Download Relationship between neurotransmitter release and cytosolic free

608th MEETING, KEELE
1077
Relationship between neurotransmitter release and cytosolic free calcium in PC12 cells
T. POZZAN,* F. DI VIRGILI0,t G. GATTIS
N. DOZIOS and J. MELDOLESIS
*Institute of General Pathology, University of Padova,
Padova. Italy, 7C.N.R. Unit for the Study of Physiology of
Mitochondria and Institute of General Pathology, University
of Padova, Padova, Italy, and $Department of Medical
Pharmacology, C.N.R . Unit for Cytopharmacology,
University of Milano, MiIano, Italy
Table 1. Relationship between [Ca2+Iiand [3H]dopamine
release
Ca2+= Krebs-Ringer medium 1 mM-CaC1, ; - Cat+ =
Krebs-Ringer medium without added Ca2++1 mM-EGTA.
[3H]Dopamine was measured in the supernatant after
spinning the cells through silicon oil. Results are expressed
as % of total content lOmin after stimulus addition. Basal
release without stimulus was 5-8%.
It is generally assumed that the release of most if not all
neurotransmitters occurs via exocytosis. A major established concept is that secretory exocytosis in nerve terminals, as well as in other cell types, is initiated when the
concentration of Cat+ is increased in cytoplasm above some
threshold level (Rubin, 1982). The rise of [Cat+],in nerve
terminals occurs physiologically when the plasma-membrane potential is depolarized and the voltage-dependent
Cat+ channel opens. This results in an increased influx of
Ca2+from the extracellular medium down its steep electrochemical gradient.
However, it is known that a number of pharmacological
agents can induce neurosecretion in the absence of extracellular Ca2+.This result has been generally interpreted as
due to mobilization of CaZ+from intracellular stores sufficient to raise [Ca2+Ii above the threshold level. Yet
direct demonstration of this Cat+ release has not been provided, due mostly to the technical difficulty of measuring
[Cat+Ii in nerve terminals. In this report we have measured
[Cat+Ii with the intracellularlytrappable fluorescentCat+ indicator quin2 (Tsien et al., 1982) in a cell line derived from a
rat pheocromocytoma, PC 12, which resembles undifferentiated sympathetoblasts. Release of previously accumulated
[3H]dopamineor quantitative electron microscopical determination of the number of secretory granules have been
utilized as a measure of exocytosis.
A number of drugs which are known, from previous
studies, to release neurotransmitters independently of
[Ca2+l,in a variety of systems have been tested.
For two of these drugs, monensin and X537A, although
we have observed that they can induce release of [3H]dopamine independently of [Cat+b, morphological evidence
suggests that the release occurs not via exocytosis, but rather
is due to swelling and disintegration of the secretory granule
membrane.
On the contrary, PMA, OAG and pHMBS (Table 1) release large amounts of [3H]dopamine without requiring a
rise in [Ca2+],,and morphological evidence indicates that
with these drugs the release is due to exocytosis: the density
of secretory granules is decreased in parallel with the release
of [3H]dopamine,and no evidence of toxicity was observed.
[3H]Dopamine release
[Ca*+Iiincrease
- &
Stimulus
+Ca*+
-CaZ+
+ C a Z + --a2+
80%
Yes
Yes*
Monensin (1 pi)
80%
60%
60%
Yes
Yes*
X537A ( 3 p i )
35%
No
No
PMA (0.1 pi)
38%
18%
No
No
DG ( 1 0 ~ ~ )
18%
Yes
No
40%
140
pHMBS (1 mM)
* Although some [Ca2+Iiincrease was observed after X537A and
monensin in the absence of Ca2+,the effects on [Ca2+liwere much
smaller than in Ca2+medium and barely significant.
Abbreviations used : [CaZ+li,cytosol free Ca2+ concentration;
[Cab, extracellular Ca2+ concentration; PMA. phorbol myristate
acetate; OAG, oleoyl acetyl glycerol; pHMBS, phydroxymercuriphenylsulphonic acid.
Vol. 12
+
+
On the basis of the pharmacological specificity of OAG
and PMA we suggest that the [Ca2+b-independentsecretion is mediated through activation of the ubiquitous Cat+and phospholipid-dependent enzyme, protein kinase c
(Nishizuka, 1983).
We have observed that the kinetics and characteristics of
[Ca2+],-independent secretion differ markedly from those
of [Ca2+],-dependent secretion : [Ca2+],-dependent secretion, as induced by Cat+ ionophores or plasma-membrane
depolarization, is rapid and complete in 0.5-1 min. [Cat+1,independent secretion is slow but lasts for several minutes.
Synergistic effects between PMA or OAG and Cat+ ionophores were observed both on the rate and extent of secretion.
The kinetics and characteristics of secretion induced by
PMA or OAG, and Cat+ ionophores resemble closely those
of the secretion elicited by the neurotoxin a-latro toxin that
we have previously investigated in detail (Meldolesi et al.,
1983, 1984). Preliminary observations indicate that a-latro
toxin can induce the breakdown of phosphatidylinositol.
This in turn suggests that in the action of this toxin both
mechanisms of secretion (protein kinase c and Ca2+)are
operative. Our results raise the possibility of a central role of
protein kinase c also in the process of neurosecretion.
Meldolesi, J., Madeddu, L., Torda, M., Gatti, G. & Minta, E.
(1983) Neuroscience 10, 997-1009
Meldolesi, J., Huttner, W.B., Tsien, R. Y. & Pozzan, T. (1984)
Proc. Natl. Acad. Sci. U.S.A. 81, 620-624
Nishizuka, Y. (1983) Trends Bwchem. Sci. 8, 13-16
Rubin, R. P. (1982) in Calcium and Cellular Secretion, Plenum
Press, New York
Tsien, R. Y., Pozzan, T. & Rink, T. J. (1982) Nature (London) 295,
68-71