Volume-stimulated,Cl−-dependent K+ efflux is highly expressed in young human red cells containing normal hemoglobin or HbS

Summary We report here that a Cl^–-dependent K^– (KCl) efflux, which is stimulated by N-ethylmaleimide, (NEM) and by increased red cell volume, exists in young red cells of individuals with normal hemoglobin A (AA) and in those homozygous for hemoglobin S (SS). We have investigated this KCl efflux in several density-defined red cell fractions obtained from Percoll-Stractan continuous density gradients. We found high activity of the NEM-stimulated KCl transport in reticulocytes and young red cells from nine sickle cell (SS) patients (43±27 mean±sd mmol K⁺/liter of cells/hr=flux units (FU)) and in the young cell fraction of three AA individuals with high reticulocytosis recuperating from nutritional anemias (41.7±10 FU). In addition, we observed significant interindividual variation of this KCl efflux in the discocyte fraction of SS blood. Cell swelling markedly stimulated the KCl efflux, in SS whole blood (9.8±7.4 FU, in SS young cells (13±13 FU), and in AA young cells (21.4±11 FU). The activity of the Na–K–Cl cotransport, as estimated by the bumetanide sensitive K⁺ efflux was not found to be cell-age dependent in either AA or SS cells.Measurements of red cell density by isopycnic gradients indicated that 27% of the young cells reduce their volume by a Cl^–-dependent process in hypotonic or low pH-induced swelling.The large volume-stimulated KCl efflux in AA young cells raises the possibility that these fluxes may be involved in the maturation of erythropoietic precursors. The high activity in the red cells of sickle cell anemia patients and its interindividual variation may have pathophysiological consequences since it reverses the decrease in the intracellular concentration of hemoglobin which occurs in response to low pH or osmolarity, an unwelcome pro-sickling event.     

Rate of activation and deactivation of K∶Cl cotransport by changes in cell volume in hemoglobin SS,CC and AA red cells

Red blood cells (RBC) of subjects homozygous for hemoglobin A (AA), C (CC) and S (SS) exhibit different cell volumes which might be related to differences in cell volume regulation. We have investigated how rapidly K:Cl cotransport is activated and deactivated to regulate the cell volume in these cells. We measured the time course of net K⁺ efflux after step changes in cell volume and determined two delay times: one for activation by cell swelling and a second for deactivation by cell shrinkage. Cell swelling induced by 220 mOsm media activated K⁺ efflux to high values (10–20 mmol/ liter cell x hr) in CC and SS; normal AA had a threefold lower activity. The delay time for activation was very short in blood with a high percentage of reticulocytes (retics): (SS, 10% retics, 1.7±0.3 min delay, n=8; AA, 10% retics, 4±1.5 min, n=3; CC, 11.6% retics, 4±0.3, n=3) and long in cells with a smaller percentage of reticulocytes: (AA, 1.5% retics, 10±1.4 min, n=8; CC whole blood 6% retics, 10±2.0 min, n=10, P<0.02 vs. SS). The delay times for deactivation by cell shrinking were very short in SS (3.6±0.4 min, n=8, P<0.02) and AA cells with high retics (2.7±1 min, n=3) and normal retics (2.8±1 min, n=3), but 8–15-fold longer in CC cells (29±2.8 min, n=9).Density fractionation of CC cells (n=3) resulted in coenrichment of the top fraction in reticulocytes and in swelling-activated cotransport (fourfold) with short delay time for activation (4±0.3 min) and long delay for deactivation (14±4 min). The delay time for activation, but not for deactivation, increased markedly with increasing cell density. These findings indicate that all CC cells do not promptly shut off cotransport with cell shrinkage and high rates of cellular K⁺ loss persist after return to isotonic conditions.In summary, (i) K:Cl cotransport is not only very active in young cells but it is also very rapidly activated and deactivated in young AA and SS cells by changes in cell volume. (ii) Delay times for cotransport activation markedly increased with RBC age and in mature cells with low cotransport rates, long delay times for activation were observed. (iii) The long delay time for deactivation exhibited even by young CC cells induces a persistent loss of K⁺ after cell shrinkage which may contribute in vivo to the uniformly low cell volume, low K⁺ and water content of CC cells.This research was supported by National Institutes of Health grants Shannon Award HL-35664, HL-42120, Sickle Cell Center grant HL-38655, and a Grant-in-Aid of the New York Branch of the American Heart Association. The technical help of Sandra M. Suzuka, M.S. is gratefully acknowledged.     

Interactions of external and internal H+ and Na+ with Na+/Na+ and Na+/H+ exchange of rabbit red cells: Evidence for a common pathway

Summary We have studied the kinetic properties of rabbit red cell (RRBC) Na⁺/Na⁺ and Na⁺/H⁺ exchanges (EXC) in order to define whether or not both transport functions are conducted by the same molecule. The strategy has been to determine the interactions of Na⁺ and H⁺ at the internal (i) and external (o) sites for both exchanges modes. RRBC containing varying Na _i and H _l were prepared by nystatin and DIDS treatment of acid-loaded cells. Na⁺/Na⁺ EXC was measured as Na _o -stimulated Na⁺ efflux and Na⁺/H⁺ EXC as Na _o -stimulated H⁺ efflux and pH _o -stimulated Na⁺ influx into acid-loaded cells.The activation of Na⁺/Na⁺ EXC by Na _o at pH _i 7.4 did not follow simple hyperbolic kinetics. Testing of different kinetic models to obtain the best fit for the experimental data indicated the presence of high (K _m 2.2 mM) and low affinity (K _m 108 mM) sites for a single- or two-carrier system. The activation of Na⁺/H⁺ EXC by Na _o (pH _i 6.6, Na _i <1 mM) also showed high (K _m 11 mM) and low (K _m 248 mM) affinity sites. External H⁺ competitively inhibited Na⁺/Na⁺ EXC at the low affinity Na _o site (K _H 52 nM) while internally H⁺ were competitive inhibitors (pK 6.7) at low Na _i and allosteric activators (pK 7.0) at high Na _i .Na⁺/H₊ EXC was also inhibited by acid pH _o and allosterically activated by H _i (pK 6.4). We also established the presence of a Na _i regulatory site which activates Na⁺/H⁺ and Na⁺/Na⁺ EXC modifying the affinity for Na _o of both pathways. At low Na _i , Na⁺/Na⁺ EXC was inhibited by acid pH _i and Na⁺/H⁺ stimulated but at high Na _i , Na⁺/Na⁺ EXC was stimulated and Na⁺/H⁺ inhibited being the sum of both pathways kept constant. Both exchange modes were activated by two classes of Na _o sites,cis-inhibited by external H _o , allosterically modified by the binding of H⁺ to a H _i regulatory site and regulated by Na _i . These findings are consistent with Na⁺/Na⁺ EXC being a mode of operation of the Na⁺/H⁺ exchanger.Na⁺/H⁺ EXC was partially inhibited (80–100%) by dimethyl-amiloride (DMA) but basal or pH _i -stimulated Na⁺/Na⁺ EXC (pH _i 6.5, Na _i 80 mM) was completely insensitive indicating that Na⁺/Na⁺ EXC is an amiloride-insensitive component of Na⁺/H⁺ EXC. However, Na⁺ and H⁺ efflux into Na-free media were stimulated by cell acidification and also partially (10 to 40%) inhibited by DMA: this also indicates that the Na⁺/H⁺ EXC might operate in reverse or uncoupled modes in the absence of Na⁺/Na⁺ EXC.In summary, the observed kinetic properties can be explained by a model of Na⁺/H⁺ EXC with several conformational states, H _i and Na _i regulatory sites and loaded/unloaded internal and external transport sites at which Na⁺ and H⁺ can compete. The occupancy of the H⁺ regulatory site induces a conformational change and the occupancy of the Na _i regulatory site modulates the flow through both pathways so that it will conduct Na⁺/H⁺ and/or Na⁺/Na⁺ EXC depending on the ratio of internal Na⁺:H⁺.     

Na+-dependent HCO 3 − transport and Na+/H+ exchange regulate pH i in human ciliary muscle cells

Summary We investigated intracellular pH (pH _i ) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4,5-dimethylfluorescein (CDMF). The steady-state pH _i was 7.09±0.04 (n = 12) in CO₂/ HCO ₃ ^– -buffered and 6.86±0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11±0.06 pH units (n = 12) in the presence of CO₂/ HCO ₃ ^– and by 0.91±0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pH _i recovery, which was almost completely amiloride-sensitive in the absence of CO₂/ HCO ₃ ^– but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25±0.02 (n = 4) in 6 min, while amiloride had no effect. The pH _i recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pH _i recovery was almost completely mediated by Na⁺/H⁺ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amilorideinsensitive pH _i recovery was observed in CO₂/HCO ₃ ^– -buffered solution which was mediated by chloride-independent and chloride-dependent Na⁺ HCO ₃ ^– cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter). was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pH _i recovery after NH₄Cl prepulse revealed V _max = 0.57 pH units/min, K _m= 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and V _max = 0.19 pH units/min, K _m= 14.3 mmol/liter extracellular sodium for the arniloride-insensitive component. We conclude that Na⁺/H⁺ exchange and chloride-independent and chloride-dependent Na⁺HCO ₃ ^– cotransport are involved in the pH _i regulation of cultured human ciliary muscle cells.The expert technical assistance of Astrid Krolik is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft grant DFG Wi 328/11.     

Outward sodium and potassium cotransport in human red cells

Summary This 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.Charge de Recherches CNRS.     

A mass spectrometry assay for detection of endogenous and lentiviral engineered hemoglobin in cultured cells and sickle cell disease mice

Sickle cell disease (SCD) results from a sequence defect in the β-globin chain of adult hemoglobin (HbA) leading to expression of sickle hemoglobin (HbS). It is traditionally diagnosed by cellulose-acetate hemoglobin electrophoresis or high-performance liquid chromatography. While clinically useful, these methods have both sensitivity and specificity limitations. We developed a novel mass spectrometry (MS) method for the rapid, sensitive and highly quantitative detection of endogenous human β-globin and sickle hβ-globin, as well as lentiviral-encoded therapeutic hβAS3-globin in cultured cells and small quantities of mouse peripheral blood. The MS methods were used to phenotype homozygous HbA (AA), heterozygous HbA–HbS (AS) and homozygous HbS (SS) Townes SCD mice and detect lentiviral vector-encoded hβAS3-globin in transduced mouse erythroid cell cultures and transduced human CD34⁺ cells after erythroid differentiation. hβAS3-globin was also detected in peripheral blood 6 weeks post-transplant of transduced Townes SS bone marrow cells into syngeneic Townes SS mice and persisted for over 20 weeks post-transplant. As several genome-editing and gene therapy approaches for severe hemoglobin disorders are currently in clinical trials, this MS method will be useful for patient assessment before treatment and during follow-up.     

H+ transport and the regulation of intracellular pH in ehrlich ascites tumor cells

Summary The intracellular pH (pH _i ) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H⁺ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pH _i of cells placed in an acidic medium (pH _o below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pH _o is acutely reduced to 5.5, pH _i falls exponentially from 7.20 ± 0.06 to 6.29 ± 0.04 with a halftime of 5.92 ± 1.37 min, suggesting a rapid influx of H⁺. The unidirectional influx of H⁺ exhibits saturation kinetics with respect to extracellular [H⁺]; the maximal flux is 15.8 ± 0.05 mmol/(kg dry wt · min) andK _m is 0.74 ± 0.09 × 10^–6 m.Steady-state cells with pH _i above 6.8 continuously extrude H⁺ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pH _i 6.3) when returned to pH _o 7.3 medium respond by transporting H⁺, resulting in a rapid rise in pH _i . The halftime for this process is 1.09 ± 0.22 min. The H⁺ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pH _i to its steady-state value.     

Mechanism of Na+/proline symport inEscherichia coli: Reappraisal of the effect of cation binding to the Na+/proline symport carrier

Summary Recently we proposed that cytoplasmic acidification of low K⁺ (LK) sheep erythrocytes may stimulate ouabain-resistant Cl^–-dependent K⁺ flux (K⁺Cl^– cotransport), also known to be activated by cell swelling, treatment with N-ethylmaleimide (NEM), or removal of cellular bivalent cations. Here we studied the dependence of K⁺ transport on intracellular and extracellular pH (pH _i , pH _o ) varied either simultaneously or independently using the Cl^–/HCO ₃ ^– exchange inhibitor 4,4, diisothiocyanatostilbene-3,2-disulfonic acid (DIDS). In both control and NEM-treated LK cells volumes were kept near normal by varying extracellular sucrose. Using DIDS as an effective pH clamp, both K⁺ efflux and influx of Rb⁺ used as K⁺ congener were strongly activated at acid pH _i and alkaline pH _o . A small stimulation of K⁺ (Rb⁺) flux was also seen at acid pH _i in the absence of DIDS, i.e., when pH _i pH _o . Anti-L _l serum, known to inhibit K⁺Cl^– cotransport, prevented the pH _i -stimulated K⁺ (Rb⁺) fluxes. Subsequent to NEM treatment at pH 6, K⁺ (Rb⁺) fluxes were activated only by raising pH, and thus were similar to the pH activation profile of K⁺ (Rb⁺) fluxes in DIDS-treated cells with pH _o varied at constant physiologic pH _i . Anti-L _l , which inhibited NEM-stimulated K⁺ (Rb⁺) fluxes, failed to do so in NEM-plus DIDS-treated cells. Thus, NEM treatment interferes with the internal but not with the external pH-sensitive site.     

Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

Summary We have investigated the kinetic properties of the human red blood cell Na⁺/H⁺ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H⁺ (H _i ) and external Na⁺ (Na _o ) in red blood cells of normal subjects.Red blood cells with different cell Na⁺ (Na _i ) and pH (pH _i ) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na⁺ influx were measured by varying pH _i (from 5.7 to 7.4), external pH (pH _o ), Na _i and Na _o and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na⁺ influx (Na _i <2.0 mmol/liter cell, Na _o = 150mm) increased sigmoidally (Hill coefficient 2.5) when pH _i fell below 7.0 and the external pH _o was 8.0, but increased linearly at pH _o 6.0. The net Na⁺ influx driven by an outward H⁺ gradient was estimated from the difference of Na⁺ influx at the two pH _o levels (pH _o 8 and pH _o 6). The H⁺-driven Na⁺ influx reached saturation between pH _i 5.9 and 6.1. TheV _max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK _m for H _i to activate H⁺-driven Na⁺ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na⁺/H⁺ exchange. The stoichiometric ratio between H⁺-driven Na⁺ influx and Na⁺-driven H⁺ efflux was 11. The dependence of Na⁺ influx from Na _o was studied at pH _i 6.0, and Na _i lower than 2 mmol/liter cell at pH _o 6.0 and 8.0. The meanK _m for Na _o of the H⁺-gradient-driven Na⁺ influx was 55±7mm.An increase in Na _i from 2 to 20 mmol/liter cell did not change significantly H⁺-driven net Na⁺ influx as estimated from the difference between unidirectional²²Na influx and efflux. Na⁺/Na⁺ exchange was negligible in acid-loaded, DIDS-treated cells. Na⁺ and H⁺ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na⁺ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na⁺/H⁺ exchanger.It is concluded that human red cell Na⁺/H⁺ exchange performs 11 exchange of external Na⁺ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H⁺ and external Na⁺ is similar to other cells, but it displays a larger variability in theV _max between individuals.     

Apical and basolateral Na+/H+ exchange in the rabbit outer medullary thin descending limb of henle: Role in intracellular pH regulation

Summary The present study was designed to investigate the apical and basolateral transport processes responsible for intracellular pH regulation in the thin descending limb of Henle. Rabbit thin descending limbs of long-loop nephrons were perfused in vitro and intracellular pH (pH _i ) was measured using BCECF. Steady-state pH _i in HEPES buffered solutions (pH 7.4) was 7.18±0.03. Following the removal of luminal Na⁺, pH _i decreased at a rate of 1.96±0.37 pH/min. In the presence of luminal amiloride (1mm), the rate of decrease of pH _i was significantly less, 0.73±0.18 pH/min. Steady-state pH _i decreased 0.18 pH units following the addition of amiloride (1mm) to the lumen (Na⁺ 140mm lumen and bath). When Na⁺ was removed from the basolateral side of the tubule, pH _i decreased at a rate of 0.49±0.05 pH/min. The rate of decrease of pH _i was significantly less in the presence of 1mm basolateral amiloride, 0.29±0.04 pH/min. Addition of 1mm amiloride to the basolateral side (Na⁺ 140mm lumen and bath) caused steady-state pH _i to decrease significantly by 0.06 pH units. When pH _i was acutely decreased to 5.87±0.02 following NH₄Cl removal (lumen, bath), pH _i failed to recover in the absence of Na⁺ (lumen, bath). Addition of 140mm Na⁺ to the lumen caused pH _i to recover at a rate of 2.17±0.59 pH/min. The rate of pH _i recovery was inhibited 93% by 1mm luminal amiloride. When 140mm Na⁺ was added to the basolateral side, pH _i recovered only partially at 0.38±0.07 pH/min. Addition of 1mm basolateral amiloride inhibited the recovery of pH _i , by 97%. The results demonstrate that the rabbit thin descending limb of long-loop nephrons possesses apical and basolateral Na⁺/N⁺ antiporters. In the steady state, the rate of Na⁺-dependent H⁺ flux across the apical antiporter exceeds the rate of Na⁺-dependent H⁺ flux via the basolateral antiporter. Recovery of pH _i following acute intracellular acidification is Na⁺ dependent and mediated primarily by the luminal antiporter.     

Alteration in lymphocyte phenotype associated with administration of adjuvant levamisole and 5-fluorouracil

Levamisole (LMS) and 5-fluorouracil (5FU) administered adjuvantly are effective in reducing the relapse rate following surgical resection of Duke's stage C colon carcinoma. It has been postulated that LMS acts to stimulate the immune system and that this is one mechanism through which this drug exerts its antitumor effects. In this study, peripheral blood mononuclear cells (PBMC) were analyzed in nine patients with surgically resected colon carcinoma prior to initiation of adjuvant LMS/5FU and at several subsequent times while patients were on therapy. Changes in lymphocyte phenotype and soluble interleukin-2 receptor (sIL-2R) between pre-study samples and samples obtained during adjuvant LMS/5FU were evaluated. Significant increases were seen in the proportion of PBMC expressing natural killer (NK) antigen CD56 (14.7±2.4% versus 18.1±2.6%;P<0.05) and surface IL-2R (CD25; 0% versus 0.42±0.15%;P<0.05), in sIL-2R (314±86 U/ml versus 736±173 U/ml;P<0.05), and in the CD4CD8 ratio (2.34±0.93 versus 3.47±1.23;P<0.01). A significant decrease in the proportion of CD8⁺ PBMC (24.7±3.8% versus 18.8±2.6%;P<0.01) and total CD8⁺ PBMC (537±118 versus 324±37;P<0.01) was seen. The increase in CD56⁺ cells correlated with sIL2R levels (r=0.46;P<0.05). No changes were noted for CD3, CD4, CD5, CD14, CD16, CD19, CDw49a, or TCR. The greatest increase in CD56⁺ cells and the smallest reduction in CD8⁺ cells were seen in the subgroup of patients who remained disease-free following adjuvant chemotherapy. This study suggests that adjuvant LMS/5FU has significant stimulatory effects on the immune system, which correlate with patient outcome and may account at least in part for its clinical efficacy.Grant support: supported by a grant from the Elsa U. Pardee Foundation (R.F.H.) and the Centers for Excellence in Cancer Research and Arthritis & Rheumatology, LSU Medical Center, Shreveport, La., USA     

31P NMR analysis of intracellular pH of Swiss mouse 3T3 cells: Effects of extracellular Na+ and K+ and mitogenic stimulation

Summary Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional³¹P and¹⁹F probes of intracellular pH (pH _c ) were found to be impracticable. Cells were therefore superfused with 1 to 4mm 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na⁺ with choline or K⁺ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pH _c on external Na⁺ concentration (c _Na ^o ). pH _c also depended on intracellular Na⁺ concentration (c _Na ^o ). Increasingc _Na ^c by withdrawing external K⁺ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducingc _Na ^o produced a larger acid shift in pH _c than with external K⁺ present. Comparison of separate preparations indicated that pH _c was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pH _c of Swiss mouse 3T3 cells using³¹P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

Na+/H+ exchange in crayfish neurons: dependence on extracellular sodium and pH

Summary Intracellular pH (pH_i) regulation was studied in crayfish neurons with pH-, and Na⁺-sensitive microelectrodes. It was confirmed to involve both a HCO ₃ ^– -dependent and a HCO ₃ ^– -independent mechanism. The latter was identified as the amiloride-sensitive Na⁺/H⁺ exchange described in vertebrate cells. Its dependence on extracellular pH (pH_e) and Na⁺ concentration ([Na⁺]_e) was studied in CO₂-free external solutions at 20°C. The steady state pH_i and the rate constant (k) of the exponential pH_i recovery following an acid load were determined. At pH_e=7.5 and [Na⁺]_e=200 mM, the average steady state pH_i was 7.09±0.12 (as compared to 7.30±0.10 in the presence of 5 mM bicarbonate). The dependence of the rate constant of recovery on [Na⁺]_e could be described by Michaelis-Menten kinetics; at pH_e=7.5 the apparentK _m andK _max were 39 mM and 1.4 mmol·l^–1·min^–1, respectively. Decreasing pH_e reduced the rate of recovery, the variations ofk with pH_e conforming to a simple titration curve with an apparent pK of 7.05±0.21. These kinetic properties of the Na⁺/H⁺ exchange in crayfish neurons are similar to those described in vertebrate cells.Preliminary results were presented at the First International Congress of Comparative Physiology and Biochemistry (Liège, Belgium, 1984)     

Amiloride-sensitive Na+ transport in human red cells: Evidence for a Na/H exchange system

Summary The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na⁺ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 m) and methazolamide (400 m). Red cells with different internal pH (pH _i =6.4, 7.0 and 7.8) were prepared and Na⁺ influx was measured at different external pH (pH _o =6.0, 7.0, 8.0). Na⁺ influx into acid-loaded cells (pH _i =6.4) markedly increased when pH _o was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na⁺/H⁺ exchange systems blocked about 60% of the H⁺-induced Na⁺ entry, while showing small inhibitory effects in the absence of pH gradients. When pH₀ was kept at 8.0, the amiloride-sensitive Na⁺ entry was abolished as pH _i was increased from 6.4 to 7.8. Moreover, measurements of H⁺ efflux into lightly buffered media indicated that the imposition of an inward Na⁺ gradient stimulated a net H⁺ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na⁺ (Na _i ⁺ =Na ₀ ⁺ =15mm,Em=+6.7 mV), an outward H⁺ gradient (pH _i =6.4, pH₀=8.0) promoted a net amiloride-sensitive Na⁺ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na⁺/H⁺ exchange system in human red cells.     

Endogenousd-glucose transport in oocytes ofXenopus laevis

Summary Endogenous glucose uptake by the oocytes ofXenopus laevis consists of two distinct components: one that is independent of extracellular Na⁺, and the other one that represents Na⁺-glucose cotransport. The latter shows similar characteristics as 2 Na⁺-1 glucose cotransport of epithelial cells: The similarities include the dependencies on external concentrations of Na⁺, glucose, and phlorizin, and on pH. As in epithelial cells, the glucose uptake in oocytes can also be stimulated by lanthanides. Both the electrogenic cotransport and the inhibition by phlorizin are voltage-dependent; the data are compatible with the assumption that the membrane potential acts as a driving force for the reaction cycle of the transport process. In particular, hyperpolarization seems to stimulat transport by recruitment of substrate binding sites to the outer membrane surface. The results described pertain to oocytes arrested in the prophase of the first meiotic division; maturation of the oocytes leads to a downregulation of both the Na⁺-independent and the Na⁺-dependent transport systems. The effect on the Na⁺-dependent cotransport is the consequence of a change of driving force due to membrane depolarization associated with the maturation process.     

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: Modes of operation,dependence on extracellular and intracellular Na+, kinetics,pH dependency and the effect of cell volume and N-ethylmaleimide

Summary The effect of extracellular and intracellular Na⁺ (Na _o ⁺ , Na _i ⁺ ) on ouabain-resistant, furosemide-sensitive (FS) Rb⁺ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na⁺+1 K⁺+2 Cl^–) cotransport system operates in two different modes: modei) promoting bidirectional 11 (Na⁺–K⁺) cotransport, and modeii) a Na _o ⁺ -independent 11 K _o ⁺ /K _i ⁺ exchange requiring Na _i ⁺ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb⁺ uptake through Rb _o ⁺ /K _i ⁺ exchange contributes about 25% to total Rb⁺ uptake in 145mm NaCl media containing 5mm RbCl at normal Na _i ⁺ (pH 7.4). Na⁺–K⁺ cotransport into the cells occurs largely additive to K⁺/K⁺ exchange. Inward Na⁺–Rb⁺ cotransport exhibits a substrate inhibition at high Rb _o ⁺ . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K⁺/K⁺ exchange (apparent pK close to pH 7.4). The apparentK _m ^Rb _o ⁺ of Na⁺–K⁺ cotransport is low (2mm) and almost independent of pH, and high for K⁺/K⁺ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na⁺+1 K⁺+2 Cl^–) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl^–-dependent K⁺ transport pathway that is independent of both Na _o ⁺ and Na _i ⁺ . This pathway differs in many properties from the basal, Na _o ⁺ -independent K⁺/K⁺ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na _o ⁺ -independent FS K⁺ transport pathway which most probably is not identical to basal K⁺/K⁺ exchange. K _o ⁺ o ⁺

o ⁺ o ²⁺ reduce furosemide-resistant Rb⁺ inward leakage relative to choline _o ⁺ .     

Fusion of cultured dog kidney (MDCK) cells: II. Relationship between cell pH and K+ conductance in response to aldosterone

Summary We have chosen the MDCK cell line to investigate aldosterone action on H⁺ transport and its role in regulating cell membrane K⁺ conductance (G _m ^K ). Cells grown in a monolayer respond to aldosterone indicated by the dose-dependent formation of domes and by the alkalinization of the dome fluid. The pH sensitivity of the plasma membrane K⁺ channels was tested in giant cells fused from individual MDCK cells. Cytoplasmic pH (pH _i ) andG _m ^K were measured simultaneously while the cell interior was acidified gradually by an extracellular acid load. We found a steep signoidal relationship between pH _i andG _m ^K (Hill coefficient 4.4±0.4), indicating multiple H⁺ binding sites at a single K⁺ channel. Application of aldosterone increased pH _i within 120 min from 7.22±0.04 to 7.45±0.02 and from 7.15±0.03 to 7.28±0.02 in the absence and presence of the CO₂/HCO ₃ ^– buffer system, respectively. We conclude that the hormone-induced cytoplasmic alkalinization in the presence of CO₂/ HCO ₃ ^– is limited by the increased activity of a pH _i -regulating HCO ₃ ^– extrusion system. SinceG _m ^K is stimulated half-maximally at the pH _i of 7.18±0.04, internal H⁺ ions could serve as an effective intracellular signal for the regulation of transepithelial K⁺ flux.     

Intracellular pH regulation in the mouse lacrimal gland acinar cells

Summary Intracellular pH (pH _i ) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pH _i was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by ±0.4 unit shifted pH _i only by ±0.08 unit. The intracellular buffering value determined by applications of 25mm NH ₄ ⁺ and bicarbonate buffer solution gassed with 5% CO₂/95% O₂ was 26 and 46mm/pH, respectively Stimulation with 1 m acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pH _i . In the presence of amiloride (0.1mm) or the absence of Na⁺, application of ACh caused a significant decrease in pH _i and removal of amiloride or replacement with Na⁺-containing saline, respectively, rapidly increased the pH _i . Pretreatment with DIDS (0.2mm) did not change the pH _i of the nonstimulated conditions; however, it significantly enhanced the increase in pH _i induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na⁺ removal from the bath solution. We suggest that both Na⁺/H⁺ and HCO ₃ ^– /Cl^– exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells.     

Activation of Na/H Exchange in Erythrocytes of Frog Rana ridibunda

Transport of Na⁺ in isolated erythrocytes of the frog Rana ridibunda was studied using radioactive isotope ^{22 22}Na. Treatment of erythrocytes with -adrenergic agonist isoproterenol (ISP) or with a combination of ISP and phosphodiesterase blocker 3-isobutyl-methyl-xanthine (IBMX) did not affect the Na⁺ transport into the cells. These data indicated that cAMP-dependent protein kinase A did not participate in regulation of the Na⁺ transport into the frog erythrocytes. Incubation of erythrocytes with protein kinase C activator phorbol ester (PMA, 0.15 µM) led to a pronounced increase of ^{22 22}Na accumulation and intracellular Na⁺ concentration. These changes of the Na⁺ transport into the cells were completely blocked in the presence of 50 µM ethyl-isopropyl-amiloride (EIPA), a selective blocker of the NHE1-isoform of Na⁺/H⁺ exchanger. Hence, PMA produced activation of Na⁺/H⁺ exchange in frog erythrocytes. The unidirectional Na⁺ influx into erythrocytes amounted, on average, to 0.99 ± 0.12 and 147 ± 9 mmol/l cells/h for control and PMA-treated cells, respectively. The EIPA concentration producing a 50% inhibition of the PMA-induced Na⁺ influx (IC₅₀) was 0.28 µM. A high sensitivity of the frog Na/H exchanger to EIPA indicates its similarity with the mammalian NHE1 isoform. The obtained data for the first time clearly indicate an important role of PKC in Na/H exchange regulation in the frog red blood cells.