Robert D. Lynch

RAPID REDUCTION OF MDCK CELL CHOLESTEROL BY METHYL-BETA -CYCLODEXTRIN ALTERS STEADY STATE TRANSEPITHELIAL ELECTRICAL RESISTANCE

The role of plasma membrane lipids in regulating the passage of ions and other solutes through the paracellular pathway remains controversial. In this study we explore the contribution of cholesterol (CH) in maintaining the barrier function of an epithelial cell line using the CH-solubilizing agent methyl beta-cyclodextrin (MBCD) to stimulate CH efflux. Inclusion of 20 mM MBCD in both apical and basolateral media reduced CH levels by 70-80% with no significant effect on cell viability. Most of that decrease occurred during the first 30 min of incubation. Recovery of CH content to initial values was nearly complete 22 h after removal of MBCD. Within 30 min of adding MBCD to the culture medium, transepithelial electrical resistance (TER) increased, reaching maximum values 30-40% above controls. This early rise in TER occurred when MBCD was added to either side of the monolayer. The later rapid decline in TER was observed only when MBCD bathed the basolateral surface from which, coincidentally, CH efflux was most rapid. Freeze fracture replicas and transmission electron microscopy of monolayers exposed to MBCD for only 30 min revealed no increase in either the average tight junction (TJ) strand number or the dimensions of the lateral intercellular space. There was a statistically significant increase in the number of TJ particles associated with the E fracture face at this time. This raises the interesting possibility that during CH efflux there is a change in the interaction between TJ particles and underlying cytoskeletal elements. There was no change in staining for occludin and ZO-1. After exposing the basolateral surface to MBCD for 2 h, TER fell below control levels. The accompanying increase in mannitol flux suggests strongly that the decrease in TER resulted from an increase in the permeability of the paracellular and not the transcellular pathway. A decrease in immuno-staining for occludin and ZO-1 at TJs, a striking accumulation of actin at tri-cellular areas as well as a decline in the number of parallel strands, as seen in freeze fracture replicas, suggest that changes in cytoskeletal organization during long incubations with MBCD had physically disrupted the TJ network. Data are presented which suggest that the observed changes in paracellular permeability during CH efflux may be related to increased levels of lipid-derived second messengers, some of which may trigger changes in the phosphorylation status of TJ proteins.

Formation and disappearance of triglyceride droplets in strain L fibroblasts. An electron microscopic study.

Abstract Formation of lipid droplets in strain L fibroblasts exposed to either oleic or linolenic acid for 5 min to 18 h was studied by ultrastructural morphometry and triglyceride analysis. Esterification of OsO4 to double bonds of unsaturated fatty acids was used to characterize morphologically the resulting triglyceride droplets. Free cytoplasmic lipid droplets were formed within 5 min of adding exogenous, fatty acid. Of these, trilinolenin droplets were smaller (1.6 × 10−2 μm3) than triolein droplets (4.7 × 10−2 μm3) but by 18 h both types of droplets had a similar mean volume (46.5 × 10−2 μm3). Initial trilinolenin droplets stained dark grey. After 40 min of linolenic acid uptake, trilinolenin droplets stained dark grey. After 40 min of linolenic acid uptake, trilinolenin droplets stained black with OsO4, corresponding to 2.8 μg trilinolenin per 106 cells. Triolein droplets stained pale grey throughout. Lipid droplets were bounded by a thin layer of osmiophilic material which lacked the bilaminar structure and was thinner than a unit membrane. Triolein droplets disappeared more rapidly than trilinolenin droplets after removal of exogenous fatty acids. A disappearance rate more rapid than could be accounted for by cell division, together with a diminution in mean droplet volume indicated the possible presence of a lipase. During the disappearance phase, lipid droplets remained free in the cytoplasm and were not associated with lysomes. By 48 h after removal of exogenous linolenic acid, trilinolenin droplets stained dark grey rather than black, corresponding to a trilinolenin content of 2.4 μg/106 cells. Strain L fibroblasts given the two fatty acids in sequence, formed droplets of intermediate staining intensity. All droplets within any given cell stained uniformly and to the same degree, indicating an exchange between droplet triglycerides and intracellular free fatty acids. Only after short sequential exposures to each fatty acid could two distinct populations of droplets be discerned within the same cell. However, this was a rare occurrence and more frequently all droplets were stained with the same intensity.

Cholesterol efflux stimulates metalloproteinase-mediated cleavage of occludin and release of extracellular membrane particles containing its C-terminal fragments.

That changes in membrane lipid composition alter the barrier function of tight junctions illustrates the importance of the interactions between tetraspan integral tight junction proteins and lipids of the plasma membrane. Application of methyl-beta-cyclodextrin to both apical and basolateral surfaces of MDCK cell monolayers for 2 h, results in an approximately 80% decrease in cell cholesterol, a fall in transepithelial electrical resistance, and a 30% reduction in cell content of occludin, with a smaller reduction in levels of claudins-2, -3, and -7. There were negligible changes in levels of actin and the two non-tight junction membrane proteins GP-135 and caveolin-1. While in untreated control cells breakdown of occludin, and probably other tight junction proteins, is mediated by intracellular proteolysis, our current data suggest an alternative pathway whereby in a cholesterol-depleted membrane, levels of tight junction proteins are decreased via direct release into the intercellular space as components of membrane-bound particles. Occludin, along with two of its degradation products and several claudins, increases in the basolateral medium after incubation with methyl-beta-cyclodextrin for 30 min. In contrast caveolin-1 is detected only in the apical medium after adding methyl-beta-cyclodextrin. Release of occludin and its proteolytic fragments continues even after removal of methyl-beta-cyclodextrin. Sedimentation and ultrastructural studies indicate that the extracellular tight junction proteins are associated with the membrane-bound particles that accumulate between adjacent cells. Disruption of the actin filament network by cytochalasin D did not diminish methyl-beta-cyclodextrin-induced release of tight junction proteins into the medium, suggesting that the mechanism underlying their formation is not actin-dependent. The 41- and 48-kDa C-terminal occludin fragments formed during cholesterol depletion result from the action of a GM6001-sensitive metalloproteinase(s) at some point in the path leading to release of the membrane particles.

Uptake of rac-glycerol 1-oleate and its utilization for glycerolipid synthesis by strain L fibroblasts

Abstract 1. 1. Strain L fibroblasts incubated with rac -glycerol 1-oleate in a medium containing 10% horse serum formed cytoplasmic lipid particles composed primarily of glycerol trioleate. This accumulation could be accounted for by the utilization of free oleic acid rapidly liberated into the medium from the added rac -glycerol 1-oleate by an horse serum esterase. 2. 2. When 5 μM paraoxon was added to the medium to inhibit this esterase, triacylglycerol synthesized by the cells from doubly labeled rac -glycerol 1-oleate derived at least 70% of its glycerol from exogenous substrate. The comparable value for cells incubated with a mixture of oleic acid and glycerol was 17%. Similar results were observed for the cellular phosphoglycerides. 3. 3. When cellular rac -glycerol 1-oleate levels were elevated, exogenous free oleic acid was not incorporated into glycerolipids via pathways utilizing endogenously generated acyl acceptors. 4. 4. Although pathways involving the initial phosphorylation of rac -glycerol 1-oleate can not be excluded, it appears that a fraction of this substrate is incorporated into glycerolipid via , a 1,3-diacylglycerol intermediate. 5. 5. Some characteristics of large lipid droplets formed in cells incubated with rac -glycerol 1-oleate are described.

The hydrolysis of rac-glycerol 1-monodecanoate by serum butyryl cholinesterase☆

Butyryl cholinesterase from horse and human sera catalyzed the hydrolysis of monoacylglycerols containing fatty acids varying in chain length from 8 to 12 carbons; maximum activity was obtained with rac-glycerol 1-monodecanoate as substrate. Neither the triacylglycerols of these fatty acids nor the monoacylglycerols of longer chain length fatty acids were hydrolyzed at measurable rates in the system used. The enzyme was eserine sensitive and indistinguishable from butyryl cholinesterase as judged by purification, response to the several inhibitors tested, and heat inactivation. Data from mixed substrate experiments suggest a possible effector role for butyryl choline in accelerating the rate of rac-glycerol 1-monodecanoate hydrolysis. Fatty acid released during the course of rac-glycerol 1-monodecanoate hydrolysis may irreversibly inactivate the enzyme.