PGF2alpha-induced signaling events in glomerular mesangial cells.
Of the various arachidonate cyclooxygenation eicosanoids synthesized in the normal and injured renal glomerular capillary, prostaglandin F2alpha (PGF2alpha) is the most abundant and potent in eliciting signaling events and biologic responses including contraction and proliferation of glomerular capillary pericytes known as mesangial cells. The regulation of PGF2alpha-induced signaling in these cells is unknown. The present studies assessed two key signaling events in response to PGF2alpha in mesangial cells; activation of phospholipase C (PLC) and protein kinase C (PKC). Mechanisms regulating PLC activation were also explored. Incubation of cultured growth arrested rat mesangial cells with PGF2alpha (1 microM) resulted in activation of a phosphatidyl inositol-specific phospholipase C (PI-PLC) assessed as increased generation of polyphosphates in myo-[3H]-inositol-labeled cells and as increased diacylglycerol (DAG) mass levels measured by a radioenzymatic assay. Generation of both inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate occurred, the former constituting 70% of total inositol trisphosphates. Enhanced generation of inositol 1,4-bisphosphate (IP2) also occurred and was greater than that of inositol 1,4,5-trisphosphate (IP3), indicating that PI-PLC utilized the phosphatidyl inositol monophosphate (PIP) to a greater extent than the phosphatidyl inositol bisphosphate (PIP2) substrate. Generation of DAG in response to PGF2alpha occurred in a biphasic pattern characterized by an early transient rise that peaked concomitantly with IP3 at 15 sec, and a late sustained increase at 2, 5, and 15 min that was not associated with an increase in IP3. PGF2alpha also activated PKC assessed as translocation of enzyme activity from cytosolic to membrane fractions. Inhibition of PKC using H-7 enhanced PGF2alpha-induced generation of IP3 at 15 sec but attenuated generation of DAG at 15 min. A more selective PKC inhibitor, Calphostin C, dose-dependently increased basal IP3 generation and also attenuated generation of DAG in response to PGF2alpha. This indicates that PKC negatively modulates PGF2alpha-induced PI-PLC activation, and that the late sustained DAG generation in response to PGF2alpha is regulated by a PKC-dependent phospholipase other than PLC. The mechanisms of PI-PLC stimulation in response to PGF2alpha were further explored using inhibitors of protein tyrosine phosphorylation and of guanine nucleotide-binding (G) protein activation. Inhibition of protein tyrosine phosphorylation using genistein had no effect on IP3 or DAG generation. ADP ribosylation of Gi using pertussis toxin (PTx) had no effect on IP3 generation in response to PGF2alpha. The inhibitor of receptor-coupled PI-PLC activation aminosteroid compound U-73122 that blocks G(PLC) was also ineffective. The observations indicate that PGF2alpha stimulates a PI-PLC which is under negative feedback regulatory control by PKC, and a phospholipase other than PLC which is under positive regulatory control by PKC. PGF2alpha-induced PI-PLC activation is independent of protein tyrosine phosphorylation and of PTx-sensitive G proteins.
Breshnahan
B A
BA
Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Kelefiotis
D
D
Stratidakis
I
I
Lianos
E A
EA
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004075:Diglycerides / Q000378:metabolism; D015237:Dinoprost / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D007295:Inositol Phosphates / Q000378:metabolism; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000494:pharmacology; D037342:Pertussis Toxin; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D004075:Diglycerides; D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D007529:Isoflavones; D007546:Isoquinolines; D009281:Naphthalenes; D010879:Piperazines; D010566:Virulence Factors, Bordetella; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D015237:Dinoprost; D019833:Genistein; D037342:Pertussis Toxin; D011493:Protein Kinase C; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; C058819:calphostin C