Inhibition of initial transport rate of basolateral organic
anion carrier in renal PT by BK and phenylephrine
Michael
Gekle1,
Sigrid
Mildenberger1,
Christoph
Sauvant1,
Dallas
Bednarczyk2,
Stephen H.
Wright2, and
William H.
Dantzler2
1 Institute of Physiology,
University of Würzburg, 97970 Würzburg, Germany; and
2 Department of Physiology,
College of Medicine, University of Arizona, Tucson,
Arizona 85721
 |
ABSTRACT |
The effect of ligands for phospholipase C-coupled
receptors and of protein kinase C (PKC) stimulation with phorbol ester
[phorbol 12-myristate 13-acetate (PMA)] or
1,2-dioctanoyl-sn-glycerol
on the activity of the basolateral organic anion transporter (OAT) in
S2 segments of single, nonperfused rabbit proximal tubules (PT) was
measured with the use of fluorescein and epifluorescence microscopy.
The initial uptake rate (25 s, OAT activity) was measured in real time
by using conditions similar to those found in vivo. Stimulation of PKC
with PMA or 1,2-dioctanoyl-sn-glycerol
led to an inhibition of OAT activity, which could be prevented by 10
7 mol/l of the
PKC-specific inhibitor bisindolylmaleimide. The
1-receptor agonist
phenylephrine as well as the peptide hormone bradykinin induced a
reversible decrease of OAT activity, which was prevented by
bisindolylmaleimide. The observed effect was not due to a decrease in
the concentration of the counterion
-ketoglutarate or to impaired
-ketoglutarate recycling, because it was unchanged in the continuous
presence of
-ketoglutarate or methyl succinate. We conclude that
physiological stimuli can inhibit the activity of OAT in rabbit PT via
PKC. The effect is not mediated by alterations in counterion
availability but by a direct action on the OAT.
kidney; isolated proximal tubule; organic anion transport; protein
kinase C; bradykinin
 |
INTRODUCTION |
RENAL ELIMINATION of a wide variety of organic anions
(OA) from the body is an essential process for human and animal health. Transport of OA into proximal cells across the basolateral membrane is
the active step in transtubular secretion (11, 17). There is a single
transport process at the basolateral membrane, the "classic" OA
transporter (OAT), that accepts a broad range of chemical structures
and for which p-aminohippurate (PAH)
and fluorescein (FL) are prototypical substrates (17, 18). Only
recently has this basolateral transporter been cloned (13, 14, 19). In accordance with the sequence data, there are consensus sites for phosphorylation of the transporter protein by protein kinase C (PKC),
thereby making it a potential target for regulation via pathways
involving the activation of PKC. Indeed, some years before the sequence
data became available, studies on the possible regulation of OAT
suggesting that phosphorylation might be important were performed. In
1994, Hohage et al. (6) observed the stimulation of PAH accumulation in
isolated S2 segments of rabbit kidney by phorbol esters. In contrast to
these data, Miller (9) as well as Takano et al. (15) reported an
inhibition of OA accumulation by PKC in killifish renal proximal
tubules (PT) and opossum kidney OK cells, respectively. In addition,
Halpin and Renfro (4) showed that OA secretion in proximal tubular
cells of winter flounder is inhibited by phorbol esters and may
underlie dopaminergic and adrenergic regulation. The reason(s) for
these apparent discrepancies is not known at present and might be
species specific or due to differences in the actual experimental
conditions, as, for example, the buffer composition.
We took advantage of an experimental protocol described recently by
Welborn et al. (18) to determine the initial transport rate (1st 25 s)
of OAT in isolated S2 segments of rabbit kidney in real time as well as
in paired experiments. As has been shown in previous studies (18), the
data obtained with this system and FL as a substrate reflect the
activity of OAT, as do measurements of PAH uptake. Our data show that
in rabbit PT, activation of PKC, either directly with 4-
-phorbol
12-myristate 13-acetate (PMA) or indirectly with ligands of receptors
coupled to the PKC pathway, inhibits the activity of OAT. This effect
is not due to changes in counterion availability but rather to a direct
inhibition of OAT.
 |
METHODS |
Chemicals.
Spectral-grade FL was purchased from Molecular Probes (Eugene, OR).
Cell-Tak was obtained from Collaborative-Biomedical Products (Bedford,
MA). All other chemicals were purchased from commercial sources and
were of the highest available purity. All compounds added to the
superfusion solution other than FL were checked for interference with
the photon counts obtained from FL under our experimental conditions.
No significant interference could be observed.
Solutions.
A modified rabbit Ringer solution, used in the experiments as a
dissection buffer, a superfusion buffer, and an uptake medium, consisted of the following (in mmol/l): 110 NaCl, 25 NaHCO3, 5 KCl, 2 Na2HPO4,
1.8 CaCl2, 1 MgSO4, 10 sodium acetate, 8.3 glucose, 5 alanine, 4 lactate, and 0.9 glycine, with an osmolality of
~290 mosmol/kgH2O. Before use,
buffer solutions were filtered (0.4-µm pore size) and aerated for 20 min with the use of 95% O2-5%
CO2, and the pH was adjusted to
7.4 with NaOH or HCl.
Animals and PT dissection.
Adult male New Zealand White rabbits were killed by intravenous
injection with pentobarbital sodium. A kidney was immediately removed,
perfused with a HEPES-sucrose buffer (250 mmol/l sucrose, 10 mmol/l
HEPES, pH 7.4 with Tris base), and transversely sliced with the use of
a single-edge razor. A kidney slice was placed in a plastic petri dish
containing ice-cold dissection buffer and aerated with 95%
O2-5%
CO2. Segments of PT were
individually dissected from the cortical zone, and a segment was
transferred to an aluminum superfusion chamber containing superfusion
buffer. The chamber floor consisted of a no. 1 glass coverslip coated with 1 µl of Cell-Tak. The chamber was transferred to the stage of an
Olympus IMT microscope and superfused with buffer at 5 ml/min. The
chamber was fitted with a water jacket, and its temperature, as well as
that of the incoming superfusion buffers, was maintained at 37°C.
With the use of two-way switching valves, superfusion buffers could be
changed in a few seconds while a constant flow rate and temperature
were maintained. A small vacuum line on the side of the chamber removed overflow.
Measuring FL uptake into rabbit PT.
Initial rates of FL uptake were calculated from measurements of
epifluorescence intensity as described previously (18). In brief, a
monochromater (Photon Technology International, Brunswick, NJ) equipped
with a 75-W xenon lamp was used to generate excitation light at 490 nm
(±1-2 nm). A 490-nm dichroic mirror (model 490DCLP; Omega
Optical, Brattleboro, VT) directed excitation light to the PT segment
through a ×40 oil-immersion fluor objective (1.3 NA, Nikon).
Emitted light passed through a 520-nm long-pass filter (Omega Optical)
before reaching a photomultiplier tube (model HC120; Hamamatsu,
Bridgewater, NJ). Photomultiplier output was recorded at 1-s intervals
with the use of LabView software (National Instruments, Austin, TX)
installed in a personal computer. Because the halftime for solution
exchange in the chamber was 1.5-2 s, the first 5-10 s of the
fluorescence record (i.e., ~5 halftimes) was discarded after the
switch to a buffer containing FL. The next 25 s of the record was
linear. The slope of this line was calculated and represents the
initial rate of FL uptake. The interval between two measurements was 10 min to allow washout of FL (18). Routinely, uptake was determined under
control conditions at least three times, and an experimental maneuver
was only performed if those three control uptakes were stable (
5%
difference). The last control uptake before starting an experimental
maneuver was normalized to 100%, and all the other uptake rates are
expressed as a percentage of this control uptake rate.
Because FL shows only weak pH dependence at physiological pH levels (a
change in pH from 8.0 to 7.2 caused a drop in FL fluorescence of only
5%), possible changes in intracellular pH would not have affected our measurements, as we have determined with the setup described here. Furthermore, stimulation of PKC is expected to activate the basolateral Na+/H+
exchange (10), thereby leading to a rise in pH. Yet, a rise in pH
would, if anything at all, enhance fluorescence and would thereby mimic
an increase in FL uptake rather than a decrease, as observed in this study.
Statistical analysis.
Unless indicated otherwise, data are means ± SE. Sample size
(n) refers to
n separate tubules. For each series,
tubules from at least four rabbits were used. Comparisons of observed
differences to determine their statistical significance at the 0.05 level were performed with the use of either Student's
t-test or analysis of variance and a
posttest, employing the Student-Newman-Keuls method.
 |
RESULTS AND DISCUSSION |
In the present study we used bicarbonate-buffered, nutrient-rich media
and physiological incubation temperatures (18). These conditions are
likely to keep the rates of transport and/or cellular metabolism under
conditions closely resembling the in vivo situation and therefore allow
the investigation of physiological regulatory processes. We tested four
different maneuvers, all known to lead to an activation of PKC in PT
(1, 3, 7): application of 1) PMA,
2)
1,2-dioctanoyl-sn-glycerol (DOG),
3) the
1-receptor agonist
phenylephrine (PE), and 4)
bradykinin (BK). To show that the observed effects were indeed
predominantly due to PKC activation, we used the PKC inhibitor
bisindolylmaleimide I (BIM) (2, 16) in a concentration at which no
other effects have been described thus far
(10
7 mol/l).
As already mentioned above, the setup used in this study allows us to
determine the initial uptake rate of FL in real time and in paired
experiments (18). Figure 1 shows a typical
experiment: after three runs under control conditions (Fig. 1,
A-C) in which the uptake rate
stayed virtually constant (Fig. 1I),
application of 10
6 mol/l PE
induced a time-dependent (Fig. 1,
D-F) and reversible inhibition
of FL (Fig. 1, G-H). During the
60 s when FL uptake was determined, PE was not present, in order to use
the same transport substrate solution during the whole experiment. This
also held true for all the other modulators of FL uptake (PMA, DOG,
BIM, and BK). Addition of the modulators to the FL solution did not alter the effects of the modulators on FL uptake (data not shown). Previously, it has been shown that FL uptake in the system used here is
virtually completely mediated by OAT (18). Thus the method applied here
is indeed suitable to detect rapid regulatory changes in the activity
of OAT in paired experiments.

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Fig. 1.
Original recordings of a typical fluorescein (FL) uptake experiment
(see METHODS for details). Control
uptake of FL was determined 3 consecutive times with intervals of 10 min (A-C). Uptake remained
virtually constant during control period (see
I). Addition of
10 6 mol/l phenylephrine
(PE) led to a time-dependent
(D-F) and reversible
(G-H) inhibition of FL uptake.
I: slopes for different uptake
measurements (A-H) are
provided; bar indicates presence of PE. s, Seconds; p, photons.
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Figure 2 shows the effects of DOG
(10
5 mol/l) or PMA (at
concentrations of 10
8 and
10
7 mol/l) on OAT activity.
Under control conditions (i.e., addition of the vehicle only after the
three control runs), the activity of OAT remained virtually constant
(there was a slight decrease to 95% of control at 60 min). However,
the addition of PMA or DOG induced a rapid and dramatic decrease in OAT
activity. At 10
7 mol/l PMA
there was already a significant reduction after 5 min, as shown in Fig.
2. PMA was added at time (t) = 25 min, and the next uptake determination was performed at
t = 30 min. In the presence of
10
7 mol/l PMA, uptake was
reduced significantly, even after this short period of exposure (5 min). These data show that PKC activation inhibits OAT in rabbit PT as
well. In four additional experiments, we determined the effect of
10
7 mol/l PMA after 45 and
55 min of exposure. Uptake was reduced to 31 ± 6% of control after
55 min, and there was no significant difference between uptake at 45 and 55 min (Fig. 2, inset). Thus the
maximum degree of inhibition by PMA is to 31% of control, a value
comparable with the human kidney PAH transporter (8). To confirm that
the effects of PMA were indeed due to PKC activation, we used the
inhibitor BIM. Although the inhibition constant of BIM for
inhibition of PKC in vitro is very low (14 nmol/l, see Ref. 16), the
50% inhibitory concentration of BIM for the inhibition of PKC-induced
effects in intact cells is ~200-250 nmol/l (2). This can be
explained, at least in part, by the fact that BIM has to compete with
high intracellular ATP concentrations for binding to PKC. Because BIM
looses its specificity for PKC at higher concentrations, we used it at
a concentration of 100 nmol/l; even so, one cannot expect a complete
inhibition of PKC under these conditions. However, the specificity of
BIM is retained. Application of BIM alone had no significant effect on
the activity of OAT under our conditions (Fig.
3), suggesting that there is no
constitutive suppression of OAT activity via PKC. As shown in Fig.
4, BIM reduced the inhibitory effect of PMA
to less than one-half of the original effect. Therefore, we can
conclude that the effect of PMA was indeed mediated by PKC stimulation.
Of course, we cannot completely rule out the possibility that part of
the action of PMA was not due to PKC activation. PKC-independent
modulation of transport activity by PMA has been proposed, for example,
for the
Na+/H+
exchanger NHE3 (5). However, the underlying mechanisms are still
unknown and possibly may involve the action of other kinases.

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Fig. 2.
Effect of protein kinase C (PKC) stimulation with phorbol 12-myristate
13-acetate (PMA) or
1,2-dioctanoyl-sn-glycerol (DOG) on
initial uptake rate of FL. All values are normalized to last control
measurement. Control, addition of vehicle in which PMA or DOG was
dissolved only (dimethyl sulfoxide; final concentration was always
1:10,000). Sample size (n) = 6 for
each value plotted. * P < 0.05 vs. last control measurement. Inset:
effect of 10 7 mol/l PMA in
4 experiments in which time of exposure was extended to 55 min; symbols
correspond to those as defined in main.
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Fig. 3.
PKC inhibitor bisindolylmaleimide I (BIM;
10 7 mol/l) did not affect
FL uptake significantly during time of exposure used in this study;
n = 6 for each value plotted.
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Fig. 4.
Change of FL uptake after 25 min vs. last control value under different
conditions. Time control shows that FL uptake was virtually constant
during period of experiments (n = 6).
PKC inhibitor BIM (10 7
mol/l) reduced effect of PMA
(10 8 mol/l,
n = 6). PMA
(10 8 mol/l) induced more
change in presence than in absence of exogenous -ketoglutarate (KG;
10 5 mol/l,
n = 6). Methyl succinate (MS;
10 3 mol/l,
n = 4) did not alter effect of PMA
(10 8 mol/l). resp,
Respective.
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Because the initial transport rate of OAT is affected by the
availability of
-ketoglutarate as a counterion, changes in OAT activity could be due to changes in metabolism that
provide
-ketoglutarate (18). To test this possibility,
we determined the effect of PMA in the continuous presence of
-ketoglutarate (10
5
mol/l) in the superfusion solution under the assumption that in this
case, changes in metabolism would not lead to significant changes in
the availability of
-ketoglutarate for OAT. As shown in Fig. 4, PMA
exerted an even greater effect on OAT in the presence than in the
absence of exogenous
-ketoglutarate. This exaggeration of the PMA
effect is most probably due to an enhanced OAT activity in the presence
of exogenous
-ketoglutarate, as shown previously (18). Thus we
conclude that the effect of PKC stimulation is not due to changes in
cellular metabolism but rather to a more direct interaction with OAT.
Furthermore, the inhibitory action of PKC activation is not due to an
interaction with the
Na+-
-ketoglutarate
cotransporter because 1) this
transport has been shown to be unaffected by PKC stimulation (12) and
2) PMA still inhibited OAT activity
when the Na+-
-ketoglutarate
cotransporter was functionally eliminated (18) by the addition of 1 mmol/l methyl succinate. In the presence of 1 mmol/l methyl succinate,
10
8 mol/l PMA reduced OAT
activity by 52 ± 5% after 25 min
(n = 4) compared with 43 ± 5% in
the absence of methyl succinate (no significant difference). Thus OAT
itself is regulated by PKC. The effect of BIM,
-ketoglutarate, and
methyl succinate on FL uptake modulation after 5, 15, and 35 min of
exposure to the different drugs was also tested. The effects were
qualitatively the same: BIM prevented inhibition, methyl succinate had
no effect on transport modulation, and
-ketoglutarate increased the
inhibitory effect of PMA slightly. For reasons of clarity, we present
only the 25-min data in Fig. 4 (see also Fig. 7).
To investigate whether PKC-mediated regulation also takes place after
physiological stimulation of receptors that are coupled to the PKC
pathway, we applied PE or BK, both of which are known to bind to
receptors in the plasma membrane
(
1-receptor and BK receptor,
respectively) that couple to the PKC pathway (1, 3, 7). As shown in
Figs. 5 and 6,
both substances led to a reversible inhibition of OAT activity.
Furthermore, the action of both substances was prevented by the PKC
inhibitor BIM (Fig. 7,
A and
B), showing that they indeed acted
via stimulation of PKC. The effects of PE and BK in the presence of
10
7 mol/l BIM were not
significantly different from time control in the presence of BIM (Fig.
3), indicating that these two substances acted exclusively via PKC. We
also tested the effect of PE in the presence of 2 × 10
7 mol/l BIM: there was no
significant effect of PE (95 ± 3% of control,
n = 4) or BK (96 ± 4% of control,
n = 3) on FL uptake compared with time
control. The reversibility of the effect also showed that PKC-mediated
inhibition of OAT was not due to a unspecific toxic action on the
cells. The rapid time course of the reversibility may be the result of
a rapid dephosphorylation of the transporter itself by endogenous
phosphatases. Yet, a direct phosphorylation of OAT has not been
demonstrated thus far. Alternatively, OAT activity may be decreased
indirectly by PKC (e.g., retrieval from the plasma membrane or
phosphorylation of regulatory proteins), and dephosphorylation of
regulatory factors accounts for the reversibility. De novo synthesis of
transport proteins can be excluded from the time course of recovery. Of
course, our data do not allow us to draw a final conclusion with
respect to the mechanisms underlying reversibility, and detailed future
studies will have to address this question. Nevertheless, OAT seems to
be under the physiological control of two regulatory systems:
circulating hormones, like BK, and the autonomic nervous system,
represented by the
1-receptor ligand phenylephrine. The in vivo importance of these effects remains
to be determined. However, the existence of such a regulation in
freshly isolated PT argues strongly in favor of the physiological relevance of the observed regulation. Additional work is needed to
determine whether OAT is phosphorylated directly by PKC or whether
there are additional events involved.

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Fig. 5.
Bradykinin (BK; 10 6 mol/l)
induced a time-dependent and reversible inhibition of FL uptake;
n = 6 for each value plotted.
* P < 0.05 vs. last control
value;
# P < 0.05 vs. last value in presence of BK.
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Fig. 6.
PE (10 6 mol/l) induced a
time-dependent and reversible inhibition of FL uptake;
n = 6 for each value plotted.
* P < 0.05 vs. last control
value.
# P < 0.05 vs. last value in presence of PE.
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Fig. 7.
PKC inhibitor BIM (10 7
mol/l) abolished effect of BK (A)
and PE (B). Effect of both
substances was not significantly different from time control (see Fig.
3 in presence of BIM).
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|
The data of the present study are in good agreement with those obtained
by Miller (9), Takano et al. (15), Halpin and Renfro (4), and Lu et al.
(8), who also reported an inhibitory action of PKC on OA transport,
albeit in other species. The reasons for the apparent discrepancy with
the data of Hohage et al. (6) are not clear at the moment. Two possible
explanations are 1) the difference
in the composition of the buffer solution, because Hohage et al. did
not use nutrient-rich media, and 2)
determination of steady-state accumulation of PAH in the tubules, not
initial transport rates, by Hohage et al. The different
regulation of OA transport by oxymetazoline (4) and PE is
most probably due to the fact that oxymetazoline interacts primarily
with
2-receptors, whereas PE is
an
1-agonist (7).
In summary, our data show that the initial transport rate of OAT in
rabbit PT is under the negative control of physiological stimuli (e.g.,
hormones or the autonomic nervous system) that act via the activation
of PKC. This action is not mediated by changes in metabolism or
counterion availability but by a direct action on the transporter itself.
 |
ACKNOWLEDGEMENTS |
We thank Kristen Evans, Theresa Wunz, Apichai Shuprisha, and Olga
Brokl for advice and assistance.
 |
FOOTNOTES |
This work was supported in part by the BRAVO! program of the University
of Arizona, the Deutsche Forschungsgemeinschaft (DFG Ge 905/3-3), and
National Institutes of Health Research Grant DK-49222.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Gekle,
Physiologisches Institut, Universität Würzburg,
Röntgenring 9, 97970 Würzburg, Germany (E-mail:
michael.gekle{at}mail.uni-wuerzburg.de).
Received 11 December 1998; accepted in final form 7 May 1999.
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0002-9513/99 $5.00
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