Vol. 282, Issue 4, R952-R959, April 2002
Enhanced vascular effects of the Ca2+ channel
agonist Bay K 8644 in pregnant rabbits
Rocco
Venuto1,
Gail
Brown2,
Marion
Schoenl1, and
György
Losonczy1,3
1 Schools of Medicine and Biomedical Science and
2 Nursing, University at Buffalo, State University
of New York, Buffalo, New York 14215; and 3 Department
of Pulmonology, Semmelweis University Medical School, Budapest 114, 1536 Hungary
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ABSTRACT |
Hemodynamic studies were
performed to determine if blunting of vascular pressor responsiveness
to vasoconstrictors during pregnancy may be due to impaired L-type
voltage-dependent calcium channels (L-VDCC). Bay K 8644 (BAY), an
L-VDCC agonist, was infused in pregnant and nonpregnant anesthetized
rabbits (10, 20, 40, and 60 µg/kg) and pregnant and nonpregnant
conscious, chronically instrumented (conscious) rabbits (10, 25, and 50 µg/kg). BAY infusions resulted in greater elevation of mean
arterial pressure in both anesthetized pregnant (n = 6)
vs. nonpregnant (n = 6) (P < 0.05) and
conscious pregnant (n = 10) vs. nonpregnant
(n = 10) rabbits (P < 0.05).
Fractional increase over baseline of total peripheral resistance index
was greater in pregnant (36 ± 5 to 78 ± 14%) vs.
nonpregnant rabbits (14 ± 4 to 52 ± 6%) (P < 0.02). Cardiac output index did not differ. There was a single
high-affinity L-VDCC antagonist aortic binding site with similar number
and affinity in pregnant (n = 7) and nonpregnant
(n = 7) rabbits. In conclusion, stimulation of L-VDCC
induces greater pressor responses in pregnant rabbits with heightened
peripheral vasoconstriction. This does not appear to be due to a change
in L-VDCC receptor parameters.
blood pressure; vascular reactivity; calcium channels; nifedipine
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INTRODUCTION |
PHYSIOLOGICAL
ALTERATIONS that occur during pregnancy in humans
(7) and experimental mammals (36) include
reductions in systemic vascular resistance and mean arterial blood
pressure (MAP). Another hallmark of gestation is the nonspecific
blunting of the hypertensive effect of pressor hormones such as ANG II, norepinephrine, and vasopressin (10, 12). On the cellular level, these pressor compounds bind with specific receptors and the
subsequent activation of a signal transduction cascade ultimately leads
to an increased concentration of free intracellular calcium in vascular
smooth muscle cells, which is currently considered as the final
mediator of vasoconstriction (33). The mechanism of the
pregnancy-induced decrease of vascular pressor responsiveness to these
hormones has not yet been clarified. Downregulation of specific
receptors (2, 13, 18), enhanced secretion of vasodilatory substances (9), and alterations of receptor-response
coupling (uncoupling) (8, 11, 28, 29, 34) have been
considered as mediators of decreased vasopressor response to these hormones.
A major portion of the intracellular calcium elevation triggering
contraction results from the opening of the L-type, voltage-dependent calcium channel (L-VDCC) (27). A gestational alteration of
the vascular L-VDCC has already been suggested. For example, activation of the channel as induced by depolarization or Bay K 8644 (BAY), a
specific L-VDCC agonist (31), was observed to be a less
potent signal for vascular constriction in aortic rings and mesenteric arteries of pregnant than nonpregnant rats (11, 28, 29, 34). In addition, chronic estrogen administration was noted to
decrease the number of L-VDCC (26). On the other hand, the activation of the L-VDCC in vitro by administration of BAY has been
shown to induce a strong hypertensive effect in the dog
(31), the male rabbit (6), and the rat
(17), and also to cause constriction of the isolated
rabbit aorta (31). We tested the hypothesis that the
nonspecific refractoriness to pressor compounds in pregnant rabbits
(10, 37) might include BAY. If so, this would indicate
impaired function of the vascular L-VDCC, which, in turn, could
underlie the nonspecific vascular pressor refractoriness developing
during gestation. Also explored was the possibility that pregnancy
might alter either the vascular receptors for BAY (the
1-subunit of the L-VDCC) (14) or the
vascular release of vasoactive prostanoids, the latter of which can
either attenuate (23) or potentiate (5) the
effects of smooth muscle contracting substances.
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METHODS |
Animals
All experiments were undertaken in New Zealand White rabbits
weighing 3.5-5 kg. Pregnant rabbits were studied within the final 8 days of the 30 ± 2 day gestation. Nonpregnant animals of
similar weight and age served as controls in all protocols. The rabbits were housed individually in cages and received standard rabbit chow and
water ad libitum. All protocols were reviewed and given approval by the
Institutional Review Boards of the University at Buffalo and Semmelweis
University, Budapest, Hungary.
Physiological Studies
Animal preparation.
Acutely studied rabbits:
The protocol followed was described previously in similar studies
(37). Briefly, general anesthesia was achieved with
intravenous pentobarbital sodium (30 mg/kg). With the use of sterile
surgical technique, the femoral artery and vein were isolated and
catheterized. The right jugular vein and right carotid artery were
isolated. A catheter was placed in the jugular vein and advanced down
the superior vena cava close to the right atrium. A thermistor
(Columbus Instruments, Columbus, OH) was placed in the right carotid
artery and advanced to the aortic arch. The animals, six pregnant and six nonpregnant controls, were studied after a stabilization period of
at least 30 min after surgery, and anesthesia was maintained with
pentobarbital sodium (10 mg/kg iv).
CONSCIOUS, CHRONICALLY INSTRUMENTED RABBITS:
The protocol followed was described previously in similar studies
(10, 19, 20). After induction of general anesthesia, the
left femoral artery was isolated and catheterized so that the catheter
tip lay in the distal aorta. The ipsilateral femoral vein was
catheterized, and the catheter was advanced to the inferior vena cava.
The carotid artery was also catheterized, and the catheter was advanced
to just before the aortic arch. Pregnant rabbits were instrumented
between the 21st and 24th days of gestation. Experiments were performed
no earlier than 72 h after instrumentation and only animals that
evidenced complete recovery of normal movement, drinking, and eating
habits were used. More than 95% of the rabbits who underwent the
~45-min surgery were employed in this study.
Animal studies.
Acute rabbit studies:
In six pregnant and six nonpregnant rabbits, MAP, heart rate (HR), and
cardiac output (CO) were measured and recorded using the Cardiomax II
System (Columbus Instruments). Baseline MAP and HR were determined
using the mean of five consecutive readings, 1 min apart. BAY was given
as an intravenous infusion over 2 min in doses of 10-60
µg · kg
1 · 1.5 ml1 · min1. The protracted
administration of BAY provided a sustained peak of blood pressure
change, enabling the measurement of CO by thermodilution technique.
Animals were allowed to recover for at least 30 min between doses. HR
and MAP were assessed continuously, while CO, stroke volume (SV), and
total peripheral resistance (TPR) were determined at baseline and at 1, 2, 5, 10, and 15 min after each injection. The animals were also
studied before and after receiving only the saline diluent. CO, TPR,
and SV were divided by the animals' weight in kilograms to determine
CO index (COI), TPR index (TPRI), and SV index (SVI). After the
experiments, rabbits were euthanized with an overdose of intravenous
pentobarbital sodium and the carcasses were examined. Pregnant rabbits
had between 6 and 10 pups, all of which appeared to be viable, with
body weights ranging from 20 to 35 g.
CONSCIOUS RABBIT STUDIES:
Before surgery and during the recovery period, the pregnant
(n = 10) and nonpregnant (n = 10)
rabbits were accustomed to a restraining cage. All experiments were
undertaken in a quiet room. MAP and HR were measured using the
Cardiomax II System (Columbus Instruments) via the arterial line
(10, 19, 20).
BAY INFUSION STUDIES:
Baseline MAP and HR were determined using the mean of five consecutive
readings, 1 min apart. Bolus doses of BAY (Sigma, St. Louis, MO)
ranging from 10 to 50 µg/kg body wt and diluted in 1 ml saline were
administered in random order, over 30 s, and the animals were
allowed to recover at least 30 min between doses. In some experiments,
only the saline diluent was administered. MAP and HR were recorded
every 15 s for the first 2 min and every min thereafter for 18 min. We report the peak change from baseline, which consistently
developed within 20-40 s after the bolus was given.
NIFEDIPINE INFUSION STUDIES:
In separate experiments performed on different days and in the same
manner as described above, 10 pregnant and 10 nonpregnant rabbits were
given the dihydropyridine calcium channel blocker nifedipine
(10-50 µg/kg body wt iv; Sigma). Also, in another series of
experiments, a dose of nifedipine (25 µg/kg) was used to attempt to
block the action of the calcium channel enhancer, BAY. This dose was
determined empirically by testing the response to a range of doses from
10 to 50 µg/kg.
Biochemical analyses.
Dihydropyridine binding in aortic membranes:
Specific binding of PN 200-110, (+)-[5-methyl-3H]
(specific activity 79.7 Ci/mM, DuPont, New England Nuclear, Boston,
MA), a 1,4-dihydropyridine calcium channel antagonist, was assessed in
aortic membranes from seven pregnant and seven nonpregnant rabbits
using a radioreceptor assay similar to that previously described
(18). Aortic membranes were prepared similar to that previously described for other vascular tissues (18). The
final pellets were suspended in 50 mM Tris · HCl, pH 7.4, and
were used immediately thereafter in the radioreceptor assay. Protein
concentrations were determined by the Lowry method (21).
Steady-state binding was attained by 120 min of incubation at 24°C
and specific binding was proportional with aortic protein concentrations to 125 µg/tube. The aortic protein concentrations used
in the radioreceptor assays were 29.7 ± 4 and 28.0 ± 2 µg/tube for pregnant and nonpregnant rabbits, respectively. PN
200-110, (+)-[5-methyl-3H]receptor equilibrium
dissociation constant (Kd) and receptor number
(Bmax) were determined by Accufit Competition nonlinear analysis of binding inhibition data (Beckman Instruments, Fullerton, CA). The binding inhibition data were generated by incubating the
aortic membranes with the radioligand (5 × 1011 M)
and varying quantities of unlabeled S-(
)BAY (5.6 × 1011 M5.6 × 107 M). The incubation
media was 50 mM Tris · HCl, pH 7.4, and the total incubation
volume was 2.5 ml. When steady-state binding was attained, bound and
free were separated by rapid Millipore filtration (Whatman GF/F glass
microfiber filters) under vacuum. Filters were rinsed with 4 ml of
ice-cold incubation buffer four times. The radioactivity trapped
on the filter was measured by liquid scintillation counting, at an
efficiency of 60%. Nonspecific binding is determined by an excess
(5.6 × 105 M) of BAY.
In vitro vascular eicosanoid responses to BAY.
Sample preparation:
Production of 6-keto PGF1
, a stable metabolite of
prostacyclin, and thromboxane B2 (TXB2), a
stable metabolite of thromboxane A2, in response to BAY was
determined similarly to that previously described (4). For
each experiment, aortic tissue was obtained from one nonpregnant rabbit
(3.9 ± 0.2 kg body wt, n = 6) or one pregnant
rabbit (day 24-day 28 of gestation, 3.9 ± 0.2 kg body wt, n = 6). Rabbits were euthanized with
intravenous pentobarbital sodium, 100 mg/kg, and the aortic tissue was
quickly removed and rinsed in ice-cold incubation buffer
(Krebs-Henseleit bicarbonate containing 25 mM HEPES and
bubbled with 95% O2-5% CO2). On an iced glass
plate, adjoining tissue was gently removed, and the vascular tissue was
sliced into 1-mm rings. The rings were apportioned by weight into
incubation tubes with 1 ml of incubation buffer and used immediately
thereafter in experimental protocols.
EXPERIMENTAL PROTOCOLS:
All incubations, except the 4°C controls, were performed in a shaking
water bath at 37°C under an environment of 95%
O2-5% CO2. The production of 6-keto
PGF1 and TXB2 attained a maximal level
between 10 and 20 min of incubation at 37°C and remained stable up to
30 min of incubation. Thereafter, the incubation time used was 20 min.
Production (pg/ml) of 6-keto PGF1 and TXB2
was proportional to the protein content of the vascular rings up to 800 µg of aortic protein. All incubations were performed in triplicate.
Aortic rings deemed basal controls were incubated at 4°C.
Simultaneously, aortic rings were incubated in the absence and presence
of BAY, 1.0-100.0 ng/ml, at 37°C for 20 min. Total incubation
volume was 1.0 ml. After incubation, all tubes were iced and
immediately centrifuged at 1,000 rpm at 5°C for 15 min. The medium
was stored in aliquots at 20°C for later determination of
eicosanoids by enzyme immunoassay kits (Cayman Chemical, Ann Arbor,
MI). The rings were also stored at 20°C for later determination of
protein content by the Lowry method. Data are reported as production and release (pg/µg protein), which is defined as the quantity in the
medium after incubation of the vascular rings at 37°C minus the
quantity in the medium after incubation at 4°C.
Statistical Analyses
Data are presented as means ± SE. Differences were
considered significant if a value of P < 0.05 was
achieved as analyzed by analysis of variance of two-factor experiments
and the least-significant difference test.
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RESULTS |
Effects of BAY in Anesthetized, Acutely Prepared Rabbits on MAP,
CO, and TPR
At baseline, pregnant rabbits had significantly lower MAP
and TPR (Table 1). Although CO was
significantly higher in pregnant, the COI (159 ± 5 ml · min1 · kg1,
pregnant vs. 142 ± 11 ml · min1 · kg1,
nonpregnant) was not significantly different in pregnant than nonpregnant rabbits. BAY induced a greater elevation of MAP in acutely
prepared pregnant (n = 6) than nonpregnant
(n = 6) rabbits (Fig.
1A). Specifically, after 40 µg/kg BAY, MAP increased by 34 ± 1 mmHg in pregnant rabbits but
only by 23 ± 5 mmHg in nonpregnant rabbits. The difference
between the fractional increases was even greater (e.g., after 40 µg/kg BAY, MAP increased by 47 ± 1% in pregnant and 25 ± 5% in nonpregnant rabbits, P < 0.01). An enhanced pressor response developed in pregnant rabbits despite the simultaneous fall of HR in these animals, by about 69 beats/min (P < 0.01 vs. nonpregnant rabbits; Fig. 1B). The cardiac
deceleration of pregnant rabbits was associated with and partially
compensated by a 20% increase in SVI. The increase of SVI in pregnant
compared with nonpregnant rabbits could not be differentiated
statistically (Fig. 2A). BAY
reduced COI in both groups and, although the magnitude of reduction
tended to be greater in pregnant rabbits (Fig. 2B), it did
not achieve statistical significance. The absolute and fractional
changes of TPRI are shown in Fig. 3,
A and B, respectively. BAY induced a
dose-dependent increase of TPRI in both groups. The absolute changes of
TPRI were only numerically greater in pregnant vs. nonpregnant rabbits,
but when fractional changes were considered, the increase of TPRI was
twice as great in pregnant than in nonpregnant rabbits
(P < 0.02, Fig. 3B). Therefore, the enhanced hypertensive effect of BAY in pregnant rabbits appeared to be
a consequence of the heightened peripheral vasoconstriction induced by
the activation of vascular L-VDCC.
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Fig. 1.
Change of mean arterial pressure (A; MAP,
mmHg) and heart rate [B; HR, beats/min (bpm)] in
anesthetized, acutely prepared pregnant (P, n = 6) and
nonpregnant (NP, n = 6) rabbits after the intravenous
infusion of various doses of Bay K 8644, the L-type voltage-dependent
calcium channel activator (mean ± SE).
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Fig. 2.
Change of stroke volume index (A; SVI, ml/kg)
and cardiac output index (B; COI,
ml · min1 · kg1) in
anesthetized, acutely prepared pregnant (n = 6) and
nonpregnant (n = 6) rabbits after the intravenous
infusion of various doses of Bay K 8644, the L-type voltage-dependent
calcium channel activator (mean ± SE).
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Fig. 3.
Absolute (A) and fractional (B)
changes of total peripheral resistance index (TPRI) in anesthetized,
acutely prepared pregnant (n = 6) and nonpregnant
(n = 6) rabbits after the intravenous infusion of
various doses of Bay K 8644, the L-type voltage-dependent calcium
channel activator (mean ± SE).
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Effects of BAY in Conscious, Chronically Instrumented Rabbits
The baseline MAP was lower (78 ± 3 vs. 89 ± 2 mmHg, P < 0.01) and the HR was higher (241 ± 9 vs. 216 ± 14 beats/min, P < 0.05) in pregnant
than in nonpregnant control rabbits, respectively. The intravenous
administration of BAY increased MAP with the peak change developing
within the first 30 s after injection. MAP increased more in
pregnant than nonpregnant rabbits after the 10- and 25-µg/kg doses
(Fig. 4A). The absolute
increase of MAP in pregnant rabbits was remarkable, because the
baseline MAP of pregnant rabbits was >10 mmHg lower than in
nonpregnant controls. HR decreased comparably in both groups of rabbits
(Fig. 4B).
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Fig. 4.
Change of MAP (A; mmHg) and HR (B; beats/min)
in conscious, chronically instrumented pregnant (n = 10) and nonpregnant (n = 10) rabbits after the
intravenous bolus injection of various doses of Bay K 8644, the L-type
voltage-dependent calcium channel activator (mean ± SE). Baseline
MAP was 78 ± 2 mmHg in P and 89 ± 2 mmHg in NP rabbits
(P < 0.01). Baseline HR was 241 ± 8 beats/min in
P and 216 ± 14 beats/min in NP rabbits (P < 0.05).
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Effects of Nifedipine, an L-VDCC Antagonist, in Conscious,
Chronically Instrumented Rabbits
The baseline MAP was lower and the HR was higher in pregnant
than in nonpregnant controls (Table 2).
Nifedipine induced a dose-dependent decrease of MAP and an increase of
HR and these effects reached maximum between the 2nd and 3rd min after
injection. The maximal decrease of MAP and increase of HR were not
different between pregnant and nonpregnant rabbits.
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Table 2.
Effect of intravenously administered nifedipine on arterial pressure
and HR in conscious, chronically instrumented nonpregnant and pregnant
rabbits
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To confirm that the effect of BAY was mediated through specific
dihydropyridine receptors, five pregnant and five nonpregnant animals
received the agonist after prior administration of approximately equimolar amounts of nifedipine. The inhibition of the pressor effect
of BAY by nifedipine was virtually complete. For example, in pregnant
rabbits, 25 µg/kg BAY (mol wt 356.3) induced 18 ± 1 mmHg
elevation of MAP when given alone; however, when given after 25 µg/kg
nifedipine (mol wt 346.3), the change was 0 ± 3 mmHg
(P < 0.05). In nonpregnant rabbits, nifedipine
similarly inhibited the pressor effect of BAY.
Dihydropyridine Binding to Aortic Membranes
PN 200-110 (+)-[5-methyl-3H] binding parameters
in aortic membranes obtained from pregnant and nonpregnant rabbits are
shown in Table 3. There was a single
class of specific, saturable binding sites in each group. The
equilibrium dissociation constants (Kd, the
inverse of receptor affinity) and receptor number (Bmax)
did not differ for binding sites in aortic membranes from the two groups of rabbits.
Eicosanoid Production in Response to BAY in Aortic Rings
Basal production and release in vascular rings are defined as
production and release at 37°C minus the quantity in medium of the
vascular rings kept at 4°C, both in the absence of BAY. The basal
production and release of 6-keto PGF1 were similar in
the two groups (12.6 ± 2.3 pg/ng protein in pregnant and 9.2 ± 2.8 pg/ng protein in nonpregnant rabbits; not significant). Basal
production and release of TXB2 were greater in rings of pregnant vs. nonpregnant rabbits (0.32 ± 0.06 and 0.20 ± 0.03 pg/ng protein, P < 0.02).
Figure 5A shows that the
production and release of 6-keto PGF1 were increased in
rings of nonpregnant rabbits in response to BAY (1-100 ng/ml),
whereas in aortic rings of pregnant rabbits there was no significant
rise in production and release in response to BAY above basal level.
The synthesis of prostacyclin in response to stimulation by 10 and 100 ng/ml BAY was less in vascular rings of pregnant rabbits compared with
nonpregnant rabbits (P < 0.05). In contrast, no
difference (P > 0.05) was evidenced between the two
groups in the BAY-induced release of TXB2 (Fig.
5B).
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Fig. 5.
Net production and release of 6-keto PGF1
(A) and thromboxane B2 (B;
TXB2) by aortic vascular tissue obtained from nonpregnant
(n = 6) and 24-28 days pregnant (n = 6) rabbits in the absence (baseline) and presence of Bay K 8644 added
in vitro. Values were determined as quantity of eicosanoid in medium
after incubation of vascular tissue at 37°C minus quantity in medium
containing vascular tissue not incubated at 37°C but kept on ice.
n, Number of animals.
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DISCUSSION |
Attempting to determine the mechanisms of the gestational
alterations of cardiovascular pressor responsiveness is relevant, because the clinical manifestation of preeclamptic hypertension is
preceded by an early, definitive increase in the sensitivity to the
pressor effect of ANG II (12). In contrast, physiological human pregnancy is associated with a significant decrease of arteriolar tone and blunting of the responsiveness to several pressor compounds (9, 12), including ANG II and norepinephrine. ANG II and norepinephrine are also less potent systemic vasoconstrictors in
pregnant rabbits (10, 37) and other pregnant laboratory animals (36). A few studies suggested that there is a
corresponding downregulation of the specific receptors (2, 13,
18), but the data are inconsistent (3, 22). Because
gestationally related blunting of the vascular responsiveness is not
specific for one particular compound, seeking for a common cause of the abrogated effect of vasopressors seems to be logical. Attempts have
been made, using isolated vascular tissue of pregnant animals, to
explore the hypothesis that there may be a unifying cause underlying the altered actions of vasoconstrictors during gestation. Conrad et al.
(8) demonstrated that the inositol phosphate cycle was impaired in isolated aortas of pregnant rats. The response of isolated
mesenteric arteries to vasopressin was found to be blunted in pregnant
rats by St. Louis et al. (34), and the authors suggested that this may have been a consequence of impaired opening of the L-VDCC.
The hypothesis that the altered vascular L-VDCC is the cause of the
nonspecific vascular hyporesponsiveness in pregnancy has not been
directly tested in a whole animal experiment. In our study, chronically
prepared conscious and anesthetized acutely instrumented pregnant and
nonpregnant rabbits were given BAY, a specific agonist of the L-VDCC.
This agent has been shown to elevate blood pressure in various species
(6, 26, 31). The hypertensive response to BAY was
enhanced, not attenuated, in pregnant rabbits. A pregnancy-induced
impairment of the vascular L-VDCC was not confirmed in these in vivo
studies. Why opposite changes were observed when testing the intact
aorta of pregnant rats in vitro (11) and the systemic
vasculature of pregnant rabbits in vivo (present study) cannot readily
be explained. Similar observations, however, have been made in pregnant
rabbits with U46619, a thromboxane analog. We found U46619 to be a more potent systemic arterial vasoconstrictor in pregnant rabbits
(20) and rats (16) than in nonpregnant
animals. More recently we compared the reactivity of the gracilis
arteriole of pregnant and nonpregnant rabbits in an in vitro system in
which resistance size vessels can be studied (15). In
contrast to the in vivo enhancement of the vasopressor effect, the in
vitro arteriolar sensitivity to U46619 was blunted (L. Ungvari, G. Brown, P. Pacher, R. Venuto, A. Koller, and G. Losonczy, unpublished
observations). Thus, in rabbit pregnancy, the in vivo change of
vascular response to U46619 is directionally opposite to the change
observed in vitro, just as they appear to be with BAY.
The blockade of the L-VDCC by nifedipine resulted in similar reductions
in MAP in pregnant and nonpregnant rabbits, which implies that the
L-VDCC contributed to the maintenance of the resting vascular tone
equally in the two groups. This supports the notion of unimpaired
L-VDCC in intact pregnant rabbits. The same conclusion could be drawn
from the biochemical studies, which indicated undiminished number and
unchanged affinity of aortic L-VDCC receptors. Although the aorta is
perceived as more of a conduit than a resistance vessel, it has
specific binding sites for BAY (28) and it contracts in
response to BAY (28, 29). Furthermore, the aorta has been
shown to develop a comparable decrease of sensitivity to pressor
compounds during pregnancy as observed in resistance vessels
(24). The Bmax of PN 200-110 estimated by
us in aortic membranes was comparable to earlier reported values in
left ventricular membranes of female rabbits (26).
The number and affinity of the vascular L-VDCC do not necessarily
parallel its functional response to specific ligands (28, 35). A potential explanation for observations of unaltered
receptor number and affinity in the presence of altered responses to
ligands (BAY and U46619) may be that there is an effect of
transmembrane potentials on receptor number or affinity
(1) that is dissipated in isolated membrane preparations.
We observed a difference in ANG II receptor number between pregnant and
nonpregnant vascular tissues when using intact glomeruli
(2), but not when using isolated vascular membrane
preparations (3). Roy et al. (29) also
reported differences in binding of a calcium channel blocker between
tissues of pregnant and nonpregnant animals when using intact aortic
rings, but not when using aortic membrane preparations (28). There is evidence that vascular tissues in pregnant
animals are hyperpolarized (25, 29, 34). Roy et al.
(28) reported that when aortic strips from pregnant and
nonpregnant rats were precontracted by KCl to the same level, but by
different concentrations of KCl, the contraction-response curves to BAY
were identical, suggesting an effect of membrane potential on the
response to BAY. It would be expected that hyperpolarization of
vascular plasma membranes would lower the sensitivity of vascular
tissue to contractile ligands. In vitro arteriolar sensitivity to BAY
and to U46619 is not enhanced in pregnant animals (28,
38). It could be reasoned that once threshold potential is
attained in excitable tissues, the in vivo contractile response to BAY
or to U46619 could be greater due to a greater store of
Ca2+ in sarcoplasmic reticulum of pregnant vs. nonpregnant
vascular tissue. This is possibly due to prolonged hyperolarization
(causing decreased sensitivity to contractile agents) of plasma
membranes. In this regard, Sagawa et al. (30) showed that
lipophilic dihydropyridine (DHP) agonists such as BAY exert their
effect in contractile tissue by actions on both the L-type
Ca2+ channel and on the sarcoplasmic reticulum. Their data
indicate that DHP agonists bind not only to L-VDCC receptors, but also to sarcoplasmic Ca2+ release channels (ryanodine
receptors). In isolated adrenal glomerulosa cells, BAY potentiated
KCl-induced Ca2+ uptake and intracellular Ca2+
concentration in cells from pregnant rats but not in cells from nonpregnant rats (32). The enhanced intracellular
Ca2+ concentration in response to BAY in cells from
pregnant rats was not inhibited by nifedipine. Their data support the
concept that calcium may be sequestered intracellularly during pregnancy.
Another potential explanation for the enhanced blood pressure
response to BAY is that this drug opens the mostly closed [or impaired
(28, 29)] L-VDCC in the resistance vasculature of pregnant rabbits. This, in turn, permits the otherwise limited action
of the much higher endogenous, circulating concentrations of ANG II and
norepinephrine that characterize pregnant rabbits (10, 37)
to fully manifest their vascular effect. Consistent with this notion,
BAY has been shown to enhance the pressor effect of phenylephrine
(28).
Pregnancy enhances vascular prostacyclin release in response to
pressor compounds (23), but the response to BAY had not been tested. We found that intact aortic rings of pregnant rabbits released less prostacyclin in response to BAY than did the tissue of
nonpregnant rabbits. It is speculated that the diminished
release of prostacyclin may provide at least a partial explanation for the enhanced pressor effect of BAY in pregnant rabbits. There may be
some danger, however, in attributing a systemic effect to a compound
better known for its local action. This alteration may be specific for
prostacyclin, because the BAY-induced release of thromboxane remained
unchanged in aortic rings of pregnant rabbits.
In summary, downregulation of the L-VDCC in rabbit pregnancy does
not appear to contribute to the nonspecific blunting of the pressor
response to ANG II or epinephrine. The increase of the peripheral
vascular resistance in response to pharmacologic activation of these
calcium channels is not only unimpaired but also enhanced in rabbit
pregnancy, at a time when the effect of most, although not all
(16, 20), pressor compounds becomes weaker. We attempted
to extrapolate the results of the pharmacologic manipulations of the
L-VDCC to the physiology of mammalian pregnancy, but such
interpretations could be misleading.
Perspectives
Systemic hypotension and refractoriness to vasoconstrictive agents
are considered hallmarks of physiological mammalian pregnancy. The
studies reported herein confirm that some prohypertensive agents have
an enhanced rather than a blunted response in intact pregnant rabbits.
Such a response could not (necessarily) have been predicted from the
results of studies employing isolated tissues. These and other data
emphasize the importance of investigation using whole animals. These
results also suggest the possibility that during pregnancy,
intracellular calcium may be in greater quantity and more readily
released from binding sites. Once a threshold stimulus is provided, the
response of these cells may be exaggerated. Such a hypothesis is
consistent with the rapid transition from hypotension to hypertension,
which occurs with potentially dire consequences in pregnant women who
develop preeclampsia.
| |
ACKNOWLEDGEMENTS |
This study was supported by The Renal Research Fund of the
University of Buffalo Foundation (to R. Venuto), Grant 9707953A of the
American Heart Association/New York State Affiliate (to R. Venuto and
G. Brown), The Hungarian National Science Fund, T025422 (to G. Losonczy), and The Western New York Kidney Foundation/Upstate NY
Transplant Services (to R. Venuto and G. Brown).
| |
FOOTNOTES |
Address for reprint requests and other correspondence:
R. C. Venuto, Nephrology Division, SUNY at Buffalo, School
of Medicine and Biomedical Science, Erie County Medical Center, 462 Grider St., Buffalo, NY 14215 (E-mail:
rvenuto{at}ecmc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 21, 2001;10.1152/ajpregu.00472.2001
Received 6 August 2001; accepted in final form 7 December 2001.
| |
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