Biomedical subjects

D Bellabarba

Publications and source records attributed to D Bellabarba.

At least 19 recordsLinked to original sources

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↗

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 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↗

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↗

PubMed1990

Effect of ACTH suppression on adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in 4-aminopyrazolopyrimidine-treated rats.

4-Aminopyrazolopyrimidine (4-APP) treatments to rats for 3 days induced 2-fold increase of circulating ACTH and 11-fold increase of adrenal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA compared to NaCl-treated controls. This in vivo model was used to study the effect of the suppression of ACTH secretion on the adrenal HMG-CoA reductase mRNA level. Dexamethasone (Dex) administration to 4-APP-treated rats caused a rapid and parallel decline of the levels of plasma ACTH and adrenal HMG-CoA reductase mRNA to 50% within 2.5 h, whereas the free and esterified cholesterol content was increased 5 and 9.4 times respectively. These changes could be counteracted by the co-administration of ACTH with Dex. Aminoglutethimide (AG) administration to 4-APP-treated rats, which increased the adrenal esterified cholesterol content (7.5 times), decreased the HMG-CoA reductase mRNA level (44%), despite plasma ACTH level remaining elevated. Moreover, the participation of newly synthesized protein(s) in the lowering of adrenal HMG-CoA reductase mRNA level induced by ACTH suppression is suggested by the fact that cycloheximide (Cyclo), when co-administered with AG, completely blocked the decrease of HMG-CoA reductase mRNA level, despite the plasma ACTH level decreasing by 68% and the free and esterified cholesterol content increasing 3.9 and 12.3 times, compared to 4-APP-treated rats. Furthermore, the specificity of these effects was established by the fact that the beta-actin mRNA level was not affected by the administration of either Dex, AG, Cyclo, or AG + Cyclo to 4-APP-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗

PubMed1990

An islet-activating protein-sensitive G-protein is involved in dopamine inhibition of both angiotensin-stimulated inositol phosphate production and human placental lactogen release in human trophoblastic cells.

In isolated human trophoblastic cells, dopamine (DA) significantly inhibited the angiotensin-II (AII)-stimulated inositol phosphate (IP) accumulation by 44 +/- 8% (EC50, 0.5 +/- 0.2 microM) and human placental lactogen (hPL) release by 85 +/- 5% (EC50, 1.0 +/- 0.8 microM). These effects were blocked by sulpiride, a specific D2 antagonist. On the contrary, scherring 23390 (a specific D1 antagonist) and propranolol (a specific beta-adrenergic antagonist) were ineffective, suggesting that these DA effects are mediated through a DA receptor of the D2 subtype. The mechanism by which DA inhibited AII-stimulated inositol phosphate production implicates a GTP-binding protein sensitive to the islet-activating-protein (IAP), since DA's effects on IP accumulation and hPL release were blocked by this toxin. To further characterize this GTP-binding protein, particulate fractions of placental cells were incubated with [alpha-32P]NAD and IAP. Solubilized extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two proteins of 40 and 41 kDa mol wt were specifically ADP ribosylated. They were probably involved in the DA inhibitory processes, since IAP treatment, known to suppress the effects of DA, also reduced the labeling of these two molecules by around 40%. The effects of AII and DA on hPL release appear to be insensitive to the external calcium concentration, since the results were not significantly different in normal (1.8 mM Ca2+) and low Ca2+ (10(-5) M Ca2+) concentrations. On the other hand, increasing the intracellular concentration of cAMP by adding forskolin did not modify the effect of DA on either IP accumulation or hPL release, suggesting that cAMP is not implicated in hPL release from freshly isolated human trophoblastic cells.

Adenosine Diphosphate↗

PubMed1990

Presence of D2-dopamine receptors in human term placenta.

We studied the binding of [3H]-spiperone on human term placental membranes. This binding reached plateau level after 30 min incubation at 37 degrees C and was reversed (t1/2 approximately 5 min) by addition of an excess of unlabeled spiperone. Scatchard analysis of saturation experiments with increasing doses of [3H]-spiperone (0-25 nM) showed one class of high affinity binding sites with a dissociation constant (Kd) of 14 +/- 2 nM and a maximal binding capacity (Bmax) of 222 +/- 9 fmoles/mg protein. The affinity of 5 competitors was determined in competitive binding assays. The D2-dopamine antagonists were the most potent inhibitors: Ki for spiperone and haloperidol were 8 +/- 2 and 56 +/- 22 nM respectively. Dopamine inhibited [3H]-spiperone binding with a Ki of 570 +/- 50 microM whereas Schering 23390 (D1 antagonist) and propranolol (beta-adrenergic antagonist) were without effect. The binding was also inhibited by 100 microM GTP gamma S (38 +/- 8% inhibition), indicating that the dopamine receptor is coupled with a GTP binding protein. These results demonstrate for the first time the presence of D2-dopamine receptors in human placenta.

Dopamine↗

PubMed1989

Estradiol receptor-nuclear interactions in aging mouse uteri: the role of DNA and nuclear matrix.

We have studied the interactions of estradiol receptor (ER) with nuclear DNA or matrix (NM) in the uteri from young, middle-aged and old mice. Recombination studies using heat-activated ER and nuclear subfractions from various age groups suggested a 30% reduction for DNA binding (P less than 0.05) but not for NM with aging. Cross-incubation studies of heat-activated ER with nuclear subfractions from mice of all three age subgroups showed that this reduced DNA binding ability followed the onset of anestrous and did not result from proteolytic degradation of ER complexes.

Aging↗

PubMed1989

Hormonal regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in the rat adrenal gland.

We studied the effect of ACTH on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase enzyme. Reductase activity and reductase mass were enhanced by 22- and 6.2-fold respectively in one series of experiments, whereas in another the levels of reductase activity, reductase mass, and reductase mRNA were increased 6.6-, 3.6- and 2.2-fold respectively, following daily administration of exogenous ACTH for 3 days. Daily injection of 4-aminopyrazolopyrimidine (4-APP) to rats for 3 days increased circulating ACTH level 5.4-fold, whereas adrenal HMG-CoA reductase activity, reductase mass and reductase mRNA levels were greatly increased 36-, 10- and 16-fold, respectively. To counteract the effect of elevated plasma ACTH, dexamethasone acetate (Dex) was administered to 4-APP treated rats. At 3 h post Dex administration, plasma ACTH and corticosteroids levels were effectively decreased by 58 and 59%, respectively. The levels of adrenal HMG-CoA reductase mRNA, reductase activity and reductase mass were also diminished by 38, 31 and 40%, respectively. Our results show that rat adrenal HMG-CoA reductase can respond rapidly to hormonal changes, presumably through variations in circulating ACTH levels.

Adenine↗

PubMed1989

The enhancing effect of adrenocorticotropin on adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase messenger ribonucleic acid level is inhibited by aminoglutethimide but not by cycloheximide.

3-Hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase activity and reductase mRNA level were determined in adrenals from hamsters treated with ACTH, with or without cycloheximide or aminoglutethimide. Both reductase activity and reductase mRNA level were similarly enhanced by ACTH administration compared to levels in NaCl-treated animals. The administration of cycloheximide with ACTH resulted in a 73% decrease in reductase activity compared to control values, but did not prevent the enhancing effect of ACTH on the reductase mRNA level. Furthermore, the administration of cycloheximide alone diminished HMG-CoA reductase activity, but enhanced by 1.1- to 1.6-fold the reductase mRNA level. Coadministration of aminoglutethimide with ACTH also resulted in a decrease (65%) in reductase activity compared to that in NaCl-treated animals. However, coadministration of aminoglutethimide, in contrast to cycloheximide, with ACTH not only prevented the reductase mRNA level increase produced by ACTH, but also resulted in a 30% decrease in the reductase mRNA level compared to that in controls injected with 0.15 M NaCl. In addition, aminoglutethimide alone resulted in 50% and 54% decreases in reductase mRNA level and reductase activity, respectively. Thus, we have shown that both cycloheximide and aminoglutethimide can prevent the enhancing effect of ACTH on HMG-CoA reductase activity, but their modes of action differ. It is likely that the aminoglutethimide inhibition could be the result of a diminution of specific reductase gene transcription, whereas cycloheximide would result in inhibition of the synthesis of specific proteins, including HMG-CoA reductase. In this respect, since the adrenal free cholesterol content was increased in groups treated with ACTH-aminoglutethimide, we postulate that free cholesterol could be one of the important components involved in the regulation of HMG-CoA reductase gene transcription. As for the ACTH-cycloheximide-treated groups, the adrenal free cholesterol content was also increased, but the effect of ACTH on the reductase mRNA level was not prevented, presumably because this drug blocked the synthesis of a putative sterol regulatory protein that is required to repress HMG-CoA reductase gene transcription.

Adrenal Glands↗

PubMed1989

Ca2+, but not membrane lipid hydrolysis, mediates human chorionic gonadotropin production by luteinizing hormone-releasing hormone in human term placenta.

We postulated a role for lipid metabolism and Ca2+ in the LHRH-induced release of hCG by human placentas. Term placental cells in suspension prelabeled with [3H]myoinositol were stimulated without or with increasing concentrations of LHRH in the presence of 10 mM LiCl, and total inositol phosphate (IP) was measured by ion exchange chromatography; a nonsignificant 0.9 +/- 0.08-fold increase over the control value was observed. In contrast, placental cells stimulated with equimolar concentrations of angiotensin II (AII) induced a 4.6 +/- 0.9-fold increase in total IP (P less than 0.01), while rat pituitary cells showed 1.9 +/- 0.2- and a 2.4 +/- 0.07-fold increases in total IP production after stimulation with LHRH or AII, respectively (P less than 0.05). These increases were blocked by coincubation with specific LHRH and AII antagonists. When 1 x 10(6) placental cells were incubated with 45Ca2+ without and with increasing doses of LHRH for 0-75 s and then filtered under negative pressure, we observed significant incorporation of 45Ca2+. This influx was linear with incubation time, significantly more pronounced in cells exposed to LHRH than in control cells, and showed a dose-response curve to LHRH that reached maximal influx rates of 3.6 +/- 0.3 nM/min.1 x 10(6) cells with 10(-5) M LHRH. This response was completely blocked by coincubation with 10(-5) M LHRH antagonists; cobalt chloride and verapamil reduced it by 60% and 80%, respectively. Compared to placental cells stimulated with LHRH alone, those coincubated with LHRH and specific LHRH or Ca2+ antagonists released from 10-100% less hCG. We conclude that Ca2+ participates in the LHRH action in human placentas, but uncoupled to PI turnover.

Angiotensin II↗

PubMed1989

Angiotensin II stimulates both inositol phosphate production and human placental lactogen release from human trophoblastic cells.

We studied the functional significance of the binding of angiotensin-II (AII) to human placentas. Human trophoblastic cell suspensions were prepared by trypsin digestion of minced tissue. Cell incubations with increasing doses of [125I](SAR1)AII, ranging from 0.01-2.5 nmol/L, were carried out for 20 min at 37 C. The results indicated the presence of specific low capacity [4300 +/- 1300 (+/- SE) sites/cell], high affinity (Kd = 0.38 +/- 0.06 nmol/L) binding sites for [125I](Sar1)AII. This binding was specific for AII analogs. When placental cells were preloaded with 40 microCi/mL [3H]myoinositol for 2 h at 37 C, AII stimulation resulted in a dose-dependent increase in inositol phosphate (InsP) production [EC50 = 1.4 +/- 0.4 (+/- SE) nmol/L], as measured by ion exchange chromatography. (Sar1)AII also stimulated InsP production, with an EC50 of 0.3 +/- 0.2 nmol/L. AII-stimulated production of InsP was completely blocked by the antagonist (Sar1,Ala8)AII. AII also stimulated human placental lactogen release from trophoblastic cells in a dose-dependent fashion. The EC50 was 18 +/- 9 pmol/L, and the stimulation was blocked by (Sar1,Ala8)AII, as found for AII-stimulated InsP production. These results suggest that stimulation of human placental lactogen release by AII may be mediated by activation of phospholipase-C, which, in turn, produces phosphoinositide breakdown. The results, therefore, provide evidence of a physiological role for the renin-angiotensin system within the human placenta.

Angiotensin II↗

PubMed1989

Basal and stimulated LHRH receptor sites in the pituitary of aging female mice.

We studied the regulation of pituitary luteinizing hormone-releasing hormone (LHRH) receptors in female mice aged 4-8 months (young), 10-14 months (middle-aged) and 15-18 months (old). In intact animals the mean +/- SE pituitary content of LHRH receptor sites ranged from 34.4 +/- 6.3 fmoles to 41.5 +/- 5.0 fmoles and remained constant throughout aging. Similarly, receptor affinity for LHRH was unaltered (range 1.0-4.0 X 10(9) M-1) by age. Ovariectomy (ovx) resulted in a 30-50% decrease in pituitary LHRH receptor sites (p less than 0.05) and the pattern of LHRH receptor fall after ovx was similar in all three age groups. Administration of estrogens (E2) at either a low (0.2 mg) or a high (2.0 mg) concentration to ovx mice prevented this fall in LHRH receptor sites and, in all three age groups, increased pituitary values to levels comparable to those measured in intact animals. We conclude that there are no significant differences in the basal and stimulated number or affinity of pituitary LHRH receptors between young, middle-aged and aged female mice.

Aging↗

PubMed1988

Aldosterone nuclear receptors in kidneys of chick embryo.

We have studied the properties of the nuclear receptors for aldosterone in kidneys of chick embryo. Aliquots of 0.4 M KCl nuclear extracts were incubated with [3H]aldosterone with or without 1 microM RU28362, a potent glucocorticoid analog. Scatchard analyses of binding data revealed two classes of binding sites with Ka of 0.26 and 0.03 X 10(9) M-1 and Nmax of 330 fmol and 620 fmol/mg DNA respectively. In presence of RU28362, however, we observed only a single class of binding sites with a Ka of 1.02 X 10(8) M-1 and a Nmax of 90 fmol/mg DNA. Competition studies performed in presence of RU28362 showed that aldosterone was the more effective competitor followed by corticosterone, progesterone, deoxycorticosterone, dexamethasone, cortisol, triamcinolone acetonide and cortisone. The nuclear complexes had a sedimentation coefficient in the area of 8 S which changed to 4-5 S in the presence of 0.4 M KCl. This effect of KCl was prevented by the addition of 10 mM sodium molybdate. Always in the presence of the glucocorticoid analog, by DEAE-c chromatography we observed a major specific aldosterone-binding fraction which was eluted with 0.2 M KCl. This fraction sedimented at 8.4 S in the absence of sodium molybdate and KCl. In the absence of RU28362, DNA-c columns retained only a small portion of the nuclear complexes which were eluted with KCl. These complexes sedimented, on sucrose gradient, at 4.6 and 3.1 S, whereas those which did not bind to DNA-c had a sedimentation coefficient of 8 S. In the presence of RU28362, the majority of bound [3H]aldosterone remained in the column flow-through fraction; when this fraction was further analyzed on DEAE-c, complexes were eluted with 0.2 and 0.3 M KCl. These data indicate that nuclear receptors for aldosterone are present in small number in kidneys of chick embryo and that they are mostly in the 8 S form.

Aldosterone↗

PubMed1988

Functional opioid receptor sites in human placentas.

We characterized the presence of opioid peptide receptor sites in plasma membranes and cells from human midterm and term placentas. Incubations with [3H]ethylketo-cyclazocine (EKC) at increasing doses revealed the presence of high affinity, low capacity, opioid peptide receptor-specific binding of the kappa-type. Scatchard analysis of the binding data showed, in the plasma membranes, linear plots at both stages of pregnancy with similar mean equilibrium association constants of 1.31 +/- 0.29 (+/- SE) X 10(9) mol/L-1 (n = 4) at midterm and 0.52 +/- 0.63 X 10(9) mol/L-1 at term (n = 4). In placental cells (n = 4) from term gestations, the binding plots were curvilinear; the first component had a Ka of 5.51 +/- 0.50 X 10(9) mol/L-1, and the second component had a Ka of 1.33 +/- 0.81 X 10(8) mol/L-1 (P less than 0.01). When standardized per mg tissue protein, the number of binding sites in plasma membranes increased from 13.8 +/- 9.8 fmol at midterm to 50.0 +/- 18.6 fmol at term (P less than 0.05). For term placental cells, the concentration of binding sites was 81.2 +/- 36.0 fmol for the high affinity sites and 713 +/- 390 fmol for the lower affinity sites. Specificity for the kappa-type of OPR was found based on the inability of mu- or delta-opioid peptides, as well as LHRH and TRH, to compete for [3H]EKC binding. Term placental cells incubated with various doses of opioid peptides had a 50% increase in placental lactogen production. The increase was significantly higher than controls only with kappa-agonists (P less than 0.05), maximal with 10(-9) mol/L EKC, and completely inhibited by 5 X 10(-6) mol/L naloxone. These results expand on previous data demonstrating the presence of opioid peptide receptor in placental plasma membranes and suggest a role for opioid peptides in regulating secretion of placental lactogen by placental cells.

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

PubMed1988

Human placental LHRH receptor: agonist and antagonist labeling produces differences in the size of the non-denatured, solubilized receptor.

Membranes of human term placenta were labeled with several photosensitive and non-photosensitive analogues of LHRH. In both groups agonistic and antagonistic peptide structures were tested. The photolabeling amino acid azidophenylalanine was placed either in positions 2, 6 or 7. All compounds were specifically displacing iodinated Buserelin from human placental membranes and from rat pituitary membranes. The iodinated photolabels were displaced by cold Buserelin, some compounds however had a high non-specific binding. Photolabeling experiments on human placental membranes with radioactive photolabels produced all a band at 58,000 daltons in SDS-gel electrophoresis. Solubilization with a non-denaturing detergent and gel filtration produced a major radioactivity peak which was attributed to the LHRH receptor. All labeling experiments with agonist labels produced Kav's indifferent from each other but significantly different from the Kav's of antagonist labeled membranes. This result was confirmed with similar experiments carried out with radioactive Buserelin and a radioactive antagonist under non-photolytic conditions. It is therefore concluded that the placental LHRH receptor contains an LHRH binding component of 58,000 daltons but that the native receptor is composed of several proteins. It is also concluded that agonist occupation of the receptor induces the association of a further component which might be involved in the transmembrane signalling system. The agonist labeled, solubilized native LHRH receptor has a Stokes radius of 52 A and the same, antagonist labeled receptor a radius of 48 A.

Affinity Labels↗

PubMed1987

Glucocorticoid receptors in chick embryos: properties and ontogeny of the nuclear renal receptor.

A putative nuclear receptor for glucocorticoids was identified in the kidney of chick embryo. This receptor was a thermolabile protein which was readily digested by proteolytic enzymes. Its sedimentation coefficient on sucrose density gradient was 3.5S and its MW approximated, according to the Svedberg formula, at 98,500 Da. Binding assays, performed with crude or purified nuclei, and nuclear extracts showed that the latter preparation was the most suitable for the binding studies since it yielded a Bmax of 8-11% with a very low nonspecific binding (1% or less). Scatchard plots performed at various days of embryogenesis revealed a single class of binding sites with an association constant (Ka) of 0.12 +/- 0.06 X 10(9) M-1 (mean +/- SD; n = 5) and a maximal binding capacity (Nmax) that rose from 3.9 +/- 1.2 fmol/micrograms DNA at Day 13 of age to 13.2 +/- 2.2 fmol/micrograms DNA at Day 16 and then rapidly fell to 1.8 +/- 1.1 fmol/micrograms DNA before hatching (means +/- SD; n = 5). Competition studies with various steroids showed that only glucocorticoids and, to a lesser degree, progesterone had an affinity for the receptor. These results demonstrate that this nuclear-binding protein had physiochemical properties similar to those attributed to other glucocorticoid receptors in target cells.

Animals↗