Platelet-activating factor stimulates multiple signaling pathways in cultured rat mesangial cells.
We have previously reported that platelet-activating factor (PAF) elevates cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded glomerular mesangial cells. To confirm that this increase in [Ca2+]i is a result of receptor-mediated activation of phospholipase C, we investigated hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) in PAF-treated mesangial cells. PAF (10(-7) M) stimulated a rapid and transient formation of inositol trisphosphate. In concomitant experiments, PAF stimulated a biphasic accumulation of 3H-arachidonate-labeled 1,2-diacylglycerol (DAG). The secondary elevation in DAG was coincident with a rise in 3H-phosphorylcholine (PC) and 3H-phosphorylethanolamine (PE) suggesting that PAF stimulates delayed phospholipase activities which hydrolyze alternate phospholipids besides the polyphosphoinositides. This PAF-stimulated elevation in 3H-water soluble phosphorylbases was seen at 5 min but not at 15 sec suggesting that the initial rise in DAG as well as the initial elevation in [Ca2+]i are due primarily to PtdIns-4,5-P2 hydrolysis. PAF also stimulated PGE2 as well as 3H-arachidonic acid and 3H-lyso phosphatidylcholine (PtdCho) formation. We suggest that arachidonate released specifically from PtdCho via phospholipase A2 is a source of this PAF-elevated PGE2. It has been postulated that anti-inflammatory prostaglandins may antagonize the contractile and proinflammatory effects of PAF via activation of adenylate cyclase. Surprisingly, exogenous PAF reduced basal and receptor-mediated cAMP concentration indicating that PAF-stimulated transmembrane signaling pathways may oppose receptor-mediated activation of adenylyl cyclase. We have taken advantage of the different sensitivities of phospholipases A2 and C(s) to PMA, EGTA, and pertussis toxin to dissociate phospholipase A2 and C activities. Acute PMA-treatment enhanced PAF-stimulated PGE2 formation, reduced PAF-induced elevations in [Ca2+]i and had no effect upon PAF-stimulated 3H-PE. We have also demonstrated that phospholipase A2, but not PtdIns-specific phospholipase C, was sensitive to external calcium concentration. The role of a GTP-binding protein to couple PAF-receptors to the PtdIns-specific phospholipase C was confirmed as GTP gamma S synergistically elevated PAF-stimulated inositol phosphate formation. We also demonstrated that pertussis toxin ADP-ribosylates a single protein of an apparent 42 kD mass and that PAF pretreatment reduced subsequent ADP-ribosylation in a time-dependent manner. However, pertussis toxin had no effect upon phospholipase C-generated water soluble phosphorylbases or inositol phosphates. In contrast, PAF-stimulated phospholipase A2 and PAF-inhibited adenylyl cyclase activities were sensitive to pertussis toxin.(ABSTRACT TRUNCATED AT 400 WORDS)
Kester
M
M
Department of Medicine, Case Western Reserve University, School of Medicine, Cleveland, Ohio.
Thomas
C P
CP
Wang
J
J
Dunn
M J
MJ
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008297:Male; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins; D011453:Prostaglandins / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000502:physiology; D043562:Receptors, G-Protein-Coupled; D015398:Signal Transduction / Q000187:drug effects*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D010716:Phosphatidylinositols; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011453:Prostaglandins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D010566:Virulence Factors, Bordetella; C039351:platelet activating factor receptor; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D037342:Pertussis Toxin; D010740:Phospholipases; D000262:Adenylyl Cyclases