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Biomedical subjects

J M Mullin

Publications and source records attributed to J M Mullin.

At least 55 records · Page 3Linked to original sources
PubMed1990

Effect of tumor necrosis factor on epithelial tight junctions and transepithelial permeability.

The serum factor inducing hemorrhagic necrosis of transplantable tumors [tumor necrosis factor (TNF)], and the macrophage hormone associated with cachexia in cancer and certain infectious diseases [cachectin] are known to be the same protein. Because an association may exist between TNF and the cachectic state, we wished to examine the effect of TNF on the permeability of epithelial barriers. We present data showing that TNF affects the tight junctional region between epithelial cells, lowering the transepithelial resistance and potential difference, and increasing the flow of solute between cells and across the epithelium. These effects are dose dependent, rapidly reversible, and inhibited by a monoclonal antibody to TNF-alpha. We suggest that the release of TNF at various sites throughout the body will cause a general breakdown in the barrier function of an epithelial cell sheet. This may relate to the cachexia observed in certain disease states. These findings are similar to our earlier published effects of phorbol esters and diacylglycerols on tight junctions, suggesting that protein kinase C activation may be involved.

Animals↗
PubMed1990

The effects of teleocidin and aplysiatoxin tumor promoters on epithelial tight junctions and transepithelial permeability: comparison to phorbol esters.

Control of transepithelial permeability by regulation of tight junctions is exerted by the non-phorbol ester tumor promoters, teleocidin and aplysiatoxin. Similar to the phorbol esters, tetradecanoylphorbol-13-acetate (TPA) and phorbol dibutyrate (PDBU), both teleocidin and aplysiatoxin cause a reversible decrease of transepithelial voltage and transepithelial resistance across LLC-PK1 renal epithelial cell sheets at concentrations as low as 10(-8) M. These compounds are effective from either side of these polar epithelial cells, i.e. apical or basolateral. The decreases in transepithelial gradients and resistance are paralleled by a rise in the transepithelial (paracellular) flux of D-mannitol between the cells (through the tight junctions). These four tumor promoters, TPA, PDBU, teleocidin and aplysiatoxin, are all known protein kinase C activators, and support the case for protein-kinase-C-mediated control of tight junctional permeability.

Carcinogens↗
PubMed1989

Na+-independent sugar transport by cultured renal (LLC-PK1) epithelial cells.

The LLC-PK1 cell line has been well characterized concerning its proximal tubule-like Na+-dependent active sugar transporter in the apical membrane. In this study, we investigated the uptake of the glucose analogue, 2-deoxy-D-glucose (2DOG), a paradigm substrate for the facilitated diffusion, Na+-independent sugar transporter in the renal basolateral membrane. The uptake of 0.1 mM 2-[14C]DOG by confluent LLC-PK1 cell sheets at 25 degrees C is linear at least to 10 min, at which time greater than 90% of intracellular radioactivity is 2DOG phosphate. The uptake of this analogue by LLC-PK1 cells is Na+ independent, and the transporter appears to be localized to the basolateral cell membrane. Phlorizin is a much less effective inhibitor than its aglycon, phloretin. Cytochalasin B is also an effective inhibitor, but it causes morphological changes in the cells at concentrations required to inhibit transport. Specificity studies indicate that this transport system requires a hexose with a free hydroxyl at C-1, and that the hydroxyls at C-3 and C-4 be preferably in the equatorial position. Glucose starvation causes an increased rate of 2DOG uptake. Subconfluent (cycling) cultures of LLC-PK1 cells have a threefold greater rate of 2DOG uptake than that seen in confluent (noncycling) LLC-PK1 cells.

Biological Transport↗
PubMed1989

Isolation of mutant renal (LLC-PK1) epithelia defective in basolateral, Na(+)-independent glucose transport.

To obtain mutant renal epithelia defective in the uptake of 2-deoxyglucose (Na(+)-independent glucose transport), LLC-PK1 renal epithelial cells were subjected to a combination of DNA alkylation, tritium-suicide with 2-[3H]deoxyglucose, and replica plating. One of the mutant sublines obtained, LLC-PK1M-7A, possesses only 40% of the 2-deoxyglucose uptake rate of the parent line, LLC-PK1M, and this defect is stable over at least 30 population doublings. Initial rate of 3-O-methylglucose transport into these mutant cells is only 20% of the parental rate, and efflux of 3-O-methylglucose from the mutant cells is correspondingly low. Glucose metabolism in the mutant cells does not appear to be altered, nor is free glucose accumulated in the cells against a concentration gradient. The uptake rate of L-leucine is the same in both mutant and parent, whereas the (Na(+)-dependent) uptake of alpha-methyl-D-glucoside and methylaminoisobutyric acid is greater in the mutant than the parent. The population doubling times of LLC-PK1M and LLC-PK1M-7A cultures are similar. LLC-PK1M-7A cell morphology is similar to LLC-PK1M when cultures are subconfluent, but on reaching confluence, LLC-PK1M-7A appear larger than LLC-PK1M cells. Their measured cell volume is then 150% of the volume of the parent cells. Future studies of the LLC-PK1M-7A mutant, and acquisition of additional mutant sublines, should elucidate the roles of transepithelial glucose transport in proximal tubular cell physiology.

3-O-Methylglucose↗
PubMed1988

Epidermal growth factor-induced mitogenesis in kidney epithelial cells (LLC-PK1).

The growth kinetics of LLC-PK1 renal epithelia is examined, particularly relating to epidermal growth factor (EGF)-induced mitogenesis in growth-arrested confluent cultures. LLC-PK1 cell proliferation is shown to be density dependent. After reaching confluence, cell number remains near constant, and addition of fresh serum-containing medium does not elicit significant increase in cell number. EGF and 12-O-tetradecanoyl-phorbol-13-acetate do however induce a marked increase in cell number, although each with different kinetics. EGF-induced mitogenesis in LLC-PK1 cells has the following characteristics: (a) DNA synthesis peaks in a defined window 10-12 h after EGF administration; (b) 10 nM EGF elicits a maximal response; (c) 10 nM EGF requires only a 30-s exposure to these cells to induce a maximal mitogenic response; (d) exposure of the basolateral membrane of these polar cells to 10 nM EGF is necessary for mitogenesis, exposure of EGF to the apical surface being ineffective. This aspect of polarity in growth factor-induced mitogenesis is a phenomenon which cannot be addressed with nonpolar fibroblastic cell cultures.

Cells, Cultured↗
PubMed1988

Effects of diacylglycerols on LLC-PK1 renal epithelia: similarity to phorbol ester tumor promoters.

As previously shown using phorbol ester tumor promoters (see Mullin and O'Brien: Am. J. Physiol., 251:C597-C602, 1986), diacylglycerols induce leakiness in LLC-PK1 renal epithelial tight junctions. The similarity between phorbol ester and diacylglycerol action includes effects on 1) cell morphology, 2) dome formation, 3) transepithelial resistance and potential difference, 4) transepithelial flux of D-mannitol, and 5) mitogenesis. Four diacylglycerols have been tested: 1,2-dioctanoylglycerol; 1,2-dicaprylglycerol; 1,2-dioleoylglycerol; and 1-oleoyl-2-acetoyl-sn-3-glycerol. Their relative effectiveness depended upon the phenomenon being observed. Unlike phorbol esters, diacylglycerol effects were reversible within hours at 37 degrees C in the continued presence of diacylglycerol, and effects were more pronounced when cell sheets were exposed to diacylglycerols from the basolateral cell surface. Overall, these findings indicate that previous results with phorbol esters may be attributed to the protein kinase C signal transduction system, and this system may therefore exert a role in transepithelial permeability.

Cell Adhesion↗
PubMed1987

The phorbol ester, TPA, increases transepithelial epidermal growth factor flux.

Exposure of cultured kidney epithelial (LLC-PK1) cell sheets to 10(-7) M TPA, a potent tumor promoter and activator of protein kinase C, initiates within minutes a drop in the transepithelial voltage across these sheets. This fall in potential difference correlates with an over 40-fold increase in the transepithelial flux of 1 mM D-mannitol, suggesting that the intercellular junctions have become leaky. Dual labeling experiments with 1 mM D-[14C]mannitol and 10 nM 125I-EGF show that after promoter treatment, a 7-fold increase in net 125I flux accompanies the increase in mannitol flux. Gel filtration and gel electrophoresis indicate that for control cell sheets only 15% of the transited 125I is actually EGF, whereas with TPA-treated cell sheets, 60% of the 125I which passed across is EGF. These percentages permitted determination of actual EGF flux values, and show that TPA treatment engenders a 35-fold increase in transepithelial EGF flux. Diacylglycerols also increase the junctional permeability of these cells, thereby suggesting the involvement of protein kinase C.

Cell Line↗
PubMed1987

Spontaneous reversal of polarity of the voltage across LLC-PK1 renal epithelial cell sheets.

While sterilely monitoring transepithelial voltage (potential difference) across LLC-PK cell sheets over a 24-hr period, we noted that the apical-negative, transepithelial voltage, a key property of the LLC-PK1 renal epithelial cell line, reverses polarity to become apical-positive. This spontaneous change of polarity of electrical potential difference (PD) across LLC-PK1 cell sheets cultured on permeable filters was observed to occur approximately 12 hr after refeeding. Unlike the apical (luminal)-negative PD, the apical-positive PD was insensitive to phlorizin and ouabain. Both were insensitive to the diuretics amiloride, furosemide, and 4-acetamido-4-isothiocynato-stilbene-2,2-disulfonic acid (SITS). A pH gradient existed across apical-positive cell sheets (apical medium more acidic by 0.3 units) but an osmotic gradient did not. Unlike the temperature-sensitive apical-negative PD, the apical positive PD was unaffected by brief exposure to 4 degrees C temperature. Junctional disruptive agents such as the tumor promotor, TPA, dissipated both types of PD with similar time courses. The formation of the apical-positive PD correlated in time with apical glucose levels falling below the reported Km of the Na+-sugar contransporter. A high glycolytic rate per se may not be essential for this PD polarity reversal since the reversal could occur in glucose-free medium with a normal time course and magnitude. The lysis with time of floating cells with consequent release of KCl into the apical compartment was also considered as a possible cause of the polarity reversal, but the turnover of even 2 X 10(6) cells in 12 hr was found not to raise apical KCl sufficiently to produce the polarity shift. Although a significant K+ gradient did not exist across cell sheets with apical-positive PD values, a sizable gradient of Cl- did exist, directed apical to basolateral. This gradient, coupled with anion-selective tight junctions, should contribute to the observed apical positive voltage. The voltage polarity shift seen in these cell cultures with time is not unlike the polarity shift occurring in the renal proximal convoluted tubule, with distance from the glomerulus.

Amino Acids↗
PubMed1987

Isolation and characterization of a glycine transport mutant in an established mammalian cell line, CHO(PEOT/1).

Glycine uptake in CHO(PEOT/1) cells is mediated by at least three systems, of which two have been identified and partially characterized in this study: (1) a low affinity "A" system that transports a number of small neutral amino acids including glycine and methylaminoisobutyric acid (MeAIB), and (2) a high-affinity system, specific for glycine and sarcosine. By a combination of tritium suicide and replica plating, we have isolated a mutant (CHY-3) with a 47% decrease in glycine transport at the standard test concentration of 2.5 microM. Uptake studies with radioactive glycine, MeAIB, and sarcosine revealed that the mutant lacks the glycine-sarcosine system, but has undergone a compensatory 30-50% increase in the A system. Thus, there appears to be a regulatory interaction between these two systems for glycine uptake by CHO cells.

Aminoisobutyric Acids↗
PubMed1986

Basal-lateral transport and transcellular flux of methyl alpha-D-glucoside across LLC-PK1 renal epithelial cells.

The characteristics of methyl alpha-D-glucoside transport by the LLC-PK1 cell line are extended by a study of the interaction of this glucose analog with the basal-lateral membrane of these cells: 1 mM methyl alpha-D-glucoside enters LLC-PK1 cells across the basal-lateral membrane 10-times more slowly than when entering across the apical membrane; neither 10 mM glucose nor 10 mM methyl alpha-D-glucoside affect the rate of methyl glucoside uptake at the basal lateral membrane; 0.1 mM phlorizin in the apical hemichamber significantly decreases the rate at which methyl glucoside enter the cell when methyl glucoside is present in the basal-lateral hemichamber; the methyl glucoside transcellular flux ratio, Ja/Jb (apical to basal vs. basal to apical) is 15, whereas Ja/Jb for D-mannitol equals 1; and basal-lateral to apical fluxes (Jb) of mannitol consistently exceed those of methyl glucoside. These results demonstrate that methyl glucoside, unlike glucose, is accumulated within these cells to a high degree because of the limited ability of methyl glucoside to exit the cells by a carrier-mediated pathway. They also raise the important caveat for any studies with glucose (and other low-molecular-weight substrates) by showing that a monosaccharide presented to one surface of these cells will traverse the cell sheet (in part) by the intercellular route and will enter the cell at the unintended cell surface. The ability of the tight junctions of this intercellular route to discriminate between open-chain molecules, such as mannitol, vs. closed ring structures, like methyl glucoside, is also described.

Animals↗
PubMed1986

Effects of tumor promoters on LLC-PK1 renal epithelial tight junctions and transepithelial fluxes.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) irreversibly dissipates the electrical potential difference (PD) across LLC-PK1 renal epithelial cell sheets in a dose-dependent manner. The promoter is equally effective when presented to either cell surface. TPA is also equally effective at dissipating the apical-negative PD or the heretofore undescribed apical-positive PD, both of which can exist spontaneously across this monolayer. Diffusion potentials arising from imposed NaCl gradients across LLC-PK1 cell sheets are likewise reduced by TPA. The non-tumor-promoting parent compound, phorbol, is ineffective, but the more hydrophilic TPA analogue, phorbol 12,13-dibutyrate (PDBU) is as effective as TPA. Unlike TPA, the effects of PDBU are reversible. By observing the (paracellular) fluxes of D-mannitol and polyethylene glycol, these effects on PD are shown to be due to increased permeability of the tight junctions. This effect is immediately reversible once PDBU is removed. These findings are discussed in light of the ramifications of the breakdown of epithelial compartments in vivo and the role this could play in the regulation of epithelial cell growth.

Carcinogens↗
PubMed1982

Anomalous patterns in cultured cell monolayers.

Gridlike patterns of differing cell density were observed in evenly seeded cell monolayers. Such patterns were obtained in five of six cell lines tested, suggesting widespread occurrence. The mechanism appears to involve small, transient temperature changes related to incubator tray structure. The very short time course of appearance of the patterns implicates attachment rather than growth as the critically affected factor. Impaired adhesion or directed sedimentation resulting from thermally induced microcurrents in the medium are the two most likely mechanisms.

Animals↗
PubMed1982

Metabolism of L-lactate by LLC-PK1 renal epithelia.

LLC-PK1 renal epithelial cells do not appreciably synthesize D-glucose or glycogen from L-lactate under the conditions described. [U-14C]L-lactate is metabolized to [14C]L-alanine and [14C]L-glutamate. Synthesis of alanine but not glutamate is decreased by the transaminase inhibitor, aminooxyacetate. LLC-PK1 cells also produce unlabeled urea in the presence of [14C]L-lactate. Evidence for net gluconeogenesis has thus not been obtained for this cell line. This is interesting because the LLC-PK1 cells possess an active sugar transport system, and both active sugar transport and gluconeogenesis are localized together in vivo in the proximal tubule.

Animals↗
PubMed1980

Sugar transport in the LLC-PK1 renal epithelial cell line: similarity to mammalian kidney and the influence of cell density.

Cells of confluent cultures of the established pig renal epithelial line, LLC-PK1, accumulate alpha-methyl-D-glucoside against a concentration gradient. This transport system is strongly inhibited by phlorizin and 6-deoxy-D-glucose, moderately inhibited by phloretin, and only weakly inhibited by 3-0-methyl-D-glucose, paralleling the situation in mammalian kidney. The time courses for the uptake of alpha-methyl-D-glucoside and for the carrier-mediated but passive uptake of 3-0-methyl-D-glucose are identical to those seen in mammalian kidney. Subconfluent cultures of LLC-PK1 cells are unable to accumulate alpha-methyl-D-glucoside, and their transport of this glucose analog is less sensitive to phlorizin inhibition than is the transport system in confluent cultures. Transmission electron micrographs show that cells from subconfluent cultures lack the microvillous surface seen in cells from confluent cultures. Cell density is thus a factor in the occurrence of structural and functional differentiated properties related to transport in these cells.

Animals↗
PubMed1980

Effects of ouabain and ortho vanadate on transport-related properties of the LLC-PK1 renal epithelial cell line.

Uptake of alpha-methyl-D-glucoside (AMG) by LLC-PK1 cells is inhibited by the uncoupler p-trifluoro-methoxyphenyl-hydrazone (FCCP) and by the absence of extracellular Na+, indicating that the transport system is energy- and Na+-dependent. We have previously demonstrated that transport of AMG by LLC-PK1 cells proceeds against a concentration gradient and is phlorizin-sensitive (Mullin et al., '80). Uptake of AMG was also inhibited by ouabain (OUA) but not by ortho-vanadate (VAN). Rubidium uptake also was affected by OUA but not by VAN. VAN, however, caused collapse of the three-dimensional domes of confluent LLC-PK1 monolayers much more rapidly and thoroughly than OUA. Since domes are presumably dependent upon the Na+ pump, yet VAN is not acting on transport-related functions of the OUA-sensitive (Na+ + K+)-ATPase, we hypothesize a direct effect of VAN on the water permeability of these cells. We also suggest that OUA does not act on these cells until domes collapse in normal course, and access of the OUA to the extracellular surface of antiluminal membranes is then achieved.

Animals↗
PubMed1977

Renal sugar transport in the winter flounder. III. Two glucose transport systems.

Teased renal tubules of the winter flounder (Pseudopleuronectes americanus) were employed to investigate the structural requirements for two pathways of D-glucose transport which take place preponderantly across the basal (antiluminal) face of renal cells. 1) An inhibition analysis of the equilibrating, Na-independent and phlorizin-sensitive transport of the nonmetabolizable methyl-alpha-D-glucoside (0.1 and 0.5 mM), with 20 glucose analogs (5 mM), was employed to establish the structural requirements for the substrate-carrier interaction: a (pyranose) ring, oxygen, or F at C1, C2-OH, C3-OH, and C4-OH (all axial, 1C model). Some interaction may also occur at C6-OH. D-Glucose shares this transport system. Hydrogen bonding between the oxygens and the carrier is suggested. 2) The phloretin- and phlorizin-sensitive, ouabain-insensitive transport of D-glucose, 2-deoxy-D-glucose, and D-mannose is associated with considerable phosphorylation. The three sugars mutually compete for a shared transport site. The specificity pattern characterizing the transport system defines the following structural requirements: a (pyranose) ring, a free C1-OH, C3-OH, and C4-OH (both axial) and possibly C6-OH. Hydrogen bonding between the carrier and the oxygens at C3, C4, and C6, and covalent bonding at C1 is suggested.

Animals↗