Afzal Z. Mehdi

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.

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.

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.

Steroid C-20 oxidoreductase activity of duck intestinal mucosa: The interrelations of the enzymatic activity with steroid binding

The binding of corticosterone and aldosterone to domestic duck (Anas platyrhynchos)-dispersed colonic mucosal cells at 37 degrees was investigated. It was found that in contrast to experiments using cell-free intestinal preparations, corticosterone was extensively metabolized and it was the metabolite, not the native steroid that became receptor bound and all the bound ligand was in the nuclear fraction. The metabolite turned out to be identical with 4-pregnene-11 beta,20 beta, 21-triol-3-one (20 beta-dihydrocorticosterone, 20 beta-DHB). Binding experiments with [3H]corticosterone yielded the following kinetic parameters: Kd = 87.6 nM, Nmax = 337,900 sites/cell. When synthetic [3H]20 beta-DHB was used as the ligand a curvilinear-binding isotherm was obtained. This could be resolved into a high affinity-low capacity (HA) and a low affinity-high capacity (LA) component with the following binding parameters: Kd,HA = 91 nM, Nmax,HA = 130,800 sites/cell; Kd,LA = 5.4 x 10(-6) M, Nmax, LA = 3.7 x 10(6) sites/cell. Binding of the metabolite to cell-free preparations, at 0 degree, gave the following results: for cytosol, linear-binding isotherm, Kd = 14.0 nM, Nmax = 26.5 fmol/mg protein; and for crude nuclei, curvilinear-binding isotherm, Kd,HA = 45.0 nM, Nmax, HA = 5.33 pmol/mg DNA; Kd,LA = 2.2 x 10(-6) M, Nmax,LA = 286.6 pmol/mg DNA. [3H]Aldosterone was also bound by the dispersed whole cells and again, this binding was only nuclear (Kd = 9.3 nM, Nmax = 10,042 sites/cell). The bound ligand was unchanged aldosterone. Competition experiments have shown that aldosterone did not compete with 20 beta-DHB for binding sites and vice versa. The intracellular 20 beta-hydroxysteroid oxidoreductase responsible for the transformation of corticosterone was found mostly in the cytoplasm. Kinetic studies with the enzyme yielded classical Michaelis-Menten kinetics (Km = 15.7 microM, Vmax = 2.6 nmol/min/mg protein). The enzyme had an apparent Mr of 35 kDa and a Rs of 25.5 A. It is believed that our results might explain the binding of aldosterone to mineralocorticoid-binding sites in the presence of overwhelming concentrations of corticosterone and that experiments with cell-free tissue preparations, performed at 0 degree, do not reflect the true cellular-binding events.

A profile of the intestinal mucosal corticosteroid receptors in the domestic duck

The corticosteroid receptor profile of the intestinal tract of the domestic duck (maintained on either a low-sodium (LS) or a high-sodium (HS) diet) was investigated. Using tritiated triamcinolone acetonide (TA), corticosterone, or aldosterone as ligands, cytoplasmic mineralocorticoid receptors (MR, type I) and glucocorticoid receptors (GR, type II) were found in the mucosal cytosol of the jejunum and colon with the following binding parameters: LS jejunum GR-Kd, 3.4 nM; Nmax, 245 fmol/mg protein; MR-Kd, 0.54 nM; Nmax, 35 fmol/ mg protein; colon GR-3.2 nM; Nmax, 531 fmol/mg protein; MR-Kd, 0.55 nM; Nmax, 113 fmol/mg protein; HS jejunum GR--Kd, 3.2 nM; Nmax, 531 fmol/mg protein; MR--Kd, 0.30 nM; Nmax, 50 fmol/mg protein; colon GR--Kd, 1.1 nM; Nmax, 572 fmol/mg protein; MR--Kd, 0.68 nM; Nmax, 221 fmol/mg protein. The diet little influenced the GR binding parameters, while the MR (aldosterone) binding parameters showed a down-regulation following LS (high circulating aldosterone) diets. The competition hierarchy of radioinert steroids on the formation of the [3H]corticosterone-receptor complex was corticosterone = cortisol = 11-deoxycorticosterone greater than aldosterone = TA = dexamethasone much greater than 11-deoxycortisol; with [3H]aldosterone, the competition was corticosterone = progesterone = 11-deoxycorticosterone greater than aldosterone = cortisol = TA = dexamethasone greater than 11-deoxycortisol greater than 11-dehydrocorticosterone. The intestinal mucosal receptor was deactivated following treatment with trypsin. On linear sucrose gradients, receptor-ligand complexes sedimented with a single peak at 8.5 S (hypotonic gradient) and 4.0-4.5 S (hypertonic gradient), respectively. Heat-activated [3H]TA- and [3H]aldosterone-receptor complexes bound avidly to DNA-cellulose and, upon ion-exchange chromatography on DEAE-Sephacel, the presence of the negatively charged unactivated and the more positively charged activated complexes could be shown. The hydrodynamic parameters, determined by gel-filtration chromatography, gave for all three ligand-receptor complexes molecular weight values from 334,000 to 351,000 and Stokes radii from 76.8 to 80.0 A. From these studies it was concluded that the duck intestinal tract possesses vertebrate-type GR and MR, though these receptors were much less specific than their mammalian counterparts. The duck intestinal corticosteroid receptor was found to be different from those of the teleost fish and anuran amphibian, establishing the possibility of a biochemical evolution in nonmammalian intestinal corticosteroid receptor conformation.

Comparative enzymology of steroid-hydroxylating enzyme systems of nonmammalian vertebrate adrenal tissue

Abstract The microsomal steroid 21-hydroxylase (EC 1. 14. 1.8) has been compared in the adrenal cortex of a bird, the domestic duck (Anas platyrhynchos), a reptile, the grass snake (Natrix sp.) and an amphibian, the American bullfrog (Rana catesbeiana). In all three species, the adrenal microsomal 21-hydroxylase, as measured by the conversion of labeled progesterone to 11-deoxycorticosterone was NADPH-dependent and had Michaelis-Menten constants (Km) in the 10−6 M range. Optimal temperature for enzyme activity was 37° in the duck, 40°C in the snake and 26°C in the frog, with respective Q10 values of 1.4, 1.5 and 1.1. In all three species, p-chloromercuribenzoic acid, carbon monoxide, Metyrapone and aminopterin inhibited hydroxylation to varying degrees. The Metyrapone inhibition was found to be noncomptitive. The presence and some characteristics of the microsomal cytochrome P450 were investigated in the above species and also in a bony fish, the European eel (Anguilla anguilla) and a cartilaginous fish, the sand skate (Raja brachyura). The spectrophotometric presence of the hemoprotein was established in the adrenal microsomal suspension of all five species. Cytochrome P450 was present in the 10−1 nanomole/mg protein range, comparable to that found in mammalian adrenal microsomes. “Affinity” constants of the hemoprotein for carbon monoxide, expressed as the time necessary for saturation of one-half of the active sites, revealed the presence of “high” and “low” affinity sites in skate, eel and duck microsomes. Similar biphasic activity curves were found in adrenal microsomes of a mammal, the rat. From these studies the tentative conclusion was drawn that in Vertebrata, microsomal 21-hydroxylation is accomplished by similar mechanisms. It is believed that the biochemical evolution of cellular steroid-hydroxylating systems antedated the phylogenetic evolution of multicellular organisms.

Product-precursor relationship during incubation of duck adrenal slices☆

Abstract Domestic duck ( Anas platyrhynchos ) adrenal slices were incubated with progesteronc-[4- 14 C] for various time intervals. Using 3 H labelled acetic anhydride the total mass of various metabolites formed was measured. The yield-time curves for the labelled products elaborated from the added 14 C precursor confirmed the previous in vitro results obtained from the duck adrenal slices. Thus labelled cpd. B‡, 18-OH-B and aldosterone were the major steroids formed at the end of 3 h incubation. However, DOC was quantitatively the major metabolite formed from endogenous substrate(s) during the course of incubation, though presence of the three above mentioned steroids was also demonstrable. At any given incubation time, the specific activities of the isolated products and the added precursor remained constant with the exception of DOC which showed a marked decline. These observations seemingly suggest that the added precursor formed a homogeneous pool with the corresponding endogenous substrate. The biosynthesis of large amounts of radio-inert DOC might be explained by the operation of the 5-ene-pathway eventually taking place in a different metabolic compartment.

Characterization of steroid receptors in the gut and kidney of the frog (Rana catesbeiana) and in the gut of the turtle (Chrysemys picta)

Paraglucocorticoid- and paramineralocorticoid-binding cytosolic receptors (pGR, pMR) were demonstrated in the intestine and kidney of the frog, Rana catesbeiana and in the intestine of the turtle, Chrysemys picta, in the presence of sodium molybdate. These receptors were of high affinity and low capacity with the following binding parameters: pGR:Kd:frog intestine (FI), triamcinolone acetonide (TA): 3.3 nM, corticosterone (B): 3.4 nM; frog kidney (FK), TA:4.3 nM, B: 9.3 nM; turtle intestine (TI), TA: 4.8 nM; Nmax: FI, TA: 357, B: 371; FK, TA: 301, B: 157; TI, TA: 350 fmol/mg protein. pMR:Kd: FI, aldosterone: 0.9 and 90 nM (biphasic curves); FK, aldosterone: 0.6 and 36 nM (biphasic curves); Nmax: FI, 13 and 147 fmol/mg protein; FK, 78 and 109 fmol/mg protein. The receptor had the following ligand affinities: pGR: FI and FK: triamcinolone acetonide greater than DOC greater than 11 beta-hydroxyprogesterone greater than progesterone greater than corticosterone greater than cortisol greater than aldosterone greater than 11-dehydrocorticosterone greater than 17 alpha-hydroxyprogesterone greater than cortisone; TI: triamcinolone acetonide greater than corticosterone greater than progesterone greater than DOC greater than cortisol greater than aldosterone; pMR: FI and FK: corticosterone greater than 11 beta-hydroxyprogesterone greater than aldosterone greater than triamcinoline acetonide = cortisol greater than DOC greater than 11-dehydrocorticosterone greater than progesterone greater than 17 alpha-hydroxyprogesterone greater than cortisone. Androgens, estrogens or 18-hydroxycorticosterone did not compete for binding in either tissue. The heat activated frog receptors did not bind to naked DNA, though the turtle receptor did. It was possible to show that cytosol receptor-ligand complexes from all tissues were bound by nuclear acceptor sites. On linear sucrose gradients, the FI TA-receptor complex sediments with a single peak (7.5S), the FK TA-receptor complex gave two peaks (8.0 and 4.4S) and the TI TA-receptor complex showed a single peak (9.0S). The hydrodynamic parameters of the pGRs were determined by gel exclusion on Sephacel S-300. The following results were obtained: Mr: FI, 265, 80, 40 kDa (multiple proteins); FK, 280, 60, 20 kDa (multiple proteins); TI, 366 kDa; Rs: FI, 6.9, 3.9 nm; FK, 6.9, 2.9 nm; TI, 7.6 nm; f/f0: FI, 1.6; FK, 1.6; TI, 1.6. It is suggested on the basis of the binding and hydrodynamic parameters that non-mammalian epithelia corticosterone receptors have undergone biochemical evolution from one class of vertebrates to another.