Identification and localisation of a type IV cytosolic phospholipase A2 in rat pancreatic beta-cells.
The liberation of arachidonic acid (AA), by phospholipase A2 (PLA2), is the rate-limiting step in a number of cell signalling pathways. In the pancreatic beta-cell, AA itself is thought to participate in the regulation of insulin secretion. Recently a Ca(2+)-sensitive, AA-selective cytosolic PLA2 (type IV cPLA2) has been isolated from the human monocyte U937 cell line. Although the DNA sequence of this enzyme implies a molecular weight of 85 kDa, the protein migrates with a molecular weight of 100-110 kDa on SDS-PAGE. In many cell types, cPLA2s which are reactive towards antibodies raised against the type IV cPLA2 have been shown to hydrolyse AA from membrane glycerophospholipids. Using a polyclonal antibody raised against a recombinant form of type IV cPLA2, we have detected an immunoreactive protein with a molecular weight of 93.5 kDa in rat islets of Langerhans. Furthermore, we have detected similar immunoreactive proteins in insulin-secreting beta-cell lines and have shown co-expression of type IV cPLA2 immunoreactivity and insulin immunoreactivity in rat pancreatic beta-cells. Under non-stimulatory conditions the 93.5 kDa immuno-reactive protein detected in rat islets of Langerhans was located predominantly in the cytosolic fraction. We have shown that immunoprecipitation of the rat immunoreactive protein from rat islet homogenates significantly decreases the total dithiothreitol/beta-mercaptoethanol-insensitive PLA2 activity by 56.4 +/- 7%. This provides further evidence that the immunoreactive rat protein is a type IV cPLA2 and is responsible for a large component of the PLA2 activity in rat islets of Langerhans. It is possible that, in the rat beta-cell, type IV cPLA2 couples the increase in intracellular Ca2+, brought about by insulin secretagogues, to the liberation of AA and the subsequent release of insulin.
Parker
K J
KJ
Biomedical Sciences Division, King's College London, UK.
Jones
P M
PM
Hunton
C H
CH
Persaud
S J
SJ
Taylor
C G
CG
Howell
S L
SL
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology*; D007150:Immunohistochemistry; D007515:Islets of Langerhans / Q000166:cytology / Q000201:enzymology*; D008297:Male; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D017207:Rats, Sprague-Dawley
D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium