Ca2+-Stimulated Catecholamine Release from alpha-Toxin Permeabilized PC12 Cells

Two possible cellular pathways of catecholamines from the
chromaffin vesicles of PC 12 cells to the surrounding medium are
explored in this study. The direct one circumventing the cytoplasm
can be activated in a-toxin-permeabilized cells with micromolar
levels of free Ca2+. Catecholamine metabolites formed in the
cytoplasm (i.e., 3,4-dihydroxyphenylacetic acid and
3,4-dihydroxyphenylethanol) are neither formed nor released from
the cells under these conditions. However, when vesicular
catecholamines were discharged into the cytoplasm by addition of
the ionophore nigericin, such metabolites are formed and released
into the medium independent of Ca2+. Both types of experiments
provide direct evidence for the operation of Ca2+-induced
exocytosis of dopamine and noradrenaline in permeabilized PC12
cells. The Ca2+ dependence of dopamine or noradrenaline release, as
measured by the determination of the endogenous catecholamines
using the high-performance liquid chromatography technique,
exhibits two different phases. One is already activated below 1 pM
free Ca2+ and plateaus at 1-5 pM free Ca2+, while a second occurs
in the presence of larger amounts of free Ca2+ (10-100 pM).
Ca2+-induced catecholamine release from the permeabilized cells can
be modulated in different ways: It is enhanced by the phorbol ester
12-0-tetradecanoylphorbol 13-acetate and the diacylglycerol 1
-oleyl-2-acetylglycerol provided Mg*+/ATP is present, and it is
inhibited by guanosine 5’-0-(3-thiotriphosphate). The latter effect
is abolished by pretreatment of the cells with pertussis toxin but
not by cholera toxin. Thus, it appears that Ca2+-induced exocytosis
can be modulated via the protein kinase C system, as well as via
GTP binding proteins.