Non-ACTH POMC fragments stimulate aldosterone production by human adrenal cells in vitro

The possibility that non-ACTH proopiomelanocortin-derived fragments may stimulate aldosterone production has previously been studied using nonhuman cells with inconsistent results. We have examined the response of aldosterone to beta-endorphin (beta-End) and joining peptide (JP) and compared these with the response to ACTH using eight cell suspensions prepared from human adrenal glands. ACTH, 10(-6), 10(-8), and 10(-10) M, consistently stimulated aldosterone accumulation above that occurring in unstimulated cells (150 +/- 83, 120 +/- 62, and 77 +/- 32 fmol/10(4) cells above basal, respectively; mean +/- SE; p < 0.05). beta-End significantly stimulated aldosterone production at 10(-6) and 10(-8) M (114 +/- 84 and 50 +/- 24 fmol/10(4) cells above basal; p < 0.05); 10(-10) M beta-End did not provide significant stimulation. Furthermore, JP stimulated aldosterone biosynthesis (41 +/- 16 fmol/10(4) cells above basal; p < 0.05), only at the highest concentration used, 10(-6) M. The addition of 10(-8) M ACTH plus 10(-6) and 10(-10) M beta-End to human adrenal cells yielded values significantly greater than those achieved with either agent alone (267 +/- 152 and 183 +/- 89 fmol/10(4) cells above basal; p < 0.05). These data indicate for the first time that beta-End and JP have the capacity to stimulate aldosterone production in human adrenal cells in vitro. The physiological and potential clinical significance of these observations has yet to be elucidated.     

Evaluating multiple alternative hypotheses for the origin of Bilateria: an analysis of 18S rRNA molecular evidence

Six alternative hypotheses for the phylogenetic origin of Bilateria are evaluated by using complete 18S rRNA gene sequences for 52 taxa. These data suggest that there is little support for three of these hypotheses. Bilateria is not likely to be the sister group of Radiata or Ctenophora, nor is it likely that Bilateria gave rise to Cnidaria or Ctenophora. Instead, these data reveal a close relationship between bilaterians, placozoans, and cnidarians. From this, several inferences can be drawn. Morphological features that previously have been identified as synapomorphies of Bilateria and Ctenophora, e.g., mesoderm, more likely evolved independently in each clade. The endomesodermal muscles of bilaterians may be homologous to the endodermal muscles of cnidarians, implying that the original bilaterian mesodermal muscles were myoepithelial. Placozoans should have a gastrulation stage during development. Of the three hypotheses that cannot be falsified with the 18S rRNA data, one is most strongly supported. This hypothesis states that Bilateria and Placozoa share a more recent common ancestor than either does to Cnidaria. If true, the simplicity of placozoan body architecture is secondarily derived from a more complex ancestor. This simplification may have occurred in association with a planula-type larva becoming reproductive before metamorphosis. If this simplification took place during the common history that placozoans share with bilaterians, then placozoan genes that contain a homeobox, such as Trox2, should be explored, for they may include the gene or genes most closely related to Hox genes of bilaterians.     

Inhibition of insulin-degrading enzyme increases translocation of insulin to the nucleus in H35 rat hepatoma cells: evidence of a cytosolic pathway

We previously demonstrated the translocation of insulin to the nucleus in several cell types and partially characterized the uptake mechanisms and pathways in H35 rat hepatoma cells. Nuclear accumulation of insulin was energy independent, time and temperature dependent, and apparently was not saturable at insulin concentrations which resulted in full receptor occupancy. We also have shown insulin could be internalized by both receptor-mediated and fluid-phase endocytosis. This study investigated subsequent steps involved in the nuclear accumulation of insulin following internalization. We examined the effects of inhibiting insulin degrading enzyme (IDE) with 1,10-phenanthroline on the nuclear accumulation of insulin in H35 cells. 1,10-phenanthroline (2 mM) which markedly inhibited insulin degradation, significantly increased nuclear accumulation of insulin without having any effects on total cell-associated and intracellular insulin. This reagent increased 125I-insulin on the cellular membrane and decreased 125iodine (125I-insulin and 125I-insulin degradation products) in the cytosolic fractions. Chemical extraction and Sephadex G-50 chromatography revealed the insulin associated with the nucleus in 1,10-phenanthroline-treated cells formed the same complex(es) with the nuclear matrix as in control cells. These results suggested that inhibition of cytosolic IDE activity resulted in increased insulin translocation from the cytosol to the nucleus. Furthermore, when IDE activity was inhibited by high cytosolic insulin concentrations, the amount of 125I-insulin in the nucleus was significantly increased. Our study suggests internalized insulin is probably released from endosomes into the cytosol where modulation of IDE activity could have significant effects on the accumulation of insulin, or insulin-cytoplasmic protein complexes, in nuclei. The IDE regulatory mechanism, by controlling the translocation of insulin to the cell nucleus, could play a crucial role in insulin's regulation of gene expression and cell proliferation.     

Inhibition of nicotinic responses by cotinine in bovine adrenal chromaffin cells

We studied the effects of cotinine, the major metabolite of nicotine, on nicotine-induced increase in [3H]phorbol dibutyrate binding, activation of protein kinase C and [3H]noradrenaline release in primary cultured bovine adrenal chromaffin cells. Cotinine (1 mM, 15 min.) and nicotine (10 microM, 5 min.) increased the [3H]phorbol binding by 100% and 150%, respectively. Both a short-term (10 min.) and a long-term (24 hr) pretreatment with cotinine inhibited the effect of nicotine. A 24 hr pretreatment with cotinine (1 mM) also reduced the nicotine-induced increase in membrane-bound protein kinase C activity. Cotinine pretreatment (10 min.) dose-dependently inhibited the release of [3H]noradrenaline induced by nicotine and dimethylphenylpiperazinium. Cotinine pretreatment did not reduce the [3H]noradrenaline release induced by high extracellular potassium (56 mM) or veratrine (10 mg l-1). The results indicate that cotinine inhibits activation of protein kinase C and noradrenaline release induced by nicotinic agonists in primary cultures of bovine adrenal chromaffin cells. The results suggest that pre-existing cotinine could modify responses to acute exposure to nicotine in neural systems.     

The role of nitric oxide in the regulation of aldosterone synthesis by adrenal glomerulosa cells

The role of nitric oxide (NO) in the regulation of aldosterone synthesis in the adrenal glomerulosa is not known. In this study, we observed that liberators of NO such as S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (Snp) and spermine nonoate (SNO) could significantly inhibit angiotensin II (AII) and ACTH-induced aldosterone synthesis in isolated rat and cultured human adrenal glomerulosa cells. To evaluate more precisely whether glomerulosa cells express NO synthase, we performed immunoblotting experiments with antibodies specific for the endothelial type ecNO synthase as well as the neuronal NO synthase. This revealed the presence of the ecNO synthase in rat adrenal capsules, in normal and in adenomatous human adrenal glomerulosa tissue, as well as in freshly dispersed rat adrenal glomerulosa cells. Furthermore, on immunohistochemical analysis, rat adrenal glomerulosa cell sections showed strongly positive staining for ecNO synthase. These results suggest that NO may be an important negative modulator of adrenal glomerulosa steroidogenesis.     

Hereditary defect in biosynthesis of aldosterone: aldosterone synthase deficiency 1964-1997

We studied two of the three patients with a hereditary defect in the biosynthesis of aldosterone originally described by Visser and Cost in 1964. All three presented as newborns with salt-losing syndrome and failure to thrive. The original biochemical studies showed a defect in the 18-hydroxylation of corticosterone. According to the nomenclature proposed by Ulick, this defect would be termed corticosterone methyl oxidase deficiency type I. We measured plasma steroids in the untreated adult patients and performed molecular genetic studies. Aldosterone and 18-OH-corticosterone were decreased, whereas corticosterone and 11-deoxycorticosterone were elevated, thus confirming the diagnosis of corticosterone methyl oxidase deficiency type I. Cortisol and its precursors were in the normal range. Genetic defects in the gene CYP11B2 encoding aldosterone synthase (P450c11Aldo) have been described in a few cases. We identified a homozygous single base exchange (G to T) in codon 255 (GAG) causing a premature stop codon E255X (TAG). This mutation destroys a Aoc II restriction site. Digestion of a PCR fragment containing exon 4 of CYP11B2 (261 bp) with this restriction enzyme revealed in the two patients homozygous for the E255X mutation only a 261-bp fragment, whereas the heterozygous parents had three fragments (261 bp from the mutant allele and 194 and 67 bp from the wild-type allele). The mutant enzyme had lost the five terminal exons containing the heme binding site, and thus there was a loss of function enzyme. We conclude that the biochemical phenotype of these prismatic cases of congenital hypoaldosteronism can be explained by the patients genotype.     

Role of tissue renin in the regulation of aldosterone biosynthesis in the adrenal cortex of nephrectomized rats

OBJECTIVE: The conventional surgical treatment of isolated critical stenosis of the left main coronary artery (LMCA) leads to the definitive occlusion of LMCA, restores only a retrograde perfusion to a rather extensive myocardial area and consumes bypass material. Direct surgical angioplasty avoids these inconveniences. METHODS: Between June 1985 and August 1996, 49 surgical angioplasties have been performed in 47 patients. LMCA was approached posteriorly in the first 11 procedures, and an anterior approach was preferred in the last 38 because of better exposure. The onlay patch consisted of saphenous vein in 37 cases; pericardium was used in 12 cases, and only for ostial stenosis. RESULTS: No technical failure occurred in the last 28 cases. 44 procedures, (90%), succeeded, but 1 patient (2.3%) died later of a massive air embolism, and 2 patients needed conventional CABG after 3 and 5 months, respectively. The 35 survivors still benefiting from a successful LMCA angioplasty on the long term are free of ischemia after a mean follow-up of 75 months (2-136). Angiographic restudy was obtained in 30 patients (70%) at an average of 38 months and revealed an excellent result in 26 (87%). In 10 patients, a late angiographic restudy at an average of 71 months (32-119) still revealed a perfect result. CONCLUSION: Provided that well-defined contra-indications (involvement of the distal bifurcation, heavy calcification) are respected, LMCA surgical angioplasty deserves a place in the array of surgical strategies.     

Choline deficiency in cultured adrenal medullary cells: effect on phosphatidylcholine biosynthesis

The gastric brushings (GBRs) and endoscopic biopsies obtained concurrently from 27 patients in whom malignant lymphoma was diagnosed or suggested by either modality were assessed to evaluate the utility of cytologic examination in the diagnosis of gastric lymphoma. GBRs provided a definitive diagnosis of lymphoma in 2 (28%) of 7 patients with a prior history of lymphoma and 10 (56%) of 18 patients with primary gastric lymphoma, yielding an overall sensitivity of 48%. All the lymphomas diagnosed cytologically were large cell type. GBRs were suspicious for a malignant neoplasm in four lymphoma patients whose concurrent biopsies were negative. GBRs obtained from two patients who had gastritis were reported as suspicious for lymphoma. There were no false-positive diagnoses.     

Inhibition of sterol biosynthesis in L cells and mouse liver cells by 15-oxygenated sterols

Described herein are the chemical syntheses of 5alpha-cholest-8(14-en-3beta-ol-15-one, 14alpha-methyl-5alpha-cholest-7-en-3beta-ol-15-one, 3beta-methoxy-14alpha-methyl-5alpha-cholest-7-en-15-one, 3beta-methoxy-14alpha-methyl-5alpha-cholest-7-en-15beta-ol, 3beta-methoxy-14alpha-methyl-15alpha-ol, and 5alpha-cholest-8(14)-en-3beta, 7xi,15xi-triol. The effects of these compounds and of 5alpha-cholest-8(14)-en-3beta,15beta-diol, 5alpha-cholest-8(14)-en-3beta,15alpha-diol, 5alpha,14beta-cholest-7-en-3beta,15beta-diol, 5alpha,14beta-cholest-7-en-3beta, 15alpha-diol, 14alpha-methyl-5alpha-cholest-7-en-3beta,15beta-diol, and 14alpha-methyl-5alpha-cholest-7-en-3beta,15alpha-diol on sterol synthesis in L cells and primary cultures of fetal mouse liver cells grown in serum-free media have been studied. With the exception of 3beta-methoxy-5alpha-cholest-7-en-15-one, all of the compounds were found to be potent inhibitors of sterol synthesis. With a few exceptions, the concentrations required to cause a 50% reduction in sterol synthesis were similar to those required to cause a 50% reduction in the level of HMG-CoA reductase activity.     

Inhibition by imipramine of ATP-evoked responses in rat pheochromocytoma cells

Determination of allantoin in bovine milk based on high-performance liquid chromatography (HPLC) is described. Following dilution and filtration, milk samples were analysed directly. Separation and quantification of allantoin was achieved using a Spherisorb 5 NH2 column (250 x 4.6 mm ID), acetonitrile-water (90:10, v/v) mobile phase at a flow-rate of 2.0 ml min(-1), temperature 20 degrees C and monitoring the effluent at 214 nm. Total analysis time was 10 min. Recovery of allantoin added to milk was 97 (+/-3.7, n = 30)%. Lowest detectable concentration was 1 micromol l(-1). Within-day and between-day variability were less than 3%. Advantages of improved retention and separation of allantoin, and less complicated sample preparation exist over current methods.     

A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis

Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-D-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-D-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli.     

Developmental expression of the peripheral-type benzodiazepine receptor and the advent of steroidogenesis in rat adrenal glands

Although the precise mechanism whereby cholesterol is transported across the outer mitochondrial membrane is uncertain, a multimeric receptor complex termed the peripheral-type benzodiazepine receptor (PBR) appears essential for this process. We therefore predicted that adrenal cells at different developmental stages would express PBR coincidentally with the advent of steroidogenesis. Adrenals of neonatal rats demonstrate greatly reduced sensitivity to ACTH that gradually increases after the first 2 weeks of life. Thus, neonates have lower circulating corticosterone levels following exposure to stress. We examined mitochondrial PBR ligand binding activity, immunoreactive (ir) PBR content, and adrenal sensitivity to ACTH in vivo and in vitro. Ontogeny of both mitochondrial PBR ligand binding capacity and irPBR directly paralleled that of ACTH-inducible steroidogenesis in isolated rat adrenal cells and in rats injected with ACTH. In addition, neonatal PBR had approximately 2-fold higher affinity for PK11195, a synthetic ligand that binds with high affinity to PBR. No correlation was observed during neonatal life between ir-steroidogenic acute regulatory (StAR) protein content and steroidogenesis. These results are consistent with the hypothesis that PBR is an absolute prerequisite for adrenocortical steroidogenesis, and suggest that the stress hyporesponsive period of neonatal rats may result from decreased PBR expression. In addition, the higher affinity of neonatal PBR and the relatively high basal expression of StAR protein in neonatal adrenals may partly explain the high constitutive steroidogenesis characteristic of neonatal rat adrenal cells.     

EGF-stimulated aldosterone secretion is mediated by tyrosine phosphorylation but not by phospholipase C in cultured porcine adrenal glomerulosa cells

We examined the effect of EGF and angiotensin II (AII) on the formation of inositol phosphates and aldosterone secretion, and observed the role of tyrosine phosphorylation in EGF or AII-mediated aldosterone secretion. As cultured glomerulosa cells were incubated with increasing concentrations of EGF (0.01-100 ng/mL), aldosterone secretion increased and reached a plateau at EGF concentration of 10-50 ng/mL. Although EGF alone did increase aldosterone secretion in glomerulosa cells, it did not enhance AII-induced aldosterone secretion when both EGF and AII were added. EGF-induced tyrosine phosphorylation peaked at around 1 min after stimulation and at a concentration of 10-50 ng/mL. AII stimulated tyrosine phosphorylation, but the stimulatory effect was less than that observed in the presence of EGF. Although the latter induced tyrosine phosphorylation of various proteins, it failed to stimulate the formation of inositol phosphates. On the other hand, AII stimulated the production of inositol phosphates in a dose-dependent manner, with maximal stimulation at 10(-8)M. The addition of 10 ng/mL EGF did not affect the AII-induced formation of inositol phosphates. In conclusion, EGF-stimulated aldosterone secretion might be mediated by tyrosine kinase. However, since EGF did not stimulate inositol phospholipid hydrolysis in cultured porcine adrenal glomerulosa cells, its effect does not seem to be mediated by phospholipase C.     

Inhibition of antigen-induced muscle contractions by inhibitors of thromboxane pathway in rat small intestine

The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (CDK) in association with cyclins. In mammalian cells the timing of activation of cyclin A-associated kinase activity coincides with the onset of DNA synthesis in S-phase. Using in vitro replication of SV40 origin-containing DNA as a model system, we have analyzed the proteins associated with DNA during initiation of DNA replication in S-phase cell extracts. This analysis reveals that, in addition to replication initiation proteins, cyclin A and cdk2 are also specifically associated with DNA. The association of cyclin A and cdk2 with DNA during initiation is cell cycle regulated and occurs specifically in the presence of SV40 origin-containing plasmid and SV40 T antigen (the viral replication initiator protein). The interactions among proteins involved in initiation play an important role in DNA replication. We therefore investigated the ability of cyclin A and cdk2 to associate with replication initiation proteins. Under replication initiation conditions, cyclin A and cdk2 from S-phase extracts specifically associate with SV40 T antigen. Further, the interaction of cyclin A-cdk2 with SV40 T antigen is mediated via cyclin A, and purified recombinant cyclin A associates directly with SV40 T antigen. Taken together, our results suggest that cyclin A and cdk2 are components of the SV40 replication initiation complex, and that protein-protein interactions between cyclin A-cdk2 and T antigen may facilitate the association of cyclin A-cdk2 with the complex.     

Mutant analysis reveals an alternative pathway for N-linked glycosylation in Drosophila melanogaster

The mas-1 gene of Drosophila melanogaster encodes Golgi mannosidase I (MAS-1), and flies homozygous for small deletions of the gene are viable. They show but few abnormalities and those have a low penetrance [Kerscher, S., Albert, S., Wucherpfennig, D., Heisenberg, M. & Schneuwly, S. (1995) Dev. Biol. 168, 613-626]. Here we sequence the N-linked oligosaccharides associated with a reporter protein, and with membrane proteins prepared from wild type and MAS-1 null organisms. The results show that the null organisms synthesise the same range of oligosaccharides as wild type, albeit with different ratios. There is an accumulation of the Man8GlcNAc2 which is one of the substrates for the MAS-1 enzyme. This supports the suggestion of Kerscher et al. that the lack of severe phenotypic disturbances in the null organisms is due to the presence of an alternative pathway(s), but it also shows that this alternative pathway(s) does not entirely compensate for the normal pathway.     

Inhibitory action of mibefradil on calcium signaling and aldosterone synthesis in bovine adrenal glomerulosa cells

Mibefradil is a new cardiovascular drug with peculiar Ca++ antagonistic properties. The most remarkable feature of mibefradil is its unique relative selectivity for T type calcium channels, a property that has been proposed to explain in part the beneficial pharmacological and clinical profiles of this drug. In adrenal glomerulosa cells, aldosterone biosynthesis and secretion in response to angiotensin II or extracellular potassium is dependent on a sustained influx of Ca++ through T type Ca++ channels. The effect of mibefradil on the steroidogenic function of glomerulosa cells was therefore investigated. Using the patch clamp technique, we found that mibefradil inhibits selectively and in a concentration-dependent manner (IC50 = 3 microM)++ T type currents in bovine glomerulosa cells. In addition to this tonic (voltage independent) inhibition, the drug also induced a shift of the steady-state inactivation curve of these channels toward hyperpolarized voltages, contributing to its efficacy to prevent Ca++ influx into the cell through T type channels. Concomitantly, mibefradil reduced the cytosolic calcium responses to potassium and angiotensin II (as assessed with fluorescent probes), without affecting the capacitative Ca++ influx, and inhibited pregnenolone and aldosterone formation. This inhibition of steroidogenesis was not exclusively due to mibefradil action on voltage-operated Ca++ channels, because this agent also partially reduced steroid synthesis induced by adrenocorticotropic hormone or forskolin, two activators of the cyclic AMP pathway. In conclusion, mibefradil is highly effective in adrenal glomerulosa cells in reducing T type channel activity and aldosterone biosynthesis, two actions that should contribute to the beneficial effect of the drug in the treatment of hypertension.     

Enhancement of potassium-, and angiotensin II-stimulated aldosterone production by the calcium chelator EGTA in bovine adrenal glomerulosa cells in vitro

OBJECTIVE: The aim of this study was to determine the influence of renal impairment on the single-dose pharmacokinetics of temocapril and its pharmacologically active metabolite, temocapril diacid. METHODS: A single oral dose of 20 mg temocapril hydrochloride was given after an overnight fast to eight healthy (control) subjects (group A, n = 8) with a mean baseline creatinine clearance (CLCR) of 115.2 ml.min-1 and to three groups of patients with decreased renal function (mean CLCR 56.9 ml in group B, n = 8, 30.0 ml.min-1 in group C, n = 8 and 15.4 ml.min-1 in group D, n = 5). RESULTS: The mean peak concentration and median time to peak concentration for both temocapril and its diacid metabolite as well as the man area under the curve (AUC0-infinity) for temocapril did not differ significantly between groups. The mean AUC0-infinity for temocapril diacid increased only two- to threefold from group A to D. The mean terminal elimination half-life (t1/2) for temocapril diacid was prolonged in subjects with impaired renal function. However, prolongation of mean t1/2 and increase in AUC0-infinity did not parallel the decrease of mean renal clearance for temocapril diacid. CONCLUSION: The results suggest the existence of an alternative pathway in addition to the renal excretion of temocapril, e.g. via the bile. This pathway substantially contributes to the elimination of the active metabolite, temocapril diacid, in patients with decreased renal function. Nonetheless, to avoid any risks, the dose of temocapril hydrochloride in patients with moderate to severe renal impairment should be reduced.