Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor

Lipoxin A4 (LXA4) triggers selective responses with human neutrophils that are pertussis toxin sensitive and binds to high affinity receptors (Kd = 0.5 +/- 0.3 nM) that are modulated by stable analogues of guanosine 5'-triphosphate (GTP). Here, we characterized [11,12-(3)]LXA4 specific binding with neutrophil granule and plasma membranes, which each display high affinity binding sites (Kd = 0.7 +/- 0.1 nM) that were regulated by GTP gamma S. Since functional LXA4 receptors are inducible in HL-60 cells, we tested orphan cDNAs encoding 7- transmembrane region receptors cloned from these cells for their ability to bind and signal with LXA4. Chinese hamster ovary (CHO) cells transfected with the orphan receptor cDNA (pINF114) displayed specific 3H-LXA4 high affinity binding (1.7 nM). When displacement of LXA4 binding with pINF114-transfected CHO cells was tested with other eicosanoids, including LXB4, leukotriene D4 (LTD4), LTB4, or prostaglandin E2, only LTD4 competed with LXA4, giving a Ki of 80 nM. In transfected CHO cells, LXA4 also stimulated GTPase activity and provoked the release of esterified arachidonate, which proved to be pertussis toxin sensitive. These results indicate that pINF114 cDNA encodes a 7-transmembrane region-containing protein that displays high affinity for 3H-LXA4 and transmits LXA4-induced signals. Together, they suggest that the encoded protein is a candidate for a LXA4 receptor in myeloid cells.     

Leukotriene B4 receptors on guinea pig alveolar eosinophils.

The existence of receptors for LTB4 on highly purified guinea pig alveolar eosinophils was investigated. Massive infiltration of eosinophils in alveolar spaces was induced in guinea pigs by i.v. injections of Sephadex beads G50 (16 mg/kg). Alveolar eosinophils (50 x 10(6) cells) were purified to approximately 98% by Percoll continuous density gradient centrifugation. The binding studies indicated that alveolar eosinophils bind LTB4 in a saturable, reversible and specific manner. Scatchard analysis indicated the existence of high-affinity binding sites (Kd1 = 1.00 +/- 0.22 nM; Bmax1 = 966 +/- 266 sites/cell) and low-affinity binding sites (Kd2 = 62.5 +/- 8.9 nM; Bmax2 = 5557 +/- 757 sites/cell). The metabolism of LTB4 by alveolar eosinophils in binding conditions was assessed by RP-HPLC and no significant degradation of [3H]LTB4 was observed. LTB4 dose-dependently stimulated eosinophil migration in both chemokinesis and chemotaxis assays with an EC50 value of 1.30 +/- 0.14 and 18.14 +/- 1.57 nM, respectively. LTB4 caused a dose-dependent increase in the production of superoxide anion with an apparent EC50 value of 50 X 10(-9) M in our experimental conditions. LTB4 also induced a dose-dependent increase in the generation of TxA2 with an EC50 value of 46.2 X 10(-9) M. Taken together, our results demonstrated that guinea pig alveolar eosinophils express two classes of specific receptors for LTB4. The high-affinity binding sites seem associated to chemokinesis and chemotaxis whereas the low-affinity binding sites seem associated to superoxide anion production and generation of TxA2. The existence of LTB4 receptors in eosinophils could explain the presence of these cells in hypersensitivity reactions.     

Identification and characterization of specific receptors for monocyte- derived neutrophil chemotactic factor (MDNCF) on human neutrophils

Specific receptors for a recently purified and cloned monocyte-derived neutrophil chemotactic factor (MDNCF) have been identified on the surface of normal human peripheral blood neutrophils using 125I-labeled recombinant human MDNCF (125I-MDNCF). Competitive binding of 125I-MDNCF to human neutrophils reached a maximal level at 1-3 h at 4 degrees C. The Scatchard analysis showed that there are approximately 20,000 receptors per cell with a single type of high affinity binding (Kd, 8 x 10(-10) M). The receptors for MDNCF are clearly distinct from the receptors for other cytokines and chemotactic agents, e.g., IL-1 alpha, TNF-alpha, and FMLP, C5a, leukotriene B4, and platelet activating factor. Based on the SDS-PAGE analysis of chemically crosslinked 125I-MDNCF receptor complex, there are two polypeptides that bind MDNCF; the molecular weight of these two MDNCF receptors were estimated to be 67,000 and 59,000. Treatment of a promyelocytic cell line, HL60, with 1.25% DMSO for 5 d in vitro increased the number of receptors up to 7,000 receptors/cell with a Kd of 1.2 x 10(-9) M.     

Adenosine: a physiological modulator of superoxide anion generation by human neutrophils

The effects of adenosine were studied on human neutrophils with respect to their generation of superoxide anion, degranulation, and aggregation in response to soluble stimuli. Adenosine markedly inhibited superoxide anion generation by neutrophils stimulated with N-formyl methionyl leucyl phenylalanine (FMLP), concanavalin A (Con A), calcium ionophore A23187, and zymosan-treated serum; it inhibited this response to PMA to a far lesser extent. The effects of adenosine were evident at concentrations ranging from 1 to 1,000 microM with maximal inhibition at 100 microM. Cellular uptake of adenosine was not required for adenosine-induced inhibition since inhibition was maintained despite the addition of dipyridamole, which blocks nucleoside uptake. Nor was metabolism of adenosine required, since both deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl) adenine did not interfere with adenosine inhibition of superoxide anion generation. The finding that 2-chloroadenosine, which is not metabolized, resembled adenosine in its ability to inhibit superoxide anion generation added further evidence that adenosine metabolism was not required for inhibition of superoxide anion generation by neutrophils. Unexpectedly, endogenously generated adenosine was present in supernatants of neutrophil suspensions at 0.14-0.28 microM. Removal of endogenous adenosine by incubation of neutrophils with exogenous adenosine deaminase (ADA) led to marked enhancement of superoxide anion generation in response to FMLP. Inactivation of ADA with DCF abrogated the enhancement of superoxide anion generation. Thus, the enhancement was not due to a nonspecific effect of added protein. Nor was the enhancement due to the generation of hypoxanthine or inosine by deamination of adenosine, since addition of these compounds did not affect neutrophil function. Adenosine did not significantly affect either aggregation or lysozyme release and only modestly affected beta-glucuronidase release by neutrophils stimulated with FMLP. These data indicate that adenosine (at concentrations that are present in plasma) acting via cell surface receptors is a specific modulator of superoxide anion generation by neutrophils.     

Chemotaxin receptor cycling in fresh and stored granulocytes

The authors reported previously that stored granulocytes (PMN) had decreased receptor affinity (Kd) for and chemotaxis (CTX) to the chemotactic peptide F-Met-Leu-Phe (FMLP), but the evidence did not favor a significant role for altered FMLP receptor affinity in causing diminished CTX of stored PMN. Since recruitment and/or recycling of FMLP receptors is required for normal CTX, the hypothesis that stored PMN might have abnormal FMLP receptor cycling was tested. The effect of storage on the proportion of high- and low-affinity FMLP receptors was also investigated. Units of PMN were tested within 4 hours of collection and after 24 and 48 hours of storage at 22 degrees C, unagitated, in 150-ml transfer packs. In comparison to fresh PMN, there was no alteration in the Kd of the high-affinity FMLP receptor of PMN stored for 24 to 48 hours; however, the Kd of the low-affinity receptor increased (fresh PMN = 36 +/- 5 nM; 24 hours = 107 +/- 19; 48 hours = 121 +/- 22; p less than 0.01 for both 24 and 48 h versus fresh PMN). Likewise, while the number of high-affinity receptors increased (fresh PMN = 25,000 +/- 6,000 receptors/PMN; 24 hours = 95,000 +/- 21,000; 48 hours = 161,000 +/- 40,000; p less than 0.01 for both 24 and 48 hours versus fresh PMN). No abnormality was found in the ability of stored PMN to down-regulate FMLP receptors in the presence of ligand or to reexpress FMLP receptors after a 15-minute incubation in the absence of ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific inhibition of leukotriene B4-induced neutrophil activation by LY223982.

LY223982, (E)-5-(3-carboxybenzoyl)-2-((6-(4-methoxyphenyl)-5- hexenyl)oxy)benzenepropanoic acid, is a newly discovered potent inhibitor of specific binding of leukotriene B4 (LTB4) to its receptor on human neutrophils. This study demonstrated that the compound is also a specific antagonist of LTB4-induced neutrophil activation under both in vitro and in vivo conditions. LY223982 was found to be 189-fold more effective in displacing [3H]LTB4 than 35S-N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) from their corresponding receptors on human neutrophils. The concentration inhibiting 50% of response (IC50) for displacement of [3H]LTB4 (13.2 nM) was only 6.8-fold higher than the value for nonradioactive LTB4. The compound inhibited the aggregation of guinea pig neutrophils caused by LTB4 more strongly than FMLP or platelet-activating factor. The IC50 for inhibition of LTB4-induced responses (74 nM) was 93- and > 135-fold lower than the IC50 for inhibition of the corresponding FMLP and platelet-activating factor-induced effects. LY223982 was also a potent antagonist of the aggregation of human neutrophils by LTB4 (IC50, 100 nM). Chemotaxis of human neutrophils induced by LTB4 was only modestly inhibited by the compound (IC50 = 6 microM) but it had even less effect on cell movement caused by FMLP. LY223982 inhibited transient leukopenia induced in rabbits with LTB4 (ED50, 3 mg/kg) but not with FMLP. It had no agonist activity in any of the test systems. In summary, the results indicate that LY223982 is a potent specific antagonist of LTB4-induced neutrophil activation.     

Monocyte-chemotactic activity of defensins from human neutrophils.

We investigated the monocyte-chemotactic activity of fractionated extracts of human neutrophil granules. Monocyte-chemotactic activity was found predominantly in the defensin-containing fraction of the neutrophil granules. Purified preparations of each of the three human defensins (HNP-1, HNP-2, HNP-3) were then tested. HNP-1 demonstrated significant chemotactic activity for monocytes: Peak activity was seen at HNP-1 concentrations of 5 X 10(-9) M and was 49 +/- 20% (mean +/- SE, n = 9) of that elicited by 10(-8) M FMLP. HNP-2 (peak activity at 5 X 10(-9) M) was somewhat less active, yielding 19 +/- 10% (n = 11). HNP-3 failed to demonstrate chemotactic activity. Checkerboard analysis of monocyte response to HNP-1 and HNP-2 confirmed that their activity was chemotactic rather than chemokinetic. Neutrophils demonstrated a low level of response to defensins but this reaction was primarily chemokinetic. Defensins may play a role in the recruitment of monocytes by neutrophils into inflammatory sites.     

Respiratory burst enzyme in human neutrophils. Evidence for multiple mechanisms of activation.

Alteration of the surface of human neutrophils with the nonpenetrating, protein-inactivating agent p-diazobenzenesulfonic acid (DASA) was found to prevent activation of the respiratory burst by some stimuli, but not others. Production of superoxide anion (O2-) stimulated by concanavalin A or the chemotactic peptide formyl-methionyl-leucyl-phenylalanine FMLP was inhibited by DASA pretreatment, whereas O2- production stimulated by phorbol myristate acetate (PMA), sodium fluoride. or the ionophore A23187 was not inhibited by DASA. Pretreatment with DASA inhibited oxygen uptake stimulated by FMLP, but not oxygen uptake stimulated by PMA. DASA reproducibly inhibited activities of two known surface enzymes Mg++-ATPase and alkaline phosphatase, by 45-55% and 60-70%, respectively. The inhibition by DASA of O2- production did not appear to be caused by interference with binding of the affected stimuli, since pretreatment with DASA did not inhibit release of the lysosomal enzymes lysozyme and myeloperoxidase induced by concanavalin A or FMLP. Membrane-rich particulate fractions from neutrophils have been shown to contain NADPH-dependent oxidative activity that is presumably responsible for the phagocytosis-associated respiratory burst of intact cells. The PMA-activated enzyme was susceptible to inhibition of directly exposed to DASA in this particulate fraction. These findings suggest that more than one mechanism exists for activation of the respiratory burst oxidase in human neutrophils, and that the neutrophil possesses at least one oxidase that is not an ectoenzyme.     

Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase.

Nitric oxide provokes vasodilation and inhibits platelet aggregation. We examined the effect of nitric oxide on superoxide anion production by three sources: activated intact neutrophils, xanthine oxidase/hypoxanthine, and the NADPH oxidase. Nitric oxide significantly inhibited the generation of superoxide anion by neutrophils exposed to either FMLP (10(-7)M) or PMA (150 ng/ml) (IC50 = 30 microM). To determine whether the effect of nitric oxide on the respiratory burst was due to simple scavenging of O2+, kinetic studies that compared effects on neutrophils and the cell-free xanthine oxidase system were performed. Nitric oxide inhibited O2+ produced by xanthine oxidase only when added simultaneously with substrate, consistent with the short half-life of NO in oxygenated solution. In contrast, the addition of nitric oxide to neutrophils 20 min before FMLP resulted in the inhibition of O2+ production, which suggests formation of a stable intermediate. The effect of nitric oxide on the cell-free NADPH oxidase superoxide-generating system was also examined: The addition of NO before arachidonate activation (t = -6 min) significantly inhibited superoxide anion production. Nitric oxide did not inhibit O2+ when added at NADPH initiation (t = 0). Treatment of the membrane but not cytosolic component of the oxidase was sufficient to inhibit O2+ generation. The data suggest that nitric oxide inhibits neutrophil O2+ production via direct effects on membrane components of the NADPH oxidase. This action must occur before the assembly of the activated complex.     

Human fibroblasts maintain the viability and augment the functional response of human neutrophils in culture.

When human neutrophils were co-cultured for 72 h with nontransformed human fibroblasts, 69 +/- 3% (n = 13) survived, as compared with survival levels of 2 +/- 1% (n = 15) and 26 +/- 6% (n = 7), respectively, for neutrophils cultured for the same time period in enriched medium alone or supplemented with 10 pM recombinant human granulocyte/macrophage colony-stimulating factor (rh GM-CSF). Conditioned medium from the human fibroblast cultures enhanced neutrophil survival in a dose-dependent fashion to the same level achieved with neutrophil/fibroblast co-cultures, and its soluble viability-sustaining activity was not inhibited by preincubation with neutralizing antiserum against rh GM-CSF. As compared with freshly isolated replicate samples, neutrophils co-cultured with human fibroblasts for 72 h exhibited augmented FMLP-stimulated superoxide production without spontaneous superoxide generation. This striking extension of survival and associated priming for a ligand response by neutrophils co-cultured with human fibroblasts suggests that fibroblasts may contribute to the proinflammatory properties of neutrophils in tissues.     

Botulinum C2 toxin ADP-ribosylates actin and enhances O2- production and secretion but inhibits migration of activated human neutrophils.

The binary botulinum C2 toxin ADP-ribosylated the actin of human neutrophils. Treatment of human neutrophils with botulinum C2 toxin for 45 min increased FMLP-stimulated superoxide anion (O2-) production 1.5-5-fold, whereas only a minor fraction of the cellular actin pool (approximately 20%) was ADP-ribosylated. Effects of botulinum C2 toxin depended on toxin concentrations, presence of both components of the toxin, and incubation time. Cytochalasin B similarly enhanced O2- production. The effects of botulinum C2 toxin and cytochalasin B were additive at submaximally, but not maximally effective concentrations and incubation time of either toxin. Botulinum C2 toxin also enhanced stimulation of O2- production by Con A and platelet-activating factor, but not by phorbol 12-myristate 13-acetate (PMA). Botulinum C2 toxin increased FMLP-induced release of N-acetyl-glucosaminidase by 100-250%; release of vitamin B12-binding protein induced by FMLP and PMA was enhanced by approximately 150 and 50%, respectively. Botulinum C2 toxin blocked both random migration of neutrophils and migration induced by FMLP, complement C5a, leukotriene B4, and a novel monocyte-derived chemotactic agent. The data suggest that botulinum C2 toxin-catalyzed ADP-ribosylation of a minor actin pool has a pronounced effect on the activation of human neutrophils by various stimulants.     

Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival.

A purified recombinant human granulocyte-macrophage colony stimulating factor (rH GM-CSF) was a powerful stimulator of mature human eosinophils and neutrophils. The purified rH GM-CSF enhanced the cytotoxic activity of neutrophils and eosinophils against antibody-coated targets, stimulated phagocytosis of serum-opsonized yeast by both cell types in a dose-dependent manner, and stimulated neutrophil-mediated iodination in the presence of zymosan. In addition, rH GM-CSF enhanced N-formylmethionylleucylphenylalanine(FMLP)-stimulated degranulation of Cytochalasin B pretreated neutrophils and FMLP-stimulated superoxide production. In contrast, rH GM-CSF did not promote adherence of granulocytes to endothelial cells or plastic surfaces. rH GM-CSF selectively enhanced the surface expression of granulocyte functional antigens 1 and 2, and the Mo1 antigen. rH GM-CSF induced morphological changes and enhanced the survival of both neutrophils and eosinophils by 6 and 9 h, respectively. These experiments show that granulocyte-macrophage colony stimulating factor can selectively stimulate mature granulocyte function.     

Characterization of an 125I-labeled thromboxane A2/prostaglandin H2 receptor agonist

Stable synthetic mimetics of thromboxane (TX) A2 and prostaglandin (PG) H2 have been synthesized and reported to stimulate platelets and vascular smooth muscle. The synthetic agonists induce aggregation of isolated platelets and contraction of vascular tissue. The tritiated agonists [3H]U46619 and [3H]U44069 have been used in radioligand binding studies to characterize platelet and vascular smooth muscle TXA2/PGH2 receptors, but have limited usefulness due to their low specific activities and variable specific binding. In an attempt to overcome these problems, we have synthesized a stable, high affinity, 125I-radiolabeled TXA2/PGH2 receptor agonist, [1S-(1 alpha, 2 beta (5Z), 3 alpha(1E,3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo - [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP). I-BOP induced shape change, increased intracellular free calcium concentrations and aggregated isolated human platelets (EC50 = 0.21 +/- 0.05 nM, n = 3; 4.1 +/- 1.1 nM, n = 4; 10.8 +/- 3 nM, n = 9, respectively). The kinetically determined Kd was 1.02 +/- 0.33 nM (kobs = 0.19 +/- 0.05 min-1, k-1 = 0.097 +/- 0.02 min-1, k1 = 0.119 +/- 0.03 min-1 M, n = 4). Equilibrium binding studies of [125I]BOP to isolated human platelets indicated one class of high affinity sites, Kd = 2.2 +/- 0.3 nM and a maximum binding of 0.028 +/- 0.002 x 10(-12) mol/10(7) platelets (1699 +/- 162 sites/platelet, n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Monophosphoryl lipid A inhibits neutrophil priming by lipopolysaccharide

It is known that lipopolysaccharides (endotoxin) prime neutrophils for oxygen radical production. Monophosphoryl lipid A is a nontoxic derivative of lipid A that protects against lethal endotoxemia. We examined the effects of Salmonella minnesota monophosphoryl lipid A on S. minnesota lipopolysaccharide-induced priming of neutrophil superoxide anion generation. Human neutrophils were preincubated with and without either lipopolysaccharide or monophosphoryl lipid A before stimulation with 10(-5) formyl-norleucyl-leucyl-phenylalanine. Neutrophil priming reached a plateau at a concentration of 100 ng/ml of lipopolysaccharide, where superoxide anion generation increased from 10.1 +/- 0.8 to 25.2 +/- 1.7 nmol superoxide anions/10(6) neutrophils/10 min (p less than 0.01). In contrast, monophosphoryl lipid A did not exhibit any priming activity. Monophosphoryl lipid A also exhibited a time-dependent inhibitory effect on lipopolysaccharide-induced priming of neutrophils, which was maximal when monophosphoryl lipid A was added 15 minutes before lipopolysaccharide. Preincubation with monophosphoryl lipid A induced a dose-dependent inhibition of neutrophil priming by 1000 ng/ml lipopolysaccharide. Neutrophil superoxide anion generation decreased by 47% from 19.0 +/- 0.6 to 10.0 +/- 0.7 nmol superoxide anions/10(6) neutrophils/10 min by 2000 ng/ml monophosphoryl lipid A (p less than 0.01). These data indicate that monophosphoryl lipid A does not enhance neutrophil superoxide generation in response to formyl-norleucyl-leucyl-phenylalanine. Monophosphoryl lipid A also inhibits lipopolysaccharide-induced priming in a dose-dependent manner that may reflect blocking of lipopolysaccharide by monophosphoryl lipid A at cellular binding sites.     

Comparison of antagonist and agonist binding to the leukotriene B4 receptor intact human polymorphonuclear neutrophils (PMN).

In the present studies, the pharmacology of the leukotriene B4 (LTB4) receptor on intact human polymorphonuclear neutrophils (PMN) was characterized using radioligand binding techniques with [3H]LTB4 and a novel LTB4 receptor antagonist radioligand [3H]CGS 23131 (LY223982). Saturation studies revealed that [3H]CGS 23131 labeled a single class of recognition sites with high affinity (Kd = 13 nM) and limited capacity (apparent Bmax = 2.8 pmol/10(7) cells). In contrast, [3H]LTB4 labeled both a set of high (Kd = 0.3 nM) and lower affinity (Kd = 5 nM) recognition sites. However, the apparent density of [3H]LTB4 binding to intact human PMN (combined Bmax = 380 fmol/10(7) cells) was approximately 14% of that observed with [3H]CGS 23131. In ligand competition studies, various LTB4 agonists and antagonists were found to inhibit the binding of [3H]CGS 23131, revealing a pharmacological profile consistent with the specific labeling of the LTB4 receptor. A positive rank order correlation (r = 0.79) was observed between the ligand competition profiles obtained with [3H]CGS 23131 and [3H]LTB4. Both LTB4 and its omega oxidation product, 20-OH-LTB4, were found to inhibit the binding of 1.0 nM [3H]CGS 23131 in a biphasic fashion consistent with the existence of multiple affinity components of the LTB4 receptor. In competing for 0.5 nM [3H]LTB4 binding, these compounds were found to produce monophasic inhibition curves, which was indicative of a selective interaction at the high-affinity LTB4 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactoferrin enhances hydroxyl radical production by human neutrophils, neutrophil particulate fractions, and an enzymatic generating system.

During phagocytosis, neutrophils take oxygen from the surrounding medium and convert it to superoxide anion (O2-) and hydrogen peroxide (H2O2). Hydroxyl radical (.OH), a particularly potent oxidant, is believed to be produced by interaction between O2- and H2O2 in the presence of iron, according to the Haber-Weiss reactions. Production of .OH by whole human neutrophils, by particulate fractions from human neutrophils disrupted after stimulation, and by a xanthine oxidase system was measured by conversion of alpha-keto-gamma-methiol butyric acid to ethylene. FeCl3 or ferric EDTA enhanced ethylene production in all three systems by 155--406% of base line at a concentration of 50--100 microM. Iron-saturated human milk lactoferrin, 100 nM, increased ethylene generation by 127--296%; and purified human neutrophil lactoferrin, 10 nM, enhanced ethylene production by 167--369%. Thus, iron bound to lactoferrin was approximately 5,000 times more effective in producing an enhancement in ethylene generation than iron derived from FeCl3 or ferric EDTA. O2- and H2O2 were required for ethylene production in the presence of lactoferrin, since superoxide dismutase inhibited ethylene formation in the three systems by 76--97% and catalase inhibited by 76--98%. Ethylene production in the presence of lactoferrin was inhibited by the .OH scavengers mannitol, benzoate, and thiourea by 43--85, 45--94, and 76--96%, respectively. Thus, most of the ethylene production could be attributed to oxidation of alpha-keto-gamma-methiol butyric acid by .OH. The ability of neutrophil lactoferrin to provide iron efficiently to the oxygen radical-generating systems is compatible with a role for lactoferrin as regulator of .OH production. As such, lactoferrin may be an important component in the microbicidal activity of neutrophils.     

Receptor-mediated regulation of superoxide production in human neutrophils stimulated by phorbol myristate acetate.

Human neutrophils contain receptors for phorbol myristate acetate (PMA), a complex lipid that induces them to generate superoxide (O (2)). Binding of PMA to these receptors displays specificity, reversibility, and high affinity. The receptor's apparent KD was approximately 0.29 nM and multiple copies (approximately 2.1 +/- 0.6 x 10(5)) were present per neutrophil. We found that the timing and magnitude of the neutrophil's respiratory burst were set independently. The onset of O (2) production occurred after a lag that was inversely proportional to the initial concentration of added PMA. The extent (rate) of O (2) production was directly proportional to the fractional occupancy of the receptor by PMA. Dual regulatory controls, such as those we noted when neutrophils were stimulated by PMA, could afford metabolic stability in the face of transient or low intensity stimuli without compromising quick and powerful responses to larger disturbances.     

Identification and characterization of presynaptic and postsynaptic beta adrenoreceptors in the longitudinal smooth muscle/myenteric plexus of dog ileum

The subcellular distribution of [125I]cyanopindolol (125I-CYP) binding in membrane fractions derived from the longitudinal smooth muscle/myenteric plexus (LSM/MP) of dog ileum followed a bimodal pattern with selective enrichments in the purified synaptosomal (S2) fraction and the purified smooth muscle plasma membrane (M2) fraction. The half-maximal saturation of binding sites in both fractions occurred at 10 pM [125I]CYP with maximal binding capacities, in femtomoles per milligram of protein, 70 +/- 10 (S2 fraction) and 110 +/- 25 (M2 fraction). Approximately 80% of binding sites in either membrane fraction exhibited a high affinity for beta-2 selective antagonist ICI 118,551 (Ki = 1.4 +/- 0.4 nM in S2 and 1.8 +/- 0.6 nM in M2; mean +/- S.D. n = 3), whereas remaining 20% of binding sites had a low affinity for this agent (Ki = 2.6 +/- 1.5 microM in S2 and 0.55 +/- 0.2 microM in M2). The beta-1-selective antagonist ICI 89,406 had a high affinity for approximately 20% of binding sites (Ki = 12 +/- 7 nM in S2 and 4 +/- 2 nM in M2) and a low affinity for approximately 80% of binding sites (Ki = 0.34 +/- 0.16 microM in S2 and 0.42 +/- 0.24 microM in M2). The displacement potencies of beta-adrenergic agonists followed the order (-)-isoproterenol greater than salbutamol (-)-epinephrine greater than (-)-norepinephrine in each membrane preparation. The inclusion of 0.1 mM 5'-guanylylimidodiphosphate into assay medium resulted in a decreased affinity of receptors for agonists and increased values of Hill coefficients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucagon-like peptide-1 stimulates luteinizing hormone-releasing hormone secretion in a rodent hypothalamic neuronal cell line.

To examine the influence of the putative satiety factor (GLP-1) on the hypothalamo-pituitary-gonadal axis, we used GT1-7 cells as a model of neuronal luteinizing hormone- releasing hormone (LHRH) release. GLP-1 caused a concentration-dependent increase in LHRH release from GT1-7 cells. Specific, saturable GLP-1 binding sites were demonstrated on these cells. The binding of [125I]GLP-1 was time-dependent and consistent with a single binding site (Kd = 0.07+/-0.016 nM; binding capacity = 160+/-11 fmol/mg protein). The specific GLP-1 receptor agonists, exendin-3 and exendin-4, also showed high affinity (Ki = 0.3+/-0.05 and 0.32+/-0.06 nM, respectively) as did the antagonist exendin-(9-39) (Ki = 0.98+/-0.24 nM). At concentrations that increased LHRH release, GLP-1 (0.5-10 nM) also caused an increase in intracellular cAMP in GT1-7 cells (10 nM GLP-1: 7.66+/-0.4 vs. control: 0.23+/-0.02 nmol/mg protein; P < 0.001). Intracerebroventricular injection of GLP-1 at a single concentration (10 microg) produced a prompt increase in the plasma luteinizing hormone concentration in male rats (GLP-1: 1.09+/-0.11 vs. saline: 0.69+/-0.06 ng/ml; P < 0.005). GLP-1 levels in the hypothalami of 48-h-fasted male rats showed a decrease, indicating a possible association of the satiety factor with the low luteinizing hormone levels in animals with a negative energy balance.