(Received for publication, July 10, 1995; and in revised form, August 14, 1995)
From the
The role of a trimeric GTP-binding protein (G-protein) in the
mechanism of vasopressin-dependent Ca inflow in
hepatocytes was investigated using both antibodies against the carboxyl
termini of trimeric G-protein
subunits, and carboxyl-terminal
-subunit synthetic peptides. An anti-G
antibody and a G
peptide (G
Ile
-Phe
), but not a G
peptide (G
Ile
-Phe
), inhibited vasopressin- and
thapsigargin-stimulated Ca
inflow, had no effect on
vasopressin-stimulated release of Ca
from
intracellular stores, and caused partial inhibition of
thapsigargin-stimulated release of Ca
. An
anti-G
antibody also inhibited vasopressin-stimulated
Ca
inflow and partially inhibited vasopressin-induced
release of Ca
from intracellular stores.
Immunofluorescence measurements showed that G
is
distributed throughout much of the interior of the hepatocyte as well
as at the periphery of the cell. By contrast, G
was
found principally at the cell periphery. It is concluded that the
trimeric G-protein, G
, is required for store-activated
Ca
inflow in hepatocytes and acts between the release
of Ca
from the endoplasmic reticulum (presumably
adjacent to the plasma membrane) and the receptor-activated
Ca
channel protein(s) in the plasma membrane.
Receptor-activated calcium channels (RACCs) ()are
present in most non-excitable and in some excitable animal cells and
are responsible for allowing the inflow of Ca
to
specific regions of the cytoplasmic space and the refilling of
intracellular Ca
stores (most likely a region of the
endoplasmic reticulum)(1, 2, 3) . For a
number of cell types it has been shown that agonist-receptor complexes
open at least two types of RACCs differing in selectivity for divalent
cations(3) . The mechanism(s) by which RACCs are opened is
poorly understood(1, 2, 3) . The hypothesis
presently favored is the store-operated (capacitative) mechanism in
which an increase in inositol 1,4,5-trisphosphate (InsP
)
and the release of Ca
from a region of the
InsP
-sensitive store are prerequisites for channel
activation(1, 2, 3) . This hypothesis is
based, in part, on the observation that thapsigargin, which inhibits
the endoplasmic reticulum (Ca
+
Mg
)-ATPase causing the release of Ca
from this organelle, leads to a stimulation of Ca
inflow(2) . The results of a variety of experimental
approaches have implicated the InsP
receptor(4) , a
mobile intracellular messenger(5, 6, 7) , a
monomeric G-protein(8, 9) , a trimeric G-protein (10, 11, 12, 13, 14) ,
protein phosphorylation(15) , and/or elements of the
cytoskeleton (16) in the mechanism that couples the release of
Ca
from the endoplasmic reticulum to activation of
the plasma membrane Ca
channels.
In hepatocytes,
agonists that employ RACCs include vasopressin, adrenaline, angiotensin
II, and epidermal growth factor(11, 12, 13) .
It has been shown previously that pretreatment of hepatocytes with
pertussis toxin, or the microinjection of GDPS, inhibits
store-operated, as well as vasopressin-stimulated, Ca
inflow. These treatments did not affect the release
Ca
from intracellular stores. This suggested that in
addition to G
, a slowly ADP-ribosylated pertussis
toxin-sensitive trimeric G-protein is required for activation of the
hepatocyte RACC(s)(11, 12, 13) . Since the
two pertussis toxin-sensitive trimeric G-proteins present in
hepatocytes at detectable levels are G
and
G
(17, 18) , it was considered likely that
one of these G-proteins is involved in activation of the hepatocyte
RACC(s) (11, 12) . The aim of the present experiments
was to identify the trimeric G-proteins involved in store-operated
Ca
inflow in hepatocytes. The approach employed used
antibodies generated against peptides corresponding to a region of the
carboxyl termini of G
(19, 20, 21) and synthetic peptides
corresponding to specific regions of the carboxyl termini of
G
(22, 23) . These antibodies and
peptides have been shown by others to inhibit the activation of
trimeric
G-proteins(19, 20, 21, 22, 23) .
Thapsigargin-stimulated Ca
inflow is inhibited by an
anti-G
antibody and by a G
peptide, indicating that the trimeric G-protein G
controls store-operated Ca
inflow in
hepatocytes. Immunofluorescence experiments indicate that the
G
polypeptide is located in the interior of the
hepatocyte and is not restricted in intracellular location to the
plasma membrane.
The peptides against which antibodies were raised,
and the peptides IKNNLKDCGLF (G
Ile
-Phe
(peptide
G
)) and IKNNLKECGLY (G
Ile
-Phe
(peptide
G
)), corresponding to the carboxyl termini of
G
and G
, respectively, were
synthesized (as free COOH) by the Merrifield solid-phase synthesis
procedure using an Applied Biosystems 430A synthesizer and were
analyzed by quantitative amino acid analysis and mass spectrometry, as
described previously(25) . Solutions of peptides G
and G
were prepared fresh each day by
dissolving the peptide in a solution of 10 mM fura-2 in 125
mM KCl to give a concentration of 12 mM peptide in
the microinjection pipette tip (estimated intracellular concentration
160 µM).
Fluorescein isothiocyanate (FITC)-conjugated rabbit IgG was obtained from Sigma-Aldrich, Castle Hill, New South Wales, Australia; and indocarbocyanine (Cy3) from Jackson, West Grove, PA. All other reagents were obtained from sources described previously(11) .
Hepatocytes loaded with anti-G
antibody exhibited a substantial inhibition of vasopressin-stimulated
Ca
inflow compared with control hepatocytes (Fig. 1a, solid line, cf.Fig. 1b). As
reported
previously(8, 11, 12, 19, 26) ,
there was some heterogeneity in the responses given by individual
hepatocytes. Details of the total number of cells tested and the
numbers of cells yielding a given type of response are set out in Table 1. In the majority of cells tested the ability of
vasopressin to release Ca
from intracellular stores
was not affected by microinjection of the antibody (Table 1).
Pretreatment of the anti-G
antibody with the
peptide against which the antibody was raised (the blocking peptide)
prevented the inhibition of vasopressin-stimulated Ca
inflow (Fig. 1a, broken line; Table 1). The anti-G
antibody also
inhibited thapsigargin-stimulated Ca
inflow and
caused some inhibition of thapsigargin-stimulated release of
Ca
from intracellular stores (Fig. 1c, Table 1). No inhibition of
vasopressin-stimulated Ca
inflow was observed in
cells loaded with an anti-G
antibody raised against a
peptide which corresponds to a region near the amino terminus of
G
(results not shown).
Figure 1:
Effects of
anti-G and anti-G
antibodies on vasopressin- and thapsigargin-stimulated
Ca
inflow. The fluorescence of single cells was
measured as described under ``Experimental Procedures.''
Vasopressin (5 nM), thapsigargin (20 µM), and
Ca
(1.3 mM) were added at the beginning of
the periods indicated by the horizontal bars.
Anti-G
antibody (estimated intracellular
concentration 30-40 µg/ml) or anti-G
antibody (estimated intracellular concentration 30 µg/ml) was
co-injected with fura-2 (estimated intracellular concentration 130
µM). a and b, inhibition by
anti-G
antibody of vasopressin-stimulated
Ca
inflow. a, solid trace,
anti-G
antibody; broken trace,
anti-G
antibody pretreated with blocking
peptide; b, no antibody present. The solid and broken traces in a are representative of the results
obtained for 1 of 14 cells (26 cells tested) and for 1 of 6 cells (8
cells tested), respectively (Table 1). The trace shown
in b is representative of the results obtained for 1 of 21
cells (26 cells tested) (Table 1). c, inhibition by
anti-G
antibody of thapsigargin-stimulated
Ca
inflow. The solid (anti-G
antibody present) and broken (no antibody) traces are representative of the results
obtained for 1 of 8 cells (10 cells tested) and 1 of 8 cells (9 cells
tested), respectively (Table 1). d, inhibition by
anti-G
antibody of vasopressin-stimulated
Ca
inflow. The solid (anti-G
antibody present) and broken (control, no antibody
present) traces are represenative of the results obtained for
1 of 5 cells (7 cells tested), and for 1 of 3 cells (3 cells tested),
respectively (Table 1).
The effects of the
anti-G antibody were compared with those of
an antibody against G
. This inhibited
vasopressin-stimulated Ca
inflow in most cells tested
and caused partial inhibition of the vasopressin-stimulated release of
Ca
from intracellular stores (Fig. 1d, Table 1), as shown previously for
hepatocytes by Yang et al.(19) . The possibility that
the microinjected anti-G
antibody was incompletely
distributed in the cytoplasmic space was tested by microinjecting
FITC-conjugated rabbit IgG to hepatocytes. The fluorescence signal
diffused evenly in recipient cells within 5 min following the
microinjection of the antibody, indicating that antibody microinjected
to an hepatocyte is distributed throughout the cell within 5 min
following its microinjection (results not shown).
The microinjection
of peptide G
(Ile
-Phe
) inhibited
vasopressin-stimulated Ca
inflow in almost all cells
tested but had no effect on the ability of vasopressin to release
Ca
from intracellular stores (Fig. 2a, Table 1). By contrast, microinjection
of peptide G
(Ile
-Phe
), at the same concentration
as that employed for peptide G
, caused no inhibition
of either Ca
inflow or Ca
release
from intracellular stores induced by vasopressin (Fig. 2a, Table 1). Neither peptide caused an
activation of Ca
inflow in the absence of vasopressin
or thapsigargin (results not shown). Thapsigargin-stimulated
Ca
inflow was also inhibited by peptide
G
. Complete inhibition was observed in 55% of the
cells tested (Fig. 2b, cf.Fig. 2c, Table 1). Peptide G
caused little or no inhibition of thapsigargin-stimulated
Ca
inflow (Fig. 2b, cf.Fig. 2c, Table 1). However, both peptides
caused some inhibition of thapsigargin-induced release of
Ca
from intracellular stores (Fig. 2b, cf.Fig. 2c, Table 1). In 7 out of 11 cells (peptide G
) and
in 3 out of the 4 cells (peptide G
) the inhibition of
the thapsigargin-induced release of Ca
from
intracellular stores by the peptide was associated with an inhibition
of thapsigargin-induced Ca
inflow. This suggests
there may be some correlation between the effects of the peptides on
the release of Ca
from intracellular stores and their
effects on Ca
inflow.
Figure 2:
The effects of G
Ile
-Phe
and G
Ile
-Phe
peptides on vasopressin- and
thapsigargin-stimulated Ca
inflow. The fluorescence
of single cells was measured as described under ``Experimental
Procedures.'' Vasopressin (5 nM), thapsigargin (10
µM), and Ca
(1.3 mM) were added
at the beginning of the periods indicated by horizontal bars.
When present, G
(Ile
-Phe
) or G
(Ile
-Phe
) (estimated intracellular
concentration 150 µM) was co-injected with fura-2. a, effects of G
and G
peptides on vasopressin-stimulated Ca
inflow.
The solid (G
peptide present) and broken (G
peptide present) traces are
representative of the results obtained for 1 of 7 cells (18 cells
tested) and for 1 of 9 cells (10 cells tested), respectively (Table 1). b and c, effects of G
and G
peptides on thapsigargin-stimulated
Ca
inflow. The solid (G
peptide present) and broken (G
peptide
present) traces in b are representative of the
results obtained for 1 of 11 cells (20 cells tested) and for 1 of 8
cells (12 cells tested), respectively (Table 1). The trace shown in c (control, no peptide present) is
representative of the results obtained for 1 of 11 cells (15 cells
tested) (Table 1).
Since the results of the
experiments conducted with anti-G antibodies and
site-specific G
peptides suggest that G
is required for the activation of Ca
inflow,
the intracellular location of G
was investigated by
immunofluorescence, using anti-G
antibody as
the primary antibody and anti-rabbit IgG antibody coupled to the
fluorescent dye Cy3. In most cells, immunofluorescence, which was
dependent on anti-G
antibody, was found to be
distributed throughout the cytoplasmic space as well as in most parts
of the cell periphery (Fig. 3a). This distribution is
seen more clearly at higher magnification (Fig. 3b).
The fluorescence signal given by anti-G
antibody was not observed when the anti-G
antibody was omitted or when this antibody was pretreated with
the blocking peptide (results not shown). A pattern of
immunofluorescence similar to that given by anti-G
antibody was observed when an anti-G
antibody
was employed as the primary antibody (results not shown). In contrast
to the results obtained with the anti-G
antibody, when anti-G
antibody was employed,
the fluorescence signal was largely confined to the periphery of the
cell, adjacent to the plasma membrane (Fig. 3, c and d). The fluorescence given by anti-G
antibody was abolished when anti-G
antibody
was omitted or when this antibody was pretreated with blocking peptide
(results not shown). The cells labeled with the anti-G
antibody exhibited a much more defined location of
immunofluorescence at the cell periphery than that exhibited by cells
labeled with the anti-G
antibody (Fig. 3c, cf.Fig. 3a). These
results indicate that while G
is distributed in the
plasma membrane and in various regions of the cytoplasmic space,
G
is located predominantly at the plasma membrane.
Figure 3:
Intracellular localization of
G and G
in hepatocytes using
immunofluorescence. Fluorescence images of cells treated with
anti-G
antibody (a and b)
and anti-G
antibody (c and d).
Freshly isolated hepatocytes were cultured for 24 h, fixed,
permeabilized, treated with the indicated primary rabbit
anti-G
polyclonal antibody and a goat anti-rabbit IgG
secondary antibody conjugated to Cy3, and the fluorescence viewed by
scanning confocal fluorescence microscopy, using a
60 (a,
c) and
100 (b, d) objective lens, as described
under ``Experimental Procedures.'' The images are from a scan
1 µm in depth in a plane located approximately half way between the
bottom and top of the cell. The scale bars represent 10
µm. The results shown are those obtained for a representative
sample of the cells observed in one of five experiments which gave
similar results.
Previous studies with hepatocytes which utilized pertussis
toxin, GTPS, and GDP
S have shown that vasopressin-dependent
Ca
inflow requires a pertussis toxin-sensitive
trimeric G-protein in addition to the pertussis toxin-insensitive
G-protein G
, which is required for the activation of
phospholipase C
and the subsequent generation of
InsP
(11, 12, 13) . The conclusion
that vasopressin- and thapsigargin-stimulated Ca
inflow requires the action of G
and/or
G
is consistent with the observation reported here
that the anti-G
antibody, but not the
anti-G
antibody treated with blocking
peptide, inhibited vasopressin- and thapsigargin-stimulated
Ca
inflow. Moreover, the observation that, when
present at the same concentration as peptide G
,
peptide G
did not inhibit vasopressin- and
thapsigargin-stimulated Ca
inflow indicates that the
observed inhibitory effects of peptide G
are most
likely due to inhibition of the action of G
. Taken in
conjunction with the observations that the only detectable pertussis
toxin-sensitive trimeric G-proteins in hepatocytes are G
and G
(16, 17) , the results
reported here indicate that the pertussis toxin-sensitive trimeric
G-protein required for activation of the hepatocyte plasma membrane
receptor-activated Ca
channel is G
.
The conclusion that G is required for store-activated
Ca
inflow in hepatocytes is consistent with the
observation that G
is distributed in regions of the
cytoplasmic space as well as at the plasma membrane, in contrast to
G
which was found at the plasma membrane of the
hepatocyte. ADP-ribosylation of G
catalyzed by
pertussis toxin is very slow (11, 19) , consistent
with a location of some G
in the cytoplasmic space.
The partial inhibition of the thapsigargin-induced release of
Ca from intracellular stores by the anti-G
antibody (which would not be expected to bind to other
intracellular proteins) and the G
and G
peptides was unexpected especially in view of the absence of an
inhibition of vasopressin-stimulated Ca
release. This
preferential inhibition of thapsigargin-induced Ca
release may reflect some form of steric interaction between
G
and the thapsigargin-sensitive (Ca
+ Mg
)-ATPase proteins.
The observations
that (a) the anti-G antibody completely
inhibited vasopressin-stimulated Ca
inflow (present
results) and (b) the only known action of G
is
to activate phosphoinositide-specific phospholipase C
(28) provide further evidence which indicates that an increase
in InsP
is a necessary prerequisite for vasopressin
activation of Ca
inflow in hepatocytes (cf. the conclusion reached previously on the basis of studies with
GDP
S and heparin(11) ). The observation that the
anti-G
antibody completely inhibits
thapsigargin-stimulated Ca
inflow as well as
vasopressin-stimulated Ca
inflow also provides
further evidence that the process of store-operated Ca
inflow is a necessary part of the mechanism of activation of the
plasma membrane Ca
channel by vasopressin in
hepatocytes (cf. the conclusion reached previously on the
basis of results with pertussis toxin which was also shown to block
both vasopressin- and thapsigargin-stimulated Ca
inflow(12, 13) ).
The failure of the
anti-G antibody to completely inhibit
vasopressin-induced release of Ca
from intracellular
stores may be due to a failure of the injected antibody to bind to all
G
molecules, possibly because the affinity of the
anti-G
antibody for G
is low or
G
is in an environment that restricts the accesses
of the antibody. Others have also reported that, relative to the
effects of anti-G
antibodies, longer incubation times
are required in order to detect the inhibition by anti-G
antibodies of phospholipase C
in intact
cells(19, 20) . Another possible explanation for
incomplete inhibition by the anti-G
antibody of
vasopressin-induced release of Ca
is that, in
hepatocytes, there is a species of phosphoinositide-specific
phospholipase C which can be activated by seven transmembrane-spanning
receptors through a mechanism which does not involve
G
(28) . However, no evidence for such a pathway
in hepatocytes has so far been reported. Furthermore, G
is
unlikely to be involved in the activation of phosphoinositide-specific
phospholipase C
in hepatocytes, since first, this function of
G
in hepatocytes has not been reported, and second, the
anti-G
antibody and the G
peptide caused no inhibition of vasopressin-stimulated release of
Ca
from intracellular stores. It is noteworthy that
in several cells the anti-G
antibody inhibited
vasopressin-stimulated Ca
inflow with little effect
on the release of Ca
from intracellular stores. One
possible explanation for this observation is that only a small region
of the intracellular Ca
stores (most likely the
endoplasmic reticulum near the plasma membrane) is involved in
activation of the plasma membrane Ca
channels.
Based on the results obtained, the sequence of events emerging for
the activation by vasopressin of RACCs in hepatocytes is likely to
include the following steps: formation of the vasopressin-receptor
complex, activation of G, activation of phospholipase
C
, an increase in InsP
at the periphery of
the cell, release of Ca
from the endoplasmic
reticulum in this region, activation of G
, and activation
of one or more RACCs. Since the anti-G
antibody and the G
peptide inhibit
thapsigargin-stimulated Ca
inflow (in the absence of
added vasopressin and hence in the absence of the formation of the
vasopressin-receptor complex) activation of G
would not
involve its interaction with a seven-transmembrane-spanning receptor
protein. One possible role of G
may be to regulate the
movement of Ca
between components of the endoplasmic
reticulum (8, 29, 30) or interaction of the
endoplasmic reticulum with the plasma membrane.
The proposed role of
G in store-activated Ca
inflow in
hepatocytes does not exclude a role for a low molecular weight
G-protein, as proposed for mast and mouse lacrimal acinar cells, in
part, on the basis of the observation that GTP
S inhibits
store-activated Ca
inflow in these cell
types(8, 9) . Indeed, other studies conducted in this
laboratory have also shown that a relatively high concentration of
GTP
S inhibits thapsigargin-stimulated Ca
inflow
in hepatocytes. (
)Furthermore, the action of G
may be complimentary to that of a Ca
influx
factor (5, 6, 7) .