Inhibition by rapamycin of PDGF- and bFGF-induced human tenon fibroblast proliferation in vitro.

PURPOSE: Fibrosis of subconjunctival tissues is a major cause of bleb failure following glaucoma filtration surgery. The aim of the present investigation was to demonstrate the effect of Rapamycin, a clinically relevant macrolide antibiotic with potent immunosuppressive properties, on human Tenon fibroblast proliferation induced by platelet-derived growth factor and basic fibroblast growth factor. METHODS: Primary Tenon fibroblast cultures were derived from patients undergoing trabeculectomies or routine cataract extractions. Rapamycin was added in concentrations of 0.1-100 ng/ml with or without 3-30 ng/ml of porcine platelet-derived growth factor or of human recombinant basic fibroblast growth factor. Two days after treatment, the cells were examined and counted. The results were expressed as the percent of cell growth in treated culture relative to its untreated control. RESULTS: Rapamycin was not cytotoxic at any of the concentrations tested. Inhibition of platelet-derived growth factor-induced Tenon fibroblast proliferation occurred with all doses of Rapamycin, the most marked effect being observed with 30 ng/ml (60% inhibition, p < 0.001). In contrast, optimal inhibition of basic fibroblast growth factor-induced proliferation was only 37% (p < 0.01), achieved with 10 ng/ml of the peptide. CONCLUSION: Rapamycin potently inhibits platelet-derived growth factor-induced fibroblast proliferation in vitro without any apparent cytotoxicity. It may eventually prove to be a useful adjunct to glaucoma filtration surgery.

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.

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.

Further studies on the corticosteroid-receptor system of the nasal gland of the domestic duck (Anas platyrhynchos).

The interaction of tritiated corticosterone with the nasal gland corticosterone receptor was investigated. Kinetic studies have shown that the association of [3H]corticosterone-receptor followed second-order reaction kinetics and the dissociation of the ligand from the receptor became "pseudo" first order in the presence of large excess of radioinert steroids at 0, 15, 25, and 35 degrees C. Similar data were obtained with an ammonium sulfate precipitate of the cytosol. Dissociation rate constants varied from 10(-5) to 10(-3) s-1 and the association rate constants varied from 0.5 X 10(4) to 3.8 X 10(5) M-1 X s-1, depending on the reaction temperature and the cytoplasmic receptor preparation. Equilibrium dissociation constants were in 10(-8)-10(-9) M range. Equilibrium dissociation constants, calculated from kinetic data (kd/ka), showed a marked temperature dependence, while those obtained by saturation analysis varied much less with the reaction temperature. Data obtained in these experiments were used to calculate some thermodynamic parameters of the binding of corticosterone to the cytoplasmic receptor. The enthalpy of dissociation was 101.5 and 79.4 kJ X mol-1 and the entropy of dissociation was 200 and 280 J X mol-1 X degree-1 for the crude cytoplasmic receptor and the ammonium sulfate precipitate, respectively. From the equilibrium dissociation constants, the enthalpy and entropy of the equilibrium binding was calculated. Polynomial fitting of Ka values versus 1/T yielded enthalpy (delta H) values from -0.9 to -88.8 kJ X mol-1, depending on the nature of the receptor preparation. Entropy values were negative for kinetically derived equilibrium association constants from the crude cytosol at all temperatures and for 0 and 15 degrees C for the precipitate. Entropy values were positive for Ka values obtained from kinetic rates at 25 and 35 degrees C and for Ka's calculated from saturation analysis. Further experiments with the precipitate confirmed our previous contention that the nasal gland cytoplasmic corticosterone receptor metabolized the bound ligand to 11-dehydrocorticosterone, though the receptor preparation was corticosterone specific. The following hydrodynamic parameters were obtained on the binding macromolecule: molecular weight, 316 000; S20W, 8.0; Stokes radius (rs), 77.3 A (1 A equal to 0.1 nm); total frictional ratio (f/f0), 1.71. The labeled receptor preparation translocated to homologous nuclear binding sites following heat activation and, at the nuclear binding sites, the ligand was almost exclusively in its oxidized form.(ABSTRACT TRUNCATED AT 400 WORDS)

Corticosteroid receptors in the avian kidney.

The binding in vitro of tritiated aldosterone to domestic duck (Anas platyrhynchos) kidney tissue has been investigated. Using tissue from animals on a normal diet, tritiated aldosterone was specifically bound to kidney cytosol with an apparent equilibrium dissociation constant of about 9 nM and number of binding sites in the 20 fmol/mg protein range. These values did not show statistically significant changes when the cytosol originated from animals with salt activated nasal glands. Kidney cytosols labeled with tritiated aldosterone sedimented with a single peak at 8S in a linear sucrose gradient (10--30%) and this peak was quenched by excess, radioinert aldosterone. Following incubation of labeled cytosols with crude nuclei, the cytosols became depleted of the label and aldosterone was translocated to the Tris-soluble and Tris-insoluble, 0.4 M KC1 soluble nuclear fractions. Kidney cytosols metabolized aldosterone extensively to a compound presumed to be 3alpha,5beta-tetrahydroaldosterone. However, only unchanged aldosterone became receptor-bound. It was concluded that the duck kidney possesses aldosterone receptors, though competition studies indicated that the specificity of these receptors might be different from those described in the mammalian kidney.