Marcel D. 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.

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).

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.

Effects of ACTH and angiotensin II on cytosolic calcium in cultured adrenal glomerulosa cells. Role of cAMP production in the ACTH effect

We have used microspectrofluorometry and video imaging techniques in order to study and compare the changes in intracellular calcium concentrations [( Ca2+]i) of individual Fura-2 loaded glomerulosa cells cultured for three days and stimulated either with angiotensin II (AT), K+, or adrenocorticotropin (ACTH). As previously demonstrated for freshly isolated cells, K+ ion induces an immediate increase in [Ca2+]i, although AT induces a biphasic response, characterized by an initial transient spike, followed by a sustained plateau. In this study, we demonstrate, for the first time, that ACTH is able to induce a [Ca2+]i increase in cultured glomerulosa cells from rat and bovine sources. Moreover, it is clear that the pattern of [Ca2+]i increase elicited by ACTH is different from that observed with AT. In most cases, addition of ACTH leads to a slow increase in [Ca2+]i after a long latency period ranging from 10-15 min, which could be correlated to cAMP time-production. The present results show that: (a) in the absence of extracellular Ca2+, ACTH does not increase [Ca2+]i; (b) the response develops slowly and cases immediately after [Ca2+]e depletion or addition of calcium channel blockers, such as nifedipine or omega-conotoxin; (c) the addition of the calcium channel agonist Bay K 8644 enhances the ACTH response; (d) the cAMP analog, 8-Br-cAMP, induces an increase in [Ca2+]i similar to that observed with ACTH, which is also dependent of the presence of calcium in the extracellular medium; (e) time-production of ACTH-induced cAMP follows quite well the increase in [Ca2+]i; (f) Bay K 8644 also enhances the 8-Br-cAMP induced increase in [Ca2+]i; and (g) ACTH-induced Cai response is inhibited by the specific protein kinase A blocker, HA1004. These observations, combined with previous results obtained on the effects of ACTH on calcium currents and action potentials, suggest that the [Ca2+]i increase induced by ACTH results from a calcium influx through dihydropyridine and omega-conotoxin sensitive calcium channels, which need to be phosphorylated by cAMP for full activation. The use of video-imaging techniques has allowed us to examine the spatial distribution of changes in [Ca2+]i in single cells. The ability to simultaneously record images of a number of cells confirm the heterogeneity of cellular responses, and corroborate results obtained through photocounting only. Our results indicate that ACTH initially increases [Ca2+]i locally beneath the cell membrane and throughout the cell thereafter, whereas angiotensin II elicits a more prominent effect in certain regions of the cell and eventually extends to the entire cell surface.

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...

VCAM-1 is internalized by a clathrin-related pathway in human endothelial cells but its α4β1 integrin counter-receptor remains associated with the plasma membrane in human T lymphocytes

Lymphocyte extravasation involves a step(s) of de‐adhesion to allow trans‐ and subendothelial migration in response to inflammatory signals. We show here that ligated VCAM‐1 was rapidly internalized (t1/2 14.5 min) in ECV 304 endothelial cells and in TNF‐α‐primed human umbilical vein‐derived endothelial cells (t1/2 11.2 min). The process required energy (ATP), intracellular Ca2+ , an intact cytoskeletal network and active protein kinases. The internalization of VCAM‐1 involved a clathrin‐dependent pathway based on the observations that 1) it was inhibited in cells treated with lysosomotropic agents or with a hypertonic concentration of sucrose, and 2) internalized VCAM‐1 colocalized with clathrin. In contrast, the cross‐linked α4β1 integrin counter‐receptor of VCAM‐1 remained associated with the plasma membrane of purified peripheral T and Jurkat cells. Our results suggest a model where VCAM‐1 would initially participate in the retention of T cells to the endothelium by binding α4β1 integrin. Lymphocyte de‐adhesion would be facilitated as a result of the internalization of VCAM‐1. The persistent cell surface expression of α4β1 integrin would allow the migrating T cells to interact with and receive signal(s) from its fibronectin ligand of the extracellular matrix.

Comparative Involvement of Cyclic Nucleotide Phosphodiesterases and Adenylyl Cyclase on Adrenocorticotropin-Induced Increase of Cyclic Adenosine Monophosphate in Rat and Human Glomerulosa Cells1

The present study investigated the role and identity of cyclic nucleotide phosphodiesterases (PDEs) in the regulation of basal and ACTH-stimulated levels of intracellular cAMP in human and rat adrenal glomerulosa cells. Comparative dose-response curves indicated that maximal hormone-stimulated cAMP accumulation was 11- and 24-fold higher in human and rat cells, compared with cAMP production obtained in corresponding membranes, respectively. Similarly to 3-isobutyl-1-methyl-xanthine, 25 microM erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA, a specific PDE2 inhibitor), caused a large increase in ACTH-stimulated cAMP accumulation; by contrast, it did not change cAMP production in membranes. Moreover, in membrane fractions, addition of 10 microM cGMP inhibited ACTH-induced cAMP production, an effect completely reversed by addition of 25 microM EHNA. These results indicate that PDE2 activity is involved in the regulation of cAMP accumulation induced by ACTH, and suggest that ACTH inhibits this activity. Indeed, time-course studies indicated that ACTH induced a rapid decrease in cGMP production, resulting in PDE2 inhibition, which in turn, contributed [with adenylyl cyclase (AC) activation] to an accumulation in cAMP for 15 min. Thereafter, cAMP content decreased, because of cAMP-stimulated PDE2, as confirmed by measurement of PDE activity that was activated by ACTH, but only after a 10-min incubation. Hence, we demonstrate that the ACTH-induced increase in intracellular cAMP is the result of a balance between activation of AC and direct modulation of PDE2 activity, an effect mediated by cGMP content. Although similar results were observed in both models, PDE2 involvement is more important in rat than in human adrenal glomerulosa cells, whereas AC is more stimulated in human than in rat glomerulosa cells.

Modulation of membrane potential and ionic currents by the AT1 and AT2 receptors of angiotensin II

Angiotensin II, the principal effector of the renin-angiotensin system, modulates various ionic currents. Its effects on potassium currents, including outward transient potassium current, the inward or outward rectifiers, as well as Ca(2+)- activated potassium currents, is well described. Other ionic currents, such as voltage-dependent calcium currents, cationic or chloride currents, are also altered by the hormone. All these effects provoke changes in membrane potential, such as modulation of action potential firing or resting membrane potential and control intracellular calcium concentration. Summarized here are the results obtained on these membrane electrical properties using electrophysiological recordings.