Overexpression of PKC-I and - contributes to higher PKC activity in the proximal - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 28 下一页

返回列表

查看: 573356\|回复: 272	go Overexpression of PKC-I and - contributes to higher PKC activity in the proximal [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 13:49 |只看该作者 |倒序浏览 |打印

作者：Mohammad Asghar, Tahir Hussain,  Mustafa F. Lokhandwala作者单位：Heart and Kidney Institute, College of Pharmacy, University of Houston, Houston, Texas 77204
   & V  c% `, \/ l% u8 v* G3 B

      6 K* r1 n& ~2 ~
                     ; ]9 ], @! X3 W0 s

         5 V# P7 F$ }0 S2 {
         % \+ S' E8 X! p! \3 m4 _
         ) k' F7 U* i$ n4 J1 B' r$ \




      【摘要】
   Previously, we reported that natriuretic and diuretic response to dopamine is diminished in old Fischer 344 rats, which is due to higher basal protein kinase C (PKC) activity and hyperphosphorylation of Na-K-ATPase in the proximal tubules (PTs) of old rats. The present study was conducted to determine whether higher PKC activity could be due to altered expression of some of the PKC isoforms in the superficial cortex (rich in PTs) of old rats. Fluorimetric measurement showed almost twofold increase in the PKC activities in homogenates and membranes of old (24 mo) compared with adult (6 mo) rats. Interestingly, in the basal state PKC- I was overexpressed in the membranes, whereas PKC- expression was increased in the cytosol of old compared with adult rats. Treatment of the cortical slices with either SKF-38393, a D1-like agonist, or PDBu, a direct activator of PKC, caused translocation of PKC- I from cytosol to membranes in adult but not in old rats. Both of these drugs caused translocation of PKC- from membranes to cytosol in adult but not in old rats. These drugs had no effect on translocation of PKC- in both adult and old rats. Both PKC- I and - coimmunoprecipiated with 1 -subunit of Na-K-ATPase in adult and old rats. These observations suggest that both SKF-38393 and PDBu differentially regulate PKC- I and - in adult but not in old rats. Also, PKC- I and - seem to interact with Na-K-ATPase in these animals. The overexpression of both PKC- I and - in old rats could be responsible for a higher basal PKC activity, which causes the hyperphosphorylation of Na-K-ATPase and contributes to the diminished inhibition of Na-K-ATPase activity by dopamine in old rats. % `( N0 G& M* }8 F3 \! B
      【关键词】 dopamine D receptors protein kinase C isoforms NaKATPase% O5 E8 u% a& J, j
   PROTEIN KINASE C (PKC) is a family of closely related isozymes of serine/threonine kinase known to play important roles in signal transduction ( 31 ). PKCs have been classified into three major groups: conventional, Ca 2  -dependent PKCs (, I, II, and ); novel, Ca 2  -independent PKCs (,,, and ); and atypical PKCs ( and ). The expression of individual PKC isoforms differs from tissue to tissue. Different parts of the nephron have been shown to express different isoforms of PKC ( 2, 24, 34 ). Proximal tubules (PTs) express PKC-, -, -, -, and - isoforms ( 12, 24 ), whereas the medullary thick ascending limb of Henle expresses PKC-, - II, -, -, and - isoforms ( 2 ).
1 K- `) [. D; b: i+ `$ J
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 ).$ u( ]' l% _3 T8 h
4 Z/ L3 C. _3 T. P* B+ ?7 O
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.6 h( `7 _  |- z  s2 X
/ U5 c0 T$ a+ W
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.
; d+ T; C5 H* R% l7 t
MATERIALS AND METHODS! r# |, E( Y5 E& V( R. ]* H

Animals. Adult (6 mo) and old (24 mo) male Fischer 344 rats were purchased from Harlan Sprague Dawley (Indianapolis, IN). The rats were housed in plastic cages in the University of Houston animal care facility and fed normal rat chow and water ad libitum. Fischer 344 rats provide an animal model that exhibits age-related changes to those documented in humans and are widely used in experimental aging research ( 29 ).. U# [- Z5 U: A: n2 n
4 Y# S2 P0 s" U4 u5 R
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 CaCl 2, 110 mM NaCl, 5.4 mM KCl, 1 mM KH 2 PO 4, 1 mM MgSO 4, 25 mM NaHCO 3, 25 mM D -glucose, and 2 mM HEPES (pH 7.6). Transverse sections of the kidneys were obtained, and superficial cortical tissue slices (400-500 µm, rich in PTs) were dissected out with a razor blade. The cortical slices were kept in cold fresh preoxygenated Kreb's buffer.
4 B# d1 }; y' H% h
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 CaCl 2, 1 DTT, 10 MgCl 2, 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 25-50 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 (0-40 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 protein -1 · min -1, 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 P O 2 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 P O 2 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.. F) ?! g  R( A( ?. b% g

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).  C# `# [( I  u
/ ?7 u/ S0 n2 r9 k
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 2 x 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.* J( @  [  E, F2 F; Q3 P, |$ D5 N
: D6 Y' x7 ]9 U, H
Measurement of proteins. Proteins were measured using BCA protein assay kit (Pierce, Rockford, IL) and BSA as standards.( R" E' {) N- i9 J7 w$ f

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.

RESULTS

Basal activity of PKC. As shown in Fig. 1, the basal PKC activity was almost twofold higher in the homogenates ( Fig. 1 A ) of superficial cortex from old compared with adult rats (adult vs. old: 2.03 ± 0.46 vs. 4.189 ± 0.39 ng peptide phosphorylated · mg protein -1 · min -1 ). Similarly, the PKC activity was also higher in the membranes ( Fig. 1 B ) from old compared with adult rats (adult vs. old: 0.88 ± 0.03 vs. 1.59 ± 0.14 ng peptide phosphorylated · mg protein -1 · min -1 ). There was no significant change in the cytosolic PKC activity between adult and old rats (adult vs. old: 5.85 ± 0.39 vs. 6.10 ± 0.42 ng peptide phosphorylated · mg protein -1 · min -1 ).
( E8 Z: h  C8 O: i2 Z
Fig. 1. Basal activity of protein kinase C (PKC) in the homogenate ( A ) and membranes ( B ) of adult and old rats. Twenty-five- to fiftymicrogram proteins were used to measure the PKC activity as its ability to phosphorylate fluorescent-tagged peptide substrate (details in MATERIALS AND METHODS ). The basal activity was higher both in the homogenate and membranes of old compared with adult rats. Values are means ± SE; n = 3 animals. *Significant difference from adult (Student's t -test, P5 [+ q2 q' w1 u9 G) x: s
4 X, F" |: [* p# P
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. 2 A ), whereas PKC- increased in the cytosol (adult vs. old: 1.28 ± 0.05 vs. 2.20 ± 0.07 density U; Fig. 2 B ) compared with adult rats.

Fig. 2. Basal expression of PKC- I and - in the membranes and cytosol of adult and old rats. A : PKC- I expression increased in the membranes of old compared with adult rats. B : PKC- expression increased in the cytosol of old compared with adult rats. Inset : representative blot for basal PKC- I ( A ) and - ( B ) expression in adult and old rats. The amount of proteins loaded on the gel for PKC- I( A ) and - ( B ) was 15 and 5 µg, respectively. B : densitometric measurement of the blots. Values are means ± SE; n = 4-5 animals. *Significant difference from adult (Student's t -test, P5 F0 ]. H! c- L# W/ x

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. 3 A ) but not old ( Fig. 3 C ) rats. Both of these drugs decreased the PKC- I expression in the cytosol of adult ( Fig. 3 B ) but not old ( Fig. 3 D ) rats. On the other hand, both SKF-38393 and PDBu caused a decrease in the membranal ( Fig. 4 A ) and an increase in the cytosolic ( Fig. 4 B ) 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.& a& k- i* h1 K8 s' I

Fig. 3. Effect of SKF-38393 and PDBu on PKC- I expression in adult ( A and B ) and old ( C and D ) rats. PKC- I expression increased in the membranes ( A ) and decreased in the cytosol ( B ) with SKF-38393 and PDBu treatment in adult rats. Both SKF-38393 and PDBu had no effect on PKC- I expression either in membranes ( C ) or cytosol ( D ) in old rats. Insets : representative blot of PKC- I expression in membranes ( A and C ) and cytosol ( B and D ) in adult and old rats. Lanes 1 - 3 in the blots represent vehicle, SKF-38393, and PDBu treatment, respectively. The amount of proteins loaded on the gel was 15 µg for both membranes and cytosol. The bar diagrams show the densitometric measurement of PKC- I proteins and are represented as a percentage of basal for adult and old rats. Values are means ± SE; n = 4-5 animals. *Significant from basal of adult rats (Student's t -test, P& m2 X) ?0 m+ @! J

Fig. 4. Effect of SKF-38393 and PDBu on PKC- expression in adult ( A and B ) and old ( C and D ) rats. PKC- expression decreased in the membranes ( A ) and increased in the cytosol ( B ) with SKF-38393 and PDBu treatment in adult rats. Both SKF-38393 and PDBu had no effect on PKC- expression either in membranes ( C ) or cytosol ( D ) in old rats. Insets : representative blot of PKC- expression in membranes ( A and C ) and cytosol ( B and D ) in adult and old rats. Lanes 1 - 3 in the blots represent vehicle, SKF-38393, and PDBu treatment, respectively. The amount of proteins loaded on the gel was 5 µg for both membranes and cytosol. The bar diagrams show the densitometric measurement of PKC- proteins and are represented as percent of basal for adult and old rats. Values are means ± SE; n = 4-5 animals. *Significantly different from basal of adult (Student's t -test, P' u4 G4 n) j; p& l% E8 U

Fig. 5. Effect of SKF-38393 and PDBu on PKC- expression in adult ( A and B ) and old ( C and D ) rats. SKF-38393 and PDBu did not affect the expression of PKC- either in membranes ( A and C ) or cytosol ( B and D ) for both adult and old rats. Insets : representative blot of PKC- expression in membranes ( A and C ) and cytosol ( B and D ) in adult and old rats. Lanes 1 - 3 in the blots represent vehicle, SKF-38393, and PDBu treatment, respectively. The amount of proteins loaded on the gel was 20 µg for both membranes and cytosol. The bar diagrams show the densitometric measurement of PKC- proteins and are represented as a percentage of basal for adult and old rats. Values are means ± SE; n = 4-5 animals.$ J3 J& y- J0 o( r" {
! f: N8 v: f* X1 w7 J2 f1 L
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. 6 A ). 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. 6 B ) and PKC- ( Fig. 6 C ) in adult and old rats.
- U$ B  o! k; d, L
Fig. 6. Coimmunoprecipitation of PKC- I, -, and Na-K-ATPase in the membranes isolated from renal cortical slices of adult and old rats. A : 1 -subunit of Na-K-ATPase antibody-immunoprecipitated samples were immunoblotted for PKC- I, PKC-, and Na-K-ATPase. Equal volumes (10 µl) of the 1 -subunit of Na-K-ATPase antibody-immunoprecipitated samples were resolved, transblotted, and immunoblotted for PKC- I, PKC-, and Na-K-ATPase. B : PKC- I antibody-immunoprecipitated samples (5 µl) were transblotted and immunoprobed for Na-K-ATPase and PKC- I. C : PKC- antibody-immunoprecipitated samples (5 µl) were transblotted and immunoblotted for Na-K-ATPase and PKC-. Lanes 1 and 2 in the blots represent samples from adult and old rats, respectively.
5 }/ k% B, P; ^) }
DISCUSSION+ I: A3 t4 U. X3 k- S0 F3 }

Aging is accompanied by the development of structural changes in the kidney, which include progressive renal sclerosis with glomerulosclerosis and interstitial fibrosis ( 10, 17 ), indicated by the accumulation of extracellular matrix proteins such as fibronectin and collagen ( 1 ). PKC plays a fundamental role in cell proliferation, differentiation, and survival ( 9, 32 ). These diverse cellular events are regulated by specific PKC isozymes that have been implicated in the pathophysiology of many diseases such as hypertension ( 42 ), diabetes ( 21 ), and lung disease ( 36 ). An altered PKC activity has been suggested for a higher phosphorylation and lower activity of Na-K-ATPase in old rats, which seemed to contribute to the defective natriuretic and diuretic response to dopamine in these animals ( 6 ). This altered PKC activity in old rats could be due to differential regulation of specific isozymes as has been reported in many diseases ( 21, 36, 42 ). Here, we provide evidence for different isozymes (PKC- I and - ) that may be responsible for a higher PKC activity and subsequent hyperphosphorylation of Na-K-ATPase in old rats.
% |3 c; H9 A* p0 T5 B" s
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. 3 A and 4 B ). 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.( s2 H3 k. t/ {# h( S

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.% x7 _9 s4 h' @1 y
: B5 ]; S7 W+ A  e
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 ).9 c+ s( c& O. l& m( O, F
: j2 N7 z( i: O7 \" G) n
DISCLOSURES

This study was supported in part by National Institutes of Health Grant AG-15031 from National Institute on Aging.! L9 c; N+ u$ t
      【参考文献】
Abrass CK, Adeoux MJ, and Raugi GJ. Aging associated changes in renal extracellular matrix. Am J Pathol 146: 742-752, 1995.* E# i, _' l4 M0 G! B% M# K  d

7 K' D% t9 V5 z- a* z
+ t9 B8 G, L. C1 `, ^; C8 T
Aristimuno PC and Good DW. PKC isoforms in rat medullary thick ascending limb: selective activation of the 3 isoforms by PGE 2. Am J Physiol Renal Physiol 272: F624-F631, 1997.- ?: s: G& a& X; }3 x* d0 p4 Y

& {' c3 {" m! T' Q1 |7 z
) s1 S) m2 E" @. Z
Armando I, Nowicki S, Aguirre J, and Barontini M. A decreased tubular uptake of dopa results in defective renal dopamine production in aged rats. Am J Physiol Renal Fluid Electrolyte Physiol 268: F1087-F1092, 1995.  V& s# G' g1 p  L; w, Q  K% A
7 _) {3 n8 U2 ]7 S: O1 n# S

Asghar M, Hussain T, and Lokhandwala MF. Higher basal serine phosphorylation of D1A receptors in proximal tubules of old Fischer 344 rats. Am J Physiol Renal Physiol 283: F350-F355, 2002.
- i, U% d2 y; K
- q8 L' \) o  U! T& [

Asghar M, Hussain T, and Lokhandwala MF. Activation of dopamine D1-like receptor causes phosphorylation of 1 -subunit of Na,K-ATPase in rat renal proximal tubules. Eur J Pharmacol 411: 61-66, 2001.5 t& M/ X$ g1 H, a
6 Z7 O! @7 a  c/ f, n: |

Asghar M, Kansra V, Hussain T, and Lokhandwala MF. Hyperphosphorylation of Na pump contributes to defective renal dopamine response in old rats. J Am Soc Nephrol 12: 226-232, 2001.' A/ ~: m4 `' a7 O2 F
- \6 D: r6 P* \: ~

2 q$ m* v& B+ _
Baines AD, Drangova R, and Ho P. Role of diacylglycerol in adrenergic-stimulated 86 Rb uptake by renal proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 258: F1133-F1138, 1990., L8 T+ H2 j/ O, F( j
- O4 |; l: t% k+ D$ H
7 U( R0 T5 b% T8 `
2 L$ j. p' m, H; e9 F/ H; J
Balaban RS, Soltoff SP, Storey JM, and Mendel LJ. Improved renal cortical tubule suspension: spectrophotometric study of O 2 delivery. Am J Physiol Renal Fluid Electrolyte Physiol 238: F50-F59, 1980.
+ a( P6 ?) i- j5 L4 i" c
  X/ V1 E' l9 I+ d
% g9 q* G1 N0 l7 k/ R5 m( Q
Barr LF, Campbell SE, and Baylin S. Protein kinase C- II inhibits cycling and decreased c-myc-induced apoptosis in small cell lung cancer cells. Cell Growth Differ 8: 381-392, 1997.
3 z' e: V' h$ G' L7 O$ V/ J

Border WA and Noble NA. Transforming growth factor- in tissue fibrosis. N Engl J Med 331: 1286-1292, 1994.
' s8 |  }5 ^3 k9 N3 W! \6 g

Chen CJ, Beach RE, and Lokhandwala MF. Dopamine fails to inhibit renal tubular sodium pump in hypertensive rats. Hypertension 21: 364-372, 1993.3 K4 |. a  \6 s. `
1 c( _) L4 X, ^; x3 H; y6 s
3 h" M8 @& L, F4 R- l& s2 K" Q% r

Dianne MB and Jeffrey LG. Age-dependent activation of PKC isoforms by angiotensin II in the proximal nephron. Am J Physiol Regul Integr Comp Physiol 281: R861-R867, 2001., O2 d- `8 E* s3 T. o9 x" I

Efendiev R, Bertorello AM, and Pedemonet CH. PKC- and PKC- mediate opposing effects on proximal tubule Na  ,K -ATPase activity. FEBS Lett 456: 45-48, 1999.
  c, r  K' {6 W2 g3 A+ a9 y3 O6 x
; T1 F: ^8 g* L' A" K

Epstein M. Aging and the kidney. J Am Soc Nephrol 7: 1106-1122, 1996.

Felder CC, Campbell T, Albrecht F, and Jose PA. Dopamine inhibits Na/H-exchanger activity in renal BBMV by stimulation of adenylate cyclase. Am J Physiol Renal Fluid Electrolyte Physiol 259: F297-F303, 1990.

Gesek FA and Schoolworth AC. Hormonal interactions with the proximal Na/H-exchanger. Am J Physiol Renal Fluid Electrolyte Physiol 258: F514-F521, 1990.8 }6 j0 M+ L; @6 I* P. e& h

) I* f! P+ E4 e: p4 J
/ e! w# c9 L8 \8 J# q# v
Goldstein RS, Tarloff JB, and Hook JB. Age-related nephropathy in laboratory rats. FASEB J 2: 2241-2251, 1988.

Houillier P, Chambrey R, Achard JM, Froissart M, Poggioli J, and Paillard M. Signaling pathways in the biphasic effect of angiotensin II on apical Na/H antiport activity in proximal tubule. Kidney Int 50: 1496-1505, 1996.
& {6 z2 ~$ ?0 o/ h9 ~* M

( h" J# y- B+ ~" C3 A; w& a' q
Hussain T and Lokhandwala MF. Renal dopamine receptor function in hypertension. Hypertension 32: 187-197, 1998.: c: c9 y* |  e

Hussain T and Lokhandwala MF. Renal dopamine DA1 receptor coupling with Gs and Gq/11 proteins in spontaneously hypertensive rats. Am J Physiol Renal Physiol 272: F339-F346, 1997.3 n# q# q% f% s
  g9 [! s  i: T; k
, m% \- ^( ~& E

Kang N, Alexander G, Park JK, Maasch C, Buchwalow B, Luft FC, and Haller H. Differential expression of protein kinase C in streptozotocin-induced diabetic rats. Kidney Int 56: 1737-1750, 1999.% U% }; `! _8 P5 ^4 q* v

Kansra V, Chen CJ, and Lokhandwala MF. Dopamine causes stimulation of protein kinase C in rat renal proximal tubules by activating dopamine D1 receptor. Eur J Pharmacol 289: 391-394, 1995.

2 m' \7 d# r! h, m: r
+ L4 S" f7 Q7 a: i
Kansra V, Hussain T, and Lokhandwala MF. Alterations in dopamine DA1 receptor and G proteins in renal proximal tubules of old rats. Am J Physiol Renal Physiol 273: F53-F59, 1997.1 r' b8 h2 d% w
! {* {& j9 K' X+ L' [
1 |6 R  @* q; _+ k! \7 O% H: [" Q1 E
0 r1 U2 |! G. t0 B
Karim Z, Defontaine N, Paillard M, and Poggioli J. Protein kinase C isoforms in rat kidney proximal tubule: acute effect of angiotensin II. Am J Physiol Cell Physiol 269: C134-C140, 1995.
+ T( W5 q, H3 M5 X5 A7 W

Kawakami Y, Kitaura J, Hartman SE, Lowell CA, Siraganian RP, and Kawakami T. Regulation of protein kinase C I by two protein-tyrosine kinases, Btk and Syk. Proc Natl Acad Sci USA 97: 7423-7428, 2000.) a; @# c# H5 y  s

Levi M and Rowe JW. Renal function and dysfunction in aging. In: The Kidney: Physiology and Pathophysiology, edited by Seldin W and Giebisch G. New York: Raven, 1992, p. 3433-3456., F( h9 g" `1 z9 L5 _- A, ^2 v, K/ F

' w3 z/ M2 ]8 y5 y/ a
Liedtke CM, Cole T, and Ikebe M. Differential activation of PKC- and - by 1 -adrenergic stimulation in human airway epithelial cells. Am J Physiol Cell Physiol 273: C937-C943, 1997.
. A+ s7 T3 [3 w( Q  ~: U  O( \& d

Logvinenko NS, Dulubova I, Fedosova N, Larsson SH, Esmann M, Greengard P, and Aperia A. Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na  ,K -ATPase affects its conformational equilibrium. Proc Natl Acad Sci USA 93: 9132-9137, 1996.

/ f0 `: x& p6 I/ ?$ S

Miller RA and Nadon NL. Principles of animal use for gerontological research. J Gerontol Biol Sci 55A: B117-B123, 2000.% h; [. w4 Z; F* ^# d

, V. q& W: j6 s, l, K3 n1 Y4 C
Miranti CK, Ohno S, and Brugge JS. Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc. J Biol Chem 274: 10571-10581, 1999.5 F" z; {: n6 ?1 [7 M* m
9 b' s. o4 m1 v) g3 t0 L

) D. ^+ v; G! i! L& _( |
Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular response. FASEB J 9: 484-496, 1995.. U6 q5 Y2 r5 t" b+ B" C$ n
9 z& r1 L4 ^  q6 }- u) ?; a4 g
, g% G* {4 q3 t6 J3 e3 q$ X0 Y

Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258: 607-614, 1992.
3 K9 {, G& n1 I% d' {

Nowicki S, Kruse MS, Brismar H, and Aperia A. Dopamine-induced translocation of protein kinase C isoforms visualized in renal epithelial cells. Am J Physiol Cell Physiol 279: C1812-C1818, 2000.4 w8 c- I# u2 c# ^& D- @

Ostlund E, Mendez CF, Jacobsson G, Fryckstedt J, Meister B, and Aperia A. Expression of protein kinase C isoforms in renal tissue. Kidney Int 47: 766-773, 1995.
' T7 X  a, m: d$ X

" ?+ Q: W4 c3 {- P. T6 E, `; Y
Pedemonte CH, Pressley TA, Lokhandwala MF, and Cinelli AR. Regulation of Na,K-ATPase transport activity by protein kinase C. J Membr Biol 155: 219-227, 1997.

Phelps DT, Ferro TJ, Higgins PJ, Shankar R, Parker DM, and Johnson A. TNF- induces peroxynitrite-mediated depletion of lung endothelial glutathione via protein kinase C. Am J Physiol Lung Cell Mol Physiol 269: L551-L559, 1995.
3 u1 J, N3 Z; J+ o- D
: _' B+ k$ I+ g
" {& m& t: \9 @+ z: K; n
Poggioli J, Karim Z, Defontaine N, Lazar G, Bodi I, and Paillard M. Protein kinase C isozymes present in brush border membrane vesicles isolated from rat kidney proximal tubules are activated by angiotensin II (Abstract). J Am Soc Nephrol 6: 743, 1995.

Qu X, Seale JP, and Donnelly R. Tissue- and isoform-specific effects of aging in rats on protein kinase C in insulin-sensitive tissues. Clin Sci (Colch) 97: 355-361, 1999.0 Q0 x) Q8 [; a7 F0 F5 _
3 E4 X* k( ?  G  |

Tsuru M, Kalagiri H, Asano T, Ohno S, Ogihara T, and Oka Y. Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC. Am J Physiol Endocrinol Metab 283: E338-E345, 2002.

/ k. w9 U6 c9 i+ i0 D1 A
: ^- {/ g1 R+ g( f% Y( L! L
Vyas SJ, Eichberg J, and Lokhandwala MF. Characterization of receptors involved in dopamine induced activation of phospholipase C in rat renal cortex. J Pharmacol Exp Ther 260: 134-139, 1992.# ?% C! \1 }2 Z6 x

8 I6 _& p0 ~  D

Wang XD, Kiang JG, Atwa MA, and Smallridge RC. Evidence for the involvement of protein kinase C isoforms in 1 adrenergic activation of phospholipase A2 in FRTL-5 thyroid cells. J Investig Med 44: 566-574, 1996.; t1 P- l2 L0 g! z+ v( s- m
( e0 L& ~5 Q' \9 U! D

Yao LP, Li XX, Yu PY, Xu J, Asico LD, and Jose PA. Dopamine D1 receptor and protein kinase C isoforms in spontaneously hypertensive rats. Hypertension 32: 1049-1053, 1998.* Z1 D9 ^4 {7 o% h/ n1 g9 C
4 |3 t" ]0 Z; L& ], b+ c

Yu PY, Asico LD, Eisner GM, and Jose PA. Differential regulation of renal phospholipase C isoforms by catecholamines. J Clin Invest 95: 304-308, 1995.

收藏0 分享0 顶0 踩0