(Received for publication, January 18, 1995; and in revised form, May 22, 1995)
From the
We have characterized the regulation of the endogenous
Na Na Few
information exists regarding the properties of the
Na
Possible
G protein regulation of Na
To study whether the X.
laevis oocyte Na
The stimulation of X.
laevis oocyte Na
Figure 1:
Effects of different protein kinase
inhibitors on GTP
It is generally accepted that the phospholipase C-PKC pathway
constitutes one route for NHE activation, with the notable exception of
the NHE-3, which is inhibited by phorbol ester-activated
PKC(7) . On the other hand, data on regulation of mammalian
Na A dual regulation of the X. laevis oocyte
Na Activation of the
oocyte endogenous G proteins by injected GTP There exists some recent
evidence for an activation of the Na In conclusion, the data presented here suggest that the
Na
/H
exchanger in Xenopus laevis oocytes by G proteins and protein kinases by measuring the
ethylisopropylamiloride-sensitive Li
uptake. Injection
of oocytes with the stable GTP analog GTP
S stimulated
Li
uptake up to almost 4-fold, an effect blocked by
coinjection with the GDP analog, guanyl-5`-yl thiophosphate. Injection
into oocytes of
subunits of the heterotrimeric G protein
transducin enhanced Li
uptake by about 3-fold. This
stimulation was blocked by transducin
subunits, which by
themselves did not influence Li
uptake. Using various
activators and inhibitors of protein kinases, it is demonstrated that
the X. laevis oocyte Na
/H
antiporter can be stimulated by activation of both protein kinase
A and C. Stimulation of Na
/H
exchanger activity by GTP
S but not that induced by
transducin
subunits was blocked by the protein kinase A
inhibitor H-89. On the other hand, transducin
subunit-stimulated activity was prevented by the protein kinase C
inhibitor, calphostin C. The non-selective protein kinase inhibitor H-7
blocked both GTP
S- and transducin
subunit-stimulated
Na
/H
exchanger activity. The results
suggest that the Na
/H
exchanger of X. laevis oocytes can be activated by G proteins and that this
activation is not direct but mediated by protein kinase A- and/or
protein kinase C-dependent pathways.
/H
exchangers (NHE)
(
)constitute a family of membrane transporters that
mediate the electroneutral exchange of Na
ions against
H
ions and that are activated by various receptors
coupled to guanine nucleotide-binding proteins (G proteins) and
receptors with tyrosine kinase activity(1) . The NHE-1 isoform
is ubiquitously expressed and mainly serves intracellular pH and cell
volume regulation(2, 3, 4, 5) . The
expression of the other hitherto cloned mammalian isoforms, referred to
as NHE-2-NHE-4, appears more restricted to the apical membranes
of epithelial cells, and their role could reside in mediating
transepithelial Na
transport (2, 3, 4, 5) . These isoforms differ
with respect to their inhibition by amiloride and its analogs (6) and with regard to their regulation by intracellular second
messengers. For example, the activity of the NHE-1 and NHE-2 expressed
in fibroblast PS120 cells is increased by protein kinase C
(PKC)-activating phorbol esters, whereas the activity of the NHE-3 is
reduced by this treatment(7) . Recent findings suggest that the
NHE-1 belongs to the family of calmodulin-binding proteins, which is
activated by a rise in cytosolic free Ca
concentration(8, 9) . Variable effects of cAMP
on mammalian NHE activity have been reported. Upon expression of NHE-1,
NHE-2, and NHE-3 in PS120 cells, no effect of cAMP on these isoforms
was found(7) . On the other hand, when studied in their natural
environment, both inhibition and stimulation of NHE activity by cAMP
have been reported (10, 11, 12) . This
variability may be due to the cell type studied and/or the NHE isoform
expressed in these cells. The
NHE isoform cloned from trout red
blood cells unambiguously differs from the mammalian isoforms as this
exchanger is phosphorylated and activated by both PKC and protein
kinase A (PKA)(13, 14, 15) .
/H
exchanger expressed in Xenopus laevis oocytes(16, 17, 18) .
In a previous study, we expressed the human NHE-1 in X. laevis oocytes, which increased the exchanger activity above the
endogenous oocyte NHE activity(18) . Interestingly, the
activity of the expressed NHE-1 was totally suppressed by low
concentrations (100 nM) of the novel isoform-selective
inhibitor HOE694(6) , whereas the endogenous NHE activity
remained unaffected even in the presence of 100 µM of this
compound. This pharmacological difference suggested that the functional
characteristics of the X. laevis oocyte
Na
/H
exchanger may also be different
from those of the mammalian transporters. In the present study, we
investigated the regulation of the Na
/H
exchanger of X. laevis oocytes by G proteins and protein
kinases. The data presented here show that the oocyte antiporter can be
activated by G proteins and that this regulation involves both PKA- and
PKC-dependent mechanisms.
Chemicals
All nucleotides were
purchased from Boehringer Mannheim. Gentamycin, streptomycin,
penicillin, calphostin C, calf intestine alkaline phosphatase,
forskolin, PKA inhibitor (type III from bovine heart), and phorbol
12-myristate 13-acetate (PMA) were from Sigma. The protein kinase
inhibitors, H-7 and H-89, and ethylisopropylamiloride (EIPA) were from
Calbiochem. S-adenosine 3`,5`-cyclic
monophosphorothioate ((S
)-cAMPS) was from Bio-Log
(Bremen, Germany). Transducin
and
subunits were
purified from illuminated bovine rod outer segments with GTP as
activating nucleotide as previously described(19) .
Reverse Transcription, PCR Analysis, and DNA
Sequencing
First strand cDNA synthesis from
oligo(dT)-selected poly(A) RNA was carried out with 25
pmol of random primers (Life Technologies, Inc.). The single strand
cDNA was then subjected to 30 cycles of PCR amplification (1 min, 94
°C; 1 min, 48 °C; 1.5 min, 72 °C), using 10 pmol of two
NHE-1- and
NHE-specific oligonucleotide primers encompassing the
region between the putative transmembrane domains Va and VIII. The
sequences of the primers were: sense 5`-GAATTCTCGGCCGTGGACCCCGTGGC-3`
(
NHE bp 667-686, NHE-1 bp 1110-1129) and antisense
5`-GAATTCCGCTCACGCTGCTCCACATC-3` (
NHE bp 1135-1116, NHE-1 bp
1578-1559). Using the EcoRI endonuclease restriction
sites of the 5`-ends of each primer, the amplification products were
subcloned into the complementary restricted pBluescript SK
plasmid (Stratagene) for sequence analysis. DNA sequencing was
carried out using fluorescent nucleotides in an automatic sequencer
(Pharmacia Biotech Inc.).
Oocyte Injection
Cytoplasmic injections
into defolliculated X. laevis oocytes were performed as
previously detailed(18) . 50 nl of nucleotides or transducin
subunits were injected to yield the indicated final intracellular
concentrations, assuming a mean oocyte volume of 1 µl. After
resealing of the plasma membrane, the oocytes were transferred to
Li-containing medium as described below. Some oocytes
were incubated for 30 min in OR-2 medium containing 1 µCi of
[
H]sorbitol (DuPont NEN), for which no endogenous
transporter in X. laevis oocytes has been described. Oocytes
were considered tight in the absence of any radioactivity above
background after extensive washing.
Determination of Na
Na/H
Exchange Activity
/H
antiporter activity was determined by measuring EIPA-sensitive
Li
uptake, i.e. Li
/H
-exchange, as described in
detail previously(18) . This method avoids the use of
radioactive Na
. In brief, oocytes were incubated for
various time periods within the linear range of Li
uptake (18) in a medium containing 65 mM LiCl,
2.5 mM lithium citrate, 8 mM MgCl
, and 5
mM Mops, pH 7.4. Thereafter, the cells were removed from the
medium, washed five times in ice-cold 0.1% (w/v) CsCl solution, and
transferred to plastic tubes containing 1 ml of 0.1% (w/v) CsCl
solution. After oocyte homogenization and removal of yolk proteins,
lithium concentration was determined in the clear supernatant by
analytical flame photometry using a Pye Unicam spectrophotometer SP9.
Assuming a mean oocyte volume of 1 µl and after correcting for
dilution, intracellular Li
concentrations were
calculated. Initial velocities of Na
/H
exchanger activity are presented here in mmol of
Li
/liter of oocyte and h. All data represent means
± S.D. performed on 10 oocytes from at least two different
animals. Data were analyzed by Student's t-test, and
differences were regarded significant at p < 0.05.
Effects of Guanine Nucleotides and G Protein
Subunits on Li
To study whether the X. laevis oocyte Na Uptake
/H
exchanger is regulated by G proteins, we first examined the
effects of injected nucleotides (5 µM, final intracellular
concentration) on Li
uptake. Injection of the stable
GTP analog GTP
S stimulated EIPA-sensitive Li
uptake by almost 4-fold (Table 1). In contrast, GTP, the
ATP analog ATP
S, and the GDP analog GDP
S had no or only
slight effects on Li
uptake. When GTP
S and
GDP
S were injected simultaneously at a molar ratio of 1:100, the
GTP
S-induced stimulation of Li
uptake was
completely suppressed. Thus, these data suggested that activated G
proteins can enhance the X. laevis oocyte
Na
/H
exchanger activity.
/H
exchanger activity was additionally studied by injecting
and
subunits (1 µM, final concentration) of the
retinal G protein transducin into oocytes. Whereas injected
subunits had no effect on Li
uptake, injection of
subunits strongly increased EIPA-sensitive Li
uptake by about 3-fold (Table 2). When
and
subunits were coinjected, no effect on Li
uptake was
observed. Thus, in addition to GTP
S, free
subunits of
transducin can stimulate the X. laevis oocyte
Na
/H
exchanger.
Effects of PKC and PKA on Li
To determine whether the stimulatory effects
of GTP Uptake
S and transducin
subunits are due to a direct G
protein activation of the X. laevis oocyte
Na
/H
antiporter or due to the
formation of second messengers and consequently of second
messenger-activated protein kinases, we first studied the possible
regulation of Li
uptake by directly activated PKC and
PKA. Pretreatment of oocytes with the phorbol ester, PMA (10
nM), increased EIPA-sensitive Li
uptake by
almost 4-fold (Table 3). This stimulation was completely
suppressed by the specific PKC inhibitor calphostin C (1
µM) (20) but only slightly reduced by H-89 (100
µM), a rather selective PKA inhibitor(21) . These
findings suggest that the Na
/H
exchanger expressed in X. laevis oocytes can be
activated by a PKC-dependent mechanism.
/H
antiporter is
also regulated by PKA, oocytes were treated with the direct PKA
activator, (S
)-cAMPS(22) , and the direct
adenylyl cyclase activator, forskolin. Both agents strongly stimulated
Li
uptake. (S
)-cAMPS increased
Li
uptake to 370, 450, and 470% of controls at 1, 10,
and 100 µM, respectively (data not shown). Forskolin (100
µM) increased EIPA-sensitive Li
uptake by
5-6-fold (Table 4). The stimulatory effect of forskolin was
completely abolished in oocytes pretreated with the PKA inhibitor, H-89
(100 µM), or injected with the bovine heart PKA inhibitor
(0.1 µM). In contrast, the PKC inhibitor, calphostin C (1
µM), had no effect on forskolin-stimulated Li
uptake. Thus, the Na
/H
exchanger expressed in X. laevis oocytes is apparently
under the control of both PKC and PKA.
/H
exchanger by
both PKA and PKC could be due to the expression of a NHE isoform
resembling the
NHE, which is stimulated by both PKA and PKC, or to
the expression of two distinct isoforms being differentially controlled
by PKA and PKC. PCR analysis of cDNA synthesized from oocyte RNA with
oligonucleotide primers homologous to both the human NHE-1 and the
NHE yielded one major reaction product of 417 bp. After subcloning
of this PCR product, 10 different clones were analyzed, which all
exhibited an identical nucleotide sequence, suggesting the expression
of only one NHE transcript in these cells. Sequence comparison with
corresponding sequences of the human NHE-1 and the
NHE indicated a
79 and 73% homology, respectively, on the nucleotide level, which was
increased to 89 and 80%, respectively, on the amino acid level.
Effects of Protein Kinase Inhibitors on G
Protein-mediated Li
Finally, we
investigated whether protein kinases, specifically PKA and PKC, are
involved in stimulation of the X. laevis Na Uptake
/H
antiporter by GTP
S and
transducin
subunits. Preincubation of oocytes with H-7 (100
µM), a non-selective protein kinase
inhibitor(21) , completely suppressed Li
uptake stimulated by either injected GTP
S or transducin
subunits (Fig. 1), suggesting the involvement of a
protein kinase-dependent mechanism(s) in stimulation of
Na
/H
antiporter by either agent. On
the other hand, the selective PKA inhibitor, H-89 (100
µM), strongly reduced Li
uptake
stimulated by GTP
S, whereas transducin
subunit-stimulated Li
uptake was not affected. Vice versa, pretreatment of oocytes with calphostin C (1
µM) completely inhibited Li
uptake
stimulated by transducin
subunits but was without effect on
stimulation by GTP
S.
S- and transducin
subunit-stimulated
Li
uptake. Shown are the effects of pretreatment of
oocytes for 6 h without (control, C) and with H-7 (100
µM), H-89 (100 µM), or calphostin C (Cal-C, 1 µM) as indicated before injection of
GTP
S (5 µM) (leftpanel) or
transducin
subunits (1 µM) (rightpanel) on Li
uptake. Each column
reflects means ± S.D. of at least 10 oocytes from at least two
different animals.
/H
exchangers by PKA-dependent
phosphorylation are rather variable, with no effects, inhibition, and
stimulation being
reported(7, 10, 11, 12) . The
Na
/H
antiporter cloned from trout red
blood cells,
NHE, is activated by agents causing an increase in
intracellular cAMP concentration and by direct PKA
activators(13, 14, 15) . The data presented
herein indicate that the Na
/H
exchanger expressed in X. laevis oocytes is stimulated
by both PKC- and PKA-dependent mechanisms, thus resembling the
regulation of the
NHE of trout red cells. In oocytes injected with
calf intestine alkaline phosphatase (0.01 units/oocyte), not only PMA-
and forskolin-stimulated but also unstimulated Li
uptake was reduced to about 50% of untreated controls (data not
shown), suggesting that even basal transport activity is maintained by
phosphorylation.
/H
exchanger was also observed by
activating oocyte endogenous G proteins with GTP
S and by injecting
G protein
subunits. Recently, evidence has been accumulated
that
subunits of heterotrimeric G proteins can directly
activate phospholipase C, preferentially the
2 and
3 isoforms
of this enzyme(23, 24) , and a phospholipase C with
homologies to the mammalian phospholipase C-
3 enzyme has been
cloned from X. laevis oocytes(25) . Transducin
subunits injected into X. laevis oocytes stimulated
NHE activity, and this stimulation was prevented by coinjected
subunits, suggesting the stimulation being due to free
subunits. Similar data were reported for phospholipase C activation by
transducin subunits(23) . However, due to the suppression by
the protein kinase inhibitors, H-7 and calphostin C, the stimulatory
effect of
subunits is unlikely due to a direct interaction
with the oocyte NHE. We rather propose that upon injection of
subunits the oocyte phospholipase C is activated, resulting
in the hydrolysis of phosphoinositides and the subsequent activation of
PKC, then activating directly or via additional kinases the oocyte
Na
/H
exchanger. Such a signal
transduction cascade could be initiated in vivo via muscarinic
or angiotensin II receptors, which, in oocytes, are coupled to
phospholipase C via pertussis toxin-sensitive G
proteins(26, 27, 28) .
S also evoked a strong
stimulation of the Na
/H
exchanger,
which was nucleotide-specific and antagonized by the GDP analog,
GDP
S. Interestingly, the stimulatory effect of GTP
S was
almost completely inhibited by the non-selective protein kinase
inhibitor, H-7, and, most notably, by the PKA inhibitor, H-89, but not
by the PKC inhibitor, calphostin C. These findings suggest that
activation of the oocyte Na
/H
exchanger by GTP
S-activated G proteins is not direct but is
mediated by a PKA-dependent mechanism most likely due to activation of
adenylyl cyclase by GTP
S-activated G
proteins.
GTP
S is a nonspecific activator of G proteins, and the cellular
response, therefore, depends on the isoform pattern and relative
amounts as well as the availability of the different G proteins and
their effectors expressed in a given cell type. Although
circumstantial, the finding that the PKC inhibitor calphostin C was
without effect on GTP
S-injected oocytes, in contrast to its strong
inhibitory effect on
subunit-injected cells, suggests that
GTP
S did not release sufficient
subunits to activate
the endogenous phospholipase-PKC pathway.
/H
exchanger by specific G protein
subunits. Expression of
mutationally activated
subunits of the G proteins G
,
G
, G
, and G
in human embryonic
kidney cells resulted in increased NHE activity by activated
and
subunits, whereas the other
activated subunits left the antiporter unaffected(29) . Whereas
activation by
was assumed to be mediated via the
stimulated phospholipase C pathway, stimulation of
Na
/H
exchanger by
was independent of the inositol phosphate and the cAMP pathways.
Expression of mutationally activated
,
,
,
, and
subunits in COS-1 cells enhanced NHE activity by
,
, and
(30) . While expression of
was without effect,
even inhibited NHE
activity. The stimulatory effect of
and
was apparently mediated by the PKC pathway, whereas the
activation by
was preserved in cells in which PKC
had been down-regulated(30) . The signaling cascade involved in
this protein kinase C-independent NHE activation by activated
remains to be elucidated. Although our experiments
have yet to yield any evidence for a direct G protein regulation of the
Na
/H
exchanger of X. laevis oocytes injected with GTP
S or G protein
subunits,
final statements on this issue require the determination of potential
effects evoked by injection of different G protein
subunits.
/H
exchanger of X. laevis oocytes can be activated by G proteins and that this activation is
most likely due to increased formation of second messengers resulting
in activation of either PKC and/or PKA. Whether this specific
Na
/H
exchanger actually contains
consensus sequences for PKA- and PKC-mediated phosphorylation has to
await the full-length cloning of this transporter, which is currently
being performed in our laboratory.
/H
exchanger; PMA, phorbol 12-myristate 13-acetate; EIPA,
ethylisopropylamiloride; GDP
S, guanyl-5`-yl thiophosphate;
GTP
S, guanosine 5`-O-(thiotriphosphate); PKA, protein
kinase A; PKC, protein kinase C; (S
)-cAMPS, S
-adenosine 3`,5`-cyclic monophosphorothioate; bp,
base pair(s); Mops, 4-morpholinepropanesulfonic acid; PCR polymerase
chain reaction; ATP
S, adenosine 5`-O-(thiotriphosphate).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.