GLUTAMATE TRANSPORT AND NOT CELLULAR CONTENT MODULATES PARACELLULAR PERMEABILITY IN LLC-PK1-F+ CELLS

Uptake of glutamate modulates two cellular processes: 1) glutamine flu x through the cellular glutaminase (GA) and 2) paracellular permeabili ty (PP). Because both responses are the result of a decreased glutamat e uptake, the present study was designed to determine whether the tran sport step or resulting fall in cellular glutamate modulates PP. To do so, advantage was taken of the ability of D-glutamate to competitivel y displace the natural L-isomer yet maintain transporter activity at o r even above that normally occurring with L-glutamate. As a consequenc e cellular L-glutamate would fall while transporter fluxes remained. A ccordingly, LLC-PK1-F+ cells were grown to confluent monolayers on por ous supports in Dulbecco's modified Eagle's medium containing 50 mu M L-glutamate and 1.8 mM: L-glutamine with and without 1 mM D-glutamate. After a 90-min exposure to D-glutamate monolayer, L-glutamate content had fallen 38%. D-Glutamate was transported in place of the L-isomer as evidenced by the accumulation of L-glutamate in the media and uptak e of the D-isomer. Although GA activation occurs as the result of the fall in cellular L-glutamate, PP did not increase; in fact, it slightl y decreased as evidenced by an increased electrical resistance (from 1 80 +/- 12 to 210 +/- 10 Ohm . cm(2), P < 0.02) and reduction in L-[C-1 4]glucose permeability (2.72 +/- 0.75 to 2.28 +/- 0.37%, P = 0.10). Th us glutamate transporter activity and associated ionic fluxes rather t han the fall in cellular glutamate and GA activation appear to play th e critical role in modulating PP.