The Phosphatidylinositol 3-Kinase Inhibitors Wortmannin and LY294002 Inhibit Autophagy in Isolated Rat Hepatocytes

Authors


A. J. Meijer, Department of Biochemistry, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
Fax:+31 20 6915519.
E-mail:a.j.meijer@amc.uva.nl

Abstract

Recent studies indicate that phosphatidylinositol 3-kinase is essential in the regulation of many processes dependent on membrane flow. Autophagy is a complex pathway in which cell material, including proteins, can be degraded. Membrane flow plays a pivotal role in this process. To find out whether phosphatidylinositol 3-kinase is also required for autophagy, we tested the effects on autophagy of two structurally unrelated phosphatidylinositol 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phe-nylchromone (LY294002).

The addition of low concentrations of each of these inhibitors to incubations of hepatocytes in the absence of amino acids resulted in a strong inhibition of proteolysis. The antiproteolytic effect of wortmannin (IC50, 30 nM) and LY294002 (IC50 10 μM) was accompanied by inhibition of autophagic sequestration and not by an increase in lysosomal pH or a decrease in intracellular ATP. No further inhibition of proteolysis by the two compounds was observed when autophagy was already maximally inhibited by high concentrations of amino acids.

3-Methyladenine, which is commonly used as a specific inhibitor of autophagic sequestration, was an inhibitor of phosphatidylinositol 3-kinase, thus providing a target for its action.

It is proposed that phosphatidylinositol 3-kinase activity is required for autophagy. 3-Methyladenine inhibits autophagy by inhibition of this enzyme.

Abbreviations
PtdIns 3-kinase

phosphatidylinositol 3-kinase

LY294002

2-(4-morpholinyl)-8-phenylchromone

Lys-Ala-NH(O-Me)Nap

lysyl-alanyl-4-methoxy-2-naphthylamide

Nap(OMe)NH2

4-methoxy-2-naphthylamine

DPP 11

dipeptidyl-peptidase II

IC50

concentration giving 50% inhibition

Me2SO

dimethylsulfoxide

PtdIns(3)P2

phosphatidylinositol 3-phosphate

PtdIns(3,4)P2

phosphatidylinositol 3,4-bisphosphate

PtdIns(3,4,5)P3

phosphatidylinositol 3,4,5-trisphosphate

Enzymes
 

1-Phosphatidylinositol 3-kinase (EC 2.7.1.137)

 

1-phosphatidylinositol 4-kinase (EC 2.7.1.67)

 

dipeptidyl-peptidase II (EC 3.4.14.2)

 

hexokinase (EC 2.7.1.1)

 

glucose-6-phosphate dehydrogenase (EC 1.1.1.49)

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