(Received for publication, February 10, 1995; and in revised form, May 12, 1995)
From the
Parathyroid hormone (PTH) and parathyroid hormone-related
peptide (PTHRP) regulate Na/H
exchanger activity in osteoblastic cells, although the signaling
components involved are not precisely defined. Since these peptide
hormones can stimulate production of diverse second messengers (i.e. cAMP and diacylglycerol) that activate protein kinase A
(PKA) and protein kinase C (PKC) in target cells, it is conceivable
that either one or both of these pathways can participate in modulating
exchanger activity. To discriminate among these possibilities, a series
of synthetic PTH and PTHRP fragments were used that stimulate adenylate
cyclase and/or PKC. In the osteoblastic cell line UMR-106, human
PTH(1-34) and PTHRP(1-34) augmented adenylate cyclase
activity, whereas PTH(3-34), PTH(28-42), and
PTH(28-48) had no effect. Nevertheless, all these peptide
fragments were found to enhance PKC translocation from the cytosol to
the membrane in a dose-dependent (10
to
10
M) manner. PTHRP(1-16), a
biologically inert fragment, was incapable of influencing either the
PKA or PKC pathway. PTH(1-34) and PTHRP(1-34), but not
PTH(3-34), PTH(28-42), PTH(28-48), or
PTHRP(1-16), elevated Na
/H
exchanger activity, implicating cAMP as the transducing signal.
In accordance with this observation, forskolin (10 µM),
which directly stimulates adenylate cyclase, also activated
Na
/H
exchanger activity. The
involvement of PKA was verified when the highly specific PKA inhibitor,
H-89, completely abolished the stimulatory effect of PTH(1-34)
and forskolin on Na
/H
exchange. In
addition, Northern blot analysis revealed the presence of only the
NHE-1 isoform of the Na
/H
exchanger
in UMR-106 cells. In summary, these results indicated that PTH and
PTHRP activate the Na
/H
exchanger
NHE-1 isoform in osteoblastic UMR-106 cells exclusively via a
cAMPdependent pathway.
Parathyroid hormone (PTH) ()and PTH-related peptide
(PTHRP) are important regulators of normal and pathological bone
remodeling(1) . In UMR-106 cells, a well characterized
osteoblastic cell line(2, 3) , both PTH and PTHRP can
interact with a common G protein-coupled receptor that is functionally
associated with both the adenylate cyclase-cAMP-protein kinase A (PKA)
and phospholipase C-diacylglycerol-protein kinase C (PKC)
pathways(4, 5) . Thus, the ability of this receptor to
couple to multiple effector systems is believed to account for the
pleiotropic actions of these peptides in bone as well as other tissues.
Nonetheless, the precise linkage of the PTH/PTHRP-receptor to these
distinct signaling pathways and various downstream molecular targets
remains poorly defined.
A recently identified target of PTH/PTHRP
action in osteoblasts is the plasma membrane
Na/H
exchanger, which plays a central
role in modulating intracellular pH
(pH
)(6, 7, 8) . In
osteoblastic cells, regulation of pH
is a critical
component of hormone-stimulated bone remodeling where local
fluctuations occur in the osseous microenvironment(9) .
Numerous studies have shown that Na
/H
exchanger activity is acutely regulated by a wide variety of
stimuli that activate diverse signal transduction systems (i.e. PKA, PKC, Ca
/calmodulin- dependent protein
kinase II, tyrosine kinases), although in many cases the precise
mechanisms have yet to be fully elucidated (reviewed in (10) ).
This extensive functional and regulatory diversity appears to be
generated by tissue-specific expression of multiple isoforms of the
Na/H exchanger as well as by variations in the signaling repertoire of
individual cell types (reviewed in (11) ). To date, four
members (NHE-1 to NHE-4) of this gene family have been identified and
characterized by cDNA cloning (11, 12, 13, 14) and functional
expression studies(15, 16, 17, 18) .
Overall, they share
40-60% amino acid identity and contain
one or more potential sites for phosphorylation by different
serine/threonine protein kinases in their putative cytoplasmic
carboxyl-terminal region. More recently, a putative fifth (19) and possibly sixth (20) isoform have been located
by chromosomal mapping in humans.
In osteoblastic cell lines, there
are conflicting reports regarding the regulation of the
Na/H
exchanger and pH
by PTH/PTHRP and second messengers. Elevation of
intracellular cAMP levels by PTH, PTHRP, forskolin, or cAMP analogues
has been reported to inhibit (6, 7, 21) or
stimulate (22) Na
/H
exchanger
activity in UMR-106 cells. Aside from methodological considerations,
the reason for this difference is unclear. In addition, phorbol
12-myristate 13-acetate (PMA treatment) also leads to stimulation of
Na
/H
exchanger activity, presumably
through activation of PKC(22) . Thus, at least two distinct
signaling pathways appear to regulate this ion transporter in bone
cells. In contrast to UMR-106 cells, PTH as well as epinephrine, a
-adrenergic agonist that also activates a G protein-linked
receptor coupled to adenylate cyclase, were found to activate the
Na
/H
exchanger independent of changes
in cAMP accumulation in rat osteosarcoma ROS 17/2.8 cells(23) .
Further studies revealed that
-adrenergic receptor-mediated
activation of the Na
/H
exchanger
involved a G protein (G
) that was not linked to
either adenylate cyclase or phospholipase C(24, 25) ,
thereby implicating the involvement of a novel third pathway. Whether
this also applies to the PTH receptor is unknown. Nonetheless, it is
conceivable that the PTH/PTHRP signal to modulate
Na
/H
exchanger activity in
osteoblasts is transduced by more than two signaling pathways. In
addition, the regulatory diversity mentioned above may also be
reflected by cellular expression of one or more isoforms of the
Na
/H
exchanger that are
differentially responsive to distinct signaling pathways. The identity
of specific Na
/H
exchanger isoforms
in osteoblastic cells has yet to be determined.
The purpose of this
study was two-fold. First, using UMR-106 cells as a model system, we
wished to examine in greater detail the reported linkage of PTH/PTHRP
to the PKA and PKC signaling pathways, and possibly to another pathway
involving G, in regulating
Na
/H
exchanger activity. 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 inhibitors
of the PKA and PKC pathways. Second, we wished to identify which
isoform(s) of the Na
/H
exchanger
is(are) present in these cells. Our results demonstrated that only the
NHE-1 isoform is expressed in UMR-106 cells and that PTH and PTHRP
selectively activate NHE-1 via a pathway involving PKA.
In experiments
where exchanger activity was to be determined from the rate of Na
influx at constant
H
concentration, pH
was
clamped by incubating the cells in medium of varying K
concentration containing the K
/H
exchange ionophore nigericin(29) . Because at equilibrium
[K
]/[K
]
=
[H
]/H
],
the desired pH
was calculated from the imposed
[K
] gradient and the extracellular pH
(pH
= 7.4), assuming an intracellular
[K
] of 140 mM. Briefly, the cell
monolayer were washed twice with Na
-saline solution
and preincubated for 15 min in KCl solution. In this study, the
pH
was set at 6.6; the pH
clamping solution
contained 142 mMN-methyl-D-glucamine
methanesulfonate, 14 mM KCl, 8 mM potassium
glutamate, 2 mM NaCl, 1 mM MgCl
, 10
µM nigericin, and 10 mM HEPES. Then, the solution
was removed and measurements were made in KCl solution supplemented
with 1 mM ouabain and 1 µCi/ml
NaCl in the
absence or presence of 1 mM amiloride.
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
UMR-106 cells. UMR-106 cells were grown to confluence in 24-well
plates. A, cells were treated with increasing concentrations
of PTH(1-34) and then assayed for adenylate cyclase activity. B, UMR-106 cells were treated with various PTH and PTHRP
fragments (each at 100 nM) and forskolin (10 µM).
Experiments were performed as described under ``Experimental
Procedures,'' and results are the mean ± S.E. of up to 6
determinations from three separate experiments. Values are reported as
the -fold stimulation of adenylate cyclase activity. Control adenylate
cyclase activity was 256 ± 21 cpm
[H]cAMP/15 min. Significance from control values
was determined by Student's t test and is indicated by
asterisks (
p < 0.01;
p < 0.001).
Figure 2:
Concentration dependence of PTH and PTHRP
peptide fragments and PMA on PKC activity of UMR-106 cells. 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 shown 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 any
agent (0.170 ± 0.003) served as the control (C) and was
normalized to a value of 1 for comparative
purposes.
Figure 3:
Determination of
Na/H
exchanger isoform expression in
UMR-106 cells. Total cellular RNA was extracted from the following cell
lines: rat UMR-106 cells; AP-1 cell transformants that stably expressed
either rat NHE-1, NHE-2, or NHE-3; and PS120 cells that stably
expressed 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:
Effect of PTH(1-34) as a function of
uptake time and concentration on Na/H
exchanger activity in UMR-106 cells. UMR-106 cells were grown to
confluence in 24-well plates. A, prior to
Na
influx measurements, the cells were
preincubated in PTH(1-34) (100 nM) 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/ml
NaCl
(carrier-free).
Na
influx was measured at
increasing time intervals in the continuing presence of
PTH(1-34). Low levels of background
Na
influx that were not inhibitable by 1 mM amiloride were
subtracted from the total influx. Na
/H
exchanger activity was expressed as initial rates of
amiloride-inhibitable
Na
influx. Each
value is the mean ± S.E. of 4-6 determinations. B, prior to
Na
influx
measurements, the cells were preincubated with increasing
concentrations of PTH(1-34) (10
to
10
M) for 15 min in isotonic NaCl solution.
The cells were then assayed for Na
/H
exchanger activity at the different concentrations of
PTH(1-34) using a 12-min uptake period as described above. Each
value is the mean ± S.E. of up to 6 determinations from three
experiments. Significance from control (C) measurements in the
absence of PTH(1-34) is indicated by an asterisk (*, p < 0.02).
Figure 5:
Influence of PTH and PTHRP fragments and
forskolin on Na/H
exchanger activity
in UMR-106 cells. Prior to measurements of
Na
/H
exchanger activity, confluent
UMR-106 cells were pretreated for 15 min with peptide fragments of PTH
and PTHRP (each at 100 nM), forskolin (10 µM), or
1,9-dideoxyforskolin (10 µM), or pretreated for 1 h with
the PKA antagonist H-89 (100 µM) to which PTH(1-34)
or forskolin were added during the last 15 min. 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 percent change in amiloride-inhibitable
Na
influx. Each value is the mean
± S.E. of 6 determinations from three 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).
The above data supported
the notion that PTH(1-34) and PTHRP(1-34) may be
selectively mediating their effects on NHE-1 activity via a
cAMP-dependent pathway. This possibility was tested by increasing
intracellular levels of cAMP with forskolin, which can directly
activate adenylate cyclase. As shown in Fig. 5, 10 µM forskolin also increased NHE-1 activity to 340% of control
values, whereas a biologically inert forskolin analogue,
1,9-dideoxyforskolin (10 µM), had no effect. The absence
of stimulation by 1,9-dideoxyforskolin indicated that forskolin-induced
activation of NHE-1 activity was biologically relevant and not a
nonspecific effect of the compound. To test this hypothesis further,
the effect of a highly selective inhibitor of PKA, H-89(36) ,
was investigated. This cell permeant compound has been shown to
effectively inhibit PKA while having no detectable effect on the
activities of PKC, protein kinase G, and
Ca
/calmodulin-dependent protein kinase II when
applied to whole cells at high micromolar concentrations(36) .
Therefore, UMR-106 cells were preincubated with 100 µM H-89, followed by coincubation in the absence or presence of 10
µM forskolin and 100 nM PTH(1-34). As
illustrated in Fig. 5, H-89 abrogated the effects of these
agents and confirmed the involvement of PKA. Furthermore, the complete
abolition of PTH(1-34) activation of NHE-1 by H-89 also indicated
that the PTH receptor did not transduce its signal to the exchanger by
a potential third pathway that was independent of adenylate cyclase and
phospholipase C.
Forskolin also accelerated the NHE-1 activity to
129 ± 4.8% (p < 0.05) of control values (100
± 6.5%) under acid-loaded, clamped pH conditions
(pH
= 6.6) using the ionophore
K
-nigericin. A similar effect was also observed for
PTH(1-34) (127 ± 3.9%; p < 0.05). Although the
percentage stimulation was lower compared to unclamped conditions, this
represented only an apparent decrease in the percentage since the
absolute rates of amiloride-inhibitable
Na influx are
greatly increased in both control and forskolin-treated cells under
acid-loaded conditions. Thus, these compounds were able to accelerate
NHE-1 activity even when the H
substrate concentration
was held constant. Mechanistically, this suggested that the effect of
both agents was not a consequence of increased metabolic production of
acid.
Because other studies (8, 22) had indicated
that Na/H
exchanger activity could be
stimulated by PKC in osteoblastic cells, it was somewhat surprising
that PTH fragments (i.e. PTH(3-34), PTH(28-42),
and PTH(28-48)) capable of increasing PKC activity were without
effect. Since the earlier studies had evaluated the influence of PKC on
Na
/H
exchanger activity by treatment
of cells with phorbol esters, this class of agents was also examined.
Consistent with the results obtained with PTH(3-34),
PTH(28-42), and PTH(28-48), nanomolar concentrations (100
nM) of PMA that significantly activated PKC (see Fig. 2) had no effect on NHE-1 activity (Fig. 6A). In contrast, 1 and 10 µM PMA
significantly increased NHE-1 activity. Interestingly, the increase in
PKC activity induced with 1 µM PMA was not significantly
different from that achieved with 0.1 µM PMA. The
biologically inactive PMA analogue, 4
-PMA (10 µM),
had no effect (Fig. 6A) and supported the view that PMA
was acting in a specific and biologically relevant manner. Thus, only
high concentrations of PMA (>0.1 µM) stimulated NHE-1
and suggested that a novel mechanism other than activation of PKC may
be involved.
Figure 6:
Effect of phorbol ester on
Na/H
exchanger activity in UMR-106
cells. A, prior to amiloride-inhibitable
Na
influx measurements, UMR-106 cells
were preincubated with increasing concentrations of PMA
(10
to 10
M) or
4
-PMA (10 µM) for 15 min in isotonic NaCl solution.
The cells were then assayed for Na
/H
exchanger activity in their continuing presence. B,
UMR-106 cells were preincubated for 1 h in the absence or presence of 1
µM chelerythrine chloride, a PKC antagonist, or 100
µM H-89, a PKA antagonist. PMA was added at the indicated
concentrations during the last 15 min of the preincubation period,
followed by measurements of amiloride-inhibitable
Na
influx. Each value is the mean
± S.E. of 6 determinations from two to three experiments.
Significance from control measurements in the absence of any agent
other than diluent was calculated using Student's t test
and is indicated by an asterisk (*, p <
0.02).
To investigate the validity of this supposition, the
effect of chelerythrine chloride, a potent and selective inhibitor of
PKC(37) , was investigated. This cell permeant compound, used
at a concentration of 1 µM, effectively inhibits the
catalytic domain of PKC but does not affect the binding of
diacylglycerol or phorbol esters to the regulatory domain(37) .
As shown in Fig. 6B, 1 µM chelerythrine
chloride had only a marginal suppressive effect (25-35%) on
1 and 10 µM PMA-mediated stimulation of NHE-1. In
contrast, 100 µM H-89 completely prevented elevation of
NHE-1 activity by 10 µM PMA. These data supported the
possibility that PMA may be modulating NHE-1 activity by a pathway
involving PKA rather than PKC. Indeed, PMA has been reported to
modulate adenylate cyclase type II activity in some cell systems via a
mechanism that is partially (
50%) independent of
PKC(38, 39) . To explore this possibility, UMR-106
cells were treated with increasing concentrations of PMA and found to
elevate adenylate cyclase activity, but only by a modest 2.5-fold at 10
µM PMA (Fig. 7). This level of stimulation does not
appear to be sufficient to activate the PKA
NHE-1 pathway, since
a minimum 20-fold increase in cAMP
by 10 nM PTH(1-34) was required to significantly stimulate NHE-1
activity (see Fig. 1A and Fig. 4B).
However, at present, one cannot exclude the possible existence of small
compartmentalized accumulations of cAMP
induced by high
concentrations of PMA that are sufficient to activate localized pools
of specific PKA subtypes (discussed below).
Figure 7:
Effect of PMA concentration on adenylate
cyclase activity of UMR-106 cells. Experiments were performed as
described under ``Experimental Procedures.'' Results are the
mean ± S.E. of 6 determinations from three separate experiments.
Control (C) adenylate cyclase activity was 146 ± 11 cpm
[H]cAMP/15 min and was normalized to a value of
100%. Significance from control measurements in the absence of PMA is
indicated by an asterisk (*, p <
0.05).
This study has identified that the
Na/H
exchanger isoform present in
UMR-106 cells is NHE-1, which is the most broadly distributed isoform
in mammalian tissues. Activation of the PTH/PTHRP receptor in these
cells stimulates both the adenylate cyclase and phospholipase C
signaling pathways and causes an increase in NHE-1 activity. However,
only PTH analogues (i.e. PTH(1-34) and
PTHRP(1-34)) capable of activating adenylate cyclase and PKA were
able to stimulate NHE-1 activity, whereas those that retained the
ability to enhance only PKC activity (i.e. PTH(3-34),
PTH(28-42), and PTH(28-48)) were ineffective.
Prior
studies examining the regulation of Na/H
exchanger activity in UMR-106 cells are conflicting, with some
reports showing that elevation of cAMP
levels by various
agents was inhibitory (6, 7, 21) or
stimulatory(22) . Our results are in complete agreement with
the latter study by Gupta and co-workers(22) . These
investigators, using the pH-sensitive fluorescent dye
2`,7`-bis-(2-carboxyethyl)-5,6-carboxyfluorescein, demonstrated that
forskolin could stimulate the rate of
ethylisopropylamiloride-inhibitable H
efflux, which is
an alternative means of assessing Na
/H
exchanger activity, in UMR-106 cells at resting pH
and following an intracellular acid load. These microflurometric
analyses of pH
complemented our measurements of forskolin-
and PTH(1-34)-induced increases in amiloride-inhibitable
Na influx under similar conditions. Since these compounds
accelerated NHE-1 activity even under an imposed acid load, it is
unlikely that their effects can be solely accounted for by a mechanism
involving cAMP enhancement of metabolic H
production
which can occur in some cell types(40) . Moreover, the kinetic
analyses of Gupta et al.(22) showed that forskolin
increased both the magnitude and H
affinity of the
Na
/H
exchanger activity in UMR-106
cells. In contrast, other studies (6, 7, 21) have observed cAMP-mediated
inhibition of Na
/H
exchanger activity
in UMR-106 cells. The reason for the discrepancy is unclear but may
reflect different experimental conditions. Both Gupta et al.(22) and our own studies were performed using adherent
cell cultures, whereas the other studies determined
Na
/H
exchanger activity in
suspensions of UMR-106 cells after enzymatic digestion of the cultures.
Cell attachment has been implicated in the regulation of pH
through the action of integrins (41) and cytoskeletal
components(42) . In view of the fact that osteoblastic and
particularly pre-osteoblastic cells in vivo(1) may
communicate via cellular processes, the maintenance of the integrity of
cell-cell communication may be important for accurately assessing
hormone-regulated Na
/H
exchanger
activity.
Additional evidence supporting a role for cAMP in the
regulation of NHE-1 is rather sparse. Human (40) and rabbit (17) NHE-1 expressed in PS120 fibroblastic cells did not
respond to cAMP analogues. In contrast, primary rat hepatocytes (43) and murine macrophages (44) showed significant
cAMP-induced stimulation of Na/H
exchanger activity. Subsequent investigations indicated that
these tissues expressed only NHE-1 mRNA(12, 13) . (
)Furthermore, preliminary data has shown that rat NHE-1
stably expressed in Chinese hamster ovary AP-1 cells is also activated
by agonists that increase cAMP accumulation. (
)In addition
to mammalian NHE-1, the trout red cell also expresses a
Na
/H
exchanger, called
NHE, that
is cAMP-activable and has a primary structure with highest identity to
that of mammalian NHE-1(40) . Thus, the diverse regulation of
NHE-1 by increasing cAMP
appears to be partly influenced by
the signaling repertoire of individual cell types and perhaps by
species variation. Further studies are in progress to define the
precise molecular mechanism by which NHE-1 is regulated by agents that
activate PKA.
Stimulation of Na/H
exchange by phorbol esters has also been reported in rat UMR-106 (22) and human osteoblastic SaOS-2 (8) cells,
presumably via activation of PKC. Our examination of the effects of PMA
in UMR-106 cells produced mixed results that were
concentration-dependent. At low concentrations of PMA (i.e. 100 nM), PKC activity was significantly increased, but no
effect on NHE-1 activity was observed. This was consistent with data
obtained using PTH analogues that exclusively activated PKC but did not
alter NHE-1 activity. However, higher pharmacological concentrations
(
1 µM) of PMA caused significant stimulation of NHE-1
and appeared to corroborate results from the previous study of UMR-106
cells (22) where a similar concentration of PMA was used. To
resolve this seemingly contradictory result, our analyses were extended
by assessing the effects of highly specific antagonists of PKA and PKC.
Surprisingly, the effects of PMA were abolished by the PKA antagonist
H-89 while being relatively unaffected by the PKC inhibitor
chelerythrine chloride. This suggested that high concentrations of PMA
could activate PKA via a nonclassical mechanism. At present, only
limited circumstantial evidence exists for PKC-independent effects
mediated by PMA. For example, in human embryonic kidney (HEK-293)
cells, PMA has been reported to modulate adenylate cyclase type II
activity in the range of 2-9-fold via a pathway that is only
partially prevented (
50%) by inhibition of
PKC(38, 39) , thereby implicating the involvement of
additional mechanisms that are PKC-independent. This may also partly
explain other observations that acute treatment of
UMR-106(45) , ROS 17/2.8(46) , or fetal rat osteoblasts (47) with phorbol esters significantly enhanced PTH-mediated
stimulation of adenylate cyclase activity. Regardless of the precise
mechanism, but assuming that PMA activated PKA by elevating
cAMP
, it remained perplexing that 10 µM PMA
could substantially elevate NHE-1 activity by raising cAMP
levels only 2.5-fold, whereas a 25-fold increase in cAMP
levels was required to achieve a similar degree of stimulation by
100 nM PTH(1-34) or PTHRP(1-34). Although
speculative, one possible explanation to account for this difference is
that PTH(1-34) and PMA may mediate their effects on NHE-1
activity in UMR-106 cells by differential activation of distinct
adenylate cyclase (48) and PKA (49) isoforms that
reside in unique subcellular regions (for review, see (50) ).
Indeed, compartmentalized increases in cAMP
have been
observed in living cells by microfluorescent techniques(51) .
Furthermore, individual hormones have been found to preferentially
activate distinct PKA isoforms in a tissue-specific manner. For
example, in UMR-106 cells, PTH and prostaglandin E2 (PGE
)
were found to preferentially activate type I PKA, whereas in normal rat
clavarial osteoblasts, PTH activated type I and II PKA to similar
extents while PGE
stimulated type II PKA almost
exclusively(52) . Regionalized membrane localization of NHE-1
has also been observed in adherent fibroblasts(42) .
Compartmentation of small changes in cAMP
below the
detection sensitivity of our assay may also explain why 1 µM PMA activated NHE-1 without causing an observable change in
adenylate cyclase activity. Thus, the magnitude and spatial
localization of increases in cAMP
resulting from different
stimuli may be important in the signaling activation of NHE-1. Further
experimentation will be required to resolve this phenomenon, but is
beyond the scope of the current study.
Notwithstanding the unique
regulation of NHE-1 by PMA in UMR-106 cells, a number of other studies
have provided convincing data that NHE-1 is regulated by PKC. Stable
expression of human (53) and rabbit (17) NHE-1 in
fibroblastic cells (PS120) has shown that this isoform is rapidly
activated following acute cell stimulation by nanomolar concentrations
of phorbol esters as well as growth factors and other mitogens.
Mechanistically, this response is associated with increased
phosphorylation of a common set of tryptic peptide fragments in the
cytoplasmic tail-region of the
exchanger(53, 54, 55) . Moreover, this
isoform contains several putative PKC consensus sequences (56) in its carboxyl-terminal region. Interestingly, this
region does not contain a classical consensus site (RRXS) for
PKA. However, since there is overlap in consensus sequence determinants
among protein kinases(56) , one cannot exclude the potential
for PKA phosphorylation of NHE-1. At present, however, it is unclear if
phosphorylation of NHE-1 in vivo is mediated directly or
indirectly by these protein kinases and, indeed, whether
phosphorylation per se is responsible for the altered
activity. It is conceivable that the true targets of protein kinases
are cell-specific ancillary factors that subsequently interact with the
Na/H
exchanger and regulate its
activity. Cell-specific expression of these factors could account for
the differential responsiveness of NHE-1 to individual protein kinases.