2 Veterans Administration and Department of Medicine1,2, University of Louisville, School of Medicine, Louisville, Kentucky 40202
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ABSTRACT |
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Parathyroid hormone (PTH) and dopamine
(DA) inhibit Na-K ATPase activity and sodium-phosphate cotransport in
proximal tubular cells. We previously showed that PTH and DA inhibit
phosphate transport in opossum kidney (OK) cells through different
signaling pathways. Therefore, we hypothesized that PTH and DA also
inhibit Na-K ATPase through divergent pathways. We measured PTH and DA inhibition of Na-K ATPase activity in the presence of inhibitors of
signaling pathways. PTH and DA inhibited Na-K ATPase in a biphasic manner, the early inhibition through protein kinase C (PKC)- and phospholipase A2 (PLA2)-dependent pathways and
the late inhibition through protein kinase A- and
PLA2-dependent pathways. Inhibition of extracellular
signal-regulated kinase (ERK) activation blocked early and late
inhibition of Na-K ATPase by PTH but not by DA. Pertussis toxin blocked
early and late inhibition by DA but not by PTH. Treatment with DA, but
not PTH, resulted in an early downregulation of basolateral membrane
expression of the -subunit, whereas total cellular expression
remained constant for both agonists. We conclude that PTH and DA
regulate Na-K ATPase by different mechanisms through activation of
divergent pathways.
protein kinase A; protein kinase C; phospholipase A2; opossum kidney cells; extracellular signal-regulated kinase
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INTRODUCTION |
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NA-K
ATPASE IS RESTRICTED to the basolateral membrane domain
of renal epithelial cells, where it provides the driving force for the
vectorial transport of various solutes and ions (28). In
renal proximal tubules, the activity of Na-K ATPase is regulated by
several signal transduction pathways, including protein kinase A (PKA),
protein kinase C (PKC), and various eicosanoids (4-16, 19-21, 23, 32, 34-37, 41). Dopamine (DA) and
parathyroid hormone (PTH) inhibit the activity of Na-K ATPase in renal
proximal tubular cells through similar and dissimilar pathways
(1-3, 12, 14, 17, 38-40, 45). DA stimulates two
classes of receptors, DA1 and DA2, which couple
to Gs and Gi (26), whereas PTH
inhibits the activity of Na-K ATPase activity by stimulation of a
single class of PTH/PTHrP receptors that are coupled to Gs
and Gq/G11 (27). DA inhibits Na-K
ATPase activity through activation of at least two parallel pathways,
the effects of which on Na-K ATPase vary depending on the duration
of treatment (1-3, 9, 17, 38). Short-term inhibition
of Na-K ATPase activity by DA is dependent on stimulation of
DA1 and DA2 receptors through activation of the
phospholipase C (PLC)-PKC cascade. However, long-term inhibition of
Na-K ATPase activity by DA occurs through a PKA-dependent pathway. PTH
also inhibits Na-K ATPase initially through activation of PKC
(14, 39-40) but through PKA-dependent pathways in the
long term (38). Both DA and PTH inhibit Na-K ATPase
activity by the activation of calcium-independent phospholipase
A2 (PLA2), which releases arachidonic acid
(AA). AA is metabolized by cytochrome P-450 to
-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE regulates rat renal
Na-K ATPase via PKC activation (34).
Our laboratory has previously shown that, in opossum kidney (OK) cells,
a model of renal proximal tubule, PTH and DA regulation of
sodium-dependent phosphate transport differ in magnitude and duration
despite activation of many similar signal transduction pathways
(29). We have attributed these differences to the fact that PTH and DA also activate some dissimilar pathways. PTH, but not
DA, regulates sodium-dependent phosphate transport through activation
of the mitogen-activated protein kinase, i.e., extracellular signal-regulated kinase (ERK) (30). DA, but not PTH,
inhibits sodium-dependent phosphate uptake by a pertussis
toxin-sensitive pathway. In preliminary experiments, we have
demonstrated that inhibition of Na-K ATPase by 1 µM ouabain for 30 min inhibits sodium-dependent phosphate uptake (21.83 ± 2.18 vs.
8.80 ± 0.14 nmol Pi · mg
protein1 · min
1, n = 2) in OK cells, confirming that Na-K ATPase activity contributes to
sodium-dependent phosphate transport. We speculated that differences in
the regulation of Na-K ATPase by PTH and DA could partially explain the
differences in mechanisms of regulation of phosphate uptake by these
agonists. The present study was undertaken to compare regulation of
inhibition of Na-K ATPase by PTH and DA in OK cells. We hypothesized
that PTH and DA would regulate Na-K ATPase through divergent signaling
mechanisms, similar to differences in regulation of phosphate transport.
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MATERIALS AND METHODS |
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Materials.
Wild-type OK cells were a generous gift from Dr. Steven Scheinman
(Health Sciences Center, Syracuse, NY). Antibody against the
-subunit of the OK cell Na-K ATPase was obtained from RBI (Natick,
MA). Phosphoserine antibody was purchased from Zymed Laboratories (San
Francisco, CA). Antibodies to PKC isoforms were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Bovine parathyroid hormone
[PTH(1---34)] was obtained from Bachem (Philadelphia, PA). DA,
PD-098059, and calphostin C were purchased from Calbiochem. H89 was
purchased from Biomol Research Laboratories (Plymouth, PA). Bromoenol
lactone (BEL) and 20-HETE were obtained from Cayman Chemicals (Ann
Arbor, MI). ATP, ouabain, AA, and ferrous sulfate were purchased from
Sigma (St. Louis, MO). Other reagents were of the highest quality available.
Cell culture. Wild-type OK cells, passages 83-89, were grown as monolayers in 175-cm2 plastic flasks (Falcon) or 3 × 10-mm tissue culture plates (Nunc) in Eagle's medium with Earle's salts (GIBCO BRL Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum, 4 mM glutamine, 100 µg/ml streptomycin, and 100 IU/ml penicillin in a humidified 5% CO2-95% air environment at 37°C. They were fed three times a week and split 1:4 once a week by trypsinization and dispersal. Cells were used for experiments at 100% confluence. They were washed the evening before use. For experiments requiring preincubation with specific inhibitor agents, these agents were added for the designated period of time at 37°C, followed by the addition of the signaling pathway activator also for the designated period of time. The inhibitor was present during the entire incubation period. The reaction was stopped by washing with ice-cold Hanks' basic salt solution (HBSS) followed by homogenization. Phosphatase inhibitors were not present.
Membrane isolation. The cells were washed twice with HBSS and homogenized in 300 mM mannitol-5 mM Tris-HEPES buffer, pH 7.6, using a 27.5-G needle. The cell lysate was centrifuged at 2,000 rpm for 10 min to remove the cell debris, and crude membranes were isolated by centrifugation of the supernatant at 17,000 rpm for 30 min. The pellet was resuspended in 300 mM mannitol-5 mM Tris-HEPES buffer, pH 7.6.
Protein determination. Protein concentration was determined by a DC protein kit (Bio-Rad) using BSA as standard.
Determination of Na-K ATPase activity. The activity of Na-K ATPase was determined in OK cell membranes by the method of Szczepanska-Konkel et al. (43). The cell membranes (50 µg protein) were incubated for 15 min at 37°C in medium containing in a final volume of 1.5 ml (final concentration in mM) 4.8 ATP, 120 NaCl, 24 KCl, 7.2 MgSO4, and 48 Tris · HCl, pH 7.6, with or without 1.2 mM ouabain. The reaction was terminated with 0.3 ml 30% TCA. The difference in the ATPase activity assayed in the absence and presence of ouabain is taken as a measure of Na-K ATPase. Na-K ATPase activity is expressed as nanomoles Pi released per milligram protein per hour.
Determination of Pi. Pi released due to the action of Na-K ATPase was determined by the method of Tausky and Shorr (44) in protein-free supernatant. One milliliter of the supernatant was diluted to 1.8 ml with glass-distilled water, and 1.2 ml of ferrous sulfate reagent [5 gm FeSO4 dissolved in 10% (wt/vol) ammonium molybdate in 10 N H2SO4] was added. A calibration curve was prepared simultaneously with the test samples, using known concentrations of KH2PO4 (9-180 nmol Pi) and 4.8 mM ATP. The blue color obtained was read at 820 nm after a 20-min incubation at room temperature in a Hewlett Packard 8453 spectrophotometer against a reagent blank.
Isolation of basolateral membranes.
The cells were grown on inserts in a six-well plate. After they reached
confluence, the cells were treated with 107 M
PTH(1---34), 10
6 M PTH(3---34), or 10
5M DA
on both sides of the inserts. The inserts were washed with Tris-buffered NaCl (154 mM). The cells were lysed in 50 mM mannitol-5 mM Tris-HEPES buffer, pH 7.0, and homogenized using a high-speed homogenizer (Powergen 125, Fisher Scientific). MgCl2 was
added in a final concentration of 10 mM to the homogenate and incubated for 20 min on ice with occasional stirring. The homogenate was centrifuged at 2,500 g for 5 min at 4°C. The pellet was
resuspended in 100 mM Mannitol-5 mM Tris-HEPES buffer, and
MgCl2 was added to a final concentration of 15 mM. The
suspension was incubated for 20 min on ice with occasional stirring and
centrifuged at 2,500 g for 5 min. The pellet was again
resuspended in 100 mM mannitol-5 mM Tris-HEPES buffer, pH 7.4, and
centrifuged at 750 g for 15 min. The supernatant was removed
and centrifuged at 48,000 g in an Ultracentrifuge (Beckman)
for 30 min. The pellet derived from centrifugation of the supernatant
was resuspended in 50% sucrose, using a dounce homogenizer. The sample
was overlaid on a discontinuous sucrose gradient, made by mixing 12 ml
38% sucrose with 5 ml 41% sucrose, and centrifuged at 88,000 g in an Ultracentrifuge (Beckman) for 3 h. The upper
layer was carefully collected and resuspended in 1 mM bicarbonate
buffer, pH 7.5, and centrifuged at 48,000 g in a Beckman
Ultracentrifuge for 30 min. The pellet was resuspended in 300 mM
mannitol-5 mM Tris-HEPES buffer, pH 7.4 (33). The
basolateral membranes were characterized by the five- to eightfold
enrichment of Na-K ATPase (data not shown).
Immunoblots. OK cell membrane proteins were solubilized in Laemmli sample buffer, separated by 10% SDS-PAGE, and transferred electrophoretically to a nitrocellulose membrane. The nitrocellulose sheet was incubated with 5% milk in 20 mM Tris and 50 mM NaCl (TBS) at room temperature for 60 min, in an appropriate dilution of primary antibody in TBS with 0.2% Tween (TTBS) for at least 60 min at room temperature or overnight at 4°C, and an appropriate concentration of horseradish peroxidase-linked goat anti-rabbit IgG (or secondary antibody of appropriate species) for 50 min at room temperature, washed, and developed by chemiluminescence assay (Biolab). The developed blots were scanned and analyzed using a Meridian densitometer and expressed as densitometric units.
Immunoprecipitation of Na-K ATPase.
The crude membranes were precleared with protein A-Sepharose beads for
2 h at 4°C and were incubated with 1 ng of rabbit polyclonal antibodies against the -subunit of Na-K ATPase (Research
Diagnostics, Flanders, NJ) overnight at 4°C. Protein A-Sepharose
beads were added and incubated for 2 h at 4°C. The beads were
washed three times with 1× PBS, and an equal volume of 2× Laemmli
sample buffer was added and boiled for 5 min. The beads were
centrifuged, and the proteins were separated by 10% SDS-PAGE,
transferred to nitrocellulose membranes, and blotted against
phosphoserine antibodies (Zymed Laboratories, San Francisco, CA).
Statistics. Data are shown as means ± SE. The n values shown represent the number of separate experiments. Each experiment was done in triplicate. The P value was calculated using SigmaStat software utilizing Student's t-test. A P value <0.05 was a priori considered as statistically significant.
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RESULTS |
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Effect of PKA and PKC activation on Na-K ATPase activity in OK
cells.
Activation of PKC decreases the activity of Na-K ATPase in proximal
tubular cells. The effect of activation of PKA in proximal tubule cells
is somewhat controversial, with some studies suggesting an increase in
activity (10, 23) whereas others suggest a decrease
(1, 9). To determine the effect of activation of PKA or
PKC on Na-K ATPase activity in OK cells, the cells were treated with
106 M 8-bromoadenosine 3',5'-cyclic monophosphate
(8-BrcAMP), a phosphodiesterase-resistant cAMP analog that directly
activates PKA, or 10
4 M phorbol 12-myristate 13 acetate
(PMA), a phorbol ester that directly activates PKC, for 15 min or
2 h. Activation of PKA by 8-BrcAMP caused a time-dependent
decrease in the activity of Na-K ATPase. Activation of PKC by PMA
caused maximum inhibition of Na-K ATPase activity after a 15-min
incubation, and the degree of inhibition did not change after 2 h
of treatment (Fig. 1, A and
B).
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PTH and DA inhibit Na-K ATPase in OK cells.
In renal proximal tubules, PTH and DA regulate Na-K ATPase through
biphasic signaling pathways. PTH inhibits Na-K ATPase activity by
15-30% after short-term (15 min) (39, 40) and by
50% after long-term (2 h) incubation (38). DA inhibits
Na-K ATPase activity by 35-40% after short- and long-term
incubation (1, 3, 9, 17, 38). To confirm that these
agonists had similar effects in OK cells, we measured Na-K ATPase
activity in OK cell membranes after 15-min and 2-h incubation with
107 M PTH(1---34) and 10
5 M DA. PTH
inhibited Na-K ATPase by 35% (21.00 ± 1.37 vs. 13.74 ± 0.46 nmol Pi released · mg
protein
1 · h
1, n = 3, P < 0.005) after 15 min and by 57% (22.45 ± 1.82 vs. 9.73 ± 0.99 nmol Pi released · mg
protein
1 · h
1, n = 3, P < 0.005) after 2-h incubation (Fig.
2, A and B). DA resulted in 32% (21.00 ± 1.37 vs. 14.25 ± 0.41 nmol
Pi released · mg
protein
1 · h
1, n = 3, P < 0.005) inhibition after 15 min and 59%
(22.45 ± 1.82 vs. 9.28 ± 0.70 nmol Pi
released · mg
protein
1 · h
1, n = 3, P < 0.005) inhibition of Na-K ATPase activity after
2 h incubation (Fig. 2, A and B).
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Signaling pathways involved in regulation of Na-K ATPase. In renal proximal tubules, PTH and DA inhibit Na-K ATPase activity through multiple signaling pathways. To examine the pathways responsible for PTH and DA inhibition of Na-K ATPase in OK cells, we measured Na-K ATPase inhibition in cells treated with specific inhibitors of several signaling pathways.
To determine whether DA and PTH regulate Na-K ATPase activity through a PKC-dependent mechanism, the cells were pretreated with 10
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Mapping agonist-stimulated pathways involved in regulation of Na-K ATPase. The previous results implicate several signaling molecules in the regulation of Na-K ATPase: PKA, PKC, and PLA2 in the case of DA and PKA, and PKC, PLA2, and ERK in the case of PTH. The requirement for PKC activation in the short-term regulation of Na-K ATPase suggests that PKC activation may be the initiating step. To map out the remainder of the pathway, OK cells were stimulated with specific activators of PKC or with PLA2 metabolites after pretreatment with specific inhibitors of the other pathways.
Figures 9, A and B, show the effects of inhibitors of PLA2 and ERK on regulation of Na-K ATPase by PKC activated by 10
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Mechanisms of short- and long-term regulation of Na-K ATPase.
Previous investigators have demonstrated that short-term
regulation of Na-K ATPase by DA in proximal tubule cells is dependent on phosphorylation of the -subunit followed by endocytosis into clathrin-coated vesicles. These processes ultimately result in decreased expression of the subunit, producing long-term inhibition of
Na-K ATPase. To determine whether PTH, like DA, decreased expression of
the
-subunit in OK cells, we immunoblotted membrane proteins from
OK cells for the
-subunit of Na-K ATPase and for
phosphoserine after treatment with PTH(1---34), PTH(3---34), DA,
20-HETE, PMA, and 8-BrcAMP. Figure
11A shows that 15-min
incubation with all agonists did not alter the expression of Na-K
ATPase. Immunoblotting with the phosphoserine antibody in Na-K
ATPase-immunoprecipitated membranes showed increased phosphorylation,
especially with PTH(1---34), PTH(3---34), DA, 20-HETE, and AA (Fig.
11B). Figure 11C shows the results of 2-h
incubation, demonstrating a marked decrease in expression of the
-subunit in response to all agonists. Consequently, the phosphoserine immunoblot shows no phosphorylation (Fig.
11D).
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Effect of PTH and DA on basolateral membrane expression of Na-K
ATPase.
For the study of the effect of PTH and DA on basolateral membrane
expression of Na-K ATPase, cells were grown on membrane inserts placed
in six-well plates to optimize cell polarization and treated with
PTH(1---34 or 3---34) and DA. Immunoblot analysis of isolated
basolateral membranes showed that DA, but not PTH, decreased the
basolateral expression of Na-K ATPase (Fig.
12) after 15 min of treatment.
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DISCUSSION |
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The function of the renal Na-K ATPase is central to the regulation of all transport processes in the proximal renal tubule. Although many hormones regulate transport processes through alterations in specific membrane transporter expression and function, several hormones exert a more global regulation of proximal renal tubule transport through alterations in the activity of the Na-K ATPase. For example, insulin (18) and norepinephrine (35) increase Na-K ATPase activity, thereby increasing proximal renal tubule sodium reabsorption and preserving extracellular fluid volume. In contrast, DA (2) and PTH (43) inhibit Na-K ATPase activity, resulting in decreased proximal renal tubule sodium reabsorption and decreased extracellular fluid volume. Not surprisingly, the pathways regulating Na-K ATPase activity are numerous, often redundant, and often antagonistic. Furthermore, regulatory mechanisms activated by several of the hormones can differ depending on the length of time of exposure to specific hormonal influences.
Previous publications investigating the regulation of Na-K ATPase in several models of proximal renal tubule, including OK cells, have yielded conflicting results. Some investigators have demonstrated an increase in Na-K ATPase activity after activation of PKA or PKC, whereas others have demonstrated a decrease. Our studies concur with the latter result. The explanation for these conflicting results is not immediately apparent, but they may be due to differences in the technique for measuring Na-K ATPase activity or differences in the several models of proximal tubule. Many of the reports showing an increase in Na-K ATPase measure activity as rubidium uptake, whereas we have measured enzyme activity directly in membrane preparations. Discrepancies in methodology, however, cannot fully explain this controversy, as the opposing effects on Na-K ATPase by PKA and PKC have been confirmed by demonstrating concurrent appropriate increases or decreases in epithelial transport. In some instances, different time points have been chosen for measurement of Na-K ATPase. We have chosen 15 min and 2 h on the basis of previous reports demonstrating the time course for phosphorylation of subunits (short-term regulation) and decreased expression of subunits (long-term regulation).
It is of interest to compare the mechanisms of DA and PTH on the
regulation of the proximal renal tubule Na-K ATPase, as both agonists
inhibit the activity of the Na-K ATPase, inhibit sodium-dependent hydrogen exchange, and inhibit sodium-dependent phosphate uptake. Because the characteristics of their inhibition of phosphate transport differ significantly, we considered the possibility that differences in
the regulation of Na-K ATPase by DA and PTH could account for some of
these differences (3, 29). In fact, we did find
significant similarities and differences in the mechanisms activated by
PTH and by DA to inhibit Na-K ATPase in OK cells. PTH and DA inhibited Na-K ATPase similarly after 15 min and 2 h. Our findings agree with previous reports showing a biphasic inhibition of Na-K ATPase activity by PTH and DA. The initial, short-term inhibition is mediated
by PKC, whereas the long-term inhibition is mediated predominantly by
PKA. For both PTH and DA, short-term inhibition is associated with an
increase in phosphoserine labeling of the Na-K ATPase -subunit,
whereas long-term inhibition is accompanied by downregulation of the
subunit expression. Our findings confirm previous reports that
short-term regulation by PTH is quantitatively less than that seen with
long-term regulation (38-40). However, previous
reports have failed to show as we have here that short-term regulation
by DA is also quantitatively less than long-term regulation (38). The reasons for this discrepancy are not clear. One
possibility is that the concentration of DA used activates
-adrenergic receptors as well as DA receptors, which might have the
opposite effects on Na-K ATPase activity (29). This would
not explain why the inhibition of Na-K ATPase activity by DA differs at
the two time points, unless the effect of
-adrenergic stimulation on
Na-K ATPase is transient. Our studies also confirm that short-term and
long-term regulation of Na-K ATPase activity by PTH and DA were
dependent on PLA2 activity (14, 34, 36) in OK
cells, a situation similar to renal tubular cells. We have demonstrated that inhibition of Na-K ATPase by DA involves both DA1 and
DA2 receptors, as has been shown in proximal renal tubule
cells (38).
PTH and DA inhibition of Na-K ATPase differed in several respects. Inhibition of Na-K ATPase activity by DA, but not PTH, was blocked by pretreatment with pertussis toxin. This finding is not surprising in view of the well-established coupling of the PTH/PTHrP receptor predominantly to Gs and Gq, with little coupling to Gi (25, 27). DA receptors couple with pertussis toxin-sensitive G proteins (26). Another significant difference between PTH and DA regulation of Na-K ATPase activity is that PTH, but not DA, regulation was dependent on ERK activity. We have previously reported that PTH inhibition of phosphate uptake is dependent, in part, on ERK activation, whereas inhibition of phosphate uptake by DA is not (30). These data suggest that PTH inhibits sodium-dependent phosphate uptake, in part, by inhibition of Na-K ATPase through an ERK-dependent pathway. ERK regulates Na-K ATPase in other cell types (22). ERK regulation of Na-K ATPase in renal cells has not been reported until now. How PTH- but not DA-stimulated ERK could play a role in the regulation of Na-K ATPase is not apparent.
Previous studies from other laboratories suggest that PKC activation is
the final step in the pathway stimulated by DA and PTH, directly
phosphorylating the -subunit, leading to its endocytosis. In this
scenario, PTH or DA activates phospholipase C, resulting in activation
of PLA2 (31). The products of PLA2
stimulation activate PKC (34, 36). We demonstrated that
inhibition of Na-K ATPase by PLA2 metabolites is blocked by
PKC inhibition. Our data, however, also suggest the possibility of a
more upstream site of action for PKC. PTH-stimulated ERK is blocked by
inhibition of PKC, suggesting that PKC activation occurs before ERK
activation (30). In this study we have shown that
inhibition of Na-K ATPase activity by PKC activation through PMA or
PTH(3---34) can be blocked by inhibitors of PLA2 and ERK,
suggesting also that PKC is an upstream event. PTH-stimulated
phospholipase C could activate PKC (4, 11-13,
19-20), leading to sequential activation of
PLA2 and/or ERK. Such a mechanism for PLA2
activation has been reported in other cell types (reviewed in Ref.
31). Activated PLA2 may then stimulate a
second PKC activation, activating either the same or a different PKC
(15, 32). Alternatively, simultaneous PKC and ERK
activation may be required for full inhibition of Na-K ATPase through
parallel additive pathways. Further analysis of the signal pathways
will be required to answer these questions.
We demonstrated that 15-min treatment with DA, but not PTH, diminishes
basolateral membrane expression of the -subunit of Na-K ATPase.
Total cellular expression remains unchanged after short-term treatment
with both agonists. After 2 h, PTH and DA both decrease expression
of the
-subunit. Previous investigators have demonstrated that DA
stimulates endocytosis of Na-K ATPase proteins into clathrin-coated
vesicles. This has not been shown for PTH. Our findings suggest
that, in all likelihood, PTH downregulates
-subunit expression
through a different pathway.
In summary, we have demonstrated that PTH and DA inhibit Na-K ATPase by some similar and some different mechanisms in proximal renal tubule cells, possibly accounting for some of the differences in PTH and DA regulation of tubular transport.
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical assistance of Nina Lesousky.
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FOOTNOTES |
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This work was supported by a grant from the Veterans Administration Merit Review Board (E. Lederer). S. J. Khundmiri is recipient of a Fellowship Award from the American Heart Association, Ohio Valley Affiliate.
Address for reprint requests and other correspondence: E. Lederer, Kidney Disease Program, Univ. of Louisville, 615 S. Preston St., Louisville, KY 40202 (E-mail: elederer{at}kdp01.kdp-baptist.louisville.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.
10.1152/ajprenal.00111.2000
Received 4 April 2000; accepted in final form 4 October 2001.
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