AN EARLY TRANSIENT INCREASE OF INTRACELLULAR NA+ MAY BE ONE OF THE FIRST COMPONENTS OF THE MITOGENIC SIGNAL - DIRECT-DETECTION BY NA-23-NMR SPECTROSCOPY IN QUIESCENT 3T3 MOUSE FIBROBLASTS STIMULATED BY GROWTH-FACTORS

Na-23-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 P-31-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 Ca2+-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.