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- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
Background: Carbonic anhydrases (CAs), which catalyze CO2 hydration to bicarbonate and protons, have been suggested to regulate potassium homeostasis and endocochlear potential in the mammalian cochlea. Sixteen mammalian CA isozymes are currently known. To understand the specific roles of CA isozymes in the inner ear, a systematic survey was conducted to reveal temporal and spatial expression patterns of all 16 CA isozymes during inner ear development. Results: Our quantitative reverse transcriptase-polymerase chain reaction results showed that different tissues express unique combinations of CA isozymes. During inner ear development, transcripts of four cytosolic isozymes (Car1, Car2, Car3, and Car13), two membrane-bound isozymes (Car12 and Car14), and two CA-related proteins (Car8 and Car11) were expressed at higher levels than other isozymes. Spatial expression patterns of these isozymes within developing inner ears were determined by in situ hybridization. Each isozyme showed a unique expression pattern during development. For example, Car12 and Car13 expression closely overlapped with Pendrin, an anion exchanger, while Car2 overlapped with Na-K-ATPase in type II and IV otic fibrocytes, suggesting functional relationships in the inner ear. Conclusions: The temporal and spatial expression patterns of each CA isozyme suggest unique and differential roles in inner ear development and function. Developmental Dynamics 242:269–280, 2013. © 2012 Wiley Periodicals, Inc.
- Top of page
- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the reversible conversion of carbon dioxide to bicarbonate ion and a proton. These enzymes, found in all types of organisms, are encoded by five independent gene families: the α-, β-, γ-, δ-, and ε-CAs (Supuran, 2008b; Xu et al., 2008). Only α-CA family members have been identified in mammals (Henry and Swenson, 2000; Esbaugh and Tufts, 2006). Thus far, 16 α-CA isozymes have been described, with different catalytic activities and subcellular localizations: CAI, II, III, VII, and XIII are cytoplasmic; CAIV, IX, XII, XIV, and XV are membrane-bound; CAVa and Vb are mitochondrial; and CAVI is secreted (Supuran, 2008a, 2008b). CAVIII, X, and XI are called CA-related proteins (CA-RPs) because they lack classical CA enzymatic activity and their biological functions remain unclear.
The importance of carbonic anhydrases has been shown in many physiological and pathological processes, including pH and CO2 homeostasis, electrolyte secretion, respiration, CO2/HCO3− transport, and bone resorption and calcification (Supuran, 2008a). In the respiratory system, CAs are involved in most CO2 transport and excretion from metabolically active tissues to red blood cells, and finally to gas exchanging organs, such as the lungs (Henry and Swenson, 2000). Cytoplasmic CAI and II are the most abundant CA isozymes in the red blood cells, while both cytoplasmic (CAII) and membrane-bound CAs (CAIV) are found in the lung (Henry and Swenson, 2000; Esbaugh and Tufts, 2006). The role of CAs has also been studied in detail in regulating renal physiology, such as acid/base homeostasis and bicarbonate reabsorption (Purkerson and Schwartz, 2007). CAs are expressed in most kidney segments. Cytoplasmic CAII accounts for most CA activity in the kidney, and different combinations of membrane-bound CAs, including CAIV, CAXII, CAXIV, and CAXV, are also expressed in different species (Purkerson and Schwartz, 2007). CAII is involved in bone physiology, such as osteoclast differentiation and bone resorption, and CAII-deficiency leads to osteopetrosis in humans and mice (Sly and Hu, 1995; Lehenkari et al., 1998; Margolis et al., 2008). In addition, various CAs expressed in the gastrointestinal canal and digestive glands are involved in ammonia detoxification, saliva production, gastric acid production, bile production, pancreatic juice production, and intestinal ion transport (Pan et al., 2007; Supuran, 2008a). Mitochondrial CAV has been associated with molecular signaling, such as insulin secretion in pancreatic β cells (Parkkila et al., 1998). Some CA isozymes are down- or up-regulated in various tumors and have been associated with oncogenesis and tumor progression (Supuran, 2008a, 2008b). Consistently, two hypoxia-inducible CA isozymes, CAIX and XII, were shown to promote tumor growth by regulating pH in an acidic and hypoxic microenvironment (Jarvela et al., 2008; Chiche et al., 2009). In the brain, CAIV and XIV were shown to regulate pH in extracellular fluid (Shah et al., 2005). Lastly, CAIII, which is highly expressed in many tissues, including skeletal muscle, has relatively low CA activity. Its physiological role is unclear because mice lacking CAIII do not have noticeable anatomical or physiological abnormalities (Kim et al., 2004). CAIII can, however, protect cells from oxidative damage by S-glutathiolation on two cysteine residues in response to oxidative stress, thereby functioning as an oxyradical scavenger (Raisanen et al., 1999; Mallis et al., 2002, 2000; Gailly et al., 2008).
CA activity in the inner ears has been demonstrated biochemically by visualizing CO2 hydration in guinea pigs, cats, and chinchillas (Erulkar and Maren, 1961; Lim et al., 1983; Hsu and Nomura, 1985; Okamura et al., 1996). Although the specific location of CA activity in the inner ear differs slightly depending on the species and detection method, CA activity is present in the organ of Corti, outer sulcus cells and their associated root processes, otic fibrocytes in the spiral ligament, as well as spiral ganglion neurons. Consistently, immunohistochemical studies have detected CAII and CAIII immunoreactivity in the otic fibrocytes, spiral limbus, and Reissner's membrane in gerbil, guinea pig, and human cochlea, with slight differences among species and detection methods.
The role of CA activity in the inner ear was first suggested by Erulkar and Maren (Erulkar and Maren, 1961). In cat inner ears, CA activity is distributed along the cochlear duct, with the highest concentrations in the apical turn, progressively lower concentrations toward the basal turns, and lowest concentrations in the vestibule (Erulkar and Maren, 1961). Administering intravenous acetazolamide, a specific CA inhibitor, considerably reduced the perilymph and endolymph volumes and pressures, and eliminated the high potassium concentration in the endolymph (Erulkar and Maren, 1961). Consistently, inhibiting CA activity affects the generation of endocochlear potential in guinea pigs and rats, suggesting a critical role of CA activity in the sound transduction (Prazma, 1978; Sterkers et al., 1984; Ikeda et al., 1987). Furthermore, CA activity has also been associated with normal otolith (otoconia) development in mice, chicken, and fish (Purichia and Erway, 1972; Kido et al., 1991; Tohse et al., 2004).
Despite the critical roles in the auditory and vestibular functions, no systematic study has been conducted to determine the expression patterns of all known CA isozymes in the mammalian inner ear. Although CAII and III localization have been described in some cochlear regions (Spicer and Schulte, 1991; Ichimiya et al., 1994; Weber et al., 2001), they do not account for all CA activities in the inner ear (Erulkar and Maren, 1961; Lim et al., 1983; Hsu and Nomura, 1985; Okamura et al., 1996). In this study, we examined the temporal and spatial expression of all known CA isozymes by quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization in mouse embryonic and postnatal inner ears. Our study provides comprehensive expression profiles for the CA isozymes and their potential role in inner ear development and function.