The mechanism of in vitro formation of irreversibly sickled cells and modes of action of its inhibitors.

When red blood cells from sickle-cell patients were exposed to repeated cycles of deoxygenation and reoxygenation (one cycle was 5 min), dehydration of the cells was observed after several cycles of the sickling-desickling process. These dehydrated cells still maintained a biconcave form after 1 h of such cycling, but they started to take the form of irreversibly sickled cells after several hours. If red cells were simply kept deoxygenated for 16 h, neither dehydrated cells nor irreversibly sickled cells were formed. The formation of dehydrated cells was inhibited either by elimination of Ca2+ from the medium, or by the increase of K+ concentration in the medium. Under conditions in which dehydrated cells were not formed, i.e., deoxygenation incubation (either in the absence or presence of Ca2+) or the deoxygenation-reoxygenation cycling in the absence of Ca2+, 15-25% of cellular K+ leaked out during 4 h of incubation. When dehydrated cells were formed in deoxygenation-reoxygenation cycling in the presence of Ca2+, 40-50% of K+ was lost in 4 h. Two different types of inhibitor were found. The first type includes inhibitors of the Ca2+-activated K+ efflux, such as quinine, quinidine or tetraethylammonium chloride. These compounds suppressed both the K+ efflux and the formation of dehydrated cells. The second type includes calmodulin-interacting drugs. For example, chlorpromazine (20 microM) inhibited the formation of dehydrated cells almost completely, even though it did not inhibit the K+ efflux remarkably. Several other calmodulin-binding drugs were found to inhibit the formation of dehydrated cells similarly, and the potency of these drugs to inhibit the formation seems to be related to the binding affinity of these drugs to calmodulin.