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

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of neuronal function is due to the direct action of α-AAA on neurons or is a secondary consequence of glial damage.

Methionine sulfoximine (MSO) exerts its toxic effect by irreversibly inhibiting glutamine synthetase (Griffith and Meister, 1978). In the presence of MSO, glutamine levels fall, and the supply of precursors for glutamate and GABA synthesis becomes limiting (Martin and Winiewski, 1996). Indeed, convulsions produced by MSO are brought on by inhibition of glutamine synthetase. Last, fluoroacetate and fluorocitrate are effective toxins that appear to act by inhibiting the TCA cycle in glial cells (Fonnum et al., 1997). Fluoroacetate is metabolized to fluorocitrate, which inhibits aconitase and blocks the mitochondrial citrate carrier. Inhibition of the TCA cycle by fluorocitrate reduces glutamine production and leads to its gliotoxic effect. Since the TCA cycle operates in both glial cells and neurons, the glial selectivity of these agents remains enigmatic.

3.3. GABA METABOLISM

GABA, a major inhibitory neurotransmitter in the retina (Yazulla, 1986), is taken up by Müller cells in mammalian retinas (see Chapter 4). Inside the Müller cell, GABA is degraded through the action of GABAtransaminase (GABA-T) and succinic semialdehyde dehydrogenase, enzymes shown histochemically to be present in Müller cells (Hyde and Robinson, 1974; Moore and Gruberg, 1974). The demonstration of GABA uptake and localization of GABA-metabolizing enzymes suggest that Müller cells are likely to participate in regulating the extracellular concentration of GABA.

The idea that Müller cells may regulate GABA levels in retina is well supported by other studies. If GABA metabolism occurs mainly in Müller cells, inhibition of GABA-T activity can be expected to result in enhanced GABA accumulation in Müller cells but not in neurons (Fig. 3.12). Indeed, GABA-immunocytochemical studies show that although little GABA is found normallyin Müllercells, there is an increase in GABA-immunoreactivity in Müller cells after the retina is treated with the GABA-T inhibitor, vigabatrin (Cubells et al., 1988; Neal et al., 1989). Since Müller cells cannot synthesize GABA, the GABA in Müller cells must come from its uptake and accumulation.

3.3.1. GABA-transaminase

GABA degradation is catalyzed by two mitochondrial enzymes: GABAtransaminase and succinic acid semialdehyde dehydrogenase (Kugler,

transamination of α -ketoglutarate by GABA-T to generate glutamate, which is subsequently converted to GABA by glutamic acid decarboxylase. Alternatively, GABA could be derived from GABA uptake. As previously described, GABA is metabolized by GABA-T to succinic acid semialdehyde. To conserve the GABA supply, transamination usually occurs when α-ketoglu- tarate is present to accept the NH2 group from GABA, thus leading to formation of glutamate. The other product of the reaction, succinic acid semialdehyde, is converted to succinic acid, and can reenter the Krebs cycle, thus completing the loop.

Although GABA taken up by neurons can be recycled, the GABA in glial cells is metabolized and cannot be converted back to GABA because glia do not have glutamic acid decarboxylase. GABA, however, can be recovered through an indirect route involving the Krebs cycle. In glia, glutamate is converted to glutamine, which is transferred back to neurons where it is converted to glutamate by glutaminase, and thus can enter the GABA shunt. The main function of the shunt appears to be in the production of GABA and not in the TCA cycle since less than 10% of the metabolic flow occurs through the shunt (Kugler, 1993). More important, the shunt is not universal since glutamic acid decarboxylase (GAD) is almost exclusively found in GABAergic neurons and never in glial cells.

In summary, there is a tight metabolic link between retinal neurons and Müller cells in the turnover and metabolism of glutamate and GABA. Müller cells contain high levels of glutamate dehydrogenase and glutamine synthetase, and their products, α-ketoglutarate and glutamine, have been proposed to be involved in supplementing the neurotransmitter pool in glutamatergic and GABAergic neurons. Fig. 3.15 gives a summary diagram of the metabolic interactions of glutamatergic and GABAergic neurons with Müller cells.

3.4. ACID-BASE REGULATION

Because of its high rate of respiration, particularly in the dark, the retina produces significant amounts of CO2 (Sickel, 1972). CO2 has a high diffusion coefficient and is very soluble; therefore, most of the CO2 generated can be expected to diffuse into adjacent retinal or choroidal vessels. Some of the CO2 diffusing into the retina will pass into the neighboring Müller cells where it is converted to bicarbonate by the action of carbonic anhydrase. Müller cells are known to contain high levels of carbonic anhydrase which is likely to be involved in CO2 fixation in the retina and the maintenance of a normal acid-base balance (Musser and Rosen, 1973; Sarthy and Lam, 1978; Linser and Moscona, 1981).

Figure 3.15. A schematic diagram of metabolic interactions among glutamatergic and GABAergic neurons and Müller cells. The enzymes aspartate aminotransferase (AAT), phosphateactivated glutaminase (PAG), and glutamate decarboxylase (GAD) are strongly enriched in neurons, whereas glutamate dehydrogenase (GDH), glutamine synthetase (GS), and GABAtransaminase (GABA-T) are strongly enriched in Müller cells.

3.4.1. Carbonic Anhydrase

The enzyme, carbonic anhydrase, catalyzes the conversion of CO2 and water to carbonic acid, which dissociates spontaneously to yield HCO3– and H+ (Fig. 3.16). Carbonic anhydrase is believed to provide H+ and HCO3– ions for rapid intracellular buffering or for exchange of other ions resulting in movement of ions and fluids across the plasma membrane (Maren, 1967).

Mammalian carbonic anhydrase exists as seven isozymes: four cytoplasmic (CAI, CAII, CAIII, and CA IV); one membrane bound (CA IV); one secretory (CAVI); and one mitochondrial (CAV). The isozymes have broad structural similarity but are distinguished by their differential tissue distribution, membrane association, and catalytic activity (Tashian et al., 1991). CAII is the predominant isozyme in retina, and other isozymes make up a smaller fraction of the total CA content (Wistrand et al., 1986; Ridderstrale et al., 1994).

According to several lines of evidence, CA is localized almost exclusively in Müller cells in the vertebrate retina (Musser and Rosen, 1973; Korhonen and Korhonen, 1965; Hansson, 1967; Sarthy and Lam, 1979a; Wistrand et al.,

Figure 3.16. Carbonic anhydrase converts CO2 to bicarbonate. The enzyme is almost exclusively localized in Müller cells in the vertebrate retina. Some of the bicarbonate is released into the vitreous through the sodium, bicarbonate exchanger. The protons generated in the reactions may also end up in the vitreous via a sodium-proton exchanger.

1986; Luten-Drecoll at al., 1983; Linser and Moscona, 1984; Ridderstrale et al., 1964). Histochemical methods as well as immunocytochemical studies using CA II-specific antibodies show that the radially oriented Müller cells are strongly labeled in many vertebrate retinas (Musser and Rosen, 1973; Linser and Moscona, 1984). Moreover, a comparison of CA levels in isolated Müller cells with values for the whole retina suggest that more than 90% of the retinal carbonic anhydrase activity can be attributed to Müller cells (Sarthy and Lam, 1978). The presence of high levels of CA in Müller cells may mean they play an active role in regulating acid-base balance in the retina.

3.4.2. Bicarbonate Exchange

There is good experimental evidence that neuronal activity leads to changes in intracellular and extracellular pH (Cheder, 1990; Brookes, 1997). The magnitude of the pH change can be expected to depend on intracellular and extracellular buffering systems as well as the activities of acid and base transporters (and exchangers) in retinal neurons and Müller cells. In CNS glial cells, a Na+/HCO3– exchanger has been implicated in regulation of internal pH (Ritchie, 1987; Cheder, 1990).

A bicarbonate exchanger, present in Müller cells, could serve a similar function. Newman (1991) demonstrated electrogenic Na+/HCO–3 exchange in isolated Müller cells from the salamander retina (Fig. 3.17). The Na+/HCO3– exchanger has a stoichiometry of ~3:1 (HCO–3 :Na+), and is predominantly localized to the endfoot region of the Müller cell. It is likely that the exchanger is involved in extruding HCO–3 from Müller cells into the vitreous body. Therefore, when CO2 is metabolized in Müller cells, a rise in [HCO–3 ]i occurs which would result in an efflux of HCO–3 into the vitreous through the exchanger (Newman and Astion, 1991).

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Bicarbonate transport is known to be mediated by anion exchangers present in the plasma membrane (Kopito, 1990). Anion exchangers (AE) belong to a multigene family involved in the regulation of intracellular pH and chloride concentration in many tissues (Kopito, 1990). In the rat retina, two different isoforms of the AE3 gene product have been described (Kobayashi et al., 1994): A 125 kDa form is expressed in horizontal cells whereas a 165 kDa form is found in Müller cells (Fig. 3.18). AE3 in Müller cells is found predominantly in the endfoot region, an observation in agreement with the localization of the Na+/HCO 3– exchanger to the Müller cell endfoot region. In developing retina, AE3 is expressed at very low levels at embryonic stages but increases steadily during postnatal development as the retina becomes functionally mature (Kobayashi et al., 1994).

If the carbonic anhydrase present in Müller cells is involved in regulating pH levels in the retina, one would expect carbonic anhydrase inhibitors, such as acetazolamide and methazolamide, to have strong effects on extracellular pH. As shown in Fig. 3.19, there is experimental evidence that in the presence of the inhibitors, there is an increase in the light-evoked acidification in the inner plexiform layer and in the subretinal space (Borgula et al., 1989; Oakley and Wen, 1989).

As mentioned before, one consequence of CO2 fixation is the lowering of intracellular pH in Müller cells. What happens to H+ inside Müller cells? It has been proposed that the excess protons inside are released into the capillaries close to Müller cells, or into the interstitial fluid, or even into the vitreous through the endfoot (Newman, 1991). A Na+/H+ exchanger involved in H+ extrusion has been localized in astrocytes (Kimelberg et al., 1979), and it seems likely that a Na+/H+ exchanger serves a similar function in the Müller cell plasma membrane.

The intimate association of Müller cells with the retinal vasculature and the influence of extracellular ionic changes on blood flow indicate that ionic activities of Müller cells may be linked to changes in retinal metabolism. In this regard, an attractive idea is that HCO–3 efflux through the

Figure 3.17. Bicarbonate transport in Müller cells. The three figures show potassium-evoked intracellular alkalinization in Müller cells isolated from salamander retina. A. The rate and extent of alkalinization is greater for larger increases in [K+]o. B. substitution of HEPES for HCO3 reduces the rate of alkalinization. C. Addition of DIDS, a Na+–HCO3– blocker, reduces the rate of alkalinization. D. pHo was recorded simultaneously beneath the endfoot and soma of a single cell in HCO3– Ringer. E. Nomarski image of aMüller cell. E Difference ratio image of pHo changes evoked by raising [K+]o from .5 to 50 mM in HCO3– Ringer. Increasing [K+]o evokes an acidification that is largest beneath the endfoot (outline) (Newman, 1996). (Copyright 1991 The Journal of Neuroscience, reprinted with permission.)

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Figure 3.19. Effect of carbonic anhydrase inhibitors on retinal pH. The figure shows pHo from inner plexiform layer and subretinal space, with the simultaneously recorded field potential (right). Control responses (top) were obtained prior to superfusion with either acetazolamide or methazolamide (Borgula et al., 1989). (Copyright 1989 Elsevier Science, reprinted with permission.)

Na+/HCO3– exchanger, H+ efflux through a Na+/H+ exchanger, and K+ efflux through potassium channels in Müller cell membrane can all serve to regulate retinal blood flow in response to variations in neuronal activity (Newman, 1991).

3.5. RETINOID METABOLISM

Vitamin A and its metabolites (retinoids) are the sine qua non for vision in all vertebrates. The essential role of vitamin A (retinol) in vision has been known since George Wald, Ruth Hubbard, and their coworkers (Wald, 1968) demonstrated that the 11-cis isomer of retinaldehyde (11-cis retinal) is the chromophore that binds to opsin within the disc membranes of rod outer segments to form the light-sensitive photopigment, rhodopsin. Light

Figure 3.18. Immunocytochemical localization of AE3 in Müller cells. Sections of rat retina (A,B) or dissociated Müller cells were stained with an antibody against AE3. A. Histological section of retina showing different layers; B. retinal section stained with anti-AE3. Immunoreactivity can be seen in the end feet, apical region and radial processes of Müller cells. C. Solitary Müller cell stained with anti-AE3 (Kobayashi et al., 1994). (Copyright 1994 The Journal of Neuroscience, reprinted with permission.)