Phe-D-Leu-Phe-D-Leu-Phe derivatives as formylpeptide receptor antagonists in human neutrophils: cellular and conformational aspects.

We synthesized several Phe-d-Leu-Phe-d-Leu-Phe analogues in which tert-butyloxycarbonyl and four different ureido substituents were included at the N-terminal of the peptides, obtained as free acid and methyl-ester derivatives. Their biological action was analysed on human neutrophil responses induced by N-formyl-Met-Leu-Phe (fMLF). Several in vitro assays were carried out: receptor binding, measurement of Ca2+ intracellular concentration, chemotaxis, superoxide anion production and enzyme release. A conformational investigation, using infrared absorption and circular dichroism, was also performed. Our results demonstrate that the compounds examined prefer an ordered conformation (beta-turn) in amphipathic environment, and are able to antagonize the neutrophil functions evoked by fMLF. Moreover, the extent of inhibition of Ca2+ intracellular enhancement, as well as of superoxide anion production and granule enzyme release, appears related to their affinity toward the formylpeptide receptor. The free acid peptide derivatives appear to be more active antagonists than the methyl-ester ones.     

Histamine modulation of human neutrophil oxidative metabolism, locomotion, degranulation, and membrane potential changes

Previous reports have suggested that histamine modulates neutrophil chemotaxis, but this has not been observed by all laboratories. We have re-addressed this controversial point and demonstrate that histamine and H1- and H2-receptor-specific agonists cause limited inhibition of chemotaxis while stimulating chemokinesis. Furthermore, using the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) as a stimulus, we demonstrate that histamine and H1/H2 agonists inhibit f-met-leu-phe-stimulated changes in membrane potential (monitored with the cyanine dye dipentyloxacarbocyanine), superoxide anion production (cytochrome c reduction), hydrogen peroxide formation (scopoletin fluorescence), and degranulation of granule contents (lysozyme and beta-glucuronidase) in a dose-dependent manner but have no effect on neutrophil functions stimulated by the secretagogues phorbol myristate acetate or A23187. All inhibitory effects of histamine and the H1/H2 agonists are reversed in a competitive manner by the H2 antagonist cimetidine. In addition, structure-activity studies using H1 and H2 receptor agonists and antagonists indicate that a single site with specificity for both H1 and H2 analogue structures modulates the various f-met-leu-phe-stimulated functions studied. Kinetic studies demonstrate that the inhibitory effects of histamine on neutrophil function are only observed when histamine is added before f-met-leu-phe and that inhibition occurs within 10 to 20 sec of histamine addition, does not persist after its removal, and is reversed by addition of cimetidine 10 to 20 sec before stimulation with f-met-leu-phe. Although the inhibitory effects of histamine are exerted early in the sequence of PMN activation by f-met-leu-phe, histamine does not affect the binding or internalization of f-met-leu-[3H]phe. The ability of histamine to modify the variety of neutrophil responses demonstrated in this report suggests an important and direct role for histamine in the regulation of inflammatory reactions in acute allergic settings or other disease states in which histamine release may occur.     

5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxy-4H-chromen-4-one (MSF-2) suppresses fMLP-mediated respiratory burst in human neutrophils by inhibiting phosphatidylinositol 3-kinase activity

Respiratory burst mediates crucial bactericidal mechanism in neutrophils. However, undesirable respiratory burst leads to pathological inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxy-4H-chromen-4-one (MSF-2), a lignan extracted from the fruit of Melicope Semecarprifolia, on fMLP-induced respiratory burst in human neutrophils and suggests a possible therapeutic approach to ameliorate disease associated with neutrophil hyperactivation. MSF-2 inhibited fMLP-induced neutrophil superoxide anion production, cathepsin G release and migration in human neutrophils isolated from healthy volunteers, reflecting inhibition of phosphatidylinositol 3-kinase (PI3K) activation. Specifically, PI3K/AKT activation results in migration, degranulation and superoxide anion production in neutrophils. MSF-2 suppresses PI3K activation and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production, and consequently inhibits downstream activation of PDK1 and AKT. Further, PI3K also stimulates respiratory burst via PLC-dependent elevation of intracellular calcium. MSF-2 reduces fMLP-mediated PLCγ2 activation and intracellular calcium accumulation notably through extracellular calcium influx in a PI3K and PLC-dependent manner. However, MSF-2 is not a competitive or allosteric antagonist of fMLP. Additionally, in an in vivo study, MSF-2 prevents fMLP-induced neutrophil infiltration and inflammation in mice. In conclusion, MSF-2 opposes fMLP-mediated neutrophil activation and inflammation by inhibiting PI3K activation and subsequent activation of AKT and PLCγ2.     

Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity

A novel monocyte-derived neutrophil-activating peptide (MONAP) produced by lipopolysaccharide- and phorbol myristate acetate-stimulated human peripheral blood monocytes was purified by sequential ion exchange-high performance liquid chromatography (HPLC), size exclusion HPLC, and reversed phase HPLC. Biologic activities of the purified cytokine were monitored by either an enzyme release assay or a chemotaxis assay, using peripheral human neutrophils. Purified MONAP was found to be homogeneous, giving a single peak on size-exclusion HPLC, reversed-phase HPLC, as well as a single 10-kDa band on silver-stained polyacrylamide gels. Purified MONAP stimulate human neutrophil chemotaxis at an estimated molarity of 5 x 10(-11) M. Half-maximal enzyme release of cytochalasin B pretreated neutrophils occurred at 2 to 3 x 10(-10) M, whereas superoxide anion production elicited by various concentrations of MONAP was found to be low. Isolated human peripheral monocytes, as well as human eosinophils, showed no chemotactic response to MONAP, indicating neutrophil specificity. MONAP activity was separated from thymocyte-stimulating activity by reversed-phase HPLC, indicating nonidentity with interleukin (IL)-1. This was further supported by heat resistance of MONAP, which is in contrast to the heat sensitivity of IL-1. In addition, IL-1 obtained as a by-product during isolation of MONAP did not stimulate human neutrophil chemotaxis.     

Biologic activity of synthetic analogues of C5a anaphylatoxin.

The C activation fragment C5a is the most potent plasma-derived chemotactic factor known. This humoral factor induces both neutrophil and macrophage activation at low nanomolar concentrations. We have synthesized a series of C-terminal C5a analogues that exhibit all of the characteristic biologic activities of C5a. These peptides apparently contain the effector site for C5a receptor-mediated cellular activation, but express only a fraction of the potency of intact C5a. We have demonstrated the following in vitro activities for these C5a peptides: 1) ileal (guinea pig) contraction; 2) platelet (guinea pig) activation; and 3) neutrophil (human) polarization and chemotaxis. The effect of C5a peptides in vivo was evaluated by measuring enhancement in vascular permeability. Although potencies of the most effective synthetic C5a analogues were on the order of 0.01 to 0.1% that of the natural factor, our biologic data confirm that the C5a peptides are full agonists of the intact factor and may be useful substitutes for intact C5a. Furthermore, our results indicate that elongation of the C5a analogues from 10 to 19 residues in length contributes little toward enhancing or decreasing potency of the synthetic C5a analogues. Replacement of residues in the effector region by D-amino acids or by introduction of a cyclic group to reduce flexibility of the backbone decreased potency of the analogues. Substitution of His 67 by Phe in the decapeptide C5a 65-74 resulted in a significant increase in potency of the C5a analogue. The marked enhancement in potency from replacing His 67 by Phe in analogue C5a peptides identifies an important hydrophobic subsite. We conclude that site-specific amino acid modifications in or near the C-terminal effector site sequence can diminish or optimize potency of the model C5a peptides. However, there apparently are subsites on folded C5a, from regions other than the C-terminal portion of the molecule, that contribute significant receptor interactions. These subsites must be identified and incorporated into C5a model peptide designs before expression of full potency by synthetic analogues of this factor will be realized.     

Nociceptin/orphanin FQ stimulates human monocyte chemotaxis via NOP receptor activation

Nociceptin/orphanin FQ (N/OFQ) produces several biological actions by activating the N/OFQ peptide receptor (NOP). It has been previously shown that N/OFQ stimulates leukocyte chemotaxis both in vitro and in vivo. In the present study we investigated the ability of N/OFQ, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP), to stimulate human neutrophil and monocyte chemotaxis and the release of lysozyme and superoxide anion (O2-) production from neutrophils. fMLP stimulated all the leukocyte functions examined. N/OFQ stimulated monocyte (pEC50 12.15) but not neutrophil chemotaxis. The production of O2- from neutrophils was not affected by N/OFQ while the release of lysozyme was increased in a concentration dependent manner (pEC50 11.00) although the maximal effects evoked by N/OFQ were about half of those of fMLP. The NOP ligands [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, Ro 64-6198, UFP-101 and the opioid antagonist naloxone were used for pharmacologically characterizing the receptor involved in the monocyte chemoattractant action of N/OFQ. [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, and Ro 64-6198 mimicked the action of N/OFQ showing similar maximal effects and the following order of potency: [Arg14, Lys15]N/OFQ (pEC50 13.22)>Ro 64-6198 (pEC50 12.96)>N/OFQ(1-13)NH2 (pEC50 12.67)>N/OFQ (pEC50 12.15). Moreover, the monocyte chemoattractant action of N/OFQ was not modified by naloxone 1 microM while antagonized by UFP-101 10 microM (pA2 7.00). Thus, the order of potency of agonists and the antagonist selectivity demonstrated that N/OFQ stimulates human monocyte chemotaxis via NOP receptor activation.     

Effects of systemic glucocorticosteroids on peripheral neutrophil functions in asthmatic subjects: an ex vivo study

In 21 asthmatic subjects, several functions of isolated peripheral neutrophils (chemokinesis and chemotaxis toward 10% E. coli; superoxide anion generation after PMA; leukotriene B(4) (LTB(4)) release from whole blood and isolated neutrophtls, before and after different stimuli) were evaluated during an acute exacerbation of asthma, and after 14 - 54 days of treatment with systemic glucocorticosteroids (GCS). During acute exacerbation, superoxide anion generation was higher in asthmatics than in eleven normal subjects (39.2 +/- 14.1 vs. 25.2 +/- 7.3 nmol, p < 0.05); there was a significant correlation between FEV(1) (% of predicted) and neutrophil chemotaxis (r = -0.52, p = 0.04). After treatment, there was no significant change in all neutrophil functions, except for a decrease in neutrophil chemotaxis in subjects who showed an FEV(1) increase > 20% after GCS treatment (from 131 +/- 18 to 117 +/- 21 mum, p = 0.005). Chemokinesis sicantly decreased in all subjects, and the changes significantly correlated with an arbitrary score of the total administered dose of GCS (r = 0.57, p < 0.05). These data suggest that neutrophil activation plays a minor role in asthma, and that treatment with GCS is not able to modify most functions of peripheral neutrophils in asthmatic subjects; chemotaxis seems to be related only to the severity of the asthma and it could reflect the improvement of the disease.     

Isopeptide bonds in chemotactic tripeptides. Synthesis and activity of lysine-containing fMLF analogs.

In order to explore the properties of chemotactic N-formylpeptides containing isopeptide bonds within their backbones, a group of lysine-containing analogs of the prototypical chemotactic tripeptide N-formylmethionyl-leucyl-phenylalanine (fMLF) was synthesized. The new analogs were designed by adding to the HCO-Met or Boc-Met residue a dipeptide fragment made up of Lys and Phe residues joined through Lys N alpha or N epsilon bonds, in all possible combinations. Thus, the following six pairs of tripeptides were synthesized and examined for their bioactivity: RCO-Met-Lys(Z)-Phe-OMe (2a, b), RCO-Met-Lys(Z-Phe)-OMe (3a, b), Z-Lys(RCO-Met)-Phe-OMe (4a, b), Z-Phe-Lys(RCO-Met)-OMe (5a, b), RCO-Met-Phe-Lys(Z)-OMe (6a, b) and Z-Lys(RCO-Met-Phe)-OMe (7a, b), with R=OC(CH3)(3 )and R=H for compounds a and b, respectively. All the new models were characterized fully and their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils determined as agonists (compounds b) and antagonists (compounds a). All N-formyl derivatives 2b-7b are less potent than fMLF-OMe as chemoattractants, but compound 7b exhibits selective activity as superoxide anion producer. Derivatives 2a-7a do not show antagonistic activity towards fMLF induced chemotaxis and O(2)(-) production, however, all these compounds except 4a antagonize lysozyme release by 60%.     

Tumor necrosis factor as an activator of human granulocytes. Potentiation of the metabolisms triggered by the Ca2+-mobilizing agonists

TNF stimulated superoxide (O2-) release directly in human granulocytes in a dose-dependent manner (1 to 1000 U/ml), although its potency was weak. TNF-induced O2- release was inhibited by cAMP agonists or ionomycin, and was not accompanied with an increase in cytoplasmic free Ca2+ [( Ca2+]i) and membrane potential changes (depolarization). These findings indicate that neither Ca2+ mobilization nor membrane depolarization is required for TNF-receptor-mediated cell activation. The pretreatment of human granulocytes with TNF enhanced O2- release and membrane depolarization in parallel stimulated by the receptor-mediated Ca2+-mobilizing agonists (FMLP, Con A, and wheat germ agglutinin) or the Ca2+ ionophore ionomycin, but not by PMA, a direct activator of protein kinase C. The optimal effect was obtained by pretreatment of cells with 100 U/ml TNF for 5 to 10 min at 37 degrees C, although the magnitude of enhancement varied according to the agonists used as subsequent stimuli. TNF did not affect an increase in [Ca2+]i stimulated by the Ca2+-mobilizing agonists, except Con A. Con A-induced increase in [Ca2+]i was enhanced by TNF in a dose-dependent manner. These diverse effects of TNF could be partly explained by the exclusive potentiation by TNF of the metabolic events triggered by an increase in [Ca2+]i.     

Chemoattractants and chemorepellents act by inducing opposite polarity in phospholipase C and PI3-kinase signaling

During embryonic development, cell movement is orchestrated by a multitude of attractants and repellents. Chemoattractants applied as a gradient, such as cAMP with Dictyostelium discoideum or fMLP with neutrophils, induce the activation of phospholipase C (PLC) and phosphoinositide 3 (PI3)-kinase at the front of the cell, leading to the localized depletion of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P(2)) and the accumulation of phosphatidylinositol-3,4,5-trisphosphate (PI[3,4,5]P(3)). Using D. discoideum, we show that chemorepellent cAMP analogues induce localized inhibition of PLC, thereby reversing the polarity of PI(4,5)P(2). This leads to the accumulation of PI(3,4,5)P(3) at the rear of the cell, and chemotaxis occurs away from the source. We conclude that a PLC polarity switch controls the response to attractants and repellents.     

GPCR-mediated PLCβγ/PKCβ/PKD signaling pathway regulates the cofilin phosphatase slingshot 2 in neutrophil chemotaxis

Chemotaxis requires precisely coordinated polymerization and depolymerization of the actin cytoskeleton at leading fronts of migrating cells. However, GPCR activation-controlled F-actin depolymerization remains largely elusive. Here, we reveal a novel signaling pathway, including Gαi, PLC, PKCβ, protein kinase D (PKD), and SSH2, in control of cofilin phosphorylation and actin cytoskeletal reorganization, which is essential for neutrophil chemotaxis. We show that PKD is essential for neutrophil chemotaxis and that GPCR-mediated PKD activation depends on PLC/PKC signaling. More importantly, we discover that GPCR activation recruits/activates PLCγ2 in a PI3K-dependent manner. We further verify that PKCβ specifically interacts with PKD1 and is required for chemotaxis. Finally, we identify slingshot 2 (SSH2), a phosphatase of cofilin (actin depolymerization factor), as a target of PKD1 that regulates cofilin phosphorylation and remodeling of the actin cytoskeleton during neutrophil chemotaxis.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or changing the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5'-(N-ethylcarboxamido)adenosine (NECA, 10 microM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 +/- 0.02 (S.E.) to 1.80 +/- 0.02 (p less than 0.001) while inosine (10 microM), a poor adenosine receptor agonist, had no effect (1.73 +/- 0.04, p = n.s. vs. control, p less than 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA greater than adenosine = N6-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 microM) did not significantly change the number or affinity of [3H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Phospholipase A2 modulates respiratory burst developed by neutrophils in patients with rheumatoid arthritis

Activated by bacterial peptides, phorbol esters, calcium ionophores and other agonists, neutrophils (PMNs) release the proinflammatory mediator, arachidonic acid (AA) via the intervention of phospholipase A(2) (PLA(2)). AA may play an essential role in activation of NADPH-oxidase, which is involved in the generation of superoxide anion by neutrophils. The present study is focused on the involvement of PLA(2) in the respiratory burst developed by PMNs isolated from patients with rheumatoid arthritis (RA). PLA(2) exists in very high levels in diseases such as rheumatoid arthritis and may cause acute inflammatory and proliferative changes in synovial structures. The respiratory burst was evaluated as superoxide anion release, using an amplified chemiluminescence method. The assays were performed using PMNs untreated or treated with different doses of stimulatory reagents (phorbol 12-myristate-13-acetate (PMA), calcium ionophore (A23187)). Our data suggested that PMA stimulated the production of superoxide anion in a dose-response manner, as compared with A23187, which did not induce a significant release of superoxide anion in PMNs-RA. The exogenous addition of AA significantly amplified the superoxide anion release by PMNs-RA stimulated with PMA and to a lesser extent, by PMNs stimulated with A23187. AA has also reversed the inhibitory effect of arachidonyl-trifluorometylketone and E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2H-pyran-2-one (BEL) on the superoxide anion release by PMNs-RA. In conclusion, the differential responses to these two agents suggested that different isoforms of PLA(2) were activated by A23187 or PMA, and support the idea that activation of these different PLA(2) served distinct functions of PMNs. Therefore, the inhibition of PLA(2) enzymes might be of great importance in the immunotherapy of rheumatoid arthritis.     

Enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor

Tumor necrosis factor (TNF) is a 17,000-Da protein which is produced by mononuclear cells upon exposure to endotoxin. Increases in adherence, phagocytosis, hydrogen peroxide release, and lysozyme secretion have been demonstrated after prolonged incubation of human neutrophils with TNF. In this study, the ability of highly purified recombinant human TNF to modulate neutrophil responses to soluble stimuli was evaluated. Tumor necrosis factor alone (0.1 to 10,000 units/ml) failed to induce neutrophil superoxide anion (O2-) production, granule release, or aggregation when incubated for up to 25 min at 37 degrees C. TNF did, however, stimulate a significant time-, dose-, and temperature-dependent increase in neutrophil F-actin content. Although exposure of neutrophils to TNF alone caused no superoxide anion production, it enhanced the O2- production in response to the chemotactic peptide, f-methionyl-leucyl-phenylalanine (FMLP) or the tumor promotor, phorbol myristate acetate, by as much as 278%. The enhancement was time-, dose-, and temperature-dependent and was due to a more rapid initial rate of O2- production. The TNF enhancement of FMLP-induced O2- production was blocked when an anti-TNF monoclonal antibody 241-1H11, is present during the preincubation period. TNF preincubation also enhanced FMLP-induced lysozyme release, but had no effect on aggregation and actin polymerization by FMLP. The kinetics of NADPH oxidase activation by arachidonic acid was unaltered by TNF. These results indicate that brief exposures to recombinant human TNF are able to enhance or prime the neutrophil oxidative burst in response to a second stimulus.     

The in vitro effects of histamine and metiamide on neutrophil motility and their relationship to intracellular cyclic nucleotide levels.

Histamine at concentrations of 1 x 10(-5) M to 5 x 10(-5) M consistently increased neutrophil movement as measured in Boyden chambers. This effect was entirely caused by stimulation of chemokinesis (stimulated random migration) and true chemotaxis was inhibited by these concentrations. This inhibition of chemotaxis could be abolished by pretreatment with metiamide, an H-2 receptor antagonist, and levamisole, but not by diphenylhydramine, an H-1 receptor antagonist. Metiamide at similar concentrations produced a mild stimulation of chemokinesis but has no effect on true chemotaxis. The histamine effects on neutrophil motility were associated with increased levels of intracellular cAMP wehreas cAMP levels were unaffected. Agents known to elevate intracellular cAMP levels produced effects on neutrophil motility similar to those of histamine. It is suggested that histamine exerts a 2-fold effect on neutrophil motility mediated via an H-2 receptor site and associated with elevated levels of cAMP.     

Cyclic AMP-elevating agents block chemoattractant activation of diradylglycerol generation by inhibiting phospholipase D activation.

Agents which elevate cellular cAMP (prostaglandin E2, theophylline, and forskolin) or mimic cAMP action (dibutyryl cAMP) are known to inhibit human neutrophil activation (superoxide generation and secretion) by receptor-linked agonists such as formyl-methionyl-leucyl-phenylalanine (fMLP). Herein, we show that these agents also markedly inhibit fMLP-stimulated diradylglycerol generation (assayed by mass methods). The magnitude of inhibition correlated with the ability of a given agent or combination of agents to elevate cAMP. Both 1,2-diacylglycerol and 1-O-alkyl,2-acyl glycerol generation were affected. Effects on the latter species, as well as a lack of effect on fMLP-stimulated inositol phosphate release, implied that cAMP affected diradylglycerol generation from a source other than phospholipase C-dependent phosphoinositide hydrolysis, since phosphatidylinositols do not contain appreciable quantities of the 1-O-alkyl linkage. In cells in which the phosphatidylcholine pool was prelabeled using 1-O-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine, prostaglandin E2 plus theophylline inhibited the fMLP-activated rapid generation of [3H]phosphatidic acid and its subsequent conversion to [3H]diradylglycerol, implying an effect at the level of phospholipase D. In the presence of ethanol, the fMLP-activated transphosphatidylation of [3H]phosphatidylcholine to generate [3H]phosphatidylethanol (a phospholipase D-dependent reaction) was also markedly inhibited. In contrast, when phorbol 12-myristate 13-acetate was used to activate cells, cAMP-related agents had no effect on phospholipase D activity, diradylglycerol generation, or superoxide generation. The data indicate an inhibitory effect of cyclic AMP on receptor-mediated phospholipase D activation at a site proximal to phospholipase D (e.g., the receptor or G protein). These studies provide a new example of "cross-talk" among signal transduction systems.     

Apical adenosine regulates basolateral Ca2+-activated potassium channels in human airway Calu-3 epithelial cells

In airway epithelial cells, apical adenosine regulates transepithelial anion secretion by activation of apical cystic fibrosis transmembrane conductance regulator (CFTR) via adenosine receptors and cAMP/PKA signaling. However, the potent stimulation of anion secretion by adenosine is not correlated with its modest intracellular cAMP elevation, and these uncorrelated efficacies have led to the speculation that additional signaling pathways may be involved. Here, we showed that mucosal adenosine-induced anion secretion, measured by short-circuit current (Isc), was inhibited by the PLC-specific inhibitor U-73122 in the human airway submucosal cell line Calu-3. In addition, the Isc was suppressed by BAPTA-AM (a Ca2+ chelator) and 2-aminoethoxydiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor blocker), but not by PKC inhibitors, suggesting the involvement of PKC-independent PLC/Ca2+ signaling. Ussing chamber and patch-clamp studies indicated that the adenosine-induced PLC/Ca2+ signaling stimulated basolateral Ca2+-activated potassium (KCa) channels predominantly via A2B adenosine receptors and contributed substantially to the anion secretion. Thus, our data suggest that apical adenosine activates contralateral K+ channels via PLC/Ca2+ and thereby increases the driving force for transepithelial anion secretion, synergizing with its modulation of ipsilateral CFTR via cAMP/PKA. Furthermore, the dual activation of CFTR and KCa channels by apical adenosine resulted in a mixed secretion of chloride and bicarbonate, which may alter the anion composition in the secretion induced by secretagogues that elicit extracellular ATP/adenosine release. Our findings provide novel mechanistic insights into the regulation of anion section by adenosine, a key player in the airway surface liquid homeostasis and mucociliary clearance.     

Studies on fMLP-receptor interaction and signal transduction pathway by means of fMLP-OMe selective analogues

For-Thp-Leu-Ain-OMe ([Thp(1), Ain(3)] fMLP-OMe) (2), for-Met-delta(z)Leu-Phe-OMe ([delta(z)Leu(2)] fMLP-OMe) (3), for-Thp-Leu-Phe-OMe ([Thp(1)] fMLP-OMe) (4), and for-Met-Leu-Ain-OMe ([Ain(3)] fMLP-OMe) (5) are for-Met-Leu-Phe-OMe (fMLP-OMe) (1) analogues which discriminate between different responses of human neutrophils. Peptides 3 and 5, similar to fMLP-OMe, enhance neutrophil cyclic AMP (cAMP) as well as calcium levels, while analogues 2 and 4, which evoke only chemotaxis, do not alter the concentration of these intracellular messengers. When we tested the peptides' ability to displace [3H]-fMLP from its binding sites, the following order of potency was observed: analogue 1 > 3 > 5 > 2 > 4. A particularly low activity at the receptor level characterized analogues 2 and 4. Their low effectiveness was not improved by the addition of cytochalasin B, by different incubation temperatures, or by the absence of endogenous guanine nucleotides, conditions known to influence fMLP receptor fate and functionality. We speculate that, in certain conditions, the fMLP receptor may undergo conformational changes that impede the binding of pure chemoattractants.     

Hybrid alpha/beta3-peptides with proteinogenic side chains. Monosubstituted analogues of the chemotactic tripeptide For-Met-Leu-Phe-OMe.

The alpha/beta3-mixed tripeptides R-CO-beta3-HMet-Leu-Phe-OMe (1a,b), R-CO-Met-beta3-HLeu-Phe-OMe (2a,b) and R-CO-Met-Leu-beta3-HPhe-OMe (3a,b) (a, R = tert-butyloxy-; b, R = H-), analogues of the potent chemoattractant For-Met-Leu-Phe-OMe, have been synthesized by classical solution methods and fully characterized. The activities of the new analogues as chemoattractants, superoxide anion producers and lysozyme releasers have been determined on human neutrophils. Whereas all of the three N-formyl derivatives are significantly less active than the parent tripeptide as chemoattractants, compound 1b has been found to be highly active as a superoxide anion producer and 3b as a lysozyme releaser. The results show that the replacement of the native Leu residue at the central position is, in each of the examined cases, the least favourable modification. The three N-Boc derivatives are, as expected, devoid of activity as agonists, but they are all good inhibitors of chemotaxis. Information on the solution conformation has been obtained by examining the involvement of the NH groups in intramolecular H-bonds using 1H NMR. The conformation of the N-Boc analogue 1a has also been determined in the crystal state by x-ray diffraction analysis. The molecule is extended at the beta3-HMet residue (phi1 = -87 degrees; theta1 = 172 degrees; psi1 = 126 degrees) and no intramolecular H-bond is present.     

Neutrophil stimulation: receptor, membrane, and metabolic events

In the neutrophil, binding of ligands to their appropriate receptors initiates a sequence of events culminating in the physiological responses of aggregation, degranulation, and superoxide anion generation. Calcium has been proposed as a second messenger in the activation sequence of the neutrophil. Increments in cytosolic free calcium are one of the first measurable events subsequent to receptor occupancy, followed by enhanced plasmalemmal permeability to calcium, a process that may serve to enhance the physiological responses. In contrast to calcium, cyclic AMP (cAMP) does not act as a signal in the activation sequence of the neutrophil. Increments in cAMP that are triggered by complete secretagogues may act as an inhibitory feedback mechanism. Protein kinases, both cAMP- and calcium/phospholipid-sensitive enzymes, may play a role in the activation sequence. Phosphorylation of proteins occurs during neutrophil activation. A role for phosphatidylinositol/phosphatidic acid turnover in calcium gating has been proposed. In addition, modulation of phospholipids could serve to activate a protein kinase C. Finally, phospholipids can serve as a source for arachidonic acid, which is metabolized by a 5-lipoxygenase pathway in the neutrophil. Products of this pathway, such as leukotriene B4, may serve to mediate or modulate the activation sequence.