Modification of chromaffin cells with pertussis toxin or N-ethylmaleimide lowers cytoskeletal F-actin and enhances Ca(2+)-dependent secretion.
In an attempt to identify proteins involved in the secretory response, bovine chromaffin cells were modified with N-ethylmaleimide (NEM). NEM concentrations less than 30 microM enhanced norepinephrine secretion evoked by nicotine or by K+ depolarization and increased Ca(2+)-dependent secretion from digitonin-permeabilized cells. Higher concentrations of NEM inhibited secretion. The protein modified by NEM which was responsible for the enhancement of secretory activity appeared to rapidly diffuse out of the digitonin-permeabilized cells. When proteins which diffuse from control digitonin-permeabilized cells were incubated with pertussis toxin and [32P]NAD, several proteins were ADP-ribosylated. However, when proteins from cells preincubated with 30 microM NEM were incubated with pertussis toxin and [32P]NAD, these GTP-binding proteins (G-proteins) were not ADP-ribosylated, which suggests that they were modified in the cell by NEM. Stimulation of norepinephrine secretion by NEM was not additive with that caused by pertussis toxin. Modification of chromaffin cells with pertussis toxin or with 30 microM NEM caused a 40-50% decrease in the amount of cytoskeletal F-actin. This decrease in cytoskeletal F-actin may account for the increase in secretory activity.
Wu
Y N
YN
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Yang
Y C
YC
Wagner
P D
PD
D000199:Actins / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000311:Adrenal Glands / Q000187:drug effects*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002417:Cattle; D002837:Chromaffin Granules / Q000187:drug effects*; D005576:Colforsin / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D015536:Down-Regulation; D005033:Ethylmaleimide / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D009638:Norepinephrine / Q000378:metabolism; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000378:metabolism; D011796:Quinacrine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D000199:Actins; D002413:Cations, Divalent; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D011493:Protein Kinase C; D011796:Quinacrine; D013755:Tetradecanoylphorbol Acetate; D005033:Ethylmaleimide; D002118:Calcium; D009638:Norepinephrine