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L Simchowitz

Publications and source records attributed to L Simchowitz.

At least 37 records · Page 2Linked to original sources
PubMed1988

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.

Amiloride↗
PubMed1988

Properties of the principal anion-exchange mechanism in human neutrophils.

As part of our ongoing studies aimed at understanding the physiology of human neutrophil function, we have been investigating the nature of the principal anion-exchange mechanism in these cells. This countertransport system functions physiologically as a Cl-/HCO3- exchanger in intracellular pH regulation from alkalinization (Simchowitz and Roos, 1985). In summarizing the results of our work, it is worthwhile to keep in mind the well-defined properties of the classic inorganic anion-exchange carrier of red blood cells, as important differences between the two are evident. Inhibitor Profile In our original articles on Cl- movements (Simchowitz and De Weer, 1986; Simchowitz et al., 1986), we had identified CHC, which suppresses monocarboxylate transport in mitochondria (Halestrap and Denton, 1975), as a relatively weak inhibitor. Several structural analogues, including alpha-methylcinnamate and alpha-phenylcinnamate, exhibited enhanced activity against anion exchange. The most potent compound thus far identified was UK-5099, which was approximately 300-fold more active than CHC. The inhibitory effect of each member of this series of compounds was strictly competitive in nature: both the drug and Cl ions appeared to bind to the same external translocation site on the exchange carrier. The inhibition by several other unrelated agents (e.g., MK-473, niflumate, flufenamate, and NAP-taurine), all weak organic acids, was also of the competitive type, which suggests that these compounds bind to the carrier by virtue of their negatively charged carboxyl or sulfonic groups. In marked contrast to erythrocytes, the anion-exchange carrier of neutrophils is rather insensitive to the disulfonic stilbenes SITS and DIDS (Simchowitz and De Weer, 1986): little or no effect can be detected in 148 mM Cl-. However, under conditions of low external Cl- (5-35 mM), SITS can in fact be shown to interact weakly with the anion binding site of the exchange carrier. In a comparable vein, several other drugs, all of which have been reported to suppress Cl- fluxes in red cells (for reviews, see Sachs et al., 1975; Gunn, 1979; Knauf, 1979; Lowe and Lambert, 1983), were either completely inactive against anion exchange in neutrophils at a concentration of 1 mM or only marginally effective at doses several orders of magnitude greater than those required in red blood cells. This list included furosemide, picrylsulfonate, maleic anhydride, 5,5'-dithio-bis(2-nitrobenzoate), salicylate, 2-methoxy-5-nitrotropone, and aldrithiol.4+he external

Anion Exchange Resins↗
PubMed1987

Intracellular accumulation of potent amiloride analogues by human neutrophils.

The mechanism of uptake of a series of amiloride derivatives by human neutrophils was investigated using [14C]amiloride and the 14C-labeled 5-(1-hexahydroazepinyl)-6-bromo analogue (BrMM) which is approximately 500-fold more potent than the parent compound at inhibiting Na+/H+ exchange. At an external concentration of 2 microM, the influx of BrMM at 37 degrees C was rapid, reaching a steady state by approximately 20 min. The rate of BrMM uptake (approximately 25 mumol/liter.min) was approximately 90-fold faster than for the same concentration of amiloride, a finding which correlates with differences in lipid partitioning of the two compounds. Uptake was unrelated to specific binding to Na+/H+ exchange transport sites: influx of either drug was nonsaturable whereas amiloride- and BrMM-mediated inhibition of Na+/H+ countertransport obeyed Michaelis-Menten kinetics with apparent Ki values of approximately 75 and approximately 0.2 microM. Entry occurred exclusively via the neutral (uncharged) forms (pK'a 8.40-8.55). Influx was markedly pH-dependent: it was enhanced by extracellular alkalinization and reduced by acidification. Influx was, however, insensitive to large changes in membrane voltage, thereby implying the protonated (charged) species to be impermeant. About 75% of the total intracellular pool of amiloride, but only approximately 25% of BrMM, is contained within the lysosomes, an expected consequence of the partitioning and subsequent trapping of a weak base within this strongly acidic subcellular compartment. With BrMM, there was a relative approximately 60-fold enrichment in the internal/external water concentration ratio of the drug; the value for amiloride was much less, approximately 4. This disparity is consistent with substantial binding of BrMM to internal constituents, presumably to proteins and/or nucleic acids. Thus, it is important to recognize that potentially large intracellular accumulations of potent analogues can occur that are not directly involved in inhibition of Na+/H+ exchange. These findings sound a cautionary note in the interpretation of results using these drugs in all cells, especially those of small size with high surface-to-volume ratios.

Amiloride↗
PubMed1987

Intracellular acidification-induced alkali metal cation/H+ exchange in human neutrophils.

Pretreatment of isolated human neutrophils (resting pHi congruent to 7.25 at pHo 7.40) with 30 mM NH4Cl for 30 min leads to an intracellular acidification (pHi congruen to 6.60) when the NH4Cl prepulse is removed. Thereafter, in 140 mM Na+ medium, pHi recovers exponentially with time (initial rate, approximately 0.12 pH/min) to reach the normal resting pHi by approximately 20 min, a process that is accomplished mainly, if not exclusively, though an exchange of internal H+ for external Na+. This Na+/H+ countertransport is stimulated by external Na+ (Km congruent to 21 mM) and by external Li+ (Km congruent to 14 mM), though the maximal transport rate for Na+ is about twice that for Li+. Both Na+ and Li+ compete as substrates for the same translocation sites on the exchange carrier. Other alkali metal cations, such as K+, Rb+, or Cs+, do not promote pHi recovery, owing to an apparent lack of affinity for the carrier. The exchange system is unaffected by ouabain or furosemide, but can be competitively inhibited by the diuretic amiloride (Ki congruent to 8 microM). The influx of Na+ or Li+ is accompanied by an equivalent counter-reflux of H+, indicating a 1:1 stoichiometry for the exchange reaction, a finding consistent with the lack of voltage sensitivity (i.e., electroneutrality) of pHi recovery. These studies indicate that the predominant mechanism in human neutrophils for pHi regulation after intracellular acidification is an amiloride-sensitive alkali metal cation/H+ exchange that shares a number of important features with similar recovery processes in a variety of other mammalian cell types.

Cesium↗
PubMed1986

Regulation of human neutrophil chemotaxis by intracellular pH.

The relationship of N-formyl-methionyl-leucyl-phenylalanine-stimulated Na+/H+ exchange to the chemotactic responsiveness of human neutrophils was investigated. The pHi changes, measured from the equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione, were correlated with the migratory behavior of the cells as assessed by the leading front method. Exposure of cells to 10 nM FMLP caused activation of Na+/H+ exchange, leading to a rise in pHi from approximately 7.25 to approximately 7.75. This intracellular alkalinization was inhibited by amiloride and by three more potent analogues. All four compounds reduced the chemotactic response to FMLP with apparent Ki values similar to those for inhibition of the pHi transients, thereby suggesting that the blocking effect of the drugs on directed cell migration was related to inhibition of Na+/H+ exchange. The effect was specific for stimulated cell locomotion: FMLP-induced chemotaxis and chemokinesis were inhibited in parallel, whereas random motility was unimpaired. The relationship of pHi to function was also studied as the pHi of FMLP-activated cells was varied between 6.8 and 8.6 by altering the chemical gradients for Na+ and H+ across the cell membrane. There was a direct, positive correlation between the pHi value attained following FMLP-stimulation and the locomotor response to a chemotactic gradient. These results indicate that the motile functions of human neutrophils can be regulated by their pHi.

Amiloride↗
PubMed1986

Chloride movements in human neutrophils. Diffusion, exchange, and active transport.

Chloride content and fluxes were measured in isolated resting human peripheral polymorphonuclear leukocytes. The intracellular Cl concentration of cells kept at 37 degrees C in 148 mM Cl media was approximately 80 meq/liter cell water, fourfold higher than expected for passive distribution at the cell's estimated membrane potential (approximately -53 mV). All intracellular Cl was rapidly exchangeable with external 36Cl. Cells lost Cl exponentially into Cl-free media, and reaccumulated it when Cl was restored to the bath; this reuptake was dependent on metabolism. One-way 36Cl fluxes in steady state cells were approximately 1.4 meq/liter X min. The bulk (approximately 70%) of these represented electrically silent Cl/Cl exchange mediated by a carrier insensitive to disulfonic stilbenes but blocked by the anion carrier inhibitor alpha-cyano-4-hydroxycinnamate (CHC). The remaining fluxes were characterized in some detail. About 20% of 36Cl influx behaved as active transport: it moved thermodynamically uphill and was absent in cells treated with 2-deoxy-D-glucose, displayed Michaelis-Menten kinetics with Km(Cl) congruent to 5 mM, Vmax congruent to 0.25 meq/liter X min, and was inhibited by CHC (Ki congruent to 1.7 mM), ethacrynate (Ki congruent to 50 microM), and furosemide (Ki congruent to 50 microM). About 30% of Cl efflux and approximately 8% of Cl influx behaved as electrodiffusion through a low-permeability pathway (PCl congruent to 4 X 10(-9) cm/s; gCl congruent to 1 microsecond/cm2; PK/PNa/PCl congruent to to 10:1:1); these fluxes were linear with concentration and strongly voltage sensitive. The putative Cl channel does not appear to be voltage gated, and gives evidence of single filing.

Biological Transport, Active↗
PubMed1986

Anion/anion exchange in human neutrophils.

Of the total one-way chloride fluxes (approximately 1.4 meq/liter cell water X min) in steady state human polymorphonuclear leukocytes bathed in 148 mM Cl media, approximately 70% behaves as self-exchange mediated by a nonselective anion carrier that is not inhibited by stilbene disulfonates. Five properties of this carrier-mediated exchange were investigated: substrate saturation is seen with respect to 36Cl influx as a function of the external Cl concentration [for normal-Cl cells, the apparent Km(Cl) is approximately 22 mM when Cl replaces para-amino-hippurate (PAH) and approximately 5 mM when Cl replaces glucuronate], and with respect to 36Cl efflux as a function of the concentration of internal Cl replacing PAH [apparent Km(Cl) congruent to 35 mM for cells bathed in 148 mM Cl]; there is trans stimulation of 36Cl influx by internal Cl (replacing PAH) with an apparent Km(Cl) congruent to 35 mM, and of 36Cl efflux by external Cl with an apparent Km(Cl) congruent to 22 mM (Cl replacing PAH) or approximately 5 mM (Cl replacing glucuronate); there is substrate competition between Cl and PAH, but the carrier appears devoid of affinity for glucuronate; influxes and effluxes mediated by the carrier are subject to competitive inhibition by extracellular alpha-cyano-4-hydroxycinnamate (CHC), with an apparent Ki congruent to 9 mM in Cl medium or approximately 1 mM in PAH medium (transport of the inhibitor itself is very slow); and internal Cl and external Cl or PAH undergo 1:1 countertransport, which is CHC sensitive. A simple equilibrium-competition model is proposed that accounts for all the extracellular ligand interactions presented for normal-Cl cells. Least-squares values of the carrier's true Michaelis constants for extracellular Cl, PAH, and CHC are 5.03 +/- 0.83, 50.3 +/- 14.9, and 0.29 +/- 0.09 mM, respectively.

Anions↗
PubMed1986

Inhibition of chemotactic factor-activated Na+/H+ exchange in human neutrophils by analogues of amiloride: structure-activity relationships in the amiloride series.

The ability of a number of analogues of the diuretic, amiloride, to inhibit chemotactic factor-stimulated Na+/H+ exchange in human neutrophils was investigated. Intracellular pH (pHi) changes were measured from the equilibrium distribution of 14C-labeled 5,5-dimethyloxazolidine-2,4-dione (DMO). Exposure of cells to 10 nm N-formyl-methionyl-leucyl-phenylalanine (FMLP) caused activation of Na+/H+ exchange: in 140 mM Na+ medium (extracellular pH 7.40), the pHi rose from a resting value of approximately 7.25 to reach a new steady state of approximately 7.75 by 10-15 min. This intracellular alkalinization was sensitive to amiloride (apparent Ki approximately 75 microM), a known inhibitor of Na+/H+ countertransport. The structure-activity relationships in the amiloride series were characterized by testing the effect of these compounds on the DMO-derived pHi changes and on the FMLP-stimulated rate of 22Na+ efflux from the cells. Substitutions of the guanidino group of amiloride resulted in relatively inactive products (Ki greater than or equal to 1 mM). Replacement of the 6-Cl group of amiloride by other halogen atoms had only modest effects on drug efficacy. However, replacement of one or both H atoms of the 5-amino group by short alkyl groups led to a 10-500-fold increase in potency for inhibition of Na+/H+ exchange. Amiloride and three of its more potent derivatives (compounds I, O, and MM, the 5-N,N-dimethyl, 5-N,N-diethyl, and 5-N,N-hexamethylene analogues, respectively) caused parallel inhibition of FMLP-activated 22Na+ efflux and the rate of intracellular alkalinization, with apparent Ki values of approximately 75, 8, 1, and 0.2 microM, respectively. In each instance, the inhibitory effects of the drugs were readily reversible on washing the cells. None of the compounds altered the binding of 3H-labeled FMLP to its cell surface receptors. The development of potent derivatives of amiloride should provide powerful tools for assessing the role of FMLP-activated Na+/H+ exchange and the resultant pHi transients on stimulated neutrophil functions.

Amiloride↗
PubMed1985

Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. I. Sodium fluxes.

The nature of Na+ fluxes in resting and in chemotactic factor-activated human neutrophils was investigated. In resting cells, ouabain-insensitive unidirectional 22Na+ in- and effluxes represented passive electrodiffusional fluxes through ion channels: they were nonsaturable and voltage-dependent (PNa = 4.3 X 10(-9) cm/s). Amiloride (1 mM) had little effect on resting 22Na+ influx (approximately 0.8 meq/liter X min), thereby suggesting a minor contribution of Na+/H+ exchange and a lack of amiloride-sensitive Na+ channels. When neutrophils were exposed to the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM), 22Na+ influx was stimulated approximately 30-fold (initial rate approximately 22 meq/liter X min). The FMLP-induced 22Na+ influx was saturable with respect to external Na+ (Km 26-35 mM, Vmax approximately 28 meq/liter X min), was electroneutral, and could be competitively inhibited by amiloride (Ki 10.6 microM). From a resting value of approximately 30 meq/liter of cell water, internal Na+ in FMLP-stimulated cells rose exponentially to reach a concentration of approximately 60 meq/liter by 10-15 min. This uptake was blocked by amiloride. FMLP also stimulated the efflux of 22Na+ which followed a single exponential time course (rate coefficient approximately 0.16 min-1). The FMLP-induced 22Na+ fluxes were similar to those observed with 10 microM monensin, a known Na+/H+ exchanging ionophore. The data indicate that FMLP activates an otherwise quiescent, amiloride-sensitive Na+/H+ exchange. Furthermore, all of the FMLP-induced 22Na+ fluxes can be satisfactorily accounted for by transport through the exchanger, leaving little room for an appreciable increase in Na+ conductance.

Amiloride↗
PubMed1985

Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. II. Intracellular pH changes.

The intracellular pH (pHi) changes resulting from chemotactic factor-induced activation of Na+/H+ exchange in isolated human neutrophils were characterized. Intracellular pH was measured from the equilibrium distribution of [14C]-5,5-dimethyloxazolidine-2,4-dione and from the fluorescence of 6-carboxyfluorescein. Exposure of cells to 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) in 140 mM Na+ medium at extracellular pH (pHo) 7.40 led to a rise in pHi along an exponential time course (rate coefficient approximately 0.55 min-1). By 10 min, a new steady-state pHi was reached (7.75-7.80) that was 0.55-0.60 units higher than the resting pHi of control cells (7.20-7.25). The initial rate of H+ efflux from the cells (approximately 15 meq/liter X min), calculated from the intrinsic intracellular buffering power of approximately 50 mM/pH, was comparable to the rate of net Na+ influx (approximately 17 meq/liter X min), an observation consistent with a 1:1 stoichiometry for Na+/H+ exchange. This counter-transport could be inhibited by amiloride (apparent Ki approximately 75 microM). When either the external ([Na+]o) or internal Na ([Na+]i) concentrations, pHo, or pHi were varied independently, the new steady-state [Na+]i and pHi values in FMLP-stimulated cells were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane. By analogy to other activated cells, these results indicate that an alkalinization of pHi in human neutrophils is mediated by a chemotactic factor-induced exchange of internal H+ for external Na+.

Amiloride↗
PubMed1985

Regulation of intracellular pH in human neutrophils.

The intracellular pH (pHi) of isolated human peripheral blood neutrophils was measured from the fluorescence of 6-carboxyfluorescein (6-CF) and from the equilibrium distribution of [14C]5,5-dimethyloxazolidine -2,4-dione (DMO). At an extracellular pH (pHo) of 7.40 in nominally CO2-free medium, the steady state pHi using either indicator was approximately 7.25. When pHo was suddenly raised from 7.40 to 8.40 in the nominal absence of CO2, pHi slowly rose by approximately 0.35 during the subsequent hour. A change of similar magnitude in the opposite direction occurred when pHo was reduced to 6.40. Both changes were reversible. Intrinsic intracellular buffering power, determined by using graded pulses of CO2 or NH4Cl, was approximately 50 mM/pH over the pHi range of 6.8-7.9. The course of pHi obtained from the distribution of DMO was followed during and after imposition of intracellular acid and alkaline loads. Intracellular acidification was brought about either by exposing cells to 18% CO2 or by prepulsing with 30 mM NH4Cl, while pHo was maintained at 7.40. In both instances, pHi (6.80 and 6.45, respectively) recovered toward the control value at rates of 0.029 and 0.134 pH/min. These rates were reduced by approximately 90% either by 1 mM amiloride or by replacement of extracellular Na with N-methyl-D-glucamine. Recovery was not affected by 1 mM SITS or by 40 mM alpha-cyano-4-hydroxycinnamate (CHC), which inhibits anion exchange in neutrophils. Therefore, recovery from acid loading is probably due to an exchange of internal H for external Na. Intracellular alkalinization was achieved by exposing the cells to 30 mM NH4Cl or by prepulsing with 18% CO2, both at a constant pHo 7.40. In both instances, pHi, which was 7.65 and 7.76, respectively, recovered to the control value. The recovery rates (0.033 and 0.077 pH/min, respectively) were reduced by 80-90% either by 40 mM CHC or by replacement of extracellular Cl with p-aminohippurate (PAH). SITS, amiloride, and ouabain (0.1 mM) were ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonium Chloride↗
PubMed1985

Intracellular pH modulates the generation of superoxide radicals by human neutrophils.

The relationship of intracellular pH (pHi) to superoxide radical (O2-) generation was investigated in chemotactic factor-stimulated human neutrophils. Exposure of cells to 100 nM N-formylmethionyl-leucyl-phenylalanine (FMLP) caused activation of Na/H exchange which, in 140 mM Na medium (pH0 7.40), led to a rise in pHi from 7.22 to 7.80. This pHi change was sensitive to amiloride (apparent Ki 78 microM), an inhibitor of Na/H countertransport. The time course of the alkalinization was similar to that of FMLP-stimulated O2- production, which was complete by 5 min. In the presence of 1 mM amiloride, which nearly blocked the pHi transient elicited by FMLP, or in the absence of external Na, where intracellular acidification was observed in FMLP-stimulated cells, O2- release was still roughly 25-45% of normal. Thus, an alkalinization cannot be an obligatory requirement for O2- generation. By independently varying either pH0, pHi, or the internal or external concentrations of Na, both the direction and magnitude of the FMLP-induced pHi transients could be altered. In each instance, the amount of O2- release correlated directly with pHi and was enhanced by intracellular alkalinization. In the absence of FMLP, a rise in pHi to 7.7-7.8 by exposure of cells to 30 mM NH4Cl, 10 microM monensin (a Na/H exchanging ionophore), or after a prepulse with 18% CO2 did not result in O2- generation. Thus, these results imply that an alkalinization per se is not a sufficient trigger. Neutrophils exposed to 4 nM FMLP exhibited a threefold slower rate of alkalinization (reaching pHi approximately 7.80 by 20-30 min) as compared to that obtained with 100 nM FMLP and did not release significant amounts of O2- under normal incubation conditions. However, these cells could be induced to generate O2- when the degree of alkalinization was enhanced by internal Na depletion or by pretreatment with 18% CO2. Together, these results indicate a modulating effect of pHi on O2- production and suggest that other functional responses of neutrophils may be regulated by their pHi.

Amiloride↗
PubMed1984

Receptor blockade as a mechanism of deactivation of human neutrophils by pepstatin and formyl-Met-Leu-Phe.

The pentapeptide pepstatin was shown to be chemotactic for human neutrophils by two techniques: ED50 for chemotaxis was found to be 3 microM by the agarose method and 0.2 microM by the Boyden chamber technique. Pepstatin also induced superoxide radical generation, release of lysosomal enzymes, and a transient increase in intercellular adenosine-3',5'-cyclic monophosphate (cAMP) levels in a dose-dependent manner. Carbobenzoxy-phenylalanyl-methionine (CBZ-PM), which competitively inhibits formyl-methionyl-leucyl-phenylalanine (FMLP) -induced neutrophil functions, also inhibited pepstatin-induced neutrophil function of superoxide generation in a dose-dependent fashion. Likewise, pepstatin inhibited the binding of [3H]FMLP to the cells. Furthermore, preincubation of neutrophils with suboptimal concentrations of FMLP or pepstatin diminished the cellular response toward either factor when tested for their chemotactic activity and for their ability to induce superoxide generation, to release granule enzymes, and to induce a transient increase in intracellular cAMP levels. The concentrations of pepstatin or FMLP tested had no effect on superoxide generation, granule enzyme release, or intracellular levels of cAMP on subsequent challenge with C5a; both of these factors, however, cross-deactivated the chemotactic response of the cells towards C5a. Similar results were observed when cells were preincubated with C5a and subsequently challenged with pepstatin or FMLP. These results suggest that FMLP and pepstatin interact with the same receptor molecules to activate human neutrophil functions. Furthermore, our data indicate that the deactivation of the neutrophil functions of superoxide production and granule enzyme release are receptor specific, but the heterologous deactivation of chemotaxis involves a postreceptor mechanism(s).

Binding, Competitive↗
PubMed1983

Evidence that the functional responses of human neutrophils occur independently of transient elevations in cyclic AMP levels.

Exposure of human neutrophils to the tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP) leads to a transient, 2-3 fold elevation of adenosine-3',5'-cyclic monophosphate (cAMP) that peaks at 5-15 seconds. This cAMP transient has been hypothesized as constituting an early activation event that may be responsible for subsequent functional responses. In order to evaluate the dependence of several FMLP-stimulated functional responses on elevated cAMP levels, we utilized 9-(tetrahydro-2-furyl)adenine (SQ 22,536), a putative inhibitor of adenylate cyclase. Pretreatment of cells with SQ 22,536 (1-1000 microM) caused dose-dependent inhibition of the FMLP (0.1 microM)-induced cAMP elevation (ID50 approximately 5 microM). Similar results were observed when cells were activated by the divalent cation ionophore A23187 (20 microM). At 1000 microM, a drug concentration which completely abolished the cAMP transient, SQ 22,536 had no effect on FMLP-stimulated superoxide radical (O2-) generation, granule enzyme release, or chemotaxis and only a modest inhibitory effect on A23187-induced O2- production. These studies strongly suggest that these FMLP- and A23187-induced responses occur independently of a transient elevation of cAMP and that, in intact human neutrophils, SQ 22,536 is a non-toxic inhibitor of adenylate cyclase.

Adenine↗