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J J Feige

Publications and source records attributed to J J Feige.

85 records · Page 5Linked to original sources
PubMed1986

Hormonal control of polyamine levels in bovine adrenocortical cells.

The implication of polyamines in cellular growth and differentiation processes and the existence of a polyamine-mediated protein phosphorylation system in adrenocortical cells suggest that polyamines may be examined as potential intracellular messengers in the pleiotypic action of ACTH. Bovine adrenocortical cells in culture exhibit a specific, energy-dependent, partly sodium-supported, inward polyamine transport system, independent of the A, L, and N aminoacid uptake systems. Steroidogenic concentrations of ACTH (10(-12) to 10(-9)M) induced a rapid activation of the polyamine uptake, resulting in a 2- to 3-fold increase in intracellular polyamine content, over 1 h. The ACTH dose-response curves for steroidogenic activity and for polyamine uptake were similar. Other adrenocortical effectors such as angiotensin II, acetylcholine, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) activated polyamine uptake with a pattern parallel to their steroidogenic potency (i.e. ACTH greater than angiotensin II greater than acetylcholine, TPA). Steroidogenic concentrations of 8-bromo-cAMP displayed no effect on the adrenocortical polyamine uptake, suggesting that cAMP does not mediate this action of ACTH. On the other hand, ACTH induced a large increase in ornithine decarboxylase (ODC) activity in bovine adrenocortical cells, after a 6- to 8-h lag period, resulting in an average 2-fold increase in cell putrescine, spermidine, and spermine content. However, when the cells were previously polyamine loaded, ACTH-dependent ODC induction was suppressed. Adrenocortical cell polyamine content thus appears to be under hormonal control. ACTH may act through two possible pathways: 1) the rapid activation of the cell polyamine accumulation from an extracellular source and 2) the delayed increase in polyamine biosynthesis secondary to induction of ODC activity, when the cells are relatively depleted of the polyamines. These observations suggest that polyamines may function as intracellular messengers for some of the ACTH effects in bovine adrenocortical cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Polyamine uptake by bovine adrenocortical cells.

Bovine adrenocortical cells of fasciculo-reticulata origin in primary culture actively accumulate polyamines from the extracellular medium in an energy-dependent process. At low extracellular concentration (e.g., 1 microM putrescine), the transport system resulted in a several-hundred-fold concentration of polyamine in the cellular compartment within 1-2 h of incubation. Putrescine uptake appeared to be the sum of a sodium-dependent, saturable process, with an apparent Km of about 10 microM and of a non-saturable, sodium-independent component. By contrast, spermine was taken up by the cells mostly in a sodium-independent manner. Cross-competition experiments suggested that both polyamines were at least partly transported by the same system. Using specific corresponding probes, it was shown that the polyamine uptake was independent of the amino acid transport systems of the A, L and N types known in a number of cell systems. Adrenocortical cell polyamine content is known to be modulated by adrenocorticotropin through induction of ornithine decarboxylase activity. The existence of a specific uptake system in these cells opens the possibility of a more rapid pathway for the regulation of cellular polyamine levels. It remains to be examined whether this polyamine transport system is under hormonal control, and whether this can support the suggestion that polyamines may represent a form of intracellular messengers in the mechanism of hormone action.

Adrenal Cortex↗
PubMed1983

Reversibility of the phosphate transfer between ATP and phosphoproteins catalysed by a cyclic nucleotide independent (G type) casein kinase.

Purified casein kinase G was found able to catalyse the synthesis of [gamma-32P]ATP in the presence of ADP, phosphocasein (previously 32P-labeled by the forward kinase reaction) and magnesium. Apparent Km values of approx. 0.5 mM for phosphocasein and 7.5 mM for ADP were calculated, these values indicating low affinities for the substrates as compared to those exhibited for casein and ATP in the forward reaction. The reverse casein kinase G activity appeared to prefer ADP and GDP as phosphate acceptors. Whereas the casein kinase G reverse reaction could be supported by casein, phosvitin and histone previously phosphorylated by the enzyme, the same proteins could not serve as a phosphate source when previously phosphorylated by the cAMP-dependent protein kinase. Forward and reverse casein kinase G reactions exhibited different optimal pH values (8.5 and 7.2, respectively) and a different sensitivity to Mg2+. Spermine, which activated the kinase activity, blocked the reverse reaction at millimolar concentrations. Although the biological significance of the casein kinase G reverse activity remains to be assessed in intact cell, the process may be useful as a tool in the characterization of phosphorylatable sites in phosphoproteins.

Adenosine Diphosphate↗
PubMed1983

Identification of the catalytic subunit of an oligomeric casein kinase (G type). Affinity labeling of the nucleotide site using 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

Identification of the catalytic subunit of a G type [using guanosine 5'-triphosphate (GTP) as well as adenosine 5'-triphosphate (ATP) as phosphate donor], oligomeric, cyclic nucleotide independent casein kinase purified from bovine lung was carried out after reaction with 5'-[p-(fluorosulfonyl)-benzoyl]adenosine (FSBA) and isolation of the subunit components of the enzyme. FSBA exhibited the major characteristics of an affinity label reacting at the nucleotide (ATP, GTP) site of the casein kinase. FSBA acted as a competitive inhibitor of ATP (and GTP), led to complete inactivation of the enzyme in a reaction showing two kinetic steps, and became irreversibly bound to the protein. After being labeled with FSBA, the casein kinase (apparent molecular weight of 140 000) was separated into its two monomeric components of apparent molecular weights 38 000 (alpha) and 27 000 (beta), respectively, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Use of radioactive FSBA showed that specific affinity labeling was limited to the alpha casein kinase subunit. This result was in agreement with the fact that casein kinase activity was found associated with the alpha monomer after electrophoretic separation of the alpha and beta subunits. It may thus be concluded that the largest (alpha) subunit contains the catalytic site of the casein kinase G. Electrophoretic analysis of purified protein kinase under denaturing conditions suggested an alpha 3 beta 2 combination for an apparent molecular weight of 130 000-140 000. However, a maximum of 2 mol of FSBA could be specifically bound to the alpha subunit per mol of enzyme, with a concomitant complete inactivation. These data would be in agreement with an alpha 2 beta 2 subunit composition for casein kinase G, as proposed by other research groups for a similar type of protein kinase of different sources. These observations suggest that the alpha subunits are functionally similar, each of them containing a nucleotide (ATP, GTP) binding site. The possible role of the beta subunit in the enzyme activity remains to be established.

Adenosine↗
PubMed1983

Catalytic and molecular properties of a highly purified G type casein kinase from bovine lung tissue.

A preparation procedure has been worked out to obtain a highly purified G type (using GTP as well as ATP) casein kinase from large quantities of bovine lung tissue. It included ion-exchange (DEAE and phosphocellulose) and affinity (casein and ATP-Sepharose) chromatography combined with a flocculation step, and yielded an apparently homogeneous preparation with a 16% yield and a purification factor of more than 1400. The purified lung casein kinase used GTP (Km 16 microM) almost as well as ATP (Km 6.7 microM) and exhibited the major catalytic properties of the casein kinase G previously described in bovine adrenal cortex (Cochet, C., Job, D., Pirollet, F. and Chambaz, E.M. (1981) Biochim. Biophys. Acta 658, 191-201). Mg2+ (30-50 mM) and spermine (2 mM) were potent activators of lung casein kinase G activity, whereas the enzyme was inhibited by heparin and quercetin. The purified enzyme underwent self-phosphorylation in the presence of ATP or GTP, serine being the only target amino acid under these conditions, whereas both serine and threonine were phosphorylated by the enzyme in casein. Lung casein kinase G exhibited an apparent molecular weight between 140 000-160 000 upon gel filtration and appeared formed by the association of two different subunits upon SDS-polyacrylamide gel electrophoresis. The two subunits of Mr 38 000 (alpha) and 27 000 (beta) exhibited a 2:1 ratio upon quantitative scanning, suggesting an alpha 3 beta 2 combination in the oligomeric native enzyme structure. Peptide mapping of the two isolated subunits following 125I-labeling and papain digestion did not disclose any common fragment. The casein kinase catalytic activity was found associated with the alpha (38 kDa) enzyme subunit after recovery from gel electrophoresis in the presence of SDS, whereas the 27 kDa (beta) subunit was the major target of the enzyme self-phosphorylation reaction. alpha and beta subunits appeared strongly associated in the oligomeric enzyme and the possible role of the beta subunit in the casein kinase G activity remains to be examined. The purified casein kinase G, which can be obtained by the present procedure, should facilitate the study of the biological significance of this phosphorylation system in the intact cell.

Animals↗
PubMed1982

Selective inhibition of a cyclic nucleotide independent protein kinase (G type casein kinase) by quercetin and related polyphenols.

The effect of quercetin and a number of structurally related phenolic compounds upon the activity of three different purified protein kinases was examined. Whereas the catalytic subunit of a cyclic AMP-dependent protein kinase and an A type (using only ATP) cyclic nucleotide-independent casein kinase (CKA) were not affected, a G type (using GTP as well as ATP) casein kinase (CKG) was selectively inhibited by several bioflavonoid structures. Kinetic studies showed that quercetin behaved as a competitive inhibitor toward the nucleotidic substrate and exhibited a high affinity for the ATP (Ki = 0.75 microM) and GTP (Ki = 0.22 microM) site of the enzyme. Considering the CKG inhibitory potency of a series of flavonoid, cinnamic acid and coumarin derivatives, it is suggested that the biological activity lays upon a common structural feature involving a phenolic ring bearing a side chain with conjugated double bonds and an oxygenated function, as found in the coumaroyl residue. These observations suggest that quercetin and related compounds may lead to a shift in intracellular protein phosphorylations by selectively inhibiting a particular type of protein kinase activity (CKG). It remains to be established whether this process may contribute to the mechanism of action of flavonoids upon cellular metabolism, particularly in the case of malignant cells.

Adrenal Cortex↗
PubMedDate not supplied

Control of glycosaminoglycan metabolism by ACTH in bovine adrenocortical cells in primary culture.

Bovine adrenocortical cells in primary culture actively incorporated [35S]sodium sulphate and [3H]glucosamine into glycosaminoglycans (GAGs). Most of the synthesized GAGs were found associated with the pericellular material and secreted in the culture medium, while less than 15% remained associated with the cell pellet. In all these fractions the major product was identified by its molecular properties and selective degradation procedures as a heparan sulphate structure. Exposure of the cells to ACTH induces a shift of the synthesized GAGs from the cellular to the pericellular compartment, an increase in the heparan sulphate labelling and the occurrence of an additional product characterized as a hyaluronate. These data suggest that GAG metabolism of the adrenocortical cell may be under hormonal control and open the possibility of a modulation of cell activity through modifications of its GAG components, especially those involved in the cell matrix architecture.

Adrenal Cortex↗
PubMedDate not supplied

Hormonally regulated components of the adrenocortical cell environment and the control of adrenal cortex homeostasis.

The extracellular matrix (ECM) strongly contributes to the regulation of cell proliferation and cell differentiation, and thereby of embryonic development and adult tissue homeostasis. We review here the ongoing characterization of the structure and functions of the extracellular matrix components secreted by adrenocortical cells and discuss their possible implication in the hormonal regulation of adrenal cortex homeostasis. Fibronectin (FN) and laminin (LN) are both major adhesive proteins for adrenocortical cells. FN is synthesized by bovine fasciculata cells in primary culture, and its synthesis is stimulated by TGF(beta)1, TGF(beta)2, and FGF-2 but is not modified by IGF-1 or by the hormones ACTH and angiotensin II. LN is also synthesized by bovine fasciculata cells and its synthesis is specifically stimulated by ACTH. Both proteins are haptotactic and chemotactic for adrenocortical cells, suggesting a physiological role in adrenocyte migration. Their distribution in the adrenal gland is quite distinct. LN is uniformly present in the steroidogenic cells from the three zones, whereas FN is abundant in the fibrovascular structures of the capsule and the cortex. ACTH treatment of adrenocortical cells strongly induces the expression and secretion of thrombospondin-2 (TSP2), a large trimeric matricellular protein. The multimodular structure of TSP2 is the support of a variety of biological functions. TSP2 promotes attachment but prevents spreading of adrenocortical cells. On the other hand, TSP2 induces the activation of latent TGFbeta through an indirect mechanism and is anti-angiogenic in vitro. The overall distribution of TSP2 in the glomerulosa and fasciculata zones of the adrenal cortex, and its absence from the reticularis zone, argue in favor of a role in the protection of adrenocortical cells against apoptosis. In the adrenal cortex, five main biological functions are potentially regulated by components of the extracellular matrix : stem cell commitment into the adrenocyte differentiation pathway, terminal differentiation toward the three distinct adrenocyte phenotypes, centripetal migration, apoptosis and the formation of the capillary network. Future studies will aim at deciphering which extracellular component(s) is involved in each of these regulations.

Adrenal Cortex↗
PubMedDate not supplied

Expression of ACTH receptors (MC2-R and MC5-R) in the glomerulosa and the fasciculata-reticularis zones of bovine adrenal cortex.

The recent cloning of a family of melanocortin receptors (MC-R) has identified five distinct G protein- and adenylate cyclase-coupled receptors. The MC2-receptor (MC2-R) preferentially binds ACTH. It is expressed in the adrenal cortex and is hence considered to be the ACTH receptor. The MC5-receptor (MC5-R) binds ACTH and alpha-MSH and is more widely expressed. The aim of this work was to study the sites of MC5-R expression in the bovine adrenal cortex and to compare the regulation of the expression of MC2-R and MC5-R in bovine adrenocortical cells in primary culture. Analysis of the expression of MC5-R was obtained by RT-PCR, using total RNA purified from glomerulosa and fasciculata zones of bovine adrenocortical tissue. MC5-R expression could be detected in RNA from the glomerulosa zone but was undetectable in the fasciculata zone. In bovine adrenocortical cells in culture, ACTH stimulates MC5-R expression in the glomerulosa and fasciculata cells. A DNA fragment, was obtained using primers based on the bovine ACTH receptor (MC2-R) sequence. This fragment was detected in RNA from the two zones. The probe was used to quantify MC2-R by Ribonuclease Protection assay and we observed that MC2-R mRNA is 3.6-fold more abundant in glomerulosa than in fasciculata-reticularis cells.

Adrenocorticotropic Hormone↗
PubMedDate not supplied

Differential implication of StAR and P450c17 in TGFbeta1-induced decrease of adrenocortical steroidogenesis.

In primary cultures of bovine adrenocortical fasciculata cells, we have previously identified two targets of TGFbeta action: StAR and P450c17. Since it is well known that adrenocortical cells tend to dedifferentiate as soon as they are placed in culture, we investigated the regulation of StAR and P450c17 expression by TGFbeta1 at different times of primary culture. On day 1, TGFbeta1 decreased the expression of basal levels of both genes. However, in the presence of ACTH, it down-regulated StAR mRNA levels but did not modify P450c17 mRNA levels. On day 4, P450c17 and StAR mRNAs were down-regulated by TGFbeta1, both in the absence and in the presence of ACTH. These observations indicate that StAR is likely to represent the major in vivo target of TGFbeta1 action in the adrenal cortex.

Adrenal Cortex↗
PubMedDate not supplied

Gastric inhibitory polypeptide (GIP) stimulates cortisol secretion, cAMP production and DNA synthesis in an adrenal adenoma responsible for food-dependent Cushing's syndrome.

We studied in vitro an adrenal tumor responsible for food-dependent, ACTH independent, Cushing's's syndrome. Cortisol secretion by isolated tumor cells was stimulated by GIP and ACTH, but not by the gut hormone glucagon-like peptide-1 (GLP-1). Both GIP and ACTH stimulated production of cAMP but not inositol 1,4,5-trisphosphate IP3). In quiescent tumor cells, GIP and ACTH stimulated [3H]-thymidine incorporation and p42-p44 MAP kinase activity. In normal human adrenocortical cells cortisol secretion and [3H]-thymidine incorporation were stimulated by ACTH but not by GIP. GIP receptor mRNA, assessed by RT-PCR, was highly expressed in the tumor, but undetectable in the adjacent hypotrophic adrenal tissue, in a normal adrenal, in two adrenal tumors responsible for food-independent Cushing's syndrome and in two hyperplastic adrenals associated with ACTH hypersecretion. Low levels of ACTH receptor mRNA were also detectable in the tumor. We conclude that abnormal expression of the GIP receptor allows adrenocortical cells to respond to food intake with an increase of cAMP that may participate in stimulation of both cortisol secretion and proliferation of the tumor cells.

Adenoma↗