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Medical Research Council Membrane and Adapter Proteins Co-operative Group, Membrane Biology Group, Department of Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom EH8 9XD; and Medical Research Council Brain Metabolism Unit, Edinburgh, United Kingdom EH8 9JZ
Address all correspondence and requests for reprints to: Dr. R. Mitchell, Medical Research Council Membrane and Adapter Proteins Co-operative Group, Membrane Biology Group, Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, United Kingdom EH8 9XD. E-mail: rory.mitchell{at}ed.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-sequestering C-terminal fragment of GRK2
(GRK2-ct) in COS7 and GH3 cells, whereas responses of
control receptors were unaffected. Blockers of receptor-activated
Ca2+ influx pathways (Co2+ and SKF 96365) also
partially inhibited VPAC2 receptor-mediated
[3H]InsP responses. This inhibition was not present in
the component of the response remaining after Ptx treatment. A range of
blockers of voltage-sensitive Ca2+ channels were
ineffective, consistent with the reported lack of these channels in
COS7 cells. The data suggest that the VPAC2 receptor may
couple to phospholipase C through both Ptx-insensitive and
Ptx-sensitive G proteins (Gq/11 and Gi/o,
respectively) to generate [3H]InsP. In addition to
G, Gi/o activation appears to require
receptor-activated Ca2+ entry. This is consistent with the
possibility that not only G
q/11-responsive and
G-responsive isoforms of phospholipase C but also
Ca2+-responsive forms may contribute to the overall
[3H]InsP response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pleiotropic effects of VIP are mediated through G protein-coupled receptors that may activate several signal transduction pathways in addition to cAMP production. Two distinct receptors for VIP have been cloned, the vasoactive intestinal polypeptide/pituitary adenylate cyclaseactivating polypeptide type 1 (VPAC1) from rat lung (6) and the VPAC2 from rat pituitary and olfactory bulb (7). The neuropeptide PACAP-38 and its 27-residue amino-terminal form PACAP-27 (which shares approximately 68% identity with VIP) are similarly potent at these receptors in stimulating cAMP production. Receptors for VIP/PACAP belong to group II of the G protein-coupled receptor family, which includes receptors for secretin, PTH, and calcitonin (8). Members of this family couple to adenylate cyclase (AC), and a number have been shown to couple additionally to phospholipase C (PLC) (9, 10).
Preliminary studies indicated that the VPAC2
receptor-mediated activation of [3H]inositol
phosphate ([3H]InsP) formation in transiently
transfected COS7 cells occurred through a mechanism that was in part
pertussis toxin (Ptx) sensitive (11). Here we investigated
the hypothesis that this response is due to the stimulation of a PLC
isoform by a mechanism involving the -subunits of the
Ptx-sensitive G proteins, Gi and
Go, and whether there is any role for
receptor-operated Ca2+ entry in the response. As
well as in transfected COS7 cells, studies were carried out in
GH3 cells, which natively express the
VPAC2 receptor at low levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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770 µCi/mmol) was
iodinated using chloramine-T and purified by HPLC similar to methods
described previously (12). Helodermin, VIP, PACAP-38, SKF
96365
(1-[-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole),
and H89
(N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulphonamide)
were supplied by Calbiochem-Novabiochem (UK) Ltd.
(Nottingham, UK). Guanosine 5'-O-(3-thiotriphosphate)
(GTPS), ionomycin, Ptx, mastoparan, cholera toxin, forskolin,
thapsigargin, methoxyverapamil, nifedipine, flunarizine, TRH,
-conotoxin MVIIC, MSH, cANP-(4–23), 5-hydroxytryptamine (5-HT)
and ATP were obtained from Sigma-Aldrich Corp. (Poole,
UK). Standard laboratory chemicals of Analar grade were obtained from
BDH Chemicals Ltd. (Poole, UK).
Transfection of COS7 cells
COS7 cells were transfected using DEAE dextran as described
previously (7, 13) or by the FuGENE 6 method
(Roche Diagnostics Ltd., Lewes, UK) and were allowed to
recover for 24 h. GH3 cells were transfected
using FuGENE 6. In the case of DEAE dextran transfections, cells were
trypsinized from flasks after 24 h and plated into 24- or 12-well
plates.
Membrane [125I]helodermin binding assay
and GTPS modulation
Confluent cells in 80-cm2 flasks were
washed once with ice-cold Earle’s balanced salt solution, scraped into
buffer A [50 mM Tris-HCl (pH 7.4), 1 mM EGTA,
1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 2 µg/ml
aprotinin, 4 µg/ml leupeptin, 2 µg/ml pepstatin, 1 mM
Na3VO4, 1 mM
NaF, and 50 µg/ml soybean trypsin inhibitor] and homogenized using a
cell homogenizer (Ystral, Dottingen, Germany) for 30 sec.
Homogenates were centrifuged at 1,000 x g for 5 min at
4 C to remove cellular debris before the supernatant was transferred to
a fresh tube and centrifuged at 12,000 x g for 30 min
at 4 C. The membrane pellet was resuspended in buffer A, and a sample
was taken for protein determination. The suspension was then
recentrifuged as previously, and the membranes were resuspended in
assay buffer [50 mM Tris-HCl (pH 7.4), 0.5 mg/ml
bacitracin, 2 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, and 1%
BSA]. Membrane aliquots were incubated at 37 C for 15 min in assay
buffer with 50 µl [125I]helodermin in the
presence of increasing concentrations of cold helodermin or with
GTPS. Nonspecific binding was determined in the presence of 10
µM helodermin.
[125I]Helodermin has been shown previously to
act as a high affinity ligand for VPAC2 receptors
with favorable binding characteristics and greater affinity than
PACAP-27 (14). The reaction was stopped by centrifugation
at 12,000 x g for 30 min at 4 C. The supernatant was
aspirated, and membrane pellets were washed once in ice-cold 500 µl
assay buffer and recentrifuged. Bound radioactivity was determined by
-counting. Protein estimations were performed on total cell
homogenate using the Coomassie protein assay reagent (Pierce Chemical Co., Rockford, IL).
cAMP production assay
Forty-eight hours after replating, cells in 24-well plates were
washed twice in MEM containing 0.25% BSA and preincubated at 37 C for
10 min in the presence of 0.5 mM isobutylmethylxanthine.
Peptides were added at the concentrations indicated in the figure
legends, and incubation was performed at 37 C for 15 min for
concentration-response studies. Assays were stopped by adding an equal
volume of ice-cold 0.2 M HCl to each well, and samples
were then stored and frozen at -40 C. cAMP levels were measured
by RIA with antibodies to cAMP provided by Dr. Ian Gow, Department of
Physiology, University of Edinburgh (Edinburgh, Scotland, UK).
[3H]InsP production assay
Cells in 12-well plates were labeled overnight with 1 µCi/ml
[3H]myo-inositol in Earle’s balanced salt
solution containing 10 mM glucose and 10 mM
HEPES, pH 7.4, in a 37 C gassed incubator. Cells were washed twice in
the same medium containing 0.2% BSA and preincubated at 37 C for 10
min with 10 mM LiCl (plus any inhibitors being tested)
before agonist stimulation for 60 min. Assays were stopped by
aspiration and addition of ice-cold 1.34 M trichloroacetic
acid. [3H]InsP were separated by anion exchange
chromatography as described previously (11), and
radioactivity was measured by scintillation counting. As the rate of
[3H]InsP production (above basal) by the
VPAC2 receptor in both COS7 cells and
GH3 cells was linear for at least 80 min (data
not shown), relatively long incubations were used to accumulate
sizeable evoked responses despite the increasing possibility of
cross-talk between signaling pathways.
RNA isolation and RT-PCR amplification of VIP receptor
subtypes
Total RNA was isolated from GH3 and COS7
cells using the SV RNA isolation kit (Promega Corp.,
Southampton UK). Cells were harvested by trypsinizing a flask
containing approximately 1 x 106 cells, and
the cells were pelleted by centrifugation. First strand complementary
DNA (cDNA) was synthesized from 5 µg total RNA with the OmniScript RT
kit (QIAGEN, Crawley, UK) and random hexamer primers
(Stratagene, Cambridge, UK). First strand cDNA was PCR
amplified with the VPAC1 receptor-specific
primers 338 (5'-CAACAGCGGGGAGATAGACC) and 339 (5'-CAGGATGGAGAGGAGGATGG)
or with the VPAC2 receptor-specific primers 9502
(5'-GAATGCCGGTTTCATCTGG) and 8670 (5'-GGAGATGAGTTCCTGGCTTG) and
Taq polymerase (Stratagene) for 40 cycles.
Amplification products were checked by gel electrophoresis.
Data analysis
All n values represent the number of separate experimental
determinations (with from two to four replicates of individual
measurements being made within each experiment). Curve fitting and
SE calculation were performed using a nonlinear regression
program, P-fit (Elsevier Biosoft, Cambridge, UK).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Multiple signaling pathways
The ability of the VPAC2 receptor to
activate AC and PLC was assessed by cAMP and
[3H]InsP production assays (Fig. 1A
). PACAP-38 caused a
concentration-dependent increase in both cAMP and
[3H]InsP production in COS7 cells transiently
expressing the rat VPAC2 receptor, with
EC50 values of 0.9 ± 0.1 and 36.9 ±
8.1 nM, respectively. The EC50 values
for VIP- and helodermin-evoked cAMP production were similar (0.5
± 0.2 and 0.4 ± 0.2 nM, respectively) to that of
PACAP-38, which is characteristic of the VPAC2
receptor (15).
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).
cANP-(4–23), a selective agonist at the natriuretic peptide clearance
receptor [which has been proposed to mediate VIP-induced
Ca2+ entry responses in gastric smooth muscle
(17)] had no effect on [3H]InsP
production in VPAC2 receptor-expressing COS7
cells from 0.1–3 µM (1.13 ± 0.11-fold of basal at
3 µM; n = 6).
To determine whether a pathway activated by the G protein
Gs could be responsible for PLC activation by the
VPAC2 receptor, we investigated whether the
Gs activator, cholera toxin, could elicit PLC
activation in the transfected COS7 cells. Incubation with 50 ng/ml
cholera toxin for 16 h caused no significant alteration in
[3H]InsP production (1.23 ± 0.17-fold of
basal) despite a 27 ± 4-fold increase in cAMP levels (mean
± SEM; n = 6). Native melanotropin receptors that
couple selectively to Gs (18) are
expressed in COS7 cells. MSH stimulated a concentration-dependent
increase in cAMP production with an EC50 of
19.3 ± 1.8 nM, but concentrations of MSH up to 3
µM caused no increase in [3H]InsP
production (Fig. 1B). Furthermore, no increase in
[3H]InsP production was measured after
treatment of the cells with a 10-µM concentration of the
adenylate cyclase activator forskolin (1.4 ± 0.12-fold of
basal levels after a 60-min stimulation; n = 6). cAMP
levels, however, were increased to 115 ± 15-fold of basal levels
under these conditions. In addition, the selective inhibitor of
cAMP-dependent protein kinase (PKA), H89, had no effect on
PACAP-38-induced [3H]InsP production in
VPAC2 receptor-expressing COS7 cells (3.03
± 0.34- and 3.27 ± 0.41-fold of basal control for 300
nM PACAP-38 and 300 nM PACAP-38 plus 30
µM H89, respectively; n = 6). Taken together, these
results indicate that the VPAC2 receptor-mediated
PLC stimulation is unlikely to be dependent on cAMP production or
Gs activation.
ATP stimulates PLC activity in COS7 cells by acting on a native
metabotropic purinoreceptor (reported as a P2
subtype that is thought to act through the Ptx-insensitive G protein
Gq/11)(19). Figure 1C shows a strong
concentration-dependent increase in [3H]InsP
production stimulated by ATP with an EC50 of
28.3 ± 0.5 µM, whereas ATP concentrations up to 300
µM caused no significant change in cAMP over basal
levels. These data stress that in contrast to examples of receptors
highly selective for adenylate cyclase activation (MSH receptor) or
for PLC activation (P2 purinergic receptor), the
VPAC2 receptor demonstrates a significant ability
to activate both of these signaling pathways.
Involvement of Ptx-sensitive G proteins and G subunits
We previously demonstrated that the activation of PLC elicited by
VIP stimulation of VPAC1 and
VPAC2 receptors expressed in COS7 cells is
partially sensitive to Ptx (11). In the present study,
[3H]InsP production evoked by PACAP-38
stimulation of the VPAC2 receptor was similarly
inhibited by 37 ± 3% after a 16-h preincubation with 100 ng/ml
Ptx (Table 1). A Ptx treatment of 200
ng/ml had no effect on basal [3H]InsP
production (100 ± 2% of control levels). When a
Gi/Go-preferring receptor,
the 5-HT1A receptor, was transfected into COS7
cells, a modest [3H]InsP response was evoked by
5-HT in a manner that appeared to be more completely blocked by Ptx
than that mediated by the VPAC2 receptor
(2.39 ± 0.21-fold of basal with 30 µM 5-HT and
1.20 ± 0.13-fold with 30 µM 5-HT plus 100 ng/ml
Ptx; n = 6). In contrast, PLC activation by two generally
Gq/11-linked receptors (the native
P2 purinergic receptor and a transfected
5-HT2A receptor) was not significantly reduced by
similar incubations with Ptx (Table 1). As PLC activation by
Ptx-sensitive G proteins is considered to occur mainly through the
-subunit rather than the -subunit (20), we
investigated whether coexpression of the G-sequestering
C-terminal fragment of GRK2 [GRK2495–689;
GRK2-ct; (21)] affected VPAC2
receptor-mediated [3H]InsP production.
Paralleling the results with Ptx, cotransfection of GRK2-ct
significantly inhibited VPAC2 but not
5-HT2A or P2
receptor-mediated PLC activation (Table 1). As GRK2-ct appeared to
reduce VPAC2 receptor-mediated PLC activation to
a greater extent than Ptx, preliminary experiments were carried out
with the combination, but there was no evidence for greater inhibition
by GRK2-ct plus Ptx than by GRK2-ct alone (data not shown).
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-subunit
(22). Figure 2A shows that
mastoparan caused a modest, concentration-dependent stimulation of
[3H]InsP production (15 µM
mastoparan caused activation to 1.34 ± 0.08-fold of basal
levels). Ptx pretreatment significantly reduced the response to 15
µM mastoparan by an average of 51 ± 14% (n =
7). These data are consistent with the idea that some stimulation of
[3H]InsP production in COS7 cells can result
from the activation of Ptx-sensitive G proteins alone.
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shows the results of experiments carried out to assess
whether GTP modulation of agonist binding to the
VPAC2 would display a detectable Ptx-sensitive
component. GTPS modulation of
[125I]helodermin binding to the
VPAC2 receptor was measured in both untreated and
Ptx-pretreated membranes prepared from COS7 cells transiently
expressing the VPAC2 receptor. The dissociation
of [125I]helodermin caused by maximally
effective levels of GTPS (3–10 µM) was significantly
reduced by 45 ± 6% (n = 6) after Ptx treatment. Binding to
membranes from cells treated with glutaraldehyde-inactivated Ptx
(23) was identical to that from untreated membranes; at 3
µM GTPS, the specific
[125I]helodermin binding levels were 72 ±
3% and 71 ± 2% of the control value, respectively (n = 6).
These observations support the idea that VPAC2
receptors may to some extent interact directly with Ptx-sensitive G
proteins.
Role of Ca2+ entry
To investigate whether elevation of Ca2+
levels might be sufficient to stimulate
[3H]InsP production, COS7 cells (which had been
transiently transfected with the VPAC2 receptor)
were treated with the Ca2+ ionophore, ionomycin.
Ionomycin stimulated concentration-dependent increases in
[3H]InsP production over a 20-min period,
ranging from 1.9 ± 0.1-fold of the basal control value at 100
nM to 7.9 ± 0.6-fold at 10 µM
(mean ± SEM; n = 6). To determine whether
Ca2+ entry might make a significant contribution
to VPAC2 receptor-mediated
[3H]InsP production in COS7 cells, the effects
of Co2+ (a blocker of divalent cation channels)
(24) were assessed. Concentration-response data for
Co2+ were obtained on PACAP-38-evoked
[3H]InsP production in both untreated and
Ptx-pretreated COS7 cells transiently expressing the
VPAC2 receptor as well as for ATP-evoked
responses in the same cells. Figure 3A shows that the
VPAC2 receptor-mediated response was
significantly inhibited in a concentration-dependent manner by
Co2+ (43 ± 6% of the control response at
100 µM Co2+), whereas the remaining
response in cells pretreated with Ptx (64 ± 4% of that in
untreated cells) was not inhibited by Co2+. The
ATP-evoked response was unaffected by Co2+
treatment (101 ± 4% of the control response at 100
µM Co2+).
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), and
similarly, the VPAC2 receptor-mediated response
was 120.2 ± 13.1% of the control value in the presence of
another selective L-channel blocker, nifedipine (10
µM; mean ± SEM; n = 6). The
selective T-type Ca2+ channel blocker,
flunarizine, was similarly without effect on
VPAC2 receptor-mediated responses (84.5 ±
13.3% of control at 10 µM; mean ± SEM;
n = 6) as was the Q, N, and P channel blocker, -conotoxin MVIIC
(97.2 ± 3.2% of control at 500 nM; mean ±
SEM; n = 6). These data provide no evidence to suggest
the involvement of any known voltage-sensitive
Ca2+ channel in the Ca2+
influx apparently necessary for the VPAC2
receptor-mediated [3H]InsP response. In
contrast, SKF 96365 (which inhibits both calcium-release activated
calcium influx and L-type Ca2+
channels (26), demonstrated a marked
concentration-dependent inhibitory effect (1–100 µM) on
PACAP-38-evoked [3H]InsP production in
VPAC2 receptor-expressing COS7 cells, whereas the
P2 receptormediated response to ATP was not
significantly altered (Fig. 3C). The effect of SKF 96365 was no
different if it was added immediately before PACAP-38 rather than 10
min previously (IC50 of 33.5 ± 2.1 and
25.2 ± 5.9 µM, respectively), suggesting that any
ability of this agent to deplete intracellular
Ca2+ pools during its 10-min preincubation did
not contribute importantly to its inhibition of PACAP-38-evoked
[3H]InsP production. When the
VPAC2 receptor-expressing cells were treated with
Ptx, the residual PACAP-38-evoked [3H]InsP
response was no longer significantly reduced by SKF 96365 (Fig. 3C).
The releaser of intracellular Ca2+ stores and
activator of the Ca2+ release-activated
Ca2+-current (Icrac),
thapsigargin (27) caused a small, concentration-dependent
production of [3H]InsP (1.30 ± 0.05-fold
of basal at 3 µM) and a significant potentiation of the
response to 100 nM PACAP-38 (from 2.75 ± 0.06- to
3.56 ± 0.30-fold of basal; n = 6; P < 0.05,
by Wilcoxon test; although the small effect on basal clearly
contributed in part to this elevation). It is therefore possible that
receptor-operated (or calcium-release-activated)
Ca2+ channels of unknown identity play a
significant role in the VPAC2 receptor-mediated
Ca2+ influx that is apparently required for its
[3H]InsP response.
VPAC2 receptor-mediated
[3H]InsP production in
GH3 cells
VIP receptors are natively expressed in pituitary somatotrophs
(28), and it has been determined that the rat
somatomammotroph GH4C1 cell
line expresses the VPAC2 receptor, but not the
VPAC1 receptor (29). Expression of
the VPAC2, but not the
VPAC1, receptor in the GH3
cell line used in this study was confirmed by RT-PCR. Figure 4A shows the results of amplifying
GH3 or untransfected COS7 cell cDNA with primers
to the VPAC1 and VPAC2
receptors. Neither receptor could be amplified from COS7 cell cDNA,
whereas the VPAC2 receptor was amplified from
GH3 cell cDNA.
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). VIP caused a concentration-dependent increase in
both cAMP and [3H]InsP production in
GH3 cells, with EC50 of
2.2 ± 0.2 and 84 ± 16 nM,
respectively. The agonist potencies in both responses were slightly
reduced compared with that in transfected COS7 cells, but the potency
ratio for the two responses in the native VPAC2
receptor-expressing cells was similar to that in COS7 host cells. We
then determined whether the VPAC2
receptor-mediated [3H]InsP response in
GH3 cells could be inhibited by Ptx and by
expression of the GRK2-ct construct, as previously shown for
transiently transfected COS7 cells. Table 2 shows that 1 µM
VIP caused a modest stimulation of [3H]InsP
production in GH3 cells, which was significantly
inhibited by either Ptx treatment or expression of the GRK2-ct
construct. However, stimulation of [3H]InsP
production by activation of the native TRH receptor (expressed at high
levels in these cells) (31), which couples to PLC
stimulation through the Ptx-insensitive Gq/11,
was not significantly inhibited by either.
[3H]Inositol phosphate production in
GH3 cells was stimulated by ionomycin
(1.60 ± 0.17-fold of basal at 100 nM;
n = 4) as in COS7 cells and additionally by raised
K+ (30 mM) medium
(1.79 ± 0.09-fold; n = 4). Furthermore, 1
µM VIP-evoked [3H]InsP
production in GH3 cells was reduced from
1.91 ± 0.20- to 1.06 ± 0.12-fold of basal in the presence
of 300 µM Co2+ (mean
± SEM; n = 4). This shows that natively
expressed VPAC2 receptors behave similarly to
those heterologously expressed in COS7 cells in terms of their ability
to elicit [3H]InsP responses that are
susceptible to a Ca2+ entry blocker, Ptx, and
GRK2-ct.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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40-fold higher
EC50 value in COS7 cells), significant
[3H]InsP generation occurs at low to medium
nM levels of VPAC2 agonists
(11), consistent with the possibility that this secondary
pathway could contribute to some physiological roles of the receptor.
Responses were compared with those of other receptors, the MSH and
P2 purinergic receptors, which appear to be
highly committed to signaling through AC and PLC activation,
respectively, and show no evidence of the dual signaling that is
characteristic of the VPAC2 receptor here. The
PLC activation response of the VPAC2 receptor is
not an artifact due to heterologous expression, nor is it due to
excessive levels of receptor expression, as essentially similar
responses were seen in GH3 cells that natively
express VPAC2 but not other VIP-responsive
receptors (29) at low levels (see Results)
(28). The potency of VPAC2
receptor-mediated [3H]InsP production in
GH3 cells was approximately 40-fold less than the
cAMP response (similar to the situation in transfected COS7 cells), and
again, significant responses were detectable at concentrations greater
than 10 nM. The inhibition by Ptx of
VPAC2 receptor-mediated PLC activation in both
transiently transfected COS7 cells and GH3 cells
indicates a role for a Ptx-sensitive G protein such as a
Gi/Go (32, 33). There is some evidence to suggest that VIP receptor-evoked
Ca2+ signaling in pancreatic acinar cells
(predominantly VPAC2) is mediated by mechanisms
involving coupling to both Gs and
Gi (34). Like
VPAC2 receptors (11), heterologously
expressed VPAC1 receptors mediate a modest,
partially Ptx-sensitive [3H]InsP response
(11, 35). Interestingly, Diehl et al.
(36) demonstrated a link between a VIP receptor (probably
VPAC1) and Gi3 in rat
lung membranes by covalent cross-linking. The reduction in
GTPS-mediated [125I]helodermin dissociation
(45%) in COS7 cell membranes expressing the
VPAC2 receptor after Ptx treatment suggests a
direct interaction of Gi/Go
with the VPAC2 receptor and that
Gi/Go constitute a sizeable
percentage of the G proteins that associate with this receptor. We were
unable to reliably measure a significant reduction in ligand
dissociation after Ptx treatment of GH3
membranes, probably because of the low levels of
[125I]helodermin binding and the low receptor
expression levels (3700 receptors/cell). However, expression of
GRK2-ct, which binds to and prevents -subunits of heterotrimeric
G proteins from activating second messenger pathways, significantly
inhibited VIP- and PACAP-38-evoked PLC activation in both
GH3 and VPAC2
receptor-transfected COS7 cells. This is consistent with the
established mechanism by which -subunits from Ptx-sensitive G
proteins can activate PLC isoforms, in particular 2 and (the more
widely distributed) 3 subtypes (20, 37). We found no
evidence that the inhibitory effect of Ptx on
VPAC2 receptor-mediated
[3H]InsP production was additive to that of
GRK2-ct, consistent with a common mechanism. In COS7 cells, the degree
of inhibition by GRK2-ct was greater than that by Ptx, which might be
explained by a contribution from G subunits other than those
derived from the Ptx targets
Gi/Go. The inhibition of
VPAC2 receptor responses by Ptx was only partial
compared with that of a largely
Gi/Go-coupled receptor (the
5-HT1A receptor), so it is possible that
Ptx-insensitive, PLC-coupling G proteins such as
Gq/11 are also activated by the
VPAC2 receptor. Any role of
Gq/11 was not directly addressed, however, and as
responses to the reportedly selective
Gi/Go activator mastoparan
were only partially sensitive to Ptx, this remains an open question. In
addition, Gs is very likely to mediate the
VPAC2 receptor’s robust cAMP response. Each of
these other G protein classes could contribute to the free G pool
upon VPAC2 receptor activation. The modest
stimulation of PLC in a Ptx-sensitive manner by mastoparan suggested
that activation of Gi/Go
alone may be sufficient for at least part of this stimulation,
independent of any requirement for activation of
Gs or any other VPAC2
receptor-associated signaling component. The idea of independence from
Gs-mediated pathways was supported by the lack of
effect of cholera toxin, forskolin, MSH receptor activation, or H89
on VPAC2 receptor-mediated
[3H]InsP production.
Ionomycin treatment of COS7 cells and either ionomycin or
K+ treatment of GH3 cells
demonstrated that PLC activity can be stimulated by
Ca2+ elevation. The potent inhibition of
VPAC2 receptor-mediated PLC stimulation by
Co2+ and by La3+ (data not
shown) indicates that a Ca2+ channel is upstream
of PLC activation. The lack of effect of Co2+ on
ATP responses rules out nonspecific toxic effects. None of the
selective blockers of the L-, T-, Q-, N-, or P-type voltage-sensitive
Ca2+ channels that were tested (i.e.
methoxyverapamil, nifedipine, flunarizine, and -conotoxin MVIIC)
affected VPAC2 receptor-mediated
[3H]InsP responses in COS7 cells (see
Results), matching the reported lack of voltage-sensitive
Ca2+ channel subunits in this cell type
(25). In contrast, SKF 96365, which is reported to block
both receptor-mediated Ca2+ entry and some routes
of voltage-dependent Ca2+ entry
(26), markedly reduced VPAC2
receptor-mediated PLC activation, with no significant effect on ATP
responses. The sensitivity of VPAC2
receptor-mediated [3H]InsP production to
Co2+ or to SKF 96365 was no longer present in the
residual part of the response after Ptx treatment. This suggests that
the component of PLC activation that requires
Ca2+ entry is due to the action of Ptx-sensitive
G proteins such as Gi/Go
and that a second component (that involves neither of these aspects)
may also be present. Such a second component could be a more
conventional coupling to PLC isoforms via Ptx-insensitive G proteins
such as Gq/G11. Consistent
with our Co2+/SKF 96353 data and with the
facilitation of VPAC2 receptor
[3H]InsP responses by thapsigargin, a component
of the VPAC2 receptor-mediated elevation of
[Ca2+]i in HIT-T15
insulinoma cells was reported to be due to activation of a
Ca2+ release-activated Ca2+
current (Icrac) (38), but
unfortunately Ptx was not tested on this system. In human neutrophils,
thapsigargin-stimulated cation entry involves a Ptx-sensitive G protein
and a nonselective cation channel sensitive to SKF 96365 and
Gd3+ (27). Furthermore,
Icrac activation in hepatocytes is dependent on
Gi2 (39). This indicates that there
may be some requirement for a Ptx-sensitive G protein in calcium
release-activated calcium influx in a number of cell types.
Receptor-mediated Ca2+ entry resulting from
emptying of intracellular Ca2+ pools
(40) could rely on PLC- isoform activation by G protein
- or -subunits. Operation of the latter mechanism (involving
Gi/Go coupling) could
account for the matching Ptx, Co2+, and SKF 96365
sensitivities of part of the VPAC2
receptor-mediated [3H]InsP production response.
Modulation of the Ca2+ entry pathway(s) by G
protein subunits may also be a possibility. Although receptor-mediated
Ca2+ entry may be enhanced or mimicked by
activation of AC/PKA in several cell types (41, 42),
neither activation nor inhibition of this pathway affected
VPAC2-mediated [3H]InsP
responses here.
Interestingly, elevations of Ca2+ concentrations
within the physiological range (0.1–10 µM) can stimulate
PLC-
1 but not PLC-1 or PLC-1 in permeabilized cells
(43). Ca2+ entry-mediated and
bradykinin receptor-mediated InsP3 production
were facilitated in PC12 cells transfected with PLC-1, and the
excess response to bradykinin was blocked by the
Icrac inhibitor SKF 96365 (44).
Although an extensive survey of the PLC isozymes expressed in these
cells is beyond the scope of this study, it is clear that there is
widespread expression of PLC- isozymes in almost all tissues and
cells, including fibroblasts and GH3 cells
(45). Such a PLC--mediated mechanism could provide
a basis for the Ca2+ entry-dependent component of
VPAC2 receptor-mediated
[3H]InsP production seen here.
In summary, the present study suggests that in addition to AC
activation, a lesser, but still potentially significant, response
mediated by the VPAC2 receptor is the activation
of PLC. We provide evidence that this is brought about by multiple
mechanisms: a Ptx-, GRK2-ct-, Co2+-, and SKF
96365-sensitive component, which may represent to some extent a
G-dependent activation of a PLC- isoform, as well as possibly
a Ptx-insensitive component, which may reflect conventional
Gq/11-mediated activation of a PLC-
isoform. Both components could potentially contribute to
Ca2+ release-activated Ca2+
entry, which may well result in further InsP3
production by way of strictly Ca2+-dependent
PLC- isoforms. From our data it seems likely that the bulk of
VPAC2 receptor-mediated
[3H]InsP production (under these conditions) is
dependent on Ca2+ entry and may therefore
potentially be brought about by a PLC- isoform.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Bioscience and Biotechnology,
University of Strathclyde, 204 George Street, Glasgow, United Kingdom
G1 1XW.
Received August 15, 2000.
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References |
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) and
[3H]InsP (•) production in COS7 cells transiently
expressing the VPAC2 receptor and stimulated by PACAP-38
(A), 
, those in the presence
of Ptx. Values are the mean ± SEM (n = 6–11).
*, Statistically significant inhibition of mastoparan-stimulated
[3H]InsP production (P < 0.05, by
Wilcoxon matched pairs signed rank test). B, Cells were pretreated with
100 ng/ml Ptx for 16 h (
) or vehicle (•) before membrane
harvesting. The maximum specific binding of
[125I]helodermin was equivalent to 13,344 ± 252
cpm/assay for the control and 13,321 ± 631 cpm/assay for Ptx
treatment. The protein concentration of the membrane preparation was
identical for the control and Ptx-treated samples (100 ± 4 and
100 ± 14 µg/ml protein, respectively). *, Statistically
significant reduction in the GTP
