Alternate Coupling of Receptors to Gs and
Gi in Pancreatic and Submandibular Gland Cells*
Xiang
Luo
,
Weizhong
Zeng
,
Xin
Xu
,
Serguei
Popov§,
Isabelle
Davignon§,
Thomas M.
Wilkie§,
Susanne M.
Mumby§, and
Shmuel
Muallem
¶
From the Departments of
Physiology and
§ Pharmacology, University of Texas Southwestern Medical
Center, Dallas, Texas 75235
 |
ABSTRACT |
Many Gs-coupled receptors can
activate both cAMP and Ca2+ signaling pathways. Three
mechanisms for dual activation have been proposed. One is receptor
coupling to both Gs and G15 (a Gq
class heterotrimeric G protein) to initiate independent signaling
cascades that elevate intracellular levels of cAMP and
Ca+2, respectively. The other two mechanisms involve
cAMP-dependent protein kinase-mediated activation of
phospholipase C
either directly or by switching receptor coupling
from Gs to Gi. These mechanisms were primarily
inferred from studies with transfected cell lines. In native cells we
found that two Gs-coupled receptors (the vasoactive
intestinal peptide and
-adrenergic receptors) in pancreatic acinar
and submandibular gland duct cells, respectively, evoke a
Ca2+ signal by a mechanism involving both Gs
and Gi. This inference was based on the inhibitory action
of antibodies specific for G
s, G
i, and
phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a
fragment of
-adrenergic receptor kinase and inhibitors of
cAMP-dependent protein kinase. By contrast,
Ca2+ signaling evoked by Gs-coupled receptor
agonists was not blocked by Gq class-specific antibodies
and was unaffected in G
15
/
knockout mice. We
conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase,
may represent a general mechanism in native cells for dual stimulation
of signaling pathways by Gs-coupled receptors.
 |
INTRODUCTION |
A family of heterotrimeric guanine nucleotide-binding proteins (G
proteins) transduces a variety of signals across the plasma membrane by
sequential interactions with receptor and effector proteins
(e.g. second messenger-generating enzymes and ion channels). These interactions result from guanine nucleotide-driven conformational changes in G protein
subunits (1). Agonist-bound receptors catalyze
the exchange of GDP for GTP on the
subunits of their cognate G
proteins to promote dissociation of
from a high affinity complex of

subunits. Dissociated subunits are competent to modulate the
activity of effectors. GTP hydrolysis ultimately returns G
to the
GDP-bound state, thus allowing reformation of inactive heterotrimer.
Sixteen distinct genes encode G protein
subunits in mammals. The
family is commonly divided into four classes based on amino acid
sequence identity and function: Gs, Gi,
Gq, and G12. Members of a newly identified
family of regulators of G protein signaling (RGS
proteins)1 have been shown to
stimulate the GTPase activity of Gi and Gq class
subunits, thus attenuating signaling (2).
One of the more thoroughly characterized examples of G protein-mediated
signal transduction is carried out by the hormone-sensitive adenylyl
cyclase system. Relevant receptors communicate with homologous G
proteins, one of which (Gs) activates adenylyl cyclase
while others (Gi) inhibit the enzyme (1). The second
messenger (cAMP) mediates diverse cellular responses, primarily by
activating cAMP-dependent protein kinase (PKA). In the case
of Ca2+-mobilizing agonists, G protein activation is
followed by stimulation of phospholipase C
(PLC
) to generate
IP3 in the cytosol, which initiates the
[Ca2+]i signal by release of Ca2+
from internal stores (1, 3). PLC
can be activated by each of the
four Gq class
subunits or by G
subunits released
from Gi class proteins (4). Only Gi-mediated
PLC
activation is inhibited by pertussis toxin (4). In this study we
sought to learn the mechanism by which Gs-coupled receptors
evoke Ca2+ signaling.
Several Gs-coupled receptors can activate dual signaling
cascades. For example, increases in both cAMP and
[Ca2+]i have been observed by histamine acting on
H2 receptors in parietal cells (5), parathyroid hormone
acting on osteoblasts (6), and isoprenaline acting on cardiac myocytes
(7) or salivary gland cells (8, 9). In contrast to the simple paradigm that each receptor molecule can activate a single class of G protein (10), activation of more than one signaling cascade could be due to
coupling of one receptor type to two classes of G proteins. This model
is supported by experiments in heterologous expression systems.
Overexpression of histaminergic H2 (11), parathyroid hormone (12), luteinizing hormone (13), P2Y11 (14), vasopressin V2,
dopamine D1A, and adenosine A2A (15) receptors resulted in stimulation
of adenylyl cyclase and PLC
. The
-adrenergic receptor (which is
considered to be a classical Gs-coupled receptor) and the
vasopressin V2, dopamine D1A, and adenosine A2A can functionally interact with the Gq family member, G15, when
both proteins are overexpressed in COS cells (15, 16).
An alternate mechanism for stimulation of Ca2+ signaling by
Gs-coupled receptors is activation of PLC
by PKA. In
several cell types, increasing cellular cAMP with forskolin (5, 8, 9) or membrane permeable cAMP analogues (5) increased
[Ca2+]i similar to stimulation of
Gs-coupled receptors. In a recent study we showed that
stimulation of submandibular gland (SMG) duct cells with forskolin
results in PLC
-mediated and IP3-dependent Ca2+ release from internal stores (9). These findings
suggest that, at least in some cell types, stimulation of PKA can
activate PLC
to generate a Ca2+ signal.
Phosphorylation-dependent switching of receptor specificity
for G proteins is another mechanism by which a single receptor could
activate more than one G protein (17). As outlined recently by
Lefkowitz (18), receptor-dependent activation of
Gs stimulates adenylyl cyclase, generates cAMP, and
activates PKA. Phosphorylation of the receptor by PKA is proposed to
switch its coupling specificity from Gs to Gi.
Receptor-dependent activation of Gi could thus release sufficient G
to activate PLC
. Activation of PLC
generates IP3 (which releases Ca2+ from
internal stores) and diacylglycerol to activate protein kinase C. Hence, PKA-dependent switching of receptor coupling to
different classes of G proteins (the Gs/Gi
switching model) is a potential mechanism for activation of multiple
signal transduction cascades by the same receptor.
In the work presented here we sought to determine if any of the above
models applied to classical Gs-coupled receptors that evoke
Ca2+ signals in cells freshly isolated from native tissues.
We used vasoactive intestinal peptide (VIP) stimulation of pancreatic acinar cells and isoprenaline (Iso) stimulation of SMG duct cells to
show that switching or augmentation of receptor coupling to Gi could account for activation of cAMP and
Ca2+ signaling systems in vivo.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Affinity purified B087, C260, and C267 polyclonal
antibodies specific for G
i1 and G
i2
(G
i1,2), G
i3, and
G
o (G
i3,0) and G
s,
respectively (19), and anti-G
q IgG (20, 21) were
prepared as described. Monoclonal antibody against PIP2 was
purchased from Preseptive Diagnostics. Pertussis toxin (PTX) (from List
Biological Laboratories) was reconstituted into distilled
H2O and diluted into a pipette solution containing 0.5 mM dithiothreitol. A glutathione-tagged fragment of
-adrenergic receptor kinase (
ARK1) was kindly provided by Dr.
Robert Lefkowitz (Duke University, Durham, NC). His-tagged RGS4 was
expressed in Escherichia coli and purified as described (22). Stock solutions of all antibodies, the
ARK1 fragment, and RGS4
were dialyzed against a solution containing 100 mM KCl and
10 mM HEPES (pH 7.2 with NaOH) and stored at
20 °C
until dilution into the pipette solution. H89 and Rp-8-CPT-cAMP-S were obtained from Biomole and BioLog, respectively. The pipette solution contained (in mM): 150 KCl, 10 HEPES (pH 7.2 with NaOH), 2 MgCl2, 1 ATP, and 0.1 EGTA. The standard bath solution A
contained (in mM): 150 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES (pH 7.2 with NaOH), and 10 glucose. When
this solution was supplemented with 10 mM pyruvate, 1 mg/ml
bovine serum albumin, and 0.02% soybean trypsin inhibitor, it was
abbreviated PSA.
Cell Preparation--
Production of G
15
(
/
)-mutant mice was described (22, 23). Single pancreatic acinar
and submandibular gland (SMG) duct cells from wild type (WT) and
G
15 (
/
)-mice were prepared by standard collagenase
and trypsin digestion procedures (24, 25). In brief, mice were
sacrificed by exposure to a methoxyflurane-saturated atmosphere. The
pancreas and SMG were removed and cleaned by injection of PSA. Minced
tissues were incubated in a PSA solution containing 0.1 mg/ml
collagenase (type CLSP, Worthington) before a short treatment with a
trypsin/EDTA solution to release single cells. The cells were washed
with PSA and kept on ice until use.
Current Recording--
The Ca2+-activated
Cl
current of pancreatic acinar and SMG duct cells was
recorded as detailed (21), using the whole cell configuration of the
patch clamp technique (26). The cells were dialyzed with the pipette
solution for 8-10 min before the first stimulation to allow
equilibration of proteins and antibodies when included in the pipette
solution. Membrane potential was held at
40 mV to record the inward
current. The output signal recorded with a pClamp 6 and DigiData 1200 interface was filtered at 20 Hz. Due to significant variations in the
current magnitude between preparations, results are given primarily as
the number of responding cells. For each protocol similar results were
obtained with cells from at least three mice.
 |
RESULTS AND DISCUSSION |
Fig. 1 summarizes the signaling
pathways by which Gs-coupled receptors (Rs) may
trigger a Ca2+ signal. Stimulation of a
Gq-coupled cholinergic receptor with carbachol
(Rq) was used as a positive control. Three mechanisms were
tested: (a) activation of G
15 by
Rs, (b) direct activation of PLC
by PKA, and
(c) switching or augmentation of coupling specificity of
Rs from Gs to Gi. We tested these
mechanisms using two Gs-coupled receptors which evoke
different types of Ca2+ signals: pancreatic acinar cells
stimulated with VIP and SMG duct cells stimulated with Iso.
Ca2+ signaling was followed by measuring the activity of
the Ca2+-activated Cl
current in each cell
type. Previous work showed that pancreatic acinar and SMG cells express
the Ca2+-activated Cl
channel (21, 25, 27)
and this current faithfully reflects changes in
[Ca2+]i (21, 27).

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Fig. 1.
Signaling pathways tested in this study.
Double slashes through arrows in the pathway
signify inhibition by the indicated agents. A positive control for
Ca2+ release was tested by carbachol stimulation of
Gq-coupled muscarinic m3 receptors (Rq), which
stimulate PLC via Gq class subunits.
Ca2+ signaling in this work is followed by measuring the
activity of Ca2+-activated Cl current.
a, Gs-coupled receptors (Rs) such as
the VIP or -adrenergic receptor might activate the Gq
class heterotrimeric G protein, G 15. A wide variety of
Gs-coupled receptors can couple to G15,
activate PLC to produce IP3, and release
Ca2+ from intracellular stores (15, 16). This potential
pathway would be absent from G 15 knockout mice.
b, Rs activation of Gs and
stimulation of adenylyl cyclase (AC) to increase production
of cAMP, activates PKA which could activate PLC . c,
agonist stimulation of Rs typically activates
Gs and stimulates AC to increase production of cAMP
(inhibited by carboxyl-terminal G s antibodies). PKA
activity is required (directly or indirectly) for generation of
*Rs, thereby eliciting a switch (17) or an augmentation to
account for activation of Gi by Gs-coupled
receptor agonists. G released by activation of Gi
could stimulate PLC activity, produce IP3, and release
Ca2+ from intracellular stores. PTX and antibodies
(Abs) to the carboxyl terminus of G i inhibit
receptor activation of Gi. PIP2 antibodies
prevent PLC hydrolysis of PIP2 and production of
IP3. The Ca2+ ionophore, A23187, bypasses the
need for IP3 production needed for Ca2+ release
from intracellular stores.
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Fig. 2a shows that stimulation
of pancreatic acinar cells with a saturating concentration of
VIP-induced [Ca2+]i oscillations which lasted for
the duration of cell stimulation, as previously reported (28). Maximal
stimulation of the Gq-coupled muscarinic m3 receptor with 1 mM carbachol in the same cells resulted in a typical
biphasic response of a spike and a plateau. This response was highly
reproducible in mouse pancreatic acinar cells; similar responses were
observed in 15/15 cells from 13 mice. Fig. 2b shows that
stimulation of SMG duct cells with the
-adrenergic agonist Iso
caused a sustained increase in the Ca2+-activated
Cl
current with no apparent oscillations. Following
removal of Iso, stimulation with carbachol caused a large biphasic
response. The Cl
current responses are similar in shape
and time course to the previously reported changes in
[Ca2+]i caused by these agonists in SMG cells (8,
9). Among cells which responded to carbachol, prior stimulation with Iso elicited a response similar to that in Fig. 2b in 19/25
SMG duct cells from 17 mice.

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Fig. 2.
Activation of
Ca2+-dependent Cl current by VIP
and Iso in cells from wild type and
G 15 ( / )-mice. Pancreatic
acinar (a and c) or SMG duct cells (b
and d) from wild type (WT, a and
b) and G 15 ( / ) (c and
d) mice were dialyzed with the standard pipette solution for
at least 10 min before cell stimulation. As indicated by the
bars, pancreatic acinar cells were stimulated by 10 nM VIP, which induced [Ca2+]i
oscillations, and then with 1 mM carbachol, which induced a
biphasic response. SMG duct cells were stimulated with 10 µM Iso and then 1 mM carbachol
(Car). Both agonists induced a biphasic response. The number
of observations under each condition is given in the text.
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-Adrenergic, vasopressin V2, dopamine D1A, and adenosine A2A
receptors overexpressed in COS cells can couple to G
15,
but not other members of the Gq class, and stimulate PLC
activity (15, 16). This would suggest that G
15 has the
unique ability to couple to receptors which are usually coupled to
Gs. Currently, there are no good biochemical tools to
specifically evaluate G
15 function in native cells.
Genetics provide an alternative approach. We measured the effect of VIP
and Iso on Ca2+ signaling in cells prepared from mutant
G
15 (
/
)-mice to rule out the possibility that
G
15 contributes to Ca2+ signaling by
Gs-coupled receptors in SMG and pancreatic acinar cells.
Fig. 2c shows that VIP- and carbachol-induced
Ca2+ signaling was completely normal in pancreatic acini
for G
15 (
/
)-mice. The same results were obtained in
six out of six experiments with acini from six mice. Fig. 2d
shows that Iso- and carbachol-induced Ca2+ signaling was
normal in SMG duct cells from G
15 (
/
)-mice. Similar
results were obtained in four out of six experiments with SMG ducts
prepared from the six mice that were used to study the response of
pancreatic acinar cells. These findings exclude coupling to
G
15 as obligatory for activation of Ca2+
signaling by the Gs-coupled receptors. Coupling of
Rs to other members of the Gq class is also
excluded by experiments with antibodies described below.
Experiments with RGS4 supplied our first evidence that activation of
Ca2+ signaling by VIP and Iso involves more than activation
of Gs. RGS4 accelerates GTP hydrolysis by Gq
and Gi class
subunits but not G
s (29,
30). In Fig. 3, infusion of 100 pM RGS4 through a patch pipette into pancreatic acinar
(Fig. 3a) or SMG duct (Fig. 3b) cells completely
inhibited the Ca2+ response to VIP and Iso, respectively.
The control shows that the response to subsequent stimulation with
carbachol was markedly reduced, as we reported recently (23).
Measurement of cAMP production in streptolysin
O-permeabilized cells showed that inhibition of Ca2+ signaling by RGS4 was not due to inhibition of cAMP
production by the Gs-coupled receptors (not shown). The
results with RGS4 exclude model b of Fig. 1 as the mechanism by which
Rs evokes a Ca2+ signal.

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Fig. 3.
Effect of RGS4 on Ca2+
signaling. Pancreatic acinar (a) and SMG duct cells
(b) were dialyzed with a pipette solution containing 100 pM recombinant RGS4. Cells were stimulated with VIP, Iso,
or carbachol (Car), as indicated.
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In the next set of experiments we systematically tested the model for
PKA-dependent Gs/Gi switching (or
augmentation) of receptor specificity shown in Fig. 1c (17,
18). We first tested if stimulation of Gs is obligatory for
launching a Ca2+ signal by the VIP and Iso receptors. This
was achieved by introducing antibodies specific for G
s
into the cells through a patch pipette. Antibodies to the carboxyl
terminus of G
s were used because they have been reported
to block receptor-mediated activation of adenylyl cyclase (31). Fig.
4 shows that the antibodies specific for G
s inhibited Ca2+ oscillations induced by
VIP stimulation of pancreatic acinar cells and the Ca2+
signal stimulated by Iso acting on SMG duct cells without affecting the
oscillations or the biphasic response evoked by stimulation of the
Gq-coupled m3 receptor with carbachol. Similar findings were observed in 4 additional acinar and 3 additional duct cells. As
discussed below, infusion of G
q specific antibodies did
not effect VIP- or Iso-evoked Ca2+ signaling. Therefore,
Gs stimulation was essential for launching a
Ca2+ signal by the two classical Gs-coupled
receptors.

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Fig. 4.
G s
antibodies inhibit effect of VIP and Iso on
[Ca2+]i. Pancreatic acinar (a) or
SMG duct (b) cells were dialyzed with a pipette solution
containing antibodies against G s for at least 10 min
before stimulation with the respective agonist as indicated by the
bars. Controls for these experiments are shown in Figs. 6,
8, and 9. The number of similar observations are indicated in the
text.
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If PKA-dependent phosphorylation were involved, then
inhibition of PKA activity should block Gs- but not
Gq-dependent signaling (Fig. 1c).
The Rs in both cell types met this criterion as shown in
Fig. 5. In control experiments,
Ca2+ oscillations were initiated by stimulation of
pancreatic acinar cells with VIP. After termination of VIP stimulation
by removing the agonist, very similar oscillations were initiated by
stimulating the same cells with low concentrations of carbachol, which
acts through the Gq-coupled muscarinic receptor. Finally,
the cell was stimulated with a supermaximal concentration of carbachol (Fig. 5). Similar results were obtained in 14 cells. In four separate experiments, the VIP response was completely abolished when pancreatic acinar cells were treated with 10 µM H89, a selective and
potent inhibitor of PKA (32), whereas the ability of a low
concentration of carbachol to induce oscillations or of a supermaximal
concentration to induce a biphasic response was unaltered (Fig.
5b). Similarly, treatment of SMG duct cells with 10 µM H89 abolished Iso-dependent [Ca2+]i increase, without affecting the
carbachol-dependent response (Fig. 5d).
Inhibition of the response to Iso was observed in all 6 SMG duct cells
treated with H89. The requirement for PKA stimulation was further
verified by testing the effect of the potent and selective inhibitor of
PKA, Rp-8-CPT-cAMP-S. Infusing the cells with 10 µM
Rp-8-CPT-cAMP-S through the pipette abolished the response to VIP
(n = 7) and Iso (n = 5) in all cells
tested (Fig. 5, c and e). Again, control
experiments in the same cells showed that all forms of
Gq-dependent responses were unaffected by
inhibition of PKA with Rp-8-CPT-cAMP-S. These inhibitory effects of the
two PKA inhibitors argue against the possibilities that unregulated VIP
or
-adrenergic receptors are coupled directly to Gi (33)
or that G
s directly modulates Ca2+ channels
(34) in these systems.

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Fig. 5.
Effect of PKA inhibitors on Ca2+
signaling. Pancreatic acinar (a-c) or SMG duct cells
(d and e) were dialyzed with the standard pipette
solution. In c and e the pipette solution
contained 10 µM Rp-8-CPT-cAMP-S. The bath was perfused
with solution A (a) that also contained 10 µM
H89 (b and d). After about 10 min incubation with
H89, pancreatic acinar cells were stimulated with 10 nM
VIP, then with the submaximal concentration of 0.5 µM
carbachol (Car) to induce
Gq-dependent oscillations, and finally with the
supermaximal concentration of 1 mM carbachol to induce a
biphasic response. SMG duct cells were stimulated with 10 µM Iso and then 1 mM carbachol. The number of
observations under each condition is given in the text.
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To directly address a role for Gi in Ca2+
signaling by VIP and Iso we measured the effect of infusing the cells
with PTX or antibodies specific for certain members of the
Gi subclass of
subunits. Preliminary studies showed
that concentrations of PTX below 20 ng/ml in the pipette solution did
not consistently inhibit VIP-induced signaling. At concentrations above
50 ng/ml, PTX rapidly caused a large, time-dependent,
nonselective increase in membrane conductance, as if PTX caused cell
permeabilization. We therefore limited our testing to the effect of 20 ng/ml PTX on Ca2+ signaling in pancreatic acinar cells.
Fig. 6 shows that treatment with PTX
inhibited VIP but not carbachol-dependent Ca2+
signaling. Similar results were obtained in four experiments. In 13 additional experiments, PTX-treated acinar cells lysed before the
experimental protocol could be completed. We were unable to find a
concentration of PTX that inhibited the Iso response in SMG duct cell
without causing cell lysis.

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Fig. 6.
PTX inhibits VIP-mediated Ca2+
signaling without affecting the response to carbachol. Pancreatic
acinar cells were dialyzed for 7 min with pipette solutions containing
0.5 mM dithiothreitol (DTT) (a and
b) and 20 ng/ml PTX (b) before stimulation with
10 nM VIP, 0.5 µM carbachol (Car),
or 1 mM carbachol as indicated by the bars. The
number of similar observations is given in the text.
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Antibodies generated against peptides representing the carboxyl termini
of G
i and G
q subunits inhibit
receptor-initiated activation of these G proteins (20, 21, 35). The
results obtained by infusing antibodies into pancreatic acinar cells
are illustrated in Fig. 7. Two types of
polyclonal antibodies against Gi were used, one recognizing
G
i3 and G
o or one specific for G
i1 and G
i2 (19). Fig. 7a
shows that infusing 17.5 µg/ml antibodies specific for
G
i3 and G
o had no effect on
Ca2+ signaling induced by Gs- or
Gq-coupled receptors. Similar results were obtained in four
cells. However, these antibodies were not without effect, as seen for
SMG cells (described below). Fig. 7b shows that infusing
pancreatic acinar cells with 9 µg/ml G
i1,i2-specific antibodies completely inhibited the response to VIP without affecting the response to carbachol. Similar results were observed in six cells.
An important control is shown in Fig. 7c. In contrast to the
effect of Gi-specific antibodies, infusing the cells with G
q,11 antibodies (at sufficient concentration to abolish
the oscillation and largely inhibit the sustained response to
carbachol) had no effect on the ability of VIP to induce oscillations.
In seven experiments with cells infused with 80 µg/ml
anti-G
q IgG the response to VIP remained normal, while
the response to the low concentration of carbachol was abolished and
the response to supermaximal concentration of carbachol was inhibited
by 83 ± 7%.

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Fig. 7.
Gi-dependent and
Gq-independent effect of VIP on
[Ca2+]i. Pancreatic acinar cells were
dialyzed for at least 10 min with pipette solutions containing 17.5 µg/ml G i3,o antibodies (a), 9 µg/ml
G i1,i2 antibodies, or 80 µg/ml IgG
anti-G q,11 common antibodies before stimulation with 10 nM VIP, 0.5 µM carbachol (Car), or
1 mM carbachol. The number of similar observations is given
in the text.
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Activation of Gi by Iso is further suggested by the results
for SMG duct cells shown in Fig. 8. In
six cells infused with G
q,11 reactive IgG, the response
to supermaximal concentrations of carbachol was reduced by 91 ± 6% while the response to Iso was not affected (Fig. 8b).
Unlike the findings in pancreatic acinar cells stimulated with VIP,
both Gi antibody preparations effectively inhibited the
response to Iso in SMG duct cells. G
i1,i2-specific antibodies, at a concentration of 9 µg/ml, completely inhibited the
Ca2+ response to Iso (Fig. 8c). Infusion of only
3.5 µg/ml G
i3,o antibodies completely inhibited the
response to Iso in two cells and partially (63 ± 14%) in three
cells (Fig. 8d). At a concentration of 7 µg/ml the
anti-G
i3,o completely inhibited the response to Iso in
five cells (Fig. 8e).

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Fig. 8.
Gi-dependent and
Gq-independent effect of Iso on
[Ca2+]i. SMG duct cells were dialyzed for at
least 10 min with standard pipette solution containing 173 µg/ml of
the IgG fraction from preimmune serum (a, control), 80 µg/ml IgG anti-G q common (b), 9 µg/ml
G i1,i2 antibodies (c), and 3.5 µg/ml
(d), or 7 µg/ml (e) G i3,o
antibodies prior to stimulation with 10 µM Iso or 1 mM carbachol (Car). The number of similar
observations is given in the text.
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The findings in Figs. 7 and 8 provide strong evidence that activation
of Ca2+ signaling by Gs-coupled receptors is
independent of members of the Gq class. The inhibitory
Gq antibodies used in the present work recognizes the
predominant Gq class
subunits expressed in these cells,
G
q, G
11, and G
14 (22).
Furthermore, these antibodies were shown to inhibit Ca2+
signaling evoked by several Gq-coupled receptors in
pancreatic (21) and other cell types (36, 37). At a concentration
inhibiting the oscillatory and the biphasic response to cholinergic
stimulation, the antibodies had no apparent effect on the response to
either VIP or Iso. This data supports the conclusion that inhibition of
VIP- and Iso-induced Ca2+ signaling by RGS4 was due to
acceleration of GTPase activity of a Gi class
subunit(s).
The use of PTX and G
i antibodies indicates that
receptor-mediated activation of Gi was required for
activation of Ca2+ signaling by VIP or Iso. It is notable
that both Gi antibody preparations inhibited Iso-stimulated
Ca2+ signaling in SMG duct cells whereas only the
G
i1,i2-specific preparation was effective for inhibiting
VIP-stimulated signaling in the pancreatic acinar cells. This minor
difference between the two systems may be attributed to cell
type-specific expression patterns of G
i isoforms or the
degree of G
i selectivity exhibited by putative
PKA-phosphorylated VIP and
-adrenergic receptors. It is puzzling
that the
-adrenergic Ca2+ response is inhibited
completely by either Gi antibody preparation. If the
-adrenergic receptor couples to all members of the Gi class, then each antibody preparation would be expected to only partially inhibit and a mixture of the antibodies to completely inhibit
signaling by these receptors. The complete inhibition of signaling by
either antibody preparation suggests that partial inhibition of
IP3 production by stimulation of the
-adrenergic receptor had reduced IP3 below a threshold level needed to
trigger Ca2+ release. This interpretation is supported by
previous work showing that Iso released Ca2+ from the
IP3 mobilizable Ca2+ pool (9) without causing a
detectable increase in global IP3 concentration (8).
In the Gs/Gi switching model, activated
receptor, phosphorylated by PKA, couples to Gi (18). This
predicts that G
released from Gi could activate
PLC
. Thus, inhibition of G
or PLC
activity is expected to
inhibit the effect of the Gs-coupled receptors on
[Ca2+]i. To test these predictions, we measured
the effect of the G
scavenging protein
ARK1 (21, 38) and of
the inhibitory PIP2 antibody (39, 40) on
VIP-dependent Ca2+ signaling. Fig.
9a shows that infusing 5 µM
ARK1 into pancreatic acinar cells completely
inhibited the response to VIP. As we (21) and others (37) reported
earlier,
ARK1 also inhibited the response to stimulation of the
Gq-coupled muscarinic receptor. Inhibition by
ARK1 was
upstream of the Ca2+ increase because elevation of
[Ca2+]i with A23187 strongly activated the
Cl
current. Results similar to those in Fig.
9a, including the positive control with A23187, were
obtained in five experiments. Fig. 9b shows that cytoplasmic
PIP2 antibodies completely inhibited the response to VIP
and reduced the response to carbachol by 88 ± 11%
(n = 7). These experiments indicate that both VIP and
carbachol stimulate PLC
to cause the hydrolysis of
PIP2.

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|
Fig. 9.
Inhibition of VIP-induced Ca2+
signaling by ARK1 and PIP2
antibodies. Pancreatic acinar cells were dialyzed with pipette
solutions containing 5 µM recombinant ARK1 fragment
(a) or PIP2 antibodies (b) prior to
stimulation with 10 nM VIP and 1 mM carbachol
(Car). As indicated, [Ca2+]i was
increased by the addition of A23187 to the cell in a. The
number of similar observations is given in the text.
|
|
In summary, our examination of the [Ca2+]i
increase triggered by Gs-coupled receptors supports a model
for switching or augmentation of receptor coupling to extend to
Gi in native cells freshly isolated from tissue. We
conclude that the pathway involves activation of Gs and
PKA, receptor stimulation of Gi, and activation of PLC
by G
(derived from Gi). We acknowledge that the PKA
substrate(s) responsible for activation of Gi are not known
but, as suggested by the switching model (17, 18), they could be the
same receptors that were initially coupled only to Gs. We
use caution, however, in referring to the Ca2+ pathway
(Fig. 1c), as a receptor switching model.
PKA-dependent phosphorylation of the VIP or
-adrenergic
receptors could allow Gi to replace Gs but the
data are also consistent with broadening of receptor coupling to
Gs plus Gi. One mode for augmentation of
receptor coupling can be envisioned if it is assumed that most
-adrenergic or VIP receptors are productively coupled to
Gs but a smaller subpopulation are poised to couple to
Gi. Effective Gi coupling would occur only when
the receptors are phosphorylated by PKA. Because expression of a mutant
(phosphorylation negative)
-adrenergic receptor prevented
PKA-dependent activation of Gi in HEK 293 cells
(17), it is unlikely that phosphorylation of proteins downstream of the
VIP or
-adrenergic receptors are responsible for activation of
Gi in pancreatic acinar or submandibular gland cells. An
alternative to the assumption that mutant receptor is unable to couple
to Gi (17) is that the mutant cannot regulate its
interaction with an RGS protein that may ordinarily suppress Gi activation stimulated by the
-adrenergic receptor. A
role for regulation of RGS protein function by receptor phosphorylation is attractive, not only because RGS proteins exhibit selectivity among
receptor signaling complexes (23, 41, 42), but also because
phosphorylation is not necessary for purified
-adrenergic receptors
to activate Gi in vitro (33). Additional
experimental tools are needed to distinguish between these and other
potential mechanisms. Independent of the mode of coupling it is clear
that in pancreatic acinar and submandibular cells
Gs-coupled receptors activate Ca2+ signaling by
coupling to Gi and this coupling requires activation of
Gs.
An equally important conclusion is that VIP and
-adrenergic receptor
regulation of Ca2+ release is completely independent of
Gq class proteins. The observation that
PKA-dependent switching/augmentation in receptor/G protein coupling occurs in two different native cell types via two different receptors (that generate different types of Ca2+ signals)
suggests a generalization of the mechanism by which Gs-coupled receptors generate a second signal to activate a
distinct signaling cascade.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Paul C. Sternweis for providing
the Gq antibodies and Dr. Robert Lefkowitz for the
ARK
fragment and our colleagues for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK38939 and DE12309 (to S. M.), GM50515 (to S. M. M.), and DK47890 (to T. M. W.), the Welch Family Foundation,
the Leukemia Association of North Central Texas, and the American Heart
Association (to T. M. W.).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.
¶
To whom correspondence should be addressed: UT Southwestern
Medical Center, Dept. of Physiology, 5325 Harry Hines Blvd., Dallas, TX
75235. Tel.: 214-648-2593; E-mail: smuall{at}mednet.swmed.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
RGS, regulator of G
protein signaling;
PIP2, phosphatidylinositol
4,5-bisphosphate;
IP3, inositol trisphosphate;
PLC
, phospholipase C
;
PKA, cAMP-dependent protein kinase;
VIP, vasoactive intestinal peptide;
Iso, isoprenaline;
ARK1,
-adrenergic receptor kinase 1;
PTX, pertussis toxin;
SMG, submandibular gland;
SLO, streptolysin O toxin;
Rs, Gs-coupled receptor;
Rq, Gq-coupled receptor;
Rp-8-CPT-cAMP-S, 8-(4-chlorophenylthio)adenosine cyclic
3',5'-phosphorothioate;
[Ca2+]i, intracellular
Ca2+.
 |
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