Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Abstract. Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stage as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stages as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7-day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/ maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

Characterization of Mg2+ efflux from human, rat and chicken erythrocytes

Net Mg2+ efflux from Mg2+-loaded, human, rat and chicken erythrocytes was measured in sucrose, NaCl and choline Cl medium. Thus, Na+-dependent (NaCl minus choline Cl) and Na+-independent Mg2+ efflux (in sucrose) were determined. Na+-dependent Mg2+ efflux amounted to 0.16, 8.9 and 1.57 mmol/l cells x 30 min, Na+-independent Mg2+ efflux amounted to 0.89, 1.55 and 0.37 mmol/l cells x 30 min for human, rat and chicken erythrocytes. Na+-dependent Mg2+ efflux was inhibited by quinidine. Na+-independent Mg2+ efflux was inhibited by SITS and Cl-. A small fraction of Na+-independent Mg2+ efflux (in choline Cl) was resistant to SITS and Cl-. Ca2+ loading increased Mg2+ efflux similar to K+ efflux (Gardos effect). This effect was differently expressed in human and chicken erythrocytes.     

Cell membrane amino acid transport processes in the domestic fowl (Gallus domesticus)

Intestinal absorption of amino acids in the chicken occurs by way of processes which are concentrative, Na+-dependent and dependent upon metabolic energy in the form of ATP. Intestinal transport is carrier-mediated, subject to exchange transport (trans-membrane effects) and is inhibitable by sugars, reagents which inactivate sulfhydryl groups, potassium ion, and by deoxpyridoxine, an anti-vitamin B6 agent. It is stimulated by phlorizin, a potent inhibitor of sugar transport, and in Na+-leached tissue by modifiers of tissue cyclic AMP levels, e.g. theophylline, histamine, carbachol and secretin. Separate transport sites with broad, overlapping specificities function in the intestinal absorption of the various classes of common amino acids. A simple model for these sites includes one for leucine and other neutral amino acids, one for proline, beta-alanine and related imino and amino acids, one for basic amino acids, and one for acidic amino acids. Absorption of amino acids appears to be widespread in occurrence in the digestive tract of the domestic fowl; transport has been reported to be present in the crop, gizzard, proventriculus, small intestine and in the colon. By the end of the first week of life post-hatch, the caecum loses its ability to transport. Similarly, the yolk sac loses its ability by the second day post-hatch. Intestinal transport was noted before hatch and was found to be maximal immediately post-hatch. A requirement for Ca2+ appears to be lost after the first week of life post-hatch. The cationic amino acids appear to be reabsorbed by a common mechanism in the kidney. Transport rates of leucine measured in the intestine or in the erythrocyte were found to cluster about discrete values when many individual chickens were surveyed; such patterns may be an expression of gene differences between individuals. Two lines of chickens have been developed, one high and the other low uptake, through selective breeding based on the ability of individual birds to absorb leucine in erythrocytes. High leucine absorbing chickens were found to be more effective in absorbing lysine and glycine, were more effectively stimulated by Na+, had greater erythrocyte Na+, K+-ATPase activity, and their erythrocytes contained about 20% less Na+ than low line erythrocytes. The underlying genetic difference between these lines may reside at the level of the Na+, K+-ATPase and (or) with a regulatory gene determining carrier copies. Amino acid transport in erythrocytes was noted to be highest in pre-hatch chicks and to diminish during post-hatch development.(ABSTRACT TRUNCATED AT 400 WORDS)     

Red-cell amino acid transport. Evidence for the presence of system ASC in mature human red blood cells.

The properties of Na+-dependent L-alanine transport in human erythrocytes were investigated using K+ as the Na+ substitute. Initial rates of Na+-dependent L-alanine uptake (0.2 mM extracellular amino acid) for erythrocytes from 22 donors ranged from 40 to 180 mumol/litre of cells per h at 37 degrees C. Amino acid uptake over the concentration range 0.1-8 mM was consistent with a single saturable component of Na+-dependent L-alanine transport. Apparent Km and Vmax. values at 37 and 5 degrees C measured in erythrocytes from the same donor were 0.27 and 0.085 mM respectively, and 270 and 8.5 mumol/litre of cells per h respectively. The transporter responsible for this uptake was identified as system ASC on the basis of cross-inhibition studies with a series of 42 amino acids and amino acid analogues. Apparent Ki values for glycine, L-alpha-amino-n-butyrate, L-serine and L-leucine as inhibitors of Na+-dependent L-alanine uptake at 37 degrees C were 4.2, 0.12, 0.16 and 0.70 mM respectively. Reticulocytes from a patient with inherited pyruvate kinase deficiency were found to have a 10-fold elevated activity of Na+-dependent L-alanine uptake compared with erythrocytes from normal donors. Separation of erythrocytes according to cell density (cell age) established that even the oldest mature erythrocytes retained significant Na+-dependent L-alanine transport activity. Amino acid transport was, however, a more sensitive indicator of cell age than acetylcholinesterase activity. Erythrocytes were found to accumulate L-alanine against its concentration gradient (distribution ratio approx. 1.5 after 4 h incubation), an effect that was abolished in Na+-free media. Na+-dependent L-alanine uptake was shown to be associated with L-alanine-dependent Na+ influx, the measured coupling ratio being 1:1.     

Amino acid transport and rubidium-ion uptake in monolayer cultures of hepatocytes from neonatal rats

Amino acid and K(+) transport during development has been investigated in hepatocyte monolayer cultures with either alpha-amino[1-(14)C]isobutyrate or (86)Rb(+) used as a tracer for K(+). Parenchymal cells from neo- and post-natal rat livers have been isolated by an improved non-perfusion technique [Bellemann, Gebhardt & Mecke (1977)Anal.Biochem.81, 408-415], and the resulting hepatocyte suspensions purified from non-hepatocytes before inoculation. In the presence of Na(+) (Na(+)-dependent component), the rates of amino acid uptake in neonatal hepatocytes were markedly enhanced compared with cells from 30-day-old rats. When Na(+) was replaced by choline (Na(+)-independent component) the accumulation of alpha-aminoisobutyrate was decreased and it was not affected by the age of the animals. Kinetic analysis of Na(+)-dependent alpha-aminoisobutyrate transport revealed the existence of a high-affinity low-K(m) component (K(m)0.91mm) with a V(max.) of 2.44nmol/mg of protein per 4min, which later declined gradually with progressive development. Rates of Rb(+) transport were concomitantly enhanced in neonatal hepatocytes and thereafter declined with postnatal age. The increased Rb(+) influx was effectively inhibited by ouabain and reflected elevated activity of the electrogenic Na(+)/K(+)-pump during early stages of development. Kinetic evaluation of the enhanced rates of Rb(+) uptake indicates multiple and co-operative binding sites of the enzyme involved in the Rb(+) uptake, and the transport system is positively co-operative (the Hill coefficient h is >1.0). In short, amino acid transport in neonatal rat hepatocytes is increased as a result of an existing low-K(m) component for the Na(+)-dependent alpha-aminoisobutyrate uptake, which endows the hepatocytes with a high capability for concentrating amino acids at low ambient values. The concomitant enhancement of K(+) transport reflects changes in the electrochemical gradient for Na(+) across the hepatocellular membrane and, along with this, presumably alterations in the membrane potential; the latter might be the driving force for the enhanced alpha-aminoisobutyrate transport in the alanine-preferring system during postnatal age.     

Amino acid transport and intracellular Na+ and K+ content of chicken erythrocytes genetically selected for high and low leucine transport activity

1. Amino acid transport and intracellular Na+ and K+ content have been studied in two lines of chickens, one high and the other low uptake, selected for their ability to transport leucine into erythrocytes. 2. Low line birds were less effective in absorbing glycine into erythrocytes than were high line birds, the difference in transport being due to a difference in maximal flux (Vmax), but not in apparent affinity for transport sites (Kt). 3. In contrast to glycine uptake, the greater ability of the high line to absorb lysine was found to be due to a difference in both Vmax and Kt. 4. High line erythrocytes were also observed to contain slightly more K+ (about 5%) and about 20% less Na+ than low line erythrocytes. 5. These results are discussed in terms of the ion dependency of amino acid transport.     

Ion transport systems in erythrocytes from paramyotonia patients

To test the hypothesis of a general defect in the ion transport systems in the cell membranes of patients with paramyotonia congenita, we measured the ouabain-inhibitable and the furosemide-inhibitable K+ influxes in erythrocytes from two patients. In the temperature range examined (15-40 degrees C), the ouabain-inhibitable flux did not differ significantly from control. Also the furosemide-inhibitable flux was well in the rather large control range. The dependence of these fluxes on the external K+ concentration was normal. Energy depletion of the erythrocytes by preincubation with desoxy-glucose led to reduced K+ influxes that were not significantly different in patients and controls. The results do not support the hypothesis of a general defect in the ion transport systems in paramyotonia; they do, however, not rule out the possibility that the ion transport systems in paramyotonic skeletal muscle are defect.     

Calcium transport in chicken leukocytes and erythrocytes

1. In the present study, Ca2+ uptake and Ca2(+)-ATPase activity of two different chicken leukocyte populations and erythrocytes isolated from 1- to 6-week-old chickens were determined. 2. The Ca2(+)-ATPase activity of the two leukocyte populations significantly increased at 3 weeks of age. Erythrocyte Ca2(+)-ATPase activity significantly increased at 2 weeks of age. 3. Calcium transport activities into the two leukocyte populations did not differ significantly with age.     

Modifications in choline transport activity as a function of membrane potential and the sodium gradient

Advantage was taken of a preparation of proteoliposomes made using Torpedo presynaptic membranes in which both the internal and external media can be controlled to investigate the effects of membrane potential and the Na+ gradient on choline transport activity. Under control conditions, Na+ outside and K+ inside, choline was concentrated by proteoliposomes and this phenomenon was sensitive to hemicholinium-3 and high levels of external choline. While proteoliposomes showed no permeability towards K+ spontaneously, in the presence of valinomycin a transmembrane potential was developed. The rate of transport was higher, the greater the inside negative potential. Both the affinity and the maximal velocity of high affinity transport rose in the presence of a potential. Likewise, the affinity and velocity of this transporter increased with increasing external Na+. Increasing internal Na+, on the other hand, caused a decrease in affinity and had little effect on the maximal velocity. The low affinity component was much less, if at all, affected by these changes. These results are consistent with a model of high affinity choline transport in which Na+ binds before choline and the carrier-Na+-choline complex is positively charged. However, these results do not provide a direct explanation for choline transport activation by nerve activity, underlining the need to study the effects of parameters other than membrane potential and the Na+ gradient on choline transport activity.     

Characterization of a Pi transport system in cartilage matrix vesicles. Potential role in the calcification process.

The mechanisms by which calcium (Ca2+) and inorganic phosphate (Pi) accumulate into matrix vesicles (MV) have not been elucidated. In the present study the characteristics of Pi uptake into MV isolated from mildly rachitic chicken growth plate cartilage have been investigated. The results indicate that Pi accumulates into MV mainly via a Na(+)-dependent Pi transport system. In the absence of NaCl in the extravesicular medium, Pi uptake was a nonsaturable process. In the presence of 150 mM NaCl, the initial rate of Pi uptake was 4.38 +/- 1.02-fold higher than with 150 mM choline chloride (mean +/- S.E., n = 8, p less than 0.005). Other cations showed partial activity to drive Pi into MV as compared to Na+:Li+ (64.4%) greater than K+ (39.8%) greater than choline (39.0%) greater than tetramethylammonium (30.0%) greater than N-methylglucamine (26.3%). Na(+)-dependent Pi transport activity displayed saturability towards increasing extra-vesicular concentrations of Na+ and Pi. The apparent Km for Pi was 0.68 +/- 0.16 mM. The Na+ concentration producing half-maximum Pi transport activity was 106.2 +/- 11.0 mM. Kinetic analysis suggests that Na+ interacts with the Pi carrier with a stoichiometry of more than one Na+ ion with one Pi molecule. In MV isolated from normal chicken growth plate cartilage, this Na(+)-dependent Pi transport system was barely expressed. In contrast to the effect on Pi uptake by MV, the activity of alkaline phosphatase was not changed when NaCl was substituted for choline chloride in the assay medium. In addition to this observation which suggests that this enzyme is not related to the Pi transport activity described in this study, levamisole, which inhibited alkaline phosphatase activity did not affect the Na(+)-dependent uptake of Pi. Both arsenate and phosphonoformic acid, two inhibitors of the epithelial Na(+)-dependent Pi transport systems, were active inhibitors of the Na(+)-dependent Pi uptake by MV with a higher potency for phosphonoformic acid. Associated with the expression of a facilitated Na(+)-coupled Pi transport in MV, in vitro calcification assessed by 45Ca2+ uptake also showed a marked dependence on extravesicular sodium. This relationship was markedly attenuated in MV isolated from normal chicken growth plate cartilage expressing a weak Na(+)-facilitated Pi transport activity. In conclusion, a saturable Na(+)-dependent Pi carrier has been characterized which facilitates Pi transport in MV. Its potential role for Ca-Pi accumulation into MV and subsequent development of vesicular calcification followed by mineralization of the osteogenic matrix is proposed and remains to be further investigated.     

The monovalent cation "leak" transport in human erythrocytes: an electroneutral exchange process.

The mechanism of the "ground permeability" of the human erythrocyte membrane for K+ and Na+ was investigated with respect to a possible involvement of a previously unidentified specific transport pathway, because earlier studies showed that it cannot be explained on the basis of simple electrodiffusion. In particular, we analyzed and described the increase in the (ouabain+bumetanide+EGTA)-insensitive unidirectional K+ and Na+ influxes as well as effluxes (defined as "leak" fluxes) observed in erythrocytes suspended in low-ionic-strength media. Using a carrier-type model and taking into account the influence of the ionic strength on the outer surface potential according to the Gouy-Chapman theory (i.e., the ion concentration near the membrane surface), we are able to describe the altered "leak" fluxes as an electroneutral process. In addition, we can show indirectly that this electroneutral flux is due to an exchange of monovalent cations with protons. This pathway is different from the amiloride-sensitive Na+/H+ exchanger present in the human red blood cell membrane and can be characterized as a K+(Na+)/H+ exchanger.     

Effect of gradients of monovalent cations on active transport of Ca2+ in the sarcoplasmic reticulum and proteoliposomes

Artificially generated K+ gradient from the sarcoplasmic reticulum vesicles enhances the ATP-dependent Ca2+ transport. The effect is not specific for K+, and is observed when K+ is replaced by Na+ or choline. Dissipation of the K+, Na+, choline gradient does not influence the ATP-dependent Ca2+ transport in proteoliposomes from asolectin and purified Ca2+-ATPase. The K gradient in the presence of valinomycin stimulates the ATP-dependent Ca2+ transport in proteoliposomes.     

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.     

Na+, K+-ATPase activity in erythrocytes after the effect of laser radiation

An in vitro single radiation of helium-neon laser (power flux density being 2 mW/cm2 exposure--1 and 3 min) does not change the concentration of Na+ and K+, activity of Na+, K+-dependent ATPase in erythrocytes and does not affect the intensity of active Na transport through their membrane in the donor blood. The 5 min laser action decreases the level of K+ and increases that of Na+ in the erythrocytes, activates Na+, K+-ATPases and intensifies the active Na+ transport.     

Effects of ATP and cyclic AMP on the (Na+ + K+ + 2Cl-)-cotransport system in turkey erythrocytes

As turkey erythrocytes were progressively depleted of ATP by preincubation with dinitrophenol, the (Na+ + K+ + 2Cl-)-cotransport system (assayed by the bumetanide-sensitive fraction of 86Rb+ influx) became less responsive to activation. The dependence upon intracellular ATP concentration was significantly steeper for transport activated by hypertonic shock (halfmaximal activity at 0.7 mM ATP) than for that activated by either epinephrine or cyclic AMP (halfmaximal activity at 1.7 mM ATP). Upon removal of epinephrine or cyclic AMP from cells that had been preincubated with those substances, bumetanide-sensitive transport activity declined sharply, even though the intracellular cyclic AMP concentration was still over 10-fold that required to maximally activate the transport system. These data are in agreement with the notion that the (Na+ + K+ + 2Cl-)-cotransport system in turkey erythrocytes is activated by cyclic AMP, presumably through the 'classical' pathway involving a protein kinase. They do however indicate that some other, as yet undefined aspect of cyclic AMP metabolism is important for the maintenance of transport activity.     

Na+ for H+ exchange in rabbit erythrocytes

The effect of a transmembrane pH gradient on the ouabain, bumetanide, and phloretin resistant H+ efflux was studied in rabbit erythrocytes. Proton equilibration was reduced by the use of DIDS (125 microM) and acetazolamide (1 mM). H+ efflux from acid loaded erythrocytes (pHi = 6.1) was measured in a K+ (145 mM) medium, pH0 = 8.0, in the presence and absence of 60 microM 5,N,N-dimethyl-amiloride (DMA). The H+ efflux rate in a K+-containing medium was 116.38 +/- 4.5 mmol/l cell X hr. Substitution of Nao+ for Ko+ strongly stimulated H+ efflux to 177.89 +/- 7.9 mmol/l cell X hr. The transtimulation of H+ efflux by Nao+ was completely abolished by DMA falling to values not different from controls with an ID50 of about 8.6 X 10(-7) M. The sequence of substrate selectivities for the external transport site were Na greater than greater than greater than Li greater than choline, Cs, K, and Glucamine. The transport system has no specific anion requirement, but is inhibited by NO3-. The DMA sensitive H+ efflux was a saturable function of [Na+]o, with an apparent Km and Vmax of about 14.75 +/- 1.99 mM and 85.37 +/- 7.68 mmol/l cell X hr, respectively. However, the Nao+-dependent and DMA-sensitive H+ efflux was sigmoidally activated by [H+]i, suggesting that Hi+ interacts at both transport and modifier sites. An outwardly directed H+ gradient (pHi 6.1, pH = 8.0) also promoted DMA sensitive Na+ entry (61.2 +/- 3.0 mmol/l cell X hr) which was abolished when pHo was reduced to 6.0. The data is therefore consistent with the presence of a Na+/H+ exchange system in rabbit erythrocytes.     

Absence of effect of aldosterone on sodium efflux catalyzed by the human erythrocyte Na+, K+-ATPase in vitro

The effect of physiological and pharmacological concentrations of aldosterone on Na+ efflux catalyzed by the human erythrocyte Na+,K+-ATPase in vitro were studied. Aldosterone had no significant effect on ouabain-sensitive Na+ efflux from fresh erythrocytes. In addition, aldosterone did not alter Na+ transport activity of stimulated Na+,K+-ATPase of Na+ loaded erythrocytes. Finally, Na+ efflux from Na+ loaded erythrocytes was not changed by preincubation of the cells with aldosterone. It is concluded that aldosterone in vitro does not modify pump activity of the human erythrocyte Na+, K+-ATPase.     

Cation-induced asymmetry of choline flux across presynaptic plasma membranes.

Highly cholinergic synaptosomes from the optic lobes of Sepia officinalis retain their ability to concentrate K+ and extrude Na+ sensitive but is not obligatorily coupled to choline metabolism, or an energy supply as shown by the action of metabolic and ion pump inhibitors. The influx and efflux and/or steady-state distributions of choline in the presence of Na+, Li+, Rb+, Cs+ and mannitol were studied. The influx studies at different cis-choline concentrations revealed two systems for choline influx with different monovalent cation sensitivity and suggested a 1 : 1 interaction of choline with both mechanisms. Choline efflux was stimulated by trans-choline. Calculations of the internal/external concentration ratio expected if choline transport were coupled to the Na+ gradient gave a maximal value of about 10(2). A secondary active transport of choline, where Na+ is the driver solute provides an explanation for the cation sensitivity of the mechanism as well as for the method of coupling of choline transport to the varying demands of the nervous system for acetylcholine.     

Role of the choline exchanger in Na(+)-independent Mg(2+) efflux from rat erythrocytes

Two types of Na(+)-independent Mg(2+) efflux exist in erythrocytes: (1) Mg(2+) efflux in sucrose medium and (2) Mg(2+) efflux in high Cl(-) media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na(+)-independent Mg(2+) efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K(+),Cl(-)- and Na(+),K(+),Cl(-)-symport, Na(+)/H(+)-, Na(+)/Mg(2+)-, Na(+)/Ca(2+)- and K(+)(Na(+))/H(+) antiport, Ca(2+)-activated K(+) channel and Mg(2+) leak flux. We suggest that, in choline Cl medium, Na(+)-independent Mg(2+) efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg(2+) efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg(2+) to the same degree. The K(d) value for inhibition of [(14)C]choline efflux and for inhibition of Mg(2+) efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg(2+) efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg(2+) efflux was reduced to the same degree by these inhibitors as was the [(14)C]choline efflux.