Phosphorylation of Na+-H+ antiporter is not stimulated by phorbol ester and acidification in granulocytic HL-60 cells

Rao Gadiparthi, C. Sardet, J. Pouyssegur, B. C. Berk

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Abstract

During differentiation of HL-60 cells into granulocyte-like cells, mRNA and protein levels for the Na+-H+ antiporter increased 10- to 15-fold. However, functional activity, as measured by recovery from an acid load [intracellular pH (pH(i)) 6.5] increased by only about twofold. In addition, basal pH(i) (measured in the absence of bicarbonate) increased from 7.15 to 7.26, suggesting an alteration in the antiporter's 'set point' during HL-60 cell differentiation. To gain insight into the role of the Na+-H+ antiporter in HL-60 cell differentiation, we studied mRNA expression of the NHE-1, NHE-3, and NHE-4 isoforms. Only the NHE-1 isoform mRNA increased during differentiation. Because it has recently been shown that the antiporter is regulated by phosphorylation, we next studied NHE-1 protein phosphorylation during HL-60 cell differentiation. Differentiation by exposure to 1 μM retinoic acid for 6 days caused a 15-fold increase in the synthesis of the NHE-1 protein. However, immunoprecipitation of 32P- labeled antiporter showed a decrease in band intensity. These data indicate that during HL-60 cell differentiation, there was a net decrease in the phosphorylation of NHE-1 despite an increase in pH(i). Nonetheless, recovery from an acid load (pH(i) 6.51) was significantly more rapid in differentiated than control cells: 62 ± 6 vs. 38 ± 8 mmol H+ · min-1 · l cells-1, respectively. However, acid loading decreased antiporter phosphorylation by twofold in differentiated and undifferentiated HL-60 cells. Phorbol 12- myristate 13-acetate did not stimulate antiporter activity in HL-60 cells and decreased antiporter phosphorylation by threefold in HL-60 cells, although it significantly increased antiporter phosphorylation in PS127A fibroblasts, which overexpress the antiporter. Our data thus indicate that the sustained increase in pH(i) in differentiated HL-60 cells is not due to increased activity of the NHE-1 antiporter or to an increase in expression of NHE-3 or NHE-4. Differentiated HL-60 cells appear to be an excellent model to study posttranslational regulation of NHE-1 activity.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume264
Issue number5 33-5
StatePublished - Jan 1 1993

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Sodium-Hydrogen Antiporter
Antiporters
HL-60 Cells
Phorbol Esters
Phosphorylation
Cell Differentiation
Acids
RNA Isoforms
Messenger RNA
Bicarbonates
Tretinoin
Immunoprecipitation
Granulocytes
Protein Isoforms
Acetates
Fibroblasts

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cell Biology

Cite this

Phosphorylation of Na+-H+ antiporter is not stimulated by phorbol ester and acidification in granulocytic HL-60 cells. / Gadiparthi, Rao; Sardet, C.; Pouyssegur, J.; Berk, B. C.

In: American Journal of Physiology - Cell Physiology, Vol. 264, No. 5 33-5, 01.01.1993.

Research output: Contribution to journalArticle

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abstract = "During differentiation of HL-60 cells into granulocyte-like cells, mRNA and protein levels for the Na+-H+ antiporter increased 10- to 15-fold. However, functional activity, as measured by recovery from an acid load [intracellular pH (pH(i)) 6.5] increased by only about twofold. In addition, basal pH(i) (measured in the absence of bicarbonate) increased from 7.15 to 7.26, suggesting an alteration in the antiporter's 'set point' during HL-60 cell differentiation. To gain insight into the role of the Na+-H+ antiporter in HL-60 cell differentiation, we studied mRNA expression of the NHE-1, NHE-3, and NHE-4 isoforms. Only the NHE-1 isoform mRNA increased during differentiation. Because it has recently been shown that the antiporter is regulated by phosphorylation, we next studied NHE-1 protein phosphorylation during HL-60 cell differentiation. Differentiation by exposure to 1 μM retinoic acid for 6 days caused a 15-fold increase in the synthesis of the NHE-1 protein. However, immunoprecipitation of 32P- labeled antiporter showed a decrease in band intensity. These data indicate that during HL-60 cell differentiation, there was a net decrease in the phosphorylation of NHE-1 despite an increase in pH(i). Nonetheless, recovery from an acid load (pH(i) 6.51) was significantly more rapid in differentiated than control cells: 62 ± 6 vs. 38 ± 8 mmol H+ · min-1 · l cells-1, respectively. However, acid loading decreased antiporter phosphorylation by twofold in differentiated and undifferentiated HL-60 cells. Phorbol 12- myristate 13-acetate did not stimulate antiporter activity in HL-60 cells and decreased antiporter phosphorylation by threefold in HL-60 cells, although it significantly increased antiporter phosphorylation in PS127A fibroblasts, which overexpress the antiporter. Our data thus indicate that the sustained increase in pH(i) in differentiated HL-60 cells is not due to increased activity of the NHE-1 antiporter or to an increase in expression of NHE-3 or NHE-4. Differentiated HL-60 cells appear to be an excellent model to study posttranslational regulation of NHE-1 activity.",
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T1 - Phosphorylation of Na+-H+ antiporter is not stimulated by phorbol ester and acidification in granulocytic HL-60 cells

AU - Gadiparthi, Rao

AU - Sardet, C.

AU - Pouyssegur, J.

AU - Berk, B. C.

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N2 - During differentiation of HL-60 cells into granulocyte-like cells, mRNA and protein levels for the Na+-H+ antiporter increased 10- to 15-fold. However, functional activity, as measured by recovery from an acid load [intracellular pH (pH(i)) 6.5] increased by only about twofold. In addition, basal pH(i) (measured in the absence of bicarbonate) increased from 7.15 to 7.26, suggesting an alteration in the antiporter's 'set point' during HL-60 cell differentiation. To gain insight into the role of the Na+-H+ antiporter in HL-60 cell differentiation, we studied mRNA expression of the NHE-1, NHE-3, and NHE-4 isoforms. Only the NHE-1 isoform mRNA increased during differentiation. Because it has recently been shown that the antiporter is regulated by phosphorylation, we next studied NHE-1 protein phosphorylation during HL-60 cell differentiation. Differentiation by exposure to 1 μM retinoic acid for 6 days caused a 15-fold increase in the synthesis of the NHE-1 protein. However, immunoprecipitation of 32P- labeled antiporter showed a decrease in band intensity. These data indicate that during HL-60 cell differentiation, there was a net decrease in the phosphorylation of NHE-1 despite an increase in pH(i). Nonetheless, recovery from an acid load (pH(i) 6.51) was significantly more rapid in differentiated than control cells: 62 ± 6 vs. 38 ± 8 mmol H+ · min-1 · l cells-1, respectively. However, acid loading decreased antiporter phosphorylation by twofold in differentiated and undifferentiated HL-60 cells. Phorbol 12- myristate 13-acetate did not stimulate antiporter activity in HL-60 cells and decreased antiporter phosphorylation by threefold in HL-60 cells, although it significantly increased antiporter phosphorylation in PS127A fibroblasts, which overexpress the antiporter. Our data thus indicate that the sustained increase in pH(i) in differentiated HL-60 cells is not due to increased activity of the NHE-1 antiporter or to an increase in expression of NHE-3 or NHE-4. Differentiated HL-60 cells appear to be an excellent model to study posttranslational regulation of NHE-1 activity.

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