Developmental changes in the response of murine cerebellar granule cells to nitric oxide.
ABSTRACT Nitric oxide is a diffusible messenger that plays a multitude of roles within the nervous system including modulation of cell viability. However, its role in regulating neuronal survival during a defined period of neurodevelopment has never been investigated. We discovered that expression of the messenger RNA for both neuronal and endothelial nitric oxide synthase increased in the early postnatal period in the cerebellum in vivo, whilst the expression of inducible nitric oxide synthase remained constant throughout this time in development. Whilst scavenging of nitric oxide was deleterious to the survival of early postnatal cerebellar granule neurons in vitro, this effect was lost in cultures derived at increasing postnatal ages. Conversely, sensitivity to exogenous nitric oxide increased with advancing postnatal age. Thus, we have shown that as postnatal development proceeds, cerebellar granule cells alter their in vitro survival responses to both nitric oxide inhibition and donation, revealing that the nitric oxide's effects on developing neurons vary with the stage of development studied. These findings have important consequences for our understanding of the role of nitric oxide during neuronal development.
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ABSTRACT: It is nearly 20 years that nitric oxide (NO) entered the scene to become an integral component in understanding physiological and pathophysiological processes ranging from fine-tuned signaling to promoting cell demise. Among multiple activities attributed to NO we find regulation of gene expression. Although there is no evidence for direct NO-responsive DNA elements within promotor regions of eukaryotic genes numerous signaling pathways exist to understand NO-regulated gene expression. A characteristic feature of may transcription factors is their redox sensitivity as well as their low protein abundance in unstressed cells due to efficient 26S proteasomal degradation. Examples comprise the hypoxia inducible factor-1alpha (HIF-1alpha) and p53 (tumor suppressor p53). It became apparent that NO is able to mimic a hypoxic response by stabilizing HIF-1alpha and/or to affect viability decisions by accumulating p53. We will review recent molecular understanding how NO affects stability regulation of HIF-1alpha and p53, considering basic chemical reactions and cellular transducing pathways. Targeting HIF-1alpha and p53 by reactive nitrogen intermediates (RNI) may help to understand a sphere of NO-evoked transcriptional regulation ranging from cellular adaptation to death, i.e. apoptosis with important implications for medicine.Toxicology 04/2005; 208(2):223-33. · 4.02 Impact Factor
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ABSTRACT: 1. We studied the effects of 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) on the activity of purified soluble guanylyl cyclase (sGC), the formation of guanosine-3':5' cyclic monophosphate (cyclic GMP) in vascular smooth muscle cells (VSMC), and on the tone of rabbit isolated aortic rings preconstricted by phenylephrine (PE). In addition, we assessed the combined effect of YC-1, and either NO donors, or superoxide anions on these parameters. 2. YC-1 elicited a direct concentration-dependent activation of sGC (EC50 18.6 +/- 2.0 microM), which was rapid in onset and quickly reversible upon dilution. YC-1 altered the enzyme kinetics with respect to GTP by decreasing KM and increasing Vmax. Activation of sGC by a combination of sodium nitroprusside (SNP) and YC-1 was superadditive at low and less than additive at high concentrations, indicating a synergistic activation of the enzyme by both agents. A specific inhibitor of sGC, 1H-(1,2,4)-oxdiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (NS 2028), abolished activation of the enzyme by either compound. 3. YC-1 induced a concentration-dependent increase in intracellular cyclic GMP levels in rat cultured aortic VSMC, which was completely inhibited by NS 2028. YC-1 applied at the same concentration as SNP elicited 2.5 fold higher cyclic GMP formation. Cyclic GMP-increases in response to SNP and YC-1 were additive. 4. YC-1 relaxed preconstricted endothelium-denuded rabbit aortic rings in a concentration-dependent manner (50% at 20 microM) and markedly increased cyclic GMP levels. Relaxations were inhibited by NS 2028. A concentration of YC-1 (3 microM), which elicited only minor effects on relaxation and cyclic GMP, increased the vasodilator potency of SNP and nitroglycerin (NTG) by 10 fold and markedly enhanced SNP- and NTG-induced cyclic GMP formation. 5. Basal and YC-1-stimulated sGC activity was sensitive to inhibition by superoxide (O-2) generated by xanthine/xanthine oxidase, and was protected from this inhibition by superoxide dismutase (SOD). YC-1-stimulated sGC was also sensitive to inhibition by endogenously generated (O-2 in rat preconstricted endothelium-denuded aortic rings. Relaxation to YC-1 was significantly attenuated in aortae from spontaneously hypertensive rats (SHR), which generated O-2 at a higher rate than aortae from normotensive Wistar Kyoto rats (WKY). SOD restored the vasodilator responsiveness of SHR rings to YC-1. 6. In conclusion, these results indicate that YC-1 is an NO-independent, O-2-sensitive, direct activator of sGC in VSMC and exerts vasorelaxation by increasing intracellular cyclic GMP levels. The additive or even synergistic responses to NO-donors and YC-1 in cultured VSMC and isolated aortic rings apparently reflect the direct synergistic action of YC-1 and NO on the sGC. The synergism revealed in this in vitro study suggests that low doses of YC-1 may be of therapeutic value by permitting the reduction of nitrovasodilator dosage.British Journal of Pharmacology 03/1997; 120(4):681-9. · 5.07 Impact Factor
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ABSTRACT: Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.Proceedings of the National Academy of Sciences 03/1990; 87(4):1620-4. · 9.74 Impact Factor