Gardos Effect Research Articles

Calcium permeability in the hereditary stomatocytosis syndrome

The syndrome of hereditary stomatocytosis includes a spectrum of mutations ranging from an overhydrated variety with typical stomatocytes and high red cell (Na + K) to a dehydrated variety with target cells and low red cell (Na + K). Haemolysis is usually mild but can be severe at the extremes of over- or under-hydration. Fluxes of Na and K are raised between 3 and 25 fold normal. In this study the specificity of the cation leak has been tested towards the divalent Ca2+ cation by 2 types of experiments. First the influx of Ca2+ was measured in erythrocytes depleted of ATP by preincubation for 90 min with inosine plus iodoacetate. This procedure inhibits the Ca pump and allows measurable uptake of Ca2+-ions when red cells are incubated in media containing 1.5 mM Ca2+. Calcium uptake into overhydrated stomatocytes was 12 nmol/ml cells/4 h and for dehydrated stomatocytes 20-50 nmol/ml cells/4 h. Control cells with comparable reticulocytosis (6-10%) allowed a Ca uptake of 20-30 nmol/ml cells/4 h while normal red cells took up 6 nmol/ml cells/4 h. The low Ca permeability of overhydrated stomatocytes was confirmed in a second type of experiment which involved measurement of K. efflux from fresh, ATP-rich cells incubated in media of varying Ca concentrations. Raising the extracellular Ca2+ from 10 to 110 mM stimulated K efflux by 3-fold in normal cells and 5.5-fold in dehydrated stomatocytes. This effect is due to internal Ca stimulating a passive K+ channel and has been termed the Gardos effect. Unexpectedly K+ efflux from overhydrated stomatocytes was unaffected by extracellular Ca concentration. However, the Ca ionophore (A23187) induced a marked efflux of K+ from overhydrated cells equal to that from normals or dehydrated stomatocytes. Thus overhydrated stomatocytes do possess a Ca-stimulated K+ channel but these cells seem impermeable to extracellular Ca ions. We conclude that overhydrated stomatocytes have a Ca permeability below that of control red cells despite Na and K fluxes which are 25-fold normal. This lack of correlation between Ca and Na permeabilities suggests that Ca enters red cells by a different pathway to the Teak’ for monovalent cations.

Stimulation of monovalent cation fluxes by electron donors in the human red cell membrane.

When human red cells are incubated at 37 degrees C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb+ (K+) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb+ (K+) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca2+ (Gardos effect). The electron donors require the presence of intracellular Ca2+ to be effective, but at levels that do not stimulate by themselves the fluxes of K+. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca2+ alone; antimycin, upcouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect. The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca2+ more effective to elicit rapid K+ movements. Plasma membrane oxidoreductase activities could be engaged in this change.

Cation Permeability Alterations During Sickling: Relationship to Cation Composition and Cellular Hydration of Irreversibly Sickled Cells

AbstractSickle erythrocytes (RBC) incubated under 100% nitrogen for 4 hr manifested marked Na gain with an equivalent K loss. There were no changes in cell total cation or water content under these conditions, and no irreversible sickle cells (ISC) were formed. In contrast, sickle RBC incubated for 24 hr under 100% nitrogen in a glucose-free Na medium containing calcium manifested marked ISC formation. ISC formed under these conditions also had elevated Na content, although K content was much more reduced, and consequently ISC were cation depleted and dehydrated. When sickle RBC were incubated 24 hr under 100% nitrogen in a glucose-free K medium containing calcium no ISC formed and there were no major changes in cation or water content. These studies indicate that the Na + K content and dehydration of ISC was not directly related to the increased cation permeability associated with sickling. Rather, the ISC changes appear to reflect the well-known Gardos effect (K and water loss occurring in ATP-depleted RBC incubated with calcium). In addition, these studies suggest that ISC formation per se is related to K and water loss, since no ISC were formed when ATP-depleted sickle RBC were deoxygenated in calcium-containing high-K media that prevented K loss and dehydration.

Cation permeability alterations during sickling: relationship to cation composition and cellular hydration of irreversibly sickled cells

Sickle erythrocytes (RBC) incubated under 100% nitrogen for 4 hr manifested marked Na gain with an equivalent K loss. There were no changes in cell total cation or water content under these conditions, and no irreversible sickle cells (ISC) were formed. In contrast, sickle RBC incubated for 24 hr under 100% nitrogen in a glucose-free Na medium containing calcium manifested marked ISC formation. ISC formed under these conditions also had elevated Na content, although K content was much more reduced, and consequently ISC were cation depleted and dehydrated. When sickle RBC were incubated 24 hr under 100% nitrogen in a glucose-free K medium containing calcium no ISC formed and there were no major changes in cation or water content. These studies indicate that the Na+K content and dehydration of ISC was not directly related to the increased cation permeability associated with sickling. Rather, the ISC changes appear to reflect the well-known Gardos effect (K and water loss occurring in ATP-depleted RBC incubated with calcium). In addition, these studies suggest that ISC formation per se is related to K and water loss, since no ISC were formed when ATP-depleted sickle RBC were deoxygenated in calcium-containing high-K media that prevented K loss and dehydration.

Enhanced sensitivity to calcium in duchenne muscular dystrophy

The ionophore A23187 causes an increase in the Ca content of human erythrocytes and a Ca-dependent increase in K efflux (Gardos effect). These changes are associated with a reduction in osmotic fragility and cell size. Treatment of erythrocytes from patients with Duchenne muscular dystrophy with A23187 results in 45Ca uptake comparable to that of erythrocytes from control subjects. However, the reduction in osmotic fragility and K content observed in dystrophic erythrocytes is twofold greater than in control erythrocytes. These results indicate that an alteration in the regulation of erythrocyte membrane function by Ca occurs in Duchenne muscular dystrophy. This alteration may be responsible for other changes in erythrocyte membrane properties observed in Duchenne muscular dystrophy.