Regulation by nerve growth factor and protein phosphorylation of Na/K/2Cl cotransport and cell volume in PC12 cells

Sara Leung, Martha E O'Donnell, Anthony Martinez, H. Clive Palfrey

Research output: Contribution to journalArticle

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Abstract

PC12 cells possess a bumetanide-sensitive Na/K/2Cl cotransport system similar to that found in other cell types. Between 10-15% of the total 86Rb influx in these cells is mediated by this pathway under normal conditions. The cotransporter has affinities of 16.5 mM for Na(o) and 0.7 mM for K(o), is absolutely dependent on Cl(o) and is loop diuretic inhibitable (benzmetanide > bumetanide > piretanide > furosemide). The cotransporter can be activated (up to 8-fold) by cell shrinkage or (up to 4-fold) by treatment with the protein phosphatase inhibitors okadaic acid (EC50 ~ 650 nM) or calyculin A (EC50 ~ 8 nM). Cell shrinkage is followed by a bumetanide-sensitive regulatory volume increase as determined in cell sizing experiments. Calyculin A rapidly elevates normal cell volume in a diuretic-inhibitable manner. Cotransport activity and cell volume are also increased by nerve growth factor (NGF) treatment. The effect of NGF an cotransport rate is biphasic, with an initial rapid ~2.5-fold increase followed by a prolonged plateau, and is blocked by pretreatment of the cells with K252a (IC50 ~ 30 nM). By contrast, agents that raise cAMP or phorbol esters lead to an inhibition of cotransport, indicating that the NGF effect is not mediated by stimulation of either cAMP-dependent protein kinase or protein kinase C. Long term NGF treatment (>2 days) leads to neurite formation and a maintained ~2- fold increase in cotransport activity. Bumetanide treatment does not affect the ability of cells to extend neurites, nor is the growth rate of cells in normal medium affected by the diuretic. These results suggest that the cotransport system in PC12 cells is acutely regulated by protein phosphorylation and dephosphorylation as well as cell shrinkage and that cotransport activity may be up-regulated during neuronotypic differentiation.

Original languageEnglish (US)
Pages (from-to)10581-10589
Number of pages9
JournalJournal of Biological Chemistry
Volume269
Issue number14
StatePublished - Apr 8 1994

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Bumetanide
Phosphorylation
PC12 Cells
Nerve Growth Factor
Cell Size
Diuretics
Proteins
Sodium Potassium Chloride Symporter Inhibitors
Okadaic Acid
Phosphoprotein Phosphatases
Furosemide
Phorbol Esters
Cyclic AMP-Dependent Protein Kinases
Neurites
Protein Kinase C
Contrast Media
Cells
Inhibitory Concentration 50
Experiments
calyculin A

ASJC Scopus subject areas

  • Biochemistry

Cite this

Regulation by nerve growth factor and protein phosphorylation of Na/K/2Cl cotransport and cell volume in PC12 cells. / Leung, Sara; O'Donnell, Martha E; Martinez, Anthony; Palfrey, H. Clive.

In: Journal of Biological Chemistry, Vol. 269, No. 14, 08.04.1994, p. 10581-10589.

Research output: Contribution to journalArticle

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