The objective of this study was to investigate the mechanism of PGE2 regulation of Cl− transport across glandular endometrial cells grown in primary culture. Most of the basal short circuit current (Isc) was inhibited by luminal addition of 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB) or glibenclamide, suggesting the presence of a basally active Cl− conductance in the apical membrane. Basolateral addition of 10 μM PGE2 increased Isc by 41 ± 3 μA. A similar response was observed when cells were treated with 8‐(4‐chlorophenylthio) adenosine 3′,5′‐cyclic monophosphate (CPT‐cAMP). Pretreatment of monolayers with NPPB and glibenclamide blocked the PGE2 and cAMP‐mediated increase in Isc, suggesting that the effects of PGE2 and cAMP were dependent on the activity of an apical NPPB‐ and glibenclamide‐sensitive conductance. Addition of 50 nM antiPGE2 antibody to the basolateral bathing solution decreased basal Isc by 20 % and shifted the threshold response to exogenous PGE2. This result suggests autocrine regulation of electrogenic Cl− transport by PGE2. Experiments with amphotericin B‐permeabilized monolayers revealed that the apical PGE2‐activated, NPPB‐ and glibenclamide‐sensitive conductance was Cl− dependent and that the current‐voltage relationship and anion permeation properties (SCN−>Br− > Cl− > I−) were characteristic of the cystic fibrosis transmembrane conductance regulator (CFTR). Cultured porcine endometrial epithelial cells were specifically labelled with an antibody to a peptide sequence within the regulatory domain of CFTR. The effect of PGE2 was blocked by basolateral addition of bumetanide and furosemide at concentrations that are selective for inhibition of Na+‐K+‐2Cl−cotransport activity. The effect of bumetanide on Isc was Cl− dependent, suggesting a role for the bumetanide‐sensitive transport pathway in Cl− secretion. PGE2 and cAMP also activated an outwardly rectifying basolateral K+ channel which presumably sustains the driving force for electrogenic Cl− efflux across the apical membrane. The concentration‐conductance and concentration‐Isc response relationships for PGE2 showed that basolateral K+ permeability was rate limiting with respect to transepithelial anion secretion and that activation of a basolateral K+ channel by PGE2 was necessary to achieve maximum rates of Cl− secretion.

The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na+ transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na+ transport two- to threefold by increasing Na+-K+ ATPase transport activity and basolateral membrane K+ conductance without increasing the apical membrane amiloride-sensitive Na+ conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na+ absorption with a further increase in Na+-K+ ATPase transport activity and an increase in apical membrane amiloride-sensitive Na+ conductance. The effect of insulin on the Na+-K+ ATPase was the result of an increase in Vmax for extracellular K+ and intracellular Na+, and an increase in affinity of the pump for Na+. Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of α-1 and α-2 isoforms, but not the α-3 isoform of Na+-K+ ATPase, which did not change in the presence of insulin. Insulin-stimulated Na+ transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)3], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na+ transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3–kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3–kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na+-K+ ATPase activation.

No abstract

The objective of this study was to investigate the mechanism of uridine 5′-triphosphate (UTP)-dependent inhibition of Na+ absorption in porcine endometrial epithelial cells. Acute stimulation with UTP (5 μM) produced inhibition of sodium absorption and stimulation of chloride secretion. Experiments using basolateral membrane–permeabilized cell monolayers demonstrated a reduction in benzamil-sensitive Na+ conductance in the apical membrane after UTP stimulation. The UTP-dependent inhibition of sodium transport could be mimicked by PMA (1 μM). Several PKC inhibitors, including GF109203X and Gö6983 (both nonselective PKC inhibitors) and rottlerin (a PKCδ selective inhibitor), were shown to prevent the UTP-dependent decrease in benzamil-sensitive current. The PKCα-selective inhibitors, Gö6976 and PKC inhibitor 20–28, produced a partial inhibition of the UTP effect on benzamil-sensitive Isc. Inhibition of the benzamil-sensitive Isc by UTP was observed in the presence of BAPTA-AM (50 μM), confirming that activation of PKCs, and not increases in [Ca2+]i, were directly responsible for the inhibition of apical Na+ channels and transepithelial Na+ absorption.

Background /Aim: Genistein, the most active isoflavone found primarily in soybeans, alters ion transport functions in intestinal and airway epithelia. The present study aims to investigate the acute effects and mechanisms of action of genistein in immortalized porcine endometrial epithelial cells. Methods: Ussing chamber technique was used for transepithelial electrical measurements. Results: Genistein increased short-circuit currents (I_sc) which were inhibited by glibenclamide, NPPB, CFTRinh-172, DIDS or bumetanide, but not amiloride. In experiments with amphotericin B-permeabilized monolayers, genistein activated the apical Cl^- current and barium-sensitive basolateral K⁺ current while inhibiting the apical K⁺ current. Genistein failed to increase the I_sc in the presence of forskolin or IBMX, but did increase the I_sc in UTP. Pretreatment with genistein also abolished the increase in the I_sc when induced by forskolin, IBMX or UTP. However, Ca²⁺-chelating BAPTA-AM did not affect the genistein-induced increase in the I_sc. The genistein-stimulated I_sc was reduced by tyrosine kinase inhibitors, tyrphostin A23 or AG490. However, vanadate, a tyrosine phosphatase inhibitor, failed to inhibit the genistein response. Estrogen receptor antagonist ICI182,780 did not alter the genistein's action. Conclusion: The soy isoflavone, genistein, stimulates Cl^- secretion in endometrial epithelial cells possibly via a direct activation of CFTR which appears to be modulated through a tyrosine kinase-dependent pathway. The present findings may be of benefit for the therapeutic application of genistein in the treatment of electrolyte transport disorders in the epithelia.