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Institute for Cardiovascular Studies, College of Pharmacy, University of Houston, Houston, Texas.
Correspondence to Dr. Mustafa F. Lokhandwala, Institute for Cardiovascular Studies, College of Pharmacy, University of Houston, Houston, TX 77204; Phone: 713-743-1253; Fax: 713-743-5678; E-mail: Mlokhandwala{at}uh.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-subunit
of Na,K-ATPase in adult but not in old rats. In adult rats, SKF 38393-mediated
phosphorylation was antagonized by SCH 23390, a D1-like receptor
antagonist. Similarly, Na,K-ATPase activity was inhibited by SKF 38393 and
PDBu in adult but not in old rats. The basal activity of Na,K-ATPase was
decreased and the basal phosphorylation state of the enzyme was increased in
old compared with adult rats. Basal activity of PKC was higher in old compared
with adult rats, and SKF 38393 and PDBu stimulated PKC activity in adult but
not in old rats. The conclusion is that the failure of D1-like
receptor agonist and phorbol ester to stimulate PKC and inhibit Na,K-ATPase
activity in old rats is due, at least in part, to the higher basal PKC
activity and Na,K-ATPase phosphorylation in old compared with adult rats. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is reported that the natriuretic response to dopamine and SKF 38393, a
D1-like receptor agonist, is reduced in spontaneously hypertensive
rats (SHR) compared with Wistar Kyoto (WKY) rats
(11,12).
Such a blunted response to D1-like receptor stimulation in SHR is
reportedly due to a defective D1-like receptor coupled signal
transduction pathway, which causes a reduced inhibition of Na,K-ATPase and
Na,H exchanger in SHR
(13,14,15).
A similar defect in D1-like receptor signaling and function was
also found in the PT of humans with essential hypertension
(16). Furthermore, this study
(16) suggested that the
defective function of D1-like receptor may be due to an increased
level of ligand-independent phosphorylation of the receptor, which leads to
its uncoupling from the G proteins. In addition to such a defect at the level
of D1-like receptor, it was found that there was a
D1-like agonist-induced differential regulation of PKC isoforms
(PKC-
and PKC-
) in SHR and WKY rats
(17), which contributed to the
failure of dopamine to stimulate PKC
(18) and inhibit Na,K-ATPase
activity (13) in the PT of
SHR. The basal PKC activity
(18) and PDBumediated
inhibition of Na,K-ATPase (13)
was not different in SHR compared with WKY rats.
The normal aging process leads to structural changes in the kidney, which
have been associated with changes in renal function such as decline in renal
blood flow and GFR
(19,20).
Renal deficiency in L-dopa uptake and its conversion to dopamine
(21) and a reduced dopamine
receptor number and its defective coupling with G proteins have been reported
in the kidney of old rats
(22). Recently, we reported
that dopamine failed to inhibit Na,K-ATPase activity in old rats, in part due
to a decreased number of D1-like receptor-binding sites and
defective D1-like receptor-G protein coupling in the PT of these
animals (22). In addition to a
defect at the level of D1-like receptor, it is likely that there
may exist alterations in other components of the signal transduction pathway,
such as PKC activity and the phosphorylation state of Na,K-ATPase itself,
which would contribute to the failure of dopamine to inhibit Na,K-ATPase
activity in old rats. Therefore, the present study was designed to examine the
potential alterations in receptor-mediated (using D1-like agonist
SKF 38393) and non-receptor-mediated (using PKC activator PDBu) activation in
PKC activity and phosphorylation of the 1-subunit of
Na,K-ATPase in adult and old rats. Our results show that there is an increase
in the basal PKC activity and subsequent phosphorylation of the Na,K-ATPase in
old rats under resting state. This abnormality beyond the level of
D1-like receptor may also contribute to the failure of receptor-
and non—receptor-mediated phosphorylation and subsequent inhibition of
Na,K-AT-Pase in old rats.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation and Enrichment of PT
Rat kidneys were used to prepare and enrich proximal tubular suspension
according to a routinely used procedure in our laboratory and described
elsewhere (13).
PKC Assay
Renal PT were incubated with SKF 38393 (1 nM to 1 µM) or PDBu (1 nM to 1
µM) for 20 min at 30°C. PKC activity was measured using a fluorescence
peptide substrate (PLSRTLSVAAK) as described in the PKC assay kit
manufacturer's manual (Promega, Madison, WI) and as we have described earlier
(8). The fluorescence peptide
phosphorylated by the activation of PKC was resolved on a 0.8% agarose gel.
The gel was photographed under ultraviolet light, and the bands were
densitometrically quantified.
Na,K-ATPase Assay
The PT were treated with PDBu (1 nM to 1 µM) or SKF 38393 (1 nM to 1
µM) for 20 min at 37°C. The incubation was terminated by rapid freezing
on dry ice-acetone and thawing. After thawing, the samples were used for
Na,K-ATPase assay using end phosphate hydrolysis of ATP and colorimetric
measurement as routinely used in our laboratory and described earlier
(13). Ouabain-sensitive ATPase
(Na,K-ATPase) was calculated as the difference between total ATPase activity
and ATPase activity in the presence of 1 mM ouabain. The sodium concentration
used in this set of Na,K-ATPase assay was 75 mM, which is a saturating (Vmax)
concentration (23).
Basal activity of Na,K-ATPase was also measured in the membranes prepared from the outer cortex of the kidneys at low sodium (5 mM) as well as saturating sodium (75 mM) concentration (23). Low sodium (5 mM) in the reaction mixture was compensated with Tris buffer to maintain osmolality. Other details of the assay are similar as described above.
Immunoprecipitation of Na,K-ATPase
Transverse sections of the kidney were obtained with the help of a razor
blade. Under magnifying glass, thin superficial cortical tissue slices
(approximately 400 to 500 µm, rich in PT) were carefully dissected using a
razor blade. The tissue slices were placed in ice-cold oxygenated buffer
containing 1.5 mM CaCl2, 110 mM NaCl, 5.4 mM KCl, 1 mM
KH2PO4, 1 mM MgSO4, 25 mM NaHCO3,
25 mM D-glucose, and 2 mM HEPES (pH 7.6). The tissue slices were preincubated
with 1 µM okadaic acid and treated with SKF 38393 (10 µM) or PDBu (1
µM) for 20 min at 30°C while oxygenating. SCH 23390 (10 µM, a
D1-like receptor antagonist) was added 5 min before the addition of
SKF 38393. The incubation was terminated by quickly freezing the tissues on
dry ice-acetone mixture. The tissues were homogenized in immunoprecipitation
buffer containing 20 mM Tris-HCl, 150 mM NaCl, 10 mM NaF, 10 mM
Na2P4O7, 2 mM ethyl enediaminetetraacetate, 2
mM ethyleneglycol-bis(ß-aminoethyl ether)-N,N'-tetraacetic
acid, 1% triton X-100, 0.1% sodium dodecyl sulfate (SDS; pH 8.0), protease
inhibitor cocktail, and 0.1 mM phenylmethylsulfonyl fluoride. Homogenate was
centrifuged at 8000 x g for 15 min, and the supernatant was
precleared with Protein A/G Plus agarose for 30 min. An aliquot of precleared
supernatant (2 mg of protein/ml) was incubated with mouse monoclonal antibody
of the 1-subunit of Na,K-ATPase
(24). Antigen
(1-subunit of Na,K-ATPase)-antibody complex was precipitated
using ProA/G-agarose. The complex was washed once with immunoprecipitation
buffer followed by three washes with buffer containing 20 mM Tris-HCl, 150 mM
NaCl, 5 mM ethylenediaminetetraacetate, 0.1% triton X-100, 0.1% SDS, and once
with 50 mM Tris-HCl (pH 8.0). All of the steps of immunoprecipitation were
carried out at 4°C. Finally, the antigen-antibody complex was dissociated
with 50 µl of 2X Laemmeli buffer (125 mM Tris-HCl, 4% SDS, 5%
ß-mercaptoethanol, 20% glycerol) by incubating at 37°C for 1 h.
Measurement of Phosphorylated 1-Subunit of
Na,K-ATPase
The immunoprecipitated sample (10 µl) was resolved by SDS/polyacrylamide
gel (8%) electrophoresis. The resolved proteins were electrophoretically
transblotted on membrane (Immobilon-P, Millipore Corp., Bedford, MA). The
membrane was blocked with 5% nonfat dry milk followed by incubation with mouse
monoclonal phosphoserine antibody for 60 min. Horseradish peroxidase
conjugated goat anti-mouse second antibody incubation was performed for 60 min
at room temperature. The membrane was treated with enhanced chemiluminescence
reagent, and the bands were visualized on x-ray film. The bands were
quantitated by densitometry analysis using the Quantity One software program
(pdi, Inc., Huntington Station, NY).
Measurement of Total 1-subunit of Na,K-ATPase
The total (phosphorylated and nonphosphorylated) contents of the
Na,K-ATPase 1-subunit in the immunoprecipitated samples and
isolated membranes were detected by Western blotting following the same
procedure described above except that the mouse monoclonal
1-subunit of Na,K-ATPase antibody was used as the primary
antibody.
Measurement of Protein
Protein in cortical homogenates and in the membranes was measured using BCA
protein assay kit (Pierce, Rockford, IL) and bovine serum albumin as
standards.
Materials
SKF 38393, SCH 23390 and PDBu were purchased from Research Biochemical
International (Natick, MA). Okadaic acid and phosphoserine antibody were
purchased from Calbiochem (San Diego, CA). Monoclonal
1-subunit Na,K-ATPase antibody was purchased from Research
Diagnostics, Inc. (Flanders, NJ). Protein A/G Plus agarose was bought from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All of the other chemicals of
the highest purity available for various buffers were purchased either from
Sigma Chemical Co. (St. Louis, MO) or from Fisher Scientific Co. (Fair Lawn,
NJ).
Statistical Analyses
Results are presented as mean ± SEM. Statistical significance was
measured using t test (within the group) and ANOVA (between the
groups). P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the cortical membranes, the basal activity of Na,K-ATPase in old rats was lower by 42% at a lower sodium concentration (5 mM) and by 25% at saturating sodium concentration (75 mM) compared with adult rats (adult versus old: at 5 mM sodium, 307 ± 25 versus 178 ± 19 nmol Pi/mg protein per min; at 75 mM sodium, 746 ± 66 versus 555 ± 14 nmol Pi/mg protein per min; Figure 2A).
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Effect of SKF 38393 or PDBu on Na,K-ATPase Phosphorylation
Mouse monoclonal 1-subunit of Na,K-ATPase antibody
recognized two protein bands in the immunoprecipitate. The lower band
(approximately 101 kD) of the doublet showed phosphorylation upon detection
with phosphoserine antibody. To confirm that 101 kD protein is the real
1-subunit, we performed Western blot analysis of the
immunoprecipitate with another monoclonal 1-subunit
antibody, 5, kindly provided by Dr. Carlos Pedemonte (University of
Houston, Houston, TX). This antibody recognized only the lower band of the
doublet, confirming it as 1-subunit (data not shown). The
antibody 5 has been shown to recognize only one protein band in various
tissues, including in the kidney
(25). Phosphorylation of
Na,K-ATPase was calculated as the ratio of arbitrary density unit of
phosphorylated Na,K-ATPase to the total Na,K-ATPase protein (lower band,
approximately 101 kD). The basal phosphorylation of Na,K-ATPase was
significantly higher in old compared with adult rats (adult versus
old: 1.47 ± 0.12 versus 2.29 ± 0.39;
Figure 2B). However, basal
abundance of Na,K-ATPase protein was the same in the cortical membranes of
both adult and old rats (adult versus old: 1.42 ± 0.02
versus 1.33 ± 0.02 arbitrary units;
Figure 2C). As shown in
Figure 3, SKF 38393 (10 µM)
or PDBu (1 µM) increased phosphorylation (by 33 and 43%, respectively) of
1-subunit of Na,K-ATPase in the cortex slices of adult rats
but not in old rats. The stimulatory effect of SKF 38393 (10 µM) on
phosphorylation in adult rats was attenuated by SCH 23390 (10 µM), a
specific D1-like receptor antagonist
(Figure 3).
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To confirm the linearity of protein immunodetection, we loaded 5, 10, and
15 µl of immunoprecipitated samples from 6-mo old rats. Western blot
analysis for both phosphorylated and total l protein was performed. The
densitometric values (for 5, 10, and 15 µl, respectively) were as follow:
0.69, 1.41, and 2.01 for phosphorylated and 0.55, 1.07, and 1.57 for total
1 protein. The ratio of phosphorylated to total
1 protein (1.25, 1.31, and 1.26) did not change with the
change in sample quantity. The data suggest that the amount used (10 µl) in
the rest of the experiments was in linear range of immunodetection of the
proteins in our samples.
Effect of SKF 38393 or PDBu on PKC Activity
SKF 38393 (1 nM to 1 µM) stimulated PKC activity in the PT of adult
rats, whereas D1-like receptor activation failed to stimulate the
enzyme activity in the old rats (Figure
4A). Similarly, PDBu (1 nM to 1 µM), a phorbol ester and direct
activator of PKC, stimulated PKC activity in the adult but not in the old rats
(Figure 4B). The basal PKC
activity (peptide phosphorylated ng/µg protein per 20 min) was
significantly higher in old (15.2 ± 2.6) compared with adult (7.3
± 1.1) rats.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study was performed to determine whether there existed a defect
at the level of Na,K-ATPase that may also have contributed to the failure of
dopamine to inhibit the enzyme activity. In support of a defect at the level
of Na,K-ATPase, we found that in addition to SKF 38393, a direct activator of
PKC, namely PDBu also failed to inhibit the Na,K-ATPase activity in old rats.
These findings are different from what has been reported in terms of defective
dopamine response in the PT of SHR compared with normotensive WKY rats
(13). We found that although
dopamine inhibits Na,K-ATPase in WKY rats but not in SHR, PDBu causes
inhibition of the enzyme activity of similar extent in both SHR and WKY rats
(13), suggesting that the
defect is located primarily at the level of D1-like receptor and
not of Na,K-ATPase. Therefore, in the present study, we conducted additional
experiments to identify the nature of the defect at the level of Na,K-ATPase
in old rats. Inasmuch as it is the phosphorylation of the
1-subunit of Na,K-ATPase that leads to the inhibition of the
enzyme activity (27), we
measured the basal phosphorylation state of the 1-subunit of
Na,K-ATPase as well as resting enzyme activity and found an elevation in the
basal phosphorylation state and a decrease in the resting enzyme activity in
old rats. We found that both SKF 38393 and PDBu did not cause any
phosphorylation or inhibition of Na,K-ATPase in old rats. The most likely
explanation for this finding is that the enzyme was already phosphorylated and
inhibited under resting state, and therefore addition of either SKF 38393 or
PDBu could not produce any further increase in phosphorylation in old rats. It
should be noted that the total immunodetectable Na,K-ATPase protein content in
the cortical membranes of old rats was not different from adult rats. This
suggests that the decrease in Na,K-ATPase activity was due not to a decrease
in Na,K-ATPase protein but rather to an increase in the basal phosphorylation
of the enzyme. It is believed that phosphorylation of Na,K-ATPase causes a
decrease in the affinity of sodium with the enzyme leading to a decrease in
its activity. The basal enzyme activity was measured in the cortical membranes
in the presence of 5 and 75 mM sodium. At 5 mM sodium, the decrease in basal
activity in old compared with adult rats was greater (42%) than the decrease
(25%) in the activity measured at 75 mM sodium (Vmax concentration). This
observation further supports the suggestion that it is the phosphorylation of
the enzyme that caused a reduction in the basal activity of Na,K-ATPase in old
rats. Phosphorylation has also been implicated as a prerequisite of
internalization and finally inhibition of the enzyme activity
(27,28).
Because we found a decrease in the basal activity of Na,K-ATPase without
alteration in its immunodetectable protein content in the membranes of old
rats, it is suggested that the increased phosphorylation may have prevented
Na,K-ATPase from further inhibition by SKF 38393 and PDBu. Whether the
reduction in the basal activity of Na,K-ATPase has physiologic significance in
the normal process of aging and maintaining electrolyte homeostasis remains to
be determined. However, while the present research was in progress, published
was a study that reported a decrease in Na,K-ATPase activity without a change
in the immunodetectable protein content in the PT of old (30 mo) compared with
adult (4 mo) Fischer 344 rats
(29).
To explore the mechanism responsible for an increased basal phosphorylation
of Na,K-ATPase in old rats, we measured PKC activity in the presence of SKF
38393 and PDBu. We found that although SKF 38393 and PDBu stimulated PKC
activity in the PT of adult rats, these agents completely failed to stimulate
the enzyme in old rats. This could be because the basal activity of PKC in old
rats was similar to the stimulated activity in adult rats. Higher basal
activity of PKC probably is responsible for the increased phosphorylation and
the decreased activity of Na,K-ATPase in old rats. As mentioned earlier, while
the activation of D1-like receptor failed to produce inhibition of
Na,K-ATPase activity in SHR, PDBu-mediated inhibition of the enzyme was not
altered compared with WKY rats
(13). In addition, the basal
activity of PKC as well as of Na,K-ATPase in the PT of SHR was similar to WKY
rats (13). As it relates to
the old rats, it is important to identify further which isoforms of PKC that
may have caused an overall increase in the basal activity of PKC in old rats
are upregulated. In SHR, although there was no difference in the basal
activity of PKC, there was a differential regulation of certain PKC isoforms
(- and -isoform), which were linked to the decreased ability of
dopamine to promote natriuresis
(17). Differential regulation
of PKC isoforms and the higher activity of PKC have been reported in other
tissues, e.g., adipose tissue affecting insulin-mediated lipolysis in
old rats (30), and in the
heart and kidney of diabetic animals, where it was suggested to be linked to
impaired function of endothelium, capillaries, and arterioles
(31).
In summary, we found that in old rats, D1-like receptor activation of PKC or direct activation of the enzyme with PDBu fails to cause the phosphorylation and subsequent inhibition of Na,K-ATPase. This phenomenon most likely is due to the increase in the basal PKC activity in old rats. We conclude that such an abnormality contributes to the failure of dopamine and D1-like receptor agonist to inhibit Na,K-ATPase and subsequently increase sodium excretion in old rats.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-adrenergic agonist. Proc Natl Acad Sci
USA 90: 21-24,1993-subunit is necessary for specific /
association. J Biol Chem 270:14332
-14339, 1995-subunit and is responsible for the decreased activity in
epithelial cells. J Biol Chem274
: 1920-1927,1999This article has been cited by other articles:
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  J. W. Horton, J. Tan, D. J. White, and D. L. Maass Burn injury decreases myocardial Na-K-ATPase activity: role of PKC inhibition Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2007; 293(4): R1684 - R1692. [Abstract] [Full Text] [PDF]
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 R. Z. Fardoun, M. Asghar, and M. Lokhandwala Role of oxidative stress in defective renal dopamine D1 receptor-G protein coupling and function in old Fischer 344 rats Am J Physiol Renal Physiol, November 1, 2006; 291(5): F945 - F951. [Abstract] [Full Text] [PDF]
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 J. L. Carrington and F. L. Bellino Developing a Research Agenda in Biogerontology: Physiological Systems Sci. Aging Knowl. Environ., June 28, 2006; 2006(10): pe17 - pe17. [Abstract] [Full Text]
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 A. C. Hakam and T. Hussain Angiotensin II Type 2 Receptor Agonist Directly Inhibits Proximal Tubule Sodium Pump Activity in Obese But Not in Lean Zucker Rats Hypertension, June 1, 2006; 47(6): 1117 - 1124. [Abstract] [Full Text] [PDF]
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 M. Asghar and M. F. Lokhandwala Antioxidant Supplementation Normalizes Elevated Protein Kinase C Activity in the Proximal Tubules of Old Rats Experimental Biology and Medicine, March 1, 2004; 229(3): 270 - 275. [Abstract] [Full Text] [PDF]
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M. Asghar, T. Hussain, and M. F. Lokhandwala Overexpression of PKC-{beta}I and -{delta} contributes to higher PKC activity in the proximal tubules of old Fischer 344 rats Am J Physiol Renal Physiol, December 1, 2003; 285(6): F1100 - F1107. [Abstract] [Full Text]
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A. A. Banday, M. Asghar, T. Hussain, and M. F. Lokhandwala Dopamine-Mediated Inhibition of Renal Na,K-ATPase Is Reduced by Insulin Hypertension, June 1, 2003; 41(6): 1353 - 1358. [Abstract] [Full Text] [PDF]
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T. Hussain and M. F. Lokhandwala Renal Dopamine Receptors and Hypertension Experimental Biology and Medicine, February 1, 2003; 228(2): 134 - 142. [Abstract] [Full Text] [PDF]
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M. Asghar, T. Hussain, and M. F. Lokhandwala Higher basal serine phosphorylation of D1A receptors in proximal tubules of old Fischer 344 rats Am J Physiol Renal Physiol, August 1, 2002; 283(2): F350 - F355. [Abstract] [Full Text] [PDF]
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significant difference between groups (t test, P <
0.05).
