Characterization of multiple Chinese hamster carbonyl reductases

Carbonyl reductase (CR) is an enzyme which can catalyze the oxidoreduction of various carbonyl compounds in the presence of NAD(P)H. With the PCR method, using primers carrying the conserved nucleotide sequence among mammalian CRs, we isolated three different cDNAs (CHCR1, CHCR2 and CHCR3) which encode a unique carbonyl reductase from the Chinese hamster. The PCR products of CHCR1 and CHCR2 were clearly isolated with Bpu1102I, BspEI and XmaI restriction enzymes. The nucleotide-sequence of CHCR3 was completely different from those of CHCR1 and CHCR2. The predicted double-wound betaalphabetaalpha-structures of the CHCRs suggests the presence of a typical NADP(+)-binding motif and is similar to the corresponding region of 3alpha,20beta-hydroxysteroid dehydrogenase and mouse lung tetrameric carbonyl reductase. The deduced amino acid sequence of CHCR1 showed a high homology to CHCR2 (>96%) and the other mammalian CRs (>81%). However, CHCR3 showed a high homology to human CBR3 (>86%) and a relatively lower homology to the other CHCRs (<76%). Bacterial recombinant CHCRs showed typical carbonyl reductase activities towards 4-benzoylpyridine, 4-nitrobenzaldehyde and pyridine 4-carboxyaldehyde. These three CRs showed not only 3-keto reductase of steroids, but also 20-keto reductase. However, these CRs did not show any activity of 17-keto reductase activity. Both CHCR1 and CHCR2 have prostaglandin 9-keto reductase and 15-hydroxyprostaglandin dehydrogenase activities towards PGE(2) and PGF(2alpha) from the analyses of enzymatic reaction products. The results of Western blotting and RT-PCR suggest these CHCRs have a tissue-dependent-distribution in the Chinese hamster.     

Characterization of multiple Chinese hamster carbonyl reductases

Carbonyl reductase (CR) is an enzyme which can catalyze the oxidoreduction of various carbonyl compounds in the presence of NAD(P)H. With the PCR method, using primers carrying the conserved nucleotide sequence among mammalian CRs, we isolated three different cDNAs (CHCR1, CHCR2 and CHCR3) which encode a unique carbonyl reductase from the Chinese hamster. The PCR products of CHCR1 and CHCR2 were clearly isolated with Bpu1102I, BspEI and XmaI restriction enzymes. The nucleotide-sequence of CHCR3 was completely different from those of CHCR1 and CHCR2. The predicted double-wound βαβα-structures of the CHCRs suggests the presence of a typical NADP⁺-binding motif and is similar to the corresponding region of 3α,20β-hydroxysteroid dehydrogenase and mouse lung tetrameric carbonyl reductase. The deduced amino acid sequence of CHCR1 showed a high homology to CHCR2 (>96%) and the other mammalian CRs (>81%). However, CHCR3 showed a high homology to human CBR3 (>86%) and a relatively lower homology to the other CHCRs (<76%). Bacterial recombinant CHCRs showed typical carbonyl reductase activities towards 4-benzoylpyridine, 4-nitrobenzaldehyde and pyridine 4-carboxyaldehyde. These three CRs showed not only 3-keto reductase of steroids, but also 20-keto reductase. However, these CRs did not show any activity of 17-keto reductase activity. Both CHCR1 and CHCR2 have prostaglandin 9-keto reductase and 15-hydroxyprostaglandin dehydrogenase activities towards PGE₂ and PGF_2α from the analyses of enzymatic reaction products. The results of Western blotting and RT-PCR suggest these CHCRs have a tissue-dependent-distribution in the Chinese hamster.     

Prostacyclin induces a surprising long-lasting motility in quiescent uterine strips (indomethacin-treated) isolated from ovariectomized rats

Dose-response curves for several prostaglandins (PGI2; PGD2; PGF2 and PGE2); BaCl2 or prostaglandin metabolites (15-keto-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha; 6-keto-PGF1 alpha and 6-keto PGE1 in quiescent (indomethacin-treated) uterine strips from ovariectomized rats, were constructed. All PGs tested as well as BaCl2, triggered at different concentrations, evident phasic contractions. Within the range of concentrations tested the portion of the curves for the metabolites of PGF2 alpha was shifted to the right of that for PGF2 alpha itself; the curve for 6-keto-PGF1 alpha was displaced to the right of the curve for PGI2 and that for 6-keto-PGE1 to the left. It was also demonstrated that the uterine motility elicited by 10(-5) M PGF2 alpha and its metabolites was long lasting (more than 3 hours) and so it was the activity evoked by PGI2;6-keto-PGF1 alpha and BaCl2, but not the contractions following 6-keto-PGE1, which disappeared much earlier. The contractile tension after PGF2 alpha; 15-keto-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha and PGI2, increased as time progressed whilst that evoked by 6-keto-PGF1 alpha or BaCl2 fluctuated during the same period around more constant levels. The surprising sustained and gradually increasing contractile activity after a single dose of an unstable prostaglandin such as PGI2, on the isolated rat uterus rendered quiescent by indomethacin, is discussed in terms of an effect associated to its transformation into more stable metabolites (6-keto-PGF1 alpha, or another not tested) or as a consequence of a factor which might protects prostacyclin from inactivation.     

Phorbol myristate acetate-stimulated release of cyclooxygenase products in rat pleural cells: derivatization of prostaglandins with 9-anthryldiazomethane for fluorometric determination by high performance liquid chromatography

White cells were collected from the wash of rat pleural cavity after exsanguination. The incubation mixture of the pleural cells with 1 microM phorbol myristate acetate (PMA) was extracted with acidified ethanol and purified with a Sep-pak C18. The resultant fraction containing prostaglandins (PG) and thromboxane (TX) was allowed to react with 9-anthryldiazomethane (ADAM). After removing contaminants and degraded reagent by silica gel Sep-pak, samples were applied to reversed phase high performance liquid chromatography of octadecylsilyl silica gel and monitored by a fluorescent detector. ADAM derivatives of the authentic PGD2, PGE2, PGF2 alpha, 6-keto-PGF1 alpha, 6-keto-PGE1, TXB2, 15-keto-PGE2, 13,14-dihydro-15-keto-PGF2 alpha and 13,14-dihydro-15-keto-PGE2 showed linear regression lines of peak heights within a range of 0.5-25 ng. By using this method PGD2, 6-keto-PGF1 alpha and TXB2 were detected in the incubation mixture of the rat pleural cells with PMA. The result clarified the origin of these PGs and TX found in the exudate of rat pleurisy induced by PMA. ADAM method for HPLC with a help of clean-up by Sep-pak could be a useful tool for detection of a series of arachidonate metabolites in biological materials.     

Tissue levels of prostaglandins and what do they mean? Studies on the guinea-pig uterus

The ratios of the concentrations of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha in guinea-pig uterine horns, which were removed and placed in ethanol in 1.5 to 2 min, were 0.3:1.0:0.6 on day 7 and 13.8:1.0:0.8 on day 15 of the oestrous cycle. Adding indomethacin (10 micrograms/ml) to the ethanol had no significant effect on the tissue levels observed. These ratios were similar to the ratios of the outputs of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha from the guinea-pig uterus (0.6:1.0:0.9 on day 7 and 7.6:1.0:1.5 on day 15), but were different (particularly on day 7, but only for 6-keto-PGF1 alpha on day 15) to the ratios of the amounts of the three PGs synthesized by homogenates of the guinea-pig uterus (7.2:1.0:2.4 on day 7 and 11.7:1.0:3.3 on day 15). Consequently, the measurement of tissue levels of PGs in the guinea-pig uterus reflects PG synthesis by intact tissue and changes in this synthesis, rather than PG synthesis by homogenates (broken cell preparations). Therefore, it appears meaningful to measure levels of PGs in the guinea-pig uterus since they reflect uterine PG output. Separation of the endometrium from the myometrium, which involved handling and mild trauma, stimulated uterine PG levels, but the ratio of the levels of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha in the endometrium was still similar to that found in the non-separated uterus.     

Patterns of prostaglandin synthesis and degradation in isolated superficial and proliferative colonic epithelial cells compared to residual colon

Prostaglandin (PG) synthesis and degradation were examined in different regions (epithelial versus non-epithelial structures) of the rat distal colon by both HPLC analysis of [14C] arachidonate (AA) metabolites and by specific radioimmunoassays. Intact isolated colonic epithelial cells synthesized mainly PGF2 alpha and TXA2, as monitored from the formation of its stable degradation product TXB2 (PGF2 alpha greater than TXB2 greater than 6-keto-PGF1 alpha, the stable degradation product of PGI2 = PGD2 = PGE2 = 13,14-dihydro-15-keto-PGF2 alpha). The profile of PG products of isolated surface epithelial cells was identical to that of proliferative epithelial cells. However, generation of PGs by surface epithelium was 2 to 3-fold higher than by proliferative cells both basally and in the presence of a maximal stimulating concentration (0.1 mM) of AA. The latter implied a greater synthetic capacity of surface epithelium, rather than differences due to endogenous AA availability. The major sites of PG synthesis in colon clearly resided in submucosal structures; the residual colon devoid of epithelial cells accounted for at least 99% of the total PGs produced by intact distal colon. The profile of AA metabolites formed by submucosal structures also differed markedly from that of the epithelium. The dominant submucosal product was PGE2. PGE2 and its degradation product 13,14-dihydro-15-keto-PGE2 accounted for 63% of the PG products formed by submucosal structures (PGE2 much greater than PGD2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGF2 alpha = TXB2 = 6-keto-PGF1 alpha greater than 13,14-dihydro-15-keto-PGF2 alpha). By contrast, epithelial cells, and particularly surface epithelium, contributed disproportionately to the PG degradative capacity of colon, as assessed from the metabolism of either PGE2 or PGF2 alpha. When expressed as a percentage, epithelial cells accounted for 71% of total colonic PGE2 degradative capacity but only 23% of total colonic protein. Approximately 15% of [3H] PGE2 added to the serosal side of everted colonic loops crossed to the mucosal side intact. Thus, at least a portion of the PGE2 formed in the submucosa reaches, and thereby can potentially influence functions of the epithelium.     

Mechanisms involved in the stimulation by cycloheximide of prostaglandin production in the guinea-pig uterus

Cycloheximide produced a large increase in prostaglandin (PG) E2 output and smaller increases in PGF2 alpha and 6-keto-PGF1 alpha when superfused over the guinea-pig uterus for 20 min. This stimulation of the outputs of these 3 PGs by cycloheximide did not require extracellular calcium. TMB-8 (an intracellular calcium antagonist) had no effect on the stimulation of PGE2 output by cycloheximide, but it completely prevented the stimulation of PGF2 alpha and 6-keto-PGF1 alpha outputs. W-7 (a calmodulin antagonist) had no effect on the stimulation of PGE2 and PGF2 alpha outputs by cycloheximide, but it partially reduced and delayed the stimulation of 6-keto-PGF1 alpha output. Neomycin (a phospholipase C inhibitor) did not prevent the increases in PGE2 and 6-keto-PGF1 alpha outputs produced by cycloheximide. However, neomycin (5 and 10 mM, but not 1 mM) inhibited the small increases in PGF2 alpha caused by cycloheximide. On its own, neomycin produced a dose-dependent, transient increase in 6-keto-PGF1 alpha output without affecting the outputs of PGF2 alpha and PGE2. It is concluded that different mechanisms are involved in the processes by which cycloheximide stimulates the syntheses of PGE2, PGF2 alpha and 6-keto-PGF1 alpha in the guinea-pig uterus.     

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.     

Effects of oestradiol, progesterone, hydrocortisone and oxytocin on prostaglandin output from the guinea-pig endometrium maintained in tissue culture

The effects of oestradiol, oxytocin, progesterone and hydrocortisone in vitro on prostaglandin (PG) output from guinea-pig endometrium, removed on days 7 and 15 of the oestrous cycle and maintained in tissue culture for 3 days, have been investigated. Oestradiol (3.7 to 3700 nM) and oxytocin (2 to 200 pM) did not stimulate endometrial PGF2 alpha output, thus not confirming the findings of a previous report (Leaver & Seawright, 1982), nor did they stimulate the outputs of PGE2 and 6-keto-PGF1 alpha. In fact, oestradiol (3700 nM) inhibited the outputs of PGF2 alpha, PGE2 and, to a lesser extent, 6-keto-PGF1 alpha. Progesterone (3.2 to 3200 nM) inhibited the outputs of PGF2 alpha and PGE2; hydrocortisone (2.8 to 2800 nM) had no effect on endometrial PG output. These findings indicate that the inhibitory effect of progesterone on endometrial PG synthesis and release in the guinea-pig is not due to progesterone having a glucocorticoid-like action. Furthermore, progesterone had no effect on 6-keto-PGF1 alpha output, suggesting that the mechanisms controlling endometrial PGI2 synthesis (as reflected by measuring 6-keto-PGF1 alpha) are different from those controlling endometrial PGF2 alpha and PGE2 synthesis.     

Basal prostaglandin synthesis by the isolated perfused rat kidney

In order to assess the main characteristics of the prostaglandin (PG) biosynthesis by the isolated perfused rat kidney, the urinary and venous outputs of PGE2, PGF2alpha, 6-keto-PGF1alpha and of thromboxane (Tx)B2 were followed during 120 min after an equilibration period of 30 min. Single pass kidneys were perfused with a Krebs-Henseleit solution added with Polygeline at a constant flow rate providing a perfusion pressure about 90 mm Hg. From the beginning of the study, major differences could be observed in the renal biosynthetic rate of the 4 PG studied which were mainly excreted into the venous effluent. During the perfusion, urinary and venous outputs of PGE2, PGF2alpha and of TxB2 remained stable whereas those of 6-keto-PGF1alpha sharply increased and were found inversely related to the glomerular filtration rate (r = -0.95; p n 0.001). Finally, the urinary and venous outputs of each of the four PGs studied were found positively related. It is concluded that the isolated perfused rat kidney is a valuable preparation for studying the biosynthesis of PGs and that, at least in thi model, the urinary excretion of PGs is a good index of their renal synthesis.     

Thymic hormones and prostaglandins. II. Synergistic effect on mouse spontaneous rosette forming cells

Prostaglandins (PGs) have been assumed to play a role in the biological activity of thymic hormones (TH). Indeed, it has been shown that type E-PGs are able to mimic the action of several TH. Moreover, indomethacin interferes in the rosette assay, which still represents the most commonly used bioassay for the evaluation of TH and, in particular thymulin levels, in biological fluids. Previously, our attempt to modulate PG production by different TH showed that none of the TH tested affect PGE2, 6-keto-PGF1 alpha, PGF2 alpha and TXB2 production by spleen cells from control and thymectomized (Tx) mice, while indomethacin was able to inhibit the spontaneous PG production. Here, we investigated a possible role for each endogenously produced PG in the experimental conditions of the rosette assay, in order to define: 1) whether or not there was a specificity of action of a given PG; and 2) to analyze the pattern of action between thymulin and the endogenously produced PGs. We demonstrated that PGE2 and 6-keto-PGF1 alpha are the PGs which are physiologically involved in the rosette assay, according to their levels of endogenous production, and that they are able to synergize with thymulin. This synergy was demonstrated in two ways: 1) by adding anti-PGE2 and anti-6-keto PGF1 alpha-antibodies, which prevent part of the thymulin effect, or 2) by simultaneous addition of PG and thymulin, at concentrations far lower than those which correspond to their thymulin-like effect. Moreover, PGE2 addition, at concentration close to that found to be endogenously produced, partially reversed the indomethacin-induced effect in the rosette assay. In conclusion, if PGs do not act as mediators of thymulin, they are able to synergize in one of its biological action.     

Altered myocardial prostaglandin synthesis in spontaneously diabetic rats

Patients with diabetes mellitus have an increased susceptibility to heart disease. The exact mechanism for this phenomenon is unclear. Abnormalities in prostaglandin (PG) production have been suggested as a possible cause. In this connection, we examined the PG synthetic capacity of cardiac microsomes from spontaneously diabetic rats. Cardiac microsomes from diabetic and control rats produced varying amounts of 6-keto-PGF1 alpha (stable degradation product of PGI2), PGE2, PGD2, PGF2 alpha, and TXB2 (stable breakdown product of TXA2). In both instances the production of 6-keto-PGF1 alpha predominated, however, microsomes from diabetic rats showed markedly greater conversion of arachidonic acid to all the PG products, especially 6-keto-PGF1 alpha. When PGF2 alpha metabolism was detected between diabetic and control heart preparations. These results show an enhanced cyclooxygenase activity in diabetic rat hearts without any change in prostaglandin dehydrogenase activity. Such a change may promote some of the cardiac alterations seen in diabetic mellitus.     

Effects of inhibitors of arachidonic acid turnover on the production of prostaglandins by the guinea-pig uterus

The supply of free arachidonic acid from phospholipids is generally regarded as the rate-limiting step for prostaglandin (PG) synthesis by tissues. Two enzymes involved in arachidonic acid uptake into, and release from, phospholipids are acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase A2 (PLA2), respectively. PGF2 alpha produced by the endometrium induces luteolysis in several species including guinea-pigs. Thimerosal, an inhibitor of ACLAT, and aristolochic acid, an inhibitor of PLA2, both reduced, in a concentration-dependent manner, the output of PGF2 alpha from guinea-pig endometrium cultured for 24 h on days 7 and 15 of the oestrous cycle. This study showed that the continual production of PGF 2 alpha by guinea-pig endometrium is not only dependent upon the activity of PLA2 for releasing free arachidonic acid for PGF2 alpha synthesis, but also on the incorporation of arachidonic acid into the phospholipid pool by the activity of ACLAT. The inhibitory effects of thimerosal and aristolochic acid on the outputs of PGE2 and 6-keto-PGF1 alpha were less marked, particularly on day 7 when the low output of PGE2 was unaffected and the output of 6-keto-PGF1 alpha was increased at the lower concentrations of thimerosal. This finding indicates that there are different pools of arachidonic acid bound as phospholipid for the syntheses of PGF2 alpha and 6-keto-PGF1 alpha by guinea-pig endometrium.     

Effect of prostaglandins against alloxan-induced cytotoxicity to insulin secreting insulinoma RIN cells in vitro

In the present study, we studied the effect of various prostaglandins (PGs) on alloxan-induced cytotoxicity to rat insulinoma (RIN) cells. Of all the PGs tested, PGE(1), PGE(2), PGI(2), PGF(1 alpha), and PGF(3 alpha) protected RIN cells from alloxan-induced cytotoxicity (P<0.05 compared to alloxan), whereas thromboxane B(2) and 6-keto-PGF(1 alpha) were not effective. PGE(1) induces a statistically significant increase in the activities of superoxide dismutase and glutathione peroxidase and decrease in lipid peroxides in alloxan-treated RIN cells (P<0.001). PGE(1) restored nitric oxide/lipid peroxide ratio to normalcy, suggesting that PGE(1) suppresses oxidant stress induced by alloxan in RIN cells in vitro. Furthermore, PGE(1) prevented DNA damage and apoptosis induced by alloxan. These results indicate that PGE(1) prevents alloxan-induced cytotoxicity to RIN cells in vitro.     

Effects of nitrogen dioxide exposure on the contents of prostaglandins and thromboxane B2 in broncho alveolar lavage

Effects of 10 ppm nitrogen dioxide (NO2) exposure on the contents of prostaglandins (PGs) and thromboxane (TX) B2 in bronchoalveolar lavage (BAL) of rats were studied. In the BAL of normal rats, the amounts of PGs and TXB2 in the whole lavage were 6-keto-PGF1 alpha (38.0 +/- 6.4 ng) greater than TXB2 (11.8 +/- 4.0 ng) greater than PGF2 alpha (5.7 +/- 1.6 ng) much greater than PGE (0.5 +/- 0.3 ng). Rats were exposed to NO2 for 1,3,5,7 and 14 days. The NO2 exposure decreased in the level of 6-keto-PGF1 alpha by about 35% throughout the exposure. The level of TXB2 was higher in the day 5 exposure group (155%). The contents of PGF2 alpha and PGE first, decreased and then transiently increased on days 3 and 5. PG 15-hydroxy-dehydrogenase activity of lung homogenate decreased correspondingly on day 3 and 5. Then the contents PGF2 alpha and PGE decreased on day 7 and 14. 6-keto-PGF1 alpha and TXB2 are stable metabolites of PGI2, a strong bronchorelaxant and TXA2, a strong bronchoconstrictor respectively. Therefore the results suggested that the decrease in 6-keto-PGF1 alpha, a major prostanoid in the BAL and the increase in TXB2 may correlate with broncho constriction by NO2 exposure.     

Enhanced prostaglandin synthesis in the parturient rat uterus and its effects on myometrial oxytocin receptor concentrations

We measure oxytocin (OT) responsiveness and prostaglandins (PGs) synthesis in uteri of 19, 20, 21 and 22-day pregnant and 2-day postpartum rats to determine whether the enhanced OT sensitivity and PG synthesis in the parturient uterus is the result of a higher cyclooxygenase activity. We also investigated the effects of suppression of PG synthesis on OT responsiveness and OT receptor in 22-day and 23-day pregnant rats. PG productions (PGE2, PGF2 alpha, 6-keto-PGF1 alpha and TXB2 in microsomal fractions were quantitated by radio- immunoassays (RIAs). OT receptor concentrations were measured in plasma membrane fractions by radioligand-receptor binding assays. Naproxen sodium was used to inhibit endogenous PG synthesis. We found a close temporal relationship between enhanced OT responsiveness and increased uterine PGE2 alpha synthesis, but no significant difference in cyclooxygenase activities among the microsomes prepared from uteri of different gestational ages. Suppression of PG synthesis attenuated OT responsiveness and markedly reduced OT binding sites, from 242 to 78 fmol/mg protein. There was no change in the binding affinity. These findings suggest that PG stimulates OT receptor formation which leads to enhanced OT responsiveness. The increase in PG production is not mediated by a higher cyclooxygenase activity.     

Enzymatic conversion of prostaglandin H2 to prostaglandin F2 alpha by aldehyde reductase from human liver: comparison to the prostaglandin F synthetase from bovine lung

The primary structure of prostaglandin (PG) F synthetase from bovine lung shows 62% similarity with that of human liver aldehyde reductase (EC 1.1.1.2) (Watanabe, K., Fujii, Y., Nakayama, K., Ohkubo, H., Kuramitsu, S., Kagamiyama, H., Nakanishi, S., and Hayaishi, O. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 11-15). We therefore purified human liver aldehyde reductase to homogeneity and compared the immunological and catalytic properties of aldehyde reductase and PGF synthetase. Although both enzymes belong to a group of aldoketoreductases and their molecular weights are essentially identical, aldehyde reductase had no cross-reactivity to anti-PGF synthetase antiserum. Furthermore, there was a difference in the substrate specificity for reduction of PGs between the two enzymes. Aldehyde reductase catalyzed the reduction of PGJ2, delta 12-PGJ2, PGH2, or PGA2, but not that of PGB2, PGD2, or PGE2, whereas PGF synthetase reduced PGD2. The optimum pH, Km value for PGH2, and the turnover number were 6.5, 100 microM, and 3.1 min-1, respectively. The PGH2 9,11-endoperoxide reductase activity of aldehyde reductase was not affected in the presence of a substrate such as p-nitrobenzaldehyde, DL-glyceraldehyde, or 9,10-phenanthrenequinone, suggesting that PGH2 9,11-endoperoxide and other substrates are reduced at different active site(s). The reaction product formed from PGH2 by this enzyme was identified as PGF2 alpha by gas chromatography/mass spectrometry. These results suggest that aldehyde reductase is not exactly identical to PGF synthetase in terms of its immunological property and substrate specificity for PGs, but that this enzyme is also involved in the direct conversion of PGH2 to PGF2 alpha similar to PGF synthetase.     

Cyclooxygenase Products of Arachidonic Acid Metabolism in Cat Cerebral Cortex After Experimental Concussive Brain Injury

Previous studies have suggested that following experimental fluid percussion brain injury, increased prostaglandin (PG) synthesis, with its concomitant production of oxygen free radicals, causes functional and morphological abnormalities of the cerebral arterioles. The purpose of this study was to chemically determine if PGs are altered following this injury. To facilitate interpretation of neurochemical measurements the cats were ventilated, blood pressure was measured, and a cranial window, for microscopic observation of pial arteriolar diameter was inserted. PG levels were determined in quick-frozen cortical tissue removed from control and 3 groups of injured cats at 1.5, 8,0, and 60 min after injury. Analysis of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha was performed by HPLC and GC/MS. The control levels of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were 216 +/- 44, 210 +/- 48, and 48 +/- 12 ng/g wet weight, respectively. Following injury, produced by a 22 ms increase in intracranial pressure, the pial arterioles dilated irreversibly and a transient hypertensive response occurred, thereby producing hyperemia. During the maximum hyperemic response, the total PGs were 75% of control. At 8 min after injury, when blood pressure returned to control level, the PGs were 158% of control and PGs fell to 111% of control at 60 min. These experiments supported our previous studies implicating increased PG synthesis in te genesis of the physiologic and morphologic sequelae of experimental concussive brain injury.     

Has cervical smooth muscle any physiological role in the human?

Strips of human cervical tissue were obtained by needle biopsy and contractile activity was registered isometrically in a tissue chamber perfused by Krebs-Ringer bicarbonate buffer. The most frequently encountered pattern of contractile activity was high frequency-short duration. Prostaglandin (PG)E2, PGI2 and 6-keto-PGF1 alpha had an inhibitory effect on the muscular activity. Cervical muscle from pregnant women was more sensitive to PGE2 than specimens from non-pregnant women. PGF2 alpha had no apparent effect on cervical contractility in non-pregnant and early pregnant patients. In late pregnancy, however, PGF2 alpha inhibited muscle contractions. The present results point to a physiological role of the cervical muscles for the control of cervical competence during pregnancy. The inhibitory effect of PGs on the muscle activity may promote cervical dilatation and retraction.     

Prostaglandins enhance parathyroid hormone-evoked increase in free cytosolic calcium concentration in osteoblast-like cells.

Prostaglandins (PGs) are autocrine or paracrine hormones that may interact with circulating hormones such as parathyroid hormone (PTH) in bone. We examined the interaction of the PGs, PGF2 alpha, PGE2, and 6-keto-PGF1 alpha with PTH to enhance the rapid, initial transient rise in free cytosolic calcium ([Ca2+]i) and cAMP levels stimulated by PTH. Pretreatment of UMR-106, MC3T3-E1, and neonatal rat calvarial osteoblast-like cells by PGs resulted in an enhancement of the early transient rise in [Ca2+]i stimulated by PTH. PGF2 alpha was approximately 100 times more potent than PGE2. PGE2 itself was more potent than 6-keto-PGF1 alpha in enhancing PTH-stimulated rise in [Ca2+]i. Near-maximal augmentation was achieved at PGF2 alpha doses of 10 nM and PGE2 of 1 microM. The degree of augmentation in [Ca2+]i by PGF2 alpha was independent of preincubation time. PGF2 alpha pretreatment did not alter the EC50 for the PTH-induced [Ca2+]i increase but only the extent of rise in [Ca2+]i at each dose of PTH. The augmented increase in [Ca2+]i was mostly due to enhanced PTH-mediated release of Ca2+ from intracellular stores. PGF2 alpha did not stimulate an increase in PTH receptor number as assessed by [125I]-PTH-related peptide binding. PG pretreatment partially reversed PTH inhibition of cell proliferation, suggesting that an increase in [Ca2+]i may play a role in tempering the anti-proliferative effect of PTH mediated by cAMP. These studies suggest a new mode by which PGs can affect cellular activity.