Phorbol ester induces phosphorylation and down-regulation of connexin 43 in WB cells

Oh, Saw Yin; Grupen, Chris G.; Murray, Andrew W.

doi:10.1016/0167-4889(91)90016-q

Cited by 114 publications

(82 citation statements)

References 20 publications

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“…Previous studies [18,19] In separate experiments using cells prelabelled with 32P as described [19], it was established that LPA stimulated the serine-phosphorylation of Cx43 (results not shown).…”

Section: Resultsmentioning

confidence: 89%

“…Both of the last two compounds have been shown to inhibit communication in WB cells [18,19]. agonists initiate their effects on communication through different receptor systems that are down-regulated by the specific ligand.…”

Section: Resultsmentioning

confidence: 99%

“…To detect Cx43 protein, cells in 100 mm dishes were washed in PBS and gap-junction-enriched pellets were prepared by alkali extraction [18]. The pellet was resuspended in 1 mM NaHCO3 containing 1 mM phenylmethanesulphonyl fluoride and solubilized in SDS sample buffer.…”

Section: Immunoblottingmentioning

confidence: 99%

“…Protein was determined by a modified Lowry procedure [25]. Samples containing 20 ,g of protein were separated on 100%-acrylamide gels, transferred to nitrocellulose (100 V, 2 h), and immunoreaction and detection were carried out as described [18]. The affinity-purified antibody to amino acids 314-322 of rat heart Cx43 was a gift from Dr. D. Gros [26].…”

Section: Immunoblottingmentioning

confidence: 99%

“…There is considerable evidence that junctional permeability can be modulated by phosphorylation of the connexin proteins (see [17][18][19] [20] and cells were cultured as described previously [18]. In Partial purification and assay of MAP kinase Cells (from 100 mm dishes) were rinsed with Ca2+/Mg2+-free PBS and incubated with LPA, PMA or EGF.…”

Section: Introductionmentioning

confidence: 99%

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Lysophosphatidic acid inhibits gap-junctional communication and stimulates phosphorylation of connexin-43 in WB cells: possible involvement of the mitogen-activated protein kinase cascade

Hill

Schmidt

et al. 1994

Biochemical Journal

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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.

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Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Immunoblottingmentioning

confidence: 99%