Cardiac glycosides are a group of compounds widely known for their action in cardiac tissue, some of which have been found to be endogenously produced (ECG). We have previously studied the effect of ouabain, an endogenous cardiac glycoside, on the physiology of epithelial cells, and we have shown that in concentrations in the nanomolar range, it affects key properties of epithelial cells, such as tight junction, apical basolateral polarization, gap junctional intercellular communication (GJIC), and adherent junctions. In this work, we study the influence of digoxin and marinobufagenin, two other endogenously expressed cardiac glycosides, on GJIC as well as the degree of transepithelial tightness due to tight junction integrity (TJ). We evaluated GJIC by dye transfer assays and tight junction integrity by transepithelial electrical resistance (TER) measurements, as well as immunohistochemistry and western blot assays of expression of claudins 2 and 4. We found that both digoxin and marinobufagenin improve GJIC and significantly enhance the tightness of the tight junctions, as evaluated from TER measurements. Immunofluorescence assays show that both compounds promote enhanced basolateral localization of claudin-4 but not claudin 2, while densitometric analysis of western blot assays indicate a significantly increased expression of claudin 4. These changes, induced by digoxin and marinobufagenin on GJIC and TER, were not observed on MDCK-R, a modified MDCK cell line that has a genetically induced insensitive α1 subunit, indicating that Na-K-ATPase acts as a receptor mediating the actions of both ECG. Plus, the fact that the effect of both cardiac glycosides was suppressed by incubation with PP2, an inhibitor of c-Src kinase, PD98059, an inhibitor of mitogen extracellular kinase-1 and Y-27632, a selective inhibitor of ROCK, and a Rho-associated protein kinase, indicate altogether that the signaling pathways involved include c-Src and ERK1/2, as well as Rho-ROCK. These results widen and strengthen our general hypothesis that a very important physiological role of ECG is the control of the epithelial phenotype and the regulation of cell-cell contacts.

Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.

Epithelial cells play a major role in animal and human homeostasis because they selectively regulate the exchange of solutes between two given media, such as blood or urine. Cardiac glycosides (CG) are a group of highly toxic compounds whose best therapeutic known effect is on heart, although recent evidence has shown that it exerts a wide range of physiological effects on cells and tissues other than the heart. Prostaglandins, on the other hand, are a group of lipids that produce diverse physiological and pathological effects among which inflammation stands out. In this chapter, we describe that cardiac glycosides modulate key features of epithelial cell physiology, including cell-cell contact junctional complexes, cilliogenesis, and gap junction-mediated intercellular communication (GJIC) in epithelial cells. Prostaglandin PGE2 also modulates GJIC through a different signaling pathway. In addition, we describe that CG induce paracrine release of prostaglandin PGE2, which in turn modulates GJIC by itself.

Ouabain is a cardiac glycoside that has been described as a hormone, with interesting effects on epithelial physiology. We have shown previously that ouabain induces gap junctional intercellular communication (GJIC) in wild, sensitive cells (MDCK-S), but not in cells that have become insensitive (MDCK-I) by modifying their Na+-K+-ATPase. We have also demonstrated that prostaglandin E2 (PGE2) is able to induce increased GJIC by a mechanism other than ouabain, that does not depend on Na+-K+-ATPase. In this work we show, by dye transfer assays, that when MDCK-S and MDCK-I are randomly mixed, to form monolayers, the latter stablish GJIC, because of stimulation by a compound released to the extracellular media, by MDCK-S cells, after treatment with ouabain, as evidenced by the fact that monolayers of only MDCK-I cells, treated with a conditioned medium (CM) that is obtained after incubation of MDCK-S monolayers with ouabain, significantly increase their GJIC. The further finding that either (1) pre-treatment with COX-2 inhibitors or (2) addition to CM of antagonists of EP2 receptor abolish CM’s ability to induce GJIC in MDCK-I monolayers indicate that PGE2 is the GJIC-inducing compound. Therefore, these results indicate that, in addition to direct stimulation, mediated by Na+-K+-ATPase, ouabain enhances GJIC indirectly through the paracrine production of PGE2.

Giardia lamblia is one of the most important worldwide causes of intestinal infections, yet little is known about its cellular physiology, especially the diversity of ionic channels that this parasite expresses. In this work, we show that injection of mRNA isolated from trophozoites of Giardia , into Xenopus laevis oocytes, induces expression of three types of chloride currents (here referred to as IC l‐G1, IC l‐G2, and IC l‐G3), which have different biophysical and pharmacological properties. IC l‐G1 currents show inward rectification and voltage dependence are enhanced by hypotonicity, show a selectivity sequence of (I > Br > Cl > F), and are inhibited by NPPB , DIDS , SITS , 9 AC , DPC , and Zinc. These findings suggest that IC l‐G1 is the result of expression of chloride channels related to ClC2. IC l‐G2 currents show outward rectification and are dependent of intracellular calcium, its selectivity sequence is (Cl > Br > I > F) and are inhibited by NPPB , DIDS , SITS , 9 AC , DPC , niflumic acid, tannic acid, and benzbromarone. These findings suggest that they are produced by calcium dependent chloride channels (Ca CC ). The third type of currents ( IC l‐G3) appears only after a hypoosmotic challenge, and has similar properties to those described for IC l‐swell, such as outward rectification, instant activation, and slow inactivation at large depolarizing voltages. They were blocked by NPPB , DIDS , 9 AC , NI f, DCPIB , and tamoxifen. Our results indicate that Giardia intestinalis has at least three types of anion conductances.