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).

Microexplant cultures from three-day-old rats were used to investigate whether angiotensin II (Ang II), through its AT 1 and AT 2 receptors, could be involved in the morphological differentiation of cerebellar cells. Specific activation of the AT 2 receptor during 4-day treatment induced two major morphological changes. The first was characterized by increased elongation of neurites. The second change was cell migration from the edge of the microexplant toward the periphery. Western blot analyses and indirect immunofluorescence studies revealed an increase in the expression of neuron-specific ␤III-tubulin, as well as an increase in expression of the microtubule-associated proteins tau and MAP2. These effects were demonstrated by co-incubation of Ang II with 1 M DUP 753 (AT 1 receptor antagonist) or with 10 nM CGP 42112 (AT 2 receptor agonist) but abolished when Ang II was co-incubated with 1 M PD 123319 (AT 2 receptor antagonist), indicating that differentiation occurs through AT 2 receptor activation and that the AT 1 receptor inhibits the AT 2 effect. Taken together, these results demonstrate that Ang II is involved in cerebellum development for both neurite outgrowth and cell migration, two important processes in the organization of the various layers of the cerebellum.A large number of studies indicate that the hormone angiotensin II (Ang II) 1 and its receptors are present in the brain (1, 2). As in the periphery, the AT 1 receptor exhibits a high affinity for the nonpeptidic antagonist DUP 753 (Losartan), whereas the AT 2 receptor has a high affinity for the antagonist PD 123319 and the agonist CGP 42112 (1, 2). Although the AT 1 receptors are detected in areas involved in the regulation of blood pressure, hydromineral balance, and thirst, no central function has yet been attributed to the AT 2 receptor. This receptor is highly expressed in the inferior olive, locus coeruleus, thalamic nuclei, medial geniculate nuclei, and the molecular layer of the cerebellum (3-5).Although several studies have been conducted on the short term effect of AT 2 receptor activation on intracellular events, a few studies focused on the physiological function of the AT 2 receptor. One well described function is its antagonistic action on cellular growth induced by neurotrophic factors (nerve growth factor) (6, 7) or by the AT 1 receptor of Ang II (8 -10). Another function recently described for the AT 2 receptor is a role in programmed cell death (11,12). Interestingly, although the expression of the AT 1 receptor either remains stable or increases with development in rats, the expression and density of the AT 2 receptor decrease dramatically with maturation from fetal to neonatal to adult, both at the periphery and in several brain nuclei (13-16). This high and transient expression of the AT 2 receptor in fetal tissues suggests that it may play a specific role during development and cellular differentiation (7,11,17,18). Indeed, in a previous study, using neuroblastoma ϫ glioma hybrid NG108 -15 cells, we have shown that a 3-da...

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.

The use of fixtures in manufacturing of automobile engines is a well established practice. The drawback of fixtures is that they must be redesigned and rebuilt whenever the part that they are supporting is modified. This operation is tedious, costly and limits the agility of the manufacturing process. In this paper, a flexible fixturing system accommodating an entire family of lightweight aluminum engine blocks is presented. The key element of this research is the use of parallel or quasi-parallel mechanisms (PMs) in the development of the fixturing system. PMs are known to have high stiffness, high payload and high precision qualities. By virtually eliminating the need for the redesign of fixtures, the flexible pallet presented in this paper may lead to significant improvements in their use in automobile manufacturing.