Role of tyrosine kinase and p44/42 MAPK in D2-like receptor-mediated stimulation of Na+, K+-ATPase in kidney

Vihang Narkar, Tahir Hussain, and Mustafa Lokhandwala

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our laboratory has shown that dopamine D2-like receptor activation causes stimulation of Na+, K+-ATPase (NKA) activity in the proximal tubules of the rat kidney. The present study was designed to investigate the cellular signaling mechanisms mediating this response to D2-like receptor activation. We measured the stimulation of NKA activity by bromocriptine (D2-like receptor agonist) in the absence and presence of PD-98059 [p44/42 mitogen-activated protein kinase (MAPK) kinase inhibitor] and genistein (tyrosine kinase inhibitor) in renal proximal tubules. Both agents inhibited bromocriptine-mediated stimulation of NKA, suggesting the involvement of p44/42 MAPK and tyrosine kinase in this response. Additionally, we found that bromocriptine increased the phosphorylation of p44/42 MAPK in the proximal tubules, which was blocked by PD-98059 and genistein. These results show that D2-like receptor activation causes stimulation of NKA activity by means of a tyrosine kinase-p44/42 MAPK pathway in the proximal tubules of the kidney.

dopamine; bromocriptine; proximal tubules; sodium-potassium-adenosine 5'-triphosphatase; mitogen-activated protein kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE PROXIMAL TUBULES OF THE kidney are responsible for reabsorbing ~60% of Na+ in the glomerular filtrate (15). In the proximal tubules, an Na+-H+ exchanger (NHE) on the apical side and Na+, K+-ATPase (NKA) on the basolateral side of the epithelial cell lining constitute major mechanisms for Na+ reabsorption (7, 9). These transporters are regulated by several hormones that stimulate or inhibit the transporters, causing antinatriuretic or natriuretic effects, respectively (4, 13).

Dopamine plays an important role in the regulation of NHE and NKA in the proximal tubules of the kidney. Dopamine and dopamine D1-like receptor agonists inhibit NHE and NKA through Gs and Gq/11 protein-mediated pathways (7, 9). The proximal tubules of the kidney also express D2-like receptors, and although dopamine predominantly inhibits NHE and NKA by means of D1-like receptors, selective activation of D2-like receptors may have different effects on these transporters. Recently, it has been shown that the D2-like receptor agonist bromocriptine did not affect NHE activity in opossum kidney (OK) cells (19). On the other hand, our laboratory has previously shown that bromocriptine causes stimulation of NKA in the proximal tubules of the rat kidney (6). This response involves D2-like receptor activation, Gi protein coupling, and a decrease in intracellular cAMP (6, 21).

Although coupling of D2-like receptors to Gi protein and the consequent decrease in cAMP seem to play an important role in stimulation of NKA, no further information is available on the downstream effectors, which may play a role in this D2-like receptor-mediated effect in the proximal tubules. Dopamine D2-like receptors are linked to several effectors such as adenylate cyclase, Ca++ channels, K+ channels, arachidonic acid metabolism, and p44/42 mitogen-activated protein kinase (MAPK) in different cell types (6, 11, 12, 14, 17, 18, 22). Recently, our laboratory found that bromocriptine caused stimulation of p44/42 MAPK in OK cells (14). This response involved D2-like receptor-mediated and Gi- or Go-mediated decreases in cAMP levels and activation of a tyrosine kinase in addition to other signaling components (14).

On the basis of our observations in rat proximal tubules and OK cells, it can be suggested that a decrease in the intracellular cAMP is the initial signal necessary for stimulation of p44/42 MAPK and NKA (by D2-like receptor activation) in kidney proximal tubules. Furthermore, it is possible that p44/42 MAPK may play a role in D2-like receptor-mediated stimulation of NKA. In addition, tyrosine kinase activation is shown to be necessary for growth factor-mediated stimulation of NKA (3) and also for D2-like receptor-mediated stimulation of p44/42 MAPK (14) in kidney proximal tubules. Thus p44/42 MAPK and tyrosine kinase may be potential candidates for D2-like receptor-mediated stimulation of NKA.

The present study was designed to test the hypothesis that p44/42 MAPK and tyrosine kinase are involved in D2-like receptor-mediated stimulation of NKA. We tested this hypothesis in proximal tubules isolated from the kidneys of Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN). First, we confirmed our laboratory's previous reports that bromocriptine causes stimulation of NKA by means of D2-like receptors in rat proximal tubules. Next, we measured the bromocriptine-mediated increase in NKA and p44/42 MAPK activities in proximal tubules in the presence and absence of tyrosine kinase, p44/42 MAPK inhibitors, and a cell-permeable cAMP analog to identify the role of these components in D2-like receptor-mediated stimulation of NKA activity.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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

Isolation of proximal tubules from kidney. The rats were anesthetized with pentobarbital sodium (50 mg/kg ip). The proximal tubules were isolated from the kidneys and enriched by using the Ficoll gradient method as previously described (6). The enriched proximal tubules were resuspended in 5 ml of modified Krebs-Henseleit buffer [KHB; (in mM) 118 NaCl, 4 KCl, 1.25 CaCl2, 1.2 MgCl2, 27.2 NaHCO3, 1 KH2PO4, 5 glucose, and 10 HEPES at pH 7.4]. For NKA assay, the proximal tubules were resuspended in KHB with no phosphate and 0.12 mM magnesium. The protein in the proximal tubular suspension was estimated by using a kit from Pierce (Rockford, IL).

NKA assay. The NKA activity was measured as previously described (6). The proximal tubular suspension (990 µl; 0.75 mg/ml protein) was incubated with vehicle (basal) or bromocriptine (10 µl to reach 0.1 µM) for 15 min at 37°C in a shaking water bath (other treatments are explained in RESULTS; see also figure legends). After incubation, the tubules were permeabilized by rapid freezing in dry ice and acetone and thawed. The freeze-thawed samples were then used in the NKA assay. The reaction mixture for the assay in its final volume (1.025 ml) contained (in mM) 37.5 immidazole buffer, 70 NaCl, 5 KCl, 1 NaEDTA, 5 MgCl2, 6 NaN3, and 75 Tris · HCl, as well as 100 µl of proximal tubular suspension (75 µg protein). The reaction was initiated by adding 4 mM ATP. Ouabain-insensitive ATPase activity was determined in parallel by using the reaction mixture containing 150 mM Tris · HCl and 1 mM ouabain instead of NaCl and KCl. The reaction was carried out at 37°C for 15 min and terminated by adding 50 µl of ice-cold 50% trichloroacetic acid. The NKA activity was measured as the function of liberated Pi. The NKA activity was calculated as the difference between total and ouabain-insensitive ATPase activity, and NKA activity is represented as percentage of basal, where basal is normalized to 100%. The average basal NKA activity was 111.2 ± 7.9 nmol Pi · mg protein-1 · min-1.

Phospho-p44/42 MAPK measurement. For this experiment, we used 2 mg/ml proximal tubular suspension in KHB. The proximal tubular suspension (990 µl) was incubated with vehicle (basal) or bromocriptine (10 µl to reach 0.1 µM) for 5 min at 37°C in a shaking water bath. After incubation, the tubules were permeabilized by rapid freezing in dry ice and acetone and thawed. Loading samples containing (in µl) 50 SDS-Lammelie (4×), 20 bromophenol blue, 100 proximal tubular lysate, and 30 H2O were prepared for Western blot analysis. Furthermore, the loading samples (25 µl) were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The phospho-p44/42 MAPK bands on the polyvinylidene difluoride membrane were detected by using an anti-phospho-p44/42 MAPK antibody kit according to the manufacturer's instructions (New England Biolabs, Beverly, MA). Total MAPK was also determined in the same membrane to confirm the same amount of protein.

Other treatments. Where indicated, the proximal tubules were incubated with domperidone (1 µM), genistein (0.001 µM), PD-98059 (10 µM), and dibutyryl-cAMP (10 mM) at 37°C for 10 min before addition of bromocriptine (0.1 µM).

Statistical analysis. Where applicable, values are means ± SE of the number of experiments. Statistical analysis was performed by using one-way ANOVA, along with the appropriate post hoc test. P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of bromocriptine on NKA activity in the absence and presence of domperidone. Bromocriptine (D2-like receptor agonist; 0.1 µM) stimulated NKA activity (117 ± 4.9% of basal). This effect of bromocriptine was blocked when the proximal tubular suspension was pretreated for 10 min with domperidone (D2-like receptor antagonist; 1 µM), thus confirming the role of D2-like receptors in stimulation of NKA (Fig. 1).


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Fig. 1.   Effect of bromocriptine on Na+, K+-ATPase (NKA) activity in the absence and presence of domperidone. The proximal tubules were isolated from the kidneys of Sprague-Dawley rats as described in METHODS. The isolated proximal tubules (0.75 mg/ml protein) were preincubated without or with domperidone (1 µM) for 10 min at 37°C, followed by incubation with bromocriptine (0.1 µM) for 15 min at 37°C. The reaction was terminated by freeze-thawing and NKA activity determined as described in METHODS. Data are represented as percentage of basal values (vehicle-treated group) from 3-4 experiments. *P < 0.05, significant difference between basal and bromocriptine-treated groups. #P < 0.05, significant difference between bromocriptine and domperidone-treated groups (1-way ANOVA; post hoc: Tukey's multiple comparison test).

Effect of bromocriptine on NKA activity in the absence and presence of various inhibitors. To examine the role of p44/42 MAPK and tyrosine kinase in the D2-like receptor-mediated stimulation of NKA, we used PD-98059 and genistein, respectively. While PD-98059 is a specific inhibitor of MAPK kinase 1/2 (2), which is the upstream activator of p44/42 MAPK, genistein (3) is a specific inhibitor for all tyrosine kinase activity. We found that both PD-98059 (10 µM) and genistein (0.001 µM) blocked bromocriptine (0.1 µM)-mediated stimulation of NKA (Fig. 2). The concentration of PD-98059 and genistein that was used did not affect the basal activity of NKA (Fig. 2). These results suggest that both p44/42 MAPK and tyrosine kinase are involved in D2-like receptor-mediated stimulation of NKA.


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Fig. 2.   Effect of bromocriptine on NKA in the absence and presence of various inhibitors. The proximal tubules were isolated from the kidneys of Sprague-Dawley rats as described in METHODS. The isolated proximal tubules (0.75 mg/ml protein) were preincubated without or with PD-98059 [mitogen-activated protein kinase (MAPK) kinase inhibitor; 10 µM], genistein (tyrosine kinase inhibitor; 0.001 µM) or dibutyryl cAMP (10 mM) for 10 min at 37°C, followed by incubation with bromocriptine (0.1 µM) for 15 min at 37°C. The reaction was terminated by freeze-thawing, and NKA activity was determined as described in METHODS. Data are represented as percentage of basal values (vehicle-treated group) from 5-8 experiments. *P < 0.05, significant difference between basal and bromocriptine-treated groups. #P < 0.05, significant difference between groups treated with bromocriptine alone and with bromocriptine and indicated agent (1-way ANOVA; post hoc: Tukey's multiple comparison test).

Effect of bromocriptine on p44/42 MAPK phosphorylation in the absence and presence of various inhibitors. To further confirm the role of p44/42 MAPK, we measured the effect of D2-like receptor activation on p44/42 MAPK phosphorylation (stimulation). We found that bromocriptine (0.1 µM) caused an increase in phosphorylation of p44/42 MAPK in 5 min. Domperidone (1 µM; Fig. 3A), PD-98059 (10 µM), and genistein (0.001 µM; Fig. 3B) blocked this effect. Thus activation of D2-like receptors causes an increase in phosphorylation of p44/42 MAPK. Moreover, because genistein blocked the effect of bromocriptine, the tyrosine kinase may lie upstream of MAPK kinase 1/2 and p44/42 MAPK.


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Fig. 3.   Effect of bromocriptine on p44/42 MAPK phosphorylation in the absence and presence of various inhibitors. The proximal tubules were isolated from the kidneys of Sprague-Dawley rats as described in METHODS. The isolated proximal tubules (2 mg/ml protein) were preincubated without or with domperidone (1 µM; A), or PD-98059 (MAPK kinase inhibitor; 10 µM; B), or genistein (tyrosine kinase inhibitor; 0.001 µM; B), or dibutyryl cAMP (10 mM; C) for 10 min at 37°C, followed by incubation with bromocriptine (0.1 µM) for 5 min at 37°C. The reaction was terminated by freeze-thawing and p44/42 MAPK phosphorylation was determined as described in METHODS. The experiment was repeated 3 times, and similar results were obtained, as shown in the representative blot.

Effect of bromocriptine on NKA and p44/42 MAPK activation in the absence and presence of dibutyryl cAMP. It was previously shown that D2-like receptor activation causes a decrease in intracellular cAMP accumulation (6, 21). Here, we tested whether this effect is involved in the activation of NKA and p44/42 MAPK. Pretreatment of the proximal tubules with dibutyryl cAMP (cell-permeable analog of cAMP; 10 mM) blocked the bromocriptine (0.1 µM)-mediated activation of NKA (Fig. 2) and p44/42 MAPK (Fig. 3C). Thus it is possible that a D2-like receptor-mediated decrease in cAMP may be important for the activation of NKA and p44/42 MAPK.

Effect of bromocriptine on NKA in the absence and presence of orthovanadate. By virtue of its inhibitory effect on tyrosine phosphatase, orthovanadate increases tyrosine kinase activity and stimulates NKA in kidney proximal tubules. We wanted to further ascertain the role of tyrosine kinases in D2-like receptor-mediated activation of NKA by investigating the additive effect of orthovanadate and bromocriptine. First, we found that orthovanadate had a biphasic effect on NKA activity (Fig. 4A). At lower concentrations (0.001 and 0.01 µM), orthovanadate stimulated NKA activity. On the other hand, higher concentrations (0.1 and 1 µM) of orthovanadate inhibited NKA (Fig. 4A). Furthermore, a combination of bromocriptine (0.1 µM) and orthovanadate (0.001 µM) did not cause an additional stimulation of NKA activity compared with bromocriptine or orthovanadate alone (Fig. 4B).


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Fig. 4.   Effect of bromocriptine on NKA in the absence and presence of orthovanadate. The proximal tubules were isolated from the kidneys of Sprague-Dawley rats as described in METHODS. A: isolated proximal tubules (0.75 mg/ml protein) were incubated with increasing concentrations of orthovanadate (0-1 µM) for 15 min at 37°C, and NKA activity was measured. *P < 0.05, significant difference between basal and drug-treated groups (1-way ANOVA). B: isolated proximal tubules (0.75 mg/ml protein) were incubated with orthovanadate (0.001 µM) and/or bromocriptine (0.1 µM) for 15 min at 37°C, and NKA activity was measured. Data are represented as percentage of basal values (vehicle-treated group) from 3-4 experiments. #P < 0.05, significant difference between basal and drug-treated groups (1-way ANOVA; post hoc: Dunnett test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have shown that D2-like receptor-mediated activation of a tyrosine kinase and p44/42 MAPK in the proximal tubules of the kidney is responsible for the stimulation of NKA caused by bromocriptine. This is supported by our observation that activation of D2-like receptors causes an increase in phosphorylation of p44/42 MAPK. Furthermore, activation of tyrosine kinase is a prerequisite for p44/42 MAPK stimulation, as genistein (tyrosine kinase inhibitor) blocked the D2-like receptor-mediated phosphorylation of p44/42 MAPK.

Our laboratory and others have reported that selective activation of D2-like receptors causes stimulation of NKA (6, 21). In terms of signaling mechanisms, a decrease in intracellular cAMP was shown to be responsible for this effect. Moreover, decreased intracellular cAMP leads to decreased protein kinase A (PKA) activity. However, this decrease in PKA activity may not stimulate NKA, as PKA does not inhibit NKA in the proximal tubules of the kidney (4). Therefore, a different effector for cAMP may play a role in D2-like receptor-mediated stimulation of NKA.

An increase in cAMP accumulation was previously shown to inhibit p44/42 MAPK, which is a member of the class of serine/threonine kinase called MAPKs (8). We have seen a similar effect in OK cells, a proximal tubular cell line (14). Usually, p44/42 MAPK is involved in the regulation of transcription and translation of genes that control mitogenesis. In addition, p44/42 MAPK may be involved in increasing the transcription and translation of other genes, leading to increased synthesis of gene product. The long-term effect of p44/42 MAPK in increasing the synthesis of NKA in alveolar epithelial cells has been reported (5). In the present study, we have identified the short-term role of p44/42 MAPK in D2-like receptor-mediated stimulation of NKA in rat proximal tubules. This is further supported by the ability of bromocriptine to increase p44/42 MAPK phosphorylation. In this situation, the increase in NKA activity may not involve an increase in NKA synthesis, as the maximum effect was observed only after 15 min of agonist (bromocriptine) treatment, which is a short period for protein synthesis. Thus D2-like receptor-mediated stimulation of NKA may involve posttranslational modification of NKA (e.g., phosphorylation) by means of a p44/42 MAPK pathway. However, the possibility that long-term activation of D2-like receptors may cause increased synthesis of NKA, by means of p44/42 MAPK, cannot be ruled out in the proximal tubules.

Genistein-sensitive tyrosine kinase activity is involved in the growth factor-mediated stimulation of NKA in the proximal tubules of the kidney (3). We found that similar genistein-sensitive tyrosine kinase activity is involved in D2-like receptor-mediated stimulation of NKA. Furthermore, orthovanadate has been shown to have a biphasic effect on NKA activity (3). At lower concentrations, orthovanadate indirectly increases tyrosine kinase activity because of its tyrosine phosphatase inhibitory effect. This increase in tyrosine kinase activity is shown to stimulate NKA by means of the increased phosphorylation of tyrosine residue in the alpha 1-subunit of NKA (3, 4). On the other hand, higher concentrations of orthovanadate directly inhibit the NKA activity by binding at the phosphorylation site and blocking the NKA under E2 confirmation (high affinity for K+) (3, 4). We found a similar biphasic effect of orthovanadate on NKA activity in proximal tubules of the kidney. Moreover, addition of bromocriptine and orthovanadate (stimulatory concentration) together did not result in an additive increase in NKA activity, suggesting that both agents use a common signaling component, which is tyrosine kinase.

Whether D2-like receptors and growth factor receptors share the same tyrosine kinase signaling pathway in stimulation of NKA activity is not known. This might depend on the growth factor receptor and the cell type. For example, in the case of insulin, p44/42 MAPK is not involved in the stimulation of NKA (16) in 3T3-L1 fibroblasts. Although insulin and EGF stimulate NKA activity in a tyrosine kinase-dependent fashion in the proximal tubules of the kidney, the role of p44/42 MAPK in this response has not been investigated (3).

As mentioned above, D2-like receptor-mediated decrease in intracellular cAMP may play a role in p44/42 MAPK and NKA activation. We have tested this hypothesis by using dibutyryl cAMP. Pretreatment of kidney proximal tubules with dibutyryl cAMP (which saturates the intracellular cAMP levels) blocked the D2-like receptor-mediated p44/42 MAPK and NKA activation. It has been shown that D2-like receptor activation decreases intracellular cAMP levels (6), and this might be important for activating p44/42 MAPK (14). As observed in OK cells, it is possible that a D2-like receptor-mediated decrease in cAMP may cause transactivation of epidermal growth factor (or other growth factor) receptors, which may further activate p44/42 MAPK in rat proximal tubules (14). This can be partially supported by our observation that genistein (tyrosine kinase inhibitor) blocks the D2-like receptor-mediated activation of p44/42 MAPK in rat proximal tubules, because the growth factor receptor uses its tyrosine kinase activity in inducing its response. On the other hand, PKA is also known to inhibit p44/42 MAPK (8). It is possible that D2-like receptors, by decreasing intracellular cAMP, may decrease PKA activity and thus activate p44/42 MAPK.

At this point, we can only speculate about the mechanism by which p44/42 MAPK can stimulate NKA activity. It is possible that D2-like receptor-mediated activation of p44/42 MAPK can increase NKA activity by several of the reported mechanisms for receptor-mediated NKA activation. Two possible mechanisms may play a role in activation of NKA in our case. First, D2-like receptor-mediated activation of p44/42 MAPK may cause the recruitment of the alpha 1-subunit of NKA from the cytosol to the membrane. Second, it is possible that D2-like receptor activation can cause p44/42 MAPK-mediated phosphorylation of tyrosine residue in the alpha 1-subunit of NKA, which leads to an increase in enzyme activity. The exact mechanism for D2-like receptor/p44/42 MAPK-mediated stimulation of NKA remains to be investigated.

The physiological significance of D2-like receptor-mediated stimulation of NKA remains to be determined. However, the role for D2-like receptors may be uncovered during pathophysiological conditions such as ischemia-reperfusion injury. Ischemia-reperfusion injury is associated with impaired Na+ and water reabsorption, which is reflected in increased fractional excretion of Na+ and polyuria (10). Because 60% of Na+ reabsorption takes place at the level of the proximal tubules, it is possible that part of the defect originates in the proximal tubules. Indeed, it was recently reported that there is a downregulation of NHE and NKA in the cortex (rich in proximal tubules) of the kidney after ischemia and reperfusion. This correlated with the increase in the fractional excretion of Na+ in the same study (10). It is possible that a D2-like receptor agonist, such as bromocriptine, may ameliorate the loss of Na+ by activating NKA during ischemia-reperfusion injury.

In summary, we have shown that activation of D2-like receptors causes stimulation of NKA in the proximal tubules of the kidney. Furthermore, this action involves a tyrosine kinase-p44/42 MAPK pathway. It remains to be determined whether this effect of bromocriptine plays a protective role in ischemia-reperfusion injury.


    ACKNOWLEDGEMENTS

This study was supported in part 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 77204-5511 (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.

10.1152/ajprenal.00126.2001

Received 18 April 2001; accepted in final form 2 November 2001.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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