The effect of ovariectomy on phenylalanine and tyrosine metabolism

Summary Although the regulatory activity of steroid hormones on amino acid metabolism has been described, no information is published on the effect of ovariectomy. We studied the influence of ovariectomy in Wistar rats determining the amino acids phenylalanine and tyrosine in liver, kidney, plasma and urine. 32 animals were used in the study, 12 animals were sham operated, 9 animals were ovariectomized and 11 rats were ovariectomized and supplemented with estradiol. No quantitative changes were detected comparing liver and kidney phenylalanine and tyrosine between the groups (sham operated rats liver phenylalanine 2,53nM/mg ± 1,07; liver tyrosine 1.95nM/mg ± 0.92; kidney phenylalanine 2.16nM/mg ± 0.53; kidney tyrosine 1.80nM/mg ± 0.39. Ovariectomized rats showed liver phenylalanine 3.07nM/mg ± 1.14; liver tyrosine 2.63nM/mg ± 1.01; kidney phenylalanine 2.30 nM/mg ± 0.74; kidney tyrosine 1.93nM/mg ± 0.63. Ovariectomized and estradiol supplemented rats presented with liver phenylalanine 2.84nM/mg ± 1.40; liver tyrosine 2.35nM/mg ± 1.28; kidney phenylalanine 1.91nM/mg ± 0.26, kidney tyrosine 1.67nM/mg ± 0.23.). When, however, the phenylalanine/tyrosine ratio in the liver was evaluated, ovariectomized rats showed a significant decrease of the quotient (p = 0.001). The phenylalanine/tyrosine ratio was restored by estradiol replacement. Our findings show that phenylalanine and tyrosine metabolism is under estradiol control. The effect on the metabolic changes could be mediated by enzyme systems as phenylalanine hydroxylase, tyrosine hydroxylase and tyrosine aminotransferase. Our results would be compatible with previous reports on the stimulatory effect of estradiol on these enzymes. The kidney phenylalanine/tyrosine ratio was unaffected by ovariectomy and/or estradiol replacement which can be easily explained by different pools, enzyme activities, filtration/reabsorption effects, etc.The urinary P/T ratio was decreased by ovariectomy and restored by estradiol replacement indicating endocrine control of renal reabsorption and secretion mechanisms.     

Liver alcohol dehydrogenase activity in the female rat: Effects of ovariectomy and estradiol administration

The effects of ovariectomy and administration of estradiol on the activity of liver alcohol dehydrogenase and on the rate of ethanol elimination were determined in female Sprague-Dawley rats. The activity of the enzyme and the rates of ethanol elimination in the female sham-operated animals were higher than obtained previously in male rats of the same age. Ovariectomy had no effect on liver alcohol dehydrogenase and on rates of ethanol elimination. Estradiol administration resulted in an increase in liver weight and in total liver alcohol dehydrogenase activity per animal in sham-operated but not in ovariectomized animals. The increase in enzyme activity after estradiol administration in sham-operated animals was not associated with a significant increase in the rate of ethanol elimination, suggesting that the enzyme activity in female rats is not rate-limiting in in vivo ethanol oxidation.     

乙醇脱氢酶Ⅰ类基因全长cDNA的克隆与表达

目的:克隆编码人Ⅰ类乙醇脱氢酶基因,并探讨Ⅰ类乙醇脱氢酶(ADH)在乙醇的肝代谢中的作用。方法:从胎儿肝,肾提取的总RNA;经RT-PCR扩增得到cDNA并克隆至pGEM-T载体。cDNA序列用Kpn I和Pst I酶切鉴定,并检测其在大肠杆菌中表达活性。通过吸光法检测酶的活性。结果:成功克隆了人Ⅰ类乙醇脱氢酶并在大肠杆菌中获得稳定表达。经检测其酶活性分别为0．81～1．31U/mg、0．09～0．15U/mg和0．76～1．11U/mg。结论:cDNA克隆成功,并发现其与肝脏中分离的酶具有相似的活性。     

Androgen induction of alcohol dehydrogenase in mouse kidney. Studies with a cDNA probe confirmed by nucleotide sequence analysis

A cDNA clone for the beta-chain of human alcohol dehydrogenase (ADH) was used to isolate several cross-hybridizing clones from a mouse liver cDNA library. Clones pADHm9 and a portion of pADHm12 were sequenced. pADHm9 coded for a sequence of 151 C-terminal amino acids and some untranslated sequences from the 3' end of its corresponding mRNA. This clone was identified as an Adh-1 cDNA clone. Consistent with the known expression of Adh-1, this gene was expressed constitutively in liver, whereas the Adh-3 gene product was found only in stomach, lung and reproductive tissues. Furthermore, the translated region of the cDNA shared 91% amino acid sequence homology with rat liver ADH. [32P]pADHm9 was used as a hybridization probe to study the mechanism of androgen induction of kidney ADH activity. Induction of A/J female mice by androgen resulted in a dramatic increase in the steady-state level of Adh-1 mRNA content which correlated with the level of enzyme induction. The size of the mRNA obtained from control or induced kidney and liver tissues was indistinguishable by Northern analysis. [32P]pADHm9 was also used to probe restriction fragments of genomic DNA obtained from several inbred mouse strains. The hybridization patterns, considered with the genetic evidence, suggested that pADHm9 recognized sequences which may be present as only a single copy in the genome. No restriction fragment length polymorphisms were observed among the several inbred mouse strains examined.     

Involvement of atrial natriuretic peptide in blood pressure reduction induced by estradiol in spontaneously hypertensive rats

The aim of the present study was to determine the involvement of atrial natriuretic peptide (ANP) in blood pressure (BP) alterations induced by estradiol treatment. Spontaneously hypertensive rats (SHR) and Wistar rats (WR) were ovariectomized and, after 3 weeks, were injected daily for 4 days with estradiol benzoate (E2; 5 microg/100 g/day) or a vehicle. One day after the last injection, the animals were decapitated, blood was collected, and both right and left atrial appendages were quickly removed for determination of ANP by radioimmunoassay (RIA), or used for ANP mRNA determination. Estradiol treatment induced a significant reduction of blood pressure in SHR, but not in WR. This reduction was correlated with the increase of plasma ANP levels that were significantly increased in E2-treated, compared with vehicle-treated, SHR. E2-treated SHR showed significant increases in ANP concentration in the right and left atria compared to the vehicle-treated animals. These observations were confirmed by ANP mRNA. In summary, the present study shows that short-term estradiol treatment reduces the blood pressure of ovariectomized SHR, but not of WR. This reduction was highly correlated with increased plasma estradiol and ANP levels. These results suggest that ANP is involved in mediating the effect of estradiol on blood pressure reduction.     

Induction of lipid peroxidation by oxalate in experimental rat urolithiasis

The function of lipid peroxidation and the antiperoxidative enzymes of rat liver and kidney were studied in stone formation induced by intraperitoneal administration of sodium oxalate (7 mg/100 g body weight). The animals sacrificed 3 and 12 h after administration of sodium oxalate had higher level of malondialdehyde in liver and kidney than control animals. A significantly pronounced release of malondialdehyde was observed in treated liver and kidney homogenates when incubated with either ferrous sulphate or hydrogen peroxide compared to control liver and kidney. Superoxide dismutase activity was increased only in liver and not in kidney in treated animals compared to the control. A highly significant decrease in catalase activity was observed in both liver and kidney of treated animals.     

Inheritance and expression of tissue-specific catalase activity during development and aging in mice

The catalase activity in the liver, kidney, lung, and blood hemolysate was measured in newborn, 21-, 70-, 175-, and greater than 400-day-old mice from the strains BALB/c, Csb, C3H/HeSnJ, C3H/S, C57BL/6J, SW, and 129/ReJ. Catalase activity was found to be highest in the liver (approximately 0.33 U/mg protein) followed by the kidney (approximately 0.13 U/mg protein), lung (approximately 0.05 U/mg protein), and blood hemolysate (approximately 0.03 U/mg protein). ANOVA analysis indicated significant differences in enzyme activity among strains and age groups studied. The developmental profiles of enzyme activity were tissue and strain specific. Catalase activity in the blood, for example, was generally higher at birth and at old age, whereas the kidney catalase activity was low at birth and increased substantially with age. Strains could be classified as normal (129/ReJ, BALB/c, C3H/HeSnJ, C3H/S), hypocatalasemic (C57BL/6J, SW), and acatalasemic (Csb) with respect to enzyme activity and it was on this basis that the inheritance of the catalase phenotype was studied using appropriate crosses. The enzyme activity level in each tissue appears to be governed by a unique set of genetic regulators/modifiers that interact with a single structural gene (Cs) or its product to produce the catalase phenotype. Some of these (e.g., Ce-1 and Ce-2) have been previously described but based on the results of various crosses reported here, more must exist that remain still uncharacterized at the molecular level. Models proposed for the inheritance of the catalase phenotype vary in complexity from single allelic differences between strains (e.g., BALB/c x Csb; blood) to a system of multiple interacting genetic determinants (e.g., BALB/c x Csb; liver) each having dominant (e.g., C57BL/6J over BALB/c; liver) and recessive components (e.g., gene(s) conferring the acatalasemic phenotype in BALB/c x Csb; blood and kidney). Such results are important and offer an interesting model to further characterize aspects of eukaryotic gene regulation.     

Courtship and copulation in prepubertally castrated male sheep (wethers) treated with 17β-estradiol,aromatizable androgens,or dihydrotestosterone

Groups of sexually inexperienced adult Clun Forest sheep (four animals per group) which had been castrated on the day after birth received one of the following treatments: testosterone propionate (TP, 20 mg/day); estradiol dipropionate (ODP, 2 mg/day); 19-hydroxy-17, 19-dipropionate (19HTP, 20 mg/day); dihydrotestosterone propionate (DHTP, 20 mg/day); or arachis oil vehicle (OIL). Treatments were in the form of sc injections given 5 days/week over a 6-week period during which time individual animals were observed in 18 tests for sexual behavior. The stimulus females used were ovariectomized ewes maintained in a state of continuous receptivity by daily injections of 15 mg of TP. Various measures of sexual and aggressive behavior were recorded during each test. Mounting was induced mainly in animals in the TP group and to a lesser extent in those receiving ODP. The extent to which precopulatory courtship was induced followed the order TP > ODP > 19HTP. Animals treated with DHTP or OIL showed negligible sexual activity.     

Ethanol metabolism in guinea pig: In vivo ethanol elimination,alcohol dehydrogenase distribution,and subcellular localization of acetaldehyde dehydrogenase in liver

Guinea pig ethanol metabolism as well as distribution and activities of ethanol metabolizing enzymes were studied. Alcohol dehydrogenase (ADH; EC 1.1.1.1) is almost exclusively present in liver except for minor activities in the cecum. All other organ tissues tested (skeletal muscle, heart, brain, stomach, and testes) contained only negligible enzyme activities. In fed livers, ADH could only be demonstrated in the cytosolic fraction (2.94 μmol/g liver/min at 38 °C) and its apparent K_m value of 0.42 mm for ethanol as substrate is similar to the average K_m of the human enzymes. Acetaldehyde dehydrogenase (ALDH; EC 1.2.1.3) of guinea pig liver was measured at low (0.05 mm) and high (10 mm) acetaldehyde concentrations and its subcellular localization was found to be mainly mitochondrial. The total acetaldehyde activity in liver amounts to 3.56 μmol/g/ min. Fed and fasted animals showed similar zero-order alcohol elimination rates after intraperitoneal injection of 1.7 or 3.0 g ethanol/kg body wt. The ethanol elimination rate of fed animals after 1.7 g ethanol/kg body wt (2.59 μmol/g liver/min) was inhibited by 80% after intraperitoneal injection of 4-methylpyrazole. Average ethanol elimination rates in vivo after 1.7 g/kg ethanol commanded only 88% of the totally available ADH activity in fed guinea pig livers. Catalase (EC 1.11.1.6), an enzyme previously implicated in ethanol metabolism, is of 3.4-fold higher activity in guinea pig (10,400 U/g liver) than in rat livers (3,100 U/g liver), but 98% inhibition by 3-amino-1,2,4-triazole did not significantly alter ethanol elimination rates. After ethanol injection, fed and fasted guinea pigs reacted with prolonged hyperglycemia.     

Effect of estrogens on phosphofructokinase activity of uterus, liver and kidney in rat and hamster

The effect of estradiol-17β on the phosphofructokinase (PFK) activity of uterus, liver and kidney in rat and hamster has been studied. 16 hours after a dose of 10 μg estradiol/100 g body weight, there are no differences in the uterotrophic responses of rats and hamsters and the increase in uterine PFK activity is similar in both animals. 48 hours after two doses of 100 μg estradiol/100 g body weight, the uterotrophic response is slightly higher in hamster than in rat, but rat uterus shows a greater increase of PFK activity than does hamster uterus.Hepatic and renal PFK activities in hamsters of both sexes are not modified 48 hours after two doses of 100 μg estradiol/100 g body weight. These results indicate that in hamster and rat, PFK is under estrogenic control in uterus and not in liver and kidney.     

Expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats: The stimulation by calcium administration

The expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb of open-reading frame). Regucalcin mRNA was expressed in the kidney cortex, and this expression was clearly increased by a single intraperitoneal administration of calcium chloride solution (5–15 mg Ca/100 g body weight) in rats; this increase was remarkable at 60–120 min after the administration. Thyroparathyroidectomy (TPTX) caused a slight decrease of regucalcin mRNA levels in the kidney cortex. However, the administration of calcium (10 mg/100 g) in TPTX rats produced a clear increase of regucalcin mRNA levels in the kidney cortex. The subcutaneous administration of calcitonin (10–100 MRC mU/100 g) or parathyroid hormone [1–34] (1–10 U/100 g) in TPTX rats which received calcium (10 mg/100 g) administration did not cause an appreciable alteration of regucalcin mRNA levels in the kidney cortex, suggesting that the mRNA expression is not stimulated by calcium-regulating hormones. The administration of trifluoperazine (TFP; 5 mg/100 g), an inhibitor of Ca²⁺/calmodulin action, completely blocked the expression of regucalcin mRNA stimulated by calcium administration. Now, calcium content in the kidney cortex was significantly elevated by a single intraperitpneal administration of calcium (10 mg/100 g) in rats. The present study clearly demonstrates that the expression of regucalcin mRNA in the kidney cortex is stimulated by calcium administration in rats. This expression may be mediated through Ca²⁺/calmodulin action in the kidney cortex.     

Expression in E. coli and tissue distribution of the human homologue of the mouse Ke 6 gene, 17β-hydroxysteroid dehydrogenase type 8

Expression of the human Ke 6 gene, 17β-hydroxysteroid dehydrogenase type 8, in E. coli and the substrate specificity of the expressed protein were examined. The tissue distribution of mRNA expression of the human Ke 6 gene was also studied using real-time PCR. Human Ke 6 gene was expressed as an enzymatically-active His-tag fusion protein, whose molecular weight was estimated to be 32.5 kDa by SDS-polyacrylamide gel electrophoresis. Expressed human Ke 6 gene effectively catalyzed the conversion of estradiol into estrone. Testosterone, 5α-dihydrotestosterone, and 5-androstene-3β,17β-diol were also catalyzed into the corresponding 17-ketosteroid at 2.4–5.9% that of estradiol oxidation. Furthermore, expressed enzyme catalyzed the reduction of estrone to estradiol, but the rate was a mere 2.3%. Human Ke 6 gene mRNA was expressed in the various tissues examined, such as brain, cerebellum, heart, lung, kidney, liver, small intestine, ovary, testis, adrenals, placenta, prostate, and stomach. Expression of human Ke 6 gene mRNA was especially abundant in prostate, placenta, and kidney. The levels in prostate and placenta were higher than that in kidney, where it is known to be expressed in large quantities.     

Tissue specific,sex and age--related differences in the 6-phosphogluconate dehydrogenase gene expression

Data presented in thid paper indicate that: (1) the age-related changes in 6-phosphogluconate dehydrogenase (6PGDH) activity depend on sex and tissue. No differences in the liver 6PGDH activity between young (1-month-old) males and females were found. In adult males, the activity was the same as in young animals but, in adult females, it reached the value twice as high as in the young. In adipose tissue (both white and brown) and kidney cortex, the enzyme activity decreased with age both in males and females. There were no differences between males and females 6PGDH activity in brain, heart and skeletal muscle. (2) The sex and age-related changes in the liver 6PGDH activity occur predominantly at the level of mRNA cellular concentration. (3) In the liver of ovariectomized rats decrease of 6PGDH activity and mRNA level was observed. Oestradiol administration to ovariectomized rats restored liver 6PGDH activity and liver 6PGDH mRNA levels to that observed in controls. No changes in 6PGDH activity and mRNA levels were found in white adipose tissue (WAT) of ovariectomized adult rats and in ovariectomized rats treated with oestradiol. (4) Oestradiol administration to males caused an increase of liver 6PGDH activity and mRNA levels to values observed in females, but was without an effect on WAT 6PGDH activity and mRNA level. (5) These results suggest that 6PGDH activity in different tissues is not regulated in coordinate fashion and that oestradiol plays an important role in the liver enzyme activity regulation.     

Melatonin Prevents Oxidative Stress in Ovariectomized Rats Treated with Aluminium

This study is designed to determine the simultaneous effect of aluminium (Al) and melatonin (Mel) treatment in intact and ovariectomized (Ovx) female rats on oxidative stress and their inter-organ relationship in the kidney and liver. Al-treated rats received an intra-peritoneal injection of solution of aluminium lactate (0.575?mg Al/100?g of body weight, three times a week), during 12?weeks. Mel groups received intra-peritoneal injections of melatonin at a dose of 10?mg/kg/day, 5?days/week, during 12?weeks. The results of this study showed that Al treatment in female rats modifies homeostasis of glutathione and the antioxidant capacity of the rat liver and kidney. The alteration of glutathione homeostasis and oxidative status was not associated with an increased lipid peroxidation in both organs with the exception of the increase observed in the liver of Ovx rats. Al also induced modifications in the activity of some enzymes related to the glutathione cycle: GSH-Px in the liver and kidney and glutathione reductase only in the kidney. Al exposure decreased CAT activity in both the kidney and liver of intact and Ovx groups. The administration of Mel in the intact and castrated females treated with Al seems to reduce oxidative changes in the liver and kidney of intact and Ovx rats.