In the present study, 3-day treatment of nondifferentiated NG108-15 cells with 100 nM angiotensin II (Ang II) induces morphological differentiation of neuronal cells characterized by the outgrowth of neurites. These morphological changes are correlated with an increase in the level of polymerized tubulin and in the level of the microtubule-associated protein, MAP2c. Mediation by the AT2 receptor may be inferred since: (a) these cells contain only AT2 receptors; (b) the effects are mimicked by CGP 42112 (an AT2 receptor agonist); (c) they are not suppressed by the addition of DUP 753 (an AT1 receptor antagonist); and (d) are abolished by co-incubation with PD 123319 (an AT2 receptor antagonist). Application of Ang II in dibutyryl cAMP-differentiated cells (which contain both types of receptors) induces neurite retraction, an effect mediated by the AT1 receptor. These results indicate that the AT2 receptor of Ang II induces neuronal differentiation, which is initiated through an increase in the levels of MAP2c associated with tubulin. Moreover, our results demonstrate that the AT1 receptor inhibit the process of differentiation induced by dibutyryl cAMP, whereas the AT2 receptors potentiate this effect, illustrating negative cross-talk interaction between the two types of Ang II receptors.

ACTH is the major regulator of adrenal cortex function, having acute and chronic effects on steroid synthesis and secretion. The precise molecular mechanisms by which ACTH stimulates steroid synthesis and secretion, as well as cell hypertrophy, survival, and migration are still poorly understood. Several studies have shown that ACTH action is mediated not only by cyclic adenosine monophosphate (cAMP), but also by calcium (Ca(2+)), both interacting closely through positive feedback loops to enhance steroid secretion. However, in spite of the evidence that ACTH could stimulate other signaling pathways, such as inositol phosphates and diacylglycerol or mitogenic-activated protein kinase pathway (MAPK), none is as potent as cAMP. Recent data indicate that duration and potency of the cAMP production could be modulated by several isoforms of adenylyl cyclases and phosphodiesterases. In addition, calcium is probably not a first second messenger per se; rather, there are several arguments indicating that its increase occurs following cAMP production. Finally, in addition to steroid secretion, ACTH, through cAMP, is a survival factor, protecting cells against apoptosis. All of the effects of ACTH are dependent on cytoskeleton integrity. In summary, after 30 years of intensive research in this field, cAMP remains the first obligatory second messenger of ACTH action. However, recent work emphasizes that cell environment (matrix and cytoskeleton) probably interacts with cAMP to coordinate functions other than steroid secretion.

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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/p44^mapk 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/p44^mapk 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).

In the present study, we show that the eicosanoid compound, 20-hydroxyeicosatetraenoic acid (20-HETE), an important arachidonic acid metabolite, activates mouse TRPC6 in a stable, overexpressing HEK293 cell line, Hek-t6.11. Application of 20-HETE rapidly induced an inward, non-selective current in whole-cell recordings, which was inhibited by N-methyl-D-glucamine, 1.8 mM Ca 2؉ , and 100 M Gd 3؉ but remained unaffected by flufenamate and indomethacin. The current-voltage relationship obtained at low concentrations of 20-HETE (1-10 M) demonstrated slight inward rectification, whereas the highest concentration of 20-HETE tested (30 M) showed outward rectification, as shown previously for these channels using 100 M 1-oleoyl-2-acetylsn-glycerol. Dose-response curves indicate that 20-HETE activated TRPC6 channels with an EC 50 ‫؍‬ 0.8 M. Single channel analysis using inside-out patches revealed that 20-HETE increased open probability of mouse TRPC6 channels ϳ3-fold, and this was in a membrane-delimited fashion. Interestingly, 20-HETE did not provoke changes in intracellular Ca 2؉ concentrations. Thus, we have identified an arachidonic acid metabolite, 20-HETE, as a novel activator for a TRP family member, TRPC6.Cationic channels thought to be responsible for capacitative Ca 2ϩ entry, as originally defined by Putney (1, 2), have been identified in the plasmalemmal membrane of a spectrum of cells (3, 4). Capacitative Ca 2ϩ entry, through store-operated channels (SOCs) 1 is triggered by the emptying of intracellular Ca 2ϩ stores by a variety of maneuvers (4 -6). SOCs differ in their ionic selectivity, conductance, and sensitivity to inorganic and organic blockers. The best characterized SOC is the calcium release-activated calcium channel, a highly selective Ca 2ϩ ion channel first described in mast cells (7) and T lymphocytes (8), whereas non-selective cationic channels activated by store depletion have also been described (9). The molecular identity of store-operated channels has not yet been firmly established despite accumulating evidence that they could be members of the TRPC family (10 -12). The transient receptor potential (dTRP) channel, found in Drosophila eye, is a storeoperated Ca 2ϩ channel, whereas the trp-like (dTRPL) protein functions as a constitutively activated, non-selective cation channel (13-15). In mammals, seven dTRP homologs, TRPC1-TRPC7 (16 -18), have been found. Overexpression studies have demonstrated that these channels show important differences in a number of properties including their mode of activation, unitary conductance, and selectivity, which may or may not be a result of the expression system used (for reviews, see Refs. 4, 17, and 19). Moreover, the formation of heteromeric TRPC channels has been reported, whose characteristics were quite distinct from those recorded for the individual, homomeric channels; such behavior was shown for coassembly of TRPC1/ TRPC3 (20) and TRPC1/TRPC5 (21). A number of studies have reported that some members of the TRPC family can be activated by produ...