Cartiotonic steroids affect monolayer permeability in lymphatic endothelial cells.

Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100 nM. Some LECs were pretreated with 1 µM L-NAME (N-Nitro-L-Arginine Methyl Ester) before adding 100 nM MBG or cinobufotalin (CINO). Expression of ß-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure ß-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1 nM) and CINO (≥ 10 nM) caused an increase (p < 0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1 µM L-NAME attenuated (p < 0.05) the MPLEC. The ß-catenin expression in LECs was downregulated (p < 0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p < 0.05) by CINO, and MLC20 phosphorylation was upregulated (p < 0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.

Hyperglycemia impairs cytotrophoblast function via stress signaling.

OBJECTIVE: Diabetes mellitus is a risk factor for preeclampsia. Cytotrophoblast (CTB) invasion is facilitated from the conversion of plasminogen to plasmin by urokinase plasminogen activator (uPA), regulated by plasminogen activator inhibitor 1 (PAI-1), and may be inhibited in preeclampsia. This study assessed signaling mechanisms of hyperglycemia-induced CTB dysfunction. STUDY DESIGN: Human CTBs were treated with 45, 135, 225, 495, or 945 mg/dL glucose for 48 hours. Some cells were pretreated with a p38 inhibitor (SB203580) or a peroxisome proliferator-activated receptor-gamma (PPAR-γ) ligand (rosiglitazone). Expression of uPA, PAI-1, and PPAR-γ levels and p38 mitogen-activated protein kinase phosphorylation were measured by Western blot in cell lysates. Messenger ribonucleic acid of uPA and PAI-1 was measured by quantitative polymerase chain reaction. Levels of interleukin-6, angiogenic (vascular endothelial growth factor [VEGF], placenta growth factor [PlGF]) and antiangiogenic factors (soluble fms-like tyrosine kinase-1 [sFlt-1], soluble endoglin [sEng]) were measured in the media by enzyme-linked immunosorbent assay kits. Statistical comparisons were performed using analysis of variance with a Duncan's post-hoc test. RESULTS: Both uPA and PAI-1 protein and messenger ribonucleic acid were down-regulated (P < .05) in CTBs treated with 135 mg/dL glucose or greater compared with basal (45 mg/dL). The sEng, sFlt-1, and interleukin-6 were up-regulated, whereas the VEGF and PlGF were down-regulated by 135 mg/dL glucose or greater. p38 phosphorylation and PPAR-γ were up-regulated (P < .05) in hyperglycemia-treated CTBs. The SB203580 or rosiglitazone pretreatment showed an attenuation of glucose-induced down-regulation of uPA and PAI-1. CONCLUSION: Hyperglycemia disrupts the invasive profile of CTB by decreasing uPA and PAI-1 expression; down-regulating VEGF and PlGF; and up-regulating sEng, sFlt-1, and interleukin-6. Attenuation of CTB dysfunction by SB203580 or rosiglitazone pretreatment suggests the involvement of stress signaling.

Interleukin-6-driven progranulin expression increases cholangiocarcinoma growth by an Akt-dependent mechanism.

BACKGROUND AND OBJECTIVES: Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. The growth factor, progranulin, is overexpressed in a number of tumours. The study aims were to assess the expression of progranulin in cholangiocarcinoma and to determine its effects on tumour growth. METHODS: The expression and secretion of progranulin were evaluated in multiple cholangiocarcinoma cell lines and in clinical samples from patients with cholangiocarcinoma. The role of interleukin 6 (IL-6)-mediated signalling in the expression of progranulin was assessed using a combination of specific inhibitors and shRNA knockdown techniques. The effect of progranulin on proliferation and Akt activation and subsequent effects of FOXO1 phosphorylation were assessed in vitro. Progranulin knockdown cell lines were established, and the effects on cholangiocarcinoma growth were determined. RESULTS: Progranulin expression and secretion were upregulated in cholangiocarcinoma cell lines and tissue, which were in part via IL-6-mediated activation of the ERK1/2/RSK1/C/EBPß pathway. Blocking any of these signalling molecules, by either pharmacological inhibitors or shRNA, prevented the IL-6-dependent activation of progranulin expression. Treatment of cholangiocarcinoma cells with recombinant progranulin increased cell proliferation in vitro by a mechanism involving Akt phosphorylation leading to phosphorylation and nuclear extrusion of FOXO1. Knockdown of progranulin expression in cholangiocarcinoma cells decreased the expression of proliferating cellular nuclear antigen, a marker of proliferative capacity, and slowed tumour growth in vivo. CONCLUSIONS: Evidence is presented for a role for progranulin as a novel growth factor regulating cholangiocarcinoma growth. Specific targeting of progranulin may represent an alternative for the development of therapeutic strategies.

Marinobufagenin is an upstream modulator of Gadd45a stress signaling in preeclampsia.

Preeclampsia (PE) is a hypertensive disorder of pregnancy, in which marinobufagenin (MBG), a circulating cardiotonic steroid, is increased. The Gadd45a stress sensor protein is an upstream modulator of the pathophysiological changes observed in PE. However, the effects of MBG on Gadd45a stress signaling remain unknown. We examined the expression of Gadd45a, the sFlt-1 receptor, and p38, as well as caspase 3 and 8 activities in placental samples from four groups of rats. These were: normal pregnant (NP, n=8); pregnant rats which received weekly injections of desoxycorticosterone acetate and 0.9% saline as their drinking water (PDS, n=9); normal pregnant rats injected with MBG (NPM, n=8); and PDS rats injected with resibufogenin (RBG), an in vivo antagonist of MBG (PDSR, n=8). Utilizing human cytotrophoblast (CTB) cells, we examined the effect of MBG on these stress signaling proteins in vitro. Placental Gadd45a expression, caspase 3 and 8 activities, sFlt-1 concentrations, and sFlt-1 receptor expression were significantly higher in PDS and NPM compared to NP and PDSR rats. Gadd45a protein was significantly upregulated in the CTB cells when MBG was present in concentrations ≥1nM. Treatment with MBG (≥1nM) also significantly arrested cell cycle progression and activated the expression of the Gadd45a-mediated stress signaling proteins. Inhibition of Gadd45a through RNAi-mediation attenuated MBG-induced CTB cell stress signaling. In conclusion, MBG is involved in the alteration in Gadd45a stress signaling both in vivo and in vitro and RBG prevents these changes when administered in vivo.

Suppression of the HPA axis during extrahepatic biliary obstruction induces cholangiocyte proliferation in the rat.

Cholestatic patients often present with clinical features suggestive of adrenal insufficiency. In the bile duct-ligated (BDL) model of cholestasis, the hypothalamic-pituitary-adrenal (HPA) axis is suppressed. The consequences of this suppression on cholangiocyte proliferation are unknown. We evaluated 1) HPA axis activity in various rat models of cholestasis and 2) effects of HPA axis modulation on cholangiocyte proliferation. Expression of regulatory molecules of the HPA axis was determined after BDL, partial BDL, and α-naphthylisothiocyanate (ANIT) intoxication. The HPA axis was suppressed by inhibition of hypothalamic corticotropin-releasing hormone (CRH) expression by central administration of CRH-specific Vivo-morpholinos or by adrenalectomy. After BDL, the HPA axis was reactivated by 1) central administration of CRH, 2) systemic ACTH treatment, or 3) treatment with cortisol or corticosterone for 7 days postsurgery. There was decreased expression of 1) hypothalamic CRH, 2) pituitary ACTH, and 3) key glucocorticoid synthesis enzymes in the adrenal glands. Serum corticosterone and cortisol remained low after BDL (but not partial BDL) compared with sham surgery and after 2 wk of ANIT feeding. Experimental suppression of the HPA axis increased cholangiocyte proliferation, shown by increased cytokeratin-19- and proliferating cell nuclear antigen-positive cholangiocytes. Conversely, restoration of HPA axis activity inhibited BDL-induced cholangiocyte proliferation. Suppression of the HPA axis is an early event following BDL and induces cholangiocyte proliferation. Knowledge of the role of the HPA axis during cholestasis may lead to development of innovative treatment paradigms for chronic liver disease.

Anandamide exerts its antiproliferative actions on cholangiocarcinoma by activation of the GPR55 receptor.

Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. It has previously been shown that the endocannabinoid anandamide exerts antiproliferative effects on cholangiocarcinoma independent of any known cannabinoid receptors, and by the stabilization of lipid rafts, thereby allowing the recruitment and activation of the Fas death receptor complex. Recently, GPR55 was identified as a putative cannabinoid receptor; therefore, the role of GPR55 in the antiproliferative effects of anandamide was evaluated. GPR55 is expressed in all cholangiocarcinoma cells and liver biopsy samples to a similar level as in non-malignant cholangiocytes. Treatment with either anandamide or the GPR55 agonist, O-1602, reduced cholangiocarcinoma cell proliferation in vitro and in vivo. Furthermore, knocking down the expression of GPR55 prevented the antiproliferative effects of anandamide. Coupled to these effects was an increase in JNK activity. The antiproliferative effects of anandamide could be blocked by pretreatment with a JNK inhibitor and the lipid raft disruptors ß-methylcyclodextrin and fillipin III. Activation of GPR55 by anandamide or O-1602 increased the amount of Fas in the lipid raft fractions, which could be blocked by pretreatment with the JNK inhibitor. These data represent the first evidence that GPR55 activation by anandamide can lead to the recruitment and activation of the Fas death receptor complex and that targeting GPR55 activation may be a viable option for the development of therapeutic strategies to treat cholangiocarcinoma.

Marinobufagenin levels in preeclamptic patients: a preliminary report.

Preeclampsia is a disorder resulting in significant fetomaternal complications with no definitive pharmacological intervention. A bufadienolide, marinobufagenin, has been implicated in the etiology of preeclampsia. We investigated both the blood and urine levels of marinobufagenin in preeclamptic and control subjects. Preeclamptic and normotensive pregnant women were recruited at various gestational age periods. Blood and urine specimens were obtained and analyzed for marinobufagenin levels and creatinine. The former determination was performed utilizing a new, novel chemifluorescent enzyme-linked immunosorbent assay. The marinobufagenin levels were higher in preeclamptics than in the controls in both serum and urine at various gestational age periods. Additionally, the mean level of marinobufagenin in the preeclamptic group was significantly greater than in controls in both blood and urine specimens ( P < 0.05). These data are consistent with a role for marinobufagenin in the etiology of preeclampsia. This study demonstrated comparable results in blood and urine samples. This suggests that subsequent studies on levels of marinobufagenin as a screening test for preeclampsia could be done utilizing urine samples, which are easier to obtain, less invasive, more cost-effective, and as accurate as the serological tests.

A chemifluorescent immunoassay for the determination of marinobufagenin in body fluids.

We describe here the development of a chemifluorescent competitive enzyme-linked immunosorbent assay (ELISA) that quantifies marinobufagenin (MBG) levels in biological fluids. Based on a polyclonal antibody raised against a novel MBG-bovine serum albumin conjugate, this assay achieved an MBG detection limit of less than 9 pg/mL. MBG levels in various rat urine and serum samples were effectively determined using this methodology. Interassay variability averaged 9.8%, while intra-assay variability averaged 1.9 and 2.5% in representative serum and urine samples, respectively. Recovery of exogenously added MBG averaged 106%, and parallelism data further established the accuracy of the assay. Employment of this assay to detect MBG abnormalities represents a powerful tool for the possible diagnosis, prevention and management of human hypertensive states, particularly preeclampsia.

Age-Dependent Decline in Neuron Growth Potential and Mitochondria Functions in Cortical Neurons.

The age of incidence of spinal cord injury (SCI) and the average age of people living with SCI is continuously increasing. However, SCI is extensively modeled in young adult animals, hampering translation of research to clinical applications. While there has been significant progress in manipulating axon growth after injury, the impact of aging is still unknown. Mitochondria are essential to successful neurite and axon growth, while aging is associated with a decline in mitochondrial functions. Using isolation and culture of adult cortical neurons, we analyzed mitochondrial changes in 2-, 6-, 12- and 18-month-old mice. We observed reduced neurite growth in older neurons. Older neurons also showed dysfunctional respiration, reduced membrane potential, and altered mitochondrial membrane transport proteins; however, mitochondrial DNA (mtDNA) abundance and cellular ATP were increased. Taken together, these data suggest that dysfunctional mitochondria in older neurons may be associated with the age-dependent reduction in neurite growth. Both normal aging and traumatic injury are associated with mitochondrial dysfunction, posing a challenge for an aging SCI population as the two elements can combine to worsen injury outcomes. The results of this study highlight this as an area of great interest in CNS trauma.

Alterations in the renin-angiotensin system in a rat model of human preeclampsia.

BACKGROUND/AIMS: Preeclampsia is a hypertensive disorder unique to pregnancy in which elevated levels of marinobufagenin (MBG) have been reported. The renin-angiotensin system (RAS) may also play a role in the pathogenesis of preeclampsia. The aim of our study was to evaluate the status of the RAS in a rat model of preeclampsia characterized by hypertension, proteinuria, excessive weight gain and intrauterine growth restriction. METHODS: We evaluated the components of the RAS in 5 groups of animals: nonpregnant control; normal pregnant (NP); pregnant rats which received injections of desoxycorticosterone acetate and 0.9% saline as their drinking water (PDS); normal pregnant rats injected with MBG (NPM), and PDS rats to which resibufogenin (RBG) had been administered (PDSR). RBG is an antagonist of MBG differing in structure from MBG only in the absence of a hydroxyl group in the beta-5 position. RESULTS: Plasma levels of active renin, renin and Ang II were significantly lower in PDS and NPM compared to NP and PDSR rats (p < 0.05). However, placental levels of these components were increased significantly in PDS and NPM compared to NP and PDSR rats (p < 0.05). Placental AT(1) receptor expression was significantly higher in PDS and NPM compared to NP and PDSR rats (p < 0.05). CONCLUSIONS: (1) The peripheral RAS is downregulated, and the uteroplacental RAS is upregulated in this rat model of preeclampsia; (2) MBG is involved in the causation of these alterations, and (3) RBG prevents these changes.

The treatment of preeclampsia in a rat model employing Digibind.

Preeclampsia (PE) is a disorder that results in significant fetomaternal morbidity and mortality with yet no definitive pharmacological intervention. It involves the development of de novo hypertension and proteinuria after 20 weeks of pregnancy. All too often, intrauterine growth restriction (IUGR) occurs. Evidence has accrued that implicates the cardiac glycosides (the cardenolides and the bufadienolides) as potentially involved in the pathophysiology of PE. These compounds act by inhibiting Na(+)/K(+) ATPase. Digibind (digoxin immune Fab) antagonizes this action of the cardenolides. It also has cross-reactivity against the bufadienolides, including marinobufagenin. This study investigated the effects of Digibind in a rat model of PE. We induced a syndrome in rats, which includes many of the phenotypic characteristics of human PE. Digibind, in escalating doses, was given on days 10 to 20 of pregnancy. Digibind produced significant lowering of the blood pressure and reduced proteinuria in our rat model of PE. However, it also did not avert IUGR. In view of these findings, in our experimental model of human PE, further studies in the quest for effective treatment of PE need to focus on pharmaceuticals that can remedy the syndrome without compromising the fetus.

Vascular leak in a rat model of preeclampsia.

BACKGROUND/AIMS: Preeclampsia is a hypertensive disorder which develops de novo in women during pregnancy. The urinary excretion of the cardiotonic steroid, marinobufagenin (MBG), is increased prior to the development of hypertension. Preeclamptic patients are volume expanded but much of the excess salt and water appears to be located primarily in the interstitial space. Therefore, 'capillary leak' syndrome has been postulated in this disorder. METHODS: We evaluated the vascular leakage in normal rats following MBG injection and in a rat model of human preeclampsia. We measured the changes in light intensity comparing that in the intravascular to the extravascular space by assessing 'leak' of fluorescein-labeled albumin (FITC-albumin) from mesenteric postcapillary venules. RESULTS: FITC-albumin extravasation continued to increase in a time-dependent fashion after MBG infusion and was significant (p < 0.05) at 60 min of observation when compared to sham rats. We also observed a significant difference in 'vascular leakage' in preeclamptic rats compared to control non-pregnant and normal pregnant groups starting at 20 min after the FITC-albumin infusion. CONCLUSION: We propose that MBG is involved in the production of a 'vascular leak' in our rat model of preeclampsia.

Suppression of aldosterone and progesterone in preeclampsia.

OBJECTIVE: Preeclampsia (preE) is a hypertensive disorder seen in 3-10% of human pregnancies and is diagnosed by de novo onset of hypertension and proteinuria. Several research groups provided evidence for reduced aldosterone (Aldo) and progesterone (Prog) availability in preE. The aim of this study was to determine the levels of Aldo and Prog in preE. METHODS: Normal pregnant (NP; n = 39) and preE (n = 30) patients were recruited to have their blood drawn between 21 and 40 weeks of pregnancy. Two groups of rats were used in this study: NP rats (n = 10) and preE rats (n = 10), which were given weekly injections of desoxycorticosterone acetate and 0.9% saline to drink. Aldo and Prog levels were assayed in plasma and urine samples by ELISA kits. RESULTS: In preE patients, the mean Aldo and Prog levels were suppressed (p < 0.05) compared to NP patients. NP patients exhibited a trend of increased levels of Aldo with an increase in gestational age; however, preE patients had the opposite trend. Both normal and preE patients exhibited a trend of increased levels of Prog with an increase in gestational age. The plasma and urinary Aldo and Prog levels were lower (p < 0.05) in preE rats compared to NP rats. CONCLUSIONS: We have demonstrated using a rat model and patients that both Aldo and Prog are suppressed in preE and thus may be used as biomarkers for preE.

A synthetic thiourea-based tripodal receptor that impairs the function of human first trimester cytotrophoblast cells.

A synthetic tripodal-based thiourea receptor (PNTTU) was used to explore the receptor/ligand binding affinity using CTB cells. The human extravillous CTB cells (Sw.71) used in this study were derived from first trimester chorionic villus tissue. The cell proliferation, migration and angiogenic factors were evaluated in PNTTU-treated CTB cells. The PNTTU inhibited the CTBs proliferation and migration. The soluble fms-like tyrosine kinase-1 (sFlt-1) secretion was increased while vascular endothelial growth factor (VEGF) was decreased in the culture media of CTB cells treated with ≥1 nM PNTTU. The angiotensin II receptor type 2 (AT2) expression was significantly upregulated in ≥1 nM PNTTU-treated CTB cells in compared to basal; however, the angiotensin II receptor, type 1 (AT1) and vascular endothelial growth factor receptor 1 (VEGFR-1) expression was downregulated. The anti-proliferative and anti-angiogenic effect of this compound on CTB cells are similar to the effect of CTSs. The receptor/ligand affinity of PNTTU on CTBs provides us the clue to design a potent inhibitor to prevent the CTS-induced impairment of CTB cells.

Resibufogenin administration prevents oxidative stress in a rat model of human preeclampsia.

BACKGROUND AND OBJECTIVES: Marinobufagenin (MBG) is a cardiotonic steroid that is increased in preeclampsia. An analog of MBG, resibufogenin (RBG), prevents the development of preeclampsia in a rat model. Oxidative stress is a concomitant of endothelial dysfunction in the latter disorder. The objective of the current studies was to evaluate the status of oxidative stress in a rat model of preeclampsia. METHODS: We measured the aortic AT(1) receptor expression and urinary excretion of 8-isoprostane (8IP) in rats rendered "preeclamptic" and compared the findings to those obtained in normal pregnant animals, pregnant rats injected with MBG, and preeclamptic rats treated with RBG. RESULTS: Aortic AT(1) receptor expression and the urinary excretion of 8IP were significantly augmented in "preeclamptic" and MBG-injected pregnant rats compared to normal pregnant animals. RBG prevented evidence of oxidative stress in "preeclamptic" rats. CONCLUSION: MBG is involved in the causation of oxidative stress in our rat model and RBG attenuates this change.

Resibufogenin prevents the manifestations of preeclampsia in an animal model of the syndrome.

BACKGROUND AND OBJECTIVES: We have developed a rat model of preeclampsia which is based upon excessive volume expansion and includes hypertension, proteinuria and intrauterine growth restriction. In this model, the urinary excretion of the circulating steroid inhibitor of Na +/ K+ ATPase, marinobufagenin, is increased prior to the development of hypertension and proteinuria. An analogue of marinobufagenin, resibufogenin, successfully treats the hypertension and proteinuria. METHODS: We administered resibufogenin early in pregnancy in this model, prior to the development of the syndrome. RESULTS: We found that resibufogenin not only prevented the advent of hypertension and proteinuria, but also the development of intrauterine growth restriction. DISCUSSION: These results may have relevance to the human condition.

Marinobufagenin causes endothelial cell monolayer hyperpermeability by altering apoptotic signaling.

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid that is involved in the inhibition of the sodium pump Na(+)/K(+)-ATPase. Increased plasma levels of MBG have been reported in patients with preeclampsia. MBG increases microvascular barrier permeability in an animal model of preeclampsia. However, the mechanism by which MBG impairs endothelial permeability is unknown. We utilized rat lung microvascular endothelial cells (RLMEC) to examine alterations in MBG-induced monolayer permeability and the effect of MBG on the phosphorylation status of ERK1/2, Jnk, and p38. Apoptosis was evaluated by examining alterations in caspases 3/7, 8, and 9 and annexin-V staining. We also examined the effect of MBG on the endothelial adherens junctions of the RLMEC monolayer. MBG inhibited the proliferation, and increased the monolayer permeability, of RLMEC. These actions of MBG were attenuated by ERK, p38, and pan caspase inhibition. MBG significantly decreased the phosphorylation of ERK1/2 and activated the phosphorylation of Jnk and p38. MBG also significantly increased the expression of caspases 3/7, 8, and 9, indicating the activation of apoptosis. MBG-induced apoptosis signaling was not observed in cells pretreated with a p38 inhibitor. MBG treatment induced the disruption of endothelial cell junctions. This effect was prevented by a pan caspase inhibitor. In conclusion, 1) MBG induced an impairment of RLMEC proliferation; 2) the bufadienolide also caused endothelial hyperpermeability; and 3) these effects of MBG were mediated by the downregulation of ERK1/2, the upregulation of Jnk and p38, by the activation of apoptosis, and by the disruption of endothelial cell junctions.

Examination of the cellular mechanisms by which marinobufagenin inhibits cytotrophoblast function.

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid involved in the inhibition of Na(+)/K(+)-ATPase. Increased plasma levels have been reported in patients with volume expansion-related hypertension. We have recently demonstrated that MBG impairs first trimester cytotrophoblast (CTB) cell proliferation, migration, and invasion, which may play a role in the development of preeclampsia. However, whether apoptosis contributes to altered CTB cell function by MBG remains unknown. Using the human extravillous CTB cell line SGHPL-4, we examined the effect of MBG and a similar Na(+)/K(+)-ATPase inhibitor, ouabain, on the phosphorylation status of Jnk, p38, and Src. Additionally, we measured apoptosis by caspase 9 and 3/7 activity and by annexin-V staining. We also investigated interleukin-6 (IL-6) secretion with or without p38 and Jnk inhibition. MBG significantly increased the phosphorylation of Jnk, p38, and Src and increased the expression of caspase 9 and 3/7 indicating the activation of apoptosis. MBG treatment also stimulated the expression of the early apoptosis marker, annexin-V, which was prevented by Jnk and p38 inhibition. MBG also stimulated the secretion of IL-6, which was attenuated by p38 inhibition. Ouabain had similar effects to those of MBG, suggesting that the apoptotic effects on CTB cells may be mediated by inhibition of Na(+)/K(+)-ATPase. In conclusion, the MBG-induced impairment of CTB function occurs via activation of Jnk, p38, and Src leading to increased apoptosis and IL-6 secretion. These observations may have clinical applicability with respect to the therapy of preeclampsia.