Downregulation of cell-to-cell communication by the viralsrc gene is blocked by TMB-8 and recovery of communication is blocked by vanadate

Abstract

The viral src gene downregulates junctional communication, closing cell-to-cell membrane channels presumably by way of the phosphoinositide signal route. We show that TMB-8 [8-N, N-(diethylamino) octyl-3,4,5-trimethoxybenzoate] counteracts this downregulation in cells transformed by temperature-sensitive mutant Rous sarcoma virus: TMB-8 (36-72 microM) raises junctional permeability when applied during activity of src protein kinase, i.e., at steady permissive temperature; and TMB-8 inhibits the fall of junctional permeability, when the activity of src protein kinase gets turned on. TMB-8 also (reversibly) inhibits the growth of the cells at permissive temperature and reverses the morphological changes associated with transformation. The morphological reversal lags several hours behind the junctional-permeability reversal. Communication recovers within a few minutes when the activity of the src protein kinase is turned off (in absence of TMB-8). Sodium orthovanadate (20 microM) prevents this recovery, but it has no major effect on junctional permeability on its own. We discuss possible modes of action of these agents on critical stages of the signal route, related to intracellular Ca2+ and protein kinase C.

Vanadium compounds promote the induction of morphological transformation of hamster embryo cells with no effect on gap junctional cell communication

“…Vanadate has previously been found to block the increase in intercellular communication after turning off a temperature sensitive src gene. (Rose et aL, 1986), but not to prevent the reversal of diacylglycerol or TPA induced inhibition of gap junctional permeability in rat liver epithelial cells (Yada et al, 1985). This difference may be caused by the fact that the inhibitory effect of vanadate is specific for tyrosine protein phosphatases.…”

Ca2+-independent form of protein kinase C may regulate Na+ transport across frog skin

“…Therefore, increasing the cytosolic Ca 2+ activity in frog skin epithelium above its baseline value [215 -+ 39 nM ] is unlikely to affect PKC stimulation of Na* transport. On the other hand, large reductions in c'~a might possibly limit the ability of PKC to phosphorylate critical modulating sites (Kojima, Kojima & Rasmussen, 1985;Sawamura, 1985;Rose, Yada & Loewenstein, 1986: Pershadsingh, Gale & McDonald, 1987. We have attempted to reduce cytosolic Ca 2+ activity in two ways: by decreasing the rate of Ca 2+ entry from the external media (with Co 2+ or Cd2-), and by lowering the rate of Ca 2" release from the endoplasmic reticulum (with TMB-8).…”

“…We have also tried to decrease a~ by inhibiting the release of Ca 2+ from the endoplasmic reticulum (Streb et al, 1984). Mobilization of intracellular Ca 2+ has been blocked in other preparations with TMB-8 (Chiou & Malagodi, 1975;Mix, Dinerstein & Villereal, 1984;Sawamura, 1985;Yada, Rose & Loewenstein, 1985;Rose et al, 1986). In these experiments, we have used only a 70-/XM concentration.…”

“…In these experiments, we have used only a 70-/XM concentration. As discussed by Rose et al (1986), at significantly higher concentrations TMB-8 may have a direct inhibitory effect on PKC (Kojima et al, 1985;Sawamura, 1985). At this concentration, TMB-8 had no significant effect on baseline Isc, when added to the external solutions bathing the experimental areas of five skins (Table 5).…”

“…To minimize this possibility, we have followed the approach of Rose et al (1986) in not exceeding a bath concentration of 70/.LM.…”