ALTERATIONS IN CELL CHOLESTEROL CONTENT MODULATE CA2-INDUCED TIGHT JUNCTION ASSEMBLY BY MDCK CELLS()

Transepithelial electrical resistance (TER), a measure of tight juncti on (TJ) barrier function, develops more rapidly and reaches higher val ues after preincubation of MDCK cells for 24 h with 2 mu M Lovastatin (lova), an inhibitor of 3-hydroxy- 3-methylglutaryl-CoA reductase. Whi le this effect was attributed to a 30% fall in cholesterol (CH), possi ble effects of lova on the supply of prenyl group precursors could not be excluded. In the current study, strategies were devised to examine effects on TER of agents that simultaneously lower CH and increase th e flux of intermediates through the CH biosynthetic pathway. Zaragozic acid, 20 mu M, an inhibitor of squalene synthase known to increase th e synthesis of isoprenoids and levels of prenylated proteins, lowered cell CH by 30% after 24 h, while accelerating development of TER in th e same manner as lova. TER was also enhanced, despite a 23% increase i n the rate of [H-3]acetate incorporation into CH, when total CH was re duced by 45% during a 2-h incubation with 2 mM methyl beta-cyclodextri n (MBCD), an agent that stimulates CH efflux from cells. The fact that the rate of TER development was diminished when cell CH content was e levated by incubation with a complex of CH and MBCD is further evidenc e that this sterol modulates development of the epithelial barrier. Ce ll associated CH derived from the complex was similar to endogenous CH with respect to its accessibility to cholesterol oxidase. Lova's effe ct on TER was diminished when 5 mu g/mL of CH was added to the medium during the last 11 h of incubation with lova.