Amino acid active transport and stimulation by substrates in the absence of a Na+ electrochemical potential gradient

Summary Uptake of -aminoisobutyric acid (AIB) was examined in Ehrlich ascites tumor cells treated with the cation-exchange ionophore nigericin (20 g/ml). Membrane voltages were measured using the voltage-sensitive dye diethyloxadicarbocyanine (DOCC). In normal phosphate-buffered media, nigericin changed the distribution ratios of Na⁺ and K⁺ (the ratio of intra- to extracellular concentrations) nearly to unity, but AIB was still accumulated to a distribution ratio of 9.0. When all but 40mm Na⁺ in the medium was replaced by choline, nigericin resulted in K⁺ loss and Na⁺ gain and both cation distribution ratios approached 2.8–3.4, as would be expected if both ions were distributing near electrochemical equilibrium with a membrane voltage in the range of –28 to –33 mV. This conclusion was supported by the observation that the addition of 5×10^–7 m valinomycin to the nigericin-treated cell suspension produced no change in DOCC absorbance. In spite of the apparent zero electrochemical potential gradients for Na⁺ and K⁺, AIB was accumulated to a distribution ratio of 5.4 in the low-Na⁺ medium. Addition of 0.1mm oubain or 50 m vanadate did not alter the extent of AIB accumulation as would have been expected if a large component of the membrane voltage were due to electrogenic operation of the (Na⁺+K⁺)-ATPase. Addition of lactate, pyruvate or glucose increased the AIB distribution ratios to 11.9, 9.4 and 15.3, respectively. The effect of glucose could be explained, at least in part, by an enhanced Na⁺ electrochemical potential gradient. However, neither lactate nor pyruvate produced any change either in membrane voltage or the intracellular Na⁺ concentration. Therefore, these results confirm the existence of a metabolic energy source which is coupled to AIB accumulation and operates in addition to the Na⁺ co-transport mechanism, and which is augmented by metabolic substrates such as lactate and pyruvate.     

H+/Ca2+ exchange in rabbit renal cortical endosomes

Summary We have examined the effect of second messengers on ATP-driven H⁺ transport in an H⁺ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1mm) had no effect on H⁺ transport. Acridine orange fluorescence in the presence of 0.5mm Ca²⁺ (+1mm EGTA) was 19±6% of control. Inhibition of ATP-driven H⁺ transport by Ca²⁺ was concentration dependent; 0.25 and 0.5mm Ca²⁺ (+1mm EGTA) inhibited acridine orange fluorescence by 50 and 80%, respectively. Ca²⁺ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca²⁺ did not affect ATP hydrolysis. ATP-dependent Br^– uptake was virtually unchanged in the presence of 0.5mm Ca²⁺ (+1mm EGTA). These vesicles were also shown to transport Ca²⁺ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca²⁺ uptake. These results are consistent with the co-existence of an H⁺ ATPase and an H⁺/Ca²⁺ exchanger on these endosomes, the latter transport system using the H⁺ gradient to energize Ca²⁺ uptake. Attempts to demonstrate an H⁺/Ca²⁺ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca²⁺ uptake could be demonstrated. A Ca²⁺-dependent increase in H⁺ permeability and an ATP-dependent Ca²⁺ uptake might have important implications for the regulation of vacuolar H⁺ ATPase activity as well as the homeostasis of cytosolic Ca²⁺ concentration.     

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.     

The sodium concentration of the lateral intercellular spaces of MDCK cells: A microspectrofluorimetric study

MDCK cell monolayers grown on glass coverslips were used to examine the Na⁺ concentration in individual lateral intercellular spaces (LIS) by video fluorescence microscopy. The LIS was filled with the Na⁺-sensitive fluorescent dye SBFO by incubation of the monolayers for 75–90 min with 250 m of the membrane impermeant form of the dye. After dye loading, the monolayers were perfused at 37°C with solutions buffered with HEPES or bicarbonate/CO₂ containing 142 mm Na⁺. Ratios of the fluorescence images after sequential excitation with 340 nm and 380 nm light were performed and in situ calibration of LIS Na⁺ was accomplished after blocking the Na⁺ pump with 5 × 10^–4 m ouabain. Measurements of Na⁺ along the basolateral-to-apical axis of the LIS at 1.0 or 1.5 m intervals did not reveal a Na⁺ gradient when the perfusate was either HEPES or bicarbonate/CO₂ solutions. In bicarbonate solutions, the mean Na⁺ concentration (mm) was 157.2 ± 2.3, 15 mm higher than the bath Na⁺ concentration. In HEPES solutions, however, the Na⁺ concentration was not different from the bath concentration (142.7 ± 3.1 mm). The time course of Na⁺ changes in LIS was investigated by rapidly switching the perfusate from 142 to 80 mm Na⁺ and measuring the Na⁺ changes at one focal plane.We would like to thank P.H. Tran and C. Gibson for their technical and computational assistance as well as Dr. B.-E. Persson (University of Uppsala, Sweden) for his contribution in the early phases of the study.     

The nature of the neutral Na+−Cl− coupled entry at the apical membrane of rabbit gallbladder epithelium: III. Analysis of transports on membrane vesicles

Summary In rabbit gallbladder epithelium, a Na⁺/H⁺, Cl^–/HCO ₃ ^– double exchange and a Na⁺–Cl^– symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na⁺, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na⁺ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (pH) was imposed ([H⁺]_in>[H⁺]_out, PD=0), the addition of Na-gluconate to the external solution caused pH dissipation at a rate that followed saturation kinetics. Amiloride (10^–4 m) reduced the pH dissipation rate. Taken together, these data indicate the presence of Na⁺ and H⁺ conductances in addition to an amiloride-sensitive, electroneutral Na⁺/H⁺ exchange.An inwardly directed [Cl^–] gradient (PD=0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl^–/OH^– exchange.When both [Na⁺] and [Cl^–] gradients (outwardly directed, PD=0) were present, fluorescence quenching reached a maximum 20–30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na⁺] gradient alone or the same [Na⁺] gradient with Cl^– at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na⁺ and Cl^– concentration gradients and hydrochlorothiazide (5×10^–4 m). The decrease was not influenced by an inhibitor of Cl^–/OH^– exchange (10^–4 m furosemide) or of Na⁺–K⁺–2Cl^– symport (10^–5 m bumetanide).We conclude that a Na⁺/H⁺ exchange and a Na⁺–Cl^– symport are present and act simultaneously. This suggests that in intact tissue the Na⁺–Cl^– symport is also likely to work in parallel with the Na⁺/H⁺ exchange and does not represent an induced homeostatic reaction of the epithelium when Na⁺/H⁺ exchange is inhibited.     

Properties of an anion/H+ contransport system in L1210 cells that utilizes phthalate as a nonphysiological substrate

Summary [¹⁴C]Phthalate is transported into L1210 cells via two separate routes, an anion exchange system whose primary substrates are folate compounds, and a second less active system which is sensitive to bromosulfophthalein. When the principal uptake component was blocked by a specific irreversible inhibitor of this system, the remaining route (at pH 7.4) appeared to be saturable and was inhibited by several anions in addition to bromosulfophthalein (K _i =2 m), including 8-anilino-1-naphthalein sulfonate (K _i =25 m), unlabeled phthalate (K _i =500 m), and chloride (K _i =3500 m). A pronounced effect by pH was also observed. Influx and total uptake of phthalate both increased progressively with decreasing pH and reached values that were 20-fold higher at pH 6.0, compared with pH 7.4. This pH-dependent increase could be blocked, however, by the addition of compounds (nigericin and carbonylcyanidem-chlorophenylhydrazone) which, in combination, collapse proton gradients. Phthalate efflux was relatively insensitive to changes in extracellular pH but could be inhibited (up to 90%) by bromosulfophthalein. Several other anions also inhibited efflux, but to a lesser extent, while chloride, phthalate, lactate, glycolate and acetate enhanced efflux up to 1.8-fold. Efflux also increased at pH 6.0, but not at pH 7.5, upon addition of nigericin and carbonylcyanidem-chlorophenylhydrazone. These results suggest that phthalate is a nonphysiological substrate for a carrier system which mediates transport via an anion/H⁺ symport mechanism. This system is not the lactate/H⁺ symport carrier of L1210 cells since: (A) phthalate and lactate influx were inhibited to differing degrees by various anions; and (B) lactic anhydride inhibited the influx and efflux of lactate but had no effect on the transmembrane movement of phthalate. The specificity of this system suggests that its primary anion substrate may be chloride.     

Solubilization and reconsitution of renal brush border Na+−H+ exchanger

Summary In order to permit future characterization and possible isolation of the Na⁺–H⁺ exchanger from the apical membrane of proximal tubular cells, studies were performed to solubilize and reconstitute this transporter. Rabbit brush border membranes were prepared by a magnesium aggregation method, solubilized with the detergent octyl glucoside, and reconstituted into artificial phospholipid vesicles. In the presence of a pH gradient (pH_in 6.0, pH_out 8.0), the uptake of 1mm ²²Na⁺ into the proteoliposomes was five- to sevenfold higher than into liposomes. Amiloride (2mm) inhibited proton gradient-stimulated uptake of sodium by 50%. As compared to proton gradient conditions, the uptake of sodium was lower in the absence of a pH gradient but was significantly higher when the outside and inside pH was 6.0 than 8.0. TheK _a for sodium in reconstituted proteoliposomes studied under pH gradient conditions was 4mm. The uptake of sodium in proteoliposomes prepared from heat-denatured membrane proteins was significantly decreased. These studies demonstrate that proteoliposomes prepared from octyl glucoside-solubilized brush border membrane proteins and asolectin exhibit proton gradient-stimulated, amiloride-inhibitable, electroneutral uptake of sodium. The ability to solubilize and reconstitute the Na⁺–H⁺ exchanger from the apical membrane of the proximal tubule will be of value in isolating and characterizing this transporter.     

Activation of potassium-dependent H+ efflux from mitochondria by cadmium and phenylarsine oxide

Addition of Cd²⁺ or phenylarsine oxide (PhAsO) to respiring rat liver mitochondria results first in acidification of the medium (H⁺ efflux) followed by disappearance of H⁺ (discharge of the pH gradient or uncoupling). The first phase of H⁺ efflux is dependent upon the presence of K⁺ in the medium, and is not seen in the presence of valinomycin, which is consistent with the conclusion that H⁺ efflux is linked to membrane potential-dependent uptake of K⁺. These effects are abolished by low levels of 2,3-dimercaptopropanol but potentiated by excess of 2-mercaptoethanol, showing involvement of a dithiol type of group in the response. Mersalyl produces only the H⁺ efflux, and subsequent addition of Cd²⁺ or PhAsO produces collapse of the pH.Abbreviations BAL British Anti-Lewisite or 2,3-dimercaptopropanol - 2-ME 2-mercaptoethanol - PhAsO phenylarsine oxide - FCCP carbonylcyanide trifluoromethoxyphenylhydrazone - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid     

Sucrose and proton cotransport in Ricinus cotyledons

In Ricinus cotyledons, evidence for proton extrusion came from observation of direct acidification of the medium in the presence of potassium salts. Increasing K⁺ influx with increasing pH suggested a link between K⁺ influx and H⁺ efflux by an H⁺ pump. The kinetics of K⁺ influx and H⁺ efflux were consistent with a 1:1 stoichiometry K⁺:H⁺, which may indicate either electrical coupling or carrier mediated exchange. The results were consistent with an H⁺ pump setting up an electrochemical potential gradient which provides the driving force for an H⁺-sucrose cotransport and the movement of K⁺. With reference to this, a model for phloem loading is suggested.     

Cl−, Na+, and H+ fluxes during the acidification of rabbit reticulocyte endocytic vesicles

The ionic fluxes associated with the ATP-dependent acidification of endocytic vesicles were studied in a preparation isolated from rabbit reticulocytes enriched for transferrin-transferrin receptor complexes. No vesicle acidification was observed in the absence of intra- and extravesicular ions (sucrose_in/sucrose_out), while maximal acidification was observed with NaCl_in/KCl_out·K _in ⁺ was a poor substitute for Na _in ⁺ , and Cl _out ^– could be replaced by other anions with the following efficacy of acidification: Cl^–>Br^–>I^–>PO ₄ ^3– >gluconate>SO ₄ ^2– . Flux studies using³⁶Cl^– and²²Na⁺ showed that the vesicles had a permeability for Cl^– and Na⁺, and that ATP-dependent H⁺ pumping was accompanied by a net influx of Cl^– and a net efflux of Na⁺ provided that there was a Na⁺ concentration gradient. After 3 mins, the time necessary to maximal acidification, the electrical charge generated by the entrance of H⁺ was countered to about 45% by the Cl^– influx and to about 42% by the Na⁺ efflux. These studies demonstrated that both Cl^– and Na⁺ fluxes are necessary for optimal endocytic vesicle acidification.     

Silver ions induce a rapid Ca2+ release from isolated intact bovine rod outer segments by a cooperative mechanism

Summary Micromolar concentrations of silver ion activate large Ca²⁺ fluxes across the plasma membrane of intact rod outer segments isolated from bovine retinas (intact ROS). The rate of Ag⁺-induced Ca²⁺ efflux from intact ROS depended on the Ag⁺ concentration in a sigmoidal manner suggesting a cooperative mechanism with a Hill coefficient between 2 and 3. At a concentration of 50 m Ag⁺ the rate of Ca²⁺ efflux was 7×10⁶ Ca²⁺/outer segment/sec; this represents a change in total intracellular Ca²⁺ by 0.7mm/outer segment/sec. Addition of the nonselective ionophore gramicidin in the absence of external alkali cations greatly reduced the Ag⁺-induced Ca²⁺ efflux from intact ROS, apparently by enabling internal alkali cations to leak out. Adding back alkali cations to the external medium restored Ag⁺-induced Ca²⁺ efflux when gramicidin was present. In the presence of gramicidin, Ag⁺-induced Ca²⁺ efflux from intact ROS was blocked by 50 m tetracaine orl-cis diltiazem, whereas without gramicidin both blockers were ineffective. Bothl-cis diltiazem and tetracaine are blockers of one kinetic component of cGMP-induced Ca²⁺ flux across ROS disk membranes. The ion selectivity of the Ag⁺-induced pathway proved to be broad with little discrimination between the alkali cations Li⁺, Na⁺, K⁺, and Cs⁺ or between Ca²⁺ and Mg²⁺. The properties of the Ag⁺-induced pathway(s) suggest that it may reflect the cGMP-dependent conductance opened in the absence of cGMP by silver ions.     

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.     

Mechanism of cAMP-induced H+-efflux ofDictyostelium cells: a role for fatty acids

AggregatingDictyostelium cells release protons when stimulated with cAMP. To find out whether the protons are generated by acidic vesicles or in the cytosol, we permeabilized the cells and found that this did not alter the cAMP-response. Proton efflux in intact cells was inhibited by preincubation with the V-type H⁺ ATPase inhibitor concanamycin A and with the plasma membrane H⁺ ATPase blocker miconazole. Surprisingly, miconazole also inhibited efflux in permeabilized cells, indicating that this type of H⁺ ATPase is present on intracellular vesicles as well. Vesicular acidification was inhibited by miconazole and by concanamycin A, suggesting that the acidic vesicles contain both V-type and P-type H⁺ ATPases. Moreover, concanamycin A and miconazole acted in concert, both in intact cells and in vesicles. The mechanism of cAMP-induced Ca²⁺-fluxes involves phospholipase A₂ activity. Fatty acids circumvent the plasma membrane and stimulate vesicular Ca²⁺-efflux. Here we show that arachidonic acid elicited H⁺-efflux not only from intact cells but also from acidic vesicles. The target of regulation by arachidonic acid seemed to be the vesicular Ca²⁺-relase channel.     

Facilitated Transport of Lactate by Rat Jejunal Enterocyte

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K _m of 39.2 ± 4.8 mm and a J _max of 8.9 ± 0.7 nmoles mg protein⁻¹ sec⁻¹. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na⁺ and H⁺ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H⁺-lactate cotransporter, whereas in the apical membrane both H⁺-lactate and Na⁺-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997     

H+/ATP stoichiometry for the gastric (K++H+)-ATPase

Summary The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150mm KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639±84 and 619±65 nmol/min×mg protein, respectively, giving an H⁺/ATP ratio of 1.03±0.7. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (<10 nmol/min×mg protein) was exhibited by vesicles in 150mm NaCl at pH 6.1 despite ATP hydrolysis of 187±46 nmol/min×mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H⁺/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K⁺ greater than 10mm. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H⁺/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K⁺+H⁺)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach.     

Characterization of Na+ transport in normal human fibroblasts and neoplastic H.Ep.2 cells and the role of inhibitin

Summary Na⁺ transport was characterized in normal human fibroblasts and neoplastic H.Ep. 2 cells in order to investigate the role of the endogenous peptidic factor inhibitin that is secreted by a variety of neoplastic cells (including H.Ep. 2) and inhibits Na⁺/Na⁺ exchange in human erythrocytes. Although active (Na⁺, K⁺-ATPase mediated) Na⁺ fluxes were similar in the two cell types, H.Ep. 2 cells maintained higher intracellular Na[su+] concentration (26mm) compared to fibroblasts (12mm). An analysis of passive Na⁺ fluxes showed a difference in the handling of Na⁺ via ouabain and bumetanide-insensitive transport between the two cell types: H.Ep. 2 cells achieved net Na⁺ influx via an amiloride-sensitive pathway that was only demonstrated in fibroblasts when 10% fetal calf serum (FCS) was present. Kinetic studies were undertaken to investigate the interaction between Na⁺ flux via Na⁺/H⁺ and Na⁺/Na⁺ exchanges. for this purpose, an outwardly directed Na⁺ gradient was created by loading the cells with Na⁺ (Na _i >100mm) to activate the reverse functioning of Na⁺/H⁺ exchange (i.e., Na _out ⁺ H _in ⁺ ). The rates of ouabain-and bumetanide-insensitive Na⁺ efflux were measured over a range of extracellular Na⁺ concentrations (Na _o ⁺ 14–140mm). In the presence of 10% FCS, the two cell types showed different responses: in fibroblasts the Na⁺ efflux rate showed an inverse correlation with extracellular Na⁺ concentration, while H.Ep. 2 cells significantly increased their rate of Na⁺ efflux as extracellular Na⁺ concentration increased. So although the thermodynamic force would direct net Na⁺ efflux when Na _i ⁺ >Na _o ⁺ , H.Ep.2 cells were under kinetic control to perform Na⁺/Na⁺ exchange.When exogenous inhibitin was tested on fibroblasts, the steady-state intracellular Na⁺ concentration increased from 14 to 19mm (p<0.01). In Na⁺-loaded fibroblasts, serum-stimulated Na⁺ efflux was partially inhibitin sensitive and the maximal inhibitory effect was seen when extracellular Na⁺ concentration was 14mm and presumably the Na⁺/H⁺ exchanger operating in the reverse mode. This study demonstrated that, in contrast to fibroblasts, H.Ep.2 cells have a modified Na⁺/H⁺ exchange system whereby it acts in the Na _in ⁺ H _out ⁺ mode without exogenous growth factor activation and resists functioning in the reversed mode. It is proposed that inhibitin, is the endogenous modifier of this transport system in H.Ep.2 cells with the result that H.Ep.2 cells maintain a higher concentration of intracellular Na⁺ compared to fibroblasts.     

Serosal Na/Ca exchange and H+ and Na+ transport by the turtle and toad bladders

Summary A Na/Ca exchange system has been described in the plasma membrane of several tissues and seems to regulate the concentration of calcium in cytosol. Replacement of extracellular Na by sucrose increases calcium uptake into and decreases calcium efflux from the cell, leading to an increase in cytosolic calcium. The effect of an increase in cytosolic calcium mediated by the Na/Ca exchange system on H⁺ and Na transport in the turtle and toad bladder was investigated by replacing serosal Na isosmotically by sucrose or choline. Replacement of serosal by sucrose was associated with a significant inhibition of H⁺ secretion or Na transport which was reversible by addition of NaCl. Replacement of mucosal Na by sucrose failed to alter H⁺ secretion. Removal of serosal Na was associated with a significant increase in⁴⁵Ca uptake which could be blocked by pretreatment with lanthanum chloride. Pretreatment with lanthanum chloride blunted the inhibitory effect of replacement of serosal Na by sucrose on H⁺ and Na transport, thus suggesting that the increase in calcium uptake and the inhibition of transport are causally related. Under anaerobic conditions the rate of H⁺ or Na transport are linked to the rate of lactate production. The inhibition of Na or H⁺ transport by removal of serosal Na was accompanied by a proportional decrease in lactate production, thus suggesting that an increase in cytosolic calcium does not inhibit transport by uncoupling glycolysis from transport. Replacement of serosal Na by sucrose did not alter the force of the H⁺ or Na pump but led to an increase in resistance of the active pathway of H⁺ and Na transport. The inhibition of Na transport by replacement of serosal Na with sucrose could be reversed by addition of amphotericin B, an agent which increases luminal permeability to Na, thus suggesting that decreased Na entry across the apical membrane is the mechanism responsible for the inhibition of Na transport. The results of the present studies strongly suggest that an increase in cytosolic calcium through the serosal Na/Ca exchange system inhibits H⁺ and Na transport in the turtle and toad bladder probably by increasing the resistance of the luminal membrane.     

Glucose inhibits Ca efflux from pancreatic β-cells also in the absence of Na-Ca countertransport

During perifusion with medium deprived of Ca²⁺, addition of glucose or omission of Na⁺ resulted in prompt and quantitatively similar inhibitions of ⁴⁵Ca efflux from β-cell rich pancreatic islets microdissected from ob / ob mice. Glucose had no additional inhibitory effect when Na⁺ was isoosmotically replaced by sucrose or choline⁺. When K⁺ was used as a substitute for Na⁺, the inhibitory effect of Na⁺ removal on ⁴⁵Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting ⁴⁵Ca efflux in the presence or absence of Na⁺ is difficult to reconcile with the postulate that the Na⁺-Ca²⁺ countertransport mechanism is a primary site of action for glucose.     

Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick

Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H⁺ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K⁺ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H⁺ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na⁺/H⁺ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na⁺ gradient. The kinetics of the Na⁺/H⁺ exchange reaction at pH 6.0 [K _t for Na⁺=57mm,V _max=42 mmol H⁺/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K _t for Na⁺=15mm,V _max=1.7 mmol H⁺/liter 3OMG space/min). Experiments involving imposed K⁺ gradients suggest that these cells have negligible K⁺/H⁺ exchange capability. They exhibit limited but measurable H⁺ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate.     

Role of the sodium pump and the background K+ channel in passive K+(Rb+) uptake by isolated cardiac sarcolemmal vesicles

Summary A simple procedure was developed for the isolation of a sarcolemma-enriched membrane preparation from homogenates of bullfrog (Rana catesbeiana) heart. Crude microsomes obtained by differential centrifugation were fractionated in Hypaque density gradients. The fraction enriched in surface membrane markers consisted of 87% tightly sealed vesicles. The uptake of⁸⁶Rb⁺ by the preparation was measured in the presence of an opposing K⁺ gradient using a rapid ion exchange technique. At low extravesicular Rb⁺ concentrations, at least 50% of the uptake was blocked by addition of 1mm ouabain to the assay medium. Orthovanadate (50 m), ADP (2.5mm), or Mg (1mm) were also partial inhibitors of Rb⁺ uptake under these conditions, and produced a complete block of Rb⁺ influx in the presence of 1mm ouabain. When⁸⁶Rb⁺ was used as a tracer of extravesicular K⁺ (Rb ₀ ⁺ 40 m K ₀ ⁺ =0.1–5mm) a distinct uptake pathway emerged, as detected by its inhibition by 1mm Ba²⁺ (K _0.5=20 m). At a constant internal K⁺ concentration (K _in ⁺ =50mm) the magnitude of the Ba²⁺-sensitive K⁺ uptake was found to depend on K ₀ ⁺ in a manner that closely resembles the K⁺ concentration dependence of the background K⁺ conductance (I _Kl) observed electrophysiologically in intact cardiac cells. We conclude that K⁺ permeates passively this preparation through two distinct pathways, the sodium pump and a system identifiable as the background potassium channel.