No abstract

Lysophosphatidic acid (LPA) was shown to be a powerful inhibitor of gap-junctional communication between cultured rat liver WB cells, as determined by the transfer of Lucifer Yellow, with 50% inhibition obtained at about 0.3 microM LPA. Inhibition of communication was rapid (5 min) and was maintained for at least 80 min. After incubation for 3 h with LPA, communication competence was partially restored and dye transfer was refractory to further addition of LPA. Communication in LPA-refractory cells retained sensitivity to inhibition by phorbol ester and by epidermal growth factor (EGF). LPA-induced inhibition was associated with phosphorylation of connexin-43 protein, as detected by slower migration of the protein detected on Western blots, which could be eliminated by incubation of samples with alkaline phosphatase. A close correspondence was observed between the time- and dose-dependency of LPA effects on communication and the induction of mitogen-activated protein kinase (MAP kinase). Activation of both the 42 kDa and 44 kDa subspecies were confirmed by mobility shifts on Western blots using an anti-(MAP kinase R1) (erk 1-III) antibody and by fractionation on Mono Q columns. Cells pretreated with phorbol ester for 24 h were insensitive to phorbol ester inhibition of communication or activation of MAP kinase, but retained their sensitivity to LPA. The results indicate that LPA initiates the activation of protein kinase cascades in WB cells that are probably independent of protein kinase C and identifies connexin-43 as one substrate for the activated kinases.

The contribution of basic fibroblast growth factor to brown adipose tissue (BAT) enlargement during cold acclimation was investigated using rat brown adipocytes in primary culture. After cold exposure (at 5 degrees C) for 28 days, the level of bFGF messenger ribonucleic acid (mRNA) in BAT of cold-acclimated rats was markedly increased with the increase in the BAT weight. In addition, the blood plasma from cold-acclimated rats considerably enhanced the expression of basic fibroblast growth factor mRNA in rat brown adipocytes. Likewise, the blood plasma from cold-acclimated rats significantly stimulated the growth of rat brown adipocyte precursor cells compared with that from warm-acclimated rats, whereas there was no difference of effect between the two blood plasmas on the growth of bovine capillary endothelial cells. Basic fibroblast growth factor, but not platelet-derived growth factor stimulated the growth of brown adipocyte precursor cells. The conditioned medium from brown adipocyte primary culture markedly stimulated the growth of bovine capillary endothelial cells and the effect was inhibited considerably by antibasic fibroblast growth factor antibody. These results suggest that some factors concerned with the growth of brown adipocyte precursor cells are present in the blood plasma from cold-acclimated rats, and that basic fibroblast growth factor produced by brown adipocytes may significantly contribute to BAT enlargement by autocrine mechanisms during cold exposure.

To study the biochemical basis of gap-junctional intercellular communication (GJIC) and its role in tumorigenesis, a mammalian cell expression vector carrying both a rat connexin 43 (Cx43) cDNA and an amplifiable dihydrofolate reductase (DHFR) gene was transfected into the GJIC-deficient rat liver mutant cell line aB1. Two stable transfectants were selected for further amplification of the transfected Cx43 gene by increasing stepwise the concentration of methotrexate (MTX) in the culture medium. The results indicate that GJIC was restored in these two Cx43 cDNA transfectants after they became highly resistant to MTX but not in the control-vector transfectants, in which the DHFR gene was similarly amplified. The amount of Cx43 DNA revealed by Southern blot analysis and the expression of Cx43 gene revealed by northern and western blot analyses were concomitantly increased in the Cx43 cDNA transfectants resistant to high concentrations of MTX. Western blot analysis, using an antipeptide antibody that specifically recognizes Cx43 protein, further revealed that an approximately 46-kDa phosphorylated Cx43 protein that was prominent in the parental GJIC-competent cells was absent in the aB1 cells. This Cx43 protein, however, reappeared in the two Cx43 cDNA transfectants after amplification. After treatment of the membrane proteins with alkaline phosphatase in vitro, the approximately 46- and 44-kDa proteins disappeared, whereas the approximately 42-kDa proteins remained with increasing intensity, indicating that the higher molecular-weight proteins were the phosphorylated Cx43. These results indicate that a defect in posttranslational phosphorylation of Cx43 protein associated with low expression of the Cx43 gene might be responsible for the GJIC deficiency in aB1 cells and that increased expression of Cx43 by gene amplification might restore this phosphorylated Cx43 protein and so reestablish GJIC.

Exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to inhibit gap junctional intercellular communication (GJIC) in many cell types in vitro. Using a scrape loading/dye transfer technique, TPA was shown to cause a dose-dependent and transient inhibition of GJIC in WB-F344, a normal rat liver epithelial cell line. Such a down-modulation of intercellular communication was found to be associated with an increase in protein kinase C (PKC) activity. Translocation of this activity to the particulate fraction occurred 10 min after exposure to 16 nM TPA and was consistent with the time course needed to inhibit GJIC. After 6 h exposure to TPA, essentially all the PKC activity was lost concurrent with the recovery of communication in these cells. During this time, the cells also became refractory to inhibition by further addition of TPA. Blockage of communication induced by TPA in WB cells was prevented by treating the cells with 23 microM palmitoyl carnitine for 1 h or 100 microM 8-N, N-(diethylamino)-octyl-3,4, 5-trimethoxybenzoate for 30 min. The results indicate that TPA transiently modulates GJIC in WB cells and PKC activation is possibly involved in blockage of communication in these cells.