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Abnormalities in intracellular Ca²⁺ regulation in fetal vascular smooth muscle in pre-eclampsia: enhanced sensitivity to arachidonic acid¹

Centre for Cardiovascular Biology and Medicine, GKT Schools of Biomedical Sciences and Medicine, King’s College London, Guy’s Campus, London SE1 1UL, UK

²Correspondence: Center for Cardiovascular Biology and Medicine, GKT School of Biomedical Sciences, King’s College London, Guy’s Campus, London SE1 1UL, UK. E-mail: ron.jacob{at}kcl.ac.uk

SPECIFIC AIMS

Pre-eclampsia (PE) is a leading cause of fetal and maternal mortality and is associated with abnormalities in maternal and fetal circulation. Our aim was to investigate whether fetal vascular smooth muscle cells exhibit abnormal regulation of free cytosolic calcium ([Ca²⁺]_i) that could be related to abnormalities in the fetal circulation. Because PE is associated with perturbed arachidonic acid (AA) metabolism and AA or its metabolites are known to affect [Ca²⁺]_i homeostasis, we focused on the [Ca²⁺]_i response to AA.

PRINCIPAL FINDINGS

1. PE cells have a potentiated [Ca²⁺]_i response to AA
Fura-2 ratio fluorescence was used as a measure of [Ca²⁺]_i in human umbilical artery smooth muscle cells (HUASMC) cultured from normal and PE deliveries. The response to 50 µM AA in PE cells was potentiated to 208% of that in normal HUASMC (P<0.01). In two-thirds of the PE cells, the rise in [Ca²⁺]_i was biphasic with the augmented [Ca²⁺]_i response being due to a delayed secondary rise in [Ca²⁺]_i (Fig. 1 ). The response in the absence of extracellular Ca²⁺ (Ca²⁺_o) was transient and < 20% of the response in the presence of Ca²⁺_o, indicating that the main effect of AA was to induce a Ca²⁺ influx. Two other fatty acids (oleic and linoleic acids) induced [Ca²⁺]_i responses smaller than those induced by AA and were monophasic even in PE cells; the nonmetabolizable AA analog eicosatetraynoic acid (ETYA) did not evoke a [Ca²⁺]_i response, suggesting that the AA response was induced by an AA metabolite. There was no correlation between the magnitude of the AA response in PE cells and gestational age. The difference in magnitude of the AA response between normal and PE cells persisted even when data were restricted to deliveries that fell within the normal gestational period of 38–40 wk. These results confirm that the augmented response in PE is due to the PE itself rather than any shortened gestational period.

View larger version (23K): [in this window] [in a new window]   Figure 1. Arachidonic acid-induced increases in [Ca2+]i in normal and PE HUASMC. A) When cells were challenged with 50 µM AA in the presence of extracellular Ca2+, AA-evoked increases in [Ca2+]i (indicated by R340/380, the ratio of fluorescence at 340 and 380 nm excitation) were significantly greater in PE cells. Basal [Ca2+]i levels (B), were not different in normal and PE cells. C) Summary of data for AA-induced [Ca2+]i increases measured at 600–800s exposure to AA. Values denote means ± SE of 3 replicate measurements from 16 normal and 19 PE cultures, **P < 0.009 vs. normal cells.

2. Inhibition of cyclooxygenase or lipoxygenase metabolism potentiates the AA response in normal cells to resemble that of PE cells
Inhibition of either the cyclooxgenase (COX) pathway with 10 µM indomethacin or the lipoxygenase pathway with 10 µM nordihydroguaiaretic acid (NDGA) had no effect on the response of PE cells to AA but potentiated the response of normal cells to resemble that of PE cells, even to the extent of inducing a delayed secondary [Ca²⁺]_i increase (Fig. 2 ).

View larger version (27K): [in this window] [in a new window]   Figure 2. [Ca2+]i measurements in normal and PE HUASMC in response to arachidonic acid in the absence or presence of indomethacin or NDGA. Normal and PE HUASMC were challenged with 50 µM arachidonic acid (AA) in Ca2+ containing solution in the presence of 10 µM indomethacin (A), an inhibitor of cyclooxygenase or 10 µM NDGA (B), an inhibitor of lipoxygenase. In normal HUASMC (), indomethacin potentiated the [Ca2+]i response to AA whereas in PE cells () the response to AA was not altered (C). A combination of both inhibitors led to similar increases of AA-evoked [Ca2+]i levels as did application of either inhibitor alone. C) Summary of data showing means ± SE of 3 replicate measurements in 4 normal and 8 PE cell cultures showing the effects of indomethacin (indo) and NDGA. *P < 0.04, **P < 0.02.

3. A monooxygenase metabolite is responsible for the augmented AA response in PE
The potentiating effect of COX and LOX inhibition on the response of normal HUASMC to AA could be due to potentiation of the effective AA concentration by inhibition of its metabolism via the COX or LOX pathways; the accumulating AA could then potentiate the Ca²⁺ response directly or via a metabolite produced as a result of diversion of AA to the monooxygenase pathway. We suspected the latter because of the lack of effect of ETYA mentioned earlier. To test this, we used three structurally dissimilar inhibitors of the monooxygenase pathway: metyrapone, isoniazid, and 17-octadecynoic acid (17-ODYA). All three reduced the response of PE cells to AA, with 10 µM 17-ODYA being the most effective, inhibiting the response by 92%. All three MOX inhibitors reduced the indomethacin-augmented response in normal cells, with 17-ODYA again being the most effective (86% inhibition, Fig. 2A ). 17-ODYA also partly inhibited the unpotentiated AA response in normal cells (56% inhibition).

CONCLUSIONS AND SIGNIFICANCE

The present study provides insight into possible abnormalities of fetal arterial smooth muscle cells isolated from pre-eclamptic pregnancies. Our findings provide the first evidence that AA-stimulated Ca²⁺ signaling is modulated differently in fetal smooth muscle cells in PE and that this alteration is linked to differences in AA metabolism in PE.

AA induces a larger Ca²⁺ influx in PE HUASMC, and this is not due to a shorter gestational period for PE pregnancies. The lack of any effect of the nonmetabolizable analog ETYA and the smaller responses to oleic and linoleic acid in the presence of Ca²⁺_o strongly suggest that AA is not acting via a nonspecific effect on membrane fluidity or a direct specific effect, but rather via a metabolite

In contrast, the response to AA in the presence of Ca²⁺_o clearly involves AA metabolism since, in normal HUASMC, the response was potentiated to resemble that seen in PE by inhibition of either the COX or LOX signaling pathways. Moreover, potentiation of Ca²⁺ influx is due to a MOX metabolite, since inhibition of the MOX pathway by structurally dissimilar compounds inhibited the indomethacin-potentiated Ca²⁺ response in normal cells as well as the enhanced [Ca²⁺]_i response seen in PE cells.

Our data establish that the MOX pathway in HUASMC can be stimulated by the increased availability of AA achieved by inhibiting AA metabolism via alternative pathways. This may seem surprising when there is an essentially unlimited provision of extracellular AA and suggests that the diffusion of extracellular AA to the MOX enzyme is a rate-limiting step. Another indication of AA accumulation and diversion for use as a substrate for MOX metabolism is the substantial delay in the onset of the secondary phase, perhaps reflecting a gradual rise in AA and attainment of a critical substrate concentration for MOX activation. Although it is apparent that the increased response in PE is due to a greater degree of metabolism of AA via MOX, it is not clear whether this is due to increased MOX activity or decreased activity by the COX and/or LOX pathways. The latter is more likely since the PE response was mimicked in normal cells by inhibiting COX or LOX. COX or LOX inhibitors did not affect the AA response of PE cells, suggesting that the combined activity of these pathways is already reduced; however, this could also arise if the ability of the MOX pathway to metabolize AA in PE cells is already fully saturated.

PE associated disturbances of Ca²⁺ regulation have been reported in endothelial cells and erythrocytes. There have been reports of altered maternal and fetal fatty acid metabolism usually reflected in altered levels of various fatty acids, including AA. Raised maternal plasma phospholipase A₂ has been reported in PE, and this could be responsible for raised concentrations of AA in the maternal circulation.

Various AA metabolites have been reported to stimulate Ca²⁺ influx including epoxyeicosatrienoic acids (EETs). Urinary levels of EETs and their hydroxylated derivatives are elevated in pregnant women and even more so in those with pregnancy-induced hypertension, so it is tempting to argue that this is a potential cause of the hypertension. However, EETs have been canvassed as a candidate for EDHF, and blocking their further metabolism by inhibiting their conversion by soluble epoxide hydrolase to the corresponding dihydroxy epoxyeicosatrienoic acids has been reported to lower blood pressure in rats with hypertension induced by angiotensin II. One possible resolution of these contradictions is that the effect of EETs may depend on the state of the muscle, since 14,15-EET produced both contraction and relaxation in porcine coronary artery rings depending on whether the rings were precontracted with acetylcholine or the thromboxane mimetic U46619, respectively.

Exogenously applied or endogenously generated AA leads to vasorelaxation, which is variously reported to be insensitive or sensitive to MOX inhibitors. In the intact vessel this is due to release of endothelial-derived prostacyclin and EDHF: prostacyclin is a COX metabolite and there is evidence that EDHF may be an AA MOX metabolite. However, it is clear that in these HUASMC AA causes a rise in [Ca²⁺]_i, which is not only likely to cause a contraction but is observed to do so in normal cells when the response is potentiated by COX inhibition. It is therefore likely that in PE, AA increases production of MOX metabolites, such as 5,6 EET, in vascular smooth muscle and this will mitigate or even predominate over any relaxing effect of prostacyclin or EDHF released from the endothelium.

Our data suggest that in pre-eclampsia there could be increased fetal vascular tone due to the increased sensitivity of vascular smooth muscle to AA. Increased fetoplacental vascular resistance is detectable by alterations in Doppler profiles and provides a prediction of worsening fetal outcome. Another important issue relating to the fetal circulation is substantial interest in the field of fetal programming of the cardiovascular system. Our previous results with fetal endothelial cells and current results with vascular smooth muscle cells demonstrate phenotypic changes that persist in culture, suggesting the possibility of adding PE to the list of intrauterine factors implicated in fetal programming. Should the altered AA metabolism we report here extend to the maternal circulation, it may contribute to the pathogenesis of the hypertension, a hallmark of PE.

View larger version (24K): [in this window] [in a new window]   Figure 3. Scheme summarizing the effects of PE. Arachidonic acid (AA) can be metabolized by three pathways: a cyclooxygenase (COX), a lipoxygenase (LOX), and a monooxygenase (MOX), which is a cytochrome P450. In PE, the balance swings from the COX and LOX pathways to the MOX pathway either because COX and LOX are down-regulated or because MOX is up-regulated. The increased production of MOX metabolites in turn stimulates Ca2+ entry. This effect can be mimicked in normal HUASMC by blocking the COX or LOX pathways and is inhibited by blocking the MOX pathway.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0507fje; to cite this article, use FASEB J. (December 17, 2002) 10.1096/fj.02-0507fje

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This Article Full Text (PDF) All Versions of this Article: 17/2/307 02-0507fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by STEINERT, J. R. Articles by JACOB, R. Search for Related Content PubMed PubMed Citation Articles by STEINERT, J. R. Articles by JACOB, R.