Diacylglycerol downregulates junctional membrane permeability. TMB-8 blocks this effect

We tested the question whether junctional cell-to-cell communication is regulated by the diacylglycerol branch of the phosphoinositide transmembrane signal pathway. Cultured epithelial rat liver cells were treated with the synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol, while their junctional permeability was probed with the microinjected 443-dalton fluorescent tracer Lucifer Yellow. The treatment reduced junctional permeability (without affecting Lucifer permeability of nonjunctional cell membrane). The effect was dose dependent, with a threshold of about 25 micrograms diacylglycerol/ml in sparse cultures and about 50 micrograms/ml in confluent cultures. The reduction of junctional permeability began within 3 min of diacylglycerol application, peaked within 20 min, and reversed spontaneously within 90 min. The phorbol ester TPA mimicked the diacylglycerol effect, but the (spontaneous) reversal was slower. We propose that cell-to-cell communication is under dual physiological control: an up-regulatory one, as exerted by the cyclic AMP signal route (Loewenstein, W.R., 1985, Biochem. Soc. Symp. London, 50: 43-58), and a downregulatory one, by the diacylglycerol signal route. TMB-8 (54-70 microM)--a blocker of intracellular Ca2+ mobilization--impeded the diacylglycerol action on junctional permeability. It prevented the effect of low diacylglycerol doses completely and it markedly reduced the effect of high doses. (It also counteracted the effect of TPA.) Ca2+ thus emerges as a possible candidate for a role in the junctional downregulation by the diacylglycerol signal route. We tentatively advance two models. In one, leaning closely on the Calcium Hypothesis of cell-to-cell channel regulation (Loewenstein, W.R., 1966, Ann. N.Y. Acad. Sci. 137:441-472), Ca2+ mediates the action of the route on the channel. In the other, Ca2+ acts farther removed from the channel, on protein kinase C. Calmidazolium (5-10 microM)--an inhibitor of calmodulinactivated proteins--did not prevent the diacylglycerol-induced reduction of junctional permeability. Nor did sodium orthovanadate (25 or 50 microM)--an inhibitor of tyrosyl phosphatase--prevent the reversal of diacylglycerol-induced (or TPA-induced) reduction of junctional permeability.