Glutamate transport and not cellular content modulates paracellular permeability in LLC-PK1-F+ cells

Uptake of glutamate modulates two cellular processes: 1) glutamine flux through the cellular glutaminase (GA) and 2) paracellular permeability (PP). Because both responses are the result of a decreased glutamate uptake, the present study was designed to determine whether the transport step or resulting fall in cellular glutamate modulates PP. To do so, advantage was taken of the ability of D-glutamate to competitively displace the natural L-isomer yet maintain transporter activity at or even above that normally occurring with L-glutamate. As a consequence cellular L-glutamate would fall while transporter fluxes remained. Accordingly, LLC-PK1-F+ cells were grown to confluent monolayers on porous supports in Dulbecco's modified Eagle's medium containing 50 microM 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 uptake 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 slightly decreased as evidenced by an increased electrical resistance (from 180 +/- 12 to 210 +/- 10 omega x cm2, P < 0.02) and reduction in L-[(14)C]glucose permeability (2.72 +/- 0.75 to 2.28 +/- 0.37%, P = 0.10). Thus glutamate transporter activity and associated ionic fluxes rather than the fall in cellular glutamate and GA activation appear to play the critical role in modulating PP.