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Previous studies have suggested a possible role for prostaglandins (PGs) in mediating alterations in nephron structure and function ensuing after renal ablation. Two isoforms of cyclooxygenase (COX) have been described: constitutive (COX-1) and inducible (COX-2). We examined expression of these isoforms following subtotal renal ablation (5/6 ablation, RA) in rats. In renal cortex, COX-2 mRNA and immunoreactive protein (IP) increased progressively compared with sham-operated littermates. In contrast, there were no significant changes in COX-1 mRNA expression. In normal kidney, cortical COX-1 IP was immunolocalized predominantly to mesangial cells and collecting tubules, whereas COX-2 IP was found in a subset of cortical thick ascending limb of Henle's loop (CTAL) cells in the region of the macula densa (MD). Following RA, significantly increased COX-2 IP was detected in the MD and surrounding CTAL cells. In addition, fainter immunoreactive COX-2 was detected in scattered visceral epithelial cells and mesangial cells of the glomerulus. Immunoblotting of isolated glomeruli demonstrated a selective increase of glomerular immunoreactive COX-2 expression following RA. No change of COX-1 expression was seen. To determine COX activity, isolated glomeruli were incubated with arachidonic acid and PGE2 measured by enzyme immunoassay (EIA). Compared with sham, glomeruli from 2 wk RA produced significantly more PGs. SC-58560, a selective COX-1 inhibitor, did not inhibit PG production in the remnant glomeruli at concentrations up to 10(-4) M, whereas SC-58236, a relatively selective COX-2 inhibitor, significantly inhibited PG production by RA glomeruli. In preliminary studies, to define mechanisms of altered expression of glomerular COX-2, rat mesangial cells were incubated with serum from sham or 2 wk RA. There were significant increases in COX-2 expression in response to 2 wk RA serum. In summary, these results indicate selective increases in renal cortical COX-2 expression following renal ablation.
Cyclooxygenase (COX), the key enzyme required for the conversion of arachidonic acid to prostaglandins was first identified over 20 years ago. Drugs, like aspirin, that inhibit cyclooxygenase activity have been available to the public for about 100 years. In the past decade, however, more progress has been made in understanding the role of cyclooxygenase enzymes in biology and disease than at any other time in history. Two cyclooxygenase isoforms have been identified and are referred to as COX-1 and COX-2. Under many circumstances the COX-1 enzyme is produced constitutively (i.e., gastric mucosa) whereas COX-2 is inducible (i.e., sites of inflammation). Here, we summarize the current understanding of the role of cyclooxygenase-1 and -2 in different physiological situations and disease processes ranging from inflammation to cancer. We have attempted to include all of the most relevant material in the field, but due to the rapid progress in this area of research we apologize that certain recent findings may have been left out.
Prostaglandin endoperoxide synthase (PGH synthase) is responsible for converting arachdonic acid to PGH2, the common precursor of prostaglandins. It has been shown previously that phorbol ester-induced differentiation of human promonocytic leukemia cell lines is accompanied by induction of PGH synthase enzyme and enhanced capacity to produce prostaglandins. However, the identity of the isoform of PGH synthase, i.e., PGH synthase-1 or -2, that is induced under these conditions has not been established. Northern and Western analyses revealed a dramatic increase in levels of PGH synthase-1 mRNA and protein levels within 24 hr after treatment of THP-1 cells with phorbol ester. No significant increase in PGH synthase-2 mRNA or protein was observed. The increases in PGH synthase-1 were accompanied by an enhanced capacity of the cells to produce PGE2. The current findings indicate that expression of PGH synthase-1 is greatly enhanced in a promonocytic cell line by treatment with an agent that induces differentiation.
Tyrosyl radicals have been detected during turnover of prostaglandin endoperoxide H synthase (PGHS), and they are speculated to participate in cyclooxygenase catalysis. Spectroscopic approaches to elucidate the identity of the radicals have not been definitive, so we have attempted to trap the radical(s) with nitric oxide (NO). NO quenched the EPR signal generated by reaction of purified ram seminal vesicle PGHS with arachidonic acid, suggesting that NO coupled with a tyrosyl radical to form inter alia nitrosocyclohexadienone. Subsequent formation of nitrotyrosine was detected by Western blotting of PGHS incubated with NO and arachidonic acid or organic hydroperoxides using an antibody against nitrotyrosine. Both arachidonic acid and NO were required to form nitrotyrosine, and tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide dismutase had no effect on nitration, indicating that peroxynitrite was not the nitrating agent. To identify which tyrosines were nitrated, PGHS was digested with trypsin, and the resulting peptides were separated by high pressure liquid chromatography and monitored with a diode array detector. A single peptide was detected that exhibited a spectrum consistent with the presence of nitrotyrosine. Consistent with Western blotting results, both NO and arachidonic acid were required to observe nitration of this peptide, and its formation was blocked by the PGHS inhibitor indomethacin. Peptide sequencing indicated that the modified residue was tyrosine 385, the source of the putative catalytically active tyrosyl radical.
Prostaglandin endoperoxide synthase (PGHS) is a heme protein that catalyzes the committed step in prostaglandin and thromboxane biosynthesis. Two isoforms of PGHS exist, a constitutive form termed PGHS-1 and an inducible form termed PGHS-2. We report here fluorescence resonance energy transfer analysis of isoform-selective inhibitors interacting with PGHS-1 and PGHS-2. By measuring fluorescence quenching due to the energy transfer of the inhibitor fluorescence to the heme prosthetic group of PGHS, we determined these inhibitors bind in the arachidonic acid substrate access channel with an R0 of 35 A for PGHS-1 with the PGHS-1 inhibitor and an R0 of 21 A for PGHS-2 with the PGHS-2 inhibitor. The observed fluorescence quenching is completely dynamic and dominated by quenching by the heme. Time-resolved results combined with molecular modeling determine the distance from the inhibitor to the heme moiety to be 20 A in PGHS-1 and 18 A in PGHS-2. Preliminary stopped-flow kinetic studies reveal that the rate of quenching is limited by a first-order protein transition, which is slow, and that bound inhibitor undergoes rapid exchange.
It has been proposed that cyclooxygenase (COX)-1 and COX-2 subserve different physiologic functions largely because of the striking differences in their tissue expression and regulation. COX-1 displays the characteristics of a "housekeeping" gene and is constitutively expressed in almost all tissues. COX-1 appears to be responsible for the production of prostaglandins (PG) that are important for homeostatic functions, such as maintaining the integrity of the gastric mucosa, mediating normal platelet function, and regulating renal blood flow. In sharp contrast, COX-2 is the product of an "immediate-early" gene that is rapidly inducible and tightly regulated. Under basal conditions, COX-2 expression is highly restricted; however, COX-2 is dramatically upregulated during inflammation. For example, synovial tissues in patients with rheumatoid arthritis (RA) express increased levels of COX-2. In animal models of inflammatory arthritis, COX-2 increases in parallel with PG production and clinical inflammation. In vitro experiments have revealed increased COX-2 expression after stimulation with proinflammatory cytokines, such as interleukin 1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha), in many cell types, including synoviocytes, endothelial cells, chondrocytes, osteoblasts, and monocytes/macrophages. Another distinguishing characteristic of COX-2 is decreased expression in response to glucocorticoids. COX-2 is also increased in some types of human cancers, particularly colon cancer. Mechanisms underlying the association between COX-2 overexpression and tumorigenic potential may include resistance to apoptosis, or programmed cell death. Upregulated COX-2 expression undoubtedly plays a role in pathologic processes characterized by increased local PG production. One would predict, based on current information regarding the differential tissue expression of COX-1 and COX-2, that highly selective inhibitors of COX-2 will provide effective antiinflammatory activity with marked reduction in toxicity.
Eicosanoids are produced throughout the gastrointestinal tract and are significant mediators of physiologic and pathophysiologic processes. Understanding the precise role(s) of specific eicosanoid metabolites remains a significant challenge, but has led to the development of new pharmacologic strategies for treating NSAID-induced gastroenteropathy and IBD. Given the complex array of arachidonic acid metabolites, the development of more specific and potent inhibitors of these cyclooxygenase isoforms is important for future studies and possible therapeutic applications. Mice have been prepared that lack expression of COX-1 or COX-2. Once these animals have been carefully evaluated, understanding of the role of various pathways of eicosanoid formation in gastrointestinal function, development, and epithelial growth regulation might be improved. Considerable progress has been made in the understanding of arachidonic acid metabolism and in eicosanoid receptor biology. The identification and characterization of an inducible cyclooxygenase isoform has led to important studies evaluating the role of this enzyme in inflammation, neoplasia, and NSAID-induced gastrointestinal injury. The demonstration that COX-2 overexpression in intestinal epithelial cells leads to specific phenotypic changes, such as increased adhesion and inhibition of apoptosis, indicates that this enzyme may alter the tumorigenic potential of epithelial cells and offers hope for the future development of improved chemopreventive agents.
A considerable amount of evidence collected from several different experimental systems indicates that cyclooxygenase-2 (COX-2) may play a role in colorectal tumorigenesis. Large epidemiologic studies have shown a 40-50% reduction in mortality from colorectal cancer in persons taking aspirin or other nonsteroidal antiinflammatory drugs on a regular basis. One property shared by all of these drugs is their ability to inhibit COX, a key enzyme in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX have been characterized, COX-1 and COX-2. COX-2 is expressed at high levels in intestinal tumors in humans and rodents. In this study, we selected two transformed human colon cancer cell lines for studies on the role of COX-2 in intestinal tumorigenesis. We evaluated HCA-7 cells which express high levels of COX-2 protein constitutively and HCT-116 cells which lack COX-2 protein. Treatment of nude mice implanted with HCA-7 cells with a selective COX-2 inhibitor (SC-58125), reduced tumor formation by 85-90%. SC-58125 also inhibited colony formation of cultured HCA-7 cells. Conversely, SC-58125 had no effect on HCT-116 implants in nude mice or colony formation in culture. Here we provide evidence that there may be a direct link between inhibition of intestinal cancer growth and selective inhibition of the COX-2 pathway.