Structural and functional analysis of Na+/Ca2+ exchange in distal convoluted tubule cells

Kenneth White, Frank A. Gesek, Peter A. Friedman

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Renal distal convoluted tubules (DCT) are a major site of hormone-regulated, active calcium absorption. Calcium exit across basolateral plasma membranes is thought to be mediated by Na+/Ca2+ exchange and a Ca2+-ATPase. In this report the presence and function of Na+/Ca2+ exchangers in DCT cells were assessed. cDNAs encoding a conserved region and the variable regions of three alternatively spliced isoforms of the Na+/Ca2+ exchanger, NACA2, NACA3, and NACA6, were isolated in a ratio of 7: 12: 1 using homology-based reverse transcription-polymerase chain reaction (RT-PCR) with RNA from an immortalized mouse DCT cell line. Northern blots probed with a 32P-labeled PCR product from a conserved region of the exchanger were positive for a single transcript of 7 kb in primary cultures of distal tubule cells (cortical ascending limb 4- DCT cells), consistent with the reported size of the exchanger in other tissues. Na+/Ca2+ exchange was assessed by measuring sodium-dependent changes of intracellular calcium ([Ca2+]i), in single cells. In the presence of an outward Na+ gradient, [Ca2+]i increased by 240%. Collapsing the Na+ gradient with monensin inhibited the rise of [Ca2+]i. Removal of extracellular Ca2+ or the addition of an Na+ ionophore inhibited the rise of [Ca2+]i. The intracellular Na+ concentration decreased upon removal of extracellular Na+ in parallel with the rise of [Ca2+]i. Western analysis performed on membranes prepared from DCT cells or primary cultures of distal tubule cells with a polyclonal antibody revealed bands at -125 and 85 kDa, consistent with reported sizes for exchanger protein. These findings show that Na+/Ca2+ exchanger transcripts, protein, and activity are present in DCT cells and that Na+-dependent Ca2+ efflux may be mediated by NACA2, NACA3, and NACA6.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Renal Physiology
Volume271
Issue number3 PART 2
StatePublished - 1996
Externally publishedYes

Fingerprint

Calcium
Distal Kidney Tubule
Monensin
Polymerase Chain Reaction
Primary Cell Culture
Calcium-Transporting ATPases
Ionophores
Northern Blotting
Reverse Transcription
Protein Isoforms
Proteins
Extremities
Complementary DNA
Sodium
Cell Membrane
Hormones
RNA
Cell Line
Membranes
Antibodies

Keywords

  • Calcium transport
  • Intracellular calcium
  • Kidney
  • Reverse transcriptionpolymerase chain reaction
  • Sodium/calcium exchanger

ASJC Scopus subject areas

  • Physiology (medical)
  • Physiology

Cite this

Structural and functional analysis of Na+/Ca2+ exchange in distal convoluted tubule cells. / White, Kenneth; Gesek, Frank A.; Friedman, Peter A.

In: American Journal of Physiology - Renal Physiology, Vol. 271, No. 3 PART 2, 1996.

Research output: Contribution to journalArticle

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abstract = "Renal distal convoluted tubules (DCT) are a major site of hormone-regulated, active calcium absorption. Calcium exit across basolateral plasma membranes is thought to be mediated by Na+/Ca2+ exchange and a Ca2+-ATPase. In this report the presence and function of Na+/Ca2+ exchangers in DCT cells were assessed. cDNAs encoding a conserved region and the variable regions of three alternatively spliced isoforms of the Na+/Ca2+ exchanger, NACA2, NACA3, and NACA6, were isolated in a ratio of 7: 12: 1 using homology-based reverse transcription-polymerase chain reaction (RT-PCR) with RNA from an immortalized mouse DCT cell line. Northern blots probed with a 32P-labeled PCR product from a conserved region of the exchanger were positive for a single transcript of 7 kb in primary cultures of distal tubule cells (cortical ascending limb 4- DCT cells), consistent with the reported size of the exchanger in other tissues. Na+/Ca2+ exchange was assessed by measuring sodium-dependent changes of intracellular calcium ([Ca2+]i), in single cells. In the presence of an outward Na+ gradient, [Ca2+]i increased by 240{\%}. Collapsing the Na+ gradient with monensin inhibited the rise of [Ca2+]i. Removal of extracellular Ca2+ or the addition of an Na+ ionophore inhibited the rise of [Ca2+]i. The intracellular Na+ concentration decreased upon removal of extracellular Na+ in parallel with the rise of [Ca2+]i. Western analysis performed on membranes prepared from DCT cells or primary cultures of distal tubule cells with a polyclonal antibody revealed bands at -125 and 85 kDa, consistent with reported sizes for exchanger protein. These findings show that Na+/Ca2+ exchanger transcripts, protein, and activity are present in DCT cells and that Na+-dependent Ca2+ efflux may be mediated by NACA2, NACA3, and NACA6.",
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AB - Renal distal convoluted tubules (DCT) are a major site of hormone-regulated, active calcium absorption. Calcium exit across basolateral plasma membranes is thought to be mediated by Na+/Ca2+ exchange and a Ca2+-ATPase. In this report the presence and function of Na+/Ca2+ exchangers in DCT cells were assessed. cDNAs encoding a conserved region and the variable regions of three alternatively spliced isoforms of the Na+/Ca2+ exchanger, NACA2, NACA3, and NACA6, were isolated in a ratio of 7: 12: 1 using homology-based reverse transcription-polymerase chain reaction (RT-PCR) with RNA from an immortalized mouse DCT cell line. Northern blots probed with a 32P-labeled PCR product from a conserved region of the exchanger were positive for a single transcript of 7 kb in primary cultures of distal tubule cells (cortical ascending limb 4- DCT cells), consistent with the reported size of the exchanger in other tissues. Na+/Ca2+ exchange was assessed by measuring sodium-dependent changes of intracellular calcium ([Ca2+]i), in single cells. In the presence of an outward Na+ gradient, [Ca2+]i increased by 240%. Collapsing the Na+ gradient with monensin inhibited the rise of [Ca2+]i. Removal of extracellular Ca2+ or the addition of an Na+ ionophore inhibited the rise of [Ca2+]i. The intracellular Na+ concentration decreased upon removal of extracellular Na+ in parallel with the rise of [Ca2+]i. Western analysis performed on membranes prepared from DCT cells or primary cultures of distal tubule cells with a polyclonal antibody revealed bands at -125 and 85 kDa, consistent with reported sizes for exchanger protein. These findings show that Na+/Ca2+ exchanger transcripts, protein, and activity are present in DCT cells and that Na+-dependent Ca2+ efflux may be mediated by NACA2, NACA3, and NACA6.

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