(Received for publication, March 17, 1997, and in revised form, May 12, 1997)
From the To elucidate the signaling events mediated by
specific somatostatin receptor (SSTR) subtypes, we expressed SSTR1 and
SSTR2 individually in rat pituitary
GH12C1 and F4C1
cells, which lack endogenous somatostatin receptors. In transfected
GH12C1 cells, both SSTR1 and SSTR2 coupled to
inhibition of Ca2+ influx and hyperpolarization of membrane
potential via a pertussis toxin (PTx)-sensitive mechanism. These
effects reflected modulation of ion channel activities which are
important for regulation of hormone secretion. Somatostatin analogs
MK678 and CH275 acted as subtype selective agonists as expected. In
transfected F4C1 cells, both SSTR1 and SSTR2
mediated somatostatin-induced inhibition of adenylyl cyclase via a
PTx-sensitive pathway. In addition, activation of SSTR2 in
F4C1 cells, but not SSTR1, stimulated
phospholipase C (PLC) activity and an increase in
[Ca2+]i due to release of Ca2+ from
intracellular stores. Unlike adenylyl cyclase inhibition, the
PLC-mediated response was only partially sensitive to PTx. To determine
the structural determinants in SSTR2 necessary for activation of PLC,
we constructed chimeric receptors in which domains of SSTR2 were
introduced into SSTR1. Chimeric receptors containing only the third
intracellular loop, or all three intracellular loops from SSTR2,
mediated inhibition of adenylyl cyclase, but failed to stimulate PLC
activity as did wild-type SSTR2. Furthermore, the C-terminal tail of
SSTR2 was not required for coupling to PLC. Thus, by expressing
individual somatostatin receptor subtypes in pituitary cells, we have
identified both overlapping and distinct signaling pathways for SSTR1
and SSTR2, and have shown that sequences other than simply the
intracellular domains are required for SSTR2 to couple to the PLC
signaling pathway.
Somatostatin (SS)1 is a tetradecapeptide which has
diverse physiological actions (1, 39). It was first
isolated and identified as an inhibitor of growth hormone (GH)
secretion from the anterior pituitary gland (2). It also inhibits
secretion of insulin, glucagon, gastrin, and secretin (1, 39). In the
central nervous system, SS acts as a neuromodulator, regulating
neuronal firing and facilitating release of neurotransmitters such as
dopamine, norepinephrine, and serotonin (3). SS acts by binding to
specific membrane receptors (4). Five SS receptor (SSTR) subtypes have been cloned and they belong to a family of receptors which couple via G
proteins to cellular effector systems (5-9). In the pituitary gland,
mRNAs for all five SSTRs are present (10). In the past, rat
pituitary GH3 and GH4C1 cells have
been used extensively to characterize endogenously expressed
somatostatin receptors (11, 12). Somatostatin was shown to activate
K+ channels and inhibit voltage-dependent
Ca2+ channels (VDCC) leading to inhibition of
Ca2+ influx (12). The secretion of GH is regulated mainly
by changes in the cytosolic Ca2+ level; thus, the action of
SS on ion channel activity is critically relevant to the physiological
role of SS as an inhibitor of secretion. Since both SSTR1 and SSTR2 are
present in GH3 and GH4C1 cells (13), the functional importance of each subtype is not known. Expression of a single SSTR subtype in heterologous cells such as COS
and CHO cells does not allow studies of coupling of receptors to those
specific ion channels that are characteristic of pituitary cells. In
the first part of this report, we describe the expression of either
SSTR1 or SSTR2 individually in pituitary GH12C1
cells, which do not contain endogenous SSTRs, to study their
independent roles in regulation of membrane ion channel activity.
Expression of SSTR1 and SSTR2 in heterologous systems such as COS and
CHO cells has given inconsistent results as to whether both subtypes
couple to inhibition of adenylyl cyclase (14-16). It is possible that
coupling of SSTRs to a particular G protein is affected by the specific
cellular environment in which the receptors are expressed. Therefore,
it is essential to study SSTR signaling pathways in a cell type in
which SSTRs are expressed physiologically. We describe in this report
that SSTR1 and SSTR2 have both overlapping and distinct patterns of
signaling in pituitary cells. In addition, we examined the structural
basis for the difference in signaling using a chimeric receptor
approach.
Pertussis toxin was purchased from List
Biologicals (Campbell, CA). MK678 was obtained from Merck Laboratories
(West Point, PA).
Des-AA1,2,5-[D-Trp8,IAmp9]SS
(CH275) was synthesized by Drs. Carl Heoger and Jean Rivier at Salk
Institute. myo-[3H]Inositol (80-120 Ci/mmol)
and [125I-Tyr11]SS (2000 Ci/mmol) were from
Amersham Inc. (Arlington Heights, IL). Cyclic AMP radioimmunoassay kits
were purchased from NEN Life Products Inc. (Boston, MA). Fura-2/AM and
bisoxonol were purchased from Molecular Probes (Eugene, OR). Lipofectin
was purchased from Life Technologies, Inc. (Grand Island, NY).
Muta-gene kits and AG1-8X Dowex columns were from Bio-Rad.
Somatostatin and other reagents were from Sigma.
GH12C1 and
F4C1 are clonal pituitary cell strains
established from rat pituitary tumors (17). Both strains lack
expression of endogenous SSTRs (4). Cells were grown in Ham's F-10
nutrient mixture supplemented with 15% horse serum and 2.5% fetal
bovine serum (F10+) at 37 °C in a humidified atmosphere
of 5% CO2, 95% air.
The cDNAs for SSTR1
from either the mouse (18) or rat (6) were used in this study, and no
differences in SS binding and receptor-mediated signaling were
detected. The cDNA for the mouse SSTR2A isoform was used throughout
this study (referred to as SSTR2). SSTR1 and SSTR2 cDNAs cloned in
the pcDNA3 vector (Invitrogen, Carlsbad, CA) were transfected into
GH12C1 and F4C1 cells
by the Lipofectin method according to the supplier's protocol, or by electroporation with a Bio-Rad electroporator. Briefly, 106
cells were suspended in 0.4 ml of F10+ medium containing 16 µg of plasmid DNA, and electroporation was performed at 260 V and 960 microfarads capacitance. In cotransfection experiments, 8 µg of human
parathyroid hormone receptor cDNA and 8 µg of SSTR cDNA were
used. For stable transfection, cells were selected in growth medium
containing 500 µg/ml G418 beginning 48 h after transfection.
After several weeks of culture in selection medium, single clones were
isolated and screened using a radioligand binding assay for SSTRs (see
below). Reverse transcriptase polymerase chain reaction using a pair of
primers from the vector and coding region, and solution RNase
protection assay using RNAs from different clones were used to confirm
the expression of only the specific transfected SSTR
isoform.2
Cells were plated
and grown in F10+ medium in 24-well dishes for at least
24 h. Cells were incubated with minimal essential medium
containing various concentrations (30 pM to 1 nM) of [125I-Tyr11]SS, 0.1%
bovine serum albumin, 0.1% bacitracin, and 2 µg/ml aprotinin for
2 h at 37 °C. The dishes were then washed rapidly 3 times with
ice-cold phosphate-buffered saline. Cells were lysed in 0.1 N NaOH and the lysates counted in a For [Ca2+]i
measurement, cells were harvested using a HEPES-buffered salt solution
(HBSS, 118 mM NaCl, 4.6 mM) KCl, 10 mM D-glucose, 20 mM HEPES, pH 7.4)
containing 0.02% EDTA and then resuspended in HBSS containing 1 mM CaCl2. Cells were loaded using 1 µM fura-2/acetoxymethyl ester (fura-2/AM) for 30 min at 37 °C. Cells were then washed 3 times in HBSS containing 1 mM CaCl2. Fluorescence was measured using a
Spex Fluorolog FIIIA spectrofluorometer at excitation wavelength of 342 nm and emission wavelength of 492 nm. The excitation and emission slit
widths were 5 nm. [Ca2+]i was calibrated from the
fluorescence signal as described previously (19). Changes in
Vm were monitored using an anionic fluorescent dye
bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (bisoxonol) (12).
Cells were harvested as described above. About 5 × 106 cells were used for each run in the presence of 40 nM bisoxonol. Fluorescence was measured at excitation
wavelength of 540 nm and emission wavelength of 580 nm. The excitation
and emission slit widths were 5 and 10 nm, respectively.
The whole cell voltage
clamp technique (20) was used to measure activity of VDCC in
GH12C1 cells. The micropipet solution contained: 110 mM CsCl, 20 mM
tetraethylammonium-Cl, 5 mM MgCl2, 5 mM Na2ATP, 4 mM BAPTA, 10 mM HEPES, pH 7.2, adjusted with Tris. The extracellular
solution contained: 130 mM tetraethylammonium-Cl, 10 mM BaCl2, 10 mM
D-glucose, 10 mM HEPES, and 100 nM
tetrodotoxin (to block voltage-dependent Na+
channels). Ba2+ was used as the cation charge carrier
because Ba2+ minimizes Ca2+ current rundown and
increases the amplitude of the Ca2+ channel current. Only
cells with total capacitance of 15-45 picofarads were used for
recording. The chamber was perfused continuously during the experiment
at a flow rate of 2 ml/min. All experiments were performed at room
temperature.
F4C1 cells were
plated in 24-well dishes after transient transfection. After 48 h,
cells were incubated with test reagents in the presence of 0.2 mM isobutylmethylxanthine for 15 min at 37 °C.
Intracellular cAMP was extracted with 50 mM HCl and
quantitated by radioimmunoassay (21).
Twenty-four h after transfection,
F4C1 cells were split into 24-well dishes at a
density of 2 × 105 cells/well and incubated in
F10+ medium containing [3H]inositol (2 µCi/ml) for 20 h. Cells were then washed with 0.5 ml of assay
medium (20 mM HEPES-buffered minimal essential medium without sodium bicarbonate) with 5 mM LiCl for 10 min and
then incubated with 0.5 ml of assay medium containing 5 mM
LiCl and somatostatin for 1 h. Cold (4 °C) formic acid (0.75 ml, 20 mM) was added to each well to lyse the cells. IP
fractions were separated by AG1-8X Dowex column chromatography (22).
Inositol polyphosphates were eluted with 2 M ammonium
formate, 20 mM formic acid and counted in a scintillation
counter.
Chimeric
receptors were constructed from parental mSSTR1 and mSSTR2 by the
polymerase chain reaction and Kunkel's method (23). The junctions for
IC3 in CH1 and CH2 (see "Results") were Leu233 and
Val276. In CH2, the residues in IC1 and IC2 of SSTR1 were
mutated one by one to the corresponding residues of SSTR2. In CH3, the
junction is Val309. DNA sequencing was performed by the
dideoxy method (24) to verify the constructs.
In control untransfected
GH12C1 cells or GH12C1
cells transfected with the pCDNA3 vector alone, no specific high
affinity receptors for SS were detected by radioligand binding assays, and SS had no effect on [Ca2+]i (Fig.
1, top panel) or membrane potential (see
below). Several stable clones of GH12C1
expressing rat SSTR1 were obtained and a single clone expressing 4,600 receptors/cell was used for further study. To obtain stable clones
expressing SSTR2, more than 200 clones were screened, and no clone with
>1,000 receptors/cell was obtained. However, several clones expressed
the transfected SSTR2 mRNA, and one of them was used in subsequent
functional experiments. In those GH12C1 clones
expressing either SSTR1 or SSTR2, SS caused a decrease in
[Ca2+]i (Fig. 1, middle
panels). Pretreatment of the cells with PTx completely abolished
the decreases in [Ca2+]i mediated by either SSTR1
or SSTR2 (Fig. 1, bottom panels).
The decreases in [Ca2+]i mediated by SSTR1
and SSTR2 (Fig. 2, top panel) were abolished
by either chelating extracellular Ca2+ with EGTA (Fig. 2,
middle panels) or by incubating the cells with the
L-type Ca2+ channel blocker nifedipine (Fig. 2,
bottom panels). Thus, the decreases in
[Ca2+]i induced by SS acting via SSTR1 and SSTR2
were mediated by inhibition of Ca2+ influx through VDCC. To
verify this conclusion, the action of SS on Ca2+ channel
activity was studied directly using the whole cell voltage clamp
technique. Consistent with the effects of SS on
[Ca2+]i, the inward Ca2+ currents
were inhibited acutely by SS in cells expressing either SSTR1 (Fig.
3B) or SSTR2 (Fig. 3C). The
inhibition was reversed by washing out SS. No inhibition by SS was
observed in GH12C1 cells transfected with
vector alone (Fig. 3A).
We next examined the ligand specificity of the action of SS on
[Ca2+]i using receptor subtype selective SS
analogs. MK678, which binds preferably to SSTR2 (18), had no effect on
[Ca2+]i in cells expressing only SSTR1, but it
induced a decrease in [Ca2+]i in cells expressing
SSTR2 (Fig. 4). A new SS analog CH275, reported to bind
with high affinity only to SSTR1 (25), induced a decrease in
[Ca2+]i in cells expressing SSTR1 but not in
cells expressing SSTR2 (Fig. 4). These results are the first
demonstration that CH275 acts as an agonist to activate SSTR1
selectively.
Bisoxonol fluorescence has been used to measure changes in membrane
potential in GH4C1 cells, and hyperpolarization
of Vm induced by SS in GH4C1
cells was caused by activation of K+ channels (12). Using
this technique, SS caused a decrease in bisoxonol fluorescence in
GH12C1 cells expressing either SSTR1 or SSTR2
(Fig. 5, middle panels), indicating that
activation of each receptor subtype caused hyperpolarization of
membrane potential. These responses were inhibited completely by
preincubation with PTx (Fig. 5, bottom panels). Thus, both
SSTR1 and SSTR2 mediate modulation of ion channel activity in
GH12C1 cells, via a PTx-sensitive G protein,
probably Go or Gi.
We next examined the coupling of SSTR1
and SSTR2 to adenylyl cyclase activity in rat pituitary
F4C1 cells, which also lack high affinity
binding sites for SS. Stable clones of F4C1
cells expressing 17,200 rat SSTR1/cell or 5,400 SSTR2/cell (determined by saturation binding assays) were used for the study of signaling properties. Inhibition of adenylyl cyclase activity by SS was determined by measuring the decrease in forskolin (Fsk)-stimulated cAMP
accumulation. SS had no effect on basal or forskolin-stimulated cAMP
levels in wild-type F4C1 cells not transfected
with SSTRs (Fig. 6, left panel). However, SS
acting via either SSTR1 or SSTR2 inhibited forskolin-stimulated cAMP
accumulation, and this process was reversed completely by pretreatment
with pertussis toxin (Fig. 6, middle and right
panels). These results demonstrate that both SSTR1 and SSTR2 can
mediate inhibition of adenylyl cyclase activity via a PTx-sensitive G
protein (possibly Gi/Go) in
F4C1 cells. In GH12C1
clones expressing either SSTR1 or SSTR2, SS did not inhibit vasoactive
intestinal peptide-stimulated cAMP accumulation, while in the same
cells carbachol, acting via endogenous acetylcholine receptors, did
inhibit the vasoactive intestinal peptide effect (data not shown).
Because several
Gi-coupled receptors can activate more than one
intracellular signaling pathway (26, 27), we examined the coupling of
SSTR1 and SSTR2 to the inositol lipid pathway. In
F4C1 cells stably expressing SSTR2, SS
stimulated total inositol polyphosphate production about 5-fold over
the basal level (Fig. 7). Stimulation was reduced about
35% by preincubation with PTx. In F4C1 cells
expressing an even higher level of SSTR1, no stimulation of IP
production was induced by SS (Fig. 7).
The functional consequences of IP production were determined by
measuring changes in [Ca2+]i. In control
F4C1 cells or F4C1
cells expressing only SSTR1, SS had no effect on
[Ca2+]i, whereas in cells expressing SSTR2, SS
induced an acute increase in [Ca2+]i (Fig.
8, top). The increase in
[Ca2+]i in cells expressing SSTR2 was not
abolished by chelation of extracellular Ca2+ with EGTA
(Fig. 8, middle panel). Preincubation of cells with 1 µM thapsigargin for 10 min, to deplete intracellular
Ca2+ pools (28), abolished the increase of
[Ca2+]i induced by SS (Fig. 8, bottom
panel), supporting the conclusion that the increase in
[Ca2+]i was due to release of Ca2+
from IP3-sensitive intracellular Ca2+ stores.
These results demonstrate that SSTR1 and SSTR2 differ in their ability
to couple to the inositol lipid/[Ca2+]i
pathway.
To identify the structural
determinants for SSTR2-mediated inositol lipid signaling, we
constructed a series of SSTR1 and SSTR2 chimeras in which specific
domains of SSTR2 were introduced into the SSTR1 backbone. From sequence
comparison, SSTR1 and SSTR2 differ in the intracellular domains,
especially the third intracellular loop (IC3) and C-terminal tail.
Because the intracellular loops, especially IC3, have been identified
as sites of interaction with G proteins for several heptahelical
receptors (29, 30), we replaced IC3 of SSTR1 with IC3 of SSTR2 in
chimeric construct CH1 (Fig. 9). In CH2, the first, second, and third
intracellular loops of SSTR1 were replaced with those of SSTR2. In
another construct (CH3), the 7th transmembrane domain and the
C-terminal tail of SSTR2 were replaced by the corresponding sequence
from SSTR1 (Fig. 9). Each chimeric receptor was tested
for its ability to express on the cell surface, to stimulate PLC
activity, and to inhibit adenylyl cyclase activity in transient
transfection assays. When transiently transfected into
F4C1 cells, CH1, CH2, and CH3 expressed at
levels comparable to wild-type SSTR1 and SSTR2 as determined by ligand
binding experiments (Fig. 10). However, CH1 and CH2 did not couple to the inositol lipid pathway in response to SS as determined by the lack of rise in IP production. In
F4C1 cells expressing CH3, SS induced formation
of IPs as it did in cells expressing the intact SSTR2 (Fig.
11). Therefore, the three intracellular loops of SSTR2
are not sufficient for coupling to the inositol lipid pathway, and the
C-terminal domain of SSTR2 is not required for this pathway. To exclude
the possibility that CH1 and CH2 are functionally impaired to couple to
any G protein, we studied their ability to inhibit adenylyl cyclase
activity. To evaluate the action of SS on ligand-stimulated cAMP
production, the human PTH receptor, a receptor known to couple to
Gs (21), was transiently co-expressed with SSTR constructs
in F4C1 cells. F4C1
cells lack functional PTH receptors, and PTH does not stimulate cAMP
accumulation in untransfected F4C1 cells (data
not shown). SS had no effect on PTH-stimulated cAMP accumulation in
F4C1 cells transfected with only the human
parathyroid hormone receptor. All three chimeric receptors as well as
wild-type SSTR1 and SSTR2 mediated inhibition of PTH-stimulated cAMP
accumulation by SS (Fig. 12). Thus, the lack of
coupling to PLC in CH1 and CH2 was not due to global nonfunctionality of these constructs. The finding that replacement of all three intracellular loops of SSTR1 with those of SSTR2 was not sufficient to
confer SSTR2-specific signaling suggests that additional domains of
SSTR2, possibly the transmembrane helices, are involved in determining
the specificity of coupling of SSTR2 to the inositol lipid signaling
pathway.
SS exerts its physiological and pharmacological actions by binding
to one or more subtypes of cell surface receptors. Different SSTR
isoforms have overlapping but distinct expression patterns (16),
raising the possibility that some of the diverse actions of SS are
mediated by different receptor subtypes. Alternatively, it is possible
that a single receptor subtype can couple to more than one effector
system and that such coupling may vary among cell types. It has been
reported that SSTR1 and SSTR2 do not couple to inhibition of adenylyl
cyclase in CHO cells (14); however, it has also been shown that SSTR1
and SSTR2 can couple, via Gi, to inhibition of adenylyl
cyclase in other cell types (15). One possible explanation for these
differences is that the G protein and effector components differ
between the cell types. In the present study, SS did not inhibit
adenylyl cyclase activity stimulated by vasoactive intestinal peptide
in GH12C1 clones expressing either SSTR1 or
SSTR2, while SS modulated ion channel activity in these cells. Both
SSTR1 and SSTR2 mediated inhibition of adenylyl cyclase activity in
F4C1 cells. Although we cannot rule out the
possibility that the lack of effect of SS on adenylyl cyclase in
GH12C1 cells was due to a lower level of
receptor expression, it is likely that SSTR coupling patterns to
adenylyl cyclase depend on the specific cellular environment.
Fujii et al. (31) reported, that in rat insulinoma RINm5F
cells, SSTR2, but not SSTR1, coupled to inhibition of VDCC, suggesting that there are subtype specific differences in regulation of ion channel activity. On the other hand, we have demonstrated that in
GH12C1 cells, both SSTR1 and SSTR2 mediate
inhibition of voltage-gated Ca2+ channels. These results
again demonstrate that SSTR isoforms couple to specific signal
transduction pathways depending on the specific type of cells in which
they are expressed.
Different signaling through the same receptor can be transduced by
different G proteins (27). Specifically, the In human embryonic kidney 293 cells expressing SSTR2, we have reported
that SS activates PLC and increases [Ca2+]i (38).
Tomura et al. (34) reported, that in COS cells expressing
SSTR2, SS stimulated PLC/[Ca2+]i, while in cells
expressing SSTR1, SS had little or no effect. In the present report, we
have shown that in pituitary F4C1 cells
expressing about 5,400 SSTR2 receptors/cell, that SS clearly activates
the PLC/[Ca2+]i pathway. Because this response
was only partially blocked by PTx, it was mediated, at least in part,
by a PTx-insensitive G protein, possibly Gq/11. SS induced
increases in [Ca2+]i have also been observed in
normal cells, including astrocytes (35) and intestinal smooth muscle
cells (36). Therefore, coupling to multiple pathways by SSTR2 cannot be
attributed to an artifact of the transfected cell systems.
The intracellular domains of a receptor serve as contact sites for
interacting with G proteins and, in many cases, determine the
specificity of coupling (30). Therefore, it was somewhat unexpected
that the introduction of the putative intracellular domains of SSTR2
into SSTR1 did not confer SSTR2-like PLC activation in response to SS
under conditions in which the chimeric receptors were expressed as well
as wild-type receptors. Thus, the specificity of G protein coupling by
SSTR2 is not determined solely by its intracellular domains. Additional
structural determinants, probably from the transmembrane domains, are
needed to enable coupling to the inositol lipid signaling pathway.
These findings are consistent with the concept that the transmembrane
domains of a G protein-coupled receptor act in concert with the
intracellular loops to affect the conformation of the contact regions
for the G protein (37). In an attempt to define the transmembrane
sequences required, we tested a series of SSTR1/SSTR2 chimeras in which
both transmembrane and intracellular domains were swapped.
Unfortunately, these chimeras all expressed poorly on the cell surface,
making it impossible to study their signaling properties. Further
studies, using chimeras with more subtle exchanges and adequate
receptor expression will be necessary to identify the critical
transmembrane and intracellular sequences needed to confer
SSTR2-specific signaling.
We thank Drs. Zhiling Xiong and Dr. Gary
Strichartz (Harvard Medical School) for their help with the
electrophysiological studies, Drs. Yun Xu and John Bruno (SUNY, Stony
Brook) for RNA solution hybridization assays of various cell clones. We
also thank Dr. Xiao-Jiang Li for rSSTR1 cDNA, Dr. Larry Suva for
human parathyroid hormone receptor cDNA, Drs. Carl Hoeger and Jean
Rivier (Salk Institute) for the SS analog CH275, and Jean Foley for
help with the preparation of this manuscript.
Department of Molecular and Cellular
Toxicology,
Department
of Biological Chemistry and Molecular Pharmacology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Materials
-counter.
Nonspecific binding was determined in the presence of 1 µM SS.
SSTR1 and SSTR2 Each Coupled to Inhibition of Ca2+
Influx through VDCC and Hyperpolarization of Membrane Potential in
GH12C1 Cells
Fig. 1.
Effect of somatostatin on
[Ca2+]i in control GH12C1
and transfected GH12C1 cells stably expressing
either SSTR1 or SSTR2. Cytosolic free Ca2+
concentration ([Ca2+]i) was measured using the
calcium probe fura-2/AM as described under "Experimental
Procedures." After washing, the cells were suspended in HBSS buffer
containing 1 mM CaCl2 during the measurement.
The traces are representative results of at least six independent
experiments for each type. Top, control
GH12C1 cells transfected with vector alone;
middle left, SSTR1-expressing clone; middle
right, SSTR2-expressing clone; bottom, cells were preincubated with pertussis toxin (180 ng/ml) for 18 h.
Arrows mark the addition of 1 µM SS; however,
the effect of SS can be observed at concentrations as low as 1 nM (data not shown).
[View Larger Version of this Image (11K GIF file)]
Fig. 2.
In GH12C1 cells
stably expressing either SSTR1 or SSTR2, SS caused inhibition of
Ca2+ influx through L-type voltage dependent
Ca2+ channels. Left panels, SSTR1; right
panels, SSTR2. Top, addition of 1 µM SS
alone; middle, addition of 1.5 mM EGTA before 1 µM SS; bottom, addition of 10 µM
nifedipine before 1 µM SS. The SS-induced decrease in
[Ca2+]i was completely abolished by either EGTA
or nifedipine. The traces are representative of at least three
independent experiments.
[View Larger Version of this Image (14K GIF file)]
Fig. 3.
Effects of SS on Ca2+ currents in
GH12C1 cells. Whole cell Ca2+
currents were recorded under voltage-clamp conditions by depolarizing pulses from 80 mV to
10 mV. Representative traces are shown. A, lack of effect of SS in control
GH12C1 cells transfected with vector alone;
B, effect of SS in GH12C1 cells
expressing SSTR1; C, effect of SS in
GH12C1 cells expressing SSTR2.
Control indicates current before addition of SS;
SS indicates currents during superfusion of the cells with 1 µM SS; and Washout indicates currents after removal of SS.
[View Larger Version of this Image (20K GIF file)]
Fig. 4.
Subtype selective SS analogs induced
[Ca2+]i responses in appropriate
GH12C1 cell clones.
[Ca2+]i was measured in
GH12C1 cells stably expressing SSTR1 (left) or SSTR2 (right). Arrows
indicate application of 100 nM MK678 or CH275. MK678 or
CH275 caused no changes in [Ca2+]i in control
GH12C1 cells lacking SSTRs (data not shown). Each trace is representative of at least three independent
experiments.
[View Larger Version of this Image (8K GIF file)]
Fig. 5.
Both SSTR1 and SSTR2 mediated membrane
hyperpolarization in GH12C1 cells. Changes
in membrane potential in GH12C1 cells were
monitored using bisoxonol as described under "Experimental Procedures." F is the change in fluorescence in
arbitrary units. Top, lack of effect of SS in control
GH12C1 cells transfected with vector alone;
middle left, SSTR1-expressing clone; middle right, SSTR2-expressing clone; bottom, cells were
preincubated with pertussis toxin (180 ng/ml) for 18 h.
Arrows mark the addition of 1 µM SS.
[View Larger Version of this Image (9K GIF file)]
Fig. 6.
Effects of SS on forskolin-stimulated cAMP
accumulation in control F4C1 cells transfected
with vector alone and in clones stably expressing SSTR1 or SSTR2.
C, basal cAMP level; F, 10 µM
forskolin; F+S, 10 µM forskolin + 1 µM SS; PTx, cells were preincubated with
pertussis toxin (180 ng/ml, 18 h). Bars and brackets represent the mean and S.D., respectively, of
triplicate measurements in a single experiment. Two additional
experiments yielded similar results.
[View Larger Version of this Image (51K GIF file)]
Fig. 7.
Effects of SS on total inositol polyphosphate
production in F4C1 cells expressing either
SSTR1 or SSTR2. SS had no effect on IP production in cells
transfected with vector alone (left panel) or cells
expressing SSTR1 (middle panel). SS caused stimulation of IP
production in cells expressing SSTR2 (right panel).
C, basal level; S, in the presence of 1 µM SS; PTx, pretreated with pertussis toxin
(180 ng/ml) for 18 h. Bars and brackets
represent the mean and S.D., respectively, of triplicate measurements
in a single experiment. Two additional experiments yielded similar results.
[View Larger Version of this Image (25K GIF file)]
Fig. 8.
Effects of SS on
[Ca2+]i in control F4C1
cells (top left), F4C1 cells
expressing SSTR1 (top middle), or SSTR2 (top right). Effect of adding 1.5 mM EGTA
(middle trace) and of pretreating the cells with 1 µM thapsigargin (Tg) for 10 min (bottom trace) on the subsequent SS response in cells expressing SSTR2 are
shown. The SS concentration was 1 µM. Traces shown are
representatives of at least three independent experiments.
[View Larger Version of this Image (13K GIF file)]
Fig. 9.
Diagram of chimeric SSTR1/SSTR2
receptors. The vertical bars represent putative
transmembrane domains. The top and bottom
portions represent the extra- and intracellular domains of each
receptor. In chimeric receptors, the dark or light
bands indicate the contribution by SSTR1 (dark) or SSTR2 (light).
The junctions for IC3 in CH1 and CH2 were Leu233 and
Val276, respectively. In CH2, the distinctive residues of
SSTR1 in IC1 and IC2 were mutated to the corresponding residues of
SSTR2. In CH3, the junction was at Val309.
[View Larger Version of this Image (17K GIF file)]
Fig. 10.
Expression level of SSTR1/SSTR2 chimeras in
F4C1 cells determined by ligand binding
assay. Forty-eight hours after transfection, cells were incubated
with minimal essential medium containing 30 pM
[125I-Tyr11]SS, 0.1% bovine serum albumin,
0.1% bacitracin, 2 µg/ml aprotinin for 2 h at 37 °C. Each
well was then rinsed rapidly three times with ice-cold
phosphate-buffered saline. T, total binding; or N, nonspecific binding was determined in the absence or
presence of 1 µM SS. Each bar represents the
mean and the brackets represent the S.D. of triplicate
measurements in a single experiment.
[View Larger Version of this Image (20K GIF file)]
Fig. 11.
Effect of SS on IP formation in
F4C1 cells transfected with various SSTR
constructs. IP formation was determined as described under
"Experimental Procedures." B, basal IP level; S, IP level in the presence of 1 µM SS. Each
bar represents the mean and the brackets the S.D.
of triplicate measurements in a single experiment. Two additional
experiments yielded similar results.
[View Larger Version of this Image (33K GIF file)]
Fig. 12.
Effect of SS on PTH-stimulated cAMP
accumulation in F4C1 cells co-transfected with
various SSTR constructs and human parathyroid hormone receptor
cDNA. Intracellular cAMP levels were determined as described
under "Experimental Procedures." B, basal cAMP level; P, cAMP level in the presence of 100 nM PTH;
P+S, cAMP level in the presence of 100 nM PTH
and 1 µM SS. Data are expressed as percentage of the
PTH-stimulated cAMP level. Each bar gives the mean value and
the brackets give the S.D. of three samples.
[View Larger Version of this Image (33K GIF file)]
1,
1, and
3
subunits of Go have been shown to mediate inhibition of Ca2+ channels by SSTRs in GH3 cells (32). In a
GH4C1 cell strain in which Go was
knocked out by overexpressing antisense mRNA (33), the effect of SS
on Ca2+ influx was no longer observed; however, the effect
of SS on membrane potential was not altered in these knockout
cells.3 Thus, membrane hyperpolarization
which is due to activation of K+ channels is not dependent
on Go and may be mediated by Gi subunits. It is
likely that in GH12C1 cells, SSTR1 and SSTR2
couple to their cognate G proteins with similar specificity.
*
This work was supported in part by a research grant from the
National Institute of Diabetes, Digestive and Kidney Diseases (DK11011), National Institutes of Health.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: Dept. of Molecular and
Cellular Toxicology, Harvard School of Public Health, 665 Huntington
Ave., Boston, MA 02115. Tel.: 617-432-1177; Fax: 617-432-1780; E-mail:
tashjian{at}hsph.harvard.edu.
¶
Current address: Dept. of Biology, Pharmacopeia, Inc., 3000 East Park Blvd., Cranbury, NJ 08512.
1
The abbreviations used are: SS, somatostatin;
SSTR, somatostatin receptor; Vm, membrane potential;
CH275,
des-AA1,2,5-[D-Trp8,IAmp9] somatostatin;
[Ca2+]i, cytosolic Ca2+
concentration; IP, inositol polyphosphate; PTx, pertussis toxin; BAPTA,
1,2-bis(2-aminophenoxy)ethane-N,N,N,N
-tetraacetic
acid; VDCC, voltage-dependent Ca2+ channel; GH,
growth hormone; CHO, Chinese hamster ovary; PTH, parathyroid hormone;
IC3, intracellular loop 3.
2
J. Bruno and L. Chen, unpublished results.
3
L. Chen, unpublished data.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.