The small GTPases Ras or Rap1 were suggested to mediate the stimulatory effect of some G protein-coupled receptors on ERK activity in neuronal cells. Accordingly, we reported here that pituitary adenylate cyclase-activating polypeptide (PACAP), whose G protein-coupled receptor triggers neuronal differentiation of the PC12 cell line via ERK1/2 activation, transiently activated Ras and induced the sustained GTP loading of Rap1. Ras mediated peak stimulation of ERK by PACAP, whereas Rap1 was necessary for the sustained activation phase. However, PACAP-induced GTP-loading of Rap1 was not sufficient to account for ERK activation by PACAP because 1) PACAP-elicited Rap1 GTP-loading depended only on phospholipase C, whereas maximal stimulation of ERK by PACAP also required the activity of protein kinase A (PKA), protein kinase C (PKC), and calcium-dependent signaling; and 2) constitutively active mutants of Rap1, Rap1A-V12, and Rap1B-V12 only minimally stimulated the ERK pathway compared with Ras-V12. The effect of Rap1A-V12 was dramatically potentiated by the concurrent activation of PKC, the cAMP pathway, and Ras, and this potentiation was blocked by dominant-negative mutants of Ras and Raf. Thus, this set of data indicated that GPCR-elicited GTP loading of Rap1 was not sufficient to stimulate efficiently ERK in PC12 cells and required the permissive co-stimulation of PKA, PKC, or Ras.Since its original establishment by Greene and Tischler (1), the PC12 pheochromocytoma cell line is a widely used model of neuronal differentiation. The first isolated neurotrophin, nerve growth factor (NGF) 1 (2), was shown to induce a neuronal like phenotype characterized by neurite outgrowth (1). The effect of NGF is dependent on a long lasting activation of the Ras-Raf-MEK-ERK pathway (3). Activation of the cAMP pathway was also reported to induce PC12 differentiation (4,5). In line with the demonstrated role of ERK activation in NGF-induced PC12 differentiation, cAMP analogues, and forskolin, a direct activator of adenylate cyclase (AC) was shown to stimulate ERK activity in this cell line (5, 6). Similarly, mobilization of calcium and/or stimulation of the diacylglycerol (DAG) production results in ERK activation and eventually neurite outgrowth (7, 8).The mechanisms responsible for the control of the ERK pathway by receptor tyrosine kinases, cAMP analogues, and calcium/DAG were intensively investigated, and Ras-like small GTP-binding proteins emerged as key elements in this pathway. The products of the H-Ras gene was shown to stimulate the activity of the MEK kinase Raf-1 following activation of receptor tyrosine kinases (9). Cyclic AMP and calcium were also suggested to control the activity of Ras in some cell types (7, 10), resulting in ERK activation. More recently, the Ras superfamily member Rap1 and the protein kinase B-Raf were suggested to link PKA activation by cAMP analogues to MEK1 stimulation in neuronal cells (11). On the other hand, several mechanisms have been proposed for calcium-induced ERK activation, including ac...

Apoptosis of cultured cerebellar granule neurons (CGNs) deprived of serum is prevented by K+ depolarization or moderate concentrations of N-methyl-d-aspartate (NMDA). Here, we have examined the role of the serine/threonine kinase Akt in these protective effects. The exposure of mouse CGNs to NMDA or K+ depolarization increased the phosphorylation of Akt, compared with that measured in cells incubated in a physiological K+ concentration. Only the NMDA-evoked response was reduced by inhibitors of phosphatidylinositol 3-kinase (wortmannin and LY294002) and mitogen-activated protein kinase (PD98059 and U0126). Similarly, the capacity of NMDA to inhibit apoptosis of CGNs deprived of serum was greatly reduced by these inhibitors as well as by the transfection of neurons with a catalytically inactive mutant of Akt, whereas the protective effect of K+ depolarization remained unaffected. These findings indicate that K+ depolarization and NMDA activate Akt through different signalling pathways in CGNs. Moreover, Akt mediates the anti-apoptotic effect of NMDA, but not that evoked by K+ depolarization.

1 Previous studies with indolyl derivatives as monoamine oxidase (MAO) inhibitors have shown the relevance of the indole structure for recognition by the active site of this enzyme. We now report a new series of molecules with structural features which determine the selectivity of MAO inhibition. 2 A benzyloxy group attached at position 5 of the indole ring is critical for this selective behaviour. Amongst all of these benzyloxy-indolyl methylamines, N-(2-propynyl)-2-(5-benzyloxyindol)methylamine FA-73 was the most potent MAO-B`suicide' inhibitor studied. 3 The K i values for MAO-A and MAO-B were 800+60 and 0.75+0.15 nM, respectively. These data represent a selectivity value of 1066 for MAO-B, being 48 times more selective than L-deprenyl (K i values of 376+0.032 and 16.8+0.1 nM for MAO A and MAO-B, respectively). The IC 50 values for dopamine uptake in striatal synaptosomal fractions from rats were 150+8 mM for FA-73 and 68+10 mM for L-deprenyl whereas in human caudate tissue the IC 50 values were 0.36+0.015 mM for FA-73 and 0.10+0.007 mM for L-deprenyl. Moreover, mouse brain MAO-B activity was 90% ex vivo inhibited by both compounds 1 h after 4 mg kg 71 adminstration, MAO-A activity was not a ected. 4 These novel molecules should provide a better understanding of the active site of monoamine oxidase and could be the starting point for the design of further selective, non-amphetamine-like MAO-B inhibitors with therapeutic potential for the treatment of neurological disorders.

Due to the flexibility of associating polymer and protein molecules, intramolecular association can have a significant affect on the thermodynamic properties and structure of associating polymer and protein solutions. The equilibrium state is determined by the minimization of the appropriate free energy with respect to intermolecular association between like and unlike species and intramolecular association. As a first step to understanding this competition between intramolecular and intermolecular hydrogen bonding, we have conducted a molecular simulation study of flexible hard chain molecules that intramolecularly associate in the absence of intermolecular association. To explain the simulation results, we have developed a new simple and accurate theory of intramolecular association. By considering the limit of total bonding, we have also developed an accurate equation of state for hard rings. The theory is in good agreement with new molecular simulation results for intramolecularly associating hard chains, rigid hard rings, and bent triatomics. 6880

Cyclic AMP (cAMP)-raising agents induce astrocytes grown in vitro to adopt a stellate morphology resembling their in vivo appearance, through the depolymerization of actomyosin stress fibres. The signalling pathways responsible for cAMP-induced astrocyte stellation have thus far remained largely elusive. We showed in this study that the neurotrophic peptide PACAP (pituitary adenylate cyclase-activating polypeptide) mimicked the effect of forskolin, a direct activator of adenylate cyclase, on the actin cytoskeleton of primary rat astrocytes. The depolymerization of stress fibres induced by PACAP or forskolin was prevented by the expression of a constitutively active mutant of RhoA, but not by a protein kinase A (PKA) blocker, indicating that cAMP-raising agents act upstream of RhoA, in a PKA-independent manner. In addition, PACAP and forskolin inhibited basal Akt phosphorylation, and basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3-kinase (PI 3-K) activities. Incubation with a PI 3-K blocker resulted in the depolymerization of stress fibres. This effect was blocked by the expression of a constitutively active mutant of RhoA, indicating that PI 3-K inhibition acted upstream of RhoA. Together, these data demonstrate for the first time that depolymerization of stress fibres, and the resulting astrocyte stellation, induced by stimulation of cAMP production involves the inhibition of the PI 3-K-RhoA pathway.