Angiotensin II Induction of Neurite Outgrowth by AT2 Receptors in NG108-15 Cells

Abstract

In the present study, 3-day treatment of nondifferen-tiated 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 AT 2 receptor may be inferred since: (a) these cells contain only AT 2 receptors; (b) the effects are mimicked by CGP 42112 (an AT 2 receptor agonist); (c) they are not suppressed by the addition of DUP 753 (an AT 1 receptor antagonist); and (d) are abolished by co-incubation with PD 123319 (an AT 2 receptor antagonist). Application of Ang II in dibutyryl cAMP-differentiated cells (which contain both types of receptors) induces neurite retrac-tion, an effect mediated by the AT 1 receptor. These results indicate that the AT 2 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 AT 1 receptor inhibit the process of differentiation induced by dibu-tyryl cAMP, whereas the AT 2 receptors potentiate this effect, illustrating negative cross-talk interaction between the two types of Ang II receptors. Pharmacological studies have clearly identified two classes of angiotensin II (Ang II) 1 receptors. The AT 1 receptor is closely associated with cardiovascular regulation, fluid volume home-ostasis, and cellular growth (1, 2). Activation of the AT 1 receptor is linked to phospholipase C activation and Ca 2 influx, effects which are mediated by G proteins (1, 2). The AT 2 receptors have been identified in many fetal tissues, including brain (3-9), and in cell lines of neuronal origin (10-12). This receptor has been cloned (13, 14), but a definitive physiological function has yet to be assigned. We have reported previously that Ang II decreases the T-type calcium current in nondifferentiated NG108-15 cells expressing only the angiotensin AT 2 receptor type (15, 16). Kang et al. (17) also reported that activation of the AT 2 receptor increased a potassium channel activity. Moreover , Xiong and Marshall (18) reported that Ang II, via the AT 2 receptor, could inhibit membrane depolarization and action potential elicited by N-methyl-D-aspartate receptors in the lo-cus coeruleus, a brain area containing only Ang II receptors of the AT 2 subtype. Considering the abundance of T-type Ca 2 channels in neurons from fetal brain (19), the crucial role of Ca 2 in neuronal differentiation (20) and the abundance of AT 2 receptors during this developmental period, it could be postulated that Ang II, via the AT 2 receptor could affect some aspects of neuronal differentiation. Neuronal differentiation is characterized by neurite extension that involves several biochemical steps directed toward promotion of the assembly of tubulin monomers into microtu-bules necessary to support the growing neurites. Several molecules play crucial roles in neurite outgrowth. Among these are the microtubule-associated proteins (MAPs) (21), which include both high molecular weight proteins, termed MAP1 to MAP5, and low molecular weight proteins, including tau (22). These proteins promote tubulin polymerization as well as stabilize microtubules and occur as embryonic and adult isoforms whose differential expression during brain development correlates with the maturation of neuronal circuitry. For example, MAP2 and tau bind to distinct populations of microtubules in adult neurons: MAP2 to somatodendritic microtubules and tau to axonal microtubules (23, 24). Expression of specific brain MAPs is critical for regulating neurite outgrowth and differentiation. Several lines of evidence have demonstrated a strong correlation between the pattern of expression of neuronal MAPs and the morphological differentiation of neurons (22). In their nondifferentiated state, the hybrid cells NG108-15 (neuroblastoma glioma) are rounded and actively dividing. Chronic exposure of NG108-15 cells to dibutyryl cAMP (db-cAMP) induces a process of differentiation that includes elaboration of neurites, development of electrical excitability, formation of functional synapses, alteration of ligand-gated channel properties, and a reduced rate of cell division. Thus, differentiated NG108-15 cells exhibit a neuronal phenotype, whereas glial properties appear to be suppressed (25, 26). Moreover, when nondifferentiated, NG108-15 cells express exclusively the AT 2 receptor subtype and mainly the T-type Ca 2 channel (15). These cells also express tau and MAP2 and are thus useful in examining factors initiating neuronal