Strenuous exercise in warm environment is associated with improved microvascular function in sickle cell trait.

PURPOSE: Sickle cell trait is characterized by the presence of both normal and abnormal haemoglobin in red blood cells. The rate of exertional collapse is increased in athletes and military recruits who carry the trait, particularly in stressful environmental conditions. The aim of the present study was to investigate microvascular function and its determinants in response to intense exercise at control and warm environmental temperatures in carriers (AS) and non-carriers (AA) of sickle cell trait. METHODS: Nine AS and 11 AA, all healthy physically active young men, randomly participated in four experimental sessions (rest at 21 °C and 31 °C and cycling at 21 °C and 31 °C). All participants performed three exercises bouts as follows: 18-min submaximal exercise; an incremental test to exhaustion; and three 30-s sprints spaced with 20-s resting intervals. RESULTS: Skin Blood Flow (SkBF) was similar at rest between AA and AS. SkBF for all participants was higher at 31 °C than 21 °C. It was significantly higher in the AS group compared to the AA group immediately after exercise, regardless of the environmental conditions. No significant differences in hemorheological parameters, muscle damage or cardiac injury biomarkers were observed between the two groups. Our data also suggest higher oxidative stress for the AS group, with high superoxide dismutase (P = 0.044 main group effect). CONCLUSION: A specific profile is identified in the AS population, with increased microvascular reactivity after maximal exercise in stressful environment and slight pro-/antioxidant imbalance.

Lactate distribution in the blood compartments of sickle cell trait carriers during incremental exercise and recovery.

Whether or not whole blood lactate concentration is the same during a ramp exercise test in subjects with sickle cell trait (AS) as in normal subjects remains a point of controversy in the literature. Some studies have shown that the ability to produce or clear circulating lactate might differ between AS and subjects with normal haemoglobin (AA). If this is indeed so, the lactate distribution in the blood compartments should also differ. To test this hypothesis, lactate concentrations in the whole blood, plasma and red blood cells of AS and AA were compared at rest and in response to exercise. Eight AS and 8 AA performed an incremental exercise test. Whole blood, plasma and red blood cell lactate concentrations, the red blood cell : plasma lactate concentration ratio, the plasma-to-red blood cell lactate gradient, haematocrit and cardiorespiratory variables were analysed at rest and during an incremental exercise test and active recovery. Maximal oxygen uptake and ventilatory thresholds were similar in the two groups. No significant difference in whole blood, plasma or red blood cell lactate concentrations was observed between the two groups at rest, during exercise, or during the immediate recovery. Neither the red blood cell : plasma lactate concentration ratio nor the plasma-to-red blood cell lactate gradient differed between groups. Lactate distribution in the blood compartments did not differ between the two groups and this finding suggests that lactate production and/or clearance is quite similar during exercise in AS and AA.

Deoxygenation of sickle cells stimulates Syk tyrosine kinase and inhibits a membrane tyrosine phosphatase.

Polymerization of hemoglobin S in sickle red cells, in deoxygenated conditions, is associated with K+ loss and cellular dehydration. It was previously reported that deoxygenation of sickle cells increases protein tyrosine kinase (PTK) activity and band 3 tyrosine phosphorylation and that PTK inhibitors reduce cell dehydration. Here, the study investigates which PTKs are involved and the mechanism of their activation. Deoxygenation of sickle cells induced a 2-fold increase in Syk activity, measured by autophosphorylation in immune complex assays, but had no effect on Lyn. Syk was not stimulated by deoxygenation of normal red cells, and stimulation was partly reversible on reoxygenation of sickle cells. Syk activation was independent of the increase in intracellular Ca++ and Mg2+ associated with deoxygenation. Lectins that promote glycophorin or band 3 aggregation did not activate Syk. In parallel to Syk stimulation, deoxygenation of sickle cells, but not of normal red cells, decreased the activity of both membrane-associated protein tyrosine phosphatase (PTPs) and membrane protein thiol content. In vitro pretreatment of Syk immune complexes with membrane PTP inhibited Syk autophosphorylation. It is suggested that Syk activation in vivo could be mediated by PTP inhibition, itself resulting from thiol oxidation, as PTPs are known to be inhibited by oxidants. Altogether these data indicate that Syk could be involved in the mechanisms leading to sickle cell dehydration.

Involvement of deoxygenation-induced increase in tyrosine kinase activity in sickle cell dehydration.

Deoxygenation of sickle (SS) cells causes cationic alterations leading to cell dehydration by various mechanisms, including activation of Ca2+-sensitive K channels and possibly of K-Cl cotransport. Since an abnormal tyrosine kinase (TK) activity exists in SS cells we investigated the possible role of tyrosine phosphorylation in SS cell dehydration. In density-fractionated SS reticulocytes and discocytes, but not in normal red cells, deoxygenation increased membrane and cytosolic TK activities and tyrosine phosphorylation of band 3, independently of external Ca2+. These effects were abolished by the TK inhibitors methyl 2, 5-dihydroxycinnamate (DiOH) or tyrphostin 47 (T47). Deoxygenation-induced Ca2+ uptake was not affected by the inhibitors and Na+ gain was reduced by T47 and not by DiOH. Both inhibitors decreased the loss of K+ and cellular dehydration. The effect of the inhibitors on K+ efflux was still observed in the absence of external Ca2+. These data indicate that the TK inhibitors do not interfere with deoxygenation-induced membrane permeabilization, but affect Ca2+-independent K+ efflux. It cannot be excluded, however, that the TK inhibitors also attenuate Ca2+-sensitive K+ efflux. Based on recent evidence from the literature, it is suggested that the diminution of K+ efflux results in part from inhibition of K-Cl cotransport activity.

Relationship between type II phosphatidylinositol 4-kinase activity and protein tyrosine phosphorylation in membranes from normal and sickle red cells.

To assess the origin of the previously reported higher type II phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity of sickle-red-cell membranes [Rhoda-Hardy-Dessources, M.D., de Neef, R.S., Mérault, G.& Giraud, F. (1993) Biochim. Biophs. Acta 1181, 90-96], we have investigated the possible involvement of protein kinase C and tyrosine kinases in the regulation of the lipid kinase activity. Both protein kinase activities were found to be markedly higher in membranes from the pathological cells. When isolated normal-red-cell or sickle-red-cell membranes were assayed, phosphatidylinositol phosphorylation activity was not significantly modified after phorbol ester modulation of protein kinase C. In contrast, stimulation (with sodium orthovanadate) or inhibiton (by tyrphostin) of tyrosine phosphorylation led respectively, to increased or decreased PtdIns 4-kinase activity in membranes from both cell types. Moreover, immunoprecipitations of membrane extracts from normal and sickle red cells types with anti-PtdIns 4-kinase antibody 4C5G, followed by immunoblotting with an anti-phosphotyrosine Ig, revealed a 56-kDa band migrating with PtdIns 4-kinase activity. Taken together, these findings indicate that PtdIns 4-Kinase in red blood cells is a phosphotyrosine-containing protein and could be regulated by a mechanism involving tyrosine phosphorylation, and the increase in PtdIns 4-Kinase activity of sickle-red-cell membranes is at least in part mediated by their intrinsic tyrosine kinase activity.

Inhibition of deoxygenation-induced membrane protein dephosphorylation and cell dehydration by phorbol esters and okadaic acid in sickle cells.

Deoxygenation (DO) of sickle cell anemia red blood cells (SS cells) induces membrane permeabilization to Ca2+, Na+, and K+ and cell dehydration mostly through the activation of the Ca(2+)-dependent K+ channels. We show that DO of both SS cells and normal red blood cells was accompanied by a nonspecific dephosphorylation of membrane proteins. After treatment with a protein kinase C activator (phorbol myristate acetate) or a phosphoprotein phosphatase inhibitor (okadaic acid), the level of membrane protein phosphorylation in deoxygenated cells was maintained higher or equal, respectively, to that of the oxygenated controls. We found that these drugs in SS cells (1) inhibited by 40% the DO-stimulated net Ca2+ uptake, without affecting the DO-stimulated Ca2+ influx, suggesting that they activated the Ca2+ efflux; (2) slightly increased the DO-induced Na+ uptake and decreased the DO-induced K+ loss; and (3) prevented the DO-induced cell dehydration. Both drugs are known to stimulate both phosphorylation and activity of the Ca pump and of the Na/H antiport. Inhibition of SS cell dehydration might be due to an activation of the Ca pump preventing [Ca2+]i elevation responsible for the stimulation of the K+ channels and/or to an activation of the Na/H exchange resulting in cell water gain.

Characterization of phosphoinositide kinases in normal and sickle anaemia red cells.

PtdIns and PtdInsP kinases from normal erythrocyte (AA) membranes and sickle cell anaemia erythrocyte (SS) membranes have been characterized. PtdIns kinase was studied in native membranes under conditions in which PtdInsP kinase and PtdInsP phosphatase do not express any activity. Kinetic analysis of the AA and SS PtdIns kinases indicate similar Km values for PtdIns and ATP but higher Vmax values for SS PtdIns kinase. PtdInsP kinase was partially purified from erythrocyte ghosts by NaCl extraction. The kinetic parameters of PtdInsP kinase determined under these conditions were similar in AA and SS NaCl extracts. These data suggest the presence of some effector of PtdIns kinase in SS cell membranes, resulting in a greater activity of the enzyme. This leads consequently, to increase the PtdIns4P pool and to activate PtdInsP kinase, in agreement with our previous observations of a greater [32P]Pi incorporation in both polyphosphoinositides in SS cells relatively to AA cells.