ADENYLATE-CYCLASE AND PROTEIN-KINASE-C MEDIATE OPPOSITE ACTIONS ON ENDOTHELIAL JUNCTIONS

To determine whether the endothelial paracellular pathway is regulated, the effect of intracellular messengers on the transendothelial flux of inert radiolabeled molecules of diverse molecular size was examined in bovine aortic endothelial cells grown on collagen-coated filters. The endothelial monolayers showed a modest electrical resistance (21 +/- 10-OMEGA.cm2;m +/- SD) and restricted the passage to C-14-sucrose, H-3-inulin, C-14-dextran (70 kDa), and I-125-polyvinyl pyrrolidone (I-125-PVP, 360 kDa) according to their molecular mass. 8-Bromoadenosine 3'-5' cyclic monophosphate (8-Br-cAMP) reduced by more than 30% the permeability coefficients of C-14-sucrose and H-3-inulin but had no effect on the permeability of I-125-PVP. The permeabilities of C-14-sucrose and of C-14-inulin were strikingly increased by activating protein kinase C(PKC) by phorbol 12-myristate 13-acetate or sn-1,2-dioctanoly-glycerol whereas the latter compound had no effect on the permeability of I-125-PVP. In addition, the permeability of C-14-sucrose was unchanged by a phorbol ester that does not activate PKC. Increasing intracellular calcium with ionomycin had not effect on the permeability of C-14-sucrose. None of these maneuvers significantly affected the protein content of the endothelial monolayers. The results indicate that 8-Br-cAMP and PKC activators modulate a pathway across the endothelial monolayers that excludes I-125-PVP (360 kDa) but readily accepts C-14-sucrose and H-3-inulin, suggesting that this pathway is the paracellular pathway. Hence, low molecular weight molecules such as sucrose and inulin can be used to probe the behavior of the paracellular pathway of endothelial monolayers grown in vitro. The results also indicate that the paracellular pathway in endothelium is regulated and suggest that endothelial junctions can be closed by stimulating adenylate cyclase and opened by stimulating protein kinase C.