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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the rat
proximal tubule, the 2B-adrenergic receptor
(
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
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
2-AR). Binding experiments indicated that PTC express
substantial amounts of
2B-AR (130 fmol/mg protein), and
only RNG transcripts were detected. In both cell types, the
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
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
2B-AR may play a positive role during tubular regeneration.
kidney; mitogen-activated protein kinase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FUNCTIONAL STUDIES,
CARRIED out in various animal species, have demonstrated that
2-adrenergic receptors (
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
2-ARs are
a heterogeneous class of receptors comprising three subtypes
(
2A,
2B, and
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 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,
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
2-agonists on the
proximal tubule are primarily due to stimulation of
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, 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
2-AR of the
2C-subtype (28). The search for alternative
pathways of signal transduction demonstrated that
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 -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
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
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.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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). [-32P]UTP (800 Ci/mmol) and
[
-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 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
2B-AR (LLC-PK1-
2B) or
the human
2A-AR (LLC-PK1-
2A)
were respectively obtained by transfection with pcDNA3 vector
containing the coding region of the RNG or
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 [-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 105 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 [-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 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-PK1-2B 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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cultured PTC having been never used for the study of
2-adrenergic receptivity, preliminary experiments were
designed to assess the level of
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
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-
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
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).
|
|
As a preliminary step in the study of the pathways of
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-
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-
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-
2B cells.
|
In isolated proximal tubules as well as in the OK cell line,
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-
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-
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
2B-AR
subtype, because a significant reduction was observed when
LLC-PK1 cells expressing
2A-AR
(LLC-PK1-
2A) were assayed. It was previously reported that
2-ARs can be linked to alternative
effectors, in addition to adenylyl cyclase. In CHO cells transfected
with
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-
2B cells, suggesting
that cPLA2 must be considered as an alternative effector in
the mediation of the
2B-adrenergic signal in the
proximal tubule.
|
|
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-PK1-2B 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
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-
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-
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
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
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
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
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
2B-AR triggers its effect through a PKC-independent pathway. Furthermore, because the activation of MAPK by several G
protein-coupled receptors, including
2A-ARs, was
previously shown to depend on Shc phosphorylation, we checked
whether this holds true for
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
2-antagonist RX-821002. A similar increase in Shc phosphorylation was observed with UK-14304.
|
|
|
|
|
|
Finally, the possibility that activation of MAPK by
2B-AR enhances cell proliferation was examined by
measuring the growth rate of PTC and
LLC-PK1-
2B cells in the presence or absence
of
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-
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).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although expression of 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
2B-AR. According
to RPA and to radioligand binding studies, cultured PTC express only
the
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
2B-AR expression in the kidney. On the
other hand, the level of receptor expression in
LLC-PK1-
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-
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-
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
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-
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
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
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
2-AR subtypes at a similar density showed
repeatedly that
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
2B-AR
(19).
Exposure of PTC or LLC-PK1-2B cells to
(
)-epinephrine or to
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 (
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
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),
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
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-
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-
2B cells are transiently phosphorylated after agonist exposure. It is thus likely that the
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-
2B cells.
In addition to MAPK activation, the treatment of PTC or
LLC-PK1-2B cells with
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,
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
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-
2B cells indicate that
MAPK activation by
2-agonists is ablated in the presence
of quinacrine, suggesting that cPLA2 is involved in the
mediation of the effect of
2B-AR. However, further
studies will be necessary to determine whether other documented mechanisms (e.g.,
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 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
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
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
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Audubert, F,
Klapisz E,
Berguerand M,
Gouache P,
Jouniaux AM,
Bereziat G,
and
Masliah J.
Differential potentiation of arachidonic acid release by rat 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
3.
Berkowitz, DE,
Price DT,
Bello EA,
Page SO,
and
Schwinn DA.
Localization of messenger RNA for three distinct 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
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
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
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
12.
Eason, MG,
and
Liggett SB.
Subtype-selective desensitization of 2-adrenergic receptors. Different mechanisms control short and long term agonist-promoted desensitization of
2C10,
2C4, and
2C2.
J Biol Chem
267:
25473-25479,
1992
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
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 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
17.
Huang, L,
Wei YY,
Momose-Hotokezaka A,
Dickey J,
and
Okusa MD.
2B-Adrenergic receptors: immunolocalization and regulation by potassium depletion in rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F1015-F1026,
1996
18.
Jansson, CC,
Marjamaki A,
Luomala K,
Savola JM,
Scheinin M,
and
Akerman KE.
Coupling of human 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
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
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
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
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 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
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
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 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 2-adrenergic receptors of the
2C subtype.
J Pharmacol Exp Ther
281:
983-991,
1997
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
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
37.
Stanko, CK,
Vandel MI,
Bose R,
and
Smyth DD.
Characterization of 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 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.
- and
-Adrenergic receptors in proximal tubules of rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
253:
F848-F856,
1987
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.
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
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
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 2B-adrenoceptors expressed by cultured rat IMCD cells.
Am J Physiol Renal Fluid Electrolyte Physiol
261:
F760-F766,
1991
47.
Zeng, DW,
Harrison JK,
D'Angelo DD,
Barber CM,
Tucker AL,
Lu ZH,
and
Lynch KR.
Molecular characterization of a rat 2B-adrenergic receptor.
Proc Natl Acad Sci USA
87:
3102-3106,
1990[Abstract].