Melatonin receptors are present in adult rat Leydig cells and are coupled through a pertussis toxin-sensitive G-protein

1997 ◽  
Vol 136 (6) ◽  
pp. 633-639 ◽  
Author(s):  
Sandra Valenti ◽  
Massimo Giusti ◽  
Roberta Guido ◽  
Giulio Giordano
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Binding Sites ◽  
Leydig Cells ◽  
Melatonin Receptors ◽  
Scatchard Analysis ◽  
Adult Rat ◽  
Testosterone Secretion ◽  
High Affinity Binding Site ◽  
17 Hydroxyprogesterone

Abstract Previous studies have suggested that melatonin (MLT) acts directly on rat Leydig cells by modulating androgen production. In the present study, the site of action of MLT was investigated. The binding of 2-[125I]iodomelatonin (125I-MLT; 7–240 pmol/l) to Leydig cell membrane fragments was tested in the presence or absence of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S; 50 μmol/l). Saturation studies and Scatchard analysis revealed the existence of a high-affinity binding site with a Bmax of 46·70± 3·50 fmol/mg protein and a Kd of 88·70±6·20 pmol/l; treatment with GTP-γ-S reduced the concentration of 125I-MLT binding sites (Bmax 34·03±4·50), while increasing the Kd to 106·5± 2·61 pmol/l. Pretreatment of the cells with pertussis toxin (PTX; 10 ng/ml for 16 h) resulted in a decreased binding of I-MLT and a lack of effect of GTP-γ-S. Moreover, the effect of MLT on testosterone secretion induced by LH (30 mIU/ml), forskolin (1 μmol/l) and LHRH (100 nmol/l) was studied after 3-h incubation of cells which had been precultured with or without PTX. The inhibition of testosterone secretion due to MLT administration was eliminated by PTX pretreatment during forskolin and LH, but not during LHRH administration. However, 17-hydroxyprogesterone levels were higher in all groups incubated in the presence of MLT, irrespective of PTX pretreatment. Our data suggest that: (a) MLT receptors are present on the membranes of adult rat Leydig cells; (b) they couple through PTX-sensitive G-protein-coupled binding sites; (c) the mechanism by which MLT blocks 17–20 desmolase enzymatic activity (thus leading to increased 17-hydroxyprogesterone levels), and testosterone secretion during LHRH stimulation is likely to depend on one or more different mechanism(s) of action. European Journal of Endocrinology 136 633–639

scholarly journals Arachidonic acid release from rat Leydig cells: the involvement of G protein, phospholipase A2 and regulation of cAMP production

2002 ◽  
Vol 172 (1) ◽  
pp. 95-104 ◽  
Author(s):  
AM Ronco ◽  
PF Moraga ◽  
MN Llanos
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Leydig Cells ◽  
Inhibitory Effect ◽  
Receptor Interaction ◽  
Dependent Manner ◽  
Camp Production

We have previously demonstrated that the release of arachidonic acid (AA) from human chorionic gonadotropin (hCG)-stimulated Leydig cells occurs in a dose- and time-dependent manner. In addition, the amount of AA released was dependent on the hormone-receptor interaction and the concentration of LH-hCG binding sites on the cell surface. The present study was conducted to evaluate the involvement of phospholipase A(2) (PLA(2)) and G proteins in AA release from hormonally stimulated rat Leydig cells, and the possible role of this fatty acid in cAMP production. Cells were first prelabelled with [(14)C]AA to incorporate the fatty acid into cell phospholipids, and then treated in different ways to evaluate AA release. hCG (25 mIU) increased the release of AA to 180+/-12% when compared with AA released from control cells, arbitrarily set as 100%. Mepacrine and parabromophenacyl bromide (pBpB), two PLA(2) inhibitors, decreased the hormone-stimulated AA release to 85+/-9 and 70+/-24% respectively. Conversely, melittin, a PLA(2) stimulator, increased the release of AA up to 200% over control. The inhibitory effect of mepacrine on the release of AA was evident in hCG-treated Leydig cells, but not in the melittin-treated cells. To determine if the release of AA was also mediated through a G protein, cells were first permeabilized and subsequently treated with pertussis toxin or GTPgammaS, a non-hydrolyzable analog of GTP. Results demonstrate that GTPgammaS was able to induce a similar level of the release of AA as hCG. In addition, pertussis toxin completely abolished the stimulatory effect of hCG on the release of AA, indicating that a member of the G(i) family was involved in the hCG-dependent release of AA. Cells treated with PLA(2) inhibitors did not modify cAMP production, but exogenously added AA significantly reduced cAMP production from hCG-treated Leydig cells, in a manner dependent on the concentration of AA and hCG. Results presented here suggest an involvement of PLA(2) and G proteins in the release of AA from hCG-stimulated Leydig cells, and under particular conditions, regulation of cAMP production by this fatty acid in these cells.

Prostaglandin E2 receptors in the heart are coupled to inhibition of adenylyl cyclase via a pertussis toxin sensitive G protein

1992 ◽  
Vol 70 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Richard W. Lerner ◽  
Gary D. Lopaschuk ◽  
Peter M. Olley
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Pertussis Toxin ◽  
Binding Sites ◽  
Cyclase Activity ◽  
Prostaglandin E ◽  
Adenylyl Cyclase Activity ◽  
Adp Ribosylation ◽  
Number Of Binding Sites ◽  
Guanine Nucleotide Binding

In previous studies we have identified and isolated a prostaglandin E2 (PGE2) receptor from cardiac sarcolemmal (SL) membranes. Binding of PGE2 to this receptor in permeabilized SL vesicles inhibits adenylyl cyclase activity. The purpose of this study was to determine if the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive guanine nucleotide binding inhibitory (Gi) protein. Incubation of permeabilized SL vesicles in the presence of 100 μM 5′-guanylamidiophosphate, Gpp(NH)p, a nonhydrolyzable analogue of GTP, resulted in a shift in [3H]PGE2 binding from two sites, one of high affinity (KD = 0.018 ± 0.003 nM) comprising 7.7% of the total available binding sites and one of lower affinity (KD = 1.9 ± 0.7 nM) to one site of intermediate affinity (KD = 0.52 ± 0.01 nM) without a significant change in the total number of PGE2 binding sites. A shift from two binding sites to one binding site in the presence of Gpp(NH)p was also observed for [3H]dihydroalprenolol binding to permeabilized cardiac SL. When permeabilized SL vesicles were pretreated with activated pertussis toxin, ADP-ribosylation of a 40- to 41-kDa protein corresponding to Gi was observed. ADP-ribosylation of SL resulted in a shift in [3H]PGE2 binding to one site of intermediate affinity without significantly changing the number of binding sites. In alamethicin permeabilized SL vesicles, 1 nM PGE2 significantly decreased (30%) adenylyl cyclase activity. Pretreatment with activated pertussis toxin overcame the inhibitory effects of PGE2. These results demonstrate that the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive Gi protein. They also demonstrate that the interaction of this Gi protein with the PGE2 receptor is important in the regulation of PGE2 binding to its receptor.Key words: prostaglandin E2, sarcolemma, heart, adenylyl cyclase, G protein.

scholarly journals Leucine-rich repeat-containing G-protein-coupled receptor 8 in mature glomeruli of developing and adult rat kidney and inhibition by insulin-like peptide-3 of glomerular cell proliferation

2006 ◽  
Vol 189 (2) ◽  
pp. 397-408 ◽  
Author(s):  
P Fu ◽  
P-J Shen ◽  
C-X Zhao ◽  
D J Scott ◽  
C S Samuel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
G Protein ◽  
Binding Sites ◽  
Renal Cortex ◽  
Mesangial Cells ◽  
Rat Kidney ◽  
Leucine Rich Repeat ◽  
G Protein Coupled Receptor ◽  
Adult Rat ◽  
G Protein Coupled

Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8, or RXFP2) is a member of the type C leucine-rich repeat-containing G protein-coupled receptor family, and its endogenous ligand is insulin-like peptide-3 (INSL3). Although LGR8 expression has been demonstrated in various human tissues, including testis, ovary, brain and kidney, the precise roles of this receptor in many of these tissues are unknown. In an effort to better understand INSL3–LGR8 systems in the rat, we cloned the full-length Lgr8 cDNA and investigated the presence and cellular localization of Lgr8 mRNA expression in adult and developing rat kidney. On the basis of these findings, we investigated the presence and distribution of renal 125I-labelled human INSL3-binding sites and the nature of INSL3–LGR8 signalling in cultured renal cells. Thus, using in situ hybridization histochemistry, cells expressing Lgr8 mRNA were observed in glomeruli of renal cortex from adult rats and were tentatively identified as mesangial cells. Quantitative, real-time PCR analysis of the developmental profile of Lgr8 mRNA expression in kidney revealed highest relative levels at late stage gestation (embryonic day 18), with a sharp decrease after birth and lowest levels in the adult. During development, silver grains associated with Lgr8 mRNA hybridization were observed overlying putative mesangial cells in mature glomeruli, with little or no signal associated with less-mature glomeruli. In adult and developing kidney, specific 125I-INSL3-binding sites were associated with glomeruli throughout the renal cortex. In primary cultures of glomerular cells, synthetic human INSL3 specifically and dose-dependently inhibited cell proliferation over a 48 h period, further suggesting the presence of functional LGR8 (receptors) on these cells (mesangial and others). These findings suggest INSL3–LGR8 signalling may be involved in the genesis and/or developmental maturation of renal glomeruli and possibly in regulating mesangial cell density in adult rat kidney.

scholarly journals A novel mechanism for the melatonin inhibition of testosterone secretion by rat Leydig cells: reduction of GnRH-induced increase in cytosolic Ca2+

1999 ◽  
Vol 23 (3) ◽  
pp. 299-306 ◽  
Author(s):  
S Valenti ◽  
S Thellung ◽  
T Florio ◽  
M Giusti ◽  
G Schettini ◽  
...  
Keyword(s):  
Leydig Cells ◽  
Calcium Concentration ◽  
Inhibitory Effect ◽  
Adult Rat ◽  
Testosterone Secretion ◽  
Gi Protein ◽  
Hydroxy Progesterone ◽  
Induced Changes ◽  
Cells Cultured

The site of inhibition, by melatonin, of GnRH-dependent testosterone secretion was investigated in adult rat Leydig cells cultured in vitro. The various effects downstream of the binding of GnRH to its own receptor were isolated and mimicked by specific drugs. Testosterone secretion was then evaluated after 3 h stimulation with GnRH, thapsigargin (1 microM), phorbol-12-myristate-13-acetate (100 nM), arachidonic acid (20 microM), and ionomycin (1 microM) in the presence or absence of melatonin (215 nM). The effect of melatonin on the GnRH-induced changes in cytoplasmic calcium concentration ([Ca(2+)](i)) was also studied, using Fura-2 as fluorescent Ca(2+) indicator. Melatonin attenuated the increase in [Ca(2+)](i) and inhibited the testosterone secretion induced by GnRH, but not that induced by ionomycin. Both ionomycin and thapsigargin potentiated GnRH-induced testosterone secretion; however, ionomycin, but not thapsigargin, partially prevented the inhibitory effect of melatonin on cells stimulated with GnRH. The effect of melatonin was probably dependent on the binding of melatonin to its Gi-protein-coupled receptor, as the inhibitory effect on GnRH-induced secretion was supressed in cells pretreated with pertussis toxin in a concentration of 180 ng/ml for 20 h. Assay of 17-hydroxy-progesterone showed that, irrespective of the treatment, cells cultured with melatonin secreted greater amounts than controls. We conclude that melatonin reduces GnRH-induced testosterone secretion by 1) decreasing [Ca(2+)](i), through impairment of the GnRH-dependent release of Ca(2+) from intracellular stores and 2) blocking 17-20 desmolase enzymatic activity, an effect that occurs irrespective of changes in [Ca(2+)](i).

Enhanced testosterone secretion in adult rams after establishment of a high-frequency, low-amplitude pattern of LH pulses in the nonbreeding season occurs without changes in the number or binding affinity of testicular LH receptors

1990 ◽  
Vol 122 (1) ◽  
pp. 55-61 ◽  
Author(s):  
Lee M. Sanford ◽  
Susan J. Baker
Keyword(s):  
Binding Affinity ◽  
Binding Sites ◽  
High Frequency ◽  
Leydig Cells ◽  
Jugular Vein ◽  
Pulse Amplitude ◽  
Nonbreeding Season ◽  
Testosterone Secretion ◽  
Low Amplitude ◽  
Amplitude Pattern

Abstract When the LH signal in the ram is changed from one of large and infrequent pulses to one of small and frequent pulses, the testes quickly become more responsive to LH and testosterone secretion is elevated, perhaps because the number and (or) binding affinity of testicular LH receptors have increased. An experiment was undertaken in the nonbreeding season (July) with 10 adult Dorset × Leicester × Suffolk rams that were about 3.5 years of age and 69 ± 2 kg in body weight. Rams were given injections into the jugular vein of either 5 μg NIH-LH-S24 (in 1 ml saline) or vehicle every 80 min for 6 days. LH treament produced a series of LH pulses that occurred three times more frequently and were 70% less in amplitude than pulses in the control rams, without causing mean LH concentration to increase. Endogenously produced LH pulses were not evident in the treated rams after LH injection began. The modified LH-pulse pattern elevated mean testosterone concentration by 150% (assessed on days 2 and 5), and caused the cumulative testosterone response to LH pulses, estimated by multiplying testosterone-pulse amplitude by frequency per 6 h, to increase progressively by 180% (days −2 through 5). Enhanced testicular steroidogenic activity, presumably due to greater enzymatic activity and cholesterol availability within Leydig cells, was not associated with increases in either the concentration or affinity of LH-binding sites in the testis (assessed on days 3 and 6).

Characterization of melatonin receptors in the ram pars tuberalis: influence of light

1990 ◽  
Vol 123 (5) ◽  
pp. 557-562 ◽  
Author(s):  
Jean Pelletier ◽  
Bertrand Castro ◽  
Georges Roblot ◽  
Renée Wylde ◽  
Marie-Madeleine de Reviers
Keyword(s):  
Median Eminence ◽  
Binding Sites ◽  
Melatonin Receptors ◽  
Scatchard Analysis ◽  
Pars Tuberalis

Abstract. The present study was conducted to assess the binding of [125I]melatonin to frozen unfixed sections of pars tuberalis/median eminence tissue from Ile-de-France rams exposed or not exposed to light before slaughter. The specificity of [125I]melatonin binding to the pars tuberalis tissue was revealed by autoradiography and the magnitude of binding as related to the pars tuberalis area was determined after incubation and counting of pars tuberalis/median eminence sections. Subsequent studies with sections incubated with [125I]melatonin indicated that 1. the binding sites were saturable; 2. binding was stable for 24 h at 20°C, but unstable at 28 or 37°C; 3. melatonin and [12 7I]melatonin had a similar potency to compete with [125I]melatonin for binding sites, whereas other ligands such as serotonin or N-acetylserotonin were devoid of activity, and 4. by Scatchard analysis, the constant affinity Ka was found to be high in the 1010 l/mol range. Rams exposed to light throughout the night prior to slaughter presented a significant increase in the apparent number of [125I]melatonin binding sites in comparison to animals maintained under darkness (2.25±0.30 vs 1.01±0.17 fmol/mm2 pars tuberalis, p<0.01), whereas Ka values were similar in both groups. These results indicate the presence of true melatonin receptors in the pars tuberalis of the ram. Furthermore, they suggest that their apparent number is light-dependent.

Pancreatic polypeptide inhibits calcium channels in rat sympathetic neurons via two signaling pathways

1995 ◽  
Vol 73 (3) ◽  
pp. 1323-1328 ◽  
Author(s):  
L. P. Wollmuth ◽  
M. S. Shapiro ◽  
B. Hille
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Pancreatic Polypeptide ◽  
Sympathetic Neurons ◽  
Whole Cell ◽  
Adult Rat ◽  
Voltage Dependent ◽  
Cervical Ganglion ◽  
Gamma S ◽  
Ca2 Channels

1. We studied modulation of N-type Ca2+ channels in adult rat superior cervical ganglion (SCG) neurons by pancreatic polypeptide (PP) using whole cell clamp. In large (> 20 pF) SCG neurons, PP inhibited ICa (35 +/- 2%, mean +/- SE) in a concentration-dependent fashion, with one-half maximal inhibition at 19 nM. 2. One-third of the inhibition was blocked by pertussis toxin, about one-half was blocked by N-ethylmaleimide (NEM) treatments, and about one-half was voltage dependent. The NEM-insensitive component of the PP inhibition was voltage independent and not significantly blocked by intracellular Ca2+ chelators. 3. The NEM-insensitive component was only weakly attenuated by GDP-beta-S, and moderately reversible with guanosine 5'-triphosphate (GTP)-gamma-S, in the whole cell pipette, leaving open the possibility that it is not mediated by a G protein. 4. Hence, PP inhibits ICa via two mechanisms: one G-protein-mediated and the other possibly G-protein independent. The former pathway is sensitive to pertussis toxin (PTX) and NEM, voltage dependent, and shared by several other transmitters in these cells. The latter pathway is PTX-and NEM-insensitive, not voltage dependent, and not affected by the presence of intracellular Ca2+ chelators.

ENDOTHELIN STIMULATES TESTOSTERONE SECRETION BY RAT LEYDIG CELLS

1993 ◽  
Vol 136 (2) ◽  
pp. R1-R4 ◽  
Author(s):  
D. Conte ◽  
P. Questino ◽  
S. Fillo ◽  
M. Nordio ◽  
A. Isidori ◽  
...  
Keyword(s):  
Binding Sites ◽  
Leydig Cells ◽  
Channel Blocker ◽  
Specific Binding ◽  
Human Chorionic Gonadotrophin ◽  
Paracrine Factor ◽  
Testosterone Secretion ◽  
Specific Binding Sites ◽  
Testicular Steroidogenesis

ABSTRACT The present study was designed to evaluate the effects of endothelin (ET) on rat testicular steroidogenesis in vitro and the involvement of prostaglandins (PG) and extracellular calcium in its mechanism of action. To this purpose we examined the effects of ET-1 and ET-3 on basal testosterone secretion, the influence of ET-1 on PGE2, release, the interaction of ET-1 and ET-3 with human chorionic gonadotrophin (hCG) and the interference of indomethacin (an inhibitor of cycloxygenase) and nifedipine (a calcium-channel blocker) in purified rat Leydig cells. The data indicate that ET-1 and ET-3 stimulate basal and hCG-induced testosterone production although the effects of ET-3 were less marked. In addition, a concomitant release of PGE2, was observed after exposure to ET-1. A sinergistic interaction between ET-1 and hCG in stimulating testicular steroidogenesis was revealed. Indomethacin was ineffective in modifying ET-1 evoked testosterone output, while in the presence of nifedipine the stimulatory effect of ET-1 was completely abolished. Since it has been shown by others that ET-1 is produced by rat Sertoli cells and specific binding sites are present in Leydig cells, the results of our study indicate that such a peptide may be regarded as a new paracrine factor able to influence steroidogenesis in Leydig cells. The action of ET-1 requires the activity of voltage-operated Ca2+ channels, while PGE2 activation is not essential for its steroidogenic effect.

scholarly journals Identification of the Ca2+-release activity and ryanodine receptor in sarcoplasmic-reticulum membranes during cardiac myogenesis

1988 ◽  
Vol 253 (3) ◽  
pp. 631-636 ◽  
Author(s):  
M Michalak
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Binding Capacity ◽  
Ryanodine Receptors ◽  
Ruthenium Red ◽  
Scatchard Analysis ◽  
Fetal Sheep ◽  
Cardiac Myogenesis ◽  
High Affinity Binding Site ◽  
Well Differentiated

Ca2+-induced Ca2+ release and pH-induced Ca2+ release activities were identified in sarcoplasmic-reticulum (SR) vesicles isolated from adult- and fetal-sheep hearts. Ca2+-induced Ca2+ release and pH-induced Ca2+ release appear to proceed via the same channels, since both phenomena are similarly inhibited by Ruthenium Red. Ca2+ release from fetal SR vesicles is inhibited by higher concentrations of Ruthenium Red than is that from adult membranes. Both fetal and adult SR vesicles bind ryanodine. Fetal SR shows higher ryanodine-binding capacity than adult SR vesicles. Scatchard analysis of ryanodine binding revealed only one high-affinity binding site (Kd 6.7 nM) in fetal SR vesicles compared with two distinct binding sites (Kd 6.6 and 81.5 nM) in the adult SR vesicles. SR vesicles isolated from fetal and adult hearts were separated on discontinuous sucrose gradients into light (free) and heavy (junctional) SR vesicles. Heavy SR vesicles isolated from adult hearts exhibited most of the Ca2+ release activities. In contrast, Ca2+-induced Ca2+ release, pH-induced Ca2+ release and ryanodine receptors were detected in both light and heavy fetal SR. These results suggest that fetal SR may not be morphologically and functionally as well differentiated as that of adult cardiac muscle and that it may contain a greater number of Ca2+-release channels than that present in adult SR membranes.

scholarly journals Endothelin-1: a new autocrine/paracrine factor in rat testis

1993 ◽  
Vol 265 (2) ◽  
pp. E267-E274 ◽  
Author(s):  
G. Fantoni ◽  
P. L. Morris ◽  
G. Forti ◽  
G. B. Vannelli ◽  
C. Orlando ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Binding Sites ◽  
Leydig Cells ◽  
Positive Staining ◽  
Paracrine Factor ◽  
High Concentration ◽  
Rat Testis ◽  
Endothelin 1 ◽  
Adult Rat ◽  
Old Rats

Cultured Sertoli cells of 20-day-old rats were found to produce and release endothelin-1-like immunoreactivity (ET-1-LI) under follicle-stimulating hormone control. The elution profile of ET-1-LI from extracts of spent Sertoli cell culture medium corresponds to that of synthetic ET-1, suggesting a testicular production of authentic ET-1. In contrast, the conditioned medium from rat Leydig cells did not contain ET-1-LI. Immunohistochemical studies confirmed that, in 20-day-old rats, the positive staining was confined to some Sertoli cells, whereas interstitial cells were negative. In the adult rat testis the positivity was not limited to the tubular compartment (Sertoli cells) but was also present in the interstitium. A high concentration (13 pmol/mg protein) of high-affinity (dissociation constant = 0.6 nM) 125I-labeled ET-1 binding sites was present in Leydig cells. These sites bind ET-1 and ET-2 with 1,000-fold higher affinity than ET-3, suggesting that they correspond to the subtype ETA of the ET receptors. Specific 125I-ET-1 binding sites are present also in Sertoli cells but are 50-fold less concentrated than in Leydig cells. Our results suggest an autocrine/paracrine role for ET-1 in rat testis.

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