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PMID: 2160809 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Evidence that the inositol phospholipids are necessary for exocytosis. Loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase C and removal of ATP.

The Biochemical journal ·Vol. 268 ·No. 1 ·1990-05-15 ·Pages 15-25

Eberhard DA, Cooper CL, Low MG, Holz RW

Abstract

We directly manipulated the levels of PtdIns, PtdInsP and PtdInsP2 in digitonin-treated adrenal chromaffin cells with a bacterial phospholipase C (PLC) from Bacillus thuringiensis and by removal of ATP. The PtdIns-PLC acted intracellularly to cause a large decrease in [3H]inositol- or [32P]phosphate-labelled PtdIns, but did not directly hydrolyse PtdInsP or PtdInsP2. [3H]PtdInsP and [3H]PtdInsP2 levels declined markedly, probably because of the action of phosphatases in the absence of synthesis. Removal of ATP also caused marked decreases in [3H]PtdInsP and [3H]PtdInsP2. The decrease in polyphosphoinositide levels by PtdIns-PLC treatment or ATP removal was reflected by the inhibition of the production of inositol phosphates upon subsequent activation of the endogenous PLC by Ca2(+)-dependent catecholamine secretion from permeabilized cells was strongly inhibited by PtdIns-PLC treatment and by ATP removal. Ca2(+)-dependent secretion was similarly correlated with the sum of PtdInsP and PtdInsP2 when the level of these lipids was changed by either manipulation. PtdIns-PLC inhibited only the ATP-dependent component of secretion and did not affect ATP-dependent secretion. Both PtdIns-PLC and ATP removal inhibited the late slow phase of secretion, but had little effect on the initial rapid phase. Although we found a tight correlation between polyphosphoinositide levels and secretion, endogenous phospholipase C activity (stimulated by Ca2+, guanine nucleotides and related agents) was not correlated with secretion. Additional experiments indicated that neither the products of the PtdIns-PLC reaction (diacylglycerol and InsP1) nor the inability to generate products by subsequent activation of the endogenous PLC is likely to account for the inhibition of secretion. Incubation of permeabilized cells with neomycin in the absence of ATP maintained the level of polyphosphoinositides and more than doubled subsequent Ca2(+)-dependent secretion. The data suggest that: (1) Ca2(+)-dependent secretion has a requirement for the presence of inositol phospholipids; (2) the enhancement of secretion by ATP results in part from increased polyphosphoinositide levels; and (3) the role for inositol phospholipids in secretion revealed in these experiments is independent of their being substrates for the generation of diacylglycerol and InsP3.

MeSH Terms
Adenosine Triphosphate/pharmacology,physiology Adrenal Medulla/drug effects,metabolism Animals Bacillus thuringiensis/enzymology Calcium/pharmacology Cattle Cell Membrane Permeability/drug effects Chromaffin System/drug effects,metabolism Digitonin/pharmacology Diglycerides/metabolism Exocytosis Guanine Nucleotides/pharmacology Kinetics Neomycin/pharmacology Norepinephrine/metabolism Phosphatidylinositol Diacylglycerol-Lyase Phosphatidylinositols/metabolism,physiology Phosphoric Diester Hydrolases/pharmacology Phosphorylation Protein Kinase C/metabolism
Chemicals
Diglycerides Guanine Nucleotides Phosphatidylinositols Adenosine Triphosphate Protein Kinase C Phosphoric Diester Hydrolases Phosphatidylinositol Diacylglycerol-Lyase Neomycin Digitonin Calcium Norepinephrine
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Eberhard D A
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
Cooper C L
Low M G
Holz R W
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2680015
Published
1990-05-15
Pages
15-25
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1131385
Subset
IM
Grants
NIADDK NIH HHS · P01AM36084 · United States
NIADDK NIH HHS · R01AM27959 · United States
NIGMS NIH HHS · R01GM35873 · United States
External Links
PubMed source DOI: 10.1042/bj2680015 PMC Full Text View journal details
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