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

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Figure 3.1. Pathways by which glucose (arrows) released from capillaries can end up in neurons. The first pathway does not involve the Müller cell. In the second pathway, glucose is taken up by the Müller cell and released for neuronal uptake. In the third pathway, glucose enters the Müller cell where it is converted to a metabolite, such as lactate (heavy arrows), and is released for use by neurons. In the final pathway, glucose is taken up by the Müller cell and converted to glycogen (dots) for storage; subsequently, the glycogen is broken down to glucose and released for uptake by neurons.

tion. How does glucose released from capillaries end up as carbon fuel in retinal neurons? Do Müller cells serve as an intermediary in this process?

There are several routes by which glucose released from capillaries can end up in neurons (Fig. 3.1; cf. Coles, 1996). One route would be direct transfer of glucose from capillaries to neurons without any Müller cell involvement; other routes can involve active participation of glial cells either as a conduit for glucose transfer or as a site for glucose storage and metabolism before release to neurons. At least four pathways can be invoked to describe glucose transfer (Coles, 1996; Fig. 3.1).

In pathways one and two, glucose does not undergo any change, whereas in pathways three and four glucose is converted to a metabolite in Müller cells. In the first pathway, glucose can diffuse away from capillaries and be taken up directly by neurons without intervention of Müller cells. In the second pathway, Müller cells can take up glucose from blood and redistribute it to neurons. In the third pathway, glucose taken up by Müller cells can be converted to a metabolite, which is released for use by neurons.

And in the fourth pathway, glucose can be transformed to glycogen in Müller cells and later broken down to provide glucose for neurons. These pathways are not mutually exclusive, Although none of the pathways can be ruled out completely, the available experimental evidence indicates that in the CNS, glucose is taken up by glial cells, converted to a metabolite, and subsequently released for neuronal use (Magistretti et al., 1999).

3.1.1. Glucose Uptake and Neuronal Activity

If Müller cells provide neurons with energy metabolites, they must first take up glucose in quantities sufficient for their own needs and for the retinal neurons they serve. The cellular sites of glucose uptake have been determined in a number of vertebrate retinas by autoradiographic studies using 3H-2-deoxyglucose (3H-2-DG), a nonmetabolizable analog of glucose. Early studies showed that photoreceptors are able to accumulate 2-DG (Basinger et al., 1979; Witkovsky and Yang, 1982; Sperling et al., 1982) irrespective of whether 3H-2-DG was supplied in an incubation medium or was injected intravitreally. Based on these data, it appeared glucose could be directly taken up by photoreceptors in some species.

In contrast, Poitry-Yamate and Tsacopoulos (1991; 1992) found that when isolated guinea pig retina was incubated with 3H-2-DG, labeling was confined to Müller cells; photoreceptors and other retinal neurons were not labeled. The authors further demonstrated that freshly dissociated Müller cells could take up and phosphorylate 2-DG (Fig. 3.2). This result, and evidence that the glucose transporters, GLUT1 and GLUT2, are localized to the apical processes of Müller cells (Mantych et al., 1993; Watanabe et al., 1994), are a good indication that Müller cells are capable of avidly taking up exogenous glucose.

Further support for the importance of glucose uptake comes from experiments showing that neuronal activity can stimulate glucose uptake by glial cells (cf. Tsacopoulos and Magistretti, 1996). How does the glial cell sense the energy needs of the neurons? There is growing experimental evidence that synaptically released glutamate serves as the signal that links neuronal activation and glial uptake of glucose. Pellerin and Magistretti (1994) have shown directly that the addition of 0.2 mM glutamate strongly stimulates glucose uptake by astrocyte cultures (Fig. 3.3).

Surprisingly, the action of glutamate in promoting glucose uptake does not involve glutamate receptors but is mediated by the glial glutamate transporters (Pellerin andMagistretti, 1994;Takahashi etal., 1995). Glutamateinduced glucose uptake appears not to be affected by glutamate receptor antagonists, but is inhibited by preincubating astrocyte cultures with the glutamate transport inhibitor, DL-threo-β-hydroxyaspartate (THA) (Pel-

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Figure 3.2. Glucose uptake by Müller cells. A. Chromatographic profile showing intracellular [3H] 2DG and [3H] 2DG-6P in Muller cells incubated with [3H] 2-deoxyglucose (2DG). Note that >80% of 3H-radioactivity is associated with 2DG-6P. B. Autoradiographic localization of 3H-radioactivity in a dissociated Muller cell (Poitry-Yamate and Tsacopoulos, 1992). (Copyright 1992 John Wiley & Sons, Inc., reprinted with permission.)

lerin and Magistretti, 1994). Uptake is also blocked by the Na+/K+ ATPase inhibitor, ouabain (Takahashi et al., 1995). Moreover, treatment with monensin, a Na+-ionophore, was shown to stimulate 2-DG uptake into astrocytes (Yarowsky et al., 1986).

The following sequence of events has been proposed to explain how glucose uptake and utilization might be triggered by the influx of glutamate. Glutamate transport, which is coupled to the inward movement of sodium ions, results in asignificantelevation in intracellular [Na+] (Kimelberg et al., 1993). The increase in [Na+]i leads in turn to activation of Na+/K+ ATPase, resulting finally in stimulation of glucose uptake and glycolysis (Fig. 3.3). Since glutamate is the neurotransmitter released by photoreceptors (Massey, 1990), it is likely that glucose uptake into Müller cells is linked to photoreceptor synaptic activity. Is the glucose taken up by glial cells transferred to neurons or is it first converted to other substrates before transfer? The fate of glucose taken up by glial cells will be considered in the following sections.

A B

C

Figure 3.3. Glutamate stimulates glucose uptake by glia. A. A concentration-response curve showing stimulation of [3H] 2DG uptake by astrocyte cultures. B. Glutamate-stimulated [3H] 2DG uptake is inhibited by ouabain. Histogram shows [3H] 2DG uptake in glutamate-treated (hatched bars) or untreated (solid bars) cultures in the absence (control) or presence of Ouabain. C. Schematic of the mechanism for glutamate-induced glycolysis in astrocytes during physiological activation. At glutamatergic synapses, glutamate depolarizes neurons by acting on specific glutamate receptors. The action of glutamate is terminated by an efficient glutamate uptake system in astrocytes. Glutamate is cotransported with Na+, resulting in an increase in [Na+]i, leading to activation of Na+–K+-ATPase. The pump fueled by ATP provided by membrane-bound glycolytic enzymes, activates glycolysis, i.e., glucose utilization and lactate production in astrocytes. Lactate, once released, can be taken up by neurons to serve as an adequate energy substrate. Direct glucose uptake into neurons under basal conditions is also shown (labeled B) (Pellerin and Magistretti, 1994). (Copyright 1994 National Academy of Sciences, U.S.A., reprinted with permission.)

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show that Müller cells synthesize and release large amounts of lactate and much smaller amounts of amino acids and Krebs cycle metabolites. Moreover, based on CO2 production, lactate was found to be a better substrate than glucose for oxidative metabolism in photoreceptors. Finally, metabolism of lactate, glutamate and glutamine is modulated by light in Müller cell–photoreceptor aggregates, indicating an association between photoreceptor activity and lactate production (Fig. 3.5). It appears, therefore, that lactate produced in the Müller cell results from the high level of glycolysis, and that the release of lactate fuels oxidative metabolism in photoreceptors.

If lactate were the substrate transferred to neurons, we would expect that neurons should have a lactate uptake system, as well as the enzymes necessary for entry of lactate into the TCA cycle. Although these requirements have been fulfilled in other neuronal tissues (Dringen et al., 1993a, b; Tholey et al., 1981; Bittar et al., 1995), they remain to be demonstrated in the retina. As previously discussed, neurons also take up and phosphorylate glucose, and much of the glucose taken up by neurons is channeled to glycolysis or the pentose phosphate pathway. The neuronal signal that links photoreceptor metabolic activity to lactate release from Müller cells has yet to be identified, but a likely candidate is glutamate, the neurotransmitter released at the photoreceptor synapse (Fig. 3.6; cf. Poitry-Yamate et al., 1995).

3.1.3. Glycogen Storage and Mobilization

The idea that Müller cells utilize glucose to synthesize glycogen as an energy store received considerable support from the early studies of Kuwabara and Cogan (1959) who used selective histochemical stains to show that enzymes involved in glycolysis and glycogen synthesis were highly concentrated in retinal Müller cells. From a survey of the glycogen content and distribution in several vertebrate retinas, they concluded that the observed variations in glycogen content in vertebrate retinas were related to the blood supply and glial cell content of the retina (Kuwabara and Cogan, 1961). For example, in the avascular rabbit and guinea pig retinas, glycogen was abundant, whereas in the highly vascularized human retina, the glycogen content was found to be comparatively modest.

It is now clear that Müller cells can synthesize, store, and metabolize glycogen rapidly (Kuwabara and Cogan, 1959a, b, 1960,1961; Kuwabara et al., 1959; Hutchinson and Kuwabara, 1962; Lessell and Kuwabara, 1964; Kuwabara, 1965; Schabadasch and Schabadasch, 1972; Goldman, 1988,1990; Babel and Stangos, 1973; Poitry-Yamate and Tsacopulos, 1991,1992; Uga and Smelser, 1973b; Magalhaes and Coimbra, 1979, 1972; Pfeiffer et al., 1994;

Figure 3.6. A scheme for lactate and amino acid trafficking between photoreceptors and Müller cells. In the presence of exogenous glucose and oxygen, lactate formed from glucose or glycogen in the Müller cell is released into the extracellular space (ECS) and fuels the photoreceptor. Also shown are the glutamate to glutamine conversion in the Müller cells and glutamate’s downstream target, the bipolar cell. C, cytoplasm; and M, mitochondrion (PoitryYamate et al., 1995). (Copyright 1995 The Journal of Nueroscience, reprinted with permission.)

Niemeyer et al., 1997; Runger-Braddle et al., 1996). In electron micrographs, glycogen granules appear as 25–30 nm, electrondense, isodiametric β particles scattered throughout the Müller cell cytoplasm (Fig. 3.7). In the rabbit retina, ischemia leads to a rapid and progressive loss of available glycogen (a decline of ~50% in 15 min) (Wassilewa et al., 1976; Johansson, 1977). Similarly, in rats with streptozotocin-induced diabetes, the glycogen content in Müller cell processes increased 2–4-fold when the blood glucose was elevated 5-fold (Sosula et al., 1974). Although many studies show that the glycogen level in situ appears to vary with changes in retinal function, there are few estimates of the glycogen content in the retina. The glycogen content of dissociated Müller cells from juvenile guinea pig retina has been estimated to be at least 175 nmol glycosyl units/mg protein (Poitry-Yamate and Tsacopoulos, 1992).

The external signal for glycogen breakdown has not been identified. Glycogen mobilization in Müller cells is likely to be regulated primarily by neuroactive substances released by retinal neurons (Table 3.1). For exam-