Christiane Plas

Ecto-alkaline phosphatase considered as levamisole-sensitive phosphohydrolase at physiological pH range during mineralization in cultured fetal calvaria cells

Alkaline phosphatase (ALP) activity expressed on the external surface of cultured fetal rat calvaria cells and its relationship with mineral deposition were investigated under pH physiological conditions. After replacement of culture medium by assay buffer and addition of p‐nitrophenyl phosphate (pNPP), the rate of substrate hydrolysis catalyzed by whole cells remained constant for up to seven successive incubations of 10 min and was optimal over the pH range 7.6–8.2. It was decreased by levamisole by a 90% inhibition at 1 mM which was reversible within 10 min, dexamisole having no effect. Values of apparent Km for pNPP were close to 0.1 mM, and inhibition of pNPP hydrolysis by levamisole was uncompetitive (Ki = 45 μM). Phosphatidylinositol‐specific phospholipase C (PI‐PLC) produced the release into the medium of a p‐nitrophenyl phosphatase (pNPPase) sensitive to levamisole at pH 7.8. The released activity whose rate was constant up to 75 min represented after 15 min 60% of the value of ecto‐pNPPase activity. After 75 min of PI‐PLC treatment the ecto‐pNPPase activity remained unchanged despite the 30% decrease in Nonidet P‐40‐extractable ALP activity. High levels of 45Ca incorporation into cell layers used as index of mineral deposition were decreased by levamisole in a stereospecific manner after 4 h, an effect which was reversed within 4 h after inhibitor removal, in accordance with ecto‐pNPPase activity variations. These results evidenced the levamisole‐sensitive activity of a glycosylphosphatidylinositol‐anchored pNPPase consistent with ALP acting as an ecto‐enzyme whose functioning under physiological conditions was correlated to 45Ca incorporation and permit the prediction of the physiological importance of the enzyme dynamic equilibrium at the cell surface in cultured fetal calvaria cells.

Role of β-GP-derived pi in mineralization via ecto-alkaline phosphatase in cultured fetal calvaria cells

The permissive effect of β‐GP on mineralization in cultured rat fetal calvaria cells was investigated in relationship with phosphohydrolase activity of ecto‐ALP at physiological pH range. β‐GP present in the culture medium for 8 days exerted a stimulatory effect on 45Ca incorporation into matrix cell layers while the ecto‐ALP activity level measured on intact cells with a saturating concentration of p cells grown either in the presence or absence of β‐GP. In both types of cultures, β‐GP addition inhibited pNPP hydrolysis in a competitive and reversible manner and increased Pi concentration in the medium. The dose dependency of the effect of β‐GP on 45Ca incorporation and generation of Pi was similar (kϕ = 3 mM). Levamisole, but not dexamisole, inhibited both pNPP and β‐GP hydrolyses, which were likely catalyzed by the same ecto‐enzyme. The rate of 45Ca incorporation into matrix cell layers, which was high (0.90 μmol/4h/mg cell protein) in cells grown in the absence of β‐GP, was inhibited by 50% by levamisole. In cells grown in the absence of β‐GP, the 45Ca incorporation rate increased progressively after β‐GP addition, reaching after 12 h the value of cultures grown in the presence of β‐GP, the increase being totally inhibited by levamisole. In both types of cells, addition of exogenous Pi at concentrations corresponding to medium levels of β‐GP‐derived Pi rapidly led to high 45Ca incorporation rate which was unaffected by levamisole. β‐GP removal from cultures grown in its presence reduced by 50% the 45Ca incorporation rate which recovered the initial value after exogenous Pi addition independently of levamisole presence. Thus, mineral deposition did not affect the level and catalytic efficiency of ecto‐ALP to hydrolyze β‐GP in cultured fetal calvaria cells, yet it influenced the β‐GP‐stimulatory effect on mineralization so as to render this process not sensitive to high medium Pi levels.

Role of Hsp70 synthesis in the fate of the insulin-receptor complex after heat shock in cultured fetal hepatocytes.

The influence of a mild heat shock on the fate of the insulin‐receptor complex was studied in cultured fetal rat hepatocytes whose insulin glycogenic response is sensitive to heat [Zachayus and Plas (1995): J Cell Physiol 162:330–340]. After exposure from 15 min to 2 hr at 42.5°C, the amount of 125I‐insulin associated with cells at 37°C was progressively decreased (by 35% after 1 hr), while the release of 125I‐insulin degradation products into the medium was also inhibited (by 75%), more than expected from the decrease in insulin binding. Heat shock did not affect the insulin‐induced internalization of cell surface insulin receptors but progressively suppressed the recycling at 37°C of receptors previously internalized at 42.5°C in the presence of insulin. When compared to the inhibitory effects of chloroquine on insulin degradation and insulin receptor recycling, which were immediate (within 15 min), those of heat shock developed within 1 hr of heating. The protein level of insulin receptors was not modified after heat shock and during recovery at 37°C, while that of Hsp72/73 exhibited a transitory accumulation inversely correlated with variations in insulin binding, as assayed by Western immunoblotting from whole cell extracts. Coimmunoprecipitation experiments revealed a heat shock‐stimulated association of Hsp72/73 with the insulin receptor. Affinity labeling showed an interaction between 125I‐insulin and Hsp72/73 in control cells, which was inhibited by heat shock. These results suggest that increased Hsp72/73 synthesis interfered with insulin degradation and prevented the recycling of the insulin receptor and its further thermal damage via a possible chaperone‐like action in fetal hepatocytes submitted to heat stress.

Consumption and production of amino acids by insulin-responsive cultured fetal rat hepatocytes: the particular case of serine.

The ability for 18-day fetal rat hepatocytes in primary culture to modify extracellular amino acid concentrations was studied between 24 and 48 h of culture. Most of the 19 amino acids tested were found to be taken up by the hepatocytes. However, serine and glutamate appeared in the 24-hour-conditioned medium to be twice as concentrated as in the fresh medium. The profile of net consumption or production of amino acids was unchanged when the medium was supplemented with essential amino acids. The use of [U-14C]glucose revealed that serine released in the medium was mainly formed from glucose. The presence of insulin (10 nM) did neither significantly modify the variations of amino acid concentrations in the medium nor 2-amino[1-14C]isobutyric acid uptake by the cells, while the hormone produced a 2-fold increase in glycogen labeling from [U-14C]glucose. This study revealed that whatever the regulatory culture conditions considered a net serine production out of the cells occurred, which appears to be specific to the fetal stage.

Compared roles of glucose, galactose and fructose as glycogen precursors during the acute response to insulin in cultured rat foetal hepatocytes.

1. The efficiency of the contribution of hexoses to basal- and stimulated-glycogenesis, when studied in cultured 18 day-old rat foetal hepatocytes in the presence of glucose, was as follows: galactose greater than glucose greater than fructose. 2. Glucose deprivation had opposite effects on the contributions of [14C]galactose (decreased) and [14C]fructose (increased) to glycogenesis, which occurred independently of insulin and were reversed by glucose concentrations as low as 30-100 microM. 3. The stimulation of glycogenesis by insulin measured with [14C]glucose (3.2-fold) was superior to that obtained with either [14C]galactose or [14C]fructose (2.7-fold in both cases), which revealed a specific beneficial effect of insulin on glucose contribution.

Differences between glucose and insulin stimulation of glycogenesis in cultured fetal hepatocytes.

The glycogenic effects of a glucose load (15 mM) and/or insulin (10 nM) were studied in 18-day-old fetal rat hepatocytes after 2 days of culture when medium contained 4 mM glucose. A glucose load led to a stimulation of [14C]glucose glycogen labelling (20 min) earlier than with insulin (30-40 min); maximal stimulations were 3-fold after 1 h for the glucose load and 5-fold after 2-3 h for insulin. Simultaneous addition of the two agents produced synergic effects. When insulin was added 4 h after a glucose load (or vice versa), a second glycogenic response was elicited: a further addition of the same glycogenic agent was ineffective. The early glycogenic effects (up to 2 h) also occurred in the presence of 10 microM cycloheximide, with, however, some decrease of insulin stimulation. The contribution of medium glucose to the glycogen formed for 2 days (67% in the absence of glycogenic agent) was clearly enhanced by a glucose load and to a lesser degree by insulin after a 4-h exposure (83 and 71%, respectively). This was accompanied by a related modification of the participation of glucogenic precursors such as fructose and galactose. Thus, acute glycogenic response to glucose and insulin appeared both synergic and independent, and quite different in several aspects in cultured fetal hepatocytes.