Cyclic nucleotide-dependent phosphorylation of endogenous proteins in bovine adrenocortical cell membranes

Activation of one or more cyclic AMP-dependent protein kinases has been suggested as an intermediate step in ACTH-stimulated adrenal cell steroidogenesis. Phosphorylation of a number of proteins from different subcellular fractions has been reported but those phosphorylation events which are relevant to the steroidogenic process have not yet been identified. In this paper we report that plasma membrane enriched fractions from bovine adrenal cortex retain the ability to phosphorylate endogenous membrane proteins and that phosphorylation of these acceptors is markedly enhanced by cyclic AMP or, to a lesser extent, by cyclic GMP. Cyclic nucleotide-dependent phosphorylation was most marked in protein acceptors of 191 000, 148 000, 138 000, 107 000, 65 000, 60 000 and 27 000 daltons. Cyclic nucleotide stimulation of phosphorylation was rapid (within 10 s), and is consistent with the rapid onset of ACTH-stimulated steroidogenesis.     

Role of polyamines in the proliferation and steroidogenic activities of bovine adrenocortical cells in culture

Difluoromethylornithine (DFMO), a selective inhibitor of ornithine decarboxylase, was used to probe the possible role of polyamines in the regulation of proliferation and steroidogenic activities of bovine adrenocortical cells in primary culture. The presence of DFMO in the culture medium not only suppressed the polyamine increase observed in proliferating control cells but resulted in a rapid depletion of the putrescine and spermidine cellular content, while spermine remained at a basal level. The proliferation of DFMO-treated cells was rapidly blocked and resumed at a normal rate upon addition of putrescine to the medium. DFMO-treated cells showed an impaired steroidogenic response to ACTH while adenylate cyclase stimulation was not altered. Thus, while ornithine decarboxylase and polyamines may be required for adrenocortical cell replication, deprivation of these compounds did not facilitate the expression of differentiated cell functions, as observed with granulosa cells.     

Effect of glucocorticoids pretreatment on steroidogenic capacity of adrenocortical cells isolated from Meishan piglets

The present in vitro experiment was designed to test whether 48 h of pretreatment with glucocorticoids, cortisol, or dexamethasone (DEX), would affect basal and corticotrophin (ACTH) stimulated (24 h) cortisol secretion from primary cultures of pig adrenocortical cells. Cells were divided into six groups: control pretreatment with or without ACTH challenge, cortisol pretreatment with or without ACTH challenge, and DEX pretreatment with or without ACTH. The culture medium and cells were collected at the end of treatment. Cortisol concentration in medium was measured by radioimmunoassay, and protein content of glucocorticoid receptor (GR) and key regulatory factors for steroidogenesis, including melanocortin type 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450scc), were detected by Western blot analysis. The results showed that glucocorticoid pretreatment did not affect cortisol secretion under basal condition without ACTH challenge, but significantly enhanced ACTH-stimulated cortisol secretion. Furthermore, the protein content of GR, MC2R, StAR, and P450scc was all increased in groups pretreated with glucocorticoids. These results indicate that adrenocortical cells pretreated with glucocorticoids display higher steroidogenic capacity under ACTH challenge, through the upregulation of GR and other steroidogenic regulatory factors.     

Modulation of auxin-binding proteins in cell suspensions

Summary Cultured cell lines from carrot (Daucus carota L.) with little or no embryogenic potential were examined for the auxin-binding capacity of their membranes. The lines belonged to different classes: (a) wild-type lines kept in culture for different periods (the longer the period, the lower being their embryogenic potential); (b) variants, isolated after mutagenesis, showing normal growth but a lack of embryogenic response; (c) auxin-resistant lines, isolated as colonies on solid media containing 45 M 2,4-d; (d) a previously described tumorous line (E9) isolated because of its resistance to hypomethylating drugs. All of these lines showed alterations in auxin-induced, auxin-binding capacity (modulation), i.e. in the non-embryogenic lines the addition of auxin increased the auxinbinding capacity to a very small degree, or removal of the hormone did not produce the proper decrease in that capacity, or both defects could be simultaneously present. Both types of defects were shown to be correctable: after treatments designed to increase the amplitude of modulation, embryogenic capacity was restored in a number of lines.     

Effect of the benzodiazepines diazepam, des-N-methyldiazepam and midazolam on corticosteroid biosynthesis in bovine adrenocortical cells in vitro; location of site of action

Diazepam and midazolam inhibited cortisol and aldosterone synthesis in bovine adrenal cells in vitro. The biologically active metabolite des-N-methyldiazepam did not. Midazolam was a more potent inhibitor (IC50: 6 micrograms/ml) than diazepam (IC50: 13 micrograms/ml) in ACTH-stimulated cells. Both compounds inhibited steroidogenesis at several points in the biosynthetic chain; the greatest effects were on 17 alpha hydroxylation and 21 hydroxylation. Diazepam had a relatively greater effect on 17 alpha hydroxylation; midazolam on 21 hydroxylation. Both were less potent inhibitors of 11 beta hydroxylation and had little apparent effect on side chain cleavage. Thus microsomal hydroxylation is more vulnerable to benzodiazepines than mitochondrial hydroxylation. It is suggested that the drugs act by competing with steroid mixed function oxidases for cytochrome P-450. The plasma concentrations required for these effects are high in relation to therapeutic levels but may be achieved, for example, during acute infusions or when they are used in combination with imidazole drugs such as cimetidine.     

Identification of bovine brain calcium binding proteins

Three peaks of calcium binding activity have been identified by the Chelex-100 calcium binding assay of the fractions from DEAE cellulose chromatography of 100,000 X g supernatant of bovine brain. These calcium binding activity peaks have been subjected to extensive purification and three novel calcium binding proteins (Mr 27,000, Mr 48,000 and Mr 63,000) and two previously characterized proteins (calcineurin and calmodulin) have been identified as components of calcium binding activity peaks. Analysis of the calcium binding properties of the novel proteins by equilibrium dialysis suggests these proteins may be intracellular calcium receptors.     

VICKZ proteins mediate cell migration via their RNA binding activity

The highly conserved, RNA binding VICKZ proteins help regulate RNA localization, stability, and translation in many eukaryotes. These proteins are also required for cell migration in embryos and cultured cells. In adults, many tumors overexpress VICKZ homologs, and it has been hypothesized that the proteins can mediate cell motility and invasion. How these proteins facilitate cell movement and, in particular, whether their ability to bind RNA plays a role in their function remain unclear. Using HPLC and mass spectrometry to identify a region of Xenopus Vg1 RBP (xVICKZ3) that binds the vegetal localization element of Vg1 RNA, we generated a deletion construct that functions in a dominant-negative manner. The construct associates with full-length xVICKZ3 and severely reduces binding to target RNAs. This dominant-negative construct phenocopies the effect of down-regulating xVICKZ3 in Xenopus embryos. A corresponding deletion in the human homolog hVICKZ1 similarly functions in a dominant-negative fashion to reduce the ability of full-length hVICKZ protein to bind RNA. Expression of the dominant-negative construct in human carcinoma cells inhibits cell movement by several criteria. We conclude that the ability of VICKZ proteins to mediate cell migration, in vitro and in vivo, requires their RNA binding activity.     

Rapid polyphosphoinositide decrease is an early event in the steroidogenic response of bovine adrenocortical fasciculata cells to angiotensin II

Addition of angiotensin II (0.3 microM) to bovine adrenal fasciculata cell suspensions prelabeled with [32P] induced a rapid (15 seconds) and marked decrease of the radioactivity from phosphatidylinositol 4,5-biphosphate (62%) and phosphatidylinositol 4-monophosphate (35%). This effect was concentration-dependent and specifically inhibited in the presence of (Sar1-Ala8)-angiotensin II; it was also completely prevented in the absence of extracellular calcium. The present data appear to illustrate the earliest biological response detectable in bovine fasciculata cells under angiotensin II challenge.     

Angiotensin binding to rat adrenal capsular cell suspensions

Adrenal cell suspensions obtained by collagenase digestion of rat adrenal capsules was demonstrated to bind tritiated angiotensin II. The binding was rapid and reversible and was temperature dependent. Saturation of binding sites of a low order of capacity could be demonstrated by the addition of unlabeled angiotensin II. Specificity for this binding was demonstrated using several peptide analogues. Specificity was also observed with respect to cell type. These studies suggest the presence of a biologically significant receptor for angiotensin in cells of the zona glomerulosa of rat adrenal glands.     

Role of albumin for the cholesterol transport and on the steroidogenic pathways in serum-free medium newborn rat adrenocortical cell cultures

[3,4-13C]cholesterol-albumin complex incubated in serum-free medium allowed to evaluate quantitatively the transfer of cholesterol in newborn rat adrenocortical cultured cells, its accumulation as free cholesterol or cholesterol esters and its transformation into steroids which were also originated (47%) from intracellular unlabelled cholesterol. Increasing concentrations of albumin up to 5 g/l enhanced the production of total steroids but in the meantime decreased the 21-hydroxylated steroid fraction. Internalization of albumin shown by using [methyl-14C]methylated-albumin as a tracer accounted only for a minor part in the cholesterol uptake but strikingly affected the steroidogenic pathways by favoring the reductive metabolism of progesterone over the corticosteroid biosynthesis.     

Endothelial binding of transferrin in fractionated liver cell suspensions

Several studies using crude liver cell suspensions incubated with labeled transferrin have led to a conclusion that hepatocytes have transferrin receptors. When a visual probe, which permits evaluation of transferrin binding to individual cells, was used, the binding was unexpectedly found to be limited to endothelial cells in liver cell suspensions. Neither hepatocytes nor Kupffer cells contained transferrin receptors. In the present study, we fractionated liver cell suspensions using metrizamide gradients and centrifugal elutriation to obtain hepatocytes, Kupffer cell and endothelial cell fractions of high purity. Incubation of these fractions with 125I- or 59Fe-labeled transferrin led to exclusive binding to endothelial cells but not hepatocytes nor Kupffer cells. Kinetic analysis demonstrated Kd of 1.9 X 10(-7) M, Bmax of 3.1 pmol/10(6) cells per min, corresponding to 2.1 X 10(5) molecules/cell per min. At 4 degrees C, the binding reached a steady-state plateau within 5 min. Comparison of our data with those of previous investigators demonstrates a consistency if we consider that crude liver cell suspensions are contaminated with 2-3% endothelial cells. Thus, the previously reported findings may be entirely due to the contamination of crude liver cell suspensions with a small number of endothelial cells.     

Modulation of soluble auxin-binding proteins in soybean cell suspensions

Rapidly growing cell suspensions of soybean were analyzed for the presence of cytoplasmic high-affinity binding sites for auxin. Cytosol preparations were studied in lag, log and early stationary phase of the growth cycle. Two binding sites were detected, which show some similarities with binding sites previously reported from etiolated pea epicotyls. While the number of both sites declined in the cytoplasm during the growth cycle, the number of one of the two sites increased at the onset of rapid cell divisions. In parallel, both sites exhibited an increase in binding affinity during the growth cycle. The data will be discussed in relation to other reports on soluble auxin binding as well as to possible physiological functions.Abbreviations CF Chromatofocusing - TCA trichloroacetic acid - sABP soluble auxin-binding protein - DIECA diethyldithiocarbamate - PEI polyethylen-imine (filter assay for ligand binding) - ASP ammonium sulphate precipitation assay - R_T number of binding sites - K_D dissociation constant - TSBS/DMSDP TRIS (0.1M)-sucrose(35%)-bromophenol blue (0.01%)-SDS-dithiothreitol-2-mercapto enthanol-SDS-DMSO-PMSF     

Effect of glucose on adrenocortical activity of newborn piglets.

Adrenocortical function was followed in newborn piglets fasted for 36 hours and in piglets given only glucose or glucoplastic amino acids during this period. The greatest increase in relative adrenal weight, blood plasma 17-hydroxycorticosteroid (17-OHCS) levels and in the production of 17-OHCS by pig adrenals in vitro as compared with suckled controls was found in fasted piglets followed by amino acid-treated animals. The latter showed no significant differences in blood glucose between the initial and final values. The administration of glucose produced a transient hyperglycemia, failed to prevent the inhibition of body growth, resulted in a reduction in liver weight similar to that seen in fasted animals, and had a marked suppressive effect on the elevation of adrenocortical function. There were no significant differences in the production of 17-OHCS by adrenals in vitro per kg body weight between the glucose-treated and suckled piglets.     

Two binding proteins for progesterone in the bovine corpus luteum

The cytosolic supernatant of bovine corpus luteum contains two proteins which bind progesterone specifically. Bovine luteal cytosol was fractionated on hydroxylapatite and the peaks of protein obtained subjected to equilibrium dialysis against progesterone. Progesterone-binding activities (Ka approx. 10(6) 1/mol) was eluted at 40 mM (Binding Protein 1) and 100 mM phosphate (Binding Protein 2). They sedimented differently (3.95 and 4.65, respectively) on sucrose gradients. In contrast to Binding Protein 1, Binding Protein 2 bound R5020 better than progesterone on sucrose gradients. Purification of the binding activity eluted by 40 mM phosphate from the hydroxylapatite column showed that it resided in a single protein (molecular weight 65,000 daltons). The function of these proteins is presently unknown, but they may participate in the biosynthesis and/or secretion of progesterone from bovine luteal cells.