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.

Dipyridamole inhibits sickling-induced cation fluxes in sickle red blood cells.

Sickling-induced cation fluxes contribute to cellular dehydration of sickle red blood cells (SS RBCs), which in turn potentiates sickling. This study examined the inhibition by dipyridamole of the sickling-induced fluxes of Na(+), K(+), and Ca(++) in vitro. At 2% hematocrit, 10 microM dipyridamole inhibited 65% of the increase in net fluxes of Na(+) and K(+) produced by deoxygenation of SS RBCs. Sickle-induced Ca(++) influx, assayed as (45)Ca(++) uptake in quin-2-loaded SS RBCs, was also partially blocked by dipyridamole, with a dose response similar to that of Na(+) and K(+) fluxes. In addition, dipyridamole inhibited the Ca(++)-activated K(+) flux (via the Gardos pathway) in SS RBCs, measured as net K(+) efflux in oxygenated cells exposed to ionophore A23187 in the presence of external Ca(++), but this effect resulted from reduced anion conductance, rather than from a direct effect on the K(+) channel. The degree of inhibition of sickling-induced fluxes was dependent on hematocrit, and up to 30% of dipyridamole was bound to RBC membranes at 2% hematocrit. RBC membrane content of dipyridamole was measured fluorometrically and correlated with sickling-induced flux inhibition at various concentrations of drug. Membrane drug content in patients taking dipyridamole for other clinical indications was similar to that producing inhibition of sickling-induced fluxes in vitro. These data suggest that dipyridamole might inhibit sickling-induced fluxes of Na(+), K(+), and Ca(++) in vivo and therefore have potential as a pharmacological agent to reduce SS RBC dehydration. (Blood. 2001;97:3976-3983)

Dehydration of mature and immature sickle red blood cells during fast oxygenation/deoxygenation cycles: role of KCl cotransport and extracellular calcium.

Sickle red blood cells (RBC) become dehydrated as a consequence of potassium loss. This process depends at least partly on deoxygenation and may be influenced by the presence of oxygenation/deoxygenation cycles and the frequency of cycling. In this study, sickle RBC were subjected to approximately 180 oxygenation/deoxygenation cycles during 4 hours to evaluate RBC dehydration with cycle periods more similar to in vivo cycles than those in previous studies. A continuous-flow, steady-state apparatus circulated a dilute RBC suspension through gas-permeable silicone tubing with segments that were exposed to either nitrogen or ambient oxygen. The percentage of sickling and partial pressure of oxygen were measured by means of sampling ports in the deoxygenation and oxygenation regions. The density increase (dehydration) of young (transferrin receptor-positive) and mature (transferrin receptor-negative) RBC and the requirements for calcium and chloride were evaluated. Density increase correlated with the percentage of sickled cells at the deoxygenation sampling port and was observed only in the presence of calcium, thereby implicating the calcium-dependent potassium channel (Gardos pathway). Density increase was not dependent on the presence of chloride, making it unlikely that KCl cotransport was an important pathway under these conditions. (Blood. 2000;95:2164-2168)