Polarization of Na(+)/H(+) and Cl(-)/HCO (3)(-) exchangers in migrating renal epithelial cells.

Cell migration is crucial for processes such as immune defense, wound healing, or the formation of tumor metastases. Typically, migrating cells are polarized within the plane of movement with lamellipodium and cell body representing the front and rear of the cell, respectively. Here, we address the question of whether this polarization also extends to the distribution of ion transporters such as Na(+)/H(+) exchanger (NHE) and anion exchanger in the plasma membrane of migrating cells. Both transporters are required for locomotion of renal epithelial (Madin-Darby canine kidney, MDCK-F) cells and human melanoma cells since their blockade reduces the rate of migration in a dose-dependent manner. Inhibition of migration of MDCK-F cells by NHE blockers is accompanied by a decrease of pH(i). However, when cells are acidified with weak organic acids, migration of MDCK-F cells is normal despite an even more pronounced decrease of pH(i). Under these conditions, NHE activity is increased so that cells are swelling due to the accumulation of organic anions and Na(+). When exclusively applied to the lamellipodium, blockers of NHE or anion exchange inhibit migration of MDCK-F cells as effectively as when applied to the entire cell surface. When they are directed to the cell body, migration is not affected. These data are confirmed immunocytochemically in that the anion exchanger AE2 is concentrated at the front of MDCK-F cells. Our findings show that NHE and anion exchanger are distributed in a polarized way in migrating cells. They are consistent with important contributions of both transporters to protrusion of the lamellipodium via solute uptake and consequent volume increase at the front of migrating cells.

K(+) channel-dependent migration of fibroblasts and human melanoma cells.

Previously, we showed that migration of transformed renal epithelial cells (MDCK-F cells) is a K(+) channel-dependent process [J Clin Invest 1994;93:1631]. In order to determine whether K(+) channel activity is a general requirement for locomotion, we extended our observations to NIH3T3 fibroblasts and human melanoma cells. Migration of both cell types and its dependence on K(+) channel activity was measured at the single cell level by time lapse photography in the absence and presence of the specific K(+) channel blocker charybdotoxin (CTX). Locomotion of both cell types is inhibited by K(+) channel blockade. CTX slows down migration of fibroblasts and of melanoma cells dose-dependently by up to 61 +/- 11%. These findings suggest that K(+) channel activity is a general prerequisite for migration. To determine whether CTX-induced inhibition of migration of fibroblasts and melanoma cells involves quantitative changes of actin filaments, we indirectly measured filamentous actin by quantitating binding of fluorescently labeled phalloidin. Whereas CTX elicits a decrease of bound phalloidin in fibroblasts there is an increase in melanoma cells. Since migration of tumor cells is required for invading surrounding tissue, we developed an assay to test whether CTX-induced inhibition of migration also impairs invasion of melanoma cells. Melanoma cells were seeded on a layer of high resistance renal epithelial cells (MDCK cells clone C7; transepithelial resistance R(te) >3,000 Omegacm(2)) and R(te) was measured daily. R(te) starts to decrease 2 days after seeding of melanoma cells onto MDCK-C7 cells. By day 7, R(te) has dropped to 24 +/- 1.5% of control. K(+) channel blockade with CTX (10 nmol/l) cannot prevent or delay this drop of R(te). R(te) reaches the same level with or without CTX. These results indicate that the disruption of an epithelial layer, unlike migration of melanoma cells, cannot be modulated by K(+) channel blockade.

Migration of transformed renal epithelial cells is regulated by K+ channel modulation of actin cytoskeleton and cell volume.

Migration of transformed renal epithelial (MDCK-F) cells depends on the polarized activity of a Ca2+-sensitive K+ channel (IK channel; Pflügers Arch 432:R87-R93, 1996). This study was aimed at elucidating the functional link between the IK channel and the actin cytoskeleton which is required for cell locomotion. We monitored migration of MDCK-F cells with video microscopy, quantified filamentous actin with phalloidin binding, and measured the intracellular Ca2+ concentration ([Ca2+]i) with the fluorescent dye fura-2/AM. We compared the effects of IK channel activation or inhibition with those of hypotonic swelling or hypertonic shrinkage. IK channel inhibition with charybdotoxin (CTX) or cell swelling (omission of up to 50 mmol/l NaCl) as well as IK channel activation with 1-ethyl-2-benzimidazolinone (1-EBIO) or cell shrinkage (addition of up to 100 mmol/l mannitol) reduce the rate of migration dose-dependently by up to 80%, i.e., to the same extent as cytochalasin D. Inhibition of migration is accompanied either by actin depolymerization (CTX and cell swelling) or by actin polymerization (1-EBIO and cell shrinkage). Changes of migration and phalloidin binding induced by CTX and cell swelling or by 1-EBIO and cell shrinkage, respectively, are linearly correlated with each other. CTX and cell swelling elicit a rise of [Ca2+]i whereas 1-EBIO and cell shrinkage induce a slight decrease of [Ca2+]i in most MDCK-F cells. Taken together IK-channel-dependent perturbations of cell volume and anisotonicity elicit virtually identical effects on migration, actin filaments and [Ca2+]i. We therefore suggest that cell volume - possibly via [Ca2+]i - is the link between IK channel activity, actin filaments and migration. We propose a model for how temporal and local changes of cell volume can support the migration of MDCK-F cells.

Phenotypically and karyotypically distinct Madin-Darby canine kidney cell clones respond differently to alkaline stress.

We isolated two cell clones from the wild-type Madin-Darby canine kidney cell line (MDCK) that resembles renal collecting duct epithelium. Morphology and karyotypes of the two cell clones were evaluated. The MDCK-C7 cell clone morphologically resembles principal cells (polygonal cell shape, flat), while the MDCK-C11 clone resembles intercalated cells (cuboidal cell shape, high). The diploid chromosome number of MDCK-C7 cells is 83.1 +/- 0.2 (n = 139); that for MDCK-C11 cells is 78.8 +/- 0.1 (n = 128). Culture of MDCK-C7 cells in alkaline medium (pH 7.7) induced irreversible phenotypical and genotypical alterations. Transformed MDCK-C7F cells are characterized by two abnormal (biarmed) chromosomes. In contrast, MDCK-C11 cells are not phenotypically altered by alkaline stress. In order to elucidate the role of intracellular pH (pHi) in the transformation process, we measured pHi under control conditions (pH 7.4), after 5 min exposure to alkaline stress ("acute experiment," pH 7.7) and after incubation of the cells in alkaline medium for two weeks ("chronic experiment," pH 7.7). Under control conditions, MDCK-C7 cells maintained pHi at 7.14 +/- 0.01 (n = 154) and MDCK-C11 cells at 7.01 +/- 0.01 (n = 147). Acute alkaline stress increased pHi of both cell types to similar steady-state values. Under chronic alkaline stress, MDCK-C7 cells were unable to maintain intracellular pH within normal limits exhibiting sustained alkalinization, whereas MDCK-C11 cells could successfully regulate pHi. We conclude that wild-type MDCK cells consist of two genetically distinct subpopulations with different morphology and function. Only the MDCK-C7 clone that resembles the principle cell type of renal collecting duct can be transformed by alkaline stress while the MDCK-C11 clone resists this treatment, due to efficient pHi control mechanisms.

Role of H+ ions in volume and voltage of epithelial cell nuclei.

Condensation of chromatin depends upon the ion composition in the cell nucleus. We tested in isolated nuclei of Madin-Darby canine kidney cells the influence of various ions on nuclear volume (i. e. DNA packing) and intranuclear voltage. After isolation, nuclei were superfused with cytosolic solutions in which Na+, K+, Ca2+ and H+ ions were varied. With video-imaging and microelectrode techniques nuclear volume and intranuclear potential were measured in response to the various ions. In control cytosolic solution, isolated nuclei exhibited an intranuclear electrical potential of -6.5 +/- 0.5 mV (relative to a reference electrode in the cytosolic solution) corresponding to a nuclear volume of 250 +/- 10 fl (n = 104). Changing the Na+, K+ or free Ca2+ concentration in the superfusate in the physiological range resulted in minor changes of volume and intranuclear potential whereas pH altered both parameters dramatically. Nuclear swelling and intranuclear negative voltage increased with alkalinization and decreased when pH was reduced. An intact nuclear envelope was found to be no prerequisite for maintaining intranuclear negativity, indicating that the composition and functional state of nuclear chromatin rather than specific ion permeabilities of the nuclear envelope determine nuclear electrical potential. We present a model that explains nuclear volume and voltage on the basis of interaction between negatively charged DNA and positively charged histones of the nuclear chromatin.

Replacement of glucose by sorbitol in growth medium causes selection of astroglial cells from heterogeneous primary cultures derived from newborn mouse brain.

Primary cultures derived from the brains of newborn mice are quantitatively dominated by astroglial cells, but contain also oligodendroglial, phagocytic and ependymal cells. When confluent cultures are fed with glucose-free growth medium containing 25 mM sorbitol for 14 days, oligodendroglial, phagocytic and ependymal cells are eliminated from the culture, as judged by morphological and immunocytochemical criteria. The remaining cells stain positively for vimentin and glial fibrillary acidic protein and, therefore, can be considered as astroglial cells. Inoculation of freshly dissociated mouse brain cells in the absence of glucose in a sorbitol-containing medium is not possible; however, feeding of the cultures from day 2 on with sorbitol instead of glucose results in a pure astroglial culture at confluency. Therefore glucose-free growth medium supplemented with sorbitol can be considered a selective medium for astroglial cells in primary mouse glial cultures.

Elevation by atrial natriuretic factors of cyclic GMP levels in astroglia-rich cultures from murine brain.

Atrial natriuretic factors, peptide hormones originally found in the heart, slowly but strongly elevate the level of cyclic GMP in primary astrocyte-rich cultures derived from brains of newborn rats or mice but not in neuron-rich cultures prepared from embryonic rat brain. In the absence of a phosphodiesterase inhibitor, a plateau level of cyclic GMP is obtained within 10 min. In the presence of the inhibitor 3-isobutyl-1-methylxanthine, the concentration of cyclic GMP continues to rise, even after 30 min. The elevation of the level of cyclic GMP in response to atrial natriuretic factor is much more pronounced in the rat cultures than the mouse cultures. Even at peptide concentrations of 1 microM, plateaus of the concentration-response curves are not yet reached. The potencies of the active peptides vary over a range of approximately 1.5 orders of magnitude, with atriopeptins II and III and auriculin A being the most potent ones. These results suggest (a) that atrial natriuretic factors may regulate functions of glial cells, most likely of astrocytes, in brain and (b) that such cultures may be useful tools in defining such astroglial functions.