Relationships between unconjugated and sulphated steroids in porcine primary Leydig cell culture

Steroidogenesis in immature porcine Leydig cells was investigated in primary culture at 48-84 h under basal conditions and in the presence of hCG. The basal accumulation of unconjugated steroids was close to linear only during the first 4 h of study, whereas the sulphate-conjugated steroids accumulated essentially linearly over the 36 h experimental period. At the last time point, 95% of the steroids measured were sulphated. Stimulation with hCG (1 ng/ml) led to a still more pronounced sulphate conjugation, and approx 99% of the steroids measured were sulphated at 36 h. Under maximal stimulation with hCG (100 ng/ml) the sulphates accounted for 74% of the total steroids measured at 36 h. Testosterone, androstenedione, dehydroepiandrosterone, 5-androstene-3 beta, 17 beta-diol and estrone were usually quantitatively the most important unconjugated steroids, and sulphated dehydroepiandrosterone, estrone, testosterone and 5-androstene-3 beta, 17 beta-diol were the most important steroid sulphates, especially following maximal stimulation of the cultures. These data emphasize the importance of steroid sulphates in porcine testicular steroid metabolism. Under stimulation with hCG, there was a rapid response in testicular steroidogenesis, initially seen as a rapid increase in the secretion of unconjugated and sulphated steroids. At approx 4-12 h, the rate of sulphate conjugation appeared to reach or even to exceed that of steroid biosynthesis, which lead to stabilisation or a decrease in the concentrations of unconjugated steroids. Only high doses of hCG, 10-100 ng/ml, were then able to lead to a net accumulation of unconjugated steroids, at 24-36 h of incubation with hCG.     

The effect of growth factors on the cytotoxicity of sulphated polysaccharides

The cytotoxicity of sulphated polysaccharides on 3T3-L1 fibroblasts was investigated. Dextran sulphate with a molecular weight of more than 100000 and with a number of sulphate groups per sugar unit (degree of sulphation) of 2.4 showed strong cytotoxicity to 3T3-L1 fibroblasts. The synthetic (1→6)-- -mannopyranan sulphate having a degree of sulphation of more than 1.0 and with a molecular weight of approx. 100000 also exhibited cytotoxicity. The results indicated that the cytotoxicity of sulphated polysaccharides strongly depended on their molecular weight and degree of sulphation. FGF-1 (aFGF) and FGF-2 (bFGF) protected the cell from the damage caused by the sulphated polysaccharide, while PDGF, EGF, and HGF had no such effect. Therefore, it is suggested that the rescuing effect is one of the special biological activities, and is shown only by FGFs.     

An adrenocortical tumor secreting weak mineralocorticoids

An adrenocortical carcinoma (15.5 g) secreting excessive amounts of steroids with weak mineralocorticoid activity in a 25-year-old woman was studied with particular reference to its in vivo and in vitro secretions of steroids. Severe hypertension, occasional low serum potassium and suppressed PRA were the major clinical findings, and were improved with removal of the tumor. In the preoperative stage, plasma levels of 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 18-hydroxycorticosterone were all increased. However, the plasma level of aldosterone was repeatedly normal. Although plasma levels of pregnenolone, 17-hydroxypregnenolone, progesterone and 17-hydroxyprogesterone were very high, those of other late step steroids, i.e. 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione and testosterone were almost normal. From these findings, a major etiological role of weak mineralocorticoids such as 11-deoxycorticosterone, 18-hydroxycorticosterone and corticosterone in her hypertension was suggested. Pregnenolone and 17-hydroxypregnenolone in tumor tissue were increased, but 11-deoxycorticosterone, corticosterone, aldosterone, cortisol and adrenal androgens such as dehydroepiandrosterone, androstenedione and testosterone were below normal or low normal. In vitro production of 11-deoxycorticosterone, aldosterone or cortisol by the tumor tissue slices was very low and scarcely responded to synthetic ACTH.     

Degradation of human gastrin and CCK by endopeptidase 24.11: differential behaviour of the sulphated and unsulphated peptides

The degradation of human sulphated heptadecapeptide gastrin (G17s) by human endopeptidase 24.11 was studied in vitro. The products of degradation were characterized by HPLC, region-specific gastrin radioimmunoassay and amino acid analysis. The enzyme cleaved G17s at four sites, Trp4-Leu5, Ala11-Tyr12, Gly13-Trp14 and Asp16-Phe17. The patterns of fragments produced when sulphated and unsulphated G17s are hydrolysed by endopeptidase 24.11 indicate that the enzyme cleaves both substrates at the same four bonds. However, the sulphated G17 was 3-times less rapidly degraded than the unsulphated G17 (G17ns). In contrast, the rate of cleavage of the octapeptide cholecystokinin (CCK8) was faster when the peptide was sulphated. The kinetic data of endopeptidase 24.11 indicated similar Km values for sulphated or unsulphated gastrin and CCK; sulphated CCK8 exhibited a 2-fold higher kcat/Km value compared to unsulphated CCK8, whereas G17s exhibited a 2-fold lower kcat/Km value compared to G17ns. The results indicate that the presence of a sulphate group causes a marked reduction in the rate of hydrolysis of gastrin by endopeptidase 24.11, whereas sulphation enhances cholecystokinin degradation by the same enzyme. They also suggest that endopeptidase 24.11 may be responsible for the difference in metabolism of sulphated and unsulphated G17, previously observed in human circulation.     

Phenol sulphotransferase and uridine diphosphate glucuronyltransferase from rat liver in vivo and in vitro. 2,6-Dichloro-4-nitrophenol as selective inhibitor of sulphation

Microsomal UDP-glucuronyltransferase and cytosolic sulphotransferase share many substrates, such as phenols and hydroxamic acids. In a search for a selective inhibitor of sulphation, several phenolic compounds were tested. 2,6-Dichloro-4-nitrophenol is introduced as a selective inhibitor of sulphation in vivo, having no effect on UDP-glucuronyltransferase activity. As substrate for both conjugating enzymes the phenolic drug harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole) was used. In the rat in vivo 2,6-dichloro-4-nitrophenol caused almost complete inhibition of harmol sulphation after a single intraperitoneal injection (26mumol/kg) for 48h; the percentage of harmol sulphated decreased from 75% in controls to 5% in the treated rats. The percentage of harmol glucuronidated increased from 25 to 95%. Pentachlorophenol was equally effective but also highly toxic. Salicylamide had only a very-short-lasting inhibitory effect on sulphation. In vitro, 2,6-dichloro-4-nitrophenol inhibited sulphation of harmol by a rat liver postmitochondrial supernatant completely at 1mum, whereas even at 100mum it had no effect on glucuronidation of harmol. It is concluded that 2,6-dichloro-4-nitrophenol is a selective inhibitor of sulphation and, further, that its long duration of action makes it suitable for studies on the regulatory role of sulphation in some biological processes.     

Variability of proteoglycan expression in the isolated rat glomerulus

The sulphation of proteoglycans in freshly isolated rat glomeruli was studied by biosynthetic labelling with [35S]sulphate. At least 75% of the observed sulphation requires de novo synthesis of core protein and proceeds at a constant rate over at least 40 h. Heparan and dermatan sulphate proteoglycans (HSPG and DSPG, respectively) are the two major species produced, with only minor amounts (less than 5%) of chondroitin sulphate labelled under these conditions. Several factors affect the population distribution of labelled material. When glomeruli were obtained from rats 6 weeks of age, HSPG accounted for 75 +/- 9% of tissue proteoglycan sulphated over 16 h. When older rats (12-14 weeks) were used, only 32 +/- 10% of label was associated with HSPG, DSPG accounting for the remainder. Production of HSPG is sulphate-dependent, increasing relative to DSPG with increasing sulphate, up to physiological concentrations. However, the net charge-density of sulphated material is conserved even at the lowest concentrations of sulphate. This may reflect the importance of electrostatic properties in the function of glomerular proteoglycans. The production of HSPG increases relative to DSPG with time following isolation and this effect is more dramatic in glomeruli from younger rats. However, reciprocal changes in production of HSPG and DSPG sustain a constant rate of sulphation. This phenomenon may arise from interdependency of the glomerular epithelial and mesangial cells with respect to regulation of proteoglycan synthesis.     

Sulphated proteins secreted by rat mammary epithelial cells

The main sulphated proteins secreted by rat mammary gland tissue have Mr of approximately 32 000, 27 000 and 25 000 Da. In addition, there are high Mr components which have a diffuse electrophoretic mobility (Mr > 200 000) and most likely corresponded to proteoglycans. The sulphate groups in the proteins with discrete Mr are most likely all linked to carbohydrates. These sulphated molecules were partially purified and identified to isoforms of rat alpha-lactalbumin for the 25-27 kDa bands and to kappa-casein for the 32 kDa band. This pattern of protein sulphation is, as far as we know, quite specific to rat mammary epithelial cells.     

Developmental regulation of galactoglycerolipid and galactosphingolipid sulphation during mammalian spermatogenesis. Evidence for a substrate-selective inhibitor of testicular sulphotransferase activity in the rat.

The synthesis of sulphatoxygalactosylacylalkylglycerol (SGG) is a differentiation marker of mammalian spermatogenesis. Maximal sulphation is observed in rat testis at about 20 days after birth and rapidly declines to low levels as the testis matures. The present data show that this decline in SGG synthesis is due to the appearance of an inhibitor of galactolipid sulphation. The inhibitor is a soluble testicular factor which is first detected at about 25 days after birth. Testicular homogenate can sulphate exogenous galactosylacylalkylglycerol (GG), galactosylceramide (GC) and lactosylceramide (LC) in vitro. The testicular inhibitor is most effective in preventing GG sulphation and inhibits GC and LC sulphation to a lesser extent; this correlates with the finding that glycolipid sulphation shifts from SGG production in 20-day-old testis to GC and LC sulphation at later stages of testicular development. The effect of the inhibitor on sulphotransferase activity from brain and kidney was also determined. The inhibitor decreased the sulphation of GG in vitro by both testis and kidney, inhibited testicular sulphation of GC less effectively and had no effect on GC sulphation by kidney and brain homogenates. A 9500-fold purification of the inhibitory activity has been obtained in a fraction isolated by h.p.l.c.     

Neuroactive steroids.

Neuroactive steroids are natural or synthetic steroids that rapidly alter the excitability of neurons by binding to membrane-bound receptors such as those for inhibitory and (or) excitatory neurotransmitters. The best-studied neuroactive steroids are a series of sedative-hypnotic 3 alpha-hydroxy ring A-reduced pregnane steroids that include the major metabolites of progesterone and deoxycorticosterone, 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydroDOC), respectively. These 3 alpha-hydroxysteroids do not interact with classical intracellular steroid receptors but bind stereoselectively and with high affinity to receptors for the major inhibitory neurotransmitter in brain, gamma-amino-butyric acid (GABA). Biochemical and electrophysiological studies have shown that these steroids markedly augment GABA-activated chloride ion currents in a manner similar (but not identical) to that of anesthetic barbiturates. Several steroids have also been observed to have convulsant or proconvulsant properties, including the synthetic amidine 3 alpha-hydroxy-16-imino-5 beta-17-azaandrostan-11-one (RU5135) and the natural sulfate esters of pregnenolone and dehydroepiandrosterone. Several of these have been shown to be bicuculline or picrotoxin-like GABAA receptor antagonists. Examples of steroids that alter neuronal excitability rapidly by augmenting or inhibiting excitatory amino acid receptor-mediated responses have also been reported. Recently, allopregnanolone and allotetrahydroDOC have also been measured in brain and plasma where their levels have been shown to fluctuate in response to stress and during the estrous and menstrual cycles of rats and humans, respectively. Although the major fraction of allopregnanolone in tissue, including brain, is of adrenal and/or ovarian origin, appreciable levels of allopregnanolone can still be measured in the brains of adrenalectomized and/or oophorectomized animals. Receptor-active neurosteroids may represent an important class of neuromodulators that can rapidly alter central nervous system excitability via novel nongenomic mechanisms.     

Increased sulphation level and altered composition of glycosaminoglycans synthesized by cultured smooth muscle cells in the presence of beta-D-xylosides

Cultured rat and bovine smooth muscle cells incorporated more 35SO4 into macromolecular glycosaminoglycans in the presence of beta-D-xylosides than in their absence. More than 90% of the xyloside-initiated glycosaminoglycans were secreted rapidly into the culture medium and were more highly sulphated than glycosaminoglycans polymerized on core protein. The increased extents of sulphation were associated with increased synthesis of dermatan sulphate and a decrease in that of nitrous acid-sensitive glycosaminoglycans.     

Effect of long-term isolation of the rat on in vitro biosynthesis of gonadal steroids from dehydroepiandrosterone.

In vitro biosynthesis of gonadal steroids from dehydroepiandrosterone was studied in isolated and in socially reared male and female rats. Acetone-dried powder of gonadal tissue incubated with dehydroepiandrosterone-4-14C yielded androstenedione, androst-5-ene-3beta, 17beta-diol, 11beta-hydroxyandrostenedione and testosterone. In the male, conversion to androstenedione was significantly increased after isolation and conversion to androst-5-ene-3beta, 17beta-diol was significantly lowered. In the female, conversion to androstenedione and androstenediol was significantly lowered by isolation. Testosterone and 11beta-hydroxyandrostenedione were not affected by isolation. Gonadal tissue of isolated and of socially reared male and female rats metabolizes dehydroepiandrosterone in a different way. These findings support the view that the conditions of housing affect the production of sex steroids.     

Delta 5-3 beta-hydroxysteroid acyl transferase activity in the rat brain.

Pregnenolone and dehydroepiandrosterone accumulate in brain as sulfate and fatty acid esters and unconjugated steroids. The steroid fatty acid ester-synthesizing activity was investigated in rat brain microsomes. Endogenous fatty acids in the microsomal fraction were used for the esterification of steroids. The enzyme system had a pH optimum of 4.5 in acetate buffer with [3H]dehydroepiandrosterone as substrate. The apparent Km was 9.2 +/- 3.1 x 10(-5) M and Vmax was 18.6 +/- 3.4 nmol/h/mg protein (mean +/- SEM). The inhibition constants of pregnenolone and testosterone were 123 and 64 microM, respectively. Results were compatible with a competitive type of inhibition. A high level of synthetic activity was found in the brain of 1- to 3-week-old male rats, which rapidly decreased with aging. Saponification of purified [3H]pregnenolone esters yielded pregnenolone and a mixture of palmitate, oleate, linoleate, stearate, and myristate as the predominant fatty acids. Contrasting with the high rates of esterification of several radioactive delta 5-3 beta-hydroxysteroids or 17 beta-hydroxysteroids, no fatty acid esters of either cholesterol, epitestosterone (with a hydroxyl group at position C-17 alpha), or corticosterone (with hydroxyl groups at C-21 and C-11 beta) were formed in the same incubation conditions.     

Increased sulphation level and altered composition of glycosaminoglycans synthesized by cultured smooth muscle cells in the presence of β-d-xylosides

Cultured rat and bovine smooth muscle cells incorporated more ³⁵SO₄ into macromolecular glycosaminoglycans in the presence of β-d-xylosides than in their absence. More than 90% of the xyloside-initiated glycosaminoglycans were secreted rapidly into the culture medium and were more highly sulphated than glycosaminoglycans polymerized on core protein. The increased extents of sulphation were associated with increased synthesis of dermatan sulphate and a decrease in that of nitrous acid-sensitive glycosaminoglycans.     

Sulphated Glycosaminoglycans in Guinea Pig Eosinophils Studied by Means of Cationic Colloidal Gold

Using bone marrow embedded in hydrophilic resin Lowicryl K4M and cationic colloidal gold pH 1.0 labelling, we studied sites of sulphation and sulphated glycosaminoglycans ultrastructurally in various maturational stages of both eosinophil granulocytes and eosinophil granules of guinea pig. Eosinophil granules reacted positively to cationic gold, the pattern of labelling varying according to the degree of cell maturation. The formation of eosinophil granules takes place throughout the myelocyte stage. Early eosinophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late ones contain a small and less active Golgi apparatus. All the immature granules were labelled positively. However, mature granules with a central crystal bar lost their affinity towards colloidal gold. Interestingly, strong colloidal gold labelling was also observed in the trans to transmost Golgi apparatus, especially in immature eosinophil granulocytes. This indicates that sulphation of glycosaminoglycans occurs in the trans to transmost Golgi apparatus of eosinophil granulocytes. Prior absorption with poly-L-lysine prevented colloidal gold labelling of tissue sections. Methylation of sections at 37°C did not alter the gold labelling, whereas the labelling disappeared after methylation at 60°C. Prior treatment with chondroitinase ABC or heparinase I abolished the majority of colloidal gold labelling in immature eosinophil granules. Taking these results together, we conclude that immature eosinophil granules contain sulphated glycosaminoglycans including chondroitin sulphate or heparan sulphate or both.     

Formation of neutral steroids from endogenous precursors in minced human fetal adrenals in vitro

The role of endogenous precursors in steroid biosynthesis in the human fetal adrenals was studied in $\text{in vitro}$ incubations with no added exogenous substrate. The identification and quantitative determination of the steroids was carried out by gas-liquid chromatography and gas chromatography - mass spectrometry. During the incubation a 10–60-fold increase in the concentration of dehydroepiandrosterone sulfate was observed. An increase was also seen in the concentrations of the other two steroid sulfates detected, pregnenolone and 17-hydroxypregnenolone sulfate. The concentrations of the corresponding free steroids were seen to decrease during the incubations. Only traces of free dehydroepiandrosterone and progesterone were detected endogenously or at any stage during the incubations. No corticoids could be found. Endogenous cholesterol was found in high concentrations (1.5 – 3.0 mg/g wet tissue) in the tissue samples studied. A small proportion of it was present as a sulfate conjugate.It is concluded that fetal adrenals can form neutral steroid sulfates of the 3β-hydroxy-5-ene series from endogenous precursors $\text{in vitro.}$ Cholesterol which was detected in high concentrations in the adrenal tissue is a possible precursor of these metabolites. The results obtained would suggest that this endogenous metabolism involves sulfated intermediates principally.     

Neurosteroid levels are increased in vivo after LPS treatment and negatively regulate LPS-induced TNF production

Neuroactive steroids such as dehydroepiandrosterone sulfate and pregnenolone inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production. Corticosteroids not only inhibit TNF production but their levels are increased in vivo after endotoxin injection, thus representing a feedback system that limits TNF production. We wondered whether the same could be true for neuroactive steroids. Thus, the possibility that neuroactive steroids might be increased concomitantly to TNF induction in vivo in mice treated with LPS was investigated. Increased plasma and hippocampal levels of allopregnanolone (but not of dehydroepiandrosterone or pregnenolone) were found 90 min after LPS injection. Allopregnanolone and progesterone (IC50 10- 7 and 10- 9 M, respectively) also inhibited TNF production by mouse peritoneal macrophages in vitro at concentrations in the range of those detected in vivo. These findings suggest that neuroactive steroids may act as endogenous inhibitors of cerebral and systemic TNF production.     

Differential immunogold localisation of sulphated and unsulphated keratan sulphate proteoglycans in normal and macular dystrophy cornea using sulphation motif-specific antibodies

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the corneal stroma for tissue organisation and transparency. Macular corneal dystrophy (MCD) is a rare, autosomal recessive disease characterised by disturbances in KS expression. MCD is caused by mutations in CHST6, a gene encoding the enzyme responsible for KS sulphation. Sulphated KS is absent in type I disease causing corneal opacity and loss of vision. Genetic studies have highlighted the mutational heterogeneity in MCD, but supportive immunohistochemical studies on corneal KS have previously been limited by the availability of antibodies mostly reactive only with highly sulphated KS epitopes. In this study, we employed four antibodies against specific KS sulphation patterns, including one against unsulphated KS, to investigate their reactivity in a case of MCD compared with normal cornea using high-resolution immunogold electron microscopy. Mutation analysis indicated type I MCD with deletion of the entire open reading frame of CHST6. Contrast enhanced fixation revealed larger PG structures in MCD than normal. Unlike normal cornea, MCD cornea showed positive labelling with antibody to unsulphated KSPG, but was negative with antibodies to sulphated KSPG. These antibodies will thus facilitate high-resolution investigations of phenotypic heterogeneity in support of genetic studies in this disease.     

Activity of sterol-sulphate sulphohydrolase in rat brain: characterization, localization and change with age

Abstract— Homogenates of rat brain hydrolysed the sulphate esters of dehydroepiandrosterone, oestrone and pregnenolone to free steroids. The pH optimum was 6.6 for all three steroid sulphates. Under similar conditions, cholesterol sulphate was not hydrolysed to a significant extent. Unlike sterol sulphatases (EC 3.1.6.2) from extraneural tissues, most of the activity in brain was found in the crude nuclear fraction. The remainder of the activity was found in the crude mitochondrial fraction and almost none was detected in microsomal or cytosol fractions. Sterol sulphatase activity was present in the foetal brain and increased rapidly with increasing postnatal age to a plateau at approx. 25 days of postnatal age. The enzymic activity did not differ significantly with the sex of the animal. The sulphatase activity was found throughout the brain, with cerebellum and brain stem exhibiting a slightly higher activity per wet wt. of tissue than other regions. Inhibition of enzymic activity occurred in the presence of sodium deoxycholate, Triton X-100, sodium dodecyl sulphate and inorganic phosphate or sulphate.     

Sulphated polysaccharide synthesis in brown algae

Summary Histochemical and autoradiographic techniques have been used to investigate the sites of synthesis, transport and location of sulphated polysaccharides in some larger brown seaweeds.The most rapid uptake of ³⁵SO₄ occurred when material was incubated in medium with 10^-4M carrier sulphate, negligable uptake occurring from seawater.Autoradiography using ³⁵SO₄ has shown that in Pelvetia sulphated material is synthesised by all cell types, particularly epidermal cells. In Laminaria spp. this activity is confined to specialized secretory cells which discharge into mucilage canals. In both instances the process of carbohydrate sulphation appears to occur in the Golgi-rich perinuclear region.     

In vitro metabolism of C19 steroids in human endometrium

In order to quantitate the extent of intracellular metabolic conversions of C19 steroids in human endometrium, specimens of proliferative and secretory tissue were superfused at a constant rate with several pairs of labeled compounds at low concentrations. About 16% of dehydroepiandrosterone sulfate interacting with endometrial cells was converted to dehydroepiandrosterone and about 3% of this compound was converted to androstenedione. Androstenedione was reversibly reduced to testosterone and the extent of this conversion was shown to be several-fold higher in secretory than in proliferative tissue. About 1% of testosterone entering the cells was reduced to 5 alpha-dihydrotestosterone. These results demonstrate that the conversion of the main circulating C19 steroids in women, i.e. dehydroepiandrosterone sulfate and androstenedione, to 5 alpha-dihydrotestosterone, the compound considered to be the true intracellular androgen, is very small. In contrast, formation of testosterone from androstenedione is extensive and increases during the luteal phase under the influence of progesterone, a hormone known to stimulate the activity of 17 beta-hydroxysteroid dehydrogenase in human endometrium.