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In a previous paper we presented evidence for a negative regulation of adenylyl cyclase activity by tyrosine protein kinase(s) in the human leukemic T cell line Jurkat. In order to examine this point in non malignant cells, we conducted the present study in human peripheral blood mononuclear cells (PBMC). In these cells, staurosporine, a broad spectrum protein kinase inhibitor, enhanced not only the receptor-mediated, induced by prostaglandin E2 (PGE2), but also the direct (forskolin-induced) stimulation of adenylyl cyclase activity. Herbimycin A, a specific protein tyrosine kinase inhibitor, reproduced only in part the effect of staurosporine, whereas bisindolylmaleimide, the most specific protein kinase C (PKC) inhibitor known at present time, was ineffective. All these observations were made both in the absence and presence of isobutylmethylxanthine, a phosphodiesterase inhibitor, indicating that the effects of staurosporine and herbimycin A on cAMP accumulation were not due to phosphodiesterase inhibition. The calcium ionophore A 23187 also enhanced the PGE2-induced cAMP accumulation, and this effect was not additive to that of staurosporine, but additive to that of herbimycin A. These results confirm and extend those obtained in Jurkat cells. Taken together, they indicate that in human PBMC the adenylyl cyclase activity is negatively regulated by tyrosine kinase(s) and not by PKC, and positively regulated by Ca2+. They also suggest that the major enhancement by staurosporine of the PGE2-induced cAMP accumulation, although chiefly mediated by protein tyrosine kinase inhibition, also depends on another, presently undetermined, effect of the drug simulating that of Ca2+.

Cross-talk between signalling pathways appears to play an important role in T-lymphocyte activation. In the present work, we have studied the effects of different inhibitors of protein tyrosine kinases or protein serine/ threonine kinases on the agonist-induced cAMP accumulation in the human T-lymphoblast cell line Jurkat. Staurosporine, a potent but nonspecific inhibitor of protein kinases, produced a ten-fold enhancement of the response to PGE2. No significant effect was obtained with two specific protein kinase C inhibitors (GF 109203X and H7), whereas herbimycin A, a specific protein tyrosine kinase inhibitor, markedly enhanced the PGE2-induced cAMP accumulation: its effect was approximately 60% that of staurosporine. It was confirmed that both staurosporine and herbimycin A inhibited by more than 90% the release of IP3 induced by ligation of the T-cell receptor, a known protein tyrosine kinase-dependent mechanism. To our knowledge, this study provides the first indication of a protein tyrosine kinase-mediated inhibition of agonist-induced cAMP accumulation. The possible targets of this inhibition are discussed.

We have examined in the human T-cell line Jurkat the interaction between the activation through the T-cell receptor/CD3 complex and the adenylate cyclase pathway. OKT3, an anti-CD3 monoclonal antibody, did not activate by itself adenylate cyclase but produced a 3-7-fold increase of the cAMP accumulation induced by indirect (chloroadenosine, PGE2) or direct (forskolin) agonists of adenylate cyclase. A more detailed study with forskolin showed that OKT3 enhanced the effect of low concentrations of the agonist without affecting the maximal capacity of cAMP synthesis of the cells. The same concentrations of OKT3 produced both the enhancement of the adenylate cyclase pathway and the activation of phospholipase C. The enhancement by OKT3 of the adenylate cyclase pathway was inhibited by 0.5 microM staurosporine, a potent inhibitor of protein kinases, including tyrosine kinases and protein kinase C, whereas it was not inhibited by H7, a specific inhibitor of PKC. Staurosporine, at the same concentration, also inhibited the OKT3-induced activation of phospholipase C, a tyrosine kinase-dependent process. Taken together, these data indicate that activation of T-cell through the T-cell receptor enhances the adenylate cyclase pathway by a tyrosine protein kinase-dependent mechanism.

Defective T cell activation has been recognized in a number of patients with primary immunodeficiencies (ID). A distal defect resulting in abnormal production of IL2, IL3, IL4 and IFN gamma was found to be associated with reduced binding of the NF-AT transcription factor to the promoters of the genes coding for these lymphokines. Defect in early steps of T cell activation have been described in primary IDs, consisting in defective calcium flux and phosphoinositides turnover following TCR/CD3 ligation. We herein report a primary ID found in a 13 year old girl. It consists in a partially defective T cell proliferation which could be related to a defective Tyrosine phosphorylation.

An experimental model of adenosine deaminase deficiency was established on the human T cell line Jurkat by using 2'-deoxycoformycin, a strong specific inhibitor of the enzyme. When deoxyadenosine was added to the inhibited cells, the nucleotide profile was modified reproducing that found in lymphocytes from adenosine deaminase-deficient children. The metabolism of phosphoinositides, analyzed by either the release of [3H]inositol phosphates or the breakdown of 32P-prelabeled phosphatidyl inositides, was compared in normal and modified cells where dATP was accumulated. No modification in 32P labeling of phosphoinositides was detectable within the 32P-loading period. However, when the cells were stimulated by phytohemagglutinin or anti-CD3 monoclonal antibody, the phosphoinositide hydrolysis was strongly reduced in the dATP-containing lymphoblasts. This decrease was correlated with the intracellular dATP concentration.

The metabolic effects of oxalate on pyruvate kinase were studied in intact human red blood cells and compared to the spontaneous modifications induced by congenital pyruvate kinase deficiency. In normal cells, oxalate (2-3 . 10(-4) M) produces a large increase of the monophosphoglycerates, phosphoenolpyruvate pool and decrease of pyruvate concentrations as a result of pyruvate kinase inhibition; it does not significantly modify 2,3-diphosphoglycerate level, ATP formation or overall glycolytic activity. Those effects of oxalate are not due to Mg2+ chelation. A similar metabolite pattern is observed in vivo in erythrocytes with congenital pyruvate kinase deficiency, in which ATP concentration and glycolytic activity are described. These cells are more sensitive to oxalate than normal ones. Results are discussed with reference to the rate-limiting character of normal or congenitally deficient pyruvate kinase.

The respective effects of 2-chloropropionate and dichloroacetate on the pyruvate metabolic crossroads, lipogenesis and ketogenesis, were compared in hepatocytes isolated from fed rats. 2-Chloropropionate acts as an exclusive pyruvate dehydrogenase activator: it increases ketogenesis, lipogenesis, Krebs cycle intermediates and mitochondrial NADH/NAD+ ratio. The effects of dichloroacetate depend on experimental conditions and the intensity of its catabolization into oxalate: the resultant action of dichloroacetate on tested parameters combines the effects of pyruvate dehydrogenase activation on the one hand, and pyruvate carboxylase inhibition by oxalate on the other. A mixture of 2-chloropropionate plus oxalate mimics the effects of dichloroacetate. In hepatocytes from fed rats, endogenous lipogenesis is correlated with the mitochondrial NADH/NAD+ ratio, irrespective of the effector added.

Oleate (0.35 and 1.5 mM) decreases, in a concentration-dependent manner, lactate and pyruvate concentrations in hepatocytes, isolated from fed rats, incubated without exogenous substrate. The glycolytic flux, estimated at 18 mM-glucose by [6-3H]-glucose detritiation and apparent production of lactate and pyruvate, is decreased by oleate. The measurement of glycolytic intermediates shows a cross-over at the phosphofructokinase level, which might result from an increased citrate concentration. All those effects are dependent on oleate oxidation in mitochondria, since they are suppressed by 1 microM-2-[5-(4-chlorophenyl)pentyl]oxiran-2-carboxylate (POCA), an inhibitor of the mitochondrial entry of oleate, but not of its uptake by hepatocytes. The decrease of lactate and pyruvate also results from an oleate-induced enhancement of pyruvate utilization by hepatocytes, as shown by the increase of 14CO2 formation from [1-14C]- and [3-14C]-pyruvate, especially at low (0.4 mM) pyruvate concentration. Those oleate effects are also suppressed by POCA. They might be due to an enhanced flux through pyruvate carboxylase and pyruvate dehydrogenase, as a result of an oleate-induced increase in the mitochondrial concentrations of pyruvate and acetyl-CoA. Thus oleate oxidation inhibits production of lactate and pyruvate in fed-rat hepatocytes, as it does in other tissues. But, in the liver, it also enhances the mitochondrial utilization of pyruvate. The physiological implications of those findings are discussed.

Two new deficient glucose-phosphate-isomerase (GPI) variants have been described in patients suffering from severe chronic hemolytic anemias. The patients' parents were consanguineous, such that the patients were true homozygotes for the mutated GPI genes. In both cases the main cause of the defect in enzyme activity was molecular instability of the mutated GPI molecules, their catalytic activity being nearly normal. GPI 'Paris' was characterized by a slow electrophoretic migration and, above all, a drastically altered affinity for the substrates glucose-6-phosphate (decreased) and fructose-6-phosphate (increased). GPI 'Enfants malades' exhibited a slightly reduced electrophoretic mobility, an abnormal curve of the activity in function of pH, and an abnormal ratio of maximal velocity in the backward direction (fructose-6-phosphate leads to glucose-6-phosphate) to that in the forward direction (glucose-6-phosphate leads to fructose-6-phosphate). No clear relation could be proved between the kinetic abnormalities of the mutant GPI variants on the one hand and the metabolic changes of the GPI-deficient red cells and the severity of hemolysis on the other. Finally we emphasized the possible role of the impairment of hexosemonophosphate pathway in the reduction of viability of the GPI-deficient red cells.

A nonspherocytic hemolytic anemia, associated with a pyruvate kinase (PK) deficiency apparently inherited as a dominant trait has been identified in a family. In the affected members, the residual PK activities were 20% that of normal controls, an unusually low level for heterozygous subjects. The anemia was mild except in the proband, a 2-year-old boy who suffered from a severe anemia. The PKs of the proband and of his both parents have been characterized as kinetically and electrophoretically normal enzymes. Immunoprecipitation tests indicated a large amount of L-type inactive protein in the proband and his father. Moreover, an M2 type PK was present in the father's hemolysate, suggesting the existence of a compensatory process that derepressed the corresponding structural gene. We suggest that the presence of one or more mutated subunits in the tetrameric forms of L-type PK leads to the inactivation of these tetramers. This hypothesis accounts for the low residual activity in affected heterozygous members of this family. The severity of the hematological symptoms in the proband, in comparison with the mild hemolysis observed in the other heterozygous members of the family points to the existence of a large spectrum of pathologic expression for an identical PK defect present in a family.

Incubation of hepatocytes from 24 h-starved rats in the presence of 0.5 mM-adenosine decreased gluconeogenesis from lactate, but not from alanine. The inhibition of gluconeogenesis was associated with a stimulation of ketone-body production and an inhibition of pyruvate oxidation. These metabolic changes were suppressed in the presence of iodotubercidin (an inhibitor of adenosine kinase), but were reinforced in the presence of deoxycoformycin (an inhibitor of adenosine deaminase); 2-chloroadenosine induced no change in gluconeogenesis from lactate. These data indicate that the inhibition of gluconeogenesis by adenosine probably results from its conversion into adenine nucleotides. In the presence of lactate or pyruvate, but not with alanine or asparagine, this conversion resulted in a decrease in the [ATP]/[ADP] ratio in both mitochondrial and cytosolic compartments. Adenosine decreased the Pi concentration with all gluconeogenic substrates.

The metabolic changes induced by the deoxycoformycin inhibition of adenosine deaminase were studied in human erythrocytes incubated with nucleosides. 1 Adenosine nucleotide levels and glycolytic rate were increased by adenosine. 2 With deoxyadenosine, the cellular ATP level was reduced when dATP increased and the glycolytic rate was similarly enhanced. 3 The hypoxanthine production was equivalent in both cases. Our data demonstrate that human red cells are able to catabolize adenine deoxynucleotides into hypoxanthine, and the control of energy metabolism is not impaired by adenosine deaminase inhibition when PO identical to 4 and NAD+ are not limiting.

A marked erythrocyte phosphofructokinase deficiency was detected in a healthy man. His enzymatic activity was only 25% that of normal controls. His father and his son had erythrocytic phosphofructokinase activities of 50-55% that of normal controls. The chromatographic separation of erythrocytic phosphofructokinase isozymes, as well as immunological studies revealed a decrease in L-type phosphofructokinase activity. The lowered erythrocytic L-type phosphofructokinase activity was not accompanied by a decreased level of L-type phosphofructokinase in proteins. The L/M subunit ratio was similar to that of normal subjects. The defect resulted from the synthesis of stable L-type mutant subunit with high electrophoretic mobility. White blood cells, which synthesize mostly the same isozyme as L-type phosphofructokinase also showed a decreased activity and a high electrophoretic mobility. In spite of this important deficiency, and of significant metabolic alterations (a slight decrease in ATP; 2,3-diphosphoglycerate; triose phosphate), hemolysis did not appear in the propositus.

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.

The molecular heterogeneity of congenital pyruvate kinase deficiencies becomes apparent from the results of immunological studies. In one case, a quantitative defect is plausible; in the second case, the most likely hypothesis is a molecular alteration of the binding site for the activator, with preservation of the antigenic specificity; in the third case an abnormal protein, extremely unstable and devoid of antigenic reactivity, carries catalytic activity. In no case can any cross-reacting material be detected.

The intensity and regulation of metabolic pathways are different depending on the sex of the source animal for hepatocytes isolated from mature rats. In cells from fed animals incubated without exogenous substrate, ATP level and ketone body production are higher in males (+25% and +100%) and lactate production is higher (+64%) in females; oleate enhances mitochondrial pyruvate oxidation in hepatocytes from fed male rats but not from fed females; in cells from starved animals oleate increases gluconeogenesis in both sexes at saturating levels of gluconeogenic substrates. However, at physiological levels (1 mM lactate and 0.1 mM pyruvate), this activation can only be detected in cells from males. In both sexes, oleate activation is abolished by adenosine which reduces in parallel the mitochondrial oxidation of pyruvate; chloroadenosine, an A2-receptor agonist, increases glycogenolysis strongly in hepatocytes from male animals (+80%) but only very slightly in female cells (+12%).

Laboratory study of 16 workers handling lead made it possible to define a state of infraclinical lead poisoning. Estimation of alpha-dehydrase and of free erythrocytic protoporphyrins is the most sensitive test for diagnostic purposes. The estimation of erythrocytic pyrimidine 5' Nucleotidase would seem to be of value in the diagnosis of mild lead poisoning. The urinary lead tolerance test confirms the diagnosis and indicates the degree of intoxication. Its repetition make it possible to treat the intoxication.

In an effort to determine its value as a biological index of lead poisoning, we investigated the red-cell pyrimidine 5'-nucleotidase (Py5N) activity, along with the classical parameters, in 22 subjects with varying degrees of lead intoxication. In all cases, the red-cell Py5N activity was found to be decreased, even when most of the other biological tests of lead intoxication remained negative. Below the level of 150 microgram of lead per 100 ml of blood, the magnitude of the red-cell Py5N decrease was roughly proportional to the level of lead in peripheral blood. We conclude that red-cell Py5N is a convenient, reliable and sensitive index of lead exposure.

The effects of oxalate on glycolysis and glucose production from trioses were studied in hepatocytes isolated from fed and fasted rats. 1--In cells from fed rats oxalate inhibited glycolysis at the pyruvate kinase step, as shown by an increased phosphoenolpyruvate concentration, a decreased lactate and pyruvate production and a reduction of the glycolytic flux estimated by the rate of detritiation of [6-3H] glucose. The plot of 1/lactate production versus oxalate concentration showed that pyruvate kinase is a limiting step of glycolysis and allowed to determine the apparent inhibition constant for oxalate: about 3035 microM which is near the physiological concentration of blood oxalate. Under conditions where both pyruvate kinase and glycolytic flux are inhibited, oxalate had no effect on the synthesis of [14C] glucose from [14C] triose. 2--In hepatocytes prepared from fasted rats and incubated with lactate and pyruvate, oxalate decreased gluconeogenesis. In cells isolated from fasted rats and incubated with dihydroxyacetone, oxalate decreased lactate and pyruvate production whereas glucose synthesis remained unchanged. It is concluded that the inhibition of pyruvate kinase cannot by itself increase the gluconeogenic flux from triose.

An increased red cell adenosine deaminase (ADA) activity (85-fold) was found in a 10-year-old male child suffering from severe hemolytic disease. Evidence is given that the excessive ADA activity in erythrocytes is due to an abnormal amount of a catalytically and immunologically normal enzyme. Metabolic studies with the patient's erythrocytes show that the low ATP concentration in these cells (64% of comparably reticulocyte-rich blood) is due both to a diminished synthesis of adenylic nucleotides from adenosine, and to an excessive catabolism of AMP.

In isolated hepatocytes, dichloroacetate directly activates pyruvate dehydrogenase whereas its biotransformation product, oxalate, inhibits pyruvate carboxylase and pyruvate kinase. Dichloroacetate, which decreases blood lactate very efficiently, has been sucessfully tested in the acute treatment of congenital lactic acidosis, but its transformation into oxalate and potential chronic toxicity prompt to replace it by 2-chloropropionate as a therapeutic agent.

In hepatocytes isolated from fed rats, physiological concentrations of oxalate lower the flux through the tricarboxylic acid cycle (-48%) and reduce the steady-state levels of oxaloacetate and other Krebs cycle intermediates. All the metabolic modifications observed are explained by pyruvate carboxylase inhibition, since oxalate hardly modifies the flux through pyruvate dehydrogenase.

A deficiency of erythrocyte pyrimidine 5'-nucleotidase was found in a 23-year-old male suffering from severe congenital hemolytic disease. Results from exhaustive metabolic exploration are given and compared with reticulocyte-rich blood from subjects with auto-immune hemolytic disease. Evidence is given that the 20% apparent P5N residual activity corresponds to a non-specific acid phosphatase.