Ординатура / Офтальмология / Английские материалы / The Retinal Muller Cell Structure and Function_Sarthy, Ripps_2001

a rise in [Ca2+]i, it did potentiate the [Ca2+]i responses elicited by electrical and mechanical stimulation as well as by application of adenosine triphosphate. These stimuli consistently initiated Ca2+ waves in astrocytes that propagated via gap junctions to neighboring astrocytes and Müller cells. Particularly noteworthy was the observation that, irrespective of the type of stimulus, the Ca2+ waves were observed in the absence of extracellular Ca2+. Thus, although unresponsive to glutamate, a rise in [Ca2+]i can be derived from intracellular stores, mediated probably by activation of inositol 1,4,5- triphosphate (IP3) receptors. Interestingly, the calcium release was not accompanied by significant changes in membrane potential and is therefore unlikely to be involved in the K+ buffering activity of Müller cells (cf. Chapter 5). It is not clear whether the very different results obtained from rat glial cells in situ as compared to those from rabbit and salamander Müller cells in culture are a consequence of differences in tissue preparation or are the result of species variation. Nevertheless, the foregoing observations again demonstrate a potentially important signal pathway linking astrocytes and Müller cells. If it can be shown that this pathway influences neuronal activity, it will be important to learn how the information is transmitted intercellularly, the targets of the signals, and in what way neuronal processes are affected by the propagation of Ca2+ waves.

4.5. 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; Szabó, 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

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+]i, 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 γ, tumor necrosis factor α, interleukin 1β, 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 (Agulló 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 α1-adrenoreceptors (Agulló 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., 1993).

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

both with NADPHd staining and NOS-selective antibodies (cf. Vaney and Young, 1988; Straznicky and Gábriel, 1991; Osborne et al., 1993; Weiler and Kewitz, 1993; Kurenni et al., 1995). Although there are significant differences among species (Fig. 4.16), NOS is expressed to a varying degree in the ellipsoids of photoreceptor inner segments, in every neuronal cell type of the inner nuclear layer, in ganglion cells, and notably, within Müller cells (Goureau et al., 1994b; Liepe et al., 1994; Darius et al., 1995; Huxlin, 1995; Kim et al., 1999). Interestingly, long before neuronal NADPHd activity was identified with NOS or associated with NO production, Toichiro Kuwabara, David Cogan, and their coworkers (Kuwabara et al., 1957; Cogan and Kuwabara, 1959) studying the retinal distribution of dehydrogenases reported that tetrazolium precipitate was present in human Müller cells under substrate conditions requiring activity of the diaphorase. In addition to cells of the neural retina, both human and bovine RPE cells produce NO in response to cytokines (Goureau et al., 1992, 1994a), a reaction that may result in inhibition of the important phagocytic activity performed by RPE cells (Becquet et al., 1994).

With so many potential sources of NO in the retina, questions arise as to which cellular functions are affected by NO, and which cells provide the NO that modulate these functions. Not surprisingly, the molecule has been implicated in a broad range of phenomena, including visual adaptation, modulation of gap junctions, and ischemic injury (reviewed by Goldstein et al., 1996). In isolated rod photoreceptors, for example, NO donors induce membrane depolarization in dark-adapted cells, and accelerate recovery of the dark current after photic stimulation (Schmidt et al., 1992; Tsuyama et al., 1993). Both reactions reflect changes in guanylate cyclase activity, cGMP production, and the opening of cGMP-gated cation channels. Activation of a cGMP-gated conductance by NO donors has been demonstrated also in ganglion (Ahmad et al., 1994) and bipolar cells (Shiells and Falk, 1992), and as illustrated in Fig. 4.17, agents that generate NO or activate endogenous guanylate cyclase close gap junction channels and significantly block electrical and dye coupling between horizontal cells (DeVries and Schwartz, 1989; Miyachi et al., 1991,1994; Pottek et al., 1997). The effect on gap-junctional coupling may vary with cell type, considering results obtained from recordings within rat supraoptic nucleus, where NO-induced cGMP production increased neuronal coupling (Yang and Hatton, 1999).

Clearly retinal cells which express NOS and contain soluble guanylyl cyclase may autoregulate their activity via NO. However, the NO directly responsible for mediating a physiological response in vivo also could reach its targets by diffusion from neighboring cells (cf. Vincent and Hope, 1992). As we have seen, Müller cells contribute to NO production in the retina of many vertebrates (Goureau et al., 1994b; Liepe et al., 1994; Darius et al.,

ronal cNOS in the “knockout” nNOS– /– mice. A more direct approach, combining NADPHd histochemistry with nNOS immunoreactivity, showed that the neuronal isoform is expressed also by Müller cells of the tree shrew retina (Cao et al., 1999).

Thus, activation of cNOS via calcium entry through the voltageand ligand-regulated Ca2+ channels of Müller cells or cytokine-induced activation of iNOS, can lead to the formation of NO. The sustained release of high levels of NO from Müller cells could potentially have profound effects on neuronal excitability, retinal blood flow, and cellular reactions to injury. Recent in vitro experiments by Goureau et al. (1999) provide evidence that such an intercellular transfer can occur. Using cocultures of mouse retinal neurons and Müller cells, they demonstrated that cytokine-induced stimulation of glial iNOS can lead to neuronal cell death. The neurotoxic reaction was not seen in cocultures in which the Müller cells were derived from NOS-2 knockout mice, nor did it occur after the production of NOS or the release of NO was suppressed.

It is evident that we will gain a better understanding of the importance of NO to the physiology and pathology of the retina from detailed in vivo studies of knockout mice. Preliminary data on the ocular effects of gene deletion indicate that retinal morphology appears to be well-preserved in NOS–null mice (Huang et al., 1993; Darius et al., 1995). However, a careful analysis of retinal function has not yet been performed on these animals. In addition, the availability of selective inhibitors of constitutive (e.g., 7-nitro- indazole) or inducible (e.g., L-N6-l-iminoethyl lysine) forms of NOS should also prove useful in further elucidating the effects of Müller cell-derived NO on retinal physiology.

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