Glutamate transport into synaptic vesicles. Roles of membrane potential, pH gradient, and intravesicular pH.

Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, is transported into bovine synaptic vesicles in a manner that is ATP dependent and requires a vesicular electrochemical proton gradient. We studied the electrical and chemical elements of this driving force and evaluated the effects of chloride on transport. Increasing concentrations of Cl- were found to increase the steady-state ATP-dependent vesicular pH gradient (delta pH) and were found to concomitantly decrease the vesicular membrane potential (delta psi). Low millimolar chloride concentrations, which cause 3-6-fold stimulation of vesicular glutamate uptake, caused small but measurable increases in delta pH and decreases in delta psi, when compared to control vesicles in the absence of chloride. Nigericin in potassium buffers was used to alter the relative proportions of delta pH and delta psi. Compared to controls, at all chloride concentrations tested, nigericin virtually abolished delta pH and increased the vesicle interior positive delta psi. Concomitantly, nigericin increased ATP-dependent glutamate uptake in 0-1 mM chloride but decreased glutamate uptake in 4 mM (45%), 20 mM (80%), and 140 mM (75%) Cl- (where delta pH in the absence of nigericin was large). These findings suggest that either delta psi, delta pH, or a combination can drive glutamate uptake, but to different degrees. In the presence of 4 mM Cl-, where uptake is optimal, both delta psi and delta pH contribute to the driving force for uptake. When the extravesicular pH was increased from 7.4 to 8.0, more Cl- was required to stimulate vesicular glutamate uptake. In the absence of Cl-, as extravesicular pH was lowered to 6.8, uptake was over 3-fold greater than it was at pH 7.4. As extravesicular pH was reduced from 8.0 toward 6.8, less Cl- was required for maximal stimulation. Decreasing the extravesicular pH from 8.0 to 6.8 in the absence of Cl- significantly increased glutamate uptake activity, even though proton-pumping ATPase activity actually decreased about 45% under identical conditions. In the absence of chloride, nigericin increased glutamate uptake at all the pH values tested except pH 8.0. Glutamate uptake at pH 6.8 in the presence of nigericin was over 6-fold greater than uptake at pH 7.4 in the absence of nigericin. We conclude from these experiments that optimal ATP-dependent glutamate uptake requires a large delta psi and a small delta pH.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Regulation of gastric acid secretion via modulation of a chloride conductance

When gastric microsomes were purified from resting and stimulated rabbit mucosae, they were found to be generally similar in (H+ + K+)-ATPase activity, peptide composition in single-dimension sodium dodecyl sulfate-gel electrophoresis, and in size. In the stimulated vesicles, optimal proton transport activity was found at pH 7.4, 20-50 mM KCl, and 1 mM ATP-Mg. However, in the case of resting vesicles, the presence of valinomycin and an inward Cl-gradient was also necessary for Mg-ATP-dependent proton transport. Measurement of K+ and Cl-diffusion potentials using 3,3-dipropylthiadicarboxocyanine iodide as a potential sensitive dye showed that both resting and stimulated vesicles developed K+ gradient-dependent potentials in the presence of an impermeant anion, but that Cl- gradient-dependent potentials were observed only in the stimulated preparation. 86Rb+ self-exchange was found in both types of vesicles, but Cl- self-exchange was confined to vesicles derived from stimulated mucosae. Putative inhibitors of anion conductance such as furosemide and anthracene 9-carboxylic acid blocked proton transport, Cl- conductance, 36Cl- uptake, and Cl- exchange. The inhibition of proton transport was overcome by valinomycin. ATPase activity in the presence of nigericin, an H+:K+ exchanger, was unaffected by these inhibitors. K+ conductance, Rb+ uptake, and Rb+ exchange were insensitive to these inhibitors. Thus, activation of acid secretion by the stimulated parietal cell appears to involve at least the appearance of a discrete Cl- conductance in the pump-associated membrane.     

Ca2+ binding kinetics of fura-2 and azo-1 from temperature-jump relaxation measurements.

The Ca2+-binding kinetics of fura-2 and azo-1 were studied using temperature-jump relaxation methods. In 140 mM KCl at 20 degrees C, the association and dissociation rate constants for fura-2 were 6.02 x 10(8) M-1s-1 and 96.7 s-1, respectively. The fura-2 kinetics were insensitive to pH over the range 7.4 to 8.4. Azo-1 was studied in 140 mM KCl, at pH 7.4, at 10 degrees and 20 degrees C. At 10 degrees C, azo-1 exhibited association and dissociation rate constants of 1.43 x 10(8) M-1s-1 and 777.9 s-1, respectively; while at 20 degrees C, the corresponding values were 3.99 x 10(8) M-1s-1 and 1,177 s-1. The kinetic results demonstrate that fura-2 and azo-1 are well suited to monitoring rapid changes in intracellular [Ca2+].     

Urea permeability of human red cells

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.     

Mechanism of activation of liver glycogen synthase by swelling.

The mechanism linking the stimulation of liver glycogen synthesis to swelling induced either by amino acids or hypotonicity was studied in hepatocytes, in gel-filtered liver extracts, and in purified preparations of particulate glycogen to which glycogen-metabolizing enzymes are bound. High concentrations of KCl, but not of potassium glutamate, were found to inhibit glycogen synthesis in permeabilized hepatocytes. Similarly, physiological concentrations (30-50 mM) of Cl- ions were also found to inhibit synthase phosphatase in vitro, whereas 10-20 mM Cl- ions, a concentration found in swollen hepatocytes, did not inhibit synthase phosphatase. Synthase phosphatase activity was more sensitive to inhibition by Cl- ions at low (0.1%) than at high (1%) concentrations of glycogen. By contrast, 10 mM glutamate and aspartate, a concentration observed in hepatocytes incubated with glutamine or proline, stimulated synthase phosphatase in vitro. Therefore, it is proposed that the fall in intracellular Cl- concentration as well as the increase in intracellular glutamate and aspartate concentrations, that are observed in swollen hepatocytes in the presence of amino acids, are responsible, at least in part, for the stimulation of synthase phosphatase and, hence, of glycogen synthesis.     

Role of separate K+ and Cl- channels and of Na+/Cl- cotransport in volume regulation in Ehrlich cells

Cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but later recover their volume with an associated KCl loss. This regulatory volume decrease (RVD) is unaffected when nitrate is substituted for Cl- or if bumetanide or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) is added. It is inhibited by quinine, Ba2+, low pH, anticalmodulin drugs, and depletion of intracellular Ca2+. It is accelerated by the Ca2+ ionophore A23187, or by a sudden increase in external Ca2+ and at high pH. A net KCl loss is also seen after addition of ionophore A23187 in isotonic medium. Similarities are demonstrated between the KCl loss seen after addition of A23187 and the KCl loss seen during RVD. It is proposed that separate conductive K+ and Cl- channels are activated during RVD by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin. After restoration of tonicity the cells shrink initially, but recover their volume with an associated KCl uptake. This regulatory volume increase (RVI) is inhibited when NO3- is substituted for Cl-, and is also inhibited by furosemide or bumetanide, but it is unaffected by DIDS. The unidirectional Cl-flux ratio is compatible with either a coupled uptake of Na+ and Cl-, or an uptake via a K+/Na+/2Cl- cotransport system. No K+ uptake was found, however, in ouabain-poisoned cells where a bumetanide-sensitive uptake of Na+ and Cl- in nearly equimolar amounts was demonstrated. Therefore, it is proposed that the primary process during RVI is an activation of an otherwise quiescent Na+/Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump. There is a marked increase in the rate of pump activity in the absence of a detectable increase in intracellular Na+ concentration.     

Selective inhibition of sodium-linked and sodium-independent bicarbonate/chloride antiport in Vero cells

The effects of compounds previously described to inhibit anion transport were tested for their ability to inhibit anion antiport in Vero cells as measured by uptake of 36Cl- by chloride self-exchange and as bicarbonate-linked uptake of 22Na+. While 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited both processes, ethacrynic acid and probenecid selectively inhibited the uptake of 36Cl-. Low concentrations of pyridoxal phosphate and picrylsulfonic acid selectively inhibited the bicarbonate linked uptake of 22Na+, while higher concentrations of these compounds also inhibited the uptake of 36Cl-. Measurements of the internal pH indicated that ethacrynic acid inhibits Na+-independent HCO-3/Cl- exchange, while it has no measurable effect on Na+-linked bicarbonate-dependent regulation of the internal pH. Conversely, picrylsulfonic acid selectively inhibits the latter process. The results indicate that anion antiport in Vero cells occurs by two independent processes.     

Effect of intracellular pH on the rate of chloride uptake and efflux in different mammalian cell lines

The effect of the internal pH on the rate of 36Cl- uptake and efflux was measured. While the rate of 36Cl- uptake by the antiport increased almost 10-fold over a narrow pH range in a number of cell lines, in other cells the pH-induced increase was considerably less. With increasing pH, the rate of 36Cl- efflux into chloride-free buffers increased strongly over a narrow pH range in all cell lines studied. Two groups of cell lines appeared, one group where the half-maximal increase in uptake and efflux rate was at pH 7.0-7.1 and another group where the corresponding values were 0.2-0.4 pH unit higher. The stoichiometry between chloride influx and chloride-stimulated efflux was close to 1:1 under various conditions.     

Modulation of ATP-dependent Ca2+ transport in rat parotid basolateral membrane vesicles by K+ + Cl- flux

In basolateral membrane vesicles (BLMV) isolated from rat parotid glands, the initial rate of ATP-dependent Ca2+ transport, in the presence of KCl, was approx. 2-fold higher than that obtained with mannitol, sucrose or N-methyl-D-glucamine (NMDG)-gluconate. Only NH4+, Rb+, or Br- could effectively substitute for K+ or Cl-, respectively. This KCl activation was concentration dependent, with maximal response by 50 mM KCl. An inwardly directed KCl gradient up to 50 mM KCl had no effect on Ca2+ transport, while equilibration of the vesicles with KCl (greater than 100 mM) increased transport 15-20%. In presence of Cl-, 86Rb+ uptake was 2.5-fold greater than in the presence of gluconate. 0.5 mM furosemide inhibited 86Rb+ flux by approx. 60% in a Cl- medium and by approx. 20% in a gluconate medium. Furosemide also inhibited KCl activation of Ca2+ transport with half maximal inhibition either at 0.4 mM or 0.05 mM, depending on whether 45Ca2+ transport was measured with KCl (150 mM) equilibrium or KCl (150 mM) gradient. In a mannitol containing assay medium, potassium gluconate loaded vesicles had a higher (approx. 25%) rate of Ca2+ transport than mannitol loaded vesicles. Addition of valinomycin (5 microM) to potassium gluconate loaded vesicles further stimulated (approx. 30%) the Ca2+ transport rate. These results suggest that during ATP dependent Ca2+ transport in parotid BLMV, K+ can be recycled by the concerted activities of a K+ and Cl- coupled flux and a K+ conductance.     

Na+, Cl− cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase)

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.     

Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+ dependent ATPase

Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation = 1.0 microM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accelerated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [gamma-32P]ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 microM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0 degrees C in the presence of 1 microM ATP and 10 microM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time = 0.1 s). The rate of EP decomposition at 0 degrees C after adding EGTA was about 6.7 times slower (rate constant: kd = 4.3 X 10(-3) s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.     

A 31P-NMR study on the recovery of intracellular pH in LLC-PK1/Cl4 cells from intracellular alkalinization

The regulation of intracellular pH (pHi) in a renal epithelial cell line, LLC-PK1/Cl4, during re-acidification from an alkaline load was studied by 31P-NMR. Intracellular alkalinization was induced by 10 mM ammonium glucuronate or by preloading with and subsequent removal of 20% CO2; the rate of re-acidification was found to be 0.047 pH units/min and 0.053 pH units/min, respectively. This rate of re-acidification was inhibited by 83% if Cl- was removed from the extracellular medium. A similar inhibition was found in the presence of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) (76% inhibition) and 1 mM bumetanide (81% inhibition). No change in recovery was found after removing sodium from the extracellular medium, indicating that LLC-PK1/Cl4 cells recover from an intracellular alkaline load by a Cl-/HCO3- exchanger, which is SITS- and bumetanide-sensitive and has no requirement for sodium. In addition, the steady-state pHi in Cl4 cells was monitored by 31P-NMR. Removal of Cl- from the extracellular medium introduced an increase in pHi by 0.33 pH units, whereas 1 mM SITS and 1 mM bumetanide caused an increase in pHi by 0.14 or 0.13 pH units. In the presence of 1 mM amiloride, an inhibitor of the Na+/H+ exchanger, the steady-state pHi did not change significantly. These results indicate that at pHo 7.4 the steady-state intracellular pH of LLC-PK1/Cl4 cells strongly depends on the activity of the Cl-/HCO3- exchanger. Under the same conditions the activity of the Na+/H+ exchanger seems to be negligible.     

Cl- transport by gastric mucosa: cellular Cl- activity and membrane permeability

The mechanism of Cl- secretion in the isolated, resting (i.e. cimetidine-treated) gastric mucosa of Necturus has been investigated with radioisotopic and electrophysiological techniques. Measurement of transepithelial 36Cl- fluxes (mucosal to serosal (M leads to S), Jms Cl-; S leads to M, Jsm Cl-) during control conditions show that at open circuit, when the transepithelial potential difference psi ms = 20 mV (S ground), Jms Cl- = Jsm Cl-, i.e. Jnet Cl- = 0, but during short-circuit current conditions Jnet Cl- = I sc = 2 mu equiv cm-2 h. Experiments with low [Cl-] solutions indicate that Cl- exchange diffusion does not contribute significantly to either Jms Cl- or Jsm Cl-. Double-barrelled, Cl- -selective microelectrodes showed that in open circuit, the cellular (C) chemical potential for Cl-, psi c Cl- = 31 mV (apparent [Cl-] = 29 mM), the electrical potential across the M membrane, psi m = -34 mV (mucosa ground) while that across the S membrane, psi s = -52 mV (serosa ground). During short-circuit current conditions, psi m = psi s = -49 mV and [Cl-]c = 30 mM. The permeability of the M membrane to Cl- (Pm Cl-) was calculated both from the tracer experiments and the electrode measurements by using the constant-field equation. Short-term (45 s) uptake of 36Cl- at [Cl]m = 96 mM during short circuit conditions gave Pm Cl- = 2.6 x 10(-5) cm s-1. Measurement of [Cl-]c by means of the electrodes when [Cl-]m was changed from 96 to 2 mM or from 2 to 96 mM gave Pm Cl- = 2.9-5.7 x 10(-5) cm s-1. Our results indicate that during open circuit conditions Cl- is accumulated across the S membrane into gastric cells in an energy-requiring step, but since Jnet Cl- = 0, Cl- must leak back into the S solution at a rate equal to the entry rate. When the tissue is short-circuited, Cl- secretion occurs (Jnet Cl- = Isc) owing to the same energy-requiring accumulation of Cl- by the cells and a passive (apparently electrodiffusive) movement across the mucosal membrane.     

Transient kinetics of adenosine 5'-triphosphate hydrolysis by covalently cross-linked actomyosin complex in water and 40% ethylene glycol by the rapid flow quench method

The initial steps of the ATPase of covalently cross-linked actomyosin subfragment 1 (acto-SF-1) were studied by the rapid flow quench method, and the results obtained were compared with those with reversible (i.e., non-cross-linked) acto-SF-1 and SF-1 under identical conditions. Cross-linked acto-SF-1 plus [gamma-32P]ATP reaction mixture milliseconds old was quenched either in a large excess of unlabeled ATP (ATP chase) or in acid (Pi burst). The conditions were pH 8 and 15 degrees C at 5 mM or 0.15 M KCl and with or without 40% ethylene glycol. In 40% ethylene glycol (5 mM KCl), as with SF-1 and reversible acto-SF-1, the ATP chase was used to titrate active sites and to study the kinetics of ATP binding. Unlike those with SF-1 or reversible acto-SF-1, saturation kinetics were not obtained. The second-order rate constant for ATP binding was 3.1 X 10(6) M-1 s-1 for cross-linked acto-SF-1, 1.8 X 10(6) M-1 s-1 for reversible acto-SF-1, and 2 X 10(6) M-1 s-1 for SF-1. In Pi burst experiments, a transient phase could not be discerned. Because of a high kcat, cross-linked acto-SF-1 was difficult to study in aqueous solution, but at 5 mM KCl, the ATP chase and Pi burst curves were similar to those obtained in 40% ethylene glycol. At 0.15 M KCl the ATP chase curve was difficult to interpret (small amplitude), and there was a small Pi burst.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Interactions of bromide, iodide, and fluoride with the pathways of chloride transport and diffusion in human neutrophils

Isolated human neutrophils possess three distinct pathways by which Cl- crosses the plasma membrane of steady state cells: anion exchange, active transport, and electrodiffusion. The purpose of the present work was to investigate the selectivity of each of these separate processes with respect to other external halide ions. (a) The bulk of total anion movements represents transport through an electrically silent anion-exchange mechanism that is insensitive to disulfonic stilbenes, but which can be competitively inhibited by alpha-cyano-4-hydroxycinnamate (CHC; Ki approximately 0.3 mM). The affinity of the external translocation site of the carrier for each of the different anions was determined (i) from substrate competition between Cl- and either Br-, F-, or I-, (ii) from trans stimulation of 36Cl- efflux as a function of the external concentrations of these anions, (iii) from changes in the apparent Ki for CHC depending on the nature of the replacement anion in the bathing medium, and (iv) from activation of 82Br- and 125I- influxes by their respective ions. Each was bound and transported at roughly similar rates (Vmax values all 1.0-1.4 meq/liter cell water.min); the order of decreasing affinities is Cl- greater than Br- greater than F- greater than I- (true Km values of 5, 9, 23, and 44 mM, respectively). These anions undergo 1:1 countertransport for internal Cl-. (b) There is a minor component of total Cl- influx that constitutes an active inward transport system for the intracellular accumulation of Cl- [( Cl-]i approximately 80 meq/liter cell water), fourfold higher than expected for passive distribution. This uptake is sensitive to intracellular ATP depletion by 2-deoxy-D-glucose and can be inhibited by furosemide, ethacrynic acid, and CHC, which also blocks anion exchange. This active Cl- uptake process binds and transports other members of the halide series in the sequence Cl- greater than Br- greater than I- greater than F- (Km values of 5, 8, 15, and 41 mM, respectively). (c) Electrodiffusive fluxes are small. CHC-resistant 82Br- and 125I- influxes behave as passive leak fluxes through low-conductance ion channels: they are nonsaturable and strongly voltage dependent. These anions permeate the putative Cl- channel in the sequence I- greater than Br- greater than Cl- with relative permeability ratios of 2.2:1.4:1, respectively, where PCl approximately 5 X 10(-9) cm/s.     

Effect of chemotactic factors on hexose transport in polymorphonuclear leucocytes

Transport of the nonmetabolizable glucose analogue, 3-O-methylglucose, was assessed in human polymorphonuclear leucocytes with or without the chemotactic peptide N-formylmethionylleucylphenylalanine (fMet-Leu-Phe). The peptide increased entry of labelled 3-O-methylglucose about 5-fold and the intracellular distribution space about 70%. The half-time of equilibration was 3 s in the treated cells. Similar effects were observed with zymosan-treated serum (containing the chemotactic factor C5a), with arachidonic acid, calcium ionophore A23187 and phorbol myristate acetate. However, the chemotactic protein, thrombin, had no effect, even though binding to high-affinity receptors was demonstrated. Km for zero-trans entry of 3-O-methylglucose was about 1 mM and fMet-Leu-Phe increased Vmax from 5 to about 25 amol.s-1.cell-1. Similar values were obtained from incubations for a few seconds with glucose and 2-deoxyglucose. The rate of 2-deoxyglucose uptake (8 min incubations) was limited by the transport step at substrate concentrations lower than approx. 0.1 mM, whereas the phosphorylation step became rate-limiting at higher concentrations. Thus, 2-deoxyglucose uptake can only be taken as a measure of transport at a tracer concentration. It is concluded that chemotactic factors can, but do not necessarily, increase the maximal transport velocity of hexoses entering the polymorphonuclear leucocyte via the glucose transporter.     

Na+-H+ exchange in isolated renal brush-border membrane vesicles in response to metabolic acidosis. Kinetic effects

Chronic metabolic acidosis increased the Na+-H+ exchange activity in isolated renal brush-border membrane vesicles. Treatment altered the initial rate of Na+ uptake by increasing Vm (acidotic, 15.3 +/- 0.7 nmol of Na+ X mg-1 X 2 s-1; normal, 11.3 +/- 0.9 nmol of Na+ X mg-1 X 2 s-1), and not the apparent affinity KNa+ (acidotic, 10.2 +/- 0.5 mM; normal 10.2 +/- 0.6 mM). Metabolic acidosis resulted in the proportional increase in 1 mM Na+ uptake at every intravesicular pH measured. A positive cooperative effect on Na+ uptake was found with increased intravesicular acidity in vesicles from both normal and acidotic rats. When the data were analyzed by the Hill equation, it was found that metabolic acidosis did not change the n (acidotic, 1.33 +/- 0.13; normal, 1.43 +/- 0.07) or the K'H+ (acidotic, 0.27 +/- 0.05 microM; normal, 0.28 +/- 0.06 microM), but increased the apparent Vm (acidotic, 1.10 +/- 0.08 nmol of Na+ X mg-1 X 2 s-1; normal, 0.81 +/- 0.07 nmol of Na+ X mg-1 X 2 s-1). The uptake of Na+ in exchange for H+ in membrane vesicles from normal and acidotic animals was not influenced by membrane potential. We conclude that metabolic acidosis leads to either an increase in the number of functioning exchangers or an increase in the turnover rate of the limiting step in the exchange.