to follow the same pattern as that observed in neuronal
tissue. In some cases, millimolar concentrations of antagonists and
agonists exhibit cytoprotection (bicuculline, norharmane, GABA, THIP),
whereas other antagonists and modulators exhibit no cytoprotection
(RU-5135, picrotoxin, t-butylbicyclophosphorothionate, flurazepam, -carboline-3-carboxylic acid N-methyl). In
addition, differences were observed in the mechanism of cytoprotection
between the agonist muscimol and the neurosteroids. Finally, the lack of a specific binding site for the most potent cytoprotectants with
specific structural determinants implies that these compounds may act
at a site other than the extracellular surface of the plasma membrane.
These observations question whether a
GABAA-like receptor is present
in RPT and plays a role in cell injury/death.
Preliminary studies by Plotkin et al. (26) and Molony et al. (21, 22)
identified the GABAA receptor
3-subunit in rat kidney cortex
and
1-subunit mRNA in rat
kidney medulla, respectively. Investigators have shown also that
GABA-catabolizing and GABA-anabolizing enzymes, and two
GABA uptake systems exist in the rat renal cortex (4, 32). Recent
studies have shown that the GABAA
receptor agonists GABA and muscimol and the antagonist bicuculline
elicit a concentration-dependent increase in the fractional excretion of both water and sodium in the isolated perfused rat kidney (23). The
differential cytoprotective effects and potencies of the
GABAA receptor
agonists/antagonists/modulators indicate that if a
GABAA-like receptor does exist in
the kidney, then it is significantly different from the neuronal form
of the receptor.
AP, PS, and muscimol completely ameliorated hypoxia/reoxygenation-induced cell lysis as determined by the prevention of LDH release. The cytoprotective effects of the neurosteroids and muscimol were not restricted to the prevention of cell lysis but also allowed the RPT to regain cellular functions. The addition of AP, PS, or muscimol to RPT subjected to hypoxia/reoxygenation improved basal oxygen consumption 1.6-fold. Furthermore, AP, PS, and muscimol increased active Na+ transport from 10% of controls in hypoxia/reoxygenation-treated RPT to 80% of controls. These data strongly suggest that the neurosteroids and muscimol not only prevent RPT cell lysis but also enable the restoration of normal mitochondrial function and ion transport and therefore are true cytoprotectants.
Numerous investigators have discussed the cascade of events that lead to cell death and the "point of no return." The idea is that once the point of no return is reached, any intervention will not prevent the cell from dying. Although our data do not specifically identify the point of no return, the observation that the neurosteroids do not block extracellular Ca2+ influx but allow the cells to regain mitochondrial function and active Na+ transport suggests that the point of no return is distal to Ca2+ influx.
In summary, this study has demonstrated that compounds that interact
with the neuronal GABAA receptor
are potent cytoprotectants. Nanomolar concentrations of the
neurosteroids are cytoprotective in RPT subjected to mitochondrial
inhibition and hypoxia with strict structural requirements for both
efficacy and potency. The mechanism of action for neurosteroid
cytoprotection does not appear to involve specific receptor binding to
the extracellular surface of the RPT plasma membrane. The neurosteroids
and muscimol are cytoprotective during the late phase of cell death and
act differently: muscimol inhibits antimycin A-induced
Ca2+ influx, whereas the
neurosteroids inhibit Cl
influx.
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ACKNOWLEDGEMENTS |
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We thank Satinder S. Sarang and Jeffrey H. Moran for technical assistance.
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FOOTNOTES |
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S. L. Waters is a recipient of an Arkansas American Heart Association Predoctoral Fellowship.
Portions of this work have been published in abstract form and were presented at the 1995 American Society of Nephrology meeting in San Diego, CA (J. Am. Soc. Nephrol. 6: 1006, 1995), at the 1996 Society of Toxicology meeting in Anaheim, CA (Fund. Appl. Toxicol. 30: 304, 1996), and at the 1996 American Society of Nephrology meeting in New Orleans, LA (J. Am. Soc. Nephrol. 7: 1836, 1996).
Current addresses: G. W. Miller, Dept. of Cell Biology, Duke Univ., Durham, NC 27710; and Michael D. Aleo, Drug Safety Evaluation, Pfizer, Groton, CT 06340.
Address for reprint requests: R. G. Schnellmann, Division of Toxicology, Slot 638, 4301 W. Markham, Little Rock, AR 72205-7199.
Received 21 January 1997; accepted in final form 24 July 1997.
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