Stimulatory guanine nucleotide binding protein in pig epidermis: transient increase of the 45KDA cholera toxin substrate (Gs alpha) in the tape stripping-induced hyperproliferative state.
Cholera toxin catalyzed the transfer of ADP-ribose from [alpha-32P] NAD to 45kDa protein in pig epidermis. Western blot analysis using anti-Gs alpha antibody identified the 45kDa protein to be Gs alpha. In contrast to pertussis toxin-catalyzed ADP-ribosylation of Gi alpha, the cholera toxin-catalyzed ADP-ribosylation was enhanced by the presence of Mg2+ in the reaction mixture. The cholera toxin-catalyzed ADP-ribosylation of the epidermal 45kDa membrane protein was significantly decreased, when samples were prepared from the cholera toxin-pretreated epidermis. The results, coupled with our previous report (Tsutsui and Iizuka 1990), indicate that pig epidermis contains functional G proteins (Gs and Gi), that affect the epidermal adenylate cyclase activity. Tape stripping-induced hyperproliferative epidermis showed an increased cholera toxin-catalyzed ADP-ribosylation of the 45kDa protein (Gs alpha) at 12-24 h following the tape stripping. Immunoblot analysis, however, showed no remarkable change in the level of Gs alpha compared with non-stripping controls. There was no significant difference in the level of the pertussis toxin-induced ADP-ribosylation of 40kDa protein (Gi alpha) in the tape-stripped epidermis. Immunoblot analysis showed no change in Gi content, either. Forskolin-induced cyclic AMP accumulation was markedly increased in the tape stripping-induced hyperproliferative epidermis. Cholera toxin-induced cyclic AMP accumulation was slightly increased, but this was not statistically significant. These results indicate that the alteration of Gs that is documented by cholera toxin-catalyzed ADP-ribosylation, is among the functional derangements of adenylate cyclase of tape stripping-induced hyperproliferative epidermis.
Tsutsui
M
M
Department of Dermatology, Asahikawa Medical College, Japan.
Tamura
T
T
Takahashi
H
H
Hashimoto
Y
Y
Iizuka
H
H
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000378:metabolism* / Q000494:pharmacology*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000078404:Epidermal Cells; D004817:Epidermis / Q000187:drug effects / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006652:Histological Techniques; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D013552:Swine
D010761:Phosphorus Radioisotopes; D009243:NAD; D005576:Colforsin; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D000242:Cyclic AMP; D019204:GTP-Binding Proteins