The activity of adenylate cyclase in the yeast Saccharomyces cerevisiae is controlled by two G-protein systems, the Ras proteins and the G␣ protein Gpa2. Glucose activation of cAMP synthesis is thought to be mediated by Gpa2 and its G-protein-coupled receptor Gpr1. Using a sensitive GTP-loading assay for Ras2 we demonstrate that glucose addition also triggers a fast increase in the GTP loading state of Ras2 concomitant with the glucoseinduced increase in cAMP. This increase is severely delayed in a strain lacking Cdc25, the guanine nucleotide exchange factor for Ras proteins. Deletion of the RasGAPs IRA2 (alone or with IRA1) or the presence of RAS2 Val19 allele causes constitutively high Ras GTP loading that no longer increases upon glucose addition. The glucose-induced increase in Ras2 GTP-loading is not dependent on Gpr1 or Gpa2. Deletion of these proteins causes higher GTP loading indicating that the two G-protein systems might directly or indirectly interact. Because deletion of GPR1 or GPA2 reduces the glucoseinduced cAMP increase the observed enhancement of Ras2 GTP loading is not sufficient for full stimulation of cAMP synthesis. Glucose phosphorylation by glucokinase or the hexokinases is required for glucose-induced Ras2 GTP loading. These results indicate that glucose phosphorylation might sustain activation of cAMP synthesis by enhancing Ras2 GTP loading likely through inhibition of the Ira proteins. Strains with reduced feedback inhibition on cAMP synthesis also display elevated basal and induced Ras2 GTP loading consistent with the Ras2 protein acting as a target of the feedback-inhibition mechanism.In Saccharomyces cerevisiae the addition of glucose or other rapidly fermentable sugars to derepressed cells (carbonstarved or growing on a non-fermentable carbon source) triggers a remarkable variety of regulatory phenomena, including many rapid changes at the post-translational and transcriptional level. Several signaling pathways are activated by glucose. One of the best studied pathways is the cAMP/PKA 1 pathway. The main component of this signaling transduction pathway is adenylate cyclase, which catalyzes the synthesis of cAMP. In S. cerevisiae adenylate cyclase activity is controlled by the Ras proteins. These proteins are members of the small GTPase superfamily, which are active in the GTP-bound form and inactive in the GDP-bound form. Recently it has been demonstrated that in S. cerevisiae adenylate cyclase activity is also controlled by a heterotrimeric G␣-protein, Gpa2 (1). A G-protein-coupled receptor, Gpr1, has been identified to be responsible for activation of the Gpa2 protein (2-4). Two triggers are known to activate the cAMP/PKA pathway: the addition of glucose to derepressed cells and intracellular acidification. The Gpr1/Gpa2 G-protein-coupled receptor system is only required for glucose activation of cAMP synthesis (2, 5). The results reported in the literature about the role played by the Ras proteins in activation of cAMP synthesis are in part contradictory. Colombo et al. (5) show...

Chronic neuroinflammation and oxidative stress contribute to the neurodegeneration associated with Alzheimer's disease and represent targets for therapy. Ferulic acid is a natural compound that expresses antioxidant and anti-inflammatory activities. Nitric oxide is also a key modulator of inflammatory responses. Grafting a nitric oxide-releasing moiety onto antiinflammatory drugs results in enhanced anti-inflammatory activity. We compared the effectiveness of ferulic acid with a novel nitric oxide-releasing derivative of ferulic acid in an animal model of chronic neuroinflammation that reproduces many interesting features of Alzheimer's disease. Lipopolysaccharide was infused into the 4th ventricle of young rats for 14 days. Various doses of ferulic acid or its nitric oxidereleasing derivative were administered daily. Both drugs produced a dose-dependent reduction in microglia activation within the temporal lobe. However, the nitric oxide-releasing ferulic acid derivative was significantly more potent. If we delayed the initiation of therapy for 14 days, we found no reduction in microglial activation. In addition, both drugs demonstrated antioxidant and hydroxyl radical scavenging abilities in in vitro studies. Overall, our results predict that a treatment using nitric oxide-releasing ferulic acid may attenuate the processes that drive the pathology associated with Alzheimer's disease if the treatment is initiated before the neuroinflammatory processes can develop.

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Background and purpose:We previously reported that NCX 2057, a compound comprising a nitric oxide (NO)-releasing moiety and the natural antioxidant, ferulic acid (FA), inhibits pro-inflammatory mediators through NO-mediated gene regulation. Here, we have assessed the activities of NCX 2057 in models of inflammatory and neuropathic pain, and characterized its effects on cyclooxygenase (COX)-1 and COX-2. ]. NCX 2057 reversed carrageenan-induced hyperalgesic responses in mice and inhibited prostaglandin E2 formation in paw exudates. Finally, NCX 2057 competitively inhibited COX-1 and COX-2 activities in whole RAW macophages (IC50 = 14.7 Ϯ 7.4 and 21.6 Ϯ 7.5 mM, respectively). None of these properties were exhibited by equivalent treatments with FA or standard NO donor compounds. Conclusions and implications:These studies indicate that NCX 2057 is effective in chronic inflammatory and neuropathic pain models, probably because of its particular combination of anti-COX, antioxidant and NO-releasing properties.

Purpose: Hypoxia of the retina and optic nerve head (ONH) is believed to be pivotal in the development of ocular vascular disorders, including diabetic macular edema (DME). Glucocorticoids are among the most effective agents for the treatment of back of the eye diseases. However, this class of compounds is highly liable to increase intraocular pressure (IOP) and does not improve ocular perfusion or tissue oxygenation. Nitric oxide (NO) has vasodilating properties and lowers IOP in experimental models and humans, suggesting that its properties might complement those of glucocorticoids. NCX 434 is an NO-donating triamcinolone acetonide (TA) that is less likely to increase IOP while targeting both the vascular and inflammatory components of DME. Methods: NCX 434 was studied in vitro with respect to its NO-releasing properties in isolated methoxamineprecontracted rabbit aortic rings and glucocorticoid-like activity in recombinant human glucocorticoid receptors. IOP and oxygen saturation in the ONH and overlaying arteries and veins were studied in the anesthetized cynomolgus monkey. Measurements were taken using, respectively, an applanation tonometer and a hyperspectral imaging system before and 7, 14, 21, 31 and 41 days after the intravitreal injection of NCX 434 (5.8 mg/ eye) or TA equimolar doses (4.0 mg/eye). Results: NCX 434 inhibited 3 H-dexamethasone-specific binding (IC 50 = 34 -5 nM) on human glucocorticoid receptors and elicited NO-dependent aortic ring relaxation (EC 50 of 0.5 -0.1 mM, E max 98.9%). In monkey eyes, NCX 434 enhanced, whereas TA did not, oxygen saturation in various ONH areas (*P < 0.05 vs. basal), decreased it in veins, and did not affect it in the overlaying arteries. Neither NCX 434 nor TA altered IOP significantly at all time points. However, at 31 days post-treatment TA appeared to start increasing IOP (D IOP = + 3.31 -0.51 mmHg, 30.8%, over baseline, NS). Conclusions: NCX 434 enhances ocular tissue oxygenation. This feature appears to depend on its NO-donating properties; thus, the compound deserves to be further investigated for the treatment of DME and other ocular disorders with impaired ocular perfusion.