Cystic fibrosis transmembrane regulator (CFTR) in human trophoblast BeWo cells and its relation to cell migration.

INTRODUCTION: ENaC and CFTR are coexpressed in epithelia and have positive or negative functional interactions. In addition, ENaC and CFTR promote migration in placental trophoblastic cells and human airway cells, respectively. Here we tested the idea if CFTR is functionally expressed in BeWo cells, a trophoblastic cell line, and if it is involved in their migratory behavior. METHODS: CFTR expression was studied in BeWo cells with RT-PCR, biotinylation and Western blot. Ion currents were analyzed with patch clamp, and cell migration with the wound healing method. RESULTS: The mature CFTR 160-kDa band was present, and its localization at the surface membrane was confirmed. Forskolin (20 µM), an adenylate cyclase activator, was used for channel activation, and subsequently CFTR(inh)-172 (2 µM) for its inhibition. The conductances in the presence of CFTR(inh)-172 plus forskolin (16.0 ± 0.7 pS/pF and 32.6 ± 1.5 pS/pF) were significantly lower than in presence of only forskolin (29.7 ± 0.9 and 47.0 ± 2.0 pS/pF). The conductance of CFTR(inh)-172 inhibited currents was 14.9 ± 0.7 pS/pF with a linear I-V relationship illustrating the nonrectifying properties of the CFTR. Cell migration was measured and covered 11.2 ± 0.4, 24.0 ± 1.7 and 13.9 ± 1.0% of the wound when cells were cultivated under control, forskolin, and forskolin plus CFTR(inh)-172, respectively. Proliferation was not changed by any of the treatments. CONCLUSIONS: Our results shows that BeWo cells functionally express the CFTR which plays a role in the wound healing increasing the cell migration process.

The migratory capacity of human trophoblastic BeWo cells: effects of aldosterone and the epithelial sodium channel.

Aldosterone is a key regulator of the epithelial sodium channel (ENaC) and stimulates protein methylation on the ß-subunit of the ENaC. We found that aldosterone (100 nM) promotes cellular migration in a wound-healing model in trophoblastic BeWo cells. Here, we tested if the positive influence of aldosterone on wound healing is related to methylation reactions. Cell migration and proliferation were measured in BeWo cells at 6 h, when mitosis is still scarce. Cell migration covered 12.4, 25.3, 19.6 and 45.1 % of the wound when cultivated under control, aldosterone (12 h), 8Br-cAMP and aldosterone plus 8Br-cAMP, respectively. Amiloride blocked the effects of aldosterone alone or in the presence of 8Br-cAMP on wound healing. Wound healing decreased in aldosterone (plus 8Br-cAMP) coexposed with the methylation inhibitor 3-deaza-adenosine (3-DZA, 12.9 % reinvasion of the wound). There was an increase in wound healing in aldosterone-, 8Br-cAMP- and 3-DZA-treated cells in the presence of AdoMet, a methyl donor, compared to cells in the absence of AdoMet (27.3 and 12.9 % reinvasion of the wound, respectively). Cell proliferation assessed with the reagent MTT was not changed in any of these treatments, suggesting that cellular migration is the main factor for reinvasion of wound healing. Electrophysiological studies showed an increase in ENaC current in the presence of aldosterone. This effect was higher with 8Br-cAMP, and there was a decrease when 3-DZA was present. AdoMet treatment partially reversed this phenomenon. We suggest that aldosterone positively influences wound healing in BeWo cells, at least in part through methylation of the ENaC.

Expression of the epithelial sodium channel sensitive to amiloride (ENaC) in normal and preeclamptic human placenta.

Aldosterone modulates the activity of the epithelial sodium channel (ENaC) through changes in its trafficking, membrane expression and open probability. Plasma levels of aldosterone are decreased in preeclampsia. Herein we postulated that if aldosterone regulates ENaC expression then its expression should be decreased in preeclampsia. We found a diminished expression of the three subunits of the ENaC in the membranes of preeclamptic placentas in comparison with the normal ones. Although the role of ENaC in placental tissues is poorly understood, these differences may have consequences for the ion transport involved in the pathophysiology of preeclampsia.

An outwardly rectifying chloride channel in BeWo choriocarcinoma cell line.

In this study, an outwardly rectifying chloride channel was characterized in the trophoblastic cell line BeWo, a human hormone-synthesizing cell which displays many biochemical and morphological properties similar to those reported for the human cytotrophoblast. Ion channel activity was recorded in the cell attached and inside-out configurations with standard patch-clamp technology. In most of the BeWo cells studied, the channel under symmetrical N-methyl-d-glucamine (NMDG-Cl) concentration (Na(+) free solution) in both sides of the membrane exhibited spontaneous activity, an outwardly rectifying current/voltage relationship and single-channel conductances of 15 pS and 48 pS for inwards and outwards currents, respectively. The channel has a low permeability for gluconate with a relative permeability P(gluconate)/P(Cl) of 0.23, and a higher permeability to I(-). The open probability (Po) of the channel exhibited dependence with the applied membrane potential with greater activity at positive pulses. The channel activity was inhibited by the sulphonylurea hypoglycemic agent glibenclamide (50 µM) or by diphenylamine-2-carboxylate (DPC, 500 µM) added to the cytoplasmic side of the patch whereas conductances remained unchanged. The blockade with glibenclamide and DPC was independent of the applied membrane potential. All these results are characteristic of the outwardly rectifying Cl channel (ORCC) found in other types of cells. Neither Po, conductances nor reversal potential (Er) values were affected by the absence of intracellular Ca(2+), suggesting that the channel is not sensitive to Ca(2+).

Functional interaction between CFTR and Cx45 gap junction channels expressed in oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel known to influence the function of other channels, including connexin channels. To further study potential functional interactions between CFTR and gap junction channels, we have co-expressed CFTR and connexin45 (Cx45) in Xenopus oocytes and monitored junctional conductance and voltage sensitivity by dual voltage clamp electrophysiology. In single oocytes expressing CFTR, an increase in cAMP caused by forskolin application induced a Cl(-) current and increased membrane conductance; application of diphenylamine carboxylic acid (CFTR blocker) readily blocked the Cl(-) current. With co-expression of CFTR and Cx45, application of forskolin to paired oocytes induced a typical outward current and increased junctional conductance (G(j)). In addition, the presence of CFTR reduced the transjunctional voltage sensitivity of Cx45 channels without affecting the kinetics of junctional current inactivation. The drop in voltage sensitivity was further enhanced by forskolin application. The data indicate that CFTR influences cell-to-cell coupling mediated by Cx45 channels.

Chloride channel blockers activate an endogenous cationic current in oocytes of Bufo arenarum.

A two-electrode, voltage-clamp technique was used to measure the effect of the Cl(-) channel blockers, 9-anthracene carboxylic acid and niflumic acid, upon the ionic currents of oocytes of the South American toad Bufo arenarum. The main results were: (1) both blockers produced a reversible increase of the outward currents on a dose-dependent manner; (2) the activated outward current was voltage dependent; (3) the 9-anthracene carboxylic acid-sensitive current was blocked with barium; and (4) the effect of 9-anthracene carboxylic acid was more pronounced in a zero-K(+) solution than in standard (2 mmol l(-1)) or high (20 mmol l(-1)) K(+) solutions, indicating that a K(+) conductance is activated. The effect of the Cl(-) channel blockers could be due to a direct interaction with endogenous cationic channels. Another possible explanation is that Cl(-) that enter the cell during depolarizing steps in control solution inhibit this cationic conductance; thus, the blockade of Cl(-) channels by 9-anthracene carboxylic acid and niflumic acid would remove this inhibition, allowing the cationic current to flow freely.

Sodium influx during action potential in innervated and denervated rat skeletal muscles.

Resting Na(+) influx (J(i)(Na)) was measured in innervated and denervated (1-6 days) rat extensor digitorum longus muscle in the absence and presence of 2 micromol/L tetrodotoxin (TTX). The mean value of Na(+) permeability (P(Na)) in innervated muscles was 49.6 +/- 2.6 pm.s(-1). At the second day postdenervation, it decreased by about 45%. This was followed, between the second and fourth days, by a sharp rise, which by the sixth day reached a steady value approximately 2.5 times greater than that of innervated muscles. This, most likely, generated the 30% increase in internal [Na(+)] concentration ([Na(+)](I)) observed at this time. Tetrodotoxin reduced P(Na) of both innervated and denervated muscles by about 25%. In 6-day denervated muscles, virtually all the TTX effect on P(Na) represents the blockage of TTX-resistant Na(+) channels. Denervation produced a depolarization of about 20 mV by the sixth day. The extra J(i)(Na) per action potential (AP) decreased monotonically with time after denervation from 20.0 +/- 3.8 in innervated to 11.1 +/- 1.0 nmol.g(-1).AP(-1) in 6-day denervated muscles. The overshoot of the AP decreased from 15 +/- 1 in innervated to 7 +/- 1 mV in 6-day denervated muscles. Likewise, the maximum rate of rise (+dV/dt), an expression of the inward Na(+) current, fell from 305 +/- 14 in innervated to 188 +/- 18 V.s(-1) in 6-day denervated muscles. The estimated 6-day denervated/innervated ratio of peak Na(+) conductance (g(Na)) was 0.67. The changes in AP parameters promoted by denervation were substantially reduced when both innervated and denervated fibers were hyperpolarized to -90 mV. These results suggest that the depolarization, mainly due to the increase in P(Na) /P(K) ratio, increases Na(+) inactivation and consequently reduces peak g(Na), in spite of the absolute increment in resting TTX-sensitive P(Na). This, in addition to the moderate reduction in the inward driving force on Na(+), decreases the inward Na(+) current and the extra J(i)(Na) per AP.

Caffeine-induced depolarization in amphibian skeletal muscle fibres: role of Na+/Ca2+ exchange and K+ release.

Caffeine (4 mM) produces a depolarization of about 10 mV in frog muscle fibres (Leptodactylus ocellatus). The aim of this work was to study the mechanisms of this effect. An approximately threefold rise in membrane resistance [Cl--free (SO(4)2-) medium] substantially increased, and both Na+-free medium and Ni2+ (5 mM) reduced, the caffeine-induced depolarization. In voltage-clamped (-60 mV) short fibres from lumbricalis muscle of the toad (Buffo arenarum), caffeine generated an inward current of 4.13 +/- 0.48 microA cm(-2). This caffeine-induced current was reduced by 60% in Na+-free medium, 44% in the presence of 5 mM amiloride and 48% by 5 mM Ni2+, suggesting that the activation of the Na+-Ca2+ exchanger in its forward mode may play a role in the observed electrical effects of the drug. Caffeine also produced a marked release of K+. Net K+ efflux increased from 3.5 +/- 0.2 (control) to 22.1 +/- 2.3 pmol s(-1) cm(-2) (caffeine). It is shown that in the presence of the drug, [K+] in the lumen of the T tubules may well increase to levels which could produce, in part, both the observed depolarization and the caffeine-induced current under voltage clamp conditions. The caffeine-induced K+ efflux was not reduced by 5 mM Ni2+. At a holding potential of 30 mV the caffeine-induced current was reversed (outward) and roughly halved by 5 mM Ni2+. The Ni2+-sensitive fraction of the caffeine-induced current, assumed to represent the Na+-Ca2+ exchanger current, had an estimated reversal potential close to 12 mV ([Na+]o = 115 mM; [Ca2+]o = 1 mM). In conclusion, the depolarizing effect of caffeine described here would be produced by two mechanisms: (a) an inward current generated by the activation of the Na+-Ca2+ exchanger in its forward mode, and (b) the rise of the external [K+] in restricted spaces like the T tubules.

Chloride channels in toad skeletal muscle fibers.

Chloride currents were measured in short lumbricalis fibers of toads (Bufo arenarum) with voltage and patch clamp techniques. For the availability of chloride currents we applied a double-pulse technique in voltage-clamped fibers. When the test pulse was preceded by a positive prepulse, the initial current was larger than with a negative prepulse and exhibited a different rate of decline to its steady-state value. At the single-channel level we found that in most of the experiments with symmetrical 110 mM NaCl solutions, two levels of conductance, 20 ("small channel") and 360 pS ("maxi channel"), occurred with the highest probabilities. The openings of the maxi channels were more frequent at potentials close to 0 mV, whereas for the small channels the openings were at negative potentials. In contrast with the results with the macroscopic currents, a change of 2 orders of magnitude in the pH, from 7.3 to 5, had only minor effects on the channels' conductance. As with some other anion channels, the selectivity of the channels described here is low, the p(Cl)/p(Na) ratio being 1.9 and 3.7 for the small and maxi Cl(-) channels, respectively. The behavior of these Cl(-) channels with a relative high Na(+) permeability could contribute to the relatively low resting membrane potential of the lumbricalis fibers measured in the standard 110 mM NaCl solution.

[Past and present of Medicina (Buenos Aires)]. / Pasado y presente de Medicina (Buenos Aires).

To celebrate the 60th anniversary of Medicina (Buenos Aires) an International Symposium was held at the National Academy of Medicine of Buenos Aires on the 6-7th of October 1999, under the title of Clinical investigation in the next millennium. This meeting was a success as evidenced by the 376 registered attendants. Sixty years of uninterrupted publication is an uncommon feat in our midst and this could be achieved on the basis of a number of factors which include, the initiative of those who founded the journal, the unfailing motivation and dedication of the Editorial Board and primarily the authors who have trusted us with their manuscripts. Of the many important papers published, we have selected a few which proved to be milestones in the development of Argentine biomedicine. It is to be hoped that the future will bring an increase in our impact index through more and even better papers eventually reflecting the authentic scientific value of our country.

Pasado y presente de Medicina (Buenos Aires). / [Past and present of Medicina (Buenos Aires)]

To celebrate the 60th anniversary of Medicina (Buenos Aires) an International Symposium was held at the National Academy of Medicine of Buenos Aires on the 6-7th of October 1999, under the title of Clinical investigation in the next millennium. This meeting was a success as evidenced by the 376 registered attendants. Sixty years of uninterrupted publication is an uncommon feat in our midst and this could be achieved on the basis of a number of factors which include, the initiative of those who founded the journal, the unfailing motivation and dedication of the Editorial Board and primarily the authors who have trusted us with their manuscripts. Of the many important papers published, we have selected a few which proved to be milestones in the development of Argentine biomedicine. It is to be hoped that the future will bring an increase in our impact index through more and even better papers eventually reflecting the authentic scientific value of our country.