Higher basal serine phosphorylation of D1A receptors in proximal tubules of old Fischer 344 rats

Mohammad Asghar, Tahir Hussain, and Mustafa F. Lokhandwala

Institute for Cardiovascular Studies, College of Pharmacy, University of Houston, Houston, Texas 77204


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dopamine (DA) and D1-like receptor agonists promote an increase in Na excretion by means of activation of the D1-like receptor signaling cascade and subsequent inhibition of the Na/H exchanger and Na-K-ATPase in renal proximal tubules. Recently, our laboratory reported that DA and the D1-like receptor agonist failed to inhibit Na-K-ATPase activity in old Fischer 344 rats because of uncoupling of D1A receptors from G proteins and that this abnormality led to a diminished natriuretic response to DA in old Fischer 344 rats. In this study, we have tested the hypothesis that the mechanism of this uncoupling may be an altered phosphorylation of D1A receptors in old rats. In experiments performed in renal cortical slices, both DA and SKF-38393, a D1-like receptor agonist, increased the serine phosphorylation of D1A receptors in adult (6 mo) but not old (24 mo) rats. Interestingly, the basal serine phosphorylation of D1A receptors was higher in old than in adult rats. Competition ligand binding ([3H]SCH-23390) experiments on the D1-like receptor in adult and old rats with fenoldopam, a D1-like receptor agonist, revealed the presence of two affinity states of the receptors. There was a rightward shift in the agonist displacement of the ligand in old compared with adult rats, as reflected in the IC50 values (adult vs. old, 7.46 × 10-9 ± 2.26 vs. 7.93 × 10-7 ± 1.33 M). Also, there was a reduction in agonist affinity in the low-affinity receptors in old compared with adult rats (IC50, adult vs. old, 5.67 × 10-5 ± 1.33 vs. 12.60 × 10-5 ± 6.50 M). Moreover, the abundance of D1A receptor proteins was ~47% lower in the membranes of old compared with adult rats. We speculate that higher basal serine phosphorylation of D1A receptors may have rendered the D1A receptor uncoupled from G protein, leading to a reduced agonist affinity state and thus diminished natriuretic response to DA in old rats.

dopamine; D1-like receptor agonist; D1A receptor; serine phosphorylation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DOPAMINE (DA) AND D1-like receptor agonists produce natriuresis and diuresis by means of activation of D1-like receptors (Ref. 14; and see also reviews in Refs. 17 and 19). The activated receptors couple to Gq/11 proteins, which results in the stimulation of the phospholipase C-linked protein kinase C (PKC) pathway and inhibition of Na-K-ATPase activity in the renal proximal tubules (5, 7, 20, 34). On the other hand, coupling of D1A receptors with Gs proteins leads to activation of the cAMP-linked protein kinase A (PKA) cascade that results in the inhibition of Na/H exchanger activity (9, 11, 16).

The pharmacological response to an agonist is attenuated by rapid phosphorylation, desensitization, and internalization of the receptors. The receptor phosphorylation is mediated by receptor-specific kinases, G protein-coupled receptor kinases (GRKs) (12, 18), or second messenger-activated kinases, such as PKC and PKA (13). The process of phosphorylation renders high-affinity receptors to a low-affinity state, a phenomenon called uncoupling of receptors from G proteins, leading to termination of agonist response. The phosphorylated receptors are internalized and then either resensitized and recycled to the plasma membrane (Refs. 24 and 28; and see also review in Ref. 22) or degraded (Refs. 10 and 27; and see also review in Ref. 32). Any alteration in the receptor desensitization/resensitization process has been linked to the development of disease. For instance, altered receptor serine phosphorylation has been attributed to the development of hypertension in both rats and humans (30, 36). In essential hypertensive patients, hyper-serine phosphorylation of DA D1 receptors leads to their uncoupling from G proteins, causing a diminished accumulation of cAMP by DA (30).

Numerous studies have shown that the aging process causes several structural and functional changes in the kidney, which include decline in renal blood flow and glomerular filtration rate (8, 25), deficiency in converting L-dopa to DA (1), reduced DA receptor numbers and their defective coupling to G proteins, and an attenuation in the natriuretic and diuretic response to DA (4, 21). Previously, our laboratory reported that DA failed to inhibit Na-K-ATPase activity in old rats, in part, because of a reduced number of D1-like receptor binding sites and defective D1-like receptor G protein coupling in the renal proximal tubules of these animals (4, 21). There is evidence that receptor G protein uncoupling has been linked to hyper-serine phosphorylation of the receptor in a diseased state (30, 36). Therefore, we hypothesized that the defective coupling of D1A receptors to G proteins in old Fischer 344 rats might be due to some abnormalities in the serine phosphorylation of D1A receptors. To test this hypothesis, we measured serine phosphorylation together with agonist affinity and protein abundance of D1A receptors in old rats and compared them with those in adult animals.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Fischer 344 rats, aged 6 (adult) and 24 (old) mo, were purchased from Harlan Sprague Dawley (Indianapolis, IN). The rats were housed in plastic cages in an animal care facility, fed with normal rat chow, and given water ad libitum. The institutional Animal Care and Use Committee approved the study protocol for the use and care of the animals.

Surgery. The rats were anesthetized with pentobarbital sodium (50 mg/kg body wt ip), abdomens were opened, and kidneys were removed and kept in ice-cold oxygenated Krebs buffer containing (in mM) 1.5 CaCl2, 110 NaCl, 5.4 KCl, 1 KH2PO4, 1 MgSO4, 25 NaHCO3, 25 D-glucose, and 2 HEPES (pH 7.6). Transverse sections of the kidneys were obtained, and superficial cortical tissue slices (400-500 µm, rich in proximal tubules) were dissected out with a razor blade. The cortical slices were kept in fresh Krebs buffer.

Drug treatment. The cortical slices were placed in 2 ml of Krebs buffer and kept on a water bath maintained at 30°C for 3 min while oxygenating. The slices were washed with warm Krebs buffer and finally placed in 2 ml of the same buffer. The slices were incubated with vehicle, DA (2 µM), and SKF-38393 (2 µM) in separate tubes in the absence or presence of SCH-23390 (2 µM) for 30 min. SCH-23390 was added 5 min before the addition of either DA or SKF-38393. All the steps of drug treatment were carried out in the presence of oxygen and at 30°C. The drug incubations were terminated with ice-cold Krebs buffer. Finally, the buffer was quickly aspirated, and the slices were frozen on a dry ice-acetone mixture.

Immunoprecipitation of DA D1A receptor. The cortical tissue slices were thawed on ice, and D1A receptors were immunoprecipiated as described (30). Briefly, the tissue slices were homogenized in immunoprecipitation (IP) buffer containing (in mM) 140 NaCl, 3 KCl, 10 Na2HPO4, 2 KH2PO4, 1 orthovanadate, and 1 phenylmethylsulfonyl fluoride (PMSF), as well as 1% NP-40, 0.5% sodium cholate, 0.1% SDS, and protease inhibitor cocktail, pH 7.4. The homogenate was centrifuged at 8,000 g for 10 min. The supernatant (1.5 mg protein/ml) was incubated with 10 µg of D1A receptor antibody (Chemicon) for 2 h to allow the formation of antigen and antibody complex (D1A receptor-antibody complex). The D1A receptor-antibody complex was incubated overnight with protein A/G covalently bound to agarose beads (protein A/G-agarose). Protein A/G has the affinity to bind to immunoglobulins (IgG), which allowed the formation of a ternary complex of D1A receptor-antibody-protein A/G-agarose. The D1A receptor-antibody-protein A/G complex attached to agarose beads was settled down and was washed once with IP buffer and finally with 50 mM Tris · HCl, pH 8.0. All the steps of IP were carried out at 4°C. Finally, the D1A receptor-antibody-protein A/G complex was dissociated with 2× Laemmeli buffer containing 125 mM Tris · HCl, 4% SDS, 5% beta -mercaptoethanol, and 20% glycerol at 37°C for 1 h. The samples were vortexed and centrifuged at room temperature, and the supernatant was used for electrophoresis.

Detection of serine phosphorylation on DA D1A receptor. The immunoprecipitated samples (10 µl) were resolved by SDS-PAGE electrophoresis, and the proteins were electrotransferred on a polyvinylidene difluoride membrane. The membrane was blocked with 4% bovine serum albumin in PBS with 0.1% Tween 20. A specific phosphoserine antibody was used to detect serine phosphorylation on D1A receptors. Horseradish peroxidase-conjugated secondary antibody was used to probe phosphoserine antibody, and the bands were visualized with an enhanced chemiluminiscence reagent kit (Alpha Diagnostics, San Antonio, TX).

Membrane preparation. Cortical tissue slices were used to prepare membranes by a standard method routinely used in our laboratory (21). Briefly, tissue slices were homogenized in a buffer containing 10 mM Tris · HCl, pH 7.6, 250 mM sucrose, 0.2 mM PMSF, and protease inhibitor cocktail and centrifuged at 2,500 g for 10 min. The supernatant was centrifuged at 24,000 g for 20 min. The upper fluffy layer of the pellet was resuspended in the same buffer, homogenized in a Dounce homogenizer, and again centrifuged at 24,000 g for 20 min. The pellet was washed with washing buffer containing 100 mM KCl, 100 mM mannitol, and 5 mM HEPES, pH 7.2, and centrifuged at 34,000 g for 30 min. Finally, the membrane pellet was resuspended in a small volume of homogenization buffer, aliquoted, and quickly stored frozen at -80°C for further use.

Competition radioligand binding assay. Fenoldopam (FD), a D1-like receptor agonist, was used to displace radioligand [3H]SCH-23390 binding on the membranes. The reaction mixture contained 4 nM radioligand, 50 µg membrane proteins, and variable concentration (10-10-10-3 M) of FD in a 250-µl assay buffer (final volume) containing (in mM) 50 Tris · HCl, 2 MgCl2, 0.2 sodium metabisulfite, and 0.2 PMSF, pH 7.4. The incubation was carried out for 120 min at 25°C. The reaction was terminated by filtration onto a glass fiber filter that was presoaked in the assay buffer. Nonspecific binding was determined by using 10 µM cold SCH-23390. All the drugs and membranes were diluted in assay buffer. The radioactivity on the filters was counted on a liquid scintillation counter (Beckman Instruments, Fullerton, CA). The data were analyzed for a two-site fit model of agonist displacement by using GraphPad Prism software.

D1A receptor protein. The same plasma membrane preparations used for binding studies were utilized to measure the protein abundance of D1A receptors by Western blotting. Different amounts (4, 6, 8, and 10 µg) of membrane proteins from adult and old rats were resolved by SDS-PAGE, transblotted on a polyvinylidene difluoride membrane, and immunoblotted for D1A receptor with an affinity-purified polyclonal D1A receptor antibody (Alpha Diagnostics) and anti-rabbit horseradish peroxidase-conjugated antibody. The bands were visualized with the enhanced chemiluminiscence reagent kit and quantitated by the Protein software program (Kodak). The specificity of the D1A receptor antibody was determined by using the peptide (used to raise the antibody)-blocked antibody, which resulted in the disappearance of the D1A protein band (data not shown).

Protein measurement. Proteins in the samples were measured with the BCA protein assay kit (Pierce, Rockford, IL) and bovine serum albumin as a standard.

Materials. D1A receptor antibody was purchased from either Chemicon (Temecula, CA) for immunoprecipitation studies or Alpha Diagnostics (San Antonio, TX) for Western blotting studies. Protein A/G-agarose was bought from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphoserine antibody was bought from Calbiochem NovaChem (San Diego, CA). Radioligand [3H]SCH-23390 was purchased from NEN Life Sciences (Boston, MA). Complete protease inhibitor cocktail tablets were bought from Roche Diagnostics (Mannheim, Germany). DA, SKF-38393, FD, SCH-23390, and all other chemicals of highest purity were bought from Sigma (St. Louis, MO).

Statistics. Results are presented as means ± SE. Statistical significance was analyzed by using either Student's t-test or ANOVA. A value of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Immunoprecipitation of D1A receptor. The D1A receptor antibody used to immunoprecipitate the D1A receptor protein was raised against a 13-residue peptide corresponding to amino acids 403-415 and has been shown to immunoprecipitate the D1A receptor (15). The peptide sequence showed no similarity with other known sequences of catecholamine receptors or with other DA receptor subtypes (6, 33). In this study, the antibody immunoprecipitated a protein of ~70 kDa.

D1A receptor serine phosphorylation. SKF-38393- and DA-mediated phosphorylation of D1A receptors at serine residues (31) was detected with a specific phosphoserine antibody, which has been used in our previous study (2). Both SKF-38393 (2 µM) and DA (2 µM) increased serine phosphorylation of D1A receptors in adult (Figs. 1A and 2A), but not in old rats (Figs. 1B and 2B). SKF-38393- and DA-mediated increase in receptor serine phosphorylation was blocked by SCH-23390 (2 µM) (Figs. 1A and 2A), a D1-like receptor antagonist. It should be noted that the basal serine phosphorylation of the D1A receptors was higher in old compared with adult rats (adult vs. old, 0.63 ± 0.03 vs. 0.84 ± 0.025 density unit; Fig. 3).


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Fig. 1.   Effect of SKF-38393 on D1A receptor serine phosphorylation. Top: representative Western blots of serine phosphorylation of D1A receptors in adult (A) and old (B) rats. Lane 1: basal. Lane 2: SKF-38393 (2 µM). Lane 3: SKF-38393+SCH-23390 (each 2 µM). Bottom: densitometric analysis of the D1A receptor serine phosphorylation in adult (A) and old (B) rats. Values are means ± SE (percentage of basal). * P < 0.05, significantly different from basal; # P < 0.05, significantly different from SKF-38393 (ANOVA, n = 3).



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Fig. 2.   Effect of dopamine (DA) on D1A receptor serine phosphorylation. Top: representative Western blot of serine phosphorylation of D1A receptors in adult (A) and old (B) rats. Lane 1: basal. Lane 2: DA (2 µM). Lane 3: DA+SCH-23390 (each 2 µM). Bottom: densitometric analysis of the D1A receptor serine phosphorylation in adult (A) and old (B) rats. Values are means ± SE (percentage of basal). * P < 0.05, significantly different from basal; # P < 0.05, significantly different from DA (ANOVA, n = 3).



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Fig. 3.   Basal serine phosphorylation of D1A receptor. Top: representative Western blot of D1A receptor serine phosphorylation in basal state in adult and old rats. Bottom: densitometric analysis of basal serine phosphorylation of D1A in adult and old rats (adult vs. old; 0.63 ± 0.03 vs. 0.84 ± 0.025 arbitrary density unit). * P < 0.05, significantly different from adult (Student's t-test, n = 4).

D1A receptor protein abundance. As shown in the protein linearity experiment, the signal for D1A receptor proteins became saturated at 10 µg membrane proteins in both adult and old rats (Fig. 4A). The abundance of D1A receptor proteins measured at 6 µg membrane proteins was reduced 47% more in old than in adult rats: adult vs. old, 6.2 ± 0.42 vs. 3.3 ± 0.05 arbitrary density unit (Fig. 4B). The difference in the abundance of the receptor protein between adult and old rats was attenuated when a higher amount of membrane proteins was used (both 8 and 10 µg; Fig. 4A). Earlier, our laboratory reported that there was no difference in the abundance of D1A receptor proteins on the basolateral or brush-border membranes (4) of adult and old rats. Most likely, the cause of this discrepancy between the present and the earlier studies is the amount of membrane proteins used in the assay. Earlier, our laboratory used 20 µg membrane proteins for the measurement of D1A receptor proteins, and our present data show that at 10 µg of membrane proteins, the D1A receptor protein signal is saturated, which attenuates the difference between adult and old rats.


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Fig. 4.   Abundance of D1A receptor protein. A, top: representative Western blot of the protein saturation experiment of the D1A receptor in the proximal tubular membrane of adult (lanes 1, 3, 5, and 7 at 4, 6, 8, and 10 µg, respectively) and old (lanes 2, 4, 6, and 8 at 4, 6, 8, and 10 µg, respectively) rats. A, bottom: densitometric analysis of the Western blot of the protein saturation experiment of the D1A receptor in adult and old rats. B, top: representative Western blot of the 6 µg membrane proteins from adult and old rats that were used to measure D1A receptor proteins. B, bottom: densitometric analysis of D1A receptor proteins in adult and old rats (adult vs. old rats: 6.2 ± 0.42 vs. 3.3 ± 0.05 arbitrary density unit). * P < 0.05, significantly different from corresponding amount of membrane protein (A) and from adult (B) (Student's t-test, n = 3).

Agonist affinity. To determine the affinity of D1A receptors with agonist, we used FD to displace [3H]SCH-23390 binding on the cortical membranes from adult and old rats. As seen in Fig. 5, the analysis of the competition binding data revealed a two-site model. The agonist affinity for high-affinity receptors was reduced by a two-log unit of FD concentration in old compared with adult rats (IC50 values for high-affinity receptors; adult vs. old, 7.46 × 10-9 ± 2.26 vs. 7.93 × 10-7 ± 1.33 M; Fig. 5, inset). The agonist affinity for low-affinity receptors was reduced to one-half in old compared with adult rats (IC50 values for low-affinity receptors; adult vs. old, 5.67 × 10-5 ± 1.33 vs. 12.60 × 10-5 ± 6.50 M; Fig. 5, inset).


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Fig. 5.   Fenoldopam displacement of [3H]SCH-23390 in adult and old rats. Fenoldopam competition for [3H]SCH-23390 binding to DA D1A receptors from proximal tubular membranes of adult and old rats (see MATERIALS AND METHODS). Total binding was determined in the absence of any drug, whereas nonspecific binding was in the presence of 10 µM SCH-23390. After accounting for nonspecific binding, the specific counts at each drug concentration were expressed as percentage of total specific binding. Competition curve was plotted with the percentage of radioligand bound values obtained as means ± SE from three different experiments. Inset: IC50 values for high- and low-affinity receptors in adult and old rats.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In previous studies, our laboratory has reported a diminished natriuretic response to DA in old Fischer 344 rats, which was attributed, in part, to a decreased number of D1A receptor binding sites and uncoupling of the receptors from Gq and Gs proteins in renal proximal tubular cells (21). In addition to this defect, our laboratory has also demonstrated a defect at the PKC level, whereby a higher basal activity of PKC led to an increase in serine phosphorylation and a decrease in the activity of Na-K-ATPase in the proximal tubules of the old animals (3). In an attempt to explore the mechanism of the defect at the D1A receptor level, we determined the basal serine phosphorylation of D1A receptors in adult and old rats. We found a higher basal serine phosphorylation of D1A receptors in old compared with adult rats. Previously, an increase in the D1A receptor phosphorylation on serine residues had been reported as the mechanism responsible for the attenuation of the natriuretic response to DA in spontaneously hypertensive rats (36). The hyper-serine phosphorylation of D1A receptors as seen in the present study could account for the uncoupling of D1A receptors from the G proteins and subsequent failure of agonists to stimulate second messengers in the proximal tubules of old rats. In this regard, it is important to mention that higher basal serine phosphorylation of D1 receptors has been reported in the proximal tubular culture from essential hypertensive patients (30). The proximal tubular culture from these patients did not respond to an FD-mediated increase in cAMP compared with that from normotensive humans (30). Therefore, it appears that an increase in the basal serine phosphorylation of D1 receptors in hypertension (30, 36) and aging, as discovered in the present study, leads to its uncoupling from the G proteins in proximal tubules.

It is known that phosphorylation of receptors by GRKs and second messenger-activated kinases (PKA and PKC) convert high-affinity receptors (capable of agonist binding) to a low-affinity state (receptor-G protein uncoupled state). Because we found a higher basal serine phosphorylation of D1A receptors in old rats, these receptors would be expected to represent those in the low-affinity state. In the present study, FD displacement of the radioligand [3H]SCH-23390 revealed a 100-fold decrease in the affinity for the agonist by high-affinity receptors in old compared with adult rats. In addition to the 50% reduction in ligand binding sites (4, 21) and D1A receptor protein abundance (Fig. 4B), such a decrease in the agonist affinity toward the remaining 50% of the D1A receptors may contribute to the inability of DA to activate the second messenger systems and inhibit Na transporters in proximal tubules of old rats.

The receptor phosphorylation and desensitization by kinases (GRK, PKC, and PKA) occur once the receptors are activated in the presence of an agonist. In the present study, we found a higher basal serine phosphorylation of D1A receptors in the absence of an agonist in old rats. There is a report showing a ligand-independent phosphorylation on serine residues and densensitization of D1 receptors, probably because of the increase of a kinase activity, in proximal tubular cells from humans with essential hypertension (30). It should be noted that the basal PKC activity is elevated in old rats (3), and PKC is reported to regulate GRK activity (23, 29, 35). Therefore, higher basal PKC activity seems likely to cause an increase in the basal serine phosphorylation of D1A receptors by means of activation of GRKs and leads to uncoupling of the receptors from the G protein in old rats. The cause or mechanism responsible for higher PKC activity in old rats is not known. However, it is likely that impairment of the D1A receptor in terms of its higher serine phosphorylation and reduced agonist affinity may be secondary to the higher basal PKC activity in old rats. The PKC-mediated regulation of GRKs, and subsequently their role in D1A receptor phosphorylation and agonist affinity, is presently under investigation in the kidneys of Fischer 344 rats. This mechanism of D1A receptor phosphorylation and loss in agonist affinity appears to be independent of the cAMP/PKA pathway, as has been suggested earlier by Lewis et al. (26). This notion is also supported by earlier studies wherein our laboratory has shown similar basal cAMP levels as well as similar basal PKA activity in the proximal tubules of adult and old rats (4).

In summary, we found 1) an increase in the basal serine phosphorylation of the D1A receptors, 2) a reduction in D1A receptor affinity to the agonist, and 3) a reduction in D1A receptor protein abundance in old compared with adult rats. We speculate that an increase in basal serine phosphorylation of D1A receptors may have caused receptor G protein uncoupling as well as downregulation of the receptors, which contribute to the inability of DA to activate the second messengers and subsequently inhibit tubular Na transporters. These abnormalities at the level of D1A receptors could account for the diminished DA-mediated natriuresis and diuresis in old rats (4, 21).


    ACKNOWLEDGEMENTS

This study was supported by National Institute on Aging Grant AG-15031.


    FOOTNOTES

Address for reprint requests and other correspondence: M. F. Lokhandwala, College of Pharmacy, Univ. of Houston, Houston, TX 77204 (E-mail: mlokhandwala{at}uh.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.

March 5, 2002;10.1152/ajprenal.00361.2001

Received 11 December 2001; accepted in final form 25 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 283(2):F350-F355
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