Báo cáo Y học: Transglutaminase-mediated polyamination of vasoactive intestinal peptide (VIP) Gln16 residue modulates VIP/PACAP receptor activity pdf

Transglutaminase-mediated polyamination of vasoactive intestinal peptide (VIP) Gln16 residue modulates VIP/PACAP receptor activity Salvatore De Maria 1 , Salvatore Metafora 2 , Vittoria Metafora 2 , Francesco Morelli 2 , Patrick Robberecht 3 , Magalı ` Waelbroeck 3 , Paola Stiuso 4 , Alfredo De Rosa 1 , Anna Cozzolino 4 , Carla Esposito 4 , Angelo Facchiano 5 and Maria Cartenı ` 1 1 Department of Experimental Medicine and Centro di Ricerca Interdipartimentale di Scienze Computazionali e Biotecnologiche, II University of Naples, Italy; 2 CNR Institute of Genetics and Biophysics ÔAdriano Buzzati TraversoÕ, Naples, Italy; 3 Department of Biochemistry and Nutrition, Medical School of Medicine, Universite ´ Libre de Bruxelles, Bruxelles, Belgium; 4 Department of Chemistry, University of Salerno, Salerno, Italy; 5 Istituto di Scienze dell¢ Alimentazione, CNR, Avellino, Italy Previous data showing an increase of receptor binding activity of [R16]VIP, a vasoactive intestinal peptide (VIP) structural analogue containing arginine at the position 16 of its amino acid sequence, have pointed out the importance of a positive charge at this site. Here, the functional charac- terization of three VIP polyaminated adducts (VIP Dap , VIP Spd , and VIP Spm ), obtained by a transglutaminase- catalysed reaction between the VIP Gln16 residue and 1,3-diaminopropane (Dap), spermidine (Spd), or spermine (Spm), is reported. Appropriate binding assays and adeny- late cyclase enzymatic determinations have shown that these VIP adducts act as structural VIP agonists, both in vitro and in vivo. In particular, their IC 50 and EC 50 values of human and rat VIP/pituitary adenylate cyclase activating peptide (PACAP) 1 and VIP/PACAP 2 receptors indicate that VIP Dap is a VIP agonist, with an affinity and a potency higher than that of VIP, while VIP Spd and VIP Spm are also agonists but with affinities lower than that of VIP. These findings suggest that the difference in adduct agonist activity reflects the differences in the positive charge and carbon chain length of the polyamine covalently linked with the VIP Gln 16 residue. In addition, the data obtained strongly suggest that the length of polyamine carbon chain could be critical for the interaction of the agonist with its receptor, even though possible hydrophobic interaction cannot be ruled out. In vivo experiments on murine J774 macrophage cell cultures have shown the ability of these compounds to stimulate the inducible nitric oxide synthase activity at the transcriptional level. Keywords: NO/iNOS; polyamines; transglutaminase; VIP agonists; VIP receptors. Vasoactive intestinal polypeptide (VIP) is a 28-amino acid long peptide that serves the function of hormone, neuro- transmitter, and immuno-modulator in mammals and other vertebrates. It belongs to the important family of brain/gut hormones including secretin, glucagon, pituitary adenylate cyclase activating peptide (PACAP), etc. [1–3]. Although originally identified on the basis of its strong vasodilating activity, VIP exerts a wide spectrum of biological effects on a number of target organs mediated by its interaction with two distinct G-protein coupled receptors (VIP/PACAP 1 and VIP/PACAP 2 or VPAC 1 and VPAC 2 ), which transduce the ligand signal through the activation of different enzymatic effector systems, such as adenylate cyclase, phospholipase C, and inducible nitric oxide synthase (iNOS) [4–9]. While work is more advanced on the mechanism of ligand binding and activation of G-protein coupled recep- tors which use relatively small molecules as their ligands, fewer results are available in the case of peptide receptors which have ligands that are much larger and which exhibit greater conformational flexibility. The detailed mechanism of signal transduction mediated by the VIP receptor and the physiological role of the different VIP receptors are currently investigated. Furthermore, the only structural information available on VIP has been mainly obtained by CD and NMR analysis [10]. Recently, a conformational study explored the theoretically preferred conformation of VIP by combining experimental information with unre- strained molecular calculation. The results of these studies showed that (a): most VIP conformations, including the global minimum, can be described as bent conformation; (b) atype1bturn involves the residues of the VIP fragment P2–5 and a different type of b-turn involves the residues of the fragment P6–11; (c) the central portion (residues 7–15) and the C-terminus (residues 19–27) are in a helical confor- mation [11,12]. Little is known on the role played by the different VIP residues in the recognition and activation of natural receptors. Structural–activity studies, performed on a Correspondence to S. Metafora, CNR International Institute of Genetics and Biophysics, Via Pietro Castellino, 111-80131 Naples, Italy. Fax: + 39 081 6132 253, Tel.: + 39 081 6132 254, E-mail: metafora@iigbna.iigb.na.cnr.it Abbreviations:MEM,minimalessentialmedium;CHO,Chinese hamster ovary; Dap, 1,3-diaminopropane; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; L-NAME, N x -nitro- L -arginine methyl ester; NO, nitric oxide; PACAP, pituitary adenylate cyclase activating peptide; Pt, putrescine; Spd, spermidine; Spm, spermine; TGase, transglutaminase; VIP, vasoactive intestinal pep- tide; VPAC 1 , VIP/PACAP 1 receptor; VPAC 2 , VIP/PACAP 2 receptor. (Received 11 February 2002, revised 14 May 2002, accepted 15 May 2002) Eur. J. Biochem. 269, 3211–3219 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02996.x number of analogues and different VIP fragments, demon- strated that full action of VIP is critically dependent on the integrity of the entire molecule [13]. The VIP N-terminal helix is known to be critical for the high affinity binding and coupling to the effector system, while the C-terminal sequence has been shown to be important for VPAC 1 and VPAC 2 discrimination [14–17]. Concerning the central region of the VIP polypeptide chain, different amino acid substitutions at this site did not affect the VIP affinity or potency, suggesting that this region is not directly involved in the recognition or activation of receptors. In contrast, Robberecht et al. demonstrated the unexpected importance of Gln16 in the central region of the secretin family peptides for its interaction with the receptor N-terminal domain [18]. On the basis of this finding, we were prompted to use the transglutaminase (TGase) to modify the primary structure of VIP in order to investigate the effect of insertion at the level of the Gln16 c-carboxyamide group of a variety of amines of different carbon chain length and positive charge on VPAC receptor activity, both in rats and humans [19– 23]. The functional characterization of three polyaminated VIP derivatives demonstrated their ability to act as agonists with an affinity and a potency higher than VIP (VIP Dap )or with an affinity lower than VIP (VIP Spd and VIP Spm )on VPAC receptors. The relevance of the polyamine carbon chain length and positive charge on receptor activation has been pointed out and the results of some experiments on murine J774 macrophage cell cultures have shown the ability of these VIP adducts to modulate in vivo the iNOS activity at the level of transcription. MATERIALS AND METHODS VIP and Ro 25-1553 synthesis These peptides were synthesized as C-terminal amides by solid phase methodology on an automated Applied Biosystem apparatus using Fmoc chemistry as described previously [24]. The peptides were cleaved and purified by RP-HPLC on an apparatus using a DBV 300A (10 · 1cm) column and by ion exchange chromatography on a Mono S HR 5/5 column. Peptide purity (95%) was assessed by capillary electrophoresis and sequence conformity was verified by direct sequencing and ion spray MS. TGase-catalysed synthesis of VIP derivatives TGase activity was preliminarily assayed by determining the Ca 2+ -dependent covalent binding of amines to the VIP peptide acting as amino acceptor substrate. Analysis of the reaction products was performed by SDS/PAGE, followed by fluorography [25,26], using radioactive putrescine (Pt), spermidine (Spd) or spermine (Spm) as amino donor substrates. Each preparation of c-(glutamyl16)-Dap-VIP (VIP Dap ), c-(glutamyl16)-Spd-VIP (VIP Spd ), and c-(glutamyl16)-Spm- VIP (VIP Spm ) was obtained by incubating for 12 h in a final volume of 200 lLat37°C50lgofnativeVIPwithTGase in 125 m M Tris/HCl buffer, pH 8.0, containing 10 m M dithiothreitol, 2.5 m M CaCl 2 , and 0.2 M Dap or Pt, or Spd, or Spm, where required; 3 lg (6.7 mU) TGase were added at the start of incubation, and the same amount of enzyme was added after 6 h. A control sample incubated in the absence of TGase was assayed simultaneously. At the end of the incubation, the reaction mixtures were centrifuged at 12 000 g for 10 min, and the resulting supernatants were used to purify the VIP analogues by HPLC. Purification and characterization of the VIP derivatives The VIP analogues present in the supernatants were purified by HPLC chromatography (Waters; Model 660 HPLC apparatus) using an analytical reversed-phase Vydac C18 column (4.6 · 150 mm; Separations Group, Hesperia, CA). The column was equilibrated with 0.01% trifluoroacetic acid and elution was performed in 35 min (flow rate 1mLÆmin )1 ) at room temperature with a 0–60% aceto- nitrile linear gradient. Fractions of 0.2 mL were collected and the absorbance peaks were pooled and evaporated to dryness. The dry samples were dissolved in distilled water and submitted to ES-MS, as described previously [27]. CHO cell line culture The recombinant Chinese hamster ovary (CHO) cells expressing the rat or human recombinant VPAC 1 and VPAC 2 receptors were prepared in P. Robberecht’s labor- atory (Department of Biochemistry and Nutrition, Medical School of Medicine, Universite ´ Libre de Bruxelles, Bel- gium). Cells were incubated at 37 °Cina-minimal essential medium (a-MEM), supplemented with 10% fetal bovine serum, 2 m ML -glutamine, 100 lgÆmL )1 penicillin and 100 lgÆmL )1 streptomycin, with an atmosphere of 95% air and 5% CO 2 . Geneticin (0.4 mgÆmL )1 ) was always present in the culture medium of stock cultures. Subcultures used for membrane purification were grown in a medium without geneticin. Membrane preparation, receptor identification, and adenylate cyclase determination An appropriate number of CHO cells was harvested with a cell scraper and pelleted by low speed centrifugation, the supernatant was discarded and the sedimented cells were lysed by addition of 1 m M NaHCO 3 and quick freezing in liquid nitrogen. After thawing, the lysate was centrifuged at 4 °C for 10 min at 400 g and the supernatant was further centrifuged at 20 000 g atthesametemperature andforthesametimelength.Thefinalpelletwas resuspended in 1 m M NaHCO 3 and used immediately as a crude membrane preparation. [ 125 I]VIP (specific radio- activity, 0.7 mCiÆmmol )1 ) was used as tracer for the identification of both rat or human VPAC 1 receptors; [ 125 I]Ro 25–1553 (specific radioactivity, 0.8 mCiÆmmol )1 ) was used as tracer for labelling the rat or human VPAC2 receptors [28]. The binding of labelled ligands to purified CHO membranes was performed as described [14]; in all cases the nonspecific binding was defined as the residual binding in the presence of 1 l M VIP. Competition curves were carried out by incubating membranes and tracer in the presence of increasing concentrations of unlabelled peptides. Peptide potency was expressed as IC 50 value, i.e. as the peptide concentration required for half maximal inhibition of tracer binding. In detail, the binding was performed at 37 °C in a buffer containing 20 m M Tris/ maleate pH 7.4, 2 m M MgCl 2 , 0.1 mgÆmL )1 bacitracin, 3212 S. De Maria et al. (Eur. J. Biochem. 269) Ó FEBS 2002 and 1% BSA; 3–30 lg protein was used per assay. The bound radioactivity was separated from the free radioac- tivity by filtration through glass fibre filters GF/C presoaked for 24 h in 0.1% polyethyleneimine and rinsed three times with a 20 m M phosphate buffer (pH 7.4) containing 1% BSA. Adenylate cyclase activity was determined by a previously published technique [29]. Membrane proteins (3–15 lg)wereincubatedinatotal volume of 60 lL containing 0.5 m M [a- 32 P]ATP, 10 l M GTP, 5 m M MgCl 2 , 0.5 m M EGTA, 1 m M cAMP, 1 m M theophylline, 10 m M phosphoenolpyruvate, 30 lgÆmL )1 pyruvate kinase, and 30 m M Tris/HCl at a final pH of 7.5. The reaction was initiated by membrane addition and was terminated after a 12-min incubation at 37 °Cby adding 0.5 mL of stop buffer (0.5% SDS, 0.5 m M ATP, 0.5 m M cAMP, 20 000 c.p.m. [8 3 H] cAMP). cAMP was separated from ATP by two successive chromatographies on Dowex 50-WX8 and neutral alumina. Macrophage cell culture The murine monocyte/macrophage cell line J774 (ATCC TIB 67) was grown as monolayers in tissue-culture flasks (75 cm 2 growth area; Falcon) in Dulbecco’s MEM sup- plemented with 10% (v/v) fetal bovine serum (Euroclone, UK), 4 m ML -glutamine, 100 unitsÆmL )1 penicillin, and 100 lgÆmL )1 streptomycin (standard culture medium). Cells were harvested by gentle scraping and passaged every 3–6 days. For use, cells were seeded into 12-well plates (Falcon) and allowed to adhere for 2 h. After this, medium was replaced with fresh medium containing either 0.01 lgÆmL )1 lipopolysaccharide (LPS; this complex molecule is a com- ponent of the Gram-negative bacteria outer membrane possessing a strong iNOS-inducing activity on murine macrophages) alone (control), or VIP and its polyaminated adducts (10 )10 )10 )6 M ), alone or in combination with LPS, and the cells were incubated at 37 °C for a further 24 h in an humidified atmosphere containing 5% CO 2 and 95% air. Cell viability was measured by both Trypan blue exclusion test and MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- nyltetrazolium bromide; Sigma Aldrich]. In specific control inhibition experiments dexamethasone (10 )6 M ;Sigma)was added to macrophages treated with either 0.01 lgÆmL )1 LPS alone, or VIP and its polyaminated adducts (10 )10 )10 )6 M ), alone or in combination with LPS. Nitric oxide measurement The NO produced by the iNOS-catalysed reaction was evaluated by measuring with the Griess reagent nitrite released by the macrophages into the culture medium [30]. Following 24 h incubation at 37 °C, 400-lL aliquots of culture medium were taken from the plates containing the cell monolayers, mixed with an equal volume of Griess reagent (0.5% sulfanilamide and 0.05% N¢-1-naphtylethy- lenediamine dihydrochloride in 2.5% phosphoric acid), and incubated at room temperature for 10 min The absorbance of the coloured solution was measured at 570 nm. The amount of nitrites released into culture medium was expressed as nmol nitrites per 5 · 10 6 cells per 24 h, using a sodium nitrite curve as a standard. Control experiments demonstrated that VIP and VIP adducts did not interfere with the Griess reaction. Evaluation of iNOS activity iNOS activity was determined in crude homogenates of J774 cells. An appropriate number of cells was incubated for 24 h in the absence or presence of either LPS (0.01 lgÆmL )1 ) or VIP or its polyaminated adducts (10 )10 )10 )6 M ), alone or in combination with LPS. After the end of the incubation time, the cells were rinsed three times with ice-cold NaCl/Pi, removed from the culture plates with a cell scraper, collected, and transferred to microcentrifuge tubes. The sedimented cells were lysed by addition of 50 lL ice-cold hypotonic homogenization buffer (1 m M EDTA, 1 m M hypotonic EGTA, 25 m M Tris/HCl pH 7.4). The iNOS activity occurring in 20 lg of homogenate proteins was evaluated by a NOS Detection Assay Kit (Stratagene) [31] according to the manufacturer’s instructions. In this assay, [ 3 H]arginine (50 CiÆmmol )1 ; Amersham) was used as substrate and the reaction mixture was incubated for 30 min at 37 °C. Two blanks were included in the assay: one was prepared by omitting the homogenate, the other by adding the iNOS inhibitor N x -nitro- L -arginine methyl ester ( L -NAME; 1 m M )tothe reaction mixture before the homogenate. The iNOS activity was expressed as citrulline pmolÆmg protein )1 Æmin )1 . Control experiments demonstrated that VIP and polyamines (Dap, Spd, Spm) did not interfere with the iNOS activity. RT-PCR Messenger RNA, isolated by the mRNA Capture Kit (Roche Diagnostics) from the J774 macrophages incuba- ted in the standard culture medium for 24 h in the presence of either 0.01 lgÆmL )1 LPS, or VIP and its polyaminated adducts (10 )10 )10 )6 M ), alone or in combi- nation with LPS, was transcribed by reverse transcriptase (Superscript II; Life Technologies) at 37 °Cfor1.5h according to the manufacturer’s protocol (final volume 20 lL). The cDNA contained in 2 lL of this reaction mixture was amplified in another reaction mixture con- taining, in a final volume of 25 lL, 10 m M Tris/HCl pH 8.3, 1.5 m M MgCl 2 , 50 m M KCl, 100 ng of both sense and antisense primers for iNOS (sense, 5¢-GTTTCT TGTGGCAGCAGC-3¢;antisense,5¢-CCTCGTGGCT TTGGGCTCCT-3¢), 100 l M deoxynucleoside triphos- phate, and 1 U Taq DNA polymerase (Roche Diagnos- tics). The reaction was carried out in a DNA thermal cycler (Promega). The PCRs were performed with 35 cycles in the exponential phase of amplification and always started with a 3-min denaturation step at 95 °Cand terminated with a final 7 min step at 72 °C. The cycle for iNOS was 95 °C, 45 s; 56 °C, 45 s; 72 °C, 45 s. The PCR products were analysed by electrophoresis on a 1.2% agarose gel in Tris/borate/EDTA [32]. The identities of the amplification products were confirmed by comparison of their sizes with the sizes expected from the known gene sequence. Coamplification of a different cDNA sequence was performed by adding into the amplification reaction mixture the b-actin gene primers (10 ng of both sense and antisense primers: sense, 5¢-CGTGGGCCGCCCTAGG CACCA-3¢;antisense,5¢-TTGGCCTTAGGGTTCA GGGGGG-3¢). No products were detectable in control amplifications performed in the absence of cDNA (data Ó FEBS 2002 VIP polyaminated agonists and receptor activity (Eur. J. Biochem. 269) 3213 not shown). The semiquantitative evaluation of the PCR products was achieved by integrating the peak area obtained by densitometry of the ethidium bromide stained agarose gels [software used: NIH image V.16; iNOS (600 bp): 109, 757, 3300, 4581, 1159, 901; b-actin (300 bp): 670, 797, 833, 963, 819, 831]. The ratio between the yield of each amplified product and coamplified b-actin (iNOS/ b-actin mRNA ratio: 0.162, 0.949, 3.961, 4.757, 1.415, 1.084) allows a relative estimate of mRNA levels in the samples analysed. Multiple alignment and charge distribution in receptor sequences The amino acid sequences of the VIP receptors analysed were derived from the SwissProt database. The following sequences were used for the multiple alignment analysis: VIPR_CARAU (VPAC 1 goldfish), VIPR_HUMAN (VPAC 1 human), VIPR_PIG (VPAC 1 pig), VIPR_RAT (VPAC 1 rat), VIPS_HUMAN (VPAC 2 human), VIPS_ MOUSE (VPAC 2 mouse), VIPS_RAT (VPAC 2 rat). The Fig.1.EffectofVPAC 1 and VPAC 2 ligands (VIP and its polyaminated agonists) on membrane binding and adenylate cyclase activity. The data reported in the figure refer to: (1) Dose-dependent inhibition of 125 I-labelled ligand ([ 125 I]VIP was used for the identification of rat or human VPAC 1 receptors, whereas [ 125 I]Ro 25-1553 was used for labelling of rat or human VPAC 2 receptors) binding (panels A, C, E, and G) to crude preparations of CHO cell membranes expressing recombinant VPAC 1 and VPAC 2 receptors, by VIP (s), VIP Dap (d),VIP Spd (h), and VIP Spm (j); the results are the means of three different determinations and are expressed as the percentage of tracer specifically bound; (2) Dose-effect curves of VIP (s), VIP Dap (d) on adenylate cyclase activation (B, D, F, and H) in crude preparations of membranes from CHO cells expressing recombinant VPAC 1 and VPAC 2 receptors; the results, expressed in percentage increase of 32 P-labelled cyclic AMP produced in the presence of 1 l M VIP, are the means of three different experiments. The cAMPase activity was evaluated by a previously published radiometric assay [29]. Further experimental details are reported in Materials and methods. 3214 S. De Maria et al. (Eur. J. Biochem. 269) Ó FEBS 2002 multiple alignment was created by the CLUSTALW software. Colours were added manually by considering the common colour-code for charged amino acids (i.e. red for acidic, and cyan/blue for basic side chains). The analysis of charge distribution in the extracellular and cytoplasmatic domains was carried out by considering the domain assignment reported in the SwissProt database. Statistical analysis The data have been reported as means ± SEM of at least three different determinations. The means were compared using analysis of variance (one-way ANOVA )plusBonfer- roni’s t-test and a P-value < 0.05 was considered significant. The software packages used for statistical analysis were GRAPHPAD INSTAT and MINITAB . The curve fitting programs used were in GRAPHPAD PRISM , GRAPHPAD INPLOT , and MINITAB . RESULTS A positively charged amino acid into position 16 modulates the VIP ability to bind its specific receptors VIP and its three polyaminated adducts (VIP Dap , VIP Spd , and VIP Spm ) possessing a different positively charged side chain at position 16, were first characterized by appropriate binding experiments to VPAC receptors, both in humans and rats. The data reported in Fig. 1 (panels A, C, E, G) and analysed in Table 1 demonstrate that the VIP Dap adduct has a higher affinity (lower IC 50 value) than VIP on both rat and human VPAC 1 receptors, and a similar affinity to VIP on VPAC 2 receptors. The VIP Spd and VIP Spm derivatives were 30–100-fold less potent than VIP. The effect of the agonists used in these experiments was tested on VPAC 1 and VPAC 2 receptors in both rat and human on the assumption that the analysis of the data obtained, associated with the knowledge of the structural differences between these two receptors and between the rat and human VPAC 1 receptors [33], could allow a better identification of the polypeptide regions involved in the ligand/receptor molecular interactions. The polyaminated VIP adducts are agonists of either higher or lower affinity and potency than VIP The effect of the three polyaminated VIP adducts on the human and rat VPAC 1 and VIPAC 2 receptor activity was evaluated by evaluating the adenylate cyclase enzymatic activity of a crude preparation of membranes. The data reported in Fig. 1 (panels B, D, F, H) and Table 1 indicate that VIP Dap has a higher apparent affinity and higher maximum effect than VIP in all the receptors tested. In contrast, VIP Spd and VIP Spm werefoundtoactwithalower apparent affinity, their EC 50 values being 3–10 times higher than the VIP value (Table 1). The data on the relative potencies of Spd- and Spm-conjugated VIP in cAMP generation assays (not shown in Fig. 1) indicate that the decrease in biological activity of these adducts reflects the apparent decrease in their binding affinity at the lowest concentrations used (10 )10 )10 )7 M ), even though at the highest concentrations (10 )7 )10 )6 M ) the biological activity improves significantly. By comparing the IC 50 and EC 50 of Table 1. IC 50 and EC 50 values (n M ) from binding experiments and adenylate cyclase assays. Experimental details are described in Materials and methods. Values are means ± SEM and are the means of at least three different determinations. IC 50 , Peptide concentration (n M ) required for 50% tracer binding inhibition; EC 50 , peptide concentration (n M ) required for half maximal stimulation of adenylate cyclase activity; IA, intrinsic activity, the ratio between the maximal stimulating effect of modified VIP and that of VIP. *P <0.05,**P < 0.01 (Bonferroni’s t-test) vs. the VIP value. Human Receptor Rat Receptor VPAC 1 VPAC 2 VPAC 1 VPAC 2 Ligand IC 50 EC 50 IA IC 50 EC 50 IA IC 50 EC 50 IA IC 50 EC 50 IA VIP 1.0 ± 0.2 2 ± 0.2 1.0 10 ± 3.0 7.0 ± 1.0 1.0 1.1 ± 0.1 0.6 ± 0.1 1.0 5 ± 1.0 1.0 ± 1.0 1.0 VIP Dap 0.3 ± 0.1* 0.8 ± 0.1* 1.3 ± 0.10 5 ± 0.8** 7.0 ± 0.8 1.5 ± 0.2 0.4 ± 0.1** 0.2 ± 0.1* 1.2 ± 0.2 3 ± 0.5* 0.6 ± 1.0 1.3 ± 0.2 VIP Spd 40 ± 3** 15 ± 4** 1.1 ± 0.15 200 ± 18** 120 ± 10** 1.0 ± 0.1 15 ± 2** 2 ± 0.5** 1.1 ± 0.1 150 ± 20** 20 ± 3** 1.1 ± 0.1 VIP Spm 200 ± 50** 90 ± 50** 0.9 ± 0.15 100 ± 15** 80 ± 12** 0.9 ± 0.1 20 ± 4** 4 ± 2** 1.0 ± 0.2 260 ± 15** 60 ± 8** 1.0 ± 0.1 Ó FEBS 2002 VIP polyaminated agonists and receptor activity (Eur. J. Biochem. 269) 3215 VIP Dap and R16VIP, their values were found to be about the same. The VIP polyamination markedly increases the ability of VIP to stimulate the NO production in J774 macrophages It is well known that VIP inhibits smooth muscle cell contractility by inducing NO production in the target cells [34]. On the other hand, it has also been demonstrated that VIP possesses the ability to modulate the humoral and cell-mediated immune response, both in vivo and in vitro, by a mechanism involving cAMP and NO [35]. Furthermore, recent findings have shown that appropriate concentrations of VIP inhibit in vitro the macrophage biochemical machinery involved in NO production [36]. In contrast with this result, we now report data that demonstrate the ability of the VIP/LPS combination to modulate the capacity of J774 macrophages to generate NO in a biphasic manner, the lower concentrations of VIP being more active (a maximum stimulation was reached at 10 )8 M ) than the higher concentrations (10 )6 M ) (Fig. 2, upper panel). Sim- ilar results were obtained with equimolar concentrations of polyaminated VIP adducts, the VIP Dap adduct being the most active. The NO production profile obtained either with VIP or polyaminated VIP adducts was similar to the iNOS activity profile induced by the same molecules (Fig. 2, upper and lower panels). In turn, the increase of iNOS activity produced by VIP or its polyaminated adducts was associ- ated with a marked increase in the expression of the gene encoding iNOS, as evaluated by semiquantitative RT/PCR (Fig. 3). DISCUSSION In this report we have shown that polyamination of Gln16 side chain significantly modulates the ability of VIP to bind and stimulate the VPAC 1 receptor. This finding supports Fig. 2. Effect of various VPAC 1 and VPAC 2 ligands (VIP and its agonists VIP Dap ,VIP Spd , and VIP Spm )onNO 2 – production (upper panel) and NO synthase activity (lower panel) in J774 murine macr- ophages. The NO produced by the iNOS-catalysed reaction was evaluated by measuring with the Griess reagent the nitrite amounts released into the culture medium by untreated or experimentally treated macrophages following a 24 h incubation at 37 °C. The amount of nitrite released was expressed as nmol nitritesÆper 5 · 10 6 cells per 24 h, using a sodium nitrite curve as a standard. iNOS activity was determined in crude homogenates of J774 cells incubated for 24 h in the absence or presence of either LPS, or VIP and its polyaminated adducts in combination with LPS. iNOS activity occurring in 20 lgof homogenate proteins was evaluated by a NOS Detection Assay Kit. In this assay, [ 3 H]arginine was used as substrate and the reaction mixture was incubated for 30 min at 37 °C. iNOS activity was expressed as citrulline pmolÆmg protein )1 Æmin )1 . Controls: cells untreated (unfilled bars) or treated with LPS alone (0.01 lgÆmL )1 ; diagonal bars, \). Experimental: cells treated with LPS (0.01 lgÆmL )1 ) in combination with different concentrations of VIP (cross hatched bars, X) or VIP agonists (VIP Dap , speckled bars; VIP Spd , diagonal bars, /) VIP Spm , horizontal bars). Further experimental details are reported in Materials and methods. Fig. 3. Expression of the gene encoding iNOS in J774 macrophages following their treatment with various VPAC 1 and VPAC 2 ligands (VIP and its agonists VIP Dap ,VIP Spd ,andVIP Spm ; used at a final concen- tration of 10 )8 M ) and LPS (0.01 lgÆmL )1 ). The expression of the iNOS gene in untreated or treated cells was evaluated by RT-PCR. The total poly(A) + messenger RNA, isolated from J774 macrophages incubated in the standard culture medium for 24 h in the presence of either LPS or VIP and its polyaminated adducts in combination with LPS, was reverse-transcribed in a reaction mixture of 20 lL. The cDNA con- tained in 2 lL of this mixture was amplified by Taq DNA polymerase in the presence of sense and antisense primers for iNOS. The PCRs were performed according to the experimental protocol reported in Materials and methods and the products were analysed by agarose gel electrophoresis. The identities of the amplification products were confirmed by comparison of their sizes with the sizes expected from the known gene sequence. Coamplification of a different cDNA sequence was performed by adding into the amplification reaction mixture the b- actin gene primers. No products were detectable in control amplifi- cations performed in the absence of cDNA. The semiquantitative evaluation of the PCR products was achieved by integrating the peak area obtained by densitometry of the ethidium bromide stained ag- arose gels. Further experimental details are reported in Materials and methods. 3216 S. De Maria et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Robberecht’s data indicating the critical role played by the presence of a positively charged amino acid (arginine, R) at position 16 of VIP polypeptide chain [18]. In addition, the possibility that the side chain length could play an important role in modulating the receptor recognition ability and activity is supported by our IC 50 and EC 50 data that show the best performance of VIP Dap activity in comparison with VIP, VIP Spd , and VIP Spm . The high affinity of VIP Dap might be related to specific interactions of this agonist with well-defined hydrophilic regions of the receptor polypeptide chain. However, the possibility that additional hydrophobic contact made by the R side-chain or by Dap may significantly contribute to the binding affinity of these agonists, cannot be ruled out on the basis of our present data. The possible existence in the receptor of different dynamic conformational states corresponding to different states of activation [37,38], allows us to hypothesize that the presence at position 16 of a positive charge, associated with Fig. 4. Distribution of electric charges in extracellular and cytoplasmic domains of seven VIP receptor amino acid sequences. The amino acid sequences of the VIP receptors analysed were obtained from the SwissProt database. The sequences used for the multiple alignment analysis are reported in Materials and methods. The multiple alignment was created by the CLUSTALW software. Colours were added manually by considering the common colour code for charged amino acids (i.e. red for acidic, cyan/blue for basic side chains). The analysis of charge distribution in the extracellular and cytoplasmatic domains was carried out by considering the domain assignment reported in the SwissProt database. Green: signal peptide; yellow: transmembrane regions; red: acidic amino acids (Asp ¼ D; Glu ¼ E); blue: basic amino acids (His ¼ H; Lys ¼ K; Arg ¼ R); black boxes: amino acids evolutionally conserved in the seven amino acid sequences analysed. The data reported in this figure (rows 1–4 refer to VPAC 1 receptors; rows 5–7 refer to VPAC 2 receptors) indicate that the extracellular domains of the analysed receptors are characterized by a predominance of acid vs. basic side chains, whereas in the cytoplasmatic domains there is a clear predominance of positively charged side chains. In addition, the N-terminal domain of VPAC 2 receptors appears to contain more acidic residues than the VPAC 1 counterpart. The contrary is true when the first extracellular loop domain is considered. No significant differences in charge distribution are found when the second and third extracellular loop domains are analysed. Ó FEBS 2002 VIP polyaminated agonists and receptor activity (Eur. J. Biochem. 269) 3217 possible hydrophobic interactions and a definite side chain length, could be effective in triggering the stabilization of the conformational state corresponding to the highest binding affinity with or without change in the receptor activity. Experiments are in progress not only to identify the receptor region/s involved in the interaction with VIP Dap , but also to define the type of receptor–ligand interaction triggered by the ligand binding process. Novel chemical modifications of a peptide ligand, similar to those reported in this paper and capable of both modulating the receptor activity and increasing the discrimination capacity between receptor subclasses, could be of the highest interest for a better control of definite biological functions. It is also interesting to note that these chemical modifications at the site 16 associated with appropriate modifications at the level of other residues in the VIP N terminus could be useful for the production of better VIP antagonists. In addition, the substitution of Arg16 in R-VIP [18] with a polyaminated derivative of glutamine (Dap) could make the modified peptide not only a better agonist or antagonist but also protect its structural integrity from a trypsin-like proteolytic attack. The hypothesis that a positive charge at the position 16 of VIP polypeptide chain could play an important role in the ligand–receptor recognition mechanism is also supported by the published data on charge distribution in VPAC receptor amino acid sequences (see Fig. 4). In fact, these data show the occurrence in the extracellular domains of VPAC receptors of a significant clustering of well conserved negative charges that could originate electrostatic interac- tions with the positive charge(s) present at position 16 of VIP adducts. Moreover, differential negative charge distri- bution in the N-terminal domain and first extracellular loop of VPAC 1 and VPAC 2 may explain the observed differences in affinity for the VPAC receptors among the modified ligands. The highest negative charge observed in the first extracellular loop of VPAC 1 in comparison with VPAC 2 is in line with the highest affinity of the modified VIP ligands for VPAC 1 and suggests that this loop is probably involved in the ligand–receptor recognition mechanism. We have also investigated the ability of VIP and its polyaminated derivatives to modulate in vivo the biochemi- cal machinery controlling NO production in the J774 macrophage cell line. The data obtained demonstrate that at low concentrations these ligands exert a marked stimu- latory effect on the macrophage NO production activity by enhancing the iNOS gene expression induced by LPS, both at protein and mRNA level. This finding is apparently in contrast with other data reported in the literature which show an inhibitory effect of VIP on macrophage ability to produce NO in vitro [36]. This discrepancy may be due to the fact that these authors used a different macrophage cell line possessing a differential expression of the two VPAC receptors and different VIP and LPS concentrations to measure the effect of VIP and other substances on an LPS- activated cell system [36]. The inhibitory effect observed at high concentrations of VIP and its polyaminated adducts is probably related to either the negative regulatory effect exerted by the relatively high levels of VIP (NF-jB inhibition by a cAMP-independent pathway [39]): or to a shedding process of membrane-bound CD14 receptors from LPS- stimulated macrophages induced by the highest concentra- tions of VIP or its adducts used in our experiments [40] or both. Experiments are in progress to elucidate the molecular mechanism at the basis of the up-regulatory effect of these ligands on iNOS gene expression. Finally, the obvious discrepancies between biological activity (evaluated in vivo on J774 macrophage cell line) and receptor binding affinity (assessed in vitro on CHO cell-derived crude membranes) of the various VIP derivatives (compare Fig. 1 with Fig. 2) might be related to the different experimental conditions in which these parameters were evaluated and to possible differences between CHO cells and J774 macrophages in membrane signal transduction mechanisms. ACKNOWLEDGEMENTS We thank P. De Neef, J. Cnudde, S. Baiano and F. Moscatiello for their skilful technical assistance. This research was supported by a Grant from ÔProgramma di Intervento per la Promozione della Ricerca Scientifica in Campania L.R. n.41-31/12/94Õ. REFERENCES 1. Campbell, R.M. & Scanes, C.G. (1992) Evolution of the growth hormone-releasing factor (GRF) family of peptides. Growth Reg. 2, 175–191. 2. Carlquist, M., McDonald, T.J., Go, V.L., Bataille, D., Johansson, C. & Mutt, V. 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