Physiological role of thiol proteases in thyroid hormone secretion

1986 ◽  
Vol 113 (2) ◽  
pp. 261-267 ◽  
Author(s):  
Mototaka Yoshinari ◽  
Alvin Taurog
Keyword(s):  
Density Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Hormone Secretion ◽  
Physiological Role ◽  
Inhibitory Effect ◽  
Thiol Proteases ◽  
Presence And Absence ◽  
Rat Thyroid

Abstract. To determine the physiological role of the thiol proteases in T4 and T3 release from thyroglobulin, experiments were performed with 131I-prelabelled rat thyroid lobes incubated in vitro in the presence and absence of leupeptin, an inhibitor of thiol proteases. Basal secretion of [131I]T4 and [131I]T3 from rat thyroid lobes prelabelled in vivo was quite low, but in the presence of 10 mU/ml bovine TSH a marked stimulatory effect was observed. The stimulatory effect of TSH was completely abolished by leupeptin. This was associated with marked inhibition of lysosomal proteolytic activity, suggesting that the inhibitory effect of leupeptin on T4 and T3 secretion could be attributed to its inhibitory action on proteolysis of thyroglobulin. Further evidence for an inhibitory effect of leupeptin on intralysosomal hydrolysis of thyroglobulin was obtained when thyroid lobes were incubated with 131I- in the presence and absence of leupeptin and TSH. The crude lysosomal preparation was fractionated on a Percoll density gradient, which separates 131I-containing particles into a dense peak containing purified lysosomes and a buoyant peak containing pinocytotic vesicles. A marked increase in the 131I-content of the dense peak was observed in the presence of TSH + leupeptin. Analysis of the 131I in the dense fraction by sucrose density gradient centrifugation and by SDS-polyacrylamide gel electrophoresis demonstrated that leupeptin inhibited degradation of 19S thyroglobulin, especially the formation of [131I]peptides of MW < 14K.

Studies on 2′,3′-cyclic nucleotide-3′-phosphohydrolase from brain

1969 ◽  
Vol 47 (10) ◽  
pp. 961-966 ◽  
Author(s):  
Robert W. Olafson ◽  
George I. Drummond ◽  
Jack F. Lee
Keyword(s):  
Density Gradient ◽  
Cyclic Nucleotide ◽  
Gradient Centrifugation ◽  
Physiological Role ◽  
Sucrose Density Gradient ◽  
Bovine Brain ◽  
Sucrose Density Gradient Centrifugation ◽  
Brain White Matter ◽  
Cyclic Phosphate

Studies on a 2′,3′-cyclic nucleotide-3′-phosphohydrolase are presented. Bovine brain white matter was fractionated by differential centrifugation and sucrose density gradient techniques. The phosphohydrolase was distributed throughout all primary fractions: nuclear, mitochondrial, and microsomal. When these fractions were subjected to sucrose density gradient centrifugation the enzyme appeared only in the myelin-containing layers. Brains of newborn rats were essentially devoid of phosphohydrolase activity and the content rose rapidly during the 12th and 25th days, coinciding precisely with the development of myelin. Mutant 'quaking' mice whose brains are partially deficient in myelin were also partially deficient in enzyme activity. The data provide evidence that the phosphohydrolase is associated with myelin.The enzyme opens the cyclic phosphodiester linkage of Ap(Ap)2A cyclic P and Ap(Ap)6A cyclic P without rupture of internucleotide bonds. The enzyme also hydrolyzed the 2′,3′-cyclic phosphorothioates of uridine and guanosine. The Km for adenosine 2′,3′-cyclic phosphate was determined to be 1.9 × 10−3 M, an inordinately high value, and since nothing is known about the physiological role of the enzyme, the possibility is raised that the true substrate may not be a cyclic nucleotide.

POSSIBLE ROLE OF 5α-ANDROSTANE-3α,17β-DIOL IN THE CONTROL OF FOLLICLE-STIMULATING HORMONE SECRETION IN THE IMMATURE FEMALE RAT

1982 ◽  
Vol 92 (1) ◽  
pp. 37-42 ◽  
Author(s):  
H. M. A. MEIJS-ROELOFS ◽  
P. KRAMER ◽  
L. GRIBLING-HEGGE
Keyword(s):  
Inhibitory Action ◽  
Combined Treatment ◽  
Hormone Secretion ◽  
Inhibitory Effect ◽  
Ovariectomized Rats ◽  
Female Rat ◽  
Immature Rat ◽  
Rat Strain ◽  
Intact Rats

A possible role of 5α-androstane-3α,17β-diol (3α-androstanediol) in the control of FSH secretion was studied at various ages in ovariectomized rats. In the rat strain used, vaginal opening, coincident with first ovulation, generally occurs between 37 and 42 days of age. If 3α-androstanediol alone was given as an ovarian substitute, an inhibitory effect on FSH release was evident with all three doses tested (50, 100, 300 μg/100 g body wt) between 13 and 30 days of age; at 33–35 days of age only the 300 μg dose caused some inhibition of FSH release. Results were more complex if 3α-androstanediol was given in combined treatment with oestradiol and progesterone. Given with progesterone, 3α-androstanediol showed a synergistic inhibitory action on FSH release between 20 and 30 days of age. However, when 3α-androstanediol was combined with oestradiol a clear decrease in effect, as compared to the effect of oestradiol alone, was found between 20 and 30 days of age. Also the effect of combined oestradiol and progesterone treatment was greater than the effect of combined treatment with oestradiol, progesterone and 3α-androstanediol. At all ages after day 20 none of the steroid combinations tested was capable of maintaining FSH levels in ovariectomized rats similar to those in intact rats. It is concluded that 3α-androstanediol might play a role in the control of FSH secretion in the immature rat, but after day 20 the potentially inhibitory action of 3α-androstanediol on FSH secretion is limited in the presence of oestradiol.

scholarly journals Effects of Kisspeptin-10 on Hypothalamic Neuropeptides and Neurotransmitters Involved in Appetite Control

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3071 ◽  
Author(s):  
Giustino Orlando ◽  
Sheila Leone ◽  
Claudio Ferrante ◽  
Annalisa Chiavaroli ◽  
Adriano Mollica ◽  
...  
Keyword(s):  
Gene Expression ◽  
Energy Homeostasis ◽  
Hormone Secretion ◽  
Reproductive Function ◽  
Inhibitory Effect ◽  
Bdnf Gene ◽  
Appetite Control ◽  
Hydroxyindoleacetic Acid ◽  
Puberty Onset

Besides its role as key regulator in gonadotropin releasing hormone secretion, reproductive function, and puberty onset, kisspeptin has been proposed to act as a bridge between energy homeostasis and reproduction. In the present study, to characterize the role of hypothalamic kisspeptin as metabolic regulator, we evaluated the effects of kisspeptin-10 on neuropeptide Y (NPY) and brain-derived neurotrophic factor (BDNF) gene expression and the extracellular dopamine (DA), norepinephrine (NE), serotonin (5-hydroxytriptamine, 5-HT), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIIA) concentrations in rat hypothalamic (Hypo-E22) cells. Our study showed that kisspeptin-10 in the concentration range 1 nM–10 μM was well tolerated by the Hypo-E22 cell line. Moreover, kisspeptin-10 (100 nM–10 μM) concentration independently increased the gene expression of NPY while BDNF was inhibited only at the concentration of 10 μM. Finally, kisspeptin-10 decreased 5-HT and DA, leaving unaffected NE levels. The inhibitory effect on DA and 5-HT is consistent with the increased peptide-induced DOPAC/DA and 5-HIIA/5-HT ratios. In conclusion, our current findings suggesting the increased NPY together with decreased BDNF and 5-HT activity following kisspeptin-10 would be consistent with a possible orexigenic effect induced by the peptide.

scholarly journals History and perspectives of pituitary folliculo-stellate cell research

2005 ◽  
Vol 153 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Wilfried Allaerts ◽  
Hugo Vankelecom
Keyword(s):  
Cell Population ◽  
Anterior Pituitary ◽  
Hormone Secretion ◽  
Physiological Role ◽  
Pituitary Cell ◽  
Stem Cell Population ◽  
Cell Group ◽  
Pituitary Hormone ◽  
Topographical Distribution

Historically, the study of folliculo-stellate (FS) cells of the anterior pituitary dates back to the onset of electron microscopical observation of the pituitary gland. The morphological and electrophysiological characteristics, topographical distribution and contribution to intercellular junctions of these FS cells have been instrumental to the understanding of their putative function. Moreover, many studies have documented the role of FS cells as a source of newly discovered peptides, growth factors and cytokines. Quantitative immunohistochemical observation of FS cells in situ and functional in vitro studies, using either cultured FS cells or cells from an immortalized FS cell line, forwarded the notion of immunophenotypical and functional heterogeneity of the FS cell group. Double immunolabeling with a classical FS cell marker (S-100 protein) and with major histocompatibility complex class II markers characteristic for dendritic cells (DC) have shown a considerable overlap of FS cells with DC. The latter cells are immunocompetent cells belonging to the mononuclear phagocyte system. In this review, the FS cell heterogeneity is discussed with respect to the question of their embryological origin and developmental fate and with respect to the physiological relevance of functionally heterogeneous subpopulations. Recent findings of a myeloid origin of part of the interstitial cells of the anterior pituitary are confronted by other developmental paradigms of pituitary cell differentiation. The possibility that FS cells represent an adult stem cell population of the pituitary is critically examined. Also the physiological role of FS cells in the interferon-γ- and nitric oxide-mediated effects on pituitary hormone secretion is discussed. New approaches for the study of this enigmatic cell group using immortalized cell lines and new markers for an hitherto unrecognized pituitary cell population, the so-called ‘side population’, are evaluated.

C-type Natriuretic Peptide Inhibits L-type Ca2+ Current in Rat Magnocellular Neurosecretory Cells by Activating the NPR-C Receptor

2005 ◽  
Vol 94 (1) ◽  
pp. 612-621 ◽  
Author(s):  
Robert A. Rose ◽  
Madhu B. Anand-Srivastava ◽  
Wayne R. Giles ◽  
Jaideep S. Bains
Keyword(s):  
Natriuretic Peptide ◽  
Action Potentials ◽  
Hormone Secretion ◽  
Inhibitory Effect ◽  
Neurosecretory Cells ◽  
Mediated Effects ◽  
Inhibitory G Protein ◽  
Whole Cell Recordings ◽  
Supraoptic Nuclei

Magnocellular neurosecretory cells (MNCs), of the paraventricular and supraoptic nuclei of the hypothalamus, secrete the hormones vasopressin and oxytocin. As a result, they have an essential role in fundamental physiological responses including regulation of blood volume and fluid homeostasis. C-type natriuretic peptide (CNP) is present at high levels in the hypothalamus. Although CNP is known to decrease hormone secretion from MNCs, no studies have examined the role of the natriuretic peptide C receptor (NPR-C) in these neurons. In this study, whole cell recordings from acutely isolated MNCs, and MNCs in a coronal slice preparation, show that CNP (2 × 10−8 M) and the selective NPR-C agonist, cANF (2 × 10−8 M), significantly inhibit L-type Ca2+ current ( ICa(L)) by ∼50%. This effect on ICa(L) is mimicked by dialyzing a Gi-activator peptide (10−7 M) into these cells, implicating a role for the inhibitory G protein, Gi. These NPR-C–mediated effects were specific to ICa(L). T-type Ca2+ channels were unaffected by CNP. Current-clamp experiments revealed the ability of CNP, acting via the NPR-C receptor, to decrease (∼25%) the number of action potentials elicited during a 500 ms depolarizing stimulus. Analysis of action potential duration revealed that CNP and cANF significantly decreased 50% repolarization time (APD50) in MNCs. In summary, our findings show that CNP has a potent and selective inhibitory effect on ICa(L) and on excitability in MNCs that is mediated by the NPR-C receptor. These data represent the first electrophysiological evidence of a functional role for the NPR-C receptor in the mammalian hypothalamus.

scholarly journals Subcellular organization of neurophysins, oxytocin, [8−lysine]-vasopressin and adenosine triphosphatase in porcine posterior pituitary lobes

1973 ◽  
Vol 132 (3) ◽  
pp. 361-371 ◽  
Author(s):  
J. C. Pickup ◽  
C. I. Johnston ◽  
S. Nakamura ◽  
L. O. Uttenthal ◽  
D. B. Hope
Keyword(s):  
Density Gradient ◽  
Adenosine Triphosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Posterior Pituitary ◽  
Neurosecretory Granules ◽  
Sucrose Density Gradient Centrifugation ◽  
Lysine Vasopressin ◽  
Neurophysin Ii

Posterior pituitary lobes from young pigs were fractionated by differential and sucrose-density-gradient centrifugation. The distributions of oxytocin and [8-lysine]-vasopressin were measured by bioassay and the distributions of neurophysin-I and -II by radioimmunoassays specific for each of these two proteins. Most of the hormone and neurophysin applied to the density gradient was localized in particles with the density expected of neurosecretory granules. However, the neurosecretory granules were separated into two bands (D and E). A close statistical correlation between the distributions of [8-lysine]-vasopressin and neurophysin-I, and of oxytocin and neurophysin-II on the gradients, suggested that in vivo porcine neurophysin-I binds [8-lysine]-vasopressin within one population of granules and porcine neurophysin-II binds oxytocin within another type of granule. However, there was no significant separation of oxytocin and vasopressin in fractions D and E. The molar ratios of hormones and neurophysins indicated that there was insufficient of either neurophysin to bind the [8-lysine]-vasopressin in the granule fractions or in the whole gland. Polyacrylamide-gel electrophoresis showed that only bands corresponding in mobility to porcine neurophysins-I, -II and -III were present in large amounts in the whole gland and in the granule fractions. The component with the mobility of neurophysin-III was, however, relatively enriched in whole young glands and granule fractions compared with adult gland extracts. It is suggested that the vasopressin that cannot be assigned to neurophysin-I may occur in (a) vesicles containing vasopressin but no neurophysin, (b) vesicles containing vasopressin and a protein that cannot be quantified by the radioimmunoassays used, such as porcine neurophysin-III, or (c) normal vasopressin–neurophysin granules which have accumulated extra vasopressin. Band E of the gradient was rich in adenosine triphosphatase activity, whereas band D possessed very little of this enzyme.

Effect of actively immunizing sheep against growth hormone-releasing hormone or somatostatin on spontaneous pulsatile and neostigmine-induced growth hormone secretion

1995 ◽  
Vol 144 (1) ◽  
pp. 83-90 ◽  
Author(s):  
E Magnan ◽  
L Mazzocchi ◽  
M Cataldi ◽  
V Guillaume ◽  
A Dutour ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Body Weight ◽  
Hormone Secretion ◽  
Growth Hormone Secretion ◽  
Physiological Role ◽  
Gh Secretion ◽  
Igf I ◽  
Plasma Gh ◽  
Hypophysial Portal

Abstract The physiological role of endogenous circulating GHreleasing hormone (GHRH) and somatostatin (SRIH) on spontaneous pulsatile and neostigmine-induced secretion of GH was investigated in adult rams actively immunized against each neuropeptide. All animals developed antibodies at concentrations sufficient for immunoneutralization of GHRH and SRIH levels in hypophysial portal blood. In the anti GHRH group, plasma GH levels were very low; the amplitude of GH pulses was strikingly reduced, although their number was unchanged. No stimulation of GH release was observed after neostigmine administration. The reduction of GH secretion was associated with a decreased body weight and a significant reduction in plasma IGF-I concentration. In the antiSRIH group, no changes in basal and pulsatile GH secretion or the GH response to neostigmine were observed as compared to controls. Body weight was not significantly altered and plasma IGF-I levels were reduced in these animals. These results suggest that in sheep, circulating SRIH (in the systemic and hypophysial portal vasculature) does not play a significant role in pulsatile and neostigmine-induced secretion of GH. The mechanisms of its influence on body weight and production of IGF-I remain to be determined. Journal of Endocrinology (1995) 144, 83–90

scholarly journals Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Andrea Gerbino ◽  
Roberta De Zio ◽  
Daniela Russo ◽  
Luigi Milella ◽  
Serena Milano ◽  
...  
Keyword(s):  
Physiological Role ◽  
Renal Medulla ◽  
Human Kidney ◽  
Inhibitory Effect ◽  
Hek293 Cells ◽  
Whole Cell Patch Clamp ◽  
New Information ◽  
Intracellular Ca ◽  
Accessory Subunit

Abstract The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl- permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl- currents time-dependently within several minutes. Of note, the same effect on Cl- currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca2+ chelator BAPTA-AM (30 μM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl- current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic.

scholarly journals Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes

1976 ◽  
Vol 155 (1) ◽  
pp. 107-115 ◽  
Author(s):  
T Noguchi ◽  
E Okuno ◽  
Y Minatogawa ◽  
R Kido
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Aminotransferase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

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