Abstract
The plasma membrane calcium ATPase (PMCA) is an ubiquitous enzyme that extrudes calcium from the cytoplasm to the extracellular space. Four PMCA genes through alternative splicing produce a large diversity of isoforms of this enzyme. We reported previously that the PMCA contained in AS-30D hepatocarcinoma cells showed significant differences in activity in comparison to normal and regenerating liver. In the present study we investigate if the difference in PMCA activity could be related to differential expression of mRNAs encoding different isoforms of PMCA. Using RT-PCR we found that variants 1b, 1x, and 4b are expressed in all liver samples. The hepatoma AS-30 and liver at 2 days of regeneration express low amounts of isoforms 2w, 4b and 4x, and do not express isoforms 4a, 4d and 4z. Fetal and neonatal liver do not express variants 4a and 4d, but they do express variants 4x and 4z. Immunoblot analysis showed a higher ratio ATPase/total protein in the hepatoma AS-30D in comparison to normal liver. Our results suggest that the Ca2+-ATPase kinetic pattern previously observed by us in the AS-30D cells, could be at least partially explained by changes in the mRNA expression of several of the PMCA isoforms expressed in the liver.
Similar content being viewed by others
References
Carafoli E, Guerini D: The plasma membrane calcium pump. In: J.R. Sotelo, J.C. Benech (eds). Calcium and Cellular Metabolism. Plenum Press, New York, 1997, pp 73-84
Pedersen PL, Carafoli E: Ion motive ATPases I: Ubiquity, properties, and significance to cell function. Trends Biochem Sci 12: 146-150, 1987
Shull GE, Greeb J: Molecular cloning of two isoforms of the plasma membrane Ca2+-transporting ATPase from rat brain. J Biol Chem 263: 8646-8657, 1988
Shull GE, Greeb J: Molecular cloning of a third isoform of the calmodulin-sensitive plasma membrane Ca2+-transporting ATPase that is expressed predominantly in brain and skeletal muscle. J Biol Chem 264: 18569-18576, 1989
Stauffer TP, Hilfiker H, Carafoli E, Strehler E: Molecular cloning of a third isoform of the calmodulin-sensitive plasma membrane Ca2+-transporting ATPase that is expressed predominantly in brain and skeletal muscle. J Biol Chem 268: 25993-26003, 1993
Zvaritch E, James P, Vorherr T, Falchetto R, Modyanov N, Carafoli E: Mapping of functional domains in the plasma membrane Ca2+ using trypsin proteolysis. Biochemistry 29: 8070-8076, 1990
Adamo HP, Penniston JT: New Ca2+ pump isoforms generated by alternative splicing of rPMCA2 mRNA. Biochem J 283: 355-359, 1992
Burk SE, Shull GE: Structure of the rat plasma membrane Ca(2+)-ATPase isoform 3 gene and characterization of alternative splicing and transcription products. Skeletal muscle-specific splicing results in a plasma membrane Ca(2+)-ATPase with a novel calmodulin-binding domain. J Biol Chem 267: 19683-19690, 1992
Keeton TP, Shull GE: Primary structure of rat plasma membrane Ca2+ ATPase isoform 4 and analysis of alternative splicing patterns at splice site A. Biochem J 306: 779-785, 1995
Heim R, Hug M, Iwata T, Strehler EE, Carafoli E: Microdiversity of human-plasma-membrane calcium-pump isoform 2 generated by alternative RNA splicing in the N-terminal coding region. Eur J Biochem 205: 333-340, 1992
Strehler EE, Zacharias DA: Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 81: 21-50, 2001
Keeton TP, Burk SE, Shull GE: Alternative splicing of exons encoding the calmodulin-binding domains and C termini of plasma membrane Ca2+ ATPase isoforms 1, 2, 3, and 4. Alternative splicing of exons encoding the calmodulin-binding domains and C termini of plasma membrane Ca2+ ATPase isoforms 1, 2, 3, and 4. J Biol Chem 268: 2740-2748, 1993
Santiago-García J, Mas-Oliva J, Saavedra D, Zarain-Herzberg A: Analysis of mRNA expression and cloning of a novel plasma membrane Ca2+ ATPase splice variant in human heart. Mol Cell Biochem 155: 173-182, 1996
Kessler F, Bennardini F, Bachs O, Serratosa J, James P, Caride AJ, Gazzoti P, Penniston JT, Carafoli E: Partial purification and characterization of the Ca2+-pumping ATPase of the liver plasma membrane. J Biol Chem 265: 16012-16019, 1990
Mas-Oliva J, Pérez-Monfort R, Cárdenas-García M, Rivas-Duro M: Altered coupling states between calcium transport and (Ca2+,Mg2+)-ATPase in the AS-30D ascites hepatocarcinoma plasma membrane. Mol Cell Biochem 100: 39-50, 1991
Howard A, Barley NF, Legon S, Walters JRF: Plasma-membrane calcium-pump isoforms in human and rat liver. Biochem J 303: 275-279, 1994
Zacharias DA, Kappen C: Developmental expression of the four plasma membrane calcium ATPase (Pmca) genes in the mouse. Biochim Biophys Acta 1428: 397-405, 1999
Knauf PA, Proverbio F, Hoffman JF: Electrophoretic separation of different phosphoproteins associated with Ca-ATPase and N-K-ATPase in human red cell ghosts. J Gen Physiol 63: 324-336, 1974
Sambrook J, Maniatis T, Fritsch EF: Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York, 1989
Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987
Higgins GM, Anderson RM: Experimental pathology of the liver. Arch Pathol 12: 186-202, 1931
Latif F, Duh FM, Gnarra J, Tory K, Kuzmin I, Yao M, Stackhouse T, Modi W, Geil L, Schmidt L, Li H, Orcutt M, Maher E, Richards FO Jr, Ferguson-Smith MA, Le Paslier D, Linchan M, Zbar B, Lerman MI: Von Hippel-Lindau syndrome. Cloning and identification of the plasma membrane Ca(++)-transporting ATPase isoform 2 gene resides in the von Hippel-Lindau gene region. Cancer Res 53: 861-867, 1993
Verma AK, Filoteo AG, Stanford DR, Wieben ED, Penniston JT, Strehler EE, Fischer R, Heim R, Vogel G, Mathews G, Strchler-Page MA, James P, Vorherr T, Krebs J, Carafoli E: Complete primary structure of a human plasma membrane Ca2+ pump. J Biol Chem 263: 14152-14159, 1988
Carafoli E: The Ca2+ pump of the plasma membrane. J Biol Chem 267: 2115-2118, 1992
Takahashi H, Yamaguchi M: Regulatory effect of regucalcin on (Ca2+-Mg2+)-ATPase in rat liver plasma membranes: Comparison with the activation by Mn2+ and Co2+. Mol Cell Biochem 124: 169-174, 1993
Takahashi H, Yamaguchi M: Activating effect of regucalcin on (Ca2+-Mg2+)-ATPase in rat liver plasma membranes: Relation to sulfhydril group. Mol Cell Biochem 136: 71-76, 1994
Jouneaux C, Audigier Y, Goldsmith P, Pecker F, Lotersztajn S: Gs mediates hormonal inhibition of the calcium pump in liver plasma membranes. J Biol Chem 268: 2368-2372, 1993
Hickie R, Kalant H: Calcium and magnesium content of rat liver in Morris hepatoma 5123tc. Can Res 27: 1053-1057, 1967
Swierenga SH, Whitfield JF, Karasaki S: Loss of proliferative calcium dependence: Simple in vitro indicator of tumorigenicity. Proc Natl Acad Sci 75: 6069-6072, 1978
Fiskum G: Intracellular levels and distribution of Ca2+ in digitonin-permeabilized cells. Cell Calcium 6: 25-37, 1985
Enyedi A, Verma AK, Heim R, Adamo H, Filoteo AG, Strehler E, Penniston JT: The Ca2+ affinity of the plasma membrane Ca2+ pump is controlled by alternative splicing. J Biol Chem 269: 41-43, 1994
Díaz-Muñoz M, Canedo-Merino R, Gutiérrez-Salinas J, Hernández-Muñoz R: Modifications of intracellular calcium release channels and calcium mobilization following 70% hepatectomy. Arch Biochem Biophys 349: 105-112, 1998
Magnino F, St-Pierre M, Lüthi M, Hilly M: Expression of intracellular calcium channels and pumps after partial hepatectomy in rat. Mol Cell Biol Res Commun 3: 374-379, 2000
Brandt P, Neve RL: Expression of plasma membrane calcium-pumping ATPase mRNAs in developing rat brain and adult brain subregions: Evidence for stage-specific expression. J Neurochem 59: 1566-1569, 1992
Guerini, D, Wang X, Li L, Genazzani A, Carafoli E: Calcineurin controls the expression of isoform 4CII of the plasma membrane Ca(2+) pump in neurons. J Biol Chem 275: 3706-3712, 2000
Cittadini A, Dani AM, Wolf F, Bossi D, Calviello G: Calcium permeability of Ehrlich ascites tumor cell plasma membrane in vivo. Biochim Biophys Acta 686: 27-35, 1982
Guerini D, Schröder S, Foletti D, Carafoli E: Isolation and characterization of a stable Chinese hamster ovary cell line overexpressing the plasma membrane Ca2+ ATPase. J Biol Chem 270: 14643-14650, 1995
Liu BF, Xu X, Fridman R, Muallem S, Huo TH: Consequences of functional expression of the plasma membrane calcium pump isoform 1a. J Biol Chem 271: 5536-5544, 1996
Kuo TH, Liu BF, Yu Y, Wuytack F, Raeymaekers L, Tsang W: Coordinated regulation of the plasma membrane calcium pump and the sarco(endo)plasmic reticular calcium pump gene expression by Ca2+. Cell Calcium 21: 399-408, 1997
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Delgado-Coello, B., Santiago-García, J., Zarain-Herzberg, A. et al. Plasma membrane Ca2+-ATPase-mRNA expression in murine hepatocarcinoma and regenerating liver cells. Mol Cell Biochem 247, 177–184 (2003). https://doi.org/10.1023/A:1024119831983
Issue Date:
DOI: https://doi.org/10.1023/A:1024119831983