Vascular Effects of Progesterone
Role of Cellular Calcium Regulation
Mario Barbagallo, Ligia J. Dominguez, Giuseppe Licata, Jie Shan, Li Bing, Edward Karpinski,
Peter K.T. Pang, Lawrence M. Resnick
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
Abstract—Vascular actions of progesterone have been reported, independently of estrogen, affecting both blood pressure
and other aspects of the cardiovascular system. To study possible mechanisms underlying these effects, we examined
the effects of P in vivo in intact rats and in vitro in isolated artery and vascular smooth muscle cell preparations. In
anesthetized Sprague-Dawley rats, bolus intravenous injections of P (100 ␮g/kg) significantly decreased pressor
responses to norepinephrine (0.3 ␮g/kg). In vitro, progesterone (10⫺8 to 10⫺5 mmol/L) produced a significant,
dose-dependent relaxation of isolated helical strips, both of rat tail artery precontracted with KCl (60 mmol/L) or
arginine vasopressin (3 nmol/L), and of rat aorta precontracted with KCl (60 mmol/L) or norepinephrine (0.1 ␮mol/L).
In isolated vascular smooth muscle cells, progesterone (5⫻10⫺7 mol/L) reversibly inhibited KCl (30 mmol/L) -induced
elevation of cytosolic-free calcium by 64.1⫾5.5% (P⬍0.05), and in whole-cell patch-clamp experiments, progesterone
(5⫻10⫺6 mol/L) reversibly and significantly blunted L-type calcium channel inward current, decreasing peak inward
current to 65.7⫾4.3% of the control value (P⬍0.05). Our results provide evidence that progesterone is a vasoactive
hormone, inhibiting agonist-induced vasoconstriction. The data further suggest that progesterone effects on vascular
tissue may, at least in part, be mediated by modulation of the L-type calcium channel current activity and, consequently,
of cytosolic-free calcium content. (Hypertension. 2001;37:142-147.)
Key Words: progesterone 䡲 intracellular calcium 䡲 vascular smooth muscle 䡲 sex steroid hormones
䡲 L-type calcium channel 䡲 hypertension 䡲 menopause
I
t is well established that premenopausal women are at
lower risk of developing hypertension and coronary heart
disease than men of the same age and that the cardiovascular
risk increases only after the cessation of ovarian function.1
Conversely, pregnancy is associated with lower blood pressure (BP), despite elevated circulating angiotensin II (Ang II)
levels and sodium retention.2 Although the mechanism(s)
underlying these observations are still poorly understood, a
critical role for dramatic changes in the sex steroid environment is widely presumed. Previous mechanistic studies have
focused on cardiovascular protective effects attributable to
estrogens, but accumulating evidence suggests that progesterone independently of estrogen also exerts a protective
influence on the vasculature.3 Thus progesterone receptors
have been localized in the myocardium4 and in peripheral
vascular tissue,5 and administration of progesterone lowers
BP in humans,3,6 blunts the pressor response to Ang II in
human pregnancy,7 and inhibits Ang II action in rats in
some,8,9 but not all, reports.10,11
We intended to further define the vascular actions of
progesterone and to study potential mechanism(s) underlying
these effects. Therefore, we evaluated the short-term effects
of progesterone in a variety of circumstances: on BP in
anesthetized rats, on vascular contractility in endotheliumdenuded isolated rat tail artery and aorta helical strips in vitro,
and on cytosolic-free calcium concentrations [Ca2⫹]i and
L-channel Ca currents in isolated vascular smooth muscle
cells (VSMC). Our results, similar to previous studies of 17-␤
estradiol,12–15 suggest that progesterone has modulatory effects on the contractility of blood vessels and that these
vascular actions may be mediated by its effects on membrane
Ca currents and thereby on [Ca2⫹]i concentrations.
Materials and Methods
Experiments were performed in the research facilities of the Department of Physiology, University of Alberta, Canada. In all experiments, male Sprague-Dawley rats obtained from Charles River
Canada (St. Foy, Quebec) were used. Experiments were conducted in
adherence with the NIH “Principles for the utilization and care of
vertebrate animals used in testing research and training.”
BP Measurements
BP was measured in anesthetized (pentobarbital sodium 65 mg/kg ip)
and cannulated rats (200 to 250 g), as previously described.15 The
Received January 28, 2000; first decision March 2, 2000; revision accepted July 17, 2000.
From the Institute of Internal Medicine and Geriatrics (M.B., L.J.D., G.L.), University of Palermo, Palermo, Italy; the Department of Physiology (J.S.,
E.K., P.K.T.P.), University of Alberta, Canada; and the Hypertension Center (L.M.R.), NY-Presbyterian Hospital/Cornell University Medical Center, New
York, NY.
Correspondence to Lawrence M. Resnick, Hypertension Center, New York Presbyterian Hospital/Cornell University Medical Center, 525 E. 68th St,
Starr 4 Pavilion, New York, NY 10021. E-mail [email protected]
© 2001 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
142
Barbagallo et al
Vascular Effects of Progesterone
143
Vascular tension was measured in tail artery and aorta helical strips,
according to the method previously described in detail.15,16 The rat
tail artery and aorta strips were suspended in Sawyer-Bartlestone
chambers containing aerated (95%O2, 5%CO2) Krebs-Henseleit solution.16 The rat tail helical strips were then contracted with KCl
(60 mmol/L) and arginine vasopressin (AVP, 3 nmol/L). The aorta
strips were contracted with KCl (60 mmol/L) and NE (0.1 mmol/L),
because it was not possible to achieve a steady tension with AVP in
this preparation. When a steady tension was achieved, a cumulative
dose-response for the vasodilatory effect of progesterone was obtained. Progesterone was added to the tissue bath to reach the
following concentrations (mol/L): 5⫻10⫺8, 1.5⫻10⫺7, 5⫻10⫺7,
1.5⫻10⫺6, 5⫻10⫺6, 1.5⫻10⫺5, and 5⫻10⫺5. The entire dose response
curve was obtained within 45 minutes, a period during which a
steady contractile effect of the vasoconstrictors was maintained. A
dose-response curve with ethanol was also performed for each type
of experiment.
containing 5 ␮mol/L fura 2-AM (Molecular Probes, Inc) at 37°C, in
a dark compartment. The cells were then gently washed 3 times and
kept in the same buffer.19 After about 5 minutes, the coverslip with
attached cells was placed in a Sykes-Moore chamber of 1-mL
volume on the stage of a Nikon (Phase Contrast-2) microscope.
Fluorometric data were obtained with a dual wavelength excitation
monochrometer spectrofluorometer (SPEX Industries Inc). Excitation wavelengths of 340 and 380 nm and an emission wavelength of
505 nm were used, and [Ca2⫹]i was calculated according to the
method described by Grynkiewicz et al,20 with the use of the
following equation: [Ca2⫹]i (nmol/L)⫽Kd⫻(R- Rmin/Rmax -R)⫻b,
where R is the ratio of fluorescence in the sample at 340 and 380 nm;
Rmax is the fluorescence ratio obtained by adding 2 ␮mol/L ionomycin; Rmin is the fluorescence ratio subsequently obtained by adding
5 mmol/L EGTA; b is the ratio of fluorescence of fura 2 at 380 nm
at zero and saturating Ca⫹⫹; Kd is the dissociation constant of fura 2
for Ca⫹⫹, 224 nmol/L.20 Control [Ca2⫹]i elevations were induced by
KCl (30 mmol/L) before the addition of the hormone. Cells showing
a lack of basal responsiveness to KCl (defined as an increase of
[Ca2⫹]i ⱖ50% of basal) were excluded from further study. No
differences in KCl responsiveness were noted in cells used from
passages 3 to 4 (the majority used) compared with those of later
passages. Progesterone (5⫻10⫺7 mol/L) was added and incubated for
10 minutes. [Ca2⫹]i was measured and compared with the control. A
second stimulus with KCl (30 mmol/L) was performed and the
response compared with the control, in absence of the hormone.
After wash-out of the hormone and a 10-minute recovery period, a
third stimulus with KCl (30 mmol/L) was then performed and the
response compared with the previous 2 KCl stimuli.
VSMC Studies
Drugs
All studies were performed on VSMC isolated from SD rat tail
artery as previously described.15,17–19 For patch-clamp (pClamp)
studies, primary culture cells were used within 18 to 36 hours of
isolation. For [Ca2⫹]i studies, cells were subcultured and used at
passages 3 to 10.
Progesterone was purchased from Sigma Chemicals, dissolved in
95% ethanol to make a stock solution of 5⫻10⫺3 mol/L, and stored
at 4°C. The same concentration of alcohol in a control solution had
no effect in any of the in vivo or in vitro assays.
arterial cannula was connected to a Statham pressure transducer.
Mean arterial pressure (MAP) was recorded continuously on a Grass
FT03 polygraph (Grass Instruments), before and for 6 hours after a
bolus injection of 100 ␮g/kg iv of progesterone. To determine the
effect of progesterone on the pressor response to norepinephrine
(NE), repeated bolus injections of NE (0.3 ␮g/kg) were administered
before and 1, 2, 3, 4, 5, and 6 hours after the initial progesterone
treatment. In the control group, solvent was injected instead of
progesterone.
Vascular Tension Studies
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L-Current Recording
The standard whole-cell version of the pClamp technique was used
to measure whole-cell inward currents, as previously described.15,17,18 In brief, cells were put on the stage of an inverted
phase-contrast microscope (Nikon). pClamp measurements were
performed with an Axopatch-1B (Axon Instruments) pClamp amplifier. Patch micropipettes were pulled from glass tubes (OD 1.2 mm,
ID 0.9 mm) with a 2-stage micropipette puller (PP83) and then
fire-polished with a micro forge. The tip diameter was ⬇1 ␮m with
a resistance of 2 to 8 M⍀ when filled with the internal solution. The
holding potential was set at ⫺40 mV. Barium currents (20 mmol/L
Ba⫹⫹ was used as the inward charge carrier) through the Ca⫹⫹
channel were elicited by 200 ms depolarization at intervals of 5
seconds. The currents were filtered with a 4-pole Bessel filter at a
cut-off frequency of 3 KHz. pClamp software and a labmaster
interface (Axon Instruments) were used to generate the test pulses
and to store and analyze data. In all cases, the peak current (leak
current corrected) was used to construct the current versus voltage
(I-V) relationship. progesterone (5⫻10⫺6 mol/L) was added to the
bath and inward Ba⫹⫹ currents were measured again (usually within
5 minutes of drug administration). L-currents were also recorded
after a 2-minute washout of progesterone (obtained with a perfusion
rate of ⬇1 mL/10s) and a 3-minute recovery period, or after the
addition of Bay K 8644 (1 mmol/L) to progesterone-treated cells.
[Ca2ⴙ]i Measurement
VSMC [Ca2⫹]i studies were conducted as previously described in
detail.15,19 Confluent cells were plated onto glass coverslips (25 mm
circle) at a density of ⬇1⫻106m/L in DMEM and kept in culture
until the cells became elongated and confluent (usually 24 to 48
hours). Cells were then incubated for 45 minutes in DMEM
Statistics
Values are expressed as means⫾SEM. The paired t test was used for
comparisons between mean values of the control and those obtained
after drug administration. In the case of multiple comparisons,
analysis of variance in connection with the Neuman-Keul’s multiplerange test was applied. A minimum of n⫽8 experiments was
performed for each of the studies. A P⬍0.05 was
considered significant.
Results
In Vivo Effects on BP Responses to NE
Bolus injection of progesterone (100 ␮g/kg) had no significant effect on the basal MAP during the 6-hour experimental
period of observation (data not shown). However, progesterone altered the BP responsiveness to NE (0.3 ␮g/kg), significantly blunting the rise in MAP (Figure 1, Top Panel). Bolus
iv injection of the control solvent (75 ␮L/kg ethanol) did not
change the pressor responses to NE up to 6 hours (Figure 1).
Heart rate did not significantly change during the 6-hour
course of the experiment (from 284⫾3bpm to 280⫾3bpm,
P⫽NS, data not shown).
In Vitro Effects on Isolated Tissues
The dose responses of precontracted aorta and rat tail artery
strips to progesterone are shown in Figure 2. Increasing doses
of progesterone produced increasing depressor responses,
starting in both preparations at the dose of 5⫻10⫺6 mol/L
(P⬍0.05). In the tail artery preparation, an almost complete
144
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January 2001
Figure 1. Bolus intravenous injection of 100 ␮g/kg progesterone significantly decreases pressor response to NE (0.3 ␮g/kg)
in anesthetized male Sprague-Dawley rats.
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relaxation was obtained at 1.5⫻10⫺5 mol/L (95.7⫾11.9%)
(P⬍0.05) for the strips precontracted with KCl and at 5⫻10⫺5
mol/L (102.9⫾4.5%) (P⬍0.05) for those precontracted with
AVP (Figure 2, Top Panel). In the aorta helical strips, the
higher concentrations tested (5⫻10⫺5 mol/L) induced a relaxation of 56.5⫾8.6% (P⬍0.05) in strips precontracted with
KCl and of 84.5⫾4.5% (P⬍0.05) in those precontracted with
NE (Figure 2, Lower Panel). Concentrations of ethanol up to
2% did not significantly change the vasoconstrictor responses
of the artery strip preparation used (⫹2.04⫾1.1%, P⫽NS at
the highest concentration, data not shown).
Effects of Progesterone on [Ca2ⴙ]i of VSMC
The effects of progesterone on [Ca2⫹]i are shown in Figure 3
(original recording in the Top Panel and summary data in the
Lower Panel). Basal [Ca2⫹]i averaged 110⫾5.4 nmol/L.
Progesterone alone (5⫻10⫺7 mol/L) did not produce any
Figure 3. Top Panel: Recording of intracellular free calcium
concentrations in a monolayer of VSMC after application of KCl
(30 mmol/L) (A), progesterone alone (5⫻10⫺7 mol/L) (B), KCl
(30 mmol/L) after progesterone (5⫻10⫺7 mol/L) (C), and after
washout (D). The 4 traces represent 4 experiments performed in
the same cell layer. Lower Panel: Effect of progesterone (5⫻10⫺7
mol/L) on intracellular free calcium induced by KCl (30 mmol/L)
in VSMC (n⫽8).
significant alteration of resting [Ca2⫹]i (Figure 3). KCl
(30 mmol/L) increased [Ca2⫹]i by 71.6⫾9.9% (mean [Ca2⫹]i
increase: ⫹78.8⫾10.9 nmol/L, P⬍0.001). However, when
KCl (30 mmol/L) was added after 10 minutes’ incubation
with progesterone (5⫻10⫺7 mol/L), the [Ca2⫹]i increase was
significantly inhibited by 64.1⫾5.3% (mean [Ca2⫹]i increase:
⫹28.2⫾4.8 nmol/L, P⬍0.05) (Figure 3 Top Panel, traces A
and C and Lower Panel). This effect of progesterone was
reversible, because after washout of progesterone and a
5-minute recovery period, KCl responsiveness (30 mmol/L)
was partially restored, [Ca2⫹]i again increasing to 87.0⫾6.8%
of the original response ([Ca2⫹]i: ⫹68.6⫾8.2 nmol/L, P⫽N.S.
versus control KCl effect) (Figure 3, Top Panel trace D versus
A and Lower Panel). Ethanol alone did not elicit any change
in basal or post-KCl [Ca2⫹]i levels (data not shown).
Effects of Progesterone on L-Currents
Figure 2. Effects of progesterone on the developed force of
helical strips of rat tail artery (top panel) contracted with KCl
(60 mmol/L) or AVP (3 nmol/L) (n⫽8), and of rat aorta (lower
panel) contracted with KCl (60 mmol/L) or NE (0.1 mmol/L)
(n⫽8).
Figure 4 (original recording in the top panel and summary
data in the lower panel) shows the whole-cell pClamp data
and I/V (current/voltage) relationship in the presence and
absence of progesterone. With the holding potential set at
⫺40 mV, L-currents were detected at a membrane potential
of ⫺20 mV and were maximal at ⫹20 mV with an apparent
Barbagallo et al
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Figure 4. Top Panel: Effect of progesterone on L-type calcium
channel current in VSMC. Left panel shows traces from a holding potential of ⫺40 mV with 3 different test potentials. Circles
represent the control traces, squares represent the traces after
treatment with progesterone (5⫻10⫺6 mol/L), and triangles represent the traces after Bay K 8644 (1 mmol/L) was added to
progesterone-treated cells. The right panel shows the current
voltage (I/V) relationship before and after the application of progesterone and Bay K 8644 to progesterone-treated cells. Lower
Panel: Effect of progesterone (5⫻10⫺6 mol/L) and of Bay K 8644
(1 mmol/L), added to progesterone-treated cells, on calcium
channel currents in VSMC (n⫽8), expressed as % of the maximal control current.
reversal potential beyond ⫹60 mV. These currents were
activated quickly and inactivated slowly (half-time inactivation ⬎150 ms). The peak amplitudes of L-current could be
maintained without significant deterioration for up to 20
minutes as previously reported.15,18,19 Progesterone significantly blunted the L-current (Figure 4). At the dose of
5⫻10⫺6mol/L, progesterone decreased the peak inward current to 65.7⫾4.3% of the control value (from 53.5⫾8.0 to
34.7⫾4.9 pA, P⬍0.05). This inhibition was reversible, because after a 2-minute washout of progesterone and a
3-minute recovery period, the peak amplitude of the L-current
returned to 86.3⫾7.6% of the control value (46.2⫾4.1 pA,
P⫽NS versus control). Ethanol alone did not elicit any
change of L-current (data not shown). In addition, the
application of the calcium ionophore Bay K 8644 (1 mmol/L)
to progesterone-treated cells increased the L-current to
120.0⫾10.0% of the basal control value (Figure 4, Top and
Lower Panels).
Discussion
Normal human pregnancy is characterized by profound
changes in BP homeostasis and electrolyte balance, including
Vascular Effects of Progesterone
145
progressive volume expansion and activation of the renin-angiotensin system. Yet the pressor response to Ang II is
blunted, and BP is usually reduced in pregnancy.2,3 Although
the biological basis underlying these observations remains
obscure, attention has been focused on estrogens, the direct
vasodilatory effects of which may be mediated by their
actions on L-channel Ca current and cytosolic-free calcium
levels.15 We wondered whether progesterone also exhibited
direct vascular effects, independently of estrogen. Since the
early work of Landau and Lugibihl, who demonstrated
natriuretic effects of P21 more than 4 decades ago, progesterone has been reported to lower BP in hypertensive men and
postmenopausal women4,7; to blunt the pressor response to
Ang II in ovariectomized rats (which estrogen does not)8,9;
and to alter adrenergic activity,22 decreasing systemic vascular resistance in normal women23,24 and increasing cardiac
output.
The present study demonstrates that progesterone, independently of estrogen, is a vasoactive hormone. This conclusion
is reinforced by the overlapping, consistent effects observed
in multiple preparations: (1) in the whole animal, progesterone blunted the pressor effects of NE. That this resulted from
direct vascular actions was suggested by (2) the parallel in
vitro action of progesterone to blunt agonist-induced tension
both in rat aorta and tail artery strips. Because these preparations were endothelium-denuded, the effects observed here
reflect direct vascular smooth muscle actions of progesterone,
consistent with previous reports of endothelium-independent
relaxant effects of progesterone in rabbit coronary arteries25
and human placental arteries and veins.26 After observing in
vitro effects of progesterone with KCl, AVP, and NE-related
stimuli, all of which are calcium-mediated, it seemed reasonable that the mechanism underlying the vascular effects of
progesterone might involve cellular calcium homeostasis.
Indeed, in VSMC, (3) progesterone directly decreased
L-current, and in association with this, (4) progesterone
blunted KCl-induced elevation of [Ca2⫹]i. Altogether, these
data not only suggest direct vascular relaxant effects of
progesterone, but also suggest that these effects are attributable, at least in part, to its actions on L-channel Ca current
and concomitant [Ca2⫹]i levels.
We hypothesize that progesterone functions to modulate
calcium channel activity, buffering the vasculature against
excessive calcium-dependent vasoconstrictor responses to a
variety of hormonal stimuli. This calcium-based mechanism
may be a common denominator that helps to explain a variety
of progesterone-induced effects observed in other tissues as
well, such as (1) the decreased production of catecholamines
by the adrenal medulla,27 of PAI-128 and of endothelin-1 by
endothelial cells,29 (2) the blunted pressor responses to
infused Ang II,7 and thus (3) the lowering of BP in normal
pregnancy, in pregnancy-associated hypertension,30 and in
hypertensive men and postmenopausal women.4 In pregnancy, progesterone also augments nifedipine-induced inhibition of uterine contractions.31
Mechanistically, under our experimental conditions, these
effects of progesterone cannot easily be attributed to a
genomic effect, because this presupposes a significant latency
period, whereas our observations, at least in the in vitro
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January 2001
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experiments, were of a more immediate type. Because some
latency period was observed in the in vivo experiments, the
potential contribution of baroreflex and other mechanisms
cannot be ruled out. As the AVP and KCl-induced vasoconstriction is independent of adrenoreceptors, interaction with
these receptors also cannot account for our data. Thus, these
calcium-related actions of progesterone may be direct cell
membrane-mediated, not involving a classic steroid/receptor
mechanism. Indeed, plasma membrane binding and biochemical effects of progesterone not inhibited by classic progesterone antagonists have been reported.32,33
Certain caveats to the interpretation of our data must be
considered. First, although circulating levels of progesterone
in pregnancy may reach 100 to 150 ng/mL (⬇10⫺6 mol/L),
similar to concentrations having vasoactive actions in our and
other in vitro systems,34 these levels are not observed physiologically outside pregnancy. However, non-pregnant levels
of 10 to 100 ng/mL result from 100 mmol/kg bolus injections
that decreased in vivo pressor responses to NE.35 Furthermore, as is true for many vasoactive substances, the doses
required to produce an effect large enough to be measured
consistently in vitro may be 1 to 2 orders of magnitude higher
than those necessary for physiological effects in vivo. This
may also reflect the lack of a normal extracellular ionic and
hormonal milieu in which many substances and hormones,
eg, estrogen, may be required for normal tissue responsiveness to progesterone.
A second caveat concerns the specificity of progesterone
action on the vasculature. Although we reported similar
Ca-related actions of estradiol15 and DHEAS,36 it is unlikely
that the effects reported here for progesterone represent
non-specific steroid action, because other steroids produce
opposite effects. Thus, 1,25 (OH)2D337 and testosterone (unpublished data) also have vasoactive properties, but in an
opposite direction, stimulating L-channel Ca currents and
increasing [Ca2⫹]i levels in VSMC. Glucocorticoids tested at
doses similar to those used in the present study do not affect
calcium transients.38
In summary, we suggest that the direct calcium-dependent
vascular relaxant effects of progesterone demonstrated here
support a physiological role for progesterone in modulating
vasoconstrictor tone in pregnancy. These direct vascular
actions of progesterone, along with its natriuretic effects and
effects on other ions such as magnesium,39 could also explain
the lower BP and increased renin system activity during the
luteal phase of the normal menstrual cycle40 if the effects
observed here also occur at the lower in vivo concentrations
of the nonpregnant state. Future studies are needed to explore
the interactions between progesterone and estrogen and also
to explore a possible link of alterations of circulating progesterone to the increased incidence of hypertension and cardiovascular disease in postmenopausal women.
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Vascular Effects of Progesterone: Role of Cellular Calcium Regulation
Mario Barbagallo, Ligia J. Dominguez, Giuseppe Licata, Jie Shan, Li Bing, Edward Karpinski,
Peter K. T. Pang and Lawrence M. Resnick
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Hypertension. 2001;37:142-147
doi: 10.1161/01.HYP.37.1.142
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