Arlette Franchi

Cytoplasmic pH, a key determinant of growth factor-induced DNA synthesis in quiescent fibroblasts

In response to growth factors, quiescent fibroblast mutants lacking Na+/H+ exchange activity fail to elevate their cytoplasmic pH (pHi) and to reinitiate DNA synthesis at neutral and acidic pHo. A pHi threshold of ~7.2 exists, below which growth factors cannot set in motion the G0 to S phase transition. Restoration of the pHi defect in mutant cells restores the wild‐type phenotype. These findings, combined with the properties of another class of mutants able to grow at very low pHo, demonstrate that pHi modulated by growth factor activation of the Na+/H+ antiporter, plays a determinant role in growth control.

Carrier activation and glucose transport in Chinese hamster fibroblasts.

Abstract Incubation of chinese hamster fibroblasts in glucose free medium, resulted in a 4 to 8 fold increase in the rate of D-glucose uptake and in a 3 to 4 fold increase in the uptake rate of glucose analogs (D-glucosamine, 2-Deoxy-D-glucose, 3-O-Methylglucose). In contrast to what is known for chick embryo fibroblasts, this increased hexose uptake activity is not blocked by cycloheximide in chinese hamster cells. The stimulation of synthesis of the Glucose Regulated Protein, GRP 95 which preceeds by 4 hours the stimulation of GRP 75 cannot account for the increase in hexose uptake-activity. Kinetic data have shown that the activation of glucose uptake activity following sugar starvation resulted only in a Vmax increase; Km for glucose remained constant at 0.6–0.7 mM. However, only the “activated” form of glucose uptake (glucose starvation) was very sensitive to N-ethylmaleimide. A mechanism of hexose “carrier activation” by glucose or a close metabolite is discussed.

The human amiloride-sensitive Na+/H+ antiporter: localization to chromosome 1 by in situ hybridization

The Na+/H+ antiporter is a ubiquitous membrane-bound enzyme involved in pH regulation of vertebrate cells. We cloned the human gene capable of complementing antiporter-deficient mouse fibroblasts and isolated an exon-containing genomic DNA fragment. Using this genomic probe, we mapped the putative structural gene of the amiloride-sensitive Na+/H+ antiporter to the human chromosome region 1p35----p36.1 by in situ hybridization.

Chapter 12 Na+-H+ Exchange and Growth Control in Fibroblasts: A Genetic Approach

Publisher Summary This chapter presents genetic and biochemical evidence establishing that the Na + –H + antiporter is a major pH i -regulating system in fibroblasts, that growth factors activate the antiporter by increasing its pH i sensitivity, and that growth factor-induced cytoplasmic alkalinization is essential for reinitiation of DNA synthesis and growth at neutral and acidic pH o . In addition, the chapter presents specific selection procedures that have led to the isolation of three classes of mutants of Na + –H + antiport system: (1) mutants partially or totally defective, (2) mutants with altered Na + or amiloride binding sites, and (3) mutants overproducing the antiporter. The first advances in genetics of Na + –H + antiport system offered new approach to analyze the physiology of pH i -regulating system in fibroblasts, the role of pH i in growth control, and the identification of Na + –H + antiport at a molecular level. Na + –H + antiporter is a major pH i -regulating system in fibroblasts. In the presence of HCO − 3 , the operation of a 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS)-sensitive and Na + -dependent Cl −- -HCO − 3 antiporter is a good substitute for Na + -H + exchange in pH, regulation. Growth factor-induced cytoplasmic alkalinization is triggered by activation of the Na + –H + antiporter. Growth factor activation results from an increased affinity of the system for internal H + . pH i exerts a control on the rate of cell entry into S phase. α-thrombin, a potent activator of polyphosphoinositide breakdown in CCL39 cells, activates as serum growth factors and phorbol esters, the Na + –H + antiporter by increasing its affinity for internal H + . The molecular identification of the antiporter molecule(s) should demonstrate if the change in pH i sensitivity reflects different states of kinase C-dependent phosphorylation.

[11] Use of tritium-labeled precursors to select mutants

Publisher Summary A killing selection is certainly a method of choice for rapid mutant isolation. However, it is very often difficult to meet the “comfortable” situation of having, for selection, a toxic compound analogous to an intermediate in the pathway of interest. This chapter illustrates with four examples that the use of tritiated-labeled precursors overcomes this difficulty and provides a powerful and general method of selection for mutants defective in membrane transport and in metabolic pathways. With these four examples of tritium suicide selection, the chapter shows that it is possible to devise a selection procedure allowing mutant isolation in a reasonable time period with either metabolizable or nonmetabolizable precursors. The tritium suicide has several advantages and is called “radiosuicide,” and was first used with bacterial genetics. This method is highly selective as the substrate itself of the pathway to “mutate” is the killing agent. The principle is based on the killing effects generated by the radiolytic decay of the incorporated labeled precursor. A killing selection is certainly a method of choice for rapid mutant isolation. However it is very often difficult to meet the “comfortable” situation of having, for selection, a toxic compound analogous to an intermediate in the pathway of interest.

Incorporation of a fatty acid containing a photosensitive group into lipids of cultured cardiac cells from chick embryo.

Radioactive 12-(4-azido-2-nitrophenoxy)-stearic acid (NAP-stearate) was synthetized; it behaves as a competitive inhibitor of long-chain fatty acids for the entry into cultured cardiac cells from chick embryo. After uptake, [3H] NAP-stearate was incorporated by an energy-dependent process into neutral and polar lipids. Photoactivation as a function of time leads to a covalent labelling of the cells: up to 31 per cent of the radioactivity was recovered in the 105 000 g cell pellet, mainly in proteins. These experiments show that fatty acids containing photosensitive groups would potentially allow to localize the proteins involved in the binding and/or in the transport of fatty acids.