Directory UMM :Data Elmu:jurnal:B:Biological Psichatry:Vol48.Issue10.2000:

Imidazoline and a2A-Adrenoceptor Binding Sites in
Postmenopausal Women before and after Estrogen
Replacement Therapy
John E. Piletz and Uriel Halbreich
Background: Platelet a2A-adrenoceptors (a2AAR) and
imidazoline binding sites (subtype I1) have been proposed
as peripheral markers of brain stem receptors that mediate sympathetic outflow and are reported to be elevated in
major depression.
Methods: In our study, p[125I]-iodoclonidine was used to
assess platelet a2AAR and I1 binding sites in healthy
postmenopausal women (n 5 34) compared with healthy
women of reproductive age (n 5 26). Receptor determinations were repeated in 19 postmenopausal women
following 59 – 60 days of estrogen replacement therapy
(ERT; 0.1 mg estradiol transdermal patches).
Results: I1 binding sites were twofold higher in platelets
of postmenopausal women compared with women of reproduction age but were down-regulated (normalized)
after 59 – 60 days of ERT. All other binding parameters,
including platelet a2AAR density, were not different between groups nor were they changed after ERT. Platelet I1
densities after 59 – 60 days of ERT were positively correlated with plasma luteinizing hormone concentrations.
Conclusions: It is suggested that increased imidazoline
binding sites might be associated with mood and behavioral changes in postmenopausal women. Biol Psychiatry

2000;48:932–939 © 2000 Society of Biological Psychiatry
Key Words: Imidazoline receptors, a2 adrenoceptors,
monoamine oxidase, estrogen, luteinizing hormone, menopause, depression

Introduction

P

ostmenopausal women are at high risk for osteoporosis
(American Medical Association 1984) and cardiovascular disease (Stampfer et al 1991). Accordingly, estrogen
From the Department of Psychiatry and Human Behavior, and Departments of
Pharmacology and Physiology, University of Mississippi Medical Center,
Jackson (JEP) and Biobehavioral Program, Departments of Psychiatry and
Gynecology and Obstetrics, State University of New York, Buffalo (UH).
Address reprint requests to Uriel Halbreich, M.D., Professor of Psychiatry,
Research Professor of Gyn/OB, Director of Biobehavioral Research, SUNY
Clinical Center, Biobehavioral Program, Room BB 170, 462 Grider Street,
Buffalo NY 14215.
Received September 23, 1999; revised February 14, 2000; accepted February 15,
2000.

© 2000 Society of Biological Psychiatry

replacement therapy (ERT) has been indicated for the
prevention of osteoporosis and cardiovascular disease in
postmenopausal women. Menopausal status may also
influence cognitive problems (Yaffe et al 1998) and
vulnerability to depression (Halbreich 1997; Halbreich
and Lumley 1993). Some studies have suggested that ERT
might improve cognition (Haskell et al 1997; Sherwin,
1988) and stabilize mood (Ditkoff et al 1991; Klaiber et al
1997) in postmenopausal women.
Clonidine is a centrally active, partial agonist for
a2-adrenoceptors (a2AR) that elicits a number of cardiovascular (Parsons and Morledge 1970), hormonal (Siever
and Uhde 1984), and cognitive effects (Ammassari-Teule
et al 1991). Clonidine’s agonistic activities at a2AR can be
discriminated from its nonadrenergic activities by induction of a G-protein “coupled” state of a2AR, yet not all of
clonidine’s effects are mediated through adrenoceptors
(Piletz et al 1994). Specifically, clonidine has nanomolar
affinity for nonadrenergic I1-imidazoline binding sites (I1

sites: Ernsberger et al 1995a).
The molecular nature and function of I1 sites has
remained a subject of debate (Ernsberger and Haxhiu
1997) even while attempts to clone I1 sites are being
reported (Ivanov et al 1998b). In radioligand binding
assays, I1 sites can be distinguished from three subtypes of
a2AR (a2A, a2B, and a2CAR) by phenylephrine and
guanidine compounds, which, in contrast to all a2AR
subtypes, lack high affinities for I1 sites (Ernsberger et al
1995a). Prolonged hypotension is induced by microinjection of imidazoline compounds, such as clonidine, into the
rostroventral lateral medulla (RVLM) region of the brain
stem (Bousquet et al 1989), the potency of which correlates with affinities of these agents at I1 sites but not at any
a2AR subtypes (Ernsberger et al 1990). Based on pharmacologic studies, I1 sites in the RVLM were proposed to
reside on imidazoline receptors that regulate sympathetic
outflow (Bousquet et al 1989; Ernsberger et al 1990).
More recently, another subtype of I-sites (I2 sites) has been
identified as a modulatory domain within monoamine
oxidase enzymes (Raddatz et al 1997; Tesson et al 1995).
The latter finding has raised the possibility that imidazo0006-3223/00/$20.00
PII S0006-3223(00)00849-0

Imidazoline Binding, Menopause, and Estrogen

lines might also mediate sympathetic outflow via the
metabolism of catecholamines in the brainstem.
There is also evidence that platelet I1 and a2AAR
clonidine-binding sites may be codysregulated in depression. This is based on reports (Piletz et al 1990, 1996a)
that I1 and a2AAR clonidine-binding sites are elevated in
depressed patients compared with healthy control subjects,
and that both sites are down-regulated following certain
types of antidepressant treatments (Piletz et al 1991,
1996b). Furthermore, platelet a2AAR and I1 sites appear to
possess identical binding properties as their counterparts
in the brain (Piletz and Sletten 1993). Elevations in brain
I1 and a2AAR clonidine-binding sites have also been
suggested (Callado et al 1998; Garcia-Sevilla et al 1996,
1999) in depressed suicide victims compared with suddendeath matched control subjects. Thus, high densities of I1
and a2AAR sites may be associated with depressed mood
(Garcia-Sevilla et al 1999).
Alpha2AR in uteri and brain have been reported to be

influenced by gonadal hormones and to regularly fluctuate
along the menstrual cycle (Bottari et al 1983; Orensanz et
al 1982; Roberts et al 1979). On the other hand, we have
found irregular fluctuations of platelet a2AAR and I1 sites
during the menstrual cycle in correlation with changes in
plasma epinephrine and norepinephrine levels (Piletz et al.
1998). Women with dysphoric premenstrual symptoms
also exhibited higher [3H]-para-aminoclonidine binding to
platelet I1 and a2AAR sites than control subjects, which
was especially pronounced during the symptomatic late
luteal phase of the disorder (Halbreich et al 1993).
To our knowledge, platelet a2AAR and I1 sites have not
previously been studied in postmenopausal women, and
the effects of ERT on these sites have not been reported.
Considering the possible role of these systems in the
modulation of blood pressure and mood, their influence by
menopause and ERT is of interest from both a clinical and
a heuristic perspective.

Methods and Materials

Subjects
Thirty-four postmenopausal women (aged 52.6 6 3.4 SD; range
45–59 years) and 26 women of reproductive age (aged 36.1 6
6.5, range 22– 45 years) participated in the baseline study. Of the
postmenopausal women, 19 were retested for radioligand binding
on days 59 – 60 of ERT (aged 52.2 6 3.4 years). Six postmenopausal women completed ERT but were unable to be rescheduled
for blood drawing until 2–3 days after estrogen (E2) discontinuation (their posttreatment data will not be included here). Nine
postmenopausal women discontinued ERT prematurely or were
lost to follow-up.
All women of reproductive age as well as postmenopausal
women were recruited, evaluated, and treated, and their samples

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2000;48:932–939

933

were drawn and processed at the same site (Biobehavioral
Program, State University of New York at Buffalo).
Eligibility of subjects was determined according to several

criteria. Postmenopausal women had not experienced menstrual
cycles for at least 2 years. They were at least 1 year after
cessation of menopausal symptoms (including hot flashes, night
sweats, and irritation by vaginal dryness) but were not older than
60 years of age. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations were within range for
menopause, and no plasma progesterone was detected on at least
two occasions before the study began. These postmenopausal
women had no medical contraindications to ERT. The subjects
weighed within 20% of ideal body weight, were physically
healthy (including normal blood pressure and pulse), and met
research diagnostic criteria (Spitzer et al 1978) for “not currently
mentally ill” for at least 2 years before the study. Ten of the
enrolled women had lifetime histories of major depressive
disorder (MDD) and were therefore treated as a subgroup.
Women were excluded if they had any active physical illnesses
or other disorders necessitating chemical or somatic treatments
or had any Axis I diagnosis of the DSM-III-R (APA 1987) at the
time of testing. Postmenopausal subjects were compared with
women of reproductive status who had regular menstrual cycles
and otherwise met the same inclusion and exclusion criteria.

Women of reproductive age were also administered the Premenstrual Assessment Form (Halbreich et al 1982) and daily rating
forms (Endicott et al 1986) to exclude subjects with dysphoric
symptoms during their midfollicular phases or those with premenstrual syndrome. This study was approved by an institutional
review committee. All subjects signed consent forms before the
study.
Clinical procedures were as follows. Before enrollment, the
women underwent a structured clinical interview with the Schedule for Affective Disorders and Schizophrenia (Endicott and
Spitzer 1978), and research diagnostic criteria were derived
(Spitzer et al 1978). Postmenopausal women had their blood
drawn before and after 59 – 60 days of Estraderm (estradiol
transdermal system) treatment (0.1 mg). Control women of
reproductive age were tested during their midfollicular phase
(days 7–10 of the menstrual cycle). Subjects monitored mood
and behavior throughout the study with daily rating forms
(Endicott et al 1986) and were confirmed to be free of mood and
behavioral abnormalities. Subjects refrained from chronic medications for at least 2 weeks before baseline blood drawings
(candidates taking any chronic medications or recreational drugs
were excluded during the screening). Alcohol, aspirin, or other
over-the-counter pain medications were also excluded for at least
2 days before giving blood. Subjects had an overnight fast, and

caffeine and cigarettes were not permitted until after the blood
drawing. None of the patients or control subjects complained of
increased anxiety or craving because of these restrictions. Symptoms were reviewed on the same day as the blood drawing
according to the Schedule for Affective Disorders and Schizophrenia (Endicott et al 1981) and the daily rating forms (Endicott
et al 1986) to ensure no changes in mental status. The women
rested supine for at least 20 min before blood drawing, which
was between 9:00 AM and 11:00 AM. The first 2–3 mL of blood
was discarded, and then 90 mL of venous blood was collected

934

J.E. Piletz and U. Halbreich

BIOL PSYCHIATRY
2000;48:932–939

slowly through an 18-gauge needle into a heparinized syringe (19
units/mL blood). An additional 5 mL of blood was collected to
obtain plasma (stored at 270°C) for hormone determinations.
The laboratory personnel were unaware of the subject’s age,

diagnosis, or treatment (single blind).

Platelet Preparations
The preparation of intact platelets began according to a slight
modification of the method of Corash (1980). Platelets were then
washed four times in sterile Ca11/Mg11-free Hank’s balanced
salt solution, pelleted, resuspended in the same solution with 0.1
mmol/L phenyl methylsulfonylflouride, a protease inhibitor, and
snap frozen at 280°C. After thawing, sonication, and centrifugation at 4°C (Piletz et al 1990), the platelet particulates were
resuspended in ice-cold reaction buffer (designated washed
lysates; formula of reaction buffer given below). Washed lysates
were then layered over a discontinuous 14.5% and 34% sucrose
gradient and centrifuged at 105,000 g for 90 min at 4°C using a
Beckman (Allendale, NJ) SW-28 rotor in a Sorvall OTD60B
ultracentrifuge (Dupont, Wilmington, DE). The interface layer,
containing the plasma membranes, was diluted in 40 mL of
ice-cold water, pelleted, and resuspended in reaction buffer
(Piletz et al 1990). The concentration of protein was determined
using a Lowry-Biuret reagent kit from Sigma Chemical Co. (St.
Louis). Samples were stored at 280°C until use.

Binding Assays
Radioligand binding with p[125I]-iodoclonidine ([125I]PIC; New
England Nuclear, Boston) was according to our previous procedure (Ernsberger et al 1995b; Piletz et al 1996b). Specific a2AAR
binding was determined from “total binding” minus “nonadrenergic binding,” as displaced in the presence of 10 mmol/L
norepinephrine. With another aliquot, I1 binding sites were
measured under a mask of 10 mmol/L NE (Piletz et al 1996b).
Specific I1 binding was then determined from “nonadrenergic
binding” minus “total nonspecific binding,” by displacement in
the presence of additional 100 mmol/L moxonidine (kindly
contributed by Dr. Siegfried Schafer, Solvay Pharma, Hanover,
Germany). In most cases, the binding assays entailed seven
concentrations of radioligand per site (Piletz et al 1996b);
however, in some cases, a low protein yield permitted only 4 – 6
concentrations of radioligand per site. All measurements were
made in hextuplicate (i.e., three total and three nonspecific values
for each concentration). Reactions were initiated by adding
0.015– 0.03 mg plasma membrane proteins (up to 0.25 mL) to the
radioligand and incubating at 21°C. The reaction buffer was 5
mmol/L HEPES, 0.5 mmol/L MgCl2, 0.5 mmol/L EGTA (adjusted to pH 7.4 with 0.1 mmol/L NaOH); with 0.1 mmol/L
ascorbic acid added on the day of the binding assay. Trapped
plasma membranes were washed three times with ice-cold wash
buffer on thick glass fiber filters (Schleicher & Schuell #32,
Keene, NH), and radioactivity was counted for 10 min per
sample in a Cobra Gamma Counter (Packard Instruments,
Meriden, CT) at 80% efficiency. Individual Bmax and KD values
were quantified using LIGAND (McPherson 1985). Data were fit
to both one-site and two-site models, but a one-site model was
always preferred by an F test.

Hormonal concentrations were determined by radioimmunoassays (RIA) with commercially available kits. 17-Beta estradiol
(E2) was assayed (Diagnostic Products, Los Angeles) with 100ml
aliquots of plasma that were incubated at room temperature for 3
hours in an antibody-coated tube with 125I-free estradiol. Bound
estradiol was counted after decantation of the supernatant. The
intra- and interassay coefficients of variation (CV) ranged from
4.0% to 8.1%; the sensitivity limit was 8 pg/mL. Plasma
progesterone determinations (Diagnostic Products) followed procedures similar to those used for estradiol. The intra- and
inter-assay CV ranged from 6% to 10%; the sensitivity limit was
0.05 ng/ml. Plasma testosterone was assayed by a solid phase
RIA (Diagnostic Products) in an aliquot of 50 mL. Interassay CV
ranged from 9%–13%, and intraassay CV ranged from 4% to 6%.
The sensitivity limit for the testosterone assay was 0.04 ng/mL.
Plasma FSH and LH were also determined with RIA reagents
purchased from Diagnostic Products in 200-mL aliquots. The
interassay CV ranged from 4% to 6% (FSH) and 2% to 7% (LH),
and the intraassay CV ranged from 2% to 4% (FSH) and 1% to
2% (LH). The sensitivity limit for FSH was 0.06 mIU/mL and for
LH was 0.1 mIU/mL.

Statistical Analyses
Subject groups were compared with each other using nonpaired
Student’s t tests. Pre- to posttreatment values were compared
with paired t tests. Pearson’s regression tests were performed for
associations of radioligand binding values with hormone values.
All p values are two tailed. The data are expressed as means 6
standard deviations.

Results
As shown in Table 1 and Figure 1, platelet I1 density was
significantly higher (pre-ERT) in postmenopausal women
(n 5 34) compared with women of reproductive status
(n 5 26; I1 Bmax t 5 5.4, p , .0001). None of the
other platelet radioligand binding parameters were significantly different between postmenopausal women and
women of reproductive status (a2AAR Bmax t 5 0.38 p
5 .7; a2AAR KD t 5 0.68, p 5 0.5; I1 site KD t 5
1.17, p 5 .25). There was no correlation between the
ages of postmenopausal women and any KD or Bmax
parameters (pretreatment rs: 5 .04 to .28, posttreatment
rs 5 2.02 to 2.24).
Following treatment with E2 for 59 – 60 days, the I1
Bmax values of postmenopausal women (n 5 19) decreased 35% (t 5 3.32, p 5 .004) to near normal levels.
A comparison of the Bmax data in postmenopausal women
that completed treatment versus women of reproductive
status (menstrual) is shown in Figure 1. The a2AAR Bmax
values of the women who completed 59 – 60 days of E2
treatment did not change (t 5 0.22, p 5 .829), as was
the case with a2AAR KD (t 5 0.55, p 5 .59). Also, I1
KD values did not change in response to treatment with E2
(t 5 0.62, p 5 .55).

Imidazoline Binding, Menopause, and Estrogen

935

BIOL PSYCHIATRY
2000;48:932–939

Table 1. Platelets Imidazoline and a2AR Binding
Baseline
Subjects
All postmenopausal women
(n 5 34)
Subjects who completed 60 days of
E2 (n 5 19)
Reproductive age women (n 5 26)

I1 Bmax

I1 KD
a

177.6 6 89.3

a2 Bmax

Post-ERT
a2 KD

4.2 6 1.2 64.6 6 59.8 0.66 6 0.53

I1 Bmax

I1 KD

a2 Bmax

a2 KD

—

—

—

—

188.3 6 63.3b 4.1 6 1.2 76.6 6 64.0 0.72 6 0.59 122.7 6 58.1 4.4 6 1.7 80.7 6 53.7 0.62 6 0.37
84.6 6 30.5

4.6 6 1.6 60.0 6 31.9 0.55 6 0.61

—

—

—

—

a

Values represent mean 6 SD. E2, 17-beta estradiol. p , .0001 vs. women of reproductive age.
b
p , .0001 vs. women of reproductive age and p 5 .004 vs. post– estrogen replacement therapy.

Before ERT, postmenopausal women had plasma E2
concentrations of 7.4 6 6.8 pg/mL, progesterone concentrations of 0.18 6 0.13 ng/mL, LH concentrations of
81.4 6 10.0 mIU/mL, FSH concentrations of 102.6 6 52.1
mIU/mL, and testosterone concentrations of 0.24 6 0.12
pg/mL. After 59 – 60 days of estrogen treatment, plasma
E2 concentrations were 63.6 6 39.7 pg/mL (p , .0001
vs. baseline), plasma LH concentrations were 9.3 6 5.7
mIU/mL (p , .0001 vs. baseline), plasma FSH concentrations were 39.2 6 11.3 mIU/mL (p , .0001 vs.
baseline), and plasma testosterone concentrations were
0.31 6 0.40 pg/mL (ns). Progesterone concentrations after
ERT were below the sensitivity of the RIA for most
women. Baseline and posttreatment concentrations of E2
or other hormones did not correlate with each other (r 5
.03, p 5 .86 for E2). Plasma E2 concentrations after 30
days of ERT (61.6 6 36.0 pg/mL) did not differ from E2
concentrations after 59 – 60 days of ERT (t 5 0.42, p 5
.63).

At baseline (pre-ERT), none of the platelet receptor
parameters were significantly correlated with any of the

plasma hormone concentrations in postmenopausal
women (r values ranged from .1 to .45, ns).
Because ERT induced a down-regulation of platelet I1
sites (Table 1 and Figure 1), it was anticipated that E2
concentrations at post-ERT might be negatively correlated
with I1 Bmax values. In fact, the opposite was observed. A
positive correlation was found between posttreatment E2
concentrations and I1 Bmax values at the p 5 .04
significance level (r 5 .49, n 5 18); however,
pre-to-post treatment I1 Bmax change scores (the magnitude of decrease) were positively correlated with posttreatment E2 concentrations (r 5 .46, p 5 .04).
It was also anticipated that plasma LH concentrations,
FSH concentrations, or both might be positively correlated
with platelet I1 Bmax values at posttreatment. This was
because all three of these measures appeared to decrease
following ERT. In accord with this prediction, plasma
concentrations of LH after 59 – 60 days of ERT were
highly positively correlated with I1 Bmax values (r 5 .73,
p 5 .001, n 5 17) (Figure 2). Furthermore, the
pre-to-post treatment I1 Bmax change scores were negatively correlated with posttreatment LH concentrations
(r 5 2.48, p 5 .04). Thus, higher E2 and lower LH
concentrations after ERT were associated with higher

Figure 1. Effect of menopause and estrogen therapy (ERT) on
platelet a2-adrenergic and imidazoline-1 sites. [125I]PIC, p[125I]iodoclonidine; prot, protein.

Figure 2. Platelet imidazoline binding site and plasma luteinizing hormone following estrogen replacement therapy in postmenopausal women. prot, protein.

Correlations between Binding Sites and Hormones

936

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2000;48:932–939

change scores (i.e., more down-regulation) of I1 Bmax
values. No other significant correlation was observed
between any of the hormone concentrations and either I1
or a2AAR parameters.
In general, there was no association between blood
pressure or pulse rate with any of the platelet I1 or a2AAR
parameters (rs: .028 –.184). A negative correlation (r 5
2.4040, p 5 .037) was observed between platelet
a2AAR affinity (KD) and pulse rate at baseline, however.

Subgroup with Lifetime History of MDD
Postmenopausal women with lifetime histories of MDD
(n 5 10) tended to have higher I1 Bmax values than those
who were never mentally ill (n 5 24), but this did not
reach statistical significance (221 6 132 vs. 159 6 69
fmol/mg protein, t 5 1.62, p 5 .12). Similarly, I1 KD
values did not differ (4.1 6 1.0 vs. 4.2 6 1.2, t 5 .3, p 5
.77), nor did a2AAR KD values differ (0.42 6 0.40 versus
0.74 6 0.63, t 5 1.39, p 5 .18) in regard to lifetime
histories of MDD. Postmenopausal women with lifetime
histories of MDD differed in their a2AAR Bmax values at
baseline, however, which were high (73.8 6 61.2 vs.
30.1 6 16.6 fmol/mg protein, t 5 2.83, p 5 .01). The
two groups (with or without lifetime MDD) did not
significantly differ in baseline hormonal concentrations.
Following 59 – 60 days of treatment with Estraderm, the
women with past MDD (n 5 7) did not differ from those
without lifetime histories of MDD on any of the I1
parameters; however, after E2 treatment, the LH concentrations of women with histories of MDD decreased to
lower concentrations than did the LH of women who were
never mentally ill (3.9 6 2.4 vs. 11.1 6 5.6 ug/L, t 5
3.23, p 5 .008).

Discussion
We report two main findings. The first is a higher
radioligand binding density of I1 sites in platelets of
postmenopausal women compared with women of reproductive age (Table 1 and Figure 1). The second is
down-regulation of platelet I1 binding sites after 59 – 60
days of ERT (Table 1 and Figure 1). These findings are
distinguished from a2AAR sites on platelets, which were
not different in postmenopausal women compared with
women of reproductive age and were not affected by
ERT.
We suggest that the high density of platelet I1 binding
sites observed in postmenopausal women might relate to
altered hormonal status, rather than to age per se. This is
for the following reasons: 1) In our study, there were no
within-group correlations between ages and I1 or a2AAR
binding parameters. 2) In our five previous studies (Hal-

J.E. Piletz and U. Halbreich

breich et al 1993; Piletz et al 1990, 1996a, 1996b, 1998),
with more than 90 subjects of varying diagnoses (male and
female subjects, ages 20 – 65), no statistically significant
associations have been found between ages and densities
of platelet I1 or a2AAR sites. 3) Even considering only
those reports (Piletz et al 1996a; 1996b, 1998) that used an
identical binding assay and where a sizeable number of
healthy female control subjects were available (n 5 44),
we could find no association between female ages (23– 63
years) and densities of platelet I1 or a2AAR sites. To our
knowledge, no other reports exist on aging effects on these
platelet sites. Because no previous reports have even
hinted at an effect of age per se on these sites, the best
explanation for the present data seems to be the postmenopausal status of women and its related hormonal state.
I1 and a2AR sites are known to be differentially
distributed in the brain (DeVos et al 1994; Kamisaki et al
1990). I1 sites are almost completely absent in the brain
stem nucleus locus coeruleus, which contains a high
density of a2AAR (Ernsberger et al 1994). The frontal
cortex is another region of high density of a2AAR, but
where I1 sites are of relatively low density (DeVos et al
1994; Kamisaki et al 1990). On the other hand, I1 sites
have been extensively studied in the RVLM brainstem
nucleus (Bricca et al 1989; Ernsberger et al 1990), where
they are associated with the hypotensive action of centrally applied imidazoline compounds. I1 sites in the
RVLM might serve a physiologic role in the tonic and
reflex control of the sympathetic nervous system (Ernsberger and Haxhiu 1997). An endogenous clonidine displacement substance has also been characterized (Atlas
1991) and identified to contain agmatine, a decarboxylated
arginine metabolite (Li et al 1994). Thus, agmatine and the
imidazoline binding sites (I1 and I2) might compose a
neurotransmitter-receptor system. Although agmatine is
now realized to be only one of several molecules that
constitute clonidine displacement substance (Piletz et al
1995; Sun et al 1995), agmatine fulfills several criteria for
a neurotransmitter (Reis and Regunathan 1999).
We are unaware of any previous study on the influence
of steroids on imidazoline binding sites (either I1 or I2);
however, monoamine oxidase B (MAO-B) encodes an I2
site (Raddatz and Lanier 1997; Tesson et al 1995) and
platelet MAO-B activity levels have been studied in
response to estrogen (Chakravorty and Halbreich 1997;
Klaiber et al 1997). Thus, the down-regulation of platelet
I1 sites after ERT could be comparable to two previous
reports (Holsboer et al 1983; Klaiber et al 1972) showing
that estrogen therapy reduces plasma MAO activity in
postmenopausal women with depressed mood. Furthermore, platelet MAO-B levels have been reported to be
negatively correlated with serum estradiol levels during a
hormone replacement therapy (Klaiber et al 1997). Al-

Imidazoline Binding, Menopause, and Estrogen

though we did not measure platelet I2 sites in our study, it
is conceivable that the platelet I1 site is physically related
to I2 sites.
Our finding of no effect of ERT on platelet a2AAR sites
in postmenopausal women is in accord with a previous
study by Best and coworkers (Best et al 1992) in which
yohimbine binding to platelet a2AAR sites was unchanged
in postmenopausal women given an estradiol implant (100
mg) for 6 weeks. By contrast, another study reported
increased platelet a2AAR sites in human male volunteers
treated with E2 (U’Prichard and Snyder 1979), and three
earlier studies of female rabbits reported that estrogen
treatment decreases a2AAR sites in platelets (Elliott et al
1980; Mishra et al 1985; Roberts et al 1979). In our study,
and in the study by Best and colleagues (Best et al 1992),
estradiol was administered for a much longer time (42– 60
days) than in the human male study (4 days) or the rabbit
studies (6 –24 days). Therefore, the length of E2 treatment
and species and gender differences might be critical
factors.
We have also reported (Halbreich et al 1993) higher
platelet a2AAR and I1 sites in women with dysphoric
premenstrual syndrome compared with healthy control
women. In that study, elevations in binding were most
pronounced during the symptomatic late luteal phase of
the disorder when plasma norepinephrine and adrenaline
concentrations typically peak (Blum et al 1992). The
literature is uniformly negative for regular, cyclic fluctuations of platelet a2AAR or I1 sites along the human
menstrual cycle (Jones et al 1983; Piletz et al 1998;
Stowell et al 1988; Sundaresan et al 1985; Theodorou et al
1987). In certain brain regions and in uteri, however,
a2AAR sites may be influenced by gonadal hormones and
appear to fluctuate along the estrus cycle (Bottari et al
1983; Orensanz et al 1982).
One potential paradox was that post-ERT I1 Bmax values
were positively associated with plasma E2 concentrations.
This would not be expected if E2 directly down-regulated
I1 sites; however, plasma E2 concentrations were found to
be positively associated with the magnitude of change in I1
Bmax values, which is in line with an E2 down-regulation
effect. We have previously reported (Piletz et al 1998)
irregular fluctuations of platelet a2AAR and I1 sites over
two menstrual cycles in healthy women, which were not
correlated with phase of the cycle but were instead
correlated with plasma concentrations of adrenaline and
norepinephrine. We have also reported (Ivanov et al
1998a) that immunoreactive I-sites on human megakaryoblastoma cells are up-regulated after 6 hours of exposure
to norepinephrine in vitro. Similar treatments with estrogen in vitro were without effect (unpublished observations). Thus, the mechanisms of the down-regulation of
platelet I1 sites in women after ERT (Table 1) is still

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937

unclear. Future studies are needed to address whether
indirect changes in plasma catecholamines might underlie
platelet I1 changes.
Another interesting observation was that post-ERT I1
Bmax values were positively correlated with plasma LH
concentrations (Figure 2). Whether these are causally
linked (i.e., LH or the I1 site exerts a regulatory effect) or
whether LH and I1 sites are independently influenced by
E2 is open to speculation. The literature suggests no effect
of clonidine on LH release (Kaufman and Vermeulen
1989). Any effect of LH on I1 binding sites therefore is
unknown and will require additional studies (in the same
way that the possible effect of LH on mood and behavior
has not been sufficiently investigated).
It is also interesting to note that the abnormal I1 BMAX
values previously reported for depressed patients (Piletz et
al 1996b) are comparable in level to those of healthy
postmenopausal women (Table 1). Of course, exact platelet I1 BMAX values are notoriously difficult to duplicate
across studies for technical reasons (Ernsberger et al
1995b). Even slight variations in platelet preparation may
be critical (Corash 1980). In fact, all the platelets in our
current study were prepared at a different site (Buffalo)
than in our previous studies with depressed patients
(Cleveland). Nevertheless, the BMAX values in our postmenopausal women (177.6 6 89.3 fmol/mg protein, n 5
34) are surprisingly close to what we previously reported
for a group of middle-aged male and female depressed
patients (161 6 66.3 fmol/mg, n 5 26) (Piletz et al
1996b). I1 densities in postmenopausal women after ERT
(122.7 6 58.1 fmol/mg, n 5 19) are also surprisingly
close to what we previously reported for healthy middleaged male and female subjects (126 6 63.6 fmol/mg, n 5
18) (Piletz et al 1996b). Furthermore, the few postmenopausal women with lifetime histories of MDD had higher
BMAX values for a2AAR (statistically significant) and I1
sites (trend) compared with women without lifetime histories of MDD. If the platelet I1 site becomes validated as
a biological marker for depression (Piletz et al 1994), the
present findings in nondepressed postmenopausal women,
as well as the down-regulation by estrogen, will have to be
taken into account.
This work was supported in part by Grant Nos. RO1-MH-45901,
RO1-MH-49248, and RO1-MH-57601. We greatly appreciate the clinical
assistance provided by Judith Vedella, Ph.D., R.N., and Barbara Bernardis, B.S., R.N., the statistical analysis provided by Khalid Bibi, Ph.D.,
and the laboratory assistance of Jianhua Shen, M.D., Guojing Yuan, and
Tammy Stefan.

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