B. M. Peskar

On the synthesis of prostaglandins by human gastric mucosa and its modification by drugs

1. Specific radioimmunoassays for the prostaglandins E2, A2 and F2alpha were used to study the synthesis of prostaglandins by gastroscopically obtained small biopsy specimens of human gastric corpus mucosa. 2. Both prostaglandin E2 and prostaglandin F2alpha were found to be synthesized from arachidonic acid by themicrosomal fraction of human gastric mucosa. The synthesis of prostaglandin E2 exceeded that of prostagladin F2alpha by a factor of about 10. 3. Synthesis of prostaglandin A2 or prostaglandin B2 was not observed under the same incubation conditions. 4. Indometacin effectively inhibited synthesis of both prostaglandin E2 (ID50 4.2 microng/ml) and prostaglandin F2alpha (ID50 1.8 microng/ml) by human gastric mucosa, while paracetamol even in a concentration of 310 microng/ml did not influence prostaglandin synthesis. The anti-ulcer agent carbenoxolone, which has been shown to inhibit prostaglandin inactivation, at the same concentration only slightly inhibited (about 20%) prostaglandin synthesis. 5. The results support the hypothesis that the gastro-intestinal effects or side effects of several drugs are mediated by an influence on the enzymes of prostaglandin synthesis or inactivation.

On the metabolism of prostaglandins by human gastric fundus mucosa

1. Specific radioimmunoassays for the prostaglandins E2, F2alpha and A2 and the metabolites 13,14-dihydro-15-keto-prostaglandin E2, 15-keto-prostaglandin F2alpha and 13,14-dihydro-15-keto-prostaglandin F2alpha were used to study the metabolism of prostaglandins by gastroscopically obtained small biopsy specimens of human gastric fundus mucosa. 2. Three prostaglandin-metabolizing enzymes were found in the 100 000 X g supernatant of human gastric fundus mucosa, 15-hydroxy-prostaglandin-dehydrogenase, delta13-reductase and delta9-reductase. The specific activity was highest for 15-hydroxy-prostaglandin-dehydrogenase and lowest for delta9-reductase. 3. Formation of prostaglandin A2 (or B2) was not observed under the same conditions. 4. None of the three enzyme activities detected in the 100 000 X g supernatant was found in the 10 000 X g and 100 000 X g pellets of human gastric fundus mucosa. 5. The results indicate that high speed supernatant derived from human gastric mucosa can rapidly metabolize prostaglandin E2 and prostaglandin F2alpha to the 15-keto and 13,14-dihydro-15-keto-derivatives. Furthermore, prostaglandin E2 can be converted to prostaglandin F2alpha, the biological activity of which, on gastric functions, differs from that of prostaglandin E2.

Radioimmunological determination of prostaglandin D2 synthesis in human thrombocytes

A specific radioimmunoassay for prostaglandin D2 was developed. Using the radioimmunoassay, prostaglandin D2 synthesis by human thrombocytes was measured. While the cyclooxygenase inhibitor indomethacin inhibits formation of prostaglandin D2, increased formation of prostaglandin D2 was observed in the presence of the thromboxane synthetase inhibitor imidazole.

Prostaglandins and prostaglandin metabolites in human gastric juice.

Human gastric juice contains higher concentrations of PG metabolites than of unmetabolized PG indicating that local metabolism might play a role in limiting the biological activity of PG in gastric mucosa and has to be considered when investigating endogenous gastric PG. A major fraction of the 15-keto-13,14-dihydro-PGE2 (KH2PGE2) formed in gastric mucosa and released into the gastric lumen seems to be rapidly dehydrated to a compound co-chromatographing with KH2PGA2, while the amounts of the bicyclic degradation product 11-deoxy-13,14-dihydro-15-keto-11,16-cyclo-PGE2 (11-deoxy-KH2-cyclo-PGE2), as measured by radioimmunoassay, in freshly extracted gastric juice are negligible. Stimulation of secretion with pentagastrin does not influence significantly the concentrations of PG and PG metabolites in human gastric juice, but total output tends to increase parallel to the increase in secretion volume. Levels of immunoreactive 6-keto-PGF1 alpha in human gastric juice are much lower than those of PGE2. Since human gastric mucosa synthesizes conciderable amounts of PGI2 and 6-keto-PGF1 alpha in vitro, the low levels of 6-keto-PGF1 alpha in gastric juice might indicate that PGI2 formed by gastric mucosa in vivo is, like PGE2 and PGF2 alpha, rapidly metabolized and/or removed preferentially via the blood stream.

Release of prostaglandins by small intestinal tissue of man and rat in vitro and the effect of endotoxin in the rat in vivo

Rat jejunal tissue in vitro synthesizes large amounts of prostaglandin (PG) D2 and smaller amounts of 6-keto-PGF1 alpha and PGE2, whereas human small intestinal mucosa synthesizes much smaller amounts of the three PG determined with about equal amounts of PGE2 and PGD2. Intraperitoneal administration of bacterial endotoxin to rats induce fluid accumulation in the small intestine and increases significantly the release of PGD2, PGE2 and 6-keto-PGF1 alpha into the small intestinal lumen in vivo. Endotoxin-induced stimulation of PG release is particularly pronounced for PGD2. Fluid accumulation and PG output are inhibited by indomethacin. It seems possible that the different total amounts of PG synthesized by small intestinal tissue of man and rat as well as the different pattern of PG released might contribute to species-specific responses of the gastrointestinal tract to various pathophysiological stimuli.

Antibodies against dehydration products of 15-keto-13,14-dihydro-prostaglandin E2

Levels of 1.5-keto-13,14-dihydro metabolites of prostaglandins (PCs) Ez and FZol in plasma reflect more accurately PG biosynthesis than levels of the primary PGs [l]. However, so far there are only few reports on the development of radioimmunoassays for the PGE, metabolite 15-keto-13,14-dihydroPGEz (KH2PGE2) [2-51. Furthermore, the reliability of these assays seems to be lower than the reliability of radioimmunoassays for the corresponding PGF,, metabolite [5,6]. This is probably a consequence of the chemical instability of KH2PGE2, which is easily dehydrated to KHzPGAz [6,7]. KHZPGA2 can bind to proteins and, under certain conditions like alkaline pH or in the presence of albumin, rearrange to the stable bicyclic degradation end product 11 -deoxy15-keto-13 ,14-dihydro-l l ,16-cycle-PGE? (DKH*cycle-PGE,) [6-81. We have described the first radioimmunoassay for KH2PGE2 [2]. Now we show that antisera obtained from rabbits immunized with a KHzPGEz-conjugate contain not only antibodies against KH,PGE,, but also against the KHzPGEz dehydration products KHIPGAz and DKH,-cycloPGE2. The three antibody populations differ in their specificities as well as in their association constants. With [3H]KHzPGEz or the tritiated dehydration products as tracers the same antisera can be used for radioimmunoassays of KH,PGE*, KH2PGAZ and DKH+yclo-PGE,, the specificity and sensitivity of the assays being dependent on the labelled ligand used. The radioimmunoassays for KH2PGE, and KH,PGA* may be used for the direct determination of these compounds under defined conditions. Thus, the radioimmunoassay for KH,PGA2 may be especially useful for the determination of the PGE2

Relationship between inhibition of prostaglandin production and gastric mucosal damage induced by anti‐inflammatory drugs may depend on type of drugs and species

the concentrations used and the DNA-berberine mixture R E F E R E N C E S (P/D = 1111) a t various concentrations up to M strictly followed Beers Law. Thus DNA-berberine fluorescence enhancement at p H 3.7 possibly reflects high quantum yield complex formation with the D N A bases, which are still closely arranged, providing the relatively hydrophobic environment for interaction (Love et a1 1978). Many drugs have been known to bind preferentially to regions in the DNA molecule (Pack & Loew 1978; Weisblum & de Haseth 1972; Jorgenson et al 1978). On interaction with the drugs, regions with different binding affinity may give different fluorescence quantum yields. Perhaps, as D N A concentration increases. berberine migrates from lower affinity binding sites to higher affinity binding sites with a consequent enhancement in fluorescence but no significant change in the u.v.-visible spectra. When the P/D ratio reaches 110/1 all drug molecules have occupied the high affinity sites. From our studies, chloroquine and quinacrine binding t o D N A did not lead to such high fluorescence enhancement over p H 2.5 to 11.5. This work was supported by the National Research Council of Thailand and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. Benesi, H. A., Hilderbrand, J. H. (1949) J. Am. Chem. SOC. 7 1 : 2703-2707 Blake, A., Peacocke, A. R. (1968) Biopolymers 6: 1225-1 253 Creasy, W. A. (1979) Biochem. Pharmacol. 28: 10811084 Davidson, M. W., Lopp, I., Alexander, S., Wilson, W. D. (1977) Nucl. Acid Res. 4: 2697-2712 Hahn, F. E., Ciak, J. (1974) in: Corcoran, J. W., Hahn, F. E. (eds) Antibiotics Vol. 111, Mechanism of Action of Antimicrobial and Antitumor Agents. SpringerVerlag, Berlin, pp 577-584 Hahn, F. E., Krey, A. K. (1971) Progr. Mol. Subcell. Biol. 2: 134-151 Jorgenson, K. F., van de Sande, J. H., Lin, C. C. (1978) Chromosoma 68 : 287-302 Krey, A. K., Hahn, F. E. (1969) Science 166: 757-758 Love, L. J. C., Upton, L. M., Ritter, 111, A. W. (1978) Anal. Chem. 50: 2059-2064 Pack, G. R., Loew, G . (1978) Biochim. Biophys. Acta 519: 163-172 Panijpan, B. (1979) Trends Biochem. Sci. 4: N2IO-211 Perrin, D. D. (1963) Aust. J. Chem. 16: 572-578 Weisblum, B., de Haseth, P. L. (1972) Proc. Natl. Acad. Sci. USA 69: 629-632 Yamagishi, H. (1962) J. Cell Biol. 15: 589-592

On the occurrence of prostacyclin metabolites in plasma and vascular tissue as determined radioimmunologically

Circulating prostaglandins (PG) of the E and F series are rapidly inactivated, mainly in the lung [ 11, by the enzymes 1 S-hydroxy-PGdehydrogenase and PG-A13-reductase [2]. Thus, levels of the 15keto13,14dihydro metabolites in plasma reflect more accurately PG biosynthesis than levels of the primary PG [2]. On the other hand, prostacyclin (PG12) has been suggested to be a circulating hormone [3,4], as its biological activity is not significantly diminished during passage through the lung [5], probably because it is not a substrate for the PG transport system across the cell membranes [6]. In aqueous solution PGIZ is hydrated to the biologically much less active 6-ketoPGFro [7], which is only slowly metabolized by 15-hydroxy-PGdehydrogenase and PG-A13-reductase [8,9]. Contrary to 6-keto-PGFro, PGIZ has a high affinity for 15-hydroxy-PGdehydrogenase of several organs [9-l l] including vascular tissue [12] in vitro. While in the rat in vivo in one study [13] no metabolism of 6-keto-PGFio via the 15-hydroxy-PGdehydrogenase pathway was observed, in other studies using different experimental conditions considerable oxidation of the C-l 5 hydroxyl group and reduction of the Al3 double bond of exogenous PGI? [6,14] and to a smaller extent of 6-keto-PGFro [ 141 have been shown to occur. The stable metabolites of PGIz resulting from these enzyme reactions followed by hydration are 6,15diketoPGFro and 6.15-diketo-13,14-dihydro-PGF,,. We describe here radioimmunoassays for these two PGIZ metabolites and determinations of the occurrence of these compounds in rat vascular tissue incubated in vitro as well as in rat and human plasma.

Advances in hapten immunoassays

SummaryThe recent advances in hapten immunoassays, especially radioimmunoassays for drugs, are reviewed. The progress in immunoassay methodology as well as the development of new radioimmunoassays for low molecular weight compounds are described.

Release of Prostaglandin D2 and Thromboxane B2 from Human Thrombocytes: Radioimmunological Determination and Modification by Drugs

Radioimmunoassays for prostaglandin D2 (PGD2) and thromboxane B2 (TXB2) were developed. Antisera were obtained from rabbits immunized with the corresponding hapten-bovine serum albumine conjugates. The conjugates were synthesized using N,N′ -carbonyldiimidazole as coupling reagent (Axen, Prostaglandins 5, 45, 1974). The radioactive tracer for the TXB2 assay was synthesized by incubation of 3H-arachidonic acid as substrate with washed human thrombocytes as enzyme source. The label for the PGD2 assay was also synthesized from 3H-arachidonic acid using tne microsomal fraction of sheep seminal vesicles as enzyme source. Both radioimmunoassays are highly specific and recognize the ring structure and the hydroxyl group at position C15 as immunodominant parts of the hapten molecules. The detection limits are 40 pg for TXB2 and 200 pg for PGD2. The radioimmunoassays were used to determine the release of TXB2 and PGD2 from washed human thrombocytes after addition of thrombin. While only small amounts of immunoreactive PGD2 were released in control incubates, the TX synthetase inhibitor imidazole (1.47 mM) increased PGD2 as well as PGF2α and PGE2 release and simultaneously reduced TXB2 release significantly. Indometacin (2.8 μM) abolished both TXB2 and PG release in response to thrombin. The PGD2 found in the incubates could be formed either enzymatically or non-enzymatically by isomerization from its endoperoxide precursor PGH2. The thrombin-induced formation of PGD2 by thrombocytes could be important in view of the potent antiaggregatory activity of PGD2.