Activation of D2-like receptors causes recruitment of tyrosine-phosphorylated NKA alpha 1-subunits in kidney

Vihang A. Narkar, Tahir Hussain, and Mustafa F. Lokhandwala

Institute for Cardiovascular Studies, College of Pharmacy, University of Houston, Houston, Texas 77204-5515


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study investigates the cellular mechanisms responsible for dopamine D2-like receptor-mediated stimulation of Na+-K+-ATPase in the proximal tubules of the kidney. Previously, we showed that D2-like receptor-mediated increase in Na+-K+-ATPase involves an increase in the maximum rate of Na+-K+-ATPase activity (Vmax). Therefore, we tested the hypothesis that D2-like receptor-mediated stimulation of Na+-K+-ATPase requires phosphorylation and recruitment of alpha 1-subunits of the enzyme from cytosol to the membrane. This hypothesis was tested by Western blotting for Na+-K+-ATPase alpha 1-subunits in proximal tubular membrane. Treatment of the proximal tubules with bromocriptine (D2-like receptor agonist) caused an increase in Na+-K+-ATPase alpha 1-subunit abundance in the membrane preparations. This effect was blocked by genistein (tyrosine kinase inhibitor), suggesting a role for tyrosine phosphorylation. Moreover, bromocriptine caused an increase in tyrosine phosphorylation of membrane-bound Na+-K+-ATPase alpha 1-subunits. This effect was blocked by bafilomycin A1 (vesicular trafficking inhibitor), which suggested that this increase was due to the recruitment of tyrosine-phosphorylated Na+-K+-ATPase alpha 1-subunits. In conclusion, we have demonstrated that activation of D2-like receptors increases Na+-K+-ATPase activity by recruitment of the tyrosine-phosphorylated alpha 1-subunits in the proximal tubules of the kidney.

dopamine; bromocriptine; bafilomycin A1; genistein; proximal tubules; sodium/potassium/adenosine 5'-triphosphatase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DOPAMINE PLAYS AN IMPORTANT role in the regulation of Na+-K+-ATPase (NKA) in the proximal tubules of the kidney. Activation of D1-like receptors is associated with the inhibition of NKA (12, 13). On the other hand, activation of D2-like receptors causes stimulation of NKA in the proximal tubules (11, 16). The inhibition or stimulation of NKA depends on several factors, such as 1) concentration of substrates (Na+, K+, Mg+), 2) covalent modification of NKA subunits, and 3) number of NKA subunits in the plasma membrane (9, 10). It has been shown that activation of D1-like receptors causes initial Ser18 phosphorylation of the NKA alpha 1-subunits and their subsequent endocytosis, which is responsible for the inhibition of NKA (3). However, the mechanism by which D2-like receptors stimulate NKA is not known. One of the mechanisms reported for activation of NKA is the recruitment of NKA alpha 1-subunits to the plasma membrane (1, 5, 7, 14). In addition, phosphorylation of Tyr10 residue in the NKA alpha 1-subunit causes activation of NKA (8).

We have previously reported that activation of D2-like receptors increases the maximum rate of NKA catalytic activity (Vmax) in the proximal tubules of the kidney (11). This observation suggests that there might be an increase in the number of NKA alpha 1-subunits in the membranes on activation of D2-like receptors. Furthermore, this D2-like receptor-mediated increase in Vmax was dependent on activation of a tyrosine kinase-p44/42 MAPK pathway (15). As tyrosine kinases increase tyrosine phosphorylation of substrate proteins, it is possible that D2-like receptor agonists may also cause the tyrosine phosphorylation of NKA alpha 1-subunits. Thus there are two potential mechanisms by which D2-like receptor activation may increase NKA activity in the proximal tubules, namely, recruitment or tyrosine phosphorylation of NKA alpha 1-subunits. Moreover, it is possible that tyrosine phosphorylation and recruitment may be interrelated phenomena, which are required together for NKA stimulation by D2-like receptor agonist.

In this study, we have tested the hypothesis that activation of D2-like receptors causes recruitment of NKA alpha 1-subunits to the membranes in the renal proximal tubules. Furthermore, we have also investigated whether activation of D2-like receptors causes tyrosine phosphorylation of NKA alpha 1-subunits in proximal tubular cell membranes as well as in the cytosol. In addition, we have asked whether tyrosine phosphorylation of NKA alpha 1-subunits plays a role in recruitment of these subunits to the plasma membranes of proximal tubular cells. All the studies were performed in freshly isolated proximal tubules of kidneys obtained from Sprague-Dawley rats. Recruitment was measured by Western blotting for NKA alpha 1-subunits in proximal tubular membranes. For tyrosine phosphorylation, NKA alpha 1-subunits were first immunoprecipitated from membranes or cytosol and then probed for tyrosine phosphorylation.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Animals. Male Sprague-Dawley rats between 12 and 15 wk old and weighing 200-250 g were used (Harlen Sprague Dawley, Indianapolis, IN). The animals were housed in plastic cages in an air-conditioned animal care facility and had access to standard rat chow (Purina Mills, St. Louis, MO) and tap water ad libitum.

Isolation of proximal tubules from the kidney. The rats were anesthetized with pentobarbital sodium (50 mg/kg ip). The proximal tubules were isolated from the kidneys of the rats and enriched by a previously described method (11). The enriched proximal tubules were resuspended in 5 ml of modified Krebs-Henseleit buffer [(in mM) 118 NaCl, 4 KCl, 1.25 CaCl2, 1.2 MgCl2, 27.2 NaHCO3, 1 KH2PO4, 5 glucose, and 10 HEPES, pH 7.4] and further used for drug treatment.

Drug treatment. All the drug treatments were done in 10 ml total volume of proximal tubular suspension (0.75 mg/ml). The proximal tubules were treated without (basal) or with 100 µl of bromocriptine to reach a final concentration of 0.1 µM at 37°C for 15 min. The reaction was terminated by rapid freezing and thawing in a dry ice-acetone mixture. The drug-treated cell lysates were then further used to prepare proximal tubular membranes. Just before freezing, 400 µl of protease inhibitor cocktail (25×; Boehringer Mannheim) (along with 4 mM sodium orthovanadate when measuring tyrosine phosphorylation) were added to each tube.

Inhibitor treatment. For inhibitor studies, the proximal tubules were incubated with PD-98059 (10 µM), genistein (0.001 µM), or bafilomycin A1 (0.02 µM), at 37°C for 10 min, before treatment with agonist.

Preparation of proximal tubular membranes. The freeze-thawed cell lysates were used for membrane preparation. The cell lysates were first centrifuged at 36,000 g at 4°C for 20 min to obtain pellets, and the supernatant was saved as cytosolic fraction. The pellets were resuspended in 5 ml of homogenization buffer (50 mM Tris · HCl and 1 mM MgCl2, pH 7.4) containing protease inhibitor cocktail and homogenized with a Wheaton homogenizer (20 strokes at setting 7). The homogenate was then centrifuged at 36,000 g at 4°C for 20 min to obtain membrane pellets. The pellet was resuspended in homogenization buffer (5 ml) and centrifuged at 36,000 g at 4°C for 10 min to obtain the membrane pellet. This washing step was repeated three times. Finally, the pellet was resuspended in buffer (250 µl) containing 50 mM Tris · HCl and 5 mM MgCl2 at pH 7.5.

Determination of NKA alpha 1-subunits in membrane preparation. Loading samples were prepared for Western blotting, from membrane samples, such that the protein concentration was 0.1 µg protein/10 µl. Samples of 10 µl/lane were loaded and separated by gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were probed for NKA alpha 1-subunits with mouse monoclonal NKA alpha 1-subunit primary antibody (1:1,000 dilution; Research Diagnostics, Flanders, NJ). Furthermore, the membranes were probed with rabbit anti-mouse secondary antibody (1:10,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and NKA alpha 1-subunit bands were detected with a chemiluminescence kit (Santa Cruz Biotechnology). Densitometric analysis of the bands was performed and data were represented in arbitrary units. In a few experiments, Commassie blue staining of the PVDF membrane was performed to confirm the similar amounts of proteins loaded.

Immunoprecipitation of NKA alpha 1-subunit from membrane and cytosolic fraction. The membrane and cytosolic proteins were dissolved in immunoprecipitation buffer [(in mM) 20 Tris · HCl, 150 NaCl, 10 NaF, 10 Na2P4O7, and 2 EDTA, as well as 1% Triton X-100 and 0.1% SDS, pH 8] containing protease inhibitor cocktail and 0.1 mM PMSF at a concentration of 250 µg protein/ml. Next, the membrane proteins were cleared with protein A/G-agarose (Santa Cruz Biotechnology). Furthermore, an aliquot (1 ml) of precleared supernatant was incubated with mouse monoclonal antibody against NKA alpha 1-subunit. Antigen (NKA alpha 1-subunit)-antibody complex was precipitated overnight with protein A/G-agarose. After precipitation, the complex was washed once with immunoprecipitation buffer followed by one wash with wash buffer [(in mM) 20 Tris · HCl, 150 NaCl, and 5 EDTA, as well as 0.1% Triton X-100 and 0.1% SDS, pH 8 at 25°C] and 50 mM Tris · HCl (pH 8). Finally, the antigen-antibody complex was dissociated with 50 µl of 2× Laemmli buffer at 37°C for 1 h. The protein A/G-agarose beads were removed by centrifugation, and the supernatant was used for Western blotting. All the immunoprecipitation steps were carried out at 4°C.

Western blotting. The immunoprecipitated samples were resolved by gel electrophoresis using SDS-PAGE and transferred onto PVDF membranes. The membranes were then probed with either mouse phosphotyrosine antibody (1:1,000 dilution; Santa Cruz Biotechnology) or mouse monoclonal NKA alpha 1-subunit primary antibody (1:1,000 dilution). The primary antibodies bound to the membranes were detected by using rabbit anti-mouse secondary antibody (1:5,000 dilution for phosphotyrosine and 1:10,000 dilution for NKA alpha 1-subunit antibody). The immunoreactivity was detected with a chemiluminescence kit. Densitometric analysis was performed on the bands, and data were represented as the ratio of phosphotyrosine to total NKA alpha 1-subunits.

Statistical analysis. Data are represented as means ± SE of several experiments. Where applicable, the data were analyzed with unpaired Student's t-test or one-way analysis of variance along with a suitable post hoc test. The difference was considered significant if P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of bromocriptine on NKA alpha 1-subunit abundance in proximal tubular membrane. Effect of bromocriptine on NKA alpha 1-subunit (the catalytic subunit) abundance in proximal tubular membranes was measured by Western blotting with mouse monoclonal antibody against the NKA alpha 1-subunits. It was found that treatment of proximal tubules with bromocriptine caused an increase in immunoreactivity for NKA alpha 1-subunit (~100 kDa) in the membranes, suggesting an increase in NKA alpha 1-subunit abundance (Fig. 1). This effect of bromocriptine was blocked by PD-98059 [10 µM, MEK1/2 inhibitor (6)], genistein [0.001 µM, tyrosine kinase inhibitor (4)], and bafilomycin A1 [0.02 µM, vesicular trafficking inhibitor (2)] (Fig. 1, A and B). The densitometric analysis of the blots showed an increase in NKA alpha 1-subunit abundance in proximal tubular membranes (Fig. 1C).


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Fig. 1.   Effect of various inhibitors on bromocriptine-mediated increase in Na+-K+-ATPase (NKA) alpha 1-subunit abundance in proximal tubular membranes. The proximal tubules were incubated without (Control) or with bromocriptine (0.1 µM) for 15 min at 37°C. The reaction was terminated by freeze-thawing and Western blotting for NKA alpha 1-subunit performed from membrane samples, as described in EXPERIMENTAL PROCEDURES. A: representative blot for effect of PD-98059 (10 µM) and genistein (0.001 µM) pretreatment (10 min) on bromocriptine-mediated increase in NKA alpha 1-subunit abundance. B: representative blot for effect of bafilomycin A1 (0.02 µM) pretreatment (10 min) on bromocriptine-mediated increase in NKA alpha 1-subunit abundance. C: data represented as average (means ± SE) density of NKA alpha 1-subunits from n = 3-6 experiments. *P < 0.05, significantly different from control; #P < 0.05, significantly different from the inhibitor-treated groups, by one-way ANOVA and, post hoc, Newman-Keuls multiple comparison test.

Effect of bromocriptine on tyrosine phosphorylation of NKA alpha 1-subunits in proximal tubular membrane in absence and presence of genistein. Next, we performed experiments to determine whether D2-like receptor activation causes tyrosine phosphorylation of NKA alpha 1-subunits in proximal tubular membranes. This was done by immunoprecipitating NKA alpha 1-subunits from the proximal tubular membranes after treating the proximal tubules with bromocriptine (0.1 µM). Furthermore, the immunoprecipitated samples were resolved by gel electrophoresis and probed with either phosphotyrosine or NKA alpha 1-subunit antibody. We found that treatment of the proximal tubules with bromocriptine caused an increase in the tyrosine phosphorylation of NKA alpha 1-subunits. Furthermore, this effect was blocked by pretreatment with genistein (0.001 µM) (Fig. 2A). In parallel experiments, it was confirmed that similar amounts of NKA alpha 1-subunits were immunoprecipitated from the membranes by checking the immunoreactivity to NKA alpha 1-subunit antibody (Fig. 2B). The densitometric ratio of phosphotyrosine to total NKA alpha 1-subunits was also increased in the bromocriptine-treated group (Fig. 2C).


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Fig. 2.   Effect of genistein on bromocriptine-mediated tyrosine phosphorylation of NKA alpha 1-subunits in proximal tubular cell membrane. The proximal tubules were incubated without (Ba, lane 1) or with bromocriptine (Br, 0.1 µM; lane 2) for 15 min at 37°C. For genistein pretreatment, the proximal tubules were incubated with genistein (0.001 µM) for 10 min before bromocriptine treatment (Br+G, lane 3). Furthermore, the proximal tubular membranes were prepared and assayed for tyrosine phosphorylation of NKA alpha 1-subunits as described in EXPERIMENTAL PROCEDURES. A: representative blot, in which the NKA alpha 1-subunits were immunoprecipitated (IP) with mouse monoclonal NKA alpha 1-subunits and detected with anti-phosphotyrosine antibody after gel electrophoresis. B: representative blot, in which the NKA alpha 1-subunits were immunoprecipitated with mouse monoclonal NKA alpha 1-subunits and detected with the same antibody after gel electrophoresis. C: densitometric ratio of phosphorylated to total NKA alpha 1-subunits. *P < 0.05, significantly different between basal and bromocriptine-treated groups; #P < 0.05, significantly different between genistein-untreated and treated groups, by one-way ANOVA and, post hoc, Tukey's multiple comparison test.

Effect of bafilomycin A1 on D2-like receptor-mediated increase in tyrosine phosphorylation of NKA alpha 1-subunit in proximal tubular cell membranes. Because D2-like receptors caused an increase in tyrosine phosphorylation of membrane NKA alpha 1-subunits, it was important to determine whether this increase was due to direct phosphorylation of membrane NKA alpha 1-subunits or recruitment of ones phosphorylated in the cytosol. To determine this, we studied the effect of bafilomycin A1 (which in our previous experiments blocked D2-like receptor-mediated recruitment of NKA alpha 1-subunits) on D2-like receptor-mediated tyrosine phosphorylation of NKA. It was found that bafilomycin A1 blocked bromocriptine-mediated tyrosine phosphorylation of NKA alpha 1-subunits in the membranes (Fig. 3A). Furthermore, the NKA alpha 1-subunits immunoprecipitated from each sample were similar (Fig. 3B). In addition, there was an increase in the ratio of phosphorylated tyrosine to total NKA alpha 1-subunit protein in the membrane (Fig. 3C). Thus the increase in membrane tyrosine phosphorylation may be due to the recruitment of tyrosine-phosphorylated alpha 1-subunits to the proximal tubular membranes.


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Fig. 3.   Effect of bafilomycin A1 on bromocriptine-mediated tyrosine phosphorylation of NKA alpha 1-subunits in proximal tubular cell membrane. The proximal tubules were incubated without (lane 1) or with bromocriptine (0.1 µM; lane 2) for 15 min at 37°C. For bafilomycin A1 pretreatment, the proximal tubules were incubated with bafilomycin A1 (0.02 µM) for 10 min before bromocriptine treatment (Br + A1, lane 3). Furthermore, the proximal tubular membranes were prepared and assayed for tyrosine phosphorylation of NKA alpha 1-subunits as described in EXPERIMENTAL PROCEDURES. A: representative blot, in which the NKA alpha 1-subunits were immunoprecipitated with mouse monoclonal NKA alpha 1-subunits and detected with anti-phosphotyrosine antibody after gel electrophoresis. B: representative blot in which the NKA alpha 1-subunits were immunoprecipitated with mouse monoclonal NKA alpha 1-subunits and detected with the same antibody after gel electrophoresis. C: densitometric analysis was performed on all the blots. Values were represented as the ratio of phosphotyrosine NKA alpha 1-subunit to total NKA alpha 1-subunit protein density. Values are means ± SE of 4 experiments. *P < 0.05, significantly different between basal and bromocriptine-treated group; #P < 0.05, significantly different between bafilomycin A1-untreated and treated groups, by one-way ANOVA and, post hoc, Tukey's multiple comparison test.

Effect of bafilomycin A1 on D2-like receptor-mediated increase in tyrosine phosphorylation of NKA alpha 1-subunit in proximal tubular cell cytosol. Furthermore, we wanted to see the effect of D2-like receptor activation on tyrosine phosphorylation of NKA alpha 1-subunits in the cytosol of the proximal tubular cells. We found that 15-min treatment of proximal tubules with bromocriptine did not have any effect on tyrosine phosphorylation of cytosolic NKA alpha 1-subunits (Fig. 4A). Surprisingly, when the proximal tubules were pretreated with bafilomycin A1 (0.02 µM), bromocriptine was able to stimulate tyrosine phosphorylation of NKA alpha 1-subunits in the cytosol (Fig. 4A), an observation reciprocal to that in the membranes. Furthermore, in bafilomycin A1-treated proximal tubules, there was an increase in the ratio of phosphorylated tyrosine to total NKA alpha 1-subunit protein in the cytosol (Fig. 4C). The immunoprecipitated NKA alpha 1-subunits were similar in all treatment groups (Fig. 4B).


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Fig. 4.   Effect of bafilomycin A1 on bromocriptine-mediated tyrosine phosphorylation of NKA alpha 1-subunits in proximal tubular cell cytosol. The proximal tubules were treated as described in Fig. 4. Furthermore, the proximal tubular cell cytosol was assayed for tyrosine phosphorylation of NKA alpha 1-subunits as described in EXPERIMENTAL PROCEDURES. A: representative blot in which the NKA alpha 1-subunits were immunoprecipitated with mouse monoclonal NKA alpha 1-subunits and detected with anti-phosphotyrosine antibody after gel electrophoresis. B: representative blot in which the NKA alpha 1-subunits were immunoprecipitated with mouse monoclonal NKA alpha 1-subunits and detected with the same antibody after gel electrophoresis. C: densitometric analysis was performed on all the blots. Values were represented as the ratio of phosphotyrosine NKA alpha 1-subunits to total NKA alpha 1-subunit protein density. Values are means ± SE of 4 experiments. *P < 0.05, significantly different between basal and bromocriptine-treated group; #P < 0.05, significantly different between bafilomycin A1-untreated and treated groups, by one-way ANOVA and, post hoc, Tukey's multiple comparison test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we have shown that dopamine D2-like receptor activation causes recruitment of NKA alpha 1-subunits in the proximal tubular cells. Furthermore, activation of D2-like receptors also causes tyrosine phosphorylation of NKA alpha 1-subunits in the proximal tubules. This effect is blocked by bafilomycin A1 (inhibitor of vesicular trafficking), which suggests that the increase in the abundance of tyrosine-phosphorylated membrane NKA alpha 1-subunits may have been caused by D2-like receptor-mediated recruitment of tyrosine-phosphorylated NKA alpha 1-subunits from cytosol. This is supported by the observation that D2-like receptor activation causes an increase in tyrosine phosphorylation of cytosolic NKA alpha 1-subunits only in the presence of bafilomycin A1 (which prevents the recruitment of these tyrosine-phosphorylated NKA alpha 1-subunits).

We had previously observed that bromocriptine (D2-like receptor agonist) caused an increase in the rate of hydrolytic activity of NKA under saturating concentrations of sodium, potassium, magnesium, and ATP (11) in the proximal tubules of the kidney. In other words, D2-like receptor activation caused an increase in Vmax of NKA enzymatic activity. Because an increase in Vmax is associated with the increase in the amount of functional enzyme, we tested the hypothesis that D2-like receptors might cause an increase in NKA abundance in the proximal tubular membranes. We tested this hypothesis with Western blotting to measure the changes in NKA alpha 1-subunits in the proximal tubular membranes on activation of D2-like receptors. We found that the NKA alpha 1-subunit antibody immunoreacted with a 100-kDa (approximate) protein. In these experiments, bromocriptine increased NKA alpha 1-subunit immunoreactivity in the proximal tubular membranes, which was blocked by PD-98059 and genistein. Thus activation of D2-like receptors causes recruitment of NKA alpha 1-subunits in the proximal tubules of the kidney, which requires activation of p44/42 MAPK and a tyrosine kinase. This finding is in concert with our previous observation that p44/42 MAPK and tyrosine kinase are required for D2-like receptor-mediated activation of NKA (15).

It has been reported that tyrosine phosphorylation of the NKA alpha 1-subunits leads to an increase in NKA activity (8, 9). Furthermore, our findings show that D2-like receptor-mediated recruitment of NKA alpha 1-subunits requires genistein-sensitive tyrosine kinase activity. Therefore, we wanted to test the possibility that activation of D2-like receptors causes tyrosine phosphorylation of NKA alpha 1-subunits, which may trigger recruitment. We found that bromocriptine caused an increase in tyrosine phosphorylation per NKA alpha 1-subunit in proximal tubular membrane. Furthermore, bafilomycin A1 blocked D2-like receptor-mediated increase in tyrosine phosphorylation per NKA alpha 1-subunit in the membrane. Because bafilomycin A1 blocked the recruitment of NKA alpha 1-subunits in our experiments, it indicates that activation of D2-like receptors might cause the recruitment of tyrosine-phosphorylated NKA alpha 1-subunits in proximal tubules.

When we measured D2-like receptor-mediated tyrosine phosphorylation of cytosolic NKA alpha 1-subunits, we found that bromocriptine alone did not increase the tyrosine phosphorylation. On the other hand, pretreatment of the proximal tubules with bafilomycin A1 resulted in a bromocriptine-mediated increase in tyrosine phosphorylation per NKA alpha 1-subunits in cytosolic preparations. This observation can be explained if the time point of measurement is considered. We measured the increase in recruitment and tyrosine phosphorylation in proximal tubules treated with bromocriptine for 15 min, when the NKA was found to be maximally stimulated (11). Therefore, it is possible that at 15 min the NKA alpha 1-subunits, which are tyrosine phosphorylated via D2-like receptors, have already been recruited to the membrane. Hence, we do not see an increase in cytosolic tyrosine phosphorylation of NKA alpha 1-subunits. However, once recruitment is blocked by bafilomycin A1 pretreatment, D2-like receptor-mediated increase in tyrosine phosphorylation of the NKA alpha 1-subunits retained in the cytosol is measurable. In other words, activation of D2-like receptors causes tyrosine phosphorylation and subsequent recruitment of NKA alpha 1-subunits in proximal tubules of the kidney.

Recruitment of NKA alpha 1-subunits seems to be a common mechanism for the receptor-mediated activation of NKA in several cell types (1, 5, 7, 14). However, the covalent modification of NKA alpha 1-subunits that drives recruitment may be varied. For example, Efendiev et al. (7) reported that simultaneous phosphorylation of Ser11 and Ser18 of the NKA alpha 1-subunits is responsible for the PKC-mediated recruitment of the NKA alpha 1-subunits to the plasma membrane. On the other hand, in alveolar epithelium, the D1-like receptor-mediated recruitment of NKA alpha 1-subunits is attributed to activation of a serine/threonine protein phosphatase 2A (14). Along the same lines, it was recently reported that aldosterone-mediated recruitment of NKA alpha 1-subunits in cortical collecting duct required a dephosphorylation of these subunits (5).

In this study, we have particularly examined the role of tyrosine phosphorylation of NKA alpha 1-subunits in its D2-like receptor-mediated recruitment. Here, we have shown that blocking tyrosine phosphorylation in the proximal tubules blocked the D2-like receptor-mediated recruitment of NKA alpha 1-subunits. This result supports our previous observation that genistein completely inhibited the stimulation of NKA via D2-like receptors (15). Whether tyrosine phosphorylation is the only requirement for recruitment of NKA alpha 1-subunits by D2-like receptors is not known. As mentioned above, serine/threonine dephosphorylation may be another mechanism for recruitment. To our knowledge, such an effect of D2-like receptors is not reported in the proximal tubules of the kidney. Nevertheless, tyrosine phosphorylation is critical for the bromocriptine-mediated activation of NKA.

In summary, we have shown that activation of the D2-like receptor causes recruitment of NKA alpha 1-subunits in a p44/42 MAPK-tyrosine kinase-dependent manner in the proximal tubules of rat kidney. Moreover, D2-like receptor-mediated recruitment requires tyrosine phosphorylation of cytosolic NKA alpha 1-subunits (Fig. 5). Therefore, the present study provides the cellular mechanisms for D2-like receptor-mediated stimulation of NKA activity in renal proximal tubules.


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Fig. 5.   Mechanism of D2-like receptor-mediated stimulation of NKA in proximal tubules of kidney. Activation of D2-like receptors with bromocriptine causes tyrosine phosphorylation of cytosolic NKA alpha 1-subunits via p44/42 MAPK and tyrosine kinase. The tyrosine-phosphorylated NKA alpha 1-subunits are subsequently recruited to the proximal tubular membranes. This mechanism was elucidated by using PD-98059 (MEK1/2-p44/42 MAPK inhibitor), genistein (tyrosine kinase inhibitor), and bafilomycin A1 (vesicular H+-ATPase inhibitor, which disrupts the vesicles and prevents their transport). Stimulation and inhibition are represented by (right-arrow) and (), respectively.


    ACKNOWLEDGEMENTS

We thank Dr. M. Asghar for suggestions relating to the immunoprecipitation experiments.


    FOOTNOTES

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

Address for reprint requests and other correspondence: M. F. Lokhandwala, College of Pharmacy, Univ. of Houston, Houston, TX 77204-5511 (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.

August 6, 2002;10.1152/ajprenal.00039.2002

Received 29 January 2002; accepted in final form 26 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Bertorello, AM, Ridge KM, Chibalin AV, Katz AI, and Sznajder JI. Isoproterenol increases Na+, K+-ATPase activity by membrane insertion of alpha -subunits in lung alveolar cells. Am J Physiol Lung Cell Mol Physiol 276: L20-L27, 1999[Abstract/Free Full Text].

2.   Bowman, EJ, Siebers A, and Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci USA 85: 7972-7976, 1988[Abstract].

3.   Chibalin, AV, Ogimoto G, Pedemonte CH, Pressley TA, Katz AI, Feraille E, Berggren PO, and Bertorello AM. Dopamine-induced endocytosis of Na+, K+-ATPase is initiated by phosphorylation of Ser-18 in the rat alpha subunit and is responsible for the decreased activity in epithelial cells. J Biol Chem 274: 1920-1927, 1999[Abstract/Free Full Text].

4.   Dean, NM, Kanemitsu M, and Boynton AL. Effects of the tyrosine kinase inhibitor genistein on DNA synthesis and phospholipid-derived second messenger generation in mouse 10T1/2 fibroblasts and rat liver T51B cells. Biochem Biophys Res Commun 165: 795-801, 1989[ISI][Medline].

5.   Djelidi, S, Beggah A, Courtois-Coutry N, Fay M, Cluzeaud F, Viengchareun S, Bonvalet JP, Farman N, and Blot-Chabaud M. Basolateral translocation by vasopressin of the aldosterone-induced pool of latent Na-K-ATPases is accompanied by alpha 1 subunit dephosphorylation: study in a new aldosterone-sensitive rat cortical collecting duct cell line. J Am Soc Nephrol 12: 1805-1818, 2001[Abstract/Free Full Text].

6.   Dudley, DT, Pang L, Decker SJ, Bridges AJ, and Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 92: 7686-7689, 1995[Abstract].

7.   Efendiev, R, Bertorello AM, Pressley TA, Rousselot M, Feraille E, and Pedemonte CH. Simultaneous phosphorylation of Ser11 and Ser18 in the alpha -subunit promotes the recruitment of Na+, K+-ATPase molecules to the plasma membrane. Biochemistry 39: 9884-9892, 2000[ISI][Medline].

8.   Feraille, E, Carranza ML, Gonin S, Be'guin P, Pedemonte C, Rousselot M, Caverzasio J, Geering K, Martin PY, and Favre H. Insulin-induced stimulation of Na+, K+-ATPase activity in kidney proximal tubule cells depends on phosphorylation of the alpha -subunit at Tyr-10. Mol Biol Cell 10: 2847-2859, 1999[Abstract/Free Full Text].

9.   Feraille, E, Carranza ML, Rousselot M, and Favre H. Modulation of Na+, K+-ATPase activity by tyrosine phosphorylation process in rat proximal tubules. J Physiol 498: 99-108, 1997[Abstract].

10.   Feraille, E, and Doucet A. Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev 81: 345-418, 2001[Abstract/Free Full Text].

11.   Hussain, T, Abdul-Wahab R, and Lokhandwala MF. Bromocriptine stimulates Na+, K+-ATPase in renal proximal tubules via the cAMP pathway. Eur J Pharmacol 321: 259-263, 1997[ISI][Medline].

12.   Hussain, T, and Lokhandwala MF. Renal dopamine receptor function in hypertension. Hypertension 32: 187-197, 1998[Abstract/Free Full Text].

13.   Jose, PA, Eisner GM, and Felder RA. Dopamine receptors in health and hypertension. Pharmacol Ther 80: 149-182, 1998[ISI][Medline].

14.   Lecuona, E, Garcia A, and Sznajder JI. A novel role for protein phosphatase 2A in the dopaminergic regulation of Na, K-ATPase. FEBS Lett 481: 217-220, 2000[ISI][Medline].

15.   Narkar, VA, Hussain T, and Lokhandwala MF. Role of tyrosine kinase and p44/42 MAPK in D2-like receptor-mediated stimulation of Na+, K+, ATPase in kidney. Am J Physiol Renal Physiol 282: F697-F702, 2002[Abstract/Free Full Text].

16.   Yamaguchi, I, Walk SF, Jose PA, and Felder RA. Dopamine D2L receptors stimulate Na+, K+-ATPase activity in murine LTK- cells. Mol Pharmacol 46: 373-378, 1996.


Am J Physiol Renal Fluid Electrolyte Physiol 283(6):F1290-F1295
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