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 delta.cm2; m +/- SD) and restricted the passage to 14C-sucrose, 3H-inulin, 14C-dextran (70 kDa), and 125I-polyvinyl pyrrolidone (125I-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 14C-sucrose and 3H-inulin but had no effect on the permeability of 125I-PVP. The permeabilities of 14C-sucrose and of 14C-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 125I-PVP. In addition, the permeability of 14C-sucrose was unchanged by a phorbol ester that does not activate PKC. Increasing intracellular calcium with ionomycin had no effect on the permeability of 14C-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 monolayer that excludes 125I-PVP (360 kDa) but readily accepts 14C-sucrose and 3H-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 simulating adenylate cyclase and opened by stimulating protein kinase C.
Department of Medicine, Columbia University, New York, New York 10032.
J A
JA
Oliver
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001011:Aorta; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003911:Dextrans / Q000378:metabolism; D004553:Electric Conductivity; D004730:Endothelium, Vascular / Q000201:enzymology / Q000502:physiology*; D007365:Intercellular Junctions / Q000502:physiology*; D007444:Inulin / Q000378:metabolism; D007700:Kinetics; D011205:Povidone / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism
D003911:Dextrans; D015124:8-Bromo Cyclic Adenosine Monophosphate; D013395:Sucrose; D007444:Inulin; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D011205:Povidone; D002118:Calcium