Hormone-stimulated Ca2+
transport in rabbit kidney: multiple sites of inhibition by
exogenous ATP
Jürgen
van Baal1,2,
Joost G. J.
Hoenderop1,2,
Maarten
Groenendijk1,
Carel H.
van
Os1,
René J. M.
Bindels1, and
Peter H. G. M.
Willems2
Departments of 1 Cell
Physiology and 2 Biochemistry,
University of Nijmegen, 6500 HB Nijmegen, The Netherlands
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ABSTRACT |
Exogenous ATP markedly reduced
1-desamino-8-D-arginine vasopressin (dDAVP)-stimulated
Ca2+ transport and cAMP
accumulation in primary cultures of rabbit connecting tubule and
cortical collecting duct cells. Similarly, ATP inhibited the
stimulatory effect of 8-bromo-cAMP. At first sight, this is in
agreement with the "classic" concept that dDAVP exerts its
stimulatory effect via cAMP. However, dDAVP-stimulated Ca2+ transport was markedly
reduced by the protein kinase C (PKC) inhibitor chelerythrine, reported
previously to inhibit the cAMP-independent pathway responsible for
parathyroid hormone-,
[Arg8]vasopressin-,
PGE2-, and adenosine-stimulated
Ca2+ transport. Chelerythrine also
inhibited the increase in Ca2+
transport evoked by the cAMP-independent
A1 receptor agonist N6-cyclopentyladenosine
(CPA). Downregulation of phorbol ester-sensitive PKC isoforms by
chronic phorbol ester treatment has been shown before to be without
effect on hormone-stimulated Ca2+
transport, indicating that the chelerythrine-inhibitable pathway consists of a phorbol ester-insensitive PKC isoform. Here, this maneuver did not affect ATP inhibition of dDAVP-stimulated
Ca2+ transport and cAMP formation,
while abolishing ATP inhibition of CPA-stimulated
Ca2+ transport. These findings
show that ATP acts via 1) a phorbol ester-sensitive PKC isoform to inhibit hormonal stimulation of Ca2+ transport at the level of the
chelerythrine-inhibitable pathway involving a phorbol ester-insensitive
PKC isoform and 2) a phorbol ester-insensitive mechanism to inhibit
V2 receptor-mediated concomitant activation of this pathway and adenylyl cyclase.
connecting tubule; cortical collecting duct; adenosine
5'-triphosphate; protein kinase C; calcium transport
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INTRODUCTION |
THE (PATHO)PHYSIOLOGICAL relevance of exogenous ATP has
been studied extensively. Under physiological conditions, ATP is
released from nerve terminals, endothelial cells, and muscle cells (7, 9). Recently, ATP release associated with the cystic fibrosis transmembrane conductance regulator has attracted a great deal of
interest (14, 22, 28, 29, 31). Certain clinical syndromes are
accompanied by the release of large quantities of ATP as a result of
tissue injury (7, 9, 19). ATP binds to specific cell-surface receptors
that are widely distributed throughout the body. Activation of these
purinoceptors requires pericellular ATP concentrations in the high
micromolar range (7, 9-11, 19, 21, 25, 30).
Purinoceptors are classified into two subtypes, designated
P1 and
P2 (9, 12, 19).
P1 purinoceptors exhibit a greater sensitivity to adenosine and AMP and are subdivided into
A1,
A2, and
A3 receptors (9, 12, 19, 24).
A1 and
A3 receptors have been
demonstrated to inhibit adenylyl cyclase and to activate phospholipase
C (23, 24), whereas A2 receptors
activate adenylyl cyclase (25, 33).
P2 purinoceptors are more
sensitive to ATP and ADP and are subdivided into ionotropic
P2X receptors and G protein-coupled P2Y receptors
(12). P2X receptors are
ligand-gated cation channels, whereas
P2Y receptors increase the
activity of phospholipase C.
Evidence for the involvement of P2
receptors in the regulation of renal electrolyte and water transport
was first provided by the observation that exogenous ATP stimulated
Na+-K+-Cl
cotransport activity in cultured A6 cells originally derived from the
distal nephron of Xenopus laevis (25).
More recently, ATP was shown to inhibit
[Arg8]vasopressin
(AVP)-stimulated water transport in perfused tubules of the rat inner
medullary collecting duct (IMCD; see Ref. 20) and rabbit cortical
collecting duct (CCD; see Ref. 30). Similarly, ATP was shown to inhibit
Na+ and
Ca2+ reabsorption in primary
cultures of rabbit connecting tubule (CNT) and CCD cells (21). The
involvement of P2Y receptors
linked to the inositol 1,4,5-trisphosphate-mediated release of
Ca2+ from the endoplasmic
reticulum was concluded from the observation that ATP readily increased
the cytosolic Ca2+ concentration
in the absence of extracellular
Ca2+. A similar observation was
reached with rat IMCD (10) and mouse cortical thick ascending limb of
Henle (27). In each case, the effect of ATP was shown to be mimicked by
UTP, which is in agreement with the involvement of UTP-preferring
P2Y receptors (12).
There is ample evidence that the distal nephron is the primary site of
active Ca2+ reabsorption (1, 8,
13). To study the hormonal regulation of this process, we established a
primary culture of immunodissected rabbit CNT and CCD cells (2-4,
16, 17, 21, 32, 33). We have shown that
Ca2+ transport across these
monolayers can be stimulated by parathyroid hormone (PTH; see Refs. 16
and 32), AVP (16, 32, 33), PGE2
(16, 32), and adenosine (16, 17). Importantly, we recently demonstrated
that the adenosine analog
N6-cyclopentyladenosine
(CPA), which acts as an agonist at the
A1 receptor, can maximally
stimulate Ca2+ transport without
detectably increasing the cytosolic cAMP concentration (17). This led
us to postulate the existence of a novel cAMP-independent pathway
leading to stimulated Ca2+
transport. Moreover, in a follow-up study, we showed that also PTH,
PGE2, AVP, and adenosine act via
this novel cAMP-independent pathway and that this pathway involves a
chelerythrine-inhibitable protein kinase C (PKC) isotype that is not
downregulated after chronic phorbol ester treatment (16).
Using this cell model, we previously reported that ATP can markedly
inhibit Ca2+ transport, and
evidence was provided for the involvement of PKC in the mechanism of
action of ATP (2, 21). However, of eminent importance for a correct
interpretation of the mechanism of action of ATP is our recent finding
that primary cultures of CNT and CCD cells produce prostanoids that
stimulate Ca2+ transport (32).
This latter finding leaves the possibility that ATP might act by
inhibiting this autostimulatory pathway.
The first aim of the present study is to assess whether the previously
reported inhibitory action of ATP on
Ca2+ transport is due to
inhibition of the autostimulatory pathway. The second aim is to
investigate whether ATP also inhibits the stimulatory effects of other
hormones such as AVP and adenosine. Finally, the third aim of the
present study is to investigate whether PKC is indeed involved in the
inhibitory actions of ATP, as suggested by our previous work, and, if
so, to elucidate the level(s) at which PKC exerts its inhibitory effects.
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MATERIALS AND METHODS |
Drugs. Collagenase A and hyaluronidase
were obtained from Boehringer (Mannheim, Germany).
1-Desamino-8-D-arginine vasopressin (dDAVP) was from Bachem
Feinchemikalien (Bubendorf, Switzerland). Pertussis toxin (PTX),
chelerythrine, and Ro-20-1724 were purchased from Research
Biochemicals International (Natick, MA). All other chemicals, including
AVP, adenosine, CPA, PGE2,
8-bromo-cAMP (8-BrcAMP), and
12-O-tetradecanoylphorbol 13-acetate
(PMA), were obtained from Sigma (St. Louis, MO).
Primary cultures of rabbit CNT and CCD
cells. New Zealand White rabbits weighing ~0.5 kg
were killed by cervical dislocation. Kidney CNT and CCD cells were
immunodissected with monoclonal antibody R2G9 and set in primary
culture on permeable filter supports (0.33 cm2; Costar, Cambridge, MA), as
described previously (32). The cells were cultured in a 1:1 mixture of
Dulbecco's modified Eagle's medium-Ham's F-12 (GIBCO, Paisley, UK),
supplemented with 5% (vol/vol) decomplemented FCS (Serva, Heidelberg,
Germany), 50 µg/ml gentamicin, 0.5% (vol/vol) of a 100× mixture of
nonessential amino acids (GIBCO), 5 µg/ml insulin, 5 µg/ml
transferrin, 50 nM hydrocortisone, 70 ng/ml
PGE1, 50 nM
Na2SeO3,
and 5 pM triiodothyronine, equilibrated with 5%
CO2-95%
O2 at 37°C. Cell monolayers
reached confluency at day 3, and
experiments were performed between 5 and 8 days after seeding the
cells. Confluency of the monolayers was routinely checked by
determining transepithelial potential difference and resistance with
two "chopstick"-like electrodes connected to a Millicell-ERS
meter (Millipore, Bedford, MA).
Measurement of transepithelial
Ca2+ transport.
Transepithelial Ca2+ transport was
measured as described previously (32). Briefly, confluent monolayers
were washed three times and preincubated in a physiological salt
solution (PSS) containing (in mM): 140 NaCl, 2 KCl, 1 K2HPO4,
1 KH2PO4,
1 MgCl2, 1 CaCl2, 5 glucose, 5 L-alanine, and 10 HEPES (adjusted to pH 7.40 with Tris) for
15 min at 37°C. Long-term treatments with drugs were performed in
culture medium. Subsequently, the monolayers were washed three times
and incubated in PSS for another 90 min at 37°C to measure
transepithelial Ca2+ transport.
Drugs and hormones were added to either the apical and/or basolateral
compartment, as indicated in the text. At the end of the incubation
period, 25-µl samples were removed in duplicate from the apical
compartment and assayed for Ca2+
with a colorimetric test kit (Boehringer).
Ca2+ transport is expressed in
nanomoles per hour per square centimeter.
Measurement of cAMP accumulation. To
assess the effect of ATP on dDAVP-stimulated adenylyl cyclase activity,
accumulation of cAMP was measured in the presence of an inhibitor of
cyclic nucleotide phosphodiesterase activity, Ro-20-1724.
Confluent monolayers were preincubated in PSS containing 0.1 mM
Ro-20-1724 for 15 min at 37°C. Subsequently, hormones were
added to either the apical and/or basolateral compartment as indicated
in the text, and the monolayers were incubated for another 15 min. At
15 min, the filters were excised and immediately transferred to
Eppendorf microtubes containing 500 µl of 5% (wt/vol)
trichloroacetic acid. The samples were vigorously mixed and rapidly
frozen in liquid nitrogen. Samples were frozen and thawed three times
and centrifuged for 4 min at 10,000 g
(Eppendorf minifuge). A 400-µl aliquot of the supernatant was removed
and extracted three times with water-saturated diethyl ether. The
aqueous phase was blown to dryness with nitrogen, after which the
residue was dissolved in 500 µl of sodium acetate buffer (pH 6.2).
Samples and standards were acetylated, and the cAMP content was
determined by RIA
([I125]cAMP Assay
System; Amersham, Arlington Heights, IL). The amount of cAMP is
expressed in nanomoles per 15 min per filter.
Statistics. Results are given as means ± SE. Overall statistical significance was determined by ANOVA and,
in the case of significance, individual groups were compared by
contrast analysis according to Scheffé.
P values <0.05 were considered significant.
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RESULTS |
ATP inhibition of Ca2+
transport stimulated by endogenously produced prostanoids.
We have shown that ATP interacts with apical and basolateral
P2Y purinoceptors to inhibit net
apical-to-basolateral Ca2+
transport across monolayers of rabbit CNT and CCD cells (21). However,
our recent finding that endogenously produced prostanoids potently
stimulate this transport process (32) urged us to investigate the
possibility that ATP might act by inhibiting this autostimulatory pathway. For maximal inhibition, ATP was added to both sides of the
monolayer (21). Figure 1 shows the
inhibitory action of ATP (100 µM; both compartments) on
Ca2+ transport
(P < 0.05;
n = 8 filters). However, ATP did not
have an effect in monolayers in which
Ca2+ transport was reduced as a
result of the inhibition of endogenous prostanoid production by the
cyclooxygenase inhibitor indomethacin (5 µM; both compartments). To
investigate whether ATP exerts its inhibitory action on the
autostimulatory pathway at or beyond the receptor level, its effect on
PGE2-stimulated
Ca2+ transport was studied in
indomethacin-treated monolayers. Figure 1 shows that ATP significantly
(P < 0.005;
n = 8 filters) reduced the stimulatory
effect of exogenous PGE2 (10 nM).
The percentage inhibition was calculated to be 51%. These findings
demonstrate that ATP inhibits the autostimulatory pathway at least at
or beyond the level of the prostanoid receptor. Subsequent experiments
were routinely performed with indomethacin-treated monolayers.

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Fig. 1.
Inhibitory effect of ATP on Ca2+
transport stimulated by endogenously produced prostanoids and exogenous
PGE2 in monolayers of rabbit
connecting tubule (CNT) and cortical collecting duct (CCD) cells.
Confluent monolayers were preincubated in the absence (open bars) and
presence (filled bars) of ATP (100 µM; both compartments) and/or
indomethacin (5 µM; both compartments) for 15 min at 37°C.
Subsequently, either vehicle or
PGE2 (10 nM; both compartments)
was added, and Ca2+ transport was
measured for another 90 min. Data presented are means ± SE of 8 filters. * Significantly different from corresponding
unstimulated monolayers (P < 0.05).
# Significantly different from corresponding monolayers incubated
(untreated) or stimulated (indomethacin treated) in the absence of ATP
(P < 0.05). & Significantly
different from corresponding monolayers incubated in the absence of
indomethacin (P < 0.05).
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ATP inhibition of AVP- and adenosine-stimulated
Ca2+ transport.
Both AVP and adenosine have been demonstrated to potently stimulate
Ca2+ transport across monolayers
of rabbit CNT and CCD cells (16, 17, 32, 33). Table
1 shows that ATP (100 µM; both
compartments) significantly (P < 0.05) reduced the stimulatory effect of AVP (1 nM; basolateral
compartment) and adenosine (10 µM; apical compartment) by 37 and
63%, respectively.
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Table 1.
Inhibitory effect of exogenous ATP on AVP- and adenosine-stimulated
Ca2+ transport across confluent monolayers of rabbit
CNT and CCD cells
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ATP inhibition of dDAVP- and CPA-stimulated
Ca2+ transport.
The stimulatory effect of AVP was shown to be mimicked by dDAVP (33),
which suggests the involvement of the
V2 receptor. Figure
2 shows that dDAVP (1 nM; basolateral
compartment) increased Ca2+
transport by 71 ± 5 nmol · h
1 · cm
2
(P < 0.05;
n = 12 filters) and that ATP (100 µM; both compartments) significantly decreased this value to 36 ± 4 nmol · h
1 · cm
2
(P < 0.05;
n = 12 filters). The percentage
inhibition was calculated to be 50%. ATP did not affect basal
Ca2+ transport. As cAMP is
generally implicated in the mechanism of action of dDAVP (5), we
investigated the effect of ATP on cAMP-stimulated Ca2+ transport. Figure 2 shows
that the membrane-permeable cAMP analog 8-BrcAMP (0.1 mM; basolateral
compartment) markedly increased Ca2+ transport by 71 ± 5 nmol · h
1 · cm
2
(P < 0.05;
n = 9 filters) and that ATP
significantly decreased this value to 39 ± 6 nmol · h
1 · cm
2
(P < 0.05;
n = 9 filters). The percentage
inhibition was calculated to be 45%. This demonstrates that, in case a
hormone acts through cAMP, ATP acts, at least in part, at or beyond the
level of protein kinase A. We recently showed that the adenosine analog
CPA, which acts as an agonist at the
A1 receptor (34), can maximally
stimulate Ca2+ transport without
detectably increasing the cytosolic cAMP concentration (17). This led
us to investigate whether ATP can also inhibit this novel
cAMP-independent stimulatory pathway. Figure 2 shows that CPA (10 µM;
apical compartment) increased Ca2+
transport by 48 ± 3 nmol · h
1 · cm
2
(P < 0.05;
n = 5 filters) and that ATP decreased
this value to 14 ± 3 nmol · h
1 · cm
2
(P < 0.05;
n = 5 filters). The percentage
inhibition was calculated to be 71%.

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Fig. 2.
Inhibitory effect of ATP on 1-desamino-8-D-arginine
vasopressin (dDAVP)-, 8-bromo-cAMP (8-BrcAMP)-, and
N6-cyclopentyladenosine
(CPA)-stimulated Ca2+ transport in
monolayers of rabbit CNT and CCD cells. Confluent monolayers were
preincubated with indomethacin (5 µM; both compartments) in the
absence (open bars) or presence (filled bars) of ATP (100 µM; both
compartments) for 15 min before stimulation with dDAVP, 8-BrcAMP, or
CPA. CPA (10 µM) was added to the apical compartment, whereas dDAVP
(1 nM) and 8-BrcAMP (100 µM) were applied basolaterally.
Ca2+ transport was determined at
90 min after the onset of stimulation. Data presented are means ± SE of 6 (CPA) and 12 (dDAVP and 8-BrcAMP) filters.
* Significantly different from corresponding unstimulated
monolayers (P < 0.05).
# Significantly different from corresponding monolayers
stimulated in the absence of ATP (P < 0.05).
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Chelerythrine inhibition of dDAVP- and CPA-stimulated
Ca2+ transport.
In a previous study, we demonstrated that chelerythrine, a potent and
selective inhibitor of PKC (15), markedly reduced PTH-,
PGE2-, AVP-, and
adenosine-stimulated Ca2+
transport (16). Table 2 shows that
chelerythrine (5 µM; both sides) abolished the stimulatory effect of
CPA (10 µM) and markedly reduced the increase in
Ca2+ transport evoked by dDAVP (10 nM) from 77 ± 4 to 37 ± 9 nmol · h
1 · cm
2
(P < 0.05;
n = 4 filters).
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Table 2.
Inhibitory effect of chelerythrine on dDAVP- and CPA-stimulated
Ca2+ transport across confluent monolayers of rabbit
CNT and CCD cells
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Effect of PKC downregulation on ATP inhibition of dDAVP-, CPA-, and
8-BrcAMP-stimulated
Ca2+ transport.
Short-term activation of PKC by the phorbol ester PMA was shown to
result in a rapid and marked inhibition of
Ca2+ transport (2). In the
continuous presence of PMA, however, Ca2+ transport gradually
recovered. This recovery reflects the disappearance or downregulation
of PMA-sensitive PKC isoforms. It was also demonstrated that ATP failed
to inhibit Ca2+ transport in
monolayers treated with PMA for a prolonged period of time (21).
Because Ca2+ transport
measurements were performed in the absence of indomethacin, the above
findings can be taken as evidence that ATP acts via one or more
PMA-sensitive PKC isoforms to inhibit the stimulatory effect of
endogenously produced prostanoids. Figure 3
shows that dDAVP increased Ca2+
transport by 67 ± 4 nmol · h
1 · cm
2
(P < 0.05;
n = 9 filters) in monolayers
pretreated with PMA (0.1 µM; both compartments) for 120 h. This value
was not different from that obtained with untreated monolayers
(P > 0.9). ATP still significantly
reduced the dDAVP-induced increase to a value of 44 ± 2 nmol · h
1 · cm
2
(P < 0.05;
n = 9 filters), and also this value
was not different from that obtained with untreated monolayers
(P > 0.6). Chronic PMA treatment did
not significantly affect the increase in
Ca2+ transport in response to
8-BrcAMP (P > 0.1) but abolished the inhibitory action of ATP thereupon. Similarly, this treatment abolished
ATP inhibition of CPA-stimulated
Ca2+ transport without altering
the response to CPA (P > 0.9).

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Fig. 3.
Effect of chronic phorbol ester treatment on ATP inhibition of dDAVP-,
8-BrcAMP-, and CPA-stimulated Ca2+
transport in monolayers of rabbit CNT and CCD cells. Confluent
monolayers were preincubated with PMA (0.1 µM; both compartments) for
120 h. Indomethacin (5 µM; both compartments) was added without (open
bars) or with (filled bars) ATP (100 µM; both compartments) 15 min
before stimulation. CPA (10 µM) was added to the apical compartment,
whereas dDAVP (1 nM) and 8-BrcAMP (100 µM) were applied
basolaterally. Ca2+ transport was
determined at 90 min after the onset of stimulation. Data presented are
means ± SE of 6 (CPA) and 12 (dDAVP and 8-BrcAMP) filters.
* Significantly different from corresponding unstimulated
monolayers (P < 0.05).
# Significantly different from corresponding monolayers
stimulated in the absence of ATP (P < 0.05).
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ATP inhibition of the dDAVP-induced increase in
adenylyl cyclase activity. To evaluate the effect of
ATP on receptor-mediated adenylyl cyclase activation, the accumulation
of cAMP was measured in the presence of the inhibitor of cyclic
nucleotide phosphodiesterase activity, Ro-20-1724 (0.1 mM; both
compartments). Under these conditions, cAMP accumulation in
unstimulated monolayers amounted to 1.6 ± 0.1 nmol · 15 min
1 · filter
1
(n = 4 filters). dDAVP (1 nM;
basolateral compartment) increased the cAMP content by 6.3 ± 0.6 nmol · 15 min
1 · filter
1
(P < 0.05;
n = 4 filters), and ATP (100 µM;
both compartments) significantly reduced this increase to 3.3 ± 0.6 pmol · 15 min
1 · filter
1
(P < 0.05;
n = 4 filters; Fig.
4). The percentage inhibition was
calculated to be 48%. ATP did not affect basal cAMP accumulation (see
also, Fig. 5).

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Fig. 4.
Effect of chronic phorbol ester treatment on ATP inhibition of
dDAVP-induced cAMP accumulation in monolayers of rabbit CNT and CCD
cells. Confluent monolayers were pretreated without (untreated) or with
(0.1 µM; both compartments)
12-O-tetradecanoylphorbol 13-acetate
(PMA) for 120 h at 37°C. Indomethacin (5 µM; both compartments)
and Ro-20-1724 (100 µM; both compartments) were added without
(open bars) or with (filled bars) ATP (100 µM; both compartments) at
15 min before stimulation with dDAVP (1 nM; basolateral compartment).
Accumulation of cAMP was measured for another 15 min. Basal cAMP
accumulation in unstimulated monolayers amounted to 1.6 ± 0.1 nmol · 15 min 1 · filter 1
(n = 4 filters). Data presented show
the increase in cAMP accumulation above basal and are means ± SE of
4 (untreated) and 9 (PMA-treated) filters. * Significantly
different from corresponding untreated monolayers
(P < 0.05).
# Significantly different from corresponding monolayers
stimulated in the absence of ATP (P < 0.05).
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Effect of PKC downregulation on ATP inhibition of
dDAVP-induced adenylyl cyclase activation. To test the
possibility that ATP acts via a phorbol ester-sensitive PKC isotype to
inhibit dDAVP-induced adenylyl cyclase activation, monolayers were
pretreated with PMA (0.1 µM; both compartments) for 120 h. Figure 4
shows that dDAVP readily increased cAMP by 4.9 ± 0.3 nmol · 15 min
1 · filter
1
and that ATP still significantly lowered this increase to 2.6 ± 0.2 nmol · 15 min
1 · filter
1
in PMA-treated monolayers (P < 0.05;
n = 9). In the absence of ATP, the
dDAVP-induced increase in cAMP was slightly but significantly (P < 0.04) reduced after chronic PMA
treatment, whereas, in the presence of ATP, no difference
(P > 0.2) between the two groups was observed.
Effect of ATP on dDAVP stimulation of
Ca2+ transport
and activation of adenylyl cyclase in PTX-treated monolayers.
To investigate the possibility that ATP might act via the inhibitory
GTP-binding protein (Gi) to
inhibit dDAVP stimulation of Ca2+
transport and activation of adenylyl cyclase, monolayers were pretreated with PTX (170 ng/ml) for 24 h. Figure
5 shows that this treatment did not
interfere with the inhibitory effect of ATP on dDAVP-stimulated
Ca2+ transport
(A; P < 0.05; n = 4 filters) and cAMP
accumulation (B;
P < 0.05;
n = 4 filters). This demonstrates that
ATP does not act via Gi to exert
its inhibitory effect. PTX did not affect basal cAMP accumulation but
significantly potentiated the stimulatory effect of dDAVP both in the
absence (P < 0.05;
n = 4 filters) and presence
(P < 0.05;
n = 4 filters) of ATP. By contrast,
PTX did not affect the dDAVP-induced increase in
Ca2+ transport either in the
absence (P > 0.8;
n = 4 filters) or presence (P > 0.9;
n = 4 filters) of ATP. This is in
agreement with the idea that cAMP does not play a role in
dDAVP-stimulated Ca2+ transport
(16). As a control, we tested the effect of PTX on CPA inhibition of
AVP-induced cAMP formation. CPA inhibited AVP-induced cAMP accumulation
by 79% (see also, Ref. 17), and PTX abolished this inhibitory effect
of CPA. PTX did not change CPA-stimulated Ca2+ transport (transport rates of
110 ± 5 and 116 ± 8 nmol · h
1 · cm
2
for untreated and PTX-treated monolayers, respectively,
n = 3 filters,
P > 0.2). This demonstrates that CPA
does not interact with a Gi
protein to activate the novel cAMP-independent pathway leading to
Ca2+ transport.


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Fig. 5.
Effect of pertussis toxin (PTX) on ATP inhibition of dDAVP-stimulated
Ca2+ transport and cAMP
accumulation in monolayers of rabbit CNT and CCD cells. Confluent
monolayers were pretreated without (open bars) or with (filled bars)
PTX (170 ng/ml) for 24 h at 37°C.
A: monolayers were preincubated with
indomethacin (5 µM; both compartments) and ATP (100 µM; both
compartments) for 15 min before stimulation with dDAVP (1 nM;
basolateral compartment). Ca2+
transport was determined at 90 min after the onset of stimulation.
B: indomethacin (5 µM; both
compartments), Ro-20-1724 (100 µM; both compartments), and ATP
(100 µM; both compartments) were added 15 min before stimulation with
dDAVP (1 nM; basolateral compartment), and accumulation of cAMP was
measured for another 15 min. Data presented are means ± SE of 4 filters. * Significantly different from corresponding
unstimulated monolayers (P < 0.05).
# Significantly different from corresponding monolayers
stimulated in the absence of ATP (P < 0.05). $ Significantly different from corresponding
monolayers not treated with PTX (P < 0.05).
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DISCUSSION |
The data obtained in the present study are incorporated in a model of
the hormonal regulation of active
Ca2+ reabsorption in rabbit CNT
and CCD. The model, schematized in Fig. 6,
includes the interaction of ATP with apical and basolateral receptors
to inhibit hormone-stimulated Ca2+
transport via a phorbol ester-sensitive PKC isoform and
vasopressin-stimulated Ca2+
transport via a phorbol ester-insensitive mechanism. Of note, the
percentage inhibition reached with a maximally effective ATP concentration in combination with a maximally effective concentration of stimulant ranged from 37% (AVP) to 71% (CPA), indicating that ATP
can only partly inhibit maximally stimulated
Ca2+ transport.

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Fig. 6.
Schematic presentation of signal pathways involved in ATP inhibition of
dDAVP- and CPA-stimulated Ca2+
transport in rabbit CNT and CCD cells. AC, adenylate cyclase; PLC,
phospholipase C; DAG, diacylglycerol;
IP3, inositol 1,4,5-trisphosphate;
CaBP, Ca2+-binding protein. For explanation, see
text.
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Inhibition of the autostimulatory
pathway. The initial observation that exogenous ATP can
markedly inhibit Ca2+ transport
across confluent monolayers of rabbit CNT and CCD cells (21) took a new
turn when it was found that basal transport was in fact increased by
the action of endogenously produced prostanoids (32). Possible
mechanisms of action of ATP are therefore
1) inhibition of the production
and/or release of the autostimulatory prostanoids and
2) inhibition of the stimulatory
action of these prostanoids. The present finding that ATP markedly
inhibited PGE2-stimulated Ca2+ transport in
indomethacin-treated monolayers is in agreement with the latter
explanation. Obviously, this finding does not exclude that ATP may also
interfere with the production and/or release of autostimulatory prostanoids.
Inhibition of AVP- and dDAVP-stimulated
Ca2+ transport.
This work shows that ATP can markedly inhibit AVP- and dDAVP-stimulated
Ca2+ transport in a primary
culture of rabbit CCD and CNT cells. Inhibitory effects of ATP have
also been reported for AVP-stimulated water transport in perfused
rabbit CCD (30) and rat IMCD (20). ATP inhibition of AVP- and
dDAVP-stimulated Ca2+ transport
was found to be paralleled by a reduction of dDAVP-stimulated cAMP
accumulation. At first sight, the latter finding supports the
"classic" concept that dDAVP interacts with
V2 receptors to stimulate
Ca2+ transport in a cAMP-dependent
fashion. However, in a recent study, we provided evidence that cAMP is
not involved in AVP-stimulated Ca2+ transport (16). Moreover, we
showed that AVP-stimulated Ca2+
transport, although insensitive to PKC downregulation by chronic phorbol ester treatment, was inhibited by chelerythrine. Similarly, chelerythrine inhibited the effect of dDAVP in the present study. These
findings suggest the involvement of a chelerythrine-inhibitable, phorbol ester-insensitive PKC isoform in the mechanism of action of AVP
and dDAVP. In this context, the present observation that ATP reduces
AVP- and dDAVP-stimulated Ca2+
transport indicates that the nucleotide inhibits the
V2 receptor-mediated activation
and/or action of this "novel" chelerythrine-inhibitable pathway.
ATP also inhibited the stimulatory effect of 8-BrcAMP on
Ca2+ transport. This demonstrates
the potential of ATP to inhibit also the classic cAMP-dependent pathway
and that inhibition occurs at a level at or beyond protein kinase A. As
far as the AVP-induced increase in cAMP is concerned, we previously
speculated that it occurs in a compartment that does not affect
Ca2+ transport, whereas a
generalized increase in cAMP, as produced by 8-BrcAMP, does stimulate
Ca2+ transport (16).
Inhibition of adenosine- and CPA-stimulated
Ca2+ transport.
We recently demonstrated that the adenosine analog CPA, which
specifically activates the A1
receptor (34), can maximally stimulate
Ca2+ transport without having an
effect on cAMP accumulation (17). Moreover, we showed that
chelerythrine completely inhibited the effect of adenosine (16).
Together with the present finding that chelerythrine abolished the
stimulatory action of CPA, this suggests that adenosine and CPA,
similar to AVP and dDAVP, exert their stimulatory effect via the
chelerythrine-inhibitable pathway. The present study shows that ATP
also partly inhibits adenosine- and CPA-stimulated
Ca2+ transport. This is in
agreement with the idea that ATP inhibits the chelerythrine-inhibitable
pathway itself and/or its activation via the adenosine
A1 receptor. Of note,
CPA-stimulated Ca2+ transport was
not affected by PTX. This excludes the possibility that a G protein of
the Gi family couples the
A1 receptor to this novel pathway.
Involvement of PKC in the mechanism of action of
ATP. In a previous study, we demonstrated that ATP acts
through apical and basolateral P2Y
receptors to inhibit Ca2+
transport (21). P2Y purinoceptors
are generally believed to couple to a PTX-insensitive G protein of the
Gq family to increase the activity
of phospholipase C (12). In agreement with this, preliminary results
revealed that PTX did not affect the ATP-induced increase in cytosolic
Ca2+ concentration, while readily
inhibiting the effect of adenosine thereupon (H. P. G. Koster, A. Hartog, C. H. Van Os, and R. J. M. Bindels, unpublished observations).
Activation of P2Y receptors leads
to the increased production of 1,2-diacylglycerol, which functions as
the physiological activator of PKC (9, 26). The ability of PKC to
inhibit Ca2+ transport was
demonstrated previously by means of the membrane-permeable diacylglycerol analog 1,2-dioctanoylglycerol (21) and the potent PKC
activator PMA (2). Both PKC activators instantaneously inhibit
dDAVP-stimulated Ca2+ transport
(J. van Baal, S. E. van Emst-de Vries, R. J. M. Bindels, and P. H. G. M. Willems, unpublished observations) and
Ca2+ transport stimulated via the
autostimulatory pathway (2). However, this inhibition was only
temporary and gradually diminished during continued PMA treatment.
Finally, we showed that ATP inhibition of the autostimulatory pathway
was abolished when phorbol ester-sensitive PKC isoforms were
downregulated by chronic PMA treatment (21). Similarly, the present
study shows that chronic PMA treatment abolished ATP inhibition of
CPA-stimulated Ca2+ transport.
These findings demonstrate that ATP acts via a phorbol ester-sensitive
PKC isoform to inhibit stimulation of
Ca2+ transport through the
prostanoid receptor and the adenosine
A1 receptor and, as can be deduced
from the instantaneous inhibition of dDAVP-stimulated
Ca2+ transport by PMA (see above),
also through the vasopressin V2 receptor. Because all three receptors act via the novel
chelerythrine-inhibitable pathway, these findings suggest that ATP acts
via the phorbol ester-sensitive mechanism to inhibit the action of this
novel pathway. However, chronic PMA treatment did not affect ATP
inhibition of dDAVP-stimulated
Ca2+ transport. This indicates
that ATP acts in addition via a phorbol ester-insensitive mechanism to
inhibit stimulation of Ca2+
transport through the vasopressin
V2 receptor.
Chronic PMA treatment did not affect ATP inhibition of dDAVP-evoked
cAMP formation. This suggests that ATP acts via a phorbol ester-insensitive mechanism to inhibit dDAVP-induced cAMP formation. It
is tempting to speculate that the same phorbol ester-insensitive mechanism attenuates both V2
receptor-mediated adenylyl cyclase activation and activation of the
chelerythrine-inhibitable pathway and therefore acts at the
V2 receptor itself. Such a
mechanism would also fit with the idea that the measured changes in
cAMP are an epi-phenomenon unrelated to the stimulatory action of dDAVP on Ca2+ transport (16).
Chronic PMA treatment abolished ATP inhibition of 8-BrcAMP-stimulated
Ca2+ transport. This indicates
that ATP acts via a phorbol ester-sensitive PKC isoform to inhibit
stimulation of Ca2+ transport via
the classic cAMP-dependent pathway at or beyond the level of protein
kinase A.
ATP inhibition does not involve a PTX-sensitive G
protein. The present work shows that PTX did not
interfere with the inhibitory action of ATP on dDAVP-stimulated cAMP
accumulation and Ca2+ transport.
This lack of effect of the toxin on dDAVP-induced cAMP formation
excludes the possibility that ATP interacts with a receptor that
couples to Gi to inhibit adenylyl
cyclase. By contrast, Rouse et al. (30) demonstrated that ATP inhibits
AVP-stimulated water transport in rabbit CCD via the activation of both
PKC and Gi.
PTX markedly potentiated dDAVP-induced cAMP accumulation without
affecting, however, dDAVP-stimulated
Ca2+ transport. This is in
agreement with the idea that dDAVP does not act via cAMP to increase
Ca2+ transport. Presently, the
mechanism underlying the potentiating effect of PTX on dDAVP-induced
cAMP accumulation is unclear. One possibility is that dDAVP not only
stimulates adenylyl cyclase via Gs
but also inhibits the enzyme via
Gi.
In conclusion, the present study shows that ATP acts primarily via a
phorbol ester-sensitive PKC isoform to inhibit hormonal stimulation of
Ca2+ transport at the level of the
novel chelerythrine-inhibitable pathway involving a phorbol
ester-insensitive PKC isoform. Presently, it is unknown at which of the
steps involved in transcellular Ca2+ transport, i.e., apical
Ca2+ influx, cytosolic diffusion
of Ca2+ bound to
Ca2+-binding protein, and
basolateral Ca2+ extrusion, the
chelerythrine-inhibitable pathway acts to exert its stimulatory effect.
However, it is tempting to speculate that the
Ca2+ influx step is rate limiting
and that regulation takes place at this level. Recent elucidation of
the primary structure of the apical
Ca2+ influx channel (18) reveals
the presence of seven potential PKC phosphorylation sites, and future
studies may decide whether or not these sites are involved in the
hormonal regulation of Ca2+
reabsorption. In addition, ATP acts via a phorbol ester-insensitive mechanism to inhibit V2
receptor-mediated concomitant activation of this novel pathway and
adenylyl cyclase. Finally, ATP acts through a phorbol ester-sensitive
PKC isoform to inhibit Ca2+
transport evoked by a generalized increase in cAMP. The latter finding
demonstrates the potential of ATP to inhibit the classic cAMP-dependent
pathway at a level at or beyond protein kinase A.
 |
FOOTNOTES |
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: R. J. M. Bindels, 162 Cell Physiology, Univ. of Nijmegen, PO Box 9101, NL-6500
HB Nijmegen, The Netherlands (E-mail: Reneb{at}sci.kun.nl).
Received 20 July 1998; accepted in final form 22 July 1999.
 |
REFERENCES |
1.
Bindels, R. J. M.
Calcium handling by the mammalian kidney.
J. Exp. Biol.
184:
89-104,
1993[Abstract/Free Full Text].
2.
Bindels, R. J. M.,
J. A. Dempster,
P. L. M. Ramakers,
P. H. G. M. Willems,
and
C. H. Van Os.
Effect of protein kinase C activation and down-regulation on active calcium transport.
Kidney Int.
43:
295-300,
1993[Medline].
3.
Bindels, R. J. M.,
A. Hartog,
S. L. Abrahamse,
and
C. H. Van Os.
Effect of pH on apical calcium entry and active calcium transport in rabbit cortical collecting system.
Am. J. Physiol.
266 (Renal Fluid Electrolyte Physiol. 35):
F620-F627,
1994[Abstract/Free Full Text].
4.
Bindels, R. J. M.,
A. Hartog,
J. A. H. Timmermans,
and
C. H. Van Os.
Active Ca2+ transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxyvitamin D3 and PTH.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F799-F807,
1991[Abstract/Free Full Text].
5.
Breyer, M. D.,
and
Y. Ando.
Hormonal signaling and regulation of salt and water transport in the collecting duct.
Annu. Rev. Physiol.
56:
711-739,
1994[Medline].
6.
Chabardes, D.,
D. Firsov,
L. Aarab,
A. Clabecq,
A. C. Bellanger,
S. Siaume-Perez,
and
J. M. Elalouf.
Localization of mRNAs encoding Ca2+-inhibitable adenylyl cyclases along the renal tubule. Functional consequences for regulation of the cAMP content.
J. Biol. Chem.
271:
19264-19271,
1996[Abstract/Free Full Text].
7.
Conigrave, A. D.,
and
L. Jiang.
Ca2+-mobilizing receptors for ATP and UTP.
Cell Calcium
17:
111-119,
1995[Medline].
8.
Costanzo, L. S.,
and
E. E. Windhager.
Renal regulation of calcium balance.
In: The Kidney: Physiology and Pathology, edited by D. W. Seldin,
and G. Giebisch. New York: Raven, 1992, p. 2375-2393.
9.
Dubyak, G. R.,
and
C. El-Moatassim.
Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides.
Am. J. Physiol.
265 (Cell Physiol. 34):
C577-C606,
1993[Abstract/Free Full Text].
10.
Ecelbarger, C. A.,
Y. Maeda,
C. C. Gibson,
and
M. A. Knepper.
Extracellular ATP increases intracellular calium in rat terminal collecting duct via a nucleotide receptor.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F998-F1006,
1994[Abstract/Free Full Text].
11.
El-Moatassim, C.,
J. Dornand,
and
J. C. Mani.
Extracellular ATP and cell signalling.
Biochim. Biophys. Acta
1134:
31-45,
1992[Medline].
12.
Fredholm, B. B.,
M. P. Abbracchio,
G. Burnstock,
G. R. Dubyak,
T. K. Harden,
K. A. Jacobson,
U. Schwabe,
and
M. Williams.
Towards a revised nomenclature for P1 and P2 receptors.
Trends Biochem. Sci.
18:
79-82,
1997.
13.
Friedman, P. A.,
and
F. A. Gesek.
Cellular calcium transport in renal epithelia: measurements, mechanisms, and regulation.
Physiol. Rev.
75:
429-471,
1995[Abstract/Free Full Text].
14.
Grygorczyk, R.,
J. A. Tabcharani,
and
J. W. Hanrahan.
CFTR channels expressed in CHO cells do not have detectable ATP conductance.
J. Membr. Biol.
151:
139-148,
1996[Medline].
15.
Hebert, J. M.,
J. M. Augereau,
J. Gleye,
and
J. P. Maffrand.
Chelerythrine is a potent and a specific inhibitor of protein kinase C.
Biochem. Biophys. Res. Commun.
172:
993-999,
1990[Medline].
16.
Hoenderop, J. G. J.,
J. J. H. H. M. De Pont,
R. J. M. Bindels,
and
P. H. G. M. Willems.
Hormone-stimulated Ca2+ reabsorption in rabbit kidney cortical collecting system is cAMP-independent and involves a phorbol ester-insensitive PKC isotype.
Kidney Int.
55:
225-233,
1999[Medline].
17.
Hoenderop, J. G. J.,
A. Hartog,
P. H. G. M. Willems,
and
R. J. M. Bindels.
Adenosine-stimulated Ca2+ reabsorption is mediated by apical A1 receptors in rabbit cortical collecting system.
Am. J. Physiol.
274 (Renal Physiol. 43):
F736-F743,
1998[Abstract/Free Full Text].
18.
Hoenderop, J. G. J.,
A. W. C. M. Van der Kemp,
A. Hartog,
S. F. J. Van de Graaf,
C. H. Van Os,
P. H. G. M. Willems,
and
R. J. M. Bindels.
Molecular identification of the apical Ca2+channel in 1,25-dihydroxyvitamin D3-responsive epithelia.
J. Biol. Chem.
274:
8375-8378,
1999[Abstract/Free Full Text].
19.
Inscho, E. W.,
K. D. Mitchell,
and
L. G. Navar.
Extracellular ATP in the regulation of renal microvascular function.
FASEB J.
8:
319-328,
1994[Abstract/Free Full Text].
20.
Kishore, B. K.,
C.-L. Chou,
and
M. A. Knepper.
Extracellular nucleotide receptor inhibits AVP-stimulated water permeability in inner medullary collecting duct.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F863-F869,
1995[Abstract/Free Full Text].
21.
Koster, H. P. G.,
A. Hartog,
C. H. Van Os,
and
R. J. M. Bindels.
Inhibition of Na+ and Ca2+ reabsorption by P2u-purinoceptors requires protein kinase C but not Ca2+ signaling.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F53-F60,
1996[Abstract/Free Full Text].
22.
Li, C.,
M. Ramjeesingh,
and
C. E. Bear.
Purified cystic fibrosis transmembrane conductance regulator (CFTR) does not function as an ATP channel.
J. Biol. Chem.
271:
11623-11626,
1996[Abstract/Free Full Text].
23.
Linden, J.
Structure and function of A1 receptors.
FASEB J.
5:
2668-2676,
1991[Abstract/Free Full Text].
24.
Linden, J.
Cloned adenosine A3 receptors: pharmacological properties, species differences and receptor functions.
Trends Pharmacol. Sci.
15:
298-306,
1994[Medline].
25.
Middleton, J. P.,
A. W. Mangel,
S. Basavappa,
and
J. G. Fitz.
Nucleotide receptors regulate membrane ion transport in renal epithelial cells.
Am. J. Physiol.
264 (Renal Fluid Electrolyte Physiol. 33):
F867-F873,
1993[Abstract/Free Full Text].
26.
Nishizuka, Y.
Protein kinase C and lipid signaling for sustained cellular responses.
FASEB J.
9:
484-496,
1995[Abstract/Free Full Text].
27.
Paulais, M.,
M. Baudouin-Legros,
and
J. Teulon.
Extracellular ATP and UTP trigger calcium entry in mouse cortical thick ascending limbs.
Am. J. Physiol.
268 (Renal Fluid Electrolyte Physiol. 37):
F496-F502,
1995[Abstract/Free Full Text].
28.
Reddy, M. M.,
P. M. Quinton,
C. Haws,
J. J. Wine,
R. Grygorczyk,
J. A. Tabcharani,
J. W. Hanrahan,
K. L. Gunderson,
and
R. R. Kopito.
Failure of the cystic fibrosis transmembrane conductance regulator to conduct ATP.
Science
271:
1876-1879,
1996[Abstract].
29.
Reisin, I. L.,
A. G. Prat,
E. H. Abraham,
J. F. Amara,
R. J. Gregory,
D. A. Ausiello,
and
H. F. Cantiello.
The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel.
J. Biol. Chem.
269:
20584-20591,
1994[Abstract/Free Full Text].
30.
Rouse, D.,
M. Leite,
and
W. N. Suki.
ATP inhibits the hydrosmotic effect of AVP in rabbit CCT: evidence for a nucleotide P2u receptor.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F289-F295,
1994[Abstract/Free Full Text].
31.
Schwiebert, E. M.,
M. E. Egan,
T. H. Hwang,
S. B. Fulmer,
S. A. Allen,
G. R. Cutting,
and
W. B. Guggino.
CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.
Cell
81:
1063-1073,
1995[Medline].
32.
Van Baal, J.,
M. D. De Jong,
F. J. Zijlstra,
P. H. G. M. Willems,
and
R. J. M. Bindels.
Endogenously produced prostanoids stimulate calcium reabsorption in rabbit cortical collecting system.
J. Physiol. (Lond.)
497:
229-239,
1996[Abstract].
33.
Van Baal, J.,
G. Raber,
J. De Slegte,
R. Pieters,
R. J. M. Bindels,
and
P. H. G. M. Willems.
Vasopressin-stimulated Ca2+ reabsorption in rabbit cortical collecting system: effects on cAMP and cytosolic Ca2+.
Pflügers Arch.
433:
109-115,
1996[Medline].
34.
Van Galen, P. J. M.,
G. L. Stiles,
G. Michaels,
and
K. A. Jacobson.
Adenosine A1 and A2 receptors: structure-function relationships.
Med. Res. Rev.
12:
423-471,
1992[Medline].
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