Thiophosphorylation causes Ca2+-independent norepinephrine secretion from permeabilized PC12 cells.
Adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) was used to examine the role of phosphorylation in the regulation of norepinephrine secretion by digitonin-permeabilized PC12 cells. While most kinases will use ATP gamma S to thiophosphorylate proteins, thiophosphorylated proteins are relatively resistant to dethiophosphorylation by protein phosphatases. Norepinephrine secretion by permeabilized PC12 cells was ATP- and Ca2+-dependent but resistant to calmodulin antagonists. Half-maximum secretion was obtained in 0.75 microM Ca2+. Permeabilized PC12 cells were incubated with ATP gamma S in the absence of Ca2+, the ATP gamma S was removed, and norepinephrine secretion was determined. Preincubation with ATP gamma S increased the amount of norepinephrine secreted in the absence of Ca2+, but it had no effect on the amount released in the presence of Ca2+. After a 15-min preincubation in 1 mM ATP gamma S, there was almost as much secretion in the absence of Ca2+ as in its presence. Inclusion of ATP in the preincubation inhibited the effect of ATP gamma S. Ca2+ stimulated the rate of modification by ATP gamma S as brief preincubations with ATP gamma S in the presence of Ca2+ resulted in higher levels of Ca2+-independent secretion than did preincubations with ATP gamma S in the absence of Ca2+. Similarly, brief preincubations of permeabilized cells with ATP in the presence of Ca2+ resulted in elevated levels of Ca2+-independent secretion. Secretion of norepinephrine from ATP gamma S-treated cells was ATP-dependent. These results suggest that norepinephrine secretion by PC12 cells is regulated by a Ca2+-dependent phosphorylation. Once this phosphorylation has occurred, secretion is still ATP-dependent, but it no longer requires Ca2+.
Wagner
P D
PD
Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892.
Vu
N D
ND
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology / Q000031:analogs & derivatives; D000310:Adrenal Gland Neoplasms / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D002463:Cell Membrane Permeability; D004072:Digitonin; D007700:Kinetics; D009638:Norepinephrine / Q000378:metabolism*; D010673:Pheochromocytoma / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats
C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004072:Digitonin; D002118:Calcium; D009638:Norepinephrine