Sandra Incerpi

Loss of GSH, Oxidative Stress, and Decrease of Intracellular pH as Sequential Steps in Viral Infection

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products. The reduced tripeptide was quantitatively recovered in the culture medium of the cells. Since the GSH loss in infected cells was not blocked by methionine, a known inhibitor of hepatocyte GSH transport, a nonspecific leakage through the plasma membrane is proposed. UV-irradiated Sendai virus gave the same results, confirming that the major loss of GSH was due to membrane perturbation upon virus fusion. Consequent to the loss of the tripeptide, an intracellular pH decrease occurred, which was due to a reversible impairment of the Na+/H+ antiporter, the main system responsible for maintaining unaltered pHi in those cells. At the end of the infection period, a rise in both pHi value and GSH content was observed, with a complete recovery in the activity of the antiporter. However, a secondary set up of oxidative stress was observed after 24 h from infection, which is the time necessary for virus budding from cells. In this case, the GSH decrease was partly due to preferential incorporation of the cysteine residue in the viral proteins and partly engaged in mixed disulfides with intracellular proteins. In conclusion, under our conditions of viral infection, oxidative stress is imposed by GSH depletion, occurring in two steps and following direct virus challenge of the cell membrane without the intervention of reactive oxygen species. These results provide a rationale for the reported, and often contradictory, mutual effects of GSH and viral infection.

Na pump and plasma membrane structure in L-cell fibroblasts expressing rat liver fatty acid binding protein

Although the intracellular fatty acid binding proteins have been investigated for nearly two decades and purified proteins are now available, little is known regarding the function of these proteins in intact cells. Therefore, L-cell fibroblasts transfected with cDNA encoding for rat liver fatty acid binding protein (L-FABP) were examined as to whether L-FABP expression in intact cells modifies plasma membrane enzyme activities, fluidity, and lipids. Plasma membrane Na/K-ATPase activity was 65.9 +/- 18.7 and 38.6 +/- 22.8 (P less than 0.001) nmol/mg protein x min for control and high-expression transfected cells, respectively. Consistent with this observation, [3H] ouabain binding to whole cells was significantly decreased from 3.7 +/- 0.3 to 2.0 +/- 0.8 pmol ouabain bound/mg cell protein in control and high-expression cells, respectively, whereas the cells affinity for ouabain was not significantly altered. Unexpectedly, Western blot analysis indicated that transfected cells had higher levels of Na+, K(+)-ATPase protein; in contrast, the activities of 5-nucleotidase and Mg-ATPase were unaltered. The effects of L-FABP expression on plasma membrane Na/K-ATPase function appeared to be mediated through alterations in plasma membrane lipids and/or structure. The plasma membrane cholesterol/phospholipid ratio decreased and the bulk plasma membrane fluidity increased in the high-expression cells. In conclusion, plasma membrane Na/K-ATPase activity in L cells may be regulated in part through expression of cytosolic L-FABP.

Insulin stimulation of Na/H antiport in L‐6 cells: A different mechanism in myoblasts and myotubes

Insulin modulation of the Na/H antiport of L‐6 cells, from rat skeletal muscle was studied in both myoblasts and myotubes using the fluorescent, pH sensitive, intracellular probe 2′,7′ bis (carboxyethyl)‐5(6)‐carboxyfluorescein. Insulin stimulated the Na/H antiport activity in L‐6 cells, showing a bell‐shaped dose response typical of other insulin responses: a maximum at 10 nM (ΔpH of 0.132 ± 0.007 and 0.160 ± 0.040 over basal value, for myoblasts and myotubes, respectively; means ± SD, n = 6–8) and smaller effects at higher and lower concentrations. Phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C, also stimulated the antiport in myoblasts but not in myotubes. Surprisingly the rapid increase in intracellular pH was not observed when insulin and PMA were added simultaneously to myoblasts; apparently these two activators mutually excluded each other. Downregulation of protein kinase C, obtained by preincubation of cells with PMA for 20 hr, totally abolished both hormone and PMA effects in myoblasts, whereas in myotubes insulin stimulation was not affected. Inhibitors of tyrosine kinase activity, such as erbstatin analog and genistein abolished insulin effect on the Na/H antiport, both in myoblasts and in myotubes. Different sensitivity to pertussis toxin in the two cell types suggests that the differentiation process leads to a change in the signal pathways involved in the physiological response to insulin. J. Cell. Physiol. 171:235–242, 1997.

Annual variations in the binding of insulin to hepatic membranes of the frog Rana esculenta

Amphibia undergo regular annual cycles of metabolic activity that are influenced by both exogenous factors and hormones. Insulin binding to crude frog hepatic membranes was studied throughout the year. The general character of insulin binding was similar to that in other vertebrates; the maximum specific binding was achieved after 4 hr at 4 degrees, the optimum pH was 7.8, half-maximal displacement of bound insulin was from 9 x 10(-10) to 1 x 10(-9) M, and insulin analogs competed for the insulin receptor in line with their relative biological potencies. A biphasic Scatchard plot and negative cooperativity of the receptor were also observed in frog liver membranes. Affinity constants from Scatchard plots revealed high and low affinity binding sites which were unchanged during the year. The seasonal cycle, however, markedly affected the binding capacity for both sites. Maximum binding occurred in May-June and the minimum in November-December for both classes of receptors. Binding capacities ranged from 1.71 to 11.33 fmol/mg protein for the high affinity sites and from 432 to 3171 fmol/mg protein for the low affinity sites. It is concluded that annual cycles of insulin binding reflect modulation of receptor number rather than receptor affinity.

Insulin-dependent release of 5′-nucleotidase and alkaline phosphatase from liver plasma membranes

Insulin treatment of isolated liver plasma membranes induced the release of 5′-nucleotidase and alkaline phosphatase. This effect was maximal at physiological hormone concentrations, being 36% and 17% for 5′-nucleotidase and alkaline phosphatase respectively, and was fully mimicked by the phosphatidylinositol specific phospholipase C (PI-PLC), thus confirming the presence of a glycosyl-phosphatidylinositol anchoring-system for these exofacial enzymatic proteins. The complete inhibition of insulin dependent enzyme release by neomycin is strongly supportive of an involvement of membrane-located PI-PLC activity. In addition, the insulin-like effect on enzyme release induced by the GTP non-hydrolysable analog, GTP-γ-S, and its sensitivity to the pertussis toxin are in favour of a mediatory role exerted by the G proteins system, in the transduction of some actions of insulin.

Insulin sensitivity of rat muscle sodium pump

The aim of the present study was to examine the possible physiological responsiveness of the sodium pump to insulin in rat muscle, an effect that has never been convincingly demonstrated. The insulin stimulation of the sodium pump was estimated by two well-established parameters: ouabain binding to pieces of soleus muscle, and Na/K-ATPase activity of purified plasma membranes. For both parameters the dose dependence of the insulin effect on the sodium pump shows the characteristic bell-shaped stimulation pattern, with a maximum in the physiological hormone concentration range. This result has not been observed in previous studies where insulin concentrations two to three orders of magnitude higher were used. It can be concluded that an effect of insulin on the regulation of the Na pump in muscle might well be operating in vivo.