Inhibition of PAH transport by parathyroid hormone in OK
cells: involvement of protein kinase C pathway
Junya
Nagai,
Ikuko
Yano,
Yukiya
Hashimoto,
Mikihisa
Takano, and
Ken-Ichi
Inui
Department of Pharmacy, Kyoto University Hospital, Faculty of
Medicine, Kyoto University, Kyoto 606-01, Japan
 |
ABSTRACT |
We have previously shown that the
p-aminohippurate (PAH)
transport system in OK kidney epithelial cell line is under the
regulatory control of protein kinase C. Parathyroid hormone (PTH) could
activate protein kinase C, as well as protein kinase A, in OK cells. In the present study, the effect of PTH on PAH transport was studied in OK
cells. PTH inhibited the transcellular transport of PAH from the basal
to the apical side, as well as the accumulation of PAH in OK cells.
Basolateral PAH uptake was inhibited by PTH in a dose- and
time-dependent manner. Protein kinase A activators did not affect the
transcellular transport or the accumulation of PAH. The PTH-induced
inhibition of the accumulation of PAH was blocked by a protein kinase C
inhibitor staurosporine. These results suggest that PTH inhibits the
PAH transport in OK cells and that the messenger system mediated by
protein kinase C, not protein kinase A, plays an important role in the
regulation of PAH transport by PTH.
organic anion transport; hormonal control; renal secretion; opossum
kidney cell
 |
INTRODUCTION |
THE ORGANIC ANION TRANSPORT system of the renal
proximal tubule has physiological roles in the excretion of a wide
variety of anionic compounds, including endogenous metabolites and
xenobiotics, into the urine (19, 20). This function is important
because many organic anions are toxic and need to be removed as
promptly as possible. The organic anion transport system has been
studied using different tissue preparations, including renal cortical slices, isolated proximal tubules, and purified brush-border and basolateral membrane vesicles (17, 20, 25). These studies have revealed
the kinetics, driving forces, and substrate specificities of the
organic anion transport system. However, little information is
available concerning the regulation of organic anion transport, especially by hormone via the receptor-mediated generation of intracellular second messengers. This might be partly due to the lack
of a good model system with which to study the regulatory factors for
organic anion transport under well-defined conditions. We reported that
OK cells, which were established from the American opossum kidney (11),
are a useful in vitro model system with which to study the organic
anion transport across intact epithelial cells (8, 16, 23). Moreover,
we showed that the transport of
p-aminohippurate (PAH), a typical
organic anion, in OK cells is regulated by protein kinase C (24).
Parathyroid hormone (PTH) has multiple effects on the kidney. In
addition to the stimulation of gluconeogenesis and 25-hydroxyvitamin D3 1
-hydroxylase activity, the
major physiological effect of PTH is on the regulation of tubular
transport processes (14). In renal proximal tubule, PTH regulates the
activities of apically located
Na+-phosphate cotransport and
Na+/H+
exchange by activating intracellular regulatory pathways (protein kinase A, protein kinase C) via PTH receptor-mediated production of
intracellular messengers [adenosine 3',5'-cyclic
monophosphate (cAMP), diacylglycerol] (3, 15). OK cells have
specific PTH receptors coupled to the protein kinase A and protein
kinase C pathways as well as various transport systems similar to those in renal proximal tubules (3, 15, 21). In addition, PTH/PTH-related peptide receptor has been cloned by COS-7 expression, using an OK cell
cDNA library (9). Thus OK cells could be useful in studying the
regulation of the organic anion transport system by PTH.
The purpose of this study is to examine whether PAH transport in OK
cells is regulated by PTH. The results show that PTH decreases the
activity of the PAH transport in OK cells via the messenger system
mediated by protein kinase C, rather than by protein kinase A.
 |
MATERIALS AND METHODS |
Cell culture. OK cells were cultured
in plastic dishes (Corning Glass Works, Corning, NY) in medium 199 (Flow Laboratories, Rockville, MD) containing 10% fetal bovine serum
(Whittaker Bioproducts, Walkersville, MD) without antibiotics, in an
atmosphere of 5% CO2-95% air at
37°C, and subcultured every 5-7 days using 0.02% EDTA and
0.05% trypsin (8). OK cells were used between passages 73 and 97.
Transport measurements. PAH transport
was measured in OK cell monolayers cultured in Transwell chambers
(Costar, Cambridge, MA). To prepare cell monolayers, cells were seeded
at a density of 4 × 105
cells/cm2 on polycarbonate
membranes (3 µm pore size) in Transwell cell chambers (4.71 cm2 surface area), which were
placed in six-well cluster plates. The volume of medium inside and
outside the chambers was 1.5 and 2.6 ml, respectively. Fresh medium was
replaced every 2 days, and the cells were used between the 5th and 7th
days after seeding. Transport was measured at 37°C in Dulbecco's
phosphate-buffered saline, containing (in mM) 137 NaCl, 3 KCl, 8 Na2HPO4,
1.5 KH2PO4, 1 CaCl2, and 0.5 MgCl2 (for PAH transport), or in
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline, containing (in mM) 137 NaCl (or for
Na+-independent transport, 137 choline chloride), 5.4 KCl, 1 CaCl2, 0.5 MgCl2, and 14 HEPES-tris(hydroxymethyl)aminomethane (for phosphate transport)
supplemented with 5 mM
D-glucose.
The transcellular transport of PAH across OK cell monolayers was
measured as described (8). To measure the cellular uptake of
[14C]PAH (15 µM) and
[32P]phosphate (100 µM), the reaction was initiated by adding each buffer containing 5 mM
D-glucose and the substrate to
either the basal or the apical side of the monolayers.
D-[3H]mannitol
(15 µM) was added simultaneously to correct for extracellular trapping and nonspecific uptake of the substrate in the PAH uptake experiments. After an incubation for a specified period, the uptake medium was aspirated and discarded, and the membrane was
rapidly washed three times with ice-cold buffer containing 5 mM
D-glucose. The cell monolayers
on the membrane were solubilized in 0.5 ml of 0.1 M sodium hydroxide,
and the amount of substrate taken up by the cells was measured by
counting the radioactivity.
Cell treatment. Stock solutions of
dibutyryladenosine 3',5'-cyclic monophosphate
(dibutyryl-cAMP), 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), forskolin, and staurosporine were prepared in dimethyl sulfoxide (DMSO). The final concentration of DMSO during
exposure was 0.25-0.5%. The compounds were applied to both the
basolateral and apical sides for a specified period. The control cells
were incubated with the same concentration of DMSO in each experiment.
Finally, the cell monolayers were washed three times with the uptake
buffer before measuring the uptake.
Analytical methods. The radioactivity
was determined in 5 ml of ACS II (Amersham International,
Buckinghamshire, UK) by liquid scintillation counting using an external
standard to correct for quenching. The appropriate crossover correction
was given to separate the radioactivities of
3H and
14C. Protein was determined by the
method of Bradford (2) with bovine
-globulin as the standard.
Statistical analysis was performed by Student's
t-test, or by the one-way analysis of
variance with the Dunnett's test for post hoc analysis
(P < 0.05 for significance).
Materials.
p-[Glycyl-1-14C]aminohippurate
(1.53 GBq/mmol),
D-[3H]mannitol
(728.9-828.8 GBq/mmol), and
KH232PO4
(37 GBq/mmol) were obtained from Du Pont-New England Nuclear (Boston,
MA). Synthetic bovine (1
34)-PTH, phorbol 12-myristate 13-acetate
(PMA), and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma
Chemical (St. Louis, MO). Dibutyryl-cAMP, forskolin, and staurosporine
were purchased from Wako Pure Chemicals (Osaka, Japan). 8-Br-cAMP was
purchased from Nacalai Tesque (Kyoto, Japan). All other chemicals used
were of the highest purity available.
 |
RESULTS |
Effect of PTH and dibutyryl-cAMP on
Na+-phosphate cotransport.
Figure 1 shows the effect of PTH and
dibutyryl-cAMP on phosphate uptake from the apical side of OK cells.
Phosphate was transported sodium dependently. PTH produced a
time-dependent decrease in Na+-phosphate cotransport. In
addition, the uptake of phosphate was inhibited by pretreatment for 3 h
with dibutyryl-cAMP (10
5
M). These findings showed the regulation coupled to PTH receptor by PTH
and the activation of protein kinase A by dibutyryl-cAMP. Therefore, OK
cells are useful in studying the regulation of PAH transport by PTH.

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Fig. 1.
Effect of parathyroid hormone (PTH) and dibutyryl-cAMP on phosphate
uptake from the apical side of OK cell monolayers. Confluent monolayers
were incubated with PTH
(10 7 M) for 30 min or 3 h
and with dibutyryl-cAMP
(10 5 M) for 3 h. After the
cells were washed,
[32P]phosphate (100 µM) was added to the apical side of the monolayers, and
[32P]phosphate uptake
for 5 min at 37°C was measured. Each column is the mean ± SE of
3 monolayers of a typical experiment.
* P < 0.05, significant
difference from control.
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|
Effect of PTH on the basal-to-apical transport and
accumulation of PAH. We examined the effect of PTH on
the transcellular transport from the basal to apical side and
accumulation of
[14C]PAH in OK cells.
PTH (final concentration
10
7 M) or its vehicle was
added at 25 min after initiation of transport measurement. The
transcellular transport activities of PAH at 30, 45, and 60 min from
start of the PAH transport (5, 20, and 35 min after the addition of PTH
or its vehicle, respectively) were measured. At 30 and 45 min, the
transcellular transport of PAH slightly decreased by PTH addition
compared with its vehicle (data not shown), and PTH significantly
inhibited the transcellular transport of PAH from the basal to apical
side at 60 min (P < 0.05) (Fig.
2A). The
simultaneously measured accumulation of PAH in OK cells at 60 min was
also significantly inhibited by PTH (Fig.
2B). The inhibition of transcellular
transport and intracellular accumulation of PAH by PTH suggested that
PTH affects, at least in part, the basolateral transport of PAH in OK
cells. Therefore, the effects of PTH on PAH uptake across basolateral
membrane of OK cells were further studied.

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Fig. 2.
Effect of PTH on basal-to-apical transport
(A) and accumulation
(B) of
p-aminohippurate (PAH) by OK cell
monolayers. [14C]PAH
(15 µM) and
D-[3H]mannitol
(15 µM) were added to the basal side of monolayers, and distilled
water (open bars, final concentration 0.25% vol/vol) or PTH (solid
bars, final concentration
10 7 M) was added at 25 min
after start of the transport measurement.
A: at 60 min, medium on apical side
was collected (100 µl), and radioactivity levels were counted to
determine transcellular transport of
[14C]PAH.
D-[3H]mannitol
was used to correct for paracellular flux. PAH transport in absence of
PTH (control) was 180.0 ± 15.1 pmol · cm 2 · 60 min 1.
B: after a 60-min transport
measurement, accumulation of
[14C]PAH in OK cells
was determined.
D-[3H]mannitol
was used to correct for extracellular trapping and nonspecific uptake.
PAH accumulation in absence of PTH (control) was 53.7 ± 4.5 pmol · mg
protein 1 · 60 min 1. Each column is the
mean ± SE of 9 monolayers of 3 separate experiments.
* P < 0.05, significant
difference from each control.
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|
Effect of PTH pretreatment concentration and time on
basolateral PAH uptake. We examined the effect of
various concentrations of PTH on the PAH uptake for 1 min from the
basal side of OK cells. The OK cells were treated with various
concentrations
(10
11-10
7
M) of PTH for 15 min, then the basolateral PAH uptake for 1 min was
measured. As shown in Fig. 3, PTH inhibited
the PAH uptake in a dose-dependent manner, and it was significant at
10
7 M
(P < 0.05). We further examined the
dose response curve after 3-h pretreatment with PTH. The dose response
curve after 3-h pretreatment slightly shifted to the left compared with
that after 15-min pretreatment, but there was no significant difference
between two experimental groups at each PTH concentration
(10
11-10
7
M) (data not shown).

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Fig. 3.
Dose-dependent effect of PTH on PAH uptake from the basal side of OK
cell monolayers. Confluent monolayers were incubated for 15 min with
various concentrations of PTH
(10 11-10 7
M). After the cells were washed,
[14C]PAH (15 µM) and
D-[3H]mannitol
(15 µM) were added to the basal side of monolayers, and
[14C]PAH uptake for 1 min at 37°C was measured. Each point is the mean ± SE of
6-7 monolayers of 3 separate experiments.
* P < 0.05, significant
difference from control.
|
|
Figure 4 shows the effect of PTH
pretreatment periods (5 to 60 min) on PAH uptake from the basal side of
OK cells. Exposure to PTH caused a time-dependent decrease in PAH
uptake, which was significant at a period of 15 min or more
(P < 0.05).

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Fig. 4.
Effect of time on PTH-induced inhibition of PAH uptake from the basal
side of OK cell monolayers. Confluent monolayers were incubated for
various periods with PTH
(10 7 M), and
[14C]PAH uptake from
the basal side of monolayers was measured as described in Fig. 3. Each
point is the mean ± SE of 8-9 monolayers of 3 separate
experiments. * P < 0.05, significant difference from control at time
0.
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Effect of protein kinase A activators on PAH
transport. OK cells have specific PTH receptors coupled
to not only the protein kinase C pathway but also the protein kinase A
pathway. We have already reported that protein kinase C activators,
such as active phorbol esters and diacylglycerols, inhibit PAH
transport in OK cells, and protein kinase C may have an important role
in the regulation of PAH transport (24). Therefore, we further analyzed the effect of protein kinase A activators on PAH transport in OK cells.
Figure 5 shows the effect of protein kinase
A activators, which are cAMP analogs, dibutyryl-cAMP and 8-Br-cAMP
(10
5 M), an adenylate
cyclase activator forskolin
(10
5 M), and a
phosphodiesterase inhibitor IBMX
(10
3 M). However, exposure
to these protein kinase A activators for 3 h had no effect on the
initial rate of PAH uptake from the basal side of OK cells. Moreover,
neither the transcellular transport from the basal to the apical side
nor the steady-state accumulation of PAH in OK cells was affected by
the incubation of dibutyryl-cAMP and forskolin
(10
5 M) for 3 h before
transport measurements (Fig. 6).

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Fig. 5.
Effect of protein kinase A activators on PAH uptake from the basal side
of OK cell monolayers. Confluent monolayers were incubated for 3 h with
cAMP analogs, forskolin
(10 5 M) and
3-isobutyl-1-methylxanthine (IBMX)
(10 3 M). After the cells
were washed, [14C]PAH
uptake from the basal side of monolayers was measured as described in
Fig. 3. Each column is the mean ± SE of 6-7 monolayers of 3 separate experiments.
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Fig. 6.
Effect of protein kinase A activators on basal-to-apical transport
(A) and accumulation
(B) of PAH by OK cells. Confluent
monolayers were incubated for 3 h without ( ) or with
10 5 M dibutyryl-cAMP ( )
or 10 5 M forskolin ( ).
After the cells were washed, transcellular transport at 15, 30, 45, and
60 min (A) and accumulation at 60 min (B) of
[14C]PAH were measured
as described in Fig. 2. Each point or column is the mean ± SE of
6-8 monolayers of 3 separate experiments.
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|
To examine whether protein kinase C and protein kinase A systems
mutually interact on the inhibition of PAH transport in OK cells by
PTH, the effects of a protein kinase C activator PMA (10
8 M) alone and in
combination with dibutyryl-cAMP
(10
5 M) were examined. PMA
inhibited PAH uptake from the basal side of OK cells as described
previously (24). However, its inhibitory effect was not affected by
coincubation with dibutyryl-cAMP (Fig. 7).

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Fig. 7.
Effect of pretreatment with phorbol 12-myristate 13-acetate (PMA) alone
or in combination with dibutyryl-cAMP on PAH uptake from the basal side
of OK cell monolayers. Confluent monolayers were incubated for 3 h in
absence or presence of PMA
(10 8 M) and/or
dibutyryl-cAMP (10 5 M).
After the cells were washed,
[14C]PAH uptake from
the basal side of monolayers was measured as described in Fig. 3. Each
column is the mean ± SE of 5 or 6 monolayers from 2 separate
experiments. * P < 0.05, significant difference from control.
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Effect of staurosporine on PTH-induced inhibition of
PAH accumulation. To clarify whether protein kinase C
activation is linked directly to the inhibition of PAH transport by
PTH, we examined the effect of staurosporine, a potent inhibitor of
protein kinase C, on the PTH-induced inhibition of PAH accumulation.
When cells were pretreated with staurosporine before adding PTH, the
inhibitory effect of PTH on the PAH accumulation was almost completely
blocked (Fig. 8).

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Fig. 8.
Effect of staurosporine on PTH-induced inhibition of PAH
accumulation of OK cell monolayers.
[14C]PAH (15 µM) and
D-[3H]mannitol
(15 µM) were added to the basal side of monolayers with staurosporine
(10 6 M) or its vehicle, and
distilled water (open bars, final concentration 1% vol/vol) or PTH
(solid bars, final concentration
10 6 M) was added at 25 min
after starting of the transport measurement. At 60 min, accumulation of
[14C]PAH in OK cells
was determined as described in Fig. 2. PAH accumulations in absence of
PTH (control) for vehicle and staurosporine were 101.5 ± 6.8 and
73.3 ± 8.5 pmol · mg
protein 1 · 60 min 1, respectively. Each
column is the mean ± SE of 7-8 monolayers of 3 separate
experiments. * P < 0.05, significant difference from each control.
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 |
DISCUSSION |
OK cells have various transport systems similar to those in renal
proximal tubules, including
Na+-coupled transport systems for
amino acids, hexoses, proton, and inorganic phosphate (12), in addition
to the PAH transport system we reported (8, 16, 23). OK cells also have
specific PTH receptors coupled to the protein kinase A and protein
kinase C pathways (3, 15). In addition, PTH/PTH-related peptide
receptor has been cloned by COS-7 expression, using an OK cell cDNA
library (9). Thus OK cells are useful in studying the effect of PTH on
transport systems in renal proximal tubules. Furthermore, several studies have shown that PTH regulates the activities of apically located Na+-phosphate cotransport,
as well as
Na+/H+
exchange, via the activation of protein kinase A and/or protein kinase C pathways (7, 21, 22). In this study, we evaluated whether PTH
regulates the PAH transport system expressed in OK cells via the
protein kinase A and/or protein kinase C pathways.
Miyauchi et al. (13) have shown that the OKH, a clonal cell line of OK
cells, is resistant to inhibition of
Na+-phosphate cotransport by PTH,
suggesting that the resistance is associated with the lack of
stimulated phospholipase C. To assess the OK cells we have used, the
effect of PTH and dibutyryl-cAMP on
Na+-phosphate cotransport was
examined. Na+-dependent phosphate
uptake from the apical side of OK cells was inhibited by dibutyryl-cAMP
and PTH, suggesting that the OK cells are useful for examining the
effect of protein kinase A activation and PTH on the PAH transport
system.
PTH inhibited both the transcellular transport and intracellular
accumulation of PAH in OK cell monolayers. These findings indicated
that the inhibitory effect of PTH is related, at least in part, to the
inhibition of PAH uptake from the basal side of OK cells. Therefore, we
further studied the effect of PTH on the basolateral uptake of PAH in
OK cells. The basolateral uptake of PAH in OK cells was inhibited by
PTH in a dose- and time-dependent manner. However, the dose-response
curve given in Fig. 3 for PTH inhibition of PAH uptake in OK cells
showed a rather low sensitivity to PTH. Quamme et al. (21) demonstrated
that half the maximally inhibitory effect of PTH on
Na+-phosphate was
10
12-10
11
M. The difference between these sensitivities to PTH remains unclear.
OK cells have PTH-responsive dual signal pathways that activate both
protein kinase A and protein kinase C. We previously demonstrated that
protein kinase C activation by phorbol esters and diacylglycerols
inhibits the PAH transport in OK cells (24). Therefore, it is likely
that the inhibition of PAH transport by PTH is due, at least in part,
to the activation of protein kinase C. The present study with
staurosporine, a protein kinase C inhibitor, has demonstrated the
involvement of protein kinase C on the inhibitory effect of PAH
transport by PTH. In contrast, various protein kinase A activators,
such as cAMP analogs (dibutyryl-cAMP and 8-Br-cAMP), an adenylate
cyclase activator (forskolin), and a phosphodiesterase inhibitor
(IBMX), had no effect on PAH transport in OK cells. Moreover, neither a
dose-dependent
(10
7-10
3
M) nor time-dependent (5-180 min) effect by
dibutyryl-cAMP was observed (data not shown). Friedman et al. (4) have
shown that activation of both protein kinase A and protein kinase C
pathways is necessary for PTH stimulation of calcium uptake in mouse
distal convoluted tubule cells. However, the inhibitory effect on the basolateral PAH uptake by a protein kinase C activator PMA was not
affected by dibutyryl-cAMP (Fig. 7). These results suggested that
protein kinase A activation is not involved in the regulation of PAH
transport in OK cells.
Several studies suggested that the protein kinase C-mediated pathway is
more important than the protein kinase A pathway in regulating
Na+-phosphate cotransport. Quamme
et al. (21) demonstrated that Na+-phosphate cotransport in OK
cells is inhibited by PTH at the concentrations with which activation
of phospholipase C occurs without increasing cAMP. In a clonal OK cell
line lacking PTH-dependent phospholipase C activation, PTH was not able
to reduce Na+-phosphate
cotransport despite a stimulation of adenylate cyclase (13). Moreover,
Hayes et al. (6) showed that the rat renal brush-border membrane
Na+-phosphate cotransporter
(NaPi-2) expressed in Xenopus laevis oocytes is inhibited by pharmacological activation of protein kinase C
but not by protein kinase A. Not only in OK cells but also in a rat
osteosarcoma cell line UMR-106, which possesses PTH-responsive dual
signal transduction systems, the protein kinase C system is involved
exclusively in the stimulation of
Na+-phosphate cotransport by PTH
without any contribution of the protein kinase A system (1).
The inhibition of PAH transport by PTH may be related to PTH-dependent
stimulation of renal gluconeogenesis. Wang and Kurokawa (26) reported
that PTH stimulated glucose production from
-ketoglutarate in
proximal tubules. The regulation of gluconeogenesis by PTH may affect
the intracellular concentration of dicarboxylates such as
-ketoglutarate. In addition, a recent report of Pritchard (18) with
rat renal cortical slices showed that PAH transport was modulated by
changes in intracellular
-ketoglutarate concentration. Therefore,
the inhibitory effect of the basolateral PAH transport by PTH in OK
cells may result from alteration of the intracellular dicarboxylate
(
-ketoglutarate) concentration.
Kippen et al. (10) studied the effect of PTH and cAMP on PAH transport
in rabbit proximal tubule suspension. In contrast to this study with OK
cells, they showed the stimulatory effect of PTH and cAMP on PAH
uptake. The reasons for this discrepancy are unclear but may be related
to methodological differences (culture cell and tubule suspension) or
species differences (opossum and rabbit). Recently, Halpin and Renfro
(5) studied the regulation of active net secretion of a xenobiotic
organic anion, 2,4-dichlorophenoxyacetic acid (2,4-D), by flounder
proximal tubule primary cultures. Activation of protein kinase C but
not protein kinase A caused a decrease in active net secretion of
2,4-D. Moreover, they demonstrated dopaminergic inhibition and
-adrenergic stimulation of 2,4-D net secretion. On the basis of
these findings, various compounds may be regulating the renal secretion
of organic anions.
In conclusion, we demonstrated that PTH inhibits PAH transport in OK
cells. In addition, we showed that the pathway via protein kinase C,
rather than protein kinase A, plays a crucial role in the regulation of
PAH transport by PTH in OK cells. The mechanism by which protein kinase
C activation induces the inhibition of PAH transport activity as well
as the physiological role of the regulation by PTH need further
studying.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, and Culture of Japan,
and by Grants-in-Aid from the Japan Health Sciences Foundation.
 |
FOOTNOTES |
Address for reprint requests: K. Inui, Dept. of Pharmacy, Kyoto Univ.
Hospital, Sakyo-ku, Kyoto 606-01, Japan.
Received 11 March 1997; accepted in final form 18 June 1997.
 |
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