Eighteen-deoxyaldosterone and other less polar forms of 18-hydroxycorticosterone as aldosterone precursors in rat adrenals

Samples containing as precursors either 18-hydroxycorticosterone (18-OH-B) in its M form, or this converted to less polar forms at pH 2 (ACM), or M or ACM enclosed in liposomes from adrenal lipids were incubated at pH 7.4, 4.8 or 3.3 in the presence or absence of quartered rat adrenals for 1 and 2 h. Optimal (10%) yields of aldosterone were obtained when (a) ACM was incubated at pH 4.8 and (b) M enclosed in liposomes was suspended in buffer and shaken without enzyme at pH 3.3. When conditions (a) were supplemented with malate and NADP, 16% of ACM was converted to aldosterone. ACM contained 80% of a fraction which, according to 13C NMR spectroscopy, was identical to 18-deoxyaldosterone (18-DAL). Experiments in which radioactivity from corticosterone (B) or M was trapped by radioinert M or 18-DAL disclosed a pathway comprising sequentially B, 18-OH-B, 18-DAL and aldosterone, and the combined evidence of this work, an enzymatic hydroxylation of 18-DAL to aldosterone.     

Convertibility of a saponifiable lipoidal derivative of 18-hydroxycorticosterone

Metabolic properties and subcellular localization of the biosynthesis of SM, a saponifiable 18-OH-B (18-Hydroxycorticosterone) derivative, were investigated. Homogenates biosynthesized SM at a nearly constant rate of 463 pmol/50 mg tissue during 30 min. This biosynthesis was more efficient at pH 7.4 than at pH 4.8. Not only 18-OH-B but also its less polar anhydride 18-DAL (18-Deoxyaldosterone) were good precursors. SM was reverted to these precursors both enzymatically and spontaneously, 4.8 being a more suitable pH for this reversion than 7.4. Trapping experiments demonstrated a sequence comprising, in this order, the following echelons: SM, 18-OH-B, 18-DAL, Aldosterone. The first two steps are reversible and the last two ones depend on proton concentrations. It is postulated that SM could be on a dead-end to which 18-OH-B could be deviated if Aldosterone biosynthesis became temporarily unnecessary. Also, that 18-OH-B may convert to either 18-DAL or SM for selective membrane transports, according to homeostatic requirements.     

Origins of the differences in function of rat adrenal zona glomerulosa cells incubated as intact tissue and as collagenase-prepared cell suspensions

While in vitro incubation of dispersed cell preparations of adrenal cell types has been widely used as an experimental model, few studies have addressed the possibility that the enzymic and mechanical treatments involved may affect tissue functions. Using rat adrenal whole capsule tissue, consisting of glomerulosa cells still attached to the connective tissue capsule together with some fasciculata cells, and dispersed glomerulosa cell preparations formed by a variety of enzymic and incubation treatments, striking differences have been demonstrated between the functions of the various preparations in vitro. Under ACTH stimulation, whole capsules produced (ng per pair ± s.e.) 405 ± 35 ng aldosterone, 650 ± 60 ng 18-hydroxycorticosterone (18-OH-B) and 850 ± 90 ng corticosterone. In cells dispersed by collagenase incubation followed by repeated pipetting and filtration, aldosterone and 18-OH-B yields under ACTH stimulation fell to values less than 10% of those produced by whole tissue, whereas corticosterone values were unchanged. Omitting the filtration step gave a less well marked decline in aldosterone and 18-OH-B to 50% of intact tissue values. When the tissue was not dispersed after collagenase incubation, aldosterone and 18-OH-B outputs were similar in the two preparations. The decline in aldosterone and 18-OH-B is not attributable to loss in cell–cell contact alone, since short term culture of collagenase dispersed cells on contracting collagen discs did not restore the capacity to produce these steroids, and a decline in their output also occurred in similar culture of intact capsule tissue. In acute incubations, hyaluronidase had similar effects to collagenase, whereas trypsin, papain and a bacterial protease evoked aldosterone release during the preincubation period, but did not affect subsequent yields of aldosterone and 18-OH-B in incubations of dispersed (but not filtered tissue) in the presence of ACTH. Chymo-trypsin had no effect on preincubation but eliminated subsequent response to ACTH in all incubation conditions. Together with previously published data on the effects of trypsin, the results support the view that in intact rat adrenal glomerulosa tissue, aldosterone and 18-OH-B are sequestered into intracellular stores in the form of novel steroid-protein complexes. These are hydrolysed by trypsin and other preoteases with consequent release of steroid, but are virtually eliminated by conventional methods of cell suspension preparations, using collagenase preincubation with subsequent mechanical dispersal and filtration.     

Analysis of biological samples by gas chromatography-mass spectrometry without a reference standard: measurement of urinary 18-hydroxytetrahydro-11-dehydrocorticosterone excretion rate in human subjects

Reference standards for some minor urinary steroid metabolites are sometimes unavailable. We describe a novel procedure to quantitate a urinary steroid metabolite of known structure and mass spectrum, using as a standard a compound which produces ions in common with it and has a similar retention time in gas chromatography-mass spectrometry. The steroid of interest was 18-hydroxy-11-dehydrotetrahydrocorticosterone (18-OH-THA), the major urinary metabolite of 18-hydroxycorticosterone (18-OH-B), a putative intermediate in the conversion of 11-deoxycorticosterone to aldosterone. The steroid used as an alternative to the authentic 18-OH-THA standard was beta-cortol which, like 18-OH-THA, produces a fragmentation ion at m/z 457. Allo-tetrahydrodeoxycorticosterone (5alpha-THDOC) was used as the internal standard. beta-Cortolone also has the fragmentation ion at m/z 449 (in common with beta-cortol) and an authentic standard is available commercially. To validate the procedure, we quantitated beta-cortolone urinary excretion rate against this alternative standard and also against authentic beta-cortolone standards. Both methods produced similar results (adjusted R(2): 0.998, P<0.001). The method was then used to measure urinary excretion of 18-OH-THA rate in healthy volunteers. The reference range obtained was 20-204 microgram/24 h (n=32). This is similar to the few results available by conventional assay. Method performance was also similar to other assays of urinary steroids. This procedure could be generally applicable for assays when authentic standards are not available but mass spectra are known or can be predicted.     

The development and application of a radioimmunoassay for 18-hydroxy-corticosterone.

A sensitive and specific radioimmunoassay has been developed for 18-hydroxy-corticosterone (18-OH-B) and applied to the measurement of this steroid in peripheral plasma. High specific activity label (3H-18-OH-B) was prepared using the incubation of 3H-corticosterone with duck adrenal mitochondria. Antisera were produced by immunisation with 18-OH-B gamma-lactone 3-oxime conjugated to bovine serum albumin. The antibodies examined showed 100% cross-reactivity with 18-hydroxy-deoxycorticosterone gamma-lactone (18-OH-DOC gamma-lactone), but minimal cross-reactivity with other steroids. Paper chromatography was used to separate 18-OH-DOC gamma-lactone from 18-OH-B gamma-lactone. The interassay precision was 7.6% and the intra-assay precision 11.0%. The accuracy of the method was confirmed by showing a linear relationship between amounts of 18-OH-B added and amounts of 18-OH-B gamma-lactone measured (y = 0.854 X +15.1, r = 0.9. p less than 0.001). The mean plasma level in normal subjects on an ad libitum sodium intake was 225 +/- 92.7 (SD) pg/ml (n = 17) when standing, and 99 +/- 38.3 (SD) pg/ml (n = 6) after lying down for 30 minutes.     

Effects of proteolytic enzymes on steroid release from rat adrenal zona glomerulosa tissue: Evidence for novel steroid-protein complexes

Trypsin (2mg/ml) added to conventional incubations of rat adrenal capsules (largely glomerulosa) reproducibly increases the amount of free extractable aldosterone (aldo) and 18-hydroxycorticosterone (18-OH-B) in incubation media, but has no effect on capsule cell suspensions formed by collagenase incubation. The effect is abolished by the addition of a trypsin inhibitor, but is still seen in the absence of $\text{de novo}$ steroidogenesis. Qualitatively similar results were obtained with capsule homogenates and high speed supernatant fractions, and chromatography of the high speed supernatant protein fraction on Sephadex G-50 gave a number of minor fractions and one major fraction which yielded free aldo on incubation with trypsin. The results indicate the existence of storage forms of aldo and 18-OH-B which are extremely tightly bound to protein. Such steroid-protein complexes appear to be of an entirely novel kind, and are quite distinct from the familiar receptor type complexes. The findings support previously proposed mechanisms for aldo synthesis and secretion.     

Two adult familial cases of selective hypoaldosteronism due to insufficiency of conversion of corticosterone to aldosterone

A 57-year-old woman (case 1) and her daughter aged 29 (case 2) with hyperkalemia exhibited subnormal plasma aldosterone (ALD) in the face of elevated plasma renin activity. Their physical findings were normal. Their arterial blood gas analysis showed that metabolic acidosis and renal function of these cases were slightly impaired. Urinary 17-OHCS and 17-KS excretions in these cases were normal. Baseline levels of corticosterone (B) and 18-hydroxycorticosterone (18-OH-B) were clearly elevated. Plasma deoxycorticosterone (DOC), B and 18-OH-B as well as cortisol remarkable increased after ACTH injection, but the increase in plasma ALD was very small. Angiotensin II infusion in case 1 resulted in a clear rise in plasma 18-OH-B but in slight depletion of B, and no increase in ALD. 9-alpha-fludrocortisone acetate treatment was performed in case 1. Serum potassium was normalized and blood pressure elevated from 82/52 to 120/78 mmHg. Arterial blood gas analysis was corrected. We concluded that these two cases with subnormal plasma ALD and hyperreninemia may exist as a congenital and familial abnormality of the final step of aldosterone boisynthesis due to the impairment of the conversion of B to ALD.     

Cellular receptors for lymphotoxin: correlation of binding and cytotoxicity in sensitive and resistant target cells.

The binding of radioactively labeled lymphotoxin (LT) to both lymphotoxin-sensitive and -resistant cell clones was examined. The sensitive clone had a low- capacity, high-affinity ("specific") binding component, the curve of which closely followed the cytotoxicity curve of the lymphocyte mediator. The capacity of this binding component was calculated to be about 600 molecules of LT/cell. In addition, there was a low-affinity, high-capacity ("nonspecific") binding component. In striking contrast, the high-affinity, low-capacity ("specific") component was absent or greatly diminished from the resistant clone, whereas the low-affinity, high-capacity ("nonspecific") component was present at a similar level as in the sensitive cells.These binding characteristics closely resemble those observed by us and other investigators working with a variety of steroid hormones in steroid-sensitive and- resistant cell lines.     

Dopaminergic control of aldosterone: modulation of the response of rat adrenal zona glomerulosa cells to alpha-Msh by pretreatment with bromocriptine or metoclopramide

Bromocriptine treatment in rats (3 mg/kg per day, 7 days) significantly reduced alpha-msh and aldosterone plasma levels 2 hrs after the final treatment in animals on low, normal and high sodium diets. Alpha-MSH dose response curves for corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC) in subsequently incubated glomerulosa cells gave stimulation at lower concentrations of alpha-MSH (10(-10) moles per litre) than in cells from untreated animals (10(-9) moles per 1). Curves for aldosterone (ald) and 18-hydroxycorticosterone (18-OH-B) were also affected in cells from animals on a low sodium diet. Fasciculata-reticularis cell responses to ACTH were unaffected. Metoclopramide (4 mg/kg per day, 7 days) elevated plasma alpha-MSH, although ald was unaffected, but inhibited the glomerulosa cell response to alpha-MSH in vitro. Acute dopaminergic responses in plasma ald may be mediated through alpha-MSH in rats, but chronically alpha-MSH may down- regulate glomerulosa cell alpha-MSH receptors. It is unlikely that alpha-MSH mediates the adrenocortical response to sodium depletion.     

Adaptation of aldosterone biosynthesis to sodium and potassium intake in the rat

The steroidogenic response of rat adrenal zona glomerulosa to stimulators is variable and depends on the activity of biosynthetic steps involved in the conversion of deoxycorticosterone (DOC) to aldosterone (Aldo). Corticosterone methyl oxidations (CMO) 1 and 2 are stimulated by sodium restriction and suppressed by potassium restriction. These slow alterations are accompanied by the appearance or disappearance of a specific zona glomerulosa mitochondrial protein with a molecular weight of 49,000. Induction of CMO 1 and 2 activities and the appearance of the 49 K protein can also be elicited in vitro by culture of rat zone glomerulosa cells in a medium with a high potassium concentration. The 49 K protein crossreacts with a monoclonal antibody raised against purified bovine adrenal cytochrome P-450(11 beta). The same antibody stains a protein with a molecular weight of 51,000 in rat zona fasciculata mitochondria and in zone glomerulosa mitochondria of rats in which CMO 1 and 2 activities have been suppressed by potassium restriction and sodium loading. The 51 K crossreactive protein was purified to electrophoretic homogeneity by chromatography on octyl-sepharose. In a reconstituted enzyme system, it converted DOC to corticosterone (B) and to 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) but not to 18-hydroxycorticosterone (18-OH-B) or Aldo. A partially purified 49 K protein preparation from zona glomerulosa mitochondria of rats kept on a low-sodium, high-potassium regimen converted DOC to B, 18-OH-DOC, 18-OH-B and Aldo. According to these results, rat adrenal cytochrome P-450(11 beta) exists in two different forms, with both of them capable of hydroxylating DOC in either the 11 beta- of the 18-position, but with only the 49 K form capable of catalyzing CMO 1 and 2. The adaptation of aldosterone biosynthesis to sodium deficiency or potassium intake in rats is due to the appearance of the 49 K form of the enzyme in zona glomerulosa mitochondria.     

Role of corticosteroids in distal acidification of amiloride-treated rats.

The role of amiloride-dependent sodium channels in the action of adrenal cortical steroids on urine-blood PCO2 (U-B PCO2) differences was studied in bicarbonate-infused and amiloride-treated adrenalectomized rats. U-B PCO2 was significantly reduced by amiloride in bicarbonate-infused control rats. Adrenalectomy further reduced U-B PCO2 in amiloride-treated, bicarbonate-infused rats (from 27.9 +/- 1.82 mmHg in sham-operated rats to 21.3 +/- 1.58 mmHg in adrenalectomized (ADX) rats) (1 mmHg = 133.322 Pa). Acute administration of corticosterone and 18-hydroxycorticosterone (18-OH-B), but not of aldosterone, caused recovery of U-B PCO2 to the level of sham-operated animals treated with amiloride. Aldosterone did not affect U-B PCO2 in the presence of amiloride (21.9 mmHg ADX group vs. 20.98 mmHg aldosterone group). Results are compatible with aldosterone affecting distal H ion secretion mostly by a sodium and potential difference dependent mechanism, while corticosterone and 18-OH-B should act by other mechanisms (e.g., increased luminal buffer level).     

Priming and triggering of tumoricidal and schistosomulicidal macrophages by two sequential lymphokine signals: interferon-gamma and macrophage cytotoxicity inducing factor 2

We describe a new lymphokine activity, macrophage cytotoxicity inducing factor 2 (MCIF2), in the T cell mitogen-induced supernatant of a murine T cell clone in long-term culture. MCIF2 has the following properties: it elutes from a Sephadex G-100 column in three m.w. forms (10, 34, and 100 KD); it is acid labile (pH 2 to 4) and heat sensitive (80 min at 56 degrees C); it is not constitutively secreted, coexists in the same supernatant with immune interferon (IFN-gamma), and synergizes with IFN-gamma for induction of tumoricidal and schistosomulicidal resident peritoneal mouse macrophages. We uncoupled this synergy and show that IFN-gamma serves as the first ("priming") and MCIF2 as the second ("triggering") signal for macrophage activation. Application of the lymphokines in the reverse order was ineffective. These data demonstrate a two-step mechanism of macrophage activation.     

Transition from cytosolic to mitochondrial thymidine kinase during development in human fetal tissues

The transition from cytosolic ("fetal") to mitochondrial ("adult") thymidine kinase, as detected by electrophoresis, was examined in six human fetal tissues of gestational ages 11-40 weeks. In all tissues there was an early period during development in which only the fetal form was detected, followed by a transitional period in which both fetal and adults forms were present, followed by a later period in which only the adult enzyme occurred. Transitional periods were 23-25 wk. gestational age for colon, 13-15 wk. for kidney, 18-20 wk. for liver, 14-18 wk. for lung, 34-36 wk. for serum, and 25-28 wk. for thyroid. In all cases, only the adult form was present by the time of birth and persisted during the first 18 months of extrauterine life. The adult form, but not the fetal form, was inhibited by dCTP.     

46,XX,r(18), + mar karyotype in the niece of a leukemic patient with trisomy 21 and cri du chat chromosome (author's transl)

The present observation concerns a female patient with a 47,XX, r(18), + mar karyotype. The size of the ring varies from mitosis to mitosis; generally it is very small. The presence of a small marker ("minute") may explain some phenotypic differences not usually observed in the "r(18) syndrome". This patient is the niece of a trisomic 21, who showed complex chromosome anomalies during the course of an acute leukemia from which he died.     

Delayed fluorescence induction transients: mathematical modelling based on the chosen kinetic models.

The paper deals with mathematical modelling of the transients obtained by fitting of delayed fluorescence (DF) induction trace. The transients are in certain, doubtless connection with electrochemical gradient (ECG) formed across thylakoid membranes upon illumination. The fitting of the C and D transients by using consecutive model for first-order reactions (A --> B --> C) showed that they might play a role of the intermediate (B), according to scheme down bellow: ("A1 state")ECG (k1(C transient))--> C transient (k2(C transient))--> products, ("A2 state")ECG (k1(D transient))--> D transient (k2(D transient))--> products. The two ECG controlled "states" (A1 & A2) are not the same, which does not exclude some sort of proportionality. On the other hand, the E band, contributing mainly to the stationary level of DF induction trace, may be fitted by parallel model of at least two first-order reactions.     

Sequence, evolution and tissue expression patterns of an epidermal type I keratin from the shark Scyliorhinus stellaris

From the shark Scyliorhinus stellaris we cloned and sequenced a cDNA encoding a novel type I keratin, termed SstK10. By MALDI-MS peptide mass fingerprinting of cytoskeletal proteins separated on polyacrylamide gels, we assigned SstK10 to a 46-kDa protein which is the major epidermal type I ("IE") keratin in this fish and is specifically expressed in stratified epithelia. In a phylogenetic tree based on type I keratin sequences and with lamprey keratins applied as outgroup, SstK10 branches off in a rather basal position. This tree strongly supports the concept that teleost keratins and tetrapod keratins resulted from two independent gene radiation processes. The only exception is human K18 because its orthologs have been found in all jawed vertebrates (Gnathostomata) studied; in the tree, they form a common, most early branch, with the shark version, SstK18, in the most basal position. Thus, the sequences of SstK10 and SstK18 also favor the classical view of vertebrate evolution that considers the cartilaginous fishes as the most ancient living Gnathostomata. To determine the overall expression patterns of epidermal ("E") and simple epithelial ("S") keratins in this shark, we furthermore tested a panel of monoclonal anti-keratin antibodies by immunofluorescence microscopy of frozen tissue sections, and in immunoblots of cytoskeletal preparations, demonstrating that immunodetection of specific keratins is a convenient method to characterize epithelial tissues in shark.     

Transport proteins of Plasmodium falciparum: defining the limits of metabolism

In this review we give an account of transport processes occurring at the membrane interface that separates the asexual stage of Plasmodium falciparum from its host, the infected erythrocyte, and also describe proteins whose activities may be important at this location. We explain the potential clinical value of such studies in the light of the current spread of parasite resistance to conventional antimalarial strategies. We discuss the uptake of substrates critical to the survival of the intracellular malaria parasite, and also the parasite's homeostatic and disposal mechanisms. The use of the Xenopus laevis expression system in the characterisation of a hexose transporter ("PfHT1") and a Ca(2+) ATPase ("PfATP4") of the parasite plasma membrane are described in detail.     

Factors affecting the trypsin induced release of aldosterone in rat adrenal zona glomerulosa tissue

The yields of aldosterone obtained during incubation of whole adrenal capsule tissue from the rat (consisting of the connective tissue capsule itself, all of the glomerulosa tissue, and some fasciculata) cannot apparently be accounted for by the gland's capacity for de novo synthesis of this steroid. Recent studies with proteolytic enzymes and inhibitors suggest that in part aldosterone output may result from the activation of proteolytic events which release aldosterone from a sequestered intraglandular pool. These proteolytic events are mimicked by the addition of trypsin to whole tissue incubations in vitro. Experiments were carried out to determine what factors may govern the size of such intraglandular steroid pools. The most remarkable effect was that prior sodium depletion greatly enhanced the yield (2-3-fold) of aldosterone on subsequent incubation of adrenal capsules with trypsin, to an extent far greater than the increase in basal (non trypsin induced) aldosterone output in this tissue. Although betamethasone (20 micrograms/ml in drinking water) and the converting enzyme inhibitor captopril (7.2 mg/day) eliminated trypsin releasable steroid in control animals, they had no effect on the enhanced levels of trypsin releasable steroid seen with sodium depletion. The data suggest that trypsin releasable steroid pools are variable in accordance with the physiological requirements of the animal, particularly in sodium depletion.     

The reoxidation of cytochrome P-450 by paraquat inhibits aldosterone biosynthesis from 18-hydroxycorticosterone

Paraquat is an artificial electron carrier that captures electrons from reduced cytochrome P-450 instead of the natural acceptors, thus decreasing the concentration of reduced mitochondrial cytochrome P-450. In the present study, paraquat inhibited the biosynthesis of aldosterone from 18-hydroxycorticosterone by mitochondria from duck adult adrenal gland, under aerobic conditions. Since paraquat did not induce any change in the absorption spectrum of highly purified cytochrome P-450 11 beta, the possibility of a displacement of steroid by the drug is ruled out. Moreover, paraquat did not affect oxidative phosphorylating chain nor did it alter by itself the chemical structure of 18-hydroxycorticosterone. In our conditions, the inhibitory role of paraquat seems restricted to a capture of electrons from reduced cytochrome P-450. Under the same conditions metopirone and spironolactone, known to bind cytochrome P-450 11 beta at the steroid binding site, also inhibited the reaction. Altogether these results show that for aldosterone synthesis from 18-hydroxycorticosterone to take place, the steroid binding site on cytochrome P-450 must be accessible to 18-hydroxycorticosterone and that the cytochrome P-450 must be the direct donor of reducing equivalents. Hence, cytochrome P-450 appears as the final linking point between 18-hydroxycorticosterone and the reducing equivalents provided by NADPH.     

Pitfalls in the etiological diagnosis of congenital adrenal hyperplasia in the early neonatal period

The plasma levels of 8 steroid hormones were studied longitudinally in 20 newborns affected with either 21-hydroxylase (21-OH), 11-hydroxylase (11-OH) or 3 beta-hydroxysteroid deshydrogenase (3 beta-ol) deficiencies during the first month of life. Comparison was also made between the patterns observed according to age at first examination (n = 13 before 8 days of age) and the type of the enzymatic block. The most striking findings were the variability in hormone levels and/or evolution with age and the difficulties in making a definite positive or etiological diagnosis at first examination. In some newborns with untreated 21-OH deficiency, 17 alpha-hydroxyprogesterone levels may start off within the normal range or not so far above it. Also, levels of unconjugated dehydroepiandrosterone and other delta 5 steroids may be so high during the first week of life in all 3 forms that an erroneous diagnosis of 3 beta-ol deficiency could easily be considered. In several cases the etiological diagnosis was only ascertained by multiple steroid determination and/or dynamic tests. These discrepancies were not found in infants studied later on in life. It thus appears that a peculiar steroid pattern is observed in the immediate postnatal period in babies with various enzyme defects, which might be related to the morphological changes occurring in the adrenal cortex at this age.