PGDH Activity Research Articles

NAD +-dependent 15-hydroxyprostaglandin dehydrogenase: Immunochemical characterization of the lung enzyme from pregnant rabbits

A polyclonal antibody was produced in guinea pig against the lung NAD +-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) purified from pregnant rabbits. Western blot analysis demonstrated that the protein identified by this antibody in the 105,000 g supernatant fraction of lung tissue from pregnant rabbits had a molecular mass of 30 kDa and comigrated with the purified PGDH. The specific activity of the lung PGDH in pregnant rabbits (25- to 28-day gestations) was 36.7 nmol NADH formed/min/mg protein compared to 0.3 nmol NADH formed/min/mg protein in nonpregnant rabbits. Although the PGDH activity in the lung cytosol of nonpregnant rabbits was inhibited by the anti-lung PGDH antibody, the 30-kDa protein was not detected by Western blot analysis. An examination of this 30-kDa protein during the gestational period indicated that the protein was present after 10 days and the amount of the protein increased from Day 10 to Day 28. This increase in the immunochemically reactive protein correlated with the marked increase in PGDH specific activity between 10 and 28 days. An immunochemically reactive protein also was observed in the ovary of 25- to 28-day pregnant rabbits and the specific activity of the ovary PGDH was 19.3 nmol NADH formed/min/mg protein. Only trace levels of the PGDH activity were detected in the ovaries of nonpregnant rabbits. A 30-kDa protein was not detected by the anti-rabbit lung PGDH in brain, kidney, bladder, uterus, liver, and heart tissue of pregnant or nonpregnant rabbits. When rabbit or human placental cytosol was examined with the anti-rabbit lung PGDH only faint 30-kDa bands were observed by Western blot analysis. A monoclonal antibody prepared against human placental PGDH did not recognize the 30-kDa band in the pregnant rabbit lung. Localization studies indicated a marked increase in immunochemical staining in pulmonary epithelial cells of pregnant rabbits as compared to nonpregnant rabbits. Lung epithelial cells but not endothelial cells were identified as containing the PGDH.

Histochemical detection of NADP-dependent prostaglandin dehydrogenase in preimplantation rabbit embryos.

Using prostaglandin (PG) E2 as the substrate, the activity of NADP-dependent 15-hydroxyprostaglandin dehydrogenase (type II PGDH) in preimplantation rabbit eggs and embryos was histochemically investigated, and the metabolism of PGE2 in them was discussed. The eggs and embryos observed were at 1-cell (unfertilized and pronuclear) to 8-cell, morula and blastocyst stages. Type II PGDH, as well as NAD-dependent PGDH (type I PGDH), was histochemically detected by the method of Nissen and Andersen (1968). The activity of the type II PGDH was strong in the eggs at 1-cell stage, strong to weak in the eggs at 2-cell stage, and weak from 4-cell eggs to the blastocyst stage. The diformazan granules showing the presence of type II PGDH activity were spread evenly throughout the cytoplasm of eggs and embryos at the stages of 1-cell through morula. Blastocysts also presented such granules spread evenly throughout the cytoplasm of both trophoblast and inner-cell-mass cells. The intensity and the distribution of the type I PGDH activity in the eggs and embryos were similar to those of the type II PGDH activity. These results thus seem to point out that rabbit eggs and embryos do have type II PGDH, as well as type I PGDH which has widely been found in different mammalian embryos.

Evidence for several fold more activity of NAD +-dependent uterine prostaglandin 15-hydroydehydrogenase in transsexual than in non-transsexual women

The activity of NAD +-dependent PGDH was measured in the cytosolic fractions (100, 000 × g) of uterine tissues obtained from transsexual, pregnant and non-pregnant women. The specific activity (mean ± SD) of the enzyme at maximum velocity of the enzyme reaction in these three groups of women was 5.5 ± 2.30, 0.53 ± 0.27 and0.54 ± 0.25 mU/mg protein respectively using PGE 2 as substrate, and with PGF 2α as substrate the respective values were 5.48 ± 2.80, 0.49 ± 0.41 and 0.51 ± 0.30 mU/mg protein. These data suggest that, with either substrate, the uterine enzyme activity in the transsexual was about 10-fold greater than in pregnant and non-pregnant women (p < 0.001). However, the Km values of the enzyme for both PGE 2 and PGF 2α were similar in all three groups, indicating the presence of same enzyme in the uterus of transsexual, pregnant and non-pregnant women. We speculate that PGDH activity was raised in the uterus of transsexual women because of the prolonged androgen therapy they received for the management of female-to-male transsexualism.

The output of uterine prostaglandins and the activity of 15-hydroxy-prostaglandin dehydrogenase are enhanced in chronic ethanol fed rats

The generation and output of prostaglandins (PGs) E 2 and F 2α into the solution suspending uterine segments from ethanol (ETOH)-fed diestrous rats and the activity of 15-OH-PG-dehydrogenase (PGDH) in uteri at diestrus, were explored and compared with normal-fed controls. Animals were fed with ETOH (35% of the total calories in a liquid diet) during 20 days before sacrifice. Paired normal-fed controls were given isocaloric quantities of dextrimaltose. It was observed that the uterine outputs of PGE 2 and of PGF 2α into the suspending solution, were significantly greater in the ETOH group. On the other hand, the PGDH activity for PGE 2 in control uterine tissue, was significantly smaller than the activity detected in preparations from animals fed with the chronic ETOH diet. Results are discussed in terms of possible mechanisms for the action of ethanol, either on the release of PG fatty acid precursors (activation of phospholipase A 2) or on the activity of PG synthesizing enzymes. Inasmuch as in the ETOH-fed group uterine PGDH activity was greater, rather than diminished, the possibility of a reduced catabolism accounting for the augmentation of PGs in the suspending medium, does not appear feasible. In fact, results suggest that the real magnitude of higher PG generation and release is even greater than that disclosed by the present study. The finding that chronic ethanol consumption augments PG production, appears relevant, in view of the unique roles played by these eicosanoids in parturition and in the development of fetuses.

New Inhibitor(s) of 15-Hydroxyprostaglandin Dehydrogenase in Human Milk

New inhibitor or inhibitors of nicotinamide-adenine dinucleotide-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) activity were found in breast milk but not in powdered milk. Milk samples were obtained manually from 23 puerperae who had delivered at 38 to 41 weeks. The inhibition pattern was competitive and noncompetitive. The percentage of inhibition of PGDH activity kept at 4.0 degrees C increased to a maximum at 12 hours after milk sampling and decreased thereafter. The percentage of inhibition of each milk sample decreased gradually with the duration postpartum. In addition, the inhibitor or inhibitors showed a smaller molecular weight than 1000 daltons and were partially resistant to heat and trypsin. Moreover, they were easily soluble in petroleum ether at various pH values, although the adsorption to C18 cartridge was inadequate. These new factor or factors may increase the benefits of breast feeding.

Human placental 15-hydroxy-prostaglandin dehydrogenase activity is under fetal genetic control

Placental 15-hydroxy-prostaglandin dehydrogenase (PGDH type I) was measured in 33 placentae obtained from 1 trizygotic, 7 dizygotic and 8 monozygotic pregnancies. PGDH activity ranged from 0.33 to 4.62 nmol PGF 2α metabolized per mg placental protein per min, which was within the range observed in singleton pregnancies. Expressing PGDH activity per mg DNA, offered no advantage over expressing it per mg total protein. PGDH activity differed significantly between the placentae of 6 of the 9 genetically non-identical placental pairs. The placentae of genetically identical twins, on the other hand, showed no difference in PGDH activity between the pairs. The data indicate that the genetic constitution of the fetus determines placental PGDH activity. They also provide us with the first evidence that the variation in prostaglandin catabolized capacity of the human placenta is not entirely dictated by the maternal endocrine environment, but is under fetal control.

Pulmonary and placental 15-hydroxyprostaglandin dehydrogenase in rat during pregnancy

15-Hydroxyprostaglandin dehydrogenase (PGDH) in rat lung and placenta during pregnancy was examined using a photometric assay. Pulmonary PGDH activity varied slightly during pregnancy, being significantly higher on days 10 and 19 of gestation than on day 22. Placental PGDH activity changed strikingly according to the day of pregnancy. Activity was relatively high on day 10 of gestation, fell to a low level on day 15, then increased to a maximum on day 22. A rapid fall in pulmonary PGDH activity and a remarkable increase of placental PGDH activity occurred on the day just before parturition. The results suggest that pulmonary and placental PGDH activity may cooperatively contribute to the continuity of pregnancy, and the process of parturition.

The Effect of 17 β-Estradiol and Estrone on Intermediary Metabolism of the Pyloric Caeca of the Asteroid Luidia clathrata (Say) Maintained under Different Nutritional Regimes.

Luidia clathrata were fed either a high or low level of food and injected with estradiol, estrone, or seawater for 16 days. In individuals maintained at a low level of food, the weight of the pyloric caeca decreased in all individuals, regardless of hormone injections. Activities of all enzymes in these individuals were lower than in individuals maintained at a high level of food. In the individuals maintained at a low level of food, the activity of GPDH was higher in the pyloric caeca of individuals injected with estradiol and estrone than in those receiving seawater injections; the activity of 6-PGDH decreased in those injected with estrone. In individuals maintained at a high level of food, the activities of glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phosphogluconate dehydrogenase (6-PGDH) were higher in individuals injected with estradiol than in those receiving seawater injections. Activities of glycerol phosphate dehydrogenase (GPDH) and pyruvate kinase (PK) were not affected. A slight decrease (P<0.1) in the activity of 6-PGDH was seen with injection of estrone. These results suggest that steroids have a role in regulating cellular metabolism in the pyloric caeca, and that nutritional condition influences the response of the pyloric caeca to steroids. In addition, these results indicate that the nutritional condition of the individual directly influences enzyme activities.

Hexose monophosphate shunt and cholesterogenesis in lead-induced kidney hyperplasia

De novo cholesterol synthesis and hexose monophosphate (HMP) shunt were studied in rat kidney stimulated to proliferate by a single administration of lead nitrate. Lead-treated rat kidneys showed an increase in DNA synthesis, as measured by [ 3H]thymidine incorporation starting at 18 h and with a maximum at 24 h. Renal DNA synthesis was preceded by an increase in de novo cholesterol synthesis and an enhancement in the activity of the HMP shunt, as indicated by increased activity of G6PDH and 6PGDH. These findings indicate that enhancement of cholesterol synthesis and of the HMP shunt is closely associated with the active proliferative process induced in the kidney by treatment with lead nitrate.

Isolation and properties of lung 15-hydroxyprostaglandin dehydrogenase from pregnant rabbits

A NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was purified to a specific activity of over 25,000 nmol NADH formed/min/mg protein with 50 μ m prostaglandin E 1 as substrate from the lungs of 28-day-old pregnant rabbits. This represented a 2600-fold purification of the enzyme with a recovery of 6% of the starting enzyme activity. The lungs of pregnant rabbits were used because a 42- to 55-fold induction of the PGDH activity was observed after 20 days of gestation. The enzyme was purified by CM-cellulose, DEAE-cellulose, Sephadex G-75, octylamino-agarose, and hydroxylapatite chromatography. The enzyme could not be purified by affinity chromatography using NAD- or blue dextran-bound resins. The purified enzyme was specific for NAD and had a subunit molecular weight of 29,000. The optimal pH range for the oxidation of prostaglandin E 1 was between 10.0 and 10.4 using 3-(cyclohexylamino)propanesulfonic acid as the buffer. The K m and V max values for prostaglandin E 1 were 33 μ m and 40,260 nmol/min/mg protein, respectively, while the K m and V max values for prostaglandin E 2 were 59 μ m and 43,319 nmol/min/mg protein, respectively. The K m for prostaglandin F 2α was four times the value for prostaglandin E 1. The PGDH activity was inhibited by p-chloromercuriphenylsulfonic acid but the enzymatic activity was restored by the addition of dithiothreitol. n-Ethylmaleimide also produced a rapid decline in enzymatic activity but when NAD was included in the incubation system, no inhibition was observed.

Localization and properties of NAD-dependent 15 hydroxyprostaglandin dehydrogenase activity in spontaneously hypertensive rat kidney.

To evaluate the pathophysiological significance of renal NAD-dependent 15 hydroxyprostaglandin dehydrogenase (PGDH) in young spontaneously hypertensive rats (SHR), the intranephron distribution of PGDH was compared with age-matched Wistar Kyoto rats (WKY). With supernatants of kidney homogenates, PGDH activities in 3 week-old SHR were significantly lower than those in WKY, and no difference in PGDH activities was found between male and female in both WKY and SHR. By using microdissected nephron segments of 3 week-old WKY and SHR, PGDH activities in both groups were found to be exclusively distributed in the proximal tubules, within which the activity of the straight portion (PST) was several fold higher than that of the convoluted one (PCT). In 3 week-old SHR, a significant decrease of PGDH activity was observed only in PST, possibly indicating a decrease of enzyme content in PST because of similar Km values in both groups. These results may suggest that intraproximal PG concentration could be regulated by the change of PGDH in PST of young SHR.

Increase in placental 15-hydroxy-prostaglandin dehydrogenase in the first half of human pregnancy

NAD-dependent 15-hydroxy-prostaglandin dehydrogenase (PGDH) activity was measured in homogenates of 25 human placentae obtained between 7 and 17 weeks of gestation. PGDH activity, expressed in nanomoles PGF 2α metabolized per min, ranged from 0.2 to 5.4 nmoles per mg placental protein and from 1.5 to 80 nmoles per g wet weight. PGDH activity per mg protein and per g weight increased significantly in function of gestational age (p<0.001). Between 7–8 weeks' gestation and 15–16 weeks mean values increased tenfold from 0.4 to 3.0 nmoles per mg protein and from 2.7 to 36.6 nmoles per g wet weight. Per unit of weight these early placentae contained less PGDH activity than term controls, but this related mainly to their high water content. Per mg placental protein PGDH activities already equalled values found at term before the end of the first trimester. The data indicate that the development of terminal villi and the migration of trophoblast into the maternal spiral arteries is associated with a substantial increase in the placental capacity for prostaglandin metabolism.

15-Hydroxyprostaglandin dehydrogenase activity in human fetal kidney tissue

Human fetal kidney tissue was found to be rich in NAD +-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) activity. The enzyme activity was localized primarily in the medullary portion of fetal kidney. Separated cortical and medullary tissue fragments were maintained in organ culture for up to 9 days. The specific activity of PGDH in cortical tissue was low at the commencement of organ culture and remained low throughout the culture period. The specific activity of PGDH in medullary tissue fell precipitously during the first 48 h in organ culture; thereafter, the specific activity of the enzyme increased steadily during the culture period and reached values of up to ten times that in starting tissue by 6–7 days in organ culture. The decline in specific activity of PGDH in medullary tissue during the first 48 h in culture could be prevented by including inhibitors of lysosomal enzyme activity, i.e., chloroquine or ammonium chloride, in the culture medium. We suggest that human fetal kidney tissue explants in organ culture may be an appropriate model for the study of the regulation of PGDH activity. Such an evaluation of the regulation of PGDH is believed to be of importance since PGDH serves to regulate the levels of and, thence, the action of the biologically active prostaglandins.

Effects of opiates and naloxone on certain enzymes of carbohydrate metabolism in human breast carcinomas.

The effects of administration of opiates and naloxone on the activities of PFK, 6PGDH and alpha-GPDH and alpha-GPDH/6PGDH ratios in human breast carcinomas were investigated in patients awaiting mastectomy. Injection of naloxone or fentanyl into an antecubital vein resulted in a statistically significant reduction in the activity of alpha-GPDH. Fentanyl was also effective in reducing the activity of 6PGDH. Injection of morphine into a branch of the internal mammary artery during mastectomy failed to induce changes in the activities of any of the enzymes but injection of naloxone resulted in a significant rise in the activity of 6PGDH. It is postulated that these alterations in the activities might not be associated with the binding of these drugs to opiate receptor proteins in the carcinoma. Furthermore opiate agonists or antagonists might not produce the required changes in the activities of any of the enzymes.

Ethanol-induced changes in the oxidative metabolism of arachidonic acid

Previous reports have suggested that ethanol (ETOH) consumption alters the metabolism of polyunsaturated fatty acids. In several models, acute ETOH exposure has been shown to stimulate the synthesis of PGE 1 from dihomogamna-linolenic acid, while chronic ETOH consumption depletes microsomal arachidonate levels and presumably lowers the availability of substrate for the synthesis of PGs and related compounds of the “2” series. Chronic ETOH has also been reported to inhibit 16-hydroxyprostaglandin dehydrogenase (PGDH) activity, the enzyme responsible for degrading the PGs. In the experiments reported here, we have measured microsomal levels of arachidonic acid, prostacyclin (PGI 2) synthetase activity, and the kinetic parameter of PGDH in male rats chronically dosed with varying amounts of ETOH. In animals matched for nutritional intake, ETOH caused a dose dependent lowering of the microsomal arachidonate content of heart, brain, and liver. Kidney microsomal arachidonate levels were significantly increased by ETOH. Total and specific PGDH activity in these kidneys were lowered by chronic exposure to ETOH. PGDH activity fell to 80% and 62% of control values at three and six weeks, respectively. In addition the catalytic efficiency of the remaining PGDH activity was significantly lowered. PGI synthesis in the stomachs and aortas of the ethanol-treated animals showed a biphasic dose response, i.e., moderate ETOH exposure stimulated PGI 2 synthesis in both aortas and stomach fundi while high ETOH levels lowered the activity. This biphasic dose response of PGI 2 synthetase mimics certain aspects of the cardiovascular response to ETOH.

Sex and age differences in the specific activity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase in human fetal kidney tissue.

The specific activity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was significantly greater (P less than 0.005) in human kidney tissue obtained from male abortuses aged 10--16 weeks after conception than in female abortuses of the same gestational age and in younger male and female abortuses. There was a significant correlation (r = 0.93, P less than 0.001) between the specific activity of PGDH in fetal kidney and testicular tissues obtained from human abortuses of 10--26 weeks gestational age. These results suggest a role for testosterone in the regulation of PGDH activity in the fetal kidney.

Renal prostaglandin excretion and metabolism in male and female New Zealand normotensive and genetically hypertensive rats.

Reduced renal 15-hydroxyprostaglandin dehydrogenase (PGDH) activity has been proposed as a cause, subsequent to elevation of intrarenal prostaglandin (PG) E2 levels, of the development or maintenance of high blood pressure (BP) in the New Zealand genetically hypertensive (NZGH) rat. To test this hypothesis, PGDH activity in homogenates of kidneys and lungs and in urine concentration and excretion of PGE2 were determined in male and female NZGH and normotensive control (NZNR) rats. Lung PGDH activities of the four groups were similar. Renal PGDH activity was 50% lower for the male NZGH than for the male NZNR, but for the female rats no difference in renal PGDH activity was found between NZGH and NZNR. In addition, there was a large sex-related difference in renal PGDH activities, values for the female rats being only 5% to 10% of the values for males. Urine PGE2 concentration and excretion were two to five times greater for the female rats than for the males, but did not differ between male NZGH and male NZNR. From these observations, it appears that neither renal PGDH activity nor urine PGE2 levels is associated with hypertension in the New Zealand genetically hypertensive strain of rats.

コイの脂質代謝に及ぼす飼料中のりん含量の影響 ＩＩ 飼料りん量がコイ肝すい臓諸酵素活性に及ぼす影響

The activities of several hepatopancreatic enzymes of carp reared at various dietary phosphorus levels were determined. Malnutrition of carp caused by the phosphorus deficiency, which accompanied accumulation of lipid in muscle and viscera, the activity of hepatopancreatic CCE, G/DH and gluconeogenic enzymes (FDPase and PEPCK) appeared to increase, and that of PK decreased remarkably. The activity ratios of both FDPase to PFK and PEPCK to PK increased. This fact shows preponderance of gluconeogenic activity. After fasting for 3 weeks, decrease in the PGDH activity and lipid of the phosphrus deficiency group was much smaller than that of the phosphorus excess group. On the contrary, decrease in PK activity was more significant in the latter. From the results, it is supposed that fatty acids synthesis via citrate from amino acids may be accelerated by the deficeency of phosphorus. In addition, recycling of pyruvate to phosphoenolpyruvate (futile cycle) activated by the dominant PEPCK acitivity, which was much higher than PK activity, would decrease in the activity of the TCA cycle, thus, energy production by the glycolytic pathway, and result in more utilization of amino acids.

Alterations in maternal and fetal prostaglandin dehydrogenase as a result of maternal ethanol consumption

Various lines of research have suggested that ethanol consumption may alter prostaglandin-related physiology. Our laboratory has reported that chronic exposure to high doses of ethanol lowers the ability of kidney and lung homogenates from male rats to catabolize prostaglandin E 2 and F 2α via 15-prostaglandin dehydrogenase (PGDH). Because of. the apparently unique role played by prostaglandins in conception, growth and development of the fetus and parturition, we have attempted to determine if the alterations observed in male rats also occur in females and if any alterations in fetal metabolism result from maternal ethanol exposure. Further, we have measured the influence of ethanol administration on renal clearance of the 15-keto metabolite of PGF 2α in an attempt to determine the in vivo significance of the enzyme inhibition observed in vitro . Initial results indicate the following. 1) Female Holtzman rats dosed at 2.0, 5.0 and 7.0 mg ethanol/kg during the first, second, and third trimesters of pregnancy, respectively, showed losses in renal PGDH activity similar to those found in males (1.52 versus 1.05 picomole/min/mg, p<0.001 by matched t-test). 2) Placental tissue (amnion) isolated from these same animals on day 21 of the pregnancies also showed a significant decrease in PGDH activity (14.79 versus 11.77 picomoles/min/ mg, p<0.01). 3) Kidney homogenates from fetuses delivered on day 21 of the pregnancies showed a significant increase in PGDH relative to pair-dosed controls (16.77 versus 12.65 picomoles/min/mg, p<0.01). 4) In a separate experiment, urinary clearance of PGF 2α metabolite was inhibited in a dose related manner up to a level of 6 gm/kg.

Variation d’Activité des Glucose-6-phosphate, 6-phospho-gluconate et Glycéraldéhyde-3-phosphate Déshydrogénases sous l’action de l’Atrazine, du Chlortoluron et du MCPA

Atrazine, chlortoluron and MCPA 1) 1) MCPA: acide, 2-méthyl-4-chlorophénoxyacétique. are applied to the roots of seventeen-day-old cucumber seedlings in the nutrient solution at a concentration of 1 ppm. After one, two, four and seven days activities of glucose-6-P-dehydrogenase (G6PDH), 6-P-gluconate dehydrogenase (6PGDH) and glyceraldehyde-3-P-dehydrogenase (G3PDH) are measured in both leaves and roots. The three herbicides do not change the activities of NAD-G3PDH and NADP-G3PDH in the leaves while the former is decreased in the roots by more than 50% after four days. Significant stimulation of G6PDH occurs in leaves of seedlings treated with atrazine and chlortoluron. This is probably due to a compensation of the blockage of photosynthetic NADPH formation. In contrast, the activity of 6PGDH is slightly lower or similar to the control. In the roots, the activities of G6PDH and 6PGDH are progressively inhibited by atrazine and chlortoluron while MCPA gives only rise to a slight inhibition after an initial activation.