Electrogenic 2 Na+/1 H+ exchange in crustanceans

Summary Hepatopancreatic brush border membrane vesicles of the freshwater prawn,Macrobrachium rosenbergii and the marine lobster,Homarus americanus exhibited²²Na uptake which was Cl-independent, amiloride sensitive, and stimulated by a transmembrane H gradient (H _i >H _o ). Sodium influx by vesicles of both species were sigmoidal functions of [Na] _o , yielding Hill coefficients that were not significantly different (P>0.5) than 2.0. Estimations of half-saturation constants (K _Na) were 82.2mm (prawn) and 280.1mm (lobster), suggesting a possible adaptation of this transporter to environmental salinity.Trans-stimulation andcis-inhibition experiments involving variable [H] suggested that the exchangers in both species possessed single internal cation binding sites (pK 6.5–6.7) and two external cation binding sites (prawn, pK 4.0 and 5.7; lobster pK 3.5 and 6.1). Similarcis inhibition studies using amiloride as a competitive inhibitor of Na uptake supported the occurrence of dual external sites (prawn,K _i 50 and 1520 m; lobsterK _i 9 and 340 m). Electrogenic Na/H exchange by vesicles from both crustaceans was demonstrated using equilibrium shift experiments where a transmembrane potential was used as the only driving force for the transport event. Transport stoichiometries of the antiporters were determined using Static Head analysis where driving forces for cation transfer were balanced using a 101 Na gradient, a 1001 H gradient, and a stoichiometry of 2.0. These electrogenic 2 Na/1 H exchangers appear thermodynamically capable of generating sufficient gastric acidification for organismic digestive activities.     

A Ca-dependent K channel in “luminal” membranes from the renal outer medulla

Summary This paper describes properties of⁸⁶Rb fluxes through K channels in luminal membrane vesicles prepared from rabbit renal outer medulla. By measuring⁸⁶Rb uptake against an opposing chemical gradient of K ions, using membranes loaded with KCl, a transient accumulation of isotope is observed, which is blocked by Ba ions. This is the behavior expected of a conductive Rb flux through a Ba-sensitive K channel. The⁸⁶Rb accumulation is driven by an electrical diffusion potential as shown in experiments using either vesicles loaded with different anions, or an outwardly directed Li gradient with a Li ionophore. The vesicles containing the channel show a cation selectivity with the order Rb > K > Cs > Li > Na > choline. The Ba-sensitive Rb flux is dependent on Ca within the vesicles, with a very high affinity estimated asK _0.5 10 to 100nm. The vesicles appear to be right-side-out. The Ba-sensitive⁸⁶Rb uptake is also inhibited by quinineK _0.5 30 m but is insensitive to tetraethyl ammonium ions and apamin. These isotope flux experiments complement electrophysiological experiments in providing independent evidence for the existence of K channels in the luminal surface of cells of this ascending limb of the loop of Henle. The very high Ca affinity suggests that cytoplasmic Ca could play an important role in regulation of transepithelial salt flux in this region of the nephron.     

Protein kinase C activates the renal apical membrane Na+/H+ exchanger

Summary Studies were performed on purified brush-border membranes from the kidney of the rabbit to examine the relation between protein kinase C and the Na⁺/H⁺ exchanger in these membranes. The brush-border membranes were transiently opened by exposure to hypotonic media and the membrane proteins phosphorylated by exposure to ATP and phorbol esters or partially purified protein kinase C. The membranes were resealed and the intravesicular space acidified by incubation in a sodium-free isotonic solution (pH 5.5). The rate of uptake of 1mm ²²Na⁺ (pH 7.5), with and without amiloride (1mm), was assayed and the proton gradient-stimulated, amiloride-inhibitable component of²²Na⁺ taken as a measure of the activity of the Na⁺/H⁺ exchanger. 12-0-tetradecanoyl phorbol-13-acetate (TPA) increased the amiloride-sensitive component of²²Na⁺ uptake TPA did not affect the amiloride-insensitive component of²²Na⁺ uptake or the equilibrium concentration of sodium. TPA also did not affect the rate of dissipation of the proton gradient in the absence of sodium or the rate of sodium-dependent or-independent uptake ofd-glucose. Other active phorbol esters stimulated the rate of Na⁺/H⁺ exchange, but phorbol esters of the 4 configuration did not. Incubation of the opened membranes in partially purified protein kinase C increased the rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake. The stimulatory effect of TPA and protein kinase C was not additive. In the absence of ATP, neither TPA nor protein kinase C affected Na⁺/H⁺ exchange transport. To determine the membrane-bound protein substrates, parallel experiments were conducted with -[³²P] ATP in the phosphorylating solutions. The reaction was stopped by SDS and the phosphoproteins resolved by PAGE and autoradiography. TPA stimulation of protein kinase C resulted in phosphorylation of approximately 13 membrane-bound proteins ranging in apparent molecule from 15,000 to 140,000 daltons. These studies indicate that activation of endogenous renal brush-border protein kinase C by phorbol esters or exposure of these membranes to exogenous protein kinase C increases the rate of proton gradient-stimulated, amiloride-inhibitable sodium transport. Protein kinase C activation also results in phosphorylation of a finite number of membrane-bound proteins.     

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.     

Ion specificity of cardiac sarcolemmal Na+/H+ antiporter

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.     

Volume- and catecholamine-dependent regulation of Na/H antiporter and unidirectional potassium fluxes in Salmo trutta red blood cells

β-Adrenergic- and volume-dependent regulation of ²²Na influx and ⁸⁶Rb influx and efflux in erythrocytes of brown trout (Salmo trutta m. lacustris) were studied. Norepinephrine (10^-6 mol·1^-1) increased the rate of ²²Na influx 10-to 20-fold via the activation of a Na/H exchanger (ethyl isopropyl amiloride inhibited component of ²²Na influx). Unlike carp erythrocytes the activity of the Na, K-pump (ouabain-inhibited ⁸⁶Rb influx) was only slightly (25–35%) increased by norepinephrine. The norepinephrine-induced increment of Na, K-pump activity was completely abolished by ethyl isopropyl amiloride thus indicating that this effect was mediated by Na/H exchanger-induced increase of intracellular Na⁺ concentration. Cell shrinkage in hyperosmotic media resulted in a several-fold activation of the Na/H exchanger. Cell swelling in hypotonic media increased both the rate of K, Cl-cotransport [((dihydroindenyl)oxy)alcanaic acidsensitive components of ⁸⁶Rb influxe and efflux] and passive permeability (leakage) of erythrocyte membranes for Na⁺ and K⁺. No volume-dependent regulation of Na, K, 2Cl-cotransport (bumetanide-sensitive components of ⁸⁶Rb fluxes) was found. It may be concluded that the regulation of monovalent cation transport in erythrocytes of fast-moving (carnivorous) brown trout differs essentially from that in slowly moving (herbivorous) carp.     

Ratio of Na:K transport in reconstituted sodium pump vesicles

The effect of ATP on the kinetics of Na and K fluxes across the membranes of reconstituted sodium pump vesicles was examined. In the absence of ATP, the active vesicles equilibrated with ⁴²K or ⁸⁶Rb within 6 hours. In contrast, the equilibration of intravesicular Na with external ²²Na was about 4 times slower. In the presence of ATP, the intravesicular K was replaced within 3 min by Na $\text{via}$ a Na:K exchange process. The total intravesicular Na pool was then labeled to the same specific radioactivity as the Na of the medium $\text{via}$ a Na:Na exchange process. The Na:K transport ratio varied with the intravesicular concentrations of Na and K.     

Ontogeny of the Na+−H+ exchanger in rat ileal brush-border membrane vesicles

Summary The developmental maturation of Na⁺–H⁺ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na⁺ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside=5.2/7.5) significantly stimulated Na⁺ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na⁺ uptake which increased with advancing age. Na⁺ uptake represented an electroneutral process.The amiloride sensitivity of the pH-stimulated Na⁺ uptake was investigated using [amiloride] 10^–2–10^–5 m. At 10^–3 m amiloride concentration, Na⁺ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed aK _m for amiloride-sensitive Na⁺ uptake of 21.8±6.4, 24.9±10.9 and 11.8±4.17mm andV _max of 8.76±1.21, 5.38±1.16 and 1.99±0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na⁺–H⁺ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters.     

Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles

Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H⁺ gradient (pH_in=6.0, pH_out=8.0)²²Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pH_in=pH_out=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K ₁ =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K⁺-stimulatedp-nitrophenyl phosphatase. In addition²²Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H⁺ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK _M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na⁺/H⁺ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.     

Further photophysical and photochemical characterization of flavins associated with single-shelled vesicles

Summary In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands ofSqualus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170mm KCl gradient was initially about five-fold higher than in the presence of a 170mm KNO₃ gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4mm bumetanide and 40% by 0.8mm N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170mm NaCl gradient than in the presence of a 170mm NaNO₃ gradient. The effect of chloride was completely abolished by 0.4mm bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound³[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparentK _D of 3.5×10^–6 m and a number of sitesn of 104 pmol/mg protein. At 0.8 m, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600mm or when 1mm bumetanide or furosemide were added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland.     

Volume-dependent regulation of ion transport and membrane phosphorylation in human and rat erythrocytes

Summary Osmotic swelling of human and rat erythrocytes does not induce regulatory volume decrease. Regulatory volume increase was observed in shrunken erythrocytes of rats only. This reaction was blocked by the inhibitors of Na⁺/H⁺ exchange. Cytoplasmic acidification in erythrocytes of both species increases the amiloride-inhibited component of²²Na influx by five- to eight-fold. Both the osmotic and isosmotic shrinkage of rat erythrocytes results in the 10- to 30-fold increase of amiloride-inhibited²²Na influx and a two-fold increase of furosemide-inhibited⁸⁶Rb influx. We failed to indicate any significant changes of these ion transport systems in shrunken human erythrocytes. The shrinking of quin 2-loaded human and rat erythrocytes results in the two- to threefold increase of the rate of⁴⁵Ca influx, which is completely blocked by amiloride. The dependence of volume-induced²²Na influx in rat erythrocytes and⁴⁵Ca influx in human erythrocytes on amiloride concentration does not differ. The rate of⁴⁵Ca influx in resealed ghosts was reduced by one order of magnitude when intravesicular potassium and sodium were replaced by choline. It is assumed that the erythrocyte shrinkage increases the rate of a nonselective Ca _o ²⁺ (Na _i ⁺ , K _i ⁺ ) exchange. Erythrocyte shrinking does not induce significant phosphorylation of membrane protein but increases the³²P incorporation in diphosphoinositides. The effect of shrinkage on the³²P labeling of phosphoinositides is diminished after addition of amiloride. It is assumed that volume-induced phosphoinositide response plays an essential role in the mechanism of the activation of transmembrane ion movements.     

Evidence for two compartments of exchangeable calcium in isolated rat liver mitochondria obtained using a45Ca exchange technique in the presence of magnesium,phosphate, and ATP at 37°C

Summary The distribution of calcium between isolated rat liver mitochondria and the extramitochondrial medium at 37°C and in the presence of 2mm inorganic phosphate, 3mm ATP, 0.05 or 1.1mm free magnesium and a calcium buffer, nitrilotriacetic acid, was investigated using a⁴⁵Ca exchange technique. The amounts of⁴⁰Ca in the mitochondria and medium were allowed to reach equilibrium before initiation of the measurement of⁴⁵Ca exchange. At 0.05mm free magnesium and initial extramitochondrial free calcium concentrations of between 0.15 and 0.5 m, the mitochondria accumulated calcium until the extramitochondrial free calcium concentration was reduced to 0.15 m. Control experiments showed that the mitochondria were stable under the incubation conditions employed. The⁴⁵Ca exchange data were found to be consistent with a system in which two compartments of exchangeable calcium are associated with the mitochondria. Changes in the concentration of inorganic phosphate did not significantly affect the⁴⁵Ca exchange curves, whereas an increase in the concentration of free magnesium inhibited exchange. The maximum rate of calcium outflow from the mitochondria was estimated to be 1.7 nmol/min per mg of protein, and the value ofK _0.5 for intramitochondrial exchangeable calcium to be about 1.6 nmol per mg of protein. Ruthenium Red decreased the fractional transfer rate for calcium inflow to the mitochondria while nupercaine affected principally the fractional transfer rates for the transfer of calcium between the two mitochondrial compartments. The use of the incubation conditions and⁴⁵Ca exchange technique described in this report for studies of the effects of agents which may alter mitochondrial calcium uptake or release (e.g., the pre-treatment of cells with hormones) is briefly discussed.     

Influence of membrane potential on the sodium-dependent uptake of gamma-aminobutyric acid by presynaptic nerve terminals: Experimental observations and theoretical considerations

Summary Sodium, potassium and veratridine were tested for their effects on the uptake of gamma-aminobutyric acid (GABA) by pinched-off presynaptic nerve terminals (synaptosomes). As noted by previous investigators, the uptake from media containing 1 m GABA (high-affinity uptake) is markedly Na-dependent; the uptake averaged 65 pmoles/mg synaptosome protein × min, with [Na]₀=145mm and [K]₀=5mm, and declined by about 90% when the external Na concentration ([Na]₀) was reduced to 13mm (Na replaced by Li). The relationship between [Na]₀ and GABA uptake was sigmoid, suggesting that two or more Na⁺ ions may be required to activate the uptake of one GABA molecule. Thermodynamic considerations indicate that with a Na⁺/GABA stoichiometry of 21, the Na electrochemical gradient, alone, could provide sufficient energy to maintain a maximum steady-state GABA gradient ([GABA] _i /[GABA]₀) of about 10⁴ across the plasma membrane of GABA-nergic terminals.In Ca-free media with constant [Na]₀, GABA uptake was inhibited, without delay, by increasing [K]₀ or by introducing 75 m veratridine; the effect of veratridine was blocked by 200nm tetrodotoxin. The rapid onset (within 10 sec) of the veratridine and elevated-K effects implies that alterations in intra-terminal ion concentrations are not responsible for the inhibition. The uptake of GABA was inversely proportional to log [K]₀. These observations are consistent with the idea that the inhibitory effects of both veratridine and elevated [K]₀ may be a consequence of their depolarizing action. The data are discussed in terms of a barrier model (Hall, J. E., Mead, C.A., Szabo, G. 1973.J. Membrane Biol. 11:75) which relates carrier-mediated ionic flux to membrane potential.     

Amiloride-blockable sodium currents in isolated taste receptor cells

Summary Isolated taste receptor cells from the frog tongue were investigated under whole-cell patch-clamp conditions. With the cytosolic potential head at –80 mV, more than 50% of the cells had a stationary inward Na current of 10 to 700 pA in Ringer's solution. This current was in some cells partially, in others completely, blockable by low concentrations of amiloride. With 110mm Na in the external and 10mm Na in the internal solution, the inhibition constant of amiloride was (at –80 mV) near 0.3 m. In some cells the amiloride-sensitive conductance was Na specific; in others it passed both Na and K. The Na/K selectivity (estimated from reversal potentials) varied between 1 and 100. The blockability bysmall concentrations of amiloride resembled that of channels found in some Na-absorbing epithelia, but the channels of taste cells showed a surprisingly large range of ionic specificities. Receptor cells, whichin situ express these channels in their apical membrane, may be competent to detect the taste quality salty. The same cells also express TTX-blockable voltage-gated Na channels.     

Characteristics of amino acid accumulation by isolated intestinal epithelial cells

Summary Accumulation of neutral amino acids by isolated chick epithelial cells has been studied and the results discussed in terms of the ion gradient model, and a model invoking a direct input of metabolic energy. The cells establish four- to eightfold concentration gradients of amino acids at an extracellular concentration of 1mm. The accumulation is sodium-dependent, inhibited by high extracellular potassium concentrations, and is sensitive to a variety of metabolic inhibitors. Also, amino acid uptake is depressed by actively transported sugars, and certain other amino acids, and is stimulated by phloridzin.Cells equilibrated with valine and loaded with 30 to 40mm intracellular sodium begin immediately to actively accumulate valine when suddenly introduced to media containing 20mm sodium. The cells establish a threefold gradient of amino acid during the interval when intracellular sodium is higher than extracellular sodium.Amino acid accumulation and²²Na efflux were monitored simultaneously in cells treated with phloridzin. While phloridzin causes a 30% stimulation of amino acid uptake, no variation in the rate of²²Na efflux or the steady-state level of²²Na maintained by the cells can be detected. Similarly, either 2.5mm glucose or 2.5mm 3-O-methylglucose cause approximately a 50% inhibition of 1mm valine uptake, but no detectable change in steady-state cellular²²Na content. Several aspects of the data seem inconsistent with concepts embodied in the ion gradient hypothesis, and it is suggested that a directly energized transport mechanism can better accommodate all of the data.     

Electrogenic and electroneutral transport modes of renal Na/K ATPase reconstituted into proteoliposomes

Summary This paper describes measurements of electrical potentials generated by renal Na/K-ATPase reconstituted into proteoliposomes, utilizing the anionic dye, oxonol VI. Calibration of absorption changes with imposed diffusion potentials allows estimation of absolute values of electrogenic potentials.ATP-dependent Na_cyt/K_exc exchange in K-loaded vesicles generates large potentials, up to 250 mV. By comparing initial rates or steady-state potentials with ATP-dependent²²Na fluxes in different conditions, it is possible to infer whether coupling ratios are constant or variable. For concentrations of Na_cyt (2–50mm) and ATP (1–1000 m) and pH's (6.5–8.5), the classical 3Na_cyt/2K_exc coupling ratio is maintained. However, at low Na_cyt concentrations (<0.8mm), the coupling ratio is apparently less than 3Na_cyt/2K_exc.ATP-dependent Na_cyt/congener_exc exchange in vesicles loaded with Rb, Cs, Li and Na is electrogenic. In this mode congeners, including Na_exc, act as K_exc surrogates in an electrogenic 3Na_cyt/2congener_exc exchange. (ATP+Pi)-dependent K_cyt/K_exc exchange in K-loaded vesicles is electroneutral.ATP-dependent uncoupled Na flux into Na- and K-free vesicles is electroneutral at pH 6.5–7.0 but becomes progressively electrogenic as the pH is raised to 8.5. The²²Na flux shows no anion specificity. We propose that uncoupled Na flux is an electroneutral 3Na_cyt/3H_exc exchange at pH 6.5–7.0 but at higher pH's the coupling ratio changes progressively, reaching 3Na/no ions at pH 8.5. Slow passive pump-mediated net K uptake into Na- and K-free vesicles is electroneutral, and may also involve K_cyt/H_exc exchange.We propose the general hypothesis that coupling ratios are fixed when cation transport sites are saturated, but at low concentrations of transported cations, e.g., Na_cyt in Na/K exchange and H_exc in uncoupled Na flux, coupling ratios may change.     

Potassium transport across the frog retinal pigment epithelium

Summary Previous experiments indicate that the apical membrane of the frog retinal pigment epithelium contains electrogenic NaK pumps. In the pressent experiments net potassium and rubidium transport across the epithelium was measured as a function of extracellular potassium (rubidium) concentration, [K] _o ([Rb] _o ). The net rate of retina-to-choroid⁴²K(⁸⁶Rb) transport increased monotonically as [K] _o ([Rb] _o ), increased from approximately 0.2 to 5mm on both sides of the tissue or on the apical (neural retinal) side of the tissue. No further increase was observed when [K] _o ([Rb] _o ) was elevated to 10mm. Net sodium transport was also stimulated by elevating [K] _o . The net K transport was completely inhibited by 10^–4 m ouabain in the solution bathing the apical membrane. Ouabain inhibited the unidirectional K flux in the direction of net flux but had not effect on the back-flux in the choroid-to-retina direction. The magnitude of the ouabain-inhibitable⁴²K(⁸⁶Rb) flux increased with [K] _o ([Rb] _o ). These results show that the apical membrane NaK pumps play an important role in the net active transport of potassium (rubidium) across the epithelium. The [K] _o changes that modulate potassium transport coincide with the light-induced [K] _o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium.     

KCl transport across and insect epithelium: I. Tracer fluxes and the effects of ion substitutions

Summary Models for active Cl transport across epithelia are often assumed to be universal although they are based on detailed studies of a relatively small number of epithelia from vertebrate animals. Epithelial Cl transport is also important in many invertebrates, but little is known regarding its cellular mechanisms. We used short-circuit current, tracer fluxes and ion substitutions to investigate the basic properties of Cl absorption by locust hindgut, an epithelium which is ideally suited for transport studies. Serosal addition of 1mm adenosine 35-cyclic monophosphate (cAMP), a known stimulant of Cl transport in this tissue, increased short-circuit current (I _sc) and net reabsorptive³⁶Cl flux (J _net ^Cl ) by 1000%. Cl absorption did not exhibit an exchange diffusion component and was highly selective over all anions tested except Br. Several predictions of Na- and HCO₃-coupled models for Cl transport were tested: Cl-dependentI _sc was not affected by sodium removal (<0.05mm) during the first 75 min. Also, a large stimulation ofJ _net ^Cl was elicited by cAMP when recta were bathed for 6 hr in nominally Na-free saline (<0.001 to 0.2mm) and there was no correlation between Cl transport rate and the presence of micromolar quantities of Na contamination. Increased unidirectional influx of³⁶Cl into rectal tissue during cAMP-stimulation was not accompanied by a comparable uptake of²²Na.J _net ^Cl was independent of exogenous CO₂ and HCO₃, but was strongly dependent on the presence of K. These results suggest that the major fraction of Cl transport across this insect epithelium occurs by an unusual K-dependent mechanism that does not directly require Na or HCO₃.     

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.     

Basolateral Cl/HCO3 exchange in rat jejunum: The effect of sodium

Basolateral membrane vesicles isolated from rat jejunum were used to characterize a Cl/HCO₃ exchange mechanism previously evidenced. Cl uptake experiments provided no evidence for Cl/OH countertransport, confirming anyhow the presence of Cl/HCO₃ antiport, which was inhibited by 2 mm furosemide and unaffected by 2 mm amiloride. An outwardly directed Na gradient stimulated Cl uptake and this effect was increased if Na was present at both vesicle surfaces. To investigate the mechanism of coupling between Na and the transport protein, we performed Na uptake experiments. Na uptake was unaffected by cis-bicarbonate and trans-Cl gradients; the reversal of anion gradients was still ineffective. Similar results were obtained when a pH difference across the membrane vesicles was imposed. This study seems to suggest that Na is not transported by the Cl/HCO₃ exchanger and that another mode of Na dependence must be taken into account.