Nicole Gallo-Payet

The angiotensin AT2 receptor modulates T‐type calcium current in non‐differentiated NG108‐15 cells

We report here that angiotensin II (AII) and the AT2 receptor‐selective ligand, CGP 42112, modulate the T‐type calcium current in non‐differentiated NG108‐15 cells, which express only AT2 receptors. Both peptides decrease the T‐type calcium current at membrane potentials above −40 mV and shift the current—voltage curve at lower potentials with maximal effect between 5 and 10 min after application. These data describe a new cellular response to AII and suggest that the AT2 receptor mediates certain neurophysiological actions of this hormone.

Nitric oxide, a new second messenger involved in the action of angiotensin II on neuronal differentiation of NG108-15 cells.

Nitric Oxide (NO) is a gas that diffuses freely through membranes of target cells to activate cGMP formation. NO is synthesised from arginine, by a family of Nitric Oxide Synthase (NOS). In the brain, NO influences synaptic plasticity, apoptosis and development. It has been recently shown that angiotensin II (Ang II) could mediate NO production by its two types of receptors, AT1 and AT2. Since we have shown that Ang II, via the AT2 receptor could induce neurite outgrowth and morphological differentiation of NG108-15 cells, the aim of the study was to investigate if NO could be one of the second messengers involved in the Ang II effect. Using the Griess colorimetric assay, we found that Ang II, by its AT2 receptor, induced nitrite formation from NO. This effect was abolished by the N-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. We also found that treatment of the cells with S-nitroso-N-acetylpenicillamine (SNAP), an exogenous source of NO, induced the same morphological differentiation. These results demonstrate that the morphological differentiation induced by the AT2 receptor is partly due to an increase in NO production.

Role of melanocortin receptor accessory proteins in the function of zebrafish melanocortin receptor type 2

In this paper, we identify three different MRAPs in zebrafish, zfMRAP1, zfMRAP2a and zfMRAP2b, and demonstrate that zfMC2R is not functional in the absence of MRAP expression. ZfMRAP1 expression was restricted to adipose tissue and the anterior kidney whereas MRAP2a and MRAP2b were expressed in all the tissues tested. Quantification of surface receptor and immunofluorescence studies indicated that the receptor is unable to translocate to membrane in the absence of MRAP isoforms. MRAP1 and MRAP2b are localized in the plasma membrane in the absence of zfMC2R expression but MRAP2b is retained in perinuclear position. MRAP1 and MRAP2a displayed an equivalent translocation capacity to the membrane of zfMC2R but only zfMRAP1 expression led to intracellular cAMP increases after ACTH stimulation. ZfMRAP2b had no effect on zfMC2R activity but both zfMRAP2 isoforms enhanced the zfMRAP1-assisted cAMP intracellular increase, suggesting an interaction between zfMRAP1 and zfMRAP2s when regulating zfMC2R activity.

Nitric Oxide and Cyclic GMP Are Involved in Angiotensin II AT 2 Receptor Effects on Neurite Outgrowth in NG108-15 Cells

In their undifferentiated state, NG108-15 cells express only the angiotensin II (Ang II) type 2 receptor (AT2). We have previously shown that Ang II induced neurite outgrowth of NG108-15 cells, a process involving sustained activation of p42/p44mapk activity. We have also shown that Ang II stimulates nitric oxide (NO) production. The aim of the present study was to investigate the role of the NO/cyclic GMP (cGMP) cascade in the signal transduction of the AT2 receptor-stimulated neurite outgrowth. Three-day treatment of cells with dbcGMP induced neurite outgrowth as did Ang II. Preincubation with an inhibitor of cGMP-dependent protein kinase, KT5823, resulted in the formation of short neurites, while in the presence of LY83583 or methylene blue, two inhibitors of guanylyl cyclase, cells resembled control cells with only one or two thin processes. Western blot analyses indicated that nNOS was present in NG108-15 cells. Immunoprecipitation with antiphosphotyrosine antibodies showed that Ang II induced NOS activity and increased cGMP production through a Gi-dependent pathway. However, neither L-NAME, KT5823, nor LY83583 affected the activation of p42/p44mapk induced by Ang II, indicating that the pathway NO/guanylyl cyclase/cGMP was not involved in Ang II-induced activation of MAPK. The present results suggest that the neurite outgrowth induced by Ang II results from at least parallel but complementary pathways, one involved in neurite elongation (through the cooperation of MAPK and PKG) and the other involved in sprouting (through cGMP).

The melanocortin receptor subtypes in chicken have high preference to ACTH‐derived peptides

Melanocortin (MC) receptors are widely distributed throughout the body of chicken, like in mammals, and participate in a wide range of physiological functions. To clarify the pharmacological impact of ligands acting in the MC system, we expressed the chicken MC1, MC2, MC3, MC4 and MC5 (cMC1–5) receptors in eukaryotic cells and performed comprehensive pharmacological characterization of the potency of endogenous and synthetic melanocortin peptides. Remarkably, the cMC receptors displayed high affinity for ACTH‐derived peptides and in general low affinity for α‐MSH. It is evident that not only the cMC2 receptor but also the other cMC receptors interact with ACTH‐derived peptide through an epitope beyond the sequence of α‐MSH. The synthetic ligand MTII was found to be a potent agonist whereas HS024 was a potent antagonist at the cMC4 receptor, indicating that these ligands are suitable for physiological studies in chicken. We also show the presence of prohormone convertase 1 (PC1) and PC2 genes in chicken, and that these peptides are coexpressed with proopiomelanocortin (POMC) in various tissues.

Cyclic AMP-independent Involvement of Rap1/B-Raf in the Angiotensin II AT2 Receptor Signaling Pathway in NG108-15 Cells

The angiotensin II (Ang II) type 2 (AT2) receptor is an atypical seven-transmembrane domain receptor. Controversy surrounding this receptor concerns both the nature of the second messengers produced as well as its associated signaling mechanisms. Using the neuronal cell line NG108-15, we have reported previously that activation of the AT2 receptor induced morphological differentiation in a p21 ras -independent, but p42/p44 mapk -dependent mechanism. The activation of p42/p44 mapk was delayed, sustained, and had been shown to be essential for neurite elongation. In the present report, we demonstrate that activation of the AT2 receptor rapidly, but transiently, activated the Rap1/B-Raf complex of signaling proteins. In RapN17- and Rap1GAP-transfected cells, the effects induced by Ang II were abolished, demonstrating that activation of these proteins was responsible for the observed p42/p44 mapk phosphorylation and for morphological differentiation. To assess whether cAMP was involved in the activation of Rap1/B-Raf and neuronal differentiation induced by Ang II, NG108-15 cells were treated with stimulators or inhibitors of the cAMP pathway. We found that dibutyryl cAMP and forskolin did not stimulate Rap1 or p42/p44 mapk activity. Furthermore, adding H-89, an inhibitor of protein kinase A, or Rp-8-Br-cAMP-S, an inactive cAMP analog, failed to impair p42/p44 mapk activity and neurite outgrowth induced by Ang II. The present observations clearly indicate that cAMP, a well known stimulus of neuronal differentiation, did not participate in the AT2 receptor signaling pathways in the NG108-15 cells. Therefore, the AT2 receptor of Ang II activates the signaling modules of Rap1/B-Raf and p42/p44 mapk via a cAMP-independent pathway to induce morphological differentiation of NG108-15 cells.

Role of MAPKs in angiotensin II-induced steroidogenesis in rat glomerulosa cells

Angiotensin II is one of the most important stimuli of rat adrenal glomerulosa cells, stimulating both steroid secretion and growth. In a previous report, we had shown that Ang II promotes cellular hypertrophy, but not proliferation, in rat adrenal glomerulosa cells maintained in primary culture for 3 days. The inhibition of proliferation and stimulation of hypertrophy induced by Ang II involves both p42/p44(mapk) and p38 MAPK activation. The increase in cell protein content induced by Ang II entails formation of a cortical actin ring and Rac-dependent activation of p42/p44(mapk) and p38 MAPK. The present study summarizes these results and provides evidences that Ang II-induced activation of p42/p44(mapk) and p38 MAPK are implicated in aldosterone secretion by enhancing expression of specific steroidogenic proteins such as StAR and 3beta-HSD.

Association of the G Proteinα q/α11-Subunit with Cytoskeleton in Adrenal Glomerulosa Cells: Role in Receptor-Effector Coupling1

In 3-day primary cultures of rat glomerulosa cells, a 30-min preincubation with either 10 μm colchicine (a microtubule-disrupting agent) or 10 μm cytochalasin B (a microfilament-disrupting agent) decreased angiotensin II (Ang II)-induced inositol phosphate accumulation by 50%. Moreover, both drugs decreased inositol phosphate production induced by fluoroaluminate (a nonspecific activator of all G proteins), indicating that both microtubules and microfilaments are essential for phospholipase C activation. Analysis of microfilament- and microtubule-enriched fractions and immunoprecipitation of actin and tubulin revealed that the αq/α11-subunit of the Gq/11 protein was associated with both structures. Ang II stimulation induced a rapid translocation ofα q/α11, microfilaments, and microtubules to the membrane and induced a time-dependent increase in the level ofα q/α11 associated with both microfilaments and microtubules. Moreover, double immunofluorescence staining clearly showed a colocalization of theα q/α...

Association of αs-subunit of the Gs protein with microfilaments and microtubules : Implication during adrenocorticotropin stimulation in rat adrenal glomerulosa cells

The aim of the present study was to investigate if and how microfilaments and microtubules could be involved in the early events of ACTH action. In primary cultures of rat glomerulosa cells, a 30-min preincubation with either 10 μm colchicine (a microtubule-disrupting agent) or 10 μm cytochalasin B (a microfilament-disrupting agent) decreased ACTH-induced cAMP production. Moreover, colchicine decreased cAMP production induced by fluoroaluminate (a nonspecific activator of all G proteins), but not of forskolin (which directly activates adenylyl cyclase). These results indicate that microtubules appear to be essential for the Gs protein activation. In contrast, cytochalasin B decreased the stimulating effect of both fluoroaluminate and forskolin, indicating that microfilaments may be involved in both Gs and adenylyl cyclase activations. Analyses of microfilament- and microtubule-enriched fractions and immunoprecipitation of actin and tubulin indicated that the αs-subunit of the Gs protein was associated wit...

Angiotensin II type 2 receptor promotes adipocyte differentiation and restores adipocyte size in high-fat/high-fructose diet-induced insulin resistance in rats.

This study was aimed at establishing whether specific activation of angiotensin II (ANG II) type 2 receptor (AT2R) modulates adipocyte differentiation and function. In primary cultures of subcutaneous (SC) and retroperitoneal (RET) preadipocytes, both AT2R and AT1R were expressed at the mRNA and protein level. Cells were stimulated with ANG II or the AT2R agonist C21/M24, alone or in the presence of the AT1R antagonist losartan or the AT2R antagonist PD123,319. During differentiation, C21/M24 increased PPARγ expression in both RET and SC preadipocytes while the number of small lipid droplets and lipid accumulation solely increased in SC preadipocytes. In mature adipocytes, C21/M24 decreased the mean size of large lipid droplets. Upon abolishment of AT2R expression using AT2R-targeted shRNAs, expressions of AT2R, aP2, and PPARγ remained very low, and cells were unable to differentiate. In Wistar rats fed a 6-wk high-fat/high-fructose (HFHF) diet, a significant shift toward larger adipocytes was observed in RET and SC adipose tissue depots. C21/M24 treatments for 6 wk restored normal adipocyte size distribution in both these tissue depots. Moreover, C21/M24 and losartan decreased hyperinsulinemia and improved insulin sensitivity impaired by HFHF diet. A strong correlation between adipocyte size area and glucose infusion rate during euglycemic-hyperinsulinemic clamp was observed. These results indicate that AT2R is involved in early adipocyte differentiation, while in mature adipocytes and in a model of insulin resistance AT2R activation restores normal adipocyte morphology and improves insulin sensitivity.