Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto, Portugal
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ABSTRACT |
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This study examined the effects of D2-like dopamine receptor activation on Na+-K+-ATPase activity while apical-to-basal, ouabain-sensitive, amphotericin B-induced increases in short-circuit current and basolateral K+ (IK) currents in opossum kidney cells were measured. The inhibitory effect of dopamine on Na+-K+-ATPase activity was completely abolished by either D1- or D2-like receptor antagonists and mimicked by D1- and D2-like receptor agonists SKF-38393 and quinerolane, respectively. Blockade of basolateral K+ channels with BaCl2 (1 mM) or glibenclamide (10 µM), but not apamin (1 µM), totally prevented the inhibitory effects of quinerolane. The K+ channel opener pinacidil decreased Na+-K+-ATPase activity. The inhibitory effect of quinerolane on Na+-K+- ATPase activity was abolished by pretreatment of opossum kidney cells with pertussis toxin (PTX). Quinerolane increased IK across the basolateral membrane in a concentration-dependent manner; this effect was abolished by pretreatment with PTX, S-sulpiride, and glibenclamide. SKF-38393 did not change IK. Both H-89 (protein kinase A inhibitor) and chelerythrine (protein kinase C inhibitor) failed to prevent the stimulatory effect of quinerolane on IK. The stimulation of the D2-like receptor was associated with a rapid hyperpolarizing effect, whereas D1-like receptor activation was accompanied by increases in cell membrane potential. It is concluded that stimulation of D2-like receptors leads to inhibition of Na+-K+-ATPase activity and hyperpolarization; both effects are associated with the opening of K+ channels.
potassium channels; dopamine type 2-like receptors; cyclic adenosine 5'-monophosphate; opossum kidney cells
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INTRODUCTION |
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DEPENDING ON THE PARTICULARITIES of the experimental model used and characteristics of the mechanisms under evaluation, it is difficult, on certain occasions, to define with precision the nature of the events observed. This may be the case in an analysis of ion transepithelial flux, namely, in conditions in which the integrity of the cell is maintained and several transporters are expected to operate in concert. In a recent study of monolayers of opossum kidney (OK) cells expressing dopamine D1- and D2-like receptors, we were able to demonstrate that dopamine and D1-like receptor agonists inhibited Na+-K+-ATPase activity, using a methodology that consists of the measurement of ouabain-sensitive transepithelial transport of Na+ (18). The technique employed consists of the measurement of the transepithelial transport of Na+ in cell monolayers placed in Ussing chambers, with the added Na+ ionophore amphotericin B, from the apical cell border, which leads to increases in the Na+ delivered to Na+-K+-ATPase at the saturating level. Under short-circuit current (Isc) conditions, the observed current is an index of basolateral membrane Na+-K+-ATPase activity (14, 38-40). The advantages of this methodology over other experimental approaches are the possibility of evaluating Na+-K+-ATPase activity under in vivo conditions, avoiding freeze-thaw cell permeabilization procedures, and independently accessing the apical and basolateral cell sides. Subsequently, it was observed that stimulation of D2-like receptors also resulted in a marked reduction in amphotericin B-induced increases in Na+ currents. D2-like receptor agonists have been reported to have no effect (12, 31, 33), to act in concert with D1-like receptor agonists to inhibit Na+-K+-ATPase activity (2, 7-9), or to stimulate the Na+ pump (1, 20, 23, 42). However, there are no reports showing that stimulation of D2-like receptors leads to inhibition of Na+-K+-ATPase activity. Thus it was decided to look in more detail at the nature of the D2-like receptor-mediated inhibition of amphotericin B-induced increases in Na+ currents.
Classically, an ouabain-sensitive and Na+-dependent amphotericin B-induced increase in Isc represents Na+ transport mediated by Na+-K+-ATPase (14). However, other mechanisms, namely, those dependent on movements of K+ across the cellular membranes, may be partially responsible for these alterations in electrogenic ion movement. For instance, it is now well established that luminal Na+ entry proceeds in concert with increases in basolateral Na+-K+-ATPase activity and increases in K+ conductance (6, 41). Na+ is extruded from the cell by the Na+-K+-ATPase at the expense of K+ entry; K+ leaves the cell again via K+ channels in the basolateral membrane. The increase in K+ conductance accompanying the stimulation of Na+-K+-ATPase activity appears to result from the opening of ATP-sensitive K+ (KATP) channels in the basolateral membrane after local reductions in ATP levels (35). The tight coordination between Na+-K+-ATPase activity and K+ channel activity (pump-leak coupling) is thought to be of considerable importance for cell volume and homeostasis during epithelial transport.
Taking these aspects into consideration along with the observation that stimulation of D2-like receptors, mainly in the central nervous system, has been reported to increase K+ condutance (11, 17, 26, 27, 32, 36), we believed it was worthwhile to evaluate in more detail the role of D2-like dopamine receptors on amphotericin B-induced Isc changes in OK cells. The present study reports on the effects of D1- and D2-like dopamine receptor activation on Na+ and K+ currents in renal OK cells and evaluated transduction pathways coupled to D2-like receptors. It is shown that activation of D2-like receptors in renal OK cells leads to stimulation of basolateral KATP channels, which appear to be under the control of pertussis toxin (PTX)-sensitive G proteins of the Gi/o class.
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MATERIALS AND METHODS |
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Cell culture. OK cells, an established cell line derived from the kidney of a female American opossum (ATCC 1840 CRL), were obtained from the American Type Culture Collection (Rockville, MD) and maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. OK cells were grown in minimum essential medium (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 (Costar, Badhoevedorp, The Netherlands). For electrophysiology 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 from the apical cell side in the presence of agents known to interfere with signal transducing pathways, namely, G proteins, such as cholera toxin (CTX) and PTX. 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 electrophysiological parameters.
Electrogenic ion transport in OK cells.
All transport experiments were conducted under short-circuit
conditions. OK cells grown on polycarbonate filters were mounted in
Ussing chambers (1.0-cm2 window area) equipped with
water-jacketed gas lifts bathed on both sides with 10 ml of
Krebs-Hensleit solution, gassed with 5% CO2-95%
O2, 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 cells were gassed with
5% CO2-95% 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. Experimental design also
required modification of the bathing solution compositions for specific experiments, and these changes are indicated in
Na+-K+-ATPase activity. After a
5-min stabilization, monolayers were continuously voltage clamped to
zero potential differences by application of an 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 a
further 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 PC by means of a
BIOPAC MP1000 data-acquisition system (BIOPAC Systems, Goleta, CA).
Data analysis was performed by using AcqKnowledge 2.0 software (BIOPAC Systems).
Na+-K+-ATPase activity. The effect of D1- and D2-like receptor agonists on Na+-K+-ATPase activity was examined in monolayers mounted in Ussing chambers bathed with the standard Krebs-Hensleit solution such 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 (14, 38). This experimental model allows the entry of apical Na+ and leads to inhibition of the Na+/H+ exchanger (18). The concentration-response relationship of the 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 added to the apical bathing solution, and the 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 over the range 0-143 mM without affecting the concentrations of other ions. Apamin, BaCl2, DIDS, glibenclamide, ouabain, and pinacidil were applied from the basolateral cell side only, whereas amiloride was applied from the apical side only. All other test drugs were applied from both apical and basolateral cell sides.
Basolateral membrane K+ conductance. To evaluate the effect of D1- and D2-like receptor agonists on the basolateral K+ conductance of OK cells, cell monolayers were mounted in Ussing chambers in the presence of an apical-to-basolateral K+ gradient (80:5 mM), while the Na+ concentration was maintained at 25 mM on both sides of the monolayers. NaCl in the apical bathing solution was replaced with KCl (75 mM), and NaCl in the basolateral bathing solution was replaced with choline chloride (75 mM). The modified Krebs-Hensleit solution contained (in mM) 25 NaCl, 5 KCl, 25 choline HCO3, 1.2 KH2PO4, 2.5 CaCl2, and 1.2 MgSO4; pH was adjusted to 7.4 after monolayers were gassed with 5% CO2-95% O2. After ~15 min, ouabain (100 µM) was added to the basolateral bath solution to inhibit Na+-K+-ATPase, and amphotericin B was added to the apical bath solution to permeabilize the apical plasma membrane. The resulting Isc is due to the movement of K+ through channels in the basolateral membrane (IK).
cAMP measurement. cAMP was determined with an enzyme immunoassay kit (Assay Designs, Ann Arbor, MI) as previously described (18). OK cells were preincubated for 15 min at 37°C in Hanks' medium [(in mM) 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 0.25 CaCl2, 1.0 MgCl2, 0.15 Tris · HCl, and 1.0 Na butyrate, pH 7.4] containing 100 µM IBMX, a phosphodiesterase inhibitor. Cells were then incubated for 15 min with test compounds. At the end of the experiment, the reaction was stopped by the addition of 0.1 M HCl. Aliquots were taken for the measurement of intracellular cAMP content.
Membrane potential assay. Changes in membrane potential were evaluated by using the bisoxonol fluorescent dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)] as previously described (19). Cells cultured on glass coverslips were rinsed twice with assay buffer [(in mM) 20 HEPES, 120 NaCl, 2 KCl, 2 CaCl2, 1 MgCl2, and 5 glucose, pH 7.4, at 25°C] containing 5 µM DiBAC4(3) and then incubated for 30 min in 500 µl buffer solution containing 5 µM DiBAC4(3) to ensure dye distribution across the cell membrane. Thereafter, 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 2.0 ml was constantly stirred and maintained at 37°C. Changes in fluorescence were monitored for 500 s by sampling every 5 s at excitation and emission wavelengths of 488 and 520 nm, respectively. Responses of drugs added to the incubation medium were corrected for any background changes in fluorescence.
Drugs.
Amphotericin B, apamin, BaCl2, chelerythrine chloride, CTX,
dopamine hydrochloride, forskolin, H-89, glibenclamide, IBMX, ouabain,
PTX, and trypan blue were purchased from Sigma. (±)-SKF-83566 hydrochloride, S()-sulpiride, (±)-SKF-38393
hydrochloride, and quinerolane hydrochloride were obtained from
Research Biochemicals International. DiBAC4(3)
was purchased from Molecular Probes (Eugene, OR). Pinacidil was a kind
gift of Leo Pharmaceuticals.
Data analysis. Arithmetic means are given with SE, or geometric means are given with 95% confidence values. Statistical analysis was done with a one-way ANOVA followed by a Newman-Keuls test for multiple comparisons. A P value <0.05 was assumed to denote a significant difference.
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RESULTS |
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In 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.
1). The maximum effect was attained at
2.0 µg/ml amphotericin B; thus, in all subsequent experiments, the
apical membrane was permeabilized with 1.0 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 (1.0 µg/ml)-induced
increase in Isc was markedly (P < 0.05) attenuated by ouabain (100 µM) and by removal of
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-induced
increase in Isc (Fig. 1B). However,
blockade of basolateral K+ channels with BaCl2
(1 mM) applied from the basolateral cell side failed to affect the
amphotericin B-induced increase in Isc (Fig.
1B). The increase in Isc induced by
amphotericin B (1.0 µg/ml) applied from the apical cell side was not
affected by the Na+/H+ exchanger inhibitior
amiloride (1 mM), the Na+-K+-Cl cotransport
inhibitor bumetanide (10 µM), or the
Na+-HCO
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Pretreatment with dopamine applied from the apical cell side
significantly reduced the effect of 1.0 µg/ml amphotericin B on
Isc, which was prevented by the
D1-like receptor antagonist SKF-83566 (1 µM) or the
D2-like receptor antagonist S-sulpiride (1 µM)
(Fig. 3A). On the other hand,
both the D1-like receptor agonist SKF-38393
(30-1,000 nM) and the D2-like receptor agonist quinerolane (100-1,000 nM) were found to attenuate, in a
concentration-dependent manner, the amphotericin B-induced increases in
Isc (Fig. 3B). The magnitude of the
inhibitory effect of 1 µM dopamine was similar to that produced by
300 nM SKF-38393 or 1 µM quinerolane. The inhibitory effect of
SKF-38393 (300 nM) on amphotericin B-induced increases in
Isc was prevented by the D1-like
receptor antagonist SKF-83566 (1 µM) (Fig. 3C). Similarly,
pretreatment with the D2-like receptor antagonist
S-sulpiride (1 µM) prevented the inhibitory effect of
quinerolane (1 µM) on amphotericin B-induced increases in
Isc (Fig. 3D).
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Next, we evaluated the involvement of G proteins in the regulation of
Na+-K+-ATPase in OK cells. CTX and PTX
ribosylate the -subunit of the Gs and Gi/o
classes of G proteins, respectively. The effect of SKF-38393 (300 nM)
on amphotericin B-induced increases in Isc was
abolished by overnight treatment of OK cells with CTX (500 ng/ml) but
not with PTX (100 ng/ml) (Fig. 4). On the
other hand, the effect of quinerolane (1 µM) on amphotericin
B-induced increases in Isc was abolished
by overnight treatment of OK cells with PTX (100 ng/ml) but not
with CTX (500 ng/ml) (Fig. 4). These results suggest that
D1-like receptors stimulated by SKF-38393 are coupled to
CTX-sensitive G proteins of the Gs class, whereas
D2-like receptors stimulated by quinerolane are coupled to
PTX-sensitive G proteins of the Gi/o class.
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Previous studies have shown that D1-like receptors in OK
cells are positively coupled to adenylyl cyclase, whereas stimulation of D2-like receptors by quinerolane failed to alter basal
levels of cAMP (18). The next series of experiments
assessed the negative coupling between D2-like receptors
and adenylyl cyclase by measuring levels of cAMP before and after
stimulation of adenylyl cyclase with forskolin. Quinerolane (1 µM)
was found to affect neither the basal levels of cAMP nor the forskolin
(3 µM)-induced increase in cAMP levels (Fig.
5). Similar results were obtained in
cells pretreated with PTX (Fig. 5). These results strongly suggest that D2-like receptors in OK cells may not be negatively coupled
to adenylyl cyclase.
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Because D2-like receptors are also known to activate
K+ channel couples via G proteins, it was believed
worthwhile to explore the role of K+ channels on the
D2-like receptor-mediated inhibition of amphotericin B-induced increases in Isc. On the other hand,
it is well known that an increase in
Na+-K+-ATPase activity resulting from enhanced
entry of apical Na+ necessarily leads to a transient
increase in K+ accumulation (6, 41). Some of
the accumulated K+ ions are then extruded out of the cell
through K+ channels located in the basolateral side of the
cell. To gain further insight into the nature of mechanisms responsible
for the quinerolane-mediated decrease in amphotericin B-induced
increases in Isc, the effects of
BaCl2 (1 mM), a nonselective K+ channel
blocker, and glibenclamide and apamin, selective blockers of ATP- and
calcium-sensitive K+ channels (10, 30),
respectively, were tested. As shown in Fig.
6, both BaCl2 (1 mM) and
glibenclamide (10 µM) prevented the inhibitory effect of quinerolane
on the amphotericin B-induced increase in Isc.
By contrast, apamin (1 µM) was not found to alter the inhibitory
effect of quinerolane. These results strongly suggest that inhibition
of amphotericin B-induced increases in Isc by quinerolane may involve the opening of KATP channels as a
result of stimulation of D2-like receptors. To confirm this
view, it was decided to evaluate the effect of a K+ channel
opener on amphotericin B-induced increases in
Isc. As shown in Fig.
7, pinacidil significantly reduced the
amphotericin B-induced increase in Isc.
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Because the experimental procedure described above does not enable one
to draw definitive conclusions as to whether stimulation of
D2-like receptors leads to the opening of KATP
channels or inhibition of Na+-K+-ATPase
activity, it was decided to implement an experimental protocol that
allows the measurement of IK across the
basolateral membrane. IK was measured in
monolayers of OK cells in conditions of an apical-to-basolateral
K+ gradient (80:5 mM) in the presence of ouabain (100 µM)
(15). The addition of amphotericin B (3 µg/ml) to the
apical side resulted in a rapid increase in IK;
this effect was markedly inhibited by the addition of the
K+ channel blocker BaCl2 (1 mM) (Fig.
8A). The amphotericin
B-induced IK in cells treated from the apical
and basolateral cell side with quinerolane (1 µM) was greater than in
controls; this was also completely abolished by the addition of
BaCl2 (1 mM) from the basolateral cell side (Fig.
8A). On the other hand, glibenclamide (10 µM) applied from
the basolateral cell side significantly attenuated the amphotericin
B-induced IK (Fig. 8A). By contrast,
apamin (1 µM) applied from the basolateral cell side failed to alter
the amphotericin B-induced IK, whereas pinacidil
increased the amphotericin B-induced IK (Fig.
8B). The potentiation by quinerolane of the amphotericin
B-induced IK was a concentration-dependent
effect that was abolished by the selective D2-like receptor
antagonist S-sulpiride (1 µM) and glibenclamide (10 µM)
(Fig. 8C). By contrast, the selective D1-like
receptor agonist SKF-38393 (1 µM) failed to alter the amphotericin
B-induced IK (Fig. 8C). Moreover, the stimulatory influence of quinerolane on the amphotericin B-induced IK was similar in the absence and presence of
SKF-38393, the D1-like receptor agonist (Fig.
8C). These findings agree with the view that stimulation of
D2-like, but not D1-like, receptors may open KATP channels.
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Previous studies have demonstrated that second- messenger pathways
thought to be involved in the renal effects of dopamine include
stimulation of protein kinase A (PKA) or C (PKC) pathways (25). This is particularly true when receptors involved
are of the D1-like type coupled to Gs and
Gq/11 proteins. By contrast, stimulation of
D2-like receptors coupled to Gi/o proteins has been demonstrated to open K+ channels (11, 17,
26) and/or inhibit adenylyl cyclase (37). The
purpose of the next series of experiments was to clarify the transduction pathways from D2-like receptor activation
downstream of the opening of KATP channels. Again,
overnight treatment of OK cells with PTX (100 ng/ml) abolished the
potentiation by quinerolane of the amphotericin B-induced
IK (Fig. 9),
suggesting that D2-like receptors stimulated by quinerolane
are coupled to PTX-sensitive G proteins of the Gi/o class.
On the other hand, selective antagonists of PKA (H-89) and PKC
(chelerythrine) (4) failed to alter the potentiation by
quinerolane of the amphotericin B-induced IK
(Fig. 9).
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Functional coupling of Na+-K+-ATPase activity
to basolateral membrane K+ conductance is crucial for
sustaining transport in the proximal tubule. Apical Na+
entry stimulates pump activity, lowering cytosolic ATP, which, in turn,
disinhibits KATP channels (28). However, this
may not explain why the opening of the basolateral KATP
channel by pinacidil and quinerolane is also accompanied by inhibition
of Na+-K+-ATPase activity, as indicated by the
reduction in ouabain-sensitive amphotericin B-induced increases in
Isc. Because the opening of KATP
channels mediates hyperpolarization of the basolateral membrane (28), we believed it was worthwhile to determine membrane
potential in cells treated with quinerolane and pinacidil and to
determine whether inhibition of Na+-K+-ATPase
activity leads to changes in membrane potential. The fluorescent dye
DiBAC4(3) was used to monitor changes in
membrane potential in OK cells treated with pinacidil, ouabain,
quinerolane, and SKF-38393. As shown in Fig.
10, addition of ouabain (100 µM) and D1-like receptor stimulation with SKF-38393 (1 µM)
produced cell membrane depolarization, as evidenced by the
time-dependent increase in fluorescence. On the other hand, pinacidil
(50 µM) and D2-like receptor stimulation with quinerolane
(1 µM) produced rapid hyperpolarization, as evidenced by the
time-dependent decrease in fluorescence (Fig. 10).
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DISCUSSION |
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The present study investigated the effects of D1- and D2-like dopamine receptor activation on ouabain-sensitive Na+ currents and ouabain-insensitive K+ currents in renal OK cells by using an in vivo method with independent accessibility to the apical and basolateral cell sides. The results presented here show that stimulation of D1-like receptors, coupled to a Gs class of G proteins, decreased ouabain-sensitive Na+ currents but failed to alter ouabain-insensitive K+ currents. In contrast, stimulation of D2-like receptors, coupled to a Gi/o class of G proteins, decreased ouabain-sensitive Na+ currents and increased ouabain-insensitive K+ currents, both events most likely resulting from the opening of K+ channels. The stimulation of the D2-like receptor was associated with a rapid hyperpolarizing effect, whereas D1-like receptor activation was accompanied by increases in cell membrane potential. Transduction mechanisms set into motion during activation of D2-like receptors in OK cells involve the activation of neither PKA nor PKC pathways.
Using the pore-forming antibiotic amphotericin B, we were able to
isolate Na+ currents and assess the effects of
D1- and D2-like receptor activation on the
basolateral membrane Na+-K+-ATPase activity in
intact OK cell monolayers. The addition of amphotericin B to the apical
cell side increased the Na+ delivered to
Na+-K+-ATPase to the saturating level, as
indicated by the fast increase in Isc. The rapid
recovery to baseline is due to the transport of Na+ across
the basolateral membrane mediated by
Na+-K+-ATPase, as indicated by complete
prevention by ouabain and removal of Na+ from the medium
bathing the apical side of the monolayer. This suggestion is also in
agreement with the finding that the
Na+-HCO
In an earlier report, we showed that OK cells expressed both D1- and D2-like receptors, with the activation of the former, but not the latter, being accompanied by stimulation of adenylyl cyclase and marked intracellular acidification as a result of inhibition of the Na+/H+ exchanger (18). The inhibitory effects of dopamine on adenylyl cyclase and the Na+/H+ exchanger were antagonized by the selective D1-like receptor antagonist SKF-83566 and mimicked by the D1-like receptor agonist SKF-38393 (18). The D2-like receptor agonist quinerolane and the D2-like receptor antagonist S-sulpiride were devoid of effects (18). In the present study, it is shown that dopamine significantly reduced amphotericin B-induced increases in Isc; this was prevented by both the D1-like receptor antagonist SKF-83566 and the D2-like receptor antagonist S-sulpiride. These effects of dopamine were mimicked, in a concentration-dependent manner, by both the D1-like receptor agonist SKF-38393 and the D2-like receptor agonist quinerolane. The D1-like receptor antagonist SKF-83566 and the D2-like receptor antagonist S-sulpiride prevented the inhibitory effect of SKF-38393 and quinerolane, respectively. This suggests that independent stimulation of both D1- and D2-like receptors may lead to inhibition of Na+-K+-ATPase, as evidenced by attenuation of amphotericin B-induced increases in Isc. The finding that inhibition of amphotericin B-induced increases in Isc by SKF-38393 was abolished by overnight treatment of OK cells with CTX, but not with PTX, suggested that D1-like receptors are coupled to a Gs class of G proteins. On the other hand, the finding that inhibition of amphotericin B-induced increases in Isc by quinerolane was abolished by overnight treatment of OK cells with PTX, but not with CTX, suggested that D2-like receptors are coupled to a Gi/o class of G proteins. These results strongly suggest that attenuation of amphotericin B-induced increases in Isc by D1- and D2-like receptor agonists results from stimulation of independent pathways coupled to these receptors.
Because the amphotericin B-induced increases in
Isc were dependent on the ouabain-sensitive
apical-to-basal Na+ gradient, it was assumed that the
increase in current measured after the addition of amphotericin B was
related to an increase in Na+ gradient driven by the
basolateral Na+-K+-ATPase. However, when
challenged with the possibility that stimulation of D1- and
D2-like receptors could lead to the activation of
independent mechanisms, we hypothesized that mechanisms set into motion
during stimulation of D2-like receptors resulted from an
indirect inhibitory effect on Na+-K+-ATPase
activity. Our first hypothesis considered that changes in the
K+ gradient as a result of stimulation of
D2-like receptors could alter
Na+-K+-ATPase activity and consequently the
amphotericin B-induced increases in Isc. In
fact, the adequate function of Na+-K+-ATPase
implies a transient increase in intracellular K+, which is
then extruded out of the cell through K+ channels located
in the basolateral membrane (6, 41). This view is in
agreement with the finding that amphotericin B-induced increases in
Isc were abolished by substitution of KCl with
cesium chloride. Another argument favoring this suggestion concerned the prevention of a quinerolane-mediated decrease in amphotericin B-induced increases in Isc by using the
nonselective K+ channel blocker BaCl2. The
observation that glibenclamide, but not apamin, prevented the
quinerolane-mediated decrease in amphotericin B-induced increases in
Isc provides evidence that stimulation of
D2-like receptors may lead to the opening of
KATP channels. With the use of another protocol that allows
measurement of IK across the basolateral
membrane, quinerolane was also found to increase the amphotericin
B-induced IK; this was completely abolished by the addition of BaCl2 and glibenclamide. The
potentiation by quinerolane of the amphotericin B-induced
IK was a concentration-dependent effect, which
was abolished by the selective D2-like receptor antagonist
S-sulpiride. By contrast, the selective D1-like
receptor agonist SKF-38393 failed to alter the amphotericin B-induced
IK. These findings strongly suggest that the
stimulation of D2-like, but not D1-like,
receptors leads to the opening of KATP channels. Taken
together, it is suggested that stimulation of D2-like
receptors coupled to a Gi/o class of G proteins decreased
ouabain-sensitive Na+ currents and increased
ouabain-insensitive K+ currents; both events most likely
resulted from the opening of KATP channels (Fig.
11). The positive coupling of
KATP channels to D2-like receptors is a
well-known characteristic, particularly in neuronal cells, which leads
to hyperpolarization and inhibition of neurotransmitter release
(11, 17, 26, 27, 32, 36). To our knowledge, this is the
first report on a positive coupling between D2-like
receptors and KATP channels in renal epithelial cells.
Another observation in line with these findings is concerned with the
stimulatory effect of dopamine on the
Na+-K+-2Cl cotransport by means
of actions on Ba2+-sensitive K+ channels
(3). Although others have previously described the presence of D2-like receptors in OK cells
(13), their function and transduction pathways have not
been reported. In the present report, we were able to demonstrate that
opening of K+ channels during stimulation of
D2-like receptors involved the coupling to a
Gi/o class of G proteins but lacked the involvement of
transduction pathways such as PKA and PKC. On the basis of these
findings, it is suggested that in OK cells, as has been demonstrated in
other cell types (36), the coupling between the G protein
and the K+ channel appears to be direct rather than
mediated by intracellular soluble second messengers. In other types of
cells, D2-like receptors have been described to be
negatively coupled to adenylyl cyclase (5, 24, 37). This
does not appear to be the case, as evidenced by the failure of
quinerolane to alter the basal and the forskolin-stimulated levels of
cAMP.
|
The mechanisms involved in quinerolane-mediated inhibition of
Na+-K+-ATPase activity during opening of the
basolateral KATP channel may involve decreases in cell
membrane potential rather than decreases in intracellular
K+. In fact, the Na+ pump is activated by
Na+ and ATP at cytoplasmic sites and by K+ at
extracellular sites (16, 34). Furthermore, K+
has been shown to act as a competitive inhibitor of Na+
binding at cytoplasmic sites (16, 34). The suggestion that Na+-K+-ATPase activity is dependent on membrane
potential agrees with the finding that cell hyperpolarization by
pinacidil was accompanied by decreases in ouabain-sensitive
Na+ currents, which fits well with the view of the voltage
dependence of Na+-K+-ATPase activity (16,
22). In fact, Na+-K+-ATPase is
electrogenic, implying that the membrane potential affects the rate
constant of the charge-translocating step of the pump cycle
(22). However, the precise relationship of
Na+-K+-ATPase activity to transmembrane voltage
depends on a number of as yet unknown kinetic parameters of the pump
cycle and the type of Na+-K+-ATPase isoform
(16). However, in renal epithelial cells that mostly
contain the 1-isoform, the pump current does increase with membrane potential within the
175 to
75 mV range
(21). The finding that quinerolane markedly reduced
membrane potential and Na+-K+-ATPase activity,
which are both effects associated with the opening of K+
channels, strongly suggests that mechanisms that decrease membrane potential negatively affect Na+-K+-ATPase
activity in OK cells (Fig. 11). This may prevent inhibition of
Na+-K+-ATPase activity set into motion by
other mechanisms, explaining why the magnitude of attenuation of
amphotericin B-induced increases in Isc by
dopamine was identical to that observed during stimulation of
D1- and D2-like receptors by selective
agonists. This contrasts with that reported before on the inhibition of
Na+-K+-ATPase, in which the inhibitory effect
has always been referred to as a cooperative action between
D1- and D2-like receptors, requiring the
simultaneous activation of both types of receptors (8).
Another discrepancy concerns reports in which activation of
D2-like receptors led to stimulation of
Na+-K+- ATPase activity (1, 20, 23,
42). However, it should be stressed that inhibition of
ouabain-sensitive Na+ currents in OK cells during
D2-like receptor stimulation most likely results from the
opening of K+ channels. In fact, in conditions in which
most K+ channels were blocked, D2-like receptor
stimulation failed to alter ouabain-sensitive Na+ currents.
This would agree with reports indicating that D2-like receptor agonists have no direct effect on
Na+-K+-ATPase activity (12, 31,
33).
In conclusion, it is demonstrated that in OK cells stimulation of D1-like receptors coupled to a Gs class of G proteins attenuates ouabain-sensitive Na+ currents but fails to alter ouabain-insensitive K+ currents, whereas stimulation of D2-like receptors coupled to a Gi/o class of G proteins inhibits ouabain-sensitive Na+ currents and increases ouabain-insensitive K+ currents; both events most likely result from the opening of K+ channels. The coupling among D2-like receptors, the G protein, and the K+ channel appears to be direct rather than mediated by intracellular soluble second messengers.
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ACKNOWLEDGEMENTS |
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This study was supported by Fundação para a Ciência e a Tecnologia Grant POCTI/35747/FCB/2000.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Soares-da-Silva, Inst. of Pharmacology and Therapeutics, Faculty of Medicine, 4200 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.00244.2001
Received 2 August 2001; accepted in final form 11 January 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abu-Jayyab, A,
and
Mahgoub A.
Role of Na+/K+-stimulated adenosine triphosphatase in the action of dopaminergic-D2 receptors of the liver in rats.
Biosci Rep
7:
839-842,
1987[ISI][Medline].
2.
Aizman, O,
Brismar H,
Uhlen P,
Zettergren E,
Levey AI,
Forssberg H,
Greengard P,
and
Aperia A.
Anatomical and physiological evidence for D1 and D2 dopamine receptor colocalization in neostriatal neurons.
Nat Neurosci
3:
226-230,
2000[ISI][Medline].
3.
Aoki, Y,
Albrecht FE,
Bergman KR,
and
Jose PA.
Stimulation of Na+-K+-2Cl cotransport in rat medullary thick ascending limb by dopamine.
Am J Physiol Regulatory Integrative Comp Physiol
271:
R1561-R1567,
1996
4.
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
5.
Bates, MD,
Senogles SE,
Bunzow JR,
Liggett SB,
Civelli O,
and
Caron M.
Regulation of responsiveness at D2 dopamine receptors by receptor desensitization and adenylyl cyclase sensitization.
Mol Pharmacol
39:
55-63,
1991[Abstract].
6.
Beck, JS,
Laprade R,
and
Lapointe JY.
Coupling between transepithelial Na transport and basolateral K conductance in renal proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F517-F527,
1994
7.
Bertorello, A,
and
Aperia A.
Both DA1 and DA2 receptor agonists are necessary to inhibit NaKATPase activity in proximal tubules from rat kidney.
Acta Physiol Scand
132:
441-443,
1988[ISI][Medline].
8.
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
9.
Bertorello, AM,
Hopfield JF,
Aperia A,
and
Greengard P.
Inhibition by dopamine of (Na(+)+K+)ATPase activity in neostriatal neurons through D1 and D2 dopamine receptor synergism.
Nature
347:
386-388,
1990[ISI][Medline].
10.
Blatz, AL,
and
Magleby KL.
Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle.
Nature
323:
718-720,
1986[ISI][Medline].
11.
Castelletti, L,
Memo M,
Missale C,
Spano PF,
and
Valerio A.
Potassium channels involved in the transduction mechanism of dopamine D2 receptors in rat lactotrophs.
J Physiol
410:
251-265,
1989[Abstract].
12.
Chen, C,
and
Lokhandwala MF.
Inhibition of Na+,K+-ATPase in rat renal proximal tubules by dopamine involved DA-1 receptor activation.
Naunyn Schmiedebergs Arch Pharmacol
347:
289-295,
1993[ISI][Medline].
13.
Cheng, L,
Precht P,
Frank D,
and
Liang CT.
Dopamine stimulation of cAMP production in cultured opossum kidney cells.
Am J Physiol Renal Fluid Electrolyte Physiol
258:
F877-F882,
1990
14.
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
15.
DuVall, MD,
and
O'Grady SM.
Regulation of K secretion across the porcine gallbladder epithelium.
Am J Physiol Cell Physiol
264:
C1542-C1549,
1993
16.
Féraille, E,
and
Doucet A.
Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control.
Physiol Rev
81:
345-418,
2001
17.
Freedman, JE,
and
Weight FF.
Single K+ channels activated by D2 dopamine receptors in acutely dissociated neurons from rat corpus striatum.
Proc Natl Acad Sci USA
85:
3618-3622,
1988[Abstract].
18.
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
19.
Gopalakrishnan, M,
Whiteaker KL,
Molinari E,
Davis-Taber R,
Scott VES,
Shieh C-C,
Buckner SA,
Milicic I,
Cain JC,
Postl S,
Sullivan JP,
and
Brioni JD.
Characterization of the ATP-sensitive potassium channels (KATP) expressed in guinea pig bladder smooth muscle cells.
J Pharmacol Exp Ther
289:
551-558,
1999
20.
Guerrero, C,
Lecuona E,
Pesce L,
Ridge KM,
and
Sznajder JI.
Dopamine regulates Na-K-ATPase in alveolar epithelial cells via MAPK-ERK-dependent mechanisms.
Am J Physiol Lung Cell Mol Physiol
281:
L79-L85,
2001
21.
Horisberger, JD,
and
Giebisch G.
Na-K pump current in the Amphiuma collecting tubule.
J Gen Physiol
94:
493-510,
1989[Abstract].
22.
Horisberger, JD,
Lemas V,
Kraehenbuhl JP,
and
Rossier BC.
Structure-function relationship of Na,K-ATPase.
Annu Rev Physiol
53:
565-584,
1991[ISI][Medline].
23.
Hussain, T,
Abdul-Wahab R,
and
Lokhandwala MF.
Bromocriptine stimulates Na+, K+-ATPase in renal proximal tubules via the cAMP pathway.
Eur J Pharmacol
321:
259-263,
1997[ISI][Medline].
24.
Johansson, MH,
and
Westlind-Danielsson A.
Forskolin-induced up-regulation and functional supersentivity of dopamine D2 long receptors expressed by Ltk- cells.
Eur J Pharmacol
269:
149-155,
1994[Medline].
25.
Jose, PA,
Eisner GM,
and
Felder RA.
Renal dopamine receptors in health and hypertension.
Pharmacol Ther
80:
149-182,
1998[ISI][Medline].
26.
Lacey, MG,
Mercuri NB,
and
North RA.
Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta.
J Physiol
392:
397-416,
1987[Abstract].
27.
Liu, LX,
Burgess LH,
Gonzalez AM,
Sibley DR,
and
Chiodo LA.
D2S, D2L, D3, and D4 dopamine receptors couple to a voltage-dependent potassium current in N18TG2 × mesencephalon hybrid cell (MES-23.5) via distinct G proteins.
Synapse
31:
108-118,
1999[ISI][Medline].
28.
Mauerer, UR,
Boulpaep BL,
and
Segal AS.
Regulation of an inwardly rectifying ATP-sensitive K+ channel in the basolateral membrane of renal proximal tubule.
J Gen Physiol
111:
161-180,
1998
29.
Pedemonte, CH,
Pressley TA,
Lokhandwala MF,
and
Cinelli AR.
Regulation of Na,K-ATPase transport activity by protein kinase C.
J Membr Biol
155:
219-227,
1997[ISI][Medline].
30.
Schmid-Antomarchi, H,
Weille JD,
Fosset M,
and
Ladzunski M.
The receptor for antidiabetic sulfonylureas controls the activity of the ATP-modulated K+ channel in insulin-secreting cells.
J Biol Chem
262:
15840-15844,
1987
31.
Shahedi, M,
Laborde K,
Azimi S,
Hamdani S,
and
Sachs C.
Mechanisms of dopamine effects on Na-K-ATPase activity in Madin-Darby canine kidney (MDCK) epithelial cells.
Pflügers Arch
429:
832-840,
1995[ISI][Medline].
32.
Sun, XD,
Lee EW,
Wong EH,
and
Lee KS.
ATP-sensitive potassium channels in freshly dissociated adult rat striatal neurons: activation by metabolic inhibitors and the dopaminergic receptor agonist quinpirole.
Pflügers Arch
440:
530-547,
2000[ISI][Medline].
33.
Takemoto, F,
Cohen HT,
Satoh T,
and
Katz AI.
Dopamine inhibits Na/K-ATPase in single tubules and cultured cells from distal nephron.
Pflügers Arch
421:
302-306,
1992[ISI][Medline].
34.
Therien, AG,
and
Blostein R.
Mechanisms of sodium pump regulation.
Am J Physiol Cell Physiol
279:
C541-C566,
2000
35.
Tsuchiya, K,
Wang W,
Giebish G,
and
Welling PA.
ATP is a coupling modulator of parallel Na,K-ATPase-K-channel activity in the renal proximal tubule.
Proc Natl Acad Sci USA
89:
6418-6422,
1992[Abstract].
36.
Uchida, S,
Akaike N,
and
Nabekura J.
Dopamine activates inward rectifier K+ channel in acutely dissociated rat substantia nigra neurones.
Neuropharmacology
39:
191-201,
2000[ISI][Medline].
37.
Vallar, L,
and
Meldosi J.
Mechanisms of signal transduction at the dopamine D2 receptor.
Trends Pharmacol Sci
10:
74-77,
1989[ISI][Medline].
38.
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].
39.
Vieira-Coelho, MA,
and
Soares-da-Silva P.
Alpha2-adrenoceptors mediate the effect of dopamine on adult rat jejunal electrolyte transport.
Eur J Pharmacol
356:
59-65,
1998[ISI][Medline].
40.
Vieira-Coelho, MA,
and
Soares-da-Silva P.
Ontogenic aspects of D1 receptor coupling to G proteins and regulation of rat jejunal Na+, K+ ATPase activity and electrolyte transport.
Br J Pharmacol
129:
573-581,
2000
41.
Welling, PA.
Cross-talk and the role of KATP channels in the proximal tubule.
Kidney Int
48:
1017-1023,
1995[ISI][Medline].
42.
Yamaguchi, I,
Walk SF,
Jose PA,
and
Felder RA.
Dopamine D2L receptors stimulate Na+/K+-ATPase activity in murine LTK- cells.
Mol Pharmacol
49:
373-378,
1996[Abstract].