Nitric Oxide

The discovery that a toxic gas such as nitric oxide (NO) can serve as a neuronal messenger (cf. Garthwaite et al., 1988; Moncada et al., 1989) has generated widespread interest in the physiological actions of this molecule. There is a large body of literature on the cellular localization of NO, the biochemical reactions that mediate its cytological effects, its role in synaptic processes, and its involvement in pathological conditions. In the brain, NO has been implicated as a mediator of neurotoxicity, a retrograde signaling molecule, a regulator of blood flow in response to neuronal activity, a potent neuromodulatory agent, and in a diversity of higher-order neuronal processes ranging from long-term potentiation to long-term depression (cf. Hibbs et al., 1988; Gally et al., 1990; Schuman and Madison, 1991; Garthwaite, 1991; Moncada et al., 1991; Dawson et al., 1991; Snyder and Bredt, 1992; Bredt and Snyder, 1994; Schuman and Madison, 1994; Szabo, 1996). Our knowledge of how NO participates in this broad spectrum of activities is still only fragmentary, but the availability of NO donors such as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenacillamine (SNAP), as well as drugs that inhibit nitric oxide synthase (e.g., analogs of L-arginine such as

L-NAME) or scavenge NO (e.g., hemoglobin) has helped provide insight into some of its actions.

4.5.1. General Features

NO, a reactive free radical with an estimated half-life of ~5-10 seconds, is a lipophilic molecule that can rapidly diffuse through cell membranes to exert its influence within a radius of ~200 ^m from the site of generation (Wood and Garthwaite, 1994). NO is generated during the conversion of L-arginine to L-citrulline (Fig. 4.15). The reaction is catalyzed by nitric oxide synthase (NOS), a cytosolic enzyme that requires as cofactor nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd). The transfer of electrons from NADPHd to tetrazolium salts, and the enzymatic reduction of soluble nitroblue tetrazolium salt to a visible formazan precipitate, provides a widely used histochemical marker for cells containing NOS (Dawson et al., 1991; Hope et al., 1991).

Figure 4.15. Intracellular levels of NOS are raised by calcium-calmodulin (CaM) activation of cNOS or a cytokine-induced expression of iNOS. NOS catalyzes the conversion of L-arginine to citrulline, a process that involves the oxidation of NADPH and the reduction of molecular oxygen. NO can diffuse intra- and intercellularly to activate a soluble guanylate cyclase (CC) which serves to catalyze the formation of cGMP.

Figure 4.15. Intracellular levels of NOS are raised by calcium-calmodulin (CaM) activation of cNOS or a cytokine-induced expression of iNOS. NOS catalyzes the conversion of L-arginine to citrulline, a process that involves the oxidation of NADPH and the reduction of molecular oxygen. NO can diffuse intra- and intercellularly to activate a soluble guanylate cyclase (CC) which serves to catalyze the formation of cGMP.

There are two distinct classes of NO synthase: a constitutive form (cNOS) and an inducible form (iNOS), both of which have been cloned and sequenced. The two forms display 50% identity and 65% similarity at the amino acid level (Bredt et al., 1991; Xie et al., 1992; Lowenstein et al.,

1992), but appear to be distinct genetically and antigenically (Lowenstein and Snyder, 1992). cNOS is Ca2+-calmodulin dependent, relatively shortlived, and produces small quantities (picomoles) of NO; its two isoforms are found predominantly in the vascular endothelium and in neurons. Neuronal cNOS tends to be inactive at resting cytosolic calcium levels (Garthwaite, 1991), but a rise in [Ca2+];, whether via entry through calcium channels or by release from intracellular stores, will enhance enzyme activity and lead to the production of NO.

iNOS, first characterized in macrophages, is typically calcium independent, and is expressed in many cell types in the presence of cytokines (Moncada et al., 1991; Nathan, 1992; Simmons and Murphy, 1992). Following its induction, iNOS can sustain NO production (in nanomolar quantities) for several hours. Agents that have proven effective in activating the iNOS gene include lipopolysaccharide, interferon y, tumor necrosis factor a, interleukin 1ß, and other cytokines (cf. Goureau et al., 1994b).

Glial cells are an important source of NO. Both the calcium-dependent and inducible forms of NOS are found in glial cells throughout the CNS (Agullo et al., 1995; Weikert et al., 1997; Simmons and Murphy, 1992; Kugler and Drenckhahn, 1996; Merrill et al., 1997). However, it is not known whether both forms co-exist in the same cell, and to what extent each contributes to NO production under physiological or pathological conditions. In the presence of L-arginine, cytokine-mediated induction of iNOS activity can continue in astrocytes for many hours, producing high levels of NO (Nomura and Kitamura, 1993). In contrast, formation of NO in astrocytes and cerebellar glia is mediated by cNOS, activated by the rise in intracellular calcium resulting from noradrenaline stimulation of a1-adrenoreceptors (Agullo et al., 1995) or Ca2+-permeable glutamate-operated ion channels (Müller et al., 1992). Regardless of its source, NO may subsequently diffuse to cerebral vessels to regulate vascular tone; pass to nerve cells, where it can disrupt essential metabolic pathways and induce cell death; or affect neuronal function through its ability to activate soluble guanylate cyclase (Fig. 4.15), the enzyme that catalyzes the production of cGMP (cf. Schmidt et al.,

4.5.2. Nitric Oxide in Retina

NADPH/diaphorase histochemistry has shown that NOS is present throughout the visual system (Sandell, 1985, 1986; Cudeiro and Rivadulla, 1999). In the retina, the cellular localization of NOS has been examined

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