臺灣博碩士論文加值系統

鈣離子在許多細胞生理反應中扮演了十分重要的角色，所以瞭解細胞如何調節其鈣離子濃度是很重要的研究課題。本實驗室一直以牛嗜鉻細胞為實驗模型系統來探討細胞調節其胞內鈣離子濃度之機制。過去之研究結果顯示在嗜鉻細胞受刺激使細胞外Ca2+進入細胞而提高胞內鈣離子濃度後，鈉鈣交換為主要將鈣離子排出細胞之機制。目前已知有兩類鈉鈣交換蛋白，NCX (3 Na+:1 Ca2+)及NCKX (4 Na+:1 Ca2++1 K+)。根據本實驗室初步的結果顯示嗜鉻細胞可能同時具有此兩類鈉鈣交換蛋白，因此本研究以螢光免疫染色及鈉鈣交換活性來偵測嗜鉻細胞上鈉鈣交換蛋白存在的種類。在免疫染色方面的結果，由於抗體可能無法辨識牛嗜鉻細胞之鈉鈣交換分子，因而結果不甚理想。而在鈉鈣交換活性方面，我們利用螢光指示劑fura-2測量反向鈉鈣交換活性，先利用carbachol增加細胞內Na+濃度，再將細胞置換到Na+-free之溶液中，以建立鈉離子梯度，然後測量此鈉離子梯度所引起之細胞內Ca2+增加量。結果顯示嗜鉻細胞上有鈉鈣交換之活性，且其活性在K+存在下有顯著之增加，因此我們推論嗜鉻細胞上同時存在兩種鈉鈣交換蛋白。

Calcium ion plays important roles in many physiological reactions. It is therefore important to understand how cells regulate their intracellular calcium concentration ([Ca2+ ]i). We have used bovine adrenal chromaffin cells as a model system to study the regulation of [ Ca2+ ]i. Previous results from our laboratory have shown that Na+/Ca2+ exchanger is the major mechanism that is responsible for removing Ca2+ from the cytosol and bringing the [ Ca2+ ]i to the resting level, after the [ Ca2+ ]i is increased by an influx of extracellular Ca2+. There are two major types of Na+-Ca2+ exchanger: NCX (3 Na+: 1 Ca2+) and NCKX (4 Na+: 1 Ca2++1 K+). Preliminary results from our laboratory show that chromaffin cells may contain both NCX and NCKX. In this study, I used Western blot, immunocytochemistry and activity assays to study the types of Na+-Ca2+ exchanger that are present in bovine chromaffin cells. Because of the antibodies used may not recognize the NCX and NCKX in bovine chromaffin cells; no conclusive results were obtained. In the study of Na+/Ca2+ exchange activity, I used the fluorescent indicator, fura-2, to measure the reverse mode of Na+/Ca2+ exchange activity. The chromaffin cells were loaded with Na+ by first treating with carbachol and then transferred to a Na+-free solution to establish a Na+-gradient. The Na+-gradient-dependent Ca2+ increase in the cells was then measured. The results show that there was Na+/Ca2+ exchanger in the chromaffin cells, and the activity was significantly increased in the presence of K+. Therefore, it appears that both NCX and NCKX exist in the chromaffin cells.

中 文 摘 要 .... 1
英 文 摘 要 .... 2
縮 寫 表...... 3
緒 論 ....... 5
材 料 與 方 法 .... 11
實 驗 結 果 .... 19
討 論 ....... 23
實 驗 圖 表 .... 27
參 考 文 獻.... 38

Berridge, M. J., Bootman, M. D. and Lipp, P. (1998). Calcium - a life and death signal. Nature 395, 645-648.
Blaustein, M. P. (1988). Calcium transport and buffering in neurons. Trends Neurosci. 11, 438-443.
Carmichael, S. W. and Winkler, H. (1988). The adrenal chromaffin cell. Sci. Am. Nov. 30-39.
Caroni, P. and Carafoli, E. (1981). The calcium pumping ATPase of heart sarcolemmal. J. Biol. Chem. 256, 3263-3270.
Cervetto, L., Lagnado, L., Perry, R. J., Robinson, D. W. and McNaughton, P. A. (1989). Extrusion of calcium from rod outer segments is driven by both sodium and potassium gradient. Nature 337, 740-743.
Chern, Y.- J., Chuen, S.- H., Lin, Y.- J., Ho, C.- M. and Kao, L.- S. (1992). Presence of Na+/Ca2+ exchange activity and its role in regulation of intracellular calcium concentration in bovine adrenal chromaffin cells. Cell Calcium 13, 99-106.
Dahan, D., Spanier, R. and Rahamimoff, H. (1991). The modulation of rat brain Na+-Ca2+ exchange by K+. J. Biol. Chem. 266, 2067-2075.
Douglas, W. W. and Rubin, R. P. (1961). The role of calcium in the secretory response of adrenal medulla to acetylcholine. J. Physiol. 159, 40-57.
Egger, M. and Niggli, E. (1999). Regulatory function of Na-Ca exchange in the heart: milestones and outlook. J. Membr. Biol. 168, 107-130.
Friedman, P. A. (1998). Codependence of renal calcium and sodium transport. Annu. Rev. Physiol. 60, 179-197.
Gabellini, N., Iwata, T. and Carafoli, E. (1995). An alternative splicing sites modifies the carboxyl-terminal trans-membrane domains of the Na+/Ca2+ exchanger. J. Biol. Chem. 270, 6917-6924.
Grover, A. K. and Samaon, S. E. (1997). Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function. Am J. Physiol. 273, C1250-C1258.
Grynkiewicz, G., Poenie, M. and Tsien, R. (1985). A new generation of Ca2+ indicatros with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440-3450.
Guerini, D., Garcia-Martin, E., Gerber, A., Volbracht, C., Leist, M., Merino, C. G. and Carafoli, E. (1999). The expression of plasma membrane Ca2+ pump isoforms in cellular granule neurons is modulated by Ca2+. J Biol. Chem. 274, 1667-1676.
Haase, W., Friese, W., Gordon, R. D., Muller, H. and Cook, N. (1990). Immunological characterization and localization of the Na/Ca-exchanger in bovine retina. J. Neurosci. 10, 1486-1494.
He, S., Ruknudin, A., Bambrick, L. L., Lederer, W. J. and Schulze, D. H. (1998). Isoform-specific regulation of the Na+/Ca2+ exchanger in rat astrocytes and neurons by PKA. J. Neurosci. 18, 4833-4841.
He, Z., Tong, Q., Quednau, B. D., Philipson, K. D. and Hilgemann, D. W. (1998). Cloning, expression, and characterization of the squid Na+-Ca2+ exchanger(NCX-SQ1). J. Gen. Physiol. 111,857-873.
Inesi, G. (1985). Mechanism of calcium transport. Annu. Rev. Physiol. 47, 573-601.
Johnson, R. G. (1988). Accumulation of biological amines into chromaffin granules: A model for hormone and neurotransmitter transport. Physiol. Rev. 68, 232-307.
Kao, L. -S. (1988). Calcium homeostasis in digitonin- permeabilized bovine chromaffin cells. J. Neurochem. 51. 221-227.
Kao, L. -S. and Cheung, N. -S. (1990). Mechanism of calcium transport across the plasma membrane of bovine chromaffin cells. J. Neurochem. 54, 1972-1979.
Kilpatrick, D. L., Ledbetter, F. H., Carson, K. A., Kirshner, A. G., Slepetis, R. and Kirshner, N. (1980). Stability of bovine adrenal medulla cells in culture. J. Neurochem. 35, 679-692.
Kim, T. S. Y., Reid, D. M. and Molday, R. S. (1998). Structure-function relationships and localization of the Na/Ca-K exchanger in rod photorecepters. J. Biol. Chem. 273, 16561-16567.
Kimura, M., Aviv, A. and Reeves, J.P. (1993). K+-dependent Na+/Ca2+ exchange in human platelet. J. Biol. Chem. 268, 6874-6877.
Kofuji, P., Lederer, W. J. and Schulze, D. H. (1994). Mutually exclusive and cassette exons underlie alternatively spliced isoforms of the Na/Ca exchanger. J. Biol. Chem. 269, 5145-5149.
Kofuji, P., Lederer, W. J. and Schulze, D. H. (1993).Na/Ca exchanger isoforms expressed in kidney. Am. J. Physiol. 265. F598-F603.
Lagnado, L. and McNaughton, P. A. (1990). Electrogenic properties of the Na:Ca exchange. J. Membr. Biol. 113, 177-191.
Landolfi, B., Curci, S., Debellis, L., Pozzan, T. and Hofer, A. M. (1998). Ca2+ homeostasis in the agonist-sensitive internal store: functional interactions between imtochondria and the ER measured in situ in intact cells. J. Cell Biol. 142, 1235-1243.
Li, Z., Matsuoka, S., Hryshko, L. V., Nicoll, D. A., Bersohn, M. M., Burke, E. P., Lifton, R. P. and Philipson, K. D. (1994). Cloning of the NCX2 isoform of the plasma membrane Na+-Ca2+ exchanger. J. Biol. Chem. 269, 17434-17439.
Liu, P. -S. and Kao, L. -S. (1990). Na+-dependent Ca2+ influx in bovine adrenal chromaffin cells. Cell Calcium 11,573-579.
Lytton, J. and Nigam, S. K. (1992). Intracellular calcium: molecules and pools. Curr. Opin. Cell Biol. 4, 220-226.
Martinez-Serrano, A., Blanco, P. and Satrustegui, J. (1992). Calcium binds to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging. J. Biol. Chem. 267, 4672-4679.
Meldolesi, J., Volpe, P. and Pozzan, T. (1988). The intracellular distribution of calcium. Trends Neurosci. 11, 449-452.
Minta, A., Kao, J. P. Y. and Tsien, R. Y. (1989). Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171-8178.
Naraghi, M., Muller, T H. and Neher, E. (1998). Two-dimensional determination of the cellular Ca2+ binding in bovine chromaffin cells. Biophy. J. 75, 1635-1647.
Neher, E. and Augustine, G. J. (1992). Calcium gradients and buffers in bovine chromaffin cells. J. Physiol. 450, 273-301.
Nicoll, D. A., Ottolia, M., Lu, L., Lu, Y. and Philipson, K. D. (1999). A new topological model of the cardiac sarcolemmal Na+-Ca2+ exchanger. J. Biol. Chem. 274, 910-917.
Nicoll, D. A., Longoni, S. and Philipson, K. D. (1990). Molecular cloning and functional expression of the cardiac sarcolemmal Na+-Ca2+ exchanger. Science 250, 562-565.
Niggli, E. and Lederer, W. J. (1991). Molecular operations of the sodium-calcium exchanger revealed by conformation currents. Nature 349, 621-624.
Ouanounou, A., Zhang, L., Charlton, M. P. and Carlen, P. L. (1999). Differential modulation of synaptic transmission by calcium chelators in young and aged hippocampal CA1 neurons: evidence for altered calcium homeostasis in aging. J. Neurosci. 19, 906-915.
Pan, C. -Y., Chu, Y. -S. and Kao, L. -S. (1998). Molecular study of the Na+/Ca2+ exchanger in bovine adrenal chromaffin cells. Biochem. J. 336, 305-310.
Pan, C. -Y. and Kao, L. -S. (1997). Catecholamine secretion from bovine adrenal chromaffin cells: the role of the Na+/Ca2+ exchanger and the intracellular Ca2+ pool. J. Neurochem. 69, 1085-1092.
Penner, R., Fasolato, C. and Hoth, M. (1993). Calcium influx and its control by calcium release. Curr. Opin. Neurobiol. 3, 368-374.
Peterson, O. H., Peterson, C. C. H. and Kasai, H. (1994). Calcium and hormone action. Annu. Rev. Physiol. 56, 297-319.
Philipson, K. D. (1985). Sodium-calcium exchange in plasma membrane vesicles. Annu. Rev. Physiol. 47, 561-571.
Philipson, K. D., Longoni, S. and Ward, R. (1988). Purification of the cardiac Na+-Ca2+ exchange protein. Biochem. Biophys. Acta 945, 298-306.
Philipson, K. D. and Nicoll, D. A. (1992). Sodium-calcium exchange. Curr. Opin. Cell Biol. 4, 678-683.
Powis, D. A., O'brien, K. J. and Von-Grafenstein, H. R. K. (1991). Calcium export by sodium-calcium exchange in bovine chromaffin cells. Cell Calcium 12, 493-504.
Reeves, J. P. (1998). Na+/Ca2+ exchange and cellular Ca2+ homeostasis. J. Bioenerg. Biomembr. 30, 151-160.
Reeves, J. P. (1992). Molecular aspects of sodium-calcium exchange. Arch. Biochem. Biophys. 292, 329-334.
Reilander, H., Achilles, A., Friedel, U., Maul, G., Lottspeich, F. and Cook, N. (1992). Primary structure and functional expression of the Na/Ca,K-exchanger from bovine rod photorecepters. EMBO. J. 11, 1689-1695.
Reuter, H. (1991). Sodium-calcium exchange - ins and outs of Ca2+ transport. Nature 349,567-568.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd ed., pp.18.2-18.88. Cold Spring Harbor Laboratory Press.
Schnetkamp, P. P. M. (1995). Calcium homeostasis in vertebrate retinal rod outer segments. Cell Calcium 18, 322-330.
Schnetkamp, P. P. M., Basu, D. K., Li, X. -B. and Szerencsei, R. T. (1991). Regulation of intracellular free Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. J. Biol. Chem. 266, 22983-22990.
Schnetkamp, P. P. M., Li, X. -B., Basu, D. K. and Szerencsei, R. T. (1991). Regulation of free cytosolic Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. J. Biol. Chem. 266, 22975-22982.
Schnetkamp, P. P. M. and Szerencsei, R. T. (1991). Effect of potassium ions and membrane potatial on the Na-Ca-K exchanger in isolated intact bovine rod outer segments. J. Biol. Chem. 266, 189-197.
Schnetkamp, P. P. M., Szerencsei, R. T and. Basu, D. K. (1991).Unidirectional Na+, Ca2+, and K+ fluxes through the bovine rod outer segment Na-Ca-K exchanger. J. Biol. Chem. 266, 198-206.
Thomas, A. P. and Delaville, F. (1991). The use of fluorescent indicators for measurements of cytosolic-free calcium concentration in cell populations and single cells, in Cellular Calcium: a practical approach. (McCormack, J. G. and Cobbold, P. H., eds), pp. 1-53. Oxford University Press, New York.
Tsoi, M., Rhee, K. -H., Bungard, D., Li, X. -F., Lee, S. -L., Auer, R. N. and Lytton, J. (1998). Molecular cloning of a novel potassium-dependent sodium-calcium exchanger from rat brain. J. Biol. Chem. 273, 4155-4162.
Ungar, A. and Philips, J. H. (1983). Regulation of the adrenal medulla. Physiol. Rev. 63, 787-843.
Verkhratsky, A. and Toescu, E. C. (1998). Calcium and neuronal ageing. Trends Neurosci. 21, 2-7.
Wilson, S. P. (1987). Purification of adrenal chromaffin cells on renografin gradients. J. Neurosci. Meth. 19, 163-171.