Partial purification of the sodium- and potassium-coupled L-glutamate transport glycoprotein from rat brain

The sodium- and potassium-coupled L-glutamate transporter from rat brain has been solubilized with cholate and 10-20-fold purified using Wheat Germ Agglutinin-Sepharose 4B. Transport activity--as determined upon reconstitution of the fraction into liposomes--was retained on the column and eluted by N-acetylglucosamine. When the glycoprotein fraction was depleted of the N-acetylglucosamine and applied to a second round of lectin-chromatography, the L-glutamate transport activity was retained and again could be eluted by the sugar. The transporter activity reconstituted from the glycoprotein fraction exhibited the same features as that in synaptic plasma membranes, including electrogenicity, an absolute dependence on external sodium and internal potassium, affinity and stereospecificity. Furthermore, efflux and exchange properties of the reconstituted preparation were also unchanged by the solubilisation and lectin-chromatography. These observations indicate that the sodium- and potassium-coupled L-glutamate transporter is a glycoprotein and is predominantly reconstituted in the 'right-side-out' conformation.     

Ion transport mechanisms in the mesonephric collecting duct system of the toad Bufo bufo: microelectrode recordings from isolated and perfused tubules

It is not clear how and whether terrestrial amphibians handle NaCl transport in the distal nephron. Therefore, we studied ion transport in isolated perfused collecting tubules and ducts from toad, Bufo bufo, by means of microelectrodes. No qualitative difference in basolateral cell membrane potential (Vbl) was observed between tubules and ducts in response to ion substitutions, inhibitor and agonist applications. Cl- substitution experiments indicated a small Cl- conductance in the basolateral membrane. The apical membrane did not have a significant Cl- conductance. Luminal [Na+] steps and amiloride application showed a small apical Na+ conductance. Arginine vasotocin depolarized Vbl. The small apical Na+ conductance indicates that the collecting duct system contributes little to NaCl reabsorption when compared to aquatic amphibians. In contrast, Vbl rapidly depolarized upon lowering of [Na+] in the bath, demonstrating the presence of a Na+-coupled anion transporter. [HCO3-] steps revealed that this transporter is not a Na+-HCO3- cotransporter. Together, our results indicate that a major task of the collecting duct system in B. bufo is not conductive NaCl transport but rather K+ secretion, as shown by our previous studies. Moreover, our results indicate the presence of a novel basolateral Na+-coupled anion transporter, the identity of which remains to be elucidated.     

Regulation of Na+/H+ and Cl-/HCO3- antiports in Vero cells

The effect of serum, phorbol-12-myristate-13-acetate (TPA), and forskolin on the activity Na+/H+ antiport and the Na(+)-coupled and Na(+)-independent Cl-/HCO3- antiport was studied in Vero cells by measuring 22Na+ and 36Cl- fluxes and changes in cytosolic pH (pHi). The Na(+)-independent Cl-/HCO3- antiport, which acts as an acidifying mechanism, is strongly pH-sensitive. In serum-starved cells it is activated at alkaline cytosolic pH, with a half-maximal activity at pHi approximately 7.20. Incubation with serum increased the activity of the Na(+)-independent Cl-/HCO3- antiport at pHi values from 6.8 to 7.2. Thus serum appeared to alter the pHi sensitivity of this antiporter such that the threshold value for activation of the antiport was shifted to a more acidic value. Na+/H+ antiport was somewhat stimulated initially by addition of serum, but further incubation with serum (greater than 45 min) decreased its activity. The activity of the Na(+)-coupled Cl-/HCO3- antiport, which is the major alkalinizing antiport in Vero cells, was not altered by short-term incubation with serum (less than 10 min) but decreased after prolonged incubation (greater than 45 min). Our findings with TPA and forskolin indicate that the effect of serum is partly mediated by the protein kinase C pathway, whereas the cyclic adenosine monophosphate pathway does not appear to play an important role. The net effect of serum on the pHi-regulating antiports was a slight decrease in intracellular pH.     

Na+, K+, and Cl- transport in resting pancreatic acinar cells

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl- ]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.     

Quercetin and NPPB-induced diminution of aldosterone action on Na+ absorption and ENaC expression in renal epithelium

In renal epithelial A6 cells, aldosterone applied for 24 h increased the transepithelial Cl- secretion over 30-fold due to activation of the Na+/K+/2Cl- cotransporter and stimulated the transepithelial Na+ absorption, activity of epithelial Na+ channel (ENaC), and alpha-ENaC mRNA expression. The stimulatory action of aldosterone on the transepithelial Na+ absorption, ENaC activity, and alpha-ENaC mRNA expression was diminished by 24h-pretreatment with quercetin (an activator of Na+/K+/2Cl- cotransporter participating in Cl- entry into the cytosolic space) or 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB) (a blocker of Cl- channel participating in Cl- release from the cytosolic space), while 24h-pretreatment with bumetanide (a blocker of Na+/K+/2Cl- cotransporter) enhanced the stimulatory action of aldosterone on transepithelial Na+ absorption. On the other hand, under the basal (aldosterone-unstimulated) condition, quercetin, NPPB or bumetanide had no effect on transepithelial Na+ absorption, activity of ENaC or alpha-ENaC mRNA expression. These observations suggest that although aldosterone shows overall its stimulatory action on ENaC (transepithelial Na+ transport), aldosterone has an inhibitory action on ENaC (transepithelial Na+ transport) via activation of the Na+/K+/2Cl- cotransporter, and that modification of activity of Cl- transporter/channel participating in the transepithelial Cl- secretion influences the aldosterone-stimulated ENaC (transepithelial Na+ transport).     

Distributions of H+,K+-ATPase and Cl–,HCO3–-ATPase in micromere-derived cells of sea urchin embryos

In cultured cells derived from isolated micromeres of sea urchin eggs, H+,K+-ATPase activity, which became detectable simultaneously with the initiation of spicule formation, was localized in the plasma membrane and the microsome fractions. Activities of marker enzymes for plasma membrane, 5'-nucleotidase, Na+,K+-ATPase, and adenylate cyclase, were found to be high in the plasma membrane fraction. Considerable activity of rotenone-insensitive NADPH-cytochrome c reductase, a marker enzyme for microsome, was detectable in the microsome fraction. These fractions exhibited barely any appreciable activity of markers for the other organellae. H+,K+-ATPase in plasma membrane probably mediates H+ release from the cells, in which H+ is produced in overall reaction to form CaCO3, the main component of spicules, from Ca2+, CO2 and H2O. Cl-,HCO3(-)-ATPase activity was also found in these two fractions before and after the initiation of spicule formation. After initiation, the skeletal vacuole fraction was obtained from subcellular structures containing spicules. Considerable activity of Cl-,HCO3(-)-ATPase was observed in this fraction, which exhibited a weak activity of UDP-galactose: N-acetylglucosamine galactosyltransferase, a marker enzyme for Golgi body. Cl-,HCO3(-)-ATPase in the skeletal vacuole membrane probably mediates HCO3- transport into the vacuoles to supply HCO3- for spicule formation.     

Functional characterization of the Betaine/gamma-aminobutyric acid transporter BGT-1 expressed in Xenopus oocytes.

Betaine is an osmolyte accumulated in cells during osmotic cell shrinkage. The canine transporter mediating cellular accumulation of the osmolyte betaine and the neurotransmitter gamma-aminobutyric acid (BGT-1) was expressed in Xenopus oocytes and analyzed by two-electrode voltage clamp and tracer flux studies. Exposure of oocytes expressing BGT-1 to betaine or gamma-aminobutyric acid (GABA) depolarized the cell membrane in the current clamp mode and induced an inward current under voltage clamp conditions. At 1 mM substrate the induced currents decreased in the following order: betaine = GABA > diaminobutyric acid = beta-alanine > proline = quinidine > dimethylglycine > glycine > sarcosine. Both the Vmax and Km of GABA- and betaine-induced currents were voltage-dependent, and GABA- and betaine-induced currents and radioactive tracer uptake were strictly Na+-dependent but only partially dependent on the presence of Cl-. The apparent affinity of GABA decreased with decreasing Na+ concentrations. The Km of Na+ also depended on the GABA and Cl- concentration. A decrease of the Cl- concentration reduced the apparent affinity for Na+ and GABA, and a decrease of the Na+ concentration reduced the apparent affinity for Cl- and GABA. A comparison of 22Na+-, 36Cl--, and 14C-labeled GABA and 14C-labeled betaine fluxes and GABA- and betaine-induced currents yielded a coupling ratio of Na+/Cl-/organic substrate of 3:1:1 or 3:2:1. Based on the data, a transport model of ordered binding is proposed in which GABA binds first, Na+ second, and Cl- third. In conclusion, BGT-1 displays significant functional differences from the other members of the GABA transporter family.     

Cloning and expression of a glycine transporter reveal colocalization with NMDA receptors.

A complementary DNA clone encoding a transporter for glycine has been isolated from rat brain, and its functional properties have been examined in mammalian cells. The transporter displays high affinity for glycine (KM approximately 100 microM) and is dependent on external Na+ and Cl-. Northern blot analysis indicates that the distribution of the mRNA encoding the glycine transporter is restricted to the nervous system. In situ hybridization data are consistent with roles for the transporter in both glycine neurotransmission and glycine modulation of N-methyl-D-aspartate (NMDA) receptors in the hippocampus. The identification of this transporter therefore opens the study of the molecular mechanisms underlying both inhibitory glycinergic transmission and NMDA-mediated excitatory transmission.     

Expression of a cloned gamma-aminobutyric acid transporter in mammalian cells.

The cDNA clone GAT-1, which encodes a Na(+)- and Cl(-)-coupled GABA transporter from rat brain, has been expressed in mammalian cells using three different systems: (1) transient expression upon transfection of mouse Ltk- cells with a eukaryotic expression vector containing GAT-1; (2) stable expression in L-cells transfected with the same vector; (3) transfection of HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase. Similar results both qualitatively and quantitatively were obtained with all systems. The GABA transporter expressed in HeLa and L-cells retains all the properties described previously for GABA transport into synaptosomes and synaptic plasma membrane vesicles. It was fully inhibited by cis-3-aminocyclohexanecarboxylic acid (ACHC) and not by beta-alanine. The KM for GABA transport and the IC50 for ACHC inhibition were similar to the presynaptic transporter. Accumulated [3H]GABA was released from transfected cells by dissipating the transmembrane Na+ gradient with nigericin or by exchange with unlabeled external GABA. Accumulation was stimulated by both Na+ and Cl- in the external medium. However, in the absence of external Cl-, a small amount of GABA transport remained which was dependent on GAT-1 transfection. Functional expression of the GABA transporter was abolished by tunicamycin. An antitransporter antibody specifically immunoprecipitates a polypeptide with an apparent molecular mass of about 70 kDa from GAT-1-transfected cells. When cells were grown in the presence of tunicamycin, only a faint band of apparent mass of about 60 kDa was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ion exchangers mediating NaCl transport in the renal proximal tubule

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. Studies of isolated brush-border membrane vesicles confirmed the presence of Na+-H+ exchange and identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanisms of uphill Cl- entry. We found that formate and oxalate each stimulate NaCl absorption in microperfused proximal tubules. Stimulation of NaCl absorption by formate was blocked by the Na+-H+-exchange inhibitor EIPA, whereas stimulation by oxalate was blocked by omission of sulfate from the perfusion solutions. These observations were consistent with recycling of formate from lumen to cell by H+-coupled formate transport in parallel with Na+-H+ exchange and recycling of oxalate by oxalate-sulfate exchange in parallel with Na+-sulfate cotransport. Using isoform-specific antibodies, we found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the loop of Henle. The inhibitor sensitivity of Na+-H+ exchange in renal brush-border vesicles and of HCO3- absorption in microperfused tubules suggested that NHE3 is responsible for most, if not all, apical membrane Na+-H+ exchange in the proximal tubule. The role of NHE3 in mediating proximal tubule HCO3- absorption and formate-dependent Cl- absorption was confirmed by studies in NHE3 null mice. Finally, we cloned and functionally expressed CFEX, an anion transporter expressed on the apical surface of proximal tubule cells and capable of mediating Cl(-)-formate exchange.     

The second intracellular loop of the glycine transporter 2 contains crucial residues for glycine transport and phorbol ester-induced regulation

Na+ and Cl(-)-coupled glycine transporters control the availability of glycine neurotransmitter in the synaptic cleft of inhibitory glycinergic pathways. In this report, we have investigated the involvement of the second intracellular loop of the neuronal glycine transporter 2 (GLYT2) on the protein conformational equilibrium and the regulation by 4alpha-phorbol 12 myristate 13-acetate (PMA). By substituting several charged (Lys-415, Lys-418, and Lys-422) and polar (Thr-419 and Ser-420) residues for different amino acids and monitoring plasma membrane expression and kinetic behavior, we found that residue Lys-422 is crucial for glycine transport. The introduction of a negative charge in 422, and to a lower extent in neighboring N-terminal residues, dramatically increases transporter voltage dependence as assessed by response to high potassium depolarizing conditions. In addition, [2-(trimethylammonium)ethyl] methanethiosulfonate accessibility revealed a conformational connection between Lys-422 and the glycine binding/permeation site. Finally, we show that the mutation of positions Thr-419, Ser-420, and mainly Lys-422 to acidic residues abolishes the PMA-induced inhibition of transport activity and the plasma membrane transporter internalization. Our results establish a new structural basis for the action of PMA on GLYT2 and suggest a complex nature of the PMA action on this glycine transporter.     

Bile-salt inhibition of sodium ion-coupled D-glucose and L-alanine accumulation by brush-border-membrane vesicles from hamster jejunum.

The effects of bile salts on Na+-coupled accumulation of D-glucose and L-alanine by brush-border-membrane vesicles isolated from hamster jejunum were investigated. The approximate percentage inhibition of Na+-coupled D-glucose accumulation produced by various bile salts at a concentration of 1 mM were: deoxycholate and chenodeoxycholate, 60%; glycine and taurine conjugates of deoxycholate and chenodeoxycholate, 40--50%; lithocholate, 45%; cholate and its glycine and taurine conjugates, less than 10%. Inhibition of Na+-coupled accumulation of D-glucose was rapid, reversible and not due to dissolution of the vesicles. Na+-coupled accumulation of L-alanine was also inhibited by deoxycholate. Deoxycholate but not cholate enhanced (1) the rate of Na+ influx, (2) the rate of influx of D-glucose and L-alanine in the absence of a Na+ gradient and (3) the rate of efflux of D-glucose and L-alanine from vesicles preloaded with this sugar or amino acid. Deoxycholate-stimulated efflux of D-glucose was not blocked by phlorizin, which completely prevented efflux in the absence of this bile salt. These results suggest that selected bile salts inhibit Na+-coupled accumulation of D-glucose and L-alanine by enhancing the rate of dissipation of the Na+ gradient required for substrate accumulation. In addition, bile salts may also decrease D-glucose and L-alanine accumulation by increasing the rate of efflux of these substrates across the brush-border plasma membrane.     

Membrane localization of H+ and HCO3- transporters in the rat pancreatic duct

The pancreatic duct secretes alkaline fluid that is rich in HCO3- and poor in Cl-. The molecular mechanisms that mediate ductal secretion and are responsible for the axial gradients of Cl- and HCO3- along the ductal tree are not well understood because H+ and HCO3- transport by duct cells have not been characterized or localized. To address these questions, we microdissected the intralobular, main, and common segments of the rat pancreatic duct. H+ and HCO3- transporters were characterized and localized by following intracellular pH while perfusing the bath and the lumen of the ducts. In intralobular ducts, Na(+)-dependent and amiloride-sensitive recovery from acid load in the absence of HCO3- was used to localize a Na+/H+ exchanger to the basolateral membrane (BLM). Modification of Cl- gradients across the luminal (LM) and BLM in the presence of HCO3- showed the presence of Cl- /HCO3- exchangers on both membranes of intralobular duct cells. Measurement of the effect of Cl- on one side of the membrane on the rate and extent of pHi changes caused by removal and addition of Cl- to the opposite side suggested that both exchangers are present in the same cell. In the presence of HCO3-, intralobular duct cells used three separate mechanisms to extrude H+: (a) BLM-located Na+/H+ exchange, (b) Na(+)-independent vacuolar-type H+ pump, and (c) BLM-located, Na(+)- dependent, amiloride-insensitive, and 4',4'-diisothiocyanatostilbene- 2,2'-disulfonic acid sensitive mechanism, possibly a Na(+)-dependent HCO3- transporter. The main and common segments of the duct displayed similar mechanisms and localization of H+ and HCO3- transporters to the extent studied in the present work. In addition to the transporters found in intralobular ducts, the main and common ducts showed Na+/H+ exchange activity in the LM. Three tests were used to exclude a significant luminal to basolateral Na+ leak as the cause for an apparent luminal Na+/H+ exchange in an HCO3- secreting cells: (a) addition of amiloride and removal of Na+ from the LM had a profound effect on Na+/H+ exchange activity on the BLM and vice versa; (b) inhibition of all transporters in the BLM by bathing the duct in the inert hydrocarbon Fluorinert FC-75 did not prevent cytosolic acidification caused by removal of luminal Na+; and (c) luminal Na+ did not activate the basolateral Na(+)-dependent HCO3- transporter. An Na(+)-independent, bafilomycin-sensitive H+ pumping activity was marginal in the absence of HCO3-.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular pH-regulatory mechanisms in pancreatic acinar cells. I. Characterization of H+ and HCO3- transporters

Rat pancreatic acini loaded with the pH sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to characterize intracellular pH (pHi) regulatory mechanisms in these cells. The acini were attached to cover slips and continuously perfused. In 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered solutions recovery from acid load (H+ efflux) required extracellular Na+ (Na+out) and was blocked by amiloride. Likewise, H+ influx initiated by removal of Na+out was blocked by amiloride. Hence, in HEPES-buffered medium the major operative pHi regulatory mechanism is a Na+/H+ exchange. In HCO3(-)-buffered medium, amiloride only partially blocked recovery from acid load and acidification due to Na+out removal. The remaining fraction required Na+out, was inhibited by H2-4,4'-diisothiocyanostilbene-2,2'-disulfunic acid (H2DIDS) and was independent of C1-. Hence, a transporter with characteristics of a Na(+)-HCO3- cotransport exists in pancreatic acini. Measurement of pHi changes due to Na(+)-HCO3- cotransport, suggests that the transporter contributes to HCO3- efflux under physiological conditions. Changing the Cl- gradient across the plasma membrane of acini maintained in HCO3(-)-buffered solutions reveals the presence of an H2DIDS-sensitive, Na(+)-independent, Cl(-)-dependent, HCO3- transporter with characteristics of a Cl-/HCO3- exchanger. In pancreatic acini the exchanger transports HCO3- but not OH- and under physiological conditions functions to remove HCO3- from the cytosol. In summary, only the Na+/H+ exchanger is functional in HEPES-buffered medium to maintain pHi at 7.28 +/- 0.03. In the presence of 25 mM HCO3- at pHo of 7.4, all the transporters operate simultaneously to maintain a steady-state pHi of 7.13 +/- 0.04.     

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.     

Changes in smooth muscle cell pH during hypoxic pulmonary vasoconstriction: a possible role for ion transporters

Hypoxic pulmonary vasoconstriction (HPV) occurs in smooth muscle cells (SMC) from small pulmonary arteries (SPA) and is accompanied by increases in free cytoplasmic calcium ([Ca2+]i) and cytoplasmic pH (pHi). SMC from large pulmonary arteries (LPA) relax during hypoxia, and [Ca2+]i and pHi decrease. Increases in pHi and [Ca2+]i in cat SPA SMC during hypoxia and the augmentation of hypoxic pulmonary vasoconstriction by alkalosis seen in isolated arteries and lungs suggest that cellular mechanisms, which regulate inward and outward movement of Ca2+ and H+, may participate in the generation of HPV. SMC transport systems that regulate pHi include the Na+ - H+ transporter which regulates intracellular Na+ and H+ and aids in recovery from acid loads, and the Na+ -dependent and Na+ -independent Cl-/HCO3- transporters which regulate intracellular chloride. The Na+ -dependent Cl-/HCO3- transporter also aids in recovery from acidosis in the presence of CO2 and HCO3-. The Na+ -independent Cl-/HCO3- transporter aids in recovery from cellular alkalosis. The Na+ - H+ transporter was present in SMC from SPA and LPA of the cat, but it seemed to have little if any role in regulating pHi in the presence of CO2 and HCO3-. Inhibiting the Cl-/HCO3- transporters reversed the normal direction of pHi change during hypoxia, suggesting a role for these transporters in the hypoxic response. Future studies to determine the interaction between pHi, [Ca2+]i and HPV should ascertain whether pHi and [Ca2+]i changes are linked and how they may interact to promote or inhibit SMC contraction.     

Isolation and functional reconstitution of a 38-kDa chloride channel protein from bovine tracheal membranes.

Secretion of chloride ions via apically located anion-selective channels in epithelia regulates fluid formation and cytosolic Cl- homeostasis. In order to understand the biochemical basis of Cl- channel function, we attempted to isolate this transporter from bovine tracheal apical membranes. Initially, peripheral polypeptides were removed from apically enriched vesicles by washing with alkaline buffer (pH 10.8) containing 2 mM CHAPS. The resulting pellet contained 50-60% of the original protein and displayed 2-fold enhanced Cl- channel activity compared to untreated vesicles. The pellet was treated with Triton X-100, and the solubilized proteins were separated on the cationic exchanger CM-cellufine. Washing the resin with a pH 8.0-8.3 buffer eluted a fraction with enriched Cl- channel activity. This fraction contained less than 5% of the total solubilized protein. A subsequent separation was performed using the anionic exchanger AM-cellufine. The highest activity was found in the fractions eluted by 80-120 mM KCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed a major 38,000-Da protein band. This band was electroeluted from the gel under nondenaturing and nonreducing conditions and reconstituted into phosphatidylcholine liposomes. KCl-loaded vesicles containing the purified 38-kDa protein transported up to 5 nmol of 125I-/mg of protein/5 min. This value was 15-fold higher than the uptake measured in vesicles reconstituted with total solubilized membrane proteins and 4-fold higher compared to the CM-cellufine-enriched fraction. The observed 125I- uptake was 90% inhibited by 100 microM 4,4-bis(isothiocyano)-2,2'-stilbenedisulfonate or 10 microM valinomycin. In summary, we have developed a biochemical protocol for the isolation of a 38 kDa protein mediating potential-dependent and 4,4-bis(isothiocyano)-2,2'-stilbenedisulfonate-sensitive Cl- channel activity.     

Stoichiometry of sodium- and chloride-coupled glycine transport in synaptic plasma membrane vesicles derived from rat brain

The stoichiometric properties of the glycine transporter were studied in synaptic plasma membrane vesicles from rat brain. The present results, together with previous data from our laboratory, allow us to suggest a stoichiometry of 2 Na+ and 1 Cl- per glycine zwitterion for the translocation cycle catalyzed by the glycine carrier. We propose a kinetic model with an ordered mechanism for the binding/debinding of solutes.     

HCO3- transport in basolateral membrane vesicles isolated from rat renal cortex

The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested.     

Phorbol ester TPA inhibits the stimulation of bumetanide-sensitive Na+/K+/Cl- transporter by different mitogens in quiescent BALB/c 3T3 mouse fibroblasts

In this study we examined the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the bumetanide-sensitive Na+/K+/Cl- transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide-sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide-sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide-sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl- cotransport activity; however it does suppress only the growth-factors-stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl- cotransport activity by growth factors.