Amiloride-sensitive sodium transport in lamprey red blood cells: evidence for two distinct transport pathways

To determine Na+/H+ exchange in lamprey erythrocyte membranes, the cells were acidified to pH(i) 6.0 using the K+/H+ ionophore nigericin. Incubation of acidified erythrocytes in a NaCl medium at pH 8.0 caused a considerable rise in 22Na+ influx and H+ efflux during the first 1 min of exposure. In addition, exposure of acidified red cells to NaCl medium was associated with rapid elevation of intracellular Na+ content. The acid-induced changes in Na+ influx and H+ efflux were almost completely inhibited by amiloride and dimethylamiloride. In native lamprey erythrocytes, amiloride-sensitive Na+ influx progressively increased as the osmolality of incubation medium was increased by addition of 100, 200, or 300 mmol/l sucrose. Unexpectedly, the hypertonic stress induced a small, yet statistically significant decrease in intracellular Na+ content in these cells. The reduction in the cellular Na+ content increased with hypertonicity of the medium. The acid- and shrinkage-induced Na+ influxes were inhibited by both amiloride and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) in a dose-dependent manner. For both blockers, the half-maximal inhibitory values (IC50) were much greater for the shrinkage-induced (44 and 15 micromol/l for amiloride and EIPA, respectively) than for the acid-induced Na+ influx (5.1 and 3.3 micromol/l, respectively). The data obtained are the first demonstration of the presence of a Na+/H+ exchanger with high activity in acidified (pH(i) 6.0) lamprey red blood cells (on average, 512 +/- 56 mmol/l cells/h, n = 13). The amiloride-sensitive Na+ influxes produced by hypertonic cell shrinkage and acid load are likely to be mediated by distinct ion transporters in these cells.     

Anion movements across lamprey (Lampetra fluviatilis) red cell membrane

Chloride and bicarbonate movements across lamprey red cell membrane were investigated. The halftime for equilibration of radioactive chloride across the red cell membrane was 2.46 h, and apparent permeability for chloride-36 was approximately 10(-9) cm X s-1, a value similar to that observed for lipid bilayers. Chloride movements were not affected by the anion exchange inhibitor, 4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid (DIDS). Furthermore, intracellular buffering is effectively isolated from the extracellular compartment, as shown by the fact that practically no pH recovery occurred in the unbuffered extracellular medium after either acidification or alkalinization. These observations show that lamprey red cell membrane is quite impermeable to bicarbonate and other acid/base equivalents.     

Sodium and calcium transport in cat red cells.

The transport of radioactive sodium and calcium in high sodium low potassium cat red blood cells has been studied under various experimental conditions. In these cells, calcium uptake was found to increase by 25-fold when cell volume was decreased from 1.0 normal to 0.55. Increasing cell volume from 1.0 to 1.1 normal was found to decrease calcium-uptake by 30%. N-ethylmaleimide, NEM, phloretin, colchicine, and vinblastine were found to inhibit Na-uptake by these cells. Among these four agents, phloretin was the most potent inhibitor of Na-influx. It appears that phloretin produces its effect through its interaction with the cell membrane and not with cell interior.     

Malonate transport in human red blood cells

Kinetic parameters of [2-¹⁴C]malonate uptake by the human erythrocyte membrane have been determined as K_m, 24 mM and turnover number, 5 × 10⁴ s^–1. The translocation of this organic dianion is concentration, pH and temperature dependent. Competitive inhibition of malonate uptake by eosin and inorganic anions, strongly implies that a common route exists for both inorganic anions and organic dianions, namely the anion-exchange Band 3 protein. ¹⁴C-Malonate which is nonmetabolized in the erythrocyte, could be a useful probe for monitoring anion-exchange in reconstituted Band 3 systems.     

The intestinal blood circulation in the River lamprey, Lampetra fluviatilis

Serial sections, injections with india ink and latex, and observations on fresh material, have been used to determine the pattern of blood circulation within the intestine of larval and adult lampreys. Attention has also been paid to resolving the variable terms previously applied to many of the blood vessels, and to the possible functional significance of the differences found between the two life cycle stages. In the larva, the main arterial supply to the intestine consists of a typhlosolar artery, while the venous return is comprised of a posterior and a left and right anterior intestinal vein that usually unite before entering the liver. Although a typhlosolar artery is also present in the adult, the main venous return of the ammocoete is replaced at metamorphosis by a newly formed typhlosolar vein. Moreover, in the ammocoete a considerable amount of blood is discharged into the haemopoietic sponge-work of the typhlosole and the arterial supply to the intestine is poorly developed. By contrast, the typhlosolar sponge-work is lost in the adult and a more efficient arterial supply is developed within the lamina propria of the various intestinal regions. Furthermore, vascular couples are developed in the adult which facilitate the flow of blood in opposite directions in the intestinal wall. Since, during both life cycle stages, the arterial blood passes into tissue spaces, there is no true capillary network in the intestine and no evidence was found for the presence of a lymphatic system. It is suggested that the changes which take place in the intestinal blood supply and the internal structure of the gut during metamorphosis result in improvements both to the vascular system and to the assimilation efficiency.     

Sodium and potassium content and membrane transport properties in red blood cells from newborn puppies

The intracellular sodium and potassium concentrations and membrane transport properties for these ions were investigated in red blood cells from newborn puppies and adult dogs. At birth the intracellular concentrations of sodium and potassium are much higher than those found in adult dog red cells. During the first few weeks of life the intracellular concentrations of these ions gradually decrease until the adult level is reached. Changes in the membrane transport properties develop concurrently. The rate of active potassium influx, as measured by ouabain-sensitivity, and the pump to leak ratio are greater in red cells from newborn puppies than in those from adult animals. No ouabain-sensitive sodium efflux could be demonstrated in red cells from older puppies or adult dogs. When either puppy or adult dog red cells are depleted of ATP (by incubation at 37°C with no substrate), potassium permeability increases, and the permeability of the membrane to sodium decreases. The addition of adenosine reverses the effect of depletion.     

Kinetic properties of sodium transport pathways in the river lamprey Lampetra fluviatilis erythrocytes

To activate Na+/H+ exchange, intracellular pH (pHi) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 to 8 using nigericin. The Na+/H+ exchanger activity was estimated from the values of amiloride-sensitive components of Na+ (22Na) inflow or of H+ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na+ and H+ transport on pHi value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9-10 mmol/l cells/min, and the H+ concentration producing the exchanger 50% activation amounted to 0.6-1.0 microM. Stimulation of H+ outcome from acidified erythrocytes (pHi 5.9) with increase of H+ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration of Na+: for the semimaximal stimulation of H+ outcome amounted to 19 mM. The obtained results indicate the presence in lamprey erythrocytes of only one binding site for H+ from the cytoplasm side and the presence of positive cooperativity in Na+ binding from the extracellular side of the Na+/H+ exchanger. Its efflux from cells in the Na+ -free medium did not change at a 10-fold increase of H+ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na+/H+ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na+ inflow on its concentration in the medium is described by Hill equation with n 1.5. The Na+ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm the concept of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

Sodium and calcium movements in dog red blood cells

Determinants of 45Ca influx, 45Ca efflux, and 22Na efflux were examined in dog red blood cells. 45Ca influx is strongly influenced by the Na concentration on either side of the membrane, being stimulated by intracellular Na and inhibited by extracellular Na. A saturation curve is obtained when Ca influx is plotted as a function of medium Ca concentration. The maximum Ca influx is a function of pH (increasing with greater alkalinity) and cell volume (increasing with cell swelling). Quinidine strongly inhibits Ca influx. Efflux of 45Ca is stimulated by increasing concentrations of extracellular Na. 22Na efflux is stimulated by either Ca or Na in the medium, and the effects of the two ions are mutually exclusive rather than additive. Quinidine inhibits Ca-activated 22Na efflux. The results are considered in terms of a model for Ca-Na exchange, and it is concluded that the system shows many features of such a coupled ion transport system. However, the stoichiometric ratio between Ca influx and Ca-dependent Na efflux is highly variable under different experimental conditions. Because the Ca fluxes may reflect a combination of ATP-dependent, outward transport and Na-linked passive movements, the true stoichiometry of an exchanger may not be ascertainable in the absence of a specific Ca pump inhibitor. The meaning of these observations for Ca-dependent volume regulation by dog red blood cells is discussed.     

Volume-activated transport systems in dog red blood cells

1. When dog red blood cells are shrunken or swollen, transport pathways are activated that do not function discernibly when the cell is at normal volume. Swelling the cells turns on two pathways, a Ca-Na exchanger and a Cl-dependent K pathway. 2. Shrinking the cells activates a Na-H antiporter. 3. The passive net flow of ions through these transporters is in such a direction as to correct the perturbation of cell volume: when the cell water content has returned to normal, the transporters turn off. 4. Recently we have investigated agents that can lock or fix the volume-responsive transporters in the activated state. Na-H exchange, for example, can be fixed in the on position with either glutaraldehyde or N-phenylmaleimide. 5. Ca-Na exchange can be locked on by the sulfhydryl-oxidizing agent, diamide. We have used these effects to investigate the relationships between cell volume and the transport mechanisms. 6. It is possible, for instance, to distinguish whether certain inhibitors act on the transporters per se or on the apparatus that perceives cell volume and communicates with the transporters. 7. Furthermore, in the case of the Ca-Na exchanger some indication of the membrane polypeptides involved in volume regulation has been possible, using radioactive compounds that bind covalently to sulfhydryl groups.     

Lithium transport pathways in human red blood cells

In human red cells, Li is extruded against its own concentration gradient if the external medium contains Na as a dominant cation. This uphill net Li extrusion occurs in the presence of external Na but not K, Rb, Cs, choline, Mg, or Ca, is ouabain-insensitive, inhibited by phloretin, and does not require the presence of cellular ATP. Li influx into human red cells has a ouabain-sensitive and a ouabain-insensitive but phloretin-sensitive component. Ouabain-sensitive Li influx is competitively inhibited by external K and Na and probably involves the site on which the Na-K pump normally transports K into red cells. Ouabain does not inhibit Li efflux from red cells containing Li concentrations below 10 mM in the presence of high internal Na or K, whereas a ouabain-sensitive Li efflux can be measured in cells loaded to contain 140 mM Li in the presence of little or no internal Na or K. Ouabain-insensitive Li efflux is stimulated by external Na and not by K, Rb, Cs, choline, Mg, or Ca ions. Na-dependent Li efflux does not require the presence of cellular ATP and is inhibited by phloretin, furosemide, quinine, and quinidine. Experiments carried out in cells loaded in the presence of nystatin to contain either only K or only Na show that the ouabain-insensitive, phloretin-inhibited Li movements into or out of human red cells are stimulated by Na on the trans side and inhibited by Na on the cis side of the red cell membrane. The characteristics of the Na-dependent unidirectional Li fluxes and uphill Li extrusion are similar, suggesting that they are mediated by the same Na-Li countertransport system.     

Glucose transport kinetics in human red blood cells

D-[14C]Glucose self exchange and unidirectional efflux from human red blood cells were studied at 20 degrees C (pH 7.2) by means of the Millipore-Swinnex filtering technique whose time resolution is greater than 1 s and the continuous flow-tube method with a time resolution of greater than 2 ms. The unidirectional efflux data were analyzed using both the method of initial rates and the integrated rate equation. Simple Michaelis-Menten kinetics apply to the results obtained under both experimental conditions. In self-exchange mode, the half-saturation constant, K1/2ex, was 10 (S.E. +/- 1) mM. In unidirectional efflux mode K1/2ue was 6.6 (S.E. +/- 0.5) mM (initial rates) or by the method of integrated rates 7.7 mM, with a range of 2.7-12.1 mM, K1/2ue increasing with an increased initial intracellular glucose concentration. Our results of K1/2ex oppose previous published values of 32 mM for self exchange (Eilam and Stein (1972) Biochim. Biophys. Acta 266, 161-173) and 25 mM for unidirectional efflux (Karlish et al. (1972) Biochim. Biophys. Acta 255, 126-132) that have been used extensively in kinetic considerations of glucose transport models. Under self-exchange conditions Jmaxex was 1.8 x 10(-10) mol cm-2s-1, and in unidirectional efflux mode Jmaxue was 8.3 x 10(-11) mol cm-2s-1 (initial rates) and 8.6 x 10(-11) mol cm-2s-1 (integrated rates). We suggest that the previous high values of Jmax and in particular K1/2 are due to the use of methods with insufficient time resolution. Our results indicate that the transport system is less asymmetric than was generally accepted, and that complicated transport models developed to account for the great difference between the determined K1/2 and J max values are redundant.     

Visual cells and visual pigments of the lamprey,Lampetra fluviatilis

Summary The long and short photoreceptors in the lamprey retina possess similar cone-like outer segments where many disks are infoldings of the outer plasmic membrane. Following the treatment by the Hartwig's (1967) method, outer segments of the long receptors are stained red, and those of the short receptor are stained blue, like the cones and rods in higher vertebrates, resp. (Fig. 1). Microspectrophotometry has shown that the short cells contain P517₁ whereas the long receptors possess P555₁ (Fig. 3). Spectral sensitivity of the dark-adapted retina measured by electroretinographic b-wave and aspartate-isolated receptor potential, corresponds to P517 (Figs. 5, 8). Judging from the receptor potential, the short receptors do not saturate at high illuminances and contribute to the retinal function in photopic conditions as well (Fig. 7). Photopic ERG is of a typical cone-dominant shape (Fig. 4).It is concluded that the long photoreceptors of the lamprey retina are cones whereas the short cells should be regarded as a peculiar kind of rods which possess cone ultrastructure and can operate in scotopic as well as in photopic conditions.Abbreviation LRP late receptor potential     

Sodium transport through the amiloride-sensitive Na-Mg pathway of hamster red cells

Previous work showed that in hamster red cells the amiloride-sensitive (AS) Na⁺ influx of 0.8 mmol/liter cells/hr is not mediated by Na-H exchange as in other red cells, but depends upon intracellular Mg²⁺ and can be increased by 40-fold by loading cells with Mg²⁺ to 10 mm. The purpose of this study was to verify the connection of AS Na⁺ influx with Na-dependent, amiloride-sensitive Mg²⁺ efflux and to utilize AS Na⁺ influx to explore that pathway.Determination of unidirectional influx of Na⁺ and net loss of Mg²⁺ in parallel sets of cells showed that activation by extracellular [Na⁺] follows a simple Michaelis-Menten relationship for both processes with a K _m of 105–107 mm and that activation of both processes is sigmoidally dependent upon cytoplasmic [Mg²⁺] with a [Mg²⁺]_0.5 of 2.1–2.3 mm and a Hill coefficient of 1.8. Comparison of V_max for both sets of experiments indicated a stoichiometry of 2 Na: l Mg. Amiloride inhibits Na⁺ influx and Mg²⁺ extrusion in parallel (K _i = 0.3 mm). Like Mg²⁺ extrusion, amiloride-sensitive Na⁺ influx shows an absolute requirement for cytoplasmic ATP and is increased by cell swelling. Hence, amiloride-sensitive Na⁺ influx in hamster red cells appears to be through the Na-Mg exchange pathway.There was no amiloride-sensitive Na⁺ efflux in hamster red cells loaded with Na⁺ and incubated with high [Mg²⁺] in the medium with or without external Na⁺, nor with ATP depletion. Hence, this is not a simple Na-Mg exchange carrier.