(Hypertension. 2000;36:1053.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Renal Protein Phosphatase 2A Activity and Spontaneous Hypertension in Rats
From the Departments of Pediatrics (P.Y., L.D.A., P.A.J.) and Physiology and Biophysics (P.A.J.), Georgetown University School of Medicine, Washington, DC; the Department of Internal Medicine (G.M.E.), Washington Hospital Center, Washington, DC; the Department of Physiology (U.H.), Case Western Reserve School of Medicine, Cleveland, Ohio; and the Department of Pathology and Medical Automation Research Center (R.A.F.), Charlottesville, Va.
Correspondence to Peiying Yu, MD, Department of Pediatrics, PHC-2, Georgetown University Medical Center, 3800 Reservoir Rd NW, Washington, DC 20007. E-mail yup{at}gunet.georgetown.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—The impaired renal paracrine function of dopamine in spontaneously hypertensive rats (SHR) is caused by hyperphosphorylation and desensitization of the renal D1 dopamine receptor. Protein phosphatase 2A (PP2A) is critical in the regulation of G-protein–coupled receptor function. To determine whether PP2A expression and activity in the kidney are differentially regulated in genetic hypertension, we examined the effects of a D1-like agonist, fenoldopam, in renal cortical tubules and immortalized renal proximal tubule cells from normotensive Wistar-Kyoto rats (WKY) and SHR. In cortical tubules and immortalized proximal tubule cells, PP2A expression and activities were greater in cytosol than in membrane fractions in both WKY and SHR. Although PP2A expressions were similar in WKY and SHR, basal PP2A activity was greater in immortalized proximal tubule cells of SHR than WKY. In immortalized proximal tubule cells of WKY, fenoldopam increased membrane PP2A activity and expression of the regulatory subunit PP2A-B56
, effects that were blocked by the
D1-like antagonist SCH23390. Fenoldopam had no
effect on cytosolic PP2A activity but decreased
PP2A-B56 expression. In contrast, in immortalized
proximal tubule cells of SHR, fenoldopam decreased PP2A
activity in both membranes and cytosol but predominantly in the
membrane fraction, without affecting PP2A-B56
expression; this effect was blocked by the D1-like
antagonist SCH23390. We conclude that renal
PP2A activity and expression are differentially regulated
in WKY and SHR by D1-like receptors. A failure of
D1-like agonists to increase PP2A activity in
proximal tubule membranes may be a cause of the increased
phosphorylation of the D1 receptor in
the SHR.
Key Words: hypertension, genetic • receptors, dopamine • dopamine • hypertension, essential • phosphatase
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Essential hypertension is a multifactorial disorder thought to arise from the interactions of environmental factors with multiple genes.1 2 Defective regulation of nerve activity, vascular reactivity, endocrine function, and ion transport in genetic hypertension has been linked to abnormalities in signal transduction.1 2 3 Dopamine synthesized by renal proximal tubule cells (RPTC) plays an important paracrine/autocrine role in the regulation of renal function during volume expansion.3 4 5 Dopamine, through different dopamine receptor subtypes, regulates cardiovascular functions by actions on the central and peripheral nervous systems, vascular smooth muscle, and adrenal glands, heart, and kidney.3 Abnormal regulation of cardiovascular function by various dopamine receptor subtypes has been implicated in the pathogenesis of genetic hypertension.2 3 6 7 In genetic hypertension, the D1 dopamine receptor, a G-protein–coupled receptor (GPCR) in renal proximal tubules, is desensitized, serine-hyperphosphorylated, and uncoupled from its G protein/effector complex.2 8 9 The functional status of GPCR is determined by the phosphorylation state, a dynamic process controlled by protein kinases and phosphatases.10 11 Receptor desensitization occurs after receptor phosphorylation and internalization.10 11 In contrast, receptor resensitization requires receptor dephosphorylation, a process mediated by a plasma membrane–associated form of protein phosphatase, a GPCR phosphatase (GRP).11 12 GPCR resensitization requires protein phosphatase 2A (PP2A) activity because okadaic acid, an inhibitor of PP2A activity, prevents receptor resensitization.13 Moreover, when phosphorylated receptors translocate from plasma membrane to cytoplasmic vesicles, they become physically associated with PP2A.12 14
Because PP2A activity is involved in the
dephosphorylation and resensitization of GPCR, it is
possible that aberrant D1 receptor regulation of
PP2A activity may be involved in the
desensitization of this GPCR in genetic hypertension.
PP2A is a heterotrimeric protein serine/threonine
phosphatase; the holoenzyme consists of a 36-kDa catalytic C subunit, a
65-kDa structural A subunit, and a variable regulatory B
subunit.15 Both the A and C subunits exist as 2 isoforms
( and ß), whereas there are 3 families of B subunits: B or PR55,
B' or B56, and B'' or PR72.16 B56 is a new gene family
that encodes B56, ß, 
, 
, and 
proteins.17
The B56ß and subunits are found predominantly in brain tissue,
the B56 and subunits are most abundant in heart and muscle, and
the subunit is expressed mainly in the testis, lung, and
brain.17 18 19 20 Of the B56 isoforms, B56 appears to be the
most abundantly expressed subunit in the
kidney.17 19 21
PP2A activity and protein levels were examined in
renal cortical tubules from spontaneously hypertensive rats (SHR) and
their normotensive controls, Wistar-Kyoto rats (WKY). To eliminate any
confounding influence of dopamine produced by renal proximal tubules
from circulating L-dihydroxyphenylalanine (L-DOPA), studies
were also performed in immortalized renal proximal tubule cells
(RPTC)22 ; RPTC cannot synthesize dopamine in the absence
of L-DOPA.23 Here we report that
PP2A activity and expression of the
PP2A regulatory subunit
PP2A-B56 were differentially affected by
D1-like receptor stimulation in the kidney of WKY
and SHR.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Materials
Reagents were obtained from the following: monoclonal anti-human PP2A-B56
(Transduction Labs), polyclonal
PP2A A subunits, A, and Aß (Santa Cruz
Biotech Inc); monoclonal anti-human PP2A
catalytic subunit (PP2AC) and Ser/Thr phosphatase
Assay Kit (Upstate Biotech Inc),24 secondary antibodies
for Western blots (Jackson ImmunoReseach Labs Inc), enhanced
chemiluminescence Western blotting detection reagents (Amersham Corp),
fenoldopam (Smith Kline Beecham Pharmaceuticals), and SCH23990
(RBI Inc).
Renal Cortical Membranes
Adult male WKY (body weight 375±43 g, mean arterial
pressure [MAP] 98±3 mm Hg, n=4) and SHR (body weight 307±33
g, MAP 161±3 mm Hg, n=4) (Taconic Farms Inc), maintained on
standard rat chow until the night before the experiment, were
anesthetized with pentobarbital (50 mg/kg body wt IP) and
tracheotomized (PE-240). MAP was monitored with Cardiomax II (Columbus
Instruments). After a 30-minute equilibration period, saline or
fenoldopam in saline (1 µg ·
kg-1 ·
min-1) was infused (1.2
mL/h) into the right renal artery through the right suprarenal artery
for 10 or 30 minutes.25 The renal cortical cytosol and
membrane fractions were prepared in TE buffer (mmol/L, 10 Tris-Cl, pH
7.4, 2 EDTA) and TBST buffer (mmol/L, 10 Tris-Cl, pH 7.4, 150 NaCl, 2
EDTA, 0.5% Triton X-100) as described previously with
modification.26 All buffers contained protease
inhibitors (mmol/L): 5 DTT, 2 EDTA, and 1 pofabloc and 10
µg/mL each of leupeptin and aprotinin.
Preparation of Brush Border Membranes
Brush border membranes (BBM) (100 µg/sample), prepared by
MnCl2 precipitation and differential
centrifugation,27 were treated with
fenoldopam (5 µmol/L) or vehicle (saline) for 30 minutes at room
temperature. The BBM were pelleted by centrifugation
for 5 minutes, suspended in Laemmli buffer, and boiled for 5 minutes
before immunoblotting.
Immortalized RPTC and Subcellular Fractions
Immortalized RPTC were maintained in DMEM/F12 supplemented with
5% fetal bovine serum, epidermal growth factor (10 ng/mL), insulin,
transferrin, and selenium (5 µg/mL each) at 37°C in humidified 5%
CO2/95% air.22 The cells were
incubated for 1 hour in serum-free DMEM/F12 medium before agonist
stimulation with the D1-like agonist fenoldopam
(5 µmol/L). The cells were washed 3 times with TBS and disrupted
by Dounce homogenizer or a syringe with a 27-gauge
needle in TE buffer. The cell lysates were centrifuged at 2000
rpm for 5 minutes to remove the nuclear fraction. The supernatants were
centrifuged at 14 000 rpm for 20 minutes and the resulting
supernatant was taken to represent cytosol. The pellets were
extracted for 30 minutes on ice with TBST lysis to obtain the membrane
fractions.26 All experiments were carried out at 4°C in
the presence of protease inhibitors described above.
Protein concentrations were determined by the Bradford method.
PP2A Activity Assay
PP2A activity was measured after
immunoprecipitation with anti-PP2A catalytic
subunit antibody, according to Gupta et al28 with
modifications. The cytosol and membrane preparations were subjected to
immunoprecipitation carried out in triplicate (see below). The immune
complexes with the protein G beads were initially washed twice with TBS
buffer and finally washed with PP2A assay buffer
(in mmol/L, 20 MOPS, pH 7.5, 60 2-mercaptoethanol, 100 NaCl, 0.1
mg/mL serum albumin). Immunoprecipitates with mouse IgG served
as negative control. The immunoprecipitates were subjected to
PP2A assay with a nonradioactive assay kit. In
brief, the immune complexes containing PP2A were
incubated for 20 minutes at 30°C in 30 µL of assay buffer
containing 200 µmol/L of phosphopeptide (KRpTIRR). The reactions
were terminated by putting the tubes on ice and immediately
centrifuging at 8000 rpm for 1 minute. The supernatants (25 µL/well)
were pipetted into 96-well microtiter plates containing malachite green
solution (100 µL/well). The absorbance was measured at optical
density of 630 nm after 15 minutes of development time.
PP2A specific activity was expressed as pmol/min
per milligram of protein. The lower limit of detection was 20 pmol of
Pi (released phosphate from the phosphopeptide).
Immunoprecipitation and Immunoblotting
The immunoprecipitation for the PP2A assay
was performed by incubation of 50 to 200 µg of protein with 2 µg of
PP2A-C monoclonal antibody and protein G beads in
TBS buffer with rocking for 2 hours in a cold room. For
immunoblotting, 50 to 100 µg of protein was loaded
onto polyacrylamide gel. The amount of protein transferred onto
the nitrocellulose membrane was verified by Ponceau-S stain.
Immunoblotting and quantification of the
immunoblots were performed as described previously with
Quantiscan.26 The results were expressed as density units
(DU).
Statistical Analysis
The data are expressed as mean±SEM. Comparison within
groups was made by ANOVA for repeated measures (or paired t
test when only 2 groups were compared), and comparison among groups (or
t test when only 2 groups were compared) was made by ANOVA
with Scheffe’s or Duncan’s test. Corresponding periods between 2
different groups were analyzed by independent t
test. A value of P<0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
D1-like receptors are expressed to a greater extent in the renal cortex (proximal tubule, distal tubule, cortical collecting) and to a lesser extent in the medulla (medullary thick ascending limb of Henle.3 Therefore, PP2A expression and activity were studied in the renal cortex.
In Vivo Studies
PP2A Expression
Basal Expression
PP2A A, the structural subunit,
PP2A-C, the catalytic subunit, and
PP2A-B56, the regulatory subunit, were
expressed to a similar extent in renal cortical tubules of WKY and SHR
(Figure 1). In agreement with data
obtained with the use of tissues other than the kidney,
PP2A was more abundant in the cytosol than in the
particulate fraction.29 In WKY rats,
PP2A subunits predominantly existed in cytosol
(PP2A-A=86.9±0.3 DU,
PP2A-C=85.2±1.5 DU,
PP2A-B56=88.1±4.0, n=5) and membrane
(PP2A-A=13.8±0.4 DU,
PP2A-C=15.2±3.5 DU,
PP2A-B56=12.2±4.0, n=5). Similar results were
obtained in SHR (data not shown). PP2A-B56
expression was greater in BBM relative to
PP2A-A and PP2A-C.
PP2A-B56 was chosen for study because this
regulatory protein is ubiquitously expressed and exists as heterotrimer
with the PP2A core dimer
PP2A-A and
PP2A-C.30 In addition, antibodies to
PP2A-B56 other than
PP2A-B56 are not commercially available.
PP2A-B55 was not detected in our samples, in
agreement with published reports.31
|
Effect of D1-Like Agonist
A 30-minute intrarenal arterial infusion of the
D1-like agonist fenoldopam (1 µg/kg body wt per
minute) has been shown previously to produce a greater natriuresis in
WKY than in SHR.32 In the current experiments, fenoldopam
infusion did not affect PP2A-B56 expression in
renal cortical homogenates in either WKY or SHR (data not
shown). Fenoldopam increased PP2A-B56
expression in BBM in WKY rats (56.7±3.5 DU versus control, 44.0±3.4
DU) (P<0.05, n=8) and decreased it in SHR (41.5±2.6 versus
control, 58.7±2.7 DU) (P<0.05, n=4) (Figure 2).
|
PP2A Activity
The majority of PP2A are heterotrimers that
contain a variable regulatory B subunit bound to the A-C
dimer.16 33 34 35 To determine specific
PP2A activity, we measured
PP2A activity in immunoprecipitates of the
specific monoclonal anti–PP2A-C subunit
antibody.28 36 The protein phosphatase activity on the
immune complex was predominantly PP2A, because
the A subunit and B56 of PP2A were also
coimmunoprecipitated with anti-C (data not shown), and the activity was
inhibited dose-dependently by a PP2A
inhibitor, okadaic acid (
50%, data not shown).
Basal Activity
Basal PP2A activity (pmol/min per milligram
of protein) in WKY was 323±97 in the cytosol and 252±45 in the
membrane fraction. The corresponding values in SHR were 385±57 and
256±38, respectively.
Effect of D1-Like Agonist
In WKY, the intrarenal arterial infusion of the
D1-like agonist fenoldopam, for 10 or 30 minutes,
produced no significant change in the cytosol. Fenoldopam also did not
change PP2A activity in renal membranes of WKY
rats after 10 minutes but did increase it after 30 minutes (12.5±5.0%
from control) (P<0.05 n=4, ANOVA, Duncan’s test). In
contrast, in SHR, fenoldopam decreased PP2A
activity in both renal cortical tubule cytosol (-17.1±1.7% at 10
minutes, -17.1±4.7% at 30 minutes) (P<0.05 versus
vehicle-treated rats) and renal cortical membrane (-14.8±2.8% at 10
minutes, -16.7±3.6% at 30 minutes, n=4) (P<0.05, ANOVA,
Duncan’s test).
In Vitro Studies
We have reported that D1-like receptor
function in immortalized RPTC is similar to that in renal proximal
tubules freshly obtained from WKY.37 38 The uncoupling of
the D1-like receptor in renal proximal tubules in
SHR persists in immortalized RPTC from SHR.9 37 38 Thus,
D1 receptor protein expression is similar in
immortalized RPTC and renal proximal tubules from WKY and
SHR.37 38 The D1-like receptor,
specifically the D1 receptor subtype, is
uncoupled from G protein subunits and effector proteins in immortalized
RPTC and renal proximal tubules of WKY and SHR.37 38
Immortalized RPTC from WKY and SHR retain characteristics of renal
proximal tubule cells,22 including expression of
-glutamyl transpeptidase, a renal proximal tubule BBM marker. These
cells express the two D1-like receptors,
D1 and D5 receptors, G
proteins, and Na+/H+
exchanger 3 protein to same extent in WKY and SHR.38
PP2A Expression
Basal Expression
As in the renal cortical tubules, in immortalized RPTC of WKY,
PP2A-B56 protein level was greater in cytosol
(WKY, 41.5±3.4 DU, n=4) than in membrane fractions (WKY, 17.1±3.8 DU,
n=4) (Figure 3A) (convert to 100% as the
sum of cytosol and membrane).
|
) and membrane (•) of RPTC from SHR in time-dependent manner; it slightly but significantly increased activity in membrane (
) of RPTC from WKY but did not change in cytosol (
) (n=4). Results are expressed as mean±SEM. #P<0.05, WKY vs SHR; *P<0.05, ANOVA for repeated measures, Scheffé’s test (vs baseline). C, Effect of D1-like antagonist SCH23390 (5 µmol/L) on PP2A activity. Cells were treated with fenoldopam (5 µmol/L) and/or SCH23390 or vehicle (control) for 30 minutes at 37°C; cell preparations were subjected to immunoprecipitation. PP2A activity was measured in immunocomplexes. Fenoldopam increased PP2A activity in membrane of RPTC from WKY and decreased activity in cytosol of RPTC from SHR. Effect of fenoldopam on PP2A activity was blocked by D1-like antagonist SCH23390. Results are expressed as mean±SEM. #P<0.05, *P<0.05, ANOVA for repeated measures, ANOVA, Scheffé’s test.
Effect of D1-Like Agonist
In membranes from immortalized RPTC of WKY, the
D1-like agonist fenoldopam (5 µmol/L)
increased PP2A-B56 protein levels with time
(Figure 3A). In cytosol of immortalized RPTC of WKY, fenoldopam
decreased PP2A-B56 protein levels in a manner reciprocal with that
seen in membranes. The increase in PP2A-B56
expression in the membranes caused by 5 µmol/L fenoldopam
(80±23% over basal, P<0.05 ANOVA, Duncan’s test) was
partially blocked by 5 µmol/L SCH23390 (47±29% over basal,
P>0.05 ANOVA, Duncan’s test), which, by itself, had no
effect (29±23% over basal, P>0.05 ANOVA, Duncan’s test).
In contrast to the WKY rats, fenoldopam produced no change in
PP2A-B56 expression in either membrane or
cytosol in immortalized RPTC of SHR (data not shown).
PP2A Activity
Basal Activity
In the absence of endogenous dopamine, basal
PP2A activity (pmol/min per milligram of protein)
in the membrane was markedly higher in immortalized RPTC of SHR
(540±46, n=3) than in immortalized RPTC of WKY (233±21, n=3)
(P<0.05, t test). However,
PP2A activity in cytosol was not different
between SHR (496±36, n=3) and WKY (473±95, n=4).
Effect of D1-Like Agonist
In immortalized RPTC membranes from WKY, fenoldopam (5
µmol/L) increased PP2A activity (pmol/min per
milligram of protein) with time, peaking at 30 minutes to 278±21
(P<0.05 versus basal activity, n=4); no effect was noted in
cytosol (Figure 3B). In contrast, in the immortalized RPTC from
SHR, fenoldopam decreased PP2A activity in both
cytosol and membrane fractions in a time-dependent manner (Figure 3B), in agreement with studies that used renal cortical tubules.
The maximum decrease occurred at 30 minutes down to 327±63 in membrane
(n=3) and 428±30 in cytosol (n=3) (P<0.05 versus basal
activity).
Effect of D1-Like Antagonist SCH23390
To determine if the effect of the D1 agonist
fenoldopam is mediated by the occupation of the
D1 receptor, studies were performed in the
presence of the D1-like antagonist
SCH23390 (5 µmol/L), a neutral antagonist.
Fenoldopam (5 µmol/L, 30 minutes (Figure 3C) increased
PP2A activity in membrane of WKY (22.6±4.4%
from basal) (P<0.05, n=4), which was blocked by SCH23390
(9.3±3.8% from basal, n=4); SCH23390, by itself (3.9±5.7% from
basal, n=5), had no effect on PP2A activity.
Fenoldopam decreased the activity in cytosol of SHR (-18.0±2.8%
versus basal, n=4, P<0.05), an effect that was blocked by
SCH23390 (-10.4±4.3% from basal, n=4 P>0.05); SCH23390,
by itself, had no effect on PP2A activity
(-8.7±3.6% from basal, n=4 to 5, P>0.05).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Several studies have shown that GRP plays a role in receptor dephosphorylation that is important in receptor resensitization.12 13 14 36 39 40 A high level of receptor phosphatase activity was reported to be associated with sequestered vesicular membranes, capable of dephosphorylating GRK-phosphorylated ß-adrenergic receptors.41 Moreover, the resensitization of GPCR was inhibited by phosphatase inhibitors.12 13 14 36 39 40 GRP has been identified as a member of the family of PP2A.12 PP2A is generally considered as a cytosolic protein.33 However, PP2A is also expressed in cell particulate fractions.12 We also found that PP2A protein level were much higher in the cytosol than in membranes from renal cortex and immortalized RPTC in both WKY and SHR. However, PP2A specific activity was not different in cytosol and membrane fractions.
In addition to GPCR resensitization, PP2A is
involved in a broad range of cellular processes, including signal
transduction, intermediary metabolism, transcriptional
regulation and control of DNA replication, and
mitosis.16 36 39 40 42 43 The diversity of
PP2A function is conferred by a variety of
targeting/regulatory subunits. Each of the B subunits consists of
numerous isoforms and splice variants.16 33 42 The
PP2A regulatory B subunits have been found to
have a number of functions, including targeting to distinct
intracellular locations, determining substrate and tissue specificity,
and acting as receptors of second messengers.17 30 Most of
the B56 family members are phosphoproteins, and different isoforms have
different targeting functions.17 30 The function of
PP2A-B56 in hypertension remains to be
determined. However, the failure of the D1-like
agonist to translocate PP2A activity from cytosol
to membrane in SHR may explain, in part, the
"hyper"-serine-phosphorylated
D1 receptor in renal membranes in genetic
hypertension.8 9 The finding of a difference in
PP2A activity between WKY and SHR in RPTC, where
dopamine is absent, but not in renal cortical tubules, where dopamine
is present, suggests that dopamine may tonically inhibit the
PP2A activity, especially in the cytosol. Because
PP2A-B56 is important in the targeting of the
PP2A holoenzyme, a defect in B56 function
could be involved in the pathogenesis of genetic hypertension. The
important finding in our study is the remarkable difference in
D1-like receptor–associated
PP2A activity in RPTC membranes between WKY and
SHR. The D1-like agonist fenoldopam increased
membranous PP2A activity in WKY but decreased it
in SHR. Fenoldopam translocated PP2A activity
from cytosol to the membrane in WKY but not in SHR. We also found that
the PP2A regulatory subunit B56 exists in the
BBM, where most of the D1 receptors are
located.43 44 It is of interest that
PP2A-B56 protein level decreased in the BBM
from SHR after fenoldopam treatment. The ability of the
D1-like antagonist to block both the
increase in PP2A activity in WKY and the opposite response
in SHR support the notion of an abnormality downstream of the
D1-like receptor. Because there is no mutation of
the D1 receptor in the SHR, this finding suggests
that a primary abnormality of PP2A-B56 may be
a cause of the discrepant response of SHR compared with WKY.
Activation of PP2A necessitates prior phosphorylation of the D1-like receptor.5 6 7 8 41 Presumably, activation of a G-protein–coupled receptor kinase (GRK) must have occurred after D1-like agonist stimulation. The phosphorylation of the D1 receptor by GRK by GRK2, GRK3, and GRK5 has been reported to play a role in the desensitization of the D1 receptor.45 Increased activity and expression of GRK2 has been reported in aortic smooth muscle and lymphocytes from SHR.46 However, these changes occurred as a consequence of the hypertension.46 Because the increased phosphorylation of the D1 receptor in renal proximal tubules is ligand independent,2 3 8 9 38 the participation of a constitutively activated GRK must have occurred. The existence of this GRK was not determined in the current studies, but GRK6 has been reported to be constitutively activated.47 We speculate that a constitutively activated GRK (eg, GRK6) causes basal phosphorylation of D1-like receptors in renal proximal tubules in both WKY and SHR. D1-like agonist occupation further increases the phosphorylation of renal D1-like receptors in WKY but not in SHR.9 PP2A dephosphorylates and resensitizes renal D1-like receptors in WKY but not in SHR, resulting in a phosphorylated and desensitized D1-like receptor in genetic hypertension.
In conclusion, we have demonstrated that after D1-like agonist activation, PP2A activity in renal proximal tubules is increased in WKY but decreased in SHR. We speculate that the increased basal levels of serine-phosphorylated D1 receptor in RPTC in hypertension may be a consequence of a defect in dephosphorylation of the phosphorylated D1 receptor. Although we have demonstrated a role of PP2A in this response, the role of other phosphatases, for example, PP2B, in the desensitization of D1-like receptors in hypertension, remains to be determined.36
|
Acknowledgments |
|---|
This project was supported in part by grants from the National Heart, Lung, and Blood Institute (HL-23081, HL-62211, HL-58536) and National Institute of Diabetes, Digestive, and Kidney Diseases (DK-39308 and DK-52612).
Received June 22, 2000; first decision July 10, 2000; accepted July 17, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Lifton RP. Molecular genetics of human blood pressure variation. Science. 1996;272:676–680.[Abstract]
2. Jose PA, Eisner GM, Felder RA. Dopaminergic mechanisms in the development of hypertension. In: McCarty R, Blizard DA, Chevalier RI, eds. Development of the Hypertensive Phenotype: Basic and Clinical Studies. In: Handbook of Hypertension. Amsterdam, Holland: Elsevier Science; 1999:1–44.
3. Jose PA, Eisner GM, Felder RA. The renal dopamine receptors in health and disease. Pharmacol Ther. 1998;80:149–182.[Medline] [Order article via Infotrieve]
4.
Hegde SS, Jadhav AL, Lokhandwala MF. Role of kidney
dopamine in the natriuretic response to volume expansion in
rats. Hypertension. 1989;13:828–834.
5.
Siragy HM, Felder RA, Howell NL, Chevalier RL, Peach
MJ, Carey RM. Evidence that intrarenal dopamine acts as a paracrine
substance at the renal tubule. Am J Physiol. 1989;257:F469–F477.
6.
Nishi A, Eklöf A-C, Bertorello AM, Aperia A.
Dopamine regulation of renal Na+,
K+-ATPase activity is lacking in Dahl
salt-sensitive rats. Hypertension. 1993;21:767–771.
7.
Hussain T, Lokhandwala MF. Renal dopamine
DA1 receptor coupling with
GS and Gq/11 proteins in
spontaneously hypertensive rats. Am J Physiol. 1997;272:F339–F346.
8.
Sanada H, Jose PA, Hazen-Martin D, Yu P-Y, Xu J, Bruns
JE, Phipps J, Carey RM, Felder RM. Dopamine-1 receptor defect in renal
proximal tubular cells in essential hypertension.
Hypertension. 1999;33:1036–1042.
9. Yu P-Y, Dirami G, Hopfer U, Carey RM, Felder RA, Jose PA. Increased serine-phosphorylation of the D1A receptor in renal proximal tubule cells in spontaneous hypertension. Pediatr Res. 1996;39:372A. Abstract.
10.
Luttrell LM, Ferguson SS, Daaka Y, Miller WE, Maudsley
S, Della Rocca GJ, Lin F, Kawakatsu H, Owada K, Luttrell DK, Caron MG,
Lefkowitz RJ. Beta-arrestin-dependent formation of beta2 adrenergic
receptor-Src protein kinase complexes. Science. 1999;283:655–661.
11. Lefkowitz RJ, Pitcher J, Kruegger K, Daaka Y. Mechanisms of beta-adrenergic receptor desensitization and resensitization. Adv Pharmacol. 1998;42:416–420.
12.
Pitcher JA, Payne ES, Csortos C, Depaoli-Roach AA,
Lefkowitz RJ. The G protein-coupled receptor phosphatase: a protein
phosphatase type 2A with a distinct subcellular distribution and
substrate specificity. Proc Natl Acad Sci U S A. 1995;92:8343–8347.
13. Garland AM, Grady EF, Lovett M, Vigna SR, Frucht MM, Krause JE, Bunnett NW. Mechanisms of desensitization and resensitization of G protein coupled neurokinin 1 and 2 receptors. Mol Pharmacol. 1996;49:438–446.[Abstract]
14.
Krueger KM, Daka Y, Pitcher JA, Lefkowitz RJ. The
role of sequestration in G protein-coupled receptor resensitization.
J Biol Chem. 1997;272:5–8.
15.
Hemmings BA, Adams-Pearson C, Maurer F, Muller P, Goris
J, Merlevede W, Hofsteenge J, Stone SR. - and ß-Forms of
65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid
repeating structure. Biochemistry. 1990;29:3166–3172.[Medline]
[Order article via Infotrieve]
16. Millward TA, Zolnierowicz S, Hemmings BA. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci. 1999;24:186–191.[Medline] [Order article via Infotrieve]
17.
McCright B, Rivers AM, Audlin S, Virshup DM. The B56
family of protein phosphatase 2A (PP2A) regulatory subunits encodes
differentiation-induced phosphoproteins that target PP2A to both
nucleus and cytoplasm. J Biol Chem. 1996;271:22081–22089.
18.
Csortos C, Zolnierowicz S, Bakó E, Durbin SD,
DePaoli-Roach AA. High complexity in the expression of the B' subunit
of protein phosphatase 2A0. J Biol
Chem. 1996;271:2578–2588.
19.
Tehrani MA, Mumby MC, Kamibayashi C. Identification of
a novel protein phosphatase 2A regulatory subunit highly expressed in
muscle. J Biol Chem. 1996;271:5164–5171.
20. Tanabe O, Nagase T, Murakami T, Nozaki H, Usui H, Nishito Y, Hayashi H, Kagamiyama H, Takeda M. Molecular cloning of a 74-kDa regulatory subunit (B'' or delta) of human protein phosphatase 2A. FEBS Lett. 1996;379:107–111.[Medline] [Order article via Infotrieve]
21. Zolnierowicz S, Csortos C, Bondor J, Verin A, Mumby MC, DePaoli-Roach AA. Diversity in the regulatory B-subunits of protein phosphatase 2A: identification of a novel isoform highly expressed in brain. Biochemistry. 1994;33:11858–11867.[Medline] [Order article via Infotrieve]
22. Woost PG, Orosz DE, Jin W, Frisa PS, Jacobberger JW, Douglas JG, Hopfer U. Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats. Kidney Int. 1996;50:125–134.[Medline] [Order article via Infotrieve]
23.
Soares-da-Silva P. Source and handling of renal
dopamine: its physiological importance. News
Physiol Sci. 1994;9:128–134.
24. Kremmer E, Ohst K, Kiefer J, Brewis N, Walter G. Separation of PP2A core enzyme and holoenzyme with monoclonal antibodies against the regulatory A subunit: abundant expression of both forms in cells. Mol Cell Biol. 1997;17:1692–1701.[Abstract]
25.
Eisner GM, Yamaguchi I, Felder RA, Asico LD, Jose PA.
Role of renal dopamine D1 receptors in
natriuresis induced by calcium channel blockers. Am J
Physiol. 1994;267:F965–F970.
26. Yu P-Y, Asico LD, Eisner GM, Jose PA. Differential regulation of renal phospholipase C isoforms by catecholamines. J Clin Invest. 1995;95:304–308.
27. Albrecht FE, Drago J, Felder RA, Printz MP, Eisner GM, Robillard JE, Sibley DR, Westphal HJ, Jose PA. Role of the D1A dopamine receptor in the pathogenesis of genetic hypertension. J Clin Invest. 1996;97:2283–2288.[Medline] [Order article via Infotrieve]
28. Gupta V, Ogawa AK, Du X, Houk KN, Armstrong RW. A model for binding of structurally diverse natural product inhibitors of protein phosphatases PP1 and PP2A. J Med Chem. 1997;40:3199–3206.[Medline] [Order article via Infotrieve]
29. Strack S, Zaucha JA, Ebner FF, Colbran RJ, Wadzinski BE. Brain protein phosphatase 2A: developmental regulation and distinct cellular and subcellular localization by B subunits. J Comp Neurol. 1998;392:515–527.[Medline] [Order article via Infotrieve]
30.
McCright B, Virshup DM. Identification of a new family
of protein phosphatase 2A regulatory subunits. J Biol
Chem. 1995;270:26123–26128.
31.
Everett AD, Xue C, Stoops T. Developmental expression
of protein phosphatase 2A in the kidney. J Am Soc
Nephrol. 1999;10:1737–1745.
32.
Yao P, Yu P-Y, Xu J, Asico LD, Jose PA. Dopamine
D1 receptor and protein kinase C isoforms in
spontaneously hypertensive rats. Hypertension. 1998;32:1049–1053.
33. Goldberg Y. Protein phosphatase 2A Who shall regulate the regulator? Biochem Pharmacol. 1999;57:321–328.[Medline] [Order article via Infotrieve]
34. Zolnierowicz S. Type 2A protein phosphatase, the complex regulator of numerous signaling pathways. Biochem Pharmacol.. 2000;60:1225–1235.[Medline] [Order article via Infotrieve]
35. Ruediger R, Brewis N, Ohst K, Walter G. Increasing the ratio of PP2A core enzyme to holoenzyme inhibits Tat-stimulated HIV-1 transcription and virus production. Virology. 1997;238:432–443.[Medline] [Order article via Infotrieve]
36.
Shih M, Lin F, Scott JD, Wang HY, Malbon CC. Dynamic
complexes of beta2-adrenergic receptors with protein kinases and
phosphatases and the role of gravin. J Biol Chem. 1999;274:1588–1595.
37.
Felder RA, Kinoshita S, Ohbu K, Mouradian MM, Sibley
DR, Monsma FJ, Minowa T, Minowa MT, Canessa LM, Jose PA. Organ
specificity of the dopamine-1 receptor/adenylyl cyclase coupling defect
in the spontaneously hypertensive rat. Am J Physiol. 1993;264:R726–R732.
38.
Xu J, Li XX, Albrect FE, Hopfer U, Carey RM, Jose PA.
Dopamine1 receptor, Gs,
and Na+/H+ exchanger interactions in the kidney
in hypertension. Hypertension.. 2000;36:395–399.
39. Ferguson SS, Barak LS, Zhang J, Caron MG. G-protein-coupled receptor regulation: role of G-protein coupled receptor kinases and arrestins. Can J Physiol Pharmacol. 1996;74:1095–1110.[Medline] [Order article via Infotrieve]
40. Krupnick JG, Benovic JL. The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annu Rev Pharmacol Toxicol. 1998;38:289–319.[Medline] [Order article via Infotrieve]
41.
Sibley DR, Strasser RH, Benovic JL, Daniel K, Lefkowitz
RJ. Phosphorylation/dephosphorylation
of the beta-adrenergic receptor regulates its functional coupling to
adenylate cyclase and subcellular distribution. Proc
Natl Acad Sci U S A. 1986;83:9408–9412.
42. Wera S, Hemmings BA. Serine/threonine protein phosphatases. Biochem J. 1995;311:17–29.
43.
O’Connell DP, Botkin SJ, Ramos SI, Sibley DR, Ariano
MA, Felder RA, Carey RM. Localization of dopamine
D1A receptor protein in rat kidneys.
Am J Physiol. 1995;268:F1185–F1197.
44. O’Connell DP, Aherne AM, Lane E, Felder RA, Carey RM. Detection of dopamine receptor D1A subtype specific mRNA in rat kidney by in situ amplification. Am J Physiol. 1998;274:F232–F241.
45.
Tiberi M, Nash SR, Bertrand L, Lefkowitz RJ, Caron MG.
Differential regulation of dopamine D1A receptor responsiveness by
various G protein-coupled receptor kinases. J Biol
Chem. 1996;271:3771–8377.
46.
Gros R, Chorazyczewski J, Meek MD, Benovic JL, Ferguson
SS, Feldman RD. G-protein-coupled receptor kinase activity in
hypertension. Hypertension. 2000;35:38–42.
47.
Hall RA, Spurney RF, Premont RT, Rahman N, Blitzer JT,
Pitcher JA, Lefkowitz RJ. G protein-coupled receptor kinase 6A
phosphorylates the
Na+/H+ exchanger regulatory
factor via a PDZ domain-mediated interaction. J Biol
Chem. 1999;274:24328–24334.
This article has been cited by other articles:
 |
|
 |
|
  C. Zeng, V. A. M. Villar, G. M. Eisner, S. M. Williams, R. A. Felder, and P. A. Jose G Protein-Coupled Receptor Kinase 4: Role in Blood Pressure Regulation Hypertension, June 1, 2008; 51(6): 1449 - 1455. [Full Text] [PDF]
|
|
|
|
|
|
C. Zeng, U. Hopfer, L. D. Asico, G. M. Eisner, R. A. Felder, and P. A. Jose Altered AT1 Receptor Regulation of ETB Receptors in Renal Proximal Tubule Cells of Spontaneously Hypertensive Rats Hypertension, October 1, 2005; 46(4): 926 - 931. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zeng, Z. Wang, U. Hopfer, L. D. Asico, G. M. Eisner, R. A. Felder, and P. A. Jose Rat Strain Effects of AT1 Receptor Activation on D1 Dopamine Receptors in Immortalized Renal Proximal Tubule Cells Hypertension, October 1, 2005; 46(4): 799 - 805. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zeng, Z. Yang, Z. Wang, J. Jones, X. Wang, J. Altea, A. J. Mangrum, U. Hopfer, D. R. Sibley, G. M. Eisner, et al. Interaction of Angiotensin II Type 1 and D5 Dopamine Receptors in Renal Proximal Tubule Cells Hypertension, April 1, 2005; 45(4): 804 - 810. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Yang, L. D. Asico, P. Yu, Z. Wang, J. E. Jones, R.-k. Bai, D. R. Sibley, R. A. Felder, and P. A. Jose D5 dopamine receptor regulation of phospholipase D Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H55 - H61. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Zeng, H. Sanada, H. Watanabe, G. M. Eisner, R. A. Felder, and P. A. Jose Functional genomics of the dopaminergic system in hypertension Physiol Genomics, November 17, 2004; 19(3): 233 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zeng, Y. Luo, L. D. Asico, U. Hopfer, G. M. Eisner, R. A. Felder, and P. A. Jose Perturbation of D1 Dopamine and AT1 Receptor Interaction in Spontaneously Hypertensive Rats Hypertension, October 1, 2003; 42(4): 787 - 792. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Kacimi and A. M. Gerdes Alterations in G Protein and MAP Kinase Signaling Pathways During Cardiac Remodeling in Hypertension and Heart Failure Hypertension, April 1, 2003; 41(4): 968 - 977. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zeng, L. D. Asico, X. Wang, U. Hopfer, G. M. Eisner, R. A. Felder, and P. A. Jose Angiotensin II Regulation of AT1 and D3 Dopamine Receptors in Renal Proximal Tubule Cells of SHR Hypertension, March 1, 2003; 41(3): 724 - 729. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hussain and M. F. Lokhandwala Renal Dopamine Receptors and Hypertension Experimental Biology and Medicine, February 1, 2003; 228(2): 134 - 142. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Efendiev, G. A. Yudowski, J. Zwiller, B. Leibiger, A. I. Katz, P.-O. Berggren, C. H. Pedemonte, I. B. Leibiger, and A. M. Bertorello Relevance of Dopamine Signals Anchoring Dynamin-2 to the Plasma Membrane during Na+,K+-ATPase Endocytosis J. Biol. Chem., November 8, 2002; 277(46): 44108 - 44114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Carey Renal Dopamine System: Paracrine Regulator of Sodium Homeostasis and Blood Pressure Hypertension, September 1, 2001; 38(3): 297 - 302. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||