Microsomal Opiate Receptors: Characterization of Smooth Microsomal and Synaptic Membrane Opiate Receptors

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.     

Epinephrine and norepinephrine syntheses are regulated by a glucocorticoid receptor-mediated mechanism in the bovine adrenal medulla

The bovine adrenal medulla was investigated regarding the presence of glucocorticoid binding protein and the increases in ornithine decarboxylase (ODC) activity and epinephrine and norepinephrine by dexamethasone. Scatchard analysis of specific cytosol [3H] dexamethasone-binding study indicated a single class of high affinity (kd, 35 +/- 5 nM) and limited binding sites (150 +/- 26 fmoles/mg protein). Competition studies of various steroids indicated a high affinity for dexamethasone and hydrocortisone. Sedimentation in sucrose density gradients revealed a 7.3 S binding peak in the cytosol. Dexamethasone caused an increase in ornithine decarboxylase (ODC) activity within 1 to 2 hours after which the norepinephrine and epinephrine contents increased 16 hours after the peak of ODC activity in a dose dependent manner of dexamethasone in bovine adrenal medullary chromaffin cells in primary monolayer culture. These data suggest that the bovine adrenal medulla is a target organ of glucocorticoid hormone and that norepinephrine and epinephrine syntheses are regulated by a glucocorticoid receptor-mediated mechanism.     

Microsomal dexamethasone binding sites identified by affinity labelling

Binding studies with [3H]dexamethasone identified a class of binding sites on male rat liver microsomes. The binding sites were glucocorticoid-dependent and specific for glucocorticoids and progestins. Scatchard binding parameters, competition studies with triamcinolone acetonide, a synthetic glucocorticoid which competes well for the glucocorticoid receptor, and immunoblotting with an antiglucocorticoid receptor antibody indicated that these sites are distinct from the cytosolic glucocorticoid receptor. Affinity labelling experiments with [3H]dexamethasone 21-mesylate revealed two specifically labelled peptides, one at approx. 66 kDa and a doublet at 45 kDa. The 66 kDa peptide had been previously identified in serum and may be present as a result of serum contamination of the microsomal preparation. The 45 kDa doublet, on the other hand, had been shown to be absent from rat serum. The characteristics of the 45 kDa peptide(s) were identical to those of the dexamethasone binding site identified in the binding studies. [3H]Dexamethasone binding characteristics and affinity labelling of microsomal subfractions, separated by isopycnic centrifugation, showed that the binding sites are located in the endoplasmic reticulum. The identification and role of the 45 kDa peptide doublet remain to be determined.     

Role of regucalcin as an activator of Ca(2+)-ATPase activity in rat liver microsomes.

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver microsomes was investigated. The presence of regucalcin (0.1-1.0 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. Thapsigargin (10(-6) M), a specific inhibitor of microsomal Ca(2+) pump enzyme (Ca(2+)-ATPase), clearly inhibited regucalcin (0.5 microM)-increased microsomal Ca(2+)-ATPase activity. Liver microsomal Ca(2+)-ATPase activity was markedly decreased by N-ethylmaleimide (NEM; 2.5 mM), while the activity was clearly elevated by dithiothreitol (DTT; 2.5 mM), indicating that the sulfhydryl (SH) group of the enzyme is an active site. The effect of regucalcin (0.5 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of NEM (2.5 mM) or digitonin (10(-2) %), a solubilizing reagent of membranous lipids. Moreover, the effect of regucalcin on enzyme activity was seen in the presence of Ca(2+) ionophore (A23187; 10(-7) M). The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver microsomes, and that the protein may act the SH groups of microsomal Ca(2+)-ATPase.     

Influence of phosphatase inhibitors and nucleotides on [3H]dexamethasone binding in cytosol of human placenta

The purpose of this investigation was to establish the properties of [3H]dexamethasone binding sites in cytosol of human placenta at term. Cytosol containing 20 mM sodium molybdate (MoO4Na2) was incubated for 120 min at 20 degrees C with 40 nM [3H]dexamethasone. The following properties were observed: (a) a single population of binding sites of high affinity and low capacity was measured by Scatchard analysis; (b) potent glucocorticoids such as dexamethasone and cortisol displaced the tritiated ligand, progesterone showed an intermediate activity, whereas cortisone, testosterone and 17 beta-estradiol were ineffective competitors; (c) ultracentrifugation on 16-41% glycerol gradients containing 20 mM MoO4Na2 yielded sedimentation values of 10.25 +/- 0.35 S (n = 4 placentas); (d) the binding sites could be differentiated from the enzyme 11 beta-hydroxysteroid dehydrogenase, as the activity of the former, but not that of the latter, was greatly dependent on the presence of MoO4Na2 in the incubation medium. Inactivation of binding sites labelled with [3H]dexamethasone by incubation at 20 degrees C was prevented by phosphatase inhibitors such as 20 mM MoO4Na2 (P less than 0.01), 20 mM sodium tungstate (WO4Na2) (P less than 0.01) and to a lower extent by 5 mM ATP and cAMP (P less than 0.05). 50 mM NaF, 5 mM GTP or cGMP had no effect. The protection afforded by MoO4Na2 and WO4Na2 was correlated with a significant inhibition of the activity of acid phosphatase, but not alkaline phosphatase. Neither ATP nor cAMP modified phosphatase activity. It is suggested that binding sites for [3H]dexamethasone in cytosol of human placenta showed properties similar to those described for glucocorticoid receptors in target cells, and that these binding sites are regulated by phosphorylation and dephosphorylation mechanisms.     

Differential effects of chelating agents on cytosolic glucocorticoid receptor stability and nuclear binding in vitro

The effect of several metal chelators (EDTA, EGTA, and 1,10 phenanthroline) on rat liver glucocorticoid receptor properties in vitro was investigated. At 4 degrees C 10 mM EDTA (unlike 10 mM EGTA and 10 mM 1,10 phenanthroline) had a significant stabilizing effect on unbound hepatic glucocorticoid receptors. At higher temperature (25 degrees C) 10 mM EGTA appeared to act as a chemical stabilizer of unbound receptors. 1,10 Phenanthroline had no stabilizing effect at either temperature. Scatchard analysis indicated that the alteration in receptor binding after incubation at 4 and 25 degrees C in the presence and absence of chelating agents was due to a change in the number of steroid binding sites rather than perturbation of receptor affinity. Unlike results obtained with unbound receptors, all three chelating agents appeared to enhance prebound glucocorticoid-receptor complex inactivation. Interestingly these chelating reagents also significantly altered glucocorticoid-receptor complex binding to isolated nuclei.     

Activation of microsomal cAMP-dependent protein kinase isoenzyme I by ACTH1-24 in bovine adrenal cells

In microsomes of bovine fasciculata reticularis cells incubated with or without 10(-8) M ACTH during 20 min, we measured covalent and non covalent cAMP binding under exchange or non-exchange conditions and cAMP-kinase activity. ACTH induced a decrease in cAMP-kinase activity and in the number of free cAMP binding sites. These results indicate an activation by ACTH of a part of microsomal cAMP-dependent protein kinase. Photoaffinity labeling of microsomal protein with 8-azido-cAMP revealed the presence of both cAMP-kinase isoenzyme I and II in this cellular fraction. Using this method, it was demonstrated that ACTH1-24 caused a preferential and nearly complete activation of microsomal protein kinase I.     

The 11beta hydroxysteroid dehydrogenase 2 exists as an inactive dimer

The 11beta-hydroxysteroid dehydrogenase types 1 and 2 enzymes (11beta-HSD1 and 11beta-HSD2), modulate glucocorticoid occupation of the mineralocorticoid and glucocorticoid receptors by interconverting corticosterone and cortisol to the inactive metabolites 11-dehydrocorticosterone and cortisone within the target cells. The NAD(+)-dependent 11-HSD 2 in the kidney inactivates corticosterone and cortisol, allowing aldosterone, which is not metabolized, access to the receptor. Studies of the kinetics of 11-HSD 2 activity in the rat kidney have produced inconsistent results. Western blots done in the absence of the reducing agent beta-mercaptoethanol showed two bands with approximate MW of 40 and 80 kDa. When beta-mercaptoethanol was used, only the 40 kDa was detected, indicating that under non-denaturing conditions a significant proportion of the 11beta-HSD 2 exists as a dimer. NAD(+)-dependent conversion of 3H-corticosterone by 20 microg of microsomal protein increased approximately 10 fold with the addition of 5 mM DTT concentration. NADP(+)-dependent activity with 20 microg of microsomal protein was very low and did not change significantly when using DTT. In the presence of DTT, the predominant 11-HSD activity in the rat kidney is NAD(+)-dependent with a K(m) of 15.1 nM, similar to that of the cloned and expressed enzyme. These data suggest that dimerization and subsequent enzyme inactivation occur when protocols promoting oxidation of this protein are used.     

Binding of [3H]triamcinolone acetonide-receptor complexes to chromatin from the B-cell leukemia line, BCL1

The binding characteristics of partially purified glucocorticoid receptor complexes from hormone sensitive, non-differentiating BCL1 cells to sequentially deproteinized BCL1 chromatin-cellulose was investigated. [3H]Triamcinolone acetonide (TA)-receptor complexes were purified (approx. 30-fold) from DEAE-cellulose columns by salt elution which allowed receptor activation only in the absence of molybdate. Addition of 10 mM molybdate completely blocked salt activation. The binding pattern of the activated [3H]TA-receptor complexes to chromatin-cellulose extracted with 0-8 M guanidine hydrochloride revealed three regions of increased binding activity (acceptor sites), at 2, 5 and 7 M guanidine hydrochloride. Acceptor site binding was markedly reduced for chromatin extracted with 3, 6 and 8 M guanidine hydrochloride. Non-activated receptor complexes demonstrated very low binding to deproteinized chromatin. It was also shown that chromatin binding required glucocorticoid receptors and that free ligand or ligand bound to other proteins did not bind significantly to chromatin. In addition, binding of [3H]TA-receptor complexes to partially deproteinized chromatin was competable by unlabeled TA-receptor complexes. Scatchard analysis demonstrated that chromatin from non-differentiating BCL1 cells possesses multiple, high-affinity binding sites which differ in their affinity for the glucocorticoid receptor. Partially deproteinized chromatin from lipopolysaccharide-stimulated BCL1 cells demonstrated a different pattern of receptor binding, i.e., receptor binding was significantly greater to chromatin previously extracted with 6-8 M guanidine hydrochloride. These results suggest that differentiation alters the state of chromatin and the interaction of non-histone protein/DNA acceptor sites with glucocorticoid receptors. These alterations may play a role in the acquisition of hormone resistance.     

Modulation of cell proliferation and senescence of WI-38 cells by hydrocortisone.

The specific binding of glucocorticoid hormones has been studied in the normal diploid human cell line WI-38. These cells were found to contain high affinity glucocorticoid binding sites whose molecular specificity showed a high correlation to that required for the stimulation of cell growth. When hydrocortisone (HC) was selectively added to or removed from parasynchronously dividing cultures, we observed that HC -enhanced stimulation of cell growth was associated with the hormone's presence in the pre-DNA synthetic period of the cell cycle. Similarly, the specific binding of [3H]dexamethasone in stimulated quiescent cells was found to increase significantly in the pre-DNA synthetic period. The concentration of specific binding sites per cell achieved in stimulated cell cultures was found to decrease with increasing in vitro age. These results suggest that the stimulation of WI-38 cell proliferation by HC involves specific glucocorticoid receptors whose concentration per cell is under cell cycle control. The age-associated decrease in specific glucocorticoid binding sites may explain, in part, our previously observed loss of responsiveness to HC in aging cell cultures.     

Properties of the MgATP and MgADP binding sites on the Fe protein of nitrogenase from Azotobacter vinelandii

Flow dialysis was used to study the binding of MgATP and MgADP to the nitrogenase proteins of Azotobacter vinelandii. Both reduced and oxidized Av2 bind two molecules of MgADP, with the following dissociation constants: reduced Av2, K1 = 0.091 +/- 0.021 mM and K2 = 0.044 +/- 0.009 mM; oxidized Av2, K1 = 0.024 +/- 0.015 mM and K2 = 0.039 +/- 0.022 mM. Binding of MgADP to reduced Av2 shows positive co-operativity. Oxidized Av2 binds two molecules of MgATP with dissociation constants K1 = 0.049 +/- 0.016 mM and K2 = 0.18 +/- 0.05 mM. Binding data of MgATP to reduced Av2 can be fitted by assuming one binding site, but a better fit was obtained by assuming two binding sites on the protein with negative co-operativity and with dissociation constants K1 = 0.22 +/- 0.03 mM and K2 = 1.71 +/- 0.50 mM. It was found that results concerning the number of binding sites and the dissociation constants of MgATP-Av2 and MgADP-Av2 complexes depend to a great extent on the specific activity of the Av2 preparation used, and that it is difficult to correct binding data for inactive protein. No binding of MgADP to Av1 could be demonstrated. Binding studies of MgADP to a mixture of Av1 and Av2 showed that Av1 did not affect the binding of MgADP to either oxidized or reduced Av2. Inhibition studies were performed to investigate the interaction of MgATP and MgADP binding to oxidized and reduced Av2. All the experimental data can be explained by the minimum hypothesis, i.e. the presence of two adenine nucleotide binding sites on Av2. MgATP and MgADP compete for these two binding sites on the Fe protein.     

Modulation by ammonium ions of gill microsomal (Na+,K+)-ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion

The modulation by Na(+), K(+), NH(4)(+) and ATP of the (Na(+),K(+))-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V=35.4+/-2.1 Umg(-1) and K(0.5)=54.0+/-3.6 nM, obeying cooperative kinetics (n(H)=3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis-Menten kinetics with K(M)=55.0+/-3.0 microM and V=271.5+/-17.2 Umg(-1). This is the first demonstration of a crustacean (Na(+),K(+))-ATPase with two ATP hydrolyzing sites. Stimulation by sodium (K(0.5)=5.80+/-0.30 mM), magnesium (K(0.5)=0.48+/-0.02 mM) and potassium ions (K(0.5)=1.61+/-0.06 mM) exhibited site-site interactions, while that by ammonium ions obeyed Michaelis-Menten kinetics (K(M)=4.61+/-0.27 mM). Ouabain (K(I)=147.2+/-7.microM) and orthovanadate (K(I)=11.2+/-0.6 microM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Na(+),K(+))-ATPase activity by ammonium ions, and the excretion of NH(4)(+) in benthic crabs are discussed.     

Phosphatidylinositol synthase from canine pancreas: solubilization by n-octyl glucopyranoside and stabilization by manganese

Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase) is active in mammalian pancreas, where it plays a role in the resynthesis of phosphatidylinositol (PI) during agonist-stimulated inositol-phospholipid metabolism. The enzyme was found to be present in relatively high specific activity [30 nmol of PI formed min-1 (mg of protein)-1] in dog pancreas microsomal membranes, and its activity in these membranes was partially characterized. The Km for myo-inositol was 0.76 mM, and the apparent Km for cytidine(5')diphospho-1,2-diacylglycerol (CDP-diacylglycerol) was 18 microM. The apparent Ka values for activation by Mn2+ and Mg2+ were respectively 42 microM and 2.5 mM. The pH optimum was 8.5-9.0. The enzyme was solubilized in stable form and in nearly quantitative yield with 40 mM n-octyl glucopyranoside (OG), with 4-6 mg of OG/mg of microsomal protein. In the presence of solubilizing levels of OG, the enzyme exhibited less than maximal activity, but full activity was restored by dilution of the OG to below its critical micelle concentration of 20-25 mM. The presence of Mn2+ was essential for stabilization of the OG-solubilized enzyme, with half-maximal stabilization at 40 microM Mn2+. The stability of the OG-solubilized enzyme was sufficient to facilitate purification of the enzyme in the presence of this detergent, with 67% of the activity remaining after 3 days at 4 degrees C. The enzyme was partially purified by OG extraction and DEAE-cellulose chromatography, in 98% yield, to a specific activity of 290 nmol of PI formed min-1 (mg of protein)-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and modulation by drugs of sheep liver microsomal flavin monooxygenase activity

The flavin monooxygenases (FMO) catalyse the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. In the present study, sheep liver microsomal FMO activity was determined by measuring the S-oxidation rate of methimazole and the average specific activity obtained from different microsomal preparations was found to be 3.8 +/- 1.5 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 7). The presence of 0.1% Triton X-100 in the reaction mixture caused an increase of specific sheep liver microsomal FMO activity towards methimazole to 6.1 +/- 1.4 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 6). Metabolism of imipramine and chlorpromazine was measured by following the oxidation of cofactor NADPH spectrophotometrically at 340 nm. Sheep liver microsomal FMO activity towards imipramine and chlorpromazine was found to be 10.7 and 12.3 nmol NADPH oxidized min(-1) mg(-1) microsomal protein, respectively. Characterization of sheep liver enzyme was carried out using methimazole as substrate and the maximum FMO enzyme activity was detected at 37 degrees C and at pH 8.0. The apparent K(m) value of sheep liver microsomal FMO for methimazole was 0.118 mM. Effects of the detergents Triton X-100, Cholate, and Emulgen 913, on FMO activity were determined and FMO activity was found to increase with the addition of detergents to the reaction medium. Sheep liver microsomal FMO-catalysed methimazole oxidation was inhibited by imipramine and chlorpromazine when these drugs were used at high concentrations. Western blot-immunochemical analysis revealed the presence of FMO3 in sheep liver microsomes.     

Characterization of cytosolic glucocorticoid receptor of fetal rat epiphyseal chondrocytes

The cytosolic glucocorticoid receptor of 21st gestational day rat epiphyseal chondrocytes has been evaluated. The receptor, a single class of glucocorticoid binding component approached saturation, utilizing [3H]triamcinolone acetonide ([3H]TA) as the radiolabeled ligand, at approximately 1.8-2.0 x 10(-8) M. The dissociation constant (Kd) reflected high-affinity binding, equaling 4.0 +/- 1.43 x 10(-9) M (n = 7) for [3H]TA. The concentration of receptor estimated from Scatchard analysis was approximately 250 fmol/mg cytosolic protein and when calculated on a sites/cell basis equalled 5800 sites/cell. The relative binding affinities of steroid for receptor were found to be triamcinolone acetonide greater than corticosterone greater than hydrocortisone greater than progesterone greater than medroxyprogesterone acetate much greater than 17 alpha-hydroxyprogesterone much greater than testosterone greater than 17 beta-estradiol. Cytosolic preparations activated in vitro by warming (25 degrees C for 20 min) were shown to exhibit an increased affinity for DNA-cellulose. 46% of the total specifically bound activated ligand-receptor complex was bound to DNA-cellulose. Cytosol maintained at 0-4 degrees C in the presence of 10 mM molybdate or activated in vitro in the presence of molybdate, bound to DNA-cellulose at 8 and 10% respectively. DEAE-Sephadex elution profiles of the nonactivated receptor were indicative of a single binding moiety which eluted from the columns at 0.4 M KCl. Elution profiles of activated receptor were suggestive of an activation induced receptor lability. The 0.4 M KCl peak was diminished, while a concomitant increase in the 0.2 M KCl peak was only modestly discernible. Evaluation of endogenous proteolytic activity in chondrocyte cytosol using [methyl-14C]casein as substrate show a temperature-dependent proteolytic activity with a pH optimum of 5.9-6.65. The proteolytic activity was susceptible to heat inactivation and was inhibitable, by 20 mM EDTA. The sedimentation coefficient of the nonactivated receptor was 9.3s (n = 6) on sucrose density gradients and exhibited steroid specificity and a resistance to activation induced molecular alterations when incubated in the presence of 10 mM molybdate. Receptor activation in vitro, in the absence of molybdate induced an increased receptor susceptibility to proteolytic attack and/or enhanced ligand receptor dissociation as evidenced by a diminution of the 9.3s binding form without a concomitant increase in 5s or 3s receptor fragments.     

A specific, high-affinity binding site for the hepta-beta-glucoside elicitor exists in soybean membranes.

The presence of a specific binding site for a hepta-beta-glucoside elicitor of phytoalexin accumulation has been demonstrated in soybean microsomal membranes. A tyramine conjugate of the elicitor-active hepta-beta-glucoside was prepared and radiolabeled with 125I. The labeled hepta-beta-glucoside-tyramine conjugate was used as a ligand in binding assays with a total membrane fraction prepared from soybean roots. Binding of the radiolabeled hepta-beta-glucoside elicitor was saturable, reversible, and with an affinity (apparent Kd = 7.5 x 10(-10) M) comparable with the concentration of hepta-beta-glucoside required for biological activity. A single class of hepta-beta-glucoside binding sites was found. The binding site was inactivated by proteolysis and by heat treatment, suggesting that the binding site is a protein or glycoprotein. Competitive inhibition of binding of the radiolabeled hepta-beta-glucoside elicitor by a number of structurally related oligoglucosides demonstrated a direct correlation between the binding affinities and the elicitor activities of these oligoglucosides. Thus, the hepta-beta-glucoside-binding protein fulfills criteria expected of a bona fide receptor for the elicitor-active oligosaccharin.     

Correlation between nuclear glucocorticoid receptor levels and casein gene expression in murine mammary gland in vitro

The relationship between nuclear binding of glucocorticoid-receptor complex and casein gene expression was studied in organ culture of the whole mammary gland of the mouse. Pyridoxal 5'-phosphate was used as a modulatory agent for measuring nuclear binding of the receptor complex. Addition of 2 mM and 5mM pyridoxal-5'-P in the medium (Waymouth's MB752/1) resulted in 4- and 12-fold increase of its concentration in the glands incubated with insulin, prolactin, and hydrocortisone. Pyridoxal-5'-P also caused a 52% and 92% inhibition of nuclear binding of [3H]dexamethasone in the glands at 2 mM and 5 mM concentration in the presence of the same hormones in the medium. Corresponding to the reduced nuclear binding of the receptor complex casein mRNA levels, measured by a specific cDNA probe was reduced 86% and over 90% in the glands exposed to 2 mM and 5 mM pyridoxal-5'-P, respectively, in presence of insulin, prolactin, and hydrocortisone in the medium. Withdrawal of pyridoxal-5'-P from the medium restored nuclear binding of the receptor complex near the level of control glands incubated only with the hormones. mRNA casein levels also increased in the gland in the pyridoxal-5'-P-free medium containing the same hormones. This indicates that pyridoxal-5'-P does not alter the specific hormone responsiveness of the mammary cells and its action mediated at the level of the glucocorticoid receptor can influence hormone-inducible expression of the casein genes. Thus, glucocorticoid plays a major role in the multiple hormone regulation of the milk protein gene(s). The findings also suggest that the breast tissue concentration of the vitamin B6 derivative may influence the physiology of lactation in nursing mothers.     

Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [3H]-leukotriene D4 [( 3H]-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, we have identified specific binding sites for [3H]-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for [3H]-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [3H]-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with [3H]-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [3H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.     

A study of the cytoplasmic receptors for glucocorticoids in intestine of pre- and postweanling rats.

Glucocorticoids cause both enzymic and morphologic changes in the rat intestine during the time of weaning. To obtain information regarding the mechanism of these actions, we examined the cytoplasmic fraction of intestines from 18-day-old rats for the presence of specific glucocorticoid-binding proteins which are characteristics of target tissues. Incubation of slices of intestine with [3H]dexamethasone in a physiological medium at 2 degrees showed the presence of a cytoplasmic binding macromolecule with high specificity for steroids having glucocorticoid activity. The binding reaction was saturable (concentration of binding sites equals 0.24 pmol per mg of protein) and of high affinity (dissociation constant equals 9.3 nM). Binding was reversible on addition of nonlabeled dexamethasone (t 1/2 equals 5.2 hours), indicating that the usual assay procedure measured both corticosterone-filled and unoccupied binding sites. Sucrose density gradient centrifugation showed that the receptor-dexamethasone complex from intestinal cytosol sedimented at the same rate as that from liver (8.2 S). The receptor-dexamethasone complex was stable at 2 degrees for at least 24 hours in intestinal slices, but in isolated cytosol fractions there was considerable loss of binding even in the presence of high concentrations of [3H]dexamethasone. Furthermore, mixing experiments showed that the presence of cytosol from intestinal mucosa (but not from the muscle layers) caused a dissociation of dexamethasone from receptors of liver cytosol. This suggested the presence of some interfering factor in isolated mucosal cytosol and meant that quantitative studies had to be confined to intact slices. Although the reasons for the instability of steroid-receptor complexes in the presence of isolated intestinal cytosol are not understood, the instability is believed to be associated with homogenization and, therefore, is believed to have no physiological significance. Finally, the ontogenesis of cytoplasmic glucocorticoid receptors in intestinal slices was examined and the pattern compared with that in liver and lung. Receptor activity was present in intestine from late fetal life through adulthood, but concentrations were significantly higher during the first two postnatal weeks than at all other times. By contrast, receptor activity detected in cytosol prepared from rat lung was high around the time of birth, while that in liver rose steadily during the first postnatal week and remained at high levels. Thus specific receptors for glucocorticoids are present in the rat intestine during periods of both responsiveness and unresponsiveness. This suggests that although corticosteroids exert their effects through the cytoplasmic receptors, this early event in glucocorticoid action may not be a controlling step for changes in responsiveness during development.