Daniel C. Tosteson

Increased sodium-lithium countertransport in red cells of patients with essential hypertension.

This paper describes experiments showing that one of the pathways of sodium transport across the red-cell membrane, sodium-lithium countertransport, is faster in patients with essential hypertension than in control subjects. This transport system accepts only sodium or lithium and is not inhibited by ouabain. The maximum rate of transport shows inherited differences. The mean maximum rate of sodium-lithium countertransport was found to be 0.55 +/- 0.02 (mean +/- S.E.M.) mmol (liter of red cells X hour)(-1) in a group of 36 patients with essential hypertension and 0.24 +/- 0.02 in 26 control subjects (P less than 0.001). The first-degree relatives of eight patients with essential hypertension and 10 control subjects had mean maximum rates of sodium-lithium countertransport of 0.54 +/- 0.05 and 0.23 +/- 0.02, respectively. Five patients with secondary hypertension had normal mean maximum rates of sodium-lithium countertransport. The relation between heritability of red-cell sodium-lithium countertransport and essential hypertension should be investigated further.

Melittin lysis of red cells.

SummaryThis paper describes experiments designed to explore interactions between human red blood cell membranes and melittin, the main component of bee venom. We found that melittin binds to human red cell membranes suspended in isotonic NaCl at room temperature, with an apparent dissociation constant of 3×10−8m and maximum binding capacity of 1.8×107 molecules/cell. When about 1% of the melittin binding sites are occupied, cell lysis can be observed, and progressive, further increases in the fraction of the total sites occupied lead to progressively greater lysis in a graded manner. 50% lysis occurs when there are about 2×106 molecules bound to the cell membrane. For any particular extent of melittin binding, lysis proceeds rapidly during the first few minutes but then slows and stops so that no further lysis occurs after one hour of exposure of cells to melittin. The graded lysis of erythrocytes by melittin is due to complete lysis of some of the cells, since both the density and the hemoglobin content of surviving, intact cells in a suspension that has undergone graded melittin lysis are similar to the values observed in the same cells prior to the addition of melittin. The cells surviving graded melittin lysis have an increased Na and reduced K, proportional to the extent of occupation of the melittin binding sites. Like lysis, Na accumulation and K loss proceed rapidly during the first few minutes of exposure to melittin but then stops so that Na, K and hemoglobin content of the cells remain constant after the first hour. These kinetic characteristics of both lysis and cation movements suggest that melittin modifies the permeability of the red cell membrane only for the first few minutes after the start of the interaction. Direct observation of cells by Nomarsky optics revealed that they crenate, become swollen and lyse within 10 to 30 sec after these changes in morphology are first seen. Taken together, these results are consistent with the idea that melittin produces lysis of human red cells at room temperature by a colloid osmotic mechanism.

Outward sodium and potassium cotransport in human red cells

SummaryThis paper reports some kinetic properties of Na−K cotransport in human red cells. All fluxes were measured in the presence of 10−4 M ouabain. We measured Na and K efflux from cells loaded by the PCMBS method to contain different concentrations of these ions into a medium that contained neither Na nor K (MgCl2-sucrose substitution) in the absence and presence of furosemide. Furosemide inhibited 30–60% of the total efflux depending on the internal ion concentration and the individual subject. We took the furosemide-sensitive fluxes to be a measure of Na−K cotransport. The ratio of Na to K cotransport was 1 over the entire range of internal Na and K concentrations studied. When Na was substituted for K as the only internal cation, cotransport was maximally activated when the Na and K concentrations were between 20 and 90 mmol/liter cells. The concentration of internal Na required to produce half-maximal cotransport was about 13±4 mmol/liter cells (n=4), while the comparable concentration of K was somewhat lower. The activation curve was definitely sigmoid in character, suggesting that at least two Na ions are involved in the transport process. The maximum of Na−K cotransport was about 0.5±0.15 mmol/liter cells × hr (n=5); it had a flat maximum in the medium at about pH 7.0, decreasing in both the acid and alkaline sides. furosemide-resistant effluxes were found to be linear functions of internal Na and K concentrations and to yield rate coefficients of 0.019±0.002 hr−1 and 0.014±0.002 hr−1 (n=7), respectively. These values are of the same order of magnitude expected of ions moving across phospholipid bilayers.

Na Countertransport and Cotransport in Human Red Cells: Function, Dysfunction, and Genes in Essential Hypertension

We describe in this paper studies on the modes of operation of ouabain-insensitive sodium transport systems in red cells of normotensive and hypertensive patients. We have extensively investigated the properties of Na countertransport and cotransport in order to clarify whether they are two different proteins or one transport protein with two modes of operation. Several criteria of discrimination between the two pathways are described: They differ in their affinity for Na and Li, sensitivity to several inhibitors, changes in cell volume, and chloride replacement by nitrate. We propose that there are two different transport systems. We have found elevated countertransport in red cells of hypertensive patients in France and in the United States. However, the cotransport system was found elevated in patients in Boston but reduced in patients in Paris. Studies of the modes of operation of the Na-K cotransport system indicate that it can promote K accumulation using an inward sodium gradient. This mode might be more efficient than Na extrusion at the physiological level of Na and K gradients. We interpret our findings of elevated Na-K cotransport in American hypertensive patients as an increased number of transport units functioning as K accumulators. It remains to be determined whether the reduced affinity for internal sodium of the outward cotransport is a defective outward cotransport or else a modulation of this transport system to favor K accumulation.

Effect of volume changes on ouabain-insensitive net outward cation movements in human red cells

SummaryThe effect of cell volume changes in human red cells on ouabain-insensitive net outward cation movements through 1) the Na−K and Li−K cotransport, 2) the Li−Na counter-transport system and 3) the furosemide-insensitive Na, K and Li pathway was studied. Cell volume was altered by changing a) the internal cation content (isosmotic method) or b) the external osmolarity of the medium (osmotic method). Na−K and Li−K cotransport were measured as the furosemide-sensitive Na or Li and K efflux into (Na, Li and K)-free (Mg-sucrose replacement) medium from cells loaded to contain approximately equal concentrations of Na and K, or a constant K/Li concentration ratio of 9∶1, respectively. Li−Na countertransport was assayed as the Na-stimulated Li efflux from Li-loaded cells and net furosemide-insensitive outfluxes in (Na, Li and K)-free media containing 1mm furosemide. Swelling of cells by the isosmotic, but not by the osmotic method reduced furosemide-sensitive Na and Li but not K efflux by 80 and 86%, respectively. Changes in cell volume by both methods had no effect on Li−Na countertransport. The effects of cell volume changes were measured on the rate constants of ouabain- and furosemide-insensitive cation fluxes and were found to be complex. Isosmotic shrinkage more than doubled the rate constants of Na and Li efflux but did not affect that of K efflux. Osmotic shrinkage increased the K efflux rate constant by 50% only in cells loaded for countertransport. Isosmotic cell swelling specifically increased the K+ efflux rate constants both in cells loaded for cotransport and countertransport assays while no effect was observed in cells swollen by the osmotic method. Thus, the three transport pathways responded differently to changes in cell volume, and, furthermore, responses were different depending on the method of changing cell water content.

Effects of palytoxin on cation occlusion and phosphorylation of the (Na+, K+)-ATPase

Palytoxin (PTX) inhibits the (Na+ + K+)-driven pump and simultaneously opens channels that are equally permeable to Na+ and K+ in red cells and other cell membranes. In an effort to understand the mechanism by which PTX induces these fluxes, we have studied the effects of PTX on: 1) K+ and Na+ occlusion by the pump protein; 2) phosphorylation and dephosphorylation of the enzyme when a phosphoenzyme is formed from ATP and from Pi; and 3) p-nitro phenyl phosphatase (p-NPPase) activity associated with the (Na+,K+)-ATPase. We have found that palytoxin 1) increases the rate of deocclusion of K+(Rb+) in a time- and concentration-dependent manner, whereas Na+ occluded in the presence of oligomycin is unaffected by the toxin; 2) makes phosphorylation from Pi insensitive to K+, and 3) stimulates the p-NPPase activity. The results are consistent with the notion that PTX produces a conformation of the Na+,K+-pump that resembles the one observed when ATP is bound to its low-affinity binding site. Further, they suggest that the channels that are formed by PTX might arise as a consequence of a perturbation in the ATPase structure, leading to the loss of control of the outside “gate” of the enzyme and hence to an uncoupling of the ion transport from the catalytic function of the ATPase.

Cation movements across mouse red blood cells

This paper describes some features of the Na and K transport systems in red cells obtained from B10.A mice. When mouse erythrocytes were incubated in a plasma-like control medium, the scillaren-sensitive Na efflux was 3.6 +/- 0.4 mmol/l red blood cells per h while the scillaren-sensitive K influx was 3.1 +/- 0.3, values not significantly different from ech other. Scillaren had no significant effect on either Na influx or K efflux. There was a large (approx. 3 mmol/l red blood cells per h) scillaren-sensitive, Na-Na exchange diffusion component present under K-free conditions. When K was present in the incubation medium, this exchange system was suppressed.

Selective ion binding and membrane activity of synthetic cyclopeptides.

Four cyclic peptides related to the membrane-active complexones PV, cyclo-(L-Pro-Lval-D-Pro-D-Val)3, and valinomycin were synthesized: (1) cyclo-(L-Pro-L-Ala-D-Val)3 or PVPA, (2) cyclo-(L-Ala-L-Val-D-Pro-D-Val)3 or PVAV, (3) cyclo-(L-Pro-L-Val-D-Pro-D-Val)2-L-Pro-D-Val or PV-10, (4) cyclo-(L-Pro-L-Val-D-Pro-D-Val)2 or PV-8. In a two-phase extraction assay the affinity of PV and PVPA for alkali picrates was about three orders or magnitude greater than that of valinomycin. It was about equal to valinomycin for PVAV and much lower for PV-10 and PV-8. PV, PVPA and PVAV showed a selectivity sequence similar to that of valinomycin, namely K+ approximately Rb+ greater than Cs+ greater than Na+ greater than Li+. In the series PV, PV-10, PV-, the preference for K+ over Na+ was 700, 5 and less than 1, respectively. Thus, it was possible to reverse the selectivity of PV for K+ over Na+ by reducing the ring size from 12 to 8 amino acid residues. In sheep red cell lipid bilayer membranes PVPA increased the membrane conductance significantly in the presence of either KCl or NaCl but it was less potent than PV. PV-10, PV-8 and PVAV on the other hand were ineffective in this assay. The inactivity of PVAV as a potassium carrier in membrane was in contrast to its high affinity for potassium picrate in two-phase assays. Such a behaviour may be observed of a compound that has too low an aqueous cation binding constant to use the solution-complexation mechanism of PV (Davis et al. (1976) Biochemistry 15, 768--774 and Pinkerton et al. (1969) Biochem. Biophys. Res. Commun. 35, 512--518) and too slow binding and release kinetics to use the interfacial-complexation mechanism of valinomycin.

Interaction of palytoxin with red cells: Structure-function studies

Palytoxin (PTX), a potent toxin isolated from the marine soft coral Palythoa tuberculosa increases the cationic permeability of red cell membranes and inhibits the (Na+,K+)-activated ATPase, effects that are completely reversed by ouabain. It has also been shown to compete with ouabain for a binding site and it has been suggested that it binds to the Na+,K(+)-pump molecule in cells. In a search for analogues of PTX that would bind covalently and could thus be used to identify and characterize the binding site, we have used compounds which differed from PTX at one end or at both ends simultaneously. In order to determine whether these derivatives could be used to replace palytoxin, we tested their potency to induce an increased cation flux, complete with ouabain for its binding site, and inhibit the isolated, purified (Na+,K(+)-ATPase). The results obtained indicate that departures from the PTX structure at one end or at both ends simultaneously substantially decrease the ability of the compounds to increase the cationic permeability of red blood cells and to inhibit the (Na+,K(+)-ATPase). These actions were found to be completely reversed by ouabain, but the analogues are two to three orders of magnitude less potent than PTX in competing with ouabain for its binding site. These results suggest that both ends of the palytoxin molecule participate in the interactions of the toxin with its receptor and that modifications in these regions of the molecule produce significant alterations in its binding and subsequent action on red cell membranes.

Effects of pH, potential, chloride and furosemide on passive Na+ and K+ effluxes from human red blood cells

SummaryOuabain-resistant effluxes from pretreated cells containing K+/Na+=1.5 into K+ and Na+ free media were measured.Furosemide-sensitive cation effluxes from cells with nearly normal membrane potential and pH were lower in NO3− media than in Cl− media; they were reduced when pH was lowered in Cl− media. When the membrane potential was positive inside furosemide increased the effluxes of Na+ and K+ (7 experiments). With inside-positive membrane potential thefurosemideinsensitive effluxes were markedly increased, they decreased with decreasing pH at constant internal Cl− and also when internal Cl− was reduced at constant pH. The correlation between cation flux and the membrane potential was different for cells with high or low internal chloride concentrations. The data with chloride≧47mm showed a better fit with the single-barrier model than with the infinite number-of-barriers model. With low chloride no significant correlation between flux and membrane potential was found. The data are not compatible with pure independent diffusion of Na+ and K+ in the presence of ouabain and furosemide.