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*
Department of Nephrology, Rambam Medical Center, B. Rappaport Faculty of
Medicine, Technion, Haifa, Israel
Department of Physiology and Biophysics, Rambam Medical Center, B.
Rappaport Faculty of Medicine, Technion, Haifa, Israel
Obstetrics and Gynecology, Rambam Medical Center, B. Rappaport Faculty of
Medicine, Technion, Haifa, Israel.
Correspondence to Dr. Jacob Green, Department of Nephrology, Rambam Medical Center, Bat-Galim Haifa 31096, Israel. Phone : +972 4 8543251 ; Fax : + 972 4 8542946 ; E-mail greeny{at}rambam.health.gov.il
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Notwithstanding the central role ascribed to the endothelium in mediating the abnormalities observed in preeclampsia, there have been very limited data related to the function and biochemical alterations in the vascular smooth muscle cell (VSMC), when studied in isolation (i.e., independent of the co-presence of overlying endothelium). Also, whether preeclamptic serum affects VSMC function in a similar manner as it does when applied to cultured endothelial cells has not been investigated. Because intracellular Ca2+ ([Ca2+]i) is a major determinant of VSMC tone and function, and in view of many reports implicating altered calcium metabolism in the pathogenesis of preeclampsia (reviewed in reference (14), we sought to determine the metabolism of [Ca2+]i in VSMC after exposure to sera from hypertensive versus normotensive pregnancies.
To address the question underlying this study, we applied sera taken from preeclamptic women to normal cultured VSMC and measured [Ca2+]i metabolism. Measurements in the preeclamptic state were compared with those made after delivery, when BP was again normal. Cytosolic Ca2+ was also measured in VSMC exposed to sera from normotensive pregnant women and pregnant women with essential hypertension as well as from non-pregnant women with and without hypertension.
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2+ by dipstick)
(Table 1). An additional
characteristic of preeclampsia found in these patients was hyperuricemia
(serum uric acid 5.9 ± 0.4 mg/dl ; normal range, 2.4 to 4.3 mg/dl).
None of the preeclamptic women manifested coagulation defects or liver
function abnormalities. Also, none of the 15 women in the preeclamptic group
had a past history of hypertension, diabetes, or renal disease before
pregnancy. Mean BP values in the preeclamptic group were 157 ± 102
mmHg.
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The women with preeclampsia were recruited from the perinatal ward of the Rambam Medical Center, where they were hospitalized because of an increase in BP in the third trimester. The patients with preeclampsia and the control subjects were following no special diet, and there was no restriction of salt intake. In all patients, blood was drawn 1 to 2 d after hospitalization. The preeclamptic women were not using antihypertensive medications before the study, nor were they given intravenous magnesium before or after the study.
The normotensive pregnant control subjects were hospitalized because of obstetrical complications at the time of the study, but were otherwise healthy ; they remained normotensive throughout their pregnancies. None of these women was taking medications other than iron or vitamins. The PE and the normotensive pregnant women were similar in age and body weight (PE group : 22 to 34 yr, mean age 27 yr, mean body weight 65 ± 5.4 kg ; normotensive pregnant women : 19 to 38 yr, mean age 25 yr, mean body weight 59 ± 6.4 kg).
The eight pregnant women with essential hypertension had mean BP values of 162/105 mmHg. The non-pregnant women with hypertension were recruited from among patients at our hypertension clinic. These patients were selected because they were taking antihypertensive drugs similar to those received by the pregnant women with essential hypertension. None of the hypertensive women (i.e., pregnant or non-pregnant women with essential hypertension) was receiving either ß blockers or angiotensin-converting enzyme inhibitors before or after the study. All medications were discontinued 12 to 24 h before the study. The non-pregnant normotensive control subjects were members of the medical and laboratory staff.
The study protocol was approved by the Institutional Review Board on Human
Investigation of the Rambam Medical Center, Technion School of Medicine, and
all of the women provided signed informed consent. In all five groups of
women, venous blood (15 to 20 ml) was drawn between 7 a.m. and 9:30 a.m., at
least 12 h after the intake of the last antihypertensive medication. Prepartum
blood samples were obtained 
24 h before delivery. In five preeclamptic
women, blood was redrawn 5 wk after delivery. For serum preparation, samples
were centrifuged at 2000 x g for 30 min and stored at -20°C
until use.
Cell Cultures
Primary Cultures of VSMC.
VSMC from Sprague Dawley rats were isolated after a well established
procedure (15). Briefly, after
dissection of the rat thoracic aortae, the vessels were cleared of fat and
then incubated for 10 min at 37°C in Hanks' balanced salt solution
containing 0.2 mg/ml elastase, 2 mg/ml collagenase, and 0.1 mg/ml soybean
trypsin inhibitor. This ensures removal of the endothelial cells. The cleaned
vessels were cut into 1- to 2-mm pieces and digested in the fresh Hanks'
balanced salt solution enzyme solution. The cell suspension was centrifuged at
200 x g for 5 min, and the pellet was resuspended in Dulbecco's
modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf
serum (FCS), 2 mmol/L L-glutamine, 100 U/ml penicillin, and 100
µg/ml streptomycin. The dispersed cells were aliquoted into tissue culture
flasks and incubated at 37°C in a humidified 5% CO2/95% air
atmosphere. Some cell aliquots were subcultured into Petri dishes (100 mm
diameter) containing 19-mm diameter type 0 glass coverslips, and the cells
were grown to conference.
All experiments were performed with cells between 6 and 12 passages. Forty-eight hours before the experiment, the cells were reincubated in serum-free medium. On the day of the experiment, confluent cells were washed once with culture media and reincubated with fresh media containing sera from the different subject groups. In preliminary experiments done in our laboratory, we used several concentrations of sera : 2, 10, and 20%. The cells were incubated with the different sera groups, at each concentration, for three time periods : 4, 8, and 24 h. We found that the maximal response to sera (as specified later) was obtained after incubation of the cells with 10% sera for 4 h. The responses at 10% sera were bigger than those obtained at 2% sera. However, 20% sera did not yield an additional increment in the magnitude of the response, when compared to 10% sera. In addition, throughout the experiments, the effects of plasma were identical to those of sera taken from the same individual subjects. Therefore, unless indicated otherwise, all experiments were done in VSMC cultured in 10% sera for 4 h.
A-10 Cells.
Rat thoracic aortic smooth muscle cells (A-10 ; American Type Culture
Collection CRL 1476, Manassas, VA) were cultured in DMEM plus 20% FCS. All
experiments were performed with cells passaged once a week for no more than 2
mo. Culture wells (35 mm diameter, 6-well Linbro plates ; Flow Laboratories,
McLean, VA) were seeded with 1 ml of medium containing 7.5 x
104 cells. The cells were allowed to grow in culture for 3 d.
Determination of
[Ca2+]i
Trypsinized VSMC.
Measurements of free [Ca2+]i were made by
incorporating the calcium-sensitive fluorescence probe fura-2 into VSMC. Cells
were released from tissue culture plates by light trypsinization and washed
twice with balanced salt solution (BSS) containing the following (in mM) : 140
NaCl, 1 MgCl2, 1.5 CaCl2, 5 KCl, 10 Hepes, 5 glucose, pH
7.4 (adjusted with Tris base). The cells were incubated with 2 µM fura-2 AM
in a shaking water bath at 37°C for 30 min and then washed and resuspended
in BSS. Aliquots of suspended cells were added to plastic cuvettes containing
2 ml of prewarmed BSS, and the cuvettes were seated in a Perkin-Elmer 650-40
spectrophotometer (Norwalk, CT). Photon emission was monitored at 510 nm with
excitation wavelengths alternating between 340 and 380 nm. Cells were
continuously stirred and kept at 37°C inside the spectrophotometer.
To calibrate the fura-2 signal, medium Ca2+ was adjusted to 2 mM and the cells were lysed with digitonin (50 µg/ml) to obtain the maximal fluorescence. Next, 10 mM ethyleneglycol-bis(ß-aminoethyl ether)-N,N'-tetra-acetic acid and sufficient NaOH to elevate the pH to 8.5 were added to obtain the minimum fluorescence. The dissociation constant for Ca2+ -fura-2 was assumed to be 220 nM, and the calculation of [Ca2+]i was similar to that described previously (16). To eliminate the effects of autofluorescence due to the cuvette medium and cells, the fluorescence was measured with an empty cuvette after addition of medium and after addition of cells without fura-2. Unloaded cells produced very minimal and almost undetectable fluorescence. Correction for the autofluorescence of the cuvette and medium was made by setting the fluorometer on autozero before each measurement.
Determination of [Ca2+]
Adherent VSMC.
Vascular smooth muscle cells (primary cultures) grown on coverslips were
loaded for 25 min at room temperature (24 to 25°C) with fura-2 AM at a
final concentration of 5 µM, in a 1:1 mixture of Tyrode's solution and a
dissociation solution containing 2% bovine albumin. Excess fura-2 was removed
by rinsing twice with Tyrode's solution. Cells were then transferred to a
nonfluorescence chamber mounted on the stage of an inverted microscope
(Diaphot 300 ; Nikon, Tokyo, Japan) and visualized with a x40 oil
immersion Neofluor objective. The chamber was perfused with Tyrode's solution
at a rate of 1 ml/min. Experiments were performed at 31 to 32°C. Fura-2
fluorescence was measured using a dual wavelength system (DeltaScan ; Photon
Technology International, South Brunswick, NJ). Briefly, light emitted from an
Xenon arc lamp was fed in parallel into two independent monochromators to
obtain quasimonochromatic light beams of two different wavelengths, exciting
the cell at 340 and 380 nm. Either a 340- or 380-nm wavelength was selected by
a rotating chopper disk at a frequency enabling ratio measurements at a rate
of 150 counts/s. The two separate monochromator outputs were collected by the
ends of a bifurcated quartz fiber optic bundle. The emitted fluorescence (510
nm) was collected by the microscope optics, passed through an interference
filter, and detected by a photomultiplier tube (710 PMT Photon Counting
Detection System ; Photon Technology International). Raw data were stored for
off-line analysis by Felix software (Photon Technology International) as 340-
and 380-nm counts, and as the ratio F340/F380. For
scaling the fluorescence ratio, cell-derived autofluorescence and non-cell
fluorescence were subtracted from the measured fluorescence.
Platelet Ca2+.
Blood samples from 10 healthy volunteers (approximately 15 ml each) were
collected in ethylenediaminetetra-acetic acid-treated Vacutainer tubes and
centrifuged at 200 x g for 15 min at 21°C for the
determination of platelet [Ca2+]i. The platelet-rich
plasma was incubated in 3 µmol of fura-2 acetoxymethyl ester (fura-2) per
liter at 37°C for 30 min and centrifuged at 650 x g for 10
min at 21°C ; the plasma and extracellular fura-2 was removed by
aspiration. The platelet pellet was then suspended in a calcium-free Hepes (10
mM) buffer (pH 7.4) and centrifuged at 650 x g ; the platelets
were suspended at a concentration of 1 x 107 cells per
milliliter in Hepes (10 mM) containing calcium (1.5 mM). Washed platelets were
then collected and incubated at 37°C with Ca2+ containing Hepes
solution mixed with 10% sera from the different subject groups.
[Ca2+]i was determined fluorometrically as described
above for VSMC in suspension. Compared with the experiments done in VSMC, the
experiments with platelets were completed within 3 h of blood sampling, as
preliminary experiments in our laboratory showed that after this time,
platelets lose membrane integrity and the basal calcium level tends to
rise.
Reagents and Hormones
Angiotensin II (AngII), vasopressin, and endothelin-1 were purchased from
Sigma Chemical Co. (St. Louis, MO). Thapsigargin was obtained from Calbiochem
and fura-2 AM was from Molecular Probes (Eugene, OR). Cell culture media and
all other chemicals were purchased from Sigma with the exception of those
specifically described.
Statistical Analyses
Comparison between the different subject groups was performed by unpaired
t test. Analysis of the differences between initial and final values
within a given group was done by paired t test and/or ANOVA. The
results are presented as means ± SD, and significant differences
correspond to a P value <0.05.
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The relative differences in [Ca2+]i responses among the various sera groups were maintained at two AngII concentrations (10-7 M, top panel of Figure 1, versus 10-8 M, bottom panel). The absolute values of peak [Ca2+]i were, however, of smaller magnitude after stimulation with 10-8 M AngII compared to 10-7 M AngII, thereby establishing a dose-response relationship to AngII. Therefore, for the rest of the experiments we used AngII 10-8 M. Time course analysis revealed that the best response generated with the different sera was obtained after exposure of the cells to the respective sera for 4 h compared with 8- and 24-h time intervals. This finding suggested to us that the longer incubation times result in receptor downregulation, thereby causing an attenuated hormonal response.
In cells preincubated with FCS alone for 4 h, the acute response to AngII (at the two doses) was comparable to the response observed in cells preexposed to serum from normotensive non-pregnant women (not shown). Preincubation for 4 h in buffer alone (i.e., serum-free media) resulted in greater [Ca2+]i response when compared with the response after incubation with FCS (peak [Ca2+]i after stimulation with 10-8 M AngII, 360 ± 15 and 424 ± 11 nM after 4-h preincubation with FCS and buffer alone, respectively). Preincubation in FCS or serum-free media did not, by themselves, alter basal [Ca2+]i.
Summary of the data related to the effect of sera on AngII (10-8 M)-induced Ca2+ transients (expressed as percent [Ca2+]i increment over baseline) showed the following : NTP (n = 22) 443 ± 22%, PE (n = 25) 184 ± 18%, pregnant EHT (n = 8) 259 ± 12%, non-pregnant EHT (n = 12) 274 ± 23%, NNP (n = 18) 255 ± 15% (P < 0.01 PE versus NTP ; P < 0.05 PE versus NNP, pregnant, and non-pregnant EHT ; P < 0.05 NTP versus NNP, pregnant, and non-pregnant EHT).
Figure 2 shows that the same qualitative results as shown above (Figure 1) for AngII-evoked Ca2+ transients in VSMC were also seen when cells were acutely stimulated with two other vasoactive hormones, vasopressin (top panel) and endothelin-1 (ET-1) (bottom panel). The qualitative differences between the effects of sera from normal pregnant women, PE women, and normotensive non-pregnant women on vasopressin and ET-1-induced [Ca2+]i responses were similar to those obtained with AngII-induced [Ca2+]i responses.
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The studies described in Figure 1 2 were performed in primary VSMC in suspension. We therefore repeated the same studies in adherent cells. Figure 3 shows results obtained in rat aortic VSMC attached to glass coverslips. The cells were preexposed to sera from the different subject groups and acutely stimulated with 10-8 M AngII. The pattern of [Ca2+]i responses was similar to that obtained with trypsinized suspended cells, i.e., marked attenuation of hormonally induced [Ca2+]i responses by PE sera compared to normotensive pregnant sera. Values in arbitrary fluorescence units (percent increment over baseline) were as follows : NTP 370 ± 14%, PE 65 ± 12%, EHT (non-pregnant) 205 ± 14%, and NNP 236 ± 16% (P < 0.004, NTP versus PE sera ; P < 0.05 PE versus NNP and EHT). Because suspended cells behave in a manner similar to adherent cells, all further experiments used suspended cells, unless otherwise specified.
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In Figure 4, we tested the effect of sera on A-10 cells, a vascular muscle cell line derived from rat thoracic aorta, and on platelets derived from healthy volunteers. Platelets were used because they bear a functional resemblance to VSMC. After determination of basal [Ca2+]i, these cells were stimulated with 10-8 M AngII, whereas A-10 cells were stimulated with vasopressin (10-7 M), since they usually do not respond to AngII (17). The results show again the same pattern as shown above in primary cultured VSMC (i.e., marked suppression of hormonally induced Ca2+ transients by PE sera, whereas pregnant sera manifests a greater response compared to normotensive, non-pregnant sera).
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Table 2 shows that the different sera affected hormonally induced Ca2+ transients both in the absence and presence of Ca2+ in the extracellular media. The results obtained in Ca2+-free media show the same qualitative differences between the various groups, as demonstrated in normal Ca2+ conditions. These data indicate that sera can affect Ca2+ mobilization from intracellular organelles after acute stimulation of the cells with an agonist.
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The release of Ca2+ from intracellular pools is dependent on the breakdown of phosphoinositols and the generation of inositol 1,4,5-trisphosphate (IP3). However, Ca2+ mobilization also depends on the size of the intracellular Ca2+ pool. We therefore evaluated whether preincubation of the cells with the different sera has any effect on the [Ca2+]i pool. To that end, cells preincubated for 4 h with different sera were acutely exposed to thapsigargin (TG), a selective inhibitor of the Ca2+ -ATPase residing on the endoplasmic reticulum membrane. This drug was added in the absence of Ca2+ in the extracellular media. Figure 5 shows that after acute exposure of cells to TG (1 µM) (Figure 5, traces A and B), there is an instantaneous rise in [Ca2+]i (from a baseline value of 82 ± 6 nM to a peak of 220 ± 12 nM). There was, however, a comparable response between cells preincubated in NTP sera and cells preincubated in PE sera. To evaluate whether alterations in AngII-mediated responses in normal and hypertensive pregnancies are dependent on TG-sensitive Ca2+ stores, cells were stimulated with AngII after treatment with TG. Under these conditions, the level of Ca2+ transients in cells preincubated in PE sera (Figure 5, trace B) was once again markedly blunted when compared with the value obtained in cells incubated with NTP sera (Figure 5, trace A) (P < 0.01). The [Ca2+]i response to AngII in cells preincubated with PE sera was the same regardless of whether TG was used (Figure 5, trace C versus trace B).
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Ca2+ Influx versus Ca2+ Release from
Intracellular Pools
To further discern between an effect of sera on hormonally induced
Ca2+ release from intracellular pools versus a possible
effect on hormonally induced Ca2+ entry across the plasma membrane,
we used manganese (Mn2+) as a surrogate for Ca2+.
Mn2+ presumably enters cells via the same pathways as
Ca2+ and has been used as a substitute for Ca2+ in
studies of Ca2+ influx. Once Mn2+ accumulates in the
cytosol, it cannot be transported by Ca2+ pumps or accumulated in
intracellular Ca2+ stores. Thus, the extent of fluorescence
quenching at the isosbestic point for fura-2 fluorescence provides an estimate
of the unidirectional Ca2+ entry
(17). In the experiment
described in Figure 6, VSMC
preincubated with either NTP sera (Figure
6, traces A and a) or with PE sera
(Figure 6, traces B and b) were
acutely (5 min) exposed to calcium-free media with
(Figure 6, traces a and b) or
without (Figure 6, traces A and
B) 10 µM MnCl2. After stabilization of the fura-2 signal, basal
[Ca2+]i stabilized at values of 75 ± 8 and 87
± 7 nM in NTP and PE sera-treated cells, respectively (P =
NS). Acute addition of AngII (10-8 M) elicited a Ca2+
transient, which, under the Ca2+ -free conditions, signifies
Ca2+ mobilization from intracellular stores. Once again, the
[Ca2+]i response was significantly attenuated by PE sera
compared to the effect of NTP sera (Figure
6, traces B and b versus traces A and a) The effect of
sera on AngII-evoked [Ca2+]i transients was the same
regardless of the absence (Figure
6, traces A and B) or the presence
(Figure 6, traces a and b) of
MnCl2. In cells exposed to MnCl2
(Figure 6, traces a and b), the
initial fast rise in fura-2 fluorescence after the addition of AngII was
followed by fluorescence quenching, signifying Mn2+ influx. As
shown in the figure, in cells pretreated with PE sera for 24 h
(Figure 6, trace b), the
magnitude of the fluorescence quench was markedly smaller when compared to the
response in cells preincubated with NTP sera
(Figure 6, trace a). It
appears, therefore, that the NTP and PE sera affect both Ca2+
mobilization from intracellular stores and the process of hormonally operated
Ca2+ influx. The Mn2+-induced fluorescence quenching
could be blocked by 10 µM La3+ (data not shown). The
Mn2+ experiments as performed with AngII were also carried out with
vasopressin (10-7 M). Similar qualitative results were obtained
(not shown).
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Because hormonally induced Ca2+ influx can result from changes in membrane potential (i.e., voltage-operated Ca2+ influx) (18), we tested the effect of the different sera groups on depolarization-activated Ca2+ channels in VSMC. In Figure 7, VSMC were acutely exposed to BaCl2 in Ca2+-free media. Barium blocks the exit of potassium from the cells, thereby causing membrane depolarization. Cell depolarization activates a voltage-gated channel through which barium (in this case, replacing Ca2+) can enter the cell. The entry of barium is detected by its binding to fura-2, which elicits a fluorescence signal. Because accurate calculation of the Kd of fura-2 to barium is very difficult, it was impossible to calibrate the barium signal. Nonetheless, Figure 7 clearly shows that NTP sera causes the most striking signal when compared to other sera, whereas PE sera brings about a markedly suppressed Ba2+ entry. The response to NNP and EHT sera is intermediate between NTP and PE. If hormones induce Ca2+ entry in VSMC through a voltage-dependent mechanism, the results presented in Figure 7 could provide a mechanistic explanation for the effect of sera from the different subject groups on hormonally induced Ca2+ entry.
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Post-Delivery Recovery from the Effect of PE Sera on
[Ca2+]i
In five preeclamptic women, none of whom was treated by antihypertensive
medications during pregnancy, blood was redrawn 5 wk after delivery at a time
when BP returned to normal and proteinuria disappeared. Serum was then applied
to VSMC (4 h incubation), and the acute effect of AngII (10-8M) on
[Ca2+]i was determined. As shown in
Figure 8, the effect of sera as
observed during the PE period disappeared completely after delivery, such that
the [Ca2+]i responses normalized and became much closer
to values obtained with sera from normal non-pregnant women. Thus, the values
for percent [Ca2+]i increments over baseline in response
to AngII (10-8M) were 184 ± 12% (PE, pre-delivery)
versus 302 ± 15% (post-delivery) (P < 0.05). These
data suggest that a circulating factor in the serum of PE women is responsible
for the attenuated [Ca2+]i responses to hormones. After
delivery, this putative factor probably disappears from the circulation, which
brings about the normalization of [Ca2+]i responses to
hormonal stimulation.
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The changes observed in PE cannot be ascribed to the hypertensive state per se. Thus, although BP values in the pregnant, essential hypertensive group were comparable to those in the PE women (162/105 versus 157/102 mmHg, respectively), sera from pregnant women with essential hypertension elicited changes in [Ca2+]i that differed significantly from PE sera and were comparable to those obtained with hypertensive non-pregnant sera. The magnitude of the latter ranged somewhere between the maximum response obtained by normotensive pregnant sera and the minimum response seen with PE sera. Therefore, we conclude that the changes in [Ca2+]i responses elicited by PE sera are over and beyond those obtained by hypertension alone.
One could argue that the changes in hormonal-induced Ca2+ transients in VSMC are attributable to differences in binding properties of the agonist to VSMC preincubated with the different sera. However, we believe that this is an unlikely possibility given that in normal pregnancy and PE the endogenous level of renin and AngII (i.e., very high in normal pregnancy, lower than normal pregnancy in PE) (19) would be expected to cause [Ca2+]i responses to acute hormonal administration, which are in contrast to the actual results obtained in our study. Furthermore, serum from PE has been recently shown to contain an antibody that binds to the AngII AT1 receptor and has agonistic activity (20). Therefore, altered AngII receptor function cannot account for the phenomena described in our study.
The effect of sera on [Ca2+]i is exerted both on [Ca2+]i mobilization from intracellular stores (assessed by the [Ca2+]i responses to hormones in the absence of Ca2+ in the extracellular buffer) and on Ca2+ influx from the extracellular media. The latter effect is clearly demonstrated by tracing fluorescence changes induced by Mn2+, which serves as a surrogate for Ca2+ entry pathways. Furthermore, because hormones stimulate Ca2+ influx through diverse mechanisms (18), we tested the possibility that a voltage-dependent Ca2+ channel (VDCC) was also affected by the different sera. In fact, we found that VDCC (assessed by using barium as a blocker of K+ exit, thereby inducing membrane depolarization) was affected by sera in the same direction as were the [Ca2+]i signals evoked by hormones. Thus, normal pregnant sera amplify, whereas PE sera blunt VDCC. It is therefore possible that the effect of sera on hormonally induced Ca2+ influx is partly attributable to their effect on voltage-dependent Ca2+ channels activated by the various agonists.
An alternative mechanism for the altered hormonally induced [Ca2+]i influx could be related to the "cross-talk" between the process of Ca2+ release from internal stores and Ca2+ influx. Calcium ions are released from intracellular stores in response to agonist-stimulated production of IP3 (21). Thus, it is possible that PE sera (compared to normotensive pregnant sera) exert an inhibitory effect on IP3 generation or cause a resistance to the effect of IP3. Furthermore, a large body of evidence has been accumulated indicating that the agonist-induced release of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane (22). The influx of Ca2+ can be recorded as a store-operated channel in the plasma membrane, which has now been shown to interact directly with IP3 receptors (23). Based on these recently gathered data, one could speculate that the reduced Ca2+ release by PE sera is directly linked to an inhibition of Ca2+ influx as well. Finally, we ruled out the possibility of altered intracellular storage pools brought about by PE sera. This was done by using TG, a potent inhibitor of Ca2+ ATPase in the endoplasmic reticulum. Thus, exposure of cells to TG produced [Ca2+]i responses in VSMC preincubated in PE sera that were comparable to those seen in cells preincubated with normal pregnant sera. Nevertheless, when AngII was acutely added to the cells after exposure to TG, the cells preincubated with PE sera manifested again a suppressed [Ca2+]i response when compared to cells treated with normotensive pregnant sera, suggesting that the different sera affect a TG-insensitive intracellular Ca2+ pool in VSMC.
The distinct effects of sera from PE and normal pregnant women on [Ca2+]i responses in VSMC, when taken together with the data showing that the effect of PE sera disappeared shortly after delivery, suggest the presence of a putative "serum factor." We speculate that in women with PE, the putative "serum factor," having a suppressive effect on [Ca2+]i in VSMC, operates as a "defense mechanism" against the intense vasoconstriction characteristic of PE. This factor may be deficient in normotensive pregnant women, which then allows for the augmentation of [Ca2+]i signals, thereby counterbalancing the reduced peripheral vascular resistance often seen in normal pregnancy.
The concept of a circulating "serum factor" in PE has been suggested by different investigators who invoked endothelial activation and dysfunction as major contributors to the pathogenesis of PE (5,6,7,8,9). It was thus postulated that an as yet unidentified factor released from the ischemic placenta into the maternal circulation is responsible for altered endothelial function (7). Interestingly, most of the studies evaluating the effect of PE sera on isolated normal endothelial cells yielded unexpected and "paradoxical" results. Thus, although the in vivo state of PE is characterized by elevated peripheral vascular resistance and exaggerated vascular response to vasoconstrictors (24), the in vitro incubation of PE sera with normal cultured endothelial cells results in reduced production of the potent vasoconstrictor endothelin (25) and augmented production of the vasodilators nitric oxide (9, 26) and prostacyclin (8, 26). Haller et al. have recently shown (13) that in normal endothelial cells preexposed to PE sera, [Ca2+]i is elevated. Again, this would suggest that PE sera is responsible for the upregulation of nitric oxide synthesis in endothelial cells, since [Ca2+]i is the main regulator of endothelial nitric oxide synthase. Furthermore, the stimulatory effect of PE sera on prostacyclin production in normal cultured endothelial cells stands in sharp contrast to the clinical observation that in PE women, reduced prostacyclin production is an early event that occurs many months before the clinical onset of PE (27).
Similar to the surprising effects of PE sera on endothelial cells, our study indicates that PE sera also exerts "paradoxical" effects on [Ca2+]i metabolism in normal cultured VSMC, when studied in isolation (i.e., denuded of endothelial cells). Along these lines, we have shown that sera from PE and normal pregnant women affect [Ca2+]i in normal platelets (often used as surrogates for VSMC) in a manner similar to that observed in VSMC. These results are in contrast to other studies showing that [Ca2+]i is elevated in platelets taken from hypertensive pregnant women, when compared to platelets from normotensive pregnant women (28,29,30). However, studies on platelets have not yielded consistent results, and the view that platelet [Ca2+]i correlates with BP values has not been universal (31, 32). Thus, similar to our data, other studies have also shown reduced (rather than augmented) agonist-stimulated Ca2+ transients in platelets from PE women compared to platelets from normotensive pregnancies (31). In addition, different studies vary in their results regarding the specific hormone to which the "hypertensive" platelets are sensitive (e.g., vasopressin versus AngII) (28, 29). The explanation for the discrepancies among the different studies is not clear. It casts doubt, however, on the validity of making trivial extrapolations from platelets to VSMC function.
Overall, based on the foregoing studies, one may conclude that the in vivo state of PE differs from the in vitro effect of PE sera on normal endothelial and vascular muscle cells. The studies on VSMC, as shown here, would suggest that PE sera harbor one or more "protective" factors, which mitigate the unfavorable hemodynamic profile characteristic of PE.
In summary, this study demonstrates that sera from normotensive pregnant women and PE women (as opposed to sera from normal-non-pregnant women and non-pregnant hypertensive women) exert distinct changes on cellular Ca2+ metabolism in normal VSMC. Whether these effects can be used as an in vitro predictor of preeclampsia is a matter for further investigation, requiring longitudinal studies throughout gestation.
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