Sodium fluxes in human fibroblasts: Effect of serum, Ca +2 , and amiloride

The polypeptide growth factors, nerve growth factor, epidermal growth factor, and platelet-derived growth factor, as well as insulin do not induce ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) unless a minimal concentration of an ornithine decarboxylase-inducing amino acid, such as asparagine, is present in the medium. The effects of the growth factors were studied in appropriately responsive cell lines: pheochromocytoma (PC12) cells for nerve and epidermal growth factors, fibroblasts (NIH 3T3) for platelet-derived growth factor, and fibroblasts and hepatoma (KRC-7) cells for insulin. The nonmetabolizable amino acid analog alpha-aminoisobutyric acid can replace asparagine, indicating that the covalent modification of the inducing amino acid is not necessary for the induction of ornithine decarboxylase by these growth factors. For the same intracellular concentration of the inducing amino acid, the presence of the growth factors induces higher levels of ornithine decarboxylase. The evidence indicates that these growth factors do not induce ornithine decarboxylase by raising the intracellular concentration of amino acids but rather act synergistically with the inducing amino acid. Evidence is provided that the induction of polyamine-dependent growth by these growth factors is mediated by amino acids. The relationship of these results to the A and N amino acid transport systems and to the Na+ influxes in relation to growth is discussed.

Section: Resultsmentioning

confidence: 99%

Induction of ornithine decarboxylase activity by insulin and growth factors is mediated by amino acids.

Rinehart¹,

Canellakis²

1985

“…Although we have used EGF and Tf in the present investigation, there is no reason to believe that the hormone-induced receptor redistribution that we have observed is unique to this combination. Indeed, many hormones have been noted to induce a, membrane "ruffling," increased secretion rate, and increased fluid phase endocytotic rate in their target cells (13,(33)(34)(35). Thus, we expect that a "compartmentation" mechanism may be a general feature of hormone action.…”

Section: Discussionmentioning

confidence: 99%

Epidermal growth factor rapidly induces a redistribution of transferrin receptor pools in human fibroblasts.

Wiley¹,

Kaplan²

1984

Treatment of human fibroblasts with epidermal growth factor (EGF) results in a rapid increase (<5 min) in the ability of the cells to bind 125I-labeled transferrin to surface receptors. Scatchard analyses of EGF-treated cells indicate that this increase was due to an increase in the number of transferrin receptors at the cell surface rather than to alterations in ligand-receptor affinity. The EGF-induced increase in transferrin receptors was transient, reaching a peak by 5 min and then declining back to near basal levels by 45 min. Increases in transferrin receptor number were observed when l1 % of the EGF receptors were occupied and were maximal at 16% occupancy. EGF treatment accelerated the rate at which previously internalized 125I-labeled transferrin-receptor complexes were returned to the cell surface. The kinetics and magnitude of the loss of intracellular transferrin receptors was sufficient to account for the increase in surface transferrin receptors. We conclude from these studies that one of the earliest effects of EGF treatment is the induced translocation of an intracellular compartment to the cell surface. This intracellular compartment contains transferrin receptors and may be part of the pathway involved in the normal recycling of cell surface proteins.Polypeptide mitogens elicit a wide range of responses in target cells. Some are induced rapidly, whereas others arise slowly. The binding of epidermal growth factor (EGF) to receptors on human cells results in a series of rapid but transient responses. Such responses include increases in protein phosphorylation (1), membrane ruffling (2), ion fluxes (3-6), phospholipid turnover (7), and fluid phase endocytosis (8, 9). These occur within 5 min of EGF addition and diminish over the subsequent 15-30 min. In contrast, the induction of DNA synthesis requires continuous exposure of cells to EGF for up to 12 hr (10-12). The relationship between the rapid and delayed hormonal responses is presently unknown. However, because of the close temporal relationship between the occupancy of the EGF receptor and the induction of the rapid responses, the biochemical mechanisms underlying their generation are presently more amenable to analysis than those responsible for the generation of the mitogenic response.

“…The re-addition of serum or purified growth factors to the culture medium results in an ordered sequence of biochemical events that lead eventually to an increased cell proliferation. Since activation of Na+/H+ exchange is one of the important early responses ofcultured fibroblasts to mitogenic stimulation (1)(2)(3)(4)(5), it is important to understand the mechanism for this activation. Our previous work suggests that in cultured human fibroblasts (HSWP cells) a rise in intracellular Ca2l activity is a necessary step in this process (2,6,7).…”

mentioning

confidence: 99%

Activation of Na+/H+ exchange in cultured fibroblasts: synergism and antagonism between phorbol ester, Ca2+ ionophore, and growth factors.

Vicentini¹,

Villereal²

1985

The effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, on Na+ influx were investigated in cultured human foreskin fibroblasts (HSWP cells). We report here that in serum-deprived HSWP cells the addition of PMA alone has no significant effect on Na+ influx. However, the addition of PMA to cells whose Na+/H+ exchanger is partially activated with a submaximal dose of the Ca2+ ionophore A23187 leads to a larger stimulation than seen with A23187 alone. These data suggest that although stimulation of protein kinase C is not a sufficient signal to activate the Na+/H' exchanger in HSWP cells or in another human foreskin line (Jackson fibroblasts) studied, there are some cooperative effects of protein kinase C activation with a rise in Ca2+ to stimulate Na+/H' exchange. In addition, we found that PMA actually inhibits the mitogen-induced stimulation of Na+ influx in HSWP and Jackson fibroblasts. This observation strengthens the argument that in these cells activation of protein kinase C is not sufficient to activate Na+/H+ exchange and suggests that there is a negative feedback control via protein kinase C that inhibits some signal that is necessary for activating Na+/H+ exchange. However, in contrast to observations in HSWP cells, we were able to activate the Na+/H+ exchanger in mouse 3T3 and human WI-38 cells with PMA alone, suggesting that there is some diversity in the mechanism for activation of Na+/H+ exchange in different types of fibroblasts.Fibroblasts grown in cell culture can be arrested in the Go/Gj phase of the cell cycle by depriving them of growth factors. The re-addition of serum or purified growth factors to the culture medium results in an ordered sequence of biochemical events that lead eventually to an increased cell proliferation. Since activation of Na+/H+ exchange is one of the important early responses ofcultured fibroblasts to mitogenic stimulation (1-5), it is important to understand the mechanism for this activation. Our previous work suggests that in cultured human fibroblasts (HSWP cells) a rise in intracellular Ca2l activity is a necessary step in this process (2, 6, 7).Early studies from our laboratory, utilizing inhibitors of calmodulin (6,8), suggested that the activation of Na+/H+ exchange in HSWP cells was a calmodulin-dependent event.However, subsequent to that study it became apparent that most ofthe calmodulin inhibitors used also can inhibit protein kinase C (9). In addition, with the recent discovery that the site of action of tumor promoters is protein kinase C (9), the early observation that the tumor promoter phorbol 12-myristate 13-acetate (PMA) would stimulate Na influx in 3T3 cells (10) suggested that protein kinase C may be involved in the activation of Na+/H+ exchange. In fact, a recent report (11) suggested that PMA stimulation of Na+/H+ exchange occurs in the absence of a rise in Ca2' activity, which led the authors to suggest that a rise in Ca2' is not a necessary event in fibroblasts for the mitogen activation of Na+/H+ exch...