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Vol. 56, Issue 5, 858-866, November 1999
1a-
and 1b-Adrenergic Receptor Subtypes
Institute of Pharmacology and Toxicology, Université de Lausanne, Lausanne, Switzerland (O.R., L.A., S.C.); Department of Chemistry, Università di Modena e Reggio Emilia, Modena, Italy (F.F.); and Pharmaceutical R&D Division, Recordati S.p.A., Milan, Italy (A.L.)
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Summary |
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 Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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We have characterized the pharmacological antagonism, i.e., neutral
antagonism or inverse agonism, displayed by a number of 
-blockers at
two 1-adrenergic receptor (AR) subtypes, 1a- and 1b-AR. Constitutively activating mutations were
introduced into the 1a-AR at the position homologous to
A293 of the 1b-AR where activating mutations were
previously described. Twenty-four -blockers differing in their
chemical structures were initially tested for their effect on the
agonist-independent inositol phosphate response mediated by the
constitutively active A271E and A293E mutants expressed in COS-7 cells.
A selected number of drugs also were tested for their effect on the
small, but measurable spontaneous activity of the wild-type
1a- and 1b-AR expressed in COS-7 cells. The results of our study demonstrate that a large number of
structurally different -blockers display profound negative efficacy
at both the 1a- and 1b-AR subtypes. For
other drugs, the negative efficacy varied at the different
constitutively active mutants. The most striking difference concerns a
group of N-arylpiperazines, including 8-[2-[4-(5-chloro-2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5] decane-7,9-dione (REC 15/3039), REC 15/2739, and REC
15/3011, which are inverse agonists with profound negative efficacy at the wild-type 1b-AR, but not at the
1a-AR.
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Introduction |
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Top
Abstract
 Introduction
 Experimental Procedures
Results and Discussion
References
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Adrenergic
receptors (ARs) mediate the functional effects of epinephrine and
norepinephrine by coupling to several of the major signaling pathways
modulated by guanine nucleotide regulatory proteins (G proteins). The
AR family includes nine different gene products: three 
(1, 2,
3), three 2 (2A, 2B, 2C), and three 1 (1a, 1b, 1d) receptor
subtypes. Like all G protein-coupled receptors (GPCR), the ARs share
seven hydrophobic regions that form a transmembrane -helical bundle
and are connected by alternating intracellular and extracellular
hydrophilic loops. Mutational analysis of the ARs has revealed that the
-helical bundle contributes to form the ligand binding site of the
receptor, whereas amino acid sequences of the intracellular regions
appear to mediate the interaction of the receptor with G proteins as
well as with different signaling and regulatory proteins (Wess, 1997
).
Both selective and nonselective antagonists for different AR subtypes
are widely used in a variety of pathological conditions, including
hypertension, heart failure, and prostate hypertrophy as well as in
mental diseases such as depression. Several studies have demonstrated
that -blockers can behave either as neutral antagonists or inverse
agonists at the wild-type 2-AR or at a constitutively active 2-AR
mutant (Samama et al., 1993b; Chidiac et al., 1994). However, inverse
agonism at other AR subtypes has been less extensively investigated. It
has been previously reported that a small range of -blockers could
inhibit the agonist-independent phospholipase C as well as
phospholipase D responses mediated by constitutively active mutants of
the 1b-AR (Lee et al., 1997). A recent study
demonstrated that some -blockers can inhibit the spontaneous
activity of the 1d-AR subtype (García-Sáinz and Torres-Padilla, 1999).
The main aim of this study was to characterize the pharmacological
antagonism, i.e., neutral antagonism or inverse agonism, displayed by a
number of -blockers at two 1-AR subtypes, 1a- and
1b-AR. To achieve this goal, constitutively activating
mutations were first introduced into the 1a-AR at the
position homologous to A293 of the 1b-AR where
activating mutations were previously described (Kjelsberg et al.,
1992). Several ligands were then screened for their effect on the
agonist-independent activity of both the wild-type 1a-
and 1b-ARs and their constitutively active mutants. Our
study provides a number of findings that might represent a solid basis
to further elucidate the activation process of the 1a-
and 1b-AR subtypes, and the mechanism of action of drugs
acting at these receptors as well as their structure-activity relationships.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Mutagenesis and Transfections.
The cDNA encoding human
1a-AR (Schwinn et al. 1995; cDNA was a kind gift from
Dr. J.P. Hieble, SmithKline Beecham, Van Nuys, CA) or hamster
1b-AR (Cotecchia et al., 1992) were mutated by polymerase chain reaction-mediated mutagenesis technique with Taq DNA polymerase. The mutated DNA fragments obtained were
digested with the appropriate enzymes and cloned into the expression
vector pRK-5 containing the wild-type 1a- or
1b-AR cDNA. Recombinant clones were isolated and
sequenced. COS-7 cells grown in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal bovine serum and gentamicin (100 µg/ml) were transfected with the diethylaminoethyl-dextran method.
The transfected DNA ranged between 0.5 and 3 µg/106 cells.
Ligand Binding.
Membranes derived from cells expressing the
1a-AR subtypes and their mutants were prepared as
previously described (Cotecchia et al., 1992). The binding was
performed at 25°C in 50 mM Tris-HCl (pH 7.4), 150 mM NCl, and 5 mM
EDTA. For saturation binding experiments of
[125I]iodo-2-[-(4-hydroxyphenyl)-ethyl-aminomethyl]tetralone
([I125]HEAT), the radioligand concentration
ranged from 12 to 400 pM (150-µl assay volume) and prazosin
(10
5 M) was used to determine nonspecific
binding. For saturation binding experiments of
[3H]prazosin, the radioligand concentration
ranged from 25 to 4400 pM (300-µl assay volume) and phentolamine
(104 M) was used to determine nonspecific
binding. In competition-binding experiments, the final concentrations
of [125I]HEAT and
[3H]prazosin were 80 and 400 pM, respectively.
In some competition-binding experiments, the concentration of
[125I]HEAT was 10 pM (500-µl assay volume).
Results of ligand binding experiments were analyzed with Prism 2.0 (GraphPAD Software, San Diego, CA).
Inositol Phosphate (IP) Measurement.
Transfected COS-7 cells
(0.15 × 106) seeded in 12-well plates were
labeled for 15 to 18 h with
myo-[3H]inositol (New England Nuclear, Boston,
MA) at 5 µCi/ml in inositol-free DMEM supplemented with 1%
fetal bovine serum. Cells were preincubated for 10 min in PBS
containing 20 mM LiCl and then treated for 45 to 100 min with different
ligands. Total IPs were extracted and separated as previously described
(Cotecchia et al., 1992).
Molecular Modeling of Ligands.
The protonated structures of
the ligands considered in this study were fully optimized by means of
semiempirical molecular orbital calculations (AM1) (Dewar et al., 1985)
with the MOPAC 6.0 (QCPE 455) program. QUANTA molecular modeling
package (release 96; Molecular Simulation Inc., Waltham, MA) was used
for building and analyzing the molecular structures.
Statistical Analysis. Statistical analysis was perfomed as indicated in the figure legends with Prism 2.0 (GraphPAD Software).
Materials.
COS-7 cells were obtained from American Type
Culture Collection (Rockville, MD). DMEM, gentamicin, fetal bovine
serum, and restriction enzymes were purchased from Life Technologies,
Inc. (Grand Island, NY). Taq polymerase was obtained from
Roche Laboratories (RotKruez, Switzerland).
[125I]HEAT,
[3H]prazosin, and
[3H]inositol were obtained from New England
Nuclear. (
)-Epinephrine and corynanthine were purchased from Sigma
Chemical Co. (St. Louis, MO). 5-Methylurapidil, prazosin, WB 4101, phentolamine, spiperone, S-(+)-niguldipine were obtained
from Research Biochemicals Inc. (Natick, MA). (+)-Cyclazosin and
()-cyclazosin were a gift from Dr. D. Giardinà (University of
Camerino, Camerino, Italy). Indoramin and AH11110A were a gift
from Dr. J.P. Hieble, (SmithKline Beecham), and BE 2254 was a gift from
Dr. D. Hoyer (Novartis, Basel, Switzerland). BMY 7378, WAY
100635, SNAP 5089, RS-17053, alfuzosin, terazosin, tamsulosin, REC
15/2739, REC 15/3039
(8-[2-[4-(5-chloro-2-methoxyphenyl)-1-piperazinyl-ethyl]-8-azaspiro[4,5]decane-7,9-dione), REC 15/2869, REC 15/3011, and REC 15/2615 were obtained from Recordati (Milano, Italy).
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Results and Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Activating Mutations of 1a- and 1b-AR
Subtypes.
One strategy to identify inverse agonists is to enhance
the basal activity of GPCR by introducing activating mutations and to
screen drugs for their ability to inhibit the agonist-independent activity of the constitutively active receptor mutants (CAM). We have
previously reported that in the 1b-AR mutations of A293 at the C-terminal end of its 3i loop with any amino acid enhanced the
constitutive activity of the receptor, and was highest when alanine was
substituted with lysine or glutamic acid (Kjelsberg et al., 1992). To
identify inverse agonists at both the 1a- and 1b-AR subtypes, we constructed CAMs of the
1a-AR by mutating A271 (homologous to A293 of the
1b-AR) to lysine or glutamic acid. As shown in Fig.
1, mutations of either A271 or A293
markedly enhanced the basal activty of 1a- and
1b-ARs, respectively, resulting in increased
agonist-independent accumulation of IPs. Saturation binding analysis of
[125I]HEAT or
[3H]prazosin indicated that the expression
levels of the wild-type and CAM receptors were good, ranging between
1.7 and 4.3 pmol/mg protein (Table 1).
Our findings support the notion previously suggested for other GPCRs
(Wess, 1997) that the C-terminal end of the 3i loop plays a crucial
role in the conformational switch underlying the transition between the
inactive (R) and active states (R*) of the 1a-AR
subtype.
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1a- and
1b-ARs. First, the agonist-independent activity of both
the wild-type 1b-AR and its CAMs was significantly
higher than that of the wild-type 1a-AR or its CAMs.
Second, for both the 1a-AR and its CAMs the
epinephrine-induced IP accumulation above basal was significantly
higher than that of the 1b-AR or its CAMs (Fig. 1). This
suggests that the agonist-occupied 1a-AR has greater efficacy in activating phospholipase C than the 1b-AR,
whereas its spontaneous or mutation-induced isomerization toward the R* is lower. Our findings are in agreement with those from a previous study (Theroux et al., 1996) describing the coupling efficiencies of
different 1-AR subtypes expressed in HEK 293 or SK-N-MC cells. In
that study, the agonist-induced IP response mediated by the 1a-AR was higher, whereas its agonist-independent
activity was lower compared with 1b-AR expressed at a
similar level.
Inhibition of Receptor-Mediated Basal IP Accumulation.
Twenty-four -blockers differing in their chemical structures were
tested for their effect on the basal activity of the constitutively active A271E and A293E mutants expressed in COS-7 cells (Fig. 2). All the ligands used in this study,
except REC 15/3039, were previously described for their structure,
binding affinity at recombinant as well as native 1-AR subtypes, and
some of their pharmacological effects in different tissues (Michel et
al., 1995; Giardinà et al., 1996; Leonardi et al., 1997; Testa et
al., 1997).
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-blockers displayed inverse
agonism as demonstrated by their ability to decrease the basal activity
of both CAMs. However, the various -blockers differed in their
negative efficacy and some of these differences depended on the 1-AR subtype.
Drugs with the highest negative efficacy (defined as 
70% inhibition
of the basal activity) at both CAMs included WAY 100635, WB 4101, all
the tested quinazolines (prazosin, terazosin, both (+)- and
()-cyclazosin, REC 15/2615, and alfuzosin), indoramin, corynanthine,
spiperone, and AH11110A (Fig. 2). For the other drugs, their negative
efficacy differed at the two CAMs.
The most striking difference concerned some
N-arylpiperazines that displayed modest negative efficacy
(e.g., 5-methylurapidil, BMY 7378, and REC 15/2869) or neutral
antagonism (e.g., REC 15/3039, REC 15/2739, and REC 15/3011) at the
A271E mutant. However, the negative efficacy of these compounds was
more pronounced at the A293E, resulting in at least 45% inhibition of
the receptor-mediated basal activity. For phentolamine, BE 2254, and
tamsulosin negative efficacy was also greater at the A293E than at the
A271E. In contrast, for S-(+)-niguldipine negative efficacy
was greater at the A293E than at the A271E mutant.
The concentration-dependence of the inhibitory effect was determined
for those ligands that displayed the most profound negative efficacy at
both the A293E and A271E receptors. The EC50
values of the compounds in inhibiting the basal activity of the CAMs (Table 2) were in the same order of
magnitude as their ligand-binding affinities (Table
3).
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-blockers observed on the CAMs
reflected their behavior at the wild-type receptors, a selected number
of drugs were tested for their effects in COS-7 cells expressing the
wild-type 1a and 1b-AR subtypes.
Overexpression of both 1-AR subtypes resulted in a small, but
measurable increase of agonist-independent accumulation of IP that was
greater for the 1b-AR than for the 1a-AR
(Fig. 3). As shown in Fig. 3, the effect
of the -blockers in cells expressing the wild-type 1-AR subtypes
displayed several similarities to that observed in cells expressing
their CAMs. First, the rank order of negative efficacy at the
1a-AR was similar to that observed at the A271E mutant, i.e., prazosin, indoramin, and WB 4101 > BE 2254 > 5-methylurapidil REC 15/2869 > REC 15/3039, REC 15/2739,
and REC 15/3011. Second, all drugs displayed pronounced negative
efficacy at the 1b-AR as previously observed for the
A293E mutant. Thus, the only ligands that did not display any inverse
agonism at the wild-type 1a-AR were REC 15/3039, REC
15/2739, and REC 15/3011, the first two being neutral, whereas REC
15/3011 displayed some nonsignificant degree of partial agonism.
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1b-AR (Fig. 3) than at its constitutively
active mutant A293E (Fig. 2). For few N-arylpiperazines,
including 5-methylurapidil and REC 15/2869 as well as for BE 2254 negative efficacy also seemed more pronounced at the wild-type
1a-AR (Fig. 3) than at its CAM A271E (Fig. 2). This
observation might find some explanation in the framework of the
allosteric ternary complex model describing GPCR activation (Samama et
al., 1993a). Activating mutations are suggested to increase the
isomerization constant (J) of the receptor allowing its transition from
the R to R* states, thus resulting in its high spontaneous activity.
Therefore, the negative effect of inverse agonists on receptor
isomerization might be larger for the wild-type receptor, which is
probably characterized by a very small J parameter, compared with its
CAM for which the value of J should be much greater. This might explain
why for compounds with some degree of negative efficacy, i.e.,
5-methylurapidil, some REC compounds, and BE 2254, inverse agonism was
more pronounced at the wild-type 1-AR subtypes than at their CAMs.
In contrast, the fact that the behavior of REC 15/3039 and REC 15/2739
was very similar at the wild-type 1a-AR compared with
its A271E mutant strongly suggests that their efficacy is truly close
to zero at the 1a-AR subtype.
Collectively, these findings identify a group of
N-arylpiperazines as -blockers that display the most
striking difference at the two 1-AR subtypes being inverse agonists
with profound negative efficacy at the wild-type 1b-AR,
but not at the 1a-AR. In particular, REC 15/3039, REC
15/2739, and REC 15/3011 are the first -blockers identified so far
that do not display inverse agonism at one of the 1-AR subtypes,
namely, the 1a.
Structure-Activity Relationships of Ligands.
A typical
-blocker contains aromatic moieties on each side of a protonated
nitrogen atom, one of these two moieties being closer to the protonated
nitrogen than the other. The protonated nitrogen atom of the ligands is
thought to interact with the conserved aspartate on helix 3 of the
receptor, i.e., D106 in the 1a-AR and D125 in the
1b-AR, according to a precise geometry dictated by a
directional charge-reinforced hydrogen bonding interaction (Cavalli et
al., 1996; De Benedetti et al., 1997). The geometry of the
electrostatic interaction between the protonated nitrogen of the ligand
and the aspartate of the receptor probably dictates the orientation of
the whole ligand molecule within the receptor binding site, thereby
generating a peculiar local perturbation that is transferred to the
receptor domains involved in G protein coupling. Thus, a conformational
link existing between the receptor binding site for various ligands and
its interaction site with the G protein might dictate the different
functional effects of ligands on distinct receptors.
1a-AR and
1b-AR differ in their "susceptibility" to inverse
agonism. Whereas various ligands displayed different effects on the
1a-AR, most of the ligands tested exerted a profound
inhibitory effect on the agonist-independent activity of the
1b-AR, independently from their structural features. This divergence might be related to the different configuration of the
binding site of the two 1-AR subtypes, as reported in our previous
molecular modeling studies (De Benedetti et al., 1997). One might
speculate that compared with 1a-AR, 1b-AR
has a larger number of "inhibitory sites", i.e., sites
mediating inverse agonist-induced receptor inactivation, or that
structurally different ligands may invariantly reach the "inhibitory
sites" because of the less flexible configuration of the receptor
binding site.
Molecular modeling of the ligands provided some interesting insight
into the structure-activity analysis of the -blockers (Fig. 5). For
simplicity, among all the -blockers used in this study (Fig.
4) only the models of those
representative of some best defined structural groups are shown in Fig.
4. The inhibitory effect observed for different ligands on the
constitutive activity of the 1a-AR and, to a lesser
extent, on that of the 1b-AR seems to be related to the
structure of a defined portion of the ligands. Our results suggest that
the geometry of the protonated nitrogen atom as well as that of the
molecular moiety closest to this nitrogen might be responsible for the
functional effect of the ligands tested (Fig. 5).
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1-AR subtypes is
observed. The quinazolines and corynanthine share a fixed distance
between the protonated nitrogen and the center of its closest
aromatic ring of ~2.8 and 3.4 Å, respectively (Fig. 5). Also the
angle between the plane of the charge reinforced hydrogen bond and
that of its closest aromatic ring is fixed in these compounds.
Second, for those ligands where the protonated nitrogen is almost in
the middle of a flexible alkylic chain and the positive charge is
almost equally distributed on two amine hydrogens, i.e., BE 2254, tamsulosin and RS-17053, the basal activity of the 1a-AR is only partially inhibited. Due to the flexibility of this class of
compounds, the distance between the protonated nitrogen and the center
of its closest aromatic ring may vary, reaching a maximum of >5 Å (Fig. 5). Thus, the peculiar functional behavior of the phenylalkylamines may be related, at least in part, to the fact that
the charge-reinforced hydrogen-bonding interaction with the receptor
can occur through either one or both amine protons. Thus, different
reciprocal orientations and distances between the charge-reinforced hydrogen bond and its closest aromatic ring are allowed during interaction with the receptor. The more pronounced inverse agonism of
WB 4101 with respect to the other phenylalkylamines may be partially
due to the fact that one of the two methylenic groups that separates
the protonated nitrogen atom from its closest aromatic moiety belongs
to a cycle, i.e., dioxane. This peculiarity may reduce the degrees of
freedom of the molecule in proximity of the protonated nitrogen,
decreasing the number of the allowed interacting modes with the receptor.
Third, the N-arylpiperazines show neutral antagonism at the
1a-AR, with the exception of WAY 100635. This class of
compounds is characterized by a fixed distance between the protonated
nitrogen and the center of its closest aromatic ring of ~5.7 Å (Fig.
5). Moreover, the angle between the plane of the charge reinforced hydrogen bond and the plane of its closest aromatic ring may vary, at
least to a small extent, in these compounds. The different behavior of
WAY 100635 may be due to the topology of the carbonylic oxygen and of
the pyridinic nitrogen. Conformational analysis showed that either one
of these two heteroatoms may alternately perform intramolecular
hydrogen bonds with the protonated nitrogen, thus stabilizing the
different local minima (results not shown). These peculiar
conformational properties may influence the docking mode of this ligand.
Forth, in S-(+)-niguldipine and SNAP 5089, the protonated
nitrogen is in proximity of two aromatic rings that lie in position 4 of the piperidinic ring. In these compounds, the distance between the
protonated nitrogen and the center of the aromatic rings in the
equatorial and axial position 4 are ~5.9 and 4.6 Å, respectively (Fig. 5). Probably, the phenyl ring in the axial position is partially responsible of the inhibitory effect of these two compounds.
Differently from all the other compounds considered in this study, the
inverse agonism of S-(+)-niguldipine and SNAP 5089 at the
1b-AR is less pronounced than at the
1a-AR.
In summary, the results of this preliminary structure-activity
relationship analysis suggest that the constitutive activity of the
1a-AR can be differently inhibited by the tested ligands in a manner that seems dependent on the structural feature of the
protonated nitrogen of the ligand and its distance from the closest
aromatic moiety. However, a more accurate structure-activity relationship analysiswould require testing a much larger number of
compounds for each structural group as well as the identification of
the docking sites of the 1a-AR and 1b-AR
for the various ligands.
Ligand Affinities at Wild-Type and CAM
Receptors
The allosteric ternary complex model of
receptor activation (Samama et al., 1993a) predicts that the transition
from the R to R* states of the receptor can be influenced by ligand
binding. The allosteric effect exerted by the ligand on the equilibrium between R and R* is given by the parameter , which is related to the
ligand efficacy. Whereas neutral antagonists ( = 1) have no
effect on this equilibrium, agonists ( > 1) and inverse
agonists ( < 1) will preferentially bind to R* and R,
respectively. Thus, activating mutations, which are suggested to
increase the stability of J for the conversion of R to R*, also will
change ligand binding affinity. CAMs are predicted to display increased
affinity for agonists and decreased affinity for inverse agonists
compared with wild-type GPCRs, whereas the affinity of neutral
antagonists should be similar (Samama et al., 1993b).
1a- and 1b-AR or their mutants A271E and
A293E, respectively. Because the unlabeled form of
[125I]HEAT is a partial inverse agonist
(indicated as BE 2254 in Fig. 2) and that of
[3H]prazosin is a full inverse agonist at both
receptor subtypes, the affinities of different ligands were measured
with both radioligands.
As shown in Table 1, the Kd values of
[125I]HEAT and
[3H]prazosin were not significantly different
at the wild-type 1a- and 1b-AR versus
their CAMs. The results of competition binding experiments with
[125I]HEAT indicated that the
Ki values of several -blockers were not
significantly different at the A271E or A293E mutants compared with
their respective wild-type receptors (Table 3). Similar findings were
obtained when the Ki values were measured
with [3H]prazosin as radioligand or with tracer
concentrations (~10 pM) of [125I]HEAT
(results not shown). In conclusion, our results indicate that
-blockers with different negative efficacies do not display significantly different binding affinities for the constitutively active forms of the 1a- and 1b-AR
subtypes.
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1a- and
1b-AR, and 0.5 and 0.09 µM for A271E and A293E,
respectively. The results are the mean of three to six independent
determinations that did not differ by >40%).
These results can find their interpretation in the framework of the
allosteric ternary complex model (Samama et al., 1993b). As predicted
by the model, activating mutations of both the 1a- and 1b-AR result in a large increase in affinity for the
full agonist epinephrine. However, changes in affinity for ligands with
different efficacy (defined by the parameter) depend on the level
of receptor isomerization (defined by the J parameter). As demonstrated
by the relationship between the difference in ligand affinity between
the CAM versus wild-type receptor and the isomerization constant J
of the receptor (Samama et al., 1993b), the mutation-induced change of
affinity can be much larger for full agonists ( > 1) than for
inverse agonists ( < 1), depending on the value of J. For
example, the affinity of isoproterenol for a constitutively active
mutant of the 2-AR was 25-fold higher, whereas
that of a full inverse agonist was 2-fold lower than for the wild-type
receptor (Samama et al., 1993b).
Computer simulations predict that if an activating mutation increases
the value of J by two orders of magnitude from 0.001 to 0.1, it can
increase the affinity of a full agonist ( = 1000) 100-fold
without changing the affinity of inverse agonists (Samama et al.,
1993b). In this context, our findings suggest that wild-type 1a-AR and 1b-AR might be characterized by
a very low isomerization level that is increased by the activating
mutations of A271 or A293 into glutamic acid, respectively. This is
consistent with the low spontaneous activity of both wild-type
receptors. However, the mutation-induced increase in the isomerization
constant J might not be sufficiently large to decrease the affinity of
the CAMs for the inverse agonists.
Our findings are also in agreement with those from a previous report
showing that the constitutive activation of the 1b-AR resulting from a single-residue mutation is not sufficient to decrease
the binding affinity of prazosin (Hwa et al., 1997). A decreased
affinity for prazosin was observed only when multiple activating
mutations were combined in the 1b-AR.
Conclusions.
Our findings suggest that CAMs carrying mutations
at the C-terminal end of the 3i loop represent a useful tool to
identify drugs that can behave as inverse agonists at wild-type
1a- and 1b-AR subtypes. This also might
be generalized to other GPCRs because mutations at the carboxyl end of
the 3i loop might alter the isomerization of the receptor to its active
forms without directly interfering with the docking process of the
ligands. However, our findings demonstrated that some partial inverse
agonists displayed more pronounced negative efficacy at wild-type
receptors than at their CAMs. This observation might be helpful to
compare results from studies in which inverse agonism has been
investigated in different experimental systems, i.e., on wild-type
receptors versus their CAMs.
-blockers, including all the tested
quinazolines are inverse agonists at both the 1a- and
1b-AR subtypes. In contrast, several
N-arylpiperazines displayed different properties at the two
1-AR subtypes being inverse agonists with profound negative efficacy
at the 1b-AR, but not at the 1a-AR. An
important finding of our study is that REC 15/3039, REC 15/2739, and
REC 15/3011 are the first -blockers identified so far that do not display inverse agonism at one of the 1-AR subtypes, namely, 1a.
The quinazolines, including prazosin, terazosin, and alfuzosin,
which are among the most commonly used -blockers, can display unwanted effects in vivo on the cardiovascular system such as orthostatic hypotension. In contrast, REC 15/2739, REC 15/2869, and REC
15/3011 are characterized by high selectivity for the urogenital
tissues and seem to have less pronounced effects on the cardiovascular
system in vivo (Testa et al., 1997). This was mainly demonstrated by
the fact that in anesthetized dogs at doses effective in inhibiting
norepinephrine-induced urethral contractions prazosin resulted in a
decrease in dyastolic blood pressure, whereas the REC compounds did
not. The pharmacological effects of REC 15/3039 in vivo or in different
tissues have not been investigated. In future studies, it will be
interesting to identify novel -blockers that are neutral antagonists
at one or more 1-AR subtypes and to assess whether these compounds,
including REC 3039, have fewer generalized cardiovascular effects
compared with full inverse agonists.
Inverse agonists of GPCRs might be of therapeutic use in pathological
conditions resulting from activating mutations of receptors (Milligan
et al., 1995). However, in most cases drugs are used to limit the
action of endogenous agonists and inverse agonists should have no
benefit over neutral antag-onists. The clinical effect of inverse
agonists could be relevant in tissues where GPCRs display high
constitutive activity. Unfortunately, because spontaneous activity of
wild-type receptors in vivo is difficult to assess, the clinical
implications for the use of inverse agonists versus neutral antagonists
remain a matter of debate. However, the distinction of receptor
blockers as inverse agonists versus neutral antagonists might allow a
better interpretation of the pharmacological effects of clinically used
drugs with respect to their mechanism of action and contribute to
assessing the therapeutic implications of inverse agonism.
The results of our work might provide an important contribution to
further elucidating the pharmacological effects of drugs acting at the
1-AR subtypes and to optimizing their therapeutic use. They also
provide useful information about the structure-activity relationships
of -blockers. In the future, mutagenesis studies aimed at
identifying the docking sites on the 1-AR subtypes for inverse
agonists and neutral antagonists might help in delineating receptor
domains crucially involved in the inhibition of receptor isomerization
and activation.
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Acknowledgments |
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We thank Drs. Tommaso Costa and Alexander Scheer for their
helpful comments and Dr. Peter Greasley for critically reading the
manuscript. We thank Dr. J.P. Hieble, (SmithKline Beecham) for
providing the cDNA encoding the human 1a-AR,
and Dr. D. Giardinà (University of Camerino, Italy) for providing
(+)-cyclazosin and ()-cyclazosin.
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Footnotes |
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Received April 21, 1999; Accepted July 28, 1999
This work was supported by the Fonds National Suisse de la Recherche Scientifique (Grant 31-51043.97) and by the European Community (Grant BMH4-CT97-2152).
Send reprint requests to: Susanna Cotecchia, M.D., Institut de Pharmacologie et de Toxicologie, 27, Rue du Bugnon, Faculté de Médecine, 1005 Lausanne, Switzerland. E-mail: susanna.cotecchia{at}ipharm.unil.ch
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Abbreviations |
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AR, adrenergic receptor;
GPCR, G
protein-coupled receptor;
DMEM, Dulbecco's modified Eagle's medium;
[125I]HEAT, [125I]iodo-2-[-(4-hydroxyphenyl)-ethyl-aminomethyl]tetralone;
IP, inositol phosphate;
CAM, constitutively active mutant;
R, inactive;
R*, active.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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