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

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 (pHi) 6.5] increased by only about twofold. In addition, basal pHi (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 microM 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 pHi. Nonetheless, recovery from an acid load (pHi 6.51) was significantly more rapid in differentiated than control cells: 62 +/- 6 vs. 38 +/- 8 mmol H+.min-1.1 cells-1, respectively. However, acid loading decreased antiporter phosphorylation by twofold in differentiated and undifferentiated HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)