Peptides (v.26, #7)

Molecular modeling and docking simulations of scorpion toxins and related analogs on human SKCa2 and SKCa3 channels by Nicolas Andreotti; Eric di Luccio; François Sampieri; Michel De Waard; Jean-Marc Sabatier (1095-1108).
The small-conductance Ca2+-activated K+ (SKCa) channels modulate cytosolic Ca2+ concentration in excitable and non-excitable tissues by regulating the membrane potential and are responsible of slow action potential after hyperpolarization that inhibits cell firing. Among these, human SKCa2 and SKCa3 channels differ in the pore region by only two residues: Ala331 and Asn367 (human small-conductance calcium-activated potassium channel, hSKCa2) instead of Val485 and His521 (hSKCa3). To design highly selective blockers of hSKCa channels, a number of known hSKCa2 and/or hSKCa3-active peptides (i.e. scorpion toxins and analogs thereof) were analyzed for their interactions and selectivities toward these channels. Molecular models of hSKCa2 and hSKCa3 channels (S5-H5-S6 portion) were generated, and scorpion toxins/peptides of unsolved three-dimensional (3D) structures were modeled. Models of toxin–channel complexes were generated by the bimolecular complex generation with global evaluation, and ranking (BiGGER) docking software and selected by using a screening method of the docking solutions. A high degree of correlation was found to exist between docking energies and experimental K d values of peptides that blocked hSKCa2 and/or hSKCa3 channels, suggesting it could be appropriate to predict K d values of other bioactive peptides. The best scoring complexes were also used to identify key residues of both interacting partners, indicating that such an approach should help the design of more active and/or selective peptide blockers of targeted ion channels.
Keywords: Molecular modeling; SKCa; Channels;

Catalytic effects of glycine on prebiotic divaline and diproline formation by Kristof Plankensteiner; Hannes Reiner; Bernd M. Rode (1109-1112).
The catalytic effects of the simple amino acid glycine on the formation of diproline and divaline in the prebiotically relevant salt-induced peptide formation (SIPF) reaction was investigated in systems of different amino acid starting concentrations and using the two enantiomeric forms of the respective amino acid. Results show an improved applicability of the SIPF reaction to prebiotic conditions, especially at low amino acid concentrations, as presumably present in a primordial scenario, and indicate excellent conditions and resources for chemical evolution of peptides and proteins on the early earth. For valine, furthermore differences in catalytic yield increase are found indicating a chiral selectivity of the active copper complex of the reaction and showing a connection to previously found enantiomeric differences in complex formation constants with amino acids.
Keywords: Origin of life; Prebiotic peptide formation; Salt-induced peptide formation reaction; Mutual catalysis; Chemical evolution; Biohomochirality;

Antifungal activity of synthetic peptides derived from Impatiens balsamina antimicrobial peptides Ib-AMP1 and Ib-AMP4 by Karin Thevissen; Isabelle E.J.A. François; Lolke Sijtsma; Aart van Amerongen; Wim M.M. Schaaper; Rob Meloen; Truus Posthuma-Trumpie; Willem F. Broekaert; Bruno P.A. Cammue (1113-1119).
Seeds of Impatiens balsamina contain a set of related antimicrobial peptides (Ib-AMPs). We have produced a synthetic variant of Ib-AMP1, oxidized to the bicyclic native conformation, which was fully active on yeast and fungal strains; and four linear 20-mer Ib-AMP variants, including two all-d forms. We show that the all-d variants are as active on yeast and fungal strains as native peptides. In addition, fungal growth inhibition nor salt-dependency of Ib-AMP4 could be improved by more than two-fold via replacement of amino acid residues by arginine or tryptophan. Native Ib-AMPs showed no hemolytic nor toxic activity up to a concentration of 100 μM. All these data demonstrate the potential of the native Ib-AMPs to combat fungal infections.
Keywords: Antifungal; Ib-AMP; Ionic strength; Toxicity; Derivative;

An antifungal peptide with a molecular mass around 7 kDa and an N-terminal sequence highly homologous to defensin was isolated from ground beans (Vigna sesquipedalis cv. ‘Ground Bean’). The peptide was adsorbed on Affi-gel blue gel and on Mono S. It exerted an antifungal action on Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola; and an antibacterial action on Escherichia coli B, Proteus vulgaris, Mycobacterium phlei and Bacillus megaterium. The antimicrobial activity was inhibited in presence of the 5 mM CaCl2 and MgCl2, but no inhibition was observed in 5 mM NaCl. The peptide exerted antiproliferative activity toward breast cancer (MCF-7) cells and leukemia M1 cells, this activity could not be inhibited by the ions mentioned above. It also exhibited some inhibitory activity toward human immunodeficiency virus-type 1 reverse transcriptase.
Keywords: Antifungal peptides; Isolation; Bean; Vigna sesquipedalis;

Interaction of antimicrobial peptides with bacterial polysaccharides from lung pathogens by Yury Herasimenka; Monica Benincasa; Maura Mattiuzzo; Paola Cescutti; Renato Gennaro; Roberto Rizzo (1127-1132).
The interaction of two cathelicidin antimicrobial peptides, LL-37 and SMAP-29, with three bacterial polysaccharides, respectively, produced by Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae, was investigated to identify possible mechanisms adopted by lung pathogens to escape the action of innate immunity effectors. In vitro assays indicated that the antibacterial activity of both peptides was inhibited to a variable extent by the three polysaccharides. Circular dichroism experiments showed that these induced an α-helical conformation in the two peptides, with the polysaccharides from K. pneumoniae and B. cepacia showing, respectively, the highest and the lowest effect. Fluorescence measurements also indicated the presence of peptide–polysaccharide interactions. A model is proposed in which the binding of peptides to the polysaccharide molecules induces, at low polysaccharide to peptide ratios, a higher order of aggregation, due to peptide–peptide interactions. Overall, these results suggest that binding of the peptides by the polysaccharides produced by lung pathogens can contribute to the impairment of peptide-based innate defenses of airway surface.
Keywords: Antimicrobial peptide; Cathelicidin; Polysaccharide; Burkholderia cepacia; Klebsiella pneumoniae; Pseudomonas aeruginosa;

Peptides from the Plasmodium falciparum STEVOR putative protein bind with high affinity to normal human red blood cells by Javier E. García; Alvaro Puentes; Hernando Curtidor; Ricardo Vera; Luis Rodriguez; John Valbuena; Ramses López; Marisol Ocampo; Jimena Cortés; Magnolia Vanegas; Jaiver Rosas; Claudia Reyes; Manuel E. Patarroyo (1133-1143).
Synthetic 20-mer long non-overlapped peptides, from STEVOR protein, were tested in RBC binding assays for identifying STEVOR protein regions having high RBC binding activity and evaluating whether these regions inhibit Plasmodium falciparum in vitro invasion. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay with high activity binding peptides (HABPs). HABP binding assays using RBCs previously treated with enzymes were carried out to study the nature of the receptor. The molecular weight of RBC surface proteins interacting with HABPs was determined by cross-linking assays and SDS-PAGE analysis. RBC binding assays revealed that peptides 30561 (41MKSRRLAEIQLPKCPHYNND60), 30562 (61PELKKIIDKLNEERIKKYIE80) and 30567 (161ASCCKVHDNYLDNLKKGCFG180) bound saturably and with high binding activity, presenting nanomolar affinity constants. HABP binding activity to RBCs previously treated with neuraminidase and trypsin decreased, suggesting that these peptides bound to RBC surface proteins and that such binding could be sialic acid dependent. Cross-linking and SDS-PAGE assays showed that the three HABPs specifically bound to 30 and 40 kDa molecular weight RBC membrane proteins. Peptides 30561, 30562 and 30567 inhibited P. falciparum in vitro invasion of red blood cells in a concentration-dependent way. Goat sera having STEVOR protein polymeric peptides antibodies inhibit parasite in vitro invasion depending on concentration. Three peptides localized in STEVOR N-terminal and central regions had high, saturable, binding activity to 30 and 40 kDa RBC membrane proteins. These peptides inhibited the parasite's in vitro invasion, suggesting that STEVOR protein regions are involved in P. falciparum invasion processes during intra-erythrocyte stage.
Keywords: P. falciparum; STEVOR; High activity binding peptides;

Synthesis and biological properties of chimeric interferon-α2b peptides by Clara Peña; Viviana C. Blank; Verónica J. Marino; Leonor P. Roguin (1144-1149).
We have previously reported the antiproliferative activity of synthetic sequences 29–35 and 122–139 of the interferon-α2b (IFN-α2b), both probably representing a common receptor recognition domain. In the search of new peptidic agonists, we designed and synthesized the linear peptide (Gly)2-122-137-Gly138-Gly29-30-35-(Gly)2, in which Gly residues replaced the 138 and 29 Cys bound through a disulfide bridge in the native cytokine. Additionally, a cyclic analog was obtained by reaction of the N- and C-terminal ends of the linear fragment. Thus, the distance that separates residues 122 and 35 in the crystalline structure of the IFN-α2b was maintained through a (Gly)4 bridge. When the influence of chimeric peptides on the proliferation of WISH cells was studied, it was shown that both derivatives significantly diminished cell growth. A more evident inhibitory effect on 125I-IFN-α2b binding to WISH cell-membrane receptors was observed for both peptides. Results indicated that chimeric IFN-α2b peptides behaved as partial agonists of the IFN-α2b molecule and may be of interest for drug design purposes.
Keywords: Interferon-α2b; Synthetic peptides; Interferon-receptor interaction; Binding properties; Antiproliferative activity;

α-Melanocyte stimulating hormone cytoprotective biology in human dermal fibroblast cells by Rebecca P. Hill; Paul Wheeler; Sheila MacNeil; John W. Haycock (1150-1158).
Alpha-melanocyte stimulating hormone (α-MSH) has been identified as a potent anti-inflammatory peptide effective in various tissues including skin. It acts by inhibiting the production and action of several pro-inflammatory stimuli including TNF-α, IL-1β and LPS in a number of cell types. The role of such stimuli in inducing cellular apoptosis is also well described; however the precise role of α-MSH in apoptosis is presently unclear, with studies reporting both anti- and pro-apoptotic activity. The present study demonstrates that cultured human dermal fibroblasts respond to serum depletion and TNF-α, IL-1β and LPS with an increase in membrane permeability, a decrease in viability and an increase in phosphatidylserine externalization (indicative of apoptosis) over 48–96 h. α-MSH (at 10−6  M, but not 10−9  M) was found to inhibit the serum free and pro-inflammatory mediated reduction in membrane permeability and cellular viability and also inhibited increases in apoptosis. In conclusion, data support a cytoprotective and anti-apoptotic role of the α-MSH peptide in human dermal fibroblast cells.
Keywords: Human dermal fibroblasts; α-MSH; Viability; Phosphatidylserine; Apoptosis;

Synthesis and biological studies of nociceptin derivatives containing the DTPA chelating group for further labeling with therapeutic radionuclides by Melinda Ligeti; Özge Gündüz; Anna Magyar; Erzsébet Kató; András Z. Rónai; Claudio Vita; Imre Varga; Ferenc Hudecz; Géza Tóth; Anna Borsodi; Sándor Benyhe (1159-1166).
Nociceptin is an endogenous anti-opiate heptadecapeptide primarily interacting with the nociceptin (NOP) receptor. This neuropeptide–receptor system is involved in pain regulation, tolerance to and dependence on opiates as well as many other physiological and pathophysiological events. The role and mechanisms of nociceptin in pathological conditions is not clearly known yet. In an attempt to have a radiopharmaceutical labeled either with 99mTc or 111In, we incorporated diethylenetriaminepentaacetic acid (DTPA) as chelator into the structure of [Arg14,Lys15]nociceptin(1–17)-NH2 at the ɛ-amino group of Lys15. Such a radiopeptide may be useful in imaging for diagnostical purposes. Preparation of the peptide ligands was carried out by solid phase synthesis. Two peptides containing DTPA were obtained and purified. The products were [Arg14,Lys(DTPA)15]nociceptin(1–17)-NH2 and its cross-linked dimer on the basis of mass spectrometric analysis. In 115In3+ binding experiments the conjugates exhibited preserved indium ion chelating properties, indicating the potential use of radiolabeled DTPA-nociceptin derivatives as radiopharmaceutical. Biological properties of these compounds were studied in rat brain membrane preparations by radioligand binding, functional biochemical [35S]GTPγS binding assays and mouse vas deferens (MVD) bioassay. Besides the similar in vitro binding characteristics to nociceptin receptor, both of the DTPA-chelated compounds were more potent and efficient than nociceptin in functional biochemical and mouse vas deferens bioassays. Our further aim is to radiolabel these compounds in order to get a radiopharmaceutical which can be used diagnostically.
Keywords: Nociceptin; NOP receptor; Radiopharmaceutical; DTPA chelate; Radioligand binding; GTPγS assay; Mouse vas deferens bioassay;

NPY-induced feeding: pharmacological characterization using selective opioid antagonists and antisense probes in rats by Y. Israel; Y. Kandov; E. Khaimova; A. Kest; S.R. Lewis; G.W. Pasternak; Y.X. Pan; G.C. Rossi; R.J. Bodnar (1167-1175).
The ability of neuropeptide Y to potently stimulate food intake is dependent in part upon the functioning of μ and κ opioid receptors. The combined use of selective opioid antagonists directed against μ, δ or κ receptors and antisense probes directed against specific exons of the MOR-1, DOR-1, KOR-1 and KOR-3/ORL-1 opioid receptor genes has been successful in characterizing the precise receptor subpopulations mediating feeding elicited by opioid peptides and agonists as well as homeostatic challenges. The present study examined the dose-dependent (5–80 nmol) cerebroventricular actions of general and selective μ, δ, and κ1 opioid receptor antagonists together with antisense probes directed against each of the four exons of the MOR-1 opioid receptor gene and each of the three exons of the DOR-1, KOR-1, and KOR-3/ORL-1 opioid receptor genes upon feeding elicited by cerebroventricular NPY (0.47 nmol, 2 ug). NPY-induced feeding was dose-dependently decreased and sometimes eliminated following pretreatment with general, μ, δ, and κ1 opioid receptor antagonists. Moreover, NPY-induced feeding was significantly and markedly reduced by antisense probes directed against exons 1, 2, and 3 of the MOR-1 gene, exons 1 and 2 of the DOR-1 gene, exons 1, 2, and 3 of the KOR-1 gene, and exon 3 of the KOR-3/ORL-1 gene. Thus, whereas the opioid peptides, β-endorphin and dynorphin A1–17 elicit feeding responses that are respectively more dependent upon μ and κ opioid receptors and their genes, the opioid mediation of NPY-induced feeding appears to involve all three major opioid receptor subtypes in a manner similar to that observed for feeding responses following glucoprivation or lipoprivation.
Keywords: μ opioid receptor; δ opioid receptor; κ opioid receptor; Naltrexone; β-Funaltrexamine; Nor-binaltorphamine; Naltrindole;

Perigestational suppression of weight gain with central leptin gene therapy results in lower weight F1 generation by Anne Lecklin; Michael G. Dube; Rita N. Torto; Pushpa S. Kalra; Satya P. Kalra (1176-1187).
The efficacy of central leptin therapy on weight homeostasis through various phases of reproduction, pregnancy outcome and postnatal, prepubertal and pubertal growth of offspring was assessed. Enhanced leptin transgene expression after a single intracerebroventricular injection of recombinant adeno-associated virus vector encoding the leptin gene (rAAV-lep) decreased calorie intake and weight in adult nulliparous female rats. rAAV-lep treated rats conceived normally, displayed unremarkable pregnancy rate, parturition and delivered normal sized litters. Significantly lower weight was maintained through gestation, lactation, and post-lactation periods. The maintenance of a modest weight reduction was accompanied by voluntarily reduced calorie intake, increased thermogenic energy expenditure, decreased adiposity as reflected by drastically reduced leptin levels, and suppressed insulin and insulin-like growth factor 1 levels through lactation and post-lactation in rAAV-lep treated dams. The offspring at birth weighed significantly less than those of controls and this lower weight range was sustained during postnatal, prepubertal, pubertal and adult (3 months old) periods, contemporaneous with metabolic circulating hormones in the normal range. For the first time we show the persistent efficacy of central leptin gene therapy to suppress weight gain through all phases of reproduction, lactation and post-lactation in dams and reveal the potential imprinting link to producing lower weight in the F1 generation.
Keywords: Weight suppression; Imprinting; Insulin; Prenatal; Postnatal; Pubertal growth;

Peripheral injection of sauvagine prevents repeated colorectal distension-induced visceral pain in female rats by Mulugeta Million; Céline Maillot; David A. Adelson; Tsukasa Nozu; Ariane Gauthier; Jean Rivier; George P. Chrousos; Alfred Bayati; Hillevi Mattsson; Yvette Taché (1188-1195).
We investigated the effects of peripheral injection of sauvagine, a CRF2  > CRF1 receptor (corticotropin-releasing factor) agonist compared with CRF, on two sets of tonic colorectal distension (CRDs 30, 40, 50 mmHg, 3-min on/off)-induced visceromotor response (VMR) measured as area under the curve (AUC) of abdominal muscle contraction in conscious female rats. Sauvagine (10 or 20 μg/kg, s.c.) abolished the 226.7 ± 64.3% and 90.4 ± 38.1% increase in AUC to the 2nd CRD compared with the 1st CRD (performed 30 min before) in female Fisher and Sprague–Dawley (SD) rats, respectively. CRF had no effect while the CRF1 antagonist, antalarmin (20 mg/kg, s.c.), alone or with sauvagine, blocked the enhanced response to the 2nd CRD, performed 60 min after the 1st CRD, and reduced further the AUC by 33.5 ± 23.3% and 63.5 ± 7.2%, respectively in Fisher rats. These data suggest that peripheral CRF2 receptor activation exerts antinociceptive effects on CRD-induced visceral pain, whereas CRF1 contributes to visceral sensitization.
Keywords: Antalarmin; Sauvagine; CRF; Visceral pain; Colorectal distension; Female Fisher rats;

Urocortin 3/stresscopin in human colon: possible modulators of gastrointestinal function during stressful conditions by Masayuki Saruta; Kazuhiro Takahashi; Takashi Suzuki; Tsuyoshi Fukuda; Akira Torii; Hironobu Sasano (1196-1206).
Urocortin 3 (Ucn 3) or stresscopin (SCP) is a new member of the corticotropin-releasing factor (CRF) neuropeptide family and is a specific ligand for CRF type 2 receptor (CRF2). CRF receptors are known to be expressed in the gastrointestinal tract and are considered to play pathophysiological roles, for example, in gastrointestinal motility under stress. We, therefore, examined Ucn 3 expression in the normal human large intestine obtained from surgery and autopsy in order to clarify this local response to stress in human intestine. Both immunohistochemistry and mRNA in situ hybridization demonstrated Ucn 3 expression in myenteric and submucosal nervous plexus, in vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) of blood vessels in subserosa, in smooth muscle layers of the large intestine, and in enterochromaffin cells. In contrast to Urocortin 1 (Ucn 1), Ucn 3 was hardly detected in lamina propria (LP) inflammatory cells in colonic mucosa. In addition, immunohistochemistry demonstrated CRF2 expression in myenteric and submucosal nervous plexus, in smooth muscle layers, in VECs, in VSMCs and in lamina propria inflammatory cells. Immunoreactive Ucn 3 was also detected in the large intestine by RIA, with high concentrations detected in the rectum (15.4 ± 9.5 pmol/g wet weight, mean ± SEM, n  = 3) and sigmoid colon (6.5 ± 3.5 pmol/g wet weight, n  = 5). Reverse-phase HPLC of the human large intestine disclosed peaks eluting in the position of synthetic Ucn 3 or SCP. These findings all suggest that Ucn 3 plays some physiological or pathological roles in the modulation of gastrointestinal functions during stressful conditions in different manners from Ucn 1.
Keywords: Urocortin 3; Stresscopin; Urocortin 1; Corticotropin-releasing factor receptor; Colon;

G17-Gly has been shown to stimulate the growth of DLD-1 human colon cancer cells in a biphasic manner via high and low affinity receptors. In the current study, the existence of heterogeneous receptor populations for G17-Gly on the HT-29 human colon cancer cell line was investigated. The effect of either N- or C-terminal peptide truncation on receptor binding and cell growth stimulation was also explored. [Leu15]G17-Gly bound to both high (nM) and low (μM) affinity sites on HT-29 cells. The peptide stimulated cell growth in a dose-dependent and biphasic manner with maximal stimulation at 10−9  M peptide concentration, suggesting that, as in the case of DLD-1 cells, it is the high affinity receptor which is responsible for the growth-promoting effects. In contrast, G17(1–12) stimulated the growth of HT-29 cells in a sigmoidal fashion with an EC50 of 4.6 × 10−9  M. Sequential N-terminal truncation of [Leu15]G17-Gly results in decreased binding to the high affinity G17-Gly receptor on DLD-1 cells. [Leu15]G17(11–17)Gly bound to the low affinity G17-Gly receptor with an affinity similar to that of the full sequence peptide but was unable to displace the radioligand from high affinity sites. G17(1–6)-NH2 was unable to displace [3H]G17-Gly from either site. These results suggest that the important residues for binding to the low affinity receptor are in the C-terminal region of the peptide while those required for interaction with the high affinity receptor lie further towards the N-terminus.
Keywords: Glycine-extended gastrin; Colon cancer; Biphasic growth response; High affinity receptor; Low affinity receptor; Gastrin fragment;

High-level expression of human TFF3 in Escherichia coli by Haibo Wang; Yuanpeng Tong; Ming Fang; Binggen Ru (1213-1218).
A strategy for expression and purification of recombinant N-terminal human trefoil factor family-domain peptide 3 (hTFF3) in Escherichia coli was established. The gene of hTFF3 was synthesized to substitute the low-usage condons with corresponding high-usage synonymous condons. At the same time, the signal peptide of DsbC was added to the N-terminus of the hTFF3 gene. The mature recombinant hTFF3 was located in the periplasm of E. coli, which can be released by sonication. The protein was further purified by a two-step cation exchange chromatography mentod. The yield is about 14–15 mg/l of culture. The biological activity of purified hTFF3 was analyzed by cell-based apoptosis assay, which shows that the recombinant hTFF3 is biologically active.
Keywords: Intestinal trefoil factor (ITF); Trefoil factor family-domain peptide 3 (TFF3); Osmotic shock; Two-step ion exchange chromatography;

Endothelin-3 applied to the brain evokes opposite effects on bile secretion mediated by a central nitric oxide pathway by Myrian R. Rodríguez; María E. Sabbatini; Gisela Santella; Paula Dabas; Alberto Villagra; Marcelo S. Vatta; Liliana G. Bianciotti (1219-1227).
We sought to establish Endothelin (ET-3) role in the central regulation of bile secretion in the rat. The intracerebroventricular (icv) injection of ET-3 evoked a cholestatic or a choleretic effect depending on the administered dose. Lower doses increased bile flow and bicarbonate excretion, whereas higher doses decreased bile flow and bile acid output. ET-3 effects were dependent on brain nitric oxide and independent of the autonomic nervous system or hemodynamic variations. A selective ETB antagonist abolished the cholestatic effect, whereas the choleretic effect was totally inhibited by either ETA or ETB selective blockade. These results show that ET-3 applied to the brain modified through a nitric oxide pathway distinct bile flow fractions depending on the administered dose and give further insights into the complexity of brain–liver interaction.
Keywords: ET-3; Bile flow; Nitric oxide; ETB receptors; ETA receptors;

Adult male Sprague–Dawley rats were treated with the anabolic androgenic steroid nandrolone decanoate (15 mg/kg day) or oil vehicle (sterile arachidis oleum) during 14 days. The effect on the densities of the neurokinin NK1 receptor in brain was examined with autoradiography. An overall tendency of attenuation of NK1 receptor density was observed after completed treatment with nandrolone decanoate. The density of the NK1 receptor was found to be significantly lower compared to control animals in the nucleus accumbens core (37% density reduction), in dentate gyrus (26%), in basolateral amygdaloid nucleus (23%), in ventromedial hypothalamic nucleus (36%), in dorsomedial hypothalamic nucleus (43%) and finally in the periaqueductal gray (PAG) (24%). In the cortex region, no structures exhibited any significant reduction of NK1 receptor density. This result provides additional support to the hypothesis that substance P and the NK1 receptor may be involved as important components that participate in mediating physiological responses including the adverse behaviors often associated with chronically administrated anabolic androgenic steroids in human.
Keywords: Anabolic androgenic steroids; Nandrolone; NK1 receptor; Substance P; Autoradiography;

Potentiation of bradykinin actions by analogues of the bradykinin potentiating nonapeptide BPP by Sylvia Mueller; Rita Gothe; Wolf-Dieter Siems; Gabriele Vietinghoff; Inge Paegelow; Siegmund Reissmann (1235-1247).
Synthetic analogues of the bradykinin potentiating nonapeptide BPP indicate significantly different structural requirements for potentiation of the bradykinin (BK)-induced smooth muscle contraction (GPI) and the inhibition of isolated somatic angiotensin I-converting enzyme (ACE). The results disprove the ACE inhibition as the only single mechanism and also the direct interaction of potentiating peptides with the bradykinin receptors in transfected COS-7 cells as molecular mechanism of potentiation. Our results indicate a stimulation of inositol phosphates (IP n ) formation independently from the B2 receptor. Furthermore, the results with La3+ support the role of extracellular Ca2+ and its influx through corresponding channels. The missing effect of calyculin on the GPI disproves the role of phosphatases in the potentiating action. These experimental studies should not only contribute to a better understanding of the potentiating mechanisms but also incorporate a shift in the research towards the immune system, in particular towards the immunocompetent polymorphonuclear leukocytes. The chemotaxis of these cells can be potentiated most likely by exclusive inhibition of the enzymatic degradation of bradykinin. Thus the obtained results give evidence that the potentiation of the bradykinin action can occur by different mechanisms, depending on the system and on the applied potentiating factor.
Keywords: Potentiation; Bradykinin; Bradykinin potentiating peptide; Angiotensin I-converting enzyme; Inositol phosphate; Arachidonic acid; Ca2+-influx; Protein phosphatases; Polymorphonuclear leukocytes; Chemotaxis; Smooth muscle contraction; Radioligand binding;

Urotensin II (UII) is a highly conserved peptide that has potent cardiovascular actions following central and systemic administration. To determine whether the cardiovascular actions of UII are mediated via β-adrenoceptors, we examined the effect of intravenous (IV) propranolol on the responses to intracerebroventricular (ICV) and IV administration of UII in conscious sheep. Sheep were surgically instrumented with ICV guide tubes and flow probes or cardiac sympathetic nerve recording electrodes. ICV UII (0.2 nmol/kg over 1 h) caused prolonged increases in heart rate (HR; 33 ± 11 beats/min; P  < 0.01), dF/dt (581 ± 83 L/min/s; P  < 0.001) and cardiac output (2.3 ± 0.4 L/min; P  < 0.001), accompanied by increases in coronary (19.8 ± 5.4 mL/min; P  < 0.01), mesenteric (211 ± 50 mL/min; P  < 0.05) and iliac (162 ± 31 mL/min; P  < 0.001) blood flows and plasma glucose (7.0 ± 2.6 mmol/L; P  < 0.05). Propranolol (30 mg bolus followed by 0.5 mg/kg/h IV) prevented the cardiac responses to ICV UII and inhibited the mesenteric vasodilatation. At 2 h after ICV UII, when HR and mean arterial pressure (MAP) were increased, cardiac sympathetic nerve activity (CSNA) was unchanged and the relation between CSNA and diastolic pressure was shifted to the right (P  < 0.05). The hyperglycemia following ICV UII was abolished by ganglion blockade but not propranolol. IV UII (20 nmol/kg) caused a transient increase in HR and fall in stroke volume; these effects were not blocked by propranolol. These results demonstrate that the cardiac actions of central UII depend on β-adrenoreceptor stimulation, secondary to increased CSNA and epinephrine release, whereas the cardiac actions of systemic UII are not mediated by β-adrenoreceptors and probably depend on a direct action of UII on the heart.
Keywords: Cardiac sympathetic nerve activity; Hyperglycemia; Propranolol; Ganglion blockade; Cardiac contractility; Regional blood flow;

Adrenomedullin induces heme oxygenase-1 gene expression and cGMP formation in rat vascular smooth muscle cells by Yong-Fen Qi; Lin-Wang Dong; Chun-Shui Pan; Jing Zhang; Bin Geng; Jing Zhao; Chao-Shu Tang (1257-1263).
Adrenomedullin (ADM) is a potent vasodilatory peptide. It regulates blood pressure by increasing cyclic adenosine monophosphate (cAMP) and guanosine-3′,5′-monophosphate (cGMP). We sought to investigate the effect of ADM on heme oxygenase-1 (HO-1) gene expression and cGMP formation in cultured rat vascular smooth muscle cells (VSMCs). ADM treatment, 10−9 and 10−8  mol/L, increased cGMP production, and it increased the intracellular cGMP content of platelets coincubated with VSMCs. It increased cGMP content by 158.8% and 273.5%, respectively; increased HO-1 activity by 49.5% and 87%, respectively; augmented HO-1 protein levels by 66% and 126%, respectively; upregulated the steady-state level of HO-1 mRNA by 73% and 159%, respectively, and increased HO-1 mRNA transcription synthesis by four- and seven-fold, respectively. These results suggest that ADM induces HO-1 gene expression and cGMP formation in rat VSMCs.
Keywords: Adrenomedullin; Vascular smooth muscle cells; Nitric oxide; Carbon monoxide; Heme oxygenase;

Phage display biopanning has been used for a number of applications including ligand generation for targeted drug delivery, targeting gene therapy vectors and identification of protein–protein interaction sites. In this study, a random phage display library was used to isolate peptide ligands to the endothelial protein C receptor (EPCR), identifying 74 different peptide sequences and several motifs. Binding to EPCR was characterized by a solid phase binding assay, demonstrating that 95% of isolated peptides were specific for EPCR. Several homologies with potential relevance to EPCR biology were identified, the most notable being leukolysin (MT-MMP6) and cerastocytin.
Keywords: Endothelial cell protein C receptor; EPCR; Peptide ligands; Phage display;

Organ-specific distribution of ACE2 mRNA and correlating peptidase activity in rodents by Florian Gembardt; Anja Sterner-Kock; Hans Imboden; Matthias Spalteholz; Franziska Reibitz; Heinz-Peter Schultheiss; Wolf-Eberhard Siems; Thomas Walther (1270-1277).
Biochemical analysis revealed that angiotensin-converting enzyme related carboxy-peptidase (ACE2) cleaves angiotensin (Ang) II to Ang-(1–7), a heptapeptide identified as an endogenous ligand for the G protein-coupled receptor Mas. No data are currently available that systematically describe ACE2 distribution and activity in rodents. Therefore, we analyzed the ACE2 expression in different tissues of mice and rats on mRNA (RNase protection assay) and protein levels (immunohistochemistry, ACE2 activity, western blot). Although ACE2 mRNA in both investigated species showed the highest expression in the ileum, the mouse organ exceeded rat ACE2, as also demonstrated in the kidney and colon. Corresponding to mRNA, ACE2 activity was highest in the ileum and mouse kidney but weak in the rat kidney, which was also confirmed by immunohistochemistry. Contrary to mRNA, we found weak activity in the lung of both species. Our data demonstrate a tissue- and species-specific pattern for ACE2 under physiological conditions.
Keywords: Angiotensin-converting enzyme 2; Enzyme activity; Peptidase; Tissue distribution; Renin–angiotensin system;