Thomas Sandor

Glucocorticoid receptors in the gill tissue of fish

The presence of glucocorticoid-binding macromolecular receptors was demonstrated in the Na2MO4 (10 mM)-stabilized gill cytosol of the American eel, Anguilla rostrata and in that of the trout, Salmo gairdneri. In all experiments, tritiated triamcinolone acetonide [( 3H]TA) was used as ligand. In the eel, the steroid was bound with a KD of 2.84 +/- 0.4 nM and an Nmax of 188 +/- 34 fmol/mg protein. The binding parameters for the trout cytosol were KD = 1.43 +/- 0.13 nM; Nmax = 271 +/- 113 fmol/mg protein. Competition studies with [3H]TA-labeled eel gill cytosol and radioinert steroids gave the following binding hierarchy: TA greater than dexamethasone greater than cortisol greater than 11-deoxycortisol greater than 21-deoxycortisol. Aldosterone, estrogens, or androgens did not complete. The eel gill receptor was deactivated by prior treatment with trypsin or mersalyl. RNase was without effect, but DNase degraded the receptor except when used in the presence of trypsin inhibitor. The eel gill TA-receptor complex sedimented on a linear (10-30%) sucrose gradient with a single peak at 7.0 S or 3.5 S, in hypotonic or hypertonic (0.4 M KCl) gradients, respectively. The eel ligand-receptor complex did not bind, following heat activation, to DNA-cellulose or phospho-cellulose, though it bound to DEAE-cellulose. In this respect, it behaved similarly to the eel intestinal mucosal TA-receptor complex, described previously. The initiation of dissociation of the eel receptor-[3H]TA complex with excess TA yielded pseudo-first-order dissociation kinetics (k-1 at 0 degree C: 2.39 X 10(-5) S-1), while the association kinetics of the receptor with the ligand was of second order (k + 1: 2.51 X 10(4) M-1 S-1). Sepharose column chromatography indicated a molecular weight of 334,690 Da. Calculation of the Stokes radius gave a value of 84.5 A and the frictional ratio, calculated from the molecular weight, was 1.84. From these data it was concluded that the gills of these two euryhaline teleosts contain tetrapod-type glucocorticoid receptors. These studies are the first to demonstrate these steroid recognition molecules in fish gill. The presence of receptors in the fish gill tissue are in agreement with the physiological action of corticosteroids in allowing adaptation of the animals to habitats of different salinity.

A comparative survey of steroids and steroidogenic pathways throughout the vertebrates

Corticosteroid hormone biosynthesis has been demonstrated in most classes of vertebrates, both Agnatha and Gnathostomata. Though comparative steroid endocrinology is still a young discipline, it is possible to group, tentatively, the different classes of animals according to the chemical nature of the corticosteroid hormones elaborated. In many instances these results were obtained in vitro and only on a limited number of representatives of a class. Thus our classification is only preliminary. Group I. Animals that secrete both 17-hydroxylated (e.g., cortisol, cortisone, 11-deoxycortisol) and 17-deoxycorticosteroids (e.g., corticosterone, 11-deoxycorticosterone, 18-hydroxycorticosterone, aldosterone). Most mammals investigated belong to this group, including subeutherian and eutherian orders. Notable exceptions from this endocrine pattern are found in the order of Rodentia, and some amphibians, both urodeles (Amphiuma) and anurans (Bufo), can be included in this group. Group II. Animals that secrete only 17-deoxycorticosteroids. The group comprises Aves, Reptilia, amphibians (Ranidae), and the rat and mouse (Order Rodentia). Group III. Animals that secrete only 17α-hydroxycorticosteroids. The few orders of Osteichthyes investigated can be classified in this group, but with some reservations, as both corticosterone and aldosterone were reported in bony fishes. Group IV. This chemically heterogeneous group comprises mostly chondrichthians. In this class adrenocortical secretion seems to vary from order to order. Thus, in the genus Raja the main, and possibly only, secretory product is 1-α-hydroxycorticosterone; the elaboration of cortisol was reported in the ratfish (Hydrolagus), and of corticosterone in dogfishes (Squalus and Mustelus). This tentative classification demonstrates that for adrenocortical secretion the same chemical building-blocks are used throughout the vertebrates. No quantitative classification has been attempted. Studies on the biosynthesis of steroid hormones in mammalian adrenals are numerous, but few reports exist on biosynthetic routes operating in the adrenals of nonmammalian vertebrates. The pathway acetate-cholesterol corticosteroids was shown to exist in birds, and the route cholesterol-corticosteroids in one species of bony fish (Anguilla). Pregnenolone and progesterone are precursors of both 17-hydroxy and 17-deoxycorticosteroids in all four groups. Aldosterone is biosynthesized from corticosterone in all aldosterone-producing species; it is accompanied in most instances by the production of 18-hydroxycorticosterone. On the intracellular level, adrenal mitochondria were shown to be the main source of steroid 18-oxygenating enzymes in Mammalia, Aves, and Amphibia. These synthetic routes are found in mammalian adrenals as well. Thus there seems to be a basic uniformity in steroid biochemistry in all hormone-producing adrenal cells. The adrenal itself shows progressive evolution from the scattered secretory cells in fishes to the encapsulated and zonated organ in mammals, but no evolutionary trend is yet apparent in the chemical nature of adrenocortical secretion. Nor can obvious connections be seen between the type of hormone synthesized and the habitat of the animal. The full potential of steroid hormone biosynthesis may have been acquired with the development of an inner supporting structure, and from this pool of possible reactions each class, order, or genus may have adopted biosynthetic routes to solve its adaptive and metabolic problems.

Studies on the biosynthesis of 18-oxygenated steroids from exogenous corticosterone by domestic duck (Anas platyrhynchos) adrenal gland mitochondria

Abstract The transformation of exogenous, isotopically labelled corticosterone to 18-hydroxycorti-costerone and aldosterone by domestic duck ( Anas platyrhynchos ) adrenal gland mitochondria was studied. The mitochondrial 18-oxygenating system was NADPH dependent. K + , Na + , Ca 2+ and Mg 2+ were necessary for maximal enzymatic activity. The enzyme present in 1 mg of mitochondrial protein became saturated with 13.2 βM of substrate (13.8 μg). Under saturation conditions the maximal production of 18-hydroxycorticosterone was found as 1.43 nmole/min/ mg protein and that of aldosterone 0.33 nmole/min/mg protein. The Michaelis constant for both 18-hydroxycorticosterone and aldosterone was 6.6 × 10 −6 M. Q 10 (average between 20°C and 40°C) was 2.16 for the reaction corticosterone → 18-hydroxycorticosterone and 1.34 for the reaction corticosterone → aldosterone. The mitochondrial enzyme system did not 18-oxygenate exogenous 11-deoxycorticosterone, 11-dehydrocorticosterone, 20β-dihydro-corticosterone. Exogenous 18-hydroxycorticosterone and aldosterone were not metabolized. The kinetics of the transformation of corticosterone to 18-oxygenated metabolites suggested a parallel pseudo-first order reaction rather than a series pseudo-first order reaction. 18-Oxygenation of corticosterone was strongly inhibited by d,1-18-hydroxycorticosterone, p-chloromercuribenzoate, carbon monoxide, metopirone (competitive inhibition; K i = 3.0 × 10 −6 M for 18-hydroxycorticosterone and 9.0 × 10 −6 M for aldosterone) and by aminopterin (noncompetitive inhibition; K i = for both metabolites 2.0 × 10 −5 M). Protein synthesis inhibitors did not have any effect. Cytochrome-P450 was shown to be present in mitochondria by spectrophotometric measurements. Addition of corticosterone. 11-deoxycorticosterone and metopirone produced type II difference spectra. The mitochondrial P450 became saturated with either corticosterone or metopirone at a concentration of 21.8 nmoles/mg protein. From these studies it was concluded that the duck adrenal mitochondrial 18-oxygenating system differed from the mammalian adrenal system previously described, especially in regard of substrate specificity. 18-Hydroxycorticosterone, either endogenous or exogenous, could not serve as substrate for aldosterone production with duck adrenal mitochondria. However, under ordinary circumstances. 18-hydroxycorticosterone synthesis was always associated with aldosterone synthesis in rather fixed proportions. Circumstantial evidence suggested the role of cytochrome-P450 in 18-oxygenation. As to the mechanism of the corticosterone → 18-hydroxycorticosterone → aldosterone reaction, this sequence could not be proven. The possibility should not be discarded that atmospheric oxygen is introduced into a hitherto unknown intermediary substance, which by enzymatic action and/or chemical rearrangement gives rise simultaneously to 18-hydroxy-corticosterone and aldosterone.

THE BIOSYNTHESIS IN VITRO OF RADIOACTIVE CORTICOSTEROIDS FROM (4-14C)PROGESTERONE BY ADRENAL SLICES OF THE DOMESTIC DUCK (ANAS PLATYRHYNCHOS).

Abstract Adrenal tissue slices originating from two female domestic ducks were incubated in a Krebs-Ringer medium using [4- 14 C]progresterone as precursor. After extracting the medium with organic solvents, the crude extract was subjected to extensive fractionation in paper partition systems. The transformation products isolated were identified by the carrier technique. The following is a list of the major [ 14 C]corticosteroids isolated and identified: aldosterone, 18-hydroxycorticosterone-20 → 18 cyclic hemiketal, corticosterone, cortisol and 11-deoxycorticosterone. 18-Hydroxy-11-deoxycorticosterone, though looked for, was not found in the mixture. The transformation rates (expressed as per cent of the precursor added) were the most important for 18-hydroxycorticosterone (15.33%); corticosterone (11.43%) and aldosterone (6.85%).

Intestinal triamcinolone acetonide receptors of the eel anguilla rostrata

The binding of [6,7-3H]triamcinolone acetonide (TA) to intestinal mucosa of freshwater-adapted silver eels was studied. The cytoplasmic preparations bound the ligand with an equilibrium dissociation constant (KD) of 2.28 +/- 0.37 nM and the maximal number of binding sites (Nmax) was 960 +/- 55 fmol/mg of protein (+/- SE, n = 13). Scatchard analysis indicated the presence of a single species of binding sites. Binding was abolished following treatment of the cytosol with trypsin, N-ethylmaleimide, or Mersalyl, but DNase or RNase treatment had little effect. The competition hierarchy of radioinert steroids on the formation of the [3H]TA-receptor complex was TA greater than dexamethasone greater than cortisol greater than 11-deoxycortisol. Aldosterone, DOC, corticosterone, 11-dehydrocorticosterone, progesterone, testosterone, or estradiol-17 beta did not compete. Sedimentation of the [3H]TA-receptor complex on a linear sucrose gradient (10-30% + 10% v/v glycerol) yielded single peaks in the absence or presence of 0.4 M KCl in the gradient (6 S or 3.5 S respectively). Following heat activation the receptor-ligand complex was freely translocated to homologous nuclei in vitro, though the activated complex did not bind to DNA-cellulose. It was concluded that the eel intestinal mucosal cytosol contains a high-affinity-low capacity steroid receptor system. This is the first instance that such a system was demonstrated in fish tissue.

Corticosteroid-binding macromolecules in the salt-activated nasal gland of the domestic duck (Anas platyrhynchos).

The interaction of tritiated corticosterone and tritiated 11-dehydrocorticosterone with salt-activated nasal glands of the domestic duck was studied. Nasal gland cytosol (105,000g supernatant) bound corticosterone and 11-dehydrocorticosterone at 0° with apparent Kd values of 10−9 and 10−11 M, respectively. The cytosols transformed [3H]corticosterone to [3H]11-dehydrocorticosterone (average transformation: 95% in 2 hr). Competition studies have shown that radioinert corticosterone is a more efficient competitor for cytosol binding sites than radioinert 11-dehydrocorticosterone. Cytosols labeled with [3H]corticosterone showed two major peaks following sucrose density gradient centrifugation: a heavy peak at 9–11S and a lighter peak at 3–4S. In sucrose gradients containing 0.4 M KCl, part of the heavier peak became transformed to the 3–4S form. Following incubation of [3H]corticosterone-labeled cytosols with crude nuclei, the cytosols became depleted of the label and the tritium activity, in the form of [3H]11-dehydrocorticosterone, accumulated in the nuclear Tris-soluble fraction and in the chromatin-bound (Tris-insoluble, 0.4 M KCl-soluble) fraction. Following the incubation of nasal gland slices with either [3H]corticosterone or [3H]11-dehydrocorticosterone, the cytosol, nuclear Tris-soluble fraction, and chromatin-bound fraction became labeled with [3H]11-dehydrocorticosterone only. Both steroids seemed to be taken up by the tissue slices at identical rates. Administration in vivo of [3H]corticosterone to a saltwater-maintained bird showed the accumulation of [3H]11-dehydrocorticosterone and, to a smaller extent, of [3H]corticosterone in the nasal gland intracellular fractions. It is suggested that the duck nasal gland is a corticosteroid target organ and the cytoplasmic protein-bound corticosterone is transported to the nucleus mostly as 11-dehydrocorticosterone in a manner similar to the intracellular transport of aldosterone in the rat and duck kidney.

Adrenal cortical function in birds. The in vitro transformation of sodium acetate-1-14C and cholesterol-4-14C by adrenal gland preparations of the domestic duck (Anas platyrhynchos) and the goose (Anser anser)☆

Abstract The in vitro incorporation of sodium acetate-l- 14 C and cholesterol-4 14 C into corticosteroids by duck adrenal preparations and the incorporation of sodium acetate-l- 14 C into corticosteroids by goose adrenal slices was investigated. Labelled sodium acetate gave rise to 14 C-labelled cholesterol, corticosterone (11β,21-dihydroxy-pregn-4-ene-3,20-dione), aldosterone (11β,21-dinydroxy-3,20-dioxo-pregn-4-ene-18-al-18-11-hemiacetal), and 18-hydroxycorticosterone (11β,18, 21-trihydroxy-3,20-dioxo-pregn-4-ene-20-18-cyclic hemiketal). In both duck and goose adrenals. Duck adrenal slices did not incorporate cholesterol-4- 14 C or cholesteryl-4- 14 C-linoleate or cholesteryl-4- 14 C-oleate. However, a fortified homogenate of duck adrenals transformed cholesterol-4- 14 C partially to corticosterone.

Metabolism of exogenous progesterone by insect tissue preparations in vitro.

Abstract The transformation in vitro of progesterone- 14 C has been studied with insect ( Orthoptera ) tissue preparations. The following two species were investigated: the hissing cockroach ( Gromphadorhina portentosa ) and the common cricket ( Gryllus assimilis ). Minces of male gonads of the cockroach transformed progesterone partially to 20β-hydroxy-pregn-4-ene-3-one. Whole homogenates of the male gonads and leg muscle of the same insect metabolized the precursor to 20α- and 20β-hydroxy-pregn-4-ene-3-one. The activity of the gonad homogenate was higher than that of the muscle homogenate. Minces of the testes of the cricket transformed progesterone partially to the above-mentioned two metabolites while a homogenate of the same tissue yielded only 20α-hydroxy-pregn-4-ene-3-one upon incubation with progesterone. The same pattern of transformation was obtained with homogenized leg muscle of the cricket. Once again, on a tissue-weight basis, the gonads exhibited a higher enzymic activity than did the muscle tissue.

The effect of melatonin on the biosynthesis of corticosteroids in beef adrenal preparations in vitro.

Abstract Melatonin inhibited corticosteroid biosynthesis by various preparations of bovine adrenal cortex in vitro . Addition of melatonin to beef adrenal slices (10.8–86.2 nmol/500 mg tissue) inhibited the transformation of carbon-labelled progesterone (2.5 nmol/500 mg tissue) to cortisol (inhibition: 33–63%) and to aldosterone (inhibition: 21–63%). The inhibition was proportional to the amounts of melatonin added though no linear dose-response relationship was obtained. Inhibition of 11β-hydroxylation by adrenal slices and by adrenal mitochondria was only slight. Further experiments with beef adrenal microsomes have shown that melatonin inhibited 17- and 21-hydroxylation. Preliminary kinetic studies seemed to show that melatonin is a non competitive inhibitor of the microsomal 17- and 21-hydroxylases. These observations suggest that melatonin may directly modulate corticosteroidogenesis.

Corticosterone receptors in the avian kidney

Abstract The binding of tritiated corticosterone to domestic duck ( Anas platyrhynchos ) kidney tissue has been investigated. Duck renal cytosols (105,000 g supernatant) contain high affinity-low capacity corticosterone-binding macromolecules ( K D : 7.3 nM for animals on a normal diet and 5.1 nM for animals with activated nasal salt glands; N max :85 fmol/mg protein and 249 fmol/mg protein respectively). Kidney cytosols labeled with [ 3 H]-corticosterone sedimented on a 10–30% linear sucrose gradient showing two peaks: one at 3.5S, the other at 10.2 S. The heavy peak was severely quenched in the presence of excess radioinert corticosterone, while in high ionic medium (0.4 M KCl), only one peak was present, at 3.7 S. Following incubation of labeled cytosols with crude nuclei, the cytosols became depleted of tritium activity and corticosterone-radioactivity was translocated to nuclear fractions. Tritiated corticosterone nuclear exchange assay showed the presence of endogenous, nuclear-bound cortico-sterone-receptor complexes ( N max R :33–44 pmol/mg DNA). Kidney cytosols did not metabolize cortico-sterone and competition studies showed that neither aldosterone nor 11-deoxycorticosterone was effective in displacing corticosterone from cytoplasmic receptors. From these studies it was concluded that the kidney of this bird possesses corticosterone-binding macromolecules which show characteristics similar to those found in mammalian kidneys and that corticosterone might have a modulating effect upon avian kidney function.