Identification of 19-nor-deoxycorticosterone in normal rat serum by enzyme immunoassay and gas chromatography-mass spectrometry

Recent reports on the isolation and identification of 19-nor-deoxycorticosterone from the urine of rats with adrenal regeneration hypertension give rise to the possibility of a role of this steroid in the pathogenesis of low renin essential hypertension. The present study was undertaken to investigate the presence of 19-nor-deoxycorticosterone in normal rat serum both by a sensitive enzyme immunoassay and by gas chromatography-mass spectrometry (GC/MS). 19-Nor-deoxycorticosterone in rat serum was separated from other steroids prior to enzyme immunoassay by using high performance liquid chromatography (HPLC). The average concentration of 19-nor-deoxycorticosterone in normal rat serum was 137 +/- 62 ng/dl (mean +/- SD, n = 32). Pooled normal rat serum (50 ml) was purified by HPLC and the purified sample was acetylated with acetic anhydride for GC/MS measurement. The retention time and m/z ions (358, 285, and 257) on the resulting mass fragmentogram coincided in position with those of authentic 21-acetoxy-19-nor-deoxycorticosterone and acetylated normal rat serum extract. The combined characteristics of HPLC elution, antigen-antibody reaction, GC retention and selected ion responses provided strongly evidence supporting the presence of 19-nor-deoxycorticosterone in normal rat serum.     

Serum level of 19-hydroxyandrostenedione during pregnancy and at delivery determined by gas chromatography/mass spectrometry

19-Hydroxyandrostenedione (19-OHA) is secreted from the adrenal glands in men and women and also from the placenta during pregnancy. It has been found to cause hypertension in animal models. We have synthesized [7,7-2H2]-19-OHA with high deuterium content and, together with [7,7-2H2]A and [9,11-2H2]estrone (E1), have developed a quantitative assay of serum level 19-OHA, A, and E1 using the gas chromatography/mass spectrometry-mass fragmentography method to monitor individual subjects throughout pregnancy. The labeled 19-OHA, used as internal standard, showed only 6.73% of unlabeled compound. Recovery of standard 19-OHA, A, and E1 (5,000 pg each) added to male plasma was 97.4 +/- 2.3%, 96.3 +/- 2.1%, and 100.1 +/- 4.1% (mean +/- SD), respectively; the intraassay coefficient of variation was 2.1%, 3.5%, and 3.8%, respectively. Ten pregnant subjects without complications and 10 pregnant subjects near term with hypertension were selected (with informed consent). The 19-OHA and E1 serum concentrations of maternal venous blood from uncomplicated pregnancies increased significantly as gestation progressed (19-OHA: first trimester, 225 +/- 72; second trimester, 656 +/- 325; third trimester, 1,518 +/- 544 pg/ml), reaching the highest level at delivery (19-OHA: 1,735 +/- 684 pg/ml). Whereas a positive correlation was found between the level of 19-OHA and E1, no apparent change of the A level was observed during pregnancy. Levels of the three steroid hormones in pregnancy complicated by hypertension in the second and third trimester were not found to be significantly different from those of normal pregnancy (19-OHA of hypertensive subjects: second trimester, 762 +/- 349; third trimester, 1,473 +/- 491 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum 11-deoxycorticosterone levels in adrenal-regeneration hypertension under conditions of quiescence and stress.

A time course study to measure adrenal cortical function was undertaken for the period prior to the development of hypertension until the onset of hypertension in the adrenal-regeneration hypertension (ARH) model. Quiescent rat kills were used so that all adrenal cortical parameters investigated would reflect basal or resting levels for controls. Thus a more accurate determination of the differences between control and experimental animals could be made. A radioimmunoassay procedure for deoxycorticosterone was developed to measure this steroid in individual rat serum samples. Elevated serum deoxycorticosterone levels were observed in rats with regenerating adrenals when they were killed under quiescent conditions. This agreed with our recently reported in vitro finding of restoration of cholesterol side chain cleavage activity while 11beta-hydroxylase activity remained imparied 25 days after adrenal enucleation. When rats were killed after ether stress, deoxycorticosterone levels were elevated in both control rats and in rats with regenerating adrenals but the difference was not significant. In contrast, after ether stress serum corticosterone levels were lower in rats with regenerating adrenals than in controls. These studies, in conjunction with our previous in vitro findings, point to the importance of deoxycorticosterone in the pathogenesis of adrenal regeneration hypertension and help to explain the anomalous corticosteroid secretion rate data found in this experimental hypertension model.     

19-Nordeoxycorticosterone synthesis by rat kidney inner medullary collecting duct cells

19-Nordeoxycorticosterone (19-nor-Doc), a potent mineralocorticoid, was found to be synthesized by the isolated rat kidney perfused by an adrenal precursor (19-oxo-Doc). To determine if this bioconversion is a function of renal tubular cells, various adrenal precursors of 19-nor-Doc were added separately to rat kidney inner medullary collecting duct cells culture media at a concentration of 10 nM. While 4.6% +/- 1.0% of 19-oxo-Doc (n = 3) and 14.4% +/- 1.4% of 19-oic-Doc (n = 3) were converted to 19-nor-Doc after 24 hours of incubation, Doc, and 19-OH-Doc were not converted. This represents further evidence that Doc has to be metabolized to 19-oxo-Doc or 19-oic-Doc (19-carboxy-Doc) before it can be converted by the kidney inner medullary collecting duct cells to 19-nor-Doc.     

Selective 19-hydroxylase inhibition by an aromatase inhibitor, 4-hydroxyandrostenedione

19-Nor-deoxycorticosterone is a newly recognized mineralocorticoid which has been associated with some forms of genetic, experimental, and human hypertension. To further examine this relationship, specific inhibitors of 19-nor-deoxycorticosterone biosynthesis must be developed. Since 19-hydroxylation is the pivotal step in both 19-nor-deoxycorticosterone biosynthesis and aromatization of androgens to estrogens, we evaluated an aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione on the inhibition of 19-hydroxylation in both rat and human adrenal mitochondria in vitro and 19-nor-deoxycorticosterone production and blood pressure in spontaneously hypertensive rats in vivo. Adrenal mitochondria from 48 male Sprague-Dawley rats and 1 patient with an aldosterone-producing adenoma were incubated in the presence of deoxycorticosterone substrate both with and without 4-hydroxyandrost-4-ene-3,17-dione. 4-Hydroxyandrost-4-ene-3,17-dione produced significant inhibition of 19-hydroxy-deoxycorticosterone production in both rat and human adrenal mitochondria, with a smaller and not significant inhibition of corticosterone and 18-hydroxy-corticosterone. 4-Hydroxyandrost-4-ene-3,17-dione given subcutaneously to spontaneously hypertensive rats lowered 19-nor-deoxycorticosterone by 69% and completely abolished hypertension compared to Wistar-Kyoto controls. These data demonstrate that 4-hydroxyandrost-4-ene-3,17-dione is a specific inhibitor of 19-hydroxylase, that it lowers 19-nor-deoxycorticosterone production and prevents hypertension in the spontaneously hypertensive rat. These studies reinforce the possible pathogenic significance of 19-nor-deoxycorticosterone in hypertension in spontaneously hypertensive rats.     

Atypical acid-fast bacteria in urine.

An antiserum was produced against the plasma of rats with adrenal regeneration hypertension. When the antiserum was adsorbed with normal rat plasma a single precipitin arc in the pre-albumin zone remained in the antiserum, indicating a specific antibody against the hypertensive factors responsible for this type of hypertension in rats. Following a single injection of the adsorbed antiserum into rats with adrenal regeneration hypertension, the level of blood pressure dropped noticeably. Histologically, the zona glomerulosa of hypertensive rats was hyperplastic, whereas that of rats receiving the antiserum showed marked atrophy.     

Differences in sleep, light, and circadian phase in offshore 18:00-06:00 h and 19:00-07:00 h shift workers

Complaints concerning sleep are high among those who work night shifts; this is in part due to the disturbed relationship between circadian phase and the timing of the sleep-wake cycle. Shift schedule, light exposure, and age are all known to affect adaptation to the night shift. This study investigated circadian phase, sleep, and light exposure in subjects working 18:00-06:00 h and 19:00-07:00 h schedules during summer (May-August). Ten men, aged 46+/-10 yrs (mean+/-SD), worked the 19:00-07:00 h shift schedule for two or three weeks offshore (58 degrees N). Seven men, mean age 41+/-12 yrs, worked the 18:00-06:00 h shift schedule for two weeks offshore (61 degrees N). Circadian phase was assessed by calculating the peak (acrophase) of the 6-sulphatoxymelatonin rhythm measured by radioimmunoassay of sequential urine samples collected for 72 h at the end of the night shift. Objective sleep and light exposure were assessed by actigraphy and subjective sleep diaries. Subjects working 18:00-06:00 h had a 6-sulphatoxymelatonin acrophase of 11.7+/-0.77 h (mean+/-SEM, decimal hours), whereas it was significantly later, 14.6+/-0.55 h (p=0.01), for adapted subjects working 19:00-07:00 h. Two subjects did not adapt to the 19:00-07:00 h night shift (6-sulphatoxymelatonin acrophases being 4.3+/-0.22 and 5.3+/-0.29 h). Actigraphy analysis of sleep duration showed significant differences (p=0.03), with a mean sleep duration for those working 19:00-07:00 h of 5.71+/-0.31 h compared to those working 18:00-06:00 h whose mean sleep duration was 6.64+/-0.33 h. There was a trend to higher morning light exposure (p=0.07) in the 19:00-07:00 h group. Circadian phase was later (delayed on average by 3 h) and objective sleep was shorter with the 19:00-07:00 h than the 18:00-06:00 h shift schedule. In these offshore conditions in summer, the earlier shift start and end time appears to favor daytime sleep.     

Cytochrome P450(11β): Structure-function relationship of the enzyme and its involvement in blood pressure regulation

Cytochrome P450(11β) is deeply involved in the final steps of biosynthesis of mineralocorticoids. This paper deals with following issues about this enzyme. (1) The structure and function of the enzymes of various animal species are discussed. By making alignment of amino acid sequences of the enzymes, we identified peptide domains essential for the enzyme actions such as a putative steroid binding domain and a heme binding region. Estimates of molecular similarity among the P450(11β) family enzymes suggested that the enzymes having both 11β-hydroxylation activity and aldosterone (ALDO) synthetic activity of certain animals such as frog, cattle and pig are more similar to the ALDO synthases of the other animals, such as rat, mouse and human, than the 11β-hydroxylases of these animals. (2) The molecular nature of the P450(11β) family enzymes of genetically hypertensive rats as well as adrenal regeneration hypertension (ARH) rats is examined. (i) Mutation was found in the P450(11β) gene of Dahl's salt-resistant normotensive rat. Steroidogenic activity expressed by the mutated gene accounted well for abnormal plasma levels of steroid hormones in this rat. (ii) 11β-, 18- and 19-Hydroxylation activities of adrenal mitochondria prepared from spontaneously hypertensive rat (SHR), Wistar-Kyoto rat (WKY), and stroke-prone (SP)-SHR were not significantly different from each other. Levels of mRNA of ALDO synthase in adrenal glands of 50-week-old SHR was significantly lower than those of 10-week-old SHR, WKY and SHR-SP. (iii) No significant difference in 19-hydroxylation activity was found between adrenal mitochondria prepared from ARH rat and those from control rat. The level of message of ALDO synthase was lower in adrenal glands of ARH rat.     

Lithium action on adrenomedullary and adrenocortical functions and serum ionic balance in different age-groups of albino rats

The aim of the current study was to investigate lithium action on adrenomedullary and adrenocortical functions and on serum ionic balance in rats. Three age-groups of male rats (juvenile: 30 days, adult: 100 days and aged: 3 years) were used. Each age-group of animal was exposed to short- (10 days) and long-term (25 days) treatments with lithium. Each age-group of rat received lithium at a dose 2mEq/kg body weight daily for 10 and 25 days. Each daily dose (2mEq) was divided equally into half (1 mEq) and each half was injected intraperitoneally twice (at 9 am and 9 pm) for both the durations of experiments. Control animals received physiological saline for similar duration of experiments. Thirty animals were used for each age-group and they were divided equally into 6 groups with 5 each. After termination of all the experiments rats were sacrificed and, adrenal glands were quickly dissected out and processed for epinephrine, norepinephrine and corticosterone estimations and, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSDH) activity of the adrenal gland. Blood was drawn from the heart of each rat and, serum was collected and stored at -20 degrees C until assayed for lithium, calcium, sodium, potassium and corticosterone concentrations. The findings revealed that lithium in both short- and long-term treatments was maintained well within the therapeutic range (0.3-0.8 mEq/l) in all the age-groups of rats. This alkali metal caused depletions of both epinephrine and norepinephrine concentrations from adrenal glands, and elevations of corticosterone in both adrenal and blood serum of each age-group of rat (juvenile, adult and aged). Additionally adrenal 3beta-HSDH activity was also increased in all the age-groups of rats irrespective of duration of the treatments. Short-term treatment of lithium elevated only serum K+ level in juvenile and adult rats and, Ca+ level only in adult animals. Significant elevations of serum K+ and Ca+ levels were observed following long-term treatments of lithium in all the age group of rats. No significant change in serum Na+ level was recorded after lithium treatment, irrespective of duration of treatments, in any age-group of rats. The findings suggest that lithium action, in respect of adrenomedullary and adrenocortical functions and, serum ionic balance, may not be largely related to the age-group of rats and that, lithium acts on adrenomedullary activity probably by stimulating the release mechanism of epinephrine and norepinephrine from the adrenal gland of rats, but stimulates adrenocortical activity by stimulating both synthesis (including 3 beta-HSDH activity) and release of corticorterone. Simultaneously, lithium disturbs normal ionic balance by elevating K+ and Ca+ levels in all the age-group of rats. Thus, the antimanic drug certainly disturbs both adrenomedullary and adrenocortical functions and, serum ionic balance in all the age-group of rats.     

Human chorionic gonadotropin and luteinizing hormone-releasing hormone reverse the blockade of ovulation in pregnant mare's serum gonadotropin-primed immature rats by the anti-androgenic drug, hydroxyflutamide

The present study was designed to examine mechanism(s) of the anti-ovulatory action of the anti-androgen, hydroxyflutamide (OH-F). Prepubertal rats were treated with 4 IU pregnant mare's serum gonadotropin (PMSG) (day -2) to induce first estrus and ovulation. They received OH-F in sesame oil or oil alone at 08:00 and 20:00 h on day 0 (the day of proestrus) and ovulations were assessed on the morning of day 1. Eighty-three percent of control animals ovulated with a mean of 7.7 +/- 1.1 corpora lutea per rat. Hydroxyflutamide blocked ovulation in all but 2 of the 12 rats receiving this drug alone. All of OH-F treated rats that received 5 and 25 IU human chorionic gonadotropin (hCG) ovulated with means +/- SEM of 9.1 +/- 0.1 and 7.3 +/- 1.4 corpora lutea per rat, respectively. The dose of 0.2 IU hCG was essentially ineffective, while the effect of 1.0 IU hCG was intermediate. At the dose of 20 ng and above (100 and 500 ng) luteining hormone-releasing hormone (LHRH) completely overcame the ovulation blockade in the OH-F treated animals, while a 4-ng dose was ineffective. At 18:00 h on the day of proestrus, serum LH levels in control animals were 17.56 +/- 2.60 ng/mL, which were 920% above basal levels (1.90 +/- 0.13) indicating a spontaneous LH surge. This surge was suppressed in OH-F treated rats. Injection of LHRH, at the dose of 20 ng and above, reinstated the LH release in OH-F treated animals. Thus, the anti-androgen, OH-F, inhibits ovulation in PMSG-treated immature rats through its interference with the preovulatory LH surge; the inhibition can be reversed by hCG or LHRH. Hydroxyflutamide does not appear to interfere at the level of the pituitary, but may have direct action at the hypothalamic and (or) extrahypothalamic sites involved in the generation of positive feedback signals that control LH release.     

Alpha-human atrial natriuretic polypeptide inhibits 19-hydroxy-androstenedione secretion by human adrenal cells

We investigated the effect of ACTH, angiotensin II (AII), and alpha-human atrial natriuretic polypeptide (alpha-hANP) which plays an important role of water-electrolytes balance, on 19-hydroxyandrostenedione (19-hydroxyandrost-4-ene-3,17-dione, 19-OH-A-dione) secretion by cultured human adrenal cells. 19-OH-A-dione in culture media was measured using a specific RIA. Basal 19-OH-A-dione secretion by adrenal cells was 0.69 +/- 0.08 ng/3h/10(6) cells and significantly rose to 1.17 +/- 0.14 ng/3h/10(6) cells in the presence of ACTH, but not in the presence of A II. These results demonstrate that 196-OH-A-dione is directly secreted from adrenal cells. alpha-hANP significantly inhibited both basal and ACTH-stimulated 19-OH-A-dione secretions, as well as aldosterone. These results demonstrate that alpha-hANP inhibits aldosterone activity by means of the inhibition of both aldosterone and 19-OH-A-dione (an aldosterone amplifier) secretion by adrenal cells.     

Endothelum-dependent and-independent relaxations in aortic rings obtained from hypertensive hooded (Aguti) rats.

Experimental hypertension studies are few in the hooded (Aguti) rat. The present study was designed to investigate the usefulness of this rat strain for experimental hypertension studies and to test the hypothesis that the hypertension may be associated with a diminution of endothelium dependent and independent relaxations. Hypertension was induced in inbred hooded rats (n=8 each) by administering 8% salt in the diet and /or 100 mg/kg/day Nomega-nitro-L-arginine-methyl-ester (L-NAME) in the drinking water for six and/or four weeks respectively. The rats were anaesthetized using a 25% urethane and 1% chloralose mixture given intraperitoneally at a dose of 5 mg/kg. Their blood pressure was measured invasively. Thereafter, relaxations of rat aortic preparations to acetylcholine, histamine and sodium nitroprusside (SNP) were assessed using standard organ bath conditions. Probabiliity level of 0.05 was taken as statistically significant. The mean arterial pressure (MAP;mm Hg) rose significantly in all test groups (Salt: 148.3 +/- 4.6; L-NAME: 181.7 +/-8.3; Salt+L-NAME:154.9 +/-8.7) compared with control (94.2 +/-6.8; [P < 0.05]. The MAP was significantly [P < 0.05] higher in the L-NAME group than in all the other groups. The heart rate fell significantly in the salt + L-NAME group compared to control [P <0.05].The IC50 of acetylcholine in aortic rings from L-NAME rats (7.9 x 10(-1) +/- 6.0 x 10(-3)) was significantly higher than in rings from control (9.4 x 10(-8) +/- 2.8 x 10(-8)), salt (7.8 x 10(-7) +/- 4.7 x 10(-7)) and salt + L-NAME (3.3 x 10 (-7) +/- 2.1 x 10(-7)) rats [P < 0.05]. The IC50 of histamine and SNP in the rings from the test groups of rats showed no significant difference from control. Also, endothelium dependent and independent relaxations were preserved in the various forms of hypertension studied except in chronic NOS inhibition where the former was attenuated in response to acetylcholine.     

Modest maternal protein restriction fails to program adult hypertension in female rats

Modest maternal dietary protein restriction in the rat leads to hypertension in adult male offspring. The purpose of this study was to determine whether female rats are resistant to developing the increased blood pressure seen in male rats after maternal protein restriction. Pregnant rats were fed a normal protein (19%, NP) or low-protein (8.5%, LP) diet throughout gestation. Renal renin protein and ANG II levels were reduced by 50-65% in male LP compared with NP pups, but were not suppressed in female LP compared with female NP. Mean arterial pressure in conscious, chronically instrumented adult female offspring (22 wk) was not different in LP (LP: 120 +/- 3 mmHg vs. NP: 121 +/- 2 mmHg), and glomerular filtration rate was also not different in LP vs. NP. The number of glomeruli per kidney was similar in adult LP and NP female offspring (LP: 26,050 +/- 2,071 vs. NP: 26,248 +/- 1,292, NP), and individual glomerular volume was also not different (LP: 0.92 +/- 0.11 10(6) microm(3), LP vs. NP: 1.07 +/- 0.11 10(6) microm(3)); the total volume of all glomeruli per kidney was also not significantly different. Thus female rats are relatively resistant to the programming for adult hypertension by perinatal protein restriction that we have described in males. This resistance may be due to the fact that modest maternal protein restriction does not reduce the number of glomeruli with which females are endowed as it does in males. The intrarenal renin-angiotensin system during development may play a key role in this protective effect of female gender.     

Long- and medium-chain triacylglycerols in nutritional support of liver regeneration of partially hepatectomized rats

An appropriate choice for a suitable diet during liver regeneration still remains an enigma. To investigate the effect of isocaloric enteral feeding with medium-chain triacylglycerols (MCT) and long-chain triacylglycerols (LCT) supplement (MCT+LCT, 40%:60% w:w) (178 kJ/kg b.w./24 h), rat liver regeneration was studied 24 and 72 h after partial hepatectomy. The liver DNA synthesis 24 h after partial hepatectomy was significantly higher in the MCT+LCT-supplemented rats (30.2+/-8.2 x 10(3) dpm/mg liver DNA) compared to MCT-treated animals (18.1+/-5.7 x 10(3) dpm/mg liver DNA). Liver protein synthesis was non-significantly elevated both 24 and 72 h after surgery in MCT+LCT-supplemented rats (13.7+/-1.1 and 10.9+/-3.1 x 10(3) dpm/mg liver protein). Seventy-two hours after partial hepatectomy, the hepatocyte mitotic activity was significantly increased in MCT+LCT- supplemented group vs. LCT- or MCT-fed rats (3.3+/-0.7 vs. 1.9+/-0.7 or 1.0+/-0.6 mitoses per 1000 hepatocytes), thus exhibiting an increased proliferative potential. The results showed a qualitative difference according to the proportion of MCT to LCT in the enteral supplements. Overfeeding with MCT decreased body weight, increased liver weight by its fatty infiltration, increased rat mortality rate and reduced spontaneous caloric intake. We conclude that the balanced supplement of MCT+LCT (40%:60% w:w) preserves liver regeneration, whereas overfeeding with MCT seems to be deleterious.     

Melatonin concentrations in patients with large goiter before and after surgery

OBJECTIVES: Surgical removal of a very large goiter may traumatize adjacent anatomical structures. The manipulations that involve superior cervical ganglia may alter melatonin secretion. To test this hypothesis we decided to study diurnal serum melatonin profiles in patients with a very large goiter before and after the surgery. MATERIAL AND METHODS:The study was performed on 10 women (mean age-46.5+/-1.6 years; mean+/-SEM; range 39-54 years) with very large non-toxic nodular goiter (mean thyroid volume-125.8+/-25.9 cm (3); mean+/-SEM; range 82.6-326.7 cm(3)). Diurnal serum melatonin profiles were estimated two days before the operation and 10 days after the surgery. Blood samples were collected at 08:00, 12:00, 16:00, 20:00, 22:00, 24:00, 02:00, 04:00, 06:00 and 08:00 h. Melatonin concentration was measured using RIA kit. RESULTS: Nocturnal serum melatonin concentrations (at 24, 02, and 04 hours) were significantly higher after the surgery than before the operation. CONCLUSIONS: Very large goiter may compress the superior cervical ganglia altering indirectly the melatonin synthesis. It cannot be excluded, however, that the presence of the large goiter in some other way affects melatonin secretion.     

19-Noraldosterone in pregnancy-induced hypertension

Pregnancy-induced hypertension (PIH) is a frequent cause of maternal and neonatal morbidity and mortality. 19-Noraldosterone, which was shown to be synthesized in the human adrenal gland, exhibits potent mineralocorticoid and hypertensive activity. To examine the role of mineralocorticoids in the pathophysiology of PIH, we studied urinary 19-noraldosterone, tetrahydroaldosterone, free cortisol, and cortisone concentrations and mineralocorticoid receptor levels in peripheral blood mononuclear leukocytes, from 17 women with PIH and 16 normal pregnant women as controls. Sequence analysis of the mineralocorticoid receptor gene in PIH patients was also done. The 24-h urinary excretion of 19-noraldosterone was significantly lower in PIH (120 +/- 38 pmol/day) than in controls (358 +/- 55 pmol/day) (P < 0.05). Urinary tetrahydroaldosterone was also decreased in PIH compared with controls. Ratios of urinary free cortisol to cortisone (a measure of 11beta-hydroxysteroid dehydrogenase 2 activity) did not differ significantly between groups. Mineralocorticoid receptor density was significantly (P < 0.05) decreased in the PIH group (133 +/- 15 binding sites/cell) compared with controls (255 +/- 21 binding sites/cell). No mutations were found in the coding region of the mineralocorticoid receptor gene in PIH. These results suggest that circulating aldosterone, 19-noraldosterone, and renal 11beta-hydroxysteroid dehydrogenase2 do not contribute to the pathogenesis of PIH. Regulatory factors that cause the down-regulation of the mineralocorticoid receptor in PIH should be clarified.     

Metabolism of C19-steroids by homogenates of normal rat and mouse adrenal tissue and of the Snell transplantable rat adrenocortical tumour 494

C(19)-steroid metabolism in homogenates of adrenal tissue from rats and mice has been studied. Production of these compounds from [7alpha-(3)H]cholesterol by rat adrenal tissue appeared to follow a route independent of pregnenolone. The major products of [7alpha-(3)H]-dehydroepiandrosterone metabolism by rat adrenal tissue were 5alpha-reduced steroids, principally androsterone, epiandrosterone and 5alpha-androstanedione. No differences in metabolism of [7alpha-(3)H]dehydroepiandrosterone or [4-(14)C]pregnenolone were detected between adrenal tissue from Sprague-Dawley, Wistar and Osborne-Mendel rats, but experiments with the Snell rat adrenocortical tumour 494 showed that this tissue had low 5alpha-reductase activity. In contrast, the major products of [7alpha-(3)H]dehydroepiandrosterone metabolism by mouse adrenal tissue were 5beta-reduced steroids. Differences were observed between LACA and NH strains of mice in that there was a lower metabolism of androstenedione by NH mouse adrenal and a considerable difference in the proportions of aetiocholanolone and epiaetiocholanolone produced.     

Acute starvation affects rat adrenal steroidogenesis

To determine how starvation affects adrenal steroidogenesis we measured the activities of 3 adrenal enzymes involved in corticosterone biosynthesis in a group of adult female rats. The animals were either starved for 7 days or fed ad libitum for the same period. Relative adrenal weight and plasma corticosterone levels were increased in the experimental group of animals compared to the control group (40 +/- 2 vs 27 +/- 1 mg/100 g body weight, P less than 0.001, and 45 +/- 4 vs 30 +/- 5 ng/dl, P less than 0.05 respectively). There were no differences in plasma ACTH levels between the groups (34 +/- 5 vs 26 +/- 4 pg/ml). 11-Hydroxylase activity was increased in the starved group of animals (18 +/- 3 vs 8 +/- 2 nmol/mg protein/min, P less than 0.01). 3 beta-Hydroxysteroid dehydrogenase and 21-hydroxylase activities were not different between the groups (19 +/- 2 vs 16 +/- 1 nmol/mg protein/min, and 100 +/- 10 vs 110 +/- 10 pmol/mg protein/min respectively). These results suggest that acute starvation in rats produces an increase in adrenal 11-hydroxylase activity.     

Insulin is a dominant suppressor of sterol 12 alpha-hydroxylase P450 (CYP8B) expression in rat liver: possible role of insulin in circadian rhythm of CYP8B

Sterol 12 alpha-hydroxylase (CYP8B) is a key enzyme for regulating the cholic acid/chenodeoxycholic acid ratio in bile acid biosynthesis. The hepatic CYP8B level was elevated in streptozotocin-induced diabetic rats, and the elevated CYP8B was suppressed by insulin administration [Ishida, H. et al. (1999) J. Biochem. 126, 19-25]. The streptozotocin-induced elevation of hepatic CYP8B mRNA concomitantly responded to the decrement of the serum insulin level. The CYP8B mRNA level in the cultivated rat hepatoma H4TG cells was strongly suppressed by insulin, although it was affected by dibutyryl cAMP or thyroxine to lesser extents. These observations demonstrate that CYP8B expression is dominantly regulated by the direct action of insulin on hepatocytes. A marked circadian rhythm (maximum at 13:00-16:00 and minimum at 1:00) was observed both on the mRNA level and the activity of CYP8B. This rhythm was shifted from that of cholesterol 7 alpha-hydroxylase, a rate-limiting enzyme of bile acid biosynthesis, showing a maximum at 22:00 and a minimum at 10:00, and this shift might oscillate the cholic acid/chenodeoxycholic acid ratio, which is increased in the late afternoon and decreased at midnight. The rhythm of CYP8B was the inverse of the circadian variation of serum insulin level and was similar to the circadian rhythm of glucose 6-phosphatase. These facts and the potent suppressive effect of insulin on CYP8B indicate that the oscillation of the serum insulin may be a factor in producing the circadian rhythm of CYP8B.     

A new inorganic vasodilator, trans-[Ru(NO)(NH(3))(4)(POEt)(3)](PF(6))(3): hypotensive effect of endothelium-dependent and -independent vasodilators in different hypertensive animals models.

The hypotensive effect of RuNO was investigated in acute and chronic hypertensive rats, as well as in normotensive rats. Acute hypertension rats were used with 30% increase on basal BP (phenylephrine, angiotensin II (Ang II), N(G)-nitro-L-arginine methyl ester (L-NAME), and adult spontaneously hypertensive rats (SHR) (basal BP 168 +/- 3 mm Hg) were used as models for chronic hypertension. Rats were implanted with catheters (iv/ia) for BP measurements and for in bolus administration of RuNO, sodium nitroprusside (SNP), and acetylcholine (Ach) (10, 20, 40 nmol/kg, iv). The principal findings of this study were: (i) The hypotensive response to RuNO was 150% higher in acutely (phenylephrine and Ang II) and chronically (SHR) hypertensive rats than in normotensive rats, except in the case of L-NAME-induced hypertension (deltaMAP = 10 +/- 1.4 mm Hg). Chronic SHR showed 60% increase (deltaMAP = 19 +/- 0.8 mm Hg) in the effect compared to normotensive rats. (ii) The hypotensive response to SNP was lower (60%) in hypertensive rats than in normotensive rats, when compared to RuNO. However, the responses were similar in L-NAME-induced hypertension (deltaMAP = 30 +/- 2 mm Hg). (iii) The vasodilator response to Ach was increased in rats with Ang II-induced hypertension (deltaMAP = 53 +/- 1 mm Hg) and in SHR (deltaMAP = 67 +/- 3 mm Hg). RuNO response was more potent than SNP in hypertensive models and the increment in relation to normotensive was observed in the phenylephrine- and L-NAME-treated rats. This response could be correlated to the different endothelial dysfunction present in each model.