Uptake of [3H]serotonin into plasma membrane vesicles from mouse cerebral cortex

Preparations of plasma membrane vesicles were used as a tool to study the properties of the serotonin transporter in the central nervous system. The vesicles were obtained after hypotonic shock of synaptosomes purified from mouse cerebral cortex. Uptake of [3H]serotonin had a Na+-dependent and Na+-independent component. The Na+-dependent uptake was inhibited by classical blockers of serotonin uptake and had a Km of 63-180 nM, and a Vmax of 0.1-0.3 pmol mg-1 s-1 at 77 mM Na+. The uptake required the presence of external Na+ and internal K+. It required a Na+ gradient ([Na+]out greater than [Na+]in) and was stimulated by a gradient of K+ ([K+]in greater than [K+]out). Replacement of Cl- by other anions (NO2-, S2O3-(2-)) reduced uptake appreciably. Gramicidin prevented uptake. Although valinomycin increased uptake somewhat, the membrane potential per se could not drive uptake because no uptake was observed when a membrane potential was generated by the SCN- ion in the absence of internal K+ and with equal [Na+] inside and outside. The increase of uptake as a function of [Na+] indicated a Km for Na+ of 118 mM and a Hill number of 2.0, suggesting a requirement of two sodium ions for serotonin transport. The present results are accommodated very well by the model developed for porcine platelet serotonin transport (Nelson, P. J., and Rudnick, G. (1979) J. Biol. Chem. 254, 10084-10089), except for the number of sodium ions that are required for transport.     

Evidence for an imipramine-sensitive serotonin transporter in human placental brush-border membranes

Serotonin is actively transported into brush-border membrane vesicles isolated from normal human term placentas and an inward-directed NaCl gradient provides the driving force for this process. Uptake is negligible if Na+ is replaced by Li+, K+, Rb+, Cs+ or choline. The presence of Cl- seems necessary for the maximal activity of this Na+-dependent uptake system. Intravesicular K+ (20-40 mM) stimulates serotonin uptake, the stimulation being considerably greater at pH 7.5 than at pH 6.5. But, in the absence of K+, uptake at pH 6.5 was twice the uptake at pH 7.5. Unlabeled serotonin and dopamine inhibit the uptake of radiolabeled serotonin and the IC50 values are 70 nM and 20 microM, respectively. Histamine and 5-hydroxytryptophan do not significantly interact with the system (IC50 greater than 1 mM). Kinetic analysis reveals that serotonin uptake in these vesicles occurs via a single, saturable, high affinity system (Kt = 51 +/- 2 nM; Vmax = 6.4 +/- 0.1 pmol/mg of protein/15 s). The transporter is highly sensitive to inhibition by imipramine (IC50 = 32 nM) and desipramine (IC50 = 160 nM) but relatively insensitive to reserpine and hydralazine.     

The high and low affinity transport systems for dipeptides in kidney brush border membrane respond differently to alterations in pH gradient and membrane potential

The principal aim of the present study was to investigate the effects of variation in proton gradient and membrane potential on the transport of glycyl-L-glutamine (Gly-Gln) by renal brush border membrane vesicles. Under our conditions of transport assay, Gly-Gln was taken up by brush border membrane vesicles almost entirely as intact dipeptide. This uptake was mediated by two transporters shared by other dipeptides and characterized as the high affinity (Kt = 44.1 +/- 11.2 microM)/low capacity (Vmax = 0.41 +/- 0.03 nmol/mg protein/5 s) and low affinity (Kt = 2.62 +/- 0.50 mM)/high capacity (Vmax 4.04 +/- 0.80 nmol/mg protein/5 s) transporters. In the absence of a pH gradient, only the low affinity system was operational, but with a reduced transport capacity. Imposing a pH gradient of 1.6 pH units increased the Vmax of both transporters. Kinetic analysis of the rates of Gly-Gln uptake as a function of external pH revealed Hill coefficients of close or equal to 1, indicating that transporters contain only one binding site for the interaction with external H+. The effects of membrane potential on Gly-Gln uptake were investigated with valinomycin-induced K+ diffusion potentials. The velocity of the high affinity system but not of the low affinity system increased linearly with increasing inside-negative K+ diffusion potentials (p less than 0.01). The Kt of neither system was affected by alterations in either pH gradient or membrane potential. We conclude that (a) the high affinity transporter is far more sensitive to changes in proton gradient and membrane potential than the low affinity transporter and (b) in the presence of a pH gradient, transport of each dipeptide molecule requires cotransport of one hydrogen ion to serve as the driving force.     

Sodium Dependence of [3H]]Paroxetine Binding and 5- [3H]]Hydroxytryptamine Uptake in Rat Diencephalon

The sodium dependence of binding of [3H]-paroxetine, a selective serotonin uptake inhibitor, to the serotonin transporter in rat diencephalon was studied in both brain membranes and tissue sections and compared with that of 5-[3H]hydroxytryptamine ([3H]5-HT) uptake by synaptosomes from the same region. Binding of [3H]-paroxetine in both the membranes and sections displayed clear sodium dependence until a plateau occurring at 60 nM NaCl, the EC50 for sodium being 8 and 25 mM, respectively. The affinity (1/KD) of [3H]paroxetine binding was a simple hyperbolic function of sodium concentration. In contrast, the density of [3H]paroxetine sites was not affected by external Na+ concentration. The uptake of [3H]5-HT showed a similar pattern of sodium dependence with an EC50 for Na+ of 25 mM. Both the affinity (1/Km) and the rate (Vmax) of [3H]5-HT uptake were dependent on external [Na+] with sodium-dependence curves fitting a rectangular hyperbola. The kinetic analysis of results indicates that one sodium ion is required for the binding of [3H]paroxetine as well as for the binding and translocation of each [3H]5-HT molecule. The results concur with a single-site model of the sodium-dependent serotonin transporter with common or overlapping domains for 5-HT and 5-HT uptake inhibitors.     

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.     

Intracellular pH and Na fluxes in barnacle muscle with evidence for reversal of the ionic mechanism of intracellular pH regulation

The ion transport mechanism that regulates intracellular pH (pHi) in giant barnacle muscle fibers was studied by measuring pHi and unidirectional Na+ fluxes in internally dialyzed fibers. The overall process normally results in a net acid extrusion from the cell, presumably by a membrane transport mechanism that exchanges external Na+ and HCO-3 for internal Cl- and possibly H+. However, we found that net transport can be reversed either by lowering [HCO-3]o and pHo or by reducing [Na+]o. This reversal (acid uptake) required external Cl-, was stimulated by raising [Na+]i, and was blocked by SITS. When the transporter was operating in the net forward direction (acid extrusion), we found a unidirectional Na+ influx of approximately 60 pmol . cm-2 . s-1, which required external HCO-3 and internal Cl- and was stimulated by cyclic AMP and blocked by SITS or DIDS. These properties of the Na+ influx are all shared with the net acid extrusion process. We also found that under conditions of net forward transport, the pHi-regulating system mediated a unidirectional Na+ efflux, which was significantly smaller than the simultaneous Na+ influx. These data are consistent with a reversible transport mechanism which, even when operating in the net forward direction, mediates a small amount of reversed transport. We also found that the ouabain-sensitive Na+ efflux was sharply inhibited by acidic pHi, being totally absent at pHi values below approximately 6.8.     

Modeling of the interaction of Na+ and K+ with the binding of dopamine and [3H]WIN 35,428 to the human dopamine transporter

Although much is known about the effects of Na+, K+, and Cl- on the functional activity of the neuronal dopamine transporter, little information is available on their role in the initial event in dopamine uptake, i.e., the recognition step. This was addressed here by studying the inhibition by dopamine of the binding of [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)[3H]tropane], a phenyltropane analogue of cocaine, to the cloned human dopamine transporter expressed in HEK-293 cells. The decrease in the affinity of dopamine (or WIN 35,428) binding affinity with increasing [K+] could be fitted to a competitive model involving an inhibitory cation site (1) overlapping with the dopamine (or WIN 35,428) domain. The K+ IC50 for inhibiting dopamine or WIN 35,428 binding increased linearly with [Na+], indicating a K(D,Na+) of 30-44 mM and a K(D,K+) of 13-16 mM for this cation site. A second Na+ site (2), distal from the WIN 35,428 domain but linked by positive allosterism, was indicated by model fitting of the WIN 35,428 binding affinities as a function of [Na+]. No strong evidence for this second site was obtained for dopamine binding in the absence or presence of low (20 mM) Cl- and could not be acquired for high [Cl-] because of the lack of a suitable substitute ion for Na+. The K(D) but not Bmax of [3H]WIN 35,428 binding increased as a function of the [K+]/[Na+] ratio regardless of total [Cl-] or ion tonicity. A similar plot was obtained for the Ki of dopamine binding, with Cl- at > or = 140 mM decreasing the Ki. At 290 mM Cl- and 300 mM Na+ the potency of K+ in inhibiting dopamine binding was enhanced as compared with the absence of Cl- in contrast to the lack of effect of Cl- up to 140 mM (Na up to 150 mM). The results indicate that Cl- at its extracellular level enhances dopamine binding through a mechanism not involving site 1. The observed correspondence between the WIN 35,428 and dopamine domains in their inclusion of the inhibitory cation site explains why many of the previously reported interrelated effects of Na+ and K+ on the binding site of radiolabeled blockers to the dopamine transporter are applicable to dopamine uptake in which dopamine recognition is the first step.     

22Na+ fluxes in thymic lymphocytes. I. Na+/Na+ and Na+/H+ exchange through an amiloride-insensitive pathway

The Na+ transport pathways of normal rat thymocytes were investigated. Na+ conductance was found to be lower than K+ conductance, which is consistent with reported values of membrane potential. In contrast, the isotopically measured Na+ permeability was greater than 10-fold higher than that of K+, which indicates that most of the flux is electroneutral. Cotransport with Cl- (or K+ and Cl-) and countertransport with Ca2+ were ruled out by ion substitution experiments and use of inhibitors. Countertransport for Na+ or H+ through the amiloride-sensitive antiport accounts for only 15-20% of the resting influx. In the presence of amiloride, 22Na+ uptake was increased in Na+-loaded cells, which suggests the existence of Na+/Na+ countertransport. Cytoplasmic pH determinations using fluorescent probes indicated that under certain conditions this amiloride-resistant system will also exchange Na+ for H+, as evidenced by an internal Na+- dependent acidification is proportional to internal [Na+] but inversely related to extracellular [Na+]. Moreover, 22Na+ uptake is inhibited by increasing external [H+]. The results support the existence of a substantial amiloride-insensitive, electroneutral cation exchange system capable of transporting Na+ and H+.     

Interaction of Na+, K+, and Cl- with the binding of amphetamine, octopamine, and tyramine to the human dopamine transporter

Little information is available on the role of Na+, K+, and Cl- in the initial event of uptake of substrates by the dopamine transporter, i.e., the recognition step. In this study, substrate recognition was studied via the inhibition of binding of [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)[3H]tropane], a cocaine analogue, to the human dopamine transporter in human embryonic kidney 293 cells. D-Amphetamine was the most potent inhibitor, followed by p-tyramine and, finally, dl-octopamine; respective affinities at 150 mM Na+ and 140 mM Cl- were 5.5, 26, and 220 microM. For each substrate, the decrease in the affinity with increasing [K+] could be fitted to a competitive model involving the same inhibitory cation site (site 1) overlapping with the substrate domain as reported by us previously for dopamine. K+ binds to this site with an apparent affinity, averaged across substrates, of 9, 24, 66, 99, and 134 mM at 2, 10, 60, 150, and 300 mM Na+, respectively. In general, increasing [Na+] attenuated the inhibitory effect of K+ in a manner that deviated from linearity, which could be modeled by a distal site for Na+, linked to site 1 by negative allosterism. The presence of Cl- did not affect the binding of K+ to site 1. Models assuming low binding of substrate in the absence of Na+ did not provide fits as good as models in which substrate binds in the absence of Na+ with appreciable affinity. The binding of dl-octopamine and p-tyramine was strongly inhibited by Na+, and stimulated by Cl- only at high [Na+] (300 mM), consonant with a stimulatory action of Cl- occurring through Na+ disinhibition.     

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism.     

The glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: transport mechanism, regulation by ATP and characterization of the glutamine/glutamate antiport

The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which external glutamine and Na+ are cotransported in exchange with internal glutamine (or other amino acids). The glutamine-Na+ cotransport occurred with a 1:1 stoichiometry. The concentration of Na+ did not influence the Km for glutamine and vice versa. Experimental data obtained by a bi-substrate analysis of the glutamine-Na+ cotransport, together with previous report on the glutamine(ex)/glutamine(in) pseudo bi-reactant analysis, indicated that the transporter catalyses a three-substrate transport reaction with a random simultaneous mechanism. The presence of ATP in the internal compartment of the proteoliposomes led to an increase of the Vmax of the transport and to a decrease of the Km of the transporter for external Na+. The reconstituted glutamine/amino acid transporter was inhibited by glutamate; the inhibition was more pronounced at acidic pH. A kinetic analysis revealed that the inhibition was competitive with respect to glutamine. Glutamate was also transported in exchange with glutamine. The external Km of the transporter for glutamate (13.3 mM) was slightly higher than the internal one (8.3 mM). At acidic pH the external but not the internal Km decreased. According with the Km values, glutamate should be transported preferentially from inside to outside in exchange for external glutamine and Na+.     

Stoichiometry and ion dependencies of the intracellular-pH-regulating mechanism in squid giant axons

The ion transport system responsible for intracellular pH (pHi) regulation in squid giant axons was examined in experiments with pH- sensitive microelectrodes and isotopic fluxes of Na+ and Cl-. In one study, axons were acid-loaded and the rate of the subsequent pHi recovery was used to calculate the acid extrusion rate. There was an absolute dependence of acid extrusion on external Na+, external HCO-3 (at constant pH), and internal Cl-. Furthermore, the dependence of the acid extrusion rate on each of these three parameters was described by Michaelis-Menten kinetics. Acid extrusion was stimulated by an acid pHi, required internal ATP, and was blocked by external 4-acetamido-4'- isothiocyanostilbene-2,2'-disulfonate (SITS). Under a standard set of conditions (i.e., [HCO-3]o = 12 mM, pHo = 8.00, [Na+]o = 425 mM, [Cl-]i = 150 mM, [ATP]i = 4 mM, pHi = 6.5, and 16 degrees C), the mean acid extrusion rate was 7.5 pmol X cm-2 X s-1. In a second study under the above standard conditions, the unidirectional Na+ efflux (measured with 22Na) mediated by the pHi-regulating system was found to be approximately 0, whereas the mean influx was about 3.4 pmol X cm-2 X s- 1. This net influx required external HCO-3, internal Cl-, and acid pHi, internal ATP, and was blocked by SITS. In the final series of experiments under the above standard conditions, the unidirectional Cl- influx (measured with 36Cl) mediated by the pHi-regulating system was found to be approximately 0, whereas the mean efflux was approximately 3.9 pmol X cm-2 X s-1. This net efflux required external HCO-3, external Na+, an acid pHi, internal ATP, and was blocked by SITS. We conclude that the pHi-regulating system mediates the obligate net influx of HCO-3 (or equivalent species) and Na+ and the net efflux of Cl- in the stoichiometry of 2:1:1. The transport system is stimulated by intracellular acid loads, requires ATP, and is blocked by SITS.     

Na/K/Cl cotransport in cultured human fibroblasts

The transport characteristics and regulation of the Na/K/Cl cotransport system were investigated in cultured human fibroblasts (HSWP cells). The existence of the system was documented by the finding that digitoxin-insensitive K+ influx was dependent upon the presence of both Na+ and Cl- in the extracellular milieu. It was found that only Br- could partially substitute for Cl-, with SCN-, I-, acetate, and gluconate being ineffective. Li+ could partially substitute for Na+; however, choline was without effect. The shape of the titration curves for K+ influx versus extracellular Cl- concentration was dependent upon the substituted anion. Furthermore, the apparent Km for Cl- at saturating [K+]o and [Na+]o, was also dependent upon the substituted anion and ranged from 30 mM (gluconate substitution) to 100 mM (acetate substitution). The titration curves for K+ influx versus extracellular Na+ concentration displayed hyperbolic kinetics and the apparent Km = 15 mM at saturating [K+]o. The curve for K+ influx versus extracellular K+ concentration was a hyperbola and the apparent Km for K+ = 3 mM at saturating [Na+]o. The digitoxin-insensitive K+ flux was found to be sensitive to related 5-sulfamoylbenzoic acid derivatives, commonly known as "loop" diuretics and to be insensitive to both: amiloride (3,5-diamino-N-(aminoiminomethyl)-6-chloropyrazinecarboxamide++ +) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. The Na/K/Cl cotransport system was not stimulated by serum, but was slightly stimulated by two peptide mitogens. Furthermore, agents which cause an elevation in cellular cyclic AMP levels were found to be potent inhibitors of cotransport.     

Active transport of alanine by the neutral amino-acid exchanger ASCT1

ASCT1 protein is a member of the glutamate transporter superfamily, which shows system ASC selectivity and properties and has been characterized as a Na+-dependent neutral amino-acid exchanger. Here, by using ASCT1-expressing oocytes, the uptake of alanine and glutamate was measured to investigate ASCT1's ability to mediate a concentrative transport of alanine, ASCT1's sodium dependence, and the influence of pH on the mutual inhibition between alanine and glutamate. Alanine uptake was measured after 30 min incubation. Kinetic analysis of the Na+ dependence of alanine uptake showed an apparent K0.5 (affinity constant) value for Na+ of 23.1 +/- 4.3 mM (mean +/- SE). Concentration dependence of alanine uptake was tested at 100 and 1 mM Na+, with apparent K0.5 values of 0.16 +/- 0.04 and 1.8 +/- 0.4 mM, respectively, at pH 7.5, and 0.21 +/- 0.06 and 1.9 +/- 0.3 mM at pH 6. Vmax was not modified between 100 and 1 mM Na+ at either pH. ASCT1 actively transports alanine and accumulates it in the cytosol even when the Na+ concentration in the medium was as low as 1-3 mM. 22Na uptake studies revealed that Na+ transport was stimulated by the presence of alanine in the medium. Our results demonstrate that ASCT1 is able to mediate a concentrative transport of alanine, which is Na+-dependent but not coupled to the Na+ gradient.     

Reconstitution into liposomes of the B degrees -like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na+ dependent [3H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K+. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [3H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na+ and internal K+. The transporter showed low if there is any tolerance towards the substitution of Na+ or K+ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na+ concentration cooperativity index close to 1 was derived, indicating that 1 Na+ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na+ transport. Optimal rate of 0.1 mM [3H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [3H]glutamine (and [3H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl2, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue alpha-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na+ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B degrees-like.     

Permeability properties and intracellular ion concentrations of epithelial cells in rat duodenum.

Effects of the K+ concentration in the bathing fluid ([K+]l) on the intracellular K+, Na+ and Cl- concentrations ([K+]i [Na+]i and [Cl-]i) as well as on the electrical potential were studied in rat duodenum. Changes in the mucosal K+ concentration ([K+]m), bringing the sum of Na+ and K+ concentrations to 147.2 mM constant, had little effect on the transmural potential difference (PDt), but did induce marked changes in the mucosal membrane potential (Vm). As [K+]m increased, Vm was depolarized gradually and obeyed the Nernst equation for a potassium electrode in the range of [K+]m greater than approx. 60 mM. Experiments of ion analyses were carried out on strips of duodenum to determine the effect of changing the external K+ concentrations on [K+] i, [Na+]i and [Cl-]i. An increase in [K+]o resulted in increases in [K+]i and [Cl-]i and a decrease in [Na+]i, [K+]i approaching its maximum at [K+]o greater than 70 mM. Such changes in [K+]i and [Na+]i seem to correlate quantitatively with the changes in [K+]o and [Na+]o. The values of the ratio of permeability coefficients, Pna+/PK+ were estimated using the Vm values and intracellular ion concentrations measured in these experiments. The results suggested that there appeared a rather abrupt increase in the PNa+/PK+ ratio from 0 to approx. 0.1, as [K+]m decreased.     

Characterisation of the serotonin efflux induced by cytosolic Ca2+ and Na+ concentration increase in human platelets.

BACKGROUND/AIM: The present study aimed at elucidating the mechanism(s) of serotonin (5-HT) efflux induced by thapsigargin from human platelets in the absence of extra-cellular Ca2+. METHODS: Efflux of pre-loaded radiolabeled serotonin was generally determined by filtration techniques. Cytosolic concentrations of Ca2+, Na+ and H+ were measured with appropriate fluorescent probes. RESULTS: 5-HT efflux from control or reserpine-treated platelets--where reserpine prevents 5-HT transport into the dense granules--was proportional to thapsigargin evoked cytosolic [Ca2+]c increase. Accordingly factors as prostacyclin, aspirin and calyculin which reduced [Ca2+]c-increase also inhibited the 5-HT efflux. Thapsigargin, which also caused a remarkable increase in cytosolic [Na+]c, promoted less 5-HT release, in parallel to lower [Na+]c and [Ca2+]c increase, when added to platelet suspensions containing low [Na+]. The Na+/H+ exchanger monensin increased the [Na+]c and induced 5-HT efflux without affecting the Ca2+ level. The 5-HT efflux induced by both [Ca2+] or [Na+]c increase did not depend on pH or membrane potential changes, whereas it decreased in the absence of extra-cellular K+, and increased in the absence of Cl- or Na+. CONCLUSION: Increases in [Ca2+]c and [Na+]c independently induce serotonin efflux through the outward directed plasma membrane serotonin transporter SERT. This event might be physiologically important at the level of capillaries or narrowed arteries where platelets are subjected to high shear stress which causes [Ca2+]c increase followed by 5-HT release which might exert vasodilatation.     

Bicarbonate/chloride antiport in Vero cells: II. Mechanisms for bicarbonate-dependent regulation of intracellular pH

The rates of bicarbonate-dependent uptake and efflux of 22Na+ in Vero cells were studied and compared with the uptake and efflux of 36Cl-. Both processes were strongly inhibited by DIDS. Whereas the transport of chloride increased approximately ten-fold when the internal pH was increased over a narrow range around neutrality, the uptake of Na+ was much less affected by changes in pH. The bicarbonate-linked uptake of 22Na+ was dependent on internal Cl- but not on internal Na+. At a constant external concentration of HCO3-, the amount of 22Na+ associated with the cells increased when the internal concentration of HCO3- decreased and vice versa, which is compatible with the possibility that the ion pair NaCO3- is the transported species and that the transport is symmetric across the membrane. Bicarbonate inhibited the uptake of 36Cl- both in the absence and presence of Na+. At alkaline internal pH, HCO3- stimulated the efflux of 36Cl- from preloaded cells, while at acidic internal pH both Na+ and HCO3- were required to induce 36Cl- efflux. We propose a model for how bicarbonate-dependent regulation of the internal pH may occur. This model implies the existence of two bicarbonate transport mechanisms that, under physiological conditions, transport OH(-)-equivalents in opposite directions across the plasma membrane.     

Cation-coupled chloride influx in squid axon. Role of potassium and stoichiometry of the transport process

Evidence is presented showing that the Cl- uptake process in the squid giant axon is tightly coupled not only to Na+ uptake but also to K+ uptake. Thus, removal of external K+ causes both Cl- and Na+ influxes to be reduced, particularly when [Cl-]i is low, that is, under conditions previously shown to be optimal for Cl-/Na+-coupled influx. In addition, there exists a ouabain-insensitive K+ influx, which depends on the presence of external Cl- and Na+, is inversely proportional to [Cl-]i, and is blocked by furosemide/bumetanide. Finally, this ouabain-insensitive K+ influx appears to require the presence of cellular ATP. The stoichiometry of the coupled transport process was measured using a double-labeling technique combining in the same axon either 36Cl and 42K or 22Na and 42K. The stoichiometry of the flux changes occurring in response either to varying [Cl-]i between 150 and 0 mM or to treatment with 0.3 mM furosemide is, in both cases, approximately 3:2:1 (Cl-/Na+/K+). Although these fluxes require ATP, they are not inhibited by 3 mM vanadate. In addition, treatment with DIDS has no effect on the fluxes.     

Properties of the Na+-dependent Cl-/HCO3- exchange system in U937 human leukemic cells

U937 cell possess two mechanisms that allow them to recover from an intracellular acidification. The first mechanism is the amiloride-sensitive Na+/H+ exchange system. The second system involves bicarbonate ions. Its properties have been defined from intracellular pH (pHi) recovery experiments, 22Na+ uptake experiments, 36Cl- influx and efflux experiments. Bicarbonate induced pHi recovery of the cells after a cellular acidification to pHi = 6.3 provided that Na+ ions were present in the assay medium. Li+ or K+ could not substitute for Na+. The system seemed to be electroneutral. 22Na+ uptake experiments showed the presence of a bicarbonate-stimulated uptake pathway for Na+ which was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate. The bicarbonate-dependent 22Na+ uptake component was reduced by depleting cells of their internal Cl- and increased by removal of external Cl-. 36Cl- efflux experiments showed that the presence of both external Na+ and bicarbonate stimulated the efflux of 36Cl- at a cell pHi of 6.3. Finally a 36Cl- uptake pathway was documented. It was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (K0.5 = 10 microM) and bicarbonate (K0.5 = 2 mM). These results are consistent with the presence in U937 cells of a coupled exchange of Na+ and bicarbonate against chloride. It operates to raise the intracellular pH. Its pHi and external Na+ dependences were defined. No evidence for a Na+-independent Cl-/HCO3- exchange system could be found. The Na+-dependent Cl-/HCO3- exchange system was relatively insensitive to (aryloxy)alkanoic acids which are potent inhibitors of bicarbonate-induced swelling of astroglia and of the Li(Na)CO3-/Cl- exchange system of human erythrocytes. It is concluded that different anionic exchangers exist in different cell types that can be distinguished both by their biochemical properties and by their pharmacological properties.