Concentrations of prostaglandin endoperoxide synthase and prostaglandin I2 synthase in the endothelium and smooth muscle of bovine aorta. D L DeWitt, … , W K Sonnenburg, W L Smith J Clin Invest. 1983;72(6):1882-1888. https://doi.org/10.1172/JCI111151. Research Article Platelets adhere to the subendothelial layer of newly deendothelialized arteries. Attachment can be reduced with exogenous prostacyclin (PGI2). Thus, the subendothelium may be unable to produce sufficient PGI2 to prevent platelet adherence and subsequent platelet-platelet interaction. Consistent with this explanation are data from an earlier report (1977. Moncada S., A. G. Herman, E. A. Higgs, and J. R. Vane. Thromb. Res. 11:323-344) indicating that the smooth muscle layer of aorta has only 10-15% of the capacity of endothelial cells to synthesize PGI2. We have measured the concentrations of PGI2 synthase and prostaglandin endoperoxide (PGH) synthase in bovine aorta and obtained results quite different from those described in this earlier report. Tandem immunoradiometric assays for PGI2 synthase and PGH synthase antigens were used to quantitate these proteins in detergent-solubilized homogenates of endothelial cells and smooth muscle tissue prepared from 10 different bovine aorta. The concentrations of PGI2 synthase in endothelial cells and smooth muscle were found to be the same. However, the concentration of PGH synthase in endothelial cells averaged greater than 20 times that of smooth muscle. Results similar to those determined by immunoradiometric assay were also obtained when PGH synthase and PGI2 synthase catalytic activities were measured in preparations of endothelial and smooth muscle cells. Furthermore, when bovine aorta and renal arteries were subjected to immunocytofluorescence staining using monoclonal […] Find the latest version: https://jci.me/111151/pdf Concentrations of Prostaglandin Endoperoxide Synthase and Prostaglandin I2 Synthase in the Endothelium and Smooth Muscle of Bovine Aorta DAVID L. DEWITT, JEFFREY S. DAY, WILLIAM K. SONNENBURG, and WILLIAM L. SMITH, Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 A B S T R A C T Platelets adhere to the subendothelial fluorescence staining of equivalent intensity was de- layer of newly deendothelialized arteries. Attachment tected in both the endothelial cells and the smooth can be reduced with exogenous prostacyclin (PGI2). muscle. Moreover, the intensity of fluorescence was Thus, the subendothelium may be unable to produce similar throughout cross-sections of vascular smooth sufficient PGI2 to prevent platelet adherence and sub- muscle, indicating that there is no gradient in PGI2 sequent platelet-platelet interaction. Consistent with synthase concentrations between the endothelium and this explanation are data from an earlier report (1977. adventitia. Our results indicate that the propensity of Moncada S., A. G. Herman, E. A. Higgs, and J. R. platelets to adhere to the subendothelium of deen- Vane. Thromb. Res. 11:323-344) indicating that the dothelialized arteries and form aggregates cannot be smooth muscle layer of aorta has only 10-15% of the attributed simply to an inability of the denuded vas- capacity of endothelial cells to synthesize PGI2. We culature to produce PGI2 from PGH2, but may be a have measured the concentrations of PGI2 synthase and consequence of the low PGH synthase activity of smooth prostaglandin endoperoxide (PGH) synthase in bovine muscle. Consistent with this concept are the results of aorta and obtained results quite different from those Eldor et al. (1981. J. Clin. Invest. 67:735-741) who described in this earlier report. Tandem immunora- reported that increases in PGH synthase activity are diometric assays for PGI2 synthase and PGH synthase associated with formation of a nonthrombogenic neoin- antigens were used to quantitate these proteins in de- tima. tergent-solubilized homogenates of endothelial cells and smooth muscle tissue prepared from 10 different bovine INTRODUCTION aorta. The concentrations of PGI2 synthase in endo- thelial cells and smooth muscle were found to be the Prostaglandin I2 (prostacyclin, PGI2)1 is a potent va- same. However, the concentration of PGH synthase in sodilator and antithrombogenic agent (1-3) formed endothelial cells averaged >20 times that of smooth from the prostaglandin endoperoxide PGH2 through muscle. Results similar to those determined by im- the action of prostacyclin synthase (4-6). Early studies munoradiometric assay were also obtained when PGH on rabbit aorta indicated that the endothelium was the synthase and PGI2 synthase catalytic activities were major site of vascular PGI2 synthesis and that smooth measured in preparations of endothelial and smooth muscle had considerably less prostacyclin synthase ac- muscle cells. Furthermore, when bovine aorta and renal tivity than endothelial cells (7). These results formed arteries were subjected to immunocytofluorescence the basis for the hypothesis that adherence of platelets staining using monoclonal antibodies to PGI2 synthase, to the vascular subendothelium resulted from a rela- tive inability of vascular smooth muscle to synthesize During the time this work was performed Dr. David L. DeWitt was the recipient of U.S. Public Health Service Traineeship HL07404. Dr. William L. Smith is an Established I Abbreviations used in this paper: BAE cells, bovine aor- Investigator of the American Heart Association. Address all tic endothelial cells; DMEM, Dulbecco's-modified Eagle's correspondence to Dr. Smith. medium; IRA, immunoradiometric assay; PGH synthase, Received for publication 31 March 1983 and in revised prostaglandin endoperoxide synthase; PGI2 synthase, pros- form 9 August 1983. taglandin I2 (prostacyclin) synthase. 1882 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. - 0021-9738/83/12/1882/07 $1.00 Volume 72 December 1983 1882-1888 PGI2 from PGH2 (2, 7). It is now clear, however, that bovine serum was used to dislodge the cells. Aliquots (0.5 ml) of the cell suspension were then applied to sterile glass arterial smooth muscle cells (8-11) cultured from a coverslips placed in 100 X 20-mm culture dishes (Corning variety of sources are capable of synthesizing PGI2 Glass Works, Corning Medical and Scientific, Corning, NY) from PGH2 at rates comparable to those obtained with and cells allowed to adhere overnight at 370C under a H20- cultured endothelial cells (10, 11). saturated 10% CO2 atmosphere. The coverslips were then We recently prepared monoclonal antibodies to washed once with Krebs buffer, pH 7.4, quick-frozen in pen- tane (-70'C) and dried. The cells were then subjected to PGI2 synthase (6). In using these reagents to determine indirect immunocytofluorescence staining for Factor VIII the subcellular location of this enzyme by immuno- antigen (14) and examined under a Leitz Orthoplan fluo- cytochemistry (12), we noted that the intensity of PGI2 rescence microscope (E. Leitz, Inc., Rockleigh, NJ). In ex- synthase-positive immunofluorescence was similar in amining cells from three separate isolates, we found that both the endothelial cells and the underlying smooth >99% of the cells examined in random fields stained for Factor VIII. This indicates that our BAE cell isolates were muscle. This observation, coupled with previous evi- essentially pure endothelial cell populations. dence for efficient PGI2 synthesis by cultured smooth Preparation of solubilized endothelial cell homogenates. muscle cells (8-11), led us to quantitate PGI2 synthase For assays of PGH synthase, BAE cells (<100 mg wet wt) in fresh vascular smooth muscle and endothelial cell isolated from single aorta were thawed and then homoge- extracts a newly developed immunoradiometric nized using a glass homogenizer in 0.5 ml of 0.1 M Tris- using chloride, pH 8.0 containing 2.5 mM diethyldithiocarbamate, assay (IRA) for this protein. In contrast to the results 5 mM EDTA, and 1% Tween 20. The sample was allowed obtained by Moncada et al. (7), our data indicate that to stand at 40C for 30 min. The solubilized BAE cell ho- the concentration of PGI2 synthase is the same in dif- mogenate was used to assay (a) PGH synthase antigen by ferent layers of the vasculature; however, the results IRA or (b) cyclooxygenase activity using the radioisotope assay described below. of parallel studies quantitating PGH synthase indicate For assays of PGI2 synthase, the isolated BAE cells were that this enzyme is present in considerably higher con- homogenized and solubilized in 0.5 ml of 0.1 M Tris-chloride centrations in endothelial cells than in smooth muscle. containing 10' M Flurbiprofen and 0.5% Triton X-100. This sample was used for both IRA and the enzyme activity assay. Preparation of solubilized smooth muscle homogenates. METHODS After removal of the endothelium, the smooth muscle was Materials. Diethyldithiocarbamate and Tween 20 were cut into 2-cm8 pieces and stored at -80'C. Before assay, obtained from Sigma Chemical Co. (St. Louis, MO) Dul- tissue (5 g) was cut into slivers and homogenized in 5 vol of becco's-modified Eagle's medium (DMEM), antibiotic-an- either (a) 0.1 M Tris-chloride, pH 8.0 containing 2.5 mM timycotic (1OOX), and fetal bovine serum were all purchased diethyldithiocarbamate and 5 mM EDTA (PGH synthase) or from Gibco Laboratories (Grand Island, NY). Collagenase (b) 0.1 M Tris-chloride, pH 8.0 containing 10' M Flurbi- (CLS II) was from Worthington Biochemical Corp. (Freehold, profen (PGI2 synthase) using a homogenizer (Polytron, NJ). [5,6,8,9,11,12,14,15-8H]Arachidonic acid (100 Ci/mmol) Brinkmann Instruments, Inc., Westbury, NY). The crude ho- and Na'"I (100 m Ci/ml) were products of New England mogenate was filtered through one layer of cheesecloth. The Nuclear (Boston, MA). Triton X-100 and rabbit anti-human fibrous matter that did not pass through the cheesecloth was Factor VIII serum were from Calbiochem-Behring Corp., resuspended in 5 vol of homogenizing buffer, rehomogenized, American Hoechst Corp. (San Diego, CA). Flurbiprofen was and refiltered as before. The two filtrates were combined and generously supplied by Dr. Udo Axen of the Upjohn Co.