CAMP MODULATES TRANSEPITHELIAL RESISTANCE RESPONSE OF LLC-PK1 RENAL EPITHELIA TO TUMOR-NECROSIS-FACTOR

For ''leaky'' epithelia the transepithelial resistance (R(t)) is an el ectrophysiological measure of the paracellular pathway within the epit helial barrier. The R(t) across a monolayer of LLC-PK1 porcine renal e pithelial cells is specifically an inverse measure of paracellular tra nsepithelial permeability and displays a multiphasic and reversible re sponse to the cytokine tumor necrosis, factor-alpha (TNF). The R(t) re sponse to TNF can be inhibited by the nonhydrolyzable adenosine 3',5'- cyclic monophosphate (cAMP) analogue, dibutyryl-cAMP. In addition, act ivation of adenylate cyclase (forskolin) or inhibition of phosphodiest erase (3-isobutyl-1-methylxanthine, Ro-20-1724, and pentoxifylline), e ach of which have been reported to elevate cellular cAMP levels, also inhibited the R(t) response to TNF. Incubation of the LLC-PK1 cell she et with N-[2(methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibito r of cAMP-dependent protein kinase (PKA), potentiated the R(t) respons e to TNF. The R(t) response to TNF was completely prevented by preincu bation of the cultures with cholera toxin, whereas pertussis toxin pre treatment had a slight but significant potentiating effect on the resp onse. Pretreatment with cholera toxin was associated with an similar t o 18-fold elevation in cAMP levels in both control and TNF-treated cul tures. Measurements of cellular cAMP content at selected intervals aft er TNF administration showed a significant elevation (P < 0.01) of 140 % above time-matched controls at 1 h after the administration of TNF t o the cell sheet. The level of cAMP then declined to approximate contr ol level within 2.5 h of TNF administration. Taken together, our data support a role for cAMP and its dependent protein kinase (PKA) as nega tive modulators within the signal transduction mechanisms underlying t he regulation of transepithelial permeability by TNF.