Ron Jacob

Environmental estrogenic pollutants induce acute vascular relaxation by inhibiting L-type Ca2+ channels in smooth muscle cells

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 17β‐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 17β‐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.

The isoflavone equol mediates rapid vascular relaxation - Ca2+-independent activation of endothelial nitric-oxide synthase/Hsp90 involving ERK1/2 and Akt phosphorylation in human endothelial cells

We recently reported that soy isoflavones increase gene expression of endothelial nitric-oxide synthase (eNOS) and antioxidant defense enzymes, resulting in improved endothelial function and lower blood pressure in vivo. In this study, we establish that equol (1-100 nm) causes acute endothelium- and nitric oxide (NO)-dependent relaxation of aortic rings and rapidly (2 min) activates eNOS in human aortic and umbilical vein endothelial cells. Intracellular Ca2+ and cyclic AMP levels were unaffected by treatment (100 nm, 2 min) with equol, daidzein, or genistein. Rapid phosphorylation of ERK1/2, protein kinase B/Akt, and eNOS serine 1177 by equol was paralleled by association of eNOS with heat shock protein 90 (Hsp90) and NO synthesis in human umbilical vein endothelial cells, expressing estrogen receptors (ER)α and ERβ. Inhibition of phosphatidylinositol 3-kinase and ERK1/2 inhibited eNOS activity, whereas pertussis toxin and the ER antagonists ICI 182,750 and tamoxifen had negligible effects. Our findings provide the first evidence that nutritionally relevant plasma concentrations of equol (and other soy protein isoflavones) rapidly stimulate phosphorylation of ERK1/2 and phosphatidylinositol 3-kinase/Akt, leading to the activation of NOS and increased NO production at resting cytosolic Ca2+ levels. Identification of the nongenomic mechanisms by which equol mediates vascular relaxation provides a basis for evaluating potential benefits of equol in the treatment of postmenopausal women and patients at risk of cardiovascular disease.

Preeclampsia is associated with altered Ca2+ regulation and NO production in human fetal venous endothelial cells

Preeclampsia (PE) is a leading cause of maternal hypertension in pregnancy, fetal growth restriction, premature birth, and fetal and maternal mortality (1). Activation and dysfunction of the maternal and fetal endothelium in PE may be the consequence of increased oxidative stress associated with circulating lipid peroxides (2–4), and in cases of severe maternal hypertension, uterine and umbilical artery waveforms are abnormal (5). We have investigated PE‐associated abnormalities in the regulation of intracellular Ca2+ ([Ca2+]i) and cyclic guanosine monophosphate (cGMP) production (index of nitric oxide [NO]) in human fetal umbilical vein endothelial cells. Basal [Ca2+]i was slightly elevated in PE cells, whereas agonist‐stimulated Ca2+ entry was reduced in cells from PE compared with normal term or age‐matched preterm pregnancies. Furthermore, PE cells exhibited a decreased permeability to Ba2+ but an increased permeability to Mn2+ and Gd3+, suggesting that PE is associated with phenotypic alterations in fetal endothelial cation channel(s). Basal and histamine‐stimulated cGMP levels were elevated in PE compared with preterm or normal cells, implying an increased NO production in PE. However, immunoblots for endothelial NO synthase (eNOS) and soluble guanylyl cyclase (sGC) revealed reduced eNOS expression in PE and preterm cells, with negligible changes in sGC levels. This study provides important and novel insights into abnormalities of fetal endothelial cells isolated from women with PE, revealing an altered cation membrane permeability and activity of eNOS‐sGC pathway. As these changes are sustained in culture in vitro, this may reflect long‐term —programming“ of the fetal cardiovascular system.

ACTIONS OF SERUM AND PLASMA ALBUMIN ON INTRACELLULAR CA2+ IN HUMAN ENDOTHELIAL CELLS

1 The effects of serum and plasma albumin on [Ca2+]i in human endothelial cells were examined using single‐cell Ca2+ imaging. Two types of endothelial cell were used: human umbilical vein endothelial cells (HUVEC) in primary culture, and the endothelial‐derived cell line ECV304. 2 Serum albumin caused a large and transient rise in [Ca2+]i, due to Ca2+ release from an IP3‐sensitive internal store, followed by a maintained elevation in [Ca2+]i attributable to Ca2+ influx from the external medium. A half‐maximal rise in [Ca2+]i was produced by a concentration of serum albumin of about 1 μg ml−1. 3 The Ca2+‐releasing action of serum albumin is abolished by methanol extraction and is therefore attributable to an attached polar lipid. A possible candidate is lysophosphatidic acid, known to be released from platelets during blood coagulation, which produced similar effects to those of serum albumin. 4 In HUVEC, plasma albumin caused a sustained decrease in [Ca2+]i from the mean resting level of 114 nm to 58 nm. No effect of plasma albumin was observed in ECV304 cells. 5 The decrease in [Ca2+]i caused by plasma albumin is due to an uptake into intracellular stores. The store loading substantially potentiates the action of Ca2+‐releasing agonists such as histamine. 6 The results show that normal plasma albumin, which carries few lipids, lowers [Ca2+]i and potentiates the actions of Ca2+‐releasing agonists by promoting Ca2+ uptake into intracellular stores. When converted to the serum form, by binding lysophosphatidic acid released during blood coagulation, albumin has a potent effect in elevating [Ca2+]i. Blood coagulation may therefore play a role in regulating vascular tone and capillary permeability.

Differential modulation of the phases of a Ca2+ spike by the store Ca2+-ATPase in human umbilical vein endothelial cells

1 Histamine‐stimulated cytosolic free Ca2+ ([Ca2+]i) oscillations in human umbilical vein endothelial cells (HUVECs) comprise repetitive spikes generated by pulsatile release from stores. We have investigated the roles of the store Ca2+‐ATPases in regulating both the upstroke and downstroke of a Ca2+ spike. 2 The sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) dramatically affected oscillations whereas inhibition of the plasma membrane Ca2+‐ATPase (PMCA) with La3+ had little effect. This and other evidence suggested that the downstroke of a spike is predominantly mediated by SERCA. 3 Artificial [Ca2+]i spiking generated by repetitive pulsatile application of 0.3 μm histamine in Ca2+‐free medium did not cause net loss of Ca2+ from the cell whereas repetitive pulsatile application of 1 and 10 μm histamine did, with the higher concentration being more effective. We conclude that there is an inverse relationship between stimulus intensity and relative SERCA activity. 4 For a Ca2+ transient, the initiation of release was suppressed by SERCA during either the lag phase or the interspike period (ISP) since: (i) the ISP was shortened by low CPA concentrations, (ii) higher concentrations of CPA stimulated an explosive Ca2+ release when applied during the ISP but not when applied in the absence of agonist, and (iii) CPA synchronized the initial Ca2+ response to a low histamine dose (even recruiting silent, histamine‐unresponsive cells). 5 Two aspects of the regenerative upstroke of a spike were differently affected by SERCA inhibition: Ca2+ wave velocity was entirely unaffected by CPA whereas the local rate of rise was increased. 6 The [Ca2+]i at which a Ca2+ spike terminated depended on SERCA since CPA dose dependently enhanced the peak [Ca2+]i. 7 We conclude that SERCA plays a powerful and dynamic role in regulating [Ca2+]i oscillations in HUVECs. SERCA differentially modulates the phases of Ca2+ release in addition to bringing about the falling phase of a Ca2+ spike.

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

Pre‐eclampsia (PE) is a leading cause of maternal and fetal mortality and morbidity. As free fatty acid metabolism is abnormally regulated in PE, we investigated the intracellular Ca2+ ([Ca2+]i) response to arachidonic acid (AA) in primary cultures of human umbilical artery smooth muscle cells (HUASMC). AA (50 μM) caused a significantly greater [Ca2+]i elevation in PE than in normal HUASMC, with many cells displaying a delayed secondary increase. The nonmetabolizable AA analog ETYA did not induce a response, suggesting that the augmented PE response depends on an AA metabolite. Inhibition of the AA metabolizing cyclooxygenase or lipoxygenase pathways did not affect the AA response of PE HUASMC but induced in normal cells the secondary rise of [Ca2+]i observed in PE cells. This potentiated response and the response in PE cells were blocked by inhibitors of the monooxygenase pathway, a third AA metabolizing pathway. We conclude that the [Ca2+]i response of HUASMC is elevated in PE because of an increased level of a monooxygenase metabolite that stimulates Ca2+ influx and that this can be mimicked in normal cells by blocking cyclooxygenase or lipoxygenase to divert AA to the monooxygenase. This and our work with fetal endothelial cells (FASEB J. 10.1096/fj.01‐0916fje) demonstrate phenotypic changes in the fetal vasculature in PE.

A PP2A-mediated feedback mechanism controls Ca2+-dependent no synthesis under physiological oxygen

Intracellular O2 is a key regulator of NO signaling, yet most in vitro studies are conducted in atmospheric O2 levels, hyperoxic with respect to the physiologic milieu. We investigated NO signaling in endothelial cells cultured in physiologic (5%) O2 and stimulated with histamine or shear stress. Culture of cells in 5% O2 (>5 d) decreased histamine‐but not shear stress–stimulated endothelial (e)NOS activity. Unlike cells adapted to a hypoxic environment (1% O2), those cultured in 5% O2 still mobilized sufficient Ca2+ to activate AMPK. Enhanced expression and membrane targeting of PP2A‐C was observed in 5% O2, resulting in greater interaction with eNOS in response to histamine. Moreover, increased dephosphorylation of eNOS in 5% O2 was Ca2+‐sensitive and reversed by okadaic acid or PP2A‐C siRNA. The present findings establish that Ca2+ mobilization stimulates both NO synthesis and PP2A‐mediated eNOS dephosphorylation, thus constituting a novel negative feedback mechanism regulating eNOS activity not present in response to shear stress. This, coupled with enhanced NO bioavailability, underpins differences in NO signaling induced by inflammatory and physiologic stimuli that are apparent only in physiologic O2 levels. Furthermore, an explicit delineation between physiologic normoxia and genuine hypoxia is defined here, with implications for our understanding of pathophysiological hypoxia.—Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. A PP2A‐mediated feedback mechanism controls Ca2+‐dependent NO synthesis under physiological oxygen. FASEB J. 31, 5172–5183 (2017). www.fasebj.org

Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels

Unregulated increases in cellular Ca2+ homeostasis are a hallmark of pathophysiological conditions and a key trigger of cell death. Endothelial cells cultured under physiologic O2 conditions (5% O2) exhibit a reduced cytosolic Ca2+ response to stimulation. The mechanism for reduced plateau [Ca2+]i upon stimulation was due to increased sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)‐mediated reuptake rather than changes in Ca2+ influx capacity. Agonist‐stimulated phosphorylation of the SERCA regulatory protein phospholamban was increased in cells cultured under 5% O2. Elevation of cytosolic and mitochondrial [Ca2+] and cell death after prolonged ionomycin treatment, as a model of Ca2+ overload, were lower when cells were cultured long‐term under 5% compared with 18% O2. This protection was abolished by cotreatment with the SERCA inhibitor cyclopiazonic acid. Taken together, these results demonstrate that culturing cells under hyperoxic conditions reduces their ability to efficiently regulate [Ca2+]i, resulting in greater sensitivity to cytotoxic stimuli.—Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels. FASEB J. 32, 2531–2538 (2018). www.fasebj.org