Stimulation of pituitary cAMP accumulation by human pancreatic GH-releasing factor-(1-44)

1984 ◽  
Vol 247 (5) ◽  
pp. E609-E615 ◽  
Author(s):  
M. D. Culler ◽  
T. Kenjo ◽  
N. Obara ◽  
A. Arimura
Keyword(s):  
Cell Cultures ◽  
Intracellular Camp ◽  
Camp Accumulation ◽  
Gh Secretion ◽  
Pituitary Growth Hormone ◽  
Order Of Magnitude ◽  
Novel Method ◽  
Intracellular Camp Accumulation ◽  
Stimulation Of ◽  
Related Increase

This study seeks to determine whether hpGRF-(1-44) stimulates pituitary growth hormone (GH) secretion and cAMP accumulation in a manner that is consistent with the concept of cAMP as an intracellular mediator of GH release. Addition of 10 nM hpGRF-(1-44) to rat anterior pituitary cell cultures stimulated a rapid elevation of intracellular cAMP that preceded or coincided with increases in GH and cAMP secretion. A dose-related increase in GH and cAMP release and in intracellular cAMP accumulation was observed in response to increasing concentrations of hpGRF-(1-44). Stimulation of cAMP accumulation and release, however, occurred over a hpGRF-(1-44) concentration range that was approximately one order of magnitude higher than required for dose-related GH release. Simultaneous addition of 0.05 nM hpGRF-(1-44) and 0.2, 0.5, or 1.0 mM 3-isobutyl-1-methylxanthine (MIX) to the cultures resulted in a significant potentiation of intracellular cAMP accumulation and release. Potentiation of GH release was not observed, however, probably due to attainment of maximal or near maximal GH release by MIX alone. The addition of increasing doses of exogenous N6-O2'-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) to cell cultures resulted in a dose-related increase in GH secretion. The results of this study are consistent with the concept of cAMP as a second messenger for hpGRF-(1-44) in stimulating GH release. Additionally, a novel method for cAMP extraction that utilizes trifluoroacetic acid is described.

scholarly journals Aminoglutethimide suppresses adrenocorticotropin receptor expression in the NCI-h295 adrenocortical tumor cell line

1998 ◽  
Vol 159 (1) ◽  
pp. 35-42 ◽  
Author(s):  
M Fassnacht ◽  
F Beuschlein ◽  
S Vay ◽  
P Mora ◽  
B Allolio ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Cdna Probe ◽  
Tumor Cell Line ◽  
Receptor Expression ◽  
Intracellular Camp ◽  
Camp Accumulation ◽  
Transcript Accumulation ◽  
Down Regulation ◽  
Stimulation Of

The adrenostatic compound aminoglutethimide (AG), a potent inhibitor of the P450 side chain cleavage enzyme, is used in the treatment of ACTH-dependent or adrenal Cushing's syndrome. Recently, AG has been shown to inhibit ACTH receptor (ACTH-R) mRNA expression in ovine adrenocortical cells in a time-dependent fashion. To investigate whether ACTH-R down-regulation will also be induced in tumor cells, we studied the effect of AG on ACTH-R expression in the human NCI-h295 adrenocortical carcinoma cell line, which expresses functional ACTH receptors and produces steroids of the glucocorticoid, mineralocorticoid and androgen pathway. The cells were incubated in triplicate with increasing doses of AG (3, 30, 300 microM) which suppressed steroid secretion dose-dependently. After 48 h, cells were harvested, and total RNA was extracted, electrophoresed, blotted and hybridized with a human ACTH-R cDNA probe. In parallel experiments, after preincubation with AG the cells were stimulated with ACTH (10 nM) for 10 min and the intracellular cAMP accumulation was determined by RIA. AG significantly suppressed the baseline ACTH-R mRNA expression in a dose-dependent fashion (300 microM AG, 5+/-1%; 30 microM AG, 64+/-1%; 3 microM AG, 108+/-19% compared with control cells, 100+/-11%). The reduced ACTH-R mRNA expression was paralleled by low ACTH-induced cAMP accumulation indicating reduced expression of the ACTH-R protein. The adrenostatic compound metyrapone, an inhibitor of 11beta-hydroxylase activity, also suppressed ACTH-R mRNA expression in a similar fashion. Stimulation of the protein kinase A pathway by simultaneous incubation of ACTH (10 nM) or forskolin (10 microM) together with AG was not able to overcome the steroid biosynthesis blockade, but reversed the inhibitory effects of AG on the ACTH-R mRNA expression. Also, cortisol (12 microM) reversed the AG-induced ACTH-R mRNA expression. We conclude that AG induces profound ACTH-R down-regulation in the NCI-h295 cell line either by affecting the gene expression or by decreasing transcript accumulation via an effect on RNA stability. This novel action of AG can be reversed by stimulation of the cAMP pathway and of the glucocorticoid-mediated signal transduction cascade. As the down-regulation occurs in vitro at concentrations which are reached during treatment with AG in humans it may contribute to its therapeutic activity in adrenal disease.

Ca2+ signaling in human fetoplacental vasculature: effect of CGRP on umbilical vein smooth muscle cytosolic Ca2+ concentration

2005 ◽  
Vol 289 (2) ◽  
pp. H960-H967 ◽  
Author(s):  
Yuan-Lin Dong ◽  
Sujatha Vegiraju ◽  
Chandrasekhar Yallampalli
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Umbilical Vein ◽  
Scatchard Analysis ◽  
Intracellular Camp ◽  
Camp Accumulation ◽  
Atpase Inhibitor ◽  
Intracellular Ca ◽  
Intracellular Camp Accumulation

CGRP is a potent vasodilator with increased levels in fetoplacental circulation during late pregnancy. We have recently demonstrated that acute CGRP exposure to fetoplacental vessels in vitro induced vascular relaxation, but the signaling pathway of CGRP in fetoplacental vasculature remains unclear. We hypothesized that CGRP relaxes fetoplacental vasculature via regulating smooth muscle cytosolic Ca2+ concentrations. In the present study, by using human umbilical vein smooth muscle (HUVS) cells (HUVS-112D), we examined CGRP receptors, cAMP generation, and changes in cellular Ca2+ concentrations on CGRP treatment. These cells express mRNA for CGRP receptor components, calcitonin receptor-like receptor, and receptor activity-modifying protein-1. Direct saturation binding for 125I-labeled CGRP to HUVS cells and Scatchard analysis indicate specificity of the receptors for CGRP [dissociation constant ( KD) = 67 nM, maximum binding capcity (Bmax) = 2.7 pmol/million cells]. Exposure of HUVS cells to CGRP leads to a dose-dependent increase in intracellular cAMP accumulation, and this increase is prevented by CGRP antagonist CGRP8–37. Using fura-2-loaded HUVS cells, we monitored the effects of CGRP on intracellular Ca2+ concentration ([Ca2+]i). In the presence of extracellular Ca2+, bradykinin (10−6 M), a fetoplacental vasoconstrictor, increases HUVS cells [Ca2+]i concentration. CGRP (10−8 M) abolishes bradykinin-induced [Ca2+]i elevation. When the cells were pretreated with glibenclamide, an ATP-sensitive potassium channel blocker, the CGRP actions on bradykinin-induced Ca2+ influx were profoundly inhibited. In the absence of extracellular Ca2+, CGRP (10−8 M) attenuated the increase of [Ca2+]i induced by a sarcoplasmic reticulum Ca2+ pump ATPase inhibitor thapsigargin (10−5 M). Furthermore, Rp-cAMPS, a cAMP-dependent protein kinase A inhibitor, blocks CGRP actions on thapsigargin-induced Ca2+ release from sarcoplasmic reticulum. Our results suggested that CGRP relaxes human fetoplacental vessels by not only inhibiting the influx of extracellular Ca2+ but also attenuating the release of intracellular Ca2+ from the sarcoplasmic reticulum, and these actions might be attributed to CGRP-induced intracellular cAMP accumulation.

scholarly journals Nonmuscle Myosin IIA (Myosin Heavy Polypeptide 9): A Novel Class of Signal Transducer Mediating the Activation of Gαh/Phospholipase C-δ1 Pathway

Endocrinology ◽  
2010 ◽  
Vol 151 (3) ◽  
pp. 876-885 ◽  
Author(s):  
Yuan-Feng Lin ◽  
Tien-Shun Yeh ◽  
Sung-Fang Chen ◽  
Yu-Hui Tsai ◽  
Chih-Ming Chou ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Ternary Complex ◽  
Tissue Transglutaminase ◽  
Spectrometric Analysis ◽  
Target Cells ◽  
Intracellular Camp ◽  
Signal Transducer ◽  
Filament Protein ◽  
Intracellular Camp Accumulation ◽  
Stimulation Of

The dimeric Gh protein is comprised of α (tissue transglutaminase) and β (Calreticulin) subunits and known to be associated with FSH-, oxytocin-, or epinephrine-receptors/functions in their respective target cells. After establishing the FSH-induced activation of Gαh/phospholipase C (PLC)-δ1 pathway in rat Sertoli cells (SCs), we have attempted to identify a possible Gαh-coupled novel FSH receptor (FSH-R). Remarkably, a protein with approximately 240-kDa molecular mass was coimmunoprecipitated with Gαh in the fractionated membrane proteins of rat SCs. The protein was identified as myosin heavy polypeptide 9 (MyH9) by mass spectrometric analysis and immunoblotting. In addition, immunoprecipitation analysis reveals that MyH9 is constitutively associated with classical Gs-coupled FSH-R and inactive GDP-bound Gαh at resting state of rat SCs, but did not interact with FSH directly as judged by Far-Western analysis. Upon the stimulation of higher levels of extracellular FSH (>1000 IU/liter), classical FSH-R induces the phosphorylation of MyH9, the dissociation of active GTP-bound Gαh from FSH-R:MyH9 complexes, and the elicitation of Gαh/PLC-δ1 pathway-dependent Ca2+-influx in rat SCs. Furthermore, the specific inhibition of MyH9 ATPase activity with Blebbistatin dose-dependently suppressed FSH-induced Gαh/PLC-δ1 signaling and Ca2+-influx, but not intracellular cAMP accumulation in rat SCs, implying that MyH9 mediates FSH-induced activation of Gαh/PLC-δ1/IP3/Ca2+-influx pathway in rat SCs. This is the first to demonstrate that the filament protein MyH9 constitutively forms a ternary complex with FSH-R and inactive GDP-bound Gαh. At higher FSH levels, this ternary complex executes an alternative signaling of classical Gs-coupled FSH-R through activating a Gs/cAMP-independent, Gαh/PLC-δ1 pathway in rat SCs.

scholarly journals Effect of Soluble Adenylyl Cyclase (ADCY10) Inhibitors on the LH-Stimulated cAMP Synthesis in Mltc-1 Leydig Cell Line

2021 ◽  
Vol 22 (9) ◽  
pp. 4641
Author(s):  
Thi Mong Diep Nguyen ◽  
Laura Filliatreau ◽  
Danièle Klett ◽  
Nong Van Hai ◽  
Nguyen Thuy Duong ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Leydig Cells ◽  
Intracellular Camp ◽  
Camp Accumulation ◽  
Adenylyl Cyclases ◽  
High Concentration ◽  
Soluble Adenylyl Cyclase ◽  
Bicarbonate Ions ◽  
Intracellular Camp Accumulation

In contrast to all transmembrane adenylyl cyclases except ADCY9, the cytosolic soluble adenylyl cyclase (ADCY10) is insensitive to forskolin stimulation and is uniquely modulated by calcium and bicarbonate ions. In the present paper, we focus on ADCY10 localization and a kinetic analysis of intracellular cAMP accumulation in response to human LH in the absence or presence of four different ADCY10 inhibitors (KH7, LRE1, 2-CE and 4-CE) in MTLC-1 cells. ADCY10 was immuno-detected in the cytoplasm of MLTC-1 cells and all four inhibitors were found to inhibit LH-stimulated cAMP accumulation and progesterone level in MLTC-1 and testosterone level primary Leydig cells. Interestingly, similar inhibitions were also evidenced in mouse testicular Leydig cells. In contrast, the tmAC-specific inhibitors ddAdo3′ and ddAdo5′, even at high concentration, exerted weak or no inhibition on cAMP accumulation, suggesting an important role of ADCY10 relative to tmACs in the MLTC-1 response to LH. The strong synergistic effect of HCO3− under LH stimulation further supports the involvement of ADCY10 in the response to LH.

The effects of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on intracellular adenosine 3′,5′-monophosphate (cAMP) levels and GH secretion in ovine and rat somatotrophs

1996 ◽  
Vol 148 (2) ◽  
pp. 197-205 ◽  
Author(s):  
D Wu ◽  
C Chen ◽  
J Zhang ◽  
C Y Bowers ◽  
I J Clarke
Keyword(s):  
Additive Effect ◽  
Intracellular Camp ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Camp Accumulation ◽  
Gh Secretion ◽  
Rat Pituitary ◽  
Rat Pituitary Cells ◽  
Camp Levels

Abstract The mechanism of action of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on GH release was investigated in ovine and rat pituitary cells in vitro. In partially purified sheep somatotrophs, GHRP-2 and GH-releasing factor (GRF) increased intracellular cyclic AMP (cAMP) concentrations and caused GH release in a dose-dependent manner; GHRP-6 did not increase cAMP levels. An additive effect of maximal doses of GRF and GHRP-2 was observed in both cAMP and GH levels whereas combined GHRP-6 and GHRP-2 at maximal doses produced an additive effect on GH release only. Pretreatment of the cells with MDL 12,330A, an adenylyl cyclase inhibitor, prevented cAMP accumulation and the subsequent release of GH that was caused by either GHRP-2 or GRF. The cAMP antagonist, Rp-cAMP also blocked GH release in response to GHRP-2 and GRF. The cAMP antagonist did not prevent the effect of GHRP-6 on GH secretion whereas MDL 12,330A partially reduced the effect. An antagonist for the GRF receptor, [Ac-Tyr1,d-Arg2]-GRF 1–29, significantly diminished the effect of GHRP-2 and GRF on cAMP accumulation and GH release, but did not affect GH release induced by GHRP-6. Somatostatin prevented cAMP accumulation and GH release responses to GHRP-2, GRF and GHRP-6. Ca2+ channel blockade did not affect the cAMP increase in response to GHRP-2 or GRF but totally prevented GH release in response to GHRP-2, GRF and GHRP-6. These results indicated that GHRP-2 acts on ovine pituitary somatotrophs to increase cAMP concentration in a manner similar to that of GRF; this occurs even during the blockade of Ca2+ influx. GHRP-6 caused GH release without an increase in intracellular cAMP levels. GH release in response to all three secretagogues was reduced by somatostatin and was dependent upon the influx of extracellular Ca2+. The additive effect of GHRP-2 and GRF or GHRP-6 suggested that the three peptides may act on different receptors. In rat pituitary cell cultures, GHRP-6 had no effect on cAMP levels, but potentiated the effect of GRF on cAMP accumulation. The synergistic effect of GRF and GHRP-6 on cAMP accumulation did not occur in sheep somatotrophs. Whereas GHRP-2 caused cAMP accumulation in sheep somatotrophs, it did not do so in rat pituitary cells. These data indicate species differences in the response of pituitary somatotrophs to the GHRPs and this is probably due to different subtypes of GHRP receptor in rat or sheep. Journal of Endocrinology (1996) 148, 197–205

γ-MSH increases intracellular cAMP accumulation and GnRH release in vitro and LH release in vivo

FEBS Letters ◽  
2003 ◽  
Vol 543 (1-3) ◽  
pp. 66-70 ◽  
Author(s):  
S.A. Stanley ◽  
S. Davies ◽  
C.J. Small ◽  
J.V. Gardiner ◽  
M.A. Ghatei ◽  
...  
Keyword(s):  
Intracellular Camp ◽  
Camp Accumulation ◽  
Gnrh Release ◽  
Lh Release ◽  
Release In Vitro ◽  
Intracellular Camp Accumulation
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