SUBCELLULAR-LOCALIZATION OF PROSTAGLANDIN ENDOPEROXIDE H SYNTHASES-1 AND -2 BY IMMUNOELECTRON MICROSCOPY

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) are th e major targets of nonsteroidal anti-inflammatory drugs like aspirin a nd ibuprofen. These enzymes catalyze the committed step in the formati on of prostanoids from arachidonic acid. Although PGHS-1 and -2 are si milar biochemically, a number of studies suggest that PGHS-1 and PGHS- 2 function independently to form prostanoids that subserve different c ellular functions. We have hypothesized that these isozymes may reside , at least in part, in different subcellular compartments and that the ir compartmentation may affect their access to arachidonic acid and se rve to separate the functions of the enzymes. To obtain high resolutio n data on the subcellular locations of PGHS-1 and -2, we employed immu noelectron microscopy with multiple antibodies specific to each isozym e. Both PGHS-1 and -2 were found on the lumenal surfaces of the endopl asmic reticulum (ER) and nuclear envelope of human monocytes, murine N IH 3T3 cells, and human umbilical vein endothelial cells. Within the n uclear envelope, PGHS-1 and -2 were present on both the inner and oute r nuclear membranes and in similar proportions. Western blotting data showed a similar distribution of PGHS-1 and -2 in subcellular fraction s, and product analysis using isozyme-specific inhibitors suggested th at both enzymes generate the same products in NIH 3T3 cells. Thus, we are unable to attribute the independent functioning of PGHS-1 and PGHS -2 to differences in their subcellular locations. Instead, the indepen dent operation of these isozymes may be attributable to subtle kinetic differences (e.g. negative allosteric regulation of PGHS-1 at low con centrations of arachidonate (500-1000 nM)). A further conclusion of im portance from a cell biological perspective is that membrane proteins such as PGHS-1 and -2, which are located on the lumenal surface of the ER, are able to diffuse freely among the ER and the inner and outer m embranes of the nuclear envelope.