Regulation of K-Cl cotransport by Syk and Src protein tyrosine kinases in deoxygenated sickle cells.

Protein tyrosine kinases (PTK) of the Src family are thought to suppress K-Cl cotransport (KCC) activity via negative regulation of protein phosphatases. However, some PTK inhibitors reduce KCC activity, suggesting opposite regulation by different PTK families. We have reported previously that deoxygenation of sickle cells stimulates KCC and activates Syk (a Syk family PTK), but not Lyn (an Src family PTK). In this study the same results were obtained when PTK activities were measured under the conditions used to measure KCC activity and which prevent any change in intracellular [Mg(2+)]. Methyl-2,5-dihydroxycinnamate (DHC), a PTK inhibitor, was more selective for Syk than Lyn, while staurosporine (ST), a broad-specificity protein kinase inhibitor, inhibited Lyn more than Syk. Deoxygenation or 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d] pyrimidine (pp2, a specific Src inhibitor) stimulated KCC independently. These effects were not additive and were inhibited by DHC. In contrast, ST-induced KCC activation was resistant to DHC, suggesting a different pathway of activation. Overall, these data indicate that Syk activity is required for KCC activation, either induced by deoxygenation of sickle cells, or mediated by Src inhibition in oxygenated cells, and that Syk and Src PTKs exert opposing and interconnected regulatory effects on the activity of the transporter.

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.

Pathophysiology of a sickle cell trait mouse model: human alpha(beta)(S) transgenes with one mouse beta-globin allele.

As a potential model for sickle cell trait (AS), we examined mice containing one normal mouse beta-globin allele in combination with a human hemoglobin S (h(alpha)beta(S)) transgene (m(beta)/hS). The mice segregated into two subpopulations containing low and high proportions of hemoglobin S (m(beta)/hS1 and m(beta)/hS2, respectively) that was associated with one or two human h(alpha)beta(S) transgenes. We noted striking kidney pathology (cortical cysts, hyperplastic tubules, and glomerulonephritis), increasing with age and with greater severity in m(beta)/hS1. mBeta/hS2 animals were largely tolerant to 5% O(2) for 1 h, whereas 80% of m(beta)/hS1 mice died, exhibiting acute sequestration of erythrocytes in spleen, liver, and heart. These pathologies appear to result from a decreased oxygen affinity of the hybrid (human alpha/mouse beta) hemoglobins with a mild beta-thalassemia phenotype. Thus, these mouse models of sickle trait seem to manifest their renal pathology and sensitivity to hypoxia by mechanisms related to low tissue oxygen delivery and are different from the human syndrome. Analyses of parameters such as P(50), red cell indices, and genetic background are necessary in establishing potential relevance of any mouse model of the sickle cell syndromes.

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.