(Received for publication, May 31, 1995; and in revised form, June 12, 1995)
From the
Parathyroid hormone (PTH) and parathyroid hormone-related
peptide (PTHRP) interact with a common G protein-coupled receptor and
stimulate production of diverse second messengers (i.e. cAMP,
diacylglycerol, and inositol 1,4,5-trisphosphate) that varies depending
on the target cell. In renal proximal tubule OK cells, PTH inhibits the
activity of the apical membrane Na/H
exchanger, although it is unclear whether the signal is
transmitted through protein kinase A (PKA) and/or protein kinase C
(PKC). To delineate the signaling circuitry, a series of synthetic PTH
and PTHRP fragments were used that stimulate the adenylate
cyclase-cAMP-PKA and/or phospholipase C-diacylglycerol-PKC pathways.
Human PTH-(1-34) and PTHRP-(1-34) stimulated adenylate
cyclase and PKC activity, whereas the PTH analogues, PTH-(3-34),
PTH-(28-42), and PTH-(28-48), selectively enhanced only PKC
activity. However, each peptide fragment inhibited
Na
/H
exchanger activity by
40-50%, suggesting that PKC and possibly PKA were capable of
transducing the PTH/PTHRP signal to the transporter. This was
corroborated when forskolin and phorbol 12-myristate 13-acetate (PMA),
direct agonists of adenylate cyclase and PKC, respectively, both
inhibited the Na
/H
exchanger. The
specific PKA antagonist, H-89, abolished the forskolin-mediated
suppression of Na
/H
exchanger
activity, but did not prevent the inhibitory effects of
PTH-(1-34) or PMA. In comparison, the potent PKC inhibitor,
chelerythrine chloride, prevented the inhibition of
Na
/H
exchanger activity mediated by
PTH-(28-48) and PMA but did not avert the negative regulation
caused by PTH-(1-34) or forskolin. However, inhibition of both
PKA and PKC prevented PTH-(1-34)-mediated suppression of
Na
/H
exchanger activity, indicating
that PTH-(1-34) acted through both signaling pathways. In
addition, Northern blot analysis revealed the presence of only the
NHE-3 isoform of the Na
/H
exchanger
in OK cells. In summary, these results demonstrated that NHE-3 is
expressed in OK cells and that activation of the PTH receptor can
stimulate both the PKA and PKC pathways, each of which can
independently lead to inhibition of NHE-3 activity.
The electroneutral transport of extracellular Na in exchange for intracellular H
is mediated by
the Na
/H
exchanger (NHE). (
)This transporter is involved in the regulation of
intracellular pH (pH
), maintenance of cell volume,
transepithelial Na
reabsorption, and
pH
-mediated cellular signaling associated with cell growth
and differentiation (for review, see (1) ). Recently several
mammalian isoforms of the Na
/H
exchanger have been identified and characterized by cDNA cloning
(NHE-1 to -4) (2, 3, 4, 5) and
chromosomal mapping (NHE-5)(6) . Based on available data, NHE-1
is an amiloride-sensitive exchanger that is expressed in most cells and
functions primarily in the regulation of pH
and
cell volume(2, 7, 8) . In polarized renal (9) and intestinal(10, 11) epithelial cells,
NHE-1 is localized to the basolateral membrane. Likewise, NHE-2, which
has a slightly reduced sensitively to amiloride analogues(12) ,
also has a wide tissue distribution (3) and exhibits similar
functional characteristics(8) . However, its membrane targeting
in polarized renal epithelial cells is controversial, with studies
reporting localization to the basolateral (13) or apical (14) membranes. In contrast, NHE-3 has a substantially lower
affinity for amiloride (7) and is found exclusively on the
apical membranes of renal (15) and intestinal (11) epithelia. Consequently this isoform is most likely
involved in transepithelial Na
reabsorption. Last,
NHE-4 is found primarily in stomach and to a much lesser extent in
other tissues(2) . At present, characterization of its
functional properties is rather limited (16) due to its poor
expression in heterologous cell systems.
Parathyroid hormone (PTH)
inhibits in vivo renal proximal tubular Na,
HCO
and phosphate reabsorption by
inhibiting the apically located Na
/H
exchanger and Na
/phosphate cotransporters
(reviewed in (17) and (18) ). Similar
results(19, 20, 21) have also been obtained
in the opossum kidney (OK) cell line, which exhibits many of the
characteristics of renal proximal tubule cells (22) . The
signaling mechanism to account for the effects of PTH in target tissues
has only recently been investigated. PTH (an 84-amino acid peptide) and
the recently identified factor, PTH-related peptide (PTHRP) (a
139-173-amino acid peptide, depending on the
species)(23) , interact with a common heterotrimeric G
protein-coupled receptor(24) . Interestingly, only 8 of the N-terminal 13 amino acids between these two peptides are
identical, whereas the remaining sequences are unique. Nonetheless,
both can bind and activate the receptor which subsequently stimulates
the adenylate cyclase and/or phospholipase C signaling
pathways(24) . In OK cells, the inhibitory effect of PTH on
apical Na
/H
exchanger activity can
also be mimicked by forskolin and phorbol esters, pharmacological
agonists of the PKA and PKC pathways,
respectively(20, 25) . Assembling these data together,
it has been inferred that the mechanism by which PTH inhibits the OK
apical Na
/H
exchanger is through
independent activation of PKA and PKC(20, 25) . While
this is a plausible interpretation, more direct evidence demonstrating
this linkage is lacking. Other signaling circuitry could account for
these observations. For example, it is quite possible that only one of
these two pathways predominates in the PTH
Na
/H
exchanger signaling cascade.
This has been observed in the SV-40 transformed murine renal cortical
proximal tubule (MCT) cell line, where PTH, forskolin, and PMA
inhibited the apical Na
/H
exchanger(26) . However, PTH was found not to elevate
cAMP
levels but rather to utilize only the
phospholipase C-PKC pathway. Aside from this scenario, other signaling
routes may also exist. It is conceivable that PTH could activate both
pathways which subsequently ``talk'' with each other such
that only one pathway ultimately leads to inhibition of the exchanger.
Cross-talk between the PKA and PKC pathways has been documented in some
cell systems where activation of PKC stimulates adenylate cyclase type
II activity and ultimately PKA(27, 28) . Indeed, there
is some evidence that PMA may act as a weak activator of cAMP
production in OK cells(29) . Last,
Na
/H
exchanger activity in human
embryonic kidney (HEK) 293 cells can be modulated by a G protein
(G
) that is not linked to either adenylate cyclase or
phospholipase C(30) . Whether this pathway is also coupled to
the PTH receptor in OK cells is unknown. Hence, the potential exists
for multiple signaling routes that can be activated by PTH or PTHRP,
with the predominant effect being dependent on the exact cellular
complement of receptor-associated G proteins and their downstream
effectors.
Therefore, in the present study, we wished to examine in
greater detail the hypothesis that PTH or PTHRP inhibits the apical
membrane Na/H
exchanger by both the
PKA and PKC pathways in the OK cell model system. This was accomplished
as follows: (i) by using a series of synthetic peptide fragments of PTH
and PTHRP that selectively activate adenylate cyclase and/or
phospholipase C and (ii) by using other downstream activators (i.e. forskolin and PMA) and selective inhibitors of the PKA and PKC
pathways. Second, we wished to establish which isoform(s) of the
Na
/H
exchanger is(are) present in
these cells. Our results demonstrated that only the NHE-3 isoform is
expressed in OK cells and that PTH and PTHRP inhibit NHE-3 via two
distinct signaling pathways that act independently of each other.
To extract the radiolabel, the cells were solubilized
in 250 µl of 0.5 N NaOH and the wells washed with 250
µl of 0.5 N HCl. Both the solubilized cell extracts and
the wash solutions were added to scintillation vials, and the
radioactivity was quantitated in a -counter. Amiloride-sensitive
Na
/H
exchanger activity was defined
as the difference between the rates of
Na
influx in the absence and presence of 1 mM amiloride.
Figure 1:
Effect of
PTH and PTHRP peptide fragments on adenylate cyclase activity in OK
cells. OK cells were grown to confluence in 24-well plates and treated
with various PTH and PTHRP fragments (each at 100 nM).
Experiments were performed as described under ``Experimental
Procedures,'' and results are the mean ± S.E. of up to six
determinations from three separate experiments. Values are reported as
the -fold stimulation of adenylate cyclase activity. Control adenylate
cyclase activity was 190 ± 20 cpm
[H]cAMP/15 min. Significance from control values
was determined by Student's t test and is indicated by
an asterisk (p <
0.002).
Figure 2:
Concentration dependence of PTH and PTHRP
peptide fragments on PKC activity of OK cells. Confluent cultures of OK
cells were treated with increasing concentrations of PTH and PTHRP
peptide fragments and then assayed for PKC activity which was defined
as the translocation of PKC from cytosol to membranes. Experiments were
performed as described under ``Experimental Procedures.''
Results are the mean ± S.E. of up to nine determinations from
three separate experiments and are presented as an index of the ratio
of PKC activity present in the membrane per PKC activity present in the
cytosol (PKC/PKC
). The ratio of
PKC
/PKC
in the absence of PTH or PTHRP
(0.057 ± 0.004) served as the control (C) and was
normalized to a value of 1 for comparative purposes. Statistical
analyses (analysis of variance) were performed on the data and all
peptide fragments, with the exception of PTHRP-(1-16), were found
to significantly stimulate PKC activity (p <
0.02).
Figure 3:
Determination of
Na/H
exchanger isoform expression in
OK cells. Total cellular RNA was extracted from the following cell
lines: oppossum renal OK cells; Chinese hamster ovary AP-1 cell
transformants that stably express either rat NHE-1, NHE-2, or NHE-3;
and Chinese hamster lung fibroblasts that stably express rat NHE-4.
Total cellular RNA (20 µg) was analyzed by Northern blot
hybridization (see ``Experimental Procedures'' for further
details).
P-Labeled isoform-specific cDNA fragments were
used as probes. The positions of the 28 and 18 S rRNAs were used as
markers and are shown on the left of the figure
panels.
Figure 4:
Concentration response profiles for
PTH-(1-34), PTH-(28-48), forskolin, and PMA on
Na/H
exchanger activity in OK cells.
OK cells were grown to confluence in 24-well plates. Prior to
Na
influx measurements, the cells were
preincubated with increasing concentrations of PTH-(1-34) (A), PTH-(28-48) (B), forskolin (C),
and PMA (D) for 15 min in isotonic NaCl solution. The cells
were rapidly washed with Na
-free isotonic choline
chloride solution and then incubated in assay medium containing 1
µCi of
NaCl (carrier free)/ml and the various agents
for a 12-min period. Control (C) cells were treated with
diluent alone. Na
/H
exchanger
activity was determined as amiloride-inhibitable
Na influx
as described under ``Experimental Procedures.'' Each value is
the mean ± S.E. of up to six determinations from three
experiments.
The above data supported the notion that PTH-(1-34)
and PTHRP-(1-34) may be mediating their effects on NHE-3 activity
via a PKC, and possibly PKA, pathway. In this regard, earlier studies (25) have demonstrated that pharmacological activation of these
two protein kinases can inhibit the apical
Na/H
exchanger in OK cells, as
assessed by measuring changes in pH
using a
pH-sensitive fluorescent dye. To corroborate these observations using
radioisotope techniques, the effects of forskolin, a direct agonist of
adenylate cyclase, and PMA, a potent activator of PKC, were examined.
Treatment of OK cells with increasing concentrations of either
forskolin (Fig. 4C) or PMA (Fig. 4D)
maximally suppressed NHE-3-mediated
Na
influx by
50-65%. In contrast, the corresponding
biologically inert analogues, 1,9-dideoxyforskolin (10 µM)
and 4
-PMA (1 µM) lacked any inhibitory effect and
supported the notion that the effects of these compounds were
biologically relevant (data not shown). To assess whether
pharmacological activation of PKA and PKC could act in a synergistic
manner, the combined effects of 10 µM forskolin and 1
µM PMA were investigated. Treatment of OK cells with both
compounds gave similar results (64 ± 4% inhibition) to those
obtained separately. Thus, these results are in accordance with
previous data(25) , suggesting that activation of either of
these two regulatory cascades, at least at this signaling juncture, can
ultimately lead to inhibition of NHE-3.
The conclusions reached
using cells at resting pH were also confirmed and
extended by complementary studies measuring exchanger activity in cells
that were acid-loaded (
pH
6.6) by an
NH
prepulse (Fig. 5). NHE-3
activity was significantly reduced by
25-50% (p < 0.05) in cells pretreated with PTH-(1-34), forskolin,
and PMA. Although the percentage inhibition was lower compared with
resting pH
conditions, this represented only an
apparent decrease in the percentage since the absolute rates of
amiloride-inhibitable
Na influx were greatly increased in
both control and treated cells under acid-loaded conditions. These data
also exclude the remote possibility that the inhibitory effects
mediated by these agents were due to a generalized reduction in
cellular metabolic activity and H
concentration.
Figure 5:
Influence of PTH-(1-34), forskolin,
and PMA on Na/H
exchanger activity in
OK cells under acidic pH
conditions. Confluent OK
cells were incubated with 25 mM NH
Cl in isotonic
saline buffer for 30 min to acidify the cells to
pH
6.6 (pH
was assessed by
microfluorometry using the dye BCECF as described
previously(8) ). During the last 15 min of the
NH
prepulse, cells were treated with
PTH-(1-34) (100 nM), forskolin (10 µM), and
PMA (1 µM). The cells were then rapidly washed with
Na
-free isotonic choline chloride solution and then
incubated in assay medium containing 1 µCi of
NaCl
(carrier free)/ml and the different agents for a 5-min period. Control
cells were treated with diluent alone. Na
/H
exchanger activity was determined as amiloride-inhibitable
Na influx. Each value is the mean ± S.E. of four
determinations from two experiments. Significance from control
measurements was calculated using the Student's t test
and is indicated by an asterisk (p <
0.05).
To
confirm the signaling circuitry involving PTH PKA/PKC
NHE-3 in greater detail, highly selective antagonists of PKA (i.e. H-89) (41) and PKC (i.e. chelerythrine chloride) (42) were used. OK cells were preincubated for 1 h with either
100 µM H-89 or 1 µM chelerythrine chloride
followed by coincubation in the absence or presence of PTH analogues
(100 nM), forskolin (10 µM), and PMA (1
µM). As illustrated in Fig. 6A, H-89
abolished the effects of forskolin but had no influence on
PTH-(1-34) or PMA. This indicated that PTH-(1-34) and PMA
could inhibit NHE-3 activity independently of PKA. This also confirmed
that the effects of both compounds were not mediated by subsequent PKC
activation of adenylate cyclase type II; such a signaling route has
been observed in some cell types(27, 28) . In
comparison, chelerythrine chloride (Fig. 6B) completely
abrogated the negative regulation mediated by PTH-(28-48) and
PMA; agents that act exclusively through PKC. However, it was
ineffective in preventing the inhibition of NHE-3 by PTH-(1-34),
as was H-89, and was unable to negate the actions of forskolin. The
inability of chelerythrine chloride to prevent the inhibitory effects
of forskolin verified that activation of the PKA pathway does not lead
to enhanced PKC activity which could subsequently inhibit NHE-3.
Furthermore, these data suggested that PTH-(1-34) was capable of
acting independently through either PKA or PKC or possibly a novel
third pathway involving G
(30) . To test this
postulation, the combined influence of H-89 and chelerythrine chloride
was assessed. However, for reasons which remain unclear, the
combination of these two protein kinase antagonists caused the cells to
detach from the culture wells during the multiple washing steps of the
Na
influx assay which did not occur when
the compounds were used separately. As an alternative protocol to
circumvent this problem, OK cells were incubated overnight (
20 h)
in the presence of PMA (400 nM) to down-regulate PKC activity (43) followed by a 1-h pretreatment with H-89. Under these
conditions, the cells remained adherent throughout the assay
procedures. Using this approach, inhibition of both PKA and PKC
prevented the suppressive effects of PTH-(1-34) on NHE-3; its
activity being 92 ± 7% (p > 0.01) of that obtained
for control cells (100 ± 5%). This provided direct evidence that
the inhibitory actions of PTH-(1-34) on NHE-3 involved both PKA
and PKC. Furthermore, this indicated that the PTH/PTHRP receptor did
not transduce its signal to NHE-3 by a potential third pathway that was
independent of adenylate cyclase and phospholipase C.
Figure 6:
Effect of PKA and PKC inhibitors on PTH
analogue-, forskolin-, and PMA-mediated inhibition of
Na/H
exchanger activity in OK cells.
Confluent OK cells were preincubated in the absence or presence of 100
µM H-89, a selective inhibitor of PKA (A) or 1
µM chelerythrine chloride (Chel), a selective
inhibitor of PKC (B), for 1 h prior to treatment with PTH
analogues (100 nM), forskolin (10 µM), and PMA (1
µM). Results are shown as the difference between initial
rates of
Na
influx in the absence and
presence of 1 mM amiloride and are expressed as a percentage
of the control value. Each value is the mean ± S.E. of six to
eight determinations from two experiments. Significance from control
measurements in the absence of any agent other than diluent was
calculated using the Student's t test and is indicated
by an asterisk (p <
0.02).
PTH inhibits apical Na/H
exchanger activity in renal proximal tubule OK
cells(19, 20) . The results from this study provide
direct evidence that activation of the PTH/PTHRP receptor in OK cells
stimulates both PKA and PKC, each of which can independently lead to
inhibition of the Na
/H
exchanger
NHE-3 isoform. Kinetically, this alteration has been demonstrated to
occur by a reduction in the V
and affinity for
protons of the Na
/H
exchanger(44) .
Previous studies had implicated the
involvement of PKA and/or PKC pathways in the PTH-mediated inhibition
of Na/H
exchanger activity in both
renal OK (20, 25) and MCT (26) cells by the
use of pharmacological agents. Our own findings in OK cells confirmed
that both forskolin, an activator of the adenylate cyclase
PKA
pathway, and PMA, a direct PKC agonist, were able to similarly inhibit
NHE-3 activity. These respective effects were only prevented with the
corresponding specific inhibitors of PKA (H-89) and PKC (chelerythrine
chloride), with no evidence for cross-talk between the two pathways.
This provided direct evidence for the involvement of PKA and PKC and is
consistent with their capacity to independently inhibit NHE-3 activity.
Similar conclusions were drawn using a series of synthetic peptide
fragments of PTH and PTHRP that differentially activated the adenylate
cyclase-cAMP-PKA and phospholipase C-DAG-PKC pathways or only the
latter signaling route. Most notably, PTH analogues such as
PTH-(1-34) triggered both signaling pathways which, at least
theoretically, could independently influence NHE-3 activity.
Antagonizing the actions of either PKA with H-89 or PKC with
chelerythrine chloride failed to inhibit the suppressive effect of
PTH-(1-34) on NHE-3 activity. This suggested a role for both
protein kinases in the action of this fragment and supported the thesis
that activation of either pathway is sufficient for similar effects on
NHE-3 activity. This was confirmed when simultaneous inhibition of PKA
and PKC prevented the actions of PTH-(1-34). Consequently, each
pathway appears independently capable of altering exchanger activity, a
conclusion consistent with previous findings in OK cells(25) .
Notwithstanding the above conclusion, it is noted that there are
significant concentration-dependent differences between
PTH-(1-34) and PTHRP-(1-34) activation of adenylate cyclase (K
10
M) (37) and that reported herein for PKC (K
10
M). Since plasma
circulating concentrations of PTH are in the picomolar range, it is
likely that PTH-mediated inhibition of renal proximal tubule NHE-3
activity is transduced primarily by the PKC pathway. However, the PKA
pathway may play a more significant role in modulating renal NHE-3
activity in certain tumorigenic malignancies associated with elevated
paracrine secretions of PTHRP that cause humoral hypercalcemia and
hypophosphatemia(23) . However, the relative importance of one
or the other pathway to PTH/PTHRP regulation of renal apical NHE-3
activity is unclear.
In addition to the regulation of renal NHE-3
cited above, other more recent studies have provided supporting
evidence that NHE-3 can be regulated by serine/threonine protein
kinases in heterologous cell expression systems. Rabbit NHE-3 stably
expressed in fibroblastic cells (PS120) is inhibited following acute
cell stimulation by phorbol esters, although elevating intracellular
cAMP had no effect(45) . In contrast, preliminary data in our
laboratory have shown that rat NHE-3 stably expressed in Chinese
hamster ovary AP-1 cells is inhibited by both phorbol esters and
agonists that increase cAMP accumulation. ()Thus, regulation
of NHE-3 exhibits species and/or cell-specific differences.
By
Northern analysis, the only NHE isoform expressed in cultured OK cells
was NHE-3. Interestingly, the size of the major NHE-3 transcript
(9 kilobases) was considerably larger than that observed in other
mammalian species which ranged from 5.4 to 5.6
kilobases(2, 46) . The reason for this discrepancy is
unknown but may reflect species variations in the size of the 5` and/or
3` mRNA untranslated regions. Although we did not detect other NHE
isoforms, we cannot exclude the possibility that small quantities were
present but below the detection threshold of this technique,
particularly since our cDNA probes were from a different species.
Nonetheless, the NHE-3 isoform clearly appears to be the most abundant
isoform of this gene family in OK cells. Moreover, NHE-3
immunoreactivity has previously been localized along the microvillar
membrane of the brush border of rabbit proximal tubule epithelia (15) and OK cells(40) . Inasmuch as OK cells are
believed to express a proximal tubule cell phenotype, our finding of
NHE-3 expression in these cells is consistent with those previous
observations.
In common with the other isoforms of the
Na/H
exchanger, NHE-3 is believed to
contain 10-12 putative transmembrane segments followed by a
hydrophilic cytoplasmic domain. The membrane-spanning region of the
exchanger is required for transport, whereas the cytoplasmic domain
appears to function in a regulatory capacity(47) . Our studies
suggest that PKA and PKC may both exert important regulatory activities
at either the same phosphorylation site or at discrete phosphorylation
sites which, nevertheless, similarly influence exchanger activity. In
this regard, the cytoplasmic domain contains consensus sequences for
PKA (R-R/K-X-S*/T*) as well as for PKC
((R/K)
, X
)-S*/T*-(X
,
R/K
)), although there is overlap in consensus
sequence determinants among protein kinases(48) . However, the
molecular signaling events that occur between protein kinase activation
and NHE-3 inhibition are unclear. At present, it is unknown whether
these protein kinases mediate their effects by direct phosphorylation
of NHE-3 or indirectly via phosphorylation-dependent ancillary
proteins. With regard to the latter, there is some in vitro evidence that PKA-mediated inhibition of the rabbit renal apical
Na
/H
exchanger requires the
involvement of a regulatory protein that is separate from the kinase
and transporter (49, 50) . Cell-specific expression of
these factors could account for the differential responsiveness of
NHE-3 to individual protein kinases. Further studies are required to
identify the precise molecular mechanisms involved.