The effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins

The influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. In order to determine what structural features of the oligosaccharide were required for fucosylation or where in the processing pathway fucosylation occurred, influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, [5,6-3H]fucose and [1-14C]mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the [14C]mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the [14C]mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the [3H]fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the [14C]mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure.     

The action of epinephrine on Madin-Darby canine kidney cells

We have used cultured monolayers of Madin-Darby canine kidney (MDCK) cells, which form epithelial layers of high transepithelial resistance, grown on Millipore filters, for transport studies. In the absence of hormones net ion transport is of small magnitude and is consistent with a net absorptive flow (apical to basal) of Na+. Epinephrine, effective only from the basolateral cell surface, stimulates a net secretion (basal to apical) of Cl-. A substantial portion of net Cl- secretion is inhibited by loop diuretics such as furosemide applied to the basolateral cell aspects. The participation of a diuretic-sensitive cotransport system for Na+, K+, and Cl-, similar to that found in other cells, in transepithelial Cl- flux is postulated. The action of catecholamines on MDCK cell adenylate cyclase and on a Ca2+-activated K+ conductance is described.     

Involvement of VIP36 in intracellular transport and secretion of glycoproteins in polarized Madin-Darby canine kidney (MDCK) cells

VIP36, an intracellular lectin that recognizes high mannose-type glycans (Hara-Kuge, S., Ohkura, T., Seko, A., and Yamashita, K. (1999) Glycobiology 9, 833-839), was shown to localize not only to the early secretory pathway but also to the plasma membrane of Madin-Darby canine kidney (MDCK) cells. In the plasma membrane, VIP36 exhibited an apical-predominant distribution, the apical/basolateral ratio being approximately 2. Like VIP36, plasma membrane glycoproteins recognized by VIP36 were found in the apical and basolateral membranes in the ratio of approximately 2 to 1. In addition, secretory glycoproteins recognized by VIP36 were secreted approximately 2-fold more efficiently from the apical membrane than from the basolateral membrane. Thus, the apical/basolateral ratio of the transport of VIP36-recognized glycoproteins was correlated with that of VIP36 in MDCK cells. Upon overproduction of VIP36 in MDCK cells, the apical/basolateral ratios of both VIP36 and VIP36-recognized glycoproteins were changed from approximately 2 to approximately 4, and the secretion of VIP36-recognized glycoproteins was greatly stimulated. In contrast to the overproduction of VIP36, that of a mutant version of VIP36, which has no lectin activity, was of no effect on the distribution of glycoproteins to apical and basolateral membranes and inhibited the secretion of VIP36-recognized glycoproteins. Furthermore, the overproduction of VIP36 greatly stimulated the secretion of a major apical secretory glycoprotein of MDCK cells, clusterin, which was found to carry at least one high mannose-type glycan and to be recognized by VIP36. In contrast to the secretion of clusterin, that of a non-glycosylated apical-secretion protein, galectin-3, was not stimulated through the overproduction of VIP36. These results indicated that VIP36 was involved in the transport and sorting of glycoproteins carrying high mannose-type glycan(s).     

Cholesterol is required for the polarized secretion of erythropoietin in Madin-Darby canine kidney cells

It has already been reported that stably expressed exogenous human wild-type EPO (wtEPO) is preferentially secreted to the apical side and one of the three N-linked carbohydrate chains critically acts as an apical sorting determinant in Madin-Darby canine kidney (MDCK) cells. It has been suggested that lipid rafts are involved in the apical sorting of membrane and secretory proteins. To investigate the involvement of lipid rafts in the apical sorting of wtEPO, we examined the effect of cholesterol depletion with methyl-beta-cyclodextrin on the secretion polarity of EPO and analyzed Triton X-100 insoluble cell extracts by sucrose density gradients centrifugation in MDCK cells. We found that wtEPO was shifted in non-polarized direction by cholesterol depletion. Most of the wtEPO was not detectable in the raft fractions by sucrose density gradients centrifugation analysis. These results indicate that apical secretion of EPO involves a cholesterol-dependent mechanism probably not involving lipid rafts.     

Endocytosis in filter-grown Madin-Darby canine kidney cells

In this paper, we have characterized the apical and basolateral endocytic pathways of epithelial MDCK cells grown on filters. The three- dimensional organization of the endocytic compartments was analyzed by confocal microscopy after internalization of a fluorescent fluid-phase marker from either side of the cell layer. After 5 min of internalization, distinct sets of apical and basolateral early endosomes were observed lining the plasma membrane domain from which internalization had occurred. At later time points, the apical and the basolateral endocytic pathways were shown to converge in the perinuclear region. Mixing of two different fluorescent markers could be detected after their simultaneous internalization from opposite sides of the cell layer. The extent of the meeting was quantitated by measuring the amount of complex formed intracellularly between avidin internalized from the apical side and biotinylated horseradish peroxidase (HRP) from the basolateral side. After 15 min, 14% of the avidin marker was complexed with the biotinylated HRP and this value increased to 50% during a subsequent chase of 60 min in avidin-free medium. We also determined the kinetics of fluid internalization, recycling, transcytosis, and intracellular retention using HRP as a marker. Fluid was internalized with the same rates from either surface domain (1.2 x 10(-4) microns 3/min per microns 2 of surface area). However, significant differences were observed for each pathway in the amounts and kinetics of marker recycled and transcytosed. The content of apical early endosomes was primarily recycled and transcytosed (45% along Bach route after 1 h internalization), whereas delivery to late endocytic compartments was favored from the basolateral early endosome (77% after 1 h). Our results demonstrate that early apical and basolateral endosomes are functionally and topologically distinct, but that the endocytic pathways converge at later stages in the perinuclear region of the cell.     

Heterogeneity of the tumorigenic phenotype expressed by Madin-Darby canine kidney cells

The mechanisms by which cells spontaneously immortalized in tissue culture develop the capacity to form tumors in vivo likely embody fundamental processes in neoplastic development. The evolution of Madin-Darby canine kidney (MDCK) cells from presumptively normal kidney cells to immortalized cells that become tumorigenic represents an example of neoplastic development in vitro. Studies of the mechanisms by which spontaneously immortalized cells develop the capacity to form tumors would benefit from quantitative in vivo assays. Most mechanistic correlations are evaluated by using single-dose tumor-induction experiments, which indicate only whether cells are or are not tumorigenic. Here we used quantitative tumorigenicity assays to measure dose-and time-dependent tumor development in nude mice of 3 lots of unmodified MDCK cells. The results revealed lot-to-lot variations in the tumorigenicity of MDCK cells, which were reflected by their tumor-inducing efficiency (threshold cell dose represented by mean tumor-producing dose; log(10) 50% endpoints of 5.2 for vial 1 and 4.4 for vial 2, and a tumor-producing dose of 5.8 for vial 3) and mean tumor latency (vial 1,6.6 wk; vial 2,2.9 wk; and vial 3,3.8 wk). These studies provide a reference for further characterization of the MDCK cell neoplastic phenotype and may be useful in delineating aspects of neoplastic development in vitro that determine tumor-forming capacity. Such data also are useful when considering MDCK cells as a reagent for vaccine manufacture.     

Lixazinone stimulates mitogenesis of Madin-Darby canine kidney cells

Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.     

Ion channels in Madin-Darby canine kidney cells.

Ion channels in Madin-Darby canine kidney cells serve transepithelial chloride transport and probably cell volume regulation. Three distinct potassium channels and one anion channel have been revealed by patch clamp studies in Madin-Darby canine kidney cells. The potassium channels are activated by an increase in intracellular calcium activity. A number of hormones activate the potassium channels by an increase in intracellular calcium activity. However, under certain conditions the hormones hyperpolarize the cell membrane without increasing intracellular calcium activity sufficiently to activate the calcium-sensitive potassium channels. Thus, the hormones may activate potassium channels via another, as yet undefined, intracellular mechanism. The anion channel is stimulated by cAMP. Another factor modifying channel activity is cell volume: cell swelling leads probably to subsequent activation of potassium and anion channels. The net result is a variable transient hyperpolarization followed by a sustained depolarization of the cell membrane.     

Effect of trifluoperazine on renal epithelioid Madin-Darby canine kidney cells

Following exposure to a number of hormones, the cell membrane in Madin-Darby Canine Kidney (MDCK) cells is hyperpolarized by increase of intracellular calcium activity. The present study has been performed to elucidate the possible role of calmodulin in the regulation of intracellular calcium activity and cell membrane potential. To this end trifluoperazine has been added during continuous recording of cell membrane potential or intracellular calcium. Trifluoperazine leads to a transient increase of intracellular calcium as well as a sustained hyperpolarization of the cell membrane by activation of calcium sensitive K+ channels. Half-maximal effects are observed between 1 and 10 mumol/L trifluoperazine. A further calmodulin antagonist, chlorpromazine, (50 mumol/L), similarly hyperpolarizes the cell membrane. The effects of trifluoperazine are virtually abolished in the absence of extracellular calcium. Pretreatment of the cells with either pertussis toxin or phorbol-ester TPA does not interfere with the hyperpolarizing effect of trifluoperazine. In conclusion, calmodulin is apparently involved in the regulation of calcium transfer across the cell membrane but not in the stimulation of K+ channels by intracellular calcium.     

Effects of foscarnet on the cell kinetics of Madin-Darby canine kidney cells

The antiherpes compound, foscarnet (trisodium phosphonoformate), showed concentration-dependent effects on the cell kinetics of Madin-Darby canine kidney cells. At 1 mM, only minor effects could be seen on cell proliferation and cell cycle distribution, as measured by flow cytometry DNA analysis. Treatment with 5 mM foscarnet resulted in an accumulation of cells in the S-phase although no complete cell cycle block was evident. At 10 mM foscarnet, cells accumulated earlier in the S phase, probably at the G1/S border. However, at both 5 and 10 mM foscarnet the block was not established until after 15 h incubation. Upon removing 10 mM foscarnet after 24 h incubation, G1 cells rapidly entered the S phase, whereas the progression through S and G2 + M was delayed considerably. The DNA synthesizing S phase seems, therefore, to be the main cell cycle phase affected by foscarnet.     

Luteinizing hormone and follicle-stimulating hormone exhibit different secretion patterns from cultured Madin-Darby canine kidney cells

LH, FSH, and chorionic gonadotropin (CG) are comprised of a common alpha subunit and a hormone-specific beta subunit. Using Madin-Darby canine kidney (MDCK) epithelial cells to examine the polarized secretion of human CG/LH, we previously reported that CG and LH were detected in the apical and basolateral compartments, respectively, and the carboxyl terminal end of the CGbeta subunit contains a strong apical signal. Here we show that the carboxyl seven amino acids in the LHbeta subunit contribute to the basolateral secretion of LH, and an LH chimera bearing the CGbeta apical signal is redirected from the basolateral to the apical compartments. Because LH and FSH are synthesized in the same cell, we also compared the secretion polarity of LH with FSH. MDCK cells expressing the FSH dimer displayed an almost equal distribution of protein into the apical and basolateral compartments. Given that the LHbeta and CGbeta carboxy terminal sequences, which differ from that in the FSHbeta subunit, occupy a pivotal role in their polarized behavior, the results support the hypothesis that pituitary exit of LH and FSH occur via different secretion pathways, and are released spatially from the pituitary via different circulatory routes.     

The effect of mannosamine on the formation of lipid-linked oligosaccharides and glycoproteins in canine kidney cells

Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with [2-3H]mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Methylation analysis also indicated that this Man5GlcNAc2 contained 1----3 linked mannose residues. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps by inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with [3H]mannose or [3H]galactose. In contrast, control cells produced complex and high-mannose structures but no hybrid oligosaccharides were detected. The inhibition by mannosamine could be overcome by adding high concentrations of glucose to the medium.     

Receptor-mediated vectorial transcytosis of epidermal growth factor by Madin-Darby canine kidney cells

Transcellular transport of a variety of ligands may be an important mechanism by which regulatory substances reach their site of action. We have studied the transcellular transport of two 6,000-mol-wt proteins, epidermal growth factor (EGF) and insulin, across polarized Madin-Darby canine kidney (MDCK) cells grown on dual-sided chambers on a nitrocellulose filter substrate. When grown on these chambers, MDCK cells are polarized and express distinct basal and apical surfaces. MDCK cells are capable of unidirectional transport of EGF from the basal-to-apical direction, 50% of bound EGF transported in 2 h. Transport was inhibited by the addition of unlabeled EGF in a dose-dependent manner. Anti-EGF receptor Ab, which inhibited binding, also inhibited transport. No transport in the apical-to-basal direction is noted. Insulin transport is not observed in either direction. Transport correlates with the presence of ligand-specific receptors on the cell surface. Hence, EGF receptors (Ro = 48,000, Kd = 3.5 X 10(-10) M) are found only on the basal surface of the MDCK cells and neither surface expresses insulin receptors. Characterization of the EGF receptors on MDCK cells, as assessed by affinity, molecular mass, and anti-receptor antibody binding reveals that this receptor has similar characteristics to EGF receptors previously described on a variety of cells. Hence, the EGF receptor can function as a transporter of EGF across an epithelial cell barrier.     

Secretion of proteases in serglycin transfected Madin-Darby canine kidney cells

Madin-Darby canine kidney (MDCK) cells, which do not normally express the proteoglycan (PG) serglycin, were stably transfected with cDNA for human serglycin fused to a polyhistidine tag (His-tag). Clones with different levels of serglycin mRNA expression were generated. One clone with lower and one with higher serglycin mRNA expression were selected for this study. 35S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography. Serglycin could also be detected in conditioned media using western blotting. To investigate the possible importance of serglycin linked to protease secretion, enzyme activities using chromogenic substrates and zymography were measured in cell fractions and serum-free conditioned media of the different clones. Cells were cultured in both the absence and presence of phorbol 12-myristate 13-acetate (PMA). In general, enzyme secretion was strongly enhanced by treatment with PMA. Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35S-labelled serglycin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthesis and secretion of serglycin and potential partner molecules.     

Stimulation by parathyroid hormone of calcium absorption in confluent Madin-Darby canine kidney cells

Parathyroid hormone (PTH) increases renal calcium absorption exclusively in cortical thick limbs and distal tubules. Lack of sufficient tissue has precluded detailed biochemical study of the mechanisms responsible for the hypercalcemic effect of PTH. Therefore, we assessed PTH action on calcium transport in Madin-Darby canine kidney (MDCK) cells, a cell line expressing distal characteristics, to determine its suitability as a model for analyzing PTH action. Calcium transport across MDCK cells grown to confluence on porous filters was measured at 37 degrees C in Ussing chambers. Mucosal-to-serosal calcium fluxes (JCa, mol/min cm-2 x 10(-9)) were measured with 45Ca at -3, -1, 5, 10, and 20 min; agonist was added at 0 min. Basal JCa averaged 0.98. PTH at 0.2 microM increased JCa by 12% (P less than 0.05) and 1 microM PTH by 70% (P less than 0.01). Calcitonin (1 microM) had no effect on JCa. The fact that high concentrations of dibutyryl cAMP (1 mM) and forskolin (10 microM) increased JCa by only 37% and 22%, respectively, suggested that cAMP-independent mechanisms may participate in PTH-stimulated JCa. Therefore we examined the effect of other putative second messengers. In the presence of 2 mM external [Ca], 10 nM A23187 increased JCa by 88%, and 10 microM A23187 increased JCa by 121%. Addition of 10 microM phorbol 12-myristate 13-acetate (PMA) increased JCa by 60%. We conclude that: 1) PTH specifically stimulates unidirectional calcium absorption in MDCK cells; 2) both adenylate cyclase-coupled and calcium-coupled receptors may participate in signaling the response to PTH; and 3) confluent MDCK cells represent a useful experimental model for elucidating the biochemical mechanisms involved in the renal hypercalcemic action of PTH.     

Toxin pharmacology of the ATP-induced hyperpolarization in Madin-Darby canine kidney cells.

The effects of Leiurus quinquestriatus hebraeus (LQH) venom, mamba venom, Buthus tamulus (BT) venom, purified apamin and synthetic charybdotoxin on the membrane hyperpolarization induced by extracellular ATP were examined in Madin-Darby canine kidney cells. For this we used a membrane potential probe (bisoxonol) to determine the potential variations. The relation between bisoxonal fluorescence and membrane potential was established by treating Madin-Darby canine kidney cells suspended in solutions containing various external sodium concentrations with gramicidin. Extracellular ATP induced a rapid hyperpolarization that was blocked by LQH venom and synthetic charybdotoxin. BT venom also blocked the response but at a much higher concentration than that of LQH. Mamba venom (Dendroaspis polylepis) and apamin did not modify the ATP-induced hyperpolarization. We concluded that the ATP induced hyperpolarization was due to the augmentation of the potassium conductance probably through Ca(2+)-activated K+ channels sensitive to charybdotoxin but not to mamba venom. The interaction previously described between charybdotoxin and dendrotoxin (the main toxin of mamba venom) was not observed in our case.     

Water permeation in Madin-Darby canine kidney cells is modulated by membrane fluidity.

Simultaneous determinations of water and antipyrine permeations in monolayers of Madin-Darby canine kidney (MDCK) cells grown on a permeant support were done to study the relationships between water transport and membrane fluidity in these epithelial cells. The changes in permeation of the lipophilic non-electrolyte antipyrine were used to probe the modifications in membrane fluidity. In controls, the apparent diffusional permeability coefficient for water (PDw) was three times higher than the antipyrine's one, PDAp (4.2.10(-5) vs. 1.4.10(-5) cm s-1). Addition of vasopressin or dibutyryl cyclic AMP to the monolayers induced a biphasic increase in antipyrine permeation with peak values at t = 2 min, 3-4-fold that of controls. Variations in water permeation were of similar amplitude and obeyed the same time course, leaving the water to antipyrine permeation ratios unchanged. Compound H7, an inhibitor of protein kinases, blunted the increase in permeation for both antipyrine and water. Finally, addition of the fluidizing agent benzyl alcohol to the monolayers resulted in a parallel increase in PDAp and PDw. These results suggest that the physical state of membrane lipids may control water permeation in MDCK cells.     

Enhancement of cell-cell contact by claudin-4 in renal epithelial Madin-Darby canine kidney cells

Claudin-4 regulates ion permeability via a paracellular pathway in renal epithelial cells, but its other physiological functions have not been examined. We found that hyperosmotic stress increases claudin-4 expression in Madin-Darby canine kidney cells. Here, we examined whether claudin-4 affects cell motility, cell association, and the intracellular distribution of endogenous junctional proteins. Doxycycline-inducible expression of claudin-4 did not change endogenous levels of claudin-1, claudin-2, claudin-3, occludin, E-cadherin, and ZO-1. Claudin-4 overexpression increased cell association and decreased cell migration without affecting cell proliferation. Doxycycline did not change cell junctional protein levels, cell association or cell migration in mock-transfected cells. The insolubility of claudin-1 and -3 in Triton X-100 was increased by claudin-4 overexpression, but that of claudin-2, occludin, ZO-1, and E-cadherin was unchanged. Immunocytochemistry showed that claudin-4 overexpression increases the accumulation of claudin-1 and -3 in tight junctions (TJs). Furthermore, claudin-4 overexpression increased the association of claudin-4 with claudin-1 and -3. These results suggest that claudin-4 accumulates claudin-1 and -3 in TJs to enhance cell-cell contact in renal tubular epithelial cells.     

Mechanism of activation of K++ channels by minoxidil-sulfate in Madin-Darby canine kidney cells

Summary We studied the mechanism of K⁺⁺ channel activation by minoxidil-sulfate (MxSO₄) in fused Madin-Darby canine kidney (MDCK) cells. Patch-clamp techniques were used to assess single channel activity, and fluorescent dye techniques to monitor cell calcium. A Ca⁺²⁺-dependent inward-rectifying K⁺⁺ channel with slope conductances of 53±3 (negative potential range) and 20±3 pS (positive potential range) was identified. Channel activity is minimal in cell-attached patches. MxSO₄ initiated both transient channel activation and an increase of intracellular Ca⁺²⁺ (from 94.2±9.1 to 475±12.6 nmol/liter). The observation that K⁺⁺ channel activity of excised inside-out patches was detected only at Ca⁺²⁺ concentrations in excess of 10 mol/liter suggests the involvement of additional mechanisms during channel activation by MxSO₄.Transient K⁺⁺ channel activity was also induced in cell-attached patches by 10 mol/liter of the protein kinase C activator 1-oleoyl-2-acetyl-glycerol (OAG). OAG (10 mol/liter in the presence of 1.6 mmol/liter ATP) increased the Ca⁺² sensitivity of the K⁺ channel in inside-out patches significantly by lowering the K _mfor Ca⁺² from 100 mol/liter to 100 nmol/liter. The channel activation by OAG was reversed by the protein kinase inhibitor H8. Staurosporine, a PKC inhibitor, blocked the effect of MxSO₄ on K⁺ channel activation. We conclude that MxSO₄-induced K⁺ channel activity is mediated by the synergistic effects of an increase in intracellular Ca⁺² and a PKC-mediated enhancement of the K⁺ channel's sensitivity to Ca⁺².A. Schwab was recipient of a Feodor-Lynen-Fellowship from the Alexander von Humboldt-Stiftung. This work was supported by NIH grant DK 17433. The authors thank Nikon Instruments Partners in Research Program for their support and generous use of equipment during the course of this study. Minoxidil-sulfate was kindly provided by Upjohn, Kalamazoo, MI.     

The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium

Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4- nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.