The Na+-K+-ATPase as self-adhesion molecule and hormone receptor.

Thanks to the homeostasis of the internal milieu, metazoan cells can enormously simplify their housekeeping efforts and engage instead in differentiation and multiple forms of organization (tissues, organs, systems) that enable them to produce an astonishing diversity of mammals. The stability of the internal milieu despite drastic variations of the external environment (air, fresh or seawater, gastrointestinal fluids, glomerular filtrate, bile) is due to transporting epithelia that can adjust their specific permeability to H(2)O, H(+), Na(+), K(+), Ca(2+), and Cl(-) over several orders of magnitude and exchange substances with the outer milieu with exquisite precision. This exchange is due to the polarized expression of membrane proteins, among them Na(+)-K(+)-ATPase, an oligomeric enzyme that uses chemical energy from ATP molecules to translocate ions across the plasma membrane of epithelial cells. Na(+)-K(+)-ATPase presents two types of asymmetries: the arrangement of its subunits, and its expression in one pole of the epithelial cell ("polarity"). In most epithelia, polarity consists of the expression of Na(+)-K(+)-ATPase towards the intercellular space and arises in part from the interaction of the extracellular segment of the ß-subunit with another ß-subunit present in a Na(+)-K(+)-ATPase molecule expressed by a neighboring cell. In addition to enabling the Na(+)-K(+)-ATPase to transport ions and water vectorially, this position exposes its receptors to ouabain and analogous cardiotonic steroids, which are present in the internal milieu because these were secreted by endocrine cells.

Control of tight junctional sealing: role of epidermal growth factor.

Epithelia can adjust the permeability of their paracellular permeation route to physiological requirements, pathological conditions, and pharmacological challenges. This is reflected by a transepithelial electrical resistance (TER) ranging from a few tenth to several thousands Omega.cm(2), depending on the degree of sealing of the tight junction (TJ). The present work is part of an effort to understand the causes and mechanisms underlying these adaptations. We observed that an extract of human urine (hDLU) increases TER in a concentration- and time-dependent manner and is more effective when added from the basolateral side of cultured monolayers of Madin-Darby canine kidney cells than from the apical one. We found that its main TER-increasing component is epidermal growth factor (hEGF), as depletion of this peptide with specific antibodies, or inhibition of its receptor with PD153035, abolishes its effect. Since the permeability of the TJ depends on the expression of several species of membrane proteins, chiefly claudins, we explored whether hDLU can affect five members of the claudin family, the three known members of the ZO family, and occludin. EGF present in hDLU decreases the content of claudins-1 and -2 as well as delocalizes them from the TJ and increases the content of claudin-4. As expected from the fact that the degree of sealing of the TJ must be a physiologically regulated parameter, besides of hEGF, we also found that hDLU appears to contain also other components that decrease TER, claudin-4 and -7, and that seem to act with different kinetics than the TER-increasing ones.

Na+,K+-ATPase and hormone ouabain:new roles for an old enzyme and an old inhibitor.

Na+,K+-ATPase and its specific inhibitor ouabain entered the 21st century with an entirely new set of properties, that are the focuses of the present review. (i) The adhesive property of the beta-subunit explains why is Na+,K+-ATPase expressed polarizedly on one side of epithelial cells, a crucial property to explain the exchange of substances between higher organisms and the environment; (ii) Ouabain was recently recognized to be a hormone. (iii) Na+,K+-ATPase is known to act as a receptor for hormone ouabain, (iv) binding of ouabain to the Na+,K+-ATPase modifies adhesion: at high concentrations the outcome is total detachment. (v) Ouabain-resistant cells and ouabain-sensitive ones establish a special type of cell-cell interaction, so that sensitive cells withstand the presence of otherwise lethal levels of ouabain. (vi) Hormone ouabain provokes relocalization of specific molecules from the submembrane scaffold to the nucleus, where these bind to promoters of genes involved in proliferation, differentiation, migration, etc. (vii) Finally, ouabain causes a retrieval of Na+,K+-ATPase from the plasma membrane. We speculate that this would reduce the driving force that operates co- and counter-transporters, which are responsible for the exchange of substances across epithelia.

Cell adhesion, polarity, and epithelia in the dawn of metazoans.

Transporting epithelia posed formidable conundrums right from the moment that Du Bois Raymond discovered their asymmetric behavior, a century and a half ago. It took a century and a half to start unraveling the mechanisms of occluding junctions and polarity, but we now face another puzzle: lest its cells died in minutes, the first high metazoa (i.e., higher than a sponge) needed a transporting epithelium, but a transporting epithelium is an incredibly improbable combination of occluding junctions and cell polarity. How could these coincide in the same individual organism and within minutes? We review occluding junctions (tight and septate) as well as the polarized distribution of Na(+)-K(+)-ATPase both at the molecular and the cell level. Junctions and polarity depend on hosts of molecular species and cellular processes, which are briefly reviewed whenever they are suspected to have played a role in the dawn of epithelia and metazoan. We come to the conclusion that most of the molecules needed were already present in early protozoan and discuss a few plausible alternatives to solve the riddle described above.

Ouabain binding to Na+,K+-ATPase relaxes cell attachment and sends a specific signal (NACos) to the nucleus.

Abstract. In previous work we described a "P-->A mechanism" that transduces occupancy of the pump ( P) by ouabain into changes in phosphorylation, stimulation of mitogen-activated protein kinase (MAPK), and endocytosis of cell-cell- and cell-substrate-attaching molecules ( A), thereby causing a release of the cell from the monolayer. In the present work we try to understand the mechanism of this effect; whether, in order to trigger the P-->A mechanism, ouabain should block the pumping activity of Na(+),K(+)-ATPase as pump, or whether it would suffice that the drug occupies this enzyme as a receptor. We assay a series of drugs known to act on the pump, such as ouabain, digoxin, digitoxin, palytoxin, oligomycin, strophanthidin, neothyoside-A, proscillaridin-A, etc. We gauge their ability to block the pump by measuring the K(+) content in the cells, and their ability to detach the cells from the monolayer by determining the amount of protein remaining in the culturing well. None of the drugs tested was able to cause detachment without stopping the pump. Ouabain also enhances phosphorylation, yet pump inhibition and signal transduction do not seem to be intimately associated in a causal chain, but to occur simultaneously. To investigate the response of the site of cell attachment, we analyze the position of beta-catenin by fluorescence confocal microscopy, and find that this adherent junction-associated molecule is sent to the nucleus, where it is known to act as a transcriptional cofactor.

Molecular physiology and pathophysiology of tight junctions. I. Biogenesis of tight junctions and epithelial polarity.

The tight junction (TJ) was first noticed through its ability to control permeation across the paracellular route, but the homologies of its molecular components with peptides that participate in tumor suppression, nuclear addressing, and cell proliferation indicate that it may be involved in many other fundamental functions. TJs are formed by a dozen molecular species that assemble through PDZ and other protein-protein clustering promoting sequences, in response to the activation of E-cadherin. The TJ occupies a highly specific position between the apical and the basolateral domains. Its first molecular components seem to be delivered to such a position by addressing signals in their molecule and, once anchored, serve as a clustering nucleus for further TJ-associated molecules. Although in mature epithelial cells TJs and E-cadherin do not colocalize, a complex chain of reactions goes from one to the other that involves alpha-, beta-, and gamma-catenins, two different G proteins, phospholipase C, protein kinase C, calmodulin, mitogen-activated protein kinase, and molecules pertaining to the cytoskeleton, which keep the TJ sensitive to physiological requirements and local conditions (notably to Ca(2+)-dependent cell-cell contacts) throughout the life of the epithelium.

Relationship between Na(+),K(+)-ATPase and cell attachment.

A prolonged ouabain blockade of the Na(+),K(+)-ATPase detaches cells from each other and from the substrate. This suggests the existence of a link between pump (P) and attachment (A). In the present work, we report that MDCK-W cells treated with ouabain increase tyrosine phosphorylation and content of active MAP kinase, redistribute molecules involved in cell attachment (occludin, ZO-1, desmoplakin, cytokeratin, alpha-actinin, vinculin and actin), and detach. Genistein and UO126, inhibitors of protein tyrosine kinase and of MAP kinase kinase, respectively, block this detachment. The content of P190(Rho-GAP), a GTPase activating protein of the Rho small G-protein subfamily, is increased by ouabain, suggesting that both the Rho/Rac and MAPK pathways are involved. Another clone of MDCK cells whose Na(+),K(+)-ATPase has a negligible affinity for the drug, show none of the effects described for MDCK-W and remain attached. Ma104 cells, a line that has a high affinity for ouabain and stops pumping, fail to modify phosphorylation, as well as the pattern of distribution of attaching molecules, and remain in the monolayer. Taken together, these results suggest that there is a mechanism (P-->A) that transduces a blockade of the pump in a detachment of the cell from neighbors and substrate, in which Ma104 cells are faulty.

Tight junctions and the experimental modifications of lipid content.

Tight junctions (TJs) are cell-to-cell contacts made of strands, which appear as ridges on P faces and complementary furrows on E faces on freeze fracture replicas. Evidences and opinions on whether these strands are composed of either membrane-bound proteins or lipid micelles are somewhat varied. In the present work we alter the lipid composition of Madin-Darby canine kidney monolayers using a novel approach, while studying (i) their transepithelial electrical resistance, a parameter that depends on the degree of sealing of the TJs; (ii) the apical-to-basolateral flux of 4 kD fluorescent dextran (JDEX), that reflects the permeability of the intercellular spaces; (iii) the ability of TJs to restrict apical-to-basolateral diffusion of membrane lipids; and (iv) the pattern of distribution of endogenous and transfected occludin, the sole membrane protein presently known to form part of the TJs. We show that changing the total composition of phospholipids, sphingolipids, cholesterol and the content of fatty acids, does not alter TER nor the structure of the strands. Interestingly, enrichment with linoleic acid increases the JDEX by 631%. The fact that this increase is not reflected in a decrease of TER, suggests that junctional strands do not act as simple resistive elements but may contain mobile translocating mechanisms.

Role of tight junctions in establishing and maintaining cell polarity.

The tight junction (TJ) is not randomly located on the cell membrane, but occupies a precise position at the outermost edge of the intercellular space and, therefore, is itself considered a polarized structure. This article reviews the most common experimental approaches for studying this relationship. We then discuss three main topics. (a) The mechanisms of polarization that operate regardless of the presence of TJs: We explore a variety of polarization mechanisms that operate at stages of the cell cycle in which TJs may be already established. (b) TJs and polarity as partners in highly dynamic processes: Polarity and TJs are steady state situations that may be drastically changed by a variety of signaling events. (c) Polarized distribution of membrane molecules that depend on TJs: This refers to molecules (mainly lipids) whose polarized distribution, although not the direct result of TJs, depends on these structures to maintain such distribution.

Activity and fluorescence changes of lactate dehydrogenase induced by guanidine hydrochloride in reverse micelles.

Denaturants activate several multimeric enzymes in reverse micelles [Garza-Ramos, G., Darszon, A., Tuena de Gómez-Puyou, M. & Gómez-Puyou, A. (1992) Eur. J. Biochem. 205, 509-517]. Here, the effect on activity and intrinsic fluorescence of pig heart lactate dehydrogenase (LDH) in reverse micelles [formed with 0.2 M cetyltrimethylammonium bromide in octane/hexanol (8.6:1, by vol.)] was explored at various water and guanidine hydrochloride (Gdn/HCl) concentrations. Emission fluorescence spectra of LDH in aqueous media and in micelles were similar. As in all aqueous media, 1.0 M Gdn/HCl in the water phase of reverse micelles produced fluorescence quenching and a blue shift of the maximal emission. In 5.0 M Gdn/HCl, instead of the red shift and significant quenching seen in water, the maximum emission further shifted to the blue and was only slightly quenched. Gdn/HCl titrations of activity and fluorescence changes of LDH in micelles with different water contents showed that at Wo ([H2O]/[surfactant]) of 6.6, 8.3, or 12.5, increasing concentrations of Gdn/HCl up to 0.6 M produced small changes in fluorescence, whereas activity increased several-fold. At higher denaturant concentrations, activity decreased with significant fluorescence changes. In reverse micelles with 1 M Gdn/HCl, Vmax but not Km of LDH decreased with time. Under these conditions, there was progressive quenching of LDH fluorescence. The results show that in reverse micelles different Gdn/HCl concentrations induce variations in activity with or without alterations of the intrinsic fluorescence of LDH. The results also indicate that in reverse micelles, concentrations of Gdn/HCl below 1.0 M cause an enhancement of protein flexibility; this is accompanied by a marked increase in activity without important changes in intrinsic fluorescence. 1.0 M Gdn/HCl produces perturbations of inter-subunit contacts that lead to fluorescence quenching and loss of catalytic activity, probably as consequence of dimerization of tetrameric LDH.

Activity of heart and muscle lactate dehydrogenases in all-aqueous systems and in organic solvents with low amounts of water. Effect of guanidine chloride.

The effect of urea and guanidine hydrochloride (GdmCl) on the activity of lactate dehydrogenases from heart and muscle was studied in standard water mixtures and in reverse micelles formed with n-octane, hexanol, cetyltrimethylammonium bromide and water in a concentration that ranged over 2.5-6.0% (by vol.). In all water mixtures GdmCl (0.15-0.75 M) and urea (0.5-3.0 M) inhibited the activity of the enzymes at non-saturating pyruvate concentrations. At concentrations of pyruvate that proved inhibitory for enzyme activity due to the formation of a ternary enzyme-NAD-pyruvate complex, GdmCl and urea increased the activity of the enzymes. This increase correlated with a decrease of the ternary complex, as evidenced by its absorbance at 320-325 nm. In the low-water system it was found that: (a) at all concentrations of pyruvate tested (0.74-30 mM), GdmCl enhanced the activity of the heart enzyme to a similar extent; (b) in the muscle enzyme, GdmCl inhibited or increased the activity through a process that depended on the concentration of pyruvate and GdmCl; (c) under optimal conditions, the activation by GdmCl was about two times lower in the muscle than in the heart enzyme, although in all-water media the activity of the muscle enzyme was twice as high. The expression of lactate dehydrogenase activity in the low-water system was higher with the heart than with the muscle enzyme compared to their activities in all-water media (about 260 and 600 mumol min-1 mg-1 in the heart and muscle enzymes respectively). Apparently for catalysis, the water requirement in the heart enzyme is lower than in the muscle enzyme. It is likely that the different response of the two enzymes to solvent is due to their distinct structural features.

Enzyme activation by denaturants in organic solvent systems with a low water content.

The effect of urea and guanidine hydrochloride (GdmCl) on the activity of heart lactate dehydrogenase, glycerol-3-phosphate dehydrogenase, hexokinase, inorganic pyrophosphatase, and glyceraldehyde-3-phosphate dehydrogenase was studied in low-water systems. Most of the experiments were made in a system formed with toluene, phospholipids, Triton X-100, and water in a range that varied over 1.0-6.5% (by vol.) [Garza-Ramos, G., Darszon, A., Tuena de Gómez-Puyou, M. & Gómez-Puyou, A. (1990) Biochemistry 29, 751-757]. In such conditions at saturating substrate concentrations, the activity of the enzymes was more than 10 times lower than in all-water media. However the activity of the first four aforementioned enzymes was increased between 4 and 20 times by the denaturants. The most marked activating effect was found with lactate dehydrogenase; with 3.8% (by vol.) water maximal activation was observed with 1.5 M GdmCl (about 20-fold); 4 M urea activated, but to a lower extent. Activation by guanidine thiocyanate was lower than with GdmCl. The activating and inactivating effects of GdmCl on lactate dehydrogenase depended on the amount of water; as the amount of water was increased from 2.0% to 6.0% (by vol.), activation and inactivation took place with progressively lower GdmCl concentrations. When activity was measured as a function of the volume of 1.5 M GdmCl solution, a bell-shaped activation curve was observed. In a low-water system formed with n-octane, hexanol, cetyltrimethylammonium bromide and 3.0% water, a similar activation of lactate dehydrogenase by GdmCl and urea was observed. The water solubility diagrams were modified by GdmCl and urea, and this could reflect on enzyme activity. However, from a comparison of denaturant concentrations on the activity of the enzymes studied, it would seem that, independently of their effect on the characteristics of the low-water systems, denaturants bring about activation through their known mechanism of action on the protein. It is suggested that the effect of denaturants is due to the release of constraints in enzyme catalysis imposed by a low-water environment.