Peter K. Lauf

A chloride dependent K+ flux induced by N-ethylmaleimide in genetically low K+ sheep and goat erythrocytes

In genetically low K+ but not in high K+ red cells of sheep and goat N-ethylmaleimide induced a ouabain insensitive K+ flux as measured by tracer influx or net efflux methods. The augmented K+ flux was observed in Cl− or Br− but not in NO3−, SO42− or PO42− media. The action of N-ethylmaleimide was distinct from that of parachloromercuribenzoate or its sulfonic acid derivative which increased both passive K+ and Na+ movements across the red cell membrane. The instantaneous selective action of N-ethylmaleimide suggests that sulfhydryl groups control a K+Cl− transport system which, associated with the low K+ gene, is apparently functionally silent in adult ruminant red cells.

K+:Cl− Cotransport: Sulfhydryls, divalent cations, and the mechanism of volume activation in a red cell

Upon exposure to moderately dilute, hyposmotic Na + media many animal cells acutely swell due to rapid water entry (osmotic phase) and subsequently regain their original volume, a process called regulatory volume decrease (RVD) [48, 49]. The phenomenon of RVD was observed first in certain bird red cells [48], and then in other cells, most notably in Ehrlich ascites tumor cells [42], fish and Amphiuma red cells [15-17, 53], lymphocytes [37, 84] and epithelial cells of the cortical collecting tubules of rabbits [20, 36] and of the gallbladder of Necturus [52, 83]. With some minor exception, the prime osmolytes extruded across the plasma membrane during RVD in hyposmotic Na + media are K (K +) and C1 (C1-) ions. The swiftness with which any of the above cells readjust their hyposmotically expanded volume back to the original volume depends largely on the transport modes utilized to lower intracellular KC1. For convenience, Fig. 1 depicts three thermodynamically possible and experimentally proven transport modes effecting RVD in a model cell suspended in hyposmotic Na § media. The comparatively slowest RVD (i.e., within 1 hr) is mediated by electroneutral K + : C1cotransport (Fig. 1A), as for example in fish red cells [11, 15, 53] and in Necturus gallbladder [52]. A much faster RVD (i.e., within 510 rain) is seen in Amphiuma red cells which possess a K+/H § antiport (Fig. 1B) in parallel with a

Thiol-dependent passive K/Cl transport in sheep red cells: I. Dependence on chloride and external K+[Rb+] ions

SummaryTreatment with 2mm N-ethylmaleimide (NEM) caused a marked increase in K+ permeability of low K+ but not of high K+ sheep red cells suspended in isosmotic Cl− media with 10−4m ouabain. The Na+ permeability was unaltered. Kinetic analysis by K+ efflux and K+ or Rb+ influx measurements suggests that NEM primarily increased the bidirectional fluxes of K+ and Rb+, since (a) no significant change in the apparent external affinities of these ions was found, and (b) below unity, the observed flux ratios were close to those calculated from the Ussing relationship. Replacement of Cl− by NO3− abolished the NEM-stimulated and reduced the basal K+ flux rates. Similarly, 10−3m furosemide inhibited Cl−-dependent K+ fluxes in both control and NEM-treated LK red cells. Exposure of LK cells to hyposmotic but not to hyperosmotic salt solutions increased the basal Cl− dependent K+ flux twofold as reported by Dunham and Ellory (J. Physiol. (London)318:511–530, 1981) but did not affect its fractional stimulation by NEM. The action of NEM is interpreted as a stimulation of a temperature-dependent and Cl−-requiring K+ transport pathway genetically preserved in adult LK but turned off in HK sheep red cells. In addition, common to both LK and HK sheep red cells was a basal K+ flux that operated in the presence of either Cl− or NO3−.

Evidence for chloride dependent potassium and water transport induced by hyposmotic stress in erythrocytes of the marine teleost,Opsanus tau

SummaryRed blood cells of the marine teleost,Opsanus tau (oyster toadfish), were characterized as to their normal hemoglobin, ion and water contents. Cells were exposed to ouabain containing, hyposmotic salt solutions (osmolarity reduced to 2/3 of normal) in which the cation or anion composition was varied. It was found that the initial cell volume expansion due to water influx was independent of the anion present. However, a secondary volume reduction was dependent on the presence of chloride or bromide anions. During volume reduction, cellular potassium and chloride ion contents fell by about equal amounts. Potassium loss was commensurate to the total amount of potassium ions detected extracellularly about 1.5h after the initial osmotic shock. No major changes were seen in the cellular sodium ion contents. When chloride ions within the cells and in the suspending medium were replaced by nitrate, iodide or thiocyanate, the cells failed to return to volumes close to those of isosmotically suspended controls, and the cellular potassium content also remained constant. In hypotonic potassium chloride the cells failed to extrude potassium chloride and water, and hence retained their expanded volume. Neither potassium loss nor volume decrease occurred in cells swollen in hypotonic sodium chloride media containing furosemide or 4,4′ diisothiocyano-2,2′-stilbene-disulfonic acid (DIDS). These two compounds are known inhibitors of monovalent cation cotransport and anion self exchange, respectively, in mammalian red cells. Hence toadfish red cells respond to osmotic swelling primarily by activation of an ouabain-insensitive, chloride dependent potassium transport system which is sensitive to inhibition by furosemide and DIDS.

Stimulation of active potassium transport in LK sheep red cells by blood group-L-antiserum

SummaryAnti-L serum prepared by immunization of a high-potassium-type (HK) (blood type MM) sheep with blood from a low-potassium-type (LK) (blood type ML) sheep contained an antibody which stimulated four- to sixfold K+-pump influx in LK (LL) sheep red cells. In long-termin vitro incubation experiments, LK sheep red cells sensitized with anti-L showed a net increase in K+ after two days of incubation at 37°C, whereas HK-nonimmune (NI)-serum-treated control cells lost K+. The antibody could be absorbed by LK (LL) sheep red cells but not by HK sheep red cells. Kinetic experiments showed that the concentration of external K+ ([K+]0) required to produce halfmaximum stimulation of the pump ([Na+]0=0, replaced by Mg++) was the same (0.25 mM) in L-antiserum-treated or untreated LK cells. LK cells with different [K+]i (Na+ replacement) were prepared by the p-chloromercuribenzene sulfonate (PCMBS) method. At [K+]0=5 mM, pump influx decreased as [K+]i increased from 1 to 70 mM in L-antiserum-treated LK cells, whereas LK cells treated with HK-NI-serum ceased to pump at [K+]i=35 mM. Exposure to anti-L serum produced an almost twofold increase in the number of pump sites of LK cells as measured by the binding of tritiated ouabain by LK sheep red cells. These findings indicate that the formation of a complex between the L-antigen and its antibody stimulates active transport in LK sheep red cells both by changing the kinetics of the pump and by increasing the number of pump sites.

Thiol-Dependent passive K/Cl transport in sheep red cells: IV. Furosemide inhibition as a function of external Rb+, Na+, and Cl−

SummaryThe effect of the loop diuretic furosemide (4-chloro-N-furfuryl-5-sulfamoyl-anthranilic acid) on the thiol-dependent, ouabain-insensitive K(Rb)/Cl transport in low K+ sheep red cells was studied at various concentrations of extracellular Rb+, Na+ and Cl−. In Rb+-free NaCl media, 2×10−3m furosemide inhibited only one-half of thiol-dependent K+ efflux. In the presence of 23mm RbCl, however, the concentration of furosemide to produce 50% K+ efflux inhibition (IC50) was 5×10−5m. In Rb+ containing NaCl media, the inhibitory effect of 10−3m furosemide was equal to that caused by NO3− replacement of Cl− in the medium. The apparent synergistic action of furosemide and external Rb+ on K+ efflux was also seen in the ouabain-insensitive Rb+ influx. A preliminary kinetic analysis suggests that furosemide binding alters both maximal K+(Rb+) transport and apparent external Rb+ affinity. In the presence of external Rb+, Na+ (as compared to choline) exerted a small but significant augmentation of the furosemide inhibition of K+(Rb+) fluxes. There was no effect of Cl− on the IC50 value of furosemide. As there is no evidence for coupled Na+K+ cotransport in low K+ sheep red cells, furosemide may modify thiol-dependent K+(Rb+/Cl flux or Rb+ (and to a slight degree Na+) modulate the effect of furosemide.

Thiol-dependent passive K+Cl- transport in sheep red blood cells: VI. Functional heterogeneity and immunologic identity with volume-stimulated K+(Rb+) fluxes.

SummaryOuabain-resistant (OR), volume-or N-ethylmaleimide (NEM)-stimulated K+(Rb+)Cl− fluxes were measured in low-K+ sheep red cells and found to be functionally separate but immunologically similar. In anisosmotic solutions both K+ effluxes and Rb+ influxes of NEM-treated and control cells were additive. In contrast to the NEM-stimulated K+Cl− flux, metabolic depletion did not reduce K+Cl− flux of normal or swollen cells. The anion preference of OR K+ efflux in NEM-treated cells was Br−>Cl−>HCO3−=F−≫I−=NO3−=CNS−, and hence consistent with a reported Br−>Cl−>NO3− sequence of the volume-dependent K+Cl− transport. Alloimmune anti-Ll antibodies known to decrease passive K+ fluxes in low K+ cells reduced by 51% both volume-and NEM-stimulated, furosemidesensitive Rb+Cl− fluxes suggesting their immunologic identity, a conclusion also supported by anti-L1 absorption studies. Since pretreatment with anti-L1 prevented the activation of Rb+ influx by NEM, and the impermeant glutathionmaleimide-I did not stimulate Rb+Cl− influx, the NEM reactive SH groups must be located apart from the L1 antigen either within the membrane or on its cytoplasmic face. A model is proposed consisting of a K+Cl− transport path(s) regulated by a protein with two functional subunits or domains; a chemically (Cs) and a volume (Vs)-stimulated domain, both interfacing with the L1 surface antigen. Attachment of alloanti-L1 from the outside reduces K+Cl− transport stimulated throughCs by NEM orVs by cell swelling.

Thiol-dependent passive K/Cl transport in sheep red cells: II. Loss of Cl− and N-ethylmaleimide sensitivity in maturing high K+ cells

SummaryA fraction of the passive, ouabain-insensitive K+ fluxes in mature low K+ (LK) but not in high K+ (HK) sheep red cells requires the presence of Cl− anions and can be stimulated by volume expansion (Dunham, P.B., Ellory, J.C.,J. Physiol. (London)318:511–530, 1981) or treatment with 2mm N-ethylmaleimide (NEM) (Lauf, P.K., Theg., B.E.,Biochem. Biophys. Res. Commun.92:1422–1428, 1980). In the present study it is shown that reticulocytes of both anemic LK and HK sheep possess the Cl−-dependent K+ transport system which subsequently remains functional in mature LK but not in HK red cells. Kinetically, Cl−-mediated K+ fluxes of reticulocytes of LK sheep are different from mature red cells only in theirVmax values as measured in Na+ or choline+ media, while there is an apparently much lower affinity for external K+ ions in reticulocytes of HK sheep. N-ethylmaleimide stimulated K+ transport in the reticulocytes of both sheep genotypes suspended in Cl− but failed to do so in NO3− media. The data are interpreted in terms of their biochemical, physiologic, and genetic implications for the HK/LK transition in sheep red cells.

Enzymatic modification of the L and M antigens in LK and HK sheep erythrocytes and their membranes : The action of neuraminidase and trypsin.

SummaryThis paper reports on the effect of two hydrolytic enzymes, neuraminidase and trypsin, on the interaction of blood group L-positive low-potassium-type (LK) and blood group M-positive high-potassium-type (HK) sheep red cells with their respective isoimmune antisera. It was found that treatment of LK and HK red cells with neuraminidase did not change the interaction of these cells with their homologous antibodies as measured by K+-pump flux, complement-mediated immune hemolysis and absorption of antibody. Similarly, trypsin pretreatment of LK and HK red cells did not interfere with the hemolytic action of anti-L and anti-M antibodies, respectively. In striking contrast, however, it was observed that pretreatment of LK cells with trypsin rendered these cells insensitive to the K+-pump stimulating antibody present in the anti-L serum.

The effect of anti-L on ouabain binding to sheep erythrocytes

SummaryBinding of3H-ouabain was studied in high potassium (HK) and low potassium (LK) sheep red cells. In particular, we investigated the effect of anti-L, an antibody raised in HK sheep against L-positive LK sheep red cells, on3H-ouabain binding and its relation to K+-pump flux inhibition in LK cells. HK cells were found to have about twice as many3H-ouabain binding sites and a higher association rate for3H-ouabain than homozygous LL-type LK cells. The number of3H-ouabain molecules bound to heterozygous LM-type LK cells is lower than that on LL cells, but the rate of ouabain binding is between that of HK and LL red cells. A close correlation was observed between the rates of3H-ouabain binding and fractional K+-pump inhibition. Exposure of LM and LL cells to anti-L did not affect the number of3H-ouabain molecules bound at saturation, but increased the rates of glycoside binding and K+-pump inhibition proportionately, so that for LK cells in the presence of anti-L, the rates of the two processes approximate those of HK cells. These data exclude the possibility that anti-L generates entirely new pump sites in LK sheep red cells, but suggest that the antibody increases the affinity of the existing Na+−K+ pumps for the glycoside.