Role of cAMP-PKA-PLC signaling cascade on
dopamine-induced PKC-mediated inhibition of renal
Na+-K+-ATPase activity
Pedro
Gomes and
P.
Soares-da-Silva
Institute of Pharmacology and Therapeutics, Faculty of Medicine,
4200 Porto, Portugal
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ABSTRACT |
We studied the molecular events set into
motion by stimulation of D1-like receptors downstream of
Na+-K+-ATPase, while measuring apical-to-basal
ouabain-sensitive, amphotericin B-induced increases in short-circuit
current in opossum kidney (OK) cells. The D1-like receptor
agonist SKF-38393 decreased Na+-K+-ATPase
activity (IC50, 130 nM). This effect was prevented by the D1-like receptor antagonist SKF-83566, overnight
cholera toxin treatment, the protein kinase A (PKA) antagonist H-89, or
the PKC antagonist chelerythrine, but not the mitogen-activated PK inhibitor PD-098059 or phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002. Dibutyryl cAMP (DBcAMP) and phorbol 12,13-dibutyrate (PDBu) both effectively reduced
Na+-K+-ATPase activity. PKA downregulation
abolished the inhibitory effects of SKF-38393 and DBcAMP but not those
of PDBu. PKC downregulation abolished inhibition by PDBu, SKF-38393,
and DBcAMP. The phospholipase C (PLC) inhibitor U-73122 prevented
inhibition by SKF-38393 and DBcAMP. However, DBcAMP increased PLC
activity. Although OK cells express both Gs
and
Gq/11
proteins, D1-like receptors are
coupled to Gs
proteins only, as evidenced by studies in
cells treated overnight with specific antibodies raised against
Gs
and Gq/11
proteins. We conclude that
PLC and Na+-K+-ATPase are effector proteins for
PKA and PKC, respectively, after stimulation of D1-like
receptors coupled to Gs
proteins, in a sequence of
events that begins with adenylyl cyclase-PKA system activation followed
by PLC-PKC system activation.
D1-like receptors; second messengers; opossum kidney
cells; protein kinase A; protein kinase C; phospholipase C; adenosine
3',5'-cyclic monophosphate; sodium-potassium
adenosinephosphatase
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INTRODUCTION |
REGULATION OF
NA+ transport across the proximal tubules occurs
through the involvement of two key proteins: the
Na+/H+ exchanger, located on the brush-border
membrane, and the Na+-K+-ATPase, located on the
basolateral membrane. These proteins have been identified as targets
for the action of dopamine, a mechanism by which the amine affects
tubular Na+ absorption and produces natriuresis
(32). The process from the activation of
D1-like dopamine receptors to the inhibition of
Na+-K+-ATPase and
Na+/H+ exchanger activity involves multiple
intracellular signaling pathways that appear to be complex and, despite
a great deal of study, are yet to be completely understood (1,
30, 31). There is evidence that signaling pathways linked to
D1-like receptors include the adenylyl cyclase-protein
kinase A (AC-PKA) and the phospholipase C (PLC)-PKC systems, through
the coupling of Gs
and Gq/11
proteins,
respectively (30, 31). The role of the PLA2-arachidonic acid-20-hydroxyeicosatetraenoic acid
(20-HETE) pathway has also been reported (14, 41) in the
cellular signaling systems involved in D1-like
receptor-mediated inhibition of Na+-K+-ATPase
activity in the proximal tubules. The proposed pathway is a
PLC/PKC-dependent mechanism, in which PKC leads to the activation of
PLA2, followed by the generation of 20-HETE, which in turn inhibits Na+-K+-ATPase activity via a
PKC-dependent pathway.
Recently, however, a considerable body of new experimental data and
alternative explanations arguing against the involvement of the AC-PKA
system on the renal effects of dopamine have been reported (for
reviews, see Refs. 22, 53). First, the
majority of the studies found a stimulatory, rather than inhibitory,
effect of cAMP on proximal tubule Na+-K+-ATPase
activity (see Refs. 22, 53). Second, the
cAMP-dependent dopamine-induced inhibition of
Na+-K+-ATPase activity in intact renal tubular
epithelial cells has been suggested to result from a decrease in apical
Na+ entry secondary to cAMP-dependent dopamine-induced
inhibition of the Na+/H+ exchanger (19,
25). Third, phosphorylation of the
Na+-K+-ATPase
-subunit by PKA is usually
detected in conditions in which there might be some denaturated enzyme
(10), but since the PKA phosphorylation site in
Na+-K+-ATPase (Ser-943) is so close to the
plasma membrane, it is unlikely to be accessible to PKA in this
conformation (52). It has also been suggested
(11) that phosphorylation of the rat
Na+-K+-ATPase at Ser-23 by PKC appears to
require prior phosphorylation at the PKA site, Ser-943. Discrepancies
concerning the involvement of PLC-PKC systems in dopamine-induced
inhibition of Na+-K+-ATPase activity have also
been reported (for reviews, see Refs. 22,
53). The inhibitory effects of dopamine and phorbol esters on Na+-K+-ATPase activity were reported
(12, 13, 42, 46) to occur through one of two mechanisms.
The first involves direct phosphorylation of the Na+ pump
at Ser-23 of the
-subunit, leading to endocytosis of pumps (12, 13). The second mechanism involves activation of
PLA2 and arachidonic acid metabolism (42, 46),
but this was found not to be the case in well-oxygenated renal tubules
(21). This led to the suggestion that phorbol
esters inhibit proximal tubule Na+-K+-ATPase
activity as a result of poor metabolic status that triggers cell-protective mechanisms (22, 53). Recently, a
correlation has been reported (34) between PKA-dependent
phosphorylation of the Na+ pump and activation of
ouabain-sensitive Rb+ uptake and
Na+-K+-ATPase activity in oxygenated, but not
hypoxic, conditions. In addition, similar to PKA, PKC's effects on
Na+-K+-ATPase activity are dependent on
Ca2+ concentration (10). On the other hand,
PKC-dependent activation of the
Na+-K+-ATPase in the proximal nephron appears
to be secondary to an increase in Na+ influx possibly via
the Na+/H+ exchanger (6), and the
activation seems to be an oxygen-dependent process (21).
Although direct phosphorylation of the
Na+-K+-ATPase by PKA and/or PKC is an
attractive and simple mechanism, the regulation of the Na+
pump is still a controversial issue. It is apparent, however, in a
review of the literature (22, 53) that some of the
difficulties arise because some of the experimental models used may not
represent the ideal conditions for the assay of
Na+-K+-ATPase activity (i.e., hypoxic
conditions, low Ca2+, changes in Na+ influx via
the Na+/H+ exchanger).
The present study investigated the molecular events set into motion by
stimulation of D1-like receptors downstream of
Na+-K+-ATPase in polarized opossum kidney (OK)
cells, while using an electrophysiological model that requires
continuous oxygenation of the medium in contact with both the apical
and basolateral cell sides. The OK cell line is frequently used as a
model of the tubular proximal epithelium and expresses characteristics useful for the study of the renal dopaminergic system (3, 4, 13,
26-28, 40, 43, 55, 56). The results reported here indicate
that inhibition of Na+-K+-ATPase activity after
stimulation of D1-like receptors involves both the AC-PKA
and the PLC-PKC systems. D1-like receptors in OK cells are
coupled to Gs
but not Gq/11
proteins, and
the chain of molecular events begins with activation of the AC-PKA
system followed by activation of the PLC-PKC system, PLC being an
effector protein for PKA.
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METHODS |
Cell culture.
OK cells, an established cell line derived from the kidney of a female
American opossum, were obtained from the American Type Culture
Collection (ATCC 1840 CRL; Rockville, MD) and maintained in a
humidified atmosphere of 5% CO2-95% air at 37°C. OK
cells were grown in MEM (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Sigma), 100 U/ml penicillin G, 0.25 µg/ml amphotericin B, 100 µg/ml streptomycin (Sigma), and 25 mM HEPES (Sigma). For subculturing, the cells were dissociated with 0.05% trypsin-EDTA (Sigma), split 1:5, and subcultured in petri dishes with a
21-cm2 growth area or six-well culture clusters (Costar,
Badhoevedorp, The Netherlands). For electrophysiological studies, the
cells were seeded onto polycarbonate filter supports (Snapwell, Costar) at a density of 13,000 cells/well. The cell medium was changed every 2 days, and the cells reached confluence after 3-5 days of initial
seeding. For 24 h before each experiment, the cell medium was free
of fetal bovine serum. Experiments were generally performed 2 days
after cells reached confluence and 4 days after initial seeding, and
each square centimeter contained ~100 µg of cell protein. In some
experiments, cells were treated overnight on the apical cell side with
agents known to interfere with signal transducing pathways, namely G
proteins, such as cholera toxin (Sigma) and specific antibodies raised
against Gs
or Gq/11
proteins (Calbiochem,
San Diego, CA). To minimize difficulties in antibodies entering the
cell, anti-Gs
and anti-Gq/11
antibodies
(1:500) were prepared in the presence of lipofectin (1%, vol/vol;
GIBCO-BRL, Grand Island, NY) and fetal bovine serum-free culture
medium. On the day of the experiment, culture medium containing the
test agents was removed, and the cells were washed with fresh medium and allowed to stabilize for at least 2 h before the start of acquisition of the electrophysiological parameters.
Electrogenic ion transport in OK cells.
All transport experiments were conducted under short-circuit
conditions. OK cells grown on polycarbonate filters (Snapwell, Costar)
were mounted in Ussing chambers (window area, 1 cm2)
equipped with water-jacketed gas lifts bathed on both sides with 10 ml
of Krebs-Hensleit solution, gassed with 95% O2 and 5%
CO2, and maintained at 37°C. The standard composition of
the apical and basolateral bathing Krebs-Hensleit solution was (in mM)
118 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 KH2PO4, 2.5 CaCl2, and 1.2 MgSO4; pH was adjusted to 7.4 after gassing with 5%
CO2 and 95% O2. The apical bathing
Krebs-Hensleit solution contained mannitol (10 mM) instead of glucose
(10 mM) to avoid entry of apical Na+ through the
Na+-dependent glucose transporter. The experimental design
also required modification of the bathing solution compositions for
specific experiments, and these changes are indicated below. After
5-min stabilization, monolayers were continuously voltage clamped to zero potential differences by application of external current, with
compensation for fluid resistance, by means of an automatic voltage
current clamp (DVC 1000, World Precision Instruments, Sarasota, FL).
Transepithelial resistance (
· cm2) was
determined by altering the membrane potential stepwise (±3 mV) and
applying the ohmic relationship. Cells were allowed to stabilize for 25 min before permeabilization with amphotericin B; this period was also
used for exposure of cells to the relevant drug treatments. The
voltage/current clamp unit was connected to a personal computer via a
BIOPAC MP1000 data-acquisition system (Goleta, CA). Data analysis was
performed using AcqKnowledge 2.0 software (BIOPAC Systems).
Na+-K+-ATPase
activity.
The effect of dopamine and D1-like receptor agonists on
Na+-K+-ATPase activity was examined in
monolayers mounted in Ussing chambers bathed with the standard
Krebs-Hensleit solution, so that the final bath Na+
concentration was 143 mM on both sides of the monolayers. The apical
membrane was then permeabilized by addition of amphotericin B to the
apical bathing solution. Under short-circuit conditions, the resulting
current is due to the transport of Na+ across the
basolateral membrane by the Na+-K+-ATPase
(15, 27, 55). This experimental model allows the entry of
apical Na+ and leads to inhibition of the
Na+/H+ exchanger (27). The
concentration-response relationship of the short-circuit current
(Isc) for bath Na+ was evaluated by
initially bathing the apical side of the monolayers mounted in Ussing
chambers with Na+-free Krebs-Hensleit solution (NaCl
replaced with choline chloride and NaHCO3 replaced with
choline bicarbonate). Amphotericin B was then administered to the
apical bathing solution, and Isc was
continuously recorded. Thereafter, the Na+ concentration
was incrementally increased by removing bathing medium from the apical
side of the monolayers and replacing it with equal volumes of normal
Krebs-Hensleit solution. Thus bath Na+ concentration was
gradually increased from 0 to 143 mM without affecting the
concentrations of other ions. In some experiments, amphotericin B was
applied from the basal cell side. All test drugs were applied to both
the apical and basolateral cell sides, with the following exceptions:
amiloride (apical only), ouabain (basolateral only), DIDS (basolateral
only), barium chloride (basolateral only), and SKF-83566 (apical only).
SKF-38393 in some experiments (indicated in text) was applied to the
basolateral cell side; in all remaining experiments, SKF-38393 was
applied to the apical cell side only.
cAMP measurement.
Intracellular cAMP was determined with an enzyme immunoassay kit (Assay
Designs, Ann Arbor, MI), as previously described (27). OK
cells were preincubated for 15 min at 37°C in Hanks' medium (medium
composition in mM: 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4,
0.25 CaCl2, 1 MgCl2, 0.15 Tris · HCl,
and 1 sodium butyrate, pH 7.4), containing 100 µM IBMX, a
phosphodiesterase inhibitor. Cells were then incubated for 15 min with
test compounds. In some experiments, cells were treated overnight from
the apical cell side in the presence of anti-Gs
and
anti-Gq/11
antibodies (1:500) prepared in the presence
of lipofectin (1%, vol/vol), as described in Cell culture.
At the end of the experiment, the reaction was stopped on ice, the
incubation medium was discarded, and the cell monolayer was added with
0.1 M HCl. Aliquots were then taken for the measurement of
intracellular cAMP content.
PLC activity.
OK cells grown in six-well culture clusters were incubated for 15 min
at 37°C with test compounds in Hanks' medium. Washing the cells
three times with ice-cold Hanks' medium terminated the incubations.
Subsequently, the cells were lysed by adding lysis buffer containing
(in mM) 20 Tris · HCl, pH 7.4, 2 EDTA, 2 phenylmethylsulfonyl fluoride (PMSF), 25 sodium pyrophosphate, and 20 sodium fluoride and 10 µg/ml each leupeptin and aprotinin. Thereafter, the cells were
centrifuged at 4,000 rpm for 20 min at 4°C, and the cytosol and
membrane fractions were separated for the assay of PLC activity. The
cytosol and membranes were assayed for PLC activity using the Amplex
Red phosphatidylcholine-specific PLC assay kit (Molecular Probes,
Eugene, OR), using a Spectramax Gemini dual-scanning fluorescence microplate reader (Molecular Devices). Briefly, PLC was monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent), a sensitive fluorogenic probe for
H2O2. Assays were performed in 96-well plates,
with 200 µl reaction volume. First, PLC converts the
phosphatidylcholine (lecithin) substrate to form phosphocholine and
diacylglycerol. After the action of alkaline phosphatase, which
hydrolyzes phosphocholine, choline is oxidized by choline oxidase to
betaine and H2O2. Finally,
H2O2, in the presence of horseradish
peroxidase, reacts with Amplex Red reagent in a 1:1 stoichiometry, to
generate the highly fluorescent product resorufin. PLC activity was
expressed as relative fluorescence units per milligram of protein.
Intracellular Ca2+
measurement.
Intracellular Ca2+ was measured as previously described
(27). At day 4 after seeding, the glass
coverslips were incubated at 37°C for 40 min with 5 µM of the
Ca2+-dependent fluorescent indicator fura-2. Coverslips
were then washed twice with prewarmed dye-free modified Krebs buffer
[buffer composition in mM: 140 NaCl, 5.4 KCl, 2.8 CaCl2,
1.2 MgSO4, 0.3 NaH2PO4, 0.3 KH2PO4, 10 HEPES, and 5 glucose (pH to 7.4 with
Tris base)] before initiation of the fluorescence recordings. Cells were mounted diagonally in a 1 × 1-cm acrylic fluorometric
cuvette and placed in the sample compartment of a FluoroMax-2
spectrofluorometer (Jobin Yvon-SPEX, Edison, NJ). The cuvette volume of
3 ml was constantly stirred and perfused at 5 ml/min with modified
Krebs buffer prewarmed to 37°C. Under these conditions, the cuvette medium was replaced within ~150 s. After 5 min, fluorescence was measured every 5 s alternating between 340- and 380-nm excitation (slit size, 2 nm) at 510-nm emission (slit size, 5 nm). The ratio of
intracellular fura 2 fluorescence at 340 and 380 nm was an index of
intracellular Ca2+.
Western blotting.
Membranes were prepared from OK cells and the kidney outer cortex of
male Wistar rats (Harlan). Both samples were added to 25 mM HEPES, pH
7.4, containing 1 mM EDTA, 1 mM dithiothreitol, and 0.2 mM PMSF,
homogenized (Diax homogeneizer, Heidolph) and centrifuged for 10 min at
500 g. The supernatant was then centrifuged at 50,000 g for 20 min, and the membrane pellet was resuspended in
HEPES buffer. Membrane proteins (30-35 µg) were solubilized in
Laemmli buffer and resolved by SDS-PAGE (10% acrylamide) together with
the molecular weight marker BenchMark prestained protein ladder (Life
Technologies). The resolved proteins were electrophoretically transferred onto nitrocellulose membrane (Hybond-C, Amersham Pharmacia Biotech). The blots were blocked with 5% skim milk in
Tween-Tris-buffered saline (0.1% Tween 20, 154 mM NaCl, and 100 mM
Tris, pH 7.5) containing 0.02% NaN3. The blots were
incubated with anti-Gs
subunit, COOH-terminal or
anti-Gq/11
subunit, COOH-terminal antibodies (1:1,000
dilution, Calbiochem) for 1 h at room temperature. The antibodies
bound to nitrocellulose were detected by incubation with a 1:1,000
dilution of an anti-rabbit IgG-alkaline phosphatase antibody (Roche,
Basel, Switzerland) and then incubation in
2-(4-iodophenyl)-3(-4-nitrophenyl)-5-phenyltetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate substrate solution (Roche).
Downregulation studies.
PKA and PKC downregulation was performed by overnight exposure to cAMP
(100 µM) or phorbol 12,13-dibutyrate (PDBu; 100 nM), respectively, as
previously described (49, 54).
Protein assay.
The protein content of monolayers of OK cells was determined by the
Bradford method (8), with human serum albumin as a standard.
Data analysis.
Arithmetic means are given with SE or geometric means with 95%
confidence values. Statistical analysis was done with one-way ANOVA
followed by a Newman-Keuls test for multiple comparisons. P < 0.05 was assumed to denote significant difference.
Drugs.
Amphotericin B, arachidonic acid, chelerythrine chloride, cholera
toxin, dibutyryl cAMP (DBcAMP), DIDS, ethoxyresorufin, forskolin, H-89, IBMX, okadaic acid, ouabain, PDBu, 4
-phorbol
12,13-didecanoate, trypan blue, and U-73122 LY-294002, PD-098059, and
wortmannin were purchased from Sigma. (±)-SKF-83566
hydrochloride and (±)-SKF-38393 hydrochloride were obtained from
Research Biochemicals (Natick, MA). Fura 2 was obtained from Molecular Probes.
 |
RESULTS |
Under conditions of 143 mM Na+ in the extracellular
medium, the addition of amphotericin B to the apical cell side induced an increase in Isc; this effect was dependent on
the concentration used (Fig.
1A and Table
1). The maximum effect was attained at 3 µg/ml amphotericin B; the effect of 5 µg/ml amphotericin B (data not shown) was similar to that obtained at a lower concentration (3 µg/ml). Thus, in all subsequent experiments, the apical membrane was
permeabilized with 1 µg/ml amphotericin B, to increase the Na+ delivered to Na+-K+-ATPase to
the half-maximal saturating level. Under these conditions, the
amphotericin B (1 µg/ml)-induced increase in
Isc was markedly (P < 0.05)
attenuated (92% reduction) by removing Na+ from the
solution bathing the apical cell border (Fig. 1B).
Similarly, removal of K+ from the solution bathing the
basolateral cell side (substitution by cesium chloride) markedly
attenuated (87% reduction) the amphotericin B (1 µg/ml)-induced
increase in Isc (Fig. 1B). As
shown in Fig. 1B, the increase in Isc
induced by amphotericin B applied to the apical cell side was not
affected by the K+ channel blocker barium chloride (1 mM)
and the Na+-HCO
cotransport inhibitor
DIDS (200 µM). In additional experiments aimed at determining the
cell border on which the driving force that generates the amphotericin
B-induced current is localized, the ionophore was applied to either the apical or basolateral cell side. As shown in Fig.
2, the increase in
Isc observed with the addition of amphotericin B
to the apical cell side was 6.6-fold that observed when amphotericin B
was applied to the basolateral cell side. The increase in
Isc elicited by apical amphotericin B was
markedly (P < 0.05) attenuated (78% reduction) by
ouabain applied to the basolateral cell side but insensitive to
amiloride applied to the apical cell side (Fig. 2A).
In contrast, the increase in Isc elicited by
basolateral amphotericin B was significantly (P < 0.05) attenuated (57% reduction) by amiloride applied to the apical
cell side but insensitive to ouabain applied to the basolateral cell
side (Fig. 2A). Taken together, these results
suggest that increases in Isc elicited by apical
amphotericin B reflect increases in the activity of Na+-K+-ATPase located in the basolateral
membrane.

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Fig. 1.
A: representative traces of the effect of
amphotericin B on short-circuit current (Isc; in
µA/cm2) in opossum kidney (OK) cells. Increasing
concentrations of amphotericin B (0.1-3 µg/ml) were applied to
the apical cell border. Traces represent means of 2-10
experiments/group. B: effect of removal of Na+
or K+ from the solution bathing the apical or basolateral
cell side, respectively, and effects of barium chloride (1 mM) and DIDS
(200 µM) on changes ( ) in Isc
(given as %control) induced by amphotericin B (1 µg/ml) applied to
the apical cell side. Values are means ± SE of 3-9
experiments/group. * P < 0.05.
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Fig. 2.
Isc changes (in
µA/cm2) in monolayers of OK cells mounted in Ussing
chambers treated with amphotericin B (1 µg/ml) applied to the apical
(A) or basolateral cell side (B) alone (control)
and amiloride (1 mM) or ouabain (100 µM). Values are means ± SE
of 3-9 experiments/group. * P < 0.05, significantly different from corresponding control value.
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Pretreatment of OK cell monolayers with dopamine (1 µM) applied to
the basolateral cell border failed to affect the amphotericin B-induced
increase in Isc (Table
2). In contrast, when dopamine (1 µM)
was applied to the apical cell border, a significant decrease (29 ± 5% reduction) in the amphotericin B-induced increase in Isc was observed, this being prevented by the
D1-like receptor antagonist SKF-83566 (1 µM) (Table 2).
The selective D1-like receptor agonist SKF-38393 (30 to
1,000 nM; apical application) was also found to attenuate, in a
concentration-dependent manner, the amphotericin B-induced increase in
Isc (Fig. 3). The
relationship between the amphotericin B-induced increase in
Isc and the concentration of extracellular
Na+ showed a Michaelis-Menten constant
(Km) of 37.6 ± 10.2 mM and a
Vmax of 49.0 ± 3.5 µA/cm2 in
control monolayers. However, in the presence of SKF-38393 (1 µM),
Vmax was significantly reduced to 31.4 ± 1.4 µA/cm2 without changes in Km
values (39.2 ± 5.0 mM) (Fig. 4).
Altogether, these results suggest that stimulation of
D1-like receptors does not alter the affinity of
Na+-K+-ATPase for Na+; instead, the
stimulation reduces the rate at which Na+ pump units
extruded intracellular Na+. The apparent affinity
of Na+-K+-ATPase for Na+ in OK
cells was in the same range of magnitude as that described previously
in colonic epithelial cells (20 mM Na+) using an identical
electrophysiological methodology (15).

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Fig. 3.
Changes in Isc (given as
%control) induced by amphotericin B (1 µg/ml) alone (control) and
increasing concentrations of SKF-38393 (30-1,000 nM; apical
application) in monolayers of OK cells mounted in Ussing chambers.
Values are means ± SE of 3-9 experiments/group.
* P < 0.05, significantly different from control
(CT) value.
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Fig. 4.
Effect of SKF-38393 (300 nM; apical application) or
vehicle on changes in Isc (in
µA/cm2) induced by amphotericin B (1 µg/ml) in the
presence of increasing Na+ concentrations. Values are
means ± SE of 2-11 experiments/group. * P < 0.05, significantly different from control value.
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Next, we evaluated the involvement of G proteins in the regulation of
Na+-K+-ATPase in OK cells. Cholera toxin was
used to maximally activate the GTP-binding protein (G protein)
Gs
and uncouple the D1-like receptor from
Gs
(37). Overnight treatment of OK cells
with cholera toxin (500 ng/ml) abolished the effect of SKF-38393 (300 nM) on the amphotericin B-induced increase in
Isc (Table 2). This was accompanied by increases
in both the basal levels of cAMP and the forskolin (3 µM)-stimulated
accumulation of cAMP (Table 3). On the
other hand, SKF-38393 (300 nM) stimulated cAMP production, this being
prevented by the specific D1-like receptor antagonist
SKF-83566 (1 µM) (Table 3).
Previous studies (32) have demonstrated that second
messenger pathways thought to be involved in D1-like
receptor-mediated inhibition of Na+-K+-ATPase
include stimulation of PKA or PKC pathways. To evaluate whether this
was the case in OK cells using electrophysiological techniques under in
vivo experimental conditions, we examined the effects of DBcAMP, a
direct activator of PKA, and PDBu, a potent activator of PKC. Treatment
of OK cells with increasing concentrations of DBcAMP (100-500
µM) (Fig. 5A) and PDBu
(10-1,000 nM) (Fig. 5B), applied from both cell sides,
effectively reduced the amphotericin B-induced increase in
Isc. The inactive phorbol ester 4
-phorbol
12,13-didecanoate (1 µM) did not affect the changes induced by
amphotericin B (data not shown). To confirm the involvement of PKA and
PKC pathways, we used selective antagonists of PKA (H-89) and PKC
(chelerythrine). As shown in Fig.
6A, H-89 (10 µM) antagonized
the inhibitory effects of both DBcAMP (200 µM) and SKF-38393 (300 nM). Similarly, chelerythrine (1 µM) antagonized the effects of both
PDBu (100 nM) and SKF-38393 (300 nM) (Fig. 6B). These
results suggest that stimulation of D1-like receptors may
lead to simultaneous activation of both PKA and PKC transduction pathways with a common point in the cascade of events, since either H-89 or chelerythrine completely prevented the effects of SKF-38393. To
confirm the involvement of both PKA and PKC in the inhibition of
Na+-K+-ATPase evoked by D1-like
receptor stimulation and clarify the sequence of events in more detail,
we performed complementary studies involving downregulation of PKA and
PKC. To promote PKA downregulation, we incubated OK cells overnight
(~16-20 h) in the presence of DBcAMP (100 µM). Under these
experimental conditions, the effects of both DBcAMP and SKF-38393 were
abolished; however, PDBu was still able to inhibit the amphotericin
B-induced increase in Isc (Fig.
7). In the application of a similar
strategy to downregulate PKC activity, cells were incubated overnight
(~16-20 h) in the presence of PDBu (100 nM). Under these
conditions, PDBu, DBcAMP, and SKF-38393 had no effect on the
amphotericin B-induced increase in Isc (Fig. 7).

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Fig. 5.
Effect of increasing concentrations of dibutyryl cAMP
(DBcAMP; 100-500 µM; A) or phorbol 12,13-dibutyrate
(PDBu; 10-1,000 nM; B) on changes in
Isc (givens as %control) induced by
amphotericin B (1 µg/ml). Values are means ± SE of 3-9
experiments/group. * P < 0.05, significantly
different from corresponding control value.
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Fig. 6.
Effect of protein kinase A (PKA) and PKC inhibitors on
SKF-38393-, DBcAMP-, and PDBu-mediated inhibition of changes in
Isc (given as %control) induced by amphotericin
B (1 µg/ml). Monolayers of OK cells were preincubated in the absence
or presence of H-89 (10 µM; A) or chelerythrine chloride
(1 µM; B) before treatment with SKF-38393 (300 nM), DBcAMP
(200 µM), or PDBu (100 nM). Values are means ± SE of 3-9
experiments/group. * P < 0.05, significantly
different from corresponding control value.
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Fig. 7.
Changes in Isc (given as
%control) induced by amphotericin B (1 µg/ml) after overnight
treatment with vehicle (active PKA/PKC), DBcAMP (100 µM; inactive
PKA), or PDBu (100 nM; inactive PKC) followed by short-term exposure to
SKF-38393 (300 nM), DBcAMP (200 µM), or PDBu (100 nM). Control values
were as follows (in µA/cm2): 47.2 ± 3.2 for active
PKA/PKC, 40.5 ± 2.1 for inactive PKA, and 19.3 ± 2.7 for
inactive PKC. Values are means ± SE of 3-9
experiments/group. * P < 0.05, significantly
different from corresponding control value.
|
|
The ability of PDBu to inhibit the amphotericin B-induced increase in
Isc in PKA downregulation and the failure of
SKF-38393 and DBcAMP to reduce the amphotericin B-induced increase in
Isc in PKC downregulation suggest that PKA may
be activated before PKC activation. One possible sequence of events
might be the activation of PLC by PKA, before activation of PKC.
Therefore, we next evaluated the involvement of PLC in the inhibitory
effects of the D1-like receptor agonist SKF-38393, DBcAMP,
and PDBu. We tested the effect of U-73122, a PLC inhibitor
(7), on the effect of SKF-38393, DBcAMP, and PDBu. As
shown in Fig. 8, U-73122 (3 µM) was
able to prevent the inhibitory effects of both DBcAMP (200 µM) and SKF-38393 (300 nM) on the amphotericin B-induced increase in
Isc (Fig. 8). However, the PLC inhibitor U-73122
(3 µM) did not affect the inhibitory effect of 100 nM PDBu (Fig. 8).
Similarly, U-73122 (3 µM) failed to alter stimulation of cAMP
production by forskolin (3 µM) (Table 3). This suggests that PLC
activation may occur downstream of AC activation. To confirm this view,
we also performed complementary studies on PLC activity in OK cells. As
shown in Fig. 9, DBcAMP (500 µM), but
not PBDu (200 nM), increased cytosolic and membrane PLC activity in OK
cells.

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Fig. 8.
Effect of phospholipase C (PLC) inhibitor on SKF-38393-,
DBcAMP-, and PDBu-mediated inhibition of changes in
Isc (given as %control) induced by amphotericin
B (1 µg/ml). Monolayers of OK cells were preincubated in the absence
or presence of U-73122 (3 µM) before treatment with SKF-38393 (300 nM), DBcAMP (200 µM), or PDBu (100 nM). Values are means ± SE
of 4-9 experiments/group. * P < 0.05, significantly different from corresponding control value.
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Fig. 9.
Effect of DBcAMP and PDBu on PLC activity in cytosol
(A) and membranes (B) from OK cells. Cells were
treated with DBcAMP (500 µM) or PDBu (200 nM) for 15 min at 37°C;
the assay for PLC activity was performed as described in
METHODS. RFU, relative fluorescence units. Values are
means ± SE of 3 experiments/group. * P < 0.05, significantly different from corresponding control value.
|
|
The signaling pathways linked to D1-like receptors include
the AC-PKA and PLC-PKC systems through the coupling of
Gs
and Gq/11
proteins, respectively
(18, 20, 57). We therefore felt it worthwhile to examine
the presence and involvement of Gs
and
Gq/11
proteins in the events after stimulation of
D1-like receptors with SK-38393. The presence of
Gs
and Gq/11
proteins in OK cells was
evaluated by Western blotting with specific antibodies from rabbit
raised against the synthetic decapeptide of the COOH terminal of
Gs
and Gq/11
proteins. As shown in Fig.
10, these antibodies recognized the
presence of both Gs
and Gq/11
proteins in
OK cells. For comparison, the rat kidney cortex was also evaluated for
the presence of Gs
and Gq/11
proteins.
The anti-Gs
protein antibody recognized a single band of
~40 kDa in OK cells and a major band of ~40 kDa and a minor band of
~45 kDa in rat renal cortex. The anti-Gq/11
protein
antibody recognized a single band of ~36 kDa in both OK cells and rat
renal cortex. The size of the
-subunits identified in this study is
comparable to the size reported in other tissues (29). To
evaluate the involvement of Gs
and Gq/11
proteins in the inhibition of Na+-K+-ATPase
evoked by D1-like receptor stimulation, further studies were performed in cells treated overnight (~16-20 h) with
antibodies raised against rat Gs
and
Gq/11
proteins. As shown in Fig.
11, the inhibitory effect of SKF-38393
on the amphotericin B-induced increase in Isc
was abolished in cells treated with the anti-Gs
antibody, but not in cells treated with the anti-Gq/11
antibody. These results agree with the view that D1-like
receptors in OK cells are coupled to AC via a Gs
type of
G protein and subsequent activation of PLC-PKC systems does not involve
a Gq/11
type of G protein but most likely results from
activation of PLC by PKA. As shown in Table 3, the dopamine-induced
increase in cAMP was abolished in cells treated with the
anti-Gs
antibody. The dopamine-induced increase in cAMP
was also completely prevented by pretreatment with the selective
D1-like receptor antagonist SKF-83566 (10 µM).

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Fig. 10.
Immunodetection of Gs and
Gq/11 in OK cells and rat kidney cortex (RK). Membrane
proteins (30-35 µg) obtained from OK cells and rat kidney cortex
were immunoblotted with anti-Gs subunit, COOH-terminal,
or anti-Gq/11 subunit, COOH-terminal antibodies
(1:1,000) for 1 h at room temperature. The results are
representative of 3 independent experiments.
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Fig. 11.
Changes in Isc (given as
%control) induced by amphotericin B (1 µg/ml) after overnight
treatment with vehicle or specific antibodies raised against
Gs or Gq/11 proteins followed by
short-term exposure to SKF-38393 (300 nM). Values are means ± SE
of 6-9 experiments/group. * P < 0.05, significantly different from corresponding control value.
|
|
Other possible interactions between PKA and PKC pathways and common
intracellular events may involve the regulation of AC. Certain AC in
some cells are stimulated by Ca2+/calmodulin, while others
are inhibited by Ca2+ (51). Another mechanism
by which AC activity can be regulated is PKC phosphorylation. The AC1,
AC2, and AC3 isoforms are significantly stimulated by PKC activation;
in contrast, the AC4, AC5, and AC6 isoforms are only modestly
stimulated (51). For these reasons, we decided to evaluate
the relationship between Ca2+, PKC activation, and cAMP
production in the OK cell line. PKC activation with PDBu (300 nM)
increased basal cAMP levels by 30%, and forskolin (3 µM) stimulated
levels by 75% on average (Table 3). Treatment of the cells with
thapsigargin (1 µM), the endosomal Ca2+-ATPase inhibitor
(38), evoked an immediate increase in intracellular Ca2+ (Fig. 12) but failed
to alter both basal and forskolin (3 µM)-stimulated cAMP levels
(Table 3). In contrast, dopamine (1 µM) and PDBu (1 µM) failed to
increase intracellular Ca2+ (Fig. 12). These results
suggest that PKC activation may stimulate AC, but this may not relate
to changes in intracellular Ca2+.

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Fig. 12.
Representative traces of the effect of thapsigargin (1 µM), dopamine (DA; 1 µM), and PDBu (1 µM) on intracellular
Ca2+ measured in monolayers of OK cells loaded with the
Ca2+-sensitive fluorophore fura 2. The duration of
perfusion with test compounds is indicated by the horizontal bar.
Traces are means of 3 independent experiments.
|
|
Data presented here suggest the involvement of PKA and PKC pathways,
Na+-K+-ATPase being ultimately an effector
protein for PKC. Recent studies (13, 36) indicate that
Na+-K+-ATPase activity may be regulated acutely
by phosphorylation/dephosphorylation mechanisms. To test this
hypothesis, we have used okadaic acid, an inhibitor of protein
phosphatases, which increases the state of phosphorylation of
Na+-K+-ATPase (36). A subthreshold
concentration of SKF-38393 (30 nM) was used. Okadaic acid (50 nM)
significantly enhanced the inhibitory effect of 30 nM SKF-38393 (Table
4).
Dopamine has also been shown to inhibit renal tubular
Na+-K+-ATPase activity via the cytochrome
P-450-monooxygenase pathway of the arachidonic acid cascade
(47). Arachidonic acid (10-1,000 nM) produced no
effect on changes in the amphotericin B-induced increase in
Isc (from 47.2 ± 3.2 to 46.3 ± 4.2 µA/cm2, n = 3-7). Ethoxyresorufin
(100 nM), a specific inhibitor of the cytochrome P-450
pathway (9), failed to prevent the inhibitory effect of
SKF-38393 (300 nM) on the amphotericin B-induced increase in
Isc (Table 4). Other studies (14)
have also reported the involvement of phosphatidylinositol 3-kinase
(PI3-kinase) during dopamine-mediated inhibition of
Na+-K+-ATPase activity. Therefore it was
decided to evaluate the effects of two inhibitors of PI3-kinase,
wortmannin and LY-294002. As shown in Table 4, both wortmannin (100 nM)
and LY-294002 (25 µM) failed to prevent the inhibitory effect of
SKF-38393 upon amphotericin B-induced increase in
Isc. The mitogen-activated PK inhibitor
PD-098059 (10 µM) also failed to prevent the inhibitory effect of
SKF-38393 (Table 4).
 |
DISCUSSION |
In the present study, while using the pore-forming antibiotic
amphotericin B to permeabilize the apical membrane, we were able to
isolate Na+ currents and assess the effects of dopamine or
D1-like receptor agonists on the basolateral membrane
Na+-K+-ATPase activity, in intact OK cell
monolayers. The polarized response to SKF-38393 (only when applied to
the apical cell side) suggests that D1-like receptors may
be absent from the basolateral membranes or not accessible to the
agonist. In the rat, D1-like receptors have been described
(18) in both the brush-border and basolateral membranes.
More recently, in the rabbit cortical collecting duct,
D2-like receptors were found (45) to be
present in the basolateral cell side only, whereas D1-like
receptors were exclusively distributed to the apical cell side.
Kinetically, the D1-like receptor-mediated decrease in
Na+ transepithelial flux in OK cells was demonstrated to
occur via a reduction in the Vmax for
Na+ without affecting the affinity of
Na+-K+- ATPase for Na+. This
finding is in agreement with previous studies (13) showing that inhibition of Na+-K+-ATPase activity in
renal epithelial cells by activation of G protein-coupled receptors is
mediated by phosphorylation of the catalytic
-subunit followed by
removal of active molecules from the plasma membrane.
Phosphorylation may serve as the triggering signal in the removal
process, but it does not affect Na+-K+-ATPase
activity while it resides in the plasma membrane (12). The
experimental conditions under which
Na+-K+-ATPase activity was assessed require a
constant oxygen supply to the preparation, and the concentration of
Ca2+ in the medium was kept to a level expected to allow
stimulation of the Na+ pump. Furthermore, under these
experimental conditions, the addition of dopamine or PDBu was not
accompanied by changes in intracellular Ca2+ concentration.
Glucose was omitted from the apical, but not the basal, bathing
solution to avoid entry of apical Na+ through the
Na+-dependent glucose transporter. However, this is not
expected to restrict metabolic requirements of OK cells. Accordingly,
the PKC-mediated inhibition of Na+-K+-ATPase
activity by dopamine, cAMP, and PDBu may not relate to hypoxic
conditions, as a result of poor metabolic status or deficient Ca2+ availability. On the other hand, the data presented
here shed some light on the possible role of the cAMP-PKA system on the dopamine-induced PKC-mediated inhibition of
Na+-K+-ATPase activity.
The results from this study also provide direct evidence that
D1-like receptor-mediated inhibition of
Na+-K+-ATPase activity involves a G protein of
the Gs
class, but not of the Gq/11
class,
positively coupled to AC. Transduction mechanisms set into motion
during activation of D1-like receptors in OK cells involve
the activation of both PKA and PKC pathways in a single sequence of
events with PKA activation occurring before PKC activation, which most
likely includes phosporylation of PLC by PKA. Both DBcAMP and PDBu were
able to inhibit Na+-K+-ATPase activity to the
same extent, with these effects being prevented by specific inhibitors
of PKA (H-89) and PKC (chelerythrine). Similarly, PKA and PKC
inhibition by H-89 and chelerythrine prevented the decrease in
Na+-K+-ATPase activity by the
D1-like receptor agonist. This suggests the involvement of
both kinases in the signal transduction pathway following
D1-like receptor activation, but does not constitute evidence that stimulation of D1-like receptors may lead to
simultaneous activation of both PKA and PKC transduction pathways. In
fact, the most likely possibility consists of a single sequence of
events with PKA activation prior to PKC activation in the signaling
cascade downstream to stimulation of D1-like
receptors. This view is compatible with the finding that PKA
downregulation abolished the inhibitory effects of both DBcAMP and
SKF-38393, but not those of PDBu, whereas PKC downregulation
abolished the effects of PDBu, SKF-38393, and DBcAMP. Furthermore, the
PLC inhibitor U-73122 prevented the inhibitory effects of both DBcAMP
and SKF-38393 on Na+-K+-ATPase activity, but
not those of PDBu. Taken together, these results strongly suggest that
downstream of D1-like dopamine receptor activation leading
to inhibition of Na+-K+-ATPase activity in OK
cells there is a chain of events comprising AC-PKA activation, followed
by activation of PLC by PKA, the product of which, diacylglycerol,
stimulates PKC (Fig. 13). This is also supported by the finding that the effects of SKF-38393 were
sensitive to cholera toxin and accompanied by increases in cAMP, which
were abolished by SKF-83566. Although regulation of
Na+-K+-ATPase activity may depend on the
expression of a particular PKC isoform (16), the most
consistent notion is that PKC is capable of phosphorylating the
-subunit and inhibiting Na+-K+-ATPase
activity in OK cells (13, 39).

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Fig. 13.
Schematic representation of the signaling pathways
linked to D1-like receptor stimulation in OK cells. AC,
adenylyl cyclase; PIP2, phosphatidylinositol
4,5-bisphosphate; DAG, diacylglycerol; IP3, inositol
1,4,5-trisphosphate; P, phosphorylation.
|
|
The dopamine D1-like receptors have been shown (33,
58) to stimulate PLC-
1 via pertussis toxin-insensitive G
proteins of the Gq family. However, our results contrast
with the previous reports (33, 58), showing that the
D1-like receptor was not positively coupled to the
Gq/11 protein. Activation of a
phosphatidylinositol-specific PLC most likely occurs as a result of
phosphorylation by PKA. This theory is based on experiments with
antibodies raised against the carboxy terminal of Gs
and
Gq/11
subunits to block interactions of G proteins with
D1-like receptors. The following observations support this
conclusion: 1) Western blot analysis revealed the presence
of both Gs
and Gq/11
proteins in OK
cells; 2) when cells were treated overnight with antibodies
raised against rat Gs
and Gq/11
proteins,
the inhibitory effect of the D1-like receptor agonist
SKF-38393 on the amphotericin B-induced increase in
Isc was abolished in cells treated with the
anti-Gs
antibody, but not in cells treated with the
anti-Gq/11
antibody; and 3) DBcAMP, but not
PDBu, significantly stimulated PLC activity in both membrane and
cytosol preparations from OK cells. Thus D1-like receptor
agonists stimulate PLC and PKC activity in renal OK cells independent
of Gq/11
proteins. However, this is not the only type of
interaction between the PKA and PKC transducing pathways in OK cells.
Although this was not directly evidenced following D1-like
receptor activation, PKC activation by PDBu was accompanied by
increases in both basal and forskolin-stimulated cAMP levels. This
indicates the presence of a positive coupling between PKC and PKA in OK
cells that involves activation of AC. Whether this mechanism
intensifies the effects of dopamine on the cell is not understood from
the experiments presented here. However, considering the modest
increase in cAMP accumulation produced by PDBu, it is unlikely that
this significantly contributes to the interaction between PKA and PKC
pathways in OK cells. On the other hand, increases in intracellular
Ca2+ by thapsigargin failed to alter both basal and
forskolin (3 µM)-stimulated cAMP levels. This would agree with the
view that AC isoforms in OK cells may be of AC4, AC5, and AC6 types,
considering their modest sensitivity to PDBu and insensitivity to
increases in intracellular Ca2+. The failure of dopamine
and PDBu to alter intracellular Ca2+ levels in OK cells
also suggests that PKC-mediated inhibition of
Na+-K+-ATPase activity in OK cells may be not
associated with marked changes in intracellular Ca2+.
To our knowledge, this chain of events constitutes a new signaling
pathway, downstream D1-like dopamine receptor activation leading to inhibition of Na+-K+-ATPase
activity. Other studies (5) have reported on
complex processes leading to inhibition of
Na+-K+-ATPase activity, namely, the requirement
of simultaneous activation of both D1-like and
D2-like receptors. Although the OK cells are endowed with
both D1-like and D2-like receptors, it is
unlikely that D2-like receptors are involved in the
generation of responses leading to direct inhibition of
Na+-K+-ATPase activity. In fact, SKF-38393 is a
rather selective D1-like receptor agonist, the effects of
which have been shown, in the OK cell line, to be insensitive to the
selective D2-like receptor antagonist
S-sulpiride (27). Another example in which the
involvement of both PKA and PKC activation was observed downstream of
dopamine receptor activation is that of LTK cells stably transfected
with the rat D1 receptor cDNA (59). Yu et al.
(59) showed that the D1-mediated stimulation
of PLC occurred as a result of PKA activation via stimulation of PKC.
This model contrasts with our proposal in OK cells (Fig. 13), the main
arguments being the lack of involvement of the Gq/11
type of G protein and the finding that inhibition of PLC by U-73122
failed to prevent inhibition of Na+-K+-ATPase
activity by PDBu. Dual coupling to AC and PLC has been reported
(44) in OK cells for parathyroid hormone (PTH).
However, it is likely that PTH signal transduction via a cAMP-dependent pathway does not involve stimulation of PLC (44). This has
been also observed (17, 23, 24, 35) for other types of
receptors, in different tissues, in which dual coupling to AC and PLC
generally involves independent pathways. Recently, however, it has been shown (48) that a new PLC and Ca2+ signaling
pathway was triggered by cAMP and mediated by a small GTPase of the Rap
family. These events, resulting from stimulation of
2-adrenoceptors in HEK-293 cells or the endogenous
receptor for PGE1 in N1E-115 neuroblastoma cells, were
caused by cAMP elevation, but independent of PKA (48).
This contrasts with our observation in OK cells that stimulation of the
D1-like receptor leads to AC-PKA activation followed by
activation of PLC by PKA (Fig. 13).
PLA2 is a potential third pathway by which the
D1-like receptor transduces its signal to
Na+-K+- ATPase in the proximal tubules
(41, 47). There is evidence suggesting this involves the
PLA2-arachidonic acid-20-HETE pathway (41,
47). 20-HETE is a cytochrome P-450 metabolite of
arachidonic acid, which appears to be of special importance for the
regulation of ion pumps and ion channels (50). The finding
that arachidonic acid failed to modify the amphotericin B-induced
increase in Isc is compatible with the view that
metabolites of PLA2 are not positively coupled to
inhibition of Na+-K+-ATPase activity in OK
cells. However, the failure of the cytochrome P-450
inhibitor ethoxyresorufin to alter the inhibitory effect of SKF-38393
on the amphotericin B-induced increase in Isc
provides further evidence that in these cells metabolites of
PLA2, namely, 20-HETE, may not participate in the
transduction pathway leading to inhibition of
Na+-K+-ATPase activity. Similarly, the lack of
effect of both wortmannin and LY-294002 on the inhibitory effect of
SKF-38393 on the amphotericin B-induced increase in
Isc suggests that PI3-kinase is not involved in
dopamine-mediated inhibition of Na+-K+-ATPase
activity. This transducing pathway was recently described (14) in rat renal proximal tubular cells, but the
involvement of PI3-kinase during inhibition of
Na+-K+-ATPase activity following
D1-like receptor stimulation appears to result from the
sequential activation of PLA2, arachidonic acid, and PKC.
The lack of involvement of the PLA2-arachidonic acid-20-HETE pathway in OK cells may explain why PI3-kinase inhibitors fail to affect the response to D1-like receptor
stimulation. It is possible this different behavior may relate to
differences between species (rat and opossum).
In conclusion, it is suggested that the D1-mediated
inhibition of Na+-K+-ATPase activity in OK
cells sequentially involves the AC-PKA system and the PLC-PKC system.
The results from this study provide direct evidence that inhibition of
Na+-K+-ATPase activity by dopamine in OK cells
involves the activation of D1-like dopamine receptors and a
G protein of the Gs
class positively coupled to AC.
Transduction mechanisms set into motion during activation of
D1-like receptors in OK cells involve the activation of
both PKA and PKC pathways in a single sequence of events, with PKA
activation prior to PKC activation, which most likely includes
activation of PLC by PKA.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Maria João Bonifácio (Department of
Research and Development, BIAL) for assistance with Western blotting.
 |
FOOTNOTES |
This work was supported by Fundação para a Ciência e
a Tecnologia Grant 35747.
Address for reprint requests and other correspondence:
P. Soares-da-Silva, Institute of Pharmacology and Therapeutics,
Faculty of Medicine, 4200-319 Porto, Portugal, (E-mail:
patricio.soares{at}mail.telepac.pt).
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 January 29, 2002;10.1152/ajprenal.00318.2001
Received 23 October 2001; accepted in final form 8 December 2001.
 |
REFERENCES |
1.
Aperia, AC.
Intrarenal dopamine: a key signal in the interactive regulation of sodium metabolism.
Annu Rev Physiol
62:
621-647,
2000[ISI][Medline].
2.
Azarani, A,
Goltzman D,
and
Orlowski J.
Parathyroid hormone and parathyroid hormone-related peptide inhibit the apical Na+/H+ exchanger Nhe-3 isoform in renal cells (OK) via a dual signaling cascade involving protein kinase A and C.
J Biol Chem
270:
20004-20010,
1995[Abstract/Free Full Text].
3.
Baines, AD,
and
Drangova R.
Does dopamine use several signal pathways to inhibit Na-Pi transport in OK cells?
J Am Soc Nephrol
9:
1604-1612,
1998[Abstract].
4.
Bates, MD,
Caron MG,
and
Raymond JR.
Desensitization of DA1 dopamine receptors coupled to adenylyl cyclase in opossum kidney cells.
Am J Physiol Renal Fluid Electrolyte Physiol
260:
F937-F945,
1991[Abstract/Free Full Text].
5.
Bertorello, A,
and
Aperia A.
Inhibition of proximal tubule Na+-K+-ATPase activity requires simultaneous activation of DA1 and DA2 receptors.
Am J Physiol Renal Fluid Electrolyte Physiol
259:
F924-F928,
1990[Abstract/Free Full Text].
6.
Bertorello, AM.
Diacylglycerol activation of protein kinase C results in a dual effect on Na+,K+-ATPase activity from intact renal proximal tubule cells.
J Cell Sci
101:
343-347,
1992[Abstract].
7.
Bleasdale, JE,
Thakur NR,
Gremban RS,
Bundy GL,
Fitzpatrick FA,
Smith RJ,
and
Bunting S.
Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils.
J Pharmacol Exp Ther
255:
756-768,
1990[Abstract].
8.
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].
9.
Burke, MD,
Thompson S,
Elcombe CR,
Halpert J,
Haaparanta T,
and
Mayer RT.
Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450.
Biochem Pharmacol
34:
3337-3345,
1985[ISI][Medline].
10.
Cheng, SX,
Aizman O,
Nairn AC,
Greengard P,
and
Aperia A.
[Ca2+]i determines the effects of protein kinases A and C on activity of rat renal Na+,K+-ATPase.
J Physiol (Lond)
518:
37-46,
1999[Abstract/Free Full Text].
11.
Cheng, XJ,
Hoog JO,
Nairn AC,
Greengard P,
and
Aperia A.
Regulation of Na+-K+-ATPase activity by PKC is modulated by state of phosphorylation of Ser-943 by PKA.
Am J Physiol Cell Physiol
273:
C1981-C1986,
1997[Abstract/Free Full Text].
12.
Chibalin, AV,
Ogimoto G,
Pedemonte CH,
Pressley TA,
Katz AI,
Feraille E,
Berggren PO,
and
Bertorello AM.
Dopamine-induced endocytosis of Na+,K+-ATPase is initiated by phosphorylation of Ser-18 in the rat alpha subunit and is responsible for the decreased activity in epithelial cells.
J Biol Chem
274:
1920-1927,
1999[Abstract/Free Full Text].
13.
Chibalin, AV,
Pedemonte CH,
Katz AI,
Feraille E,
Berggren PO,
and
Bertorello AM.
Phosphorylation of the catalyic alpha-subunit constitutes a triggering signal for Na+,K+- ATPase endocytosis.
J Biol Chem
273:
8814-8819,
1998[Abstract/Free Full Text].
14.
Chibalin, AV,
Zierath JR,
Katz AI,
Berggren PO,
and
Bertorello AM.
Phosphatidylinositol 3-kinase-mediated endocytosis of renal Na+, K+-ATPase alpha subunit in response to dopamine.
Mol Biol Cell
9:
1209-1220,
1998[Abstract/Free Full Text].
15.
DuVall, MD,
Guo Y,
and
Matalon S.
Hydrogen peroxide inhibits cAMP-induced Cl
secretion across colonic epithelial cells.
Am J Physiol Cell Physiol
275:
C1313-C1322,
1998[Abstract/Free Full Text].
16.
Efendiev, R,
Bertorello AM,
and
Pedemonte CH.
PKC-beta and PKC-zeta mediate opposing effects on proximal tubule Na+,K+-ATPase activity.
FEBS Lett
456:
45-48,
1999[ISI][Medline].
17.
Fargin, A,
Yamamoto K,
Cotecchia S,
Goldsmith PK,
Spiegel AM,
Lapetina EG,
Caron MG,
and
Lefkowitz RJ.
Dual coupling of the cloned 5-HT1A receptor to both adenylyl cyclase and phospholipase C is mediated via the same Gi protein.
Cell Signal
3:
547-557,
1991[ISI][Medline].
18.
Felder, CC,
Blecher M,
and
Jose PA.
Dopamine-1-mediated stimulation of phospholipase C activity in rat renal cortical membranes.
J Biol Chem
264:
8739-8745,
1989[Abstract/Free Full Text].
19.
Felder, CC,
Campbell T,
Albrecht F,
and
Jose PA.
Dopamine inhibits Na+-H+ exchanger activity in renal BBMV by stimulation of adenylate cyclase.
Am J Physiol Renal Fluid Electrolyte Physiol
259:
F297-F303,
1990[Abstract/Free Full Text].
20.
Felder, CC,
Jose PA,
and
Axelrod J.
The dopamine-1 agonist, SKF-82526, stimulates phospholipase-C activity independent of adenylate cyclase.
J Pharmacol Exp Ther
248:
171-175,
1989[Abstract].
21.
Féraille, E,
Carranza ML,
Buffin-Meyer B,
Rousselot M,
Doucet A,
and
Favre H.
Protein kinase C-dependent stimulation of Na+-K+-ATPase in rat proximal convoluted tubules.
Am J Physiol Cell Physiol
268:
C1277-C1283,
1995[Abstract/Free Full Text].
22.
Féraille, E,
and
Doucet A.
Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control.
Physiol Rev
81:
345-418,
2001[Abstract/Free Full Text].
23.
Force, T,
Bonventre JV,
Flannery MR,
Gorn AH,
Yamin M,
and
Goldring SR.
A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C.
Am J Physiol Renal Fluid Electrolyte Physiol
262:
F1110-F1115,
1992[Abstract/Free Full Text].
24.
Francesconi, A,
and
Duvoisin RM.
Role of the second and third intracellular loops of metabotropic glutamate receptors in mediating dual signal transduction activation.
J Biol Chem
273:
5615-5624,
1998[Abstract/Free Full Text].
25.
Gesek, FA,
and
Schoolwerth AC.
Hormonal interactions with the proximal Na+-H+ exchanger.
Am J Physiol Renal Fluid Electrolyte Physiol
258:
F514-F521,
1990[Abstract/Free Full Text].
26.
Glahn, RP,
Onsgard MJ,
Tyce GM,
Chinnow SL,
Knox FG,
and
Dousa TP.
Autocrine/paracrine regulation of renal Na+-phosphate cotransport by dopamine.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F618-F622,
1993[Abstract/Free Full Text].
27.
Gomes, P,
Vieira-Coelho MA,
and
Soares-da-Silva P.
Ouabain-insensitive acidification by dopamine in renal OK cells: primary control of the Na+/H+ exchanger.
Am J Physiol Regulatory Integrative Comp Physiol
281:
R10-R18,
2001[Abstract/Free Full Text].
28.
Guimaraes, JT,
Vieira-Coelho MA,
Serrao MP,
and
Soares-da-Silva P.
Opossum kidney (OK) cells in culture synthesize and degrade the natriuretic hormone dopamine: a comparison with rat renal tubular cells.
Int J Biochem Cell Biol
29:
681-688,
1997[ISI][Medline].
29.
Hussain, T,
and
Lokhandwala MF.
Renal dopamine DA1 receptor coupling with Gs and Gq/11 proteins in spontaneously hypertensive rats.
Am J Physiol Renal Physiol
272:
F339-F346,
1997[Abstract/Free Full Text].
30.
Hussain, T,
and
Lokhandwala MF.
Renal dopamine receptor function in hypertension.
Hypertension
32:
187-197,
1998[Abstract/Free Full Text].
31.
Jose, PA,
Eisner GM,
and
Felder RA.
Renal dopamine receptors in health and hypertension.
Pharmacol Ther
80:
149-182,
1998[ISI][Medline].
32.
Jose, PA,
Raymond JR,
Bates MD,
Aperia A,
Felder RA,
and
Carey RM.
The renal dopamine receptors.
J Am Soc Nephrol
2:
1265-1278,
1992[Abstract].
33.
Jose, PA,
Yu PY,
Yamaguchi I,
Eisner GM,
Mouradian MM,
Felder CC,
and
Felder RA.
Dopamine D1 receptor regulation of phospholipase C.
Hypertens Res
18, Suppl1:
S39-S42,
1995[Medline].
34.
Kiroytcheva, M,
Cheval L,
Carranza ML,
Martin PY,
Favre H,
Doucet A,
and
Féraille E.
Effect of cAMP on the activity and the phosphorylation of Na+,K+-ATPase in rat thick ascending limb of Henle.
Kidney Int
55:
1819-1831,
1999[ISI][Medline].
35.
Kuhn, B,
Schmid A,
Harteneck C,
Gudermann T,
and
Schultz G.
G proteins of the Gq family couple the H2 histamine receptor to phospholipase C.
Mol Endocrinol
10:
1697-1707,
1996[Abstract].
36.
Li, D,
Cheng SXJ,
Fisone G,
Caplan MJ,
Ohtomo Y,
and
Aperia A.
Effects of okadaic acid, calyculin A, and PDBu on state of phosphorylation of rat renal Na+-K+-ATPase.
Am J Physiol Renal Physiol
275:
F863-F869,
1998[Abstract/Free Full Text].
37.
Liu, YF,
Civelli O,
Zhou QY,
and
Albert PR.
Cholera toxin-sensitive 3',5'-cyclic adenosine monophosphate and calcium signals of the human dopamine-D1 receptor: selective potentiation by protein kinase A.
Mol Endocrinol
6:
1815-1824,
1992[Abstract].
38.
Lytton, J,
Westlin M,
and
Hanley MR.
Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.
J Biol Chem
266:
17067-17071,
1991[Abstract/Free Full Text].
39.
Middleton, JP,
Khan WA,
Collinsworth G,
Hannun YA,
and
Medford RM.
Heterogeneity of protein kinase C-mediated rapid regulation of Na/K-ATPase in kidney epithelial cells.
J Biol Chem
268:
15958-15964,
1993[Abstract/Free Full Text].
40.
Nash, SR,
Godinot N,
and
Caron MG.
Cloning and characterization of the opossum kidney cell D1 dopamine receptor: expression of identical D1A and D1B dopamine receptor mRNAs in opossum kidney and brain.
Mol Pharmacol
44:
918-925,
1993[Abstract].
41.
Nowicki, S,
Chen SL,
Aizman O,
Cheng XJ,
Li D,
Nowicki C,
Nairn A,
Greengard P,
and
Aperia A.
20-Hydroxyeicosa-tetraenoic acid (20-HETE) activates protein kinase C. Role in regulation of rat renal Na+,K+-ATPase.
J Clin Invest
99:
1224-1230,
1997[Abstract/Free Full Text].
42.
Ominato, M,
Satoh T,
and
Katz AI.
Regulation of Na-K-ATPase activity in the proximal tubule: role of the protein kinase C pathway and eicosanoids.
J Membr Biol
152:
235-243,
1996[ISI][Medline].
43.
Pedemonte, CH,
Pressley TA,
Cinelli AR,
and
Lokhandwala MF.
Stimulation of protein kinase C rapidly reduces intracellular Na+ concentration via activation of the Na+ pump in OK cells.
Mol Pharmacol
52:
88-97,
1997[Abstract/Free Full Text].
44.
Pfister, MF,
Forgo J,
Ziegler U,
Biber J,
and
Murer H.
cAMP-dependent and -independent downregulation of type II Na-Pi cotransporters by PTH.
Am J Physiol Renal Physiol
276:
F720-F725,
1999[Abstract/Free Full Text].
45.
Saito, O,
Ando Y,
Kusano E,
and
Asano Y.
Functional characterization of basolateral and luminal dopamine receptors in rabbit CCD.
Am J Physiol Renal Physiol
281:
F114-F122,
2001[Abstract/Free Full Text].
46.
Satoh, T,
Cohen HT,
and
Katz AI.
Different mechanisms of renal Na-K-ATPase regulation by protein kinases in proximal and distal nephron.
Am J Physiol Renal Fluid Electrolyte Physiol
265:
F399-F405,
1993[Abstract/Free Full Text].
47.
Satoh, T,
Cohen HT,
and
Katz AI.
Intracellular signaling in the regulation of renal Na-K-ATPase. II. Role of eicosanoids.
J Clin Invest
91:
409-415,
1993[ISI][Medline].
48.
Schmidt, M,
Evellin S,
Weernink PA,
von Dorp F,
Rehmann H,
Lomasney JW,
and
Jakobs KH.
A new phospholipase-C-calcium signaling pathway mediated by cyclic AMP and a Rap GTPase.
Nat Cell Biol
3:
1020-1024,
2001[ISI][Medline].
49.
Schumacher, M,
Schwarz M,
and
Brandle W.
Desensitization of the cAMP system in mouse Leydig cells by hCG, cholera toxin, dibutyryl cAMP and cAMP: localization of the 'lesion' to the guanine nucleotide regulatory protein-adenylate cyclase complex.
Mol Cell Endocrinol
34:
67-80,
1984[ISI][Medline].
50.
Schwartzman, ML,
Ferreri NR,
Carroll MA,
Songu-Mize E,
and
McGiff JC.
Renal cytochrome P-450-related metabolite inhibits (Na+-K+) ATPase.
Nature
314:
620-622,
1985[ISI][Medline].
51.
Sunahara, RK,
Dessauer CW,
and
Gilman AG.
Complexity and diversity of mammalian adenylyl cyclases.
Annu Rev Pharmacol Toxicol
36:
461-480,
1996[ISI][Medline].
52.
Sweadner, KJ,
and
Feschenko MS.
Predicted location and limited accessibility of protein kinase A phosphorylation site on Na-K-APTase.
Am J Physiol Cell Physiol
280:
C1017-C1026,
2001[Abstract/Free Full Text].
53.
Therien, AG,
and
Blostein R.
Mechanisms of sodium pump regulation.
Am J Physiol Cell Physiol
279:
C541-C566,
2000[Abstract/Free Full Text].
54.
Turner, NA,
Walker JH,
and
Vaughan PF.
The effect of down-regulation and long-term inhibition of protein kinase C on noradrenaline secretion in the neuroblastoma cell line SH-SY5Y.
Biochem Soc Trans
24:
426S,
1996[Medline].
55.
Vieira-Coelho, MA,
Gomes P,
Serrao MP,
and
Soares-da-Silva P.
D1-like dopamine receptor activation and natriuresis by nitrocatechol COMT inhibitors.
Kidney Int
59:
1683-1694,
2001[ISI][Medline].
56.
Vieira-Coelho, MA,
and
Soares-da-Silva P.
Apical and basal uptake of L-dopa and L-5-HTP and their corresponding amines, dopamine and 5-HT, in OK cells.
Am J Physiol Renal Physiol
272:
F632-F639,
1997[Abstract/Free Full Text].
57.
Vyas, SJ,
Eichberg J,
and
Lokhandwala MF.
Characterization of receptors involved in dopamine-induced activation of phospholipase-C in rat renal cortex.
J Pharmacol Exp Ther
260:
134-139,
1992[Abstract].
58.
Yu, PY,
Asico LD,
Eisner GM,
and
Jose PA.
Differential regulation of renal phospholipase C isoforms by catecholamines.
J Clin Invest
95:
304-308,
1995[ISI][Medline].
59.
Yu, PY,
Eisner GM,
Yamaguchi I,
Mouradian MM,
Felder RA,
and
Jose PA.
Dopamine D1A receptor regulation of phospholipase C isoform.
J Biol Chem
271:
19503-19508,
1996[Abstract/Free Full Text].
Am J Physiol Renal Fluid Electrolyte Physiol 282(6):F1084-F1096
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