Effects of a 12-month moderate weight loss intervention on insulin sensitivity and inflammation status in nondiabetic overweight and obese subjects.

Weight loss intervention is the principal non-pharmacological method for prevention and treatment of type 2 diabetes. However, little is known whether it influences insulin sensitivity directly or via its anti-inflammatory effect. The aim of this study was to assess the independent role of changes in inflammation status and weight loss on insulin sensitivity in this population.Overweight and obese nondiabetic participants without co-morbidities underwent a one-year weight loss intervention focused on caloric restriction and behavioral support. Markers of inflammation, body composition, anthropometric para-meters, and insulin sensitivity were recorded at baseline, 6, and 12 months. Insulin sensitivity was assessed with frequently sampled intravenous glucose tolerance test and Minimal Model. Twenty-eight participants (F: 15, M: 13, age 39±5 years, BMI 33.2±4.6 kg/m(2)) completed the study, achieving 9.4±6.9% weight loss, which was predominantly fat mass (7.7±5.6 kg, p<0.0001). Dietary intervention resulted in significant decrease in leptin, leptin-to-adiponectin ratio, hs-CRP, and IL-6 (all p<0.02), and improvement in HOMA-IR and Insulin Sensitivity Index (SI) (both p<0.001). In response to weight loss IL-1ß, IL-2, leptin, and resistin were significantly associated with insulin, sensitivity, whereas sICAM-1 had only marginal additive effect. Moderate weight loss in otherwise healthy overweight and obese individuals resulted in an improvement in insulin sensitivity and in the overall inflammation state; the latter played only a minimal independent role in modulating insulin sensitivity.

Expression and characterization of the human erythrocyte anion exchanger in a baculovirus/Sf-9 cell system.

The human erythrocyte anion-exchange protein (HAE1) has been expressed in insect Sf-9 cells using a recombinant baculovirus. We subcloned the full-length cDNA encoding HAE1 into the baculovirus expression vector pVL1392 and cotransfected Sf-9 cells with the recombinant vector and wild-type AcMNPV DNA to obtain recombinant baculovirus. The expressed protein was targeted to the Sf-9 plasma membrane at an apparent density of approximately 0.5 x 10(6) copies/cell as determined by quantitative autoradiography using an HAE1-specific monoclonal antibody. Unlike native HAE1, the expressed protein was not glycosylated. Transport studies with HAE1-recombinant-infected Sf-9 cells showed saturable [Km(Cl-) = 44 mM; Vmax(Cl-) = 48 mEq/liter of cell waters min] and H2DIDS-inhibitable (K(O.5) = 34 microM) 36Cl- uptake that was not present in uninfected cells. We also found that extracellular SO4(2-) reduced 36Cl- influx [K(0.5)((SO4)2-) = 26 mM], presumably through substrate competition as in erythrocytes. Finally, we observed that H2DIDS-inhibitable 36Cl- efflux was reduced by 77% in the nominal absence of a suitable counter-anion in the external solution (HCO3(-)-free, all-glucuronate medium), thereby providing strong evidence for an obligatory exchange mechanism. We conclude that there is high-level expression of + ++HAE1 functional activity in recombinant baculovirus-infected Sf-9 cells and that this system will prove useful for kinetic and structural analyses of the HAE1 protein.

Carrier-mediated transport of lipoxin A4 in human neutrophils.

Lipoxins and other eicosanoids display potent and selective biological effects on leukocytes. In this study, we utilized radiolabeled lipoxin A4 ([3H]LXA4) to investigate whether carrier-mediated transport of LXA4 might occur in human neutrophils. At a concentration of 5 nM, uptake of [3H]LXA4, above that due to specific binding to receptors, amounted to approximately 0.6 fmol.10(6) cells-1.min-1. This influx was sensitive to a number of anionic inhibitors, including 3,5-diiodosalicylic acid (K0.5 12 microM), pentachlorophenol (K0.5 25 microM), alpha-cyano-beta-(1-phenylindol-3-yl) acrylic acid, and the organomercurial agents mersalyl (K0.5 110 microM) and p-hydroxy-mercuribenzoate. Influx, which was Na+ and membrane voltage independent, exhibited a striking dependence on pH (negative log of dissociation 5.9), results compatible with an H+ + LXA4 anion cotransport system. The LXA4 carrier did not appear to interact with arachidonic acid, prostaglandin E2, 15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid, or the leukotrienes B4, C4, and D4. Moreover, transport activity was not observed in human erythrocytes, lymphocytes, or platelets, but it was inducible in HL-60 cells on differentiation by exposure to retinoic acid. These findings represent the identification and initial characterization of a novel carrier-mediated pathway in human neutrophils that facilitates transport of LXA4 into cells.

Whole cell Cl- currents in human neutrophils induced by cell swelling.

The properties of the conductive Cl- transport pathway underlying regulatory volume decrease (RVD) in human neutrophils were investigated using the whole cell patch-clamp technique. Cell swelling was induced during whole cell recordings by making the patch pipette solution hyperosmotic (approximately 20%) relative to the bath by addition of sucrose. Immediately after establishment of the whole cell configuration, no measurable Cl- currents were evident. Over a period of several minutes the outwardly rectifying Cl- current that developed displayed no apparent voltage dependence of activation and did not inactivate with time during voltage steps over the range of -80 to +80 mV. Reduction of Cl- currents by application of suction to the interior of the pipette implied that the swelling-induced Cl- channels are activated by membrane stretch. Based on reversal potential measurements, the volume-induced Cl- conductance was found to discriminate poorly among Cl-, Br-, I-, and NO3-, to possess a finite permeability to glucuronate (Pglucuronate/PCl approximately 0.1) and to be impermeable to cations. Single-channel conductance was estimated to be 1.5 pS from analysis of the variance of membrane current fluctuations. The activated Cl- currents were blocked by 100 microM of the compound MK-447 analogue A (inhibitor constant Ki = 37 microM) and by 200 microM 3,5-diiodosalicylate, 500 microM 4-acetamido-4'-iodothiocyanostilbene-2,2'-disulfonic acid, and 200 microM UK-5099. These results suggest that the initial event triggering RVD in neutrophils may be activation of stretch sensitive Cl- channels in the plasma membrane.

Cell volume regulation in human neutrophils: 2-(aminomethyl)phenols as Cl- channel inhibitors.

When subjected to hypotonic stress, human peripheral neutrophils initially swell due to rapid water entry and thereafter recover toward the normal cell size (approximately 330 microns 3). Neutrophils do not behave as perfect osmometers: when resuspended in half-isotonic medium (150 mosM), they swell by only approximately 40% rather than doubling in size as predicted for ideal behavior. As with lymphocytes, restoration to the normal cell size involves the net loss of K+ and Cl- from the cytosol through independent conductance pathways. Volume regulation is sensitive to 0.4-1 mM of quinine, UK-5099, 3,5-diiodosalicylate (DISA), MK-473 (an indanyloxyacetate derivative), and to MK-447 [a 2-(aminomethyl)phenol]. From correlation of drug effects on the time course of cell volume recovery and the associated volume-activated 86Rb+ and 36Cl- fluxes, it was evident that quinine blocked only K+ channels, whereas MK-447 acted as a selective inhibitor of Cl- channels. In contrast, UK-5099, DISA, and MK-473 were nonspecific in that the compounds displayed comparable suppressive effects on all three parameters. Structure-activity relationships in the MK-447 series revealed the critical elements of the molecule responsible for drug potency. In particular, the importance of the neighboring ionizable 1-hydroxyl and 2-aminomethyl groups and the formation of secondary ring structures for biological activity is emphasized. The most potent derivative thus far identified, termed analogue A [inhibitor constant (Ki) approximately 16 microM], had a potency approximately sixfold greater than that of the parent compound (Ki approximately 90 microM). These findings define the mechanism of action of a relatively new class of agents that behave as inhibitors of swelling-activated Cl- channels in these cells.

Enhancement of wound healing by the alkaloid taspine defining mechanism of action.

Taspine (mol wt 369,000) is an alkaloid extracted from trees of Croton (family Euphorbiaceae) of the western Amazon region that has been used by natives and others as a vulnerary agent. Taspine was purified from tree sap to test its healing properties using different topical concentrations in the paired rat surgical incision model. Wound tensile strength and histology were evaluated. Samples treated with 250 micrograms, but not those treated with 50 micrograms or 10 micrograms, had significant higher values for MBS than paired controls (26%, P < 0.005, and 30%, P < 0.001, by Days 5 and 7, respectively). Taspine did not modify MBS at Day 12. Sample treated with 250 micrograms had significantly greater mononuclear cellular infiltration at Days 5 and 7 but not at Day 12. To better understand the effect of taspine as an enhancer of wound healing, we conducted in vitro studies in cell cultures. Taspine stimulated chemotaxis for fibroblasts. Taspine did not have an effect on specific assays for macrophage chemotaxis, neutrophil activation, fibroblast proliferation, or matrix assembly. Taken together, the data suggest that taspine promotes early phases of wound healing in a dose-dependent manner with no substantial modification thereafter. Its mechanism of action is probably related to its chemotactic properties on fibroblasts and is not mediated by changes in extracellular matrix.

Substrate and inhibitor specificity of the lactate carrier of human neutrophils.

The substrate and inhibitor specificity of the lactic acid (Lac) transport system of human neutrophils was investigated. The ability of a variety of compounds to inhibit the influx of [14C]lactate, presumably reflecting competition by substrate analogues for binding at the external translocation site, was taken as an index of affinity for the Lac carrier. pH-state techniques were utilized to assess transportability. Results indicate a relatively low order of selectivity, the neutrophil H+(+)lactate- cotransport system demonstrating a broad acceptance of short-chain unsubstituted and substituted alkyl monocarboxylates as well as aromatic monocarboxylates. There was a slight preference for oxo, Cl, and OH substituents over other groups at the two-position of short chain alkyl fatty acids: all were readily transported across the plasma membrane at rates approaching that of L-lactate itself. Aromatic acids were not transported inward by the carrier although these compounds did permeate via simple nonionic diffusion. The neutrophil Lac carrier can be blocked by a number of cyanocinnamate derivatives, the classical inhibitors of monocarboxylate transport in mitochondria, and by dithiol compounds and sulfhydryl-reactive agents. This constellation of biochemical properties is similar to the features that characterize other well described H+(+)lactate- cotransport systems in red blood cells, Ehrlich ascites tumor cells, hepatocytes, and cardiac sarcolemmal vesicles, although significant differences exist when comparisons are made to the Na(+)-dependent lactate transporter of the kidney proximal tubule.

Lactic acid secretion by human neutrophils. Evidence for an H+ + lactate- cotransport system.

The pathway by which L-lactate (Lac) crosses the plasma membrane of isolated human neutrophils was investigated. The influx of [14C]Lac from a 2 mM Lac, 145 mM Cl-, 5.6 mM glucose medium was approximately 1.5 meq/liter of cell water.min and was sensitive to the organomercurial agent mersalyl (apparent Ki approximately 20 microM), to alpha-cyano-4-hydroxycinnamate (CHC), the classical inhibitor of monocarboxylate transport in mitochondria, and to UK-5099 (apparent Ki approximately 40 microM), a more potent analogue of CHC. Transport was also strongly blocked (greater than 80%) by 1 mM of either 3,5-diiodosalicylic acid, MK-473 (an indanyloxyacetate derivative), or diphenyl-amine-2-carboxylate, and by 0.4 mM pentachlorophenol, but not by 1 mM ethacrynic acid, furosemide, or the disulfonic stilbenes SITS or H2DIDS. One-way [14C]Lac efflux from steady-state cells amounted to approximately 6 meq/liter.min and was likewise affected by the agents listed above. Influx, which was membrane potential insensitive and Na+ independent, displayed a strong pH dependence: extracellular acidification enhanced uptake while alkalinization inhibited the process (pK' approximately 5.7 at 2 mM external Lac). The rate of [14C]Lac influx was a saturable function of external Lac, the Km being approximately 7 mM. Steady-state cells exhibited an intracellular Lac content of approximately 5 mM and secreted lactic acid into the bathing medium a a rate of approximately 4 meq/liter.min. Secretion was completely suppressed by 1 mM mersalyl which inactivates the carrier, leading to an internal accumulation of Lac. That the Lac carrier truly mediates an H+ + Lac- cotransport (or formally equivalent Lac-/OH- exchange) was documented by pH-stat techniques wherein an alkalinization of poorly buffered medium could be detected upon the addition of Lac; these pH changes were sensitive to mersalyl. Thus, the Lac carrier of neutrophils possesses several features in common with other monocarboxylate transport systems in erythrocytes and epithelia.

Use of tributyltin to probe contribution of Cl(-)-HCO3- exchange to regulation of steady-state pHi in human neutrophils.

Organotin derivatives represent a class of artificial ionophores that mediate Cl(-)-OH- exchange and thereby facilitate the chemical equilibrium distribution of Cl- and H+ across biological membranes. Imposing different pH and Cl- gradients by varying extracellular pH (pHo) and extracellular [Cl-] in the presence of 1 microM tributyltin validated the above assumptions in human neutrophils. Under relatively alkaline conditions [intracellular pH (pHi) greater than or equal to 7.10 and pHo greater than or equal to 7.40], the cell's natural Cl(-)HCO3- exchanger mimicked the actions of the tributyltin compound and was the principal factor controlling steady-state pHi. However, with increasing extracellular acidification, there was a progressive deviation from the predicted equilibrium distribution in the case of the normal Cl(-)-HCO3- transport system, whereas tributyltin-treated cells followed theoretical expectations. Exposure of neutrophils to a number of inhibitors of Cl(-)-HCO3- exchange led to a fall in pHi, apparently confirming the impression that a net HCO3- influx through Cl(-)-HCO3- countertransport was chiefly responsible for maintaining steady-state pHi. However, this intracellular acidification could be satisfactorily ascribed to proton movements through a parallel pathway, namely nonionic diffusion of the free acid form of the drugs. These results imply that Cl(-)-HCO3- exchange is the dominant pH regulatory device only under relatively alkaline conditions and that other mechanisms in addition to Na(+)-H+ exchange are likely to play an important role in recovery from acidification and in maintaining steady-state pHi. The possibility that the lactate carrier may function in this capacity is discussed.

Internal alkalinization by reversal of anion exchange in human neutrophils: regulation of transport by pH.

When steady-state human neutrophils bathed in 148 mM Cl- are transferred to a Cl(-)-free medium containing 0.5 mM HCO3- and 148 mM glucuronate or aspartate as nominally inert replacement ions, there is a rapid efflux of 36Cl- from the cells. The accelerated loss of Cl- is accompanied by an intracellular alkalinization of 0.7-0.9 pH units. Both the Cl- efflux and intracellular pH (pHi) transient are dependent on extracellular HCO3- and are sensitive to inhibition by SITS and alpha-cyano-4-hydroxycinnamate, which block anion exchange, thereby indicating that these processes are due to the countertransport of internal Cl- for external HCO3-. Rate of anion exchange is strongly influenced by pH, which functions to regulate carrier activity; alkalinization stimulates the transport velocity, whereas acidification inhibits it. The relationship to pHi follows a Hill equation with pK' approximately 7.40 and Hill coefficient of 3.3, thereby suggesting that approximately 3 HCO3- may be required to bind to the modifier site. Neutrophils placed in glucuronate medium progressively shrink during the first 7.5 min of incubation due to the net loss of osmotically active particles through Cl(-)-HCO3- exchange. However, between 7.5 and 30 min, cells regain their normal cell size. This volume recovery phase correlates with the time course of 22Na+ and [14C]glucuronate influxes, whose kinetics can be dissociated from that of anion exchange. Uptake of glucuronate is largely Na+ dependent (whereas Cl(-)-HCO3- exchange is not), is resistant to amiloride, and can be blocked by furosemide, which suggests that glucuronate probably enters via a volume-activated pathway such as Na+ + glucuronate cotransport.

Intracellular pH recovery from alkalinization. Characterization of chloride and bicarbonate transport by the anion exchange system of human neutrophils.

The nature of the intracellular pH-regulatory mechanism after imposition of an alkaline load was investigated in isolated human peripheral blood neutrophils. Cells were alkalinized by removal of a DMO prepulse. The major part of the recovery could be ascribed to a Cl-/HCO3- counter-transport system: specifically, a one-for-one exchange of external Cl- for internal HCO3-. This exchange mechanism was sensitive to competitive inhibition by the cinnamate derivative UK-5099 (Ki approximately 1 microM). The half-saturation constants for binding of HCO3- and Cl- to the external translocation site of the carrier were approximately 2.5 and approximately 5.0 mM. In addition, other halides and lyotropic anions could substitute for external Cl-. These ions interacted with the exchanger in a sequence of decreasing affinities: HCO3- greater than Cl approximately NO3- approximately Br greater than I- approximately SCN- greater than PAH-. Glucuronate and SO4(2-) lacked any appreciable affinity. This rank order is reminiscent of the selectivity sequence for the principal anion exchanger in resting cells. Cl- and HCO3- displayed competition kinetics at both the internal and external binding sites of the carrier. Finally, evidence compatible with the existence of an approximately fourfold asymmetry (Michaelis constants inside greater than outside) between inward- and outward-facing states is presented. These results imply that a Cl-/HCO3- exchange mechanism, which displays several properties in common with the classical inorganic anion exchanger of erythrocytes, is primarily responsible for restoring the pHi of human neutrophils to its normal resting value after alkalinization.

Polyvalent cations inhibit human neutrophil chemotaxis by interfering with the polymerization of actin.

The effect of a series of di- and trivalent cations on the locomotor response of human neutrophils to the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) was investigated. Migration was assessed by the leading front method. The cations inhibited FMLP-stimulated chemotaxis in the rank order: Ni2+ approximately Co2+ greater than Sr2+ greater than Zn2+ greater than Mn2+ approximately La3+ greater than Cd2+ approximately Ba2+ much greater than Mg2+. Benzamil, which blocks Na+/Ca2+ exchange, did not alter chemotaxis by itself but prevented the suppressive effects of each of the polyvalent cations on motility. The ion selectivity sequence and the lack of activity of benzamil are strikingly different than for O(-2) generation, thereby implying different modes of action in the two functional expressions. The F-actin content of the cells was monitored by the fluorescence of rhodamine-phalloidin. Each of the cations displayed comparable efficacy in blocking the polymerization of actin in FMLP-activated cells. Likewise, benzamil exhibited a protective effect, completely overcoming the inhibitory action of the polyvalent cations. The results indicate that these foreign ions gain access to the cell interior via a benzamil-sensitive pathway, namely Na+/Ca2+ exchange. Upon entry into the cytosol, they then interfere with the formation of filaments from actin monomers. These studies help to shed light on the interaction of divalent cations with cytoskeletal and contractile elements in cell motility.

A role for Na+/Ca2+ exchange in the generation of superoxide radicals by human neutrophils.

A Na+/Ca2+ exchange mechanism has been recently described in human neutrophils that constitutes the principal pathway for Ca2+ influx into resting cells. The potential role of this system in regulating the respiratory burst in response to activation by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine was explored. In the presence of 1 mM Ca2+, a variety of di- and trivalent cations suppressed the generation of O(-2) radicals in a series of decreasing efficacy: La3+ approximately Zn2+ much greater than Sr2+ approximately Cd2+ greater than Ba2+ greater than Co2+ greater than Ni2+ approximately Mg2+. This sequence is similar to their rank order of activity in inhibiting 45Ca2+ influx via Na+/Ca2+ counter-transport. Benzamil, phenamil, and 2',4'-dichlorobenzamil, analogues of amiloride which selectively block Na+/Ca2+ exchange in neutrophils, likewise suppressed the release of O(-2) with apparent Ki values of approximately 30 microM. The effect of the cations was competitive with Ca2+, while the interaction between the benzamil derivatives and Ca2+ appeared to be noncompetitive in nature. Both the divalent cations and benzamil also inhibited the rise in cytoplasmic Ca2+ as monitored by fura-2 fluorescence: these agents reduced peak cytosolic Ca2+ levels after N-formyl-methionyl-leucyl-phenylalanine stimulation to values seen in the absence of extracellular Ca2+. These results are compatible with the hypothesis that the influx of Ca2+ via Na+/Ca2+ exchange contributes to the transient elevation in intracellular free Ca2+. The polyvalent cations block the entry of critical Ca2+ ions by competing with Ca2+ for binding to the translocation site on the exchange carrier, while benzamil acts by lowering the maximal transport rate. These studies emphasize that Na+/Ca2+ exchange through its effects on cytoplasmic Ca2+ plays a major regulatory role in activation of the respiratory burst in chemotactic factor-stimulated neutrophils.

Sulfate transport in human neutrophils.

The mechanism by which SO4(2-) is transported across the plasma membrane of isolated human neutrophils was investigated. Unlike the situation in erythrocytes, SO4(2-) and other divalent anions are not substrates for the principal Cl-/HCO3- exchange system in these cells. At an extracellular concentration of 2 mM, total one-way 35SO4(2-) influx and efflux in steady-state cells amounted to approximately 17 mumol/liter of cell water per min. The intracellular SO4(2-) content was approximately 1 mM, approximately 25-fold higher than the passive distribution level. Internal Cl- trans stimulated 35SO4(2-) influx. Conversely, 35SO4(2-) efflux was trans stimulated by external Cl- (Km approximately 25 mM) and by external SO4(2-) (Km approximately 14 mM), implying the presence of a SO4(2-)/Cl- countertransport mechanism. The exchange is noncompetitively inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2' -disulfonate (SITS) (Ki approximately 50 microM) and competitively blocked by alpha-cyano-4-hydroxycinnamate (Ki approximately 230 microM) and by ethacrynate (Ki approximately 7 microM); furosemide and probenecid also suppressed activity. The carrier exhibits broad specificity for a variety of monovalent (NO3- approximately Cl- greater than Br- greater than formate- greater than I- approximately p-aminohippurate-) and divalent WO4(2-) greater than oxalate2- greater than SO4(2-) greater than MoO4(2-) greater than SeO4(2-) greater than AsO4(2-) anions. There was little, if any, affinity for HCO3-, phosphate, or glucuronate. The influx of SO4(2-) is accompanied by an equivalent cotransport of H+, the ion pair H+ + SO4(2-) being transported together in exchange for Cl-, thereby preserving electroneutrality. These findings indicate the existence of a separate SO4(2-)/Cl- exchange carrier that is distinct from the neutrophil's Cl-/HCO3- exchanger. The SO4(2-) carrier shares several properties in common with the classical inorganic anion exchange mechanism of erythrocytes and with other SO4(2-) transport systems in renal and intestinal epithelia, Ehrlich ascites tumor cells, and astroglia.

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.

Lithium movements in resting and chemotactic factor-activated human neutrophils.

The ability of the chemotactic factor-activated Na+-H+ exchange system of human neutrophils to bind and transport other cations of the alkali metal series was investigated. After exposure of cells to the tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP), the influx of Li+ was measured by flame photometry and correlated with changes in intracellular pH (pHi) derived from the equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione. In Na+-depleted cells, Li+ (Km approximately equal to 14 mM) could substitute effectively for Na+ (Km approximately equal to 23 mM) at the external translocation site of the carrier, though the maximal transport rate for Li+ (approximately 8 meq.l-1.min-1) was roughly half that for Na+ (approximately 15 meq.l-1.min-1). On the other hand, the carrier lacked appreciable affinity for K+, Rb+, and Cs+. The influx of Li+ from the external solution was accompanied by an equivalent counterefflux of H+ from the internal milieu. The H+ efflux thus induced led to an intracellular alkalinization of approximately 0.7 units, the pHi rising from approximately 7.20 to approximately 7.90. The influx of Li+, as well as the increase in pHi in 140 mM Li+ medium, was competitively inhibited by amiloride (Ki approximately equal to 9 microM). Extracellular H+ also behaved as a competitive inhibitor of Li+ with a Ki of approximately 30 nM (pK'a approximately 7.50). These studies indicate that the FMLP-activated alkali metal cation-H+ exchange mechanism of neutrophils shares a number of features in common with those of Na+-H+ exchangers in a variety of different cells.