Interaction between insulin and androgen signalling in decidualization,cell migration and trophoblast invasion in vitro

Finely tuned decidualization of endometrial stromal fibroblasts into decidual cells is crucial for successful implantation and a healthy pregnancy. Both insulin and androgens are known to modulate decidualization, however, their complex effect on this process has not been fully elucidated. As hyperinsulinemia and hyperandrogenism are associated in clinical conditions, we aimed to investigate the interaction between insulin and androgens on decidualization. Primary human endometrial stromal cells were decidualized in vitro in the presence of insulin and/or androgens (dihydrotestosterone (DHT), testosterone). Gene or protein expressions of decidualization markers were measured, and cells size characteristics were determined. Migration of decidualizing endometrial stromal cells and invasion of HTR-8/SVneo trophoblast spheroids were assessed. We found that insulin and androgens in combination enhanced the upregulation of several decidualization markers including prolactin, tissue factor, tissue inhibitor of matrix metalloproteinase 3 and connexin-43, and also interacted in modulating cell size characteristics resulting in enlarged decidualizing cells. However, insulin and DHT together restricted the migration of decidualizing cells and invasion of trophoblast spheroids. Our findings suggest that insulin and androgens interact to potentiate the process of decidualization. On the other hand, inhibited cell migration and trophoblast invasion might negatively impact the function of decidualizing endometrial stromal cells.     

Dihydrotestosterone potentiates insulin to up-regulate prokineticin-1 in decidualizing human endometrial stromal cells

Prokineticin 1 (PROK1) is a key regulator of embryo implantation and placentation, and its dysregulation is associated with pregnancy complications, such as pre-eclampsia and foetal growth restriction. We have previously shown that insulin strongly enhances the expression of PROK1 in human decidualizing stromal cells. Here, we demonstrate that dihydrotestosterone (DHT), but not testosterone, potentiates insulin to up-regulate PROK1 in these cells. However, the androgens alone do not influence the expression of PROK1. Our findings suggest that insulin and androgens both are involved in the regulation of PROK1 that could have implications for normal and pathological pregnancies.     

Gonadal steroids regulate the expression of aggrecanases in human endometrial stromal cells in vitro

The human endometrium undergoes cyclic change during each menstrual cycle in response to gonadal steroids. Proteolysis of endometrial extracellular matrix (ECM) is necessary to prepare this dynamic tissue for pregnancy. Proteolytic enzymes such as matrix metalloproteinase (MMP) and closely related a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) have been assigned key roles in the highly regulated cyclic remodelling of the endometrial ECM. We have previously shown that ADAMTS‐1 undergoes spatiotemporal changes in human endometrial stromal cells under the regulation of gonadal steroids. This suggests that other ADAMTS subtypes, known as aggrecanases, may contribute to the ECM remodelling events that occur in female physiological cycles and in preparation for pregnancy. To determine whether progesterone (P4), 17β‐estradiol (E2), or dihydrotestosterone (DHT), alone or in combination, are capable of regulating ADAMTS‐4, ‐5, ‐8 or ‐9 expression in human endometrial stromal cells in vitro. Real‐time quantitative PCR and Western blot analysis were used to measure ADAMTSs mRNA and protein levels in primary cultures of human endometrial stromal cells (n = 12). P4, DHT but not E2 have regulatory effects on ADAMTS‐8, ‐9 and ‐5 expression. Combined treatment with gonadal steroids did not show any synergistic or antagonistic effects. However, the synthetic steroid antagonists RU486 and hydroxyflutamide specifically inhibited the P4‐ or DHT‐mediated regulatory effects on ADAMTS expression. These studies provide evidence that the regulation of aggrecanases by gonadal steroids in human endometrial stromal cells may play an important role during decidualization.     

Analysis of interferon‐γ receptor IFNGR1 and IFNGR2 expression and regulation at the maternal‐conceptus interface and the role of interferon‐γ on endometrial expression of interferon signaling molecules during early pregnancy in pigs

It has long been known that pig conceptuses produce interferon‐γ (IFNG) at the time of implantation, but the role of IFNG and its mechanism of action at the maternal‐conceptus interface are not fully understood. Accordingly, we analyzed the expression and regulation of IFNG receptors IFNGR1 and IFNGR2 in the endometrium during the estrous cycle and pregnancy in pigs. Levels of IFNGR1 and IFNGR2 messenger RNA (mRNA) expression changed in the endometrium, with the highest levels during mid pregnancy for IFNGR1 and on Day 12 of pregnancy for IFNGR2. The expression of IFNGR1 and IFNGR2 mRNAs was also detected in conceptuses during early pregnancy and chorioallantoic tissues during mid to late pregnancy. IFNGR1 and IFNGR2 mRNAs were localized to endometrial epithelial and stromal cells and to the chorionic membrane during pregnancy. IFNGR2 protein was also localized to endometrial epithelial and stromal cells, and increased epithelial expression of IFNGR2 mRNA and protein was detectable during early pregnancy than the estrous cycle. Explant culture studies showed that estrogen increased levels of IFNGR2, but not IFNGR1, mRNAs, while interleukin‐1β did not affect levels of IFNGR1 and IFNGR2 mRNAs. Furthermore, IFNG increased levels of IRF1, IRF2, STAT1, and STAT2 mRNAs in the endometrial explants. These results in pigs indicate that IFNGR1 and IFNGR2 are expressed in a stage of pregnancy‐ and cell‐type specific manner in the endometrium and that sequential cooperative action of conceptus signals estrogen and IFNG may be critical for endometrial responsiveness to IFNs for the establishment of pregnancy in pigs.     

Lefty inhibits in vitro decidualization by regulating P57 and cyclin D1 expressions

Endometrial decidualization is highly important for successful construction and maintenance of embryo implantation and pregnancy. Lefty gene at different menstrual cycle phases has different expressions, indicating its regulatory significance. To study the mechanism of Lefty in decidualization, human endometrial stromal cells (hESCs) were cultured and induced with medroxyprogesterone acetate (MPA) and 8‐bromoadenosine‐cAMP (8‐Br‐cAMP) in vitro as a research model. Our results showed that Lefty1 overexpression inhibited MPA‐ and 8‐Br‐cAMP‐induced hESC decidualization and significantly reduced the secretion of prolactin (PRL) and insulin‐like growth factor‐binding protein 1 (IGFBP‐1). With the inhibition of Lefty1 expression, hESC decidualization induced by MPA and 8‐Br‐cAMP became more remarkable, and the secretions of PRL and IGFBP‐1 were higher too. Further tests indicated that during the process of decidualization, P57 expression increased, whereas cyclin D1 expression decreased. Although Lefty1 overexpression did not significantly change the expressions of P57 and cyclin D1, inhibition of Lefty1 expression resulted in more evident changes in P57 and cyclin D1 expressions. Meanwhile, cell cycle examination showed that Lefty1 overexpression reduced the cell cycle arrest at G1/S phase in the in vitro hESC decidualization model. Therefore, Lefty1 could regulate the cell cycle via modulating the expressions of P57 and cyclin D1 and then inhibit the decidualization in vitro. Copyright © 2014 John Wiley & Sons, Ltd.     

Long non‐coding RNA MALAT1 mediates hypoxia‐induced pro‐survival autophagy of endometrial stromal cells in endometriosis

Endometriosis is a common gynecological disease characterized by diminished apoptosis, sustained ectopic survival of dysfunctional endometrial cells. Hypoxia has been implicated as a crucial microenvironmental factor that contributes to endometriosis. It has been reported that long non‐coding RNA MALAT1 (lncRNA‐MALAT1) highly expressed in endometriosis and up‐regulated by hypoxia. Hypoxia may also induce autophagy, which might act as cell protective mechanism. However, the relationship between lncRNA‐MALAT1 and autophagy under hypoxia conditions in endometriosis remains unknown. In the present study, we found that both lncRNA‐MALAT1 and autophagy level were up‐regulated in ectopic endometrium from patients with endometriosis, and its expression level correlates positively with that of hypoxia‐inducible factor‐1α (HIF‐1α). In cultured human endometrial stromal cells, both lncRNA‐MALAT1 and autophagy were induced by hypoxia in a time‐dependent manner and lncRNA‐MALAT1 up‐regulation was dependent on HIF‐1α signalling. Our analyses also show that knockdown of lncRNA‐MALAT1 suppressed hypoxia induced autophagy. Furthermore, inhibiting autophagy with specific inhibitor 3‐Methyladenine (3‐MA) and Beclin1 siRNA enhanced apoptosis of human endometrial stromal cells under hypoxia condition. Collectively, our findings identify that lncRNA‐MALAT1 mediates hypoxia‐induced pro‐survival autophagy of endometrial stromal cells in endometriosis.     

Warburg-like Glycolysis and Lactate Shuttle in Mouse Decidua during Early Pregnancy

Decidualization is an essential process of maternal endometrial stromal cells to support pregnancy. Although it is known that enhanced glucose influx is critical for decidualization, the underlying mechanism in regulating glucose metabolism in decidua remains insufficiently understood. Here, we demonstrate that aerobic glycolysis-related genes and factors are all substantially induced during decidualization, indicating the existence of Warburg-like glycolysis in decidua. In vitro, progesterone activates hypoxia-inducible factor 1α (Hif1α) and c-Myc through Pi3k-Akt signaling pathway to maintain aerobic glycolysis in decidualizing cells. Knocking down of pyruvate kinase M2 (Pkm2) attenuates the induction of decidual marker gene. Decidual formation in vivo is also impaired by glycolysis inhibitor 3-bromopyruvate. Besides, lactate exporter monocarboxylate transporter 4 (Mct4) is induced in newly formed decidual cells, whereas lactate importer Mct1 and proliferation marker Ki-67 are complementarily located in the surrounding undifferentiated cells, which are supposed to consume lactate for proliferation. Hif1α activation is required for lactate-dependent proliferation of the undifferentiated cells. Inhibition of lactate flux leads to compromised decidualization and decelerated lactate-dependent proliferation. In summary, we reveal that Warburg-like glycolysis and local lactate shuttle are activated in decidua and play important roles for supporting early pregnancy.     

Endogenous hormone 2‐methoxyestradiol suppresses venous hypertension‐induced angiogenesis through up‐ and down‐regulating p53 and id‐1

Brain arteriovenous malformations (AVMs) which associate with angiogenesis due to local hypertension, chronic cerebral ischaemia and tissue hypoxia usually lead to haemorrhage, however, the therapeutic medicine for the disease is still lacking. 2‐methoxyestradiol (2‐ME) has been shown effective in the anti‐angiogenic treatment. This study was conducted to examine whether and how 2‐ME could improve the vascular malformations. Intracranial venous hypertension (VH) model produced in adult male Sprague‐Dawley rats and culture of human umbilical vein endothelial cells (HUVECs) at the anoxia condition were used to induce in vivo and in vitro angiogenesis, respectively. Lentiviral vectors of ID‐1 and p53 genes and of their siRNA were intracranially injected into rats and transfected into HUVECs to overexpress and down‐regulate these molecules. 2‐ME treatment not only reduced the in vivo progression of brain tissue angiogenesis in the intracranial VH rats and the in vitro increases in microvasculature formation, cellular migration and HIF‐1α expression induced by anoxia in HUVECs but also reversed the up‐regulation of ID‐1 and down‐regulation of p53 in both the in vivo and in vitro angiogenesis models. All of the anti‐angiogenesis effects of 2‐ME observed in VH rats and anoxic HUVECs were abrogated by ID‐1 overexpression and p53 knockdown. Our data collectively suggest that 2‐ME treatment inhibits hypoxia/anoxia‐induced angiogenesis dependently on ID‐1 down‐regulation and p53 up‐regulation, providing a potential alternative medical treatment for un‐ruptured AVM patients.     

Up‐regulated basigin‐2 in microglia induced by hypoxia promotes retinal angiogenesis

Retinal microglia cells contribute to vascular angiogenesis and vasculopathy induced by relative hypoxia. However, its concrete molecular mechanisms in shaping retinal angiogenesis have not been elucidated. Basigin, being involved in tumour neovasculogenesis, is explored to exert positive effects on retinal angiogenesis induced by microglia. Therefore, we set out to investigate the expression of basigin using a well‐characterized mouse model of oxygen‐induced retinopathy, which recapitulated hypoxia‐induced aberrant neovessel growth. Our results elucidate that basigin is overexpressed in microglia, which accumulating in retinal angiogenic sprouts. In vitro, conditioned media from microglia BV2 under hypoxia treatment increase migration and tube formation of retinal capillary endothelia cells, compared with media from normoxic condition. The angiogenic capacity of BV2 is inhibited after basigin knockdown by small interfering RNAs. A new molecular mechanism for high angiogenic capacity, whereby microglia cells release basigin via up‐regulation of PI3K‐AKT and IGF‐1 pathway to induce angiogenesis is unveiled. Collectively, our results demonstrate that basigin from hypoxic microglia plays a pivotal pro‐angiogenic role, providing new insights into microglia‐promoting retinal angiogenesis.     

PV1 down‐regulation via shRNA inhibits the growth of pancreatic adenocarcinoma xenografts

PV1 is an endothelial‐specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumours. On the basis of in vitro data, PV1 is thought to actively participate in angiogenesis. To test whether or not PV1 has a function in tumour angiogenesis and in tumour growth in vivo, we have treated pancreatic tumour‐bearing mice by single‐dose intratumoural delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 down‐regulation by shRNAs inhibits the growth of established tumours derived from two different human pancreatic adenocarcinoma cell lines (AsPC‐1 and BxPC‐3). The effect observed is because of down‐regulation of PV1 in the tumour endothelial cells of host origin, PV1 being specifically expressed in tumour vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumours treated or not with PV1 shRNA, and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumour angiogenesis is not impaired. Together, our data argue that down‐regulation of PV1 in tumour endothelial cells results in the inhibition of tumour growth via a mechanism different from inhibiting angiogenesis.     

Osteopontin Is Expressed in the Mouse Uterus during Early Pregnancy and Promotes Mouse Blastocyst Attachment and Invasion In Vitro

Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. In this study, we showed that Opn mRNA levels are up-regulated in the mouse uterus on day 4 and at the implantation sites on days 5 and 8 of pregnancy. Immunohistochemistry localized the OPN protein to the glandular epithelium on day 4 and to the decidual zone on day 8 of pregnancy. OPN mRNA and proteins are induced by in vivo and in vitro decidualization. OPN expression in the endometrial stromal cells is regulated by progesterone, a key regulator during decidualization. As a secreted protein, the protein level of OPN in the uterine cavity is enriched on day 4, and in vitro embryo culturing has indicated that OPN can facilitate blastocyst hatching and adhesion. Knockdown of OPN attenuates the adhesion and invasion of blastocysts in mouse endometrial stromal cells by suppressing the expression and enzymatic activity of matrix metalloproteinase-9 in the trophoblast. Our data indicated that OPN expression in the mouse uterus during early pregnancy is essential for blastocyst hatching and adhesion and that the knockdown of OPN in mouse endometrial stroma cells could lead to a restrained in vitro trophoblast invasion.     

Decreased ANGPTL4 impairs endometrial angiogenesis during peri‐implantation period in patients with recurrent implantation failure

Insufficient endometrial angiogenesis during peri‐implantation impairs endometrial receptivity (ER), which contributes to recurrent implantation failure (RIF) during in vitro fertilization and embryo transfer (IVF‐ET). Angiopoietin‐like protein 4 (ANGPTL4) acts as a multifunctional secretory protein and is involved in the regulation of lipid metabolism and angiogenesis in various tissues including the endometrium. Herein, we found decreased ANGPTL4 expression in endometrial tissue and serum during peri‐implantation period in 18 RIF‐affected women with elevated uterine arterial impedance (UAI) compared with the pregnancy controls. ANGPTL4 and peroxisome proliferator‐activated receptor gamma (PPARγ) expression were up‐regulated upon decidualization on human endometrial stromal cells (HESCs). Rosiglitazone promoted the expression of ANGPTL4 in HESCs and human umbilical vein endothelial cells (HUVECs) via PPARγ. ANGPTL4 promoted the proliferation, migration and angiogenesis of HUVECs in vitro. Our results suggest that decreased abundance of ANGPTL4 in endometrial tissues impairs the endometrial receptivity via restraining endometrial angiogenesis during decidualization; while rosiglitazone‐induced ANGPTL4 up‐regulation in hESCs and HUVECs through PPARγ. Therefore, ANGPTL4 could be a potential therapeutic approach for some RIF‐affected women with elevated UAI.     

Internalization of nanopolymeric tracers does not alter characteristics of placental cells

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA‐NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV‐MSC). We report that PMMP‐NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP‐NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP‐NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS‐treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP‐NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP‐NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV‐MSC in preclinical models of inflammatory‐driven diseases.     

Bone marrow mesenchymal stem cells increase motility of prostate cancer cells via production of stromal cell‐derived factor‐1α

Prostate cancer frequently metastasizes to the bone, and the interaction between cancer cells and bone microenvironment has proven to be crucial in the establishment of new metastases. Bone marrow mesenchymal stem cells (BM‐MSCs) secrete various cytokines that can regulate the behaviour of neighbouring cell. However, little is known about the role of BM‐MSCs in influencing the migration and the invasion of prostate cancer cells. We hypothesize that the stromal cell‐derived factor‐1α released by BM‐MSCs may play a pivotal role in these processes. To study the interaction between factors secreted by BM‐MSCs and prostate cancer cells we established an in vitro model of transwell co‐culture of BM‐MSCs and prostate cancer cells DU145. Using this model, we have shown that BM‐MSCs produce soluble factors which increase the motility of prostate cancer cells DU145. Neutralization of stromal cell‐derived factor‐1α (SDF1α) via a blocking antibody significantly limits the chemoattractive effect of bone marrow MSCs. Moreover, soluble factors produced by BM‐MSCs greatly activate prosurvival kinases, namely AKT and ERK 1/2. We provide further evidence that SDF1α is involved in the interaction between prostate cancer cells and BM‐MSCs. Such interaction may play an important role in the migration and the invasion of prostate cancer cells within bone.     

Natural Selection of Human Embryos: Impaired Decidualization of Endometrium Disables Embryo-Maternal Interactions and Causes Recurrent Pregnancy Loss

Background

Recurrent pregnancy loss (RPL), defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs) into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation.

Methodology/Principal Findings

Analysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL) but increased levels of prokineticin-1 (PROK1), a cytokine that promotes implantation. These in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP) intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered “superfertile”, defined by a mean TTP of 3 months or less.

Conclusions

Impaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL.     

The apolipoprotein A‐I mimetic peptide,D‐4F,restrains neointimal formation through heme oxygenase‐1 up‐regulation

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, possesses distinctly anti‐atherogenic effects. However, the biological functions and mechanisms of D‐4F on the hyperplasia of vascular smooth muscle cells (VSMCs) remain unclear. This study aimed to determine its roles in the proliferation and migration of VSMCs. In vitro, D‐4F inhibited VSMC proliferation and migration induced by ox‐LDL in a dose‐dependent manner. D‐4F up‐regulated heme oxygenase‐1 (HO‐1) expression in VSMCs, and the PI3K/Akt/AMP‐activated protein kinase (AMPK) pathway was involved in these processes. HO‐1 down‐regulation with siRNA or inhibition with zinc protoporphyrin (Znpp) impaired the protective effects of D‐4F on the oxidative stress and the proliferation and migration of VSMCs. Moreover, down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) abolished the activation of Akt and AMPK, the up‐regulation of HO‐1 and the anti‐oxidative effects of D‐4F. In vivo, D‐4F restrained neointimal formation and oxidative stress of carotid arteries in balloon‐injured Sprague Dawley rats. And inhibition of HO‐1 with Znpp decreased the inhibitory effects of D‐4F on neointimal formation and ROS production in arteries. In conclusion, D‐4F inhibited VSMC proliferation and migration in vitro and neointimal formation in vivo through HO‐1 up‐regulation, which provided a novel prophylactic and therapeutic strategy for anti‐restenosis of arteries.     

Hypoxia induces BMP‐2 expression via ILK,Akt, mTOR,and HIF‐1 pathways in osteoblasts

It has been shown that hypoxia stimulation regulates bone formation, maintenance, and repair. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. However, the effects of hypoxia exposure on BMP‐2 expression in cultured osteoblasts are largely unknown. Here we found that hypoxia stimulation increased mRNA and protein levels of BMP‐2 by qPCR, Western blot and ELISA assay in osteoblastic cells MG‐63, hFOB and bone marrow stromal cells M2‐10B4. Integrin‐linked kinase (ILK) inhibitor (KP‐392), Akt inhibitor (1L‐6‐hydroxymethyl‐chiro‐inositol‐2‐[(R)‐2‐O‐methyl‐3‐O‐octadecylcarbonate]) or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) inhibited the potentiating action of hypoxia. Exposure to hypoxia increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, hypoxia also increased the stability and activity of HIF‐1 protein. The binding of HIF‐1α to the HRE elements after exposure to hypoxia was measured by EMSA assay. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for hypoxia‐induced HIF‐1α activation and subsequent BMP‐2 up‐regulation. Taken together, our results provide evidence that hypoxia enhances BMP‐2 expression in osteoblasts by an HIF‐1α‐dependent mechanism involving the activation of ILK/Akt and mTOR pathways. J. Cell. Physiol. 223:810–818, 2010. © 2010 Wiley‐Liss, Inc.