channels in smooth muscle cells - The FASEB Journal

Environmental estrogenic pollutants induce acute vascular relaxation by inhibiting L-type Ca2/ channels in smooth muscle cells DIETRICH O. RUEHLMANN,* JOERN R. STEINERT,* MIGUEL A. VALVERDE,† RON JACOB,* AND GIOVANNI E. MANN*,1 *Vascular Biology Research Centre, Biomedical Sciences Division, King’s College London, London W8 7AH, U.K.; and †Neuroscience Research Centre, Biomedical Sciences Division, King’s College London, Strand, London WC2R 2LS, U.K.

There is an ongoing scientific debate concerning the potential threat of environmental estrogenic pollutants to animal and human health (1– 5). Pollutants including the detergents 4-octylphenol and p-nonylphenol and chlorinated insecticides have recently been reported to modulate sexual differentiation by interacting with nuclear steroid receptors (6–8). So far, the focus has been on reproductive organs, but sex steroids have far more widespread actions. The lower incidence of cardiovascular disease in women has been attributed to estrogens (9– 14), yet no information is available on the vascular actions of environmental estrogenic pollutants. In the present study we have investigated the effects of acute exposure to 17b-estradiol, the antiestrogen ICI 182,780, and estrogenic pollutants on coronary vascular tone as well as on intracellular Ca2/ levels ([Ca2/]i) and Ca2/ and K/ channel activity in vascular smooth muscle cells. We report here that 4-octylphenol, p-nonylphenol, o.p*-DDT, and the antiestrogen ICI 182,780 inhibit L-type Ca2/ channels in vascular smooth muscle cells and evoke a rapid and endothelium-independent relaxation of the coronary vasculature similar to that induced by 17b-estradiol. Thus, inhibition of Ca2/ influx via L-type Ca2/ channels in vascular smooth muscle cells may explain the acute, nongenomic vasodilator actions of environmental estrogenic pollutants.—Ruehlmann, D. O., Steinert, J. R., Valverde, M. A., Jacob, R., Mann, G. E. Environmental estrogenic pollutants induce acute vascular relaxation by inhibiting L-type Ca2/ channels in smooth muscle cells. FASEB J. 12, 613– 619 (1998) ABSTRACT

Key Words: environmental pollutants · DDT · nonylphenol · octylphenol · estrogen · antiestrogen · ICI 182,870 · Ca2/ and K/ channels · vascular smooth muscle cells · coronary vascular tone · heart

GENDER DIFFERENCES in cardiac morbidity and mortality have been attributed to the difference in hormonal status between men and women (9–14).

Women are less susceptible to cardiovascular disease until the onset of menopause, when cessation of ovarian hormone production is accompanied by an increased incidence of coronary heart disease (10). The beneficial effects of hormone replacement therapy are now well recognized, and activation of genomic estrogen receptors results in inhibition of vascular smooth muscle cell proliferation and up-regulation of constitutive nitric oxide (NO)2 synthase in endothelial cells (13, 14). In addition to the genomic actions of steroid hormones in the vasculature, recent evidence confirms that 17b-estradiol acutely restores impaired coronary blood flow in postmenopausal women (11). However, the cellular and molecular mechanisms mediating the acute vasodilator responses to 17b-estradiol remain to be elucidated. The consensus of several studies is that acute vascular relaxation induced by 17b-estradiol is predominantly endothelium independent (15) and mediated by the inhibition of Ca2/ influx through Ltype Ca2/ channels, resulting in decreases in myosin light chain kinase phosphorylation and contraction of smooth muscle (16–19). However, in addition to inhibiting voltage-operated Ca2/ channels in smooth muscle cells, 17b-estradiol has also been reported to increase Ca2/-activated K/ currents (20). Thus, activation of genomic and nongenomic steroid receptors by 17b-estradiol modulates vascular function. There is an ongoing scientific debate concerning the potential threat of environmental estrogenic pollutants to animal and human reproductive health (1– 5), yet there is no information on the effects of these compounds on steroid receptors in the vasculature. Although alkylphenolic detergents (4-octylphenol, p1 Correspondence: Vascular Biology Research Centre, Biomedical Sciences Division, King’s College London, Campden Hill Road, London W8 7AH, U.K. E-mail: [email protected] 2 Abbreviations, intracellular calcium, [Ca2/]i; BSS, balanced salt solution; BSA, bovine serum albumin; L-NAME, NGnitro-L-arginine methyl ester; CPP, coronary perfusion pressure; o.p*-DDT, 1,1,1-trichloro-2-[o-chlorophenyl]-2-[pchlorophenyl]ethane; NO, nitric oxide.

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nonylphenol) and the organochloride pesticide o.p*DDT (1,1,1-trichloro-2-[o-chlorophenyl]-2-[pchlorophenyl]ethane) are known to modulate genomic steroid receptors (6–8), there is no information on their interaction with nongenomic receptors. In the present study we have correlated the acute vasodilator actions of 4-octylphenol, p-nonylphenol, o.p*-DDT, and the antiestrogen ICI 182,720 in the isolated and perfused rat heart with alterations in intracellular Ca2/ levels and Ca2/ and K/ channel activity in rat vascular smooth muscle cells in culture. METHODS Perfusion of the isolated rat heart Male Sprague-Dawley rats (ú250 g) were killed by cervical dislocation; the heart was excised and the aorta was cannulated retrogradely. The coronary vasculature was perfused at constant flow (10 ml · min01) with oxygenated, warmed (377C) Krebs Henseleit buffer (mM: 118 NaCl, 25 NaHCO3, 11 Dglucose, 4.7 KCl, 1.2 MgSO4, 1.1 KH2PO2, 1.2 CaCl2). Coronary perfusion pressure was initially elevated by continuous infusion of the thromboxane A2 mimetic U46619 (5 nM) or the NO synthase inhibitor NG-nitro-L-arginine methyl ester (LNAME, 50 mM). Vasodilator responses to close-arterial bolus injections of bradykinin (0.1 nmol) and sodium nitroprusside (10 nmol) served as markers of endothelial and smooth muscle cell function (21). Drugs and vehicle controls (100% ethanol) were injected as a 10 ml bolus. Changes in coronary perfusion pressure (CPP) served as an index of vascular resistance and were recorded continuously on a Lectromed pen recorder. Fluorescence measurements of intracellular calcium ([Ca2/]i) in A7r5 smooth muscle cells For [Ca2/]i determinations, confluent rat fetal aortic A7r5 smooth muscle cell monolayers were cultured on glass coverslips and loaded for 1 h at room temperature with 4 mM Fura2AM in HEPES-buffered Dulbecco’s modified Eagle’s medium. After loading, A7r5 cells were kept in a balanced salt solution (BSS, mM: 145 NaCl, 5 KCl, 1 MgSO4, 10 Hepes, 10 D-glucose, pH 7.4) containing 1% bovine serum albumin (BSA) for up to 2 h. To measure [Ca2/]i in cell populations, coverslips were transferred to a cuvette containing 2 ml BSS / 0.1% BSA at 377C. Cells were depolarized with high K/ BSS (150 K//0 Na/), yielding a nominal 80 mM K/ extracellular concentration. Fluorescence was measured as described previously (22, 23) using a rotating wheel spectrophotometer (Cairn, Sittingbourne, Kent, U.K.) with excitation at 340, 360, and 380 nm and emission detected at ú500 nm. Autofluorescence and background fluorescence were estimated by quenching the Fura2 fluorescence by adding 2 mM Mn2/ at the end of an experiment. The ratio of fluorescences at 340 and 380 nm excitation was used as a measure of [Ca2/]i. In some experiments, subconfluent coverslips were mounted on an inverted microscope, with excitation at 350 and 380 nm, using a LEP dual-filter wheel (Ludl, Hawthorne, N.Y.). The emission was detected at ú470 nm by an extended Isis M camera (Photonic Sciences, Robertsbridge, East Sussex, U.K.) and digitized by a Pixel Grabber board (Perceptics, Knoxville, Tenn.), hosted by a Macintosh Quadra 800 using Ionvision Software (Improvision, Coventry, U.K.). A pair of images was obtained at approximately 3 s intervals. 614

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Whole-cell patch clamp analysis of ion channels in A7r5 smooth muscle cells Ionic currents were measured using the whole-cell recording mode of the patch clamp technique (24) with an Axopatch 200A (Axon, U.S.A.) or an RK-400 (Biologic, France) amplifier, as described previously (25). Recording pipettes (2–5 MV were filled with an intracellular solution containing (mM): 140 KCl, 1.2 MgCl2, 0.5 CaCl2, 1 EGTA, 10 HEPES, 2 ATP, 1 GTP at pH 7.4. The bathing solution contained (mM): 140 Nmethyl D-glucamine chloride, 0.5 MgCl2, 1.3 CaCl2, 10 HEPES, 20 mannitol at pH 7.4. Barium currents through Ca2/ channels were measured with intracellular solutions containing (mM) 110 CsCl, 30 tetraethylammonium, 1 MgCl2, 10 HEPES, pH 7.4, 2 ATP, and 1 GTP as well as extracellular solutions containing (mM) 50 BaCl2, 45 NaCl, 1 MgCl, 10 HEPES, pH 7.4. To prevent the spontaneous activation of swelling-activated chloride currents (26), the osmolality of the pipette and bathing solutions was adjusted with D-mannitol to 283 mOsm and 300 mOsm, respectively. Voltage pulse generation, data acquisition, and analysis were performed using software written by J. Dempster (University of Strathclyde, Glasgow, U.K.). All traces shown were leak subtracted. Reagents o.p*-DDT was obtained from Chem Service, Chester, Pa.; fura2 acetoxymethyl ester (Fura2-AM) was from Calbiochem, Nottingham, U.K.; and ICI 182,780 was a gift from Zeneca Pharmaceuticals, Macclesfield, U.K. All other drugs, chemicals, and culture media were obtained from Sigma, Poole, Dorset, U.K.

RESULTS The acute effects of 17b-estradiol and environmental estrogenic pollutants on the vasculature were investigated using whole-organ and cellular techniques. In the isolated and perfused heart, CPP was elevated with the thromboxane A2 mimetic U46619 (5 nM). Close-arterial bolus injections of 17b-estradiol and 4octylphenol caused a rapid, transient, and dose-dependent fall in CPP (Fig. 1a, b), with a maximal decrease at 100 nmol of 35 { 3 mmHg (mean { SE, nÅ36 preparations) and 38 { 3 mmHg (nÅ6), respectively. Similar responses were observed with the detergent p-nonylphenol (21{7 mmHg, nÅ3), the chlorinated insecticide o.p*-DDT (38{8 mmHg, nÅ7), and the antiestrogen ICI 182,780 (26{7 mmHg, nÅ3; Faslodex; see ref 27). Continuous perfusion of isolated hearts with 17b-estradiol or 4octylphenol caused a rapid, sustained, and concentration-dependent decrease in coronary vascular tone (Fig. 1c, d). As acute vascular relaxation evoked by 17b-estradiol may involve both endothelium-dependent and independent mechanisms (13, 15), we investigated whether the endothelium-derived vasodilator NO was involved in acute coronary vasodilator responses to 17b-estradiol and 4-octylphenol. When isolated hearts were perfused with the NO synthase inhibitor

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Figure 1. Acute effects of 17b-estradiol and 4-octylphenol on coronary vascular tone in the isolated, perfused rat heart. a) Trace of coronary perfusion pressure (CPP) in an isolated heart perfused continuously with U46619 (5 nM) before and after close arterial bolus injections of 17b-estradiol (1–100 nmol). The dose-response curve for the fall in CPP in three to six preparations (mean { SE) is also shown. Bolus injections of vehicle did not cause a significant fall in CPP (1{1 mmHg, nÅ47). b) Closearterial bolus injections of 4-octylphenol (1–100 nmol) caused a transient and dose-dependent coronary vasodilation. c, d) Continuous perfusion of isolated hearts with either 17b-estradiol or 4-octylphenol evoked a concentration-dependent and sustained fall in CPP. e, f ) 17b-estradiol or 4-octylphenol induced a significant coronary vasodilation even in the presence of LNAME (50 mM), an inhibitor of endothelial cell nitric oxide synthase.

L-NAME (50 mM), basal coronary perfusion pressure increased, confirming the involvement of NO in the maintenance of basal coronary vascular tone (28). The acute vasodilator response of hearts to either 17b-estradiol or 4-octylphenol infusion was unaffected by the presence of L-NAME (Fig. 1e, f ), suggesting that estrogenic compounds do not acutely modulate NO production. Similar results were obtained for o.p*-DDT (data not shown). Thus, environmental estrogenic pollutants cause acute endothelium-independent vasodilatation, presum-

ably due to their direct action on coronary vascular smooth muscle cells, as demonstrated previously for 17b-estradiol (13, 15, 19, 20). To further elucidate the cellular mode of action of these environmental pollutants, we compared the effects of 17b-estradiol and estrogenic pollutants on [Ca2/]i in the vascular smooth muscle cell line A7r5. Smooth muscle cells were depolarized with 80 mM K/ in nominally Ca2/-free solutions, and the rise in [Ca2/]i after addition of 1 mM Ca2/ was monitored in the absence or presence of select estrogenic com-

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pounds (Fig. 2a–c). 17b-estradiol, 4-octylphenol, pnonylphenol, o.p*-DDT, ICI 182,780, and the L-type Ca2/ channel blocker isradipine (data not shown) inhibited the rise in [Ca2/]i (Fig. 2d). To investigate the homogeneity of this effect, experiments were performed using a single-cell fluorescence imaging system. In the presence of extracellular Ca2/, cells responded to depolarization with a sharp increase in [Ca2/]i, followed by reduction to a sustained plateau (Fig. 2e). Peak and plateau responses were both attenuated by 10 mM 17b-estradiol (Fig. 2f ) compared to the preceding response (peak: 63{2%, plateau 54{1%, mean { SE, nÅ46). Similar results were obtained with 4-octylphenol (Fig 2g) at 10 mM (peak: 38{2%, plateau 39{1%, nÅ46). These results suggest that environmental estrogenic compounds either increase K/ channel activity, thereby hyperpolarizing the cell membrane and closing Ca2/ channels (29), and/or directly inhibit Ca2/ channels. Both

effects have been reported for 17b-estradiol in cultured smooth muscle cells (16, 17, 20). To resolve whether estrogenic pollutants exert their effect by interacting with K/ channels and/or Ca2/ channels, we simultaneously measured the activity of Ca2/ and K/ channels in A7r5 vascular smooth muscle cells in response to 17b-estradiol and environmental estrogenic pollutants. Figure 3 shows whole-cell measurements of Ca2/ and K/ currents in single A7r5 vascular smooth muscle cells. Addition of 17b-estradiol induced rapid inhibition of the inward Ca2/ current and an increase in the outward K/ current (seven of eight cells) (Fig. 3a). Similar increases in K/ currents were obtained in three of four cells exposed to ICI 182,780 (data not shown). To further resolve the inhibition of Ca2/ currents by 17b-estradiol, which could be masked by the activation of K/ currents, Ba2/ currents flowing through Ca2/ channels were measured in the absence of intracellular

Figure 2. Acute effects of 17b-estradiol and estrogenic pollutants on [Ca2/]i in A7r5 vascular smooth muscle cells. A7r5 cells were depolarized by raising [K/]o to 80 mM, after which 1 mM Ca2/ was added to the cuvette (denoted by arrows). This caused a rise in [Ca2/]i that was substantially inhibited by isradipine (1 mM, data not shown). 17b-estradiol (10 mM) (a), 4-octylphenol (10 mM) (b), and ICI 182,780 (10 mM) (c) reduced the rise in [Ca2/]i. d) Average [Ca2/]i responses to vehicle (s), 17b-estradiol (h), 4-octylphenol (n), o.p*-DDT (L), p-nonylphenol (pentagon), and ICI 182,780 (w), all at 10 mM. Responses are plotted relative to an extrapolation of the initial trace before addition of [Ca2/]o in order to correct for the baseline slope (mean { SE; nÅ3–6 coverslips). e) Effect of consecutive depolarizations on [Ca2/]i measured by fluorescence imaging in single A7r5 smooth muscle cells bathed in Ca2/-containing solutions. Depolarizations in the presence of [Ca2/]o resulted in a reproducible effect comprised of a transient peak, followed by a sustained plateau. 17b-estradiol (10mM) ( f ) and 4-octylphenol (10 mM) (g) inhibited the peak and plateau phases of this response. 616

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Figure 3. Modulation of K/ and Ca2/ currents by environmental estrogenic pollutants in A7r5 vascular smooth muscle cells. a) Increase in K/ currents and inhibition of high-threshold, L-type Ca2/ currents by 10 mM 17b-estradiol in a single A7r5 cell. b) Inhibition of Ba2/ currents by 17b-estradiol (10 mM). c) Inhibition of Ca2/ currents by 10 mM 4-octylphenol. Cells were clamped at 080 mV, and membrane currents in response to 400 ms pulses from 040 to /40 mV (a, c) or /20 mV (b) were measured.

K/ (Fig. 3b). Under these conditions, a complete inhibition of the otherwise stable Ba2/ current by 17bestradiol was observed within 45 s. The short time courses for the inhibition of Ca2/ currents and activation of K/ currents by 17b-estradiol (Fig. 3a, b) suggest mechanisms of action different from the well-known genomic effect of steroids (30, 31) and similar to other nongenomic actions of 17b-estradiol and antiestrogens described by us (32) and other groups (16, 19, 20; see ref 33). In contrast to 17bestradiol and ICI 182,780, the environmental estrogenic pollutants tested selectively inhibited Ca2/ currents without affecting the K/ current (Fig. 3c). The inhibitory effects of estrogenic pollutants on Ba2/ currents subsequently were further characterized (Fig. 4). Ba2/ currents were activated at potentials positive to 020 mV, inhibited by 1 mM isradipine, and thus identified as high-threshold, dihydropyridine-sensitive L-type currents (34). Figures 4a, b show the concentration-dependent inhibitory effects of o.p*-DDT and 4-octylphenol, which were equally ef-

Figure 4. Concentration-dependent inhibition of Ba2/ currents by estrogenic pollutants in A7r5 vascular smooth muscle cells. Ba2/ currents recorded in single A7r5 cells were inhibited in a concentration-dependent manner by o.p*-DDT (a), 4-octylphenol (b), and the antiestrogen ICI 182,780 (c). Ba2/ currents were elicited by a voltage pulse to /20 mV from a holding potential of 080 mV. Current traces in the presence of drugs were recorded 2 min after the addition of each drug at the concentrations specified.



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fective inhibitors of the Ba2/ currents (40{5% and 48{5% at 100 pM, mean { SE, nÅ3), whereas ICI 182,780 produced a 60 { 6% inhibition at the same concentration (Fig. 4c). In contrast to the high potency of estrogenic pollutants and ICI 182,780 as inhibitors of L-type Ca2/ currents, 17b-estradiol did not inhibit this current at subnanomolar concentrations (data not shown). DISCUSSION Our results provide the first direct evidence that the environmental estrogenic pollutants p-nonylphenol, 4-octylphenol, and o.p*-DDT acutely inhibit Ca2/ influx through L-type Ca2/ channels in vascular smooth muscle cells, which may explain their rapid vasodilator actions in the coronary circulation. Unlike the selective inhibition of L-type Ca2/ channels by these estrogenic pollutants, we demonstrated in single smooth muscle cells that 17b-estradiol simultaneously stimulates outward K/ currents and inhibits L-type Ca2/ channels. Thus, the acute coronary vascular relaxation induced by estrogenic pollutants may be mediated predominantly by their inhibition of L-type Ca2/ channels rather than by activation of K/ channels or release of endothelium-derived NO. In view of the potential threat of estrogenic pollutants to animal and human reproductive health (1, 2, 6, 7), our findings have identified another acute mode action involving yet unidentified nongenomic steroid receptors, probably localized in the plasma membrane. The acute effects of the estrogenic compounds on L-type Ca2/ channels, together with the reported actions of 17b-estradiol and other steroids on L-type Ca2/ channels and Ca2/-activated K/ channels, imply the involvement of receptor-mediated events rather than alterations in lipid solubility (33). In fact, estrogen membrane receptors have been characterized in different cell types (35, 36), although the physiological role of these steroid binding sites remains to be defined. In our whole-cell patch clamp experiments, o.p*-DDT and alkylphenolic detergents selectively reduced L-type Ca2/ channel currents without altering outward K/ currents. The potency of these estrogenic pollutants on L-type Ca2/ activity was much higher than that of 17bestradiol (data not shown) and contrasts with the Ç1000-fold lower potency of o.p*-DDT and 4-octylphenol in activating nuclear estrogen receptors (7). Our findings in the isolated perfused heart demonstrate that estrogenic pollutants also cause an acute coronary vasodilatation similar to that evoked by 17bestradiol. Although higher concentrations of estrogenic pollutants may be required to mimic the genomic effects induced by 17b-estradiol, we have shown that lower concentrations modulate L-type Ca2/ channel activity in vascular smooth muscle cells. 618

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Thus, environmental pollutants inhibit L-type Ca2/ channel activity at approximately nanomolar concentrations, whereas vasodilatory responses in the perfused heart were detected only at micromolar concentrations (compare Fig. 1 and Fig. 4). Lipid-soluble environmental pollutants, including organochloride insecticides and other estrogenic compounds, accumulate to high concentrations in human tissues (37, 38) and have been reported to impair or to have limited effects on reproductive development in rodents (39, 40). Plasma concentrations of organochloride pesticides have been reported to reach approximately micromolar levels (37, 38), high enough potentially to modulate L-type Ca2/ channel activity. If estrogenic pollutants are not bound as effectively as 17b-estradiol to plasma steroid binding proteins, then lower plasma concentrations could elicit short-term nongenomic biological effects. Our study has demonstrated novel acute effects of estrogenic pollutants on Ca2/ influx via L-type Ca2/ channels and vascular tone. It is conceivable that Ltype Ca2/ channels in other tissues, such as islets of Langerhans and neurons expressing nongenomic steroid receptors, will be affected similarly. As inhibitors of L-type Ca2/ channel activity are currently used therapeutically in the treatment of hypertension (41), it is interesting to speculate whether estrogenic pollutants would act as effective inhibitors of L-type Ca2/ channels in hypertension. Although o.p*-DDT and 4-octylphenol inhibited L-type Ca2/ channel activity at significantly lower concentrations than needed to induce coronary vascular relaxation, we cannot comment on whether humans are exposed to effective concentrations of these environmental pollutants. This study demonstrates that environmental estrogenic pollutants and the estrogen antagonist ICI 182,780 acutely modulate vascular tone by mimicking some of the actions of 17b-estradiol. As ICI 182,780 acts as a pure antagonist when interacting with the nuclear estrogen receptor (27), our findings have considerable implications for elucidating the structure of the nongenomic estrogen receptor and the therapeutic use of this class of compounds. The differential mode of action of 17b-estradiol and estrogenic pollutants in regulating vascular smooth muscle tone may provide an additional experimental tool to unravel the signaling mechanisms associated with nongenomic steroid receptors and extend the ongoing debate as to their role in human health and disease. This work was supported by grants from the Wellcome Trust (G.E.M., R.J.), Tommy’s Campaign (G.E.M., R.J.), British Heart Foundation (R.J.), Cystic Fibrosis Trust (M.A.V.), and Royal Society (M.A.V.). D.O.R. was the recipient of a Wellcome Trust Ecotoxicology Prize Ph.D. studentship in G.E.M.’s

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laboratory. We thank Paulo Cesare, Dr. Chris Smith, Prof. Peter McNaughton, and Prof. Jeremy Pearson for their helpful discussions, Dr. A. E. Wakeling (Zeneca Pharmaceuticals, Macclesfield, U.K.) for the initial supply of ICI 182,780, and Dr. M. Schachter (St. Mary’s Hospital London) for the A7r5 cell line.

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