Activity and Expression of the Na+/H+ Exchanger in the Microvillous Plasma Membrane of the Syncytiotrophoblast in Relation to Gestation and Small for Gestational Age Birth

Abstract

The effect of gestational age, low birth weight, and umbilical plasma pH on the activity and expression of the Na ϩ /H ϩ exchanger in the microvillous plasma membrane (MVM) of the placental syncytiotrophoblast was investigated. MVM were isolated from placentas of fetuses delivered in the first and second trimesters and from appropriately grown for gestational age (AGA) and small for gestational age (SGA) babies born at term. 48 [0.92-1.66] nmol/mg protein/30s, respectively, n ϭ 6, 12, and 9). As regards exchanger isoforms, Western blotting showed that NHE1 expression did not change across gestation, but NHE2 and NHE3 expression were lower (p Ͻ 0.01) in the first and second trimesters than in term AGA MVM. There were no differences in Na ϩ /H ϩ exchanger activity or in NHE1-3 expression in term AGA MVM versus SGA (n ϭ 11) MVM. There was no correlation between exchanger activity and umbilical artery or vein plasma pH, although with a relatively small number of samples (n ϭ 12 and 15, respectively). We conclude that there is differential regulation of the activity and expression of Na Abbreviations SGA, small for gestational age MVM, microvillous plasma membrane BM, basal membrane IUGR, intrauterine growth restricted AGA, appropriately grown for gestational age SGAD, small for gestational age babies with abnormal systolic/diastolic ratios on umbilical Doppler ultrasound assessment.Fetal and neonatal acidemia are associated with intrapartum hypoxia, a known risk factor for poor neurologic outcome of affected babies (1). Fetal acidemia is more common in SGA babies, especially those with compromised umbilical artery blood flow (2, 3). The role of the placenta in regulating the pH of the fetus is not fully understood. Undoubtedly, proton and bicarbonate may be rapidly lost, or gained, across the placenta in the form of carbon dioxide and water. Furthermore, the placenta has a high paracellular permeability to hydrophilic solutes including ions (4, 5) so that simple diffusion of proton and bicarbonate via this route might also be important. In addition to this, Na ϩ /H ϩ and Cl Ϫ /HCO 3 Ϫ exchangers have been identified in the MVM and BM of the syncytiotrophoblast (6 -10), the transporting epithelium of the placenta. Although these might be involved in the homeostasis of intracellular syncytiotrophoblast pH, they also afford the possibility of a transcellular mechanism of transplacental transfer, which might contribute to fetal pH regulation. A role of the exchangers in either of these processes would provide a mechanism for acute, hormonal (e.g. epidermal growth factor, angiotensin II) control of H ϩ transport, by analogy to other tissues (11). The Na ϩ /H ϩ exchanger is found in most cells and contributes to intracellular pH homeostasis and, depending on the organ, transepithelial transport (11). Six different isoforms of this transport protein, designated NHE, have now been cloned and sequenced (11). Na

Placental Phenotypes of Intrauterine Growth

“…These two critical membrane transport systems are involved in pH regulation, vectorial Na ϩ transport and maintenance of the Na ϩ gradient that drives the transport of other vital nutrients such as amino acids. Alterations in MVM NHE activity appear to be related to the severity of IUGR: transporter activity is not changed in less severe IUGR cases, primarily at term (22,30,32). Some ions appear to be regulated quite differently.…”

Epidermal growth factor and sphingosine-1-phosphate stimulate Na+/H+exchanger activity in the human placental syncytiotrophoblast

“…There are nine members of the NHE protein family (25), and at least three NHE isoforms have been identified in the human placenta, localized to both the microvillous (MVM, maternal facing) and basal (fetal facing) plasma membranes (BM) of the syncytiotrophoblast. Reported NHE isoform distribution to these membranes varies between studies (14,16,26,33), but we have shown by immunoblotting that NHE 1 protein is localized to the MVM and BM, while NHE 3 is only found on the MVM (33). However, NHE 1 activity predominates on MVM, with no evidence of NHE 3 activity under our normal conditions of experimentation (33).…”

“…The stimulatory effect of EGF is completely blocked by amiloride at 500 M, which preferentially blocks the activation of NHE1 but also potentially blocks activation of NHE2 and 3 (23). NHE2 activity is not present in the syncytiotrophoblast (33); however, NHE2 protein is present in the syncytiotrophoblast (14), and mRNA is present in the placenta throughout gestation (19), and absence of its activity cannot be confirmed without a specific inhibitor able to distinguish between NHE1 and NHE2. This is currently not available.…”

Regulation of Expression and Localisation of the Na+/H+ Exchanger (NHE) 3 and the NHE Regulatory Factor 2 in Baboon Placental Syncytiotrophoblast by Oestrogen

“…Briefly, MVM were centrifuged at 15,000 × g, washed with 500 μl ice-cold intravesicular buffer (IVB; pH 6.5) comprised of 25 mM 2[N-morpholino] ethanesulphonic acid (MES; Sigma) and 150 mM KCl, and incubated for 30 min (37°C) in 200 μl IVB containing 10 μM 2′,7′bis(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM; Molecular Probes). After washing with IVB, samples were incubated for 15 min (4°C) in 600 μl IVB without/with amiloride (Sigma) at a concentration (0.5 mM) shown to inhibit NHE activity by >98% [7]. MVM vesicles (200 μg protein/600 μl IVB) were then placed into one chamber of a KinTek SF 2001 Stopped Flow spectrofluorimeter (KinTek Laboratories, La Marque, TX) and 2 ml of extravesicular buffer (EVB; pH 9.5) comprised of 10 mM HEPES (Sigma) and 150 mM NaCl added to a second chamber.…”

“…Moreover, the specific concentrations of NHE1 and its regulatory factor NHERF1, both localized primarily to the microvillus membrane (MVM), were similar at mid and late gestation in association with a 4-fold increase in MVM alkaline phosphatase activity and presumably microvillus surface area as shown in human placenta [5,6]. Despite the physiologic importance of the placental NHE system [3,[7][8][9], our understanding of the factors controlling NHE/NHERF expression and localization in the primate syncytiotrophoblast is incomplete.…”