NCX1 Na/Ca exchanger inhibition by antisense oligonucleotides in mouse distal convoluted tubule cells

Kenneth White, F. A. Gesek, R. F. Reilly, P. A. Friedman

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Background. Plasma membrane NCX1 Na+/Ca2+ exchangers mediate cellular Ca2+ efflux. Renal distal convoluted tubule (DCT) cells express transcripts encoding three alternatively spliced NCX1 isoforms: NACA2 (exons B, C, D), NACA3 (exons B and D), and NACA6 (exons A, C, D). We used antisense oligodeoxynucleotides (ODNs) to determine the function of these NACA isoforms on Na+/Ca2+ exchanger activity and expression in DCT cells. Methods. Sense and antisense ODNs targeting exchanger transcripts were introduced into DCT cells permeabilized with streptolysin O. Na+/Ca2+ exchange activity was assessed by measuring Na+-dependent changes of free intracellular Ca2+ concentration (Δ[Ca2+](i)), in single cells, when the electrochemical gradient for Na+ was reversed. Results. The change of [Ca2+](i) in cells treated with antisense ODNs to a downstream or upstream region common to all NCX1 isoforms was 173 nM (-66%) to the downstream region located in the putative ninth transmembrane domain, and 226 nm (-39%) with ODNs to an upstream region located 5' to the variable portion of the intracellular loop. Antisense ODNs to exon B, present in both NACA2 and NACA3, decreased Δ[Ca2+](i) by 209 nM (-44%), while antisense ODNs specific for NACA6 (exon A) were without effect. Antisense ODNs specific for exon C, present in NACA2 and NACA6, decreased Δ[Ca2+](i) by 226 nM (-39%). Northern analysis of mRNA prepared from primary cultures of distal tubule cells revealed exon B- but not exon A-containing transcripts. Immunofluorescence analysis using a potyclonal antibody that recognizes NCX1 confirmed that protein expression was inhibited after treatment with the exon B antisense ODNs. Conclusion. These findings show that Na+-dependent cellular Ca2+ efflux in DCT cells is primarily mediated by NACA2 and NACA3.

Original languageEnglish (US)
Pages (from-to)897-906
Number of pages10
JournalKidney International
Volume54
Issue number3
StatePublished - 1998
Externally publishedYes

Fingerprint

Antisense Oligonucleotides
Oligodeoxyribonucleotides
Exons
Protein Isoforms
Distal Kidney Tubule
Fluorescent Antibody Technique
Cell Membrane
Messenger RNA
Antibodies

Keywords

  • Antisense oligonucleotides
  • Calcium transport
  • Immunofluorescence
  • Intracellular calcium
  • Sodium-calcium exchange

ASJC Scopus subject areas

  • Nephrology

Cite this

NCX1 Na/Ca exchanger inhibition by antisense oligonucleotides in mouse distal convoluted tubule cells. / White, Kenneth; Gesek, F. A.; Reilly, R. F.; Friedman, P. A.

In: Kidney International, Vol. 54, No. 3, 1998, p. 897-906.

Research output: Contribution to journalArticle

White, Kenneth ; Gesek, F. A. ; Reilly, R. F. ; Friedman, P. A. / NCX1 Na/Ca exchanger inhibition by antisense oligonucleotides in mouse distal convoluted tubule cells. In: Kidney International. 1998 ; Vol. 54, No. 3. pp. 897-906.
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abstract = "Background. Plasma membrane NCX1 Na+/Ca2+ exchangers mediate cellular Ca2+ efflux. Renal distal convoluted tubule (DCT) cells express transcripts encoding three alternatively spliced NCX1 isoforms: NACA2 (exons B, C, D), NACA3 (exons B and D), and NACA6 (exons A, C, D). We used antisense oligodeoxynucleotides (ODNs) to determine the function of these NACA isoforms on Na+/Ca2+ exchanger activity and expression in DCT cells. Methods. Sense and antisense ODNs targeting exchanger transcripts were introduced into DCT cells permeabilized with streptolysin O. Na+/Ca2+ exchange activity was assessed by measuring Na+-dependent changes of free intracellular Ca2+ concentration (Δ[Ca2+](i)), in single cells, when the electrochemical gradient for Na+ was reversed. Results. The change of [Ca2+](i) in cells treated with antisense ODNs to a downstream or upstream region common to all NCX1 isoforms was 173 nM (-66{\%}) to the downstream region located in the putative ninth transmembrane domain, and 226 nm (-39{\%}) with ODNs to an upstream region located 5' to the variable portion of the intracellular loop. Antisense ODNs to exon B, present in both NACA2 and NACA3, decreased Δ[Ca2+](i) by 209 nM (-44{\%}), while antisense ODNs specific for NACA6 (exon A) were without effect. Antisense ODNs specific for exon C, present in NACA2 and NACA6, decreased Δ[Ca2+](i) by 226 nM (-39{\%}). Northern analysis of mRNA prepared from primary cultures of distal tubule cells revealed exon B- but not exon A-containing transcripts. Immunofluorescence analysis using a potyclonal antibody that recognizes NCX1 confirmed that protein expression was inhibited after treatment with the exon B antisense ODNs. Conclusion. These findings show that Na+-dependent cellular Ca2+ efflux in DCT cells is primarily mediated by NACA2 and NACA3.",
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T1 - NCX1 Na/Ca exchanger inhibition by antisense oligonucleotides in mouse distal convoluted tubule cells

AU - White, Kenneth

AU - Gesek, F. A.

AU - Reilly, R. F.

AU - Friedman, P. A.

PY - 1998

Y1 - 1998

N2 - Background. Plasma membrane NCX1 Na+/Ca2+ exchangers mediate cellular Ca2+ efflux. Renal distal convoluted tubule (DCT) cells express transcripts encoding three alternatively spliced NCX1 isoforms: NACA2 (exons B, C, D), NACA3 (exons B and D), and NACA6 (exons A, C, D). We used antisense oligodeoxynucleotides (ODNs) to determine the function of these NACA isoforms on Na+/Ca2+ exchanger activity and expression in DCT cells. Methods. Sense and antisense ODNs targeting exchanger transcripts were introduced into DCT cells permeabilized with streptolysin O. Na+/Ca2+ exchange activity was assessed by measuring Na+-dependent changes of free intracellular Ca2+ concentration (Δ[Ca2+](i)), in single cells, when the electrochemical gradient for Na+ was reversed. Results. The change of [Ca2+](i) in cells treated with antisense ODNs to a downstream or upstream region common to all NCX1 isoforms was 173 nM (-66%) to the downstream region located in the putative ninth transmembrane domain, and 226 nm (-39%) with ODNs to an upstream region located 5' to the variable portion of the intracellular loop. Antisense ODNs to exon B, present in both NACA2 and NACA3, decreased Δ[Ca2+](i) by 209 nM (-44%), while antisense ODNs specific for NACA6 (exon A) were without effect. Antisense ODNs specific for exon C, present in NACA2 and NACA6, decreased Δ[Ca2+](i) by 226 nM (-39%). Northern analysis of mRNA prepared from primary cultures of distal tubule cells revealed exon B- but not exon A-containing transcripts. Immunofluorescence analysis using a potyclonal antibody that recognizes NCX1 confirmed that protein expression was inhibited after treatment with the exon B antisense ODNs. Conclusion. These findings show that Na+-dependent cellular Ca2+ efflux in DCT cells is primarily mediated by NACA2 and NACA3.

AB - Background. Plasma membrane NCX1 Na+/Ca2+ exchangers mediate cellular Ca2+ efflux. Renal distal convoluted tubule (DCT) cells express transcripts encoding three alternatively spliced NCX1 isoforms: NACA2 (exons B, C, D), NACA3 (exons B and D), and NACA6 (exons A, C, D). We used antisense oligodeoxynucleotides (ODNs) to determine the function of these NACA isoforms on Na+/Ca2+ exchanger activity and expression in DCT cells. Methods. Sense and antisense ODNs targeting exchanger transcripts were introduced into DCT cells permeabilized with streptolysin O. Na+/Ca2+ exchange activity was assessed by measuring Na+-dependent changes of free intracellular Ca2+ concentration (Δ[Ca2+](i)), in single cells, when the electrochemical gradient for Na+ was reversed. Results. The change of [Ca2+](i) in cells treated with antisense ODNs to a downstream or upstream region common to all NCX1 isoforms was 173 nM (-66%) to the downstream region located in the putative ninth transmembrane domain, and 226 nm (-39%) with ODNs to an upstream region located 5' to the variable portion of the intracellular loop. Antisense ODNs to exon B, present in both NACA2 and NACA3, decreased Δ[Ca2+](i) by 209 nM (-44%), while antisense ODNs specific for NACA6 (exon A) were without effect. Antisense ODNs specific for exon C, present in NACA2 and NACA6, decreased Δ[Ca2+](i) by 226 nM (-39%). Northern analysis of mRNA prepared from primary cultures of distal tubule cells revealed exon B- but not exon A-containing transcripts. Immunofluorescence analysis using a potyclonal antibody that recognizes NCX1 confirmed that protein expression was inhibited after treatment with the exon B antisense ODNs. Conclusion. These findings show that Na+-dependent cellular Ca2+ efflux in DCT cells is primarily mediated by NACA2 and NACA3.

KW - Antisense oligonucleotides

KW - Calcium transport

KW - Immunofluorescence

KW - Intracellular calcium

KW - Sodium-calcium exchange

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