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Failure of 2 Hydroxyestradiol to interact with dopamine inhibition of human prolactin secretion in vitro and with dopamine receptors of prolactin-secreting adenomas.

The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors.

Adenoma↗

In vitro study of frog (Rana ridibunda Pallas) neurointermediate lobe secretion by use of a simplified perifusion system. IV. Interaction between dopamine and thyrotropin-releasing hormone on alpha-melanocyte-stimulating hormone secretion.

The interaction between dopamine and TRH on alpha-melanocyte-stimulating hormone (MSH) release from the intermediate lobe of amphibian pituitary has been studied in vitro using the perifusion model. Dopamine (10(-10) to 10(-6) M) was responsible for a dose-related inhibition of alpha-MSH secretion. The inhibitory effect of dopamine (10(-8) and 3.16 X 10(-8) M) was completely abolished in the presence of haloperidol (10(-5) and 10(-6) M, respectively). It has been previously established that, in amphibians, TRH stimulated alpha-MSH release in vitro and that the action of TRH was not mediated via an inhibition of the release of endogenous dopamine (M. C. Tonon, P. Leroux, M. E. Stoeckel, S. Jégou, G. Pelletier, and H. Vaudry, 1986, Endocrinology 112, 133-141). In the present study we demonstrate that TRH (10(-7) M) reverses the inhibitory effect of dopamine (for concentrations ranging from 3.16 X 10(-8) to 10(-6) M) on alpha-MSH secretion and that the effects of TRH and dopamine are additive. Thus, these results indicate that the intracellular events associated with TRH-induced stimulation and dopamine-induced inhibition of alpha-MSH release are not linked together.

Animals↗

Role of steroids in secretion--modulating effect of triamcinolone and estradiol on protein synthesis and secretion from the rat exocrine pancreas.

Following adrenalectomy of male rats, or adrenalectomy and ovariectomy of females, there was marked depletion of zymogen granules in acinar cells of the pancreas. Within 9 h after treatment with either triamcinolone or 17 beta-estradiol, complete restoration of these secretory vesicles was observed. This repletion was not inhibited by actinomycin-D. Supernatant fractions (100,000 g, 60 min) of rat pancreas, from both normal and surgically altered animals, contained proteins that bound [3H]-triamcinolone and [3H]-estradiol, suggesting that the action of these hormones is exerted directly on the pancreas. Binding of both steroid hormones required the presence of an additional coligand referred to as accessory factor. In addition, the binding proteins for [3H]-triamcinolone and [3H]-estradiol eluted in similar positions after Sephadex G-200 and CM Affi-gel Blue chromatography. It is uncertain, however, whether a single protein binds both steroid hormones since they had different binding isotherms. Scatchard analysis of binding of [3H]-estradiol yielded a single straight line of negative slope from which it was calculated that there were about 4.4 pmol of binding sites per mg protein, having an average apparent Kd of about 5 X 10(-8) M. Similar analysis of the data for [3H]-triamcinolone yielded a straight line of zero slope indicating nonsaturable binding of hormone at concentrations as high as 10 microM. Since both [14C]-L-leucine incorporation into protein and amylase secretion were affected markedly by the steroid-hormonal status of the animal, it is presumed that steroid-bound complexes in acinar cells of the pancreas modulate synthesis and secretion of protein.

Adrenalectomy↗

Adrenalectomy increases beta-lipotropin secretion over beta-endorphin secretion from anterior pituitary corticotrophs.

Previous studies have indicated that acute stress in vivo or ovine corticotropin releasing hormone (oCRH) in vitro, releases both beta-lipotropin (beta-LPH) and beta-endorphin (beta-END) from the anterior lobe, with beta-END predominating over beta-LPH by 2:1. However, repeated stress shifts this ratio to proportionately more beta-LPH released with re-stress or oCRH in vitro. Alternative hypotheses were that the glucocorticoids released during stress altered the processing of proopiomelanocortin (POMC) or that the increased biosynthetic drive resulted in an inability of the processing enzymes to keep pace with biosynthesis. To distinguish between these alternatives, adrenalectomy studies were performed. Following removal of glucocorticoid negative feedback there is greatly increased secretion of beta-END-IR from anterior lobe corticotrophs with a subsequent increase in biosynthetic drive. Under these conditions of increased biosynthetic drive in the absence of steroids, the corticotroph secretes primarily beta-LPH, suggesting that increased biosynthetic drive alters the posttranslational processing rate of POMC.

Adrenal Glands↗

Calpain-induced down-regulation of protein kinase C inhibits dense-granule secretion in human platelets. Inhibition of platelet aggregation or calpain activity preserves protein kinase C and restores full secretion.

The relationship between platelet aggregation, calpain activation, PKC activities and the secretory response have been examined in PMA-and ionomycin-stimulated platelets. Co-addition of PMA and ionomycin resulted in a maximal synergistic secretion of [14C]5-hydroxytryptamine ([14C]5-HT) from platelet dense granules. However, prior addition of PMA for 5 or 10 min resulted in a reduction of this secretory response. Inclusion of either RGDS (to inhibit platelet aggregation) or E64-d (to inhibit calpain activity) resulted in full restoration of the secretory response. In experiments to determine the activity status of PKC, PMA was found to induce a loss in cytosolic and total PKC activity without an increase in membrane-associated activities during this time period. Inhibition of either platelet aggregation or calpain activity resulted in preservation of total and cytosolic activities with a measurable increase in membrane translocated activity. PMA-induced phosphorylation of a number of PKC substrates was measured in 32P-labelled platelets. PMA induced potent phosphorylation of the 45 and 20 kDa species and also proteins of the molecular masses 66, 80, 97 and 119 kDa. Phosphorylation was maximal at either 1 or 2 min after which dephosphorylation occurred. Inclusion of either RGDS or E64-d resulted in a reduction of the dephosphorylation rates, and sustained phosphorylation of the 66, 80, 97 and 119 kDa proteins. These studies suggest that the activity status of PKC is an important factor in the level of secretion obtained and that platelet aggregation is involved in calpain-initiated down-regulation of PKC.

Blood Platelets↗

Discrimination between constitutive secretion and basal secretion from the regulated secretory pathway in GH3 cells.

The present experiments were undertaken to characterize basal release from vesicles of the regulated secretory pathway. In transfected GH3 cells, progastrin was released by the constitutive route, and mature, bioactive, amidated gastrin by the regulated secretory pathway. Studies using brefeldin A and bafilomycin A1 which inhibit progression through the Golgi complex suggested that both basal and stimulated release of amidated gastrin originated from mature secretory granules. Basal, but not stimulated, secretion of amidated gastrin was strongly inhibited at 22 degrees C. Mature secretory vesicles therefore support both basal and evoked secretion although the mechanisms underlying the two processes differ in their temperature sensitivity.

Amides↗

Increase in insulin secretion with age: its clinical importance in evaluating abnormal secretions focused on diabetes type II and obesity.

The urinary C-peptide excretion was measured in a healthy standardized population sample of 160 subjects from 20 to 90 years of age, homogeneously distributed by age and sex. Urinary C-peptide excretion corresponded to 7% of the total amount released. The daily C-peptide excretion was 61.23 +/- 2.2 (S.E.) microg in the whole sample which corresponds to 41.9 +/- 1.5 IU of insulin secreted/day (I(CP)d), without sex differences. There is an increase of the I(CP)d value from the young to the healthy middle-aged person, but when the results were corrected for standard amounts of excreted creatinine (1 g) and urea (22 g) the age-dependent increase is to be observed during the whole adult life span. Assuming that cross-sectionally observed data are representative of the individual changes, it is concluded that age alone increases insulin secretion. The results which may be useful as reference values for clinical application were as follows: (A) in 5 diabetes type II patients in which the I(CP)d value was measured several times a week, the intraindividual variation coefficient was 10.9 +/- 7.2%;(B) in a sample of 47 type II diabetic patients of both sexes, between 51 and 70 years of age, a clear correlation was found between I(CP)d and the results of the glucagon stimulation test, mainly regarding the relationship between I(CP)d and the planimetrically measured area under the curve (r = 0.7, P < 0.0001); (C) in 7 obese non-diabetic individuals of similar ages the influence of the hypocaloric diet on the I(CP)d value was more evident than the use of C-peptide blood determinations before or after glucagon. Finally, the I(CP)d values of type II diabetes patients with insulin requirement (n = 27) were significantly lower than in the healthy control group (31.1 +/- 24.0 vs. 45.0 +/- 20.4), while diabetic patients without insulin requirement showed significantly higher values (73.0 +/- 33.0) (n = 27). These clinical studies primarily focused on the physiology of human ageing justify the measurement of C-peptide urinary excretion for evaluating daily insulin secretion in patients with type II diabetes.

Journal Article↗

Inhibitory effect of phentolamine on diazepam-induced growth hormone secretion and lack of effect of diazepam on prolactin secretion in man.

Phentolamine, a postsynaptic noradrenergic (NA) receptor blocker, inhibits diazepam-induced growth hormone (GH) secretion in man. In order to study the effect of phentolamine on six healthy subjects with a diazepam (10 mg i.v.)-induced GH increase greater than 7.5 ng/ml, it was necessary to test 11 subjects. The six diazepam GH responders showed a significantly higher (p less than 0.01) GH stimulation after diazepam alone than after diazepam plus phentolamine (60 mg i.v.). The inhibitory effect of phentolamine suggests that NA alpha receptors are involved in the diazepam-induced GH increase. Prolactin secretion after administration of diazepam (10 mg i.v.) was only slightly elevated in comparison with placebo (i.v.), suggesting that diazepam does not have an agonistic effect on dopamine (DA) receptors.

Diazepam↗

Localization of the mouse gene for secreted phosphoprotein 1 (Spp-1) (2ar, osteopontin, bone sialoprotein 1, 44-kDa bone phosphoprotein, tumor-secreted phosphoprotein) to chromosome 5, closely linked to Ric (Rickettsia resistance).

We have used a cDNA probe for mouse secreted phosphoprotein 1 (Spp-1, also known as 2ar, osteopontin, bone sialoprotein 1, 44-kDa bone phosphotein, tumor-secreted protein) to find a restriction fragment length polymorphism in the gene from C57BL/6J and DBA/2J mice. The strain distribution pattern in 25 BXD recombinant inbred lines is identical with that previously determined for the dominant, autosomal gene, Ricr (Rickettsia resistance). This places Spp-1 on mouse chromosome 5 with a 95% confidence limit of being within 4.32 cM of Ric. Evidence supporting the possibility of allelism between Spp-1 and Ric is briefly discussed.

Animals↗

The short chain sugar acid, 2-buten-4-olide, activates oxytocin-secreting neurons but not arginine vasopressin-secreting neurons in the hypothalamus of rats.

The effects of intraperitoneal (i.p.) administration of 2-buten-4-olide (2-B4O), an endogenous sugar acid, on the hypothalamo-neurohypophyseal system were examined in rats. Plasma oxytocin (OXT) levels were significantly increased 15-60 min after i.p. administration of 2-B4O (100 mg/kg), whereas plasma arginine vasopressin (AVP) did not change. Dual immunostaining revealed that Fos-like immunoreactivity (LI) was predominantly observed in OXT-secreting neurons in the paraventricular (PVN) and the supraoptic nuclei (SON) 120 min after i.p. administration of 2-B4O. In addition, many Fos-LI neurons were observed in the nucleus of the tractus solitarius (NTS) after i.p. administration of 2-B4O. These results suggest that a peripherally administered high dose of 2-B4O activates OXT-secreting neurons in the hypothalamus through activation of NTS neurons, possibly as a result of a stress response.

4-Butyrolactone↗

Gonadotropin-releasing hormone antagonists do not influence the secretion of steroid hormones but affect the secretion of vascular endothelial growth factor from human granulosa luteinized cell cultures.

OBJECTIVE: To evaluate the secretion of E(2), P, and vascular endothelial growth factor (VEGF) in human granulosa luteinized cell cultures with the presence of a gonadotropin-releasing hormone (GnRH) agonist or antagonist. DESIGN: In vitro cell culture study. SETTING: Research laboratory of a university hospital. PATIENTS: Granulosa luteinized cells were obtained from 24 patients undergoing ovarian stimulation for IVF treatment. INTERVENTIONS: Granulosa cells were cultured for 48 hours with 1 nM of cetrorelix or leuprorelide. For a further 48 hours, granulosa cells were cultured with or without the combination of cetrorelix plus leuprorelide. MAIN OUTCOMES: At the end of each culturing period, the concentrations of E(2), P, and VEGF were measured in culture supernatants by immunoassays. RESULTS: Estradiol and P concentrations were similar between the culture supernatants from controls and treatment groups. The VEGF concentrations in supernatants from cultures with cetrorelix (2,315.1 +/- 1,565.5 pg/mL) were moderately, but significantly, lower than in controls (2,604.3 +/- 1,907.1 pg/mL) or cultures with leuprorelide (2,558.8 +/- 1,403.1 pg/mL). CONCLUSIONS: The GnRH analogues do not affect steroidogenesis in human granulosa luteinized cell cultures. The GnRH antagonists moderately affect the secretion of VEGF from human granulosa luteinized cells.

Adult↗

ACSD labelling and magnetic cell separation: a rapid method of separating antibody secreting cells from non-secreting cells.

Several new ways of selecting cells have recently been developed. These include magnetic separation of cells by labelling with magnetic beads against the recombinant product, gel microdrop technology which encapsulates the cells in gelatine beads and matrix-based secretion assays. Affinity capture surface display (ACSD) is a matrix-based assay for the enrichment of high producing cells and relies on the strong affinity between biotin and avidin derivatives. Matrix-based assays have previously only been used for the enrichment of recombinant cells. Here, we have optimised this assay and developed a method of separating antibody producing cells from non-producing cells in a recombinant myeloma cell line using ACSD combined with MACS magnetic separation. The method is rapid, simple enough to become routine and adaptable to many different secreted products from recombinant mammalian cells.

Animals↗

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells.

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semi-quantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs.

Animals↗

Inhibition of prohormone convertase 1 (PC1) expression in cholecystokinin (CCK) expressing At-T20 cells decreased cellular content and secretion of CCK and caused a shift in molecular forms of CCK secreted.

Two different RNAi methods were used to inhibit the expression of prohormone convertase 1 (PC1) in At-T20 cells. Transient transfection of double stranded RNA and stable expression of a vector expressing hairpin-loop RNA targeting PC1 reduced cholecystokinin (CCK) secretion from At-T20 cells. PC1 mRNA and protein were also decreased in the vector transfected cells. This treatment caused a shift in the forms of cholecystokinin (CCK) secreted, decreasing CCK 22 and increasing CCK 8. Stable expression of RNAi effectively decreased PC1 expression. The observed decrease in CCK seen with these RNAi treatments further supports a role for PC1 in CCK processing in these cells.

Amino Acid Sequence↗

Type II secretion: a protein secretion system for all seasons.

In Gram-negative bacteria, type II secretion (T2S) is one of five protein secretion systems that permit the export of proteins from within the bacterial cell to the extracellular milieu and/or into target host cells. An analysis of numerous sequenced genomes now reveals that T2S genes are common, but by no means universal, in Gram-negative bacteria. Recent functional studies indicate that T2S can promote the virulence of human, animal and plant pathogens, as well as the physiology of various environmental bacteria. Thus, it is an opportune time to highlight the new and different ways in which T2S serves bacterial function.

Bacterial Proteins↗

Gonadotropin secretion in rats bearing a prolactin-secreting pituitary tumor: effects of naloxone administration.

In this study the effects of the implantation of the transplantable rat prolactin (PRL)-secreting pituitary tumor 7315a on gonadotropin secretion were investigated. Hyperprolactinemia was accompanied by suppressed plasma levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The total PRL content of the host's pituitary gland was decreased, but the glands of tumor-bearing animals contained greatly increased amounts of LH and FSH. Chronic administration of naloxone to tumor-bearing rats for 5 days further diminished the already suppressed total PRL content of the pituitary gland, normalized the total LH content, and did not affect the FSH content. The pituitary glands from tumor-bearing rats given naloxone showed a higher ability to release LH in vitro. Hyperprolactinemia in rats is accompanied by an increased total gonadotropin content of the pituitary gland with lowered circulating gonadotropin levels. Some of the PRL-induced changes on LH synthesis and release are mediated by opioid receptors in the hypothalamus, as naloxone administration reversed some of these effects.

Animals↗

Plasma protein synthesis and secretion in the visceral yolk sac of the fetal rat: gene expression, protein synthesis and secretion.

This report compares the relative levels of messenger RNA species coding for plasma proteins in rat visceral yolk sac and fetal liver from 12.5 days to 21.5 days gestation. Transthyretin, retinol-binding protein, transferrin and alpha 1-fetoprotein mRNAs were detected in both tissues, although relative levels were much higher in the yolk sac compared to fetal liver, in early gestation. Messenger RNA coding for the positive acute phase proteins thiostatin, fibrinogen, alpha 2-macroglobulin and alpha 1-antitrypsin were detected at a low but significant level in yolk sac, while the levels in fetal liver steadily increased from 16.5 days gestation and, with the exception of alpha 1-antitrypsin, reached levels higher than those found in adult liver just prior to birth. Albumin, inter-alpha 1-trypsin inhibitor, alpha 1-acid glycoprotein, haptoglobin, vitamin D-binding protein and ceruloplasmin messenger RNA levels were either very low or undetectable in yolk sac and fetal liver. Secretion of proteins by yolk sac endoderm occurred largely across the basolateral surface, i.e. towards the fetal compartment. These data support the hypothesis that one function of the yolk sac in the rat is the synthesis and secretion of a select group of plasma proteins to maintain homeostasis in the fetal compartment in the period before the fetal liver has matured sufficiently to carry out this function.

Albumins↗