Laboratoire de Neurobiologie et Pharmacologie Cardiovasculaire,
Faculté de Medecine, Université Louis Pasteur, Strasbourg,
France (H.G., C.M., F.M., C.R., P.B., M.D.); Dipartimento di Scienze
Chimiche, Universita di Camerino, Camerino, Italy (W.Q., M.G., M.P.);
Dipartimento di Scienze Farmaceutiche, Universita di Modena, Modena,
Italy (L.B.); and Faculté de Pharmacie, Illkirch-Graffenstaden,
France (P.R., C.L.)
Clonidine and benazoline are two structurally related imidazolines.
Whereas clonidine binds both to 
2-adrenoceptors
(2R) and to I1 imidazoline receptors
(I1R), benazoline showed a high selectivity for imidazoline
receptors. Although the 2R are negatively coupled to
adenylate cyclase, no effect on cAMP level by activation of
I1R has been reported so far. We therefore aimed to compare the effects of clonidine and benazoline on forskolin-stimulated cAMP
levels in cell lines expressing either I1R only (PC12
cells), 2R only (HT29 cells), or I1R and
2R together (NG10815 cells). Clonidine proved able to
decrease the forskolin-stimulated cAMP level in the cells expressing
2R and this effect could be blocked by rauwolscine. In
contrast, in cells lacking these adrenoceptors, clonidine had no
effect. On the other hand, benazoline and other I1
receptor-selective imidazolines decreased forskolin-stimulated cAMP
level in the cells expressing I1R, in a rauwolscine- and pertussis toxin-insensitive manner. These effects were antagonized by
clonidine. According to these results, we demonstrated that 1)
2R and I1R are definitely different entities
because they are expressed independently in different cell lines; 2)
2R and I1R are both implicated in the cAMP
pathway in cells (one is sensitive to pertussis toxin and the other is
not); and 3) I1R might be coupled to more then one
transduction pathway. These new data will be essential to further
understand the physiological implications of the I1R and
the functional interactions between I1 receptors and
2-adrenoceptors.
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Introduction |
Most imidazolines and related
compounds bind both to -adrenoceptors and to imidazoline-specific
receptors (for review, see Regunathan and Reis, 1996
). During the last
decade, imidazoline-binding proteins different from adrenoceptors have
been characterized by extensive binding studies, photoaffinity
labeling, purification procedures in different tissues, and
immunological analysis with specific antibodies. All these results,
taken together, support the existence of a heterogeneous family of
imidazoline-specific binding sites/proteins resolved in at least three
different subtypes, defined as I1,
I2, and
non-I1/non-I2 imidazoline
receptors (for review, see Regunathan and Reis, 1996).
I1 receptors corresponding to clonidine
high-affinity binding sites have been implicated in blood pressure
regulation (Ernsberger and Haxhiu, 1997) as well as in ocular pressure
decrease and catecholamine release from chromaffin cells (Regunathan
and Reis, 1996). Such I1 high-affinity binding
sites have been detected with tritiated clonidine or iodinated paraiodoclonidine in several models, including bovine brainstem membranes (Heemskerk et al., 1998), human brainstem membranes (Dontenwill et al., 1999), bovine chromaffin cells (Molderings et al.
1993), PC12 cells (Separovic et al., 1996), canine prostate (Felsen et
al., 1994), and human platelets (Piletz and Sletten, 1993). Moreover,
the subcellular localization of I1 receptors to
the plasma membrane has been assessed in the bovine brainstem (Ernsberger and Shen, 1997; Heemskerk et al., 1998), in the human platelets (Piletz and Sletten, 1993) and in the PC12 cells (Ernsberger et al., 1995; Separovic et al., 1996).
Identification of the transduction pathway associated to the
stimulation of I1 receptors has been approached
in different ways. The coupling of I1 receptors
to G proteins has been suggested by the sensitivity of the
imidazoline-specific binding to GTP or nonhydrolysable analogs in the
canine prostate (Felsen et al., 1994), in the chromaffin cells
(Molderings et al., 1993; Ernsberger et al., 1995), and in the bovine
brainstem (Ernsberger and Shen, 1997).
Effects of imidazolines on classical second messenger systems of G
protein-coupled receptors, either cAMP or inositol-phosphates and
diacylglycerol (DAG), have been studied in various models, including
rat adrenal glands, bovine chromaffin cells, and rat brain. No effect
on Pi turnover could be detected with moxonidine and clonidine in adrenal glands or chromaffin cells (Regunathan et al.,
1990, 1991). Recently, however, an increase of DAG through activation
of a specific phosphatidylcholine-phospholipase C (PC-PLC) was shown
for moxonidine in PC12 cells. This effect of moxonidine was blocked by
efaroxan, a putative I1 receptor antagonist
(Separovic et al., 1996). Whether this PC-PLC activation is associated
with G proteins remains to be determined.
The decrease of cAMP that was observed with clonidine, rilmenidine, or
moxonidine in rat brain cortex (Regunathan and Reis, 1994; Regunathan
et al., 1995), was clearly caused by the stimulation of
2-adrenoceptors; however, these imidazolines
had no effect on cAMP in tissues that only express
I1 receptors, such as adrenal glands or
chromaffin cells (Regunathan et al., 1990, 1991). In the rat brainstem,
where 2-adrenoceptors (Guyenet et al., 1994) and I1 receptors coexist (Kamisaki et al., 1990),
an inhibitory interaction between the two types of receptor was
suggested, because no effect on cAMP could be seen with moxonidine or
rilmenidine in this tissue (Regunathan and Reis, 1994; Regunathan et
al., 1995).
All these studies were performed with hybrid imidazoline ligands
(clonidine, rilmenidine, and moxonidine) able to bind to imidazoline
receptors and 2-adrenoceptors. Therefore, we
reassessed the capability of imidazolines to affect the cellular cAMP
turnover using a highly selective imidazoline receptor ligand,
benazoline (Pigini et al., 1997). Three different cell lines expressing
either I1 receptors alone (PC12 cells),
I1 receptors and
2-adrenoceptors together (NG10815 cells), or
2-adrenoceptors alone (HT29 cells) were used
to compare the effects of benazoline and clonidine on forskolin-stimulated cAMP values. In cells expressing
2-adrenoceptors (NG10815 cells and HT29
cells), clonidine elicited a decrease of forskolin-stimulated cAMP
level by a rauwolscine and pertussis toxin (PTX)-sensitive mechanism as
expected for an 2-adrenoceptor agonist. In
contrast, benazoline proved able to dose dependently decrease
forskolin-stimulated cAMP content only in cells expressing I1 receptors (PC12 cells and NG10815 cells). This
effect was rauwolscine- and PTX-insensitive. Other
I1 receptor ligands exhibited properties similar
to those of benazoline, whereas clonidine acted as an antagonist. We
propose, therefore, that the I1 receptors are
negatively coupled to the cAMP pathway.
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Experimental Procedures |
Cell Cultures.
PC12 cells were obtained from Dr. G. Rebel (IRCAD, Strasbourg, France). They were cultured in
75-cm2 flasks at 37°C with 10%
CO2 in Dulbecco's modified Eagle's
medium (DMEM; 1000 mg/ml glucose) supplemented with 10%
heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. When the cells reached confluence (3 to 4 days
after plating), they were harvested by 1-min exposure to 0.25% trypsin
at 37°C. For binding assays, after removing the medium, cells at
confluence were frozen in the flasks at 
20°C until use to prepare membranes.
NG10815 cells were obtained from Dr. B. Kieffer (ESBS, Illkirch,
France). They were cultured in 75-cm2 culture
flasks at 37°C with 10% CO2 in DMEM (4500 mg/ml glucose) with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and HAT medium (0.1 µM hypoxanthine, 4 µM
aminopterin, and 16 µM thymidine). After reaching confluence, cells
were harvested for passaging by gentle shaking. For binding assays,
cells were harvested at confluence after 24-h incubation in DMEM
without FBS, and membranes were prepared immediately.
HT29 cells were obtained from Dr H. Paris (INSERM U338, Toulouse,
France) and cultured in 75-cm2 culture flasks at
37°C with 10% CO2 in DMEM (4500 mg/ml glucose) with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were harvested at confluence after 48-h incubation in fresh DMEM without FBS, and membranes were prepared immediately.
Membrane Preparations.
Frozen PC12 cells were scraped into
cold Tris-HEPES buffer (5 mM Tris-HEPES, pH 7.7, 0.5 mM EDTA, 0.5 mM
EGTA, and 0.5 mM MgCl2) and homogenized with a
Potter homogenizer. After centrifugation at 75,000g for 20 min, the pellet was washed in cold Tris-HEPES buffer and centrifuged
again. Pellets were resuspended in Tris-HEPES buffer at 2 to 4 mg
protein/ml and used immediately for binding assays.
NG10815 and HT29 cell membrane preparations were obtained after
homogenization of the cells in 50 mM cold Tris·HCl buffer containing
5 mM EDTA with a Polytron homogenizer. The homogenate was then
centrifuged at 65,000g for 25 min and the pellet was washed
thrice with Tris·HCl buffer without EDTA. Membrane preparations were
stored at 80°C until use.
Binding Assays.
Binding assays on PC12 cell membranes were
performed with [125I]paraiodoclonidine (PIC).
Incubation was initiated by the addition of membranes (200 µg of
protein/400-µl final volume) and were carried out at 25°C during 30 min. For saturation experiments, concentrations of
[125I]PIC ranging from 0.05 to 5 nM were used.
For competition experiments, increasing concentrations of drugs
(10
10 to 104 M)
were added with 0.5 nM [125I]PIC (corresponding
to the KD value of the radioligand). To
stop the incubation, samples were filtered very quickly through GF/B glass fiber filters, incubated for 3 h in 0.03% polyethylenimine with a Brandel harvester, and filters washed twice with 3 ml of 50 mM
cold Tris·HCl buffer, pH 7.7. Radioactivity retained on the dried
filters was determined in a Minaxi gamma counter (Packard, Meriden,
CT). NG10815 membrane binding assays were performed as described in
Greney et al. (1999). HT29 membrane binding assays were performed with
0.5 nM [125I]PIC or with 5 nM
[3H]clonidine. Incubation was initiated by the
addition of membranes (100 µg protein/assay) and were carried out at
25°C during 45 min in a total volume of 400 µl. Assays were then
processed as described above and radioactivity retained on the filters
determined in a beta TriCarb counter (Packard) or in a Minaxi gamma
counter. Nonspecific binding was defined with 10 µM phentolamine for
[3H]clonidine and
[3H]PIC binding in HT29 cells, using 1 mM
phentolamine for [3H]clonidine binding in
NG10815 cells and 10 µM BDF6143 for [125I]PIC
binding in PC12 cells. Phentolamine is able to bind to both I1 imidazoline binding sites and
2-adrenoceptors and was therefore chosen to
define nonspecific binding in NG10815 cells and in HT29 cells. BDF6143
was chosen to define nonspecific binding in PC12 cells according to
Separovic et al. (1996). Because of the low level of imidazoline
binding sites in the PC12 cell membranes (Bmax = 20 fmol/mg of protein) compared
with the NG10815 cell membranes (Bmax = 320 fmol/mg of protein, Greney et al., 1999), we used
[125I]PIC as the radioligand to detect the
imidazoline receptors in the former cells.
cAMP Experiments.
PC12 cells and HT29 cells at confluence
were harvested by mild trypsinization and NG10815 cells were harvested
by gentle shaking and centrifuged at 200g for 5 min. In a
series of experiments, cells were treated with PTX (200 ng/ml culture
medium) for 24 h before harvesting. They were washed thrice with
DMEM containing 50 mM HEPES without FBS. Cells
(3-5 × 105 cells/assay) were incubated for 10 min at 37°C in 200 µl of DMEM-HEPES containing 250 µM
3-isobutyl-1-methylxanthine (a nonselective phosphodiesterase
inhibitor), 10 µM forskolin, and increasing concentrations of drugs
(109 to 103 M). The
reaction was stopped by 800 µl of ice-cold methanol/formic acid
(95:5, v/v) and cells were then sonicated for 5 min. Pellets obtained
after centrifugation at 2000g for 15 min were discarded and
supernatants were used to determine cAMP levels.
Dosage of cAMP was determined by a radioimmunoassay using specific
rabbit anti-succinylated cAMP antibodies. Briefly, cAMP contained in
the samples was submitted to succinylation by addition of succinic
anhydride and incubated with the antibodies (diluted 1:8000) and
125I-cAMP for 18 to 24 h at 4°C. Free
radioactivity was separated from bound by absorption on ice-cold
activated charcoal (2 mg/ml in 0.1 M phosphate buffer, pH 6.3, in the
presence of 2.5 mg/ml BSA) and centrifugation for 20 min at
2000g. Radioactivity in the supernatant was counted in a
Minaxi gamma counter. Results were extrapolated from a standard curve
of cAMP constructed with increasing concentrations of cold cAMP (0.48 to 624 fmol). Alternatively, a radioreceptor assay kit for dosage of
cAMP (Amersham, Orsay, France) was used according to the
instructions of the manufacturer with similar results.
For experiments with PC12 cell membranes, membranes were prepared
according to Hide et al. (1991). Adenylate cyclase assays were
conducted in a total volume of 350 µl containing 50 mM Tris·HCl, pH
7.4, 10 µM GTP, 1 U of adenosine deaminase, 5 mM creatinine phosphate, 0.4 mg of creatinine kinase, 0.01 µM cAMP, 250 µM
3-isobutyl-1-methylxanthine, 30 µg BSA, and 5 mM
MgCl2. Benazoline was added from stock solution in 50 mM Tris·HCl, pH 7.4. Incubations were conducted for 10 min at
37°C and were initiated by the addition of PC12 cell membranes (about
10 µg) to reaction mixture that had been preincubated for 10 min at
37°C. Reactions were stopped by addition of 1 ml of ice-cold 65%
methanol (v/v) solution. After drying the samples in a SpeedVac, dosage
of cAMP was effected with a radioreceptor assay kit (Amersham).
Dosage of Phosphodiesterase Activity.
Cytosolic cyclic
nucleotide phosphodiesterase (PDE) isoforms (PDE1, PDE3, PDE4, and
PDE5) were isolated from media layer of bovine aorta by a modification
of the methods of Lugnier et al. (1986). Cytosolic PDE2 was isolated
from cultured bovine aortic endothelial cells as described previously
(Lugnier and Schini, 1990). PDE activities were measured by
radioenzymatic assay at a substrate concentration of 1 µM cAMP or
cGMP in the presence of 15,000 cpm [3H]cAMP or
[3H]cGMP, respectively, as a tracer. The buffer
solution was of the following composition: 50 mM Tris·HCl, pH 7.5, 2 mM magnesium acetate, and 1 mM EGTA. PDE1 was assayed in basal state
(in presence of 1 mM EGTA) and calmodulin-activated states (with 10 µM CaCl2 and 18 nM calmodulin) using
[3H]cGMP as substrate. PDE2 was assayed in
basal (without cGMP) and cGMP-activated states (with 5 µM cGMP) using
[3H]cAMP. To prevent the influence of
cross-contamination between isolated PDE3 and PDE4, the studies
performed with [3H]cAMP as substrate were
always carried out in the presence of 10 µM rolipram or 100 µM
cGMP, respectively. PDE5 was assayed using
[3H]cGMP as substrate. Dose-effect curves of
PDE activity were made using six concentrations and
IC50 values were determined using a nonlinear
regression analysis with the computer program Prism 2.01 (GraphPAD
Software, San Diego, CA). The results are expressed as percentage of
inhibition of substrate hydrolysis.
PC-PLC Experiments.
PC12 cells were seeded at about
1 × 106 cells/well in a six-well plate in DMEM
containing 10% FBS. After 12-h culture at 37°C with 8%
CO2, wells were rinsed thrice with serum-free
DMEM and drugs (106 M) were added for 1 min. The time of stimulation and the drug concentrations were chosen
according to the method of Separovic et al. (1996). After three washes
with serum- and drug-free DMEM, cells were lysed by addition of 1.5 ml
of ice-cold 3 mM 1,4-piperazinediethanesulfonic acid (PIPES), 0.6 mM
EDTA, 0.03% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) and frozen at 20°C. After thawing at room temperature, lysed cells were scraped out of the wells. Phosphocholine levels were
measured according to the protocol of the Amplex Red
phosphatidylcholine-specific PLC assay kit (Molecular Probes,
Interchim, France). In this enzyme-coupled assay, PC-PLC activity is
monitored indirectly by using 10-acetyl-3,7-dihydrophenoxazine (Amplex
red reagent), a sensitive fluorogenic probe for
H2O2. First PC-PLC converts
the phosphatidylcholine substrate to form phosphocholine and DAG. After
the action of alkaline phosphatase, which hydrolyzes phosphocholine,
choline is oxidized by choline oxidase to betaine and
H2O2. Finally,
H2O2 in the presence of horseradish peroxidase, reacts with Amplex red reagent in a 1:1 stoichiometry to generate the highly fluorescent product resorufin. Thus 100 µl of a solution containing 400 µM Amplex red reagent, 2 U/ml horseradish peroxidase, 8 U/ml alkaline phosphatase, and 0.2 U/ml
choline oxidase was added to each assay tube and fluorescence red in a
fluorometer (Perkin-Elmer Cetus, Norwalk, CT) using excitation at 560 nm and emission detection at 590 nm. Fluorescence was recorded from 20 to 40 min after 30-min incubation at room temperature of the samples
with the enzyme cocktail. Linear regression was used to determine the
fluorescence of each sample at exactly 30-min incubation to accurately
compare the values. Basal levels of phosphocholine were determined in
samples run in parallel without adding drugs for the 1-min stimulation.
Use of the Amplex Red phosphatidylcholine-specific PLC assay kit with
cellular extracts can, however, also measure the free choline
presumably generated by a PC-PLD. However, the activation of a PC-PLD
by imidazoline receptors in PC12 cells has been ruled out by Separovic
et al. (1996; see discussion).
Fluorimetric Measurement of Relative Internal Free
Ca2+ Concentration in PC12 Cells.
For determination of
changes in internal free Ca2+ concentration
([Ca2+]i) in PC12 cells,
cells were harvested and washed with Ringer's solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM
MgCl2, 10 mM HEPES, and 11 mM glucose, pH 7.4. The cells were then loaded with 10 µM fura-red (a fluorescent calcium
indicator; Molecular Probes, Eugene, OR) for 45 min at 37°C using the
acetoxymethyl ester (AM) derivative of the dye, washed and resuspended
in Ringer's solution. Measurements of changes in
Ca2+ levels in stirred cell suspensions were made
using a Perkin-Elmer model LS50B luminescence spectrometer and were
expressed as fluorescence emitted at 640 nm in response to excitation
at 488 nm (data sampling interval, 0.5 s).
Materials.
DMEM medium, FBS, penicillin, and streptomycin
were obtained from Life Technologies (Cergy-Pontoise, France).
Benazoline was synthetized by Prof. Pigini (Camerino, Italy), BDF6143
was kindly provided by Beiersdorf-Lilly (Hamburg, Germany). Clonidine
and rauwolscine were purchased from Research Biochemicals (Bioblock, Strasbourg, France). [125I]PIC (2200 Ci/mmol)
and [3H]clonidine (66.5 Ci/mmol) were purchased
from New England Nuclear (Paris, France). All other chemicals were from
Sigma Chemical (L'Isle d'Abeau Chesnes, France).
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Results |
Binding Characteristics of Imidazolines for I1
Receptors and 2-Adrenoceptors.
PC12 cells express
imidazoline binding sites corresponding to the I1
subtype (Separovic et al., 1996). Because this cell line was shown to
be very labile according to cell culture conditions, we attempted to
characterize more extensively the clone used in our laboratory. For
this purpose, saturation experiments were performed with
[125I]PIC on cell membrane preparations. In
fact, specific binding to membrane receptors was saturable and of high
affinity. The specific binding defined by 10 µM BDF6143 exhibited a
KD value of 0.5 nM and a
Bmax value of about 20 fmol/mg of protein.
On the other hand, no specific binding could be obtained with 10 µM
rauwolscine (an 2-adrenoceptor antagonist) to
define nonspecific binding. Competition experiments with rauwolscine
confirmed the absence of 2-adrenoceptors in
this cell line because a Ki value as high
as 70 µM (n = 2) was obtained for this drug.
Imidazolines completely displaced the specific binding of
[125I]PIC to PC12 cell membranes. Clonidine and
BDF6143 exhibited Ki values of 125 ± 75 nM (n = 4) and 28 ± 6 nM (n = 3), respectively. The competition curves of benazoline were better
resolved by two compartments, one with a high affinity
(Ki = 1.3 nM, 48% of the total sites) and
the other with an affinity of 2800 nM (n = 6) (Fig.
1A). Moxonidine also proved able to
displace [125I]PIC binding sites with two
affinities (34 ± 5 nM and 24 ± 10 µM, respectively;
n = 5). The high-affinity binding sites accounted for
59% of the total sites. Efaroxan behaved like moxonidine and benazoline in binding assays; its competition curves appeared biphasic
and led to determination of two binding affinities, 144 ± 170 nM
(33% of total sites) and 100 µM (n = 5). We checked
the effect of a nonhydrolyzable GTP analog, GTP
S, on the competition curve of benazoline (Fig. 1B). In the presence of 100 µM GTPS, the
competition curve of benazoline was better resolved by one compartment
with an affinity of 1090 ± 1000 nM (n = 4).
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Fig. 1.
A, concentration-dependent inhibition of specific
I1R and 2A-R binding by benazoline.
Increasing concentrations of benazoline (1010 to
103 M) were incubated with membrane preparations of HT29
cells ( , 2A-R), NG10815 cells ( , I1R),
and PC12 cells ( , I1R) in the presence of 5 nM
[3H]clonidine, 20 nM [3H]clonidine with 10 µM rauwolscine, and 0.5 nM [125I]PIC, respectively.
Nonspecific bindings were determined by 10 µM phentolamine, 1 mM
phentolamine, and 10 µM BDF6143, respectively. Total and nonspecific
binding were, respectively, 14634 ± 3147 and 6434 ± 2384 dpm on PC12 membranes, 4240 ± 770 and 2170 ± 680 dpm on
NG10815 membranes, and 10230 ± 6 and 780 ± 25 dpm on HT29
membranes. Data points are means ± S.D. of three to nine
experiments performed in triplicate. B, effect of 100 µM GTPS on
competition curve of benazoline for I1 receptors in PC12
cells. Binding assays were performed as described under
Experimental Procedures except that 100 µM GTPS was
included in each tube. Each point represents the mean of six
experiments, each conducted in triplicate. In this set of experiments,
high-affinity binding sites of benazoline accounted for 30% of total
binding sites in the absence of GTPS and were completely lost in the
presence of 100 µM GTPS.  , control; , 100 µM GTPS.
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HT29 cells express well characterized
2A-adrenoceptors (Bylund et al., 1988). To
check the presence of imidazoline binding sites in this cell line,
competition experiments were performed with clonidine and noradrenaline
on [3H]clonidine and
[125I]PIC total binding. Clonidine and
noradrenaline proved able to displace the total binding of either
radioligand to a similar extent, with IC50 values
of 1.8 ± 0.8 and 44 ± 10 nM, respectively, on
[3H]clonidine binding sites and 51 ± 9 and 153 ± 91 nM on [125I]PIC binding
sites. These results demonstrate that specific imidazoline binding
sites of the I1 receptor subtype, different from
adrenoceptors, are not expressed in these cells. Competition curve of
benazoline on [3H]clonidine-specific binding to
2A-adrenoceptors in this cell line led to the
determination of a Ki of 3500 ± 2700 nM (n = 3) (Fig. 1A).
NG10815 cells expressed imidazoline receptors and
2B-adrenoceptors. In these cells,
[3H]clonidine proved able to label
high-affinity I1 receptors when the experiments
were performed in the presence of 10 µM rauwolscine to mask the
2B-adrenoceptors (Greney et al., 1999).
Benazoline completely displaced this imidazoline-specific binding. The
competition curve was better resolved by two compartments with
Ki values of 2.3 ± 1.8 nM (30% of
the sites) and 5700 ± 500 nM, respectively (n = 9) (Fig. 1A).
Effect of Imidazolines on cAMP Level in the Cells.
The PC12
cells were incubated with increasing concentrations of imidazolines
after stimulation by 10 µM forskolin. Benazoline produced a
dose-dependent decrease in forskolin-stimulated cAMP content of the
cells. The dose-response curve of benazoline appeared biphasic
(P = .02 for the comparison of fits) with
EC50 values of 2.2 ± 2.1 nM (20% of
maximal effect) and 27 ± 18 µM, respectively (n = 8), with a maximal inhibition of 51 ± 11%. Moxonidine and BDF6143 dose dependently decreased the forskolin-stimulated cAMP level
in the cells with EC50 values of 35 ± 34 nM
(n = 5) and 78 ± 8 nM (n = 3),
respectively. Maximal inhibition values recorded with moxonidine and
BDF6143 were 12 ± 2 and 27 ± 2%, respectively. Efaroxan
dose-response curves were better resolved by two compartments (P < .001) with EC50 values of
0.4 ± 0.1 nM (14% of maximal effect) and 270 µM, respectively,
and maximal inhibition of 56 ± 8%. Conversely, clonidine proved
unable to modify significantly (n = 5) the
forskolin-stimulated cAMP concentrations under the same conditions
(Fig. 2). Clonidine (1 µM),
which had no effect on its own, antagonized the decrease of cAMP
induced by 1 µM benazoline (n = 4) (Fig.
3). Same antagonism on the effect of 10 µM benazoline was obtained with 10 µM clonidine. As expected from
the results mentioned above, efaroxan did not antagonise the benazoline
effect (n = 4) (Fig. 3).
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Fig. 2.
Dose-dependent inhibition of forskolin-stimulated
cAMP production in the PC12 cells by imidazolines. Inhibition curves of
forskolin-stimulated cAMP production in PC12 cells by benazoline (),
efaroxan (), BDF6143 ( ), moxonidine (), and clonidine ()
are shown. Results from three to eight experiments performed in
triplicate were averaged and expressed as percentage of control (in the
presence of forskolin alone) ± S.E.  , nonlinear least-squares
fit, determined as described under Experimental
Procedures. Basal cAMP production was 118 ± 16 fmol/min/105 cells; forskolin-stimulated cAMP production
was 2358 ± 508 fmol/min/105 cells.
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Fig. 3.
Effect of clonidine on forskolin-stimulated cAMP
level in response to benazoline in PC12 cells. To measure the effect of
benazoline, cells (105) were incubated with benazoline and
forskolin for 10 min at 37°C. For coincubation experiments, cells
(105) were preincubated with 1 µM clonidine or 100 µM
efaroxan in the presence of forskolin for 10 min at 37°C before
addition of 1 µM benazoline and a subsequent incubation for 10 min at
37°C. In control experiments of the latter, clonidine and efaroxan
were added for 20 min and cAMP content was measured. Results are
expressed as percentage ± S.E.M. change from matched controls
containing forskolin only. Bars, mean values from at least four
separate experiments. * P < .05, paired
t test; statistically different from control.
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When adenosine (20 µM) instead of forskolin was used to stimulate
adenylate cyclase in the PC12 cells, no inhibition was obtained with
increasing concentrations of benazoline (Fig.
4), suggesting the involvement of
specific adenylate cyclase isoforms.
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Fig. 4.
Effect of benazoline on adenosine-stimulated cAMP
level in PC12 cells. Cells (105) were incubated with
benazoline (107 to 105 M) and adenosine (20 µM) for 10 min at 37°C and cAMP measured as described. Results are
expressed as percentage ± S.E. of control containing adenosine
only. Bars, mean values of three experiments performed in triplicate.
Basal cAMP level was 45.3 ± 0.5 fmol/105 cells/min
and adenosine-stimulated cAMP production was 252 ± 14 fmol/105 cells/min.
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To confirm that the modulation of the forskolin-stimulated cAMP
accumulation obtained by imidazolines in the PC12 cells involved membrane receptors, we next examined whether such a modulation could
also occur in membrane preparations instead of whole cells. In this
case, a monophasic dose-response curve was obtained for benazoline
(Fig. 5) with an
EC50 value of 0.7 ± 0.3 nM
(n = 3) and a maximal inhibition of 27 ± 4%.
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Fig. 5.
Effects of benazoline on forskolin-stimulated cAMP
accumulation in PC12 cell membranes. PC12 cell membranes (10 µg) were
incubated with increasing concentrations of benazoline as described
under Experimental Procedures. Data were analyzed using
a nonlinear regression program (GraphPad). Values are mean ± S.E.
of three experiments. Basal value of cAMP was 45 ± 16 pmol/mg
protein/min and forskolin- stimulated value was 90 ± 14 pmol/mg
protein/min.
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To ascertain whether I1 receptor-mediated cAMP
decrease was dependent on an increase in intracellular calcium
level, PC12 cells were loaded with the fluorescent calcium indicator
fura-red, which displays a decrease in fluorescence upon binding of
calcium. Application of 10 µM benazoline induced a small but
significant increase in the relative level of
[Ca2+]i (Fig.
6). However, at lower concentrations, no
significant effect could be observed.
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Fig. 6.
Effect of benazoline on
[Ca2+]i in PC12 cells. PC12 cells were loaded
with fura-red for 45 min at 37°C. Fluorescence emitted was recorded
at 640 nm in response to excitation at 488 nm. One representative
experiment of four is shown. Benazoline [10 µM (filled symbol) or
100 nM (open symbol)] was added to stirred cells at 60 s of
incubation.
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To confirm the effects of imidazolines on cAMP levels we tested them in
NG10815 cells known to express I1 imidazoline
receptors as well as 2B-adrenoceptors. As
shown in Fig. 7A, benazoline decreased the forskolin-stimulated cAMP content in a dose-dependent manner with an EC50 value of 25 ± 0.7 µM
and a maximal inhibition of 60 ± 6% (n = 5).
Clonidine also decreased forskolin-stimulated cAMP level in these cells
with an EC50 value of 15 ± 18 nM and a
maximal inhibition of 38 ± 3% (Fig. 7B). Because
2-adrenoceptors were present in these cells,
we checked whether the clonidine- and benazoline-induced effects were
caused by any activation of these receptors. Rauwolscine (10 µM), an
2-adrenergic blocking drug, did not
significantly modify the effects of benazoline on cAMP levels, but it
shifted the dose-response curve of clonidine to the right
(EC50 = 350 µM; n = 2) (Fig. 7,
A and B, respectively).
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Fig. 7.
Inhibition of forskolin-stimulated cAMP production in
the NG10815 cells. Inhibition of forskolin-stimulated cAMP production
by benazoline (A) and by clonidine (B) is shown. A, cells were
incubated with increasing concentrations of benazoline only () or in
the presence of 10 µM rauwolscine (). B, cells were incubated with
increasing concentrations of clonidine only () or in the presence of
10 µM rauwolscine (). Results from two to five experiments
performed in triplicate were averaged and expressed as percentage of
control (with forskolin only) ± S.E. , nonlinear least-squares
fit, determined as described under Experimental
Procedures. Basal cAMP production was 433 ± 186 fmol/min/105 cells; forskolin-stimulated cAMP production
was of 8600 ± 2300 fmol/min/105 cells.
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These results confirmed that clonidine behaved as an
2-adrenoceptor agonist in the NG10815 cells on
the one hand and that benazoline elicited its effect on the cAMP
content of the cells by an
2-adrenoceptor-independent mechanism on the
other hand.
Because 2A-adrenoceptors are naturally
expressed in HT29 cells without coexpression of imidazoline receptors,
we used this cell line to further confirm the above results. In these
cells, no significant effect on forskolin-stimulated cAMP accumulation could be recorded with increasing concentrations of benazoline (Fig.
8A). Moreover, the addition of 10 µM rauwolscine did not unmask any effect of benazoline (Fig. 8A).
Clonidine decreased the forskolin-stimulated cAMP level with an
EC50 value of 10 ± 3 nM and a maximal
inhibition of 50 ± 5% (n = 3). Rauwolscine (10 µM) shifted the dose-response curve of clonidine to the right (EC50 = 40 ± 16 µM, maximal inhibition of
70 ± 8%; n = 2), demonstrating that the effect
of clonidine was obviously mediated by the activation of
2A-adrenoceptors (Fig. 8B).
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Fig. 8.
Inhibition of forskolin-stimulated cAMP production in
HT29 cells. Inhibition of forskolin-stimulated cAMP production in HT29
cells by benazoline (A) or clonidine (B). A, cells were incubated with
increasing concentrations of benazoline only () or in the presence
of 10 µM rauwolscine (). B, cells were incubated with increasing
concentrations of clonidine only () or in the presence of 10 µM
rauwolscine (). Results from two to five experiments performed in
triplicate were averaged and are expressed as percentage of control (in
the presence of forskolin only) ± S.E. , nonlinear
least-squares fit, determined as described under Experimental
Procedures. Basal cAMP production was 49.6 ± 7.9 fmol/min/105 cells; forskolin-stimulated cAMP production
was of 3526 ± 1200 fmol/min/105 cells.
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The 2-adrenoceptors are coupled to
PTX-sensitive G proteins. When NG10815 cells were pretreated with PTX
(200 ng/ml) for 24 h, the dose-response curve of benazoline was
shifted to the left [EC50 = 25 µM without PTX
(n = 5) and 2.8 µM with PTX (n = 3)]
without change of the maximal cAMP decrease (maximal inhibition with
PTX pretreatment, 57 ± 5%), suggesting that this effect did not
depend on Gi/o activation (Fig.
9A). On the other hand, similar PTX
pretreatment markedly affected the effect of the
2-adrenoceptor agonist clonidine in these
cells (Fig. 9A). When PC12 cells were pretreated with PTX (200 ng/ml)
during 24 h, the dose-response curve of benazoline was not
significantly changed (Fig. 9B), confirming that this effect was
independent of Gi/o protein activation.
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Fig. 9.
Effect of imidazolines on forskolin-stimulated cAMP
production after PTX pretreatment of the cells. Inhibition of
forskolin-stimulated cAMP production by benazoline and clonidine
without (, ) or with (, ) PTX (200 ng/ml during 24 h)
pretreatment in NG10815 cells (A) or in PC12 cells (B). Results are
from three experiments performed in triplicate and expressed as the
percentage of control values (with forskolin only). In NG10815 cells,
basal values of cAMP were 22 ± 7 fmol/min/105 cells
and 74 ± 33 fmol/min/105 cells for control and
PTX-treated cells, respectively, and forskolin-stimulated cAMP values
were 1900 ± 640 fmol/min/105 cells and 1900 ± 780 fmol/min/105 cells for control and PTX-treated cells,
respectively. In PC12 cells, basal values of cAMP were 126 ± 23 fmol/min/105 cells and 113 ± 27 fmoles/min/105 cells for control and PTX-treated cells,
respectively, and forskolin-stimulated cAMP values 1130 ± 73 fmol/min/105 cells and 940 ± 126 fmol/min/105 cells for control and PTX-treated cells,
respectively.
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To preclude a direct effect of benazoline on PDE enzymes, experiments
were conducted on purified PDE isoforms. Table
1 shows that in contrast with their
respective specific inhibitors, the various PDE isoforms were not
significantly activated or inhibited by 100 µM benazoline. Similar
results were obtained with 10 µM benazoline (data not shown).
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TABLE 1
Effect of 100 µM benazoline on purified PDE isoforms
Experiments have been conducted as described under Experimental
Procedures. The results are given in percentage inhibition
compared with the inhibitory effects (EC50) of the specific
inhibitors of each PDE isoform. Results are the mean of three
experiments. The standard error was <15%.
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It has been shown (Separovic et al., 1996, 1997) that moxonidine
activated a PC-PLC in the PC12 cells, leading to an increase of DAG and
phosphocholine levels and that these effects involved activation of
I1 receptors, because they were blocked by
efaroxan. We aimed to determine whether this transduction pathway was
also associated to the I1 receptors in the cell
line used in the present study. In fact, similar results were obtained
as moxonidine (1 µM) increased (after 1 min of stimulation) the
phosphocholine content of the cells (37 ± 8% above control,
n = 4), although efaroxan and BDF6143 proved inactive
(Fig. 10). Moreover, benazoline (1 µM) significantly increased the phosphocholine level in these cells
(57 ± 23% above control, n = 4), suggesting
that, like moxonidine, it behaved as an agonist on this pathway (Fig.
10).
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Fig. 10.
Effect of imidazolines on the accumulation of
phosphocholine in the PC12 cells. Cells were incubated with drugs
(106 M) for 1 min. The results were determined from four
experiments, each performed with separate cell cultures, and are shown
as values of fluorescence (excitation at 560 nm and emission detection
at 590 nm) red after 30-min development of the enzymatic reaction as
described under Experimental Procedures.
*P < .05 by nonparametric paired Mann-Whitney
U test; significantly different from vehicle-treated
control. 1, basal; 2, moxonidine; 3, efaroxan; 4, BDF6143; 5, benazoline.
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Discussion |
The aim of this study was to get further insight into the
transduction pathway(s) of I1 receptors. We took
advantage of the availability of a newly described selective
imidazoline receptor ligand, benazoline, to reexamine the influence of
imidazolines on the cAMP pathway in cells. Regunathan et al. (1991)
clearly showed that clonidine, although able to bind to
I1 imidazoline receptors and to
2-adrenoceptors, was unable to modulate cAMP levels in bovine adrenal chromaffin cells expressing
I1 receptors without coexpression of
2-adrenoceptors. Nevertheless, we examined the
effect of benazoline in PC12 cells (derived from a rat adrenal pheochromocytoma) and in two other cell lines, the NG10815 and HT29
cells on this pathway. In addition, we tested the capability of other
imidazolines (including clonidine) known to interact with
I1 binding sites to modulate the cAMP pathway in
these cells. All the results presented here confirm the hypothesis that
benazoline can negatively modulate the forskolin-stimulated cAMP
accumulation through the activation of I1
imidazoline receptors and that clonidine behaves in this pathway as an antagonist.
Imidazoline and Adrenergic Receptors Expressed by the Different
Cell Lines Used in This Study.
We have shown for the first time in
this study that HT29 cells, expressing the
2A-subtype of human adrenoceptors only (Bylund et al., 1988) did not express simultaneously the
I1 subtype of imidazoline binding sites labeled
either by tritiated clonidine or iodinated PIC. It was previously
reported that no I2 imidazoline binding sites
could be detected in these cells with
[3H]idazoxan (Cantiello and Lanier, 1989).
Therefore, this cell line represents a uniquely suitable model to study
the 2A-adrenoceptors in the absence of the two
subtypes of imidazoline receptors.
The second cell line used in this study, the NG10815 cells, expressed
2B-adrenoceptors (Bylund et al.,1988),
I1 receptors, and I2
binding sites as demonstrated by binding studies using [3H]rauwolscine,
[3H]clonidine in the presence of 10 µM
rauwolscine, and [3H]idazoxan, respectively, as
the radioligands (Greney et al., 1999).
PC12 cells were described previously as cells lacking
2-adrenoceptors in binding assays using
p-[125I]iodoclonidine (Separovic et
al., 1996) and in hybridization studies with adrenoceptor cDNA probes
(Duzic and Lanier, 1992). We confirmed that it was also the case in the
PC12 cell line used in this study, because rauwolscine, an
2-adrenoceptor antagonist, did not displace
the p-[125I]iodoclonidine-specific
binding with high affinity. On the other hand, imidazolines proved able
to completely displace
p-[125I]iodoclonidine binding with
high affinities. A high affinity for I1
imidazoline receptors of PIC was described in the bovine brainstem
(Heemskerk et al. 1998) and in the human platelets (Piletz and Sletten,
1993). The KD and
Bmax values determined in our study for PIC
were in close agreement with those described previously by Separovic et
al. (1996) in the same cell line. The imidazoline binding sites
detected in the PC12 cells were clearly different from
I2 binding sites, because clonidine, moxonidine,
efaroxan, and BDF6143 displayed high affinities in this model, as was
the case for I1 receptors in human platelets
(Piletz and Sletten, 1993) and in bovine chromaffin cells (Molderings
et al., 1993), although they were only weak ligands for
I2 binding sites (Bricca et al., 1993; Piletz and
Sletten, 1993). On the other hand, no specific high-affinity
[3H]idazoxan I2 binding
sites could be detected in the PC12 cells (Steffen et al., 1995). Thus
PC12 cells represent an interesting model with which to study the
I1 receptors in the absence of
2-adrenoceptors and of
I2 binding sites. The existence of cell lines
expressing 2-adrenoceptors in the absence of
imidazoline receptors (HT29 cells) on the one hand and
I1 imidazoline receptors in the absence of
2-adrenoceptors (PC12 cells) on the other hand
definitely confirms that these receptors are different molecular entities.
We also confirmed that benazoline is a ligand that is highly selective
for imidazoline receptors over
2-adrenoceptors, as shown previously (Pigini
et al., 1997). In addition, competition curves obtained on
I1 receptor bindings with benazoline, moxonidine, and efaroxan were best resolved in two compartments, one displaying a
high affinity and the other a low affinity for these drugs. Resolution
of I1 binding sites in two compartments were also
shown in human platelets (Piletz et al., 1996) and in bovine chromaffin cells (Molderings et al., 1993) defined either by
p-[125I]iodoclonidine binding or by
[3H]clonidine binding, respectively. However,
our results differed from those of Separovic et al. (1996) in that they
detected only the first high-affinity binding site for moxonidine and
efaroxan in their PC12 cells. This discrepancy could be attributable to the fact that we used whole-cell membrane preparations in contrast with
the purified plasma membranes in their binding studies.
I1 Receptors Are Coupled to the cAMP Pathway.
Clonidine decreased forskolin-stimulated cAMP in the cell lines
expressing either 2A- or
2B-adrenoceptors (HT29 and NG10815 cells,
respectively) as described previously (Bouscarel et al., 1985; Convents
et al., 1989). In PC12 cells, which express I1 receptors able to bind clonidine (Ki = 125 ± 75 nM) but did not express
2-adrenoceptors, this drug had no influence on
the forskolin-stimulated cAMP accumulation. Similar results were
reported previously in the rat adrenal gland (Regunathan et al., 1990)
and bovine chromaffin cells (Regunathan et al., 1991).
In contrast, benazoline decreased forskolin-stimulated cAMP levels in
the cell lines expressing I1 receptors. These
effects can hardly be attributed to the stimulation of
2-adrenoceptors, which are negatively coupled
to adenylate cyclase through the activation of
Gi/o proteins, because 1) rauwolscine, an
2-adrenoceptor antagonist, was unable to
antagonize the effects of benazoline, although it antagonized those of
clonidine in NG10815 cells and in HT29 cells; and 2) pretreatment of
cells by PTX, which inhibited Gi/o proteins by
ADP-ribosylation, had no effect on cAMP decrease elicited by
benazoline, although such a pretreatment abolished the activity of the
2-adrenoceptor agonist clonidine; and,
finally, 3) similar effects of benazoline could be recorded in NG10815 cells, which express 2-adrenoceptors, and in
PC12 cells, which do not.
In addition, benazoline is a ligand selective for imidazoline
receptors, and its ability to dose dependently decrease the forskolin-stimulated cAMP level was observed only in cell lines expressing I1 receptors. In fact, in cells
lacking the I1 receptors (HT29 cells), such a
decrease was never observed. It was tempting, therefore, to propose
that benazoline acted through the activation of
I1 receptors. This hypothesis was further
confirmed by four lines of evidence: 1) other ligands (moxonidine,
efaroxan, and BDF6143), shown to be I1-selective
ligands looked like benazoline; 2) clonidine (a high-affinity
I1 receptor ligand), which was devoid of any
effect by itself, behaved as an antagonist in the PC12 cells; 3)
benazoline activity took place in I1 receptors
containing membrane preparations; and 4) benazoline acted as an agonist
on the PC-PLC pathway described as an I1 receptor
transduction mechanism (Separovic et al.,1996, 1997). In addition, the
EC50 values recorded for the drugs on cAMP
accumulation fit with the IC50 values obtained for the high-affinity binding sites (at least in the PC12 cells).
Another transduction pathway for the I1 receptors
in the PC12 cells has been proposed previously (Separovic et al., 1996, 1997). These authors clearly showed that moxonidine increased the DAG
and phosphocholine levels in the cells by activation of a PC-PLC. We
confirmed these results in the cell line used in our laboratory,
because moxonidine increased the level of phosphocholine, which was
formed by hydrolysis of PC through activation of PC-PLC. Although we
cannot completely exclude an activation of a PC-PLD by benazoline (see
Experimental Procedures), benazoline behaved like moxonidine
in these experiments. These results are strongly in favor of the
association of two transduction pathways with the
I1 receptors (again, at least in the PC12 cells).
Interestingly, in the case of the I1 receptor,
although benazoline and moxonidine were agonists for both pathways,
some agonists for the cAMP pathway (efaroxan) had antagonist effects on
the PC-PLC pathway (Separovic et al., 1996). Recently, similar data
were reported for the 
3-adrenoceptor (Gerhardt et al., 1999). Our
data open a new field of investigations aiming to determine the
existence of a cross talk between the different transduction pathways
and to identify their respective contributions to the physiological
roles of the I1 receptors.
We further characterized the cAMP transduction pathway associated with
the I1 receptors in the PC12 cells with
benazoline. One of the questions addressed was the coupling of the
I1 receptors to G proteins. It has been shown
that I1 binding sites were sensitive to
nonhydrolyzable analogs of GTP (Ernsberger and Shen, 1997). We
confirmed these data and showed that benazoline behaved as a G
protein-coupled receptor agonist in binding assays. In addition, benazoline also decreased cAMP in membrane preparations, suggesting that the cAMP signaling passed through the activation of an inhibitory G protein insensitive to PTX (Ho and Wong, 1998). Further work is
needed to explore this hypothesis.
An intracellular target for imidazolines implicated in the cAMP pathway
in whole cells might also exist. Benazoline and efaroxan decreased the
cAMP level in whole PC12 cells with biphasic dose-response curves,
although in membrane preparations, the second low-affinity compartment
was lost. We showed that this putative intracellular target could not
be the PDE enzymes. One explanation for the biphasic dose- response
curves obtained with some ligands might be the existence of a cross
talk between the cAMP and PC-PLC pathways using the intracellular
machinery, leading to complex effects in whole cells. Alternatively,
the increase of intracellular Ca2+ observed with
high concentrations of benazoline might be involved in the second part
of the dose- response curves. Additional work is required to understand
these mechanisms.
In conclusion, we showed in this study that some ligands selective for
I1 receptors decreased forskolin-stimulated cAMP
content in cells expressing these receptors and that clonidine proved able to antagonize these effects. The present results strongly support
the hypothesis according to which I1 receptors
might be negatively linked with the cAMP pathway and that such a
pathway coexists with the activation of a PC-PLC. Although a decrease of the cAMP level in cells can also be achieved by activation of
2-adrenoceptors, we clearly demonstrated that
the two receptors are different molecular entities acting through
different mechanisms and in different cell lines. Our work may open new
perspectives for the understanding of the transduction mechanism(s) and
the physiological implications of the I1 receptors.
We are grateful to Dr. B. Bucher (UMR Centre National de
la Recherche Scientifique 7519, Strasbourg, France), who provided us
with the specific anti-cAMP antibodies.
DAG, diacylglycerol;
PC, phosphatidyl choline;
PL, phospholipase;
PTX, pertussis toxin;
DMEM, Dulbecco's modified
Eagle's medium;
FBS, fetal bovine serum;
PIC, paraiodoclonidine;
PDE, phosphodiesterase;
PIPES, 1,4-piperazinediethanesulfonic acid;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
[Ca2+]i, intracellular free calcium
concentration.
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Abstract
Introduction
