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From the
Vasopressin is known to activate two types of cell surface
receptors; V
AVP caused a rapid increase in electrogenic
sodium transport, reflected by the transepithelial potential difference (V
These results indicate that AVP-stimulated Na
Vasopressin (AVP)
AVP can bind and activate two
types of cell surface receptors: V
The transepithelial potential
difference (V
The
method used to measure Ca
Monolayers of A6 cells,
grown to confluence on Falcon membranes, were treated for 30 min with
AVP and were then immediately placed on ice and washed with ice-cold
phosphate-buffered saline (pH 7.2). The phosphate-buffered saline was
aspirated from the apical side and replaced with 250 µl of 20
mM Tris-HCl buffer (pH 7.4), containing 10 µM leupeptin, 25 µg/ml aprotinin, 1 mM disodium
pyrophosphate, 1 mM 1,4-dithiothreitol, and 1 mM EGTA. Cells were scraped from the membrane with a rubber policeman
and homogenized by probe sonication for 15 s (Kontes, model KT-50 Micro
Ultrasonic Cell Disrupter). Samples were aliquoted and stored at
-70 °C until assayed.
The PKA assay reaction was run with
6-9 µg of homogenate protein, 1 mM ATP, 10
mM MgCl
After agarose electrophoresis of all
samples, the relevant gel pieces were excised, brought to a volume of
0.5 ml with deionized water, and heated in boiling water until the gel
was liquified. Fluorescence was then determined (Perkin-Elmer LS-5
spectrofluorometer), using an excitation wavelength of 568 nm and an
emission wavelength of 592 nm. Calculated PKA-specific enzyme
activities are expressed in fluorescence units generated per 10 µg
of cellular protein/15 min.
Dihydroxychloropromazine, highly specific for PKC with a K
The percent of PKA activation (-cAMP/+cAMP) in control
monolayers was 44 ± 7%, as shown in Fig. 7. Addition of 1
µM AVP increased the percent activation of PKA to 62
± 4% (p < 0.05) an increase of 41% above control.
This change is consistent with other reports regarding PKA stimulation
by high concentrations of other cAMP-elevating agonists; isoproterenol
and prostaglandin E
In control cells
(vehicle alone) the percent of PKA activation was 16 ± 0.5%,
whereas in the AVP-treated group the activated fraction was 24 ±
2%, an increase of 50% (since data are the mean of two separate
experiments, the variance is shown as error bars). In cells preloaded
with IP
To examine the ability of cAMP
analogues to stimulate Na
Although it is known that AVP regulates various biological
responses by at least two classes of second messenger systems, one
involving the cyclic nucleotide cAMP and the other
Ca
The present study in cultured A6 cells demonstrates that AVP
stimulates an immediate increase in I
We have also begun
to explore the possible role of calcium-dependent effectors in the
calcium mobilizing transduction system mediating Na
The demonstration of Ca
We
found that the specific antagonists DDA and IP
We also
examined the cellular action of forskolin and externally applied
8-CPT-cAMP, since their actions to stimulate Na
Since our studies indicate that AVP stimulation of Na
In summary, this study indicates that AVP
stimulation of electrogenic Na
Experiments were performed 90 min after chilling cells at
-20 °C to load IP
Volume 270,
Number 27,
Issue of July 07, pp. 16082-16088, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-mobilizing Signal Mechanism (*)
coupled to adenylate cyclase, and V
linked to a Ca
-dependent transduction system.
We investigated whether arginine vasopressin (AVP) stimulation of
electrogenic sodium transport in A6 cells, derived from Xenopus
laevis, is mediated by activation of either one or both types of
AVP-specific receptors.
) and equivalent short circuit current (I) measurements. AVP also rapidly increased
intracellular Ca
(Ca
)
and total inositol trisphosphate. The increase in I was dependent on the rise in (Ca
),
because
1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic
acid (BAPTA) dose-dependently inhibited the I response. There was no evidence, however, that activation of
adenylate cyclase mediated AVP-stimulated I
;
transport was not inhibited after AVP-induced activation of adenylate
cyclase was abolished by 2`,5`-dideoxyadenosine or when cAMP-dependent
protein kinase (PKA) activity was abolished by the specific PKA
inhibitor IP
. Further studies showed that although both
forskolin and 8-(4-chlorophenylthio)-cAMP stimulated I
, this occurred by mechanisms independent of PKA
activation.
transport is mediated by a V
receptor and a
Ca- dependent mechanism.
()stimulates hydraulic
water transport and electrogenic sodium transport in the collecting
duct epithelial cells of mammalian kidney and in some amphibian
epithelia(1) . Pioneering studies by Orloff and Handler (2) suggested that cAMP acts as a second messenger for both of
these effects. Subsequent studies confirmed that activation of
adenylate cyclase, and thus the generation of cAMP, mediates AVP
stimulation of water transport, because inhibition of the enzyme with
2`,5`-dideoxyadenosine (DDA) abolishes the response(3) .
However, the role of cAMP in AVP stimulation of electrogenic
Na transport remains in question, for there are as yet
no studies demonstrating a direct dependence of Na
transport on cAMP generation.
receptors, present in
renal epithelial cells and some amphibian epithelia, are coupled to
adenylate cyclase via a cholera toxin-sensitive G protein, whereas
V receptors, present mainly in smooth muscle, mesangial
cells, hepatocytes, and central nervous system neurons, are linked to a
calcium-dependent transduction system(4) . Recent studies,
however, have demonstrated V receptors in rabbit cortical
collecting tubule cells (5). In addition, we have reported that a
calcium-dependent mechanism mediates aldosterone(6) -,
adenosine(7) -, and insulin-stimulated (8) electrogenic
sodium transport in amphibian renal epithelial (A6) cells. We have
therefore investigated whether AVP stimulation of Na transport is similarly linked to a Ca
-dependent
messenger system via activation of a V
or
V-like receptor.
Na
Experiments were
performed on a clone of A6 cells derived from the kidney of Xenopus
laevis. These cells, provided by Gregory Grillo, Walter Reed
Research Institute, were obtained from the American Tissue Type
Collection and cloned by limiting dilution (clone A6-S2). The methods
employed for cell culture, measurement of net sodium transport, and
study of inhibitory agents have been reported recently(7) . In
brief, cells were grown to confluence on porous membranes (Millicell-HA
cups, Millipore Corp., Bedford, MA) and studied when fully
differentiated 10-14 days after subculture. Net Na Transport
transport was determined from the equivalent short circuit (I
), calculated from the open circuit
measurements of V
and R using
Ohm's law, which represents the net current associated with
active ion transport when V = O mV. Data
are expressed as the change in I above the
initial level observed in control and experimental monolayers at 30 min
after addition of agonist or control vehicle. The agonist or vehicle
was added to media on the basal surface of monolayers in a volume that
was 1% of the media volume. Although basal I
varied between different passages, agonist-induced changes were
proportional to basal values.
) was used to estimate the time of
onset of agonist-induced changes in Na transport,
because this parameter is more sensitive to rapid changes than
measurement of I
. V
changes
in parallel with I. Continuous recordings were
made with a strip recorder before and after addition of AVP. To avoid
transient disturbances in culture media, culture cups were maintained
in an incubator at 27 °C.
Intracellular Ca
Ca (Ca
i)
i was measured in suspended cells which had been grown to
confluence on porous Falcon membranes with an active surface of 4.5
cm
. These larger membranes were used to obtain a yield
appropriate for the assay. Previous studies demonstrated that basal and
hormone-stimulated changes in I were comparable
when cells were grown on Millipore HA culture cups or on Falcon
membranes (data not shown). Porous membranes were employed, because
cells grown on a nonporous surface, such as glass or culture plastic,
did not generate cAMP in response to AVP, suggesting that such growth
conditions altered receptor expression and/or availability. However,
the transparent porous membranes were found to be unsuitable for
measurement of Ca
, because
autofluorescence prohibited adequate signal to noise ratios. Therefore,
suspended cells were used, recovered by gentle scraping in
phosphate-buffered saline with 5 mM EGTA (no trypsin).
with Fura-2/AM
was recently reported(7) . The use of suspended cells prohibited
rapid removal of Fura-2 leaked to the external bathing solution which
was detectable after about 5 min. Continuous recordings of
Ca, therefore, did not exceed
approximately 5 min.
Inositol Trisphosphate (IP
The
methods used to measure total cellular IP
) were previously
reported(7) . Cells were grown to confluence on Falcon
membranes. In brief, total IP was measured by high pressure
liquid chromatography from cells labeled with myo-[H]inositol (50 µCi/ml) for 48 h
in inositol free amphibian Ringer's solution. Preliminary studies
demonstrated that under this condition basal and hormone-stimulated I values were comparable with those in normal
culture media (data not shown). LiCl was not added to the external
bathing solution.
Cyclic AMP
Total cellular cAMP was measured as
described (7) with a radioimmunoassay kit obtained from Biomedical
Technologies Inc. (Stoughton, MA). Confluent cells grown on Falcon
membranes were exposed to the phosphodiesterase inhibitor RO-201724
(100 µM) for 15 min before addition of agonist. Following
a 10-min treatment with agonist, the reactions were terminated by
addition of cold 6% trichloroacetic acid. After centrifugation to
remove precipitated material, trichloroacetic acid was extracted by the
Freon/tri-N-octylamine technique(9) . Results are
expressed as total cAMP accumulated at 10 min/mg of protein.
cAMP-dependent Protein Kinase (PKA)
Measurements
The activity of PKA was determined with a
commercial kit (Promega, Inc., Madison, WI) that measures the
phosphorylation of a fluorescently labeled substrate peptide,
Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide)(10) . Subsequent
electrophoresis in an 0.8% agarose gel provides clear separation of the
substrate species: the phosphorylated species migrates toward the
anode, whereas the unphosphorylated remainder migrates toward the
cathode. The measured fluorescence of the phosphorylated band,
visualized under UV light, is directly proportional to the amount of
peptide phosphorylated by active kinase.
, 20 mM Tris-HCl (pH 7.4), and 60
µM fluorescently tagged Kemptide (PepTag A1 peptide) in a
final volume of 25 µl. Samples were incubated at 30 °C for 15
min. Each cell extract was assayed in duplicate in both the absence and
presence of 1 µM cAMP to determine the fraction of total
enzyme (+cAMP) that was endogenously activated (-cAMP). In
parallel samples, the inclusion of the PKA-specific inhibitor IP (5 µM) (11) enabled calculation of true PKA
activity: non-inhibitable (non-PKA) phosphorylation was subtracted from
total measured phosphorylation.
In Situ Inhibition of PKA
Experiments were also
performed to directly inhibit PKA in intact A6 cells with the highly
selective antagonist IP. To increase cell membrane
permeability, and thus increase cellular uptake of the applied
polypeptide, monolayers of A
cells were exposed to 5
µM IP while chilled, but not frozen, at
-20 °C for 10 min (suggested by Dr. Adrian Katz).
Subsequently, cells were allowed to recover in the same bathing
solution for 90 min in a 27 °C incubator before experiments were
performed. The effects of chilling on the basal properties of the
monolayers and on hormone responsiveness were determined by comparison
with cells from the same seeding that were not subject to the chilling
procedure.
Reagents
AVP, 8-(4-chlorophenylthio)-cAMP
(8-CPT-cAMP), trifluoperazine, Freon, and tri-N-octylamine
were obtained from Sigma. Additional chemicals were obtained from the
following vendors: chelerythrine, Alomone Laboratories (Jerusalem,
Israel); forskolin and 1,9-dideoxyforskolin, Calbiochem; DDA, Pharmacia
Biotech Inc.; RO-201724, Biomol Research Laboratory, Inc. (Plymouth
Meeting, PA); Fura-2/AM and 5,5`-dimethyl BAPTA/AM, Molecular Probes
(Eugene, OR); amiloride was a gift from Merck Sharp and Dohme; and
7,8-dihydroxychloropromazine was provided by Research Biochemical,
Inc., as part of the Chemical Synthesis Program of the National
Institute of Mental Health, Contract 278-90-007(BS).
Statistical Analysis
Statistical comparisons were
made using the unpaired Student's t test. The Dunnett
test was used when multiple experimental groups were compared with a
single control group. A p < 0.05 was regarded as denoting
statistical significance.
Effect of AVP on Electrogenic Sodium
Transport
Addition of AVP to medium bathing the basal surface of
A6 monolayers stimulated I at a minimal
concentration of 1 nM (Fig. 1A). Higher
concentrations progressively increased I
up to
10-fold at 1 µM. AVP was found to increase V
within 6 s (Fig. 1B). The
response persisted for about 3.5 h, but decayed progressively after the
first hour. To our knowledge, this is the first demonstration of an
AVP-induced increase in I in less than 1 min. The
stimulation of I
was abolished by 100 µM amiloride in the apical solution, indicating that I
reflected electrogenic sodium transport:
control, 1.6 ± 0.1 µA
cm; AVP, 3.1
± 0.1; AVP + amiloride, 0.4 ± 0.2 (mean ±
S.E.).
Figure 1:
Effects of vasopressin (AVP)
on I in A6 cells. A, dose-dependent
stimulation of electrogenic Na
transport (I
) by AVP added to the basal solution (5 culture
cups per group). Values are mean ± S.E. B, time course
analysis of stimulation; transepithelial potential difference (V
) was measured over 3 h following exposure of
cells to 1 µM AVP. Results of this experiment are
representative of five experiments. The initial effect is shown in the inset.
Role of Intracellular Calcium in the Action of
AVP
Addition of AVP to A6 cells also resulted in an immediate
dose-dependent rise in Ca, as indicated
by Fura-2 fluorescence (Fig. 2). The time and dose dependencies
of the Ca response were similar to those
of the I response (Fig. 1, A and B). AVP appeared not to stimulate Ca
influx
during the period of observation, because addition of 100 µM MnCl
did not quench intracellular Fura-2 fluorescence
during stimulation; Mn enters cells via activated
Ca
channels and displaces Ca
from
Fura-2(12) .
Figure 2:
Effect of AVP on
Ca. AVP induced a dose-dependent increase
in Ca, reflected by an increase in the
fluorescence ratio (expressed in arbitrary units) of Fura-2 at an
emission of 510 nm, during dual excitation at 340 and 380 nm (A-C). These data obtained in a single experiment are
representative of 10 separate experiments.
To determine whether AVP-stimulated sodium
transport was dependent on this increase in Ca monolayers were preloaded (2.5 h) with the calcium chelator
5,5`-dimethyl BAPTA. Fig. 3shows that BAPTA dose-dependently
inhibited subsequent stimulation of I by 1
µM AVP, with an apparent K
of approximately 10 µM. These results therefore
imply that AVP stimulation of I
is mediated by an
increase in Ca
.
Figure 3:
Effect of Ca chelation by BAPTA on AVP-induced I.
Dose-dependent inhibition of I
in cells preloaded
with 5,5` dimethyl BAPTA/AM, at indicated concentrations. There were 5
culture cups in each group. Values are means ±
S.E.
Since the above
experiments suggested that elevated Ca was entirely due to release from intracellular Ca stores, we tested whether AVP increased phosphoinositide
turnover. Initially, total IP
was measured before and after
addition of 1 µM AVP. As shown in Fig. 4A,
an increase in IP was observed at 10 s, reached a maximum
at 30 s, and persisted for at least 300 s. Subsequently, a
dose-response analysis was performed by measuring IP levels
at 30 s after addition of various AVP concentrations. Fig. 4B shows that AVP increased IP in the range of 10 nM to 1 µM. The lack of a progressive rise in IP with high concentrations of AVP may have resulted from increased
rates of degradation (uninhibited because LiCl was not used).
Figure 4:
AVP
stimulation of total inositol trisphosphate (IP). A, time-dependent effect of 1 µM AVP. B,
dose-dependent stimulation of IP by AVP. Data are derived
from two separate experiments, each with two samples for each time
interval or concentration. Values are mean ±
S.E.
These
studies together provide strong evidence that the action of AVP to
stimulate electrogenic Na transport depends on an
increase in Ca
via turnover of membrane
bound phospholipids and IP-induced release of
Ca from non-mitochondrial intracellular stores.
The role of Calcium-dependent Effectors in AVP-stimulated I
The demonstration that AVP causes a rise in
IP implies that diacylglycerol, an endogenous activator of
protein kinase C (PKC), is also produced. Studies were performed to
determine whether PKC appears to play a role in Na transport stimulation by AVP. In these experiments the effect of
three PKC antagonists on AVP-stimulated I
were
examined.
of 8 µM for PKC (K
values for other kinases > 50
µM)(13) , dose-dependently inhibited AVP
stimulation of I
(Fig. 5A), with
an apparent K
of approximately 12
µM. The second selective antagonist, chelerythrine, is a
benzophenanthridine alkaloid that exhibits an apparent K
for PKC of 0.7 µM in rat
brain tissue under in vitro conditions (K
values for other kinases > 100
µM)(14) . Chelerythrine half-maximally inhibited
AVP-stimulated I
in intact cells at approximately
3 µM (Fig. 5B). In addition,
trifluoperazine, which inhibits calmodulin kinase as well as PKC,
reduced the action of AVP by 50% at a K
of approximately 25 µM (Fig. 5C), a value that corresponds to the K
for PKC inhibition in reports for other
types of intact cells(15, 16) . Taken together, these
results suggest that the onset of AVP stimulation of I
is dependent on PKC activation.
Figure 5:
Effects
of specific kinase inhibitors on AVP-stimulated I. A, dose-dependent effect of the PKC
inhibitor dihydroxychloropromazine (DHCP). B,
inhibitory profile of the PKC inhibitor, chelerythrine (Chel),
on AVP-stimulated I
. C, effect of
trifluoperazine (TFP), a PKC and calmodulin inhibitor, on I
. Values are mean ± S.E. There were 5
culture cups in each control and experimental group (A-C).
As reported previously (7) these antagonists did not affect either basal transport or R in the concentrations employed in these
experiments, suggesting that they were not toxic to the cultured cells.
Role of Adenylate Cyclase and cAMP in the Action of
AVP
Addition of AVP to the basal medium also resulted in a
dose-dependent increase in cAMP generation (Fig. 6A)
that paralleled the stimulation of I. The cAMP
level, in the presence of the phosphodiesterase inhibitor RO-201724,
increased 8-fold from the control value of 19 ± 1.6 pmol/mg
protein to 138 ± 12 at 1 µM AVP. However, after
preloading cells with the adenylate cyclase inhibitor DDA, which binds
to the ``P'' site on the catalytic subunit of the
enzyme(17) , AVP failed to induce cAMP generation at any
concentration that was previously shown to stimulate I
(1 nM to 1 µM, Fig. 1A).
Figure 6:
The effect of DDA on the AVP-induced
accumulation of cAMP and I. A,
AVP-induced accumulation of cAMP in the absence and presence of DDA
(100 µM), a specific inhibitor of adenylate cyclase. These
data obtained in a single experiment are representative of three
separate experiments. B, the action of DDA on AVP-stimulated I
, with 5 culture cups in each group. Values are
mean ± S.E.
The corresponding effect of DDA on AVP stimulation of I is shown in Fig. 6B. Despite its
abolition of cAMP generation, DDA did not significantly reduce the
ability of AVP to stimulate I
, indicating that
AVP stimulation of Na
transport was independent of
cAMP generation.
Role of Protein Kinase A
PKA is the major
intracellular target for cAMP. Activation of this enzyme is therefore
expected to occur as a result of AVP-induced cAMP generation. However,
one would predict that DDA would abolish such PKA activation if it were
completely effective in inhibiting adenylate cyclase. Further
experiments were, therefore, performed to test these predictions.
, for example, increased fractional
activation of true PKA by 50% in tracheal smooth muscle(18) . As
predicted, preloading A6 cells with the adenylate cyclase inhibitor DDA
abolished AVP stimulation of PKA; the fractional PKA activation was
then only 46 ± 5% (p = not significant, compared
with control).
Figure 7:
The effect of AVP on PKA activity in the
absence or presence of DDA (100 µM). Shown is PKA-specific
kinase activity in A6 cells, expressed as a percent of maximal
activity. Data were derived from five separate experiments performed in
duplicate. Results are mean ± S.E. The asterisk indicates p < 0.05, compared with
control.
In Situ Inhibition of PKA
Taken together, our
findings indicated that DDA completely blocked AVP stimulation of the
cAMP pathway, but did not affect stimulation of I. Nevertheless, an additional study was
performed to rule out PKA in mediating the action of AVP. To directly
suppress the enzyme in situ intact cells were loaded with the
specific PKA inhibitor IP
, added to the external bathing
solution at a concentration of 5 µM. This experiment was
prompted by reports that sufficient cellular loading of the polypeptide
could be achieved in renal proximal tubules to inhibit
dopamine-induced, cAMP-mediated inhibition of Na-K-ATPase (19) or the apically located Na:H antiporter(20) .
Monolayers were chilled briefly at -20 °C for 10 min to
enhance the cellular uptake of the polypeptide by increasing cell
membrane permeability. After recovery at a normal temperature, cells
were exposed to either control vehicle or 1 µM AVP for 30
min and, then, allocated either for assay for PKA activity or
measurement of electrogenic sodium transport.
before treatment with AVP the percent activation
was 18 ± 0.1%, indicating that intracellular IP
completely blocked the action of AVP to stimulate PKA. Transport
studies verified that cold exposure did not result in injury to the
cell monolayer. Ninety minutes after the chilling process, neither
basal resistance nor basal I
of monolayers were
reduced compared with control values, shown in . In
addition, indicates that abolition of PKA activation by
IP
did not inhibit AVP-stimulated I
,
compared with control.
Mechanism of Forskolin- and 8-CPT-cAMP-mediated
Na
The observation that
forskolin and/or analogues of cAMP induce a biological response is
often used as evidence that the effect in question is mediated by cAMP.
Since these reagents have recently been shown to stimulate various
transport processes by cAMP-independent mechanisms, studies were
performed to determine their action mechanisms in stimulating
electrogenic NaTransport
transport. Forskolin is a naturally
occurring diterpene that activates adenylate cyclase after binding to
the catalytic subunit, or G
-catalytic subunit complex (21).
Application of forskolin to A6 cells caused an increase in cAMP
accumulation at 100 nM and above (Fig. 8A).
Preloading cells with DDA abolished cAMP generation at 100 nM forskolin and reduced it by approximately 60% at 1
µM. Despite complete or partial inhibition of cAMP
production, DDA did not significantly reduce forskolin stimulation of I (Fig. 8B).
Figure 8:
The
effect of DDA on forskolin-induced cAMP generation and I. A, forskolin-induced accumulation of
cAMP in the absence or presence of DDA (100 µM). B, the effect of DDA on forskolin-stimulated I
. C, the effect of 1,9-dideoxyforskolin
on I
. There were 5 culture cups in each control
and experimental group (A-C). Values are mean ±
S.E.
In further support
of a cAMP-independent mechanism, the forskolin analogue
1,9-dideoxyforskolin, was found to also stimulate Na transport, at concentrations of 10 µM and above (Fig. 8C). This compound does not bind to the catalytic
subunit of adenylate cyclase, even in concentrations as high as 100
µM(22) .
transport, we employed the
highly permeant and phosphodiesterase-resistant analogue 8-CPT-cAMP.
Since synthetic derivatives of cAMP exhibit different binding
affinities for the regulatory subunits of PKA(23) , the
concentrations needed for half-maximal and maximal activation of PKA by
cAMP and 8-CPT cAMP were first compared in homogenates of A6 cells. The
dose-response curves (Fig. 9A) indicate similar
potencies for both reagents: half-maximum values of approximately 100
nM and maximum, approximately 400 nM. This in
vitro experiment suggests that, intracellularly, 8-CPT-cAMP should
have a potency that is similar to that of cAMP. Our data are consistent
with other reports that cAMP activates PKA at half-maximum and maximum
values of approximately 50 nM and 200-400 nM,
respectively(23) . By this in vitro data, we can then
predict that an externally applied equipotent cAMP analogue, to be
similarly biologically effective, should yield intracellular
concentrations of about 50-100 nM. Fig. 9B shows the effect of 8-CPT-cAMP applied externally to intact cells
on PKA activity. In fact PKA activation increased approximately 50 and
100% at external 8-CPT-cAMP concentrations of 1 and 10 µM,
respectively. Thus, the intracellular concentrations of 8-CPT-cAMP are
probably about 10% of the external concentration.
Figure 9:
Effects
of 8-CPT-cAMP on PKA activity and I. A,
comparison of the dose dependencies of cAMP and 8-CPT-cAMP in A6 cell
homogenates. B, comparison of the dose-dependent effect of
8-CPT-cAMP on intact cells to increase the percent activation of PKA (light bars) and to stimulate I
(dark bars). Data on PKA activation are representative
of two separate experiments. There were 4 culture cups in each group in
the transport study. The asterisk indicates that the
experimental group exceeds control by the Dunnett Test, with a
confidence limit of 95%.
These data further
indicate that if the stimulation of Na transport were
dependent upon PKA activation by 8-CPT-cAMP, it would occur at external
concentrations of less than 10 µM. However, Fig. 9B reveals that 8-CPT-cAMP stimulation of I
required concentrations between 1 and 3 orders
of magnitude higher than those necessary to activate PKA. Therefore, as
found with forskolin, 8-CPT-cAMP stimulation of I
is mediated by a PKA-independent mechanism. Furthermore, it is
seen that an increase in PKA activity per se does not
stimulate Na
transport.
, it is generally accepted that AVP stimulation of
electrogenic Na
transport is mediated by cAMP.
Evidence for this proposition is based largely on the demonstration
that AVP induces the generation of cAMP at concentrations that
stimulate sodium transport (2) and the belief, until recently, that AVP
target cells with the capability for electrogenic Na
transport possess only V
receptors coupled to
adenylate cyclase(24) . Recent reports have shown, however, that
AVP acts to increase Cai, as well as cAMP,
in cultured cells from rabbit cortical collecting tubule (5, 25) and toad bladder(26) . That observation,
together with reports that activation of receptors which are not linked
to adenylate cyclase, including receptors for aldosterone (6) and insulin(8) , and the A
adenosine
receptor(7) , cause a calcium-dependent stimulation of
Na transport, prompted this examination of the action
of AVP.
,
Ca
and IP at concentrations
of 10 nM and above. The dependence of AVP-stimulated I on increases in Ca
was shown in experiments in which chelation of
Ca with the EGTA derivative BAPTA
dose-dependently blocked I. Previous studies have
indicated that intracellular BAPTA inhibits agonist-stimulated
increases in Ca
, although basal levels
are unaffected(6) . These results therefore show that AVP
stimulates electrogenic Na transport by a
calcium-mobilizing second messenger system, due primarily to release of
Ca
from intracellular stores.
transport. The findings in the present study provide evidence
that PKC is at least one such effector. First, the demonstration of
AVP-stimulated IP
implies a rise in diacylglycerol
production, the natural PKC activator. Second, specific antagonists of
PKC dose-dependently inhibited AVP-stimulated I at concentrations near to those that inhibit the purified enzyme in vitro. Further studies, using more direct experimental
approaches, will be required to confirm an effector role for PKC in
Na
transport.
dependence, however, did not exclude the possibility of an
additional cAMP-mediated transduction mechanism acting in a redundant
or additive manner. Further experiments were therefore performed to
examine the potential role of cAMP in mediating Na
transport. First, we sought to determine whether AVP stimulation
of Na
was dependent upon cAMP production, that is
whether Na
transport was abolished or reduced when
adenylate cyclase was inhibited. Second, we examined the effect of
inhibiting PKA on electrogenic Na
transport, since
this enzyme is thought to be the sole, or at least major, substrate for
cAMP. And, finally, we probed the effect of experimentally induced
increases in cAMP on Na
transport, by loading intact
cells with either forskolin or the cAMP analogue 8-CPT-cAMP.
, when
loaded into intact cells, were effective in abolishing hormone
stimulation of cAMP production and PKA activation, respectively, but
did not alter AVP-stimulated Na
transport. This
dissociation between cAMP generation and Na
transport
was confirmed by experiments in which the cAMP analogue was applied
externally; full stimulation of PKA did not result in increased
Na
transport. Together, our results provide strong
evidence that electrogenic sodium transport is not mediated by a
cAMP-dependent system. It should be noted that this finding is not
unprecedented, because there are now numerous other examples of
biological responses, initially thought to involve cAMP as a second
messenger, which have been shown to involve transduction mechanisms
that are unrelated to adenylate cyclase
activation(27, 28, 29, 30) .
transport have been regarded as evidence for a cAMP-mediated
messenger system. Both reagents were found to stimulate Na
transport by mechanisms that were independent of cAMP production
or activation of PKA, a finding that invalidates their use as markers
for a cAMP-mediated mechanism without further direct confirmation. This
observation extends other reports that both forskolin and externally
applied cAMP can modulate transport proteins by diverse mechanisms
other than activation of the cAMP second messenger system. Both
forskolin and 1,9-dideoxyforskolin can directly modulate a number of
transport proteins, including the glucose transporter(31) , the
nicotinic acetylcholine receptor(32, 33) , and several
types of voltage-dependent Na
and K
channels (see Ref. 34 for review). Externally applied cAMP or
analogues of cAMP can modify voltage-dependent ion channels by a
PKA-independent mechanism in cardiac pacemaker cells(28) ,
cardiac myocytes(30) , and olfactory receptor cells(29) .
transport depends on mobilization of Ca
,
mention should be made of previous reports suggesting that
experimentally induced increases in Ca
actually inhibit Na transport(35, 36) . Interpretation of such
evidence is confounded, we believe, by the nature of the agents
utilized. The Ca
-elevating agonists clearly also
activate several signaling pathways other than Ca
which may exhibit inhibitory effects. Increases in
Ca
obtained with calcium ionophores are
difficult to control and probably cannot mimic the highly organized
temporal and spatial patterns of incremental Ca which are induced by physiological agonists, as reviewed
elsewhere(37) .
transport is mediated
by a Ca
-mobilizing signal transduction
system. Contrary to the prevailing dogma and despite extensive probing
no evidence was obtained that cAMP acts as a second messenger for the
stimulation of Na transport. Along with previous
reports showing that the actions of aldosterone, insulin, and adenosine
to stimulate Na
transport are mediated by the second
messenger Ca
(6, 7, 8) , these
findings imply that multiple agonists that stimulate electrogenic
Na
transport all utilize a single primary transduction
system.
Table: Demonstration of the effects of cold exposure
and IP loading on transepithelial resistance
(R
) and AVP-stimulated I
, while controls were not
chilled. There were 5 cups in each group and values are mean ±
SEM.
, inositol trisphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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