魚類胚胎或仔魚必須進行酸鹼調節以應付水中酸鹼變動。斑馬魚仔魚體表上的富含氫幫浦細胞(HRCs)是主要魚體排酸的細胞。本實驗目的是探討斑馬魚仔魚排酸的短期調節機制。利用掃瞄式電子顯微鏡和共軛焦顯微鏡來觀察和量化HRCs的頂膜開口型態和密度。並利用掃瞄離子電極技術(SIET)來測量魚體內酸鹼值和魚體排出氫離子的能力。結果顯示當仔魚由pH 7轉移到pH 4環境30分鐘後，魚體內氫離子濃度有顯著增加。仔魚馴養在pH 4環境中，HRCs的頂膜開口面積和密度顯著大於馴養在pH 7環境下的仔魚。當仔魚由pH 4轉移到pH 7環境10分鐘之內，HRCs頂膜開口面積顯著下降，同時可以觀察到頂膜型態皺縮和內吞現象。然而，當仔魚由pH 7轉移到pH 4環境4小時後，HRCs頂膜開口面積才會逐漸增加。本研究證實斑馬魚仔魚具有短期調節排酸的機制。

Acid-base regulation is crucial for fish to cope with environmental pH changes particularly during embryonic and larval development. H+-ATPase-rich cells (HRCs) in the skin of zebrafish larvae are the major sites for H+ secretion. In this study, I examined the short-term (in hours) regulation of H+ secretion in zebrafish subjected to ambient pH changes. Scanning electron microscopy (SEM) and confocal microscopy were used to observe and quantify the apical surface (membrane) of HRCs. Whole body pH and H+ secreting by the skin of larvae were measured with scanning ion-selective electrode technique (SIET). Results showed that whole body H+ concentration significantly increased in the larvae transferred from pH 7 to pH 4 for 30 min. The apical surface area and density of HRCs were higher in pH 4 than in pH 7 acclimated larvae. As early as 10 min after being transferred from pH 4 to pH 7, the apical surface area dramatically decreased, meanwhile morphological change and internalization of the apical surface were observed. Down-regulation of H+ secretion was also found after the transfer. In contrast, when the larvae were transferred from pH 7 to pH 4, the apical surface gradually increased after 4 h.

Al-Awqati, Q. (1996). Plasticity in epithelial polarity of renal intercalated cells: targeting of the H+-ATPase and band 3. Am. J. Physiol. 270, C1571-1580.
Breton, S. and Brown, D. (2007). New insights into the regulation of V-ATPase-dependent proton secretion. Am. J. Physiol. Renal. Physiol. 292, F1-10.
Breton, S., Wiederhold, T., Marshansky, V., Nsumu, N. N., Ramesh, V. and Brown, D. (2000). The B1 subunit of the H+-ATPase is a PDZ
domain-binding protein. Colocalization with NHE-RF in renal
B-intercalated cells. J. Biol. Chem. 275, 18219-18224.
Brown, D. and Breton, S. (2000). H+-V-ATPase-dependent luminal acidification in the kidney collecting duct and the epididymis/vas deferens: vesicle recycling and transcytotic pathways. J. Exp. Biol. 203, 137-145.
Brown, P. (1992). Gill chloride cell surface-area is greater in fresh-water-adapted adult sea trout (Salmo trutta L) than those adapted to seawater. J. Fish Biol. 40, 481-484.
Chang, I. C., Lee, T. H., Yang, C. H., Wei, Y. Y., Chou, F. I. and Hwang, P. P. (2001). Morphology and function of gill mitochondria-rich cells in fish acclimated to different environments. Physiol. Biochem. Zool. 74, 111-119.
Chang, I. C., Wei, Y. Y., Chou, F. I. and Hwang, P. P. (2003). Stimulation of Cl- uptake and morphological changes in gill mitochondria-rich cells in freshwater tilapia (Oreochromis mossambicus). Physiol. Biochem. Zool. 76, 544-552.
Choe, K. P., O'Brien, S., Evans, D. H., Toop, T. and Edwards, S. L. (2004). Immunolocalization of Na+/K+-ATPase, carbonic anhydrase II, and vacuolar H+-ATPase in the gills of freshwater adult lampreys, Geotria australis. J. Exp. Zoolog. A. Comp. Exp. Biol. 301, 654-665.
Claiborne, J. B. (1998). Acid-base regulation. In: Evans DH, editor. The physiology of fishes. Boca Raton: CRC Prese, p 179-200.
Claiborne, J. B., Compton-McCullough, D. and Walton, J. S. (2000). Branchial acid-base transfers in the euryhaline oyster toadfish (Opsanus tau) during expose to dilute seawater. J. Fish Biol. 56, 1539-1544.
Claiborne, J. B., Edwards, S. L. and Morrison-Shetlar, A. I. (2002). Acid-base regulation in fishes: cellular and molecular mechanisms. J. Exp. Zool. 293, 302-319.
Esaki, M., Hoshijima, K., Kobayashi, S., Fukuda, H., Kawakami, K. and Hirose, S. (2007). Visualization in zebrafish larvae of Na+ uptake in mitochondria-rich cells whose differentiation is dependent on foxi3a. Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R470-480.
Evans, D. H., Piermarini, P. M. and Choe, K. P. (2005). The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation and excretion of nitrogenous waste. Physiol. Rev. 85, 97-177.
Galvez, F., Wong, D. and Wood, C. M. (2006). Cadmium and calcium uptake in isolated mitochondria-rich cell populations from the gills of the freshwater rainbow trout. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, R170-176.
Goss, G. G., Laurent, P. and Perry, S. F. (1992). Evidence for a morphological component in the regulation of acid-base balance in hypercapnic catfish (Ictalurus nebulosus). Cell Tissue Res. 268, 539-552.
Goss, G. G., Laurent, P. and Perry, S. F. (1994a). Gill morphology during hypercapnia in brown bullhead (I. nebulosus): role of chloride cells and pavement cells in acid-base regulation. J. Fish Biol. 45, 705-718.
Goss, G. G. and Perry, S. F. (1993). Physiological and morphological regulation of acid-base status during hypercapnia in rainbow trout (Oncorhynchus mykiss). Can. J. Zool. 71, 1673-1680.
Goss, G. G. and Perry, S. F. (1994b). Different mechanisms of acid-base regulation in rainbow trout (Oncorhynchus mykiss) and American eel (Anguilla rostrata) during NaHCO3 infusion. Physiol. Zool. 67, 381-406.
Goss, G. G., Perry, S. F. and Laurent, P. (1995). Gill morphology and acid-base regulation. In Fish Physiology, ed. CM Wood, TJ Shuttleworth, 14, 257-284. New York: Academic.
Goss, G. G., Wood, C. M., Laurent, P. and Perry, S. F. (1994c). Morphological responses of the rainbow trout (Oncorhynchus mykiss) gill to hyperoxia, base (NaHCO3) and acid (HCl) infusions. Fish Physiol. Biochem. 12,465-477.
Greco, A. M., Fenwick, J. C. and Perry, S. F. (1996). The effects of softwater acclimation on gill morphology in the rainbow trout, Oncorhynchus mykiss. Cell Tissue Res. 285, 75-82.
Grosell, M., Laliberte, C. N., Wood, S., Jensen, F. B. and Wood, C. M. (2001). Intestinal HCO3- secretion in marine teleost fish: evidence for an apical rather than a basolateral Cl-/HCO3- exchanger. Fish Physiol. Biochem. 24, 81-95.
Heisler, N. (1986a). Acid-base regulation in fishes. In Heisler N, editor. Acid-base regulation in animals. Amsterdam: Elsevier. p 309-356.
Heisler, N. (1986b). Comparatively aspects of acid-base regulation. In: Heisler N, editor. Acid-base regulation in animals. Amsterdam: Elsevier. p 397-450.
Heisler, N. (1993). Acid-base regulation. In: Evans DH, editor. The physiology of fishes. Boca Raton: CRC Press, p 343-378.
Hirata, T., Kaneko, T., Ono, T., Nakazato, T., Furukawa, N., Hasegawa, S., Wakabayashi, S., Shigekawa, M., Chang, M. H., Romero, M. F. and Hirose, S. (2003). Mechanism of acid adaptation of a fish living in a pH 3.5 lake. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284, R1199-1212.
Hiroi, J., Kaneko, T. and Tanaka, M. (1999). In vivo sequential
changes in chloride cell morphology in the yolk-sac membrane of mozambique tilapia (Oreochromis mossambicus) embryos and larvae during seawater adaptation. J. Exp. Biol. 202, 3485-3495.
Hiroi, J., McCormick, S. D., Ohtani-Kaneko, R. and Kaneko, T. (2005). Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia (Oreochromis mossambicus) embryos, by means of triple immunofluorescence staining for Na+/K+-ATPase, Na+/K+/2Cl- cotransporter and CFTR anion channel. J. Exp. Biol. 208, 2023-2036.
Hiroi, J., Yasumasu, S., McCormick, S. D., Hwang, P. P. and Kaneko, T. (2008). Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish. J. Exp. Biol. 211, 2584-2599.
Horng, J. L. and Lin, L. Y. (2008). Expression of the Na-K-2Cl cotransporter in branchial mitochondria-rich cells of mozambique tilapia (Oreochromis mossambicus) subjected to varying chloride conditions. Zool. Stud. 47, 733-740.
Horng, J. L., Lin, L. Y., Huang, C. J., Katoh, F., Kaneko, T. and Hwang, P. P. (2007). Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio). Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R2068-2076.
Hwang, P. P. and Lee, T. H. (2007). New insights into fish ion regulation and mitochondrion-rich cells. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 148, 479-497.
Hwang, P. P., Lee, T. H., Weng, C. F., Fang, M. J. and Cho, G. Y. (1999). Presence of Na-K-ATPase in mitochondria-rich cells in the yolk-sac epithelium of larvae of the teleost, Oreochromis mossambicus. Physiol. Biochem. Zool. 72, 138-144.
Jonz, M. G. and Nurse, C. A. (2006). Epithelial mitochondria-rich cells and associated innervation in adult and developing zebrafish. J. Comp. Neurol. 497, 817-832.
Kaneko, T., Shiraishi, K., Katoh, F., Hasegawa, S. and Hiroi, J. (2002). Chloride cells during early life stages of fish and their functional differentiation. Fisheries Sci. 68, 1-9.
Katoh, F., Hasegawa, S., Kita, J., Takagi, Y. and Kaneko, T. (2001). Distinct seawater and freshwater types of chloride cells in killifish, Fundulus heteroclitus. Can. J. Zool. 79, 822-829.
Katoh, F., Hyodo, S. and Kaneko, T. (2003). Vacuolar-type proton pump in the basolateral plasma membrane energizes ion uptake in branchial mitochondria-rich cells of killifish, Fundulus heteroclitus, adapted to a low ion environment. J. Exp. Biol. 206, 793-803.
Laurent, P., Hobe, H. and Dunel-Erb, S. (1985). The role of environmental sodium chloride relative to calcium in gill morphology of freshwater salmonid fish. Cell Tissue Res. 240, 675-692.
Laurent, P. and Perry, S. F. (1995). Morphological basis of acid-base and ionic regulation in fish. In advances in comparative and environmental physiology. Mechanisms of systemic regulation: acid-base regulation, ion transfer and metabolism, ed. N Heisler, pp. 91-118. Heidelberg: Springer-Verlag
Lee, T. H., Hwang, P. P. and Feng, S. H. (1996a). Morphological studies of gill and mitochondria-rich cells in the stenohaline cyprinid teleosts, Cyprinus carpio and Carassius auratus, adapted to various hypotonic environments. Zool. Stud. 35, 272–278.
Lee, T. H., Hwang, P. P., Feng, S. H. and Huang, F. L. (1996b). The gill structure and branchial mitochondria-rich cells of the medaka, Oryzias latipes. Acta. Zool. Taiwanica. 7, 43-50.
Lee, T. H., Hwang, P. P., Lin, H. C. and Huang, F. L. (1996c). Mitochondria-rich cells in the branchial epithelium of the teleost, Oreochromis mossambicus, acclimated to various hypotonic environments. Fish Physiol. Biochem. 15, 513-523.
Lin, L. Y., Horng, J. L., Kunkel, J. G. and Hwang, P. P. (2006). Proton pump-rich cell secretes acid in skin of zebrafish larvae. Am. J. Physiol. Cell. Physiol. 290, C371-378.
Lin, L. Y. and Hwang, P. P. (2001). Modification of morphology and function of integument mitochondria-rich cells in tilapia larvae (Oreochromis mossambicus) acclimated to ambient chloride levels. Physiol. Biochem. Zool. 74, 469-476.
Lin, L. Y. and Hwang, P. P. (2004). Mitochondria-rich cell activity in the yolk-sac membrane of tilapia (Oreochromis mossambicus) larvae acclimatized to different ambient chloride levels. J. Exp. Biol. 207, 1335-1344.
Pan, T. C., Liao, B. K., Huang, C. J., Lin, L. Y. and Hwang, P. P. (2005). Epithelial Ca2+ channel expression and Ca2+ uptake in developing zebrafish. Am. J. Physiol. 289, R1202-R1211.
Parks, S. K., Tresguerres, M. and Goss, G. G. (2007). Interactions between Na+ channels and Na+-HCO3- cotransporters in the freshwater fish gill MR cell: a model for transepithelial Na+ uptake. Am. J. Physiol. Cell. Physiol. 292, C935-944.
Parks, S. K., Tresguerres, M. and Goss, G. G. (2008). Theoretical considerations underlying Na+ uptake mechanisms in freshwater fishes. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 148, 411-418.
Pastor-Soler, N., Beaulieu, V., Litvin, T. N., Da Silva, N., Chen, Y., Brown, D., Buck, J., Levin, L. R. and Breton, S. (2003). Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling. J. Biol. Chem. 278, 49523-49529.
Paunescu, T. G., Da Silva, N., Russo, L. M., McKee, M., Lu, H. A., Breton, S. and Brown, D. (2008). Association of soluble adenylyl cyclase with the V-ATPase in renal epithelial cells. Am. J. Physiol. Renal. Physiol. 294, F130-138.
Perry, S. F., Beyers, M. L. and Johnson, D. A. (2000). Cloning and molecular characterisation of the trout (Oncorhynchus mykiss) vacuolar H+-ATPase B subunit. J. Exp. Biol. 203, 459-470.
Perry, S. F., Furimsky, M., Bayaa, M., Georgalis, T., Shahsavarani, A., Nickerson, J. G. and Moon, T. W. (2003). Integrated responses of Na+/HCO3- cotransporters and V-type H+-ATPases in the fish gill and kidney during respiratory acidosis. Biochim. Biophys. Acta. 1618, 175-184.
Perry, S. F., Goss, G. G. and Laurent, P. (1992). The interrelationships between gill chloride cell morphology and ionic uptake in four freshwater teleosts. Can. J. Zool 70, 1737-1742.
Perry, S. F. and Laurent, P. (1989). Adaptational responses to rainbow trout to lowered external NaCl concentration: contribution of the branchial chloride cell. J. Exp. Biol. 147, 147-168.
Pisam, M., Le Moal, C., Auperin, B., Prunet, P. and Rambourg, A. (1995). Apical structures of "mitochondria-rich" alpha and beta cells in euryhaline fish gill: their behaviour in various living conditions. Anat. Rec. 241, 13-24.
Reid, S. D., Hawkings, G. S., Galvez, F. and Goss, G. G. (2003). Localization and characterization of phenamil-sensitive Na+ influx in isolated rainbow trout gill epithelial cells. J. Exp. Biol. 206, 551-559.
Seidelin, M., Brauner, C. J., Jensen, F. B. and Madsen, S. S. (2001). Vacuolar-type H+-ATPase and Na+, K+-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water. Zool. Sci. 18, 1199-1205.
Shih, T. H., Horng, J. L., Hwang, P. P. and Lin, L. Y. (2008).
Ammonia excretion by the skin of zebrafish (Danio rerio) larvae. Am. J. Physiol. Cell. Physiol. 295, 1625-1632.
Sullivan, G., Fryer, J. and Perry, S. (1995). Immunolocalization of proton pumps (H+-ATPase) in pavement cells of rainbow trout gill. J. Exp. Biol. 198, 2619-2629.
Van der Heijden, A. J., van der Meij, J. C., Flik, G. and Wendelaar Bonga, S. E. (1999). Ultrastructure and distribution dynamics of chloride cells in tilapia larvae in fresh water and sea water. Cell Tissue Res. 297, 119-130.
Wilson, J. M., Laurent, P., Tufts, B. L., Benos, D. J., Donowitz, M., Vogl, A. W. and Randall, D. J. (2000). NaCl uptake by the branchial epithelium in freshwater teleost fish: an immunological approach to ion-transport protein localization. J. Exp. Biol. 203, 2279-2296.
Wilson, R., Gilmour, K. M., Henry, R. P., Wood, C. M. (1996). Intestinal base excretion in the seawater-adapted rainbow trout: a role in acid-base balance? J. Exp. Biol. 199, 2331-2343.
Wood, C. M., Milligan, C. L. and Walsh P. J. (1999). Renal responses of trout to chronic respiratory and metabolic acidosis and metabolic alkalosis. Am. J. Physiol. 46, R482-R492.
Yan, J. J., Chou, M. Y., Kaneko, T. and Hwang, P. P. (2007). Gene expression of Na+/H+ exchanger in zebrafish H+-ATPase-rich cells during acclimation to low-Na+ and acidic environments. Am. J. Physiol. Cell. Physiol. 293, C1814-1823.