Methylation of 2-Hydroxyestradiol in Isolated Organs
Lefteris C. Zacharia, Raghvendra K. Dubey, Zaichuan Mi, Edwin K. Jackson
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Abstract—Vascular smooth muscle and glomerular mesangial cells in culture express a biochemical pathway that
methylates 2-hydroxyestradiol (17␤-estradiol metabolite) to produce 2-methoxyestradiol, a cell growth inhibitor that
may mediate the cardiorenal protective effects of 17␤-estradiol. Whether this pathway exists in intact organ systems is
currently unclear. Accordingly, the purpose of the present investigation was to characterize the methylation of
2-hydroxestradiol in intact organs from both male and female rats. No significant differences were detected in the ability
of male and female tissues to methylate 2-hydroxyestradiol. In isolated hearts, kidneys, and mesenteries perfused with
Tyrode’s solution, Km values for 2-hydroxyestradiol methylation were 0.175⫾0.021, 0.387⫾0.054, and 0.495⫾0.089
␮mol/L, respectively, and Vmax values were 21.0⫾1.58, 24.9⫾1.49, and 1.01⫾0.148 pmol 2-methoxyestradiol · min⫺1
· ml⫺1 per gram, respectively. The catalytic efficiency (Vmax/Km) was greatest in the heart compared with the kidney and
mesentery (132⫾14.3, 78.4⫾15.1, and 2.30⫾0.263 pmol 2-methoxyestradiol · min⫺1 · mL⫺1 · ␮mol/L⫺1 per gram,
respectively). In the kidney, the catechol-O-methyltransferase inhibitor quercetin and norepinephrine (10 ␮mol/L)
reduced methylation of 2-hydroxyestradiol by approximately 90% and 41%, respectively. Importantly, methylation in
the kidney was inhibited by an average of 16.6⫾1.80% by endogenous norepinephrine released by renal artery nerve
stimulation. Our results indicate that a robust 2-hydroxyestradiol methylation pathway exists in the kidney and heart, but
not in the mesentery, and that this pathway is mediated by catechol-O-methyltransferase. Our findings also suggest that
catecholamines may interfere with 2-hydroxyestradiol methylation and thereby attenuate the cardiorenal protective
effects of 17␤-estradiol. (Hypertension. 2003;42:82-87.)
Key Words: vascular diseases 䡲 coronary artery disease 䡲 sympathetic nervous system 䡲 renal disease 䡲 catecholamines
B
lood vessels are protected from disease and the progression of disease by 17␤-estradiol,1–3 but by not conjugated equine estrogens.4 –5 Apparently, the vascular protective
effects of 17␤-estradiol are mediated in part via the antiproliferative effects of 17␤-estradiol on vascular smooth muscle
cells.2,3
Our findings support the concept that the antiproliferative
effects of 17␤-estradiol on vascular smooth muscle cells are
not mediated by estrogen receptors, but rather are due to the
direct effects of metabolites of 17␤-estradiol on molecular
processes regulating cell proliferation.6 –9 In this regard, in
vascular smooth muscle cells, 17␤-estradiol is converted to
2-hydroxyestradiol (2OHE) by cytochrome P450s, and 2OHE
is methylated by catechol-O-methyltransferase (COMT) to
form 2-methoxyestadiol (2MeOE). The order of potency for
inhibition of vascular smooth muscle cell growth is 2MeOE
⬎2OHE ⬎17␤-estradiol, whereas the order of potency at
estrogen receptors is 17␤-estradiolⰇ2OHEⰇ2MeOE (essential inactive). Moreover, blockade of cytochrome P450s
inhibits the antiproliferative effects of 17␤-estradiol, but not
2OHE or 2MeOE, and blockade of COMT inhibits the
antiproliferative effects of both 17␤-estradiol and 2OHE, but
not 2MeOE. These findings support the conclusion that the
2OHE methylation pathway plays a key role in determining
the vascular protective effects of 17␤-estradiol and 2OHE.
Catecholamines are physiological substrates of COMT10
and therefore may inhibit the 2OHE methylation pathway. In
support of this hypothesis, increased synthesis of catecholamines under pathological conditions increases the risk of
vaso-occlusive and renal disorders.11
Considering the importance of the 2OHE methylation
pathway as indicated by our in vitro studies, the objectives of
this study were to (1) determine whether this metabolic
pathway exists in intact and physiologically relevant vascular
beds, specifically, the perfused kidney, heart (coronary circulation), and mesentery; (2) determine and compare the
kinetic parameters for the 2OHE methylation pathway in
these organs; and (3) examine whether exogenous and endogenous catecholamines block this pathway in the intact
kidney.
Materials and Methods
Animals
Male and female Sprague-Dawley rats (nⱖ5 for each group) obtained from Charles River (Wilmington, Mass) were housed at the
Received December 16, 2002; first decision January 14, 2003; revision accepted April 21, 2003.
From the Center for Clinical Pharmacology (L.C.Z., R.K.D., Z.M., E.K.J.), Departments of Medicine (L.C.Z., R.K.D., Z.M., E.K.J.) and Pharmacology
(E.K.J.), University of Pittsburgh Medical Center, Pittsburgh, Penn; and Clinic for Endocrinology, Department of Obstetrics and Gynecology, University
Hospital Zurich (R.K.D.), Zurich, Switzerland.
Correspondence to Dr Edwin K. Jackson, University of Pittsburgh Medical Center, Center for Clinical Pharmacology, 623 Scaife Hall, 3550 Terrace
Street, Pittsburgh, PA 15261. E-mail edj⫹@pitt.edu
© 2003 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000074702.06466.27
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2-Hydroxyestradiol Methylation Pathway
83
University of Pittsburgh Animal Facility and fed Prolab RMH 3000
(PMI Feed Inc). All studies received prior approval by the University
of Pittsburgh Animal Care and Use Committee. Animals were
anesthetized with an intraperitoneal injection of 45 mg/kg
pentobarbital.
Organ Perfusion
All organs were prepared and perfused at a rate of 5 mL/min and
equilibrated for 40 minutes with Tyrode’s solution, followed by 20
minutes with Tyrode’s solution containing 0.57 mmol/L ascorbic
acid. Ascorbic acid was essential to prevent oxidation of the
substrate 2OHE.12
Preparation of Kidneys for Perfusion
Rat kidneys from male and female rats were prepared for perfusion
as previously described.13
Figure 1. Line graphs showing the conversion of
2-hydroxyestradiol (2OHE; 0.1 to 3 ␮mol/L) to
2-methoxyestradiol (2MeOE) in the isolated perfused male (A)
and female (B) kidney, heart, and mesentery. After equilibration
as described in Methods, rat organs were perfused with 0.1,
0.3, 1, and 3 ␮mol/L 2OHE. For each concentration the organs
were perfused for 10 minutes for equilibration before collecting
perfusate for another 5 minutes (male n⫽6, female n⫽6).
Preparation of Hearts for Perfusion
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Rat hearts from male and female rats were perfused using the Langendorff method. The thoracic cavity was opened, and the heart was excised
and immediately placed in cold Tyrode’s solution (4°C). A
polyethylene-240 tube was placed in the aorta while the heart was in the
cold solution. The tube was secured in place, and the heart was
connected to a perfusion pump. A second polyethylene-240 tube was
placed in the left ventricle and secured in place to ensure that only
perfusate from the coronary circulation was collected.
Preparation of Mesenteries for Perfusion
Rat superior mesenteric vascular beds from male and female rats
were prepared for perfusion as previously described.14
Concentration-Dependent Metabolism of 2OHE
Organs were perfused with 0.3 to 10 ␮mol/L 2OHE in the
presence and absence of 10 ␮mol/L quercetin (a COMT inhibitor)
as appropriate. Kinetic parameters were calculated using GraphPad Prism 3.0.
Inhibition of 2OHE Methylation by
Exogenous Catecholamines
Kidneys were perfused with Tyrode’s solution containing 2OHE (0.3
␮mol/L for male, 0.05 ␮mol/L for female) and 0.57 mmol/L
ascorbic. Male kidneys were perfused in the presence and absence of
10 to 100 ␮mol/L norepinephrine or epinephrine, and female kidneys
were perfused in the presence and absence of 0.5 ␮mol/L epinephrine. The perfusate was collected and analyzed.
Inhibition of 2OHE Methylation by
Endogenous Catecholamines
An electrode was placed around the renal artery, and stimulation was
initiated at 7 Hz for a few seconds to ensure that the renal
sympathetic nerves were active. Activity was determined by measuring the change in perfusion pressure using a polygraph. The
kidney was perfused with Tyrode’s solution containing 2OHE (0.1
␮mol/L), a re-uptake inhibitor, desipramine (10 ␮mol/L), and a
monoamine oxidase inhibitor, pargyline (500 ␮mol/L), to allow
norepinephrine to diffuse in the kidney. Perfusate samples were then
collected in unstimulated kidneys (control) or kidneys stimulated at
12 Hz for 8 minutes.
Sample Preparation and High-Performance Liquid
Chromatography Analysis
Internal standard was added (16␣-hydroxyestradiol) to each sample
before processing. Samples were applied to a Waters™ Sep-Pak®
column (reverse phase; C18), and the column was washed with 5 mL
of 20% methanol. 2MeOE was eluted with 2 mL of 100% methanol.
The eluted samples were further concentrated under vacuum, and the
samples were extracted in methylene chloride 3 times. Samples were
dried under vacuum and redissolved in water and methanol, and
2MeOE was quantified as previously described.7
Statistics
All experiments were conducted with at least 5 subjects. Results are
presented as mean⫾SEM. Statistical analyses were performed using
ANOVA, paired Student t test, or Fisher least significant difference
test, as appropriate. A value of P⬍0.05 was considered statistically
significant.
Results
In both male and female rats, 2OHE, in a rapid transit pass
through the coronary and renal circulation, was methylated in
a concentration-dependent manner (0.1 to 3 ␮mol/L) (Figure
1). Concentrations of 2OHE as low as 0.1 ␮mol/L were
methylated in measurable amounts during a rapid pass
through the kidney and heart. In male and female kidneys, a
concentration of 0.1 ␮mol/L 2OHE produced an average of
0.881⫾0.052 and 1.12⫾0.236 nmol 2MeOE/5 min, respectively, and in male and female hearts, an average of
1.07⫾0.070 and 0.857⫾0.138 nmol 2MeOE/5 min, respectively. At a concentration of 3 ␮mol/L 2OHE, in male and
female kidneys, this amount increased to a maximum of
4.48⫾0.240 and 4.26⫾0.783 nmol 2MeOE/5 min, respectively, and in male and female hearts, this amount increased
to a maximum of 2.69⫾0.190 and 2.82⫾0.167 nmol
2MeOE/5 min, respectively.
The concentration-response data were employed to calculate the kinetic parameters for the methylation of 2OHE
(Figure 2). In the male heart, the apparent Km for 2OHE was
0.148⫾0.018 ␮mol/L versus 0.215⫾0.038 ␮mol/L in the
female heart (not significant). In the male heart, the Vmax was
19.2⫾1.73 pmol 2MeOE.min⫺1 · mL⫺1 per gram versus
23.8⫾2.62 pmol 2MeOE · min⫺1 · mL⫺1 per gram in the
female heart (not significant). In the male heart, the Vmax/Km
was 137⫾15.3 pmol 2MeOE · min⫺1 · mL⫺1 · ␮mol/L⫺1 per
gram versus 126⫾30 pmol 2MeOE · min⫺1 · mL⫺1 · ␮mol/L⫺1
per gram in the female heart (not significant).
In the male kidney, the Km was 0.407⫾0.028 ␮mol/L
versus 0.356⫾0.138 ␮mol/L in the female kidney (not
significant). The Vmax in the male kidney was 22.4⫾1.10
pmol 2MeOE · min⫺1 · mL⫺1 per gram versus 28.5⫾2.49
pmol 2MeOE · min⫺1 · mL⫺1 per gram in the female kidney
(small but significant difference). In the male kidney, the
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July 2003
Figure 2. Comparison of the apparent Km,
Vmax, and Vmax/Km ratio values for the metabolism of 2OHE in the 3 perfused organs in male
and female rats. Values represent mean⫾SEM
(male n⫽6, female n⫽5). *P⬍0.05 vs heart;
#P⬍0.05 vs mesentery.
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Vmax/Km was 56.2⫾4.30 pmol 2MeOE · min⫺1 · mL⫺1 ·
␮mol/L⫺1 per gram versus 112⫾32.3 pmol 2MeOE · min⫺1 ·
mL⫺1 · ␮mol/L⫺1 per gram in the female kidney (not
significant).
The mesenteric vascular bed had little ability to methylate
2OHE (Figure 1). At 0.1 ␮mol/L 2OHE, the amount of
2MeOE produced was only 0.104⫾0.009 and 0.179⫾0.03
nmol 2MeOE/5 min in the male and female, respectively. In
the male mesenteric vascular bed, the Km was 0.530⫾0.110
␮mol/L versus 0.438⫾0.167 ␮mol/L in the female mesenteric vascular bed (not significant). The Vmax in the male
mesentery was 1.19⫾0.19 pmol 2MeOE · min⫺1 · mL⫺1 per
gram versus 0.726⫾0.168 pmol 2MeOE · min⫺1 · mL⫺1 per
gram in the female mesentery (P⬍0.05 vs male and female
hearts and kidneys). The Vmax/Km in the male mesentery was
2.46⫾0.320 pmol 2MeOE · min⫺1 · mL⫺1 · ␮mol/L⫺1 per
gram versus 2.07⫾0.483 pmol 2MeOE · min⫺1 · mL⫺1 ·
␮mol/L⫺1 per gram in the female (P⬍0.05 vs male and
female hearts and kidneys).
When expressed as conversion per total organ, the kidney
had the greatest capacity to methylate 2OHE, with a Vmax of
41.0⫾2.70 and 36.4⫾7.27 pmol 2MeOE · min⫺1 · mL⫺1 in the
male and female, respectively, followed by the heart, which
had a Vmax of 25.2⫾2.60 and 24.3⫾1.51 pmol 2MeOE · min⫺1
· mL⫺1 in the male and female, respectively. The male and
female mesentery had a Vmax of 3.99⫾0.60 and 2.40⫾0.558
pmol 2MeOE · min⫺1 · mL⫺1.
Disregarding urinary excretion of 2MeOE and combining
both males and females, the efficiency of 2OHE methylation
was as high as 41% in the coronary circulation and 38% in the
kidney, with the lower concentration of 2OHE used (0.1
␮mol/L). At higher concentrations of 2OHE, the efficiency of
conversion was either the same in the heart and the kidney or
slightly higher in the kidney (Table). The percentage conversion of 2OHE to 2MeOE decreased with increasing concen-
trations of 2OHE. For example, in the heart at concentrations
of 0.3, 1, and 3 ␮mol/L 2OHE, the percentage conversion
was 28.8⫾2.79, 10.9⫾0.919, and 3.74⫾0.198, respectively,
and in the kidney at 0.3, 1, and 3 ␮mol/L 2OHE, the
percentage conversion was 28.9⫾2.16, 13.2⫾1.16, and
5.79⫾0.445, respectively. In the mesentery the percentage
conversion was very low compared with the percentage
conversion in the coronary circulation (heart) and kidney, and
even at 0.1 ␮mol/L of 2OHE, the percentage conversion was
only 5.17⫾0.610, about 7 times less than in the kidney and 8
times less than the heart. At 0.3, 1, and 3 ␮mol/L, the
percentage conversion in the mesentery was 2.83⫾0.315,
1.30⫾0.132, and 0.642⫾0.079, respectively.
Our previous in vitro experiments demonstrate that 2OHE
methylation is COMT-dependent. We therefore examined
whether in the male kidney 2OHE is mainly methylated via a
COMT-dependent pathway. Methylation of 2OHE was
concentration-dependent for 0.1 to 10 ␮mol/L 2OHE. At
these concentrations, methylation was blocked by quercetin
(10 ␮mol/L), a competitive inhibitor of COMT (Figure 3).
Quercetin blocked the methylation of 2OHE by 51.8⫾7.50%,
86.3⫾2.60%, 88.9⫾1.00%, 87.2⫾1.50%, and 84.8⫾1.30%
for the 0.1, 0.3, 1, 3, and10 ␮mol/L concentrations of 2OHE,
respectively (P⬍0.05).
Catecholamines are endogenous substrates of COMT and
could potentially inhibit the metabolism of 2OHE in the
kidney. We therefore examined in the male and female
kidney whether exogenous catecholamines block the 2OHE
methylation pathway in the kidney. Indeed, in the male
kidney, norepinephrine at concentrations of 10, 30, and 100
␮mol/L inhibited methylation of 0.3 ␮mol/L 2OHE by
40.9⫾2.40%, 47.8⫾4.40%, and 57.5⫾1.80%, respectively
Efficiency of Conversion of 2OHE to 2MeOE in the Combined
Male and Female Perfused Kidney
Concentration of
2OHE Infused
Heart
Kidney
Mesentery
0.1 ␮mol/L
40.6⫾2.90†
38.5⫾3.91†
0.3 ␮mol/L
28.8⫾2.79†
28.9⫾2.16†
2.83⫾0.315
1 ␮mol/L
10.9⫾0.919†
13.2⫾1.16†
1.30⫾0.132
3 ␮mol/L
3.74⫾0.198†
5.79⫾0.445†*
5.17⫾0.610
0.642⫾0.079
The efficiency of conversion of 2OHE to 2MeOE is expressed as a percentage
of infused substrate in the combined male and female perfused kidney and
heart, and in the mesentery at 0.1–3 ␮mol/L 2OHE. Data are mean⫾SEM
(n⫽10).
*P⬍0.05 vs heart; †P⬍0.05 vs mesentery.
Figure 3. Concentration-dependent metabolism of 2OHE (0.1 to
10 ␮mol/L) and inhibition by quercetin (10 ␮mol/L) in the perfused male kidney. After equilibration, rat kidneys were perfused
with Tyrode’s solution containing 0.1, 0.3, 1, 3, and 10 ␮mol/L
2OHE with or without 10 ␮mol/L quercetin. For each concentration, the kidney was perfused for 10 minutes for equilibration
before collecting perfusate for another 10 minutes. Values represent mean⫾SEM (n⫽6). *P⬍0.05 vs quercetin.
Zacharia et al
2-Hydroxyestradiol Methylation Pathway
85
minutes. In one experiment the inhibition was as high as 40%
during the first 2 minutes of nerve stimulation.
Discussion
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Figure 4. Bar graphs showing inhibition of 2OHE methylation by
norepinephrine (NE), and epinephrine (EPI) in perfused kidneys.
A, Inhibition of 0.3 ␮mol/L 2OHE methylation by NE and EPI in
male kidney. B, Inhibition of 0.05 ␮mol/L 2OHE methylation by
0.5 ␮mol/L EPI in the female kidney. After equilibration with
Tyrode’s solution, male kidneys were first perfused with
Tyrode’s solution containing 0.3 ␮mol/L 2OHE and 0.57 mmol/L
ascorbic acid for 20 minutes, and the last 10 minutes of perfusate was collected (baseline). The perfusion solution was then
switched to 0.3 ␮mol/L 2OHE and 0.57 mmol/L ascorbic acid,
with or without 10 ␮mol/L norepinephrine or epinephrine, and
the kidney was perfused for another 20 minutes, again collecting the last 10 minutes of perfusate. The same procedure was
repeated for the 30 and 100 ␮mol/L concentrations of EPI or
NE. Female kidneys were similarly perfused, but with 0.05
␮mol/L 2OHE and 0.5 ␮mol/L EPI. Values represent mean⫾SEM
(for n⫽5 to 6 observations). *P⬍0.05 vs treated.
(P⬍0.05). Epinephrine at the same concentrations had effects
similar to norepinephrine (Figure 4A). At 10, 30, and 100
␮mol/L epinephrine, methylation of 2OHE was inhibited by
52.4⫾5.03%, 53.9⫾8.00%, and 66.6⫾5.05%, respectively
(P⬍0.05). In the female kidney (Figure 4B), a concentration
of epinephrine of 0.5 ␮mol/L inhibited methylation of 0.05
␮mol/L 2OHE by 23.7⫾3.98% (P⬍0.05).
To examine the physiologically relevant effect of catecholamines as inhibitors of the 2OHE methylation pathway,
endogenous norepinephrine was released by electrical stimulation of the renal artery in the perfused male kidney.
Continuous stimulation of the nerves in the renal artery at 12
Hz inhibited methylation of 0.1 ␮mol/L 2OHE by an average
of 16.6⫾1.80% over the 8-minute period of stimulation
(Figure 5). The inhibition was persistent for at least 8
Figure 5. Inhibition of 2OHE (0.1 ␮mol/L) methylation in the perfused male kidney by electrical stimulation of renal artery nerve
at 12 Hz for 8 minutes (n⫽6). Values represent mean⫾SEM.
*P⬍0.05 vs stimulated. The kidney was perfused with Tyrode’s
solution containing 2OHE (0.1 ␮mol/L), desipramine (10 ␮mol/L),
and pargyline (500 ␮mol/L). For the control kidneys, after a
10-minute equilibration with 2OHE/desipramine/pargyline,
2-minute samples were collected for the duration of the experiments. For stimulated kidneys, after baseline sample collection,
the electrode was stimulated at a frequency of 12 Hz continuously and samples collected every 2 minutes for 8 minutes.
Cardiovascular and renal diseases are mediated in part by
inappropriate cell proliferation caused by growth stimuli such
as mechanical injury, oxidative stress, or pro-proliferative
biomolecules. Consequently, appropriate concentrations of
endogenous antimitogenic molecules to prevent cell proliferation are essential to attenuate the development and progression of cardiovascular and renal diseases.
2MeOE is a potent endogenous antimitogenic molecule
produced via methylation of 2OHE, thus rendering this
pathway an important growth regulatory mechanism. In this
regard, previous findings in our laboratory indicate that
glomerular mesangial, vascular smooth muscle, and endothelial cells (coronary and aortic) are capable of methylating
2OHE.8,15 Moreover, our data indicate that this pathway
inhibits cell proliferation and collagen synthesis and is
blocked by classical COMT inhibitors and catecholamines.6,7
In the present study, we examined the 2OHE methylation
pathway in the intact kidney, heart (coronary circulation), and
mesentery. Our results suggest that this pathway is a robust
and efficient process in the heart and kidney. Further, our
results indicate that there are no differences between male
and female with respect to the efficiency of the 2OHE
methylation pathway in the heart and kidney. In contrast to
the heart and kidney, the mesenteric vascular bed has little
capacity to convert 2OHE to 2MeOE.
At the lowest and more physiologically relevant 2OHE
concentration used (0.1 ␮mol/L), the percentage conversion
of 2OHE to 2MeOE was highest in the heart and kidney (41%
in heart vs 38% in kidney vs 5% in the mesentery). The
higher efficiency of conversion in the heart and the kidney
compared with the mesentery is predictable given the lower
Km in the heart and kidney and the higher Vmax/Km ratio value
of the heart and kidney compared with the mesentery.
Expressed per whole organ, the kidney maintains higher
capacity to metabolize 2OHE, as indicated by the higher Vmax.
The results of the present study confirm our previous findings
in coronary and aortic smooth muscle cells and endothelial
cells in culture. In this regard, COMT in coronary cells has a
lower Km for 2OHE and a higher Vmax/Km than aortic cells,
and coronary cells are thus more efficient in metabolizing
2OHE.8
The 2OHE methylation pathway in the kidney is blocked
by quercetin (a known inhibitor of COMT) by nearly 90%,
indicating a COMT-dependent methylation of 2OHE. The
catecholamines, norepinephrine and epinephrine, also inhibit
methylation of 2OHE in the kidney, with the 2 catecholamines showing about the same order of potency as in
cultured cells.7 Stimulation of periarterial nerves with the
consequent release of endogenous norepinephrine also causes
a marked inhibition of 2OHE methylation. This is of notable
physiological significance because we show not only that
exogenous catecholamines can inhibit 2OHE methylation, but
also that local release of norepinephrine in the neuroeffector
junction can inhibit 2OHE methylation.
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In the present study, we employed a monoamine oxidase
inhibitor (pargyline) and a norepinephrine re-uptake inhibitor
(desipramine) to enhance diffusion of endogenous norepinephrine through the kidney. In this regard, detection of
inhibition of 2OHE methylation by periarterial nerve stimulation in the absence of these inhibitors would have been
possible only with very low concentrations of 2OHE because
the interaction between 2OHE and norepinephrine at COMT
is competitive. Because of the sensitivity limitations of our
high-performance liquid chromatography method, lower concentrations of 2OHE could not have been used. Importantly,
the re-uptake inhibitor desipramine is an ␣1-adrenoreceptor
antagonist. Consequently, on renal artery stimulation and
release of norepinephrine, there were no changes in perfusion
pressure or renal vascular resistance. Therefore, blockade of
the 2OHE methylation pathway by sympathetic nerve stimulation in the kidney was not secondary to changes in renal
hemodynamics but most likely was mediated by competition
between 2OHE and endogenous norepinephrine for COMT.
The fact that endogenous norepinephrine can inhibit 2OHE
methylation, together with the fact that increased catecholamines are related to renal diseases,16 –18 provides evidence
that one possible mechanism by which catecholamines accelerate renal disease is by inhibiting methylation of 2OHE, thus
abrogating the antiproliferative effect of 2MeOE.
Our findings that methylation of 2OHE is inhibited by
catecholamines may be of pathophysiological relevance.
Evidence suggests that catecholamines contribute to the
process of glomerulosclerosis16 –18 and vaso-occlusive disorders.11,19 –21 In a model of chronic renal failure induced by
renal ablation, renal afferent denervation by dorsal rhizotomy
retarded the progression of glomerulosclerosis/proliferation.17 Furthermore, in rats with reduced renal mass, administration of moxonidine at doses that do not cause hypotension but inhibit sympathetic nerve traffic reduced
glomerulosclerosis.18 The sympathetic nervous system is
activated in patients with the nephrotic syndrome (normal
creatinine clearance) compared with normal individuals.
These patients not only have higher plasma norepinephrine
but also have increased renal excretion of norepinephrine.16
Premenopausal women have lower incidence of both renal
and cardiovascular diseases.22–24 This suggests that the presence of an active 2OHE methylation pathway in the kidney
and heart in premenopausal women may be responsible for
protection against renal and vascular damage. Further,
women under chronic stress, which results in increased
catecholamines, may not be protected by 17␤-estradiol to the
same extent as women without stress. The fact that endogenous catecholamines can inhibit the 2OHE methylation pathway in the kidney is physiological evidence that increased
stress resulting in high catecholamine levels can inhibit this
pathway.
Importantly, the nerve stimulation experiment did not
account for circulating catecholamines. This implies that, in
physiological situations, inhibition of the 2OHE methylation
pathway by stress may be even more significant because both
norepinephrine released in the vascular bed locally and
additional circulating catecholamines would inhibit this pathway. Physiologically, inhibition of this pathway might be
even higher because epinephrine is even more potent than
norepinephrine in inhibiting COMT and 2OHE methylation.7
Because circulating catecholamines include both norepinephrine and epinephrine, our hypothesis is feasible.
The mesentery was not an efficient vascular bed compared
with the kidney or the coronary circulation with respect to
methylating 2OHE. The low amount of 2MeOE produced
indicates low levels of COMT in the mesentery because the
Km in the mesentery was comparable to that of the kidney.
The differences in conversion efficiencies may be a physiological adaptation for 17␤-estradiol–induced protection of
organs, such as the heart and the kidney, that are the most
susceptible to vaso-occlusive diseases. The differences in the
COMT activities in these vascular beds may be the result of
differences in developmental origins of the cells in these
beds.25–27 Different arteries and even different segments of
the same artery are composed of smooth muscle cells that
differ greatly in their embryonic lineage and developmental
history,25 and smooth muscle cells from different lineage
backgrounds may not have identical functional and growth
regulatory mechanisms.25,26 Smooth muscle cells cultured
from different vascular beds, as well as from different
sections of a common vessel, grow differentially in response
to a common stimulus.26 One of the main factors contributing
to these different effects is the phenotype of the cells and its
embryonic lineage.27 This may explain the different COMT
activities in these vascular beds, because coronary vessels
develop completely independently of the systemic
vasculature.26 –28
The mesentery is not as frequently affected by severe
vaso-occlusive disorders as is the heart and kidney, which
may explain why high levels of COMT activity in the
mesentery are not necessary. The heart and kidney have
higher capacity to metabolize 2OHE, and this could be a
potential mechanism for estradiol-induced cardiovascular and
renoprotection during the reproductive age in women. The
low Km in the heart assures that even low concentrations of
2OHE will be metabolized and confer protection. This may
further explain the low rates of coronary heart disease in
women during the reproductive years.
Our findings that local release of catecholamines in the
kidney inhibit 2OHE methylation may be of broader pathophysiological relevance. In this regard, our results imply that
activation of the sympathetic nerves in other innervated
vascular beds could also inhibit 2OHE metabolism. Because
the 2OHE methylation pathway is an active antimitogenic,
antifibrotic pathway,6 our results suggest that the initiation/
progression of vaso-occlusive disorders in other vascular
beds may, in part, be mediated through decreased methylation
of 2OHE, and hence, reduced protection from cell proliferation. In addition, in this study the inhibition was the result of
norepinephrine diffusing to the whole kidney, as a result of
the use of monoamine oxidase (MAO) and re-uptake inhibitors. This implies that the high levels of norepinephrine
normally achieved in neuroeffector junctions would effectively inhibit 2OHE methylation in the junction.
The high methylation of 2OHE in the heart may ensure that
not only coronary arteries are protected but also cardiac
tissue. The high amounts of 2MeOE produced in the coronary
Zacharia et al
arteries may diffuse to inhibit cardiac fibroblast growth and
extracellular matrix deposition. Patients with heart failure
have high levels of catecholamines and increased sympathetic
activity, and high plasma norepinephrine levels are associated
with increased left ventricular mass and dysfunction.19 –21 Our
results indicate that increased catecholamines in these patients may further worsen heart failure by abrogating the
antimitogenic and antifibrotic effects of 2OHE by preventing
its metabolism.
In summary, we provide evidence that the 2OHE methylation
pathway is present in the kidney, heart, and mesentery. This
pathway is efficient in the heart and the kidney, but inefficient in
the mesentery. Furthermore, we provide evidence that exogenous and endogenous catecholamines inhibit methylation of
2OHE in the kidney. Given the adverse effects of catecholamines in the cardiovascular and renal systems, our results imply
that inhibition of this pathway by catecholamines may contribute
to the progression of cardiorenal diseases.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Perspectives
Our results suggest that the conversion of 17␤-estradiol to
2OHE and then to 2MeOE may be an important determinant
of the cardiorenal protective effects of circulating 17␤estradiol and that individual differences in the local formation
of 2MeOE could influence a woman’s risk of renal and
cardiovascular disease. Thus, genetic, acquired, or tissuespecific differences in COMT activity may determine the
benefits a woman receives from either endogenous 17␤estradiol in the premenopausal state or exogenous 17␤estradiol from replacement therapy in the postmenopausal
state. The fact that local release of catecholamines inhibits the
conversion of 2OHE to 2MeOE by competing for COMT
suggests that pathological increases in catecholamine release
may also influence an individual woman’s risk of renal and
cardiovascular disease. Specifically, chronic stress or other
disease states that elevate catecholamines may limit methylation of 2OHE, and this may worsen the progression of renal
and cardiovascular proliferative diseases. Future studies
should examine the relationship between catecholamine levels and the production rate of 2MeOE in women.
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Methylation of 2-Hydroxyestradiol in Isolated Organs
Lefteris C. Zacharia, Raghvendra K. Dubey, Zaichuan Mi and Edwin K. Jackson
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Hypertension. 2003;42:82-87; originally published online May 27, 2003;
doi: 10.1161/01.HYP.0000074702.06466.27
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