Relative contributions of the slippage and tunneling mechanisms to anion net efflux from human erythrocytes

The rates of anion net efflux from gramicidin-treated erythrocytes in the presence of a K gradient were measured at 25 degrees C, pH 7.8, as rates of loss of Ki. The experiments served to estimate the relative contributions of two hypothetical mechanisms to Cl net efflux at low extracellular Cl concentrations. Cl, Br, and NO3 net effluxes were measured into media of different Cl, Br, or NO3 concentrations, respectively, to determine and compare the relative rates of the extracellular anion-inhibitable components. They were 48, 160, and 230 mmol/(kg Hb X min), respectively, at a membrane potential of about -90 mV. This indicates that the anion-inhibitable efflux is not due solely to the return translocation of the empty transport site ("slippage") because slippage should be independent of the chemical nature of the anion. Cl net efflux was also measured as a function of the intracellular Cl concentration into media containing either 0 or 50 mM Cl. Under both conditions, net efflux was linearly dependent on Cli between 30 and 300 mM Cli and was 0 when back-extrapolated to 0 Cli. This observation is not compatible with the slippage process, which under these conditions would have been expected to be independent of Cli above 15 mM Cli. It was concluded that slippage contributes negligibly to Cl net efflux even at low extracellular anion concentrations and that the alternative process of "tunneling"--that is, movement of the anion through the anion transporter without a conformational change in a channel-type behavior--is the major, if not the sole, mechanism underlying Cl conductance.     

Relationship of net chloride flow across the human erythrocyte membrane to the anion exchange mechanism

The parallel effects of the anion transport inhibitor DIDS (4,4'- diisothiocyanostilbene-2,2'-disulfonate) on net chloride flow and on chloride exchange suggest that a major portion of net chloride flow takes place through the anion exchange system. The "slippage" model postulates that the rate of net anion flow is determined by the movement of the unloaded anion transport site across the membrane. Both the halide selectivity of net anion flow and the dependence of net chloride flux on chloride concentration over the range of 75 to 300 mM are inconsistent with the slippage model. Models in which the divalent form of the anion exchange carrier or water pores mediate net anion flow are also inconsistent with the data. The observations that net chloride flux increases with chloride concentration and that the DIDS- sensitive component tends to saturate suggest a model in which net anion flow involves "transit" of anions through the diffusion barriers in series with the transport site, without any change in transport site conformation such as normally occurs during the anion exchange process. This model is successful in predicting that the anion exchange inhibitor NAP-taurine, which binds to the modifier site and inhibits the conformational change, has less effect on net chloride flow than on chloride exchange.     

Calcium distribution and exchange in the pregnant and post partum rabbit uterus

Calcium content and distribution of the 25-day pregnant (PR) and post partum (PP) rabbit uterus was studied by atomic absorption spectrophotometry and 45Ca determination. Total Ca content [2.28 +/- 0.28 (PR) and 2.19 +/- 0.12 (PP) mM/kg wet wt] extracellular [1.21 +/- 0.09 (PR) and 1.25 +/- 0.11 (PP) mM/kg wet wt] cellular [1.07 +/- 0.08 (PR) and 0.94 +/- 0.09 (PP) mM/kg wet et], total exchangeable [1.86 +/- 0.11 (PR) and 1.84 +/- 0.09 (PR) mM/kg wet wt] and inexchangeable [0.43 +/- 0.05 (PR) and 0.35 +/- 0.04 (PP) mM/kg wet wt] Ca fractions were identical in the two extreme endocrinological conditions. In contrast compartment size and rate constant of different exchangeable Ca fractions determined by kinetic analysis of 45Ca desaturation "urves (curve-peeling tecnique and computer method), revealed significant differences between PR and PP uteri. Two exchangeable phases could be identified in both endocrinological states. The rate constants of both phases of efflux were significantly higher in the PP (alpha 1 = 0.173 +/- 0.02 min-1; alpha 2 = 0.023 +/- 0.001 min-1) than in the PR uterus (alpha 1 = 0.099 +/- 0.01 min-1; alpha 2 = 0.018 +/- 0.01 min-1). Compartment size of phase 1 (fast component) was significantly higher in the PR (1.13 +/- 0.1 mM/kg wet wt) than in the PP uterus (0.77 +/- 0.06 mM/kg wet wt). In contrast, compartment size of phase 2 (slow component) was significantly smaller in PR than in PP uterine strips (0.74 +/- 0.06 and 1.08 +/- 0.11 mM/kg wet wt). The last portion of desaturation curves represents efflux from one homogenous compartment. The present results suggest that endocrinological control of the rabbit myometrium is linked to the regulation of the binding of a superficial exchangeable Ca fraction.     

Chloride-stimulated sulfate efflux in Ehrlich ascites tumor cells: evidence for 1:1 coupling

The kinetics of Cl-SO4-(2) exchange in Ehrlich ascites tumor cells was investigated in an attempt to determine the stoichiometry of this process. When tumor cells, equilibrated in Cl--free, 25 mM SO4-(2) medium are placed in SO4-(2)-free, 25 mm Cl-medium, both the net amount and rate of Cl-uptake far exceeds SO4-(2) loss.. Addition of the anion transport inhibitor SITS (4-acetamido-4,-isothiocyano-stilbene-2,2'-disulfonic acid) greatly reduces sulfate efflux (97%), but has no measurable effect on chloride uptake. Addition of furosemide, a Cl-transport inhibitor, reduces chloride uptake 94% but is without effect on sulfate efflux. These findings suggest that a chloride permeability pathway exists distinct from that utilized by SO4-(2). SITS, when added to furosemide treated cells, further reduces chloride uptake as well as inhibiting sulfate efflux, and under these experimental conditions, a linear relationship exists between SITS-sensitive, net chloride uptake and sulfate loss. The slope of this line is 1.05 (correlation coefficient = 0.996) which suggests the stoichiometry of Cl-SO4-(2) exchange is 1:1. Assuming a 1:1 stoichiometry, measurement of the initial chloride influx and initial sulfate efflux indicate that 92% of net chloride uptake is independent of sulfate efflux. Taken altogether, these results support the contention that the tumor cell possesses a permeability pathway which facilitates the exchange of one sulfate for one chloride. Under conditions where anion transport is not inhibited, this coupling is obscured by a second and quantitatively more important pathway for chloride uptake. This pathway is SITS-insensitive, although partially inhibited by furosemide.     

The relationship between anion exchange and net anion flow across the human red blood cell membrane

The conductive (net) anion permeability of human red blood cells was determined from net KCl or K2SO4 effluxes into low K+ media at high valinomycin concentrations, conditions under which the salt efflux is limited primarily by the net anion permeability. Disulfonic stilbenes, inhibitors of anion exchange, also inhibited KCl or K2SO4 efflux under these conditions, but were less effective at lower valinomycin concentrations where K+ permeability is the primary limiting factor. Various concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) had similar inhibitory effects on net and exchange sulfate fluxes, both of which were almost completely DIDS sensitive. In the case of Cl-, a high correlation was also found between inhibition of net and exchange fluxes, but in this case about 35% of the net flux was insensitive to DIDS. The net and exchange transport processes differed strikingly in their anion selectivity. Net chloride permeability was only four times as high as net sulfate permeability, whereas chloride exchange is over 10,000 times faster than sulfate exchange. Net OH-permeability, determined by an analogous method, was over four orders of magnitude larger than that of Cl-, but was also sensitive to DIDS. These data and others are discussed in terms of the possibility that a common element may be involved in both net and exchange anion transport.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells. Evidence for separate modes of operation of the transporter

(1) Unidirectional K+ (86Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K+ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 . 10(-3) M ouabain. (3) Measurements of net K+ loss and Na+ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K+ (Cl-) outward net flux uncoupled from net Na+ movement. Net K+ (Cl-) outward flux was half-maximally inhibited by 2 microM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K+ influx/efflux were essentially unchanged when compared to untreated controls.     

3-O-methyl-D-glucose transport in rat red cells: effects of heavy water.

Transport of 3-O-methyl-D-glucose (3-OMG) in rat red blood cells (RBCs) has been examined at 24 degrees C. The Km and Vm of zero-trans net uptake are 2.3 +/- 0.48 mM and 0.055 +/- 0.003 mumol (ml cell water)-1) min-1, whereas the Km and Vm for net exit are 2.1 +/- 0.12 mM and 0.12 +/- 0.01 mumol (ml cell water)-1 min-1. The Km and Vm for infinite-trans exchange uptake are 2.24 +/- 0.14 mM and 0.20 +/- 0.04 mumol (ml cell water)-1 min-1. In agreement with Whitesell et al. (Abumrad, N.A., Briscoe, P., Beth, A.H. and Whitesell, R.R. (1988) Biochim. Biophys. Acta 938, 222-230), we find that there is no significant acceleration of the rate of exchange exit over net exit. Substitution of D2O for water results in an increase in the Vm for zero-trans net uptake to 0.091 +/- 0.004 mumol (ml cell water)-1 min-1. There is no change in the Vm or Km for exchange uptake or net or exchange exit. Counterflow experiments indicate, in agreement with Helgerson and Carruthers (1989) Biochemistry 28, 4580-4594), that there is some compartmentalization of 3-OMG within the cells, perhaps resulting from slow complexation of the sugar with some intracellular component. The data can be simulated by assuming that transport across the membrane is mediated by either a fixed 2-site, or an alternating 1-site symmetrical transporter. With both models the observed asymmetries in net and exchange kinetics and in counterflow can be ascribed entirely to the complexation reaction of the sugar to an intracellular component. Also the D2O effects can entirely be attributed to an increase in the rate of sugar movement between bound and free compartments.     

Na+-independent Mg2+ efflux from Mg2+-loaded human erythrocytes

Net Mg2+ efflux from Mg2+-loaded human erythrocytes was maximal after reincubation in sucrose. Net Mg2+ efflux was not inhibited by furosemide or bumetanide and, therefore, was not performed by the (Na,K,Cl)- or (K,Cl)-cotransport system. A component of net Mg2+ efflux was inhibited by extracellular NaC1, KCl, LiCl, choline Cl and SITS, in analogy to the inhibition of net Cl- and SITS. Therefore, it was concluded that net Mg2+ efflux is dependent on net Cl- efflux for charge compensation. Cl- -dependent net Mg2+ efflux was inhibited by amiloride. Only 10% of the maximal net Mg2+ efflux may depend on extracellular Na+.     

Mechanism of the increase in cation permeability of human erythrocytes in low-chloride media. Involvement of the anion transport protein capnophorin

When human erythrocytes are suspended in low-Cl- media (with sucrose replacing Cl-), there is a large increase in both the net efflux and permeability of K+. A substantial portion (greater than 70% with Cl- less than 12.5 mM) of this K+ efflux is inhibited by the anion exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). This inhibition cannot be explained as an effect of DIDS on net Cl- permeability (Pcl) and membrane potential, but rather represents a direct effect on the K+ permeability. When cells are reacted with DIDS for different times, the inhibition of K+ efflux parallels that of Cl- exchange, which strongly indicates that the band 3 anion exchange protein (capnophorin) mediates the net K+ flux. Since a noncompetitive inhibitor of anion exchange, niflumic acid, has no effect on net K+ efflux, the net K+ flow does not seem to involve the band 3 conformational change that mediates anion exchange. The data suggest that in low-Cl- media, the anion selectivity of capnophorin decreases so that it can act as a very low-conductivity channel for cations. Na+ and Rb+, as well as K+, can utilize this pathway.     

Calcium control of passive permeability to calcium in sarcoplasmic reticulum vesicles

Calcium efflux from sarcoplasmic reticulum vesicles that have been equilibrated with 1-100 mM CaCl2 in the absence of ATP has two apparently first order components. The initial calcium content of each component increases with the total Ca content of the sarcoplasmic reticulum, which reaches 5, 24, and 80 nmol/mg of protein after equilibration with 1, 10, and 100 mM CaCl2, respectively. Initial rates of Ca efflux into a medium containing 10 mM EGTA increase in proportion to Ca in the loading medium up to 20 mM. Above 20 mM, efflux from the slow component clearly saturates, whereas efflux from the fast component continues to increase. The rate constant for the smaller, faster component to efflux (k congruent to 0.5 min-1) is not affected by changing the concentration of Ca either inside or outside the vesicles. The rate constant of the larger, slower component (k congruent to 0.05 min-1) is also unaffected by changes in internal Ca concentration. However, external [Ca2+] diminishes the rate constant of the slow component 6-10-fold. Inhibition by external [Ca2+] is characterized by cooperative interaction between two sites with an apparent Kd of 5.3 X 10(-6) M. The two components may represent two populations of sarcoplasmic reticulum vesicles that differ 10-fold in passive permeability to Ca when external [Ca2+] is less than 10(-6) M, and 60-100-fold when external [Ca2+] is greater than 10(-5) M. The passive permeability in one of these populations seems to be regulated by external, high affinity Ca binding sites.     

Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso- thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS- insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.     

The change from symmetry to asymmetry of a sodium transport system in red cell membranes

A study has been made with human red cells of sodium movements that are sensitive to the drug furosemide. The aim was to see if furosemide-sensitive movements that are symmetrical (exchange) became asymmetrical (net transport) on replacement of chloride with nitrate as the major external anion. Cells were incubated for 4 h at 37 degrees C with 140 mM sodium, and chloride or nitrate as the principal anion. Under a variety of conditions (presence and absence of ouabain or furosemide, or both) the cell sodium concentration was always higher when chloride was replaced with nitrate. The cells became leakier to sodium. Tracer studies indicated that, in contrast to the results in chloride medium, the decrease in sodium influx was greater than the fall in efflux when furosemide was added to cells in nitrate medium. The results confirm that the sensitivity of sodium efflux to furosemide depended on chloride. However, influx showed a different sensitivity in that furosemide still inhibited in cells incubated in nitrate medium. The stimulation of sodium influx with nitrate medium was independent of external potassium (10-50 mM) and the furosemide-sensitive influx was also constant. It is concluded that symmetrical transmembrane sodium movements with cells in chloride medium became downhill asymmetrical in nitrate medium, giving a net gain of cell sodium that was insensitive to ouabain and sensitive to furosemide. The drug thus partly retarded the gain of cell sodium that otherwise occurred in the somewhat leaky cells.     

A component of fluid absorption linked to passive ion flows in the superficial pars recta

We studied salt and water absorption in isolated rabbit superficial proximal straight tubules perfused and bathed with solutions providing oppositely directed transepithelial anion gradients similar to those which might obtain in vivo. The perfusing solution contained 138.6 mM Cl- 3.8 mM HCO-3 (pH 6.6) while the bathing solution contained 113.6 mM Cl- and 25 mM HCO-3 (pH 7.4); the system was bubbled with 95% O2-5% CO2. At 37 degrees C, net volume absorption (Jv nl min-1 mm-1) was 0.32 +/- 0.03 (SEM); Ve, the transepithelial voltage (millivolts; lumen to bath), was +3.1 +/- 0.2. At 21 degrees C, Ve rose to +3.7 +/- 0.1 and Jv fell to 0.13 +/- 0.01 (significantly different from zero at P less than 0.001); in the presence of 10(-4)M ouabain at 37 degrees C, Ve rose to +3.8 +/- 0.1 and Jv fell to 0.16 +/- 0.01 (P less than 0.001 with respect to zero). In paired experiments, the ouabain- and temperature-insensitive moieties of Jv and Ve became zero when transepithelial anion concentration gradients were abolished. Titrametric determinations net chloride flux at 21 degrees C or at 37 degrees C with 10(-4) M ouabain showed that chloride was the sole anion in an isotonic absorbate. And, combined electrical and tracer flux data indicated that the tubular epithelium was approximately 18 times more permeable to Cl- than to HCO-3. We interpret these results to indicate that, in these tubules, NaCl absorption depends in part on transepithelial anion concentration gradients similar to those generated in vivo and in vitro by active Na+ absorption associated with absorption to anions other than chloride. A quantitative analysis of passive solute and solvent flows in lateral intercellular spaces indicated that fluid absorption occurred across junctional complexes when the osmolality of the lateral intercellular spaces was equal to or slightly less than that of the perfusing and bathing solutions; the driving force for volume flow under these conditions depended on the fact that sigmaHCO3 exceeded sigmaCl.     

A role for membrane transport in modulation of intramuscular free glutamine turnover in streptozotocin diabetic rats.

We wished to examine the effects of diabetes on muscle glutamine kinetics. Accordingly, female Wistar rats (200 g) were made diabetic by a single injection of streptozotocin (85 mg/kg) and studied 4 days later; control rats received saline. In diabetic rats, glutamine concentration of gastrocnemius muscle was 33% less than in control rats: 2.60 +/- 0.06 mumol/g vs. 3.84 +/- 0.13 mumol/g (P < 0.001). In gastrocnemius muscle, glutamine synthetase activity (Vmax) was unaltered by diabetes (approx. 235 nmol/min per g) but glutaminase Vmax increased from 146 +/- 29 to 401 +/- 94 nmol/min per g; substrate Km values of neither enzyme were affected by diabetes. Net glutamine efflux (A-V concentration difference x blood flow) from hindlimbs of diabetic rats in vivo was greater than control values (-30.0 +/- 3.2 vs. -1.9 +/- 2.6 nmol/min per g (P < 0.001)) and hindlimb NH3 uptake was concomitantly greater (about 27 nmol/min per g). The glutamine transport capacity (Vmax) of the Na-dependent System Nm in perfused hindlimb muscle was 29% lower in diabetic rats than in controls (820 +/- 50 vs. 1160 +/- 80 nmol/min per g (P < 0.01)), but transporter Km was the same in both groups (9.2 +/- 0.5 mM). The difference between inward and net glutamine fluxes indicated that glutamine efflux in perfused hindlimbs was stimulated in diabetes at physiological perfusate glutamine (0.5 mM); ammonia (1 mM in perfusate) had little effect on net glutamine flux in control and diabetic muscles. Intramuscular Na+ was 26% greater in diabetic (13.2 mumol/g) than control muscle, but muscle K+ (100 mumol/g) was similar. The accelerated rate of glutamine release from skeletal muscle and the lower muscle free glutamine concentration observed in diabetes may result from a combination of: (i), a diminished Na+ electrochemical gradient (i.e., the net driving force for glutamine accrual in muscle falls); (ii), a faster turnover of glutamine in muscle and (iii), an increased Vmax/Km for sarcolemmal glutamine efflux.     

Catecholamine-stimulated ion transport in duck red cells. Gradient effects in electrically neutral [Na + K + 2Cl] Co-transport

The transient increase in cation permeability observed in duck red cells incubated with norepinephrine has been shown to be a linked, bidirectional, co-transport of sodium plus potassium. This pathway, sensitive to loop diuretics such as furosemide, was found to have a [Na + K] stoichiometry of 1:1 under all conditions tested. Net sodium efflux was inhibited by increasing external potassium, and net potassium efflux was inhibited by increasing external sodium. Thus, the movement of either cation is coupled to, and can be driven by, the gradient of its co-ion. There is no evidence of trans stimulation of co- transport by either cation. The system also has a specific anion requirement satisfied only by chloride or bromide. Shifting the membrane potential by varying either external chloride (at constant internal chloride) or external potassium (at constant internal potassium in the presence of valinomycin and DIDs [4,4'-diisothiocyano- 2,2'-disulfonic acid stilbene]), has no effect on nor-epinephrine- stimulated net sodium transport. Thus, this co-transport system is unaffected by membrane potential and is therefore electrically neutral. Finally, under the latter conditions-when Em was held constant near EK and chloride was not at equilibrium-net sodium extrusion against a substantial electrochemical gradient could be produced by lowering external chloride at high internal concentrations, thereby demonstrating that the anion gradient can also drive co-transport. We conclude, therefore, that chloride participates directly in the co- transport of [Na + K + 2Cl].     

Evidence of multiple operational affinities for D-glucose inside the human erythrocyte membrane.

1. The Michaelis-Menten parameters of labelled D-glucose exit from human erythrocytes at 2 degrees C into external solution containing 50 mM D-galactose were obtained. The Km is 3.4 +/0 0.4 mM, V 17.3 +/- 1.4 MMOL . 1(-1) cell water . min-1 for this infinite-trans exit procedure. 2. The kinetic parameters of equilibrium exchange of D-glucose at 2 degrees C are Km = 25 +/- 3.4 mM, V 30 +/- 4.1 mmol . 1(-1) cell water . min-1. 3. The Km for net exit of D-glucose into solutions containing zero sugar is 15.8 +/- 1.7 mM, V 9.3 +/- 3.3 mmol . 1(-1) cell water . min-1. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126--132] that there are sites with both high and low operational affinities for D-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.     

Regulation of Cl/HCO3 exchange in gastric parietal cells.

Microspectrofluorimetry of the fluorescent indicators 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein and 6-methoxy-N-(3-sulfopropyl)-quinolinium was used to measure intracellular pH (pHi), intracellular Cl (Cli), and transmembrane fluxes of HCO3 and Cl in single parietal cells (PC) in isolated rabbit gastric glands incubated in HCO3/CO2-buffered solutions. Steady-state pHi was 7.2 in both resting (50 microM cimetidine) and stimulated (100 microM histamine) PCs. Transmembrane anion (HCO3 or Cl) flux rates during Cl removal from or readdition to the perfusate were the same in resting and stimulated PCs. These rates increased at alkaline pHi, though this pHi dependence was small in the physiological range. Maximum velocity (Vmax) for Cl influx or HCO3 efflux was 80-110 mM/min at pHi 7.6-7.8, and the Km for extracellular concentrations of Cl (Clo) was 25 mM; in the physiological range (pHi 7.1-7.3), Vmax for anion fluxes was approximately 50 mM/min. Steady-state Cli in the unstimulated PC was 62 +/- 5 mM, but on histamine stimulation, Cli decreased rapidly to 25 mM and then increased back to a steady-state level of 44 mM. HCO3 fluxes due to Cl removal or readdition were completely blocked by 0.5 mM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS), but Cl fluxes were only inhibited by 80%. H2DIDS did not inhibit the decrease in Cli that occurred with histamine treatment. Diphenylamine carboxylate (0.5 mM) inhibited Cl flux by only 50% and caused no additional inhibition of Cl flux when used in conjunction with H2DIDS. Transmembrane anion fluxes during solution Cl removal or readdition occurred 80% through the anion exchanger at the basal membrane and 20% through other pathway(s), presumably the Cl channel in the apical membrane. We conclude that the increase in transport activity via the Cl/HCO3 exchanger that occurs during histamine-induced increases in HCl secretion is due mostly to the decrease in Cli. In the resting cell with Cli = 62 mM, Clo = 120 mM, pHi = 7.2, and extracellular pH = 7.4, the anion exchanger is poised near its thermodynamic equilibrium. During histamine stimulation Cli drops from 62 mM to 44 mM, the thermodynamic equilibrium of the anion exchanger at the basolateral membrane is disturbed, and the anion exchanger then exchanges cellular HCO3 for extracellular Cl. Cli serves a crucial regulatory role in stimulus-secretion coupling in the PC.     

Kinetics of lactate and pyruvate transport in cultured rat myotubes

Skeletal muscle transport of lactate and pyruvate was studied in primary cultures of rat myotubes, applying the pH-sensitive fluorescent indicator 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The initial rate of decrease in intracellular pH (pHi) upon lactate or pyruvate incubation was used to determine total transport (carrier mediated and diffusion). Both lactate and pyruvate transport could be inhibited by a combination of 0.5 mM 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid, 5 mM mersalyl and 10 mM alpha-cyano-4-hydroxycinnamate. The kinetic parameters, Km and Vmax, for carrier-mediated transport of lactate were 9.9+/-1.1 mM and 0. 69+/-0.02 mmol l-1 s-1, respectively. For pyruvate, Km and Vmax were 4.4+/-1.3 mM and 0.30+/-0.05 mmol l-1 s-1, respectively. The diffusion component of the total transport was 0.0040+/-0.0005[S] (n=4) and 0.0048+/-0.0003[S] (n=4) for lactate and pyruvate, respectively. Furthermore, it was observed that the two monocarboxylate transporter isoforms present in mature skeletal muscles, MCT1 and MCT4 (formerly called MCT3 (M.C. Wilson, V.N. Jackson, C. Heddle, N.T. Price, H. Pilegaard, C. Juel, A. Bonen, I. Montgomery, O.F. Hutter, A.P. Halestrap, Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3, J. Biol. Chem. 273 (1998) 15920-15926)), were also expressed in primary culture of myotubes.     

Effect of epinephrine on net lactate uptake by contracting skeletal muscle.

The purpose of this study was to determine the effect of epinephrine on net lactate (La(-)) uptake at constant elevated blood La(-) concentration and steady level metabolic rate (O(2) uptake) in the canine gastrocnemius-plantaris muscle in situ. Infusion of La(-)/lactic acid (pH 3.5) established a mean arterial blood La(-) concentration of ~10 mM while normal blood-gas and pH status were maintained as the gastrocnemius-plantaris was stimulated with tetanic trains at a rate of one contraction every 4 s. After steady-state control measures, epinephrine was infused for 35 min at rates that produced a high physiological concentration with (Pro; n = 6) and without (Epi; n = 6) beta-adrenergic-receptor blockade via propranolol. Net La(-) uptake values during the control conditions were not significantly different between trials (Epi: 0.756 +/- 0.043; Pro: 0.703 +/- 0.061 mmol. kg(-1). min(-1)). Steady level O(2) uptake averaged approximately 69.5 ml. kg(-1). min(-1) for both control conditions and did not significantly change over the course of the experiments in either set of trials. Epi experiments resulted in a significantly reduced net La(-) uptake (0.346 +/- 0.088 mmol. kg(-1). min(-1) after 5 min of infusion) compared with control value at all sample times measured. However, net La(-) uptake was not significantly different from control at any time during Pro (0.609 +/- 0.052 mmol. kg(-1). min(-1) after 5 min of infusion). When the change from the respective control values for net La(-) uptake was compared across time for both series of experiments, Epi resulted in a significantly greater change from control than did Pro. This study suggests that epinephrine can have a profound effect on net La(-) uptake by contracting muscle and that these effects are elicited through beta-adrenergic-receptor stimulation.     

Cyclic variations in nitrogen uptake rate of soybean plants: effects of external nitrate concentration

Net uptake of NO3- by non-nodulated soybean plants [Glycine max (L.) Merr. cv. Ransom] growing in flowing hydroponic cultures containing 0.5, 1.0 and 10.0 mol m-3 NO3- was measured daily during a 24-d period of vegetative development to determine if amplitude of maximum and minimum rates of net NO3- uptake are responsive to external concentrations of NO3-. Removal of NO3- from the replenished solutions during each 24-h period was determined by ion chromatography. Neither dry matter accumulation nor the periodicity of oscillations in net uptake rate was altered by the external NO3- concentrations. The maxima of the oscillations in net uptake rate, however, increased nearly 3-fold in response to external NO3- concentrations. The maxima and minima, respectively, changed from 4.0 and 0.6 mmol NO3- per gram root dry weight per day at an external solution level of 0.5 mol m-3 NO3- to 15.2 and -2.7 mmol NO3- per gram root dry weight per day at an external solution level of 10.0 mol m-3 NO3-. The negative values for minimum net uptake rate from 10.0 mol m-3 NO3- solutions show that net efflux was occurring and indicate that the magnitude of the efflux component of net uptake was responsive to external concentration of NO3-.