Nitric Oxide: Foe or Friend to the Injured Brain?

Over the past 6 years or so, the reactive gas nitric oxide (NO) has become established as a diffusible messenger mediating cell-cell interactions throughout the body, including immune cell-mediated cytotoxicity, inhibition of platelet aggregation, smooth muscle relaxation, and neuronal signaling (1). Appreciation that NO serves as a neuronal messenger was triggered by the discoveries that cultured cerebellar neurons release an NO-like muscle relaxing factor, and that brain neurons contain a constitutive form of the enzyme responsible for synthesizing NO from arginine, nitric oxide synthase (cNOS) (2, 3). Neuron-derived NO regulates smooth muscle tone and the behavior of other central neurons as an "aberrant transmitter" (3), differing from more conventional neurotransmitters in being able to diffuse freely from the point of synthesis to intracellular target sites in neighboring cells and being independent of vesicular release, membrane receptors, or lipid cell boundaries. While the normal neuronal actions of NO have not been fully delineated, NO has been implicated specifically in some forms of synaptic plasticity, including both longterm potentiation and long-term depression (4, 5). A key role for NO has also been postulated in the pathogenesis of brain damage following acute insults, such as hypoxia-ischemia (6). This hypothesis represents a consequential new twist on an older premise that the cytotoxic overstimulation of neuronal glutamate receptors, especially of the N-methyl-Daspartate (NMDA) type, contributes to hypoxic-ischemic neuronal loss (7, 8). Such NMDA receptor-mediated "excitotoxicity" depends on extracellular Ca2+ and is likely initiated by excess Ca2+ influx through the NMDA receptor-gated channel (9). The linkage of neuronal NMDA receptor stimulation to activation of neuronal cNOS in an extracellular Ca2+and calmodulin-dependent fashion (10, 11) led Dawson et al. (12) to propose that NO formation by neuronal cNOS mediates NMDA receptor-mediated neurotoxicity. This idea received strong support from their experiments showing that NMDA receptor-mediated neuronal death in cortical cell cultures could be blocked by four different maneuvers: (i) pharmacological inhibition of NOS; (ii) addition of hemoglobin, which complexes with free NO; (iii) deletion of the NO precursor, L-arginine, from the bathing medium; and (iv) prior selective destruction of the small neuronal subpopulation containing high concentrations of cNOS, previously referred to as "NADPH-diaphorase"containing cells. The idea was reinforced by the finding that the glutamate receptor-mediated neurotoxicity of the human immunodeficiency virus type 1 coat protein gp120 (13) could also be blocked by inhibition of NO formation (14). Most recently, the same laboratory has reported that calcineurin inhibition by the immunosuppressant FK506 results in increased phorbol ester-induced cNOS phosphorylation, reduced cNOS catalytic activity, and reduced neuronal vulerability to NMDA-induced neurotoxicity (15). This latest observation may have broad implications for understanding the normal regulation of cNOS activity. As predicted by its sequence, neuronal cNOS can be phosphorylated by protein kinases, specifically protein kinase C, Ca2+/calmodulin-dependent protein kinase, and cAMP-dependent protein kinase, resulting in an inhibition of catalytic activity (3). Dawson et al. (15) proposed that NMDA receptor-mediated Ca2+ influx may activate calcineurin to dephosphorylate cNOS, acting synergistically with Ca2+/calmodulin to enhance cNOS activation. They cited unpublished data indicating that cGMP-dependent protein kinase can also phosphorylate cNOS. Although guanylyl cyclase has a macroscopically different distribution in brain than cNOS, perhaps enough guanylyl cyclase is present in cNOScontaining neurons to provide a negative feedback loop: NO formation inducing local cGMP formation, cNOS phosphorylation, and resultant cNOS inhibition. Additional work will be needed to establish that calcineurin-induced cNOS dephosphorylation regulates neuronal cNOS activity in vivo. The study of Dawson et al. (15) was carried out in 293 human kidney cells overexpressing cloned neuronal cNOS; extension to primary cultured neurons or brain slices and eventually to the intact brain will be desirable. Furthermore, the authors caution that FK506 has known actions other than calcineurin inhibition and increases the phosphorylation of proteins other than NOS. If calcineurin-induced cNOS dephosphorylation does indeed enhance brain neuronal cNOS activity in vivo, calcineurin inhibition might be an attractive therapeutic goal in the setting of focal brain ischemia (e.g., stroke) and other forms of brain injury involving NMDA receptormediated excitotoxicity. As Dawson et al. point out, both FK506 and another immunosuppressant, cyclosporin A, are already used in humans to suppress organ rejection after transplant surgery. One study has raised the possibility that FK506 may reduce global ischemic brain injury in liver transplant patients (16), although the small size of this study and the paucity of other evidence supporting a role of NMDA receptor-mediated neurotoxicity in animal models of global ischemia (17) are caveats. A second therapeutic strategy might be to enhance cNOS phosphorylation by one or another kinase, although some indirect data has implicated kinases in the pathogenesis of ischemic brain injury. It may turn out that manipulating the cNOS phosphorylation state cannot be accomplished selectively enough to form a practical therapeutic strategy. Even so, the protective effect of FK506 in vitro provides additional rationale for examining other means of attenuating neuronal cNOS activity as a treatment for focal brain ischemia. Encouraging results have been reported with competitive NOS inhibitors in some animal models of stroke (18-20). However, as highlighted by the study of Wink et al. (21) in the present issue, not all available data support a role of neuronal NO formation in pathological neuronal death. Wink et al. describe the use of spontaneous NO-releasing compounds, NONOates, to elucidate a cytoprotective effect of NO against cell death induced by exposure to hydrogen peroxide and superoxide in transformed fibroblast cell cultures or primary cultures of mesencephalic neurons. These are exciting, provocative observations; follow-up experiments will be needed to determine their generality. Studies will be needed to compare results