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Sodium-divalent cation exchange in erythroleukemia cells

University of British Columbia

Magnesium and calcium are required for many cell functions. Accordingly, it is important that their intracellular levels be closely regulated for normal metabolism. Our understanding of the cellular control of these two cations is not completely clear. In this study we examined two separate transport systems, Na7Mg2 + exchange and Na7Ca2 + exchange, in three erythroleukemia cell lines. Intracellular free magnesium, [Mg2+]i 5 is in the order of 0.50 mM, allowing for Mg2 + to enter cells passively down an electrochemical gradient. Mg2 + exit from the cell, however, must be active and is thought to be mediated by a sodium-dependent mechanism. We developed an expression system to test this notion. Since most studies regarding Na+/Mg2 + exchange have been performed in red blood cells, where Mg2 + affects volume regulatory processes, we used mRNA isolated from erythroleukemia cells for these expression studies. Poly (A)+ RNA was microinjected into Xenopus laevis oocytes and incubated for 36-48 hours prior to assaying transport. Na7Mg2 + exchange was determined either by quantitation of radioisotopic 2 2Na or by atomic absorption measurement of magnesium to assess influx and efflux, respectively. The observed transport was dependent on the amount of mRNA injected, with 50 ng resulting in maximal 2 2Na influx. Magnesium efflux was dependent on the concentration gradient for magnesium across the oocyte membrane. Sodiumdependence of magnesium efflux was demonstrated by inhibition with amiloride and quinidine. These studies indicate that genetically encoded Na+-dependent Mg2 + transport can be expressed in Xenopus oocytes. This approach may be employed to expression clone the cDNA coding the Na+/Mg2 + exchanger protein. The Na7Ca2 + exchanger plays an important role in maintaining cytosolic Ca2 + concentration. Reports are inconclusive as to whether erythrocytes express a Na7Ca2 + exchanger. The human (K- 562 and HEL) and mouse (GM979) erythroleukemia cell lines have been extensively used as model systems for studying intracellular Ca2 + involvement in cellular proliferation and differentiation. The present studies were designed to provide molecular evidence for the presence of Na7Ca2 + exchanger in these cells and to identify the molecular isoforms expressed. The cDNA coding the Na7Ca2 + exchanger contains an alternatively spliced site which determines, in part, tissue specific expression. The cDNA encodes seven different alternatively spliced isoforms containing combinations of exons A-F. Oligonucleotide primers were designed from conserved regions flanking the alternatively spliced region of the Na7Ca2 + exchanger. Homology based RT-PCR was then performed with mRNA from the three erythroleukemia cell lines. Cloning and sequencing of RT-PCR products from all three cell lines demonstrated the presence of a ~ 280 bp cDNA which represented the NACA3 isoform of the Na7Ca2 + exchanger, consisting of exons B and D. The B and D exons in K- 562 and HEL cells were identical with those of the human B and D exons whereas the expressed exons identified in GM979 mouse cells shared 98% nucleotide and 95% amino acid sequence identity with the B and D exons of rat kidney cDNA. These results demonstrate the presence of Na7Ca2 + exchanger transcripts in human and mouse erythroleukemia cell lines and show that the alternatively spliced isoform expressed in these cells consists of exons B and D. The role of these exchangers in diseases such a sickle cell disease and abnormal erythroid differentiation is unknown but would be better understood by identifying the protein(s) involved with transport.

Thesis/Dissertation

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2009-03-10

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http://hdl.handle.net/2429/5848

Experimental Medicine

University of British Columbia

UBCV

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