From the
Imidazoline binding sites (IBS) were proposed to be responsible
for some of the pharmacological and therapeutic activities of
imidazoline and related compounds and have been classified into two
subtypes, I
In the present study, by combining biochemical and
molecular biology approaches, we show that 1) microsequencing of
I
These results show definitely that I
The pharmacological and therapeutic effects of several
imidazoline and guanidinium derivatives, such as clonidine, guanabenz,
and idazoxan, have been related to their specific interaction with
The
identification of the functional activity of I
Hydroxylapatite-agarose chromatography was
performed in a 1
Size exclusion
chromatography was performed on a Superose 12 fast protein liquid
chromatography column. Fractions from hydroxylapatite-agarose column
displaying [
The I
To investigate the involvement of
I
The molecular
relationship between I
We thank C. Bouchier and her colleagues from
Généthon (France) for their contribution in sequence
analysis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
BS and I
BS. Convergent studies
attribute a role in central blood pressure regulation to the
I
BS. In contrast, the function of I
BS remains
unknown.
BS purified from rabbit kidney mitochondria allowed the
recovery of four peptide sequence stretches displaying up to 85.7%
similarity with human, rat, and bovine monoamine oxidases (MAO)-A and
-B; 2) I
BS and MAO displayed identical biophysical
characteristics as their activities, measured by
[
H]idazoxan binding and
[
C]tyramine oxidation, respectively, could not
be separated using various chromatographic procedures; and 3)
heterologous expression of human placenta MAO-A and human liver MAO-B
in yeast, inherently devoid of I
BS and MAO activities, led
to the co-expression of [
H]idazoxan binding sites
displaying ligand-recognition properties typical of I
BS.
BS is located on
both MAO-A and -B. The fact that I
BS ligands inhibited MAO
activity independently of the interaction with the catalytic region
suggests that I
BS might be a previously unknown MAO
regulatory site.
-adrenergic receptors
(1, 2, 3) . However, during the last 10 years,
several studies showed that some of these effects, including the
centrally mediated decrease in blood pressure
(4) and the
proconvulsant
(5) and the anxiogenic-like
(6) activities, were partially independent of the stimulation of
-adrenergic receptors. This suggested the involvement
of putative imidazoline receptors in mediating the
``nonadrenergic'' activities of the imidazoline and
guanidinium derivatives. The existence of imidazoline binding sites
(IBS)
(
)
has been further supported by numerous
binding and autoradiography studies showing that three imidazoline
-adrenergic tritiated ligands, clonidine,
p-aminoclonidine, and idazoxan, label binding sites that
display a high affinity for imidazoline derivatives and are not
recognized by catecholamines
(7, 8, 9, 10) . To date, two major
subclasses of IBS have been identified based on their high
(I
) or low (I
) affinity for clonidine
(11) . Convergent studies
(4, 12) attribute a
role in blood pressure regulation to the I
BS. In contrast,
the function of I
BS remains largely unknown.
BS has been
hindered mostly by the lack of a purified endogenous ligand as well as
by the difficulty to define the agonist or antagonist properties of
their synthetic ligands. To address the functional role of
I
BS we used an alternative approach consisting of the
characterization of their subcellular localization and structural
properties. By this strategy, we showed that I
BS is a
60-kDa protein
(13) located in the outer membrane of
mitochondria
(14) and selectively regulated by K
and H
in vitro (13) .
Thereafter, the major mitochondrial localization of I
BS has
been confirmed in different tissues and species
(15, 16) . By comparing the structural and regulatory
properties of I
BS with those of other outer membrane
mitochondrial proteins, we observed that I
BS share with
monoamine oxidases (MAO), enzymes metabolizing endogenous active
substances ( i.e. adrenaline, noradrenaline, serotonin, and
dopamine) and exogenous amines, the same molecular weight, subcellular
localization, and ion regulation. This prompted us to investigate the
structural and functional relationships between I
BS and
MAO. In the present study, we demonstrate that I
BS is
located on both MAO-A and MAO-B. In addition, the fact that
I
BS ligands inhibited MAO activity independently of the
interaction with the catalytic region suggests that I
BS
might be involved in the regulation of the enzyme activity.
Materials
[H]Idazoxan
(40-60 Ci/mmol) was obtained from Amersham International.
[
C]Tyramine (50-60 mCi/mmol) was from
DuPont NEN. [
C]Phenylethylamine was purchased
from Isotopchim (Ganagobie, France). (±)-Idazoxan was supplied
by Dr. Malen from I.D.R. Servier (Suresnes, France). Cirazoline was a
gift from Synthelabo (Paris, France). Clonidine was obtained from
Boehringer-Ingelheim (Ridgefield, CT). Rauwolscine was purchased from
Roth (Karlsruhe, Germany). Moxonidine was a gift from Laboratoire
Therapeutique Moderne. Hydroxylapatite-agarose Ultrogel was obtained
from I.B.F. (Paris, France). Superose-12 and phenyl-Sepharose 4B gels
were purchased from Pharmacia Biotech Europe (St. Quentin en Yvelynes,
France). All remaining drugs were obtained from Sigma (Paris, France).
Microsequencing
The IBS was purified
from rabbit kidney mitochondrial membranes to apparent homogeneity by
using sequential chromatofocusing and hydroxylapatite chromatographies
(13) . The purified fraction was further analyzed by
SDS-polyacrylamide gel electrophoresis and transferred onto a
polyvinylidene difluroride membrane. The 60-kDa band corresponding to
I
BS was detected by staining with Amido Black, excised, and
subjected to on-membrane digestion with trypsin. The generated peptides
were separated by narrow-bore reversed-phase high performance liquid
chromatography, as described previously
(17, 18) .
Eluting peptides were collected manually and stored at
20 °C
until further processing. Amino acid sequencing was carried out with an
Applied Biosystems Sequencer (model 477A), on-line connected with a
phenylthiohydantoin amino acid analyzer (model 120A).
Yeast Transformation
The Saccharomyces
cerevisiae W303 cells ( MAT
a, leu2, his3, ura3,
ade2-1, can, cyr+) were
transformed by pYeDP60 plasmid (control yeast), or the expression
plasmids pHMAOAV60 and pHMAOBV60 constituted, respectively, by human
placental MAO-A and human liver MAO-B open reading frames cloned into
pYeDP60 expression plasmid, as described previously
(19) .
Spheroplasts, obtained by enzymatic digestion of yeast cell walls, and
crude mitochondrial fractions were prepared as previously described
(20) .
Chromatographic Procedures
Preparations and
solubilization of mitochondrial membranes from rabbit renal cortex were
performed as described previously
(13) . Hydrophobic
chromatography was carried out in a glass chromatography column (1
30 cm) filled with 25 ml of phenyl-Sepharose 4-B gel and
equilibrated with 10 column volumes of the following buffer: 0.1%
digitonin, 300 mM NaCl, 50 mM Tris-HCl, pH 7.4 at 20
°C. Thirteen ml of solubilized extract were diluted in 10 volumes
of a buffer containing 300 mM NaCl, 50 mM Tris-HCl,
pH 7.4, at 20 °C to adjust the digitonin concentration at 0.1%.
Solubilized material was applied to the phenyl-Sepharose 4-B column at
a constant flow rate of 0.5 ml/min, at 20 °C. The column was then
washed with 3 volumes of buffer containing 0.1% digitonin, 50
mM sodium phosphate, pH 7.4. Elution was performed by applying
a linear gradient (0-2 mM) of CHAPS, and 1-2-ml
fractions were collected.
30-cm chromatography column filled with 25 ml
of hydroxylapatite-agarose-Ultrogel. The gel was equilibrated with 10
column volumes of buffer A containing 0.1% digitonin, 100 mM
NaCl, 50 mM Tris-HCl, pH 7.4, at 20 °C. Thirty ml of
solubilized material (protein concentration, 1-2 mg/ml) were
applied to the column at a constant flow rate of 0.5 ml/min, at 20
°C. Washing and elution were performed as described previously
(13) . Fractions of 0.8 ml were collected and tested for sodium
phosphate concentration. Prior to MAO and I
BS activities
assays, eluted fractions from hydroxylapatite-agarose columns were
desalted by centrifugation on UNISEP Ultracent-30 (Bio-Rad) to adjust
the sodium phosphate concentration at 10 mM.
H]idazoxan binding and MAO activities
were pooled, desalted, concentrated to 200 µl, and applied to the
Superose 12 column. The elution was performed using buffer A at a flow
rate of 0.3 ml/min. Fractions of 0.5 ml were collected.
Binding Studies
Mitochondrial membranes from
rabbit renal cortex or transformed yeast cells (50 µg of protein)
were incubated at 20 °C for 45 min in a final volume of 250 µl
of 50 mM Tris-HCl, pH 7.4, 2 mM EGTA, 1 mM
MgCl, unless otherwise mentioned, with the required
concentrations of [
H]idazoxan in the presence of
10
M rauwolscine to mask any
-adrenergic receptor. Nonspecific binding was defined
in the presence of 1 µM cirazoline. At the end of the
incubation, bound radioligand was separated from free by vacuum
filtration as described previously
(13) . Filters were placed in
5 ml of Ready-Safe scintillation fluid (Beckman), and the radioactivity
was counted in a liquid scintillation spectrometer (Packard, model
Tri-Carb 300) at 48% efficiency. [
H]Idazoxan
binding on solubilized extracts was performed in the same experimental
conditions except that incubation was ended by precipitation with
bovine
-globulin/polyethylene glycol followed by vacuum filtration
over glass fiber filters (Whatman GF/B). Binding data were analyzed
using a nonlinear least-square curve fitting procedure
(PRISM
, GraphPAD Software, San Diego, CA).
Monoamine Oxidase Activity Measurement
Membranes
(50 µg of protein) or solubilized extracts were incubated at 37
°C for 20 min, unless otherwise mentioned, in a final volume of 250
µl of sodium phosphate buffer, 50 mM, pH 7.5, with the
required concentrations of [C]tyramine or
[
C]phenylethylamine. Pargyline (10
M) was used to define monoamine oxidase-specific
activity. The reaction was ended by addition of 1 ml of HCl, 2
N, 4 °C. The reaction product was extracted by addition of
2 ml of ethyl acetate/toluene (v/v), and the radioactivity contained in
the organic phase was counted in a liquid scintillation spectrometer at
97% efficiency. Steady-state kinetic parameters were calculated using a
nonlinear least-square curve fitting procedure (PRISM
).
BS was purified from rabbit kidney
mitochondrial membranes to apparent homogeneity by using sequential
chromatofocusing and hydroxylapatite chromatographies
(13) .
Microsequencing of selected tryptic peptides allowed for the recovery
of four peptide sequence stretches. Amino acid sequences of such
peptides displayed 50-85.7% similarity with human, rat, and
bovine MAO-A and MAO-B (Fig. 1). These data, along with other
studies showing that I
BS and MAO are co-localized in
different areas of rabbit and human brain
(20, 21) ,
further supported the hypothesis of structural relationship between
I
BS and MAOs.
Figure 1:
Partial amino
acid sequences of rabbit IBS: comparison with the sequences
of human, rat, and bovine MAOs. Partial sequences of purified
I
BS were obtained by microsequencing, as described under
``Experimental Procedures.'' Amino acid sequence portions of
human, rat, and bovine MAO-A and MAO-B were retrieved from the
SwissProt data base and compared with I
BS partial sequences
using the FASTA search program. Percentages of identity are given in
parentheses. Identical residues are indicated by
``:'' and similar residues by ``.'' marks,
respectively. X, nonidentified amino acid
residues.
To address this issue, we combined
biochemical and molecular biology approaches. First, we investigated
whether IBS and MAO could be discriminated through their
biophysical properties. Treatment of mitochondria from rabbit kidney
with increasing digitonin concentrations allowed a dose-dependent
solubilization of both I
BS and MAO activities. The
recoveries of [
H]idazoxan binding and
[
C]tyramine degradation rate at the different
digitonin concentrations were significantly correlated ( r = 0.987, p < 0.01), suggesting a similar
insertion of I
BS and MAO in the mitochondrial membrane
matrix. Using hydrophobic, hydroxylapatite, and size exclusion
chromatography, I
BS and MAO activities were systematically
eluted in the same fractions (Fig. 2), and the recovery of
[
H]idazoxan binding and
[
C]tyramine degrading activity were almost
identical after each chromatographic procedure (data not shown). These
data indicate that I
BS and MAO share structural properties
preventing their separation by purification procedures.
Figure 2:
Elution profiles of IBS and
MAO activities after separation of solubilized rabbit renal cortex
mitochondrial membranes by phenyl-Sepharose 4-B ( A),
hydroxylapatite-agarose ( B), and Superose 12 ( C)
chromatography. Chromatographic procedures were performed as described
under ``Experimental Procedures.'' For each chromatography,
the elution profile was recorded by a UV detector set at 280 nm.
Fractions were tested for I
BS and MAO activities in a final
volume of 125 µl. The I
BS density (
) was measured
by binding studies with 20 nM
[
H]idazoxan using 10
M cirazoline to define nonspecific binding. MAO activity
(
) was defined by measuring the specific oxidation of 1
mM [
C]tyramine (isotopic dilution,
1:100) for 20 min, in presence or absence of 10
M pargyline. Solid lines, optical
density.
To assess
whether IBS and MAO are a single entity or two co-purified,
tightly associated proteins, we transformed yeast with recombinant cDNA
encoding for human placenta MAO-A or human liver MAO-B
(22) .
Then, we verified if I
BS could be co-expressed with the
enzyme activity. As shown in Fig. 3, I
BS and MAO were
not expressed in wild-type and control plasmid transformed yeast. In
contrast, expression of MAO-A or MAO-B gave rise simultaneously to
[
H]idazoxan binding in yeast mitochondria.
[
H]Idazoxan binding was saturable, of high
affinity and reversible. Indeed, Scatchard analysis of saturation
experiments showed that [
H]idazoxan interacted
with a homogeneous population of high affinity binding sites in yeast
cells expressing MAO-A ( B
1.81 ± 0.41
pmol/mg of protein, K
94.4 ± 13.3
nM) or MAO-B ( B
1.39 ± 0.13
pmol/mg of protein, K
25.7 ± 1.7
nM). In addition, [
H]idazoxan-specific
binding was completely dissociated after the addition of a large excess
of cold ligand to the mitochondrial preparations ( t
= 2.32 min). The [
H]idazoxan binding
sites co-expressed with MAO-A and MAO-B display ligand recognition
properties consistent with those described for I
BS. Indeed,
competition studies showed that, in yeast expressing MAO-A or MAO-B,
[
H]idazoxan binding was inhibited in a
dose-dependent manner and with similar affinities by the
I
BS ligands cirazoline ( K
5.08 ± 1.73 nM; 2.97 ± 1.09 nM
for MAO-A and -B, respectively) and guanabenz
( K
35.2 ± 9.5 nM; 41.8
± 8.2 nM for MAO-A and -B) and poorly affected by
clonidine or moxonidine, selective ligands for I
BS, and by
the
antagonist rauwolscine ( K
> 10 µM). Taken together, these data clearly show
that I
BS is located on both MAO-A and MAO-B.
Figure 3:
Expression of MAOs and IBS in
yeast cells. I
BS density and MAO activity in wild-type,
control, and transformed yeast cells expressing MAO-A ( upper
panel) or MAO-B ( lower panel). I
BS densities
( white bars) and MAO activities ( black bars) in
mitochondrial membranes were evaluated on 50 µg of protein by
saturation studies of [
H]idazoxan binding (using
[
H]idazoxan concentration from 1 to 150
nM) and by applying the radiochemical assay with
[
C]tyramine for MAO-A ( upper panel) or
[
C]phenylethylamine for MAO-B ( lower
panel), respectively. Results were the mean ± S.E. of four
experiments.
Next, to
define the localization of IBS with respect to the binding
sites of classical MAO inhibitors, we tested the effect of clorgyline,
deprenyl, pargyline, Ro 411049, and Ro 196327 on
[
H]idazoxan binding. In yeast expressing MAO-A or
MAO-B, MAO inhibitors failed to inhibit
[
H]idazoxan binding at concentration up to 5
µM. These findings, in addition to previous results
obtained from [
H]idazoxan binding studies in
rabbit cerebral
(20) and renal cortex
(
)
and rat cerebral cortex
(23) , indicate that
I
BS are not associated to MAO regions interacting with
classical MAO inhibitors.
BS in the regulation of MAO activity, we studied the
effect of imidazoline and related compounds on MAO-dependent
[
C]tyramine oxidation in mitochondria from
rabbit kidney and transformed yeast cells. As observed for
[
H]idazoxan binding, MAO activity was inhibited
dose-dependently by cirazoline and guanabenz but not by clonidine,
moxonidine, or rauwolscine (Fig. 4 A). The mechanism of
MAO inhibition by cirazoline and guanabenz was distinct from the
``suicide inhibition'' previously reported for various MAO
inhibitors
(24) . Indeed, MAO activity was fully recovered after
removing of cirazoline or guanabenz by extensive membrane washing (98
± 2% versus control). To determine the type of MAO
inhibition by imidazoline derivatives, we studied the effect of
cirazoline on [
C]tyramine oxidation in
mitochondria from rabbit kidney. The double reciprocal plot of MAO
velocity versus substrate concentration curve showed that
cirazoline acts as a noncompetitive MAO inhibitor as it decreased
V
and did not affect K
(Fig. 4 B). Thus, inhibition of MAO activity by
imidazoline derivatives occurs by a mechanism distinct from the direct
interaction with the MAO catalytic site. Comparison of results from
radioligand and enzyme assays showed that inhibition constants of
imidazoline derivatives were lower for
[
H]idazoxan binding to I
BS than for
MAO activities. Multiple factors may account for discrepancy. First, in
competition studies, we found that [
H]idazoxan
binding to mitochondrial I
BS was inhibited by the MAO
substrates tyramine, serotonin, and phenylethylamine, at concentrations
close to the lowest required for the measure of MAO activity
(Fig. 5). Thus, competition of ligand binding to I
BS
by the MAO substrates may be responsible, in part, for the decrease in
the potency of imidazoline derivatives to inhibit the enzyme activity.
Second, previous studies have shown that I
BS and MAO
activities are regulated by different factors including K
(25) and H
(13)
,
protein(s) and various endogenous ligands isolated in the central
nervous system and in the periphery
(13, 24, 26, 27) . It is conceivable
that the presence or the absence of one of these regulatory factors
could modulate the effects of I
BS on MAO activity. This is
particularly true considering that I
BS ligands bind to a
MAO regulatory domain and do not interact directly with the catalytic
site. Finally, two observations indicate that the high affinity state
of I
BS may be associated to a small population of MAOs: 1)
in different human tissues and transformed yeast cells, the density of
I
BS is much lower than the number of MAO molecules
(14, 15, 22, 28, 29) and 2)
within the same tissue, various conformational states of MAOs can be
expressed depending on their assembling in the mitochondrial membranes
and their interaction with membrane or cytosolic factors
(30) .
Thus, the effects of I
BS ligands on a subpopulation of MAOs
may not be precisely quantified by the classical enzyme assay, which
measures the total MAO activity. At present, further studies are
necessary to define the role of I
BS in the inhibition of
MAO activity. However, the involvement of I
BS in the
regulation of MAO activity is supported by biochemical and functional
studies: first, the order of potency for inhibition of
[
H]idazoxan binding and MAO activities are
identical; second, both ligand binding to I
BS and
inhibition of MAO activity by imidazoline derivatives are fully
reversible processes; finally, I
BS ligands, independently
of their interaction with
-adrenergic receptors,
regulate centrally mediated activities where MAO are implied
(5, 6, 31) .
Figure 4:
A, inhibition of MAO activity by
imidazoline and related compounds. Mitochondrial membranes (50 µg)
from yeast expressing MAO-A ( left panel) or from rabbit kidney
( right panel) were incubated in 50 mM sodium
phosphate buffer, pH 7.5, for 20 min at 37 °C, in presence of
increasing concentrations of cirazoline (), guanabenz (
),
clonidine (
), moxonidine (
), or rauwolscine (
). MAO
activity was tested in presence of [
C]tyramine
at K values around 150-200 µM. The results
shown are the average of duplicate determination and are representative
of three to four separate experiments. B, double reciprocal
plot of MAO velocity versus substrate concentration curve in
the presence or in absence of cirazoline. Rabbit kidney mitochondrial
membranes (50 µg) were incubated in 50 mM sodium phosphate
buffer, pH 7.5, for 20 min at 37 °C, in the absence (
) or in
the presence of 10
(
) or 10
M (
) cirazoline prior to enzyme assay. MAO
activity was tested in presence of 5-1000 µM
[
C]tyramine. Pargyline (10
M) was used to define monoamine oxidase specific
activity. (Control values: V
= 7.3
± 1.5 nmol/min/mg of protein; K = 119.9 ±
12.5 µM). The figure is representative of four independent
experiments.
Figure 5:
Inhibition of
[H]idazoxan binding by the MAO substrate
tyramine, serotonin, and phenylethylamine. Mitochondrial membranes from
rabbit kidney were incubated with [
H]idazoxan
(1.5 nM) and increasing concentrations of tyramine, serotonin,
or phenylethylamine. The plot is representative of four separate
experiments.
In conclusion, our results
definitively show that IBS is located on both MAO-A and
MAO-B. Indirect evidence for a relationship between I
BS and
MAOs has been previously suggested by other studies showing that their
density increases simultaneously in human brain in the elderly
(21) and Alzheimer's disease
(32) , and the
I
BS density is down-regulated by rat chronic treatment with
MAO inhibitors clorgyline and pargyline
(23) .
BS and MAOs being clearly defined,
our results should contribute to characterize definitively the
functional activity of I
BS and to identify a novel
mechanism of MAO regulation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.