Epidermal growth factor regulates the transition from basal sodium absorption to anion secretion in cultured endometrial epithelial cells.

The objective of this study was to investigate acute and long-term effects of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) on basal ion transport activity of glandular endometrial epithelial cells in primary culture. The effects of EGF on insulin-dependent regulation of Na+ transport across this epithelium was also investigated. Addition of 1.6 nM EGF or 2 nM TGFalpha to the basolateral, but not the apical, solution inhibited both basal and insulin-stimulated Na+ transport with a maximum response within 45-60 min. This effect was mimicked by the calcium ionophore ionomycin. Incubation with EGF for 4 days inhibited insulin-stimulated Na absorption in a concentration-dependent fashion with an IC(50) value of 0.3 nM. Experiments using amphotericin B-permeabilized monolayers demonstrated that EGF inhibited Na transport by decreasing apical membrane Na conductance without affecting insulin-dependent stimulation of the Na+-K+ ATPase. Addition of EGF or TGFalpha for 24 h resulted in increased basal Cl- secretion in addition to inhibition of Na absorption. The EGF-induced increase in Cl- secretion was inhibited in part by indomethacin, suggesting that long-term regulation by EGF involves stimulation of arachidonic acid synthesis and prostaglandin release. The EGF-induced increase in indomethacin-insensitive Cl- secretion was prevented by the protein synthesis inhibitor cyclohexamide, and by the DNA transcription inhibitor actinomycin D indicating that EGF-stimulated anion secretion required DNA transcription and protein synthesis. The results of these studies demonstrated that the basal transport properties of endometrial epithelial cells are differentially regulated by EGF, TGFalpha, and insulin.

Insulin stimulates transepithelial sodium transport by activation of a protein phosphatase that increases Na-K ATPase activity in endometrial epithelial cells.

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 V(max) 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 alpha-1 and alpha-2 isoforms, but not the alpha-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.

Regulation of chloride secretion across porcine endometrial epithelial cells by prostaglandin E2.

1. The objective of this study was to investigate the mechanism of PGE2 regulation of Cl- transport across glandular endometrial cells grown in primary culture. 2. 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. 3. Basolateral addition of 10 microM PGE2 increased Isc by 41 +/- 3 microA. 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. 4. 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. 5. 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). 6. Cultured porcine endometrial epithelial cells were specifically labelled with an antibody to a peptide sequence within the regulatory domain of CFTR. 7. 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. 8. 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. 9. 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.

Na absorption across endometrial epithelial cells is stimulated by cAMP-dependent activation of an inwardly rectifying K channel.

Previous studies in our laboratory have shown that Na absorption across the porcine endometrium is stimulated by PGF2alpha and cAMP-dependent activation of a barium-sensitive K channel located in the basolateral membrane of surface epithelial cells. In this study, we identify and characterize this basolateral, barium-sensitive K conductance. Porcine uterine tissues were mounted in Ussing chambers and bathed with KMeSO4 Ringer solution. Amphotericin B (70 microM) was added to the luminal solution to permeabilize the apical membrane and determine the current-voltage relationship of the basolateral K conductance after activation by 100 microM CPT-cAMP. An inwardly rectifying current was identified which possessed a reversal potential of -53 mV when standard Ringer solution was used to bathe the serosal surface. The K:Na selectivity ratio was calculated to be 12:1. Administration of 5 mM barium to the serosal solution completely inhibited the current activated by cAMP under these conditions. In addition to these experiments, amphotericin-perforated whole cell patch clamp recordings were obtained from primary cultures of porcine surface endometrial cells. The isolated cells displayed an inwardly rectifying current under basal conditions. This current was significantly stimulated by CPT-cAMP and blocked by barium. These results together with our previous studies demonstrate that cAMP increases Na absorption in porcine endometrial epithelial cells by activating an inwardly rectifying K channel present in the basolateral membrane. Similar patch clamp experiments were conducted using cells from a human endometrial epithelial cell line, RL95-2. An inwardly rectifying current was also identified in these cells which possessed a reversal potential of -56 mV when the cells were bathed in standard Ringer solution. This current was blocked by barium as well as cesium. However, the current from the human cells did not appear to be activated by cAMP, indicating that distinct subtypes of inwardly rectifying K channels are present in endometrial epithelial cells from different species.

Barakol: a potential anxiolytic extracted from Cassia siamea.

The behavioural effects of an extract of Cassia siamea, a plant used in Thai traditional medicine, and barakol, its active chemical, were studied on an elevated plus-maze compared with diazepam. An aqueous extract of C. siamea (1, 6, and 12 g/kg body wt., orally) produced a small increase in the percentage of the open: total number of arm entries and time, time spent on the end of the open arms, total number of arm entries, and number of rears/min. Barakol [10 mg/kg, intraperitoneally (IP)] significantly increased all of these behavioural parameters in a manner similar to diazepam (1 mg/kg, IP, 30 or 60 min before testing), except that barakol and not diazepam increased both the number of rears and total arm entries. Barakol at 25 and 50 mg/kg increased the percentage of the open: total number of arm entries and time and number of rears. The results indicate that barakol has anxiolytic properties similar to diazepam but differs from diazepam in that it also increases exploratory and locomotor behaviour, as shown by the number of rears and total arm entries.

Biliary calcium and bile acid secretion in intact and TPTX rats with varying plasma calcium concentration.

Investigations of the effects of plasma calcium concentration on the relationship between biliary secretion of bile acid and calcium were performed in normocalcemic, calcium gluconate-induced hypercalcemic, thyroparathyroidectomy-induced hypocalcemic (TPTX) rats, and TPTX rats that received calcium gluconate to maintain normocalcemia. Studies were done at normal bile flow and at sodium taurocholate-stimulated bile flow. The results showed that biliary calcium secretion, which could occur in the absence of parathyroid hormone and calcitonin, was dependent mainly on plasma calcium concentration and was only partly influenced by bile acid secretion. Concerning the route of biliary calcium secretion, 80% was by the transcellular pathway and 20% was by the paracellular pathway. During theophylline-stimulated bile-acid-independent bile flow, the increase in bile-acid-independent calcium was found to be secreted by both pathways.