Heart and Kidney Institute, College of Pharmacy, University of Houston, Houston, Texas 77204
Submitted 27 May 2003 ; accepted in final form 30 July 2003
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ABSTRACT |
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dopamine; D1 receptors; protein kinase C isoforms; Na-K-ATPase
Numerous studies demonstrated a differential regulation of different PKC isoforms by hormones such as norepinephrine, ANG II, and dopamine. Norepinephrine induced the translocation of PKC-, -
I, -
II, -
, -
, and -
but not PKC-
or -
from cytosol to membrane in a thyroid cell line (41).
1-Adrenoceptor stimulation produced a sustained translocation of cytosolic PKC-
and transient translocation of PKC-
to the membrane in airway epithelial cells (27), and ANG II increased the expression of PKC-
and -
in rat PTs (24, 37). Additionally, differential regulation of different PKC isoforms has also been reported in disease states. In genetic hypertensive rat models, D1-like agonists have been reported to increase the membranous expression of PKC-
and -
in the PTs of spontaneously hypertensive rats (SHRs) compared with normotensive Wistar-Kyoto (WKY) rats (42). In streptozotocin-induced diabetic rats, PKC-
expression increased, whereas -
decreased in kidney and heart (21). PKC-
expression has been reported to increase in the PT and decrease in the myocardium in diabetic rats (21).
Dopamine produces natriuresis and diuresis via activation of D1-like receptors and inhibition of Na-K-ATPase and Na/H exchanger activities (16, 19). Dopamine-mediated inhibition of Na-K-ATPase activity in the PTs has been attributed to the activation of PKC (22, 40), whereas the inhibition of Na/H exchanger activity is linked to the stimulation of PKA (15, 20). The inhibitory effect of dopamine on Na-K-ATPase activity has also been shown to occur via the stimulation of PKC in proximal tubular epithelial cell line derived from opposum kidney (13). In contrast, norepinephrine and ANG II increase PKC, Na-K-ATPase (7), and Na/H exchanger activities (18). The mechanism through which these receptors produce opposite effects in the kidney may relate to differential regulation of PKC isoform expression and activity.
The biological process of aging initiates various structural and functional changes within the kidney such as decline in renal blood flow and glomerular filtration rate (14, 26). Previously, deficiency in L-dopa uptake and its conversion to dopamine (3) and a reduced dopamine receptor number and its defective coupling with G proteins have been shown in the kidneys of old (24 mo) rats (23). Recently, we reported that dopamine failed to inhibit Na-K-ATPase activity in the PTs of old compared with adult (6 mo) rats, in part, due to a reduced number of D1-like receptor binding sites and defective receptor G protein coupling because of higher phosphorylation of D1A receptors (4, 23). Together with these defects at the level of D1 receptor, we also found an altered PKC activity in the PTs of old rats (6). The altered (higher) PKC activity was linked to hyperphosphorylation of Na-K-ATPase, which led to a basal low activity of the transporter in the PTs of old rats (6). Therefore, we hypothesized that the altered PKC activity in old rats could be due to altered expression of some of the PKC isoforms, a phenomenon that occurs in many disease states (21, 42). To test this hypothesis, we measured the D1-like receptor-dependent (SKF-38393 induced) and -independent (PDBu induced) cellular translocation of some of the PKC isoforms in the kidney slices (superficial cortex rich in PTs) of adult and compared them with old Fischer 344 rats. We also measured the basal PKC activity in the superficial cortical slices (homogenates, membranes, and cytosol) from adult and old rats.
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MATERIALS AND METHODS |
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Surgery. The rats were anesthetized with pentobarbital sodium (50 mg/kg ip), a midline incision was made in the abdomen, and kidneys were removed and kept in ice-cold oxygenated Kreb's 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). Transverse sections of the kidneys were obtained, and superficial cortical tissue slices (400500 µm, rich in PTs) were dissected out with a razor blade. The cortical slices were kept in cold fresh preoxygenated Kreb's buffer.
Measurement of basal PKC activity. Superficial cortical slices from adult and old rats were homogenized in a buffer containing (in mM) 50 Tris · HCl, pH 7.4, 5 EDTA, 10 EGTA, 0.4 DTT, 10 benzamidine, 1 PMSF, and protease inhibitor cocktail and subjected to centrifugation at 2,000 g for 10 min. An aliquot of the supernatant was saved to measure total PKC activity. The rest of the supernatant was centrifuged at 100,000 g for 30 min, and the second supernatant (100,000 g) was collected and considered cytosolic fraction. The pellet (100,000 g) was washed twice with homogenization buffer and resuspended in the same buffer and considered membrane fraction. The PKC activity in the homogenate, cytosol, and membrane fractions was measured following the protocol supplied by the manufacturer (V5330, Promega, Madison, WI). Briefly, the PKC activity was carried out in a reaction volume of 25 µl containing (in mM) 20 HEPES, pH 7.4, 1.3 CaCl2, 1 DTT, 10 MgCl2, 1 ATP, 0.05% Triton X-100, 0.2 mg/ml phosphatidylserine, and 2 µg fluorescent PepTag C1 peptide substarte (P-L-S-R-T-L-S-V-A-A-K, amino acid sequence). The hot pink fluorescence is imparted by a dye molecule attached to the peptide substrate and has excitation and emission maxima at 568 and 592 nm, respectively. The reaction was started by the addition of 2550 µg proteins and carried out for 30 min at 30°C. The reaction was terminated by heating at 95°C for 10 min. The phosphorylated PepTag C1 peptide was separated from the nonphosphorylated peptide by electrophoresis on 0.8% agarose. The phosphorylated peptide was cut under UV light, agarose was melted, and the fluorescence intensity was recorded using excitation and emission wavelengths 568 and 592 nm, respectively, in a Luminescence Spectrometer LS 50 (PerkinElmer, Beconsfield, Buckinghamshire, UK). The fluorescence intensity was read on a standard curve prepared by using pure PKC enzyme (040 ng) as standard supplied with the kit. The PKC enzyme (V5261, Promega) supplied was purified from rat brain and is greater than 90% pure as determined by SDS-PAGE, which constitutes primarily of -,
-, and
-isoforms with lesser amount of
- and
-isoforms.
Drug treatment. The cortical slices were placed in 2 ml of Kreb's buffer and kept on a water bath maintained at 30°C for 3 min while oxygenating. The slices were washed with preoxygenated warm Kreb's buffer and finally placed in 2 ml of the same buffer. The slices were incubated with vehicle, SKF-38393 (1 µM), and PDBu (1 µM) in separate tubes for 20 min. This time point was chosen because earlier we showed that both SKF-38393 and PDBu increased the PKC-mediated serine phosphorylation of 1-subunit of Na-K-ATPase (5). To confirm that the longer incubation time (20 min) may not have driven the steady state of PKC to activation, we measured the PKC activity at lower incubation time (1 and 5 min). We found that PDBu (1 µM) treatment significantly increased the membranal PKC activity at 5 min in cortical slices from adult rats (vehicle vs. PDBu 1 min vs. PDBu 5 min: 0.96 ± 0.03 vs. 0.94 ± 0.04 vs. 1.34 ± 0.02 ng peptide phosphorylated · mg protein1 · min1, n = 3 animals). All the steps, starting from isolation of the kidneys to the drug treatment, were carried out in the presence of oxygen. We supplied oxygen continuously to ensure that PO2 of reaction bath remained in excess at all time. We took this precaution because cortical slice has shown to be oxygen deficient even at bath PO2 of greater than 570 mmHg (8). The drug incubations were terminated with ice-cold Kreb's buffer. Finally, the buffer was quickly aspirated and the slices were frozen on dry ice-acetone mixture.
Preparation of cytosol and membranes from kidney slices (superficial cortex). The cytosol and membranes from the cortical tissue slices were prepared using a previously described method (43) with some modifications. Briefly, the slices were thawed on ice and equilibrated with cold homogenization buffer containing (in mM) 50 Tris · HCl, pH 7.4, 1 EDTA, 0.1 DTT, 100 NaCl, 250 sucrose, 1 PMSF, and protease inhibitor cocktail. The tissues were homogenized and subjected to a low-spin centrifugation at 2,000 g for 10 min to remove nuclei and cell debris. The supernatant was centrifuged at 100,000 g for 30 min. The 100,000-g supernatant represented the cytosolic fractions, and the pellet obtained was washed twice with a buffer containing (in mM) 50 Tris · HCl, pH 7.4, 1 EDTA, 200 KCl; resuspended in homogenization buffer; and considered membrane fractions. All the steps of cytosol and membrane preparation were carried out at 4°C. Aliquots of cytosol and membrane fractions were made and kept at 70°C for further use.
Western immunoblotting. Equal amount of proteins from cytosol and membrane fractions was resolved on 8% SDS-PAGE electrophoresis. The resolved proteins were electro-transblotted on PVDF (Immobilon) membrane and blocked with 5% milk in phosphate-buffered saline with 0.05% Tween 20 (PBST). Specific PKC isoforms such as -, -
I, -
, -
, -
, and -
were probed with their respective antibodies. Anti-PKC-
was raised in mouse, whereas the rest of the anti-PKC isoform-specific antibodies were raised in rabbit. Horse radish peroxidase (HRP)-conjugated goat-anti-mouse antibody was used to immunodetect anti-PKC-
, whereas goat-anti-rabbit conjugated with HRP was used to probe the rest of the PKC antibodies raised in rabbit. Finally, the specific bands for PKC isoforms were visualized using enhanced chemiluminescent reagent kit (Alpha Diagnostics, San Antonio, TX). The specificity of the antibodies for different PKC isoforms was detected using the peptide, used as an immunogen to raise the antibodies, which resulted in the disappearance of the specific protein bands (data not shown).
Coimmunoprecipitation of Na-K-ATPase and PKC isoforms. Monoclonal antibody raised against the 1-subunit of Na-K-ATPase was used to immunoprecipitate Na-K-ATPase as we described earlier (5, 6). Briefly, the membrane fractions (1 mg/ml) were added in immuoprecipitation (IP) buffer containing (in mM) 50 Tris · HCl, pH 8.0, 150 NaCl, 1 EDTA, 1 EGTA, 1 DDT, 1 PMSF, 1% Triton X-100, and protease inhibitor cocktail and incubated overnight with antibody of
1-subunit of Na-K-ATPase. The antigen (
1-subunit of Na-K-ATPase)-antibody complex thus formed was incubated with Protein A/G-agarose for 2 h. The ternary complex of antigen-antibody-Protein A/G-agarose was settled down by centrifugation and washed once with IP buffer and then with a buffer containing (in mM) 50 Tris · HCl, pH 8.0, 250 NaCl, 1 EDTA, and 0.1% Triton X-100. The complex was finally washed with another buffer containing (in mM) 50 Tris · HCl, pH 8.0, 250 NaCl. The proteins were dissociated in 2x Laemmeli buffer (125 mM Tris · HCl, 4% SDS, 5%
-mercaptoethanol, 20% glycerol) at 37°C and subjected to SDS-PAGE. The resolved proteins were transblotted on PVDF (Immobilon) membranes and immunoblotted for specific PKC isoforms as described above. PKC-
I and -
antibodies were used to immunoprecipitate PKC-
I and -
, respectively, following the same protocol described above. Finally, the respective immunoprecipitates were probed for respective PKC-isozyme and Na-K-ATPase. The antibodies for PKC-
I and -
used in our studies have been shown to immunoprecipitate PKC-
I (25) and -
(30), respectively.
Measurement of proteins. Proteins were measured using BCA protein assay kit (Pierce, Rockford, IL) and BSA as standards.
Materials. Protein A/G Plus-agarose and the antibodies specific for PKC isoforms, except for PKC-, used in the study were bought from Santa Cruz Biotechnology (Santa Cruz, CA). PKC-
antibody was purchased from GIBCO BRL (Life Technologies, Gaithersburg, MD). Monoclonal
1-subunit Na-K-ATPase antibody was bought from Research Diagnostics (Flanders, NJ). SKF-38393, PDBu, and all other chemicals of highest purity available for various buffers were purchased from Sigma (St. Louis, MO).
Statistics. Results are means ± SE. Data within the groups were analyzed by unpaired Student's t-test. A value of P < 0.05 was considered to be significant.
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RESULTS |
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Basal expression of PKC isoforms. Because we found a higher basal PKC activity in old rats, we wanted to determine which specific PKC isoforms were responsible for a higher basal PKC activity in old rats. Figure 2 shows the basal expression of PKC-I and -
in the cytosol and membranes of adult and old rats. In old rats, the expression of PKC-
I increased in the membranes (adult vs. old: 1.90 ± 0.08 vs. 2.97 ± 0.10 density U; Fig. 2A), whereas PKC-
increased in the cytosol (adult vs. old: 1.28 ± 0.05 vs. 2.20 ± 0.07 density U; Fig. 2B) compared with adult rats.
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D1 receptor-mediated and nonreceptor-mediated regulation of PKC. We adopted two approaches to activate PKC either indirectly (dopamine D1 receptor mediated), using a D1-like agonist SKF-38393, or directly, using a phorbol ester PDBu. As shown in Fig. 3, both SKF-38393 and PDBu caused translocation of PKC-I in the membranes of adult (Fig. 3A) but not old (Fig. 3C) rats. Both of these drugs decreased the PKC-
I expression in the cytosol of adult (Fig. 3B) but not old (Fig. 3D) rats. On the other hand, both SKF-38393 and PDBu caused a decrease in the membranal (Fig. 4A) and an increase in the cytosolic (Fig. 4B) PKC-
expression in adult but not old (Fig. 4, C and D) rats. The levels of PKC-
were not affected in membranes (Fig. 5, A and C) or cytosol (Fig. 5, B and D) in response to either of these two drugs in both adult (Fig. 5, A and B) and old (Fig. 5, C and D) rats.
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Coimmunoprecipitation of Na-K-ATPase and PKC isoforms. Earlier we reported that SKF-38393 and PDBu increased phosphorylation of Na-K-ATPase in adult but not old rats and the enzyme was hyperphosphorylated in old compared with adult rats (4). In this study, we found that both SKF-38393 and PDBu regulated PKC-I (Fig. 3) and -
(Fig. 4) in adult but not old rats and both of these isoforms were overexpressed in old rats (Fig. 2, A and B). It is known that PKC can directly phosphorylate and inhibit the activity of Na-K-ATPase (26, 31). Therefore, we wanted to determine potential interaction between these isoforms and Na-K-ATPase. To test this,
1-subunit of Na-K-ATPase antibody immunoprecipitates was probed for PKC-
I and -
. We found the presence of both PKC-
I and -
in adult and old rats (Fig. 6A). To further substantiate this phenomenon, we probed for Na-K-ATPase in the immunoprecipitates of PKC-
I and -
separately. We found that Na-K-ATPase coimmunoprecipitated with PKC-
I (Fig. 6B) and PKC-
(Fig. 6C) in adult and old rats.
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DISCUSSION |
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In this study, we employed antibodies to PKC-, -
I, -
, -
, -
, and -
to determine their expression in terms of contributing to the higher PKC activity seen in old rats. Of these isoforms, we did not find a difference in the basal expression of PKC-
in adult and old rats (data not shown) and PKC-
and -
proteins were not detected in our studies. However, we have been able to detect PKC-
I, -
, and -
in the superficial cortical tissues of adult and old rats. Yao et al. (42) did not find the expression of PKC-
in the PTs of WKY and SHR rats. However, other studies reported the expression of PKC-
in the PTs of Sprague-Dawley rats (12). The discrepancy in the expression of PKC-
in the above and our studies could be due to difference in strains as well as the age of the animals.
We found higher basal PKC activities both in the homogenates and membranes of old compared with adult rats (Fig. 1, A and B), which are in agreement with our previous findings of increased PKC activity reported in the PTs of old rats (6). To determine the role of PKC isoforms, which may be responsible for a higher basal PKC activity in old rats, we measured the basal expression of PKC-I and -
in the cytosol and membranes isolated from superficial cortical tissues of adult and old rats. Interestingly, we found that PKC-
I and -
expressions were increased in the membranes and cytosol, respectively, in old rats. The same pattern of increased expression of PKC-
I in the membranes and -
in the cytosol was observed upon activation either with PDBu or SKF-38393 in adult rats (Figs. 3A and 4B). These results suggest that both PKC-
I and -
are already activated and hence both PDBu and SKF-38393 could not produce their effects in old rats as they did in adult rats. It is worth mentioning that increased membranal translocation of PKC-
I, -
II, and -
has been linked to 2.5-fold increase in the activity of PKC in the adipose tissue and a role suggested for insulin resistance in normal aging (38). It is likely that increased expression of PKC-
I in the membranes was a contributing factor toward the elevated PKC activity seen in old rats.
Both receptor- and nonreceptor-mediated activation of PKC caused translocation of PKC-I from cytosol to membranes in adult but not in old rats. It is generally believed that once activated, certain PKC isoforms translocate from cytosol to membranes to regulate many short-term as well as long-term events, such as proliferation and differentiation (32). As shown in the present study, PKC-
I translocates from cytosol to membranes under the drug treatment in the cortical tissues of adult but not old rats. On the other hand, we found that activated PKC-
translocates from membranes to cytosol in adult but not old rats. A similar translocation pattern of PKC-
from membranes to cytosol upon activation with D1-like agonists has been reported in the PTs of normotensive WKY rats (42). In another study, PKC-
was mainly localized in the membranes of LLCPK-1 cells, proximal tubular cell line from pig kidney, in resting state (33). However, the authors were not able to detect the effect of dopamine on the translocation of PKC-
in those cells. This could be due to the shorter time of dopamine incubation (3 min) used in their study compared with the infusion time used by Yao et al. (42) (10 and 60 min) and in our study (20 min). Taken together, it seems that activated PKC-
I translocates from cytosol to membranes, whereas activated PKC-
follows just the reverse route from membranes to cytosol in the renal cortical slices.
We were unable to detect a regulation of PKC- either with PDBu (phorbol ester) or SKF-38393 both in adult and old rats. Our findings that PDBu or SKF-38393 did not affect the regulation of PKC-
either in adult and old rats are in agreement with the notion that PKC-
is insensitive toward phorbol ester stimulation (39). On the other hand, dopamine and D1-like agonist-mediated regulation of PKC-
have been shown in PTs and in cell line from proximal tubular cells (13, 42). An increased expression of PKC-
in the membranes of PTs of SHRs by D1-like agonists has been linked to a defective natriuresis and diuresis in these animals (42). A role for PKC-
has been suggested for dopamine-mediated inhibition of Na-K-ATPase in OK cells, a cell line from opossum kidney (13). However, another report suggests the role of PKC-
and -
but not -
in dopamine-mediated inhibition of Na-K-ATPase in LLCPK cells (33). It seems that the same ligand may not activate the similar PKC isoform in animals of two different species. In the same way, D1-like agonists are able to regulate PKC-
in WKY and SHR but not in Fischer 344, a rat strain different from WKY and SHR. Apart from rat strain, age could also be a factor for the discrepancy in D1-like agonist-mediated regulation of PKC-
seen in WKY, SHR, and Fischer 344 rats. We used Fischer 344 rats at 6 and 24 mo of age, whereas Yao et al. (42) employed WKY and SHR rats aged between 2.5 and 4 mo in their study.
PKC activation is known to directly phosphorylate and inhibit the activity of Na-K-ATPase (28, 35). Previously, we reported that receptor (SKF-38393)- and nonreceptor-mediated (PDBu) activation of PKC leads to an increase in phosphorylation and inhibition of Na-K-ATPase activity in adult but not old rats (6). The reasons for these drugs to be ineffective in old rats were suggested to be due to higher basal PKC activity, hyperphosphorylation, and low activity of Na-K-ATPase (6). On the basis of the present findings, it can be speculated that an interaction among Na-K-ATPase, PKC-I, and -
may be involved in the phosphorylation and inhibition of Na-K-ATPase in the PTs of Fischer 344 rats.
Our findings suggest that D1 receptor-mediated (SKF-38393) and nonreceptor-mediated (PDBu) activation of PKC differentially regulates specific isoforms (PKC-I and -
) in the cortical tissues (rich in PTs) of adult but not old rats. Both PKC-
I and -
seem to interact with Na-K-ATPase in the cortical tissues of these animals. Furthermore, overexpression of PKC-
I and -
could be responsible for a higher basal PKC activity, which might be contributing to the hyperphosphorylation of Na-K-ATPase as we previously reported (6) and to a diminished natriuretic and diuretic response to dopamine in old rats (23).
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DISCLOSURES |
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FOOTNOTES |
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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.
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REFERENCES |
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