cPLA2alpha-evoked formation of arachidonic acid and lysophospholipids is required for exocytosis in mouse pancreatic beta-cells.
Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2 (cPLA2alpha) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. cPLA2alpha dose dependently (EC50 = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca2+ current or cytoplasmic Ca2+ levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70-80 to 280-300. cPLA2alpha-stimulated exocytosis was antagonized by the specific cPLA2 inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3 or an antisense oligonucleotide against cPLA2alpha. The action of cPLA2alpha was mimicked by a combination of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl- uptake through ClC-3 Cl- channels. Accordingly, the stimulatory action of cPLA2alpha was inhibited by the Cl- channel inhibitor DIDS and in cells pretreated with ClC-3 Cl- channel antisense oligonucleotides. We propose that cPLA2alpha has an important role in controlling the rate of exocytosis in beta-cells. This effect of cPLA2alpha reflects an enhanced transgranular Cl- flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine.
Juhl
Kirstine
K
Laboratory of Islet Cell Physiology, Novo Nordisk, Bagsvaerd, Denmark.
Høy
Marianne
M
Olsen
Hervør L
HL
Bokvist
Krister
K
Efanov
Alexander M
AM
Hoffmann
Else K
EK
Gromada
Jesper
J
D000818:Animals; D016718:Arachidonic Acid / Q000096:biosynthesis*; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D015220:Calcium Channels / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005260:Female; D054513:Group IV Phospholipases A2; D066298:In Vitro Techniques; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008246:Lysophospholipids / Q000096:biosynthesis*; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D016376:Oligonucleotides, Antisense; D018408:Patch-Clamp Techniques; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013268:Stimulation, Chemical
D015220:Calcium Channels; D018118:Chloride Channels; C115376:ClC-3 channel; D016859:Lipoxygenase Inhibitors; D008244:Lysophosphatidylcholines; D008246:Lysophospholipids; D016376:Oligonucleotides, Antisense; D016718:Arachidonic Acid; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; D054467:Phospholipases A2; D002118:Calcium