Experimental hyperthyroidism in rats suppresses in vitro prostaglandin metabolism in lung and kidney

Metabolism of prostaglandin (PG) F2α and PGE2 was depressed 40–62% in 100,000 g cytoplasmic supernatants of lungs and kidneys prepared from rats made hyperthyroid by 18 daily L(-) thyroxine injections (200μg,s-c). These hyperthyroid rats had elevated serum thyroxine levels, cardiac hypertrophy and thyroid atrophy. There were no differences in soluble protein concentrations, NAD⁺ utilisation by endogenous enzymes and substrates, or in the NAD⁺ dependence of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) between the supernatants prepared from hyperthyroid rats and saline-injected controls. Thyroxine did not inhibit PG metabolism in vitro up to 260 μM. These results suggest that thyroxine specifically decreases intracellular levels of PG-metabolising enzymes, especially of the rate-limiting 15-PGDH. Metabolism of PGF2α and PGE2 by 15-PGDH was faster in smaller rats and declined with increasing animal weight. These studies imply that some of the clinical features of hyperthyroidism in man might be caused by deficiencies in PG metabolism.     

Indomethacin increases 15-PGDH mRNA expression in HL60 cells differentiated by PMA

We previously reported an induction of 15-hydroxyprostaglandin dehydrogenase type I mRNA (15-PGDH) expression accompanied by a decrease in prostaglandin E2(PGE2) levels during cord blood monocytes differentiation into preosteoclastic cells by 1,25 dihydroxyvitamin D3 (1,25 (OH)2D3). These results suggested a role of prostaglandin (PG) enzymes in adhesion and/or differentiation of monocytes.In the present work, we studied modulation of gene expression of PG metabolism enzymes mRNAs in HL60 cells differentiated by phorbol myristate acetate (PMA) into the monocyte/macrophage lineage. We showed that adhesion of HL60 induced by PMA causes an increase of cyclooxygenase 2 (COX 2) and 15-PGDH mRNAs. When adding indomethacin, a non steroidal antiinflammatory drug known to inhibit COX activity, the cells remained attached and expressed large amounts of 15-PGDH mRNA while COX 2 mRNA expression remained unchanged. Indomethacin, in association with PMA can consequently exert a dual control on key enzymes of PGE2 metabolism without modifying adhesion of the cells.     

Threonine 188 is critical for interaction with NAD+ in human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in the inactivation pathway of prostaglandins. It is a member of the short-chain dehydrogenase family of enzymes. A relatively conserved threonine residue corresponding to threonine 188 of 15-PGDH is proposed to be involved in the interaction with the carboxamide group of NAD+. Site-directed mutagenesis was used to examine the important role of this residue. Threonine 188 was changed to alanine (T188A), serine (T188S) or tyrosine (T188Y) and the mutant proteins were expressed in E. coli. Western blot analysis showed that the expression levels of mutant proteins were similar to that of the wild type protein. Mutants T188A and T188Y were found to be inactive. Mutant T188S still retained substantial activity and the Km value for PGE2 was similar to the wild enzyme; however, the Km value for NAD+ was increased over 100 fold. These results suggest that threonine 188 is critical for interaction with NAD+ and contributes to the full catalytic activity of 15-PGDH.     

15-Hydroxyprostaglandin dehydrogenase is down-regulated in colorectal cancer

Prostaglandin E2 (PGE2) can stimulate tumor progression by modulating several proneoplastic pathways, including proliferation, angiogenesis, cell migration, invasion, and apoptosis. Although steady-state tissue levels of PGE2 stem from relative rates of biosynthesis and breakdown, most reports examining PGE2 have focused solely on the cyclooxygenase-dependent formation of this bioactive lipid. Enzymatic degradation of PGE2 involves the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The present study examined a range of normal tissues in the human and mouse and found high levels of 15-PGDH in the large intestine. By contrast, the expression of 15-PGDH is decreased in several colorectal carcinoma cell lines and in other human malignancies such as breast and lung carcinomas. Consistent with these findings, we observe diminished 15-Pgdh expression in ApcMin+/- mouse adenomas. Enzymatic activity of 15-PGDH correlates with expression levels and the genetic disruption of 15-Pgdh completely blocks production of the urinary PGE2 metabolite. Finally, 15-PGDH expression and activity are significantly down-regulated in human colorectal carcinomas relative to matched normal tissue. In summary, these results suggest a novel tumor suppressive role for 15-PGDH due to loss of expression during colorectal tumor progression.     

C-Terminal region of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase is involved in the interaction with prostaglandin substrates.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S) hydroxyl group of prostaglandins to a 15-keto group resulting in a significant reduction of the biological activities of prostaglandins. Although the key residues involved in NAD+ binding and in catalytic activity have been partially identified, the sites of interaction of the enzyme with the prostaglandin substrates are yet to be determined. Homology analysis of the primary structures of 15-PGDH from human, mouse and rat indicates that the sequences are almost homologous except for two regions near the C-terminus. The involvement of the C-terminal region in catalytic activity was examined by studies on C-terminally truncated enzymes and on human/rat chimeric enzymes. When three to four amino acids were removed successively from the C-terminal end of human 15-PGDH, the truncated enzymes exhibited decreasing Vmax/Km ratios and increasing Km values for PGE2 as the chain was shortened. Similarly, when the C-terminal 14 amino acids of human 15-PGDH were replaced by the C-terminal 14 amino acids of rat 15-PGDH or vice versa, the Vmax/Km ratios and the Km values for prostaglandin E2 of the chimeric enzymes were in between those of the two wild-type enzymes. This indicates that the catalytic effectiveness of human 15-PGDH decreases as the C-terminal region is gradually removed or replaced by rat sequences. The C-terminal region appears to be more important for the interaction of the enzyme with the prostaglandin substrates than with the coenzyme.     

15-Hydroxyprostaglandin-dehydrogenase is involved in anti-proliferative effect of non-steroidal anti-inflammatory drugs COX-1 inhibitors on a human medullary thyroid carcinoma cell line

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin (PG) synthesis enzymes, the cyclooxygenases (COX-1 and 2). It is suggested that these enzymes are not their only targets. We reported that in tumoral TT cell, indomethacin, in vivo and in vitro, decreases proliferation and increases activity of 15-hydroxyprostaglandin-dehydrogenase (15-PGDH), the PG catabolism key enzyme. Here, we show that the COX-1 inhibitors, selective or not, and sulindac sulfone, a non-COX inhibitor, increased 15-PGDH activity and reduced PGE2 levels. This increase was negatively correlated to the decrease in cell proliferation and suggested that 15-PGDH could be implicated in NSAIDs anti-proliferative effect. Indeed, the silencing of 15-PGDH expression by RNA interference using 15-PGDH specific siRNA enhanced TT cell proliferation and abolished the anti-proliferative effect of a representative non-selective inhibitor, ibuprofen. Moreover, a specific inhibitor of 15-PGDH activity, CAY 10397, completely reversed the effect of ibuprofen on proliferation. Consequently our results demonstrate that, at least in TT cells, 15-PGDH is implicated in proliferation and could be a target for COX-1 inhibitors specific or not. NSAIDs defined by their COX inhibition should also be defined by their effect on 15-PGDH.     

Involvement of the constitutive prostaglandin E synthase cPGES/p23 in expression of an initial prostaglandin E2 inactivating enzyme, 15-PGDH

We previously showed that cytosolic prostaglandin (PG) E synthase (cPGES/p23) which isomerizes PGH(2) to PGE(2), is essential for fetal mouse development. Embryonic fibroblasts derived from cPGES/p23 knockout mice generated higher amounts of PGE(2) in culture supernatants than wild-type-derived cells. In order to elucidate this apparent conflict that absence of PGE(2) synthetic enzyme caused facilitation of PGE(2) biosynthesis, we examined expression of the PGE(2) degrading enzyme in embryonic fibroblasts. We report here that embryonic fibroblasts deficient in cPGES/p23 decreased the expression of the PGE(2) degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which catalyzes the inactivating conversion of the PGE(2) 15-OH to a 15-keto group, compared with that of wild-type. In addition, rat fibroblastic 3Y1 cells harboring cPGES/p23 siRNA exhibited lower 15-PGDH expression than mock-transfected cells. Furthermore, forcible expression of cPGES/p23 in 3Y1 cells resulted in facilitation of 15-PGDH promoter activity. These results suggest that the PGE(2)-inactivating pathway is controlled by the PGE(2) biosynthetic enzyme, cPGES/p23.     

Stimulation of prostaglandin synthesis in rabbit gastric antral mucosal slices by desferrioxamine in vitro.

Desferrioxamine is an iron-chelating agent used in the treatment of iron overload. It is a powerful inhibitor of iron-dependent radical reactions. The effect of desferrioxamine of prostaglandin (PG) synthesis and metabolism in rabbit gastric antral mucosal slices has been examined. Desferrioxamine significantly enhanced the production of PGE2 and PGF2 alpha. The formation of 13,14-dihydro-15-keto PGE2 and 13,14-dihydro-15-keto PGF2 alpha was also increased slightly by desferrioxamine. The addition of Fe3+ or Al3+ blocked the stimulatory action of desferrioxamine on PGE2 and PGF2 alpha production. Desferrioxamine appears to be stimulating the activity of PG cyclooxygenase through the removal of endogenous antral mucosal iron. These results suggest that desferrioxamine has the potential to increase the PG levels in gastric mucosa by primarily stimulating PG biosynthesis. The possibility that desferrioxamine may be of therapeutic value in the treatment of ischemic injury in the stomach is discussed.     

Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins. Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme. Site-directed mutagenesis was used to examine the important roles of these residues. Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography. Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP+ but still retained substantial activity with NAD+. Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD+ and NADP+ with an increase in activity nearly 3-fold over that of the wild type. However, approximately 30-fold higher in K(m) for NADP+ than that of the wild type enzyme for NAD+ was found for mutants Q15K-W37R and Q15R-W37R. Similarly, the K(m) values for PGE(2) of mutants were also shown to increase over that of the wild type. Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP+ but not with NAD+. The triple mutants showed a decrease in K(m) for NADP+ but an increase in K(m) for PGE(2). Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K(m) values for both NADP+ and PGE(2) to levels comparable to those of the wild type. These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD+ to NADP+ by changing a few critical residues near the N-terminal end.     

Effective use of rabbit gastric antral mucosal slices in prostaglandin synthesis and metabolism studies

Intact slice preparations of rabbit stomach (antral mucosa, corporal mucosa, antral muscle and corporal muscle) were incubated and the released prostaglandins (PGs) were measured by reverse-phase high-performance liquid chromatography using 9-anthryldiazomethane for derivatization. With respect to total PG production, the highest amounts were generated by antral mucosal slices. Antral mucosal slices produced PGE2, 6-keto PGF1 alpha, thromboxane B2, PGF2 alpha and PGD2 (in descending order of magnitude) and possessed a high capacity for producing 13,14-dihydro-15-keto derivatives of both PGE2 and PGF2 alpha. Studies utilizing aspirin, EGTA or Ca2+ revealed that PG release by antral mucosal slices in the present in vitro system reflects a composite of the activities of phospholipase A3, PG cyclooxygenase and PG-metabolizing enzymes. These results show that antral mucosal slices will be useful in physiological and pharmacological studies on PG synthesis and metabolism of the stomach.     

Increased Expression of 15-Hydroxyprostaglandin Dehydrogenase in Spinal Astrocytes During Disease Progression in a Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive, and fatal neurodegenerative disease caused by selective loss of motor neurons. Both ALS model mice and patients with sporadic ALS have increased levels of prostaglandin E2 (PGE2). Furthermore, the protein levels of microsomal PGE synthase-1 and cyclooxygenase-2, which catalyze PGE2 biosynthesis, are significantly increased in the spinal cord of ALS model mice. However, it is unclear whether PGE2 metabolism in the spinal cord is altered. In the present study, we investigated the protein level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin metabolism, in ALS model mice at three different disease stages. Western blotting revealed that the 15-PGDH level was significantly increased in the lumbar spinal cord at the symptomatic stage and end stage. Immunohistochemical staining demonstrated that 15-PGDH immunoreactivity was localized in glial fibrillary acidic protein (GFAP)-positive astrocytes at the end stage. In contrast, 15-PGDH immunoreactivity was not identified in NeuN-positive large cells showing the typical morphology of motor neurons in the anterior horn. Unlike 15-PGDH, the level of PGE2 in the spinal cord was increased only at the end stage. These results suggest that the significant increase of PGE2 at the end stage of ALS in this mouse model is attributable to an imbalance of the synthetic pathway and 15-PGDH-dependent scavenging system for PGE2, and that this drives the pathogenetic mechanism responsible for transition from the symptomatic stage.     

Understanding human 15-hydroxyprostaglandin dehydrogenase binding with NAD+ and PGE2 by homology modeling, docking and molecular dynamics simulation

Homology modeling, molecular docking, and molecular dynamics simulation have been performed to determine human 15-hydroxyprostaglandin dehydrogenase (15-PGDH) binding with its NAD+ cofactor and prostaglandin E2 (PGE2) substrate. The computational studies have led to a three-dimensional (3D) model of the entire 15-PGDH-NAD+-PGE2 complex, demonstrating the detailed binding of PGE2 with 15-PGDH for the first time. This 3D model shows specific interactions of the protein with the cofactor and substrate in qualitative agreement with available experimental data. Our model demonstrates the PGE2-binding cavity of the protein for the first time. The model further leads to an interesting prediction that the catalytic activity of 15-PGDH should also significantly be affected by Gln148, in addition to the previously known three catalytic residues (Ser138, Tyr151, and Lys155). The reported 3D model of 15-PGDH-NAD+-PGE2 complex might be valuable for future rational design of novel inhibitors of 15-PGDH.     

PRESENCE OF 15-HYDROXY PROSTAGLANDIN DEHYDROGENASE, PROSTAGLANDIN-Δ13-REDUCTASE AND PROSTAGLANDIN E-9-KETO(α)-REDUCTASE IN THE FROG SPINAL CORD

Metabolism of prostaglandin E₁ (PGE₁) and F_1α (PGF_1α) was studied in the frog spinal cord, using a hemisected preparation in vitro and tissue homogenates (whole honiogenate and tissue fractions). In the intact tissue, PGE, was converted to three Metabolites, 1 to 111, whereas only Metabolites 11 and 111 werc detected in experiments with PGF_1α. Work with tissue homogenatcs confirmed that PG transformation is enzymatic, and endproducts were identified as PGF_1α (Metabolite 1), 15-kcto metabolite (Metabolite 11) and 15-keto-13,14-dihydro metabolite (Metabolite 111). The 15-keto-13,14-dihydro metabolite was formed via the 15-keto metabolite which is consistent with findings elsewhere. These results establish the presence in the frog spinal cord of two pathways for PG metabolism, consisting one of the 15-hydroxy prostaglandin dehydrogenase (15-PGDH) and the prostaglandin-A13- reductase (13-PGR), the other of the prostaglandin E 9-keto(α)-reductase (9K-PGR). 9K-PGR is regarded as an inactivating enzyme because amphibian spinal neurons are less responsive to PGF, than to PGE₁. In the intact or in the homogenized tissue, PGE, is metabolized more efficiently by the 15-PGDH/13-PGR than by the 9K-PGR route. The 15-PGDH metabolizes PGE, more readily than PGF_1α. The present findings, together with the previous demonstration of active PG synthesis in the tissue and the potent actions of exogenous PGs, strongly suggest that the PGs play an important role in the function of neurons in the frog spinal cord.     

Role of glutamine 148 of human 15-hydroxyprostaglandin dehydrogenase in catalytic oxidation of prostaglandin E2

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins. This enzyme oxidizes the C-15 hydroxyl group of prostaglandins and lipoxins to produce 15-keto metabolites which exhibit greatly reduced biological activities. A three-dimensional (3D) structure of 15-PGDH based on the crystal structures of the levodione reductase and tropinone reductase-II was generated and used for docking study with NAD+ coenzyme and PGE2 substrate. Three well-conserved residues among SDR family which correspond to Ser-138, Tyr-151, and Lys-155 of 15-PGDH have been shown to participate in the catalytic reaction. Based on the molecular interactions observed from 3D structure of 15-PGDH, we further propose that Gln-148 in 15-PGDH is important in properly positioning the 15-hydroxyl group of PGE2 by hydrogen bonding with the side-chain oxygen atom of Gln-148. This residue is found to be less conserved and replaceable by glutamyl, histidinyl, and asparaginyl residues in SDR family. Accordingly, site-directed mutagenesis of Gln-148 of 15-PGDH to alanine, glutamic acid, histidine, and asparagine (Q148A, Q148E, Q148H, and Q148N) was carried out. The activity of mutant Q148A was not detectable, whereas those of mutants Q148E, Q148H, and Q148N were comparable to or higher than the wild type. This indicates that the side-chain oxygen or nitrogen atom at position 148 of 15-PGDH plays an important role in anchoring C-15 hydroxyl group of PGE2 through hydrogen bonding for catalytic reaction.     

Inhibition of 15-hydroxy prostaglandin dehydrogenase and increase of prostaglandin E2: effect of sofalcone on rat gastric mucosa

The effect of sofalcone, an anti-ulcer agent, on gastric mucosal prostaglandin (PG) metabolism was studied. Gastric mucosal PGE2 was determined in rats in which PGE2 synthesis was inhibited by preadministration of indomethacin. Oral administration of sofalcone at doses of 200 and 400 mg/kg significantly inhibited the PG metabolizing enzyme, 15-hydroxy-PG-dehydrogenase (15-OH-PG-DH) activity and increased PGE2 contents in the rat gastric mucosa. The inhibition of 15-OH-PG-DH activity was accompanied by an increase of PGE2 contents up to 6 hours after the administration of sofalcone. These changes, however, were not observed 12 hours after its administration. Intraperitoneally administered sofalcone also inhibited 15-OH-PG-DH activity and increased PGE2 content. The inhibition of 15-OH-PG-DH activity by sofalcone was noncompetitive and uncompetitive against substrates NAD and PGE1, respectively. These results suggest that the increase of the gastric PGE2 level is mainly due to the inhibition of 15-OH-PG-DH activity, and this increase in PGE2 may be involved in the anti-ulcer effect of sofalcone.     

Prostaglandin catabolism in fetal and maternal tissues--a study of 15-hydroxyprostaglandin dehydrogenase and delta 13 reductase with specific assay methods

Recent studies have demonstrated that extraductal tissues such as lung are important sources of prostaglandin E2 which maintains the patency of ductus arteriosus in fetuses and prematurely-born infants. Also, organs such as lung are known to be active in the catabolism of PGE2. Earlier studies of enzymes involved in the catabolism of PGE2 such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and delta 13 reductase all used non-specific methods. In the present report, we studied 15-PGDH in fetal and maternal rat lung, kidney, and fetal lamb lung, kidney and ductus arteriosus with the use of a specific substrate (15-S)-[15(3)H-PGE2]. In addition, we measured the activity of delta 13 reductase in these tissues by measuring the conversion of [1-14C]-15-keto PGE2 to [1-14C]-15-keto-13,14-dihydro PGE2. The results from these studies demonstrated that in fetal rat lung and kidney, 15-PGDH activities increased rapidly while delta 13 reductase remained unchanged during late gestation. Ductus arteriosus possessed little 15-PGDH activities. These results strongly suggest that extraductal regulation of PGE2 metabolism is important in determining ductal caliber in fetuses and prematurely delivered neonates.     

Mechanical stress and prostaglandin E2 synthesis in cartilage

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE2) is one of the main catabolic factors involved in OA in which metalloproteinase (MMP) is crucial for cartilage degradation. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy-prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE2. Among the isoforms described, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. We investigated the regulation of the COX, PGES and 15-PGDH and MMP-2, MMP-9 and MMP-13 genes by mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) from 2 to 24 h. After determination of the PGE2 release in the media, mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blot respectively. Mechanical compression of cartilage explants significantly increased PGE2 production in a time dependent manner. This was not due to the synthesis of IL-1, since pretreatment with IL1-Ra did not alter the PGE2 synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE2 synthesis after 18 hours suggesting that PGE2 synthesis could be altered by the induction of 15-PGDH expression. MAPK are involved in signaling, since specific inhibitors partially inhibited COX-2 and mPGES-1 expressions. Lastly, compression induced MMP-2, -9, -13 mRNA expressions in cartilage. We conclude that dynamic compression induces pro-inflammatroy mediators release and matrix degradating enzymes synthesis. Notably, compression increases mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

Novel prostaglandin dehydrogenase in rat skin.

Present evidence suggests that skin is an important organ of prostaglandin metabolism. To clarify its role, the basic kinetics of 15-hydroxyprostaglandin dehydrogenase (PGDH) from rat skin were investigated with either NAD+ of NADP+ as co-substrate. Prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) were used as substrates and preliminary studies were made of the inhibitory effects of the reduced co-substrates NADH and NADPH. A radiochemical assay was used in which [3H]PGF2 alpha or [14C]PGE2 were incubated with high-speed supernatant of rat skin homogenates. The substrate and products were then extracted by solvent partition, separated by t.l.c. and quantified by liquid-scintillation counting. At linear reaction rates and at an NAD+ concentration of 10 mM the mean apparent Km for PGF2 alpha was 24 microM with a mean apparent Vmax. of 9.8 nmol/s per litre of reaction mixture. For PGE2 the mean apparent Km was 8 microM, with a mean apparent Vmax, of 2.7 nmol/s per litre of reaction mixture. With NADP+ as a co-substrate at a concentration of 5 mM a mean apparent Km of 23 microM was obtained for PGF2 alpha with a mean apparent Vmax. of 5.2 nmol/s per litre. For PGE2 values of 7.5 microM and 3.0 nmol/s per litre were obtained respectively. These results show that skin contains NAD+- and NADP+-dependent PGDH. An important finding was that the NADP+-linked enzyme gave Km values for PGE2 that were considerably lower than those reported for NADP+-linked PGDH from other tissues. Furthermore, preliminary inhibition studies with the NAD+-linked PGDH system indicate that this enzyme is not only inhibited by NADH, but also by NADPH, a property not previously reported for NAD+-linked PGDH derived from other tissues.