American Journal of Physiology - Cell Physiology
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CXCL12-induced glioblastoma cell migration requires intermediate conductance Ca<sup>2+</sup>-activated K<sup>+</sup> channel activity The activation of ion channels is crucial during cell movement, including glioblastoma cell invasion in the brain parenchyma. In this context, we describe for the first time the contribution of intermediate conductance Ca2+ -activated K (IKCa ) channel activity in the chemotactic response of human glioblastoma cell lines, primary cultures, and freshly dissociated tissues to CXC chemokine ligand 12 (CXCL12), a chemokine whose expression in glioblastoma has been correlated with its invasive capacity. We show that blockade of the IKCa channel with its specific inhibitor 1-[(2-chlorophenyl) diphenylmethyl]-1 H-pyrazole (TRAM-34) or IKCa channel silencing by short hairpin RNA (shRNA) completely abolished CXCL12-induced cell migration. We further demonstrate that this is not a general mechanism in glioblastoma cell migration since epidermal growth factor (EGF), which also activates IKCa channels in the glioblastoma-derived cell line GL15, stimulate cell chemotaxis even if the IKCa channels have been blocked or silenced. Furthermore, we demonstrate that both CXCL12 and EGF induce Ca2+ mobilization and IKCa channel activation but only CXCL12 induces a long-term upregulation of the IKCa channel activity. Furthermore, the Ca2+ -chelating agent BAPTA-AM abolished the CXCL12-induced, but not the EGF-induced, glioblastoma cell chemotaxis. In addition, we demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway is only partially implicated in the modulation of CXCL12-induced glioblastoma cell movement, whereas the phosphoinositol-3 kinase (PI3K) pathway is not involved. In contrast, EGF-induced glioblastoma migration requires both ERK1/2 and PI3K activity. All together these findings suggest that the efficacy of glioblastoma invasiveness might be related to an array of nonoverlapping mechanisms activated by different chemotactic agents.
American Journal of Physiology - Cell Physiology - Tập 299 Số 1 - Trang C175-C184 - 2010
Imaging of filter-grown epithelial cells: MDCK Na(+)-H+ exchanger is basolateral We report a simple method for growing epithelial cells on permeable supports and for imaging the cells from the apical side using an inverted microscope. Madin-Darby canine kidney (MDCK) cells were either seeded onto the conventional side of Millipore-CM filters or onto “inverted” filters. The peak transepithelial resistance of confluent monolayers was the same with either system. Cells on inverted filters that were stained with various dyes and imaged by epifluorescence exhibited more distinct intercellular spaces, cell margins, nuclei, and subapical vesicles. We also perfused both sides of inverted filters with HCO3/CO2-free saline and measured intracellular pH (pHi) using 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and digital imaging. The intrinsic buffer capacity of MDCK cells increased exponentially as pHi decreased. After an NH4Cl load, the H+ extrusion rate (JH+) in control saline was 2.42 +/- 0.62 mM/min. JH+ was completely blocked by 1 mM basolateral amiloride. In contrast, 1 mM apical amiloride had no effect. We conclude that 1) growth of epithelial cells on an inverted filter system is useful for the microspectrofluorimetric determination of pHi in single cells and for the imaging of apical/subapical structures, and 2) the Na(+)-H+ exchanger of MDCK cells is functionally polarized to the basolateral membrane.
American Journal of Physiology - Cell Physiology - Tập 260 Số 4 - Trang C868-C876 - 1991
Effect of adenosine 3',5'-cyclic monophosphate on volume and cytoskeleton of MDCK cells We examined the effect of N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) on the volume and cytoskeleton of confluent cultures of Madin-Darby canine kidney (MDCK) cells. A 90-min exposure to 1 mM DBcAMP resulted in a 20% reduction in volume as measured by [14C]-urea water space. The volume in cells exposed to isobutylmethylxanthine (IBMX, 0.1 mM) was reduced by 24%. In control cultures F-actin, revealed by staining with nitrobenzoxadiazole-phallacidin, was found at the base of the cell as fibers, at the junctional region as a circumferential band, and on the apical cell surface as a mottled fluorescence. A dense pattern of microtubules, revealed by indirect immunofluorescence, was seen throughout the cell. Exposure to DBcAMP for 90 min resulted in a change of F-actin fibers into dense bundles near the periphery of the cell. This effect was even more striking when cells were exposed to IBMX. Cytochalasin B disrupted F-actin and resulted in a volume reduction similar to that in DBcAMP. Neither DBcAMP nor IBMX affected the distribution of microtubules. Moreover, colchicine, which completely disrupted the microtubules, did not change MDCK cell volume. The results suggest that DBcAMP and F-actin play a role in volume control in MDCK cells.
American Journal of Physiology - Cell Physiology - Tập 250 Số 2 - Trang C319-C324 - 1986
Growth and differentiated properties of a kidney epithelial cell line (MDCK) The MDCK dog kidney epithelial cell line has been shown to retain the capacity for vectorial salt and fluid transport, sensitivity to growth regulation, and the ability to regenerate kidney tubular-like structures when injected into athymic nude mice. MDCK cells grown in tissue culture or in baby nude mice have the morphological properties of distal tubular cells, form tight and gap junctions, lack proximal tubular enzyme markers, and possess appreciable activities of Na+-K4-ATPase, ectoleucine aminopeptidase, and ectoalkaline phosphatase. Adenylate cyclase in intact cells is responsive to vasopressin, prostaglandins E1 and E2, and glucagon. Two Na+ transport systems have been characterized: a Na+-H+ antiport system, sensitive to amiloride inhibition, and a NaCl-KCl cotransport system, dependent on metabolic energy and sensitive to furosemide inhibition. Genetic techniques have been used to modify the properties of the cells. The results suggest that the MDCK cell line has retained the differentiated properties of the kidney epithelial cells of origin and that a clonally isolated cell possesses the receptor, transmission, and target enzyme systems necessary for the regulation of transcellular salt and fluid transport.
American Journal of Physiology - Cell Physiology - Tập 240 Số 3 - Trang C106-C109 - 1981
Volume-activated calcium uptake: its role in cell volume regulation of Madin-Darby canine kidney cells Immediately after osmotic swelling of Madin-Darby canine kidney (MDCK) cells, a transient (1-2 min) increase in Ca2+ influx and internal Ca2+ (Ca2+i) is observed. The normal Ca2+ influx appears to be mediated by the 3Na(+)-Ca2+ exchange system [Borle et al. Am. J. Physiol. 259 (Cell Physiol. 28): C19-C25, 1990], but the swelling-induced component is different in 1) Na+ dependence, 2) affinity for Ca2+, 3) inhibition by La3+, and 4) direction of net flux at low external Ca2+. Swelling appears to activate an uncoupled Ca2+ flow, perhaps through cation-nonspecific stretch-activated channels. The regulatory volume decrease (RVD) is dependent on the swelling-induced pulse of Ca2+ influx and associated rise in Ca2+i. Swelling also induces a biphasic change in membrane potential, a hyperpolarization followed by depolarization, reflecting sequential increases in K+ and Cl- permeabilities. The time dependence of the former corresponds closely with the transient peak in Ca2+i, but the latter does not. Ca2+i appears to have a direct activating effect on K+ channels but an indirect effect on Cl- channels, mediated via other Ca(2+)-triggered systems. The sequence of events following cell swelling appears to be transient increases in Ca2+ permeability, Ca2+ influx, Ca2+i, K+ permeability, followed by triggering of a mediating system that increases Cl- permeability. The net result is KCl, osmotic water loss, and volume adjustment.
American Journal of Physiology - Cell Physiology - Tập 262 Số 2 - Trang C339-C347 - 1992
Activation of potassium channels in renal epithelioid cells (MDCK) by extracellular ATP Extracellular ATP has been shown to stimulate transepithelial chloride transport in confluent Madin-Darby canine kidney (MDCK) cell layers and to enhance potassium conductance in subconfluent MDCK cells. The present study has been performed to test for the effect of extracellular ATP on channel activity in patches from subconfluent MDCK cells. Within 8 s, addition of extracellular ATP (10 mumol/l) leads to a sustained, but fully reversible, appearance of potassium-selective channels in cell-attached patches [increase of open probability from 0.03 +/- 0.02 (n = 10) to 0.50 +/- 0.07 (n = 6)]. With the use of pipettes filled with 145 mmol/l KCl, inwardly rectifying property of the channels is disclosed with a single-channel conductance of 65.7 +/- 3.1 pS (n = 9) at zero potential difference between pipette and bath and with a reversal potential of 75.4 +/- 2.0 mV (n = 5; pipette negative vs. reference in the bath). The open probability of the channels is not significantly modified by altering pipette potential from -50 mV, pipette positive, to 50 mV, pipette negative. At extracellular calcium activities of less than 10 nmol/l, ATP leads to a transient activation of channels. In conclusion, extracellular ATP activates inwardly rectifying potassium channels in the cell membrane of subconfluent MDCK cells. A sustained activation of the channels requires the presence of extracellular calcium and is probably mediated by increases in intracellular calcium.
American Journal of Physiology - Cell Physiology - Tập 256 Số 5 - Trang C1016-C1021 - 1989
Regulation of Na(+)-K(+)-ATPase expression in cultured renal cells by incubation in hypertonic medium To determine whether alterations in cell volume affect Na(+)-K(+)-adenosinetriphosphatase (ATPase) expression, a subclone of the Madin-Darby canine kidney (MDCK) cell line was incubated in anisotonic serum-free medium and alpha- and beta-subunit mRNA, Na(+)-K(+)-ATPase activity, and active K+ transport were measured. In medium adjusted to 500 mosmol/kgH2O by adding NaCl, the alpha-subunit mRNA concentration was 2.93 +/- 0.14 (SE) times control and beta-mRNA was 1.93 +/- 0.27 times control. When sucrose was added to increase osmolality, alpha-subunit mRNA increased to 1.85 +/- 0.18 times control. Na(+)-K(+)-ATPase activity of homogenates from cells incubated in 500 mosmol/kgH2O medium for 24 h increased to 2.62 +/- 0.52 times control when NaCl was added and 2.31 +/- 0.34 times control when sucrose was added. Active K+ transport increased between 60 and 90% after cells were incubated in 450 mosmol/kgH2O medium with either NaCl or sucrose added. Stimulation of Na(+)-K(+)-ATPase expression in renal cells facing hypertonic stress may represent a long-term mechanism that allows cells to maintain cation gradients in a hypertonic environment.
American Journal of Physiology - Cell Physiology - Tập 262 Số 4 - Trang C845-C853 - 1992
Anion channels for amino acids in MDCK cells Large losses of amino acids by diffusion were previously observed in Madin-Darby canine kidney (MDCK) cells during volume regulation. Also, an outward rectifying anion channel was activated. Because this channel was not selective among anions, it was suggested that it could be permeable to amino acids. Its permeability to aspartate, glutamate, and taurine was studied using the patch-clamp technique in the inside-out configuration. Solutions containing 500 mM aspartate or glutamate were used on the cytoplasmic side of excised patches to detect single-channel currents carried by these anions. Permeability ratios were estimated in two different ways: 1) from the shift in reversal potential of current-voltage curves after anion replacement in the bath solution and 2) from comparisons of amplitudes of single-channel currents carried by tested anions and chloride, respectively. The values of aspartate-to-chloride and glutamate-to-chloride permeability ratios obtained with both methods were quite consistent and were of the order of 0.2 for both amino acids. Taurine in solutions at physiological pH 7.3 is a zwitterionic molecule and bears no net charge. To detect single-channel currents carried by taurine, solutions containing 500 mM taurine at pH 8.2 were used in inside-out experiments. Under these conditions 120 mM of negatively charged taurine was present in the solutions bathing the cytoplasmic side of excised patches. The permeability ratio estimated from the shift in reversal potential was 0.75. These results showed that some of the organic compounds released by cells during regulatory volume decrease could diffuse through this outwardly rectifying anionic channel.
American Journal of Physiology - Cell Physiology - Tập 263 Số 6 - Trang C1200-C1207 - 1992
Repolarization of Na+-K+ pumps during establishment of epithelial monolayers Madin-Darby canine kidney (MDCK) cells plated at confluence and incubated for 20 h in low (5 microM) Ca2+ have no tight junctions (TJs), and their Na+-K+-ATPase is randomly distributed over the surface. On transfer to normal Ca2+ levels (1.8 mM) ("Ca2+ switch"), TJs and transepithelial resistance develop quickly, trapping a considerable fraction (35%) of the surface Na+-K+-ATPase on the apical (incorrect) side. This misplaced enzyme is subsequently removed from this region or inactivated, demonstrating that polarization proceeds despite TJs. Simultaneously, the amount of Na+-K+-ATPase on the basolateral side increases in a higher proportion (125%), than could be accounted for by relocation of the misplaced apical enzyme. This incorporation is prevented by cycloheximide, ammonium chloride, primaquine, or chloroquine, suggesting that Na+-K+-ATPase originates in an intracellular pool and that its surface insertion requires synthesis of new enzyme or of a protein factor, since it is carried to the surface membrane through a mechanism of exocytosis. In summary, asymmetric distribution of ion pumps depends 1) on polarized insertion of Na+-K+-ATPase as well as 2) on removal or inactivation of misplaced enzyme.
American Journal of Physiology - Cell Physiology - Tập 257 Số 5 - Trang C896-C905 - 1989
Inhibition of ion conductances by osmotic shrinkage of Madin-Darby canine kidney cells Osmotic swelling of Madin-Darby canine kidney (MDCK) cells enhances the ion conductances of the cell membrane, which allows release of cellular ions and subsequent regulatory cell volume decrease. The present study has been performed to test whether cell shrinkage similarly affects the ion conductances of MDCK cell membranes. Increase of extracellular osmolarity by addition of 50 mM NaCl or 100 mM mannitol leads within 3 min to a hyperpolarization of the cell membrane, a marked increase of cell membrane resistance [by 223 +/- 38% (n = 8) and 228 +/- 21% (n = 5), respectively], as well as a moderate increase of the K+ selectivity of the cell membrane (by 37 +/- 13%, n = 9). Thus exposure to hypertonic extracellular fluid decreases the cell membrane conductances including the K+ conductance. Cell volume measurements reveal a regulatory cell volume increase, which is sensitive to both furosemide and dimethylamiloride. Extracellular ATP (10 microM), which activates calcium-sensitive K+ channels, hyperpolarizes the cell membrane close to the K+ equilibrium potential. The respective values are -69.9 +/- 3.1 mV (n = 9) in isotonic fluid, -79.4 +/- 1.8 mV (n = 9) within 3 min, and -76.4 +/- 1.8 mV (n = 7) within 16-h exposure to hypertonic extracellular fluid. This observation points to a sustained increase of intracellular K+ activity after exposure to hypertonic extracellular fluid.
American Journal of Physiology - Cell Physiology - Tập 261 Số 4 - Trang C602-C607 - 1991
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