Volume-sensitive K transport in human erythrocytes

Studies have been carried out on human erythrocytes to examine the alterations of K transport induced by swelling or shrinking the cells by osmotic and isosmotic methods. Hypotonic swelling of erythrocytes (relative cell volume, 1.20) resulted in a striking, four- to fivefold augmentation in the ouabain-resistant K influx over the value obtained at a normal cell volume. Shrinking the cells in hypertonic media resulted in a small but statistically significant reduction in K influx. Three different methods of varying cell volume gave similar results. These include the addition of sucrose and of NaCl to hypotonic media and the isosmotic (nystatin) method. The major fraction of the K influx in swollen cells is specific in its requirement for Cl or Br and is not supported by thiocyanate, iodide, nitrate, methylsulfate, or acetate. Bumetanide (0.1 mM), MK-196 (0.2 mM), and piretanide (1 mM) are poorly effective in suppressing K uptake in swollen cells, but at higher concentrations, bumetanide (1 mM) inhibits 80% of the Cl-dependent K influx in swollen cells. The bumetanide concentration required to inhibit 50% of the Cl-dependent K influx is 0.17 mM. The volume-sensitive K influx is independent of both extracellular and intracellular Na, so that the (Na + K + 2Cl) cotransport pathway is not a likely mediator of the volume-sensitive K transport. A variety of inhibitors of the Ca-activated K channel are ineffective in suppressing swelling-induced K influx. Like K uptake, the efflux of K is also enhanced by cell swelling. Swelling-activated K efflux is Cl dependent, is independent of extracellular and intracellular Na, and is observed with both hypotonic and isosmotic methods of cell swelling. The activation of K efflux by cell swelling is observed in K-free media, which suggests that the volume-sensitive K transport pathway is capable of net K efflux. The addition of external K to hypotonic media resulted in an increase in K efflux compared with the efflux in K-free media, and this increase was probably due to K/K exchange. Thus, hypotonic or isosmotic swelling of human erythrocytes results in the activation of a ouabain-resistant, Cl-dependent, Na-independent transport pathway that is capable of mediating both net K efflux and K/K exchange.     

Cholinergic stimulation of glucose transport in human erythrocytes

The effects of cholinergic stimulation on glucose equilibrium exchange rate have been studied in human erythrocytes. Carbamylcholine increases the V of equilibrium exchange by 20% but has no significant effect on K_m. The cholinergic effect is abolished by the muscarinic antagonist atropine or by alterations in intracellular calcium concentrations induced by the calcium ionophore A23187.     

Activation of sodium transport in human erythrocytes by beta-adrenoceptor stimulation in vivo

Beta-adrenoceptor stimulation in vivo shifts potassium into the cells. To examine whether human erythrocytes participate in this process, we measured, along with serum or plasma potassium, the concentrations of potassium and sodium in erythrocytes. Beta-adrenoceptor stimulation was obtained by infusion of either fenoterol or hexoprenaline into 6 volunteers at rest or by endogenous amines provoked in 14 volunteers during ergometric exercise. Metabolic effects were followed at rest on serum insulin, C-peptide, and growth hormone levels, and during exercise on pH on lactate concentration in blood. The potassium concentration (mean +/- S.E.M.) dropped (p less than 0.01) in serum from 4.64 +/- 0.37 to 3.19 +/- 0.43 mmol x l-1 in the first hour at rest and in plasma from 5.70 +/- 0.93 to 4.63 +/- 0.45 in 90 sec directly after exercise. The concentration of erythrocyte sodium dropped (p less than 0.001) from 9.68 +/- 0.73 to 8.81 +/- 0.62 mmol x l-1 in cells and from 9.62 +/- 1.16 to 8.55 +/- 1.24 during exercise for 90 s, respectively. Changes in the concentration ratio of cellular sodium to potassium confirmed this sodium shift. An increased sodium transport in erythrocytes due to beta-adrenoceptor stimulation in vivo appears to complement a shift of serum potassium into the cells and may be mediated by the membrane-bound sodium, potassium ATPase.     

K(+) transport in red blood cells from human umbilical cord

The current study was designed to characterise K(+) transport in human fetal red blood cells, containing mainly haemoglobin F (HbF, and termed HbF cells), isolated from umbilical cords following normal parturition. Na(+)/K(+) pump activity was comparable to that in normal adult human red cells (which contain HbA, and are termed HbA cells). Passive (ouabain-resistant) K(+) transport was dominated by a bumetanide (10 microM)-resistant component, inhibited by [(dihydroxyindenyl)oxy]alkanoic acid (100 microM), calyculin A (100 nM) and Cl(-) removal, and stimulated by N-ethylmaleimide (1 mM) and staurosporine (2 microM) - all consistent with mediation via the K(+)-Cl(-) cotransporter (KCC). KCC activity in HbF cells was also O(2)-dependent and stimulated by swelling and urea, and showed a biphasic response to changes in external pH. Peak activity of KCC in HbF cells was about 3-fold that in HbA cells. These characteristics are qualitatively similar to those observed in HbA cells, notwithstanding the different conditions experienced by HbF cells in vivo, and the presence of HbF rather than HbA. KCC in HbF cells has a higher total capacity, but when measured at the ambient PO(2) of fetal blood it would be similar in magnitude to that in fully oxygenated HbA cells, and about that required to balance K(+) accumulation via the Na(+)/K(+) pump. These findings are relevant to the mechanism by which O(2) regulates membrane transporters in red blood cells, and to the strategy of promoting HbF synthesis as a therapy for patients with sickle cell disease.     

Anaerobic stimulation of sugar transport in avian erythrocytes

The kinetic parameters of the sugar transport in avian erythrocytes were evaluated under aerobic and anaerobic conditions. In anaerobic cells, transport measurements with $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose resulted in a set of similar dissociation-like constants. Thus the Michaelis constants of $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose entry and exit, $\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>si</mtext>}}$, were 8 and 7 mM, respectively. The equilibrium exchange constant, $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$, and the counterflow constant, $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$, were 9 and 11 mM, respectively. The activity constant for $\text{3-O-}\text{methylglucose}$ transport, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, defined as $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$, was 4 ml/h per g. This set of kinetic constants was compatible with a symmetrical mobile-carrier model. In contrast, the Michaelis constant for glucose entry, $\text{K}{}_{\mathrm{<mtext>go</mtext>}}$, was 2 mM and less than the counterflow constant, $\text{R}{}_{\mathrm{<mtext>g</mtext>}}$ (8 mM). This result could be accounted for by slower movement of the glucose-carrier complex than the free carrier. The activity constant for glucose transport, $\text{F}{}_{\mathrm{<mtext>g</mtext>}}$, was 5 ml/h perg.Under aerobic conditions, two of the dissociation-like constants ($\text{K}{}_{\mathrm{<mtext>si</mtext>}}$ and $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$) for $\text{3-O-}\text{methylglucose}$ transport were significantly larger than those obtained in anaerobic cells, but the remaining two ($\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$) remained unchanged. The values, for $\text{K}{}_{\mathrm{<mtext>so</mtext>}}\text{, K}{}_{\mathrm{<mtext>si</mtext>}}\text{, B}{}_{\mathrm{<mtext>s</mtext><mtext>\; and</mtext>}}\text{R}{}_{\mathrm{<mtext>s</mtext>}}$ were 8, 26, 20 and 8 mM, respectively. The activity constant, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, decreased to 2 ml/h per g. These changes in kinetic constants were consistent with the hypothesis that anoxia accelerated sugar transport by releasing free carrier that was previously sequestered on the inside of the cell membrane.     

cGMP and glutathione-conjugate transport in human erythrocytes.

The nature of cGMP transport in human erythrocytes, its relationship to glutathione conjugate transport, and possible mediation by multidrug resistance-associated proteins (MRPs) have been investigated. MRP1, MRP4 and MRP5 are detected in immunoblotting studies with erythrocytes. MRP1 and MRP5 are also detected in multidrug resistant COR-L23/R and MOR/R cells but at greatly reduced levels in the parent, drug sensitive COR-L23/P cells. MRP4 is detected in MOR/R but not COR-L23/R cells. Uptake of cGMP into inside-out membrane vesicles prepared by a spontaneous, one-step vesiculation process is shown to be by a low affinity system that accounts for more than 80% of the transport at all concentrations above 3 micro m. This transport is reduced by MRP inhibitors and substrates including MK-571, methotrexate, estradiol 17-beta-d-glucuronide, and S(2,4-dinitrophenyl)glutathione (DNP-SG) and also by glibenclamide and frusemide but not by the monoclonal Ig QCRL-3 that inhibits high-affinity transport of DNP-SG by MRP1. It is concluded that the cGMP exporter is distinct from MRP1 and has properties similar to those reported for MRP4. Furthermore the evidence suggests that the protein responsible for cGMP transport is the same as that mediating low-affinity DNP-SG transport in human erythrocytes.     

The effect of ACTH and adrenal steroids on K transport in human erythrocytes

The effect of ACTH and adrenal steroids on K transport in human erythrocytes has been studied. A new method of calculation has revealed that in normal human erythrocytes the K transport is not independent of external K concentration as had previously been thought. The equation describing the relationship is, K influx (m.eq./liter cells hour) = [K]_pi/(0.697 + 0.329 [K]_pi) in which [K]_pi refers to the plasma K concentration at the beginning of the experiment. At the physiological plasma K concentration of 4.65 m.eq./liter, K influx is 2.09 m.eq./liter cells hour; K efflux is 1.95 m.eq./liter cells hour and is independent of plasma K concentration. The effect of the infusion of ACTH and adrenal steroids on the K content of the erythrocytes was also studied. Infusions of ACTH or cortisone do not cause the expected loss in erythrocyte K content and may well cause a gain. Infusions of ACTH and cortisone decrease the rate of K influx and efflux slightly at all stages of the infusion, as measured in vitro in blood samples drawn at various times during and following the infusion. However, the erythrocytes incubated in vitro do not exhibit the same changes in K content as are found in vivo. Hydrocortisone added to normal cells in vitro also decreases both influx and efflux of K, without affecting the K content of the cells.     

39K nuclear magnetic resonance and a mathematical model of K+ transport in human erythrocytes

³⁹K nuclear magnetic resonance was used to measure the efflux of K⁺ from suspensions of human erythrocytes [red blood cells (RBCs)], that occurred in response to the calcium ionophore, A23187 and calcium ions; the latter activate the Gárdos channel. Signals from the intra- and extracellular populations of ³⁹K⁺ were selected on the basis of their longitudinal relaxation times, T ₁, by using an inversion- recovery pulse sequence with the mixing time, τ₁, chosen to null one or other of the signals. Changes in RBC volume consequent upon efflux of the ions also changed the T ₁ values so a new theory was implemented to obviate a potential artefact in the data analysis. The velocity of the K⁺ efflux mediated by the Gárdos channel was 1.19±0.40 mmol (L RBC)⁻¹ min⁻¹ at 37°C.     

Perturbation of the pump-leak balance for Na(+) and K(+) in malaria-infected erythrocytes

In humanerythrocytes infected with the mature form of the malaria parasitePlasmodium falciparum, the cytosolic concentration ofNa⁺ is increased and that of K⁺ is decreased.In this study, the membrane transport changes underlying thisperturbation were investigated using a combination of⁸⁶Rb⁺, ⁴³K⁺, and²²Na⁺ flux measurements and a semiquantitativehemolysis technique. From >15 h postinvasion, there appeared in theinfected erythrocyte membrane new permeation pathways (NPP) that causeda significant increase in the basal ion permeability of theerythrocyte membrane and that were inhibited by furosemide (0.1 mM). The NPP showed the selectivity sequenceCs⁺ > Rb⁺ > K⁺ > Na⁺, with the K⁺-to-Na⁺permeability ratio estimated as 2.3. From 18 to 36 h postinvasion, the activity of the erythrocyte Na⁺/K⁺ pumpincreased in response to increased cytosolic Na⁺ (aconsequence of the increased leakage of Na⁺ via the NPP)but underwent a progressive decrease in the latter 12 h of theparasite's occupancy of the erythrocyte (36-48 h postinvasion). Incorporation of the measured ion transport rates into a mathematical model of the human erythrocyte indicates that the induction of the NPP,together with the impairment of the Na⁺/K⁺pump, accounts for the altered Na⁺ and K⁺levels in the host cell cytosol, as well as predicting an initial decrease, followed by a lytic increase in the volume of the host erythrocyte.

     

Ozone alteration of transport of cations and the Na+/K+-ATPase in human erythrocytes.

We have purified glutaminase 65-fold from cow brain; the final specific activity is 24 μmol/min/mg. The enzyme is stable between pH 7.5 and 9.0 and has maximal activity at pH 8.8. It requires P_i for activity. The dependence of activity on P_i concentration is sigmoidal; 50 mmP_i gives half-maximal velocity at pH 8.8. At 0.2 mP_i, pH 8.8, the dependence of activity on glutamine concentration is hyperbolic; the observed K_Gln was 30 mm. Increasing P_i concentrations increase the apparent V_m and decrease the apparent K_Gln. NH₄⁺ does not inhibit at concentrations up to 0.1 m. Glutamic acid inhibits competitively with respect to glutamine; at 0.2 mP_i pH 8.8, K_Gln was 30 mm and K_Glu was 19 mm. The results are consistent with a model in which NH₄⁺ is released irreversibly from the enzyme-substrate complex and is the first product released. The activity of glutaminase appears to be independent of the nature of the buffer with which it is equilibrated before being assayed.     

Adrenergic stimulation of membrane protein phosphorylation in human erythrocytes.

Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocytes. Micromolar norepinephrine increased 32P incorporation into Band 2 by 70%, and into Band 3 by 40%. Phosphorylation levels observed with a series of specific agonists and antagonists suggest that an alpha-adrenergic receptor is involved in this effect. The mechanism of linkage between this receptor and protein phosphorylation does not appear to involve modulation of intracellular concentrations of ATP, cyclic AMP, or cyclic GMP.     

Beta-adrenergic stimulation of volume-sensitive chloride transport in lamprey erythrocytes

We measured the effects of a beta-adrenergic agonist, isoproterenol, on chloride transport and volume regulation of lamprey (Lampetra fluviatilis) erythrocytes in isotonic (288 mosm L(-1)) and hypotonic (192 mosm L(-1)) medium. Isoproterenol at a high concentration (10(-5) M) did not influence chloride transport in isotonic medium but markedly increased chloride fluxes in hypotonic conditions: unidirectional flux increased from 100 mmol kg dcw(-1) h(-1) in the absence to 350 mmol kg dcw(-1) h(-1) (dcw=dry cell weight) in the presence of isoproterenol. Simultaneously, the half-time for volume recovery decreased from 27 to 9 min. Isoproterenol caused an increase in cellular cyclic AMP (cAMP) concentration. The stimulation of chloride transport in hypotonic conditions could be induced by application of the permeable cAMP analogue, 8-bromo-cyclic AMP, suggesting that the effect of beta-adrenergic stimulation on chloride transport occurs downstream of cAMP production. As isoproterenol did not affect unidirectional rubidium fluxes in hypotonic conditions, the transport pathway influenced by beta-adrenergic stimulation is most likely the swelling-activated chloride channel. Because the beta-adrenergic agonist only influenced the transport in hypotonic conditions despite the fact that cAMP concentration also increased in isotonic conditions, the activation may involve a volume-dependent conformational change in the chloride channel.