Biomedical subjects

J G Lehoux

Publications and source records attributed to J G Lehoux.

At least 19 recordsLinked to original sources

PubMed1994

Both low sodium and high potassium intake increase the level of adrenal angiotensin-II receptor type 1, but not that of adrenocorticotropin receptor.

Angiotensin-II (AII), a component of the renin-angiotensin system, is the major factor that regulates the formation of aldosterone in the adrenal cortex zona glomerulosa (ZG). The activity of this system is increased by an increase in potassium intake or a decrease in sodium intake. Using immunoblotting analysis, we determined whether these ions affect the expression of type 1 AII receptors (AT1) and compared the results thus obtained with the AT1 receptor mRNA levels. We also studied the interrelation among AII, AT1 receptors, cytochrome P450 aldosterone synthase (P450c18), and plasma aldosterone levels in rats fed a normal diet or a low sodium or high potassium diet with or without captopril, an inhibitor of angiotensin-converting enzyme, for 7 days. The effects of ions on the level of ACTH receptor mRNA were also analyzed. We found that a low sodium intake increased plasma aldosterone levels from 5.5 to 236 ng/dl and led to 2.3- and 3.7-fold increases in the levels of adrenal ZG AT1 receptor protein and AT1 receptor mRNA, whereas a 11.8-fold increase was found in the level of P450c18 mRNA. Captopril almost completely reversed these effects. We have shown that a high potassium intake increased plasma aldosterone levels to 25.9 ng/dl and also led to 1.84- and 1.95-fold increases in the level of ZG AT1 receptor protein and AT1 receptor mRNA, whereas the ZG P450c18 mRNA level was increased 3.5-fold. The plasma aldosterone level of animals fed a high diet of potassium and captopril was still higher than that in control animals at 16.6 ng/dl, and the levels of ZG AT1 receptor and P450c18 mRNAs were only slightly less than those of the high potassium groups, indicating that captopril did not efficiently block aldosterone formation under these conditions. ACTH receptor mRNA levels remain unaffected by either low sodium or high potassium intake. Collectively, these results indicate that the increased aldosterone secretion induced by low sodium or high potassium intake involves concomitant increases in AT1 receptor and P450c18 mRNAs, which are effectively translated into their respective proteins, and that the expression of both proteins is mediated in part by AII.

Aldosterone↗

PubMed1994

Labelling of D2-dopaminergic and 5-HT2-serotonergic binding sites in human trophoblastic cells using [3H]-spiperone.

We previously reported that dopamine (DA) inhibited the release of human placental lactogen (hPL) from human placental cells. We also demonstrated the presence of D2-dopamine receptors in membrane preparations of human term placenta. The aim of the present study was to characterize these D2 receptors on freshly isolated human trophoblastic cells. The binding of [3H]-spiperone to these cells showed a curvilinear Scatchard plot suggesting the presence of two classes of binding sites (Kd1 = 1.26nM; Kd2 = 44.3nM). Competition experiments showed the following inhibitory binding potencies: serotonin-2 (5-HT2) > or = D2 >>> alpha-adrenergic, beta-adrenergic, D1-dopamine, thus suggesting the presence of 5-HT2 binding sites. We have examined this possibility by blocking [3H]-spiperone binding to 5-HT2 receptors in the presence of 50nM ketanserin, a selective antagonist of 5-HT2 sites. Under this condition, the linear Scatchard plot obtained suggested a single population of homogeneous binding sites for [3H]-spiperone with a Kd of 0.55nM. To further characterize placental D2 receptors we conducted binding experiments with [3H]-raclopride, an more selective D2 antagonist. The linear Scatchard plot obtained with this ligand suggested one class of binding sites for [3H]-raclopride (Kd = 6nM) with the following inhibitory potencies: D2 >>> beta-adrenergic >> 5-HT2, D1, alpha-adrenergic. These results suggest an important paracrine function for DA in human placenta and show for the first time that [3H]-spiperone binds putative 5-HT2 receptors in human placenta.

Humans↗

PubMed1993

The modulation of placental lactogen release by opioids: a role for extracellular calcium.

We previously reported that kappa opioids stimulated the release of human placental lactogen (hPL) from trophoblastic cells and that this effect was prevented by co-incubation with naloxone. We also reported that adenylate cyclase was not directly involved in this process. In order to understand the post-receptor events mediating hPL release by opioids in the human placenta, we studied the role of extracellular calcium. Human trophoblastic cells obtained by trypsin digestion were cultured for 48 h in Ham's F-10 medium supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 micrograms/ml streptomycin. 45Ca2+ influx was then measured by filtration on glass-fiber filters. We observed a time- and dose-dependent stimulation of 45Ca2+ influx by ethylketocyclazocine (EKC) with an EC50 of 0.5 nM and a maximal stimulation of 196% over control. This effect was completely blocked by naloxone, a non-specific opioid antagonist, and by nor-binaltorphimine, a specific kappa antagonist. We also demonstrated that U-50,488 (kappa agonist) had the same stimulatory effect as EKC (221 +/- 25% of control). D-Ala2,NMe-Phe4,Gly-ol5)-enkephalin (DAGO) (mu agonist) slightly stimulated Ca2+ influx (128 +/- 5% of control, p > 0.05) whereas D-Ser2,Leu,Thr6)-enkephalin (DSLET) (delta agonist) had no effect. Pre-incubation of trophoblastic cells with pertussis toxin (PTX) did not affect the EKC-induced 45Ca2+ influx, suggesting that this placental opiate effect is not coupled with PTX-sensitive G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1993

A role for extracellular calcium in the regulation of placental lactogen release by angiotensin-II and dopamine in human term trophoblastic cells.

We previously reported that angiotensin-II (AII) stimulated and dopamine (DA) inhibited the release of human placental lactogen (hPL) from trophoblastic cells. The mechanisms of action involved in these endocrine regulations are poorly known. In this study, we investigated the role of Ca2+ as a potential cellular mediator of the effects of AII and DA. Incubation of freshly isolated human term trophoblastic cells with DA led to a dose-dependent inhibition of 45Ca2+ influx, with a maximum of 55 +/- 5% and an EC50 of 10 +/- 3 mumol/L. This DA-inhibited Ca2+ influx was reversed by spiperone, a D2-dopamine receptor antagonist. Preincubation of cells with pertussis toxin completely blocked the inhibitory effect of DA on placental 45Ca2+ influx. Nifedipine (10(-5) mol/L), like DA, inhibited 45Ca2+ influx (41 +/- 3% inhibition). Moreover, nifedipine decreased hPL release (57 +/- 10%; EC50, 0.25 +/- 0.09 mumol/L). Coincubation of DA and nifedipine did not enhance the inhibitory effects of these agents on either 45Ca2+ influx or hPL release. The incubation of trophoblastic cells with [Sar1]AII, a potent agonist of AII, led to a dose-dependent stimulation of 45Ca2+ influx. The maximal stimulation was 221 +/- 37% of the control value, with an EC50 of 50 +/- 15 nmol/L. This stimulation was inhibited by coincubation with the AII antagonist [Sar1,Ala8]AII. [Sar1]AII-stimulated Ca2+ influx was blocked by preincubation with pertussis toxin. Bay K 8644 also stimulated 45Ca2+ influx (238 +/- 41% of the control). Moreover, Bay K 8644 stimulated hPL release. The maximal stimulation was 180 +/- 22% of the control value, with an EC50 of 0.40 +/- 0.30 mumol/L. Coincubation of Bay K 8644 and AII did not led to additional stimulation of either 45Ca2+ influx or hPL release. These results suggest that Ca2+ influx is one mechanism that mediates AII and DA regulation of hPL release in human term trophoblastic cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1992

Direct expression of mature bovine adrenodoxin in Escherichia coli.

Site-directed mutagenesis was utilized to enable direct expression of the mature form of bovine adrenodoxin cDNA using the pKK223-3 expression vector in Escherichia coli. Expression was under control of the "tac" promoter and resulted in a direct expression of soluble mature bovine adrenodoxin (greater than 15 mg per liter). Chromatographic behavior of recombinant adrenodoxin did not differ from that reported for mature native adrenodoxin. The purified recombinant protein was identical to native mitochondrial adrenodoxin on the basis of molecular weight, NH2 terminal sequencing and immunoreactivity. E. coli lysates were brown in color, and the purified protein possessed a visible absorbance spectra identical to native bovine adrenodoxin consistent with incorporation of a [2Fe-2S] cluster in vivo. Recombinant bovine adrenodoxin was active in cholesterol side-chain cleavage when reconstituted with adrenodoxin reductase and cytochrome P450scc and exhibited kinetics reported for native bovine adrenodoxin. The presence of the adrenodoxin amino terminal presequence does not appear to be essential for correct folding of mature recombinant adrenodoxin in E. coli. This expression system should prove useful for overexpression of adrenodoxin mutants in future structure/function studies. The approach described herein can potentially be used to directly express the mature form of any protein in bacteria.

Adrenal Glands↗

PubMed1992

3,5,3'-Triiodothyronine binding sites in synaptosomes from brain of chick embryo. Properties and ontogeny.

In this study we have demonstrated the presence of specific 3,5,3'-L-triiodothyronine (T3) binding sites in the synaptosomes of chick embryo cerebral cortex and described their ontogeny. Scatchard analyses of binding data obtained with synaptosomal preparations from 17-day-old embryos revealed two T3 binding sites. The first site (N1) had a high affinity and low capacity since its dissociation constant (Kd) was 68 +/- 1.3 nM T3 (mean +/- S.D.; n = 3-5) and its maximal binding capacity (Bmax) was 8.63 +/- 1.59 ng T3/mg of protein, whereas the second site (N2) had a higher Kd of 5.04 +/- 0.5 microM T3 and a larger Bmax of 405 +/- 49 ng T3/mg of protein. The relative affinity of the synaptosomal fraction for T3 and other analogs was the following: T3 greater than T4 (thyroxine) greater than D-T3 (3,5,3'-D-triiodothyronine) = TRIAC (triiodothyroacetic acid) greater than rT3 (reverse T3). Gel chromatography of the [125I]T3 labeled fraction revealed a partially saturable peak with an estimated MW of more than 100 kDa. The ontogenic pattern showed a progressive increase of Kd and Bmax of N1, occurring mainly between the 12 and 19 days of incubation, and a marked fall, particularly of the Bmax, after hatching. The second site did not show any important variation during the embryogenesis. These data indicate the existence of specific T3 binding sites in synaptosomes from cerebral cortex of chick embryo, whose properties and ontogeny are completely different from those of the nuclear receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Characterization of 3,5,3'-triiodothyronine receptors in primary cultures of hepatocytes and neurons from chick embryo.

We have detected the presence of nuclear 3,5,3'-triiodothyronine (T3) receptors in primary cultures of chick embryo hepatocytes and neurons. Hepatocytes were isolated from livers of embryos of 12, 16 and 19 days by treatment with 0.2% collagenase and hyaluronidase. They were plated at a density of 3-4 x 10(5)/35-mm petri dish in Ham's F-10 medium containing fetal calf serum, tryptose phosphate, and antibiotics. Cells were used for the binding assay at Day 3 of culture. Neurons from 8-day-old embryo brains were cultured in a serum-free medium at a density of 1.2 x 10(6) cells/35-mm petri dish and used for the binding assay after 7 days of culture. Biological activity of hepatocytes was determined by measuring insulin binding, inositol phosphate formation, and 5'-monodeiodinase activity. Neurons or glial cells in culture were identified by immunostaining with anti-neurofilaments and anti-glial fibrillary acidic protein antisera. Binding assay was performed with isolated nuclei and 0.4 M NaCl nuclear extracts. With the latter preparation, the Scatchard analysis showed, in both cells, a single, high-affinity, low-capacity T3 receptor. In the hepatocytes of 12-, 16-, and 19-day-old embryos association constants (Ka) were, respectively, 0.93 +/- 0.02, 0.74 +/- 0.03, and 0.56 +/- 0.04 nM-1, whereas the maximal binding capacities (MBC) were 2.26 +/- 0.2, 2.72 +/- 0.33, and 1.83 +/- 0.19 fmol/microgram DNA (mean +/- SE, n = 3). In neurons Ka was 1.25 +/- 0.53 nM-1 and MBC 0.59 +/- 0.14 fmol/microgram DNA (n = 3). The receptor had a sedimentation coefficient of 3.4 S, an estimated Mr of 59 kDa, and the following relative affinity for thyroid hormone analogues: TRIAC greater than L-T3 greater than L-T4. These data indicate that cultured hepatocytes and neurons of chick embryo contained T3 receptors with properties similar to those described in intact tissues from this and other species. Only the MBC of neurons was 50% lower than that observed in whole brain of embryo, but was comparable to values observed in cultured neurons from other species.

Animals↗

PubMed1992

Influence of captopril on adrenal cytochrome P-450s and adrenodoxin expression in high potassium or low sodium intake.

To investigate the role of the renin-angiotensin system in steroidogenic enzyme expression, the angiotensin-I converting enzyme inhibitor captopril was administered in conjunction with high potassium (K+) or low (Na+) intake to rats for a 7-day period. Northern blot analysis of adrenocortical zona glomerulosa RNA revealed that sodium restriction markedly increased mRNA production of P-450scc (3.1-fold) and P-450(11 beta) (3.4-fold) as well as of the electron donor adrenodoxin (2.0-fold). Captopril combined to the low Na+ diet led to suppression of these effects and, as also seen with captopril alone, further diminished P-450(11 beta) mRNA levels below controls. These responses were accompanied by parallel changes in respective protein levels of the enzymes as indicated by Western blot analyses. Captopril was also shown to inhibit the K(+)-stimulated levels of P-450(11 beta) mRNA (3.3-fold) and protein (1.4-fold) beneath control values (0.6- and 0.8-fold, respectively). On the other hand, increased P-450scc mRNA and protein levels by K+ loading were not affected by captopril treatment. No response was observed in any steroidogenic enzyme expression in zona fasciculata-reticularis following either diet with or without captopril. Thus, the inhibitory effect of captopril on stimulated steroidogenesis seemed to be mediated in part through transcriptional regulation of P450s. In addition, it appeared that P-450(11 beta) expression might be under the control of the renin-angiotensin system in both high K+ and low Na+ diets as opposed to the K+ stimulation of P-450scc where other mechanisms seemed to be involved.

Adrenal Cortex↗

PubMed1992

The differential regulation of aldosterone output in hamster adrenal by angiotensinII and adrenocorticotropin.

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 +/- 1.9 ng and 8.0 +/- 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K+ concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K+. AngiotensinII (AII) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10(-8) M AII, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10(-8) M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of AII and ACTH on adrenal cytochrome P-450(11 beta) involved in the last steps of aldosterone formation were evaluated by combined in vivo and in vitro experiments. The P-450(11 beta) mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and AII differentially regulate P-450(11 beta). It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.

Adrenal Cortex↗

PubMed1992

Dietary potassium supplementation and sodium restriction stimulate aldosterone synthase but not 11 beta-hydroxylase P-450 messenger ribonucleic acid accumulation in rat adrenals and require angiotensin II production.

Increasing evidence indicates that the adrenal cortex of most mammalian species expresses distinct forms of cytochrome P-450(11 beta), a steroidogenic enzyme that catalyses the terminal steps in the biosynthesis of both glucocorticoids and mineralocorticoids. In the human, mouse, and rat, two genes have been isolated, designated CYP11B1 and CYP11B2. The product of CYP11B2 (aldosterone synthase) is required for the successive 11 beta-, 18-hydroxylations and 18-oxidation of deoxycorticosterone that lead to the production of aldosterone in the zona glomerulosa. In contrast, the product of CYP11B1 (11 beta-hydroxylase) mediates only the 11 beta-hydroxylation of deoxycorticosterone and 11-deoxycortisol. The recent identification of these two P-450(11 beta) isozymes mandates further analysis of their expression in different zones of the adrenal cortex, both under basal conditions and in response to conditions known to alter mineralocorticoid biosynthesis. To evaluate the expression of the two isozymes in different adrenocortical zones, we performed Northern blotting analyses with specific oligonucleotide probes that discriminated between the two forms of rat P-450(11 beta). The transcripts detected by the two probes were of similar size (2.7 kilobase), but differed in their zonal distribution: aldosterone synthase P-450 messenger RNA (mRNA) was detected only in zona glomerulosa, whereas 11 beta-hydroxylase P-450 was expressed in both zona fasciculata-reticularis and zona glomerulosa. Next, we analyzed the response of these two genes to various physiological and pharmacological interventions known to affect aldosterone biosynthesis. High potassium or low sodium diet given to rats for 1 week increased aldosterone synthase P-450 mRNA levels by approximately 5- and 6-fold, respectively. These increases, moreover, were significantly attenuated by treatment with captopril, an inhibitor of angiotensin-converting enzyme. In contrast, neither dietary manipulation significantly affected 11 beta-hydroxylase P-450 mRNA levels in any zone. Thus, stimulation of the terminal steps of aldosterone biosynthesis by variations in dietary intake of monovalent cations involves regulation of aldosterone synthase P-450 mRNA levels. Finally, captopril inhibited potassium induction of aldosterone synthase P-450 mRNA levels despite the presence of low plasma renin activity in the potassium-treated rats. This finding implicates intraadrenal angiotensin II formation in the effect of potassium on mineralocorticoid production.

Adrenal Glands↗

PubMed1991

Regulation of rat adrenal messenger RNA and protein levels for cytochrome P-450s and adrenodoxin by dietary sodium depletion or potassium intake.

The effect of low sodium and high potassium intake on rat adrenal zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR) were studied during a 7-day period, by analyzing mRNA and protein levels of various enzymes involved in aldosterone synthesis. In ZG significant increases in cytochrome P-450scc, P-450c21, P-450(11 beta), adrenodoxin mRNA and protein levels were observed after 2 days with either diet, and at day 7 these levels were further increased. The largest mRNA induction was observed at day 7 in sodium-depleted rats for P-450(11 beta), with a 4-fold increase, followed by 2.7- and 2.0-fold increases for P-450scc and P-450c21, respectively. A pattern similar to those of P-450scc and P-450(11 beta) was observed for adrenodoxin with a 2.1-fold increase after 7 days of Na+ restriction. In K(+)-loaded rats mRNA levels for P-450scc, P-450(11 beta), P-450c21, and adrenodoxin were also increased by 2.2-, 2.1-, 1.5-, and 1.9-fold respectively. Protein levels of these enzymes were also measured in ZG and showed increases similar to those of their respective mRNAs for both treatments. On the other hand, mRNA levels of P-450scc, P-450(11 beta), P-450c21, and adrenodoxin in ZFR were found significantly lower than in ZG, although they were slightly increased for both treated groups of rats as compared with controls. In addition, ZFR protein levels of corresponding enzymes did not fluctuate significantly under both ionic regimens. In conclusion, both low sodium and high potassium intakes act primarily on ZG. Their action on plasma aldosterone seems to be mediated by increasing both mRNA and protein and levels of steroidogenic enzymes, especially at the early step (cytochrome P-450scc) and even more at the late steps (cytochrome P-450(11 beta]. In addition, a close relationship appears to exist between the two mitochondrial P-450s and their electron donor adrenodoxin, since their mRNA and protein levels were similarly enhanced for both diets used.

Adrenal Cortex↗

PubMed1991

Mitogenic effects of insulin and insulin-like growth factors on PA-III rat prostate adenocarcinoma cells: characterization of the receptors involved.

Four transplantable cell lines (PA-I, II, III, and IV) derived from four Lobund-Wistar (L-W) rats that manifested spontaneous prostate cancer have demonstrated metastatic capacity in visceral organs. Interestingly, PA-III cells, when deposited over the scapula or calvarium of the Lobund-Wistar rat, could produce lytic and blastic reactions on rat skeleton. Since growth factors and growth factor receptors have been implicated in bone remodeling, cancer biology, and metastatic growth of cancer cells, we have examined 1) the effects of insulin and insulin-like growth factors (IGF-I and IGF-II) on the proliferation of PA-III cells; and 2) the presence of specific receptors for these peptides. IGF-I (0.5 to 100 ng/ml), IGF-II (0.5 to 100 ng/ml), and insulin (0.5 to 10 micrograms/ml) stimulated tritiated thymidine uptake and increased the number of PA-III cells in culture. Receptor studies demonstrated the presence of specific bindings sites for IGF-I and II but not for insulin. The number and affinity of the receptor sites were: IGF-I (nb = 675 fmol/100 g protein, Kd = 0.56 nmol) and IGF-II (nb = 225 fmol/100 g protein, Kd = 0.71 nmol). Molecular characterization of IGF binding sites by polyacrylamide gel electrophoresis under denaturing conditions indicated only the presence for the type I IGF receptor. The presence of the IGF-I receptor and the absence of IGF-II and insulin receptors are discussed in relation to the capacity of PA-III cells to produce bone lesions on the L-W rat.

Adenocarcinoma↗

PubMed1991

Adenosine 3':5'-cyclic monophosphate (cAMP) is not the mediator of kappa opiate effect on human placental lactogen release.

We previously reported that kappa opiates stimulated the release of human placental lactogen (hPL) from human placental cells. In this study, we investigated the role of adenylate cyclase as a potential cellular mediator of such an effect. Incubations with ethylketocyclazocine (EKC) led to a time- and dose-dependent inhibition of adenylate cyclase activity. The maximal inhibition was 45 +/- 5% of control value after 15 min exposure to 10(-7)M EKC. This inhibition was reversed by opiate antagonist naloxone and was specific to kappa opiate type. Preincubation of human trophoblastic cells with 0.1 microgram/ml Islet-Activating-Protein (IAP; also called pertussis toxin) did not modify basal adenylate cyclase activity but abolished the inhibition of adenylate cyclase activity by EKC, indicating that the effect of opiates on cAMP production was mediated by an IAP-sensitive GTP binding protein. Also, IAP stimulated basal hPL release; the control levels were 22.4 ng/ml and 46.5 ng/ml without and with IAP respectively. However, the EKC-stimulated hPL levels were unchanged by preincubation with IAP. This difference in cAMP and hPL response in IAP-treated cells suggested that the opiate receptors are not directly coupled to adenylate cyclase. This hypothesis was confirmed by 1) experiments on placental membranes showing that in absence of the cytoplasmic elements (membranes only), EKC had no effect on membrane adenylate cyclase and 2) experiments on placental cells showing that dibutyryl-cAMP (dbcAMP) stimulated hPL release.

Adenylate Cyclase Toxin↗

PubMed1991

The protein kinase C content is increased in the nuclear fraction of rat adrenal zona glomerulosa following long-term ACTH administration.

It is well known that the adrenal zona glomerulosa is transformed to zona fasciculata-reticularis in rats exposed chronically to ACTH. This model was used to study the intracellular distribution of protein kinase C, which is known to be involved in differentiation processes. Under basal conditions, in zona glomerulosa, 70, 23, and 7% of the protein kinase C was located in the cytosol, membrane and nuclear fractions, respectively. At 30 min after ACTH administration to rats, the protein kinase C content remained unchanged in the nuclear fraction, whereas that of the cytosolic fraction was decreased to 43% while in the membranes it was increased to 48%. After 2 days of ACTH treatment, we observed a significant increase, up to 25%, of protein kinase C in the nuclear fraction, a decrease to 47% in the cytosol, whereas the membrane fraction content had returned to its basal value. The intracellular distribution of inner zones was 17% in nuclear fraction, 47% in cytosol and 36% in membranes. ACTH treatments did not change these proportions. The total protein kinase C content of ACTH-treated groups was not different than that of their respective controls, in zona glomerulosa and in inner zones respectively. The cytosolic protein kinase C formed complexes with detergent-treated nuclei; this association was saturable, and could be measured by the ability of the kinase to bind [3H]PDBu ([20(n)-3H]phorbol-12,13-dibutyrate). The number of nuclear 'acceptor sites' thus measured was calculated to be 5245 fmol/mg DNA in the zona glomerulosa; this did not change significantly following a 3-day administration of ACTH. Protein kinase C prepared from the adrenal inner zones also bound zona glomerulosa detergent-treated nuclei but occupied fewer sites than the protein kinase C from the zona glomerulosa. In conclusion, the effects of chronic ACTH treatment on rat adrenal zona glomerulosa could be mediated by an increased level of protein kinase C in the nuclear fraction and possibly through its binding to specific 'acceptor sites'.

Adrenocorticotropic Hormone↗

PubMed1991

The effects of age on the postreceptor regulation of luteinizing hormone secretion by gonadotropin-releasing hormone.

We studied the effects of age on the roles of phosphoinositide (PI) and protein kinase C (PKC) in luteinizing hormone (LH) release by gonadotropin-releasing hormone from mouse pituitaries. Pituitary cells from intact and 14-day ovariectomized (OVX) mice aged 4-8 months, 10-12 months and 14-18 months were cultured at a dilution of 3 x 10(5) cells/ml of M199-bovine serum albumin medium for 3 days prior to stimulation with either buserelin or phorbol ester (phorbol myristate acetate, PMA), while LH was assayed by radioimmunoassay using anti-rat LH antibody (NIDDK-5-10). In intact young mice, buserelin and PMA specifically induced time- and dose-dependent increases in LH release with specific mean ED50 of 0.82 x 10(-11) M (buserelin) and of 1.6 x 10(-8) M (PMA) and a maximal LH release of 138 +/- 15 ng/10(6) cells after a 3 h stimulation period. Age did not affect the ED50 of either agonist but significantly reduced their ability to release LH. This reduction was more pronounced for buserelin than for PMA and was evident as early as middle-age. OVX resulted in a significant increase in both basal and stimulated LH release, but did not affect the age-related reduced secretion rate of LH by either agonist. Buserelin stimulated the incorporation of [3H]inositol into [3H]inositol phosphates (IP) in a dose-dependent manner, which was unaffected by either age or OVX. We conclude that, with aging, there occurs a reduced LH release rate to both buserelin and PKC stimulations, uncoupled to changes in PI-IP cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1991

Short-term effects of ACTH on the low-density lipoprotein receptor mRNA level in rat and hamster adrenals.

Low-density lipoprotein (LDL) receptor mRNA was found in both rat and hamster adrenals. Within 30 min after ACTH administration a significant increase in the levels of both LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) mRNAs was observed in rat adrenals; these levels remained increased for up to 240 min. The increase in the levels of LDL receptor and HMG-CoA reductase mRNAs produced by ACTH was reduced by co-administration of aminoglutethimide while, at the same time, the adrenal cholesterol content of rats treated with both aminoglutethimide and ACTH was significantly increased compared with that in groups treated with ACTH alone. Cycloheximide also induced increased levels of rat adrenal mRNAs for LDL receptor and HMG-CoA reductase, but this effect was not additive with that of ACTH. These results suggest that, in the rat, the short-term effect of ACTH on the levels of mRNAs for the LDL receptor and HMG-CoA reductase is similarly controlled and might be mediated through changes in the adrenal cholesterol content. In the hamster adrenal, however, no significant fluctuations were found in the level of LDL receptor mRNA, although a marked increase was found in the level of HMG-CoA reductase mRNA, 2 h after ACTH administration. This indicates that an important effect of ACTH on cholesterol metabolism in the hamster adrenal is at the level of HMG-CoA reductase. In the hamster, therefore, where the main source of cholesterol for the adrenal gland is de-novo synthesis, it seems that a complex mechanism is involved in the control of LDL receptor gene expression.

Adrenal Glands↗

PubMed1990

Phosphorylation and nuclear processing of the androgen receptor.

Although transformed androgen receptor (AR) complexes derived from cytosol and nuclear AR complexes have been shown to bind with high affinity to nuclei and DNA, we have shown that the binding characteristics of the two receptor populations to rat ventral prostate nuclei are different. To account for these differences, we investigated the possibility that the two receptor populations differed in phosphorylation status. Significantly, an anti-phosphotyrosine antibody immunoprecipitated androgen binding from the nuclear AR preparation but not from the transformed cytosolic receptor preparation. These studies suggest that (i) further processing of the AR complex takes place after it has become transformed, and (ii) phosphorylation of the complex is one modification which occurs during the processing of the nuclear receptor.

Androgens↗

PubMed1990

Hypothalamic-pituitary axis during reproductive aging in mice.

We correlated the content of hypothalamic (HT) GnRH and pituitary (PT) GnRH receptor sites with PT and plasma gonadotropin levels throughout aging in C57BL/6J mice. Female mice of 4-6 months (young), 10-12 months (middle-age) or 15-18 months (old) of age were studied either intact or 15 days post-ovariectomy (OVX) with or without E2 therapy. In intact mice, HT GnRH content increased twofold during aging while GnRH receptor sites in PT remained unchanged. PT content of both FSH and LH gradually rose during aging while plasma concentration rose even more. OVX resulted in a significant decrease in both HT GnRH content and PT receptor sites and no age difference was observed. OVX also resulted in a significant increase in both PT content and plasma levels of gonadotropin in young and middle-age mice while old mice showed a blunted response. After E2 therapy for 7 days, HT GnRH content and PT GnRH receptor sites returned to normal levels in all age groups. By contrast, E2 therapy resulted in no change in PT content of FSH:LH in any age group. Whereas plasma FSH:LH levels returned to intact levels in young mice, they remained elevated to OVX levels in middle-age and old ones. Our results demonstrate an age related dichotomy in the PT production of FSH:LH unrelated to changes in either HT GnRH content or its PT receptor sites, thus suggesting cellular defects in post-receptor binding events within the pituitary.

Aging↗