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Abstract  Red blood cells (RBCs) from patients with sickle cell disease (SCD) lyse in deoxygenated isosmotic non-electrolyte solutions. Haemolysis has features which suggest that it is linked to activation of the pathway termed Psickle. This pathway is usually described as a non-specific cationic conductance activated by deoxygenation, HbS polymerisation and RBC sickling. The current work addresses the hypothesis that this haemolysis will provide a novel diagnostic and prognostic test for SCD, dependent on the altered properties of the RBC membrane resulting from HbS polymerisation. A simple test represented by this haemolysis assay would be useful especially in less affluent deprived areas of the world where SCD is most prevalent. RBCs from HbSS and most HbSC individuals showed progressive lysis in deoxygenated isosmotic sucrose solution at pH 7.4 to a level greater than that observed with RBCs from HbAS or HbAA individuals. Cytochalasin B prevented haemolysis. Haemolysis was temperature- and pH-dependent. It required near physiological temperatures to occur in deoxygenated sucrose solutions at pH 7.4. At pH 6, haemolysis occurred even in oxygenated samples. Haemolysis was reduced in patients on long-term (>5 months) hydroxyurea treatment. Several manoeuvres which stabilise soluble HbS (aromatic aldehydes o-vanillin or 5-hydroxymethyl, and urea) reduced haemolysis, an effect not due to increased oxygen affinity. Conditions designed to elicit HbS polymerisation in cells from sickle trait patients (deoxygenated hyperosmotic sucrose solutions at pH 6) supported their haemolysis. These findings are consistent with haemolysis requiring HbS polymerisation and support the hypothesis that this may be used as a test for SCD.

The abnormally high cation permeability in red blood cells (RBCs) from patients with sickle cell disease (SCD) occupies a central role in pathogenesis. Sickle RBC properties are notably heterogeneous, however, thus limiting conventional flux techniques that necessarily average out the behaviour of millions of cells. Here we use the whole-cell patch configuration to characterise the permeability of single RBCs from patients with SCD in more detail. A non-specific cation conductance was reversibly induced upon deoxygenation and was permeable to both univalent (Na+, K+, Rb+) and also divalent (Ca2+, Mg2+) cations. It was sensitive to the tarantula spider toxin GsMTx-4. Mn2+ caused partial, reversible inhibition. The aromatic aldehyde o-vanillin also irreversibly inhibited the deoxygenation-induced conductance, partially at 1mM and almost completely at 5mM. Nifedipine, amiloride and ethylisopropylamiloride were ineffective. In oxygenated RBCs, the current was pH sensitive showing a marked increase as pH fell from 7.4 to 6, with no change apparent when pH was raised from 7.4 to 8. The effects of acidification and deoxygenation together were not additive. Many features of this deoxygenation-induced conductance (non-specificity for cations, permeability toCa2+ andMg2+, pH sensitivity, reversibility, partial inhibition by DIDS and Mn2+) are shared with the flux pathway sometimes referred to as Psickle. Sensitivity to GsMTx-4 indicates its possible identity as a stretch-activated channel. Sensitivity to o-vanillin implies that activation requires HbS polymerisation but since the conductance was observed in whole-cell patches, results suggest that bulk intracellular Hb is not involved; rather a membrane-bound subfraction is responsible for channel activation. The ability to record P(sickle)-like activity in single RBCs will facilitate further studies and eventual molecular identification of the pathway involved.

K(+)-Cl(-) cotransporters (KCCs) play a fundamental role in epithelial cell function, both in the context of ionic homeostasis and also in cell morphology, cell division and locomotion. Unlike other ubiquitously expressed KCC isoforms, expression of KCC2 is widely considered to be restricted to neurons, where it is responsible for maintaining a low intracellular chloride concentration to drive hyperpolarising postsynaptic responses to the inhibitory neurotransmitters GABA and glycine. Here we report a novel finding that KCC2 is widely expressed in several human cancer cell lines including the cervical cancer cell line (SiHa). Membrane biotinylation assays and immunostaining showed that endogenous KCC2 is located on the cell membrane of SiHa cells. To elucidate the role of KCC2 in cervical tumuorigenesis, SiHa cells with stable overexpression or knockdown of KCC2 were employed. Overexpression of KCC2 had no significant effect on cell proliferation but dramatically suppressed cell spreading and stress fibre organization, while knockdown of KCC2 showed opposite effects. In addition, insulin-like growth factor 1 (IGF-1)-induced cell migration and invasiveness were significantly increased by overexpression of KCC2. KCC2-induced cell migration and invasion were not dependent on KCC2 transport function since overexpression of an activity-deficient mutant KCC2 still increased IGF-1-induced cell migration and invasion. Moreover, overexpression of KCC2 significantly diminished the number of focal adhesions, while knockdown of KCC2 increased their number. Taken together, our data establish that KCC2 expression and function are not restricted to neurons and that KCC2 serves to increase cervical tumourigenesis via an ion transport-independent mechanism.

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum-infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole-cell configuration of the patch-clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole-cell recordings in malaria-infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4-fold and inward currents by approximately 2-fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.

A recent study on malaria-infected human red blood cells (RBCs) has shown induced ion channel activity in the host cell membrane, but the questions of whether they are host- or parasite-derived and their molecular nature have not been resolved. Here we report a comparison of a malaria-induced anion channel with an endogenous anion channel in Plasmodium falciparum-infected human RBCs. Ion channel activity was measured using the whole-cell, cell-attached and excised inside-out configurations of the patch-clamp method. Parasitised RBCs were cultured in vitro, using co-cultured uninfected RBCs as controls. Unstimulated uninfected RBCs possessed negligible numbers of active anion channels. However, anion channels could be activated in the presence of protein kinase A (PKA) and ATP in the pipette solution or by membrane deformation. These channels displayed linear conductance (~15 pS), were blocked by known anion channel inhibitors and showed the permeability sequence I(-) > Br(-) > Cl(-). In addition, in less than 5 % of excised patches, an outwardly rectifying anion channel (~80 pS, outward conductance) was spontaneously active. The host membrane of malaria-infected RBCs possessed spontaneously active anion channel activity, with identical conductances, pharmacology and selectivity to the linear conductance channel measured in stimulated uninfected RBCs. Furthermore, the channels measured in malaria-infected RBCs were shown to have a low open-state probability (P(o)) at positive potentials, which explains the inward rectification of membrane conductance observed when using the whole-cell configuration. The data are consistent with the presence of two endogenous anion channels in human RBCs, of which one (the linear conductance channel) is up-regulated by the malaria parasite P. falciparum.

1. This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells. 2. Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. 3. The activation of swelling-activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling-activated Cl- channel. 4. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl- channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase. 5. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and down-regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen-activated/ERK-activating kinase (MEK) activity. 6. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume-regulatory mechanism of human cervical cancer cells.

This study investigated the volume-regulated anion channel (VRAC) of human cervical cancer SiHa cells under various culture conditions, testing the hypothesis that the progression of the cell cycle is accompanied by differential expression of VRAC activity. Exponentially growing SiHa cells expressed VRACs, as indicated by the presence of large outwardly rectifying currents activated by hypotonic stress with the anion permeability sequence I- > Br- > Cl-. VRACs were potently inhibited by tamoxifen with an IC50 of 4.6 [mu]M. Fluorescence-activated cell sorting (FACS) experiments showed that 59 +/- 0.5, 5 +/- 0.5 and 36 +/- 1.1% of unsynchronized, exponentially growing cervical cancer SiHa cells were in G0/G1, S and G2/M stage, respectively. Treatment with aphidicolin (5 [mu]M) arrested 88 +/- 1.4% of cells at the G0/G1 stage. Arrest of cell growth in the G0/G1 phase was accompanied by a significant decrease of VRAC activity. The normalized hypotonicity-induced current decreased from 48 +/- 5.2 pA pF-1 at +100 mV in unsynchronized cells to 15 +/- 2.6 pA pF-1 at +100 mV in aphidicolin-treated cells. After removal of aphidicolin, culturing in medium containing 10% fetal calf serum triggered a rapid re-entry into the cell cycle and a concomitant recovery of VRAC density. Pharmacological blockade of VRACs by tamoxifen or NPPB caused proliferating cervical cancer cells to arrest in the G0/G1 stage, suggesting that activity of this channel is critical for G1/S checkpoint progression. This study provides new information on the functional significance of VRACs in the cell cycle clock of human cervical cancer cells.

The active Ca2+ transport properties of malaria-infected, intact red blood cells are unknown. We report here the first direct measurements of Ca2+ pump activity in human red cells infected with Plasmodium falciparum, at the mature, late trophozoite stage. Ca2+ pump activity was measured by the Co2+-exposure method adapted for use in low-K+ media, optimal for parasitised cells. This required a preliminary study in normal, uninfected red cells of the effects of cell volume, membrane potential and external Na+/K+ concentrations on Ca2+ pump performance. Pump-mediated Ca2+ extrusion in normal red cells was only slightly lower in low-K+ media relative to high-K+ media despite the large differences in membrane potential predicted by the Lew-Bookchin red cell model. The effect was prevented by clotrimazole, an inhibitor of the Ca2+-sensitive K+ (KCa) channel, suggesting that it was due to minor cell dehydration. The Ca2+-saturated Ca2+ extrusion rate through the Ca2+ pump (Vmax) of parasitised red cells was marginally inhibited (2-27 %) relative to that of both uninfected red cells from the malaria-infected culture (cohorts), and uninfected red cells from the same donor kept under identical conditions (co-culture). Thus, Ca2+ pump function is largely conserved in parasitised cells up to the mature, late trophozoite stage. A high proportion of the ionophore-induced Ca2+ load in parasitised red cells is taken up by cytoplasmic Ca2+ buffers within the parasite. Following pump-mediated Ca2+ removal from the host, there remained a large residual Ca2+ pool within the parasite which slowly leaked to the host cell, from which it was pumped out.

1. The sodium-dependent amino acid transport systems responsible for proline, glycine and glutamine transport, together with the sodium-independent systems for leucine and tryptophan, have been investigated in isolated bovine chondrocytes by inhibition studies and ion replacement. Each system was characterized kinetically. 2. Transport via system A was identified using the system-specific analogue alpha-methylaminoisobutyric acid (MeAIB) as an inhibitor of proline, glycine and glutamine transport. 3. Uptake of proline, glycine and glutamine via system ASC was identified by inhibition with alanine or serine. 4. System Gly was identified by the inhibition of glycine transport with excess sarcosine (a substrate for system Gly) whilst systems A and ASC were inhibited. This system, having a very limited substrate specificity and tissue distribution, was also shown to be Na+ and Cl- dependent. Evidence for expression of the system Gly component GLYT-1 was obtained using the reverse transcriptase-polymerase chain reaction (RT-PCR). 5. System N, also of narrow substrate specificity and tissue distribution, was shown to be present in chondrocytes. Na+-dependent glutamine uptake was inhibited by high concentrations of histidine (a substrate of system N) in the presence of excess MeAIB and serine. 6. System L was identified using the system specific analogue 2-aminobicyclo(2,2, 1)heptane-2-carboxylic acid (BCH) and D-leucine as inhibitors of leucine and tryptophan transport. 7. The presence of system T was tested by using leucine, tryptophan and tyrosine inhibition. It was concluded that this system was absent in the chondrocyte. 8. Kinetic analysis showed the Na+-independent chondrocyte L system to have apparent affinities for leucine and tryptophan of 125 +/- 27 and 36 +/- 11 microM, respectively. 9. Transport of the essential amino acids leucine and tryptophan into bovine chondrocytes occurs only by the Na+-independent system L, but with a higher affinity than the conventional L system.

1. K+ influx and efflux were measured in normal (HbA) and sickle (HbS) red blood cells to investigate the interaction of swelling, H+ ions and urea with O2 (0 to 150 mmHg O2) in the presence of ouabain and bumetanide (both 100 microM). 2. In HbA cells, K+-C1- cotransport was O2 dependent. At low oxygen tensions (PO2s) the transporter was inactive and refractory to low pH, swelling or urea. 3. C1--independent K+ influxes in sickle cells were elevated at low PO2s, as previously reported. C1--dependent K+ influxes were large at both high and low PO2s, whether stimulated by swelling, H+ ions or urea. In the absence of O2, C1--dependent K+ influxes were similar in magnitude to those measured at high PO2s. The minimum for C1--dependent K+ influx was observed at PO2s of about 40-70 mmHg. 4. K+ efflux from HbS cells was stimulated by the addition of urea (500 mM). The rate constants were of similar magnitude whether measured at high PO2 or in the absence of O2, and were predominantly C1- dependent under both conditions. 5. In HbS red blood cells, reduction of extracellular Ca2+, addition of 1 mM Mg2+ or nitrendipine (10 microM) to the saline had no effect. Inhibitors of K+-C1- cotransport, [(dihydroindenyl)oxy] alkanoic acid (DIOA; 100 microM) or calyculin A (0.1 microM), inhibited influxes by a similar magnitude to C1- substitution. 6. Results are significant for the pathophysiology of sickle cell disease. Low pH and urea are able to stimulate KC1 loss from sickle cells, leading to cellular dehydration, even in regions of low PO2.

1. Pressure induced a 4- to 5-fold stimulation of the residual (i.e. oubain-bumetanide insensitive) 86Rb+ influx across the human red cell membrane. This enhancement showed a broad pHo dependence with a maximum stimulation around pHo 7. 2. At atmospheric pressure, the protein kinase inhibitors staurosporine and chelerythrine stimulated a normally silent component of 86Rb+ influx in a dose-dependent manner with a half-maximum stimulatory concentration at about 550 nM and 140 microM, respectively. The component stimulated by staurosporine was entirely Cl- dependent, but part of the chelerythrine effect was Cl- independent. 3. Staurosporine (3 microM), chelerythrine (200 microM) and N-ethylmaleimide (1 mM) stimulated further the increased residual 86Rb+ influx in cells at high pressure. 4. The serine/threonine protein phosphatase inhibitors okadaic acid, cantharidin and calyculin A inhibited the stimulatory pressure effect in a dose-dependent manner with half-maximum inhibitory concentrations of 70 nM, 2.5 microM and 3.3 nM, respectively. In contrast, deltamethrin, a specific protein phosphatase type 2B inhibitor, did not affect the stimulation by pressure, up to a concentration of 10 microM. 5. Decreasing the internal ionized magnesium concentration ([Mg2+]i) with A23187 and EDTA stimulated the increased residual 86Rb+ influx in cells at high pressure. On the other hand, increasing the [Mg2+]i nearly abolished the stimulatory pressure effect. 6. Decreasing the [Mg2+]i produced a marked change in the pHo dependence curve, with a linear increase of the 86Rb+ influx at higher pHo values. 7. We demonstrate that high pressure stimulates the normally silent component of 86Rb+ influx by modifying the phosphorylation/dephosphorylation ratio of the KCl cotransporter.

1. Suspending human red blood cells in isotonic sucrose (low ionic strength, LIS) medium induces a significant increase in membrane transport of glutamine, glutamate, lactate, histidine, taurine, glycine, serine, choline and carnitine but not sorbitol or sucrose. 2. Progressive lowering of ionic strength by sucrose or NaCl replacement gave a similar activation profile for taurine influx as found earlier for residual K+(86Rb+) flux. 3. The induced taurine transport could be measured as enhanced influx and efflux. Influx was linear with external concentration up to 10 mM, largely insensitive to alteration in cell volume, and did not vary with red blood cell age. 4. Unlike previous results for residual K+ transport, altering transmembrane potential with gluconate or glucuronate media led to an increase in taurine influx similar to that observed in LIS media. Varying medium pH confirmed the effect was not due to alteration in pH. 5. The LIS-induced flux was sensitive to a variety of 'classical' anion transport inhibitors in the order of potency DNDS, DIDS, NPPB, DIOA, niflumic acid, furosemide (frusemide), glibenclamide, nitrendipine and bumetanide. 6. The taurine flux showed a temperature dependence similar to that of the LIS-induced residual K+ flux. High hydrostatic pressure (40 MPa), however, inhibited taurine flux but stimulated residual K+ influx in LIS media. 7. A significant enhanced taurine flux could be demonstrated in red blood cells of other species, including horse, cattle, pig and high and low potassium type sheep. 8. It is concluded that lowering ionic strength activates a transport pathway for organic molecules sharing some similarities with background Cl- channels and LIS-induced residual K+ fluxes. In the latter context, however, there are certain significant differences (effect of transmembrane potential; volume; pressure sensitivity; species distribution) which may be important, and the unequivocal identity of the two transport processes remains to be confirmed.

1. The effect of low ionic strength media on the residual, i.e. (ouabain + bumetanide + Ca2+)-insensitive, K+ influx was characterized in human red blood cells. 2. This K+ flux was enhanced significantly in isotonic solutions of low ionic strength using sucrose to maintain constant osmolarity. This effect was found for fresh red blood cells as well as for stored (bank) red blood cells. However, the absolute magnitude of K+ influx in solutions of low ionic strength was halved for stored red blood cells. 3. Anion replacement of Cl- by CH3SO4- did not affect residual K+ fluxes, showing that Cl- -dependent transport pathways (e.g. the KCl co-transporter) are not involved in the low ionic strength effect. 4. The enhanced K+ influx in low ionic strength media was reversible when the cells were resuspended in a solution of physiological ionic strength. 5. K+ influx measured in light and dense fractions of erythrocytes (separated by centrifugation and corresponding to samples enriched with either 'young' or 'mature' red cells) showed that the low ionic strength effect does not change markedly with cell age. 6. Low ionic strength media elevated residual, i.e. (ouabain + bumetanide + Ca2+)-insensitive, influx of both K+ and Na+ by about the same amount. In both cases the flux was linear with concentration in the range investigated (0.25-10 mM). No significant increase in the uptake of the cations Ca2+ and lysine in low ionic strength solutions could be found. 7. In CH3SO4- -containing solutions of physiological ionic strength the residual K+ influx was almost independent of cell volume, whereas this flux in CH3SO4- -containing solutions of low ionic strength declined as cell volume was increased. 8. K+ flux measurements in solutions of different external pH, where NaCl was replaced by sodium gluconate or sodium glucuronate, showed that the reduced ionic strength is of more importance for the enhanced residual K+ influx than the changed transmembrane potential or the changed intracellular pH. However, a small pH dependence could be found, the K+ flux passing through a minimum around pHi 7.3. 9. Hydrostatic pressure enhanced the residual K+ flux in media of low ionic strength synergistically, so that very large fluxes (greater than 10 mmol (1 cells)-1 h-1) were obtained at 40 MPa. The apparent activation volumes (delta V*) for the pressure-sensitive K+ flux were -108 and -69 ml mol-1 in low ionic strength or physiological ionic strength solutions respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Thallium (Tl) inhibits the ouabain-sensitive K influx in human red cells in high-Na medium. At 1 mM external K concentration [K(o)], the ouabain-sensitive K influx decreases steadily with increasing Tl concentration, up to 0.9 mM outside; at 0.17 mM-K(o), however, Tl stimulates the ouabain-sensitive K influx below 0.1 mM-Tl(o) and inhibits it at higher concentrations.2. In a K-free medium in which all except 5 mM-Na is replaced by choline, and into which red cells show zero control ouabain-sensitive Na efflux, Tl is able to support ouabain-sensitive Na efflux up to 2.1 m-mole/l. cells.hr following a sigmoid activation curve which is half-maximal between 0.03 and 0.05 mM-Tl(o) and that follows two-site kinetics up to 0.1 mM-Tl(o). Beyond 0.15 mM-Tl(o), the Tl-activated ouabain-sensitive Na efflux attained is inhibited slightly.3. When the ouabain-sensitive Na efflux is measured at 5 mM-Na(o) and 5 mM-K(o), increasing concentrations of Tl have little effect on it, 0.9 mM-Tl(o) inhibiting by some 14%; in similar conditions, the ouabain-sensitive K influx is inhibited by about 40%.4. The dependence of ouabain-sensitive K influx on external K concentration at 5 mM-Na(o), which follows a slightly sigmoid curve in the absence of Tl, changes to hyperbolic at 0.06 mM-Tl(o) at the same time that ouabain-sensitive K influx is inhibited. The fitted V(max) values for ouabain-sensitive K influx are the same in the presence and in the absence of 0.06 mM-Tl(o).5. In high-Na cells, loaded by nystatin treatment, the ouabain-sensitive K influx measured at 0.2 mM-Na(o) follows a hyperbolic curve between 0.05 and 0.4 mM-K(o), and is inhibited by Tl in a strictly competitive fashion.6. The effects of Tl on ouabain-sensitive Na efflux and ouabain-sensitive K influx are interpreted in terms of a high-affinity substitution for K at the external K sites of the Na pump and suggest that in human red cells Tl can be actively transported inwards in exchange for internal Na.7. Thallium can inhibit about 25% of the ouabain-insensitive Na efflux into 5 mM-Na(o) and part of this inhibition occurs with a high Tl-affinity; the ouabain-insensitive K influx is inhibited by Tl both in high-Na and in 5 mM-Na medium, but with a different concentration dependence than the ouabain-insensitive Na efflux.

1. Glycine transport in human erythrocytes was resolved into five separate components of uptake. The first and major component of uptake was transport by a high-affinity (apparent Km 25 microM) Na+- and Cl- -dependent system. This system was specific for glycine, sarcosine and proline; Br- but not I- was able to substitute for Cl-. Uptake by this route was inhibited less than 20% by the loop diuretics, bumetanide and furosemide (10(-4) M), suggesting that it was distinct from the Cl- -dependent system responsible for Na+/K+ transport. Its properties closely resembled those of the gly transport system described previously in avian erythrocytes. 2. The second uptake route was transport by the Na+-dependent, Cl- -independent small neutral amino acid transport system (designated ASC). Neither NA+-dependent uptake route was present in sheep erythrocytes. 3. Two Na+-independent uptake mechanisms were also identified; first, uptake by the amino acid transport system (designated L), and secondly, SITS-sensitive uptake by the anion-exchange (band 3) transport mechanism (SITS is 4-acetamido-4'-iso-thiocyanatostilbene-2,2'-disulphonic acid, an effective inhibitor of anion transport by this route). Uptake by the latter route was increased markedly when fluxes were measured in isotonic SO42- medium or when the pH was increased. 4. At 0.2 mM extracellular glycine, the relative contributions of each of these uptake routes to the total glycine flux were 42, 11, 15 and 16% for the gly, ASC, L and band 3 systems, respectively. 5. Finally, there was a residual Na+-independent component of glycine uptake which contributed 16% of the total flux. With the exception of the gly system, all uptake routes showed a linear concentration dependence up to 2 mM-glycine.

1. The activation by Na ions and the effect of the anti-L antibody on the sodium pump of low-potassium type (LK) erythrocytes, have been studied by measuring ouabain-sensitive ATPase activity of red cell membranes of LK goats. The experimental data were first corrected for incomplete occupation of the external K sites of the pump, using a saturation function obtained from influx experiments.2. Double-reciprocal plots of the corrected rates against Na concentration at various fixed K concentrations, yield a pattern of competitive K inhibition when it is assumed that three equivalent sodium sites take part in the internal activation of LK-(Na+K)-ATPase. The dissociation constant of Na at each site (K(m)) lies between 10 and 20 mM and that of K as competitive inhibitor (K(i)), between 1.5 and 4.5 mM.3. The maximal rate of hydrolysis of LK goat (Na + K)-ATPase is not different from those usually obtained with the high-potassium type (HK) red cell enzyme. Then, the low pumping rate of LK erythrocytes in physiological conditions is only reflecting the poor Na affinity, both absolute and relative, at the internal Na sites of their sodium pumps.4. The stimulation of the ouabain-sensitive ATPase activity by sensitization of the membranes with anti-L serum, is mediated by a threefold reduction of the K(m)/K(i) ratio at each site. K(m) decreases by a factor of 10, but there is also a smaller diminution of K(i). The maximal rate of hydrolysis, however, is unchanged by the anti-L treatment. The least-squares fitting of the pooled data by the rate equation, converges better with less than three and more than two equivalent sodium sites.5. The affinity sequence at two external K sites of the LK goat erythrocyte sodium pump, determined in the presence of 100 mM external Na, is Rb > K > Cs. It is obtained from the concentration dependence in influx experiments, and is the same as reported for human red cells.6. Cubic-root Dixon plots of the corrected ouabain-sensitive ATPase activity against the concentration of K and its congeners, show the sequence Tl > K > Rb > Na > Cs for the affinities at the internal cation sites of the LK sodium pump. Anti-L treatment decreases the relative magnitude of Na and Cs selectivities, it being not certain whether a Rb-Na transition then occurs.7. The results are discussed in terms of possible mechanisms whereby the sodium pump of LK and HK red cells may adjust the properties of their cation sites upon translocation of monovalent cations.

The influx and efflux of Na and K across the human red cell membrane by the bumetanide-sensitive (Na-K co-transport) and residual (ouabain- and bumetanide-insensitive) routes were inhibited by increasing concentrations of external Mg. Ca, Sr, Ba, Mn and Co also inhibited bumetanide-sensitive and residual K transport. External Mg inhibited choline uptake and the Na-dependent fractions of L-alanine and L-serine uptakes. External Mg reduced the maximal rate (app. Vmax) but not the affinity (app. Km) of the bumetanide-sensitive K and Na influxes when they were measured as functions of external K and Na respectively. The inhibitory effect of Mg was not due to a small reduction in zeta potential since much larger reductions in zeta potential produced by neuraminidase did not affect transport. Internal Mg stimulated the ouabain-sensitive K influx but inhibited the co-transport and residual components of K influx. Bumetanide was a poor co-transport inhibitor in red cells pre-treated with A23187 and EDTA. It was concluded that the inhibitory effects of external Mg were probably not due to changes in the ionic composition of the diffuse double layer adjacent to the cell membrane. Mg and other divalent cations should not be used as 'inert' ionic substitutes in human red cell Na and K transport studies.

1. Internal microinjection of the Ca-sensitive photoprotein aequorin or the isotope (45)Ca have been used to assess Ca movements in single muscle fibres from the barnacle Balanus nubilus and the crab Maia squinado.2. Progressive isosomotic replacement of external Na by Li, choline, sucrose or Tris was associated with a rapid increase in the level of light emission from internally injected aequorin. This response was dependent upon the presence of external Ca. The light output was maximal for Na concentrations < 50 mM in the replaced salines, while Na concentrations > 350 mM produced no apparent increase in the resting light emission.3. If the Ca concentration in the external saline was altered, no effect was observed in Na replaced salines when the Ca concentration was < 1 mM, but maximal effects were observed at concentrations of Ca of ca. 100 mM.4. The increased light emission from aequorin in Na-replaced salines was substantially inhibited by 1 mM-La(3+) applied externally, but not by N-ethyl maleimide (NEM), propanolol or D-600.5. Following microinjection of (45)Ca and 2 hr equilibration, the residual efflux of Ca was shown to be sensitive to the removal of external Ca and Na, the extent of each component being variable.6. The Na-sensitive Ca efflux was partially inhibited by external La(3+) (1 mM), but not by high concentrations of Mg(2+), Ca(2+), Mn(2+) or Co(2+). It was stimulated by NEM and ethacrynic acid, which was considered to be due to an indirect effect upon the sarcoplasmic reticulum.7. It is concluded that Ca movements in these single crustacean muscle fibres involve Na gradients and a Ca: Ca exchange and from the relative insensitivity of Ca movements to various inhibitors, in contrast to the Mg system, it seems that there may be two independent modes for divalent cation transport in this preparation.

The major pathway of passive K influx (ouabain-insensitive) was characterized in low-K type (LK) red cells of sheep. 1. Passive K transport in these cells was highly sensitive to variations in cell volume; it increased threefold or more in cells swollen osmotically by 10%, and decreased up to twofold in cells shrunken 5-10%. Active K influx was insensitive to changes in cell volume. Three different methods for varying cell volume osmotically all gave similar results. 2. The volume-sensitive pathway was specific for K in that Na influx did not vary with changes in cell volume. 3. The volume-sensitive K influx was a saturable function of external K concentration. It was slightly inhibited by Na, whereas K influx in shrunken cells was unaffected by Na. 4. Passive K influx was dependent on the major anion in the medium in that replacement of Cl with any of six other anions resulted in a reduction of K influx by 50-80% (replacement of Cl by Br caused an increase in K influx). The activation of K influx by Cl followed sigmoid kinetics. 5. Passive K influx is inhibited by anti-L antibody. The antibody affected only that portion of influx which was Cl-dependent and volume-sensitve. Of the subfractions of the antibody, it is anti-L1 which inhibits passive K transport. 6. Pretreatment of cells with iodoacetamide reduced the sensitivity of K influx to cell volume in that the influx was reduced in swollen IAA-treated cells and increased in shrunken IAA-cells. 7. Intracellular Ca has no role in altering passive K transport in LK sheep cells. Therefore, the major pathway of passive K transport in LK sheep red cells is sensitive to changes in cell volume, specific for K, dependent on Cl, and inhibited by anti-L1 antibody, The minor pathway, observed in shrunken cells, has none of these properties.

1. Treatment of low K goat red cells with trypsin stimulated the Na-K pump more than twofold. Dose dependence and time course experiments indicated a half-maximal stimulation at 1.6 mg trypsin/ml. (37 degrees C, 3 hr), and a maximum effect after 5 hr (10 mg/ml). 2. Trypsin had only a small and variable effect on the ouabain-insensitive component of K influx. 3. The Na-K pump activity of high K goat red cells was not affected by trypsinization. 4. When intracellular K was varied by the PCMBS technique, it was found that the trypsin stimulation was greatest (2-5-fold) in cells with the highest K (40 m-mole/1. cells) and lowest (1.1-fold) in cells with low K (<1 m-mole/1. cells). 5. The trypsin effect was reversed by nystatin treatment or hypotonic lysis. 6. Trypsin did not increase the number of ouabain-binding sites. 7. It is concluded that trypsinization modifies the L antigen in low K goat red cells to decrease the apparent internal affinity for K of the Na-K pump in these cells.

1. In single striated muscle fibres from the barnacle Balanus nubilus tension development following axial injection of caffeine (50 mM in 150 mM-KCl, pH 7.1) was used as an index of releasable Ca. It was shown that fibres incubated in 0 Ca (Na replaced) salines for up to 400 min gave ca. 15% of the control tension response. Inclusion of 1 mM-La in the 0 Ca (Na) saline significantly reversed this decline.2. Estimates of the total fibre calcium assayed under the same experimental conditions indicated a 67% loss of fibre Ca in 0 Ca, and only a 37% loss in La-0 Ca media. No correction was made for the loss of calcium from the extracellular space.3. Experiments with (45)Ca indicated that the efflux of calcium from this preparation was inhibited by 1 mM-La externally and that this effect was still significant even in the presence of 0 Ca (Na) salines. The caffeine-stimulated efflux of Ca was also reduced by ca. 70% in the presence of 1 mM La saline externally.4. The influx and efflux of (14)C caffeine were shown to be rapid, and apparently passive. The diffusion coefficient for caffeine following intracellular injection was 2.4 +/- 0.2 x 10(-6) cm(2) sec(-1) at 18-22 degrees C.5. There was no significant loss of (140)La over a period of 2 hr following axial micro-injection into the fibres.6. In (140)La uptake experiments there was a progressive increase in the La space over 10.5 hr, in contrast to the results with [(3)H]inulin, whose uptake saturated in ca. 1.5 hr. The probability of surface binding and the precipitation of La salts in the extensive extracellular space was suggested as an explanation.7. It is concluded that internal Ca within the sarcoplasmic reticulum, and not cleft or extracellular Ca is the most significant source for these caffeine-induced contractions. Fluxes across the surface membrane can however alter the internal Ca stores over longer periods of time.

1. The short-term uptake of Na by the goldfish mucosa was compared using both inulin and choline as markers of extracellular space. The results were virtually identical, the distribution of both choline and inulin increasing rapidly to measure a space at 1 min which then remained nearly constant during a following 4 min incubation.2. Using inulin as space marker, the uptake of various alkali metal cations was determined from a 1 min contact with the mucosa. The relative rates of uptake were Tl > K > Rb > Cs > Na > Li, with a low selectivity ratio, the range of permeabilities being no greater than 5.3. The selectivity sequence was the same in both salt and fresh-water adapted fish. Of the alkali metal cations tested, only Na showed a significantly decreased uptake on adaptation to salt.4. Isolated intestinal preparations from salt-adapted fish showed a reduced short-circuit current compared with fish adapted to fresh water, the values being 12.3 +/- 1.2 and 35.7 +/- 1.5 muA cm(-2) respectively. In both cases the short-circuit current was equivalent to the net transport of Na measured isotopically.5. In Krebs-Henseleit medium, the measured tissue resistance was approximately 100 Omega cm(-2) for both salt and fresh-water adapted fish.6. It is concluded that regulation of cation transport in goldfish intestinal epithelium is specific for Na and mediated primarily through cellular rather than extracellular pathways.

1. In the large single muscle fibres from the barnacle Balanus nubilus, the total fibre Mg concentration was estimated as 15.1 m-mole/kg wet wt., of which about 3-3.5 m-mole/kg wet wt. was extracellular. The diffusible Mg, measured by internal sampling, was 11.5 m-mole/kg wet wt., of which at least half may be complexed to larger diffusible molecules. The free ionized Mg level was estimated as < 5 m-mole/kg wet wt.2. The loss of [(28)Mg]MgCl(2) from both Maia and Balanus muscle fibres following axial micro-injection approximated to first-order kinetics. The maximum rate constant for the loss was 1.51 +/- 0.20 (S.E.) x 10(-5) sec(-1) for Balanus (sixty-seven fibres) and 1.06 +/- 0.46 (S.E.) x 10(-5) sec(-1) for Maia (seven fibres) at 20-25 degrees C.3. The calculated Mg efflux was in the range 6-12 p-mole/cm(2).sec based on this rate constant, assuming isotopic equilibration internally and that the surface area of the fibres approximated to that of a simple cylinder. If account was taken of the area of the cleft system the efflux was reduced by about fifteen times.4. The diffusion coefficient for injected (28)Mg was estimated as 2-3 x 10(-6) cm(2) sec(-1), about half the value in free solution. Injections of 2 M-MgCl(2) or 200 mM-EDTA subsequent to the injection of the isotope caused about a 30% reduction in the tracer efflux.5. External application of salines containing 100 mM-Ca or Mg caused a rapid but reversible inhibition of the magnesium efflux. Similar effects were observed with salines containing 32 mM-Co or Mn chlorides or 1-2 mM-La or Gd chlorides. Polyarginine (200 mug/ml.) had no effect.6. The Mg efflux had a Q(10) of 3-4 over the temperature range of about 5-20 degrees C. It was irreversibly inhibited by the sulphydryl reagent NEM (1 mM), but PCMBS (0.2-2 mM) had no effect. Contractile agents (5 mM caffeine or 200 mM-K salines) and a variety of inhibitors of ion movement or active transport had no appreciable effect on the Mg efflux. Lowering the pH of the saline from 7 to 5 produced a 70% reduction in the efflux which was reversible over short periods of application.7. Replacement of external Na, but not Ca or Mg, with Li, choline or sucrose caused a rapid and partially reversible reduction of the Mg efflux, but increasing the internal Na by micro-injection in zero Na salines had no consistent effect. It is suggested that the extrusion of Mg from these muscle cells is largely dependent upon the inward movement of Na.

1. The uptake and transflux of sodium by goldfish intestines has been compared under different experimental conditions. Uptake was measured from a 1 min contact of the intestinal mucosa with radioactive sodium chloride solution. Transflux was measured over a period of 2 hr using the everted sac technique.2. Keeping goldfish in saline reduced the transflux of sodium to one quarter the value found for fresh-water fish. The uptake of sodium was halved by this treatment. Cortisol injected previously into saline-adapted fish changed neither the transflux nor the uptake of sodium measured subsequently.3. In fresh-water fish hypophysectomy reduced sodium transflux while leaving the uptake of sodium unchanged. Injection of cortisol restored sodium transflux to control levels without producing any additional effects on the uptake of sodium.4. It is suggested that adaptation to saline involves regulation of sodium movement across the microvillar membrane of the mucosal cell. Cortisol would appear to play no part in this type of regulation.5. The presence of cortisol, or possibly other steroids with similar actions, has however been shown to be essential for the normal operation of sodium transport in this tissue. It is not clear exactly how cortisol exerts this effect. What evidence there is suggests that cortisol exerts a metabolic control rather than changing directly the membrane permeability of the cell.