Modification of cysteine residues within Go and other neuronal proteins by exposure to nitric oxide

doi:10.1016/0028-3908(94)90028-0

Neuropharmacology

Volume 33, Issue 11, November 1994, Pages 1283-1292

https://doi.org/10.1016/0028-3908(94)90028-0 Get rights and content

Abstract

Nitric oxide (NO), a free-radical gas produced endogenously by some neurons, functions as a diffusible intercellular messenger and appears to play a role in activity-dependent modification of synaptic efficacy in the mammalian CNS. The molecular targets and mechanisms of action of NO in neurons remain largely uncharacterized. Employing in vitro brain slices and isolated synaptosomes, we show here that exposure to exogenous or endogenously generated NO results in the modification of cysteine residues within neuronal proteins, as revealed by reduced binding of agents which react with cysteine sulfhydryls. In particular, exposure of synaptosomes to NO inhibits subsequent thiol-linked ADP-ribosylation of the heterotrimeric G-protein, G_o, by pertussis toxin. Our results demonstrate directly that NO may exert its neuronal effects through modification of protein cysteine thiols, and identify G_o as a potential synaptic target of NO.

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Citation Excerpt :
NO could activate a cGMP-dependent phosphorylation pathway (Pineda et al., 1996; Xu et al., 1998), which would directly affect μ-opioid receptor/G-protein signalling (Pfeifer et al., 1995; Trujillo, 2002) or indirectly counteract the opioid inhibition of non-selective cation channels (Pineda et al., 1996; Nestler and Aghajanian, 1997). Alternatively, μ-opioid receptor/G-protein signalling may be blocked by NO in a cGMP-independent manner as a consequence of enhanced ADP ribosylation of G-proteins (Schuman et al., 1994) or increased oxidation of protein residues (Hess et al., 1994; Christopoulos and El-Fakahany, 1999; Liu and Anand, 2001). The activation of the NOS system after morphine administration in the brain and spinal cord seems to limit the analgesic efficacy of morphine (Leza et al., 1995, 1996; Yamaguchi and Naito, 1996; Machelska et al., 1997a, 1997b; Bhargava et al., 1998; Wong et al., 2000; Li and David Clark, 2000).
Nitric oxide (NO) has been reported to be involved in the mechanisms of pain generation throughout the nervous system. We examined the effects of intrathecally (i.t.) administered nitric oxide synthase (NOS) inhibitors on the antinociceptive effects of morphine and endomorphin-1 during acute pain and in chronic constriction injury (CCI)-exposed rats. We used N^G-nitro-l-arginine methyl ester (l-NAME), a non-selective NOS inhibitor; 7-nitroindazole (7-NI) or 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM), selective inhibitors of neuronal NOS (NOS1); and 1400W dihydrochloride, a selective inhibitor of inducible NOS (NOS2). Morphine (0.5–2.5 μg) and endomorphin-1 (2.5–20 μg) in acute pain and morphine (10–40 μg) and endomorphin-1 (5–20 μg) after CCI-injury were combined with NOS inhibitors. For acute pain, the ED₅₀ for endomorphin-1 (7.1 μg) was higher than that of morphine (1.3 μg) in the tail-flick test. For neuropathic pain, the ED₅₀ value for morphine was much higher (43.2 μg) than that of endomorphin-1 (9.2 μg) in von Frey test. NOS inhibitors slightly influenced pain thresholds in both pain models. Moreover, in neuropathic pain, the effects of morphine were more potentiated by l-NAME, TRIM, 7-NI and 1400W (12×, 8.6×, 4.1× and 5.3×, respectively) than were the effects of endomorphin-1 (2.7×, 4.3×, 3.4× and 2.1×, respectively) in the von Frey test. Minocycline which is known to enhance the efficiency of morphine in neuropathic pain, decreased the mRNA expression of NOS1 in the DRG and NOS2 and C1q in the spinal cord after CCI. Both NOS2 and IBA-1 protein levels in the spinal cord and NOS1, NOS2 and IBA1 protein levels in DRG decreased after minocycline administration. In conclusion, our results provide evidence that both neuronal and non-neuronal NOS/NO pathways contribute to the behavioural pain responses evoked by nerve injury. The NOS inhibitors regardless of the type of pain enhanced morphine antinociception and, to a lesser extent, altered the effects of endomorphin-1, an opioid ligand with a peptidergic structure.
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Oxidants are produced as a by-product of aerobic metabolism, and organisms ranging from prokaryotes to mammals have evolved with an elaborate and redundant complement of antioxidant defenses to confer protection against oxidative insults. Compelling data now exist demonstrating that oxidants are used in physiological settings as signaling molecules with important regulatory functions controlling cell division, migration, contraction, and mediator production. These physiological functions are carried out in an exquisitely regulated and compartmentalized manner by mild oxidants, through subtle oxidative events that involve targeted amino acids in proteins. The precise understanding of the physiological relevance of redox signal transduction has been hampered by the lack of specificity of reagents and the need for chemical derivatization to visualize reversible oxidations. In addition, it is difficult to measure these subtle oxidation events in vivo. This article reviews some of the recent findings that illuminate the significance of redox signaling and exciting future perspectives. We also attempt to highlight some of the current pitfalls and the approaches needed to advance this important area of biochemical and biomedical research.
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Nitric oxide (NO) participates in the regulation of the daily activities of cells as well as in cytotoxic events. Elucidating the mechanism(s) by which NO carries out its diverse functions has been the goal of numerous laboratories. In the cardiovascular system, evidence indicates that NO mediates its effects via an activation of soluble guanylyl cyclase (sGC). In other tissues, it is not clear if sGC is an exclusive target for NO or what the functions of cGMP might be. It is also unlikely that the diversity of NO actions is explained solely by changes in cGMP. This review focuses on the evidence that NO modulates cAMP signalling, with specific attention to the effects of NO on adenylyl cyclase (AC) as the target of NO regulation.
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Sepsis is a heterogeneous class of syndromes caused by a systemic inflammatory response to infection. Septic shock, a severe form of sepsis, is associated with the development of progressive damage in multiple organs, and is a leading cause of patient mortality in intensive care units. Despite important advances in understanding its pathophysiology, therapy remains largely symptomatic and supportive. A decade ago, the overproduction of nitric oxide (NO) had been discovered as a potentially important event in this condition. As a result, great hopes arose that the pharmacological inhibition of NO synthesis could be developed into an efficient, mechanism-based therapeutic approach. Since then, an extraordinary effort by the scientific community has brought a deeper insight regarding the feasibility of this goal. Here we present in summary form the present state of knowledge of the biological chemistry and physiology of NO. We then proceed to a systematic review of experimental and clinical data, indicating an up-regulation of NO production in septic shock; information on the role of NO in septic shock, as provided by experiments in transgenic mice that lack the ability to up-regulate NO production; effects of pharmacological inhibitors of NO production in various experimental models of septic shock; and relevant clinical experience. The accrued evidence suggests that the contribution of NO to the pathophysiology of septic shock is highly heterogeneous and, therefore, difficult to target therapeutically without appropriate monitoring tools, which do not exist at present.
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Ha-Ras is modified by isoprenoid on Cys¹⁸⁶ and by reversibly attached palmitates at Cys¹⁸¹ and Cys¹⁸⁴. Ha-Ras loses 90% of its transforming activity if Cys¹⁸¹ and Cys¹⁸⁴ are changed to serines, implying that palmitates make important contributions to oncogenicity. However, study of dynamic acylation is hampered by an absence of methods for acutely manipulating Ha-Ras palmitoylation in living cells. S-nitrosocysteine (SNC) and, to a more modest extent, S-nitrosoglutathione were found to rapidly increase [³H]palmitate incorporation into cellular or oncogenic Ha-Ras in NIH 3T3 cells. In contrast, SNC decreased [³H]palmitate labeling of the transferrin receptor and caveolin. SNC accelerated loss of [³H]palmitate from Ha-Ras, implying that SNC stimulated deacylation and permitted subsequent reacylation of Ha-Ras. SNC also decreased Ha-Ras GTP binding and inhibited phosphorylation of the kinases ERK1 and ERK2 in NIH 3T3 cells. Thus, SNC altered two important properties of Ha-Ras activation state and lipidation. These results identify SNC as a new tool for manipulating palmitate turnover on Ha-Ras and for studying requirements of repalmitoylation and the relationship between palmitate cycling, membrane localization, and signaling by Ha-Ras.
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Citation Excerpt :
In the absence of extracellular CaCl2, the addition of 200 μM p-CMBA, which weakly stimulates [3H]NA release alone, significantly inhibited the effect of 200 μM SNC (Table 4), and the concentration–response curve of SNC shifted to the right in the presence of HgCl2 (100 μM) or p-CMBA (200 μM) (Fig. 1). It has been reported that radioactivity is released from [32P]NAD-labeled glyceraldehyde-3-phosphate dehydrogenase in the presence of a NO donor by HgCl2in vitro[48] and that [32P]ADP-ribosylation of the cysteine residue of the α subunit of the GTP binding protein (Goα) by pertussis toxin is inhibited by the addition of HgCl2 and NO donors in vitro[49]. These reports and findings suggest the possibility that NO and p-CMBA interact with the same cysteine residues on proteins.
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Modification of cysteine residues within Go and other neuronal proteins by exposure to nitric oxide

Abstract

Brain Res. Rev.

Eur. J. Pharmac.

Neuron

J. Biol. Chem.

J. Biol. Chem.

Eur. J. Pharmac.

Trends Neurosci.

Brain Res.

J. Biol. Chem.

Neuron

Neuron

Neuron

Neuron

J. Biol. Chem.

J. Biol. Chem.

Meth. Enzym.

J. Biol. Chem.

Trends Pharmac. Sci.

J. Biol. Chem.

Different types of ADP-ribose protein bonds formed by botulinum C2 toxin, botulinum ADP-ribosyltransferase C3 and pertussis toxin

Biochem. Biophys. Res. Commun.

Nitric oxide activates guanylate cyclase and increases guanosine 3',5'-cyclic monophosphate levels in various tissue preparations

Presynaptic mechanisms of long-term potentiation

Nature

G proteins in signal transduction

A. Rev. Pharmac. Toxic.

Regulation of cyclic GMP formation by soluble guanylate cyclase: stimulation by NO-containing compounds

Adv. Cyclic Nucleotide Protein Phosphorylation Res.

Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum

Ionic channels and their regulation by G protein subunits

A. Rev. Physiol.

Temporally distinct pre- and post-synaptic mechanisms maintain long-term potentiation

Nature

Modification of cysteine residues within G_o and other neuronal proteins by exposure to nitric oxide