Characterization of NA+/CA2+ exchange protein and cdna in cultured human retinal pigment epithelial cells

N. J. Mangini, J. Valle, L. Haugh-Scheidt, Brian Kennedy

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Abstract

Purpose To assay Na+/Ca2+ exchange (NCX) activity in cultured human retinal pigment epithelial cells (HRPE), and determine the nucleotide sequence of HRPE NCX mRNA transcripts Methods Regulation of intracellular free Ca2+ concentration ([Ca ]i) by an Na+/Ca2+ exchanger in HRPE monolayers was measured by digital imaging in cells loaded with fiira-2 or fluo-3. Northern blot analysis of total HRPE RNA using a restriction fragment cRNA probe coding for the canine cardiac NCX was used to identify HRPE NCX mRNA transcripts. RT-PCR of cultured HRPE RNA employing NCX gene-specific primers was used to synthesize HRPE NCX cDNAs. Analysis of PCR DNAs was by direct sequencing. Results. Resting [Ca2+], in control solution (containing 1 mM Ca2+ and 140 mM NaCl) was, on average, - 200 nM Removal of [Na+]0 (Na+ replaced by N-methyl-D-glucamine) caused [Ca2+], to rise rapidly. The [Na+]0-freeinduced rise in [Ca2+], was abolished by the removal of [Ca2+]0, and was not blocked by nifedipine (blocks Ca2+ channels) or thapsigargin (inhibits Ca2+ sequestration by internal stores). Western and Northern blot analyses, respectively, confirmed thç presence of NCX protein and mRNA in cultured HRPE, and indicated that the HRPE NCX is of the cardiac type. RT-PCR of cultured HRPE yielded two 1.5 kb cDNAs that together covered 91% of the HRPE NCX coding region. Direct sequencing of the PCR cDNAs, and analysis with PCGENE software confirmed homology of the HRPE cDNAs to cardiactype NCXs Conclusions A Na+/Ca2+ exchanger of the cardiac type is present in cultured HRPE and plays a role in regulating [Ca2+], in this tissue.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume38
Issue number4
StatePublished - 1997
Externally publishedYes

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Retinal Pigments
Epithelial Cells
Proteins
Complementary DNA
Polymerase Chain Reaction
Northern Blotting
Messenger RNA
RNA
Complementary RNA
Thapsigargin
Nifedipine
Canidae
Software

ASJC Scopus subject areas

  • Ophthalmology

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Characterization of NA+/CA2+ exchange protein and cdna in cultured human retinal pigment epithelial cells. / Mangini, N. J.; Valle, J.; Haugh-Scheidt, L.; Kennedy, Brian.

In: Investigative Ophthalmology and Visual Science, Vol. 38, No. 4, 1997.

Research output: Contribution to journalArticle

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abstract = "Purpose To assay Na+/Ca2+ exchange (NCX) activity in cultured human retinal pigment epithelial cells (HRPE), and determine the nucleotide sequence of HRPE NCX mRNA transcripts Methods Regulation of intracellular free Ca2+ concentration ([Ca ]i) by an Na+/Ca2+ exchanger in HRPE monolayers was measured by digital imaging in cells loaded with fiira-2 or fluo-3. Northern blot analysis of total HRPE RNA using a restriction fragment cRNA probe coding for the canine cardiac NCX was used to identify HRPE NCX mRNA transcripts. RT-PCR of cultured HRPE RNA employing NCX gene-specific primers was used to synthesize HRPE NCX cDNAs. Analysis of PCR DNAs was by direct sequencing. Results. Resting [Ca2+], in control solution (containing 1 mM Ca2+ and 140 mM NaCl) was, on average, - 200 nM Removal of [Na+]0 (Na+ replaced by N-methyl-D-glucamine) caused [Ca2+], to rise rapidly. The [Na+]0-freeinduced rise in [Ca2+], was abolished by the removal of [Ca2+]0, and was not blocked by nifedipine (blocks Ca2+ channels) or thapsigargin (inhibits Ca2+ sequestration by internal stores). Western and Northern blot analyses, respectively, confirmed th{\cc} presence of NCX protein and mRNA in cultured HRPE, and indicated that the HRPE NCX is of the cardiac type. RT-PCR of cultured HRPE yielded two 1.5 kb cDNAs that together covered 91{\%} of the HRPE NCX coding region. Direct sequencing of the PCR cDNAs, and analysis with PCGENE software confirmed homology of the HRPE cDNAs to cardiactype NCXs Conclusions A Na+/Ca2+ exchanger of the cardiac type is present in cultured HRPE and plays a role in regulating [Ca2+], in this tissue.",
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