An early transient increase of intracellular Na + may be one of the first components of the mitogenic signal. Direct detection by 23Na-NMR spectroscopy in quiescent 3T3 mouse fibroblasts stimulated by growth factors

Abstract

23Na-NMR spectroscopy was designed to allow for continuous recording of intracellular Na + in 3T3 fibroblasts stimulated by serum growth-factors in the presence of ion transport inhibitors. The metabolic state of cells at rest and following stimulation was monitored by 31P-NMR spectra of ATP and related high-energy phosphates. The study demonstrates that early activation of ion transporters by addition of serum is marked by the appearance of transient increase of the intracellular Na +, beginning 3 min after addition of serum to quiescent culture and lasting approx. 20 min. The initial rise in cellular Na + results from an increased activity of the bumetanide-sensitive Na +/K +/Cl − cotransport and of the amiloride-sensitive Na +/H + antiport. It is suppressed by any one of these inhibitors. Subsequent activation of the ouabain-sensitive Na +/K +-ATPase results in an increased Na + efflux, leading to a return of intracellular Na + to its initial baseline. Previous work had shown that the early activation of bumetanide-sensitive and amiloride sensitive ion-transporters by growth-factors was essential for induction of cell division, at least in some cell types. Preventing ion activation by adding ion-transport inhibitors lead to the inhibition of DNA synthesis 18 h later. This process was reversible upon elimination of these inhibitors. Even though alternative non-specific effects of these inhibitors cannot be ruled out, the observed transient peak in intracellular Na + may be one of the earliest components of the mitogenic signal. On the basis of previous works, its effect seems to be related to the activation of Ca 2+-dependent and cyclic AMP second messenger pathways. The different mechanisms whereby the activated Na +/K +/Cl − cotransport and the Na +/H + antiport contribute to this signal need to be further investigated.