(Received for publication, September 19, 1996, and in revised form, December 12, 1996)
From the Departments of Medicine and
§ Biochemistry and Molecular Biology, Medical University of
South Carolina and Veterans Affairs Medical Centers, Charleston, South
Carolina 29425 and the ¶ Department of Medicine, 5-HT1A receptors couple to many
signaling pathways in CHO-K1 cells through pertussis toxin-sensitive G
proteins. The purpose of this study was to determine which members of
the Gi/o/z family mediate 5-HT1A
receptor-activated Na+/H+ exchange as measured
by microphysiometry of cell monolayers. The method was extensively
validated, showing that proton efflux was sodium-dependent,
inhibited by amiloride analogs, and activated by growth factors,
phorbol ester, calcium ionophore, and hypertonic stress. 5-HT and the
specific agonist
(±)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide
rapidly stimulated proton efflux that was blocked by a specific
receptor antagonist, amiloride analogs or pertussis toxin. The
activation by 5-HT depended upon extracellular sodium and could be
demonstrated under conditions of imposed intracellular acid load, as
well as in the presence and absence of glycolytic substrate.
Acceleration of proton efflux was not inhibited by sequestration of G
protein Electroneutral Na+/H+ exchangers
(NHEs)1 are expressed at the surface of all
mammalian cells, subserving diverse functions including regulation of
intracellular pH, cell volume, mitogenesis, and vectorial and
nonvectorial transepithelial transport of Na+ and acid-base
equivalents (1, 2). The NHE-1 exchanger subtype is widely expressed and
can be activated by a number of stimuli that include growth factors,
hyperosmolarity, and ligands that act through G protein-coupled
receptors (1-4). Surprisingly little is known about the intermediary
signals and effectors involved in stimulation of NHE-1 activity,
particularly the pathways activated by receptors that act through
heterotrimeric G proteins.
A number of receptors that are classically linked to the inhibition of
adenylyl cyclase have been shown to inhibit or stimulate NHE activity
through pathways that do not involve cAMP. Unexpectedly, in many cases
those receptors couple to the regulation of NHE through pathways that
are not sensitive to pertussis toxin. Hence, G proteins other than
Gi For example, D2 dopamine receptors, and the closely related
D3 and D4 dopamine receptors, have been shown
to stimulate NHE activity in CHO cells through pertussis
toxin-sensitive G proteins (13, 14). Indeed, angiotensin 1 receptors in
opossum kidney cells (15) and endothelin receptors in renal cortical
slices (16) were also shown to increase NHE activity through pertussis toxin-sensitive G proteins. Finally, supportive evidence for the involvement of Gi or Go proteins in the
regulation of NHE was provided by recent studies that showed an
association between increased NHE activity and pertussis
toxin-sensitive second messenger generation in immortalized B
lymphoblasts derived from hypertensive patients when compared with
those derived from normotensive patients (17, 18). Therefore, the role
of pertussis toxin-sensitive G proteins in the regulation of NHE
activity remains unresolved.
The purpose of the current study was to determine whether members of
the Gi/o/z family of G proteins regulate NHE-1 activity in
CHO-K1 cells, and if so, which ones. We chose CHO-K1 fibroblast cells,
because they have previously proven useful in elucidating some of the
pathways involved in platelet-derived growth factor receptor (4, 19)-
and dopamine receptor (13, 14)-stimulated NHE-1 activity. To have a
means by which pertussis toxin-sensitive G proteins could be
selectively activated, we transfected CHO-K1 cells with the human
5-HT1A receptor, a prototypical Gi The basic strategy in the current study was to neutralize endogenous
pertussis toxin-sensitive G proteins, then to attempt to rescue NHE
activation by transfecting at high efficiency either Gz CHO-K1 cells expressing 5-HT1A
receptors ( Na+/H+ exchange
activity was measured in real time as the rate of decrease in
extracellular pH in intact cells placed in an eight chamber
CytosensorTM microphysiometer (Molecular Devices Corp., Sunnyvale, CA)
(36, 37). The microphysiometer uses a light addressable silicon sensor
to detect extracellular protons, which can be derived primarily from
Na+/H+ exchange and glycolysis, and from other
metabolic pathways (36, 37). Rate data transformed by a personal
computer running CytosoftTM version 2.0 (Molecular Devices Corp.) were
presented as the extracellular acidification rate (ECAR) in
microvolts/s, which roughly correspond to millipH units/min (Nernst
equation). To facilitate comparison of data between two channels,
values were expressed as a percentage of the base line as determined by
computerized analysis of the five data points prior to exposure of the
cell monolayers to a test substance. CHO-K1 cells expressing the
5-HT1A receptor were grown in Ham's F-12 medium
supplemented with fetal bovine serum (10%), penicillin (100 units/ml),
and streptomycin (100 µg/ml). The night prior to experimentation,
cells were replated onto polycarbonate membranes (3 µ pore size, 12 mm size) at a density of 300,000 cells per insert. The day of the
study, cells were washed with serum-free, bicarbonate-free Ham's F-12
medium, placed into the microphysiometer chambers, and perfused at
37 °C with the same medium or balanced salt solutions as described
in figure legends or text. For most studies, the pump cycle was set to
perfuse cells for 60 s, followed by a 30-s "pump-off" phase,
during which proton efflux was measured from the 6th through the 28th
second. Cells were exposed to the test agent for two or three cycles
(180-270 s). Valve switches (to add or remove test agents) were
performed at the beginning of the pump cycle. In some cases where the
response was expected to be both rapid and very transient
(i.e. recovery from sodium-free conditions) the valve switch
was performed 55 or 58 s into the perfusion phase, allowing 7-10
s for solution mixing prior to rate measurements. Data points were then
acquired every 90 s. The peak effect during stimulation was
expressed as percentage increase from average basal ECARs from five
rate measurements prior to application of the test agent(s). Typical
basal ECARs in glucose-containing solutions were in the For some experiments, intracellular pH (pHi) was fixed by a
method similar to that used by Azarani et al. (38) to
measure NHE activity by 22Na+ uptake. That
method employs nigericin, a K+/H+ exchange
ionophore. In using this tool, two major assumptions were made. (i) At
equilibrium the desired pHi could be calculated from the
[K+] gradient and pHo using the following
formula: [K+i]/[K+o] = [H+i]/[H+o]. (ii) The
intracellular [K+] was assumed to be 140 mM.
Briefly, cells were perfused with a low-sodium balanced salt solution
containing (in mM) 118 choline chloride, 20 NaCl, 10 glucose, 5 KCl, 1.3 CaCl2, 0.8 K2HPO4, 0.5 MgCl2, and 0.1 KH2PO4, pH Cells grown on coverslips were loaded with 7 µM BCECF-AM in a Hepes-buffered, bicarbonate-free
balanced salt solution at 37 °C for 1 h. Cells were washed and
loaded into cuvettes that were perfused at 37 °C. Measurements were
made with a Perkin-Elmer LS50 fluorescence spectrometer using an
emission wavelength of 530 nM and excitation wavelengths of
500 and 440 nm. Periodic measurements were made to calculate the
excitation ratio (500/440), which were compared with a calibration
curve that was generated by clamping pHi with nigericin as
described above.
Cells were
transfected with 1 µg of pRK- All G
protein constructs were subcloned into the pCDNA3 vector, which
incorporates a G418 resistance motif. Stable expression of
Gz MAPK activity was measured in anti-MAPK
immunoprecipitates using myelin basic protein as a phosphorylation
substrate as described previously (27, 39).
High affinity binding was
defined as the amount of [3H]8-OH-DPAT (1 nM)
displaced by 100 µM GTP, and the assays were performed as
described previously (40). Under these conditions, each tube contained
about 3,000 cpm of specifically bound [3H]8-OH-DPAT, and
GTP-displaced binding was about 90% of the specific binding (displaced
by 10 µM 5-HT).
To establish the ability of the CytosensorTM to detect changes in
Na+/H+ exchange activity under our experimental
conditions, cells were perfused with a sodium-free balanced salt
solution (138 mM choline chloride, 10 mM
glucose, 5 mM KCl, 1.3 mM CaCl2,
0.8 mM K2HPO4, 0.5 mM
MgCl2, and 0.1 mM
KH2PO4, pH 7.35) for 20-30 min, then switched
to the same solution where NaCl was substituted for choline chloride.
Fig. 1A shows that a rapid burst of proton
efflux occurred when cells were exposed to sodium. Under identical
conditions in which cells were exposed to amiloride analogs EIPA (5, 10, or 20 µM) or MIA (10 µM) during the
choline sodium-free perfusion, the burst of proton efflux was
completely blocked upon exposure to sodium (Fig. 1A). Those
studies confirm the presence of an amiloride-inhibitable
sodium-dependent proton efflux pathway in CHO-K1 cells. We
next wanted to establish that the ECAR could be acutely increased by
maneuvers that are known to activate fibroblast NHE as measured by
other techniques. Such measures include hypertonic stress, phorbol
12-myristate, 13-acetate acting through protein kinase C, maneuvers
that increase intracellular Ca2+ levels, and growth factors
such as fibroblast growth factors (FGF) and thrombin (41-44). Fig.
1B demonstrates that exposure to hypertonic solutions (270 mM NaCl or 170 mM sucrose + 100 mM NaCl) resulted in brisk increases in ECAR, as would be expected if the
proton efflux is mediated through NHE-1 (5, 6). Fig. 1C
shows that phorbol 12-myristate, 13-acetate, and the calcium ionophore
A23187, each rapidly activate ECAR in CHO cells, as do the growth
factors basic FGF (100 pg/ml) and thrombin (1 unit/ml) (Fig.
1D). In aggregate, the studies presented in this Fig. 1 show
that ECAR as measured by microphysiometry corresponds very closely with
what is known about the activity and properties of NHE.
It was important to next demonstrate that the G protein-coupled
5-HT1A receptor can activate ECAR in these cells. This
evidence was provided by demonstrating that the agonists 5-HT and
8-OH-DPAT (at 1 µM) stimulate ECAR in Ham's F-12 by
42 ± 3% (n = 22) and 38 ± 3%
(n = 19), respectively. Those effects could be blocked effectively by co-incubation with 1 µM of the
5-HT1A receptor antagonist (S)-UH-301, which
itself had no effect (Figs. 2A). Moreover,
there was no stimulation of ECAR by 5-HT or 8-OH-DPAT in cells that
were not transfected with the 5-HT1A receptor, confirming that the increase in ECAR is indeed mediated through that receptor (n = 12).
It was next necessary to provide evidence that the receptor-mediated
increases in ECAR were due to activation of NHE. Fig. 2, C
and D, support that hypothesis by demonstrating that 5-HT increased ECAR in a sodium-containing balanced salt solution and that
the amiloride analog EIPA (10 µM) attenuated the 5-HT (1 µM)-mediated increase in acidification rate by
The data in Fig. 2F document the dependence of 5-HT-mediated
increases in ECAR upon [Na+o]. The ability of
5-HT to increase ECAR depended upon [Na+o], being
half-maximal at about 10 mM, which is quite consistent with
half-maximal values for NHE as determined by other methods (1, 2, 44).
Consequently, the data provided in Figs. 1 and 2 validate the
microphysiometric technique for measuring NHE activity in monolayers of
CHO-K1 cells.
We next explored potential pathways by which the 5-HT1A
receptor increased NHE, with particular emphasis upon identifying the G
proteins involved in the process. Fig. 3A
shows that pertussis toxin treatment (200 ng/ml overnight) completely
abolished the ability of 5-HT1A receptors to increase ECAR
without altering the ability of ATP (acting through an endogenous
Gq
The final series of studies was performed to assess the role of the
We next assessed whether the transfected G proteins were capable of
interacting with the 5-HT1A receptor. This was accomplished after transfection by measuring high affinity agonist binding ([3H]8-OH-DPAT), which is a gauge of the ability of
agonist-bound receptors to couple to G proteins. Transfected cells were
treated with pertussis toxin or vehicle, and the ability of each
construct to "rescue" high affinity binding after pertussis toxin
treatment was measured. In cells transfected with empty vector,
pertussis toxin eliminated all detectable high affinity binding as
determined by the ability of GTP to suppress binding of
[3H]8-OH-DPAT. The pertussis toxin-resistant G
proteins were able to restore high affinity binding in varying degrees
as follows and as depicted in Fig. 3D: Go The expression of any of the G protein constructs did not significantly
alter the basal ECAR (not shown) or the extent of 5-HT-mediated
increases in ECAR (Fig. 3E, open bars). In contrast, pertussis toxin treatment reduced the basal ECAR by almost 50% in
cells transfected with each of the constructs, as it did in nontransfected cells (not shown). There were clear differences among
the individual constructs in the ability to rescue 5-HT-mediated increases of ECAR after treatment with pertussis toxin. Whereas 5-HT
did not increase ECAR in pertussis toxin-treated cells that were
transfected with empty vector (pcDNA3), Gz Thus, although we were able to document detectable expression of all
five pertussis toxin-insensitive G protein constructs, only
Gi The current study was undertaken to define some of the G proteins
involved in the short term activation of NHE activity by a prototypical
cell surface receptor that couples exclusively to pertussis
toxin-sensitive G proteins. Three major findings are presented within
this report. First, a clear role for pertussis toxin-sensitive G
proteins in the activation of NHE in CHO fibroblast cells was
established. In the current study, a transfected receptor, which
couples primarily or exclusively to Gi/o/z proteins, was shown to activate ECAR. The response was due to the transfected 5-HT1A receptor because (i) it was not present in
nontransfected cells, (ii) it was initiated by a specific receptor
agonist, and (iii) was blocked by a specific receptor antagonist.
Furthermore, the increase in ECAR was mainly due to activation of NHE
based on the following evidence: (i) dependence on
[Na+o] with a half-maximal effect which
approximates that described previously for NHE proteins in various
systems; (ii) activation of proton efflux by hypertonic exposure,
growth factors, calcium ionophore, and phorbol ester; and (iii)
inhibition by amiloride analogs. The stimulation of NHE was not
secondary to metabolic production of protons because (i) 5-HT did not
decrease pHi, (ii) the stimulation occurred when glycolytic
substrate was removed from the perfusate, and (iii) the stimulation
occurred when pHi was fixed at The second major finding was an apparent lack of involvement of G
protein The involvement of Gi/o/z proteins in acute
agonist-regulated NHE activity has been controversial. On the one hand,
although many receptors that classically couple to Gi/o/z
proteins are capable of regulating NHE activity, this effect has most
frequently been shown to be insensitive to pertussis toxin (5-9).
Additionally, studies using constitutively activated G proteins have
not supported a role for pertussis toxin-sensitive G proteins in NHE
regulation (10, 11). On the other hand, some more recent studies have indicated a role for pertussis toxin-sensitive G proteins in the activation of NHE by various agonists (13-16). What, then, might account for the wide variety of findings? The differences likely involve factors related to the specific cell types and the different protocols used to measure NHE activity in those studies. Clearly, the
complement of G proteins expressed within a cell might directly influence whether a given receptor modulates NHE activity and whether
that modulation occurs in a positive or negative direction. Alternatively, different types of NHE with distinct regulatory characteristics might be expressed in the various cell types. As
pointed out by Azarani et al. (46), many aspects of the
experimental protocols could influence the observed results. Cell
attachment has been identified as a major factor in the regulation of
pHi and responsiveness to regulation of NHE activity (44). Most of the previous studies were performed in nonadherent cells shortly after being detached by calcium chelation or enzymatic digestion. In
the current study, and in several others that used a microphysiometer (8, 13, 14), cells were studied in relatively undisturbed monolayers.
An additional difference is that most of the prior studies used
fluorescent dyes and measured the rate of recovery of cells after an
imposed acid load, whereas neither dye nor acid loading is required for
microphysiometric measurement. In a real sense, because
microphysiometry allowed the preservation of cell to cell contact and
the examination of NHE activity absent any acid loading protocols, it
may more accurately preserve features that mimic agonist exposure of
cells within functioning organs.
What of the differences between the current studies and those that
utilized activated G protein mutants? Prior studies, which used
constitutively active versions of the G proteins, would be expected to
exert their effects over long periods of time (hours), whereas in the
current studies, short term (minutes) effects were examined. Moreover,
those studies examined the rate of recovery from an acid load absent
any agonist stimulation, whereas the current studies examined the
effects of brief exposure to agonists in nonacid-loaded cells.
The finding that The current studies are remarkable in that they demonstrate a high
degree of specificity for activation of NHE among the members of the
Gi/o/z family of G proteins (when activated by a single receptor type), whereas the degree of specificity of those same G
proteins to couple to the 5-HT1A receptor appears to be
much less constrained. Gi Should we interpret these findings as evidence that only
Gi In summary, the current work provides evidence that a fibroblast
Na+/H+ exchange activity, putatively the
ubiquitously expressed NHE-1, can be rapidly stimulated through the
transfected human 5-HT1A receptor. The activation pathway
involves pertussis toxin-sensitive G protein We thank Drs. S. Pitchford and J. Owicki of
Molecular Devices Corp. (Sunnyvale, CA) for useful suggestions, Dr. R. J. Lefkowitz for encouragement, lively discussions and for critiquing
the manuscript prior to submission, and Drs. W. Koch, R. Taussig, E. Peralta, T. Hunt, S. E. Senogles, P. J. Casey, and K. Peppel for
providing critical reagents.
Howard Hughes Medical Institute,
Durham, North Carolina 27710
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-subunits, a maneuver that blocked 5-HT1A
receptor activation of mitogen-activated protein kinase. Transfection
of Gz
and pertussis toxin-resistant mutants of
Go
and Gi
1 did not reverse the blockade
induced by pertussis toxin. In contrast, pertussis toxin-resistant
mutants of Gi
2 and Gi
3 "rescued" the
ability of 5-HT to increase proton efflux after pertussis toxin
treatment. These experiments demonstrate clearly that
Gi
2 and Gi
3 can specifically mediate
rapid agonist-induced acceleration of Na+/H+
exchange.
or Go
are usually thought to convey
the modulatory signal. For example, the D2 dopamine
receptors expressed in pituitary lactotroph cells cause intracellular
acidification by inhibiting NHE activity (5). Likewise, somatostatin
receptors expressed in pituitary lactotrophs, Ltk
fibroblasts and HEK293 cells also inhibit NHE in a non-pertussis toxin-sensitive manner (6, 7). In contrast, D2 dopamine receptors expressed in mouse L cells and C6 glioma cells
activate NHE (8), as do
2-adrenergic,
muscarinic, and
-opiate receptors expressed in NG108-15 hybridoma
cells (9), and all act through pertussis toxin-insensitive mechanisms.
The common thread in all of those studies (5-9) is that receptors that
inhibit adenylyl cyclase through pertussis toxin-sensitive G proteins
do not use those G proteins to modulate NHE activities. That notion has
been supported by recent studies in which constitutively active G
protein
-subunits were transiently expressed in mammalian host
cells. Those studies showed clearly that Gq
,
G12
, and G13
can increase NHE activity,
the former two through a protein kinase C-dependent
(phorbol ester-sensitive) pathway, and the latter through a protein
kinase C-independent (phorbol ester-insensitive) pathway (10-12).
However, in those studies, constitutively active Gi
proteins failed to increase the rate of recovery from an acid load.
Thus, a number of careful studies have failed to show a role for
Gi proteins in regulating NHE activity (5-12).
Nevertheless, there has been mounting contrary evidence that supports
some role for pertussis toxin-sensitive G proteins in the short term
regulation of NHE activity.
-linked receptor. That receptor has been shown to modulate a large number of
signaling pathways in CHO-K1 cells (20-23) exclusively through pertussis toxin-sensitive pathways. Therefore, the 5-HT1A
receptor serves as a very useful and specific tool to achieve short
term activation of Gi/o proteins. Gi
2 and
Gi
3 are the major known pertussis toxin-sensitive G
protein subtypes in CHO-K1 cells (23-26), although a measurable but
far smaller amount of Go
is also present (27).
Importantly for the current study, the 5-HT1A receptor has
already been shown to efficiently activate all three Gi
subtypes, Go
, and Gz
(26, 28-30). Because most cells endogenously express two or more subtypes of pertussis toxin-sensitive G proteins, it has been very difficult to
assign specific downstream regulatory functions to individual
-subunit types.
or various pertussis toxin-insensitive mutants of Gi
and
Go
subunits. Each of those constructs, all characterized previously (31-33), was created by mutating the cysteine four residues from the carboxyl terminus of each G protein to remove the ADP-acceptor site for the ribosylation catalyzed by pertussis toxin. By transfecting those constructs and subsequently neutralizing the endogenous Gi
and Go
subunits, we were able to study
the effects of each G protein
-subunit individually. Thus, this
model system allowed us to examine the specific contribution of
Gi/o/z proteins to the regulation of NHE-1 activity.
Materials
1 pmol of receptor/mg of protein) were obtained as
described previously (18). Cell culture supplies were obtained from
Life Technologies, Inc., the Comprehensive Cancer Center at Duke
University, or Corning Costar (Cambridge, MA). EIPA, MIA, 5-HT,
8-OH-DPAT and (S)-UH-301 were from Research Biochemicals
International (Natick, MA). [3H]8-OH-DPAT was from
Amersham Corp. Constructs of pertussis toxin-resistant G proteins
incorporating cysteine to serine mutations four positions from the
carboxyl terminus were obtained from Dr. R. Taussig
(Go
PT) (31), and Drs. E. Peralta and T. Hunt
(Gi
2PT, Gi
3PT) (32), and a construct
incorporating a cysteine to glycine mutation of Gi
1
(Gi
1PT) was from Dr. S. E. Senogles (33).
Gz
and antiserum P960 were from Dr. P. Casey. Minigene
constructs encoding carboxyl-terminal residues 495-689 of bovine
-adrenergic receptor kinase 1 (
ARK-1) were generously provided by
Drs. Karsten Peppel and Wally Koch (Duke University), as was an
antibody specific for
ARK-1. The constructs were either in a
mammalian expression vector (pRK-
ARK-1-(495-689) (34) or packaged
in a replication-deficient adenoviral vector (35)
(
ARK-1-(495-689-adeno)). Specific G protein antibodies were
obtained from the following sources: Gz
, Dr. P. Casey;
Go
, Upstate Biotechnologies, Inc.; and
Gi
1, Gi
2, Gi
3,
Calbiochem.
120-200
µV/s range, regardless of the presence of sodium. In glucose-depleted
pyruvate-containing solutions, basal rates were in the range of
25-60 µV/s.
7.4. One or two ECAR
readings were obtained after stimulation with 1 µM 5-HT,
then cells were perfused for 10 min with the same salt solution
supplemented with 10 µM nigericin except that the
[K+o] was adjusted to provide a calculated
pHi of 6.6. The choline content was adjusted such that the sum
of choline chloride and KCl equaled 123 mM. To achieve a
pHi of 7.0, [K+o] and pHo were
increased accordingly. After perfusion with nigericin, cells were
quenched with the same buffer supplemented with 0.5% bovine serum
albumin (minus nigericin) to remove free nigericin, then exposed to 1 µM 5-HT. The presence or absence of the bovine serum
albumin did not affect the basal ECAR, nor did it affect
5-HT-stimulated ECAR.
ARK-1 Minigene in CHO-K1 Cells
ARK-1-(495-689) or empty pRK vector
in the presence of LipofectAMINETM liposomes or infected with 50 plaque-forming units/cell of
ARK-1-(495-689)-adeno or a control
virus. Transfections for microphysiometry were performed with 1 µg of
DNA/300,000 cells and for immunoblot with 2 µg of DNA/1,000,000
cells. After 40-48 h, microphysiometry was performed as described
above. For immunoblots, cells were scraped into Laemmli buffer, boiled
for 3 min, and subjected to SDS-polyacrylamide gel electrophoresis
under reducing conditions with 4-20% precast gels (Novex, San Diego,
CA). After semidry transfer to polyvinylidine difluoride membranes,
proteins were subjected to immunoblot with anti-
ARK-1 rabbit serum
(1:8,000) (39), and immunoreactive bands were visualized using an
enhanced chemiluminescence technique (ECL, Amersham Corp.). We used
several marker constructs including a 12CA5 epitope-tagged
5-HT1A receptor and
-galactosidase staining to estimate
the transfection efficiency of both protocols. Lipopolyamine-mediated gene transfer resulted in expression of the marker in at least 50-80%
of cells at 48 h, and adenovirus-mediated transfer resulted in
expression in essentially 100% of the cells.
was achieved by co-transfection with the cDNA
encoding the human 5-HT1A and a hygromycin resistance
motif; selection in the presence of both G418 and hygromycin produced
several clones, one of which contained
800 fmol of
5-HT1A receptor/mg of protein and expressed
Gz
by immunoblot, was used for the current study.
Transient expression of other constructs was achieved in those cells by
transfection (2 µg of each G protein cDNA/one well of a six-well
dish) in the presence of LipofectAMINETM (2 h in serum- and
antibiotic-free medium). Further studies were performed as described
above.
Fig. 1.
Characteristics of ECAR in CHO-K1 cells.
A, cells were preincubated in choline chloride-containing
(Cho-Cl) balanced salts for at least 30 min, then switched
to an identical solution in which sodium replaced choline. Measurements
were taken 7 or 10 s after the valve switch because of the very
rapid nature of the response. The sodium-dependent proton
efflux could be blocked by 10 µM MIA (452 ± 76 versus 25 ± 7% increase in the absence or presence of
10 µM MIA; n = 6) or by 5, 10, or 20 µM EIPA (592 ± 55% versus no
stimulation in the presence of EIPA; n = 4-12). B, hypertonic stress activates ECAR (tracings are
representative of at least 20 experiments). C, phorbol
12-myristate, 13-acetate (PMA) and A23187 increase ECAR.
D, growth factors thrombin (1 unit/ml) and basic FBF (100 pg/ml) activate ECAR. The response to all of the stimulating agents
(B-D) was rapid and relatively short-lived after removal of
the agent, with the exception of basic FGF, which intiated a slower and
less abrupt increase in ECAR, which nevertheless persisted long after
the stimulus was removed (>30 min). Shaded areas represent
times during which cells were exposed to test agents.
[View Larger Version of this Image (54K GIF file)]
Fig. 2.
Characteristics of 5-HT-mediated increases in
ECAR. A, nonspecific (5-HT) and specific
(8-OH-DPAT) agonists of the 5-HT1A receptor
activate extracellular acidification. Those responses were blocked by
the specific antagonist (S)-UH-301. Experiments were
performed in Ham's F-12 medium (bicarbonate- and serum-free) three
different times in at least four wells for each condition. B, 5-HT does not cause intracellular acidification as
measured by BCECF-AM fluorescent technique. C, EIPA greatly
attenuates 5-HT-mediated increases in ECAR in sodium and glucose
containing poorly buffered balanced salt solution (n = 7). D, the same experiment as described in C was
repeated in salt solution with pyruvate substituted for glucose to
minimize the glycolytic component of the acidification response.
E, 5-HT activates ECAR in the presence of intracellular acid
load imposed by nigericin. F, ability of 5-HT to increase
ECAR depends upon the extracellular concentration of Na+.
Typical basal ECARs were 120-200 µV/s, with the exception of
D, which was
25-60 µV/s. All tracings are
representative examples, with the exception of that shown in
D, which was derived from the pooled values of four chambers
at one sitting. Shaded areas represent times during which
cells were exposed to test agents.
[View Larger Version of this Image (45K GIF file)]
70-80%. Because hormones and growth factors can have complex
cellular effects, including activation of metabolic pathways
(glycolysis and mitochondrial respiration) which can lead to
intracellular accumulation of protons (37, 38), the increase in ECAR
could either be due to a primary activation of the NHE molecules
through a regulatory pathway or secondarily to a decrease in
pHi leading to activation of NHE through a proton-sensing site
located within the protein itself (44). To address these possibilities,
we performed three sets of experiments. First, pHi was measured
before and after treatment with 5-HT. The resting pHi of
nonacid-loaded CHO-K1 cells in monolayer at 80% confluence was
determined to be 7.23 ± 0.03 by the BCECF-AM fluorescence method,
and this was not decreased by treatment with 5-HT (Fig. 2B,
n = 5). Second, glucose was removed from the perfusion
medium, and pyruvate was substituted as the carbon source for some
microphysiometry studies. Removal of glucose rapidly and dramatically
reduced ECAR by nearly 80% within one pump cycle (minutes); ECAR
returned quickly to the previous base line (several minutes) after
restoring the glucose to the perfusate solution (not shown). Thus,
CHO-K1 cells appeared to rely quite heavily on glucose for maintaining
metabolism and ECAR under our conditions. Therefore, if the increase in
ECAR mediated by 5-HT was mediated solely through augmented glycolysis, removal of glucose would be expected to abrogate the stimulation of
ECAR by 5-HT. In the pyruvate-containing, glucose-free solution, 5-HT
increased acidification rate by 28 ± 4% (n = 6 in duplicate or triplicate), and that stimulation was almost completely
blocked by 10 µM EIPA (n = 12) (Fig.
2D). In a third set of experiments, the pHi was
clamped either to 6.6 or 7.0 by manipulating the
[K+o] and [pHo] in the presence of
nigericin. Under those conditions, 5-HT was still able to increase ECAR
by 25 ± 3% and 29 ± 4%, respectively, confirming that the
increase in ECAR can occur under a condition (low pHi) in which
a further reduction in pHi would be unlikely to affect the
proton-sensing site of the NHE molecule (44). The experiments in Fig.
2, C-E, provide consistent evidence against a passive mechanism of activation of ECAR that is secondary to decreased pHi resulting from increased metabolic activity.
-linked purinergic receptor) to do the same. Because
pertussis toxin shows a selective uncoupling of the 5-HT1A
receptor from downstream effectors, its effects are not due to
nonspecific cellular toxicity. Moreover, it provided a null background
against which the effects of the various pertussis toxin-insensitive G
proteins could be studied. Fig. 3B shows that a minigene
construct encoding the carboxyl terminus of
ARK-1, which is a known
sequesterer of G protein
-subunits (34, 39), did not alter the
ability of 5-HT to increase the rate of acidification. However, that
treatment on the same batch of cells completely blocked the ability of
5-HT to stimulate the mitogen-activated protein kinases, ERK1 and -2 (Fig. 3C), confirming both high transfection efficiency and
functional expression of the sequestering minigene. The lack of effect
upon NHE activity was apparent whether the minigene was delivered by liposome-mediated transfection or by infection with
replication-deficient adenovirus. Regardless of the delivery system,
ample expression of a 24-kDa
ARK-1 carboxyl terminus fragment
(
1-CT) was documented by immunoblot. The apparent densitometric
immunoreactivity was at least 20-fold greater for
1-CT than for the
endogenous
80-kDa
ARK-1 holoprotein (not shown). Those results
suggest a lack of critical involvement of
G protein subunits and
are consistent with the necessary involvement of pertussis
toxin-sensitive G protein
-subunits in the activation of NHE by the
5-HT1A receptor.
Fig. 3.
Effects of G proteins on ECAR. A,
pertussis toxin treatment (200 ng/ml × 18 h) abrogates
5-HT-increased ECAR without any effect on ATP-activated ECAR
(n = 6 for each). Pertussis toxin reduced the basal
ECAR by about 50% (not shown). B, -sequestering reagent
1-CT does not affect 5-HT-activated ECAR, but markedly decreases the ability of 5-HT to activate MAPK (390 ± 41%
versus 20 ± 20% increase; n = at
least 6 for each) (C). D, expression of various
pertussis toxin-resistant G proteins proteins (Gz
, Go
PT, Gi
1PT, Gi
2PT, and
Gi
3PT) and empty plasmid (pCDNA3) on P960
immunoreactivity (striped bars) and on high affinity agonist
binding in pertussis toxin-treated cells (speckled bars) (n = 2 in duplicate for both assays). E,
effects of expression of various pertussis-resistant G proteins and
empty plasmid on ability of 5-HT to increase ECAR after pertussis toxin
treatment). Pertussis toxin reduced the basal ECAR by about 50% in
each condition (not shown). All of these experiments were performed at
least five times in duplicate, with the exception of those with
pCDNA3, which were performed three times.
[View Larger Version of this Image (57K GIF file)]
-subunits of specific members of the Gi/o/z family of G
proteins in conveying the stimulatory signal from the
5-HT1A receptor to the NHE molecule. In that respect, we
limited our studies entirely to members of the family to which the
5-HT1A receptor has been shown previously to couple, those
being Gi
1, Gi
2, Gi
3,
Go
, and Gz
(26, 28-30). Transfection of
each of the constructs encoding a specific pertussis toxin-resistant G
protein
-subunit resulted in increased expression of that subunit as
detected by immunoblot (Figs. 3D and 4).
Although it is difficult to quantify the exact amount of a given G
protein in membrane fractions due to issues of antibody specificity and
sensitivity, we probed membranes derived from cells transfected with
the cDNAs of the various pertussis toxin-resistant G proteins with
a polyspecific antiserum (P960, Fig. 3D) and more specific
sera (Fig. 4). P960 is a rabbit serum raised against the common GTP
binding region of G
(45) and is capable of interacting
with all of the known heterotrimeric G protein
-subunits.
Transfection with all of the plasmids except the empty pcDNA3
vector increased the immunoreactivity detected by P960 from 130-210%
(Fig. 3D). Confirmation that G proteins were expressed after
transfection with each plasmid was obtained (Fig. 4) with more specific
antisera (23, 26). Because different sera were used for each blot, one
cannot directly compare the level of expression of each construct.
Nevertheless, the results clearly document detectable expression of all
of the G protein constructs which were tested in this work.
Fig. 4.
Heterologous expression of G proteins in
CHO-K1 cells as determined by specific antibodies. Transfection
and immunoblot were performed as described under "Experimental
Procedures." All lanes show about 25 µg of membrane protein probed
with the specific antibodies that correlated with the corresponding
constructs. T indicates transfected cells, and N
indicates nontransfected cells.
[View Larger Version of this Image (33K GIF file)]
,
37%; Gi
1, 42%; Gi
2, 55%;
Gi
3, 43%; and Gz
, 13%. Thus, the
transfected constructs were shown to be functional in their ability to
couple to the 5-HT1A receptors in CHO cells with a rank
order of Gi
2
Gi
3 = Gi
1 = Go
> Gz
in our assay system. These results are similar to those previously published for
5-HT1A receptors interacting with nonmutant G proteins in
nonmammalian expression systems (28, 30).
PT,
Go
PT, or Gi
1PT, it did substantially
increase ECAR in cells transfected with Gi
2PT (22.0 ± 3.8%, n = 5) or Gi
3PT (29.8 ± 7.8%, n = 5) when compared with the same cells that
had not been treated with pertussis toxin (36.4 ± 6.7% and
40.5 ± 10.4%, respectively; Fig. 3E) or when compared
with cells transfected with empty vector and not treated with pertussis
toxin (31.7 ± 5.2%, n = 3).
2PT and Gi
3PT were able to restore the
activation of NHE by the 5-HT1A receptor. Moreover, because
sequestration of G protein
-subunits had no effect, the
-subunits themselves are likely to directly convey the stimulatory
signal.
6.6 and 7.0 with the
K+/H+ ionophore, nigericin.
-subunits in the signaling pathway connecting the
Gi-coupled receptor to activation of NHE. This conclusion was based on the inability of a G protein
-sequestering reagent (
1-CT) to inhibit NHE activation despite effectively blocking activation of MAPK in the same cells. The third finding was that very
specific members of the Gi/o/z
-subunit family, namely
Gi
2 and Gi
3, convey the stimulatory
signal to NHE. Each of these findings is highly significant in light of
previous work.
-subunits of Gi proteins are not
critically involved in conveying the stimulatory signal from the
5-HT1A receptor dissociates the regulation of the
growth-associated NHE activity (
-subunit-mediated) from regulation
of MAPK activity (
-subunits) at a very proximal location in the
signaling pathways. The lack of involvement of
in activating NHE
was also somewhat surprising in light of previously published data.
Busch et al. (47) demonstrated that transducin
-subunits activate an endogenous NHE activity when microinjected
into oocytes derived from Xenopus laevis, whereas
microinjection of transducin
-subunits, which are closely related to
Gi
subunits, had no effect (47). That finding led us to
study the role of
-subunits of specific members of the
Gi/o/z to which the 5-HT1A receptor is known to couple. The members of the Gi/o/z family showed a high
degree of specificity for conveying the signal from the receptor to
activation of NHE, despite the fact that the receptor has been shown to
activate all of the subtypes studied here. Only Gi
2 and
Gi
3, and not Gz
, Go
, or
Gi
1, were shown to be involved. Those differences cannot
be accounted for by varying degrees of efficiency of expression or
transfection of the various G proteins, because transfection efficiency
was very high, and because increased expression of all of the target G
proteins was significantly increased as determined by immunoblot.
Moreover, all of the G proteins were capable of coupling to the
5-HT1A receptor as assessed by high affinity binding,
albeit Gz
much less so than the other constructs.
2 and Gi
3 are
the two most abundant Gi/o/z family proteins in CHO cells,
being expressed at 4.8 and 0.6 pmol/mg of membrane protein,
respectively (48), and it is interesting to note that their pertussis
toxin-resistant mutants were best able to reconstitute the activation
of ECAR. Taken together, those results support a primary role for
endogenous Gi
2 and Gi
3 in CHO cells in
activating ECAR through the 5-HT1A receptor.
2 and Gi
3, but not Gz
,
Go
, or Gi
1, are capable of short term
activation of NHE? We think not. It is important to understand that
membrane preparations were used in the previous studies in which the
5-HT1A receptor was shown to activate all five of the G
proteins (26, 28-30), as well as in the current study (Fig.
3D). By creating broken cells, those protocols may have
removed important physical or functional constraints (such as
cytoskeleton or membrane fences) that would have normally prevented the
receptors from freely interacting with the entire array of G proteins
contained within the cell (49). For example, one study showed that
tubulin binds specifically to Gs
and Gi
1, but not to Go
, Gi
2, or Gi
3
(50), thus providing one possible selective means of sequestering
Gi
1 away from the 5-HT1A receptor. Rather
than proving that Gz
, Go
, and
Gi
1 are not capable of regulating NHE, our findings in intact cells might suggest that similar subtle constraints prevented the receptors from effectively interacting in situ with each
or all of Gz
, Go
, and Gi
1.
Clearly, further studies will be needed to resolve this important
issue. It is critical to stress that the current studies do not
definitively rule out a role for Gz
, Go
,
and Gi
1 in the regulation of NHE activity, and that
these results should not be generalized to other cell types or
receptors.
-subunits
Gi
2 and Gi
3, but not
-subunits or
Gz
, Go
, or Gi
1, this
despite the fact the receptor can couple efficiently to each of those
subunits.
*
This work was supported in part by United States Public
Health Service Grants NS30927, DK52448, and DK42486, a VA Merit Award, a shared equipment grant from the Department of Veterans Affairs, and
monies from the Division of Nephrology Research Fund at Duke University. The laboratory of J. R. R. is supported by an endowment jointly administered by the MUSC Division of Nephrology and Dialysis Clinics, Inc.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
Recipient of a postdoctoral award from the Alberta Heritage
Foundation during the course of these studies.
To whom correspondence should be addressed: Rm. 829E Clinical
Sciences Bldg., 171 Ashley Ave., Charleston, SC 29425. Tel.: 803-792-4123; Fax: 803-792-8399.
1
The abbreviations used are: NHE,
Na+/H+ exchange; NHE-1, ubiquitously expressed
Na+/H+ exchange protein; BCECF-AM,
2,7-biscarboxyethyl-5(6)-carboxyfluorescein acetoxymethyl ester;
1-CT, peptide derived from the carboxyl terminus of
-adrenergic
receptor kinase 1; ECAR(s), extracellular acidification rate(s); EIPA,
5-(N-ethyl-N-isopropyl)-amiloride; ERK,
extracellular signal-regulated kinase; FGF, fibroblast growth factor; G
protein, guanine nucleotide-binding regulatory protein; Gi
,
-subunit of G proteins that inhibits adenylyl
cyclase activity; Go
,
-subunit of G proteins that
inhibits Ca2+ channel activity; Gq
,
-subunit of G proteins that activates phospholipases;
Gz
,
-subunit of G proteins that has no currently assigned function; 8-OH-DPAT,
(±)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide;
MAPK, mitogen-activated protein kinase; MIA,
5-(N-methyl-N-isobutyl)-amiloride; Na+i, intracellular sodium;
Na+o, extracellular sodium; pHi,
intracellular pH; pHo, extracellular pH; (S)-UH-301,
(
)-(S)-5-fluoro-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.