Interactions of lithium and protons with the sodium-proton exchanger of dog red blood cells

Passive movements of Li in dog red blood cells (RBC) ar like those of Na and protons in being stimulated by osmotic cell shrinkage and inhibited by amiloride. Li and protons have similar asymmetrical effects on Na-H exchange. When the intracellular fluid is made rich in Li or protons, Na-H exchange is stimulated. When the extracellular fluid is enriched in Li or protons, Na-H exchange is inhibited. In the case of protons, these effects can override alterations in driving force that are created by the experimental conditions. For example, acidification of the cytoplasm stimulates outward Na movements, while acidification of the medium inhibits Na efflux. Thus, protons (and, by analogy, Li) can interact with the Na-H exchanger not only as substrates but also as modulators. In previous experiments, the only way to activate the Na-H exchanger in dog RBC was to shrink the cells in hypertonic media. The influences of Li or protons, however, are so strong as to preempt the volume effects, so that the pathway can be activated even in swollen cells and deactivated in shrunken ones.     

pH regulatory Na/H exchange by Amphiuma red blood cells

In Amphiuma red blood cells, the Na/H exchanger has been shown to play a central role in the regulation of cell volume following cell shrinkage (Cala, P. M. 1980. Journal of General Physiology. 76:683- 708.) The present study was designed to evaluate the existence of pH regulatory Na/H exchange in the Amphiuma red blood cell. The data illustrate that when the intracellular pHi was decreased below the normal value of 7.00, Na/H exchange was activated in proportion to the degree of acidification. Once activated, net Na/H exchange flux persisted until normal intracellular pH (6.9-7.0) was restored, with a half time of approximately 5 min. These observations established a pHi set point of 7.00 for the pH-activated Na/H exchange of Amphiuma red blood cell. This is in contrast to the behavior of osmotically shrunken Amphiuma red blood cells in which no pHi set point could be demonstrated. That is, when activated by cell shrinkage the Na/H exchange mediated net Na flux persisted until normal volume was restored regardless of pHi. In contrast, when activated by cell acidification, the Na/H exchanger functioned until pHi was restored to normal and cell volume appeared to have no effect on pH-activated Na/H exchange. Studies evaluating the kinetic and inferentially, the molecular equivalence of the volume and pHi-induced Amphiuma erythrocyte Na/H exchanger(s), indicated that the apparent Na affinity of the pH activated cells is four times greater than that of shrunken cells. The apparent Vmax is also higher (two times) in the pH activated cells, suggesting the involvement of two distinct populations of the transporter in pH and volume regulation. However, when analyzed in terms of a bisubstrate model, the same data are consistent with the conclusion that both pH and volume regulatory functions are mediated by the same transport protein. Taken together, these data support the conclusion that volume and pH are regulated by the same effector (Na/H exchanger) under the control of as yet unidentified, distinct and cross inhibitory volume and pH sensing mechanisms.     

Effect of cell age and phenylhydrazine on the cation transport properties of rabbit erythrocytes

We studied the effect of cell age on the cation transport systems of rabbit erythrocytes by increasing the proportion of circulating young erythrocytes with either repeated bleeding or with phenylhydrazine (PHZ) treatment. We found that when the reticulocyte content of rabbit blood is increased by bleeding (from 1 to 40–50% of the circulating red cells), the response of the various transport pathways differs. The largest increase (fivefold) was found in the activity of K-CI contransport which peaked 3 days after the last bleeding. The Na-K pump activity peaked at a similar time, but the % increase was twofold less than the K-CI contransport. There was very small increase in the activity of the Na-Li exchange, whereas the Na-H exchange reached peak values 10 days after the last bleeding (twofold increase), when activities of K-Cl contransport and Na-K pump had returned to almost normal levels. In vivo PHZ treatment resulted in anemia and marked reticulocytosis (80–90% of circulating cells). Transport rates were markedly increased (Na-K pump 9.6-fold, Na-H exchange 6.8-fold, Na-Li exchange 2.75-fold; K-CI contransport: 10–20-fold). When blood from PHZ-treated rabbits was incubated in vitro for 24–48 hour, red cell volume and K content decreased. This process was associated with a 70% reduction in the activity of the K-CI contransport after 24 hours and a 90% reduction after 48 hours. The activity of the other systems also declined and approached baseline values after 48 hours. Loss of transport activity was not affected by 10 μM E-64, whereas 10 mM methylamine reduced the inactivation of the Na-H exchange and of the Na-Li exchange. PHZ treatment of rabbit red cells in vitro resulted in marked increase of the K-CI contransport and inhibition of Na-K pump, Na-H exchange, and Na-Li exchange. These effects were abolished by DTT, with the exception of the Na-K pump inhibition, which was DTT insensitive. Thus both cell age and oxidative damage are important determinants of cation transport in rabbit red cells. © 1993 Wiley-Liss, Inc.     

Multiple receptors coupled to adenylate cyclase regulate Na-H exchange independent of cAMP

We have previously determined that beta-adrenergic and somatostatin receptors stimulate and inhibit, respectively, Na-H exchange independent of changes in cAMP accumulation (Barber, D.L., McGuire, M.E., and Ganz, M.B. (1989) J. Biol. Chem. 264, 21038-21042). The present study extends our work on the beta-adrenergic receptor (beta AR) by investigating receptor activation of Na-H exchange in multiple cell types that either endogenously express the beta AR or that have been transfected with cDNA of the hamster lung beta 2AR or the turkey erythrocyte beta AR. Exchanger activity was determined by monitoring intracellular pH in cell populations loaded with the pH-sensitive dye BCECF (2,7-biscarboxyethyl-5(6)-carboxyfluorescein). In addition to the action of the beta AR, activation of prostaglandin E1 and parathyroid hormone receptors induced an intracellular alkalinization by stimulating a Na(+)-dependent amiloride-sensitive Na-H exchange. In contrast, activation of D2-dopaminergic receptors induced an intracellular acidification by inhibiting Na-H exchange. beta-Adrenergic, prostaglandin E1, and parathyroid hormone receptors activated Na-H exchange independent of changes in intracellular cAMP accumulation and independent of a cholera toxin-sensitive stimulatory GTP regulatory protein. D2-dopaminergic receptors inhibited exchanger activity independent of a pertussis toxin-sensitive inhibitory GTP regulatory protein. We suggest that these receptors are functionally coupled to adenylate cyclase and Na-H exchange through divergent signaling mechanisms.     

Polarization of Na(+)/H(+) and Cl(-)/HCO (3)(-) exchangers in migrating renal epithelial cells

Cell migration is crucial for processes such as immune defense, wound healing, or the formation of tumor metastases. Typically, migrating cells are polarized within the plane of movement with lamellipodium and cell body representing the front and rear of the cell, respectively. Here, we address the question of whether this polarization also extends to the distribution of ion transporters such as Na(+)/H(+) exchanger (NHE) and anion exchanger in the plasma membrane of migrating cells. Both transporters are required for locomotion of renal epithelial (Madin-Darby canine kidney, MDCK-F) cells and human melanoma cells since their blockade reduces the rate of migration in a dose-dependent manner. Inhibition of migration of MDCK-F cells by NHE blockers is accompanied by a decrease of pH(i). However, when cells are acidified with weak organic acids, migration of MDCK-F cells is normal despite an even more pronounced decrease of pH(i). Under these conditions, NHE activity is increased so that cells are swelling due to the accumulation of organic anions and Na(+). When exclusively applied to the lamellipodium, blockers of NHE or anion exchange inhibit migration of MDCK-F cells as effectively as when applied to the entire cell surface. When they are directed to the cell body, migration is not affected. These data are confirmed immunocytochemically in that the anion exchanger AE2 is concentrated at the front of MDCK-F cells. Our findings show that NHE and anion exchanger are distributed in a polarized way in migrating cells. They are consistent with important contributions of both transporters to protrusion of the lamellipodium via solute uptake and consequent volume increase at the front of migrating cells.     

Activation of ion transport pathways by changes in cell volume.

Swelling-activated K+ and Cl- channels, which mediate RVD, are found in most cell types. Prominent exceptions to this rule include red cells, which together with some types of epithelia, utilize electroneutral [K(+)-Cl-] cotransport for down-regulation of volume. Shrinkage-activated Na+/H+ exchange and [Na(+)-K(+)-2 Cl-] cotransport mediate RVI in many cell types, although the activation of these systems may require special conditions, such as previous RVD. Swelling-activated K+/H+ exchange and Ca2+/Na+ exchange seem to be restricted to certain species of red cells. Swelling-activated calcium channels, although not carrying sufficient ion flux to contribute to volume changes may play an important role in the activation of transport pathways. In this review of volume-activated ion transport pathways we have concentrated on regulatory phenomena. We have listed known secondary messenger pathways that modulate volume-activated transporters, although the evidence that volume signals are transduced via these systems is preliminary. We have focused on several mechanisms that might function as volume sensors. In our view, the most important candidates for this role are the structures which detect deformation or stretching of the membrane and the skeletal filaments attached to it, and the extraordinary effects that small changes in concentration of cytoplasmic macromolecules may exert on the activities of cytoplasmic and membrane enzymes (macromolecular crowding). It is noteworthy that volume-activated ion transporters are intercalated into the cellular signaling network as receptors, messengers and effectors. Stretch-activated ion channels may serve as receptors for cell volume itself. Cell swelling or shrinkage may serve a messenger function in the communication between opposing surfaces of epithelia, or in the regulation of metabolic pathways in the liver. Finally, these transporters may act as effector systems when they perform regulatory volume increase or decrease. This review discusses several examples in which relatively simple methods of examining volume regulation led to the discovery of transporters ultimately found to play key roles in the transmission of information within the cell. So, why volume? Because it's functionally important, it's relatively cheap (if you happened to have everything else, you only need some distilled water or concentrated salt solution), and since it involves many disciplines of experimental biology, it's fun to do.     

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.     

Red cell volume regulation: the pivotal role of ionic strength in controlling swelling-dependent transport systems.

A volume increase of trout erythrocytes can be induced either by beta-adrenergic stimulation of a Na+/H+ antiport in an isotonic medium (isotonic swelling) or by suspending red cells in an hypotonic medium (hypotonic swelling). In both cases cells regulate their volume by a loss of osmolytes via specific pathways. After hypotonic swelling several volume-dependent pathways were activated allowing K+, Na+, taurine and choline to diffuse. All these pathways were fully inhibited by furosemide and inhibitors of the anion exchanger (DIDS, niflumic acid), and the K+ loss was mediated essentially via a 'Cl(-)-independent' pathway. After isotonic swelling, the taurine, choline and Na+ pathways were practically not activated and the K+ loss was strictly 'Cl(-)-dependent'. Thus cellular swelling is a prerequisite for activation of these pathways but, for a given volume increase, the degree of activation and the degree of anion-dependence of the K+ pathway depend on the nature of the stimulus, whether hormonal or by reduction of osmolality. It appears that the pattern of the response induced by hormonal stimulation is not triggered by either cellular cAMP (since it can be reproduced in the absence of hormone by isotonic swelling in an ammonium-containing saline) or by the tonicity of the medium in which swelling occurs since after swelling in an isotonic medium containing urea, the cells adopt the regulatory pattern normally observed after hypotonic swelling. We demonstrated that the stimulus is the change in cellular ionic strength induced by swelling: when ionic strength drops, the cells adopt the hypotonic swelling pattern; when ionic strength increases, the isotonic swelling pattern is activated. To explain this modulating effect of ionic strength a speculative model is proposed, which also allows the integration of two further sets of experimental results: (i) all the volume-activated transport systems are blocked by inhibitors of the anion exchanger and (ii) a Cl(-)-dependent, DIDS-sensitive K+ pathway can be activated in static volume trout red cells (i.e., in the absence of volume increase) by the conformational change of hemoglobin induced by the binding of O2 or CO to the heme.     

Volume regulation of Chinese hamster ovary cells in anisoosmotic media

Chinese hamster ovary (CHO) cells when suspended in anisoosmotic media regulate their volumes by the activation of specific ion transport pathways. In hypoosmotic media the cells first swell and then return to their isoosmotic volumes by the loss of cellular KCl and osmotically obliged water. This regulatory volume decrease (RVD) is insensitive to ouabain or bumetanide but is blocked by quinine, cetiedil and oligomycin C. Based on cell volume and membrane potential measurements under various experimental conditions, we conclude that hypoosmotic shock activates independent, conductive transport pathways for K+ and for Cl-, respectively. The anion pathway can also transport NO3- and SCN- but not gluconate- anions. Osmotic shrinkage of CHO cells does not produce a regulatory volume increase (RVI) unless the cells have previously undergone a cycle of RVD. RVI is a Na+-dependent, amiloride-sensitive, but ouabain- and oligomycin-insensitive process, probably involving a Na+-H+ exchange system. Internal acidification of isoosmotic cells by addition of a permeable weak acid also activates an amiloride-sensitive Na+-H+ exchange, producing a volume increase. Both RVD and RVI in CHO cells seem to involve molecular mechanisms similar to those described for the volume regulation of lymphocytes, indicating the prevalence of these phenomena in nucleated mammalian cells. Cultured CHO cell lines may provide a basis for a genetic characterization of the volume-regulatory transport pathways.     

Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2

Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na+-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na+-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na+-independent pathways, possibly mediated by an organic anion transporter, and Na+-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na+-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na+-dependent transport of succinate by NaDC1.     

Positive feedback between Cdc42 activity and H+ efflux by the Na-H exchanger NHE1 for polarity of migrating cells

A fundamental feature of cell polarity in response to spatial cues is asymmetric amplification of molecules generated by positive feedback signaling. We report a positive feedback loop between the guanosine triphosphatase Cdc42, a central determinant in eukaryotic cell polarity, and H(+) efflux by Na-H(+) exchanger 1 (NHE1), which is necessary at the front of migrating cells for polarity and directional motility. In response to migratory cues, Cdc42 is not activated in fibroblasts expressing a mutant NHE1 that lacks H(+) efflux, and wild-type NHE1 is not activated in fibroblasts expressing mutationally inactive Cdc42-N17. H(+) efflux by NHE1 is not necessary for release of Cdc42-guanosine diphosphate (GDP) from Rho GDP dissociation inhibitor or for the membrane recruitment of Cdc42 but is required for GTP binding by Cdc42 catalyzed by a guanine nucleotide exchange factor (GEF). Data indicate that GEF binding to phosphotidylinositol 4,5-bisphosphate is pH dependent, suggesting a mechanism for how H(+) efflux by NHE1 promotes Cdc42 activity to generate a positive feedback signal necessary for polarity in migrating cells.     

Macromolecular crowding and volume perception in dog red cells

To differentiate whether the primary volume signal in dog red cells arises from a change in cell configuration or the concentration and dilution of cell contents, we prepared resealed ghosts that had the same surface area and hemoglobin concentration as intact cells but less than 1/3 their volume. Shrinkage of both intact cells and resealed ghosts triggered Na/H exchange. Activation of this transporter in the two preparations correlated closely with cytosolic protein concentration but not at all with volume. The Na/H exchanger was more sensitive to shrinkage in albumin-loaded resealed ghosts than in intact cells or ghosts containing only hemoglobin. Similar results were obtained for the swelling-induced [K-Cl] cotransporter. We believe perception of cell volume originates with changes in cytoplasmic protein concentration. We think the kinases and phosphatases that control the activation of membrane transporters in response to cell swelling or shrinkage are regulated by the mechanism of macromolecular crowding.     

Fish red blood cells: characteristics and physiological role of the membrane ion transporters

Several membrane ion transporters playing a role in gas transport and exchanges, cell volume regulation and intracellular acid-base regulation have been identified in fish red blood cells (RBCs). This short review focuses on Na+/K+ATPase and its role in establishing the ionic gradients across the membrane, on the Cl-/HCO3- exchanger and its key role in respiration and possibly in inducing a chloride conductance, on the Na+/H+ exchanger and the recent advances on its molecular mechanisms of activation and regulation, on the different types of K-Cl cotransports, the different hypotheses and suggested models and their role in cell volume regulation. There is no evidence in the literature for ionic channels in fish RBCs. We present original data obtained with the patch-clamp technique that shows for the first time the existence of a DIDS-sensitive chloride anionic conductance measured in whole cell configuration and the presence of a stretch-activated nonselective cationic channel recorded in cell-attached and excised inside-out configuration. The part played by these ionic conductances is discussed in relation with their possible involvement in volume regulation.     

Differential localization and operation of distinct Mg(2+) transporters in apical and basolateral sides of rat liver plasma membrane

Upon activation of specific cell signaling, hepatocytes rapidly accumulate or release an amount of Mg(2+) equivalent to 10% of their total Mg(2+) content. Although it is widely accepted that Mg(2+) efflux is Na(+)-dependent, little is known about transporter identity and the overall regulation. Even less is known about the mechanism of cellular Mg(2+) uptake. Using sealed and right-sided rat liver plasma membrane vesicles representing either the basolateral (bLPM) or apical (aLPM) domain, it was possible to dissect three different Mg(2+) transport mechanisms based upon specific inhibition, localization within the plasma membrane, and directionality. The bLPM possesses only one Mg(2+) transporter, which is strictly Na(+)-dependent, bi-directional, and not inhibited by amiloride. The aLPM possesses two separate Mg(2+) transporters. One, similar to that in the bLPM because it strictly depends on Na(+) transport, and it can be differentiated from that of the bLPM because it is unidirectional and fully inhibited by amiloride. The second is a novel Ca(2+)/Mg(2+) exchanger that is unidirectional and inhibited by amiloride and imipramine. Hence, the bLPM transporter may be responsible for the exchange of Mg(2+) between hepatocytes and plasma, and vice versa, shown in livers upon specific metabolic stimulation, whereas the aLPM transporters can only extrude Mg(2+) into the biliary tract. The dissection of these three distinct pathways and, therefore, the opportunity to study each individually will greatly facilitate further characterization of these transporters and a better understanding of Mg(2+) homeostasis.     

Cation transport and cell volume changes in maturing rat reticulocytes

During maturation, reticulocytes lose membrane material,including transporters, and this is accompanied by a loss of cell waterand volume. Here we determined a possible role of ion transport inadjusting cell volume during maturation. Reticulocytes and red bloodcells of different ages were prepared from erythropoietin-treated ratsby density gradient fractionation. Cell volume and ion transport weremeasured in freshly prepared cells and in reticulocytes during in vitromaturation. Reticulocytes had an increased K content and cell volume,whereas intracellular Na was decreased. All parameters approached wholeblood values after 2 days in culture. Na-K pump was elevated inreticulocytes and decreased during maturation. Na-K-2Cl cotransport(NKCC) activity was lower in reticulocytes and was activated 8- and20-fold by shrinkage and okadaic acid, respectively, whereasstimulation was barely detectable in high-buoyant density red bloodcells. The ouabain- and bumetanide-insensitive Na flux in reticulocytesdecreased on maturation. Most of it was inhibited by amiloride,indicating the presence of Na/proton exchange. Our results show that,although the Na-K-pump activity in reticulocytes is very muchincreased, the enhanced capacity of NKCC is essentially cryptic untilstimulated. Both types of capacities (activities) decrease duringmaturation, indicating a possible loss of transport protein. Thedecrease was constrained to the period of reticulocyte maturation. Lossof transport capacity appears to exceed the loss of membrane surfacearea. Reticulocyte age-related changes in the net electrochemicaldriving force indicate that the increasing NKCC activity mightcontribute to the reduction in cell water.

     

Swelling activation of K-Cl cotransport in LK sheep erythrocytes: a three-state process

K-Cl cotransport in LK sheep erythrocytes is activated by osmotic swelling and inhibited by shrinkage. The mechanism by which changes in cell volume are transduced into changes in transport was investigated by measuring time courses of changes in transport after osmotic challenges in cells with normal and reduced Mg concentrations. When cells of normal volume and normal Mg are swollen, there is a delay of 10 min or more before the final steady-state flux is achieved, as there is for swelling activation of K-Cl cotransport in erythrocytes of other species. The delay was shown to be independent of the extent of swelling. There was also a delay after shrinkage inactivation of cotransport. Reducing cellular Mg concentration activates cotransport. Swelling of low-Mg cells activates cotransport further, but with no measurable delay. In contrast, there is a delay in shrinkage inactivation of cotransport in low-Mg cells. The results are interpreted in terms of a three-state model: [formula see text] in which A state, B state, and C state transporters have relatively slow, intermediate, and fast transport rates, respectively. Most transporters in shrunken cells with normal Mg are in the A state. Swelling converts transporters to the B state in the rate-limiting process, followed by rapid conversion to the C state. Reducing cell Mg also promotes the A-- >B conversion. Swelling of low-Mg cells activates transport rapidly because of the initial predominance of B state transporters. The results support the following conclusions about the rate constants of the three-state model: k21 is the rate constant for a Mg-promoted process that is inhibited by swelling; k12 is not volume sensitive. Both k23 and k32 are increased by swelling and reduced by shrinkage; they are rate constants for a single process, whereas k12 and k21 are rate constants for separate processes. Finally, the A-->B conversion entails an increase in Jmax of the transporters, and the B-->C conversion entails an increase in the affinity of the transporters for K.     

Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2

Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na⁺-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na⁺-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na⁺-independent pathways, possibly mediated by an organic anion transporter, and Na⁺-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na⁺-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na⁺-dependent transport of succinate by NaDC1.     

Cytosolic protein concentration is the primary volume signal in dog red cells

It is not known whether the activation of Na/H exchange by shrinkage in dog red cells is due to the packing of cell contents or a change in cell configuration. To make this distinction we prepared resealed ghosts that resembled intact cells in hemoglobin concentration and surface area, but had one-third their volume. A shrinkage-induced, amiloride-sensitive Na flux in the ghosts was activated at a much smaller volume in the ghosts than in the intact cells, but at the same concentration (by weight) of dry solids in both preparations. Na/H exchange in ghosts containing a mixture of 40% albumin and 60% hemoglobin (weight/weight) was activated by osmotic shrinkage at a dry solid concentration similar to that of intact cells or of ghosts containing only hemoglobin. We conclude that the process of Na/H exchange activation by cell shrinkage originates with an increase in the concentration of intracellular protein and not with a change in membrane configuration or tension. The macromolecular crowding that accompanies the reduction in cell volume probably alters the activities of key enzymes that in turn modulate the Na/H exchanger.     

Na+ /H+ Exchanger in Tissues of Lower Vertebrates

Na⁺/H⁺ exchange is one of the major pathways of ion transport in cells of pro- and eukaryots and plays an important role in intracellular pH and cell volume regulation, in cell division, proliferation, as well as in epithelial transport processes. Since 1989, investigations on the molecular nature of this transporter have revealed six isoforms (NHE1–NHE6) in mammalian tissues. Most works on studies of properties of the Na/H antiporter and regulation of its activity have been carried out on mammalian tissues. This review summarizes results of studies on the Na⁺/H⁺ exchange in tissues of lower vertebrates. Of the greatest interest are investigations on the rainbow trout, whose erythrocytes were found to contain a Na⁺/H⁺ exchanger activated by catecholamines. This carrier in trout erythrocytes has been cloned and called beta-NHE ( ;NHE). Another exchanger isoform, atNHE, was isolated from the red blood cells of the giant salamander Amphiuma tridactulum. Isoforms of antiporter isolated from oocytes (XL-NHE) and renal cells of the clawed frog Xenopus laevis (XNHE) have also been described.     

Role of Na-H exchangers and vacuolar H+ pumps in intracellular pH regulation in neonatal rat osteoclasts

Osteoclasts resorb bone by pumping of H+ into a compartment between the cell and the bone surface. Intracellular pH (pHi) homeostasis requires that this acid extrusion, mediated by a vacuolar-type H+ ATPase, be complemented by other acid-base transporters. We investigated acid- extrusion mechanisms of single, freshly isolated, neonatal rat osteoclasts. Cells adherent to glass coverslips were studied in the nominal absence of CO2/HCO3-, using the pH-sensitive dye BCECF and a digital imaging system. Initial pHi averaged 7.31 and was uniform throughout individual cells. Intrinsic buffering power (beta 1) decreased curvilinearly from approximately 25 mM at pHi = 6.4 to approximately 6.0 mM at pHi = 7.4. In all polygonally shaped osteoclasts, and approximately 60% of round osteoclasts (approximately 20% of total), pHi recovery from acid loads was mediated exclusively by Na-H exchange. In these pattern-1 cells, pHi recovery was 95% complete within 200 s, and was blocked by removing Na+, or by applying 1 mM amiloride, 50 microM ethylisopropylamiloride (EIPA), or 50 microM hexamethyleneamiloride (HMA). The apparent K1/2 for HMA ([Na+]o = 150 mM) was 49 nM, and the apparent K1/2 for Na+ was 45 mM. Na-H exchange, corrected for amiloride-insensitive fluxes, was half maximal at pHi 6.73, with an apparent Hill coefficient for intracellular H+ of 2.9. Maximal Na-H exchange averaged 741 microM/s. In the remaining approximately 40% of round osteoclasts (pattern-2 cells), pHi recovery from acid loads was brisk even in the absence of Na+ or presence of amiloride. This Na(+)-independent pHi recovery was blocked by 7-chloro- 4-nitrobenz-2-oxa-1,3-diazol (NBD-Cl), a vacuolar-type H+ pump inhibitor. Corrected for NBD-Cl insensitive fluxes, H+ pump fluxes decreased approximately linearly from 96 at pHi 6.8 to 11 microM/s at pHi 7.45. In approximately 45% of pattern-2 cells, Na+ readdition elicited a further pHi recovery, suggesting that H+ pumps and Na-H exchangers can exist simultaneously. We conclude that, under the conditions of our study, most neonatal rat osteoclasts express Na-H exchangers that are probably of the ubiquitous basolateral subtype. Some cells express vacuolar-type H+ pumps in their plasma membrane, as do active osteoclasts in situ.