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. 1990 Nov 1;271(3):649–653. doi: 10.1042/bj2710649

Expression of endoplasmic-reticulum Ca2(+)-pump isoforms and of phospholamban in pig smooth-muscle tissues.

J A Eggermont 1, F Wuytack 1, J Verbist 1, R Casteels 1
PMCID: PMC1149611  PMID: 2244871

Abstract

The expression of the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2(+)-pump isoforms (SERCA2a and SERCA2b) and of phospholamban was studied in pig smooth muscle of the stomach, longitudinal ileum, pulmonary artery and aorta. mRNA levels were determined using an RNAase protection assay. The SERCA2 isoforms and phospholamban were tested on Western blots with a panel of antibodies, some of which were isoform-specific. The pig smooth-muscle tissues all contained comparable SERCA2 mRNA levels, but these levels were 10-20-fold lower than SERCA2 mRNA levels in cardiac muscle. Of the SERCA2 mRNAs in smooth muscle, 72-81% encoded the non-muscle isoform (SERCA2b), and Western blot analysis with isoform-specific antibodies confirmed that the SERCA2b isoform is the predominant endoplasmic-reticulum Ca2(+)-pump in smooth muscle. In contrast with SERCA2 mRNA levels, phospholamban mRNA levels varied by 12-fold between the different pig smooth-muscle tissues, with low and very low levels in the pig pulmonary artery and the pig aorta respectively. The differential expression of phospholamban was also confirmed on Western blots. The finding that the phospholamban content varied between the different smooth-muscle tissues whereas the SERCA2 expression remained rather constant indicates that, in pig smooth muscle, the expression of phospholamban is not coupled with that of SERCA2.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brandl C. J., Green N. M., Korczak B., MacLennan D. H. Two Ca2+ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell. 1986 Feb 28;44(4):597–607. doi: 10.1016/0092-8674(86)90269-2. [DOI] [PubMed] [Google Scholar]
  2. Brandl C. J., deLeon S., Martin D. R., MacLennan D. H. Adult forms of the Ca2+ATPase of sarcoplasmic reticulum. Expression in developing skeletal muscle. J Biol Chem. 1987 Mar 15;262(8):3768–3774. [PubMed] [Google Scholar]
  3. Burk S. E., Lytton J., MacLennan D. H., Shull G. E. cDNA cloning, functional expression, and mRNA tissue distribution of a third organellar Ca2+ pump. J Biol Chem. 1989 Nov 5;264(31):18561–18568. [PubMed] [Google Scholar]
  4. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  5. Eggermont J. A., Vrolix M., Raeymaekers L., Wuytack F., Casteels R. Ca2+-transport ATPases of vascular smooth muscle. Circ Res. 1988 Feb;62(2):266–278. doi: 10.1161/01.res.62.2.266. [DOI] [PubMed] [Google Scholar]
  6. Eggermont J. A., Wuytack F., Casteels R. Characterization of the mRNAs encoding the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2+ pump in pig smooth muscle. Biochem J. 1990 Mar 15;266(3):901–907. [PMC free article] [PubMed] [Google Scholar]
  7. Eggermont J. A., Wuytack F., De Jaegere S., Nelles L., Casteels R. Evidence for two isoforms of the endoplasmic-reticulum Ca2+ pump in pig smooth muscle. Biochem J. 1989 Jun 15;260(3):757–761. doi: 10.1042/bj2600757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fujii J., Lytton J., Tada M., MacLennan D. H. Rabbit cardiac and slow-twitch muscle express the same phospholamban gene. FEBS Lett. 1988 Jan 18;227(1):51–55. doi: 10.1016/0014-5793(88)81412-1. [DOI] [PubMed] [Google Scholar]
  9. Gaetani S., Smith J. A., Feldman R. A., Morimoto T. Preparation of rough microsomes and membrane-bound polysomes that are active in protein synthesis in vitro. Methods Enzymol. 1983;96:3–24. doi: 10.1016/s0076-6879(83)96005-6. [DOI] [PubMed] [Google Scholar]
  10. Gunteski-Hamblin A. M., Greeb J., Shull G. E. A novel Ca2+ pump expressed in brain, kidney, and stomach is encoded by an alternative transcript of the slow-twitch muscle sarcoplasmic reticulum Ca-ATPase gene. Identification of cDNAs encoding Ca2+ and other cation-transporting ATPases using an oligonucleotide probe derived from the ATP-binding site. J Biol Chem. 1988 Oct 15;263(29):15032–15040. [PubMed] [Google Scholar]
  11. James P., Inui M., Tada M., Chiesi M., Carafoli E. Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum. Nature. 1989 Nov 2;342(6245):90–92. doi: 10.1038/342090a0. [DOI] [PubMed] [Google Scholar]
  12. Jones L. R., Cala S. E. Biochemical evidence for functional heterogeneity of cardiac sarcoplasmic reticulum vesicles. J Biol Chem. 1981 Nov 25;256(22):11809–11818. [PubMed] [Google Scholar]
  13. Komuro I., Kurabayashi M., Shibazaki Y., Takaku F., Yazaki Y. Molecular cloning and characterization of a Ca2+ + Mg2+-dependent adenosine triphosphatase from rat cardiac sarcoplasmic reticulum. Regulation of its expression by pressure overload and developmental stage. J Clin Invest. 1989 Apr;83(4):1102–1108. doi: 10.1172/JCI113989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Korczak B., Zarain-Herzberg A., Brandl C. J., Ingles C. J., Green N. M., MacLennan D. H. Structure of the rabbit fast-twitch skeletal muscle Ca2+-ATPase gene. J Biol Chem. 1988 Apr 5;263(10):4813–4819. [PubMed] [Google Scholar]
  15. Krieg P. A., Melton D. A. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 1987;155:397–415. doi: 10.1016/0076-6879(87)55027-3. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Leberer E., Härtner K. T., Brandl C. J., Fujii J., Tada M., MacLennan D. H., Pette D. Slow/cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban mRNAs are expressed in chronically stimulated rabbit fast-twitch muscle. Eur J Biochem. 1989 Oct 20;185(1):51–54. doi: 10.1111/j.1432-1033.1989.tb15080.x. [DOI] [PubMed] [Google Scholar]
  18. Lee J. J., Costlow N. A. A molecular titration assay to measure transcript prevalence levels. Methods Enzymol. 1987;152:633–648. doi: 10.1016/0076-6879(87)52070-5. [DOI] [PubMed] [Google Scholar]
  19. Lompre A. M., de la Bastie D., Boheler K. R., Schwartz K. Characterization and expression of the rat heart sarcoplasmic reticulum Ca2+-ATPase mRNA. FEBS Lett. 1989 May 22;249(1):35–41. doi: 10.1016/0014-5793(89)80010-9. [DOI] [PubMed] [Google Scholar]
  20. Lytton J., MacLennan D. H. Molecular cloning of cDNAs from human kidney coding for two alternatively spliced products of the cardiac Ca2+-ATPase gene. J Biol Chem. 1988 Oct 15;263(29):15024–15031. [PubMed] [Google Scholar]
  21. Lytton J., Zarain-Herzberg A., Periasamy M., MacLennan D. H. Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase. J Biol Chem. 1989 Apr 25;264(12):7059–7065. [PubMed] [Google Scholar]
  22. MacLennan D. H., Brandl C. J., Korczak B., Green N. M. Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature. 1985 Aug 22;316(6030):696–700. doi: 10.1038/316696a0. [DOI] [PubMed] [Google Scholar]
  23. Nagai R., Zarain-Herzberg A., Brandl C. J., Fujii J., Tada M., MacLennan D. H., Alpert N. R., Periasamy M. Regulation of myocardial Ca2+-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2966–2970. doi: 10.1073/pnas.86.8.2966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Raeymaekers L., Eggermont J. A., Wuytack F., Casteels R. Effects of cyclic nucleotide dependent protein kinases on the endoplasmic reticulum Ca2+ pump of bovine pulmonary artery. Cell Calcium. 1990 Apr;11(4):261–268. doi: 10.1016/0143-4160(90)90002-c. [DOI] [PubMed] [Google Scholar]
  25. Raeymaekers L., Hofmann F., Casteels R. Cyclic GMP-dependent protein kinase phosphorylates phospholamban in isolated sarcoplasmic reticulum from cardiac and smooth muscle. Biochem J. 1988 May 15;252(1):269–273. doi: 10.1042/bj2520269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Raeymaekers L., Jones L. R. Evidence for the presence of phospholamban in the endoplasmic reticulum of smooth muscle. Biochim Biophys Acta. 1986 Jun 19;882(2):258–265. doi: 10.1016/0304-4165(86)90163-7. [DOI] [PubMed] [Google Scholar]
  27. Raeymaekers L., Wuytack F., Casteels R. Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum. Biochim Biophys Acta. 1985 May 28;815(3):441–454. doi: 10.1016/0005-2736(85)90372-4. [DOI] [PubMed] [Google Scholar]
  28. Tada M., Kadoma M. Regulation of the Ca2+ pump ATPase by cAMP-dependent phosphorylation of phospholamban. Bioessays. 1989 May;10(5):157–163. doi: 10.1002/bies.950100505. [DOI] [PubMed] [Google Scholar]
  29. Verboomen H., Wuytack F., Eggermont J. A., De Jaegere S., Missiaen L., Raeymaekers L., Casteels R. cDNA cloning and sequencing of phospholamban from pig stomach smooth muscle. Biochem J. 1989 Aug 15;262(1):353–356. doi: 10.1042/bj2620353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wuytack F., Eggermont J. A., Raeymaekers L., Plessers L., Casteels R. Antibodies against the non-muscle isoform of the endoplasmic reticulum Ca2(+)-transport ATPase. Biochem J. 1989 Dec 15;264(3):765–769. doi: 10.1042/bj2640765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wuytack F., Kanmura Y., Eggermont J. A., Raeymaekers L., Verbist J., Hartweg D., Gietzen K., Casteels R. Smooth muscle expresses a cardiac/slow muscle isoform of the Ca2+-transport ATPase in its endoplasmic reticulum. Biochem J. 1989 Jan 1;257(1):117–123. doi: 10.1042/bj2570117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. de la Bastie D., Wisnewsky C., Schwartz K., Lompré A. M. (Ca2+ + Mg2+)-dependent ATPase mRNA from smooth muscle sarcoplasmic reticulum differs from that in cardiac and fast skeletal muscles. FEBS Lett. 1988 Feb 29;229(1):45–48. doi: 10.1016/0014-5793(88)80794-4. [DOI] [PubMed] [Google Scholar]

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