»-Glutamine GDH

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

Na+/H+ exchanger (?) —* vitreous Carbonic anhydrase J

Na+/HCO3" exchanger----- vitreous

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

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 (Ko-bayashi 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- exchanger to the Müller cell end-foot 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 aNa+/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- 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+-HCO- blocker, reduces the rate of alkalinization. D. pHo was recorded simultaneously beneath the endfoot and soma of a single cell in HCO- 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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