alpha 2B-Adrenergic receptors activate MAPK and modulate proliferation of primary cultured proximal tubule cells

Daniel Cussac1, Stéphane Schaak1, Céline Gales2, Christodoulos Flordellis3, Colette Denis1, and Hervé Paris1

Institut National de la Santé et de la Recherche Médicale 1 Unit 388 and 2 Unit 531, Institut L. Bugnard, Centre Hospitalier Universitaire Rangueil, 31403 Toulouse Cedex 4, France; and 3 Department of Pharmacology, School of Medicine, University of Patras, 26110 Rio Patras, Greece


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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In the rat proximal tubule, the alpha 2B-adrenergic receptor (alpha 2B-AR) enhances Na+ reabsorption by increasing the activity of Na+/H+ exchanger isoform NHE3. The mechanisms involved are unclear, and inhibition of cAMP production remains controversial. In this study, we reinvestigated alpha 2B-AR signaling pathways using rat proximal tubule cells (PTC) in primary culture and LLC-PK1 cells permanently transfected with the RNG gene (rat nonglycosylated alpha 2-AR). Binding experiments indicated that PTC express substantial amounts of alpha 2B-AR (130 fmol/mg protein), and only RNG transcripts were detected. In both cell types, the alpha 2B-AR is coupled to G protein, and its stimulation by dexmedetomidine, but not by UK-14304, provoked a significant inhibition of the accumulation of cAMP induced by forskolin or parathyroid hormone. Exposure to alpha 2-agonists increased arachidonic acid release and caused extracellular signal-regulated kinase (ERK)1/2 phosphorylation, which correlated with enhanced mitogen-activated protein kinse (MAPK) activity and nuclear translocation. MAPK phosphorylation was blunted by pertussis toxin but not by protein kinase C desensitization, and it coincided with transient phosphorylation of Shc. Finally, treatment with UK-14304 accelerated cell growth. Further studies will be necessary to clarify the precise mechanism of MAPK activation, but the present data suggest that alpha 2B-AR may play a positive role during tubular regeneration.

kidney; mitogen-activated protein kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FUNCTIONAL STUDIES, CARRIED out in various animal species, have demonstrated that alpha 2-adrenergic receptors (alpha 2-ARs) are of predominant importance for the mediation of the regulatory effects of catecholamines on several renal functions, including renin release, glomerular filtration, and Na+ and water excretion (8, 27, 38). Pharmacological characterization and molecular cloning have provided evidence that alpha 2-ARs are a heterogeneous class of receptors comprising three subtypes (alpha 2A, alpha 2B, and alpha 2C) encoded by distinct genes (33). All three subtypes have been detected as mRNA in the human and rat kidney (3, 15, 26). However, the respective abundance, precise anatomic location, and specific roles of the corresponding proteins remain somewhat controversial.

In the rat proximal tubule, the stimulation of alpha 2-ARs induces an augmentation of Na+ and bicarbonate reabsorption by increasing the activity of the Na+/H+ exchanger (30). This increase is primarily due to the activation of the NHE3 isoform that is expressed in the brush-border membrane of proximal tubule cells (PTC). Additionally, alpha 2-agonists were found to enhance the activity of the Na+/K+-ATPase located in the basal membrane (2). Thus both mechanisms may coordinately contribute to the acceleration of Na+ reabsorption. According to binding and immunolocalization studies (17), it is clear that the effects of alpha 2-agonists on the proximal tubule are primarily due to stimulation of alpha 2B-AR, the expression of which is particularly high in this segment of the nephron. However, the molecular mechanisms responsible for the activation of NHE3 and Na+/K+-ATPase are not fully understood.

In almost every known cell type, alpha 2-ARs are coupled to Gi/Go proteins, and their stimulation causes inhibition of cAMP production. In agreement with this view, (-)-epinephrine was found to inhibit parathyroid hormone (PTH)-induced cAMP accumulation in microdissected proximal tubule (42) as well as in opossum kidney (OK) cells, a model for proximal tubule cells that express an alpha 2-AR of the alpha 2C-subtype (28). The search for alternative pathways of signal transduction demonstrated that alpha 2B-AR stimulates protein kinase C (PKC) activity and inositol trisphosphate production in the distal convoluted tubule (14). On the other hand, studies in transfected Chinese hamster ovary (CHO) cells showed increased cystolic phospholipase A2 (cPLA2) activity (1). However, none of these effects has ever been demonstrated in the proximal tubule.

Originally identified as a signaling pathway activated by tyrosine kinase receptors, extracellular signal-regulated kinases (ERKs) are now well documented to be also stimulated by a large variety of receptors coupled to G proteins. As shown by experiments initially performed in transfected cells (21, 44), Gi-coupled receptors activate ERK via release of beta gamma -subunits and subsequent tyrosine phosphorylation of the adaptor protein Shc (25). Phosphorylation of Shc provokes its association with Grb2 and Sos, which leads to increased GTP binding to ras and consecutive activation of raf, mitogen-activated protein kinase (MAPK)-ERK kinase (MEK), and MAPK. Recently, activation of ERK was reported after stimulation of the alpha 2C-AR in OK cells (22, 23). The occurrence of such an effect in rat proximal tubule also remains to be proved.

The purpose of the present study was to investigate the alpha 2B-AR signaling pathways in cultured PTC. We found that receptor stimulation caused inhibition of cAMP production, activation of cPLA2, and a transient increase of ERK phosphorylation. This latter effect resulted in augmentation of MAPK activity, translocation of ERK2 to the nucleus, and acceleration of cell proliferation.


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Drugs and reagents. [3H]RX-821002 (59 Ci/mmol), [3H]arachidonic acid (202 Ci/mmol), and an enhanced chemiluminescence (ECL) Western blotting system were from Amersham Pharmacia Biotech (Courtaboeuf, France). [3H]MK-912 (81 Ci/mmol) and Polyscreen polyvinylidene difluoride (PVDF) membranes were from NEN Life Science (Boston, MA). [alpha -32P]UTP (800 Ci/mmol) and [gamma -32P]ATP (5,000 Ci/mmol) were purchased from ICN (Costa Mesa, CA). Phentolamine was donated by Ciba-Geigy (Basel, Switzerland), dexmedetomidine by Orion Pharma (Turku, Finland), and RX-821002 by Reckitt and Colman Laboratories (Kingston-upon-Hull, UK). UK-14304 and prazosin hydrochloride were generous gifts from Pfizer (Sandwich, UK). Collagenase, insulin, dexamethasone, transferrin, epidermal growth factor (EGF), triiodothyronine, PTH, oxymetazoline, forskolin, pertussis toxin, 5'-guanylylimidodiphosphate (GppNHp), phorbol 12-myristate 13-acetate (PMA), and all other chemicals were from Sigma (St. Louis, MO). Fetal calf serum (FCS) was purchased from GIBCO BRL (Cergy Pontoise, France). Quinacrine, PD-98059, and genistein were obtained from Calbiochem (La Jolla, CA). Anti-ERK1 and anti-ERK2 polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-active MAPK was from Promega (Madison, WI). RIA kits for cAMP determination were from Immunotech (Luminy, France).

Culture of LLC-PK1 cells and generation of alpha 2-AR transfectants. The renal tubular cell line LLC-PK1 was routinely grown in DMEM containing 25 mM glucose, 100 µg/ml streptomycin, 100 IU/ml penicillin, and 5% FCS. LLC-PK1 cells permanently expressing the rat alpha 2B-AR (LLC-PK1-alpha 2B) or the human alpha 2A-AR (LLC-PK1-alpha 2A) were respectively obtained by transfection with pcDNA3 vector containing the coding region of the RNG or alpha 2C10 genes. Cells were transfected using the calcium-phosphate method. Two days posttransfection, they were subcultured in the presence of G418-sulfate (1 mg/ml), and antibiotic resistant cells were selected.

Proximal tubule preparation and culture. PTC were isolated as previously described by Vinay et al. (43). Male Sprague-Dawley rats (4 wk of age, mean weight 40 g) were purchased from Harlan-France (Gannat, France). The animals were killed by intraperitoneal injection of pentobarbital sodium (50 mg/kg). The kidneys were rapidly removed, decapsulated, and placed in ice-cold Hanks' balanced saline solution (HBSS) buffered with 10 mM HEPES (pH 7.4). Cortex slices were incubated for 30 min at 37°C in HBSS containing 0.3 mg/ml collagenase and 1 mg/ml BSA. The resulting cell suspension was centrifuged for 2 min at 500 g, and the particulate fraction was washed twice in HBSS containing 1 mg/ml BSA. The final pellet was taken up in 30 ml of 42% Percoll made isotonic with 10× Krebs-Henseleit buffer. After centrifugation (20,000 g, 30 min at 4°C), the layer corresponding to proximal tubules was retrieved and washed twice in DMEM-Ham's F-12 medium (1:1). The protein concentration was determined using BSA as a standard (5), and the isolated proximal tubules were plated (20 µg cellular protein/cm2) on collagen-coated plastic dishes in culture medium consisting of DMEM-Ham's F-12 mixture containing 5% FCS, 10 µg/ml insulin, 10 ng/ml EGF, 5 µg/ml transferrin, 0.1 µM dexamethasone, and 5 µM triiodothyronine. The cells were maintained undisturbed under a 5% CO2 atmosphere at 37°C for 48 h, after which the medium was exchanged for a serum-free medium.

RNA extraction and RNase protection assay. Cellular RNAs were extracted using the guanidinium isothiocyanate/phenol-chloroform method (6). The probe used in the RNase protection assay (RPA) was obtained by cloning the PstI/PstI fragment corresponding to nucleotides 628-897 of the ORF of the RNG gene (47) into pKS+ (Stratagene, La Jolla, CA). Labeled antisense RNA was synthesized from the linearized matrix using T3 RNA polymerase (Promega) in the presence of [alpha -32P]UTP. Assays were performed as described previously (34); RNA were taken up in 30 µl of hybridization buffer (80% deionized formamide, 0.4 M NaCl, 1 mM EDTA, 40 mM PIPES, pH 6.7) containing an excess of [32P]-labeled riboprobe. The samples were heated to 95°C for 5 min and then immediately placed at 55°C for 14 h. Nonhybridized probe was eliminated by the addition of 0.3 ml of RNase A (40 µg/ml) and RNase T1 (2 µg/ml) in 300 mM NaCl, 5 mM EDTA, and 10 mM Tris · HCl (pH 7.5). After 2 h at 37°C, digestion was stopped by addition of 5 µl of proteinase K (10 mg/ml), and the samples were further incubated for 15 min at 37°C. Carrier tRNA (10 µg) and 0.3 ml of 4 M guanidinium isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.1 M 2-mercaptoethanol, and 0.5% sarkosyl (solution D) were added to each tube, and protected hybrids were precipitated with isopropyl alcohol. Pellets were washed with 70% ethanol, air-dried, taken up in sample buffer (97% deionized formamide, 0.1% SDS, 10 mM Tris · HCl, pH 7.0) and run on a 5% polyacrylamide gel containing 7 M urea. The gels were fixed, dried, and exposed for 48 h at -80°C to X-ray film for autoradiography.

Membrane preparation and radioligand binding studies. Frozen cells were harvested in 50 mM Tris · HCl buffer (pH 7.5) containing 5 mM EDTA and centrifuged at 27,000 g for 10 min (4°C). The pellet was taken up in 50 mM Tris · HCl and 0.5 mM MgCl2, pH 7.5 (TM buffer) and centrifuged again. The final pellet was suspended in the appropriate volume of TM buffer and immediately used for binding experiments as described previously (34). Briefly, membranes were incubated at 25°C in a 400-µl final volume of TM buffer containing the 3H-labeled antagonist. After a 45-min period of incubation, membrane-bound radioactivity was separated from free by rapid filtration through a Whatman GF/C filter. Retained radioactivity was counted by liquid scintillation spectrometry, and specific binding was calculated as the difference between total and nonspecific binding determined in the presence of 10-5 M phentolamine. For saturation experiments, the final radioligand concentration ranged from 0.5 to 16 nM for [3H]RX-821002 and from 0.25 to 8 nM for [3H]MK-912. The values of maximal binding (Bmax), the dissociation constant (Kd), and the inhibition constant (Ki) were calculated from computer-assisted analysis of the data using GraphPad Prism (GraphPad Software, San Diego, CA).

Measurement of intracellular levels of cAMP. The measurement of intracellular levels of cAMP was carried out in cells at day 3 of culture. Cells were placed for 6 h in HEPES-buffered DMEM containing 0.2 mM 3-isobutyl-1-methylxanthine. The experiments were started by adding the appropriate concentration of hormone and/or drug to be tested. After a 20-min incubation period at 37°C, the medium was aspirated off and the reaction was terminated by adding 4 ml of methanol/acetic acid mixture (95:5). After 30 min of extraction, the cell layer was scraped off, sonicated, and centrifuged (2,500 g, 15 min). Aliquots of the supernatant were evaporated, and cAMP content was determined by RIA.

Arachidonic acid release. Cells were labeled with 1 µCi/ml [3H]arachidonic acid for 1 h at 37°C. They were carefully washed in DMEM containing 10 mM HEPES and 0.2% fatty acid-free BSA and then exposed to the drug to be tested. Aliquots of the incubation medium were collected every 10 min over a period of 30 min, centrifuged (20,000 g, 10 min, 4°C), and the radioactivity was measured in the supernatant.

Detection of ERK1/2 and Shc. Three days postseeding, cells were placed for 24 h in culture medium free of serum and hormone. Cell layers were then exposed to the compound to be tested, rapidly rinsed with ice-cold PBS, and harvested in 1 ml of RIPA buffer [10 mM Tris · HCl, pH 7.4, 1% Triton X-100, 1% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM Na-orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM aprotinin]. Soluble proteins were extracted by centrifugation (15,000 g, 15 min at 4°C), separated by SDS-PAGE, and blotted onto a Polyscreen PVDF membrane (NEN-Life Sciences). Phosphorylated forms of MAPK were revealed by chemiluminescence (ECL Western blotting system, Amersham Pharmacia Biotech) using anti-active MAPK antibody (Promega). Equal protein loading was assessed by reprobing the membrane using a mixture of anti-ERK1 and anti-ERK2 antibodies (Santa Cruz Biotechnology). Shc phosphorylation was determined after immunoprecipitation. Briefly, 500 µl of cell lysate were incubated overnight at 4°C with 5 µg of rabbit polyclonal Shc-antibody (Upstate Biotechnology, Lake Placid, NY) and 50 µl of protein A-agarose beads. Immune complexes were extensively washed with ice-cold RIPA, dried, and denatured in Laemmli buffer. Samples were subjected to SDS-PAGE, transferred onto a Polyscreen PVDF membrane, and probed with an anti-phosphotyrosine-horseradish peroxidase-conjugated monoclonal antibody. As above, the membrane was then stripped of Ig and reprobed using anti-Shc antibody to verify equal loading.

Measurement of MAPK activity. MAPK activity was measured as previously described (13). Briefly, the cells were rapidly rinsed in 10 ml of ice-cold PBS and harvested in 1 ml of 25 mM HEPES buffer (pH 7) containing 5 mM EDTA, 50 mM NaF, 0.1 mM Na-orthovanadate, 1 mM PMSF, and 10 µg/ml leupeptin. Lysates were further homogenized by passing repeatedly through a 25-gauge needle, and insoluble material was eliminated by ultracentrifugation (100,000 g, 20 min at 4°C). Aliquots of the cytosolic extracts were incubated for 10 min at 37°C with 25 µg of a synthetic peptide substrate corresponding to amino acids 95-98 of myelin basic protein (APRTPGGRR) in 25 mM Tris · HCl buffer (pH 7.4) containing 10 mM MgCl2, 1 mM dithiothreitol, 40 µM ATP, 2 mM protein kinase inhibitor peptide, and 2 µCi of [gamma -32P]ATP. The reaction was terminated by the addition of 125 mM cold ATP in 50% formic acid. An aliquot of the reaction mixture was spotted onto 4-cm2 Whatman P81 paper, extensively washed in 180 mM phosphoric acid, rinsed in ethanol, and counted.

Immunofluorescence microscopy. Cells plated on glass coverslips were grown, rendered quiescent, and exposed or not to the alpha 2-agonist, as indicated above. The cells were fixed (15 min) in 4% paraformaldehyde and then treated (10 min) with 50 mM NH4Cl in PBS. They were permeabilized first in buffer consisting of PBS containing 0.05% saponin and 0.2% BSA (15 min) and then in methanol (10 min at -20°C). All subsequent steps were carried out in permeabilization buffer and were separated by several washes. Samples were incubated with ERK2 polyclonal antibody (1:40, Santa Cruz Biotechnolgy) and then with fluorescein-conjugated goat anti-rabbit IgG (1:400, Nordic Immunology). The coverslips were washed in PBS, mounted in fluorescent mounting medium (Dako, Carpinteria, CA), and examined under epifluorescence illumination. Digital images were captured using CoolSNAP software (Roper Scientific, Munich, Germany) and processed with Adobe Photoshop 4 (Adobe Systems, San Jose, CA).

Cell proliferation assay. PTC were seeded in six-well plates and grown for 2 days in DMEM-Ham's F-12 medium (1:1) supplemented with 5% FCS. They were deprived of serum for 1 day and then treated or not with 1 µM UK-14304. Culture plates were collected at the indicated time, and DNA content per well was measured by the fluorometric method using DAPI. LLC-PK1-alpha 2B were seeded in six-well plates, grown for 2 days in DMEM supplemented with 5% FCS and then treated or not with 1 µM-UK14304. At the indicated time, the cells were harvested by trypsin/EDTA treatment and counted.

Statistical analysis. Results are expressed as means ± SE for the number of experiments (n) indicated. The data were analyzed using Student's t-test, and a P value <0.05 was considered statistically significant.


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Cultured PTC having been never used for the study of alpha 2-adrenergic receptivity, preliminary experiments were designed to assess the level of alpha 2-AR expression under primary culture conditions. Results from binding experiments carried out with [3H]RX-821002 and [3H]MK-912 in membranes prepared from confluent PTC (5-day postseeding) are summarized in Table 1. Analysis of saturation isotherms revealed that these two specific alpha 2-antagonists labeled a single class of sites with high affinity. With both radioligands, binding site number was fairly similar, Bmax values being 127 ± 11 and 118 ± 16 fmol/mg protein for [3H]RX-821002 and [3H]MK-912, respectively. As also shown in Table 1, the level of receptor expression in LLC-PK1-alpha 2B was about sixfold higher than in PTC (Bmax for [3H]RX-821002 = 730 ± 51 fmol/mg protein). Prazosin inhibited [3H]RX-821002 binding with a slightly higher potency than oxymetazoline (data not shown), suggesting that the binding sites exhibited the pharmacological properties expected for an alpha 2B-AR. This conclusion was confirmed by RPA with riboprobes specific for the different receptor subtypes. Indeed, RNG but not RG10 or RG20 transcripts were found in PTC (Fig. 1).

                              
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Table 1.   Measurement of alpha 2-AR density in PTC and LLC-PK1-alpha 2B cells



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Fig. 1.   Characteristics of proximal tubule cells (PTC) in culture. A: phase-contrast microscopy of PTC monolayer. B: detection of RNG transcripts. An excess of labeled RNG probe was hybridized with either 100 µg of yeast tRNA (-), 5 µg of RNA from COS-7 cells transfected with pcDNA3-RNG (+), 100 µg of RNA from rat kidney (lane K), or 80 µg of RNA extracted from PTC in primary culture (lane PTC). Samples were digested with a mixture of RNases and resistant hybrids analyzed by electrophoresis. A typical autoradiogram is presented. P, undigested probe.

As a preliminary step in the study of the pathways of alpha 2B-AR signal transduction, we examined the ability of the receptor to interact with G proteins by analyzing its propensity to exist under a high-affinity state for agonists (Fig. 2). In both PTC and LLC-PK1-alpha 2B, inhibition of [3H]RX-821002 binding by the physiological amine (-)-epinephrine yielded shallow competition curves, and data were better fitted with a two-site model. From three independent experiments, the Ki values of (-)-epinephrine for the high- and low-affinity state receptor (Ki H and Ki L) were 4.1 ± 2.3 nM and 1.22 ± 0.55 µM, respectively, in PTC. The proportion of receptor under the high-affinity conformation for agonist represented 59 ± 8% of the whole population. As expected, this fraction was abolished in the presence of GppNHp/Na+ or in membranes prepared from cells treated with pertussis toxin (data not shown). Fairly similar results were obtained with LLC-PK1-alpha 2B cells, the Ki H and Ki L values of (-)-epinephrine being, respectively, 3.7 ± 1.7 nM and 0.63 ± 0.23 µM. The only difference is that the fraction of receptor under high-affinity conformation was lower (33 ± 5%). This is probably the consequence of higher receptor expression in LLC-PK1-alpha 2B cells.


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Fig. 2.   Analysis of receptor coupling to G proteins. Membranes prepared from PTC (A) and LLC-PK1-alpha 2B cells (B) were incubated in the presence of [3H]RX-821002 (8 nM) and increasing concentrations of (-)-epinephrine. Inhibition experiments were carried out in binding buffer containing 0.1 mM 5'-guanylylimidodiphosphate (GppNHp) plus 100 mM NaCl () or not (open circle ).

In isolated proximal tubules as well as in the OK cell line, alpha 2-ARs are negatively coupled to adenylyl cyclase. The next series of experiments was designed to determine whether this mechanism of signal transduction is also effective in our models. As summarized in Table 2, PTC and LLC-PK1-alpha 2B were similarly responsive to forskolin exposure. In both cell types, UK-14304 had a tendency to inhibit the forskolin-induced cAMP accumulation, but the effect was marginal and not statistically significant. By contrast, an inhibitory action was found with dexmedetomidine (20 and 17% decrease of cAMP level in PTC and LLC-PK1-alpha 2B, respectively). The difference of efficacy between the two compounds was further confirmed in PTC treated with PTH. Indeed, dexmedetomidine caused a 43% inhibition of PTH-induced cAMP production, whereas UK-14304 did not. At this point, it is worth mentioning that the failure of UK-14304 to lower cAMP was likely due to a particularity of alpha 2B-AR subtype, because a significant reduction was observed when LLC-PK1 cells expressing alpha 2A-AR (LLC-PK1-alpha 2A) were assayed. It was previously reported that alpha 2-ARs can be linked to alternative effectors, in addition to adenylyl cyclase. In CHO cells transfected with alpha 2B-AR, agonist exposure stimulates cPLA2 activity (1). In the distal segment of the rat nephron, an enhancement of PKC activity subsequent to an increase in diacylglycerol formation and to an elevation of intracellular Ca2+ concentration has been demonstrated (14). These observations raise the possibility that cAMP-independent mechanisms of signal transduction also exist in PTC. To investigate in this direction, PLA2 activity was examined. As shown in Fig. 3, the exposure of PTC to UK-14304 resulted in an increase of [3H]arachidonic acid release. This effect was potentiated by the addition of the Ca2+ ionophore A-23187, and it was blocked in the presence of RX-821002 or quinacrine. Similar data were obtained in LLC-PK1-alpha 2B cells, suggesting that cPLA2 must be considered as an alternative effector in the mediation of the alpha 2B-adrenergic signal in the proximal tubule.

                              
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Table 2.   Effect of UK-14304 on cAMP production induced by forskolin or PTH



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Fig. 3.   Effect of UK-14304 on arachidonic acid release. PTC (A) and LLC-PK1-alpha 2B cells (B) were loaded with [3H]arachidonic acid (AA; 1 µCi/ml) for 1 h. They were extensively washed in HEPES-buffered DMEM containing 0.2% fatty acid-free BSA and treated with 1 µM A-23187 (A-23187), 1 µM UK-14304 (UK), 1 µM A-23187 plus 1 µM UK-14304 (A-23187+UK), 1 µM UK-14304 plus 10 µM RX-821002 (UK+RX), or 1 µM UK-14304 plus 100 µM quinacrine (UK+quin). Arachidonic acid release was measured as described in MATERIALS AND METHODS. Results are means ± SE from 4 independent experiments with triplicate determinations expressed as percentage of control. *P < 0.05 and **P < 0.02, values significantly different from control.

The consequence of receptor stimulation on MAPK was investigated by estimating the extent of ERK phosphorylation as well as by measuring their enzymatic activity in cellular extracts and following their nuclear translocation in intact cells. In both PTC and LLC-PK1-alpha 2B cells, the anti-active MAPK antibody preferentially recognized a protein that, when revealed with ERK1/2 antibody, corresponded to ERK2. As expected, exposure of PTC to PMA for 5 min resulted in a very marked increase in ERK2 phosphorylation (Fig. 4). A significant rise in the extent of ERK2 phosphorylation was also observed after treatment with UK-14304. The effect of the alpha 2-agonist was clear as early as 5 min after the start of treatment. It was maximal at 10 min and persisted for at least 30 min. Similar kinetics were observed when PTC were exposed to (-)-epinephrine or dexmedetomidine (Fig. 4), and MAPK phosphorylation was also observed when the three agonists were assayed in LLC-PK1-alpha 2B cells (Fig. 5). In agreement with the Western blot analysis data, a 5-min exposure to UK-14304 caused a threefold increase in MAPK enzymatic activity in PTC (Fig. 6) and resulted in massive translocation of ERK2 to the nucleus in LLC-PK1-alpha 2B cells (Fig. 7). The change in MAPK activity matched the change in the extent of ERK2 phosphorylation. Indeed, both processes were blunted by the addition of the alpha 2-antagonist RX-821002 and by prior treatment of the cells with pertussis toxin, indicating that the effects on MAPK are primarily due to activation of alpha 2-AR and require Gi/Go integrity. Complementary experiments (Fig. 8) showed that the effect of UK-14304 was also totally abolished by the addition of genistein (25 µM), the inhibitor of protein tyrosine kinases, or the addition of PD-98059 (50 µM), the inhibitor of MEK1/2. The stimulation of alpha 2-AR was previously reported to increase PKC activity in collecting ducts (14). As treatment of PTC with PMA causes a huge increase in MAPK phosphorylation (see Fig. 4), we wondered whether PKC activation is involved in the mediation of UK-14304 effects. The consequences of exposure to the alpha 2-agonist were therefore studied after PKC desensitization induced by long-term treatment with PMA. As expected, such pretreatment totally abolished any further response to the phorbol ester (data not shown). By contrast, it did not affect the response to UK-14304, suggesting that alpha 2B-AR triggers its effect through a PKC-independent pathway. Furthermore, because the activation of MAPK by several G protein-coupled receptors, including alpha 2A-ARs, was previously shown to depend on Shc phosphorylation, we checked whether this holds true for alpha 2B-AR in PTC (Fig. 9). The direct use of anti-Shc antibody on cellular extracts demonstrated that the 46- and 52-kDa isoforms of Shc are predominant in this cell type. Treatment with 1 µM (-)-epinephrine led to increased phosphorylation of both isoforms of Shc within the minutes after exposure. As is the case for MAPK phosphorylation, this effect was transient and was totally abolished in the presence of the alpha 2-antagonist RX-821002. A similar increase in Shc phosphorylation was observed with UK-14304.


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Fig. 4.   Measurement of mitogen-activated protein kinase (MAPK) phosphorylation in PTC. PTC were grown, rendered quiescent, and treated as described in MATERIALS AND METHODS. Soluble proteins were extracted by centrifugation, separated by SDS-PAGE, and blotted onto a Polyscreen polyvinylidene difluoride (PVDF) membrane. Top: MAPK phosphorylation was revealed using anti-active MAPK antibody. Bottom: membranes were stripped of Ig and probed with anti-extracellular signal-regulated kinase (ERK) antibody to assess equal protein loading. Left: cells were treated with 1 µM UK-14304 for 2, 5, 10, 20, 30, and 60 min (UK) and 2 µM phorbol 12-myristate 13-acetate (PMA) for 5 min. Center: cells were treated with 1 µM (-)-epinephrine (Epi) for 2, 5, and 10 min. Right: cells were treated with 1 µM dexmedetomidine (Dxm) for 2, 5, and 10 min.



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Fig. 5.   Measurement of MAPK phosphorylation in LLC-PK1-alpha 2B. LLC-PK1-alpha 2B cells were grown and rendered quiescent as described in MATERIALS AND METHODS. Then, they were treated for 5 min with 1 µM UK-14304 (UK), 1 µM Dxm, 1 µM Epi, or 2 µM PMA. Soluble proteins were extracted by centrifugation, separated by SDS-PAGE, and blotted onto a Polyscreen PVDF membrane. Top: MAPK phosphorylation was revealed using anti-active MAPK antibody. Bottom: membranes were stripped of Ig and probed with anti-ERK antibody to assess equal protein loading



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Fig. 6.   Measurement of MAPK activity. PTC were grown and rendered quiescent as described in MATERIALS AND METHODS. They were then treated with 1 µM UK-14304 for 5 min (UK). The effect of the alpha 2-agonist was tested in the presence of 10 µM RX-821002 (RX+ UK) or in cells pretreated for 16 h with 100 ng/ml pertussis toxin (PTX+UK). A: soluble proteins were separated by SDS-PAGE and blotted onto a Polyscreen PVDF membrane. The phosphorylated forms of MAPK were revealed using anti-active MAPK antibody (top). Membranes were stripped of Ig and probed with anti-ERK antibody to assess equal protein loading (bottom). B: cell extracts were assayed for MAPK activity using a synthetic substrate as described in MATERIALS AND METHODS. Activities are expressed as a percentage of that in untreated cells. Values are means ± SE from 3 independent experiments with quadrate determinations. **P < 0.02, significantly different from control.



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Fig. 7.   Subcellular redistribution of ERK2 following alpha 2-agonist treatment. LLC-PK1-alpha 2B cells plated on glass coverslips were grown, rendered quiescent, and exposed (B) or not to UK-14304 (A) for 15 min as described in MATERIALS AND METHODS. The cells were fixed and permeabilized, and the location of ERK2 was assessed by immunofluorescence using anti-ERK2 polyclonal antibody. Panels are representative of 3 independent experiments. Bars, 10 µM.



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Fig. 8.   Effect of protein kinase C (PKC) desensitization, genistein, and PD-98059. PTC were grown and rendered quiescent as described in MATERIALS AND METHODS. They were treated for various periods of time with the indicated compound. Soluble proteins were separated by SDS-PAGE and blotted onto a Polyscreen PVDF membrane. MAPK phosphorylation was revealed using anti-active MAPK antibody. The cells were treated for 20 h with 2 µM PMA (PMA 20 h) or for 30 min with either 25 µM genistein (Genist) or 50 µM PD-98059 before exposure (+) or not (-) to 1 µM UK-14304 for 5 min.



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Fig. 9.   Effect of Epi on Shc phosphorylation. Left: identification of the isoforms of Shc expressed in PTC. Protein extracts from PTC and A431 cells were subjected to SDS-PAGE, and Shc isoforms were revealed by Western blot analysis using anti-Shc antibody. Right: effect of Epi on Shc phosphorylation. PTC were grown and rendered quiescent as described in MATERIALS AND METHODS. They were then treated for 2, 5, and 10 min with 1 µM Epi or for 5 min with 1 µM Epi+10 µM RX-821002 (Epi+RX). Shc proteins were immunoprecipitated (IP) with anti-Shc antibody, and their phosphorylation was estimated using anti-phosphotyrosine antibody (top). The membrane was stripped of Ig and reprobed using anti-Shc antibody to assess equal protein loading (bottom). IB, immunoblot.

Finally, the possibility that activation of MAPK by alpha 2B-AR enhances cell proliferation was examined by measuring the growth rate of PTC and LLC-PK1-alpha 2B cells in the presence or absence of alpha 2-agonist. As shown in Fig. 10, UK-14304 accelerated the growth rate of PTC cultured in medium deprived of FCS and of LLC-PK1-alpha 2B cells cultured in the presence of 5% FCS. The proliferative response to UK-14304 was blunted by the cotreatment of PTC with RX-821002, and it was not observed in wild-type LLC-PK1 cells (not shown).


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Fig. 10.   Effect of UK-14304 on cell proliferation. A: PTC were seeded in 6-well plates, grown for 2 days in DMEM-Ham's F-12 medium (1:1) supplemented with 5% FCS, and placed for 1 day in serum-free medium. Serum-deprived cells were treated with vehicle (open circle ), 1 µM UK-14304 () or 1 µM UK-14304 plus 10 µM RX-821002 (black-triangle). Culture plates were collected at the indicated times, and the DNA content was determined. Values are means ± SE from 5 independent experiments with triplicate determinations expressed as percentage of increase relative to time 0. B: LLC-PK1-alpha 2B cells were seeded in 6-well plates, grown for 2 days in DMEM supplemented with 5% FCS, and then treated with 1 µM UK-14304 () or not treated (). Culture plates were collected at the indicated time, and the number of cells was determined. Values are means ± SE from 3 independent experiments with triplicate determinations expressed as percentage of increase relative to time 0. *P < 0.05 and **P < 0.02, values significantly different from control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although expression of alpha 2B-AR in the rat proximal tubule has been known for a long time (37), the precise roles and the molecular mechanisms whereby this receptor exerts its actions on NHE3 are not completely understood. The purpose of the present study was to reinvestigate this using rat PTC in primary culture and a cell line of proximal tubule origin permanently transfected with the gene encoding rat alpha 2B-AR. According to RPA and to radioligand binding studies, cultured PTC express only the alpha 2B-AR subtype at a density of ~130 fmol/mg protein. This value is fairly similar to that previously reported in freshly isolated proximal tubules using [3H]rauwolscine as radioligand (39). It is significantly higher than in cells cultured from inner medullary collecting duct (46), a difference that agrees with the fact that the proximal tubule is the major site of alpha 2B-AR expression in the kidney. On the other hand, the level of receptor expression in LLC-PK1-alpha 2B cells was ~sixfold that in PTC. In both models, a substantial proportion of the receptor was under the high-affinity state for an agonist, suggesting efficient coupling to G proteins. In support of this conclusion, dexmedetomidine attenuated cAMP production. The extent of inhibition was rather weak (~20%) in LLC-PK1-alpha 2B cells or PTC stimulated with forskolin, but it was >40% in PTH-treated PTC. A similar amplitude of inhibition (35%) has been previously reported in microdissected tubule using (-)-epinephrine as an agonist (42). Unexpectedly, UK-14304 had a negligible action on cAMP level. The lack of effect in LLC-PK1-alpha 2B is somewhat at variance with previous findings, demonstrating that this agonist is able to strongly inhibit forskolin-induced cAMP accumulation in another clone of LLC-PK1 cells transfected with alpha 2B-AR (31). The reasons for this discrepancy are unclear. The clone used in that study expressed the receptor at a higher density (~2 pmol/mg of protein) than did our LLC-PK1-alpha 2B cells (0.73 pmol/mg protein). In addition, the inhibitory effect was measured in polarized cell layers vs. nondifferentiated cells in the present study. As already found for other processes, it is possible that the lack of inhibition is due to nonoptimal assemblage of the alpha 2B-AR signaling pathway in nonpolarized cells. A larger decrease in forskolin-induced cAMP accumulation and no difference between UK-14304 and dexmedetomidine efficiency was observed in LLC-PK1 cells expressing alpha 2A-AR, suggesting that the data obtained under our experimental conditions also reflect intrinsic differences between the two subtypes. Regarding this point, it is noteworthy that experiments in CHO cells or on S115 mouse mammary tumor cells expressing the different alpha 2-AR subtypes at a similar density showed repeatedly that alpha 2B-AR was less efficient than other subtypes in inhibiting adenylyl cyclase (12, 18). Furthermore, measurement of pertussis toxin-sensitive GTPase in transfected CHO cells showed that UK-14304, but not dexmedetomidine, acts as a partial agonist at the alpha 2B-AR (19).

Exposure of PTC or LLC-PK1-alpha 2B cells to (-)-epinephrine or to alpha 2-agonists (UK-14304, dexmedetomidine) caused an increase in the phosphorylation state of p42/44 MAPK. As expected, this effect was blocked by the addition of RX-821002 (alpha 2-antagonist) and resulted in an enhancement of MAPK activity and translocation to the cell nucleus. The kinetics of MAPK phosphorylation were rapid and similar to those in other cell systems in which the alpha 2A-AR was found to exert mitogenic effects (4, 35). ERKs are expressed in the different segments of the nephron (41), and their pattern of expression changes with the stage of renal development (32). The activation of ERKs was demonstrated to play a crucial role in tubular cell regeneration after oxidative injury (9) and to be induced by various growth factors, cytokines, and vasoactive substances (16, 24). With regard to proximal tubule-derived cells, other G protein-coupled receptors already shown to activate ERKs include serotonin 1B receptor (5-HT1B), alpha 2C-AR, and lysophosphatidic acid receptor in OK cells (10, 22) as well as angiotensin II and PTH receptors in OK and rabbit proximal tubule cells (7, 20, 36, 40). It is now clearly established that the mechanisms responsible for the activation of ERKs by G protein-coupled receptors is highly dependent on the particular receptor or cell type examined. Phosphorylation of ERKs may be due to receptor interaction with Gq or Gi/Go and be triggered by direct activation of Raf by PKC or by indirect recruitment of p21-Ras via the formation of the Shc-Grb2-Sos complex. Because alpha 2-ARs were reported to stimulate PKC and to increase intracellular Ca2+ in renal epithelial cells (14), we investigated whether PKC is involved. As expected, short-term exposure of PTC or LLC-PK1-alpha 2B cells to PMA caused a marked increase in the phosphorylation of ERK1/2, but the action of UK-14304 was unaffected by PKC desensitization. Conversely, the effect of the agonist was totally abolished by PTX treatment or by the prior addition of genistein or PD-98059, showing that it depends on Gi proteins and requires the activity of tyrosine kinases and MEK1/2. This view was also supported by measurement of Shc phosphorylation. Indeed, the two forms of Shc (46 and 52 kDa) found in PTC or LLC-PK1-alpha 2B cells are transiently phosphorylated after agonist exposure. It is thus likely that the alpha 2B-AR stimulates MAPK through a pathway comprising activation of Gi proteins, phosphorylation of Shc, recruitment of the Grb2-Sos complex, and subsequent activation of the p21-Ras-MAPK cascade. Measurement of cell growth in different culture conditions indicated that activation of MAPK resulted in accelerated proliferation of PTC and LLC-PK1-alpha 2B cells.

In addition to MAPK activation, the treatment of PTC or LLC-PK1-alpha 2B cells with alpha 2-agonists induced a significant increase in arachidonic acid release that is potentiated by the Ca2+ ionophore A-23187. In this respect, our results match recent observations showing that, in transfected CHO cells, alpha 2B-AR was much more efficient than other subtypes in stimulating cPLA2 (1). The relationship between MAPK and cPLA2 was not investigated in the present study. Although it is known that cPLA2 is a substrate for MAPK (29), in CHO cells, phosphorylation of cPLA2 does not correlate with its activation by alpha 2B-AR, suggesting the involvement of another mechanism (1). Alternatively, activation of MAPK could be the consequence of arachidonic acid release. In this respect, it is noteworthy that arachidonic acid was recently demonstrated to mediate the effects of angiotensin II on MAPK in primary culture of proximal tubule cells from rabbist (11). The action of arachidonic acid was dependent on the production of epoxy derivative, and it involved activation of p21ras, subsequent to Shc phosphorylation and association with Grb2 (20). Preliminary results obtained in LLC-PK1-alpha 2B cells indicate that MAPK activation by alpha 2-agonists is ablated in the presence of quinacrine, suggesting that cPLA2 is involved in the mediation of the effect of alpha 2B-AR. However, further studies will be necessary to determine whether other documented mechanisms (e.g., alpha 2-AR internalization, EGF receptor "transactivation," c-Src recruitment) should also be considered.

Depending on the cell type considered and on the duration of their activation, ERKs may have opposite effects on cell growth and result either in proliferation or in arrest of the cell cycle. For instance, activation of MAPK by angiotensin II has no mitogenic effect but induces hypertrophy in cultured murine PTC or in LLC-PK1 cells (45). According to recent reports, this effect depends on activation of NADPH-oxidase, generation of reactive oxygen species, and consecutive induction of p27kip, an inhibitor of cyclin-dependent kinases. Interestingly, our results demonstrate that alpha 2B-AR stimulation enhances cell growth. Although the involvement of other factors cannot be ruled out, it is thus likely that activation of ERK is primarily responsible for the increased rate of proliferation.

In conclusion, the present work shows that the alpha 2B-AR activates the ERK pathway and stimulates the proliferation of epithelial cells derived from the proximal tubule of rat and pig. Activation of ERK by alpha 2B-AR is independent of adenylyl cyclase inhibition or PKC stimulation, but it correlates with increased cPLA2 activity. Regardless of the precise mechanism responsible for ERK phosphorylation, it appears that the action of catecholamines on alpha 2B-AR may play a role in the adaptive response to acute renal tissue injury in the rat.


    ACKNOWLEDGEMENTS

The authors thank E. Fonta and F. Quinchon for excellent technical assistance.


    FOOTNOTES

This study was supported by the BIOMED 2 Programme PL963373 (European Commission, Brussels, Belgium) and by a grant from the Fondation pour la Recherche Médicale (Paris, France).

Address for reprint requests and other correspondence: H. Paris, INSERM Unit 388, Institut Louis Bugnard, CHU Rangueil, Bat. L3, 31403 Toulouse Cedex 4, France (E-mail: paris{at}toulouse.inserm.fr).

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.

First published August 8, 2001;10.1152/ajprenal.0108.2001

Received 3 April 2001; accepted in final form 7 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Audubert, F, Klapisz E, Berguerand M, Gouache P, Jouniaux AM, Bereziat G, and Masliah J. Differential potentiation of arachidonic acid release by rat alpha 2-adrenergic receptor subtypes. Biochim Biophys Acta 1437: 265-276, 1999[ISI][Medline].

2.   Beach, RE, Schwab SJ, Brazy PC, and Dennis VW. Norepinephrine increases Na+-K+-ATPase and solute transport in rabbit proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 252: F215-F220, 1987[Abstract/Free Full Text].

3.   Berkowitz, DE, Price DT, Bello EA, Page SO, and Schwinn DA. Localization of messenger RNA for three distinct alpha 2-adrenergic receptor subtypes in human tissues. Evidence for species heterogeneity and implications for human pharmacology. Anesthesiology 81: 1235-1244, 1994[ISI][Medline].

4.   Bouloumie, A, Planat V, Devedjian JC, Valet P, Saulnier-Blache JS, Record M, and Lafontan M. Alpha2-adrenergic stimulation promotes preadipocyte proliferation. Involvement of mitogen-activated protein kinases. J Biol Chem 269: 30254-30259, 1994[Abstract/Free Full Text].

5.   Bradford, MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

6.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

7.   Cole, JA. Parathyroid hormone activates mitogen-activated protein kinase in opossum kidney cells. Endocrinology 140: 5771-5779, 1999[Abstract/Free Full Text].

8.   de Leeuw, PW, and Birkenhager WH. Alpha-adrenoceptors and the kidney. J Hypertens Suppl 6: S21-S24, 1988.

9.   di Mari, JF, Davis R, and Safirstein RL. MAPK activation determines renal epithelial cell survival during oxidative injury. Am J Physiol Renal Physiol 277: F195-F203, 1999[Abstract/Free Full Text].

10.   Dixon, RJ, and Brunskill NJ. Lysophosphatidic acid-induced proliferation in opossum kidney proximal tubular cells: role of PI 3-kinase and ERK. Kidney Int 56: 2064-2075, 1999[ISI][Medline].

11.   Dulin, NO, Alexander LD, Harwalkar S, Falck JR, and Douglas JG. Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II. Proc Natl Acad Sci USA 95: 8098-8102, 1998[Abstract/Free Full Text].

12.   Eason, MG, and Liggett SB. Subtype-selective desensitization of alpha 2-adrenergic receptors. Different mechanisms control short and long term agonist-promoted desensitization of alpha 2C10, alpha 2C4, and alpha 2C2. J Biol Chem 267: 25473-25479, 1992[Abstract/Free Full Text].

13.   Flordellis, CS, Berguerand M, Gouache P, Barbu V, Gavras H, Handy DE, Bereziat G, and Masliah J. Alpha2-adrenergic receptor subtypes expressed in Chinese hamster ovary cells activate differentially mitogen-activated protein kinase by a p21ras independent pathway. J Biol Chem 270: 3491-3494, 1995[Abstract/Free Full Text].

14.   Gesek, FA. Alpha2-adrenergic receptors activate phospholipase C in renal epithelial cells. Mol Pharmacol 50: 407-414, 1996[Abstract].

15.   Handy, DE, Flordellis CS, Bogdanova NN, Bresnahan MR, and Gavras H. Diverse tissue expression of rat alpha 2-adrenergic receptor genes. Hypertension 21: 861-865, 1993[Abstract].

16.   Heasley, LE, Senkfor SI, Winitz S, Strasheim A, Teitelbaum I, and Berl T. Hormonal regulation of MAP kinase in cultured rat inner medullary collecting tubule cells. Am J Physiol Renal Fluid Electrolyte Physiol 267: F366-F373, 1994[Abstract/Free Full Text].

17.   Huang, L, Wei YY, Momose-Hotokezaka A, Dickey J, and Okusa MD. alpha 2B-Adrenergic receptors: immunolocalization and regulation by potassium depletion in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 270: F1015-F1026, 1996[Abstract/Free Full Text].

18.   Jansson, CC, Marjamaki A, Luomala K, Savola JM, Scheinin M, and Akerman KE. Coupling of human alpha 2-adrenoceptor subtypes to regulation of cAMP production in transfected S115 cells. Eur J Pharmacol 266: 165-174, 1994[Medline].

19.   Jansson, CC, Pohjanoksa K, Lang J, Wurster S, Savola JM, and Scheinin M. Alpha2-adrenoceptor agonists stimulate high-affinity GTPase activity in a receptor subtype-selective manner. Eur J Pharmacol 374: 137-146, 1999[ISI][Medline].

20.   Jiao, H, Cui XL, Torti M, Chang CH, Alexander LD, Lapetina EG, and Douglas JG. Arachidonic acid mediates angiotensin II effects on p21ras in renal proximal tubular cells via the tyrosine kinase-Shc-Grb2-Sos pathway. Proc Natl Acad Sci USA 95: 7417-7421, 1998[Abstract/Free Full Text].

21.   Koch, WJ, Hawes BE, Allen LF, and Lefkowitz RJ. Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras. Proc Natl Acad Sci USA 91: 12706-12710, 1994[Abstract/Free Full Text].

22.   Kribben, A, Herget-Rosenthal S, Lange B, Erdbrugger W, Philipp T, and Michel MC. Alpha2-adrenoceptors in opossum kidney cells couple to stimulation of mitogen-activated protein kinase independently of adenylyl cyclase inhibition. Naunyn Schmiedebergs Arch Pharmacol 356: 225-232, 1997[ISI][Medline].

23.   Kribben, A, Herget-Rosenthal S, Lange B, Michel MC, and Philipp T. Stimulation of mitogen activated protein kinase and cellular proliferation in renal proximal tubular cells. Renal Fail 20: 229-234, 1998[ISI][Medline].

24.   Lal, MA, Proulx PR, and Hebert RL. A role for PKC epsilon and MAP kinase in bradykinin-induced arachidonic acid release in rabbit CCD cells. Am J Physiol Renal Physiol 274: F728-F735, 1998[Abstract/Free Full Text].

25.   Luttrell, LM, Daaka Y, Della Rocca GJ, and Lefkowitz RJ. G protein-coupled receptors mediate two functionally distinct pathways of tyrosine phosphorylation in rat 1a fibroblasts. Shc phosphorylation and receptor endocytosis correlate with activation of Erk kinases. J Biol Chem 272: 31648-31656, 1997[Abstract/Free Full Text].

26.   Meister, B, Dagerlind A, Nicholas AP, and Hokfelt T. Patterns of messenger RNA expression for adrenergic receptor subtypes in the rat kidney. J Pharmacol Exp Ther 268: 1605-1611, 1994[Abstract].

27.   Michel, MC, and Rump LC. Alpha-adrenergic regulation of human renal function. Fundam Clin Pharmacol 10: 493-503, 1996[ISI][Medline].

28.   Murphy, TJ, and Bylund DB. Characterization of alpha 2-adrenergic receptors in the OK cell, an opossum kidney cell line. J Pharmacol Exp Ther 244: 571-578, 1988[Abstract].

29.   Nemenoff, RA, Winitz S, Qian NX, Van Putten V, Johnson GL, and Heasley LE. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J Biol Chem 268: 1960-1964, 1993[Abstract/Free Full Text].

30.   Nord, EP, Howard MJ, Hafezi A, Moradeshagi P, Vaystub S, and Insel PA. Alpha2-adrenergic agonists stimulate Na+-H+ antiport activity in the rabbit renal proximal tubule. J Clin Invest 80: 1755-1762, 1987[ISI][Medline].

31.   Okusa, MD, Huang L, Momose-Hotokezaka A, Huynh LP, and Mangrum AJ. Regulation of adenylyl cyclase in polarized renal epithelial cells by G protein-coupled receptors. Am J Physiol Renal Physiol 273: F883-F891, 1997[Abstract/Free Full Text].

32.   Omori, S, Hida M, Ishikura K, Kuramochi S, and Awazu M. Expression of mitogen-activated protein kinase family in rat renal development. Kidney Int 58: 27-37, 2000[ISI][Medline].

33.   Ruffolo, RR, Jr, Nichols AJ, Stadel JM, and Hieble JP. Pharmacologic and therapeutic applications of alpha 2-adrenoceptor subtypes. Annu Rev Pharmacol Toxicol 33: 243-279, 1993[ISI][Medline].

34.   Schaak, S, Cayla C, Blaise R, Quinchon F, and Paris H. HepG2 and SK-N-MC: two human models to study alpha 2-adrenergic receptors of the alpha 2C subtype. J Pharmacol Exp Ther 281: 983-991, 1997[Abstract/Free Full Text].

35.   Schaak, S, Cussac D, Cayla C, Devedjian JC, Guyot R, Paris H, and Denis C. Alpha2-adrenoceptors regulate proliferation of human intestinal epithelial cells. Gut 47: 242-250, 2000[Abstract/Free Full Text].

36.   Sneddon, WB, Liu F, Gesek FA, and Friedman PA. Obligate mitogen-activated protein kinase activation in parathyroid hormone stimulation of calcium transport but not calcium signaling. Endocrinology 141: 4185-4193, 2000[Abstract/Free Full Text].

37.   Stanko, CK, Vandel MI, Bose R, and Smyth DD. Characterization of alpha 2-adrenoceptors in the rat: proximal tubule, renal membrane and whole kidney studies. Eur J Pharmacol 175: 13-20, 1990[ISI][Medline].

38.   Strandhoy, JW. Role of alpha 2-receptors in the regulation of renal function. J Cardiovasc Pharmacol 7, Suppl8: S28-S33, 1985[ISI][Medline].

39.   Sundaresan, PR, Fortin TL, and Kelvie SL. alpha - and beta -Adrenergic receptors in proximal tubules of rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 253: F848-F856, 1987[Abstract/Free Full Text].

40.   Terada, Y, Tomita K, Homma MK, Nonoguchi H, Yang T, Yamada T, Yuasa Y, Krebs EG, and Marumo F. Sequential activation of MAP kinase cascade by angiotensin II in opossum kidney cells. Kidney Int 48: 1801-1809, 1995[ISI][Medline].

41.   Terada, Y, Yamada T, Takayama M, Nonoguchi H, Sasaki S, Tomita K, and Marumo F. Presence and regulation of Raf-1-K (kinase), MAPK-K, MAP-K, and S6-K in rat nephron segments. J Am Soc Nephrol 6: 1565-1577, 1995[Abstract].

42.   Umemura, S, Marver D, Smyth DD, and Pettinger WA. alpha 2-Adrenoceptors and cellular cAMP levels in single nephron segments from the rat. Am J Physiol Renal Fluid Electrolyte Physiol 249: F28-F33, 1985[ISI][Medline].

43.   Vinay, P, Gougoux A, and Lemieux G. Isolation of a pure suspension of rat proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 241: F403-F411, 1981[Abstract/Free Full Text].

44.   Winitz, S, Russell M, Qian NX, Gardner A, Dwyer L, and Johnson GL. Involvement of Ras and Raf in the Gi-coupled acetylcholine muscarinic m2 receptor activation of mitogen-activated protein (MAP) kinase kinase and MAP kinase. J Biol Chem 268: 19196-19199, 1993[Abstract/Free Full Text].

45.   Wolf, G, Zahner G, Mondorf U, Schoeppe W, and Stahl RA. Angiotensin II stimulates cellular hypertrophy of LLC-PK1 cells through the AT1 receptor. Nephrol Dial Transplant 8: 128-133, 1993[Abstract].

46.   Yasuda, G, Sun L, Umemura S, Pettinger WA, and Jeffries WB. Characterization of prazosin-sensitive alpha 2B-adrenoceptors expressed by cultured rat IMCD cells. Am J Physiol Renal Fluid Electrolyte Physiol 261: F760-F766, 1991[Abstract/Free Full Text].

47.   Zeng, DW, Harrison JK, D'Angelo DD, Barber CM, Tucker AL, Lu ZH, and Lynch KR. Molecular characterization of a rat alpha 2B-adrenergic receptor. Proc Natl Acad Sci USA 87: 3102-3106, 1990[Abstract].


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