Учебники / Auditory Trauma, Protection, and Repair Fay 2008

50 P. Wangemann

2.1 Cellular Energy Production and Redox Homeostasis

Glucose is the major energy substrate of the cochlea although other substrates can support cochlear function (Kambayashi et al. 1982). Glucose is supplied via the bloodstream, enters perilymph by facilitated diffusion, and is taken up into cells independent of insulin (Ferrary et al. 1987; Wang and Schacht 1990). Several glucose transporters have been identified: SLC2A51 (GLUT5) has been localized to outer hair cells (Nakazawa et al. 1995) and SLC2A1 (GLUT1) appears to be the main uptake mechanism in stria vascularis (Ito et al. 1993; Nakazawa et al. 1995; Takeuchi and Ando 1997). As an additional fuel, marginal cells of stria vascularis can take up pyruvate and lactate via the monocarboxylate transporters SLC16A1 (MCT1) and convert lactate to pyruvate (Shimozono et al. 1997; Okamura et al. 2001). Glucose and pyruvate are metabolized to CO2 to yield energy mainly in the form of ATP (Fig. 3.1).

Several key enzymes and intermediates of glycolysis and the citric acid cycle had been analyzed in some of the earliest biochemical assays of microdissected structures of the cochlea (Thalmann et al. 1970). Oxygen consumption and metabolic activity are higher in stria vascularis than in the organ of Corti, which supported the concept that the cochlear transducer is powered by stria vascularis and that stria vascularis is the structure that generates the endocochlear potential (Thalmann et al. 1972; Marcus et al. 1978b; Ryan et al. 1982). In addition to glycolysis and the citric acid cycle, stria vascularis uses the pentose phosphate pathway (Marcus et al. 1978a) to generate large quantities of NADPH (Fig. 3.1) required in defense of free radical stress associated with high metabolic rates.

Free radicals, generated in controlled amounts, can serve as signaling molecules and are part of the cellular redox homeostasis. Unalleviated free radical stress, however, leads to redox imbalance, uncontrolled oxidation of proteins and lipids, and causes cell damage and unwanted cell death. Free radicals are a byproduct of an inefficient “leaky” electron transfer in the mitochondrial electron transport chain and of the mitochondrial cytochrome P450 systems. Incomplete reduction of O2 generates the free radical superoxide anion •O−2 . In addition, •O−2 can be generated by mitochondrial xanthine oxidases, by NADPH oxidases (Banfi et al. 2004), and by the cyclooxygenase pathway that is part of the arachidonic acid metabolism (Ziegler et al. 2004). Superoxide anion •O−2 dismutates to hydrogen peroxide H2O2 (see reaction 3.1), which in the presence of Fe2+ or •O−2 gives rise to the formation of the extremely aggressive hydroxyl radial •OH− (Fenton reaction, see reactions 3.2 and Haber-Weiss reaction, see reaction 3.3). Alternatively, •O−2 can react with nitric oxide radical •NO to form peroxynitrate ONOO− (reaction 3.4) that under acidic conditions (reactions 3.5 and 3.6) or in the presence of CO2 (reactions 3.7 and 3.8) causes nitration of proteins, lipids

1Throughout this chapter, proteins are identified by their human gene names according the HUGO human genome nomenclature committee (http://www.gene.ucl.ac.uk/ nomenclature/). Commonly used alternative names are given in brackets.

Figure 3.1. Metabolic energy production. Metabolism of glucose to CO2 via glycolysis, oxidative decarboxylation, the citric acid cycle, and oxidative phosphorylation yields energy mainly in the form of ATP. Alternatively, glucose is metabolized by the pentose phosphate pathway to yield the reducing equivalent NADPH.

and nucleic acids (represented as R) via formation of the very aggressive nitrate radical •NO2 (Beckman et al. 1990; Lymar et al. 1996).

Maintenance of redox homeostasis is accomplished by cellular defenses against free radical stress, which include the prevention of the formation of free radicals and detoxification of free radicals. Given that iron is a catalyst for free radicals, the availability of free iron is carefully controlled. Iron is chelated by transferrin, a protein dimer that binds 2 iron atoms. Iron bound to transferrin is taken up into cells via transferrin receptor. In the cytosol, iron is either bound to transferrin or confined by ferritin, a cannonball-like protein multimer that houses up to 4500 iron atoms (Eisenstein 2000). The role of iron homeostasis for inner ear function is evident from the findings that ototoxicity is enhanced by iron and alleviated by the administration of iron chelators (Song et al. 1998; Conlon and Smith 1998). Control of tissue pH, CO2, and •NO are additional homeostatic mechanisms intertwined with the defense against free radicals. Acidification aggravates and alkalinization alleviates ototoxicity (Tanaka et al. 2004), and inhibition of nitric oxide synthase alleviates ischemia reperfusion injury to the inner ear (Tabuchi et al. 2001).

Direct defense mechanisms against free radicals aim to scavenge radicals or convert aggressive radicals to less toxic radicals. The first line of defense is superoxide dismutase that converts superoxide anion •O−2 to hydrogen peroxide H2O2 (see reaction 3.1). Although H2O2 is potentially harmful, this reaction is of benefit, as long as H2O2 is rapidly detoxified before it can give rise to the formation of the more harmful •OH radicals.

Catalase and glutathione peroxidase are the second line of defense catalyzing the conversion of hydrogen peroxide to O2 and H2O (see reactions 3.10 and 3.11). Glutathione peroxidase oxidizes glutathione (GSH) to glutathione disulfide (GSSG) in this process. GSH is a tripeptide composed of glutamate, cysteine,

and glycine that is present in the cytosol in millimolar concentration and can convert hydrogen peroxide to O2 and H2O even in the absence of glutathione peroxidase, albeit more slowly. GSH is restored by reduction of GSSG into 2 moles of GSH in a reaction catalyzed by glutathione reductase, an enzyme that uses NADPH as a cofactor (see reaction 3.12).

Stria vascularis and organ of Corti contain high activities of superoxide dismutase, catalase, and glutathione peroxidase (Spector and Carr 1979; Pierson and Gray 1982). In the lateral wall, GSH immunoreactivity is preferentially distributed in basal cells and intermediate cells of stria vascularis, as well as in fibrocytes and capillary endothelial cells of the spiral ligament (Usami et al. 1996). Glutathione does not only play a role in free radical defense but also in detoxification reactions in which denatured proteins or xenobiotics are conjugated to glutathione in a reaction catalyzed by glutathione S-transferases (GSTs). Several isoforms of GST have been found in intermediate cells and the basal cells of the stria vascularis and various types of fibrocytes in the spiral ligament that may be serve the need to detoxify metabolic waste products (El Barbary et al. 1993; Takumi et al. 2001.

Defenses against free radical stress are not static but adaptable. Moderate noise stimulation, which may lead to low levels of free radicals, has been shown to enhance defense mechanisms and cause protection against noise induced hearing loss (Jacono et al. 1998). Similarly, preadministration of ototoxic drugs at nontoxic concentrations provides protection (Oliveira et al. 2004). Defense mechanisms, however, can be overwhelmed by ischemia reperfusion, intense noise stimulation, in homeostatic disorders, and by ototoxic drugs including aminoglycoside antibiotics and platinum-based chemotherapeutic agents (see Henderson et al., Chapter 7; Rybak et al., Chapter 8).

2.2 Cellular Na+, K+, and Cl− Homeostasis

Cells use much of the energy available in form of ATP for the maintenance of steep Na+ and K+ gradients across the plasma membrane. Typical cytosolic concentrations of 5–15 mM Na+ and 100–120 mM K+ are 10to 40-fold different from typical extracellular concentrations of 150 mM Na+ and 3–4 mM K+. These steep Na+ and K+ gradients that are established by Na+/K+-ATPases in virtually all cells and serve as sources of energy for cellular homeostasis and for cell signaling. Na+-coupled transporters, which use the steep Na+ gradient as energy source, include cotransporters and exchangers (Fig. 3.2). Some of these transporters are electrogenic and contribute to the membrane potential or use the membrane potential as an additional source of energy. The steep K+ gradient in conjunction with K+-selective channels defines the resting membrane potential in most cells. A notable exception are strial marginal cells and vestibular dark cells that maintain their membrane potential predominantly by a basolateral Cl− conductance (Wangemann and Marcus 1992; Takeuchi and Irimajiri 1994).

Cytosolic concentrations for Cl− vary greatly between cell types ranging between 5 and 50 mM. Uptake of Cl− can be mediated in exchange for metabolically

54 P. Wangemann

Figure 3.2. Establishment and use of Na+ and K+ gradients. The Na+/K+-ATPases establishes steep Na+ and K+ gradients that are used by cotransporters and exchangers as energy sources. The K+ gradient in conjunction with K+ channels defines in most cells the cytosolic side negative membrane potential.

generated HCO−3 or driven by the Na+ gradient via a Na+/2Cl−/K+ cotransporter or a Na+/Cl− cotransporter (Fig. 3.2). Elimination of Cl− can be driven by the membrane potential via Cl− channels or by the K+ gradient via KCl cotransporters. Homeostasis of the cytosolic salinity, which is defined mainly by the concentrations of K+, Cl−, and HCO−3 ensures the maintenance of a suitable environment for proteins, lipids, and nucleic acids.

2.3 Cell Volume Regulation

Cellular function depends on a normal cell volume that ensures appropriate proximities between molecules in an environment of normal ionic strength and osmolarity, and on factors that affect protein configuration, protein–protein interactions, and enzyme activity (Lang et al. 1998). Challenges to the constancy of cell volume can originate with changes in the osmolarity or electrolyte composition of the extracellular environment, with mismatches between uptake and release of osmolytes such as salts and sugars, and with catabolic formation of osmolytes from osmotically inactive macromolecules or the anabolic fixation of osmolytes into macromolecules. In response to excessive cell swelling, cells respond with a regulatory volume decrease that may consist of the release of osmolytes. Most commonly, cells release KCl via K+ or nonselective cation channels and Cl− channels or via KCl cotransporters (Fig. 3.3). Conversely, in responsetoexcessivecellshrinking,cellsrespondwitharegulatoryvolumeincrease that commonly consists of the uptake of NaCl via activation of Na+/2Cl−/K+ cotransporters or parallel activation of Na+/H+ and Cl−/HCO−3 exchangers.

Cell volume, however, is not just a parameter that is kept constant. Mechanisms of cell volume regulation have been incorporated into normal cell function,

Figure 3.3. Cell volume regulation. Cell swelling is corrected by a regulatory volume decrease (RVD) and cell shrinking by a regulatory volume increases (RVI). Mechanisms of RVD include release of osmolytes via K+ and Cl− channels or KCl cotransporters. Mechanisms of RVI include uptake of osmolytes via the Na+/2Cl−/K+ cotransporter.

where cell volume plays the role of a cellular signaling mechanisms much like a second messenger. Cell volume can communicate changes in ion transport across the basolateral membrane to the apical membrane and vice versa. In vestibular dark cells, for example, cell volume communicates changes in the rate of transport across the basolateral membrane to the apical membrane. The apical K+ channel in vestibular dark cells is activated by cell swelling and the basolateral Na+/2Cl−/K+ cotransporter by cell shrinkage (Wangemann and Shiga 1994; Wangemann et al. 1995b). Cell volume couples the two cell membranes; increased uptake of ions across the basolateral membrane causes cell swelling, which provides the signal to activate K+ secretion across the apical membrane.

2.4 Cellular pH Regulation

The cytosolic pH is another critical variable in the cytosolic environment that affects many fundamental homeostatic mechanisms. For example, cytosolic acidification inhibits protein synthesis and favors the generation of toxic free radicals. Cells are under a constant thread of acidification since energy metabolism generates CO2 as an end product (Fig. 3.1), which causes cellular acidification through the generation of H+ (See reaction 3.13).

The spontaneous conversion of CO2 to HCO−3 and H+ occurs very slowly but can be facilitated by carbonic anhydrases. Metabolically active tissues as well as epithelia engaged in acid and base transport express large quantities of carbonic anhydrases to maintain the equilibrium between CO2, HCO−3 , and

The steep H+ gradient across the vesicular membrane is used by neurotransmitter containing vesicles to drive the uptake of neurotransmitters. Lysosomes use the low luminal pH for the activation of enzymes and vesicles containing internalized receptors require the low luminal pH for the dissociation of ligand receptor complexes. The expression of vacuolar H+-ATPases is not limited to cellular vesicles and organelles but occurs also in the plasma membrane where vacuolar H+-ATPases mediate cytosolic acid extrusion and participate in the vectorial transport of acids or bases.

In the cochlea prominent acid extrusion mechanisms exist in the stria vascularis and spiral ligament, in the outer sulcus, in outer hair cells, and in interdental cells of the spiral limbus. Strial marginal cells contain vacuolar H+-ATPase in their apical membrane, which was identified by the subunit ATP6V1E, and K+/H+- ATPase in their basolateral membrane (Stankovic et al. 1997; Shibata et al. 2006). Notably, marginal cells lack the nonessential H+-ATPase subunit ATP6V1B1 (Karet et al. 1999b). Functional evidence for H+-ATPases in marginal cells has not yet been obtained. Further, marginal cells contain the Na+/H+ exchanger SLC9A1 (NHE1) in their basolateral membrane, which likely plays a major role in acid extrusion (Wangemann et al. 1996a; Bond et al. 1998). Interestingly, although marginal cells are metabolically highly active and thus can be expected to generate notable amounts of CO2, they appear to lack carbonic anhydrase (Lim et al. 1983; Yamashita et al. 1992; Okamura et al. 1996). Several cells adjacent cells, however, contain this enzyme including erythrocytes in the bloodstream, strial intermediate, and basal cells as well as fibrocytes of the spiral ligament (Lim et al. 1983; Okamura et al. 1996). In fact, erythrocytes as well as type I and III fibrocytes bordering stria vascularis and bone contain the highly active carbonic anhydrase isoform CA2 (Spicer and Schulte 1991). In addition, several HCO−3 transporters have been identified in fibrocytes of the spiral ligament as well as in outer sulcus and spiral prominence epithelial cells including the Cl−/HCO−3 exchangers SLC4A2 (AE2) and SLC26A4 (pendrin) and the Na+/HCO−3 cotransporter SLC4A7 (NBC3) (Stankovic et al. 1997; Bok et al. 2003; Wangemann et al. 2004). It is conceivable that intermediate cells and basal cells of stria vascularis in conjunction with spiral ligament fibrocytes provide a buffer system between sources and sinks for CO2. Metabolically active strial marginal cells as well as certain fibrocytes may be seen as sources of CO2 whereas plasma, endolymph, and perilymph may be seen as sinks.

Outer hair cells contain in their basolateral membrane the Na+/H+ exchanger SLC9A1 (NHE1), which most likely functions as an acid extrusion mechanism (Ikeda et al. 1992a; Bond et al. 1998). Carbonic anhydrase activity has been found to be limited in outer hair cells to the area of the cuticular plate (Okamura et al. 1996). In addition, outer hair cells contain the Cl−/HCO−3 exchanger SLC4A2 (Zimmermann et al. 2000). It is unclear whether SLC4A2 functions as an acid extrusion mechanism because it is unclear whether outer hair cells maintain an outwardly directed Cl− gradient that could drive the uptake of HCO−3 . It may be more likely that SLC4A2 participates in the maintenance of cell volume (Cecola and Bobbin 1992).

58 P. Wangemann

Interdental cells of the spiral limbus express in their apical membrane vacuolar H+-ATPase, which was identified by two subunits, ATP6V1E and ATP6V1B1 (Stankovic et al. 1997; Karet et al. 1999b). Further, interdental cells express the cytosolic carbonic anhydrases CA1 and CA3 and the Cl−/HCO−3 exchangers SLC4A2 in their basolateral membrane (Yamashita et al. 1992; Stankovic et al. 1997). In the absence of functional data, the significance of these transporters is currently unclear although it is conceivable that interdental cells are engaged in H+ secretion and HCO−3 reabsorption and the maintenance of pH in endolymph.

2.5 Cellular Ca2+ Regulation

is both a key second messenger involved in cell signaling as well as a cytotoxin. The cytosolic Ca2+ concentration under resting conditions of approximately 100 nM is approximately 10,000-fold lower than the interstitial Ca2+ concentration of 1 mM. This enormous gradient is carefully controlled to prevent ambiguity in cell signaling and cell death due to Ca2+ overload (Fig. 3.5). Increases in the cytosolic Ca2+ concentration are used to translate mechanical signals such as cellular deformation and chemical signals such as hormones, neurotransmitters, and growth factors into a variety of cellular actions such as regulation of enzyme activities, neurotransmitter release, salt and water secretion, contraction, proliferation, and cell death. Increases in the cytosolic

Figure 3.5. Cellular Ca2+ regulation. Cellular Ca2+ regulation consists mainly of Ca2+ export, sequestration in Ca2+ stores and Ca2+ buffering. Ca2+ extrusion can be mediated by PMCA Ca2+-ATPases that pump Ca2+ out of the cell or by SERCA Ca2+-ATPases that pump Ca2+ into cytosolic stores. Alternatively, Ca2+ extrusion can be driven by the Na+ gradient established by the Na+/K+-ATPase and mediated by Na+/Ca2+ exchangers. Ca2+ mediated cell signaling entails increases in the cytosolic Ca2+ concentration. Ca2+ increases can be mediated by voltageor receptor-gated Ca2+ channels in the plasma membrane or by plasma membrane receptors that cause release of Ca2+ from cytosolic stores via IP3 receptors (IP3R). Increases in the cytosolic Ca2+ concentration can be enhanced by Ca2+-induced Ca2+ release via ryanodine receptors (RyR).