PGF(2-ALPHA)-INDUCED SIGNALING EVENTS IN GLOMERULAR MESANGIAL CELLS

Of the various arachidonate cyclooxygenation eicosanoids synthesized i n the normal and injured renal glomerular capillary, prostaglandin F-2 alpha (PGF(2 alpha)) is the most abundant and potent in eliciting sig naling events and biologic responses including contraction and prolife ration of glomerular capillary pericytes known as mesangial cells. The regulation of PGF(2 alpha)-induced signaling in these cells is unknow n. The present studies assessed two key signaling events in response t o PGF(2 alpha) 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 c ells with PGF(2 alpha) (1 mu M) resulted in activation of a phosphatid yl inositol-specific phospholipase C (PI-PLC) assessed as increased ge neration of polyphosphates in myo-[H-3]-inositol-labeled cells and as increased diacylglycerol (DAG) mass levels measured by a radioenzymati c assay, Generation of both inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate occurred, the former constituting 70% of total ino sitol trisphosphates. Enhanced generation of inositol 1,4-bisphosphate (IP2) also occurred and was greater than that of inositol 1,4,5-trisp hosphate (IP3), indicating that PI-PLC utilized the phosphatidyl inosi tol monophosphate (PIP) to a greater extent than the phosphatidyl inos itol bisphosphate (PIP2) substrate, Generation of DAG in response to P GF(2 alpha) occurred in a biphasic pattern characterized by an early t ransient 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 a n increase in IP3. PGF(2 alpha) also activated PKC assessed as translo cation of enzyme activity from cytosolic to membrane fractions. Inhibi tion of PKC using H-7 enhanced PGF(2 alpha)-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 gen eration and also attenuated generation of DAG in response to PGF(2 alp ha). This indicates that PKC negatively modulates PGF(2 alpha)-induced PI-PLC activation, and that the late sustained DAG generation in resp onse to PGF(2 alpha) is regulated by a PKC-dependent phospholipase oth er than PLC, The mechanisms of PI-PLC stimulation in response to PGF(2 alpha) were further explored using inhibitors of protein tyrosine pho sphorylation 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 pertuss is toxin (PTx) had no effect an IP3 generation in response to PGF(2 al pha). The inhibitor of receptor-coupled PI-PLC activation aminosteroid compound U-73122 that blocks G(PLC) was also ineffective. The observa tions indicate that PGF(2 alpha) stimulates a PI-PLC which is under ne gative feedback regulatory control by PKC, and a phospholipase other t han PLC which is under positive regulatory control by PKC. PGF(2 alpha )-induced PI-PLC activation is independent of protein tyrosine phospho rylation and of PTx-sensitive G proteins.