scholarly journals Autocrine role of adrenomedullin in the human adrenal cortex

2001 ◽  
Vol 170 (1) ◽  
pp. 259-265 ◽  
Author(s):  
LM Thomson ◽  
S Kapas ◽  
M Carroll ◽  
JP Hinson
Keyword(s):  
Cell Line ◽  
Second Messenger ◽  
Cell Types ◽  
Zona Glomerulosa ◽  
Adrenocortical Cell ◽  
Gene Encoding ◽  
Calcitonin Gene ◽  
H295r Cells ◽  
Glomerulosa Cells

Previous studies from our laboratory have reported that adrenomedullin is synthesised in rat zona glomerulosa cells. In the present studies, it was found that the human adrenocortical cell line H295R expresses the gene encoding adrenomedullin, and that immunoreactive adrenomedullin is released into the culture medium. Furthermore, it was found that secretion of adrenomedullin is regulated by angiotensin II and forskolin. Studies on the actions of adrenomedullin and calcitonin gene-related peptide (CGRP) revealed a stimulatory effect of adrenomedullin, but not of CGRP, on aldosterone and cortisol secretion. These data suggest that adrenomedullin is not acting by a CGRP receptor-mediated mechanism in the H295R cell line. Adrenomedullin was also found to increase cAMP production, suggesting that in the adrenal, as in other cell types, cAMP is a second messenger for adrenomedullin action. However, the effects of adrenomedullin were not fully mimicked by forskolin, possibly suggesting a role for an additional second messenger. The presence of mRNA encoding both the putative adrenomedullin receptors, L1 and calcitonin receptorlike receptor/receptor-associated modulatory protein 2 (CRLR/RAMP-2), was demonstrated in H295R cells, but RAMP-1 was not detected, suggesting that these cells do not express the CGRPI receptor CRLR/RAMP-1. Taken together, these data have demonstrated that adrenomedullin is synthesised and secreted by H295R cells. The observed rate of adrenomedullin synthesis suggests that this peptide exerts a paracrine/autocrine effect in this adrenocortical cell line, probably acting through a specific adrenomedullin receptor, to stimulate steroidogenesis and increase aldosterone synthase expression.

scholarly journals Thapsigargin inhibits voltage-activated calcium channels in adrenal glomerulosa cells

1993 ◽  
Vol 296 (2) ◽  
pp. 309-312 ◽  
Author(s):  
M F Rossier ◽  
C P Python ◽  
M M Burnay ◽  
W Schlegel ◽  
M B Vallotton ◽  
...  
Keyword(s):  
Cell Types ◽  
Inhibitory Effect ◽  
Zona Glomerulosa ◽  
High Threshold ◽  
Patch Clamp Technique ◽  
Dual Effect ◽  
Blocking Voltage ◽  
Current Voltage ◽  
Glomerulosa Cells ◽  
Ca2 Channels

Thapsigargin, an inhibitor of the microsomal Ca2+ pumps, has been extensively used to study the intracellular Ca2+ pool participating in the generation of the agonist-induced Ca2+ signal in various cell types. A dual effect of this agent was observed in bovine adrenal zona glomerulosa cells. At nanomolar concentrations, thapsigargin stimulated a sustained Ca2+ influx, probably resulting from Ca(2+)-store depletion. In contrast, when added at micromolar concentrations, thapsigargin prevented the rise in cytosolic free Ca2+ concentration ([Ca2+]c) induced by K+. This inhibitory effect of thapsigargin on voltage-activated Ca2+ channels was confirmed by measuring Ba2+ currents by the patch-clamp technique. Both low-threshold (T-type) and high-threshold (L-type) Ca2+ channels were affected by micromolar concentrations of thapsigargin. Analysis of the current-voltage relationship for T-type channels revealed that thapsigargin did not modify the sensitivity of these channels to the voltage, but decreased the maximal current flowing through the channels. In conclusion, thapsigargin appears to exert a dual effect on adrenal glomerulosa cells. At lower concentrations, this agent induces a sustained Ca2+ entry, whereas at higher concentrations it decreases [Ca2+]c by blocking voltage-activated Ca2+ channels.

Identification of human thrombin-activatable fibrinolysis inhibitor in vascular and inflammatory cells

2011 ◽  
Vol 105 (06) ◽  
pp. 999-1009 ◽  
Author(s):  
Joellen Lin ◽  
Mathieu Garand ◽  
Branislava Zagorac ◽  
Steven Schadinger ◽  
Corey Scipione ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Umbilical Vein ◽  
Inflammatory Cells ◽  
Cell Types ◽  
Rt Pcr ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Gene Encoding ◽  
Fibrinolysis Inhibitor

SummaryTAFI (thrombin-activatable fibrinolysis inhibitor) is a carboxypeptidase zymogen originally identified in plasma. The TAFI pathway helps to regulate the balance between the coagulation and fibrinolytic cascades. Activated TAFI (TAFIa) can also inactivate certain pro-inflammatory mediators, suggesting that the TAFI pathway may also regulate communication between coagulation and inflammation. Expression in the liver is considered to be the source of plasma TAFI. TAFI has also been identified in platelets and CPB2 (the gene encoding TAFI) mRNA has been detected in megakaryocytic cell lines as well as in endothelial cells. We have undertaken a quantitative analysis of CPB2 mRNA and TAFI protein in extrahepatic cell types relevant to vascular disease. Using RT-PCR and quantitative RT-PCR, we detected CPB2 mRNA in the human megakaryoblastic cell lines MEG-01 and Dami, the human monocytoid cell line THP-1 as well as THP-1 cells differentiated into a macrophage-like phenotype, and in primary human umbilical vein and coronary artery endothelial cells. CPB2 mRNA abundance in MEG-01, Dami, and THP-1 cells was modulated by the state of differentiation of these cells. Using a recently developed TAFIa assay, we detected TAFI protein in the lysates of the human hepatocellular carcinoma cell line HepG2 as well as in MEG-01 and Dami cells and in the conditioned medium of HepG2 cells, differentiated Dami cells, and THP-1 macrophages. We have obtained clear evidence for extrahepatic expression of TAFI, which has clear implications for the physiological and pathophysiological functions of the TAFI pathway.

scholarly journals cAMP-dependent protein kinase activation inhibits proliferation and enhances apoptotic effect of tumor necrosis factor-α in NCI-H295R adrenocortical cells

2004 ◽  
Vol 33 (2) ◽  
pp. 511-522 ◽  
Author(s):  
J Liu ◽  
X-D Li ◽  
A Ora ◽  
P Heikkilä ◽  
A Vaheri ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Line ◽  
Adrenocortical Cell ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
H295r Cells ◽  
Induced Apoptosis ◽  
Factor Α

Adrenocorticotropin is the major regulator of adrenocortical development and function. It acts mainly through the cAMP-dependent protein kinase A (PKA) pathway. Our aim was to study the interaction of tumor necrosis factor-α (TNFα) and the PKA pathway in adrenocortical cell proliferation and apoptosis. The PKA activator Dibutyryl cAMP ((Bu)2cAMP) strongly induced differentiation and inhibited proliferation in the human adrenocortical cell line NCI-H295R (H295R). TNFα induced apoptosis of H295R cells. Interestingly, (Bu)2cAMP treatment clearly enhanced TNFα-induced apoptosis in H295R cells, but not in another human adrenocortical cell line SW-13, the mouse adrenocortical Y-1 cell line or the human HeLa cell line. This synergistic effect was not due to the (Bu)2cAMP-induced glucocorticoid secretion since dexamethasone had no significant effect on the TNFα-induced apoptosis. (Bu)2cAMP treatment rapidly increased the expression of the proto-oncogene c-myc in H295R cells, but not in SW-13, Y-1 or HeLa cells. In transient c-myc transfection assay, c-myc expression associated with decreased expression of the proliferation marker Ki-67 in H295R cells. In conclusion, cAMP-dependent protein kinase activation reduced proliferation and augmented TNFα-induced apoptosis in adrenocortical H295R cells, and these effects were associated with increased c-myc expression.

Both TASK-3 and TREK-1 two-pore loop K channels are expressed in H295R cells and modulate their membrane potential and aldosterone secretion

2008 ◽  
Vol 295 (6) ◽  
pp. E1480-E1486 ◽  
Author(s):  
Tanja Brenner ◽  
Kevin M. O'Shaughnessy
Keyword(s):  
Membrane Potential ◽  
Cell Line ◽  
Potassium Current ◽  
Dominant Negative ◽  
Aldosterone Secretion ◽  
K2p Channel ◽  
Selective Permeability ◽  
K2p Channels ◽  
H295r Cells ◽  
Glomerulosa Cells

The rate of aldosterone synthesis by adrenal glomerulosa cells relies on the selective permeability of the glomerulosa cell to K+ ions. In rodent and bovine adrenal glomerulosa cells, this background potassium current is provided by a two-pore loop potassium (K2P) channel: largely TASK-3 in the rat and TREK-1 in the cow. The nature of the K2P channel in the human adrenal cortex is not known, and we have addressed this issue here using the H295R human adrenal cell line. We show that these cells express mRNA and protein for both TASK-3 and TREK-1 K2P channels. Using a potentiometric dye (FMP), we also show that TASK-3 and TREK-1 channel modulators can affect the membrane potential of H295R cells. Transfecting H295R cells with TASK-3 or TREK-1 dominant-negative mutants (TASK-3 G95E or TREK-1 G144E) produced depolarization of H295R cells and altered K-stimulated aldosterone secretion. Finally, transfection of a constitutively active mutant of Gαq into H295R cells (GTPase-deficient Gαq-QL) depolarized them and increased basal aldosterone secretion. Taken together, our data support both TASK-3 and TREK-1 as being functionally operational in the H295R cell line. This suggests that human adrenal glomerulosa cells may utilize both of these K2P channels for their background potassium current.

scholarly journals Control of IntracellularFrancisella tularensisby Different Cell Types and the Role of Nitric Oxide

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Sarah L. Newstead ◽  
Amanda J. Gates ◽  
M. Gillian Hartley ◽  
Caroline A. Rowland ◽  
E. Diane Williamson ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Line ◽  
Rational Design ◽  
Cell Types ◽  
No Production ◽  
Macrophage Cell Line ◽  
Macrophage Cell ◽  
Schu S4 ◽  
Intracellular Infections

Reactive nitrogen is critical for the clearance ofFrancisella tularensisinfections. Here we assess the role of nitric oxide in control of intracellular infections in two murine macrophage cell lines of different provenance: the alveolar macrophage cell line, MH-S, and the widely used peritoneal macrophage cell line, J774A.1. Cells were infected with the highly virulent Schu S4 strain or with the avirulent live vaccine strain (LVS) with and without stimuli. Compared to MH-S cells, J774A.1 cells were unresponsive to stimulation and were able to control the intracellular replication of LVS bacteria, but not of Schu S4. In MH-S cells, Schu S4 demonstrated control over cellular NO production. Despite this, MH-S cells stimulated with LPS or LPS and IFN-γwere able to control intracellular Schu S4 numbers. However, only stimulation with LPS induced significant cellular NO production. Combined stimulation with LPS and IFN-γproduced a significant reduction in intracellular bacteria that occurred whether high levels of NO were produced or not, indicating that NO secretion is not the only defensive cellular mechanism operating in virulentFrancisellainfections. Understanding howF. tularensisinteracts with host macrophages will help in the rational design of new and effective therapies.

scholarly journals Agonist-Induced Phosphorylation of the Endogenous AT1 Angiotensin Receptor in Bovine Adrenal Glomerulosa Cells

1998 ◽  
Vol 12 (5) ◽  
pp. 634-644 ◽  
Author(s):  
Roger D. Smith ◽  
Albert J. Baukal ◽  
Annamaria Zolyomi ◽  
Zsuzsanna Gaborik ◽  
Laszlo Hunyady ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Receptor Activation ◽  
Zona Glomerulosa ◽  
Target Cells ◽  
Ang Ii ◽  
Kinase C ◽  
Glomerulosa Cells ◽  
At1a Receptor ◽  
G Protein Coupled

Abstract A polyclonal antibody was raised in rabbits against a fusion protein immunogen consisting of bacterial maltose-binding protein coupled to a 92-amino acid C-terminal fragment of the rat AT1b angiotensin II (Ang II) receptor. The antibody immunoprecipitated the photoaffinity-labeled bovine AT1 receptor (AT1-R), but not the rat AT2 receptor, and specifically stained bovine adrenal glomerulosa cells and AT1a receptor-expressing Cos-7 cells, as well as the rat adrenal zona glomerulosa and renal glomeruli. The antibody was employed to analyze Ang II-induced phosphorylation of the endogenous AT1-R immunoprecipitated from cultured bovine adrenal glomerulosa cells. Receptor phosphorylation was rapid, sustained for up to 60 min, and enhanced by pretreatment of the cells with okadaic acid. Its magnitude was correlated with the degree of ligand occupancy of the receptor. Activation of protein kinase A and protein kinase C (PKC) also caused phosphorylation of the receptor, but to a lesser extent than Ang II. Inhibition of PKC by staurosporine augmented Ang II-stimulated AT1-R phosphorylation, suggesting a negative regulatory role of PKC on the putative G protein-coupled receptor kinase(s) that mediates the majority of AT1-R phosphorylation. The antibody should permit further analysis of endogenous AT1-R phosphorylation in Ang II target cells.

scholarly journals Type 2 Iodothyronine Selenodeiodinase Is Expressed throughout the Mouse Skeleton and in the MC3T3-E1 Mouse Osteoblastic Cell Line during Differentiation

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 195-200 ◽  
Author(s):  
Cecilia H. A. Gouveia ◽  
Marcelo A. Christoffolete ◽  
Clarissa R. Zaitune ◽  
José Miguel Dora ◽  
John W. Harney ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Cell Line ◽  
Bone Cells ◽  
Bone Development ◽  
Cell Types ◽  
Osteoblastic Cell ◽  
Dependent Manner ◽  
Osteoblastic Cell Line ◽  
Serum T3

Thyroid hormone affects multiple aspects of bone metabolism, but little is known about thyroid hormone deiodination in bone cells except that cultures of skeletal cells and bone organ express types 1 and 2 iodothyronine deiodinases (D1 and D2) mRNAs. In the present study, outer ring deiodination (ORD) activity was detected in bone extracts of multiple sites of the mouse skeleton, bone marrow, and the MC3T3-E1 osteoblastic cell line. In all tissues, ORD was detected using 125I-rT3 or 125I-T4 as substrates and was found to be 6-n-propylthiouracil insensitive, display a Michaelis constant (T4) of approximately 1 nm, increase about 3-fold in hypo- and virtually disappear in thyrotoxicosis. Extracts of calvaria had the lowest ORD activity, whereas tibial and femoral extracts had roughly three times as much. The absence of ORD activity in bone extracts from mice with targeted disruption of the Dio2 gene confirms the principal role of D2 in this tissue. In the MC3T3-E1 osteoblasts, D2 activity increased in a time-dependent manner after plating, and with the content of selenium in the media, reaching a maximum 5–7 d later as cells attained more than 90% confluence. In these cells D2 half-life is about 30–40 min, which is further accelerated by exposure to substrate and stabilized by the proteasome inhibitor, MG132. Treatment with vitamin D [1,25(OH)2VD]-induced D2 activity by 2- to 3-fold as early as 24 h, regardless of the level of cell confluence, but estradiol, PTH, forskolin, leptin, TNFα, TGFβ, and dexamethasone did not affect D2. Given the role of D2 in other cell types and processes, it is likely that bone ORD not only plays a role in bone development and adult bone T3 homeostasis but also contributes to extrathyroidal T3 production and maintenance of serum T3.

Neuropeptide Y modulates the sensitivity of the rat adrenal cortex to stimulation by ACTH

1995 ◽  
Vol 145 (2) ◽  
pp. 283-289 ◽  
Author(s):  
J P Hinson ◽  
L A Cameron ◽  
S Kapas
Keyword(s):  
Angiotensin Ii ◽  
Neuropeptide Y ◽  
Adrenal Cortex ◽  
Mammalian Species ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Steroid Secretion ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

Abstract Neuropeptide Y (NPY) has been identified in nerves supplying the adrenal cortex of several mammalian species, although its function in this tissue is unknown. The present studies, employing adrenocortical cells prepared by collagenase digestion, have shown that NPY, in the absence of other stimulants, has no effect on steroid secretion by the rat adrenal over a range of peptide concentrations (10−11 to 10 −6 mol/l). However, in the presence of physiological concentrations of ACTH, which are submaximal for the stimulation of aldosterone secretion, NPY (10−6 mol/l) significantly enhanced the secretion rate of aldosterone by rat zona glomerulosa cells in response to ACTH. This effect was specific to the rat zona glomerulosa as NPY had no effect on the response to ACTH in rat zona fasciculata cells. The effect of NPY appears to be biphasic, however, as NPY significantly attenuated the steroidogenic response to supramaximal ACTH concentrations: in rat zona glomerulosa cells the aldosterone response to 10 −8 mol ACTH/l was significantly inhibited by NPY. The effect of NPY on the ACTH response appeared to be mediated by changes in the cAMP response. NPY had no effect on the steroidogenic response to potassium ions (K+), but enhanced the response to angiotensin II. NPY (10 −6 mol/l) significantly stimulated inositol 1,4,5-trisphosphate (InsP3) production although this concentration of peptide had no effect on steroid secretion. The effects of NPY on InsP3 production were additive with those of angiotensin II. These results suggest that the role of NPY in the adrenal cortex may be to regulate the sensitivity of the zona glomerulosa to peptide stimulation. Journal of Endocrinology (1995) 145, 283–289

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