Binding of thyroid hormone to the goat testicular Leydig cell induces the generation of a proteinaceous factor which stimulates androgen release

1994 ◽  
Vol 143 (3) ◽  
pp. 549-556 ◽  
Author(s):  
N R Jana ◽  
S Bhattacharya
Keyword(s):  
Protein Synthesis ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Actinomycin D ◽  
Competitive Inhibition ◽  
Binding Capacity ◽  
Cell Protein ◽  
Scatchard Analysis ◽  
Cell Nuclei ◽  
Stimulation Of

Abstract Leydig cells isolated from goat testis were sonicated and pure nuclear preparations obtained for 125I-3,5,3′-triiodothyronine (T3)-binding assay. Under optimum assay conditions of pH 7·2 at 37 °C and 90 min of incubation, binding of 125I-T3 to Leydig cell nuclei reached saturation at 1·2 nmol/l concentration. A Scatchard analysis of T3 binding exhibited a Kd of 0·535 × 10−9 mol/l and a maximum binding capacity of 1·25 pmol/mg DNA. Competitive inhibition studies showed T3 binding to be analogue specific. The physiological relevance of T3 binding to goat Leydig cell was examined by adding increasing concentrations of T3 to the Leydig cell incubation (1×10 cells/incubation). T3 (10, 25 and 50 ng/ml or 4, 10 and 20 ng/incubation) resulted a dose dependent increase in androgen release and in all cases stimulation of androgen release was statistically significant (P<0·01) compared with control. Stimulation of Leydig cell androgen release by T3 was significantly inhibited by actinomycin-D (P<0·01) and cycloheximide (P<0·01). T3 had additive stimulatory effects on LH-augmented androgen release from Leydig cells. T3 (50 ng/ml or 20 ng/incubation) effected a more than twofold increase in Leydig cell protein synthesis compared with control and both actinomycin-D and cycloheximide (50 μg/ml) inhibited it completely. The data indicated that the stimulatory effect of T3 on androgen release is mediated via T3-induced protein(s). Sub-cellular fractions obtained from T3-treated Leydig cells showed an increase in protein synthesis in mitochondrial and soluble supernatant fractions (100 k sup) and it was only 100 k sup which stimulated androgen release from Leydig cells in separate incubations. Treatment of 100 k sup with trypsin or heat abolished its stimulatory effect. Incubation of Leydig cells with T3 for different times showed an increase in protein synthesis prior to the stimulation of androgen release. The results therefore indicated that T3 binding to Leydig cells induced the generation of a proteinaceous factor(s) which in turn stimulated androgen release. Journal of Endocrinology (1994) 143, 549–556

scholarly journals Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

1978 ◽  
Vol 170 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Felix H. A. Janszen ◽  
Brian A. Cooke ◽  
Maria J. A. Van Driel ◽  
Henk J. Van Der Molen
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Leydig Cells ◽  
Actinomycin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phosphodiesterase Inhibitor ◽  
Dibutyryl Cyclic Amp ◽  
Rat Testis ◽  
Stimulation Of

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [35S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of35S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.

Electrically stimulated contraction accelerates protein synthesis rates in adult feline cardiocytes

1993 ◽  
Vol 265 (2) ◽  
pp. H666-H674 ◽  
Author(s):  
C. T. Ivester ◽  
R. L. Kent ◽  
H. Tagawa ◽  
H. Tsutsui ◽  
T. Imamura ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Electrical Stimulation ◽  
Total Protein ◽  
Actinomycin D ◽  
Electrical Field Stimulation ◽  
Cell Protein ◽  
Electrical Field ◽  
Butanedione Monoxime ◽  
H Protein ◽  
Stimulation Of

Cardiocytes were induced to contract via electrical field stimulation with an 8 V/cm electrical square-wave pulse of 5 ms at 0.125-2.0 Hz for up to 6 h. Protein synthesis rates were measured as rate of incorporation of [3H]-phenylalanine into total cell protein. Rates of protein synthesis were accelerated 43 +/- 4%, P < 0.001, by 4 h. The acceleration of total protein synthesis showed a frequency dependence between 0.125 and 0.5 Hz. In addition to accelerating rates of total protein synthesis, electrical stimulation of contraction accelerated fractional rates of synthesis of myosin heavy chain by 42 +/- 8%, P < 0.05. Protein synthesis rates were not accelerated upon electrical stimulation using subthreshold voltages. Addition of 100 ng/ml of actinomycin D had no effect on the ability of electrical stimulation of contraction to accelerate protein synthesis. To uncouple excitation-contraction coupling, 2,3-butanedione monoxime (BDM) was used to block actin-myosin cross-bridge interactions. BDM significantly decreased the ability of electrical stimulation to accelerate protein synthesis rates.

Insulin action in pancreatic acini from streptozotocin-treated rats. II. Binding of 125I-insulin to receptors

1981 ◽  
Vol 240 (1) ◽  
pp. G63-G68 ◽  
Author(s):  
H. Sankaran ◽  
Y. Iwamoto ◽  
M. Korc ◽  
J. A. Williams ◽  
I. D. Goldfine
Keyword(s):  
Protein Synthesis ◽  
Binding Sites ◽  
Insulin Binding ◽  
Diabetic Rats ◽  
Cell Protein ◽  
Scatchard Analysis ◽  
Pancreatic Acini ◽  
Maximal Stimulation ◽  
Major Class ◽  
Stimulation Of

The binding of 125I-insulin to its receptors was investigated with isolated pancreatic acini obtained from diabetic rats under incubation conditions identical to those used to study the effects of insulin on acinar cell protein synthesis. Binding was specific, time dependent, reversible, and linearly related to the acinar protein content. Degradation of insulin after 30 min of incubation was less than 10% of the total hormone present in the incubation medium. 125I-insulin dissociated from acini with a one-half time of 9 min. Unlabeled insulin at 83.5 nM accelerated the rate of dissociation of labeled insulin. 125I-insulin binding to acini was competitively inhibited by insulin and its analogues in proportion to their biological potencies. Scatchard analysis revealed a major class of insulin-binding sites with a Kd of 1.6 nM; maximal stimulation of protein synthesis was observed when > 90% of these high-affinity receptors were occupied. These studies indicate, therefore, that insulin binding to receptors on pancreatic acini can be correlated with subsequent regulation of biological functions.

EFFECT OF ACTINOMYCIN D ON TSH-INDUCED STIMULATION OF THYROIDAL PROTEIN SYNTHESIS IN THE RAT

1974 ◽  
Vol 77 (1) ◽  
pp. 64-70 ◽  
Author(s):  
Gustav Wägar
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
The Other ◽  
Leucine Incorporation ◽  
Short Term ◽  
Other Hand ◽  
Thyroid Glands ◽  
Translational Level ◽  
Stimulation Of

ABSTRACT Whether the short-term regulation of thyroidal protein synthesis by TSH occurs at the transcriptional or the translational level was tested by measuring the effect of actinomycin D (act D) on the TSH-induced stimulation of L-14C-leucine incorporation into the thyroidal proteins of rats. TSH was injected 6 h before the rats were killed. The thyroid glands were then removed and incubated in vitro in the presence of L-14C-leucine for 2 h. The pronounced stimulation of leucine incorporation in the TSH-treated animals was depressed as compared with controls but still significant even when the animals had been pre-treated with 100 μg act D 24 and 7 h before sacrifice. On the other hand, act D strongly decreased incorporation of 3H-uridine into RNA. Short-term regulation of thyroidal protein synthesis by TSH appears to be partly but not wholly dependent on neosynthesis of RNA. Hence regulation may partly occur at the translation level of protein synthesis.

BINDING OF HUMAN CHORIONIC GONADOTROPHIN BY RAT TESTIS: EFFECT OF SEXUAL MATURATION, CRYPTORCHIDISM AND HYPOPHYSECTOMY

1975 ◽  
Vol 64 (1) ◽  
pp. 59-66 ◽  
Author(s):  
JOACHIM FROWEIN ◽  
WOLFGANG ENGEL
Keyword(s):  
Dissociation Constant ◽  
Sexual Maturation ◽  
Leydig Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Scatchard Analysis ◽  
Rat Testis ◽  
Relative Binding

SUMMARY The specific binding of 125I-labelled human chorionic gonadotrophin (HCG) by rat testicular homogenate as compared with isolated Leydig cells differs with respect to total binding capacity but not to the dissociation constant (KD) as revealed by Scatchard analysis. The maximal binding capacity for [125I]HCG of crude testicular homogenate was 95 ng/g rat testis. Hypophysectomy causes a decline in binding capacity within the first three days but on the 20th and 30th day after hypophysectomy the relative binding capacity no longer differs from that of controls. Binding capacity is enhanced in cryptorchid testes relative to normal, and increases during sexual maturation to a peak shortly before puberty.

scholarly journals Leydig Cell Protein Synthesis and Steroidogenesis in Response to Acute Stimulation by Luteinizing Hormone in Rats1

1998 ◽  
Vol 59 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Lindi Luo ◽  
Haolin Chen ◽  
Douglas M. Stocco ◽  
Barry R. Zirkin
Keyword(s):  
Protein Synthesis ◽  
Luteinizing Hormone ◽  
Leydig Cell ◽  
Cell Protein

Rapid response of protein synthesis to insulin in 3T3 cells: Effects of protein kinase C depletion and differences from the response to serum repletion

1986 ◽  
Vol 6 (9) ◽  
pp. 797-804 ◽  
Author(s):  
John E. Hesketh ◽  
Gillian P. Campbell ◽  
Peter J. Reeds
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Actinomycin D ◽  
Calf Serum ◽  
Cell Protein ◽  
Foetal Calf Serum ◽  
3T3 Cells ◽  
Kinase C

Quiescent 3T3 cells grown in media containing 4% foetal calf serum showed different responses to insulin and to serum repletion (to 12%). Insulin stimulated protein synthesis within 1 h and this early response was insensitive to actinomycin D. The later insulin response showed progressive sensitivity to actinomycin D. The serum response was slower, not occurring until 1 h, and was inhibited by actinomycin D. Depletion of cell protein kinase C by pre-treatment with phorbol ester caused a total block of the immediate response to insulin but had little effect on the response to serum or the later response to insulin. Acute phorbol ester treatment stimulated protein synthesis.

A localized stimulation of lens protein synthesis by actinomycin D

1966 ◽  
Vol 114 (2) ◽  
pp. 428-430 ◽  
Author(s):  
John Papaconstantinou ◽  
James A. Stewart ◽  
Paul V. Koehn
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
Lens Protein ◽  
Stimulation Of

STUDIES ON THE MECHANISM OF ACTION OF ANGIOTENSIN ON FLUID TRANSPORT BY THE MUCOSA OF RAT DISTAL COLON

1972 ◽  
Vol 54 (3) ◽  
pp. 483-492 ◽  
Author(s):  
N. T. DAVIES ◽  
K. A. MUNDAY ◽  
B. J. PARSONS
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Rna Synthesis ◽  
Fluid Transport ◽  
Actinomycin D ◽  
Distal Colon ◽  
Rat Colon ◽  
Colon Mucosa ◽  
Rat Distal Colon ◽  
Stimulation Of

SUMMARY A study was made of the effects of cyclic AMP, theophylline, cycloheximide, puromycin and actinomycin D on the stimulation by angiotensin of fluid transport by sacs of rat colon mucosa. Cyclic AMP and theophylline, added together or separately, had no effect on fluid transport by colon sacs, suggesting that the stimulation of fluid transport after the application of angiotensin is not mediated through cyclic AMP. Cycloheximide and puromycin (used at concentrations which block colon protein synthesis by 50–90%) had no effect on fluid transport by control colon sacs, but completely blocked the stimulatory response of the colon to angiotensin. In contrast, actinomycin D (at a concentration which significantly inhibits RNA synthesis) did not affect fluid transport in control or angiotensin-stimulated colon sacs. The results are discussed in relation to the possibility that protein synthesis, at the stage of translation, is involved in the action of angiotensin on fluid transport by the colon.

scholarly journals Stimulation of Na+/H+ exchange is not required for induction of hypertrophy of renal cells in vitro.

1992 ◽  
Vol 3 (5) ◽  
pp. 1124-1130 ◽  
Author(s):  
M Mackovic-Basic ◽  
L G Fine ◽  
J T Norman ◽  
E J Cragoe ◽  
I Kurtz
Keyword(s):  
Growth Factors ◽  
Protein Content ◽  
Competitive Inhibition ◽  
Cell Protein ◽  
Renal Cells ◽  
Wild Type ◽  
Control Value ◽  
Mutant Cells ◽  
Stimulation Of

Hypertrophy of renal proximal tubular cells is associated with an early increase in Na+/H+ antiport activity both in vivo and in vitro. The purpose of the study presented here was to determine whether functioning Na+/H+ antiport activity is required for hypertrophy to occur. LLC-PK1 cells deficient in Na+/H+ antiport activity were prepared by the "proton-suicide" method. Mutant cells had 28 to 40% of the normal Na+/H+ antiport activity. The addition of 50 nM methylisobutylamiloride to these cells decreased the antiport activity to less than 5% of the control value. In the mutant cells, steady-state intracellular pH was normal as was the protein content. After exposure of the wild-type cells for 72 h to 10(-6) M insulin and 10(-9) M insulin-like growth factor 1, cell protein content increased significantly. The increase in protein content induced by these growth factors in the mutant cells did not differ significantly from the response of the wild-type cells. Lowering the Na+/H+ exchange further by the addition of methylisobutylamiloride (50 nM) to less than 5% of the control value did not blunt the hypertrophic response in the mutant cells. These studies indicate that hypertrophy can be induced in LLC-PK1 cells by growth factors when basal Na+/H+ antiport activity is reduced to low levels by selective mutation or by competitive inhibition. The results suggest that stimulation of the Na+/H+ antiporter is not an essential prerequisite for the induction of hypertrophy in renal cells.

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