Newly synthesized Na,K-ATPase alpha-subunit has no cytosolic intermediate in MDCK cells

Recently published data indicate that the alpha-subunit of Na,K-ATPase, a transmembrane protein of animal cell plasma membranes, is synthesized as a soluble precursor. In the present experiments we demonstrate that an apparent "soluble" form can indeed be detected in crude cytosolic fractions prepared by centrifugation from MDCK cells disrupted by sonication. We find, however, that this form has no precursor-product relationship with membrane-associated alpha-subunit. The quantity of unsedimentable alpha-subunit can be greatly diminished by increasing the centrifugal field employed to remove membranous vesicles from the cytosol fraction. Sonication of membrane pellets generates alpha-subunit which, like the "soluble" form, resists pelleting. Finally, cytosol fractions prepared from cells disrupted by sonication contain membrane-bound vesicles which can be immunoadsorbed on Staphylococcus aureus cells coated with a monoclonal antibody directed against alpha-subunit. We find, therefore, that the previously observed soluble precursor of alpha-subunit is actually a component of small unpelleted membrane vesicles generated by harsh disruption conditions. When cells are disrupted by less violent techniques no newly synthesized alpha-subunit can be detected in the cytosol fraction. We calculate that to escape detection under our experimental conditions a bona fide soluble precursor of alpha-subunit must have a cytosolic t1/2 less than 20 s. We conclude that the alpha-subunit is most probably inserted into the bilayer cotranslationally.     

Regulation of EGF signaling by cell polarity in MDCK kidney epithelial cells.

Although the presence of a dominant basolateral sorting signal ensures that the majority of newly synthesized epidermal growth factor (EGF) receptors are delivered directly to the basolateral surface in polarized epithelial cells, a fraction of the receptors are also delivered to the apical surface. Similar to most basolateral membrane proteins, the EGF receptor has an additional signal(s) that selectively targets molecules lacking a dominant basolateral signal to the apical surface. Although the physiological relevance of signal hierarchy is not known, alternative targeting may occur in different epithelial cell types or during development. The goal of this study, therefore, was to determine the effect of membrane domain location on EGF receptor function, focusing on EGF-induced MAP kinase signaling and DNA synthesis. Whereas ligand responsiveness was restricted to the basolateral domain in Madin-Darby canine kidney (MDCK) cells expressing a normal complement of receptors, apical ligand was effective if apical receptor density was increased by overexpression of an exogenous wild-type human gene. Unexpectedly, cells expressing apically localized, cytoplasmically truncated receptors, which behave as dominant negative mutations in other cell types, were also responsive to apical EGF. The cytoplasmically truncated molecules appear to have at least two effects: first, to increase the local concentration of ligand at the apical cell surface; and second, to facilitate activation of the relatively few native EGF receptors normally located at the apical surface. These results indicate that cell context is a critical determinant of receptor mutant protein phenotype.     

Molecular mechanisms of membrane polarity in renal epithelial cells

Exciting discoveries in the last decade have cast light onto the fundamental mechanisms that underlie polarized trafficking in epithelial cells. It is now clear that epithelial cell membrane asymmetry is achieved by a combination of intracellular sorting operations, vectorial delivery mechanisms and plasmalemma-specific fusion and retention processes. Several well-defined signals that specify polarized segregation, sorting, or retention processes have, now, been described in a number of proteins. The intracellular machineries that decode and act on these signals are beginning to be described. In addition, the nature of the molecules that associate with intracellular trafficking vesicles to coordinate polarized delivery, tethering, docking, and fusion are also becoming understood. Combined with direct visualization of polarized sorting processes with new technologies in live-cell fluorescent microscopy, new and surprising insights into these once-elusive trafficking processes are emerging. Here we provide a review of these recent advances within an historically relevant context.     

The GTP-binding protein RhoA mediates Na,K-ATPase exocytosis in alveolar epithelial cells

The purpose of this study was to define the role of the Rho family of small GTPases in the beta-adrenergic regulation of the Na,K-ATPase in alveolar epithelial cells (AEC). The beta-adrenergic receptor agonist isoproterenol (ISO) increased the Na,K-ATPase protein abundance at the plasma membrane and activated RhoA in a time-dependent manner. AEC pretreated with mevastatin, a specific inhibitor of prenylation, or transfected with the dominant negative RhoAN19, prevented ISO-mediated Na,K-ATPase exocytosis to the plasma membrane. The ISO-mediated activation of RhoA in AEC occurred via beta2-adrenergic receptors and involved Gs-PKA as demonstrated by incubation with the protein kinase A (PKA)-specific inhibitors H89 and PKI (peptide specific inhibitor), and Gi, as incubation with pertussis toxin or cells transfected with a minigene vector for Gi inhibited the ISO-mediated RhoA activation. However, cells transfected with minigene vectors for G12 and G13 did not prevent RhoA activation by ISO. Finally, the ISO-mediated Na,K-ATPase exocytosis was regulated by the Rho-associated kinase (ROCK), as preincubation with the specific inhibitor Y-27632 or transfection with dominant negative ROCK, prevented the increase in Na,K-ATPase at the plasma membrane. Accordingly, ISO regulates Na,K-ATPase exocytosis in AEC via the activation of beta2-adrenergic receptor, Gs, PKA, Gi, RhoA, and ROCK.     

Cytomatrix synthesis in MDCK epithelial cells

Detailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak (J. Biol. Chem., 256:4863-4870, 1981), was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with [14C]leucine over several days and then pulse-labeled for 4 hours with [3H]leucine. FSRs (as percent per hour) were calculated from the 3H/14C ratio of cell extracts or individual proteins separated by two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered-Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of alpha- and beta-tubulin diverged in quiescent cells with alpha-tubulin turnover exceeding beta-tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and alpha-actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form. The results suggest that metabolic coupling between individual cellular filament systems is not strict. The data are, however, consistent with models that predict that assembly of a subcellular structure influences the turnover of its component proteins.     

Regulation of inositol 1,4,5-trisphosphate receptor-mediated calcium release by the Na/K-ATPase in cultured renal epithelial cells

It is known that the Na/K-ATPase alpha1 subunit interacts directly with inositol 1,4,5-triphosphate (IP(3)) receptors. In this study we tested whether this interaction is required for extracellular stimuli to efficiently regulate endoplasmic reticulum (ER) Ca(2+) release. Using cultured pig kidney LLC-PK1 cells as a model, we demonstrated that graded knockdown of the cellular Na/K-ATPase alpha1 subunit resulted in a parallel attenuation of ATP-induced ER Ca(2+) release. When the knockdown cells were rescued by knocking in a rat alpha1, the expression of rat alpha1 restored not only the cellular Na/K-ATPase but also ATP-induced ER Ca(2+) release. Mechanistically, this defect in ATP-induced ER Ca(2+) release was neither due to the changes in the amount or the function of cellular IP(3) and P2Y receptors nor the ER Ca(2+) content. However, the alpha1 knockdown did redistribute cellular IP(3) receptors. The pool of IP(3) receptors that resided close to the plasma membrane was abolished. Because changes in the plasma membrane proximity could reduce the efficiency of signal transmission from P2Y receptors to the ER, we further determined the dose-dependent effects of ATP on protein kinase Cepsilon activation and ER Ca(2+) release. The data showed that the alpha1 knockdown de-sensitized the ATP-induced ER Ca(2+) release but not PKCepsilon activation. Moreover, expression of the N terminus of Na/K-ATPase alpha1 subunit not only disrupted the formation of the Na/K-ATPase-IP(3) receptor complex but also abolished the ATP-induced Ca(2+) release. Finally, we observed that the alpha1 knockdown was also effective in attenuating ER Ca(2+) release provoked by angiotensin II and epidermal growth factor.     

Molecular polarity dependence of the transformed steroid effect on Na,K-ATPase

The transformed steroids containing an additional cycle E (delta-lactone or 16.23-pyranone) or 23-carbethoxy side chain (1.10(-5) M) inhibit Na,K-ATPase from pig kidney medulla. The steroid structure has a noticeable effect on ATPase inhibiton varying from 9 to 35%. The data obtained suggest that the position, stereochemistry and the uneven distribution of polar substituents in the steroid molecule are essential for ATPase inhibition by steroid aglycons.     

Oxidative Resistance of Na/K-ATPase

1. Oxidative modification of Na/K-ATPase from brain and kidney has been studied. Brain enzyme has been found to be more sensitive than kidney enzyme to inhibition by both H₂O₂ and NaOCl.2. The inhibition of Na/K-ATPase correlates well with the decrease in a number of SH groups, suggesting that the latter belong mainly to ATPase protein and are essential for the enzyme activity. We suggest that the differences in the number, location, and accessibility of SH groups in Na/K-ATPase isozymes predict their oxidative stability.3. The hydrophilic natural antioxidant carnosine, the hydrophobic natural antioxidant -tocopherol, and the synthetic antioxidant ionol as well as the ferrous ion chelating agent deferoxamine were found to protect Na/K-ATPase from oxidation by different concentrations of H₂O₂. The data suggest that these antioxidants are effective due to their ability to neutralize or to prevent formation of hydroxyl radicals.     

Terahertz vibration modes in Na/K-ATPase

Mechanical vibration in the Terahertz range is believed to be connected with protein functions. In this paper, we present the results of a normal-mode analysis (modal analysis) of a Na/K-ATPase all-atom model, focusing the attention on low-frequency vibration modes. The numerical model helps in the interpretation of experimental results previously obtained by the authors via Raman spectroscopy of Na/K-ATPase samples, where several unassigned peaks were found in the sub-500 cm^?1 range. In particular, vibration modes corresponding to peaks at 27, 190 and 300 cm^?1, found experimentally, are confirmed here numerically, together with some other modes at lower frequencies (wavenumbers) that were not possible to observe in the experimental test. All the aforementioned modes correspond to vibrations involving the protein ends, i.e. portions directly related to the operating mechanism of the sodium-potassium pump.     

Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells

The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin-Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)-expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells.     

Assessment of heterologous membrane protein polarity in transiently transfected MDCK cells

We have evaluated transient transfection of MDCK cells by the DEAE-dextran/chloroquine method as a rapid method for study of heterologous plasma membrane protein polarity. Transiently transfected cells reseeded onto permeable supports formed confluent monolayers with normal tight junctions and normal distribution of endogenous apical and basolateral surface markers. Transfected monolayers reseeded onto opaque polycarbonate filters attained cell heights 3 times greater than on transparent filters. Conventional and confocal immunofluorescence microscopy were used to assess polarity of transient expression of heterologous proteins previously defined in stably transfected cell lines as apical (DAF-CD55), basolateral (VSV-G), and nonpolarized (CD7) in distribution. Through each transiently expressed protein exhibited a polarity phenotype in most cells which resembled the stable phenotype, consistency of polarized localization was less than in stably transfected cells. Similar results were obtained by lipofection. We conclude that transient transfection of MDCK cells may be useful as a rapid screen, but is not sufficiently reliable for definitive assessment of heterologous membrane proein polarity.Abbreviations CD55-DAF CD55-Decay-accelearating factor - DMSO Dimethylsulfoxide - FBS Fetal bovine serum - FITC Fluorescein isothiocyanate - MDCK Madin Darby canine kidney cells - PBS Phosphate-buffered saline - TER Transepithelial resistance - VSVG Vesicular stomatis virus G protein     

Structural and functional membrane polarity in cultured monolayers of MDCK cells

Summary MDCK cells form monolayers which have many of the properties usually found in transporting epithelia. The present article is devoted to the study of the structural and functional polarization of MDCK cells, which is one of the central features of transporting epithelia. The results show: (i) that MDCK monolayers transport 2.6 mol hr^–1 cm^–2 of sodium in the apical to basolateral direction; (ii) the passive flux of this ion is relatively large (20.3 mole hr^–1 cm^–2), which is a characteristic of leaky epithelia; (iii) a large fraction of the penetration of sodium into the cells proceeds through an amiloride-sensitive channel, and the exit is operated mainly by a ouabain-sensitive pump; (iv) the net transport of sodium from the apical to the basolateral side agrees with the asymmetric labeling of the pumps with³H-ouabain; (v) this asymmetric labeling agrees, in turn, with a higher concentration of intramembrane particles (IMPs) in freeze-fracture replicas of the basolateral side of the plasma membrane; (vi) the structural polarization of confluent MDCK cells is also revealed by the location of microvilli, occluding junctions, and pinocytotic vesicles; and (vii) the presence of a continuous ring formed by actin microfilaments visualized by immunofluorescence under the lateral aspect of the plasma membrane that may be related to the distribution of the occluding junctions, which act as barriers separating apical from basolateral membrane components.     

Na,K-ATPase inhibition alters tight junction structure and permeability in human retinal pigment epithelial cells

Na,K-ATPase regulates avariety of transport functions in epithelial cells. In cultures ofhuman retinal pigment epithelial (RPE) cells, inhibition of Na,K-ATPaseby ouabain and K⁺ depletion decreased transepithelialelectrical resistance (TER) and increased permeability of tightjunctions to mannitol and inulin. Electrophysiological studiesdemonstrated that the decrease in TER was due to an increase inparacellular shunt conductance. At the light microscopy level, thisincreased permeability was not accompanied by changes in thelocalization of the tight junction proteins ZO-1, occludin, andclaudin-3. At the ultrastructural level, increased tight junctionpermeability correlated with a decrease in tight junction membranecontact points. Decreased tight junction membrane contact points andincreased tight junction permeability were reversible inK⁺-repletion experiments. Confocal microscopy revealed thatin control cells, Na,K-ATPase was localized at both apical andbasolateral plasma membranes. K⁺ depletion resulted in alarge reduction of apical Na,K-ATPase, and after K⁺repletion the apical Na,K-ATPase recovered to control levels. Theseresults suggest a functional link exists between Na,K-ATPase and tightjunction function in human RPE cells.

     

Activation of glutamate receptors inhibits Na/K-ATPase of cerebellum granule cells

Na/K-ATPase prepared from cerebellum granule cells of 10-12-day-old mice is inhibited by glutamate and its agonists, NMDA (ligand for ionotropic receptors) and ACPD (ligand for metabotropic receptors). The inhibition is specific and prevented by subsequent antagonists (MK-801 for ionotropic NMDA-receptors and MCPG for metabotropic receptors). The inhibiting effect of NMDA is significantly reversed by cysteine and that of ACPD by chelerythrine or indolyl maleimide. It is concluded that ionotropic receptors inhibit Na/K-ATPase because of intracellular production of reactive oxygen species, and metabotropic receptors mediate their effect via protein kinase C.     

Immunolocalization of Na,K-ATPase in blowfly photoreceptor cells

The Na,K-ATPase (sodium pump) plays a central role in the physiology of arthropod photoreceptors as it re-establishes gradients for Na⁺ and K⁺ after light stimulation. We have mapped the distribution of the Na,K-ATPase in the photoreceptors of the blowfly (Calliphora erythrocephala) by immunofluorescent and immunogold cytochemistry, and demonstrate that the distribution pattern is more complex than previously presumed. High levels of sodium pumps have been detected consistently in all photoreceptors R1-8 on the nonreceptive surface, but no sodium pumps are found on the microvillar rhabdomere. Within the nonreceptive surface of the cells R1-6, however, the sodium pumps are confined to sites juxtaposed to neighboring photoreceptor or glial cells; no sodium pumps have been detected on the parts of the nonreceptive surface exposed to the intra-ommatidial space. In R7 and R8, the sodium pumps are found over the entire nonreceptive surface. The cytoskeletal protein spectrin colocalizes with the sodium pumps suggesting that linkage of the pump molecules to the spectrin-based submembrane cytoskeleton contributes to the maintenance of the complex pattern of pump distribution.     

Polarity of the Forssman glycolipid in MDCK epithelial cells

To determine whether epithelial plasma membrane glycolipids are polarized in a manner analogous to membrane proteins, MDCK cells grown on permeable filters were analyzed for the expression of Forssman ceramide pentasaccharide, the major neutral glycolipid in these cells. In contrast to a recent report which described exclusive apical localization of the Forssman glycolipid (Hansson, G.C., Simons, K. and Van Meer, G. (1986) EMBO J. 5, 483-489), immunofluorescence and immunoelectron microscopic staining revealed the Forssman glycolipid on both the apical and basolateral surfaces of polarized cells. Immunoblots indicated that the Forssman antigen was detectable only on glycolipids and not on proteins. Analysis of metabolically labeled glycolipids released into the apical and basal culture medium, either as shed membrane vesicles or in budding viruses, also demonstrated the presence of the Forssman glycolipid on both apical and basolateral membranes of polarized cells. Quantitation of the released glycolipid indicated that the Forssman glycolipid was concentrated in the apical membrane. These results are consistent with previous reports which described quantitative enrichment of glycolipids in the apical domain of several epithelia.