Prematurity has no impact on the quality of parents' sleep 6 months after birth.

AIM: Due to the stress that is classically associated with the premature birth of a child, these parents may be prone to sleep disorders. The aim of this study was to compare sleep quality of preterm infants' parents with that of term infants' parents. METHODS: Prospective observational cohort study conducted at the University Hospital of Brest between January 2019 and January 2021. The primary outcome criterion was the score obtained by the parents on the Pittsburg Sleep Quality Index (PSQI) 6 months after their child's birth. Each parent was recruited in the days following their child's birth and completed the PSQI online. RESULTS: Overall, 316 parents were included. The median gestational age at birth was 34.3 (31.6-35.5) weeks in the preterm infant group and 39.7 (38.6-40.7) weeks in the term infant group. Of the 948 expected questionnaires, 771 were completed and collected. On average, 59% of the parents obtained a PSQI global score >5. Six months after birth, no differences were reported between parents of preterm and full-term infants. CONCLUSION: This study did not reveal any difference between sleep quality of preterm infants' parents and term infants' parents.

Comparison of the outcomes of the pediatric kidney transplantation between recipients below and above 15 kg: a single center retrospective study.

BACKGROUND: The renal transplantation is the best treatment for end-stage renal disease in children. We present the findings of an analysis of our institution's paediatric transplant outcomes comparing recipients under 15 kg, who represent this potentially higher risk group, to those above 15 kg. METHODS: We retrospectively identified consecutive paediatric kidney transplants from a prospectively collected database for analysis. We included all recipients under the age of 18 years at the time of transplant between 2006 and 2018 without any exclusion criteria. The primary outcome was death-censored graft survival at 1 year, 5 years and 10 years. RESULTS: 109 paediatric kidney transplants were performed in 100 children. Graft survival in the all population was 98%, 96% and 76% at 1 year, 5 years and 10 years, respectively. Recipient weight below 15 kg was not found to be a risk factor of graft loss. Overall, we found no individual factor to be statistically significantly associated with renal graft lost. The overall complication rate was 16% (18/109) with 12 early complications (11%) and 6 late ones (5%). CONCLUSION: Kidney transplantation in children weighing < 15 kg seems safe and offers the same patient and graft survival outcomes as in other (> 15 kg) pediatric recipients with equally low complication rates.

Structure Elucidation of Helical Aromatic Foldamer-Protein Complexes with Large Contact Surface Areas.

The development of large synthetic ligands could be useful to target the sizeable surface areas involved in protein-protein interactions. Herein, we present long helical aromatic oligoamide foldamers bearing proteinogenic side chains that cover up to 450 Å2 of the human carbonic anhydrase II (HCA) surface. The foldamers are composed of aminoquinolinecarboxylic acids bearing proteinogenic side chains and of more flexible aminomethyl-pyridinecarboxylic acids that enhance helix handedness dynamics. Crystal structures of HCA-foldamer complexes were obtained with a 9- and a 14-mer both showing extensive protein-foldamer hydrophobic contacts. In addition, foldamer-foldamer interactions seem to be prevalent in the crystal packing, leading to the peculiar formation of an HCA superhelix wound around a rod of stacked foldamers. Solution studies confirm the positioning of the foldamer at the protein surface as well as a dimerization of the complexes.

Structural evidence of a phosphoinositide-binding site in the Rgd1-RhoGAP domain.

Phosphoinositide lipids recruit proteins to the plasma membrane involved in the regulation of cytoskeleton organization and in signalling pathways that control cell polarity and growth. Among those, Rgd1p is a yeast GTPase-activating protein (GAP) specific for Rho3p and Rho4p GTPases, which control actin polymerization and stress signalling pathways. Phosphoinositides not only bind Rgd1p, but also stimulate its GAP activity on the membrane-anchored form of Rho4p. Both F-BAR (F-BAR FCH, and BAR) and RhoGAP domains of Rgd1p are involved in lipid interactions. In the Rgd1p-F-BAR domain, a phosphoinositide-binding site has been recently characterized. We report here the X-ray structure of the Rgd1p-RhoGAP domain, identify by NMR spectroscopy and confirm by docking simulations, a new but cryptic phosphoinositide-binding site, comprising contiguous A1, A1' and B helices. The addition of helix A1', unusual among RhoGAP domains, seems to be crucial for lipid interactions. Such a site was totally unexpected inside a RhoGAP domain, as it was not predicted from either the protein sequence or its three-dimensional structure. Phosphoinositide-binding sites in RhoGAP domains have been reported to correspond to polybasic regions, which are located at the unstructured flexible termini of proteins. Solid-state NMR spectroscopy experiments confirm the membrane interaction of the Rgd1p-RhoGAP domain upon the addition of PtdIns(4,5)P2 and indicate a slight membrane destabilization in the presence of the two partners.

Self-Assembled Protein-Aromatic Foldamer Complexes with 2:3 and 2:2:1 Stoichiometries.

The promotion of protein dimerization using the aggregation properties of a protein ligand was explored and shown to produce complexes with unusual stoichiometries. Helical foldamer 2 was synthesized and bound to human carbonic anhydrase (HCA) using a nanomolar active site ligand. Crystal structures show that the hydrophobicity of 2 and interactions of its side chains lead to the formation of an HCA2-23 complex in which three helices of 2 are stacked, two of them being linked to an HCA molecule. The middle foldamer in the stack can be replaced by alternate sequences 3 or 5. Solution studies by CD and NMR confirm left-handedness of the helical foldamers as well as HCA dimerization.

Multivalent Interactions between an Aromatic Helical Foldamer and a DNA G-Quadruplex in the Solid State.

Quinoline-based oligoamide foldamers have been identified as a potent class of ligands for G-quadruplex DNA. Their helical structure is thought to target G-quadruplex loops or grooves and not G-tetrads. We report a co-crystal structure of the antiparallel hairpin dimeric DNA G-quadruplex (G4 T4 G4 )2 with tetramer 1-a helically folded oligo-quinolinecarboxamide bearing cationic side chains-that is consistent with this hypothesis. Multivalent foldamer-DNA interactions that modify the packing of (G4 T4 G4 )2 in the solid state are observed.

Structure of a complex formed by a protein and a helical aromatic oligoamide foldamer at 2.1 Å resolution.

In the search of molecules that could recognize sizeable areas of protein surfaces, a series of ten helical aromatic oligoamide foldamers was synthesized on solid phase. The foldamers comprise three to five monomers carrying various proteinogenic side chains, and exist as racemic mixtures of interconverting right-handed and left-handed helices. Functionalization of the foldamers by a nanomolar ligand of human carbonic anhydrase II (HCA) ensured that they would be held in close proximity to the protein surface. Foldamer-protein interactions were screened by circular dichroism (CD). One foldamer displayed intense CD bands indicating that a preferred helix handedness is induced upon interacting with the protein surface. The crystal structure of the complex between this foldamer and HCA could be resolved at 2.1 Å resolution and revealed a number of unanticipated protein-foldamer, foldamer-foldamer, and protein-protein interactions.

Mutant ferritin L-chains that cause neurodegeneration act in a dominant-negative manner to reduce ferritin iron incorporation.

Nucleotide insertions that modify the C terminus of ferritin light chain (FTL) cause neurodegenerative movement disorders named neuroferritinopathies, which are inherited with dominant transmission. The disorders are characterized by abnormal brain iron accumulation. Here we describe the biochemical and crystallographic characterization of pathogenic FTL mutant p.Phe167SerfsX26 showing that it is a functional ferritin with an altered conformation of the C terminus. Moreover we analyze functional and stability properties of ferritin heteropolymers made of 20-23 H-chains and 1-4 L-chains with representative pathogenic mutations or the last 10-28 residues truncated. All the heteropolymers containing the pathogenic or truncated mutants had a strongly reduced capacity to incorporate iron, both when expressed in Escherichia coli, and in vitro when iron was supplied as Fe(III) in the presence of ascorbate. The mutations also reduced the physical stability of the heteropolymers. The data indicate that even a few mutated L-chains are sufficient to alter the permeability of 1-2 of the 6 hydrophobic channels and modify ferritin capacity to incorporate iron. The dominant-negative action of the mutations explains the dominant transmission of the disorder. The data support the hypothesis that hereditary ferritinopathies are due to alterations of ferritin functionality and provide new input on the mechanism of the function of isoferritins.

Peripheral markers of oxidative stress in Labrador retrievers with copper-associated hepatitis.

OBJECTIVES: To evaluate biomarkers of oxidative stress in dogs with copper-associated hepatitis (CAH) as compared with healthy controls, and to evaluate if these markers correlate with hepatic copper concentrations and hepatic histopathologic features. MATERIALS AND METHODS: Prospective study. Plasma reactive metabolite concentrations, plasma antioxidant potential, and plasma and urine isoprostane concentrations were determined in Labrador retrievers with copper-associated hepatitis (n=9) as well as in breed- and sex-matched (n=9) and age- and sex-matched (n=9) healthy control populations. Possible correlations between markers of oxidative stress and hepatic histopathological features also were investigated. RESULTS: Reactive metabolites (median, range) were over twofold greater in dogs with copper-associated hepatitis (87.2 RFU/µL, 60.9 to 185.6 RFU/µL) as compared to breed- and sex-matched (38.2 RFU/µL, 22.4 to 116.8 RFU/µL) and age- and sex-matched controls (32.0 RFU/µL, 18.5 to 127.4 RFU/µL). Antioxidant potential was decreased in copper-associated hepatitis dogs (6.5 TE/µL, 5.1 to 7.7 TE/µL) as compared to breed- and sex-matched controls (8.2 TE/µL, 5.3 to 11.8 TE/µL). Both reactive metabolite concentrations and the reactive metabolite to antioxidant potential ratio were positively correlated with hepatic copper concentrations. Plasma and urine isoprostanes were variable and not significantly different between populations. CLINICAL SIGNIFICANCE: Labrador retrievers with copper-associated hepatitis have altered oxidant status. Plasma reactive metabolite concentrations and the reactive metabolite to antioxidant potential ratio could be useful biomarkers. However, neither plasma nor urine isoprostanes were useful biomarkers for copper-associated hepatitis.

Structure and epimerase activity of anthocyanidin reductase from Vitis vinifera.

Together with leucoanthocyanidin reductase, anthocyanidin reductase (ANR) is one of the two enzymes of the flavonoid-biosynthesis pathway that produces the flavan-3-ol monomers required for the formation of proanthocyanidins or condensed tannins. It has been shown to catalyse the double reduction of anthocyanidins to form 2R,3R-flavan-3-ols, which can be further transformed to the 2S,3R isomers by non-enzymatic epimerization. ANR from grape (Vitis vinifera) was expressed in Escherichia coli and purified. Unexpectedly, RP-HPLC, LC-MS and NMR experiments clearly established that the enzyme produces a 50:50 mixture of 2,3-cis and 2,3-trans flavan-3-ols which have been identified by chiral chromatography to be 2S,3S- and 2S,3R-flavan-3-ols, i.e. the naturally rare (+)-epicatechin and (-)-catechin, when cyanidin is used as the substrate of the reaction. The first three-dimensional structure of ANR is described at a resolution of 2.2 A and explains the inactivity of the enzyme in the presence of high salt concentrations.

Production of 3,4-cis- and 3,4-trans-Leucocyanidin and Their Distinct MS/MS Fragmentation Patterns.

(+)-2,3-trans-3,4-cis-Leucocyanidin was produced by acidic epimerization of (+)-2,3-trans-3,4-trans-leucocyanidin synthesized by reduction of (+)-dihydroquercetin with NaBH4, and structures of the two stereoisomers purified by C18- and phenyl-reverse-phase high-performance liquid chromatography (HPLC) were confirmed by NMR spectroscopy. We confirm that only 3,4-cis-leucocyanidin is used by leucoanthocyanidin reductase as substrate. The two stereoisomers are quite stable in aqueous solution at -20 °C. Characterization of the two stereoisomers was also performed using electrospray ionization tandem mass spectrometry (ESI-MS/MS), and we discuss here for the first time the corresponding MS/MS fragmentation pathways, which are clearly distinct. The main difference is that of the mode of dehydration of the 3,4-diol in positive ionization mode, which involves a loss of hydroxyl group at either C3 or C4 for the 3,4-cis isomer but only at C3 for the 3,4-trans isomer. Tandem mass spectrometry therefore proves useful as a complementary methodology to NMR to identify each of the two stereoisomers.

Pharmacokinetics and Relative Bioavailability of Orally Administered Innovator-Formulated Itraconazole Capsules and Solution in Healthy Dogs.

BACKGROUND: Itraconazole is commonly used for treatment of systemic and cutaneous mycoses in veterinary medicine. Two formulations, capsule and solution, are used interchangeably in dogs. However, marked differences in bioavailability have been reported in other species. Similar investigations have not been performed in dogs. OBJECTIVE: To determine and compare pharmacokinetics of itraconazole in dogs after oral administration of commercially available capsule and solution formulations intended for use in humans. ANIMALS: Eight healthy, adult, purpose-bred dogs. METHODS: Dogs received approximately 10 mg/kg of innovator-formulated itraconazole solution and capsule PO in randomized, crossover design with a 10-day washout period. To ensure maximal absorption, solution was administered to fasted dogs, whereas capsules were co-administered with food. Blood samples were collected at predetermined time points, and plasma drug concentrations were measured using high-pressure liquid chromatography. Pharmacokinetic parameters were determined with compartmental analysis. RESULTS: The mean relative bioavailability of the capsule was 85% that of the solution, but drug absorption was variable, and overall drug concentrations were similar between formulations. Mean elimination half-lives of both formulations were nearly identical at approximately 33 hours. Regardless of formulation, simulations suggest that a loading dose of 20 mg/kg, followed by 10 mg/kg once every 24 hours, will result in plasma concentrations considered to be adequate in most dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Contrary to findings reported in other species, overall drug exposures after capsule and solution administration are not substantially different in dogs. Despite some pharmacokinetic differences between itraconazole capsule and solution, formulation-specific dosages do not appear to be necessary.

Evaluation of Basal Serum or Plasma Cortisol Concentrations for the Diagnosis of Hypoadrenocorticism in Dogs.

BACKGROUND: Previous studies that included limited numbers of affected dogs have suggested basal cortisol concentrations ≤55 nmol/L (2 µg/dL) are sensitive, but nonspecific, for a diagnosis of hypoadrenocorticism. A detailed assessment of the diagnostic utility of basal cortisol concentrations is warranted. HYPOTHESIS/OBJECTIVES: To evaluate the utility of basal cortisol concentrations for the diagnosis of hypoadrenocorticism in a large number of dogs, including those with and without serum electrolyte abnormalities. ANIMALS: Five hundred and twenty-two dogs, including 163 dogs with hypoadrenocorticism, 351 dogs with nonadrenal gland illness, and 8 dogs with equivocal results. METHODS: Retrospective study. Basal and post-ACTH cortisol concentrations and sodium and potassium concentrations were collected from medical records. A receiver operating characteristic (ROC) curve was constructed for basal cortisol concentrations by standard methodologies. Sensitivity, specificity, and predictive values were determined for various cut-points. RESULTS: The area under the ROC curve was 0.988 and was similarly excellent regardless of serum electrolyte concentrations. At the most discriminatory cut-point of 22 nmol/L (0.8 µg/dL), sensitivity and specificity were 96.9 and 95.7%, respectively. A basal cortisol concentration of ≤55 nmol/L (2 µg/dL) resulted in a sensitivity of 99.4%. Conversely, a basal cortisol concentration of ≤5.5 nmol/L (0.19 µg/dL) resulted in a specificity of 99.1%. CONCLUSIONS AND CLINICAL IMPORTANCE: Similar to findings in previous studies, basal cortisol concentrations >55 nmol/L (2 µg/dL) are useful in excluding a diagnosis of hypoadrenocorticism. Interestingly, excellent specificities and positive predictive values were observed at lower cut-point cortisol concentrations.

Crystal structure and biochemical properties of the human mitochondrial ferritin and its mutant Ser144Ala.

Mitochondrial ferritin is a recently identified protein precursor encoded by an intronless gene. It is specifically taken up by the mitochondria and processed to a mature protein that assembles into functional ferritin shells. The full mature recombinant protein and its S144A mutant were produced to study structural and functional properties. They yielded high quality crystals from Mg(II) solutions which diffracted up to 1.38 Angstrom resolution. The 3D structures of the two proteins resulted very similar to that of human H-ferritin, to which they have high level of sequence identity (approximately 80%). Metal-binding sites were identified in the native crystals and in those soaked in Mn(II) and Zn(II) solutions. The ferroxidase center binds binuclear iron at the sites A and B, and the structures showed that the A site was always fully occupied by Mg(II), Mn(II) or Zn(II), while the occupancy of the B site was variable. In addition, distinct Mg(II) and Zn(II)-binding sites were found in the 3-fold axes to block the hydrophilic channels. Other metal-binding sites, never observed before in H-ferritin, were found on the cavity surface near the ferroxidase center and near the 4-fold axes. Mitochondrial ferritin showed biochemical properties remarkably similar to those of human H-ferritin, except for the difficulty in renaturing to yield ferritin shells and for a reduced ( approximately 41%) rate in ferroxidase activity. This was partially rescued by the substitution of the bulkier Ser144 with Ala, which occurs in H-ferritin. The residue is exposed on a channel that connects the ferroxidase center with the cavity. The finding that the mutation increased both catalytic activity and the occupancy of the B site demonstrated that the channel is functionally important. In conclusion, the present data define the structure of human mitochondrial ferritin and provide new data on the iron pathways within the H-type ferritin shell.

Production, in Pichia pastoris, of a recombinant monomeric mapacalcine, a protein with anti-ischemic properties.

Mapacalcine is a small homodimeric protein of 19 kDa with 9 disulfide bridges extracted from the Cliona vastifica sponge (Red Sea). It selectively blocks a calcium current insensitive to most calcium blockers. Specific receptors for mapacalcine have been described in a variety of tissues such as brain, smooth muscle, liver, and kidney. Previous works achieved on hepatocytes and nervous cells demonstrated that this protein selectively blocks a calcium influx triggered by an ischemia/reperfusion (I/R) shock and efficiently protects cells from death after I/R. The aim of this work was to produce the recombinant mapacalcine in the yeast Pichia pastoris. Mass spectrometry, light scattering analysis and biological characterization demonstrated that the recombinant mapacalcine obtained was a monomeric form with 4 disulfide bridges which retains the biological activity of the natural protein.

Regulation of guanine nucleotide binding regulatory proteins in cultured adrenal cells by adrenocorticotropin and angiotensin-II.

In addition to their steroidogenic effect on cultured bovine adrenal fasciculata cells ACTH and angiotensin-II (A-II) have a long term effect on the ability of these cells to respond to subsequent hormonal stimulation. The present work explores the effects of a 72-h pretreatment of adrenal cells with both hormones on the first steps of the mechanism of action of ACTH and A-II and on the amounts of the alpha-subunits of guanine nucleotide binding proteins Gs and Gi. ACTH but not A-II increased acute ACTH or cholera toxin-induced cAMP production. Moreover, ACTH but not A-II enhanced the amount of alpha S protein evaluated by cholera toxin ADP ribosylation, whereas both hormones elevated immunoblotted alpha S. Both hormones increased A-II induced phosphoinositide breakdown and Ca2+ uptake without modification of the A-II potentiating effect on ACTH-induced cAMP production. Treatment of cells with pertussis toxin (PT, 0.5 micrograms/ml) for the last 24 h reduced by 27% the A-II induced phosphoinositide breakdown in A-II pretreated cells but had no significant effect in ACTH-pretreated cells. No effect of PT was observed on A-II induced Ca2+ uptake or on its potentiating action on ACTH-induced cAMP production in ACTH as well as A-II-pretreated cells. Moreover, both hormones increased Gi proteins (40-41 kDa) evaluated by PT ADP ribosylation. Immunoblot analysis revealed that ACTH preferentially enhanced alpha i3, whereas the stimulatory effect of A-II was more marked on alpha i1 and alpha i2. These results indicate that in bovine adrenal fasciculata cells, peptide hormones settle target cell responsiveness not only by regulating the membrane-bound receptors, but also by modulating the level of G proteins coupling these receptors to the intracellular signals.

Stimulatory effect of insulin and insulin-like growth factor I on Gi proteins and angiotensin-II-induced phosphoinositide breakdown in cultured bovine adrenal cells.

Recent data have shown that pretreatment of bovine adrenal fasciculata cells with insulin-like growth factor I (IGF-I) or insulin enhances the steroidogenic response to angiotensin II (A-II). In the present work we have studied the effects of both peptides on the first steps of the mechanism of action of A-II and on the amounts of pertussis toxin (PT)-sensitive guanine nucleotide binding proteins (Gi proteins). Both peptides increased A-II-induced phosphoinositide breakdown without modification of either A-II-induced Ca2+ uptake or the A-II-potentiating effect on ACTH-induced cAMP production. The effects of IGF-I at a nanomolar concentration were higher than those induced by insulin at a micromolar concentration, which in turn was higher than those induced by a nanomolar concentration of this peptide. Treatment of cells with pertussis toxin (0.5 microgram/ml) for 24 h reduced by 25% of the A-II-induced phosphoinositide breakdown in control cells and 32% and 28% in cells pretreated with insulin at nanomolar and micromolar concentrations, respectively, but had no significant effect in cells pretreated with IGF-I. No effect of pertussis toxin was observed on A-II-induced Ca2+ uptake or on its potentiating action on ACTH-induced cAMP production. Moreover, both IGF-I and insulin enhanced the amounts of Gi protein(s) evaluated by pertussis toxin ADP-ribosylation or immunoblotting. Again, the effects of insulin at nanomolar concentrations were lower than those induced by the same concentrations of IGF-I or insulin at micromolar concentrations. These results suggest that, in bovine adrenal fasciculata cells, A-II receptors are coupled to the phosphoinositide pathway through pertussis toxin sensitive and insensitive Gp protein(s). Moreover, the findings also indicate that the enhanced A-II responsiveness of IGF-I or insulin treated cells is in part mediated through an increase in the amount of G protein(s).

Regulation by adrenocorticotropin (ACTH), angiotensin II, transforming growth factor-beta, and insulin-like growth factor I of bovine adrenal cell steroidogenic capacity and expression of ACTH receptor, steroidogenic acute regulatory protein, cytochrome P450c17, and 3beta-hydroxysteroid dehydrogenase.

The purpose of this study was to evaluate the time-course effect of a 36-h treatment with ACTH (10(-8) M), transforming growth factor-beta1 (TGFbeta1; 10(-10) M), angiotensin II (AngII; 10 (-7) M), and insulin-like growth factor I (IGF-I; 10(-8) M) on the steroidogenic capacity of bovine adrenocortical cells (BAC) and on messenger RNA (mRNA) levels of ACTH receptor, cytochrome P450c17, 3beta-hydroxysteroid dehydrogenase (3betaHSD), steroidogenic acute regulatory protein (StAR), and StAR protein. ACTH and IGF-I enhanced, in a time-dependent manner, the acute 2-h ACTH-induced cortisol production, whereas TGFbeta 1 and AngII markedly reduced it. ACTH, IGF-I, and AngII increased ACTH receptor mRNA, but the opposite was observed after TGFbeta1 treatment. ACTH and IGF-I increased P450c17 and 3betaHSD mRNAs, whereas AngII and TGFbeta1 had the opposite effects. However, the effects of the four peptides on ACTH-induced cortisol production appeared before any significant alterations of the mRNA levels occurred. The most marked and rapid effect of the four peptides was on StAR mRNA. The stimulatory effect of ACTH was seen within 1.5 h, peaked at 4-6 h, and declined thereafter, but at the end of the 36-h pretreatment, the levels of StAR mRNA and protein were higher than those in control cells. IGF-I also enhanced StAR mRNA levels within 1.5 h, and these levels remained fairly constant. The effects of AngII on StAR mRNA expression were biphasic, with a peak within 1.5-3 h, followed by a rapid decline to almost undetectable levels of both mRNA and protein. TGFbeta1 had no significant effect during the first 3 h, but thereafter StAR mRNA declined, and at the end of the experiment the StAR mRNA and protein were almost undetectable. Similar results were observed when cells were treated with ACTH plus TGFbeta1. A 2-h acute ACTH stimulation at the end of the 36-h pretreatment caused a higher increase in StAR mRNA and protein in ACTH- or IGF-I-pretreated cells than in control cells, which, in turn, had higher levels than cells pretreated with TGFbeta1, ACTH plus TGFbeta1, or AngII. These results and the fact that the stimulatory (IGF-I) or inhibitory (AngII and TGFbeta1) effects on ACTH-induced cortisol production were more pronounced than those on the ability of cells to transform pregnenolone into cortisol strongly suggest that regulation of StAR expression is one of the main factors, but not the only one, involved in the positive (IGF-I) or negative (TGFbeta1 and AngII) regulation of BAC for ACTH steroidogenic responsiveness. A high correlation between steady state mRNA level and acute ACTH-induced cortisol production favors this conclusion.

Regulation by growth factors of angiotensin II type-1 receptor and the alpha subunit of Gq and G11 in bovine adrenal cells.

In the present work we have investigated the effects of several growth factors on the expression of Angiotensin II (A-II) receptors subtype AT1 and their pertussis toxin-insensitive coupling to G-proteins in bovine adrenal fasciculata-reticularis cells (BAC). Insulin, Insulin-like growth factor and basic Fibroblast growth factor increased AT1 receptors (mRNA and binding sites) as well as the alpha subunit of Gq (mRNA and protein) and G11 (protein). These changes were associated with an enhanced A-II-induced inositol phosphate accumulation and cortisol production. In contrast, Transforming growth factor beta 1, which reduced slightly AT1 binding sites, but not the level of alpha q or alpha 11 proteins, did not change the A-II-induced inositol phosphate accumulation. However, this factor, as previously reported, markedly reduced cortisol production.

Angiotensin II potentiates agonist-induced 3',5'-cyclic adenosine monophosphate production by cultured bovine adrenal cells through protein kinase C and calmodulin pathways.

Interactions between signal transducing systems may be important in the integrated control of cellular processes in basal and hormonally regulated cells. The cultured bovine adrenal fasciculata cell provides a model to study the interactions between the cAMP and calcium-sensitive phospholipid dependent protein kinase C. In this study, angiotensin II (A-II) and phorbol ester (PMA) potentiated the stimulatory actions of ACTH in a dose-dependent manner on cAMP production. At maximal concentrations, A-II and PMA also potentiated the effects of cholera toxin and forskolin on cAMP production. Both staurosporine, a protein kinase C inhibitor, and desensitization of protein kinase C by a 24-h pretreatment with PMA blunted the effect of PMA, but only partially inhibited (34%) the effect of A-II. Neither nifedipine, a specific calcium channel antagonist, nor pretreatment of cells with pertussis toxin modified the amplifying effects of A-II or PMA. In contrast, trifluoperazine, a calmodulin inhibitor, reduced the potentiating effect of A-II by about 35%, but association with staurosporine blunted its effects. Moreover, the steroidogenic effects of ACTH plus A-II were more than additive, but this synergism was blunted in the presence of both inhibitors. In conclusion, PMA and A-II potentiated agonist-induced cAMP production by bovine adrenal fasciculata cells. The data suggest that the effects of PMA were mediated exclusively by protein kinase C, whereas those of A-II were mediated by both protein kinase C and calmodulin.