High glucose and advanced glycation end products induce phospholipid hydrolysis and phospholipid enzyme inhibition in bovine retinal pericytes.
In the present study, we investigated the possible role of oxidative stress and the modulation of phospholipid turnover in two related models of pericyte injury, i.e., treatment with high glucose or advanced glycation end products (AGEs). Growing microcapillary pericytes from bovine retinas in culture were incubated, for 3 weeks, with 20-50 mM glucose or 2-20 microM AGEs, and peroxidation parameters (malondialdehyde, conjugated diene, hydroperoxide, glutathione (GSH) levels and lactate dehydrogenase (LDH) release) were evaluated. Arachidonate (AA) and choline release from membrane phospholipids was determined in pericytes prelabeled with [1-(14)C]arachidonate and [Me-(3)H]choline, respectively, and stimulated with elevated glucose or AGEs for 30 min or 2 h. [1-(14)C]arachidonate and [Me-(3)H]choline incorporation into phospholipids, for 2 h and 3 h respectively, was also studied in conditioned and serum-starved cultures. Finally, lysates of treated and control cells were assayed for cytosolic phospholipase A(2) (cPLA(2)), acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (AT), CTP:phosphocholine cytidylyltransferase (CT) and microsomal choline phosphotransferase (CPT) enzyme activities. We found that high glucose and AGEs caused neither significant production of reactive oxygen species nor cell toxicity or death, unlike other cell types. Both agents had no significant effect on the cellular ultrastructure, evaluated by light and electron microscopy, AA incorporation and release, cytosolic phospholipase A(2) (cPLA(2)) and AT activities. On the contrary, choline incorporation into phosphatidylcholine, CT and CPT activities were significantly reduced either by 50 mM glucose or 20 microM AGEs. Simultaneously, [Me-(3)H]choline release was significantly stimulated by both agents. We conclude that prolonged treatments with high glucose or AGEs are not able to induce oxidative injury in bovine retinal capillary pericytes. Nevertheless, they do induce phospholipid hydrolysis and phospholipid enzyme activity inhibition.
Assero
G
G
Department of Biochemistry, Faculty of Medicine, University of Catania, Viale Andrea Doria 6, 95125, Catania, Italy.
Lupo
G
G
Anfuso
C D
CD
Ragusa
N
N
Alberghina
M
M
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology* / Q000633:toxicity; D017127:Glycation End Products, Advanced / Q000494:pharmacology* / Q000633:toxicity; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D015227:Lipid Peroxidation; D008854:Microscopy, Electron; D018384:Oxidative Stress; D020286:Pericytes / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D010743:Phospholipids / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012171:Retinal Vessels / Q000187:drug effects* / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology
D017127:Glycation End Products, Advanced; D010743:Phospholipids; D017382:Reactive Oxygen Species; D012710:Serum Albumin, Bovine; D016718:Arachidonic Acid; D007770:L-Lactate Dehydrogenase; D053148:Caspase 3; D020169:Caspases; D005947:Glucose; D002794:Choline