There are two isozymes of prostaglandin endoperoxide H synthase called PGHS-1 and PGHS-2. Both enzymes catalyze the same two reactions: (a) a cyclooxygenase reaction in which arachidonic acid is converted to the prostaglandin endoperoxide PGG-2 and (b) a peroxidase reaction in which PGG-2 is reduced to PGHS-2. PGHS-1 is a constitutive enzyme whose expression appears to be regulated developmentally, PGHS-2 is an inducible enzyme which is expressed transiently in response to growth factors, tumor promoters, and cytokines. We have recently obtained immunocytochemical and histochemical evidence suggesting (a) that PGHS-1 functions on the luminal surface of the endoplasmic reticulum (ER) and the contiguous outer membrane of the nuclear envelope (NE); (b)that part of the PGHS-2 molecules also function on the ER and outer membrane of the NE; and (c) that a portion of the PGHS-2 molecules synthesize prostanoids on the luminal surface of the inner membrane of the nuclear envelope (NE). We hypothesize that PGHS-1 and the subset of PGHS-2 molecules which colocalize with PGHS-1 represent a biosynthetic system which forms prostanoids that act extracellularly, whereas there is a subset of PGHS-2 molecules located on the inner membrane of the NE which produce prostanoids that function in the nucleus to subserve differentiative/replicative functions. The three general goals of our proposed studies are: (a) to determine if PGHS-2 is uniquely located and functions on the inner membrane of the NE; we will employ in these studies a combination of subcellular fractionation procedures, immunoelectron microscopy, and a new histofluorescence assay for PGHS activity; (b) to determine if prostacyclin synthase and/or thromboxane synthase are present on the nucleoplasmic surface of the NE under conditions in which PGHS-2 is expressed; these studies will employ subcellular fractionation procedures and immunoelectron microscopy; and (c) to identify structural domains of PGHS-I and PGHS-2 involved in targeting these isozymes to the ER and NE, respectively; these studies will utilize molecular biological procedures for preparing and expressing modified forms of the enzymes in which various domains within the proteins are altered or deleted. PGHSs are of considerable therapeutic interest because of their involvement in inflammation and hemostasis, and perhaps colon cancer and Alzheimer's disease. The long range goal of the proposed research is to determine the cellular functions of the two PGHS isozymes.