Ex Vivo Smooth Muscle Pharmacological Effects of a Novel

Ex Vivo Smooth Muscle Pharmacological Effects of a Novel Bradykinin-Related Peptide, and Its Analogue, from Chinese Large Odorous Frog, Odorrana livida Skin Secretions Xiang, J., Wang, H., Ma, C., Zhou, M., Wu, Y., Wang, L., Guo, S., Chen, T., & Shaw, C. (2016). Ex Vivo Smooth Muscle Pharmacological Effects of a Novel Bradykinin-Related Peptide, and Its Analogue, from Chinese Large Odorous Frog, Odorrana livida Skin Secretions. Toxins, 8(10). https://doi.org/10.3390/toxins8100283

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Article Ex Vivo Smooth Muscle Pharmacological Effects of a Novel Bradykinin-Related Peptide, and Its Analogue, from Chinese Large Odorous Frog, Odorrana livida Skin Secretions

Jie Xiang 1,†, Hui Wang 2,†, Chengbang Ma 1, Mei Zhou 1, Yuxin Wu 1,*, Lei Wang 1,*, Shaodong Guo 3, Tianbao Chen 1 and Chris Shaw 1

1 Natural Drug Discovery Group, School of Pharmacy, Queen’s University Belfast, Belfast BT9 7BL, Northern Ireland, UK; [email protected] (J.X.); [email protected] (C.M.); [email protected] (M.Z.); [email protected] (T.C.); [email protected] (C.S.) 2 School of Pharmaceutical Sciences, China Medical University, Shenyang 110001, China; [email protected] 3 Department of Nutrition and Food Science, College of Agriculture and Life Sciences, Texas A&M University, 123A Cater Mattil Hall, 2253 TAMU, College Station, TX 77843, USA; [email protected] * Correspondence: [email protected] (Y.W.); [email protected] (L.W.); Tel.: +44-28-9097-2361 (Y.W. & L.W.); Fax: +44-28-9094-7794 (Y.W. & L.W.) † These authors contributed equally to this work.

Academic Editor: R. Manjunatha Kini Received: 3 September 2016; Accepted: 21 September 2016; Published: 27 September 2016

Abstract: Bradykinin-related peptides (BRPs) are one of the most extensively studied frog secretions-derived peptide families identified from many amphibian species. The diverse primary structures of BRPs have been proven essential for providing valuable information in understanding basic mechanisms associated with drug modification. Here, we isolated, identified and characterized a dodeca-BRP (RAP-L1, T6-BK), with primary structure RAPLPPGFTPFR, from the skin secretions of Chinese large odorous frogs, Odorrana livida. This novel peptide exhibited a dose-dependent contractile property on rat bladder and rat ileum, and increased the contraction frequency on rat uterus ex vivo smooth muscle preparations; it also showed vasorelaxant activity on rat tail artery smooth muscle. In addition, the analogue RAP-L1, T6, L8-BK completely abolished these effects on selected rat smooth muscle tissues, whilst it showed inhibition effect on bradykinin-induced rat tail artery relaxation. By using canonical antagonist for bradykinin B1 or B2 type receptors, we found that RAP-L1, T6-BK -induced relaxation of the arterial smooth muscle was very likely to be modulated by B2 receptors. The analogue RAP-L1, T6, L8-BK further enhanced the bradykinin inhibitory activity only under the condition of co-administration with HOE140 on rat tail artery, suggesting a synergistic inhibition mechanism by which targeting B2 type receptors.

Keywords: bradykinin related peptide (BRP); B2 receptor; rat tail artery; agonist and antagonist; smooth muscle

1. Introduction Bradykinin (BK) is a nonapeptide hormone first discovered in 1949 by Rocha et al. upon the basis of its inherent capability to dilate vessels [1]. It has subsequently been reported that, in mammals, endogenous BK is generated via the effect of kallikrein on kininogen, which was derived mostly from, but not restricted to, liver tissues [2,3]. Since the first amphibian canonical BK was reported in the defensive skin secretions systems in 1965 from Rana temporaria [4], numerous bradykinin-related peptides (BRPs), with extraordinary diverse primary structure flanked by C/N-terminal extension,

Toxins 2016, 8, 283; doi:10.3390/toxins8100283 www.mdpi.com/journal/toxins Toxins 2016, 8, 283 2 of 15 were isolated and identified from the Ascaphidae, Hylidae, Ranidae and Bombinatatoridae families. Of note is that mammals, reptiles, birds, and even fish were found to produce structurally discrete BRPs [5]. The research that focused on elucidating the hypervariable primary structures of BRPs across vertebrates was initially conducted by Conlon and his colleagues, and found that the diverse BRPs presented in unique vertebrate taxa all have identical counterparts in certain amphibian species [3,6]. Following on from this, the hypothesis was proposed that taxon-specific BRPs might be the most effective ligands for activating taxon-specific receptor mediated signal pathways, and for the improper BRP-ligands, they were believed to act as antagonists of the receptors to suppress the downstream signalling. DNA sequences that encode a wide range of BRPs have been cloned from frog skin secretions of different genus and species, many of which have been proved to have myotropic activities by using diverse mammalian smooth muscle preparations [5]. BK triggers its effect mainly through two types of receptors, B1 and B2, which all belong to the G protein-coupled receptors family. Compared with B2 receptors, which are generally expressed in multiple tissues, B1 receptors are only constitutively expressed in certain tissues, such as spinal cord, brain, and artery endothelial cells in the aorta and lung [7]. While upon inflammatory stimulus, B1 receptor expressions in a variety of tissues are upregulated in response to NF-κB (extensively studied transcription factors involved in immune response and infection) activation [8]. Stimulation or inhibition of B1 or B2 receptors is thought to be related with many pathophysiological disorders. Therefore, development of agonists or antagonists for either B1 or B2 receptors or even both would provide researchers significant implications no matter in the fields of pharmacology or clinical therapy. Here, we reported a novel BRP, RAP-L1, T6-BK, which was isolated and identified from the skin secretion of the Chinese large odorous frog Odorrana livida by using 30RACE and 50RACE “shotgun” cloning technique. The primary sequence was further confirmed via the MS/MS fragmentation sequencing approach. The synthetic replicates of this novel peptide and its analogue RAP- L1, T6, L8-BK, were characterized by multiple organ-bath based ex vivo rat smooth muscle tissue probes. The wild-type RAP-L1, T6-BK was found to stimulate rat bladder, ileum, uterus contractile and tail artery relaxant responses. By contrast, the analogue RAP-L1, T6, L8-BK completely abrogated these functions, and it showed strong inhibition effect upon the BK-induced rat tail artery relaxation. Further pharmacological analysis revealed that BK B2 receptors are highly likely to be involved in the rat tail artery related effects caused by this novel BRP and its analogue.

2. Results

2.1. Molecular Cloning of cDNA Encoding the Biosynthetic Precursor of the Novel Bradykinin-Related Peptide The preprobradykinin-like peptide encoding cDNA was consistently cloned from the Odorrana livida skin secretion-constructed cDNA library, the open-reading frame of this novel BRP precursor consists of 61 amino acids, and the architecture of translated open-reading frame can be divided into four domains. The 50 N-terminus begins with a putative signal peptide with 22 amino acid residues followed by a 21 acidic residue-rich spacer, the putative 12-mer mature peptide is preceded by a propeptide convertase processing site -VK-, and it was followed by a C-terminal extension peptide (Figure1).

2.2. Isolation and Structure Characterization of the Novel BRP Reverse-phase HPLC chromatogram of the Odorrana livida skin secretion is shown in Figure2. The fraction with the same mass of the peptide deduced from the molecular cloning (with calculated molecular mass 1355.59 Da) by using the speciﬁc BRP degenerate primer, which was described in Section 4.2, was identiﬁed by MS/MS fragmentation sequencing using the electrospray ion-trap mass spectrometer, the observed molecular mass was 1355.78 Da (Figure3). Together with the result of the molecular cloning, the primary structure of the novel bradykinin-related peptide is unambiguously Toxins 2016, 8, 283 3 of 15

ToxinsL1, T6,2016 L8, 8,‐ 283BK (with calculated molecular mass 1320.59 Da) was confirmed by observation 3of of its 15 molecular mass of 1321.01 Da by using MALDI‐TOF (Figure S2). determinedToxins as RAPLPPGFTPFR,2016, 8, 283 which is named as RAP-L1, T6-BK. Meanwhile, the analogue3 of 15 RAP-L1, T6, L8-BK (with calculated molecular mass 1320.59 Da) was conﬁrmed by observation of its molecular mass of 1321.01L1, T6, DaL8‐BK by (with using calculated MALDI-TOF molecular (Figure mass S2).1320.59 Da) was confirmed by observation of its molecular mass of 1321.01 Da by using MALDI‐TOF (Figure S2).

Figure 1. The nucleotide sequence and open‐reading frame amino acid sequence of full length FigureFigure 1.1. The nucleotide sequence and open-readingopen‐reading frame amino acid sequence of fullfull lengthlength preprobradykinin-likepreprobradykinin peptide‐like peptide encoding encoding cDNA cDNA from from Chinese Chinese Large Large frog, frog,Odorrana Odorrana lividalivida. .The The putative preprobradykininputative signal‐like peptidepeptide is doubleencoding‐underlined. cDNA The from mature Chinese peptide isLarge single ‐underlined.frog, Odorrana The stop livida . The signalputative peptide signalcodon is ispeptide indicated double-underlined. isby doublean asterisk.‐underlined. The mature The peptide mature is peptide single-underlined. is single‐underlined. The stop The codon stop is indicatedcodon is indicated by an asterisk. by an asterisk.

Figure 2. Region of reverse phase HPLC chromatogram of Odorrana livida skin secretion with arrow indicating the retention times (at 90 min) of the novel peptide RAP‐L1, T6‐BK. The detection wavelength was 214 nm with a flow rate of 1 mL/min in 240 min.

FigureFigure 2.2. RegionRegion ofof reversereverse phasephase HPLCHPLC chromatogramchromatogram ofof OdorranaOdorrana lividalivida skinskin secretionsecretion withwith arrowarrow indicatingindicating the the retention retention times times (at 90(at min) 90 ofmin) the novelof the peptide novel RAP-L1,peptide T6-BK.RAP‐L1, The T6 detection‐BK. The wavelength detection waswavelength 214 nm withwas 214 a ﬂow nm rate with of a 1 flow mL/min rate of in 1 240 mL/min min. in 240 min.

Toxins 2016, 8, 283 4 of 15 Toxins 2016, 8, 283 4 of 15

(a)

(b)

Figure 3. ThermoquestThermoquest LCQ™ LCQ™ fragmentfragment scan scan spectrum spectrum derived from ions ions corresponding corresponding in molecular mass to RAP-L1,RAP‐L1, T6-BKT6‐BK ((aa)) andand electrosprayelectrospray ion-trapion‐trap MS/MSMS/MS fragmentation dataset (b) Expected singly-singly‐ and doubly-chargeddoubly‐charged b-ionsb‐ions andand y-ionsy‐ions arisingarising fromfrom MS/MSMS/MS fragmentation were predicted using the MS Product programme available through Protein Prospector on-line.on‐line. Truly observed ions are indicated in bold-typefacebold‐typeface and underlined.

2.3. Bioinformatic Analysis of Novel BRP RAP‐L1, T6‐BK

Toxins 2016, 8, 283 5 of 15

2.3. Bioinformatic Analysis of Novel BRP RAP-L1, T6-BK

A BLASTToxins 2016 search, 8, 283 of this structure using the National Center for Biotechnological5 of Information 15 (NCBI) on line portal, revealed that the full length open reading frame of novel RAP-L1, T6-BK A BLAST search of this structure using the National Center for Biotechnological Information (OL SBN54116) peptide displayed relative high amino acid sequence identity (Query Cover: 100%; (NCBI) on line portal, revealed that the full length open reading frame of novel RAP‐L1, T6‐BK (OL E value =SBN54116) 0.001; Identity: peptide 64%–90%, displayed relative including high amino the Best acid Hits) sequence with identity the BRPs (Query precursor Cover: 100%; sequences E from Amolops species.valueToxins 2016= 0.001;, 8 The, 283 Identity: highly-conserved 64%–90%, including domain the Best includes Hits) with the the ﬁrst BRPs residue precursor Arginine sequences from5 of of 15N -terminal Amolops species. The highly‐conserved domain includes the first residue Arginine of N‐terminal extension andA -PPGFTPFR- BLAST search inof thethis structure canonical using BRP the section National (Figure Center 4for). DetailedBiotechnological alignment Information was analysed extension and ‐PPGFTPFR‐ in the canonical BRP section (Figure 4). Detailed alignment was analysed using the AlignX(NCBI) on program line portal, of revealed the Vector that the NTI full Bioinformatics length open reading Suite frame (Informax) of novel RAP (Figure‐L1, T6‐4BK). (OL using the AlignX program of the Vector NTI Bioinformatics Suite (Informax) (Figure 4). SBN54116) peptide displayed relative high amino acid sequence identity (Query Cover: 100%; E value = 0.001; Identity: 64%–90%, including the Best Hits) with the BRPs precursor sequences from Amolops species. The highly‐conserved domain includes the first residue Arginine of N‐terminal extension and ‐PPGFTPFR‐ in the canonical BRP section (Figure 4). Detailed alignment was analysed using the AlignX program of the Vector NTI Bioinformatics Suite (Informax) (Figure 4).

Figure 4. AlignmentFigure 4. Alignment of cDNA-deduced of cDNA‐deduced RAP RAP-L1,‐L1, T6‐BK T6-BK precursor precursor sequence and sequence BRPs from and Amolops BRPs species. from Amolops species. SubstitutionsSubstitutions are arehighlighted highlighted in grey. in Asterisks grey. designate Asterisks identical designate amino identicalacid residues. amino Accession acid residues. numbers are given in parentheses: (1) Putative N‐terminal signal peptide domains; (2) acid spacer peptide Accession numbers are given in parentheses: (1) Putative N-terminal signal peptide domains; (2) acid domains; (3) mature peptide domains; and (4) C‐terminal extension peptide domains. OL: Odorrana livida ; spacer peptideAR: Amolops domains; ricketti; (AT:3) matureAmolops torrentis peptide; AM: domains; Amolops mantzorum and (4); AW:C-terminal Amolops wuyiensis extension; AL1: peptide Amolops domains. Figure 4. Alignment of cDNA‐deduced RAP‐L1, T6‐BK precursor sequence and BRPs from Amolops species. OL: Odorranalifanensis livida; AG:; AR: AmolopsAmolops granulosus ricketti; AD: ;Amolops AT: Amolops daiyunensis torrentis; AL2: Amolops; AM: loloensisAmolops. * represents mantzorum the highly; AW: Amolops Substitutions are highlighted in grey. Asterisks designate identical amino acid residues. Accession wuyiensis;conserved AL : Amolops residues. lifanensis; AG: Amolops granulosus; AD: Amolops daiyunensis; AL : Amolops numbers1 are given in parentheses: (1) Putative N‐terminal signal peptide domains; (2) acid spacer peptide2 loloensis 2.4.. Preliminary *domains; represents (3 )Study mature the of highly peptide the Inherent conserveddomains; Cytotoxicity and residues.(4) C‐ terminalof Novel extension BRPs peptide domains. OL: Odorrana livida; AR: Amolops ricketti; AT: Amolops torrentis; AM: Amolops mantzorum; AW: Amolops wuyiensis; AL1: Amolops The human microvessel endothelial cells (HMECs) were used to evaluate inherent cytotoxicity 2.4. Preliminarylifanensis Study; AG: of Amolops the Inherent granulosus Cytotoxicity; AD: Amolops daiyunensis of Novel; AL BRPs2: Amolops loloensis. * represents the highly of twoconserved BRPs in residues.this study. After incubation for 24 h, the viability of cells was evaluated using the MTT The humanassay. The microvessel synthetic replicates endothelial of the cells BRPs (HMECs) exhibited were no significant used to evaluate cytotoxic inherenteffects at cytotoxicity the of concentrations2.4. Preliminary employed Study of the (10 Inherent−5–10−11 CytotoxicityM) in this assayof Novel (Figure BRPs 5), which is coincident with the molar two BRPsconcentrations in this study. used After in pharmacological incubation for experiments 24 h, the (Figures viability 6–11). of cells was evaluated using the MTT assay. The syntheticThe human replicates microvessel of the endothelial BRPs exhibited cells (HMECs) no signiﬁcant were used to cytotoxic evaluate effectsinherent at cytotoxicity the concentrations of two− 5BRPs− in11 this study. After incubation for 24 h, the viability of cells was evaluated using the MTT employedassay. (10 The–10 syntheticM) in replicates this assay of (Figurethe BRPs5), exhibited which is no coincident significant withcytotoxic the molareffects concentrationsat the used in pharmacologicalconcentrations employed experiments (10−5–10−11 (Figures M) in this6– assay11). (Figure 5), which is coincident with the molar concentrations used in pharmacological experiments (Figures 6–11).

Figure 5. Assessment of cytotoxicity of BRPs. Dose–response curves of BRPs on human microvessel

endothelial cells after a 24 h incubation. Each column represents the mean ± SEM of three replicates. Toxins 2016, 8, 283 6 of 15

Figure 5. Assessment of cytotoxicity of BRPs. Dose–response curves of BRPs on human microvessel endothelial cells after a 24 h incubation. Each column represents the mean ± SEM of three replicates. Toxins 2016, 8, 283 6 of 15 Toxins 2016, 8, 283 6 of 15 2.5. Smooth Muscle Pharmacology of Two Synthetic BRPs Figure 5. Assessment of cytotoxicity of BRPs. Dose–response curves of BRPs on human microvessel 2.5. SmoothPrior Muscleendothelial to the Pharmacology constructionscells after a 24 h of incubation. Twoof kinins Synthetic Each dose–response column BRPs represents curves,the mean ±the SEM eNOS of three inhibitor,replicates. L‐NIO, was employed to exclude the endothelial‐derived NO triggered artery relaxation. The results revealed thatPrior there2.5. toSmooth is the no Muscle constructions significant Pharmacology difference of of kinins Two Synthetic(p dose–response value BRPs 0.7562, two curves,‐way theANOVA) eNOS on inhibitor, tissue responsiveness L-NIO, was employed to exclude the endothelial-derived NO triggered artery relaxation. The results revealed between BKPrior‐induced to the constructions artery relaxation of kinins and dose–response L‐NIO pre ‐curves,administrated the eNOS BK inhibitor,‐induced L‐NIO, artery was relaxation, that there is no signiﬁcant difference (p value 0.7562, two-way ANOVA) on tissue responsiveness whichemployed confirmed to exclude that the the NO endothelial was not‐derived likely toNO be triggered involved artery in therelaxation. effects Theof vasodilation results revealed under such betweenrat tailthat BK-inducedartery there smoothis no significant artery muscle relaxation difference preparation and (p value L-NIOcircumstances 0.7562, pre-administrated two‐ way(Figure ANOVA) 6). BK-induced on tissue responsiveness artery relaxation, which conﬁrmedbetween BK that‐induced the NO artery was relaxation not likely and to L be‐NIO involved pre‐administrated in the effects BK‐induced of vasodilation artery relaxation, under such rat tail arterywhich smooth confirmed muscle that preparation the NO was not circumstances likely to be involved (Figure in6 the). effects of vasodilation under such rat tail artery smooth muscle preparation circumstances (Figure 6).

FigureFigure 6. Assessment 6. Assessment of of the the effects effects ofof eNOS eNOS on on the the smooth smooth muscle muscle effects effects of BK. Dataof BK. represent Data representthe the Figure 6. Assessment of the effects of eNOS on the smooth muscle effects of BK. Data represent the meanmean ± SEM ± SEM of sixof six replicates. replicates. ThereThere werewere no no significant significant effects effects (p value (p value0.7562, 0.7562,two‐way two ANOVA)‐way ANOVA) mean ± SEM of six replicates. There were no signiﬁcant effects (p value 0.7562, two-way ANOVA) presentedpresented in thein the presence presence ( ●(●)) oror absence ( ▲(▲) of) ofthe the eNOS eNOS inhibitor, inhibitor, L‐NIO, L in‐NIO, each inconcentration each concentration presentedcompared in the to presence controls. ( ) or absence (N) of the eNOS inhibitor, L-NIO, in each concentration comparedcompared to controls.to controls. The novel BRP RAP‐L1, T6‐BK exhibited a dose‐dependent activation of contraction in rat urinary Thebladder novel with BRP EC50 RAP = 3.54‐ L1,± 0.83 T6 μ‐MBK compared exhibited with a doseBK (EC‐dependent50 = 61.78 ± 0.93 activation nM) (Figure of contraction7a). For rat ileum, in rat urinary The novel BRP RAP-L1, T6-BK exhibited a dose-dependent activation of contraction in rat urinary bladderthe withdose‐ dependentEC50 = 3.54 contractile ± 0.83 μ Meffects compared of RAP‐ L1,with T6 BK‐BK (ECand 50BK = were61.78 EC ± 0.9350 = 70.95 nM) ± (Figure0.98 nM and7a). ECFor50 rat ileum, bladder with EC50 = 3.54 ± 0.83 µM compared with BK (EC50 = 61.78 ± 0.93 nM) (Figure7a). For rat the dose= 2.95‐dependent ± 0.12 nM, respectivelycontractile (Figure effects 7b). of RAPOn rat‐L1, uterus T6‐ smoothBK and muscle BK were preparations, EC50 = 70.95 RAP ±‐L1, 0.98 T6 ‐nMBK and EC50 ileum, the dose-dependent contractile effects of RAP-L1, T6-BK and BK were EC = 70.95 ± 0.98 nM = 2.95significantly ± 0.12 nM, increasedrespectively the (Figurenumber 7b).of contractions On rat uterus during smooth 5 min muscle intervals preparations, when50 the peptide RAP‐L1, T6‐BK and ECconcentration= 2.95 ± 0.12raised nM, to 10 respectively−6 M and 10−5 M, (Figure with EC7b).50 = 6.82 On ± rat 0.75 uterus μM versus smooth BK EC muscle50 = 51.02 preparations, ± 1.05 significantly50 increased the number of contractions during 5 min intervals when the peptide RAP-L1,nM T6-BK (Figure signiﬁcantly 7c). For rat tail increasedartery, BK exerted the number more potency of contractions in relaxation effect during with 5 IC min50 = 1.408 intervals ± 0.15 when concentration raised to 10−6 M and 10−5 M, with EC50 = 6.82 ± 0.75 μM versus BK EC50 = 51.02 ± 1.05 nM versus RAP‐L1, T6‐BK with IC50 =− 34.046 ± 2.35 nM− (Figure5 7d). the peptide concentration raised to 10 M and 10 M, with EC50 = 6.82 ± 0.75 µM versus BK nM (Figure 7c). For rat tail artery, BK exerted more potency in relaxation effect with IC50 = 1.408 ± 0.15 EC50 = 51.02 ± 1.05 nM (Figure7c). For rat tail artery, BK exerted more potency in relaxation effect nM versus RAP‐L1, T6‐BK with IC50 = 34.04 ± 2.35 nM (Figure 7d). with IC50 = 1.408 ± 0.15 nM versus RAP-L1, T6-BK with IC50 = 34.04 ± 2.35 nM (Figure7d).

(a) (b)

Figure 7. Cont.

ToxinsToxins 2016,, 8,, 283 77 of of 15 15

Toxins 2016, 8, 283 7 of 15

(c) (d) (c) (d) FigureFigure 7. 7. Dose–responseDose–response curve curve of of BK BK (black) (black) and and RAP RAP-L1,‐L1, T6 T6-BK‐BK (red) (red) induced induced contractile contractile effects effects on on ratFigurerat bladder 7. Dose–response (a(a);); and and ileum ileum curve (b );(b relaxant );of relaxant BK (black) effects effects onand tail RAPon artery tail‐L1, preparationsarteryT6‐BK preparations(red) induced (d); and ( contractioncontractiled); and contraction effects frequency on frequencyraton uterusbladder preparationson ( auterus); and preparationsileum (c). For (b); rat relaxant ( tailc). arteryFor effectsrat treatment, tail onartery tail RAP-L1, treatment,artery T6-BKpreparations RAP dose–response‐L1, T6 (d‐BK); and dose–response curves contraction in the − −6 − −6 curvesfrequencypresence in the of on 10 presence uterus6 M R715 preparationsof 10 (green) M R715 and (c (green)). 10 For6 Mrat and HOE140 tail 10 artery M (violet) HOE140 treatment, were (violet) presented RAP were‐L1, presented accordingly.T6‐BK dose–response accordingly. curves in the presence of 10−6 M R715 (green) and 10−6 M HOE140 (violet) were presented accordingly. To further examine the preliminary vasodilation mechanism of RAP‐L1, T6‐BK, specific BK To further examine the preliminary vasodilation mechanism of RAP-L1, T6-BK, specific BK subtype receptor antagonists (R715 for typical BK B1 receptor antagonist; HOE140 BK B2 receptor subtypeTo further receptor examine antagonists the preliminary (R715 for typical vasodilation BK B1 receptor mechanism antagonist; of RAP HOE140‐L1, T6‐BK, BK specific B2 receptor BK antagonist) were pre‐treated alone or in combination with RAP‐L1, T6‐BK in rat tail artery smooth subtypeantagonist) receptor were antagonists pre-treated alone(R715 orfor in typical combination BK B1 withreceptor RAP-L1, antagonist; T6-BK HOE140 in rat tail BK artery B2 receptor smooth muscle preparations. BK B2 receptor antagonist, HOE‐140, significantly inhibited the RAP‐L1, T6‐BK antagonist)muscle preparations. were pre‐treated BK B2 receptor alone or antagonist, in combination HOE-140, with significantlyRAP‐L1, T6‐ inhibitedBK in rat thetail RAP-L1, artery smooth T6-BK induced relaxation of rat tail artery (p = 0.0002, n = 5), whereas specific BK B1 receptor antagonist muscleinduced preparations. relaxation of BK rat tailB2 receptor artery (p antagonist,= 0.0002, n =HOE 5), whereas‐140, significantly specific BK inhibited B1 receptor the antagonistRAP‐L1, T6 R715‐BK R715 did not show any attenuation effect on RAP‐L1, T6‐BK induced rat tail artery relaxation (p = induceddid not show relaxation any attenuation of rat tail artery effect on(p = RAP-L1, 0.0002, n T6-BK = 5), inducedwhereas ratspecific tail artery BK B1 relaxation receptor (antagonistp = 0.1222, 0.1222, n = 5). In addition, the combined pre‐treatment of HOE 140 and R715 exhibited no significant R715n = 5). did In addition,not show the any combined attenuation pre-treatment effect on RAP of HOE‐L1, 140 T6‐ andBK R715induced exhibited rat tail no artery significant relaxation difference (p = difference with the singular application of HOE 140 (p = 0.7931, n = 5) (Figure 8). These data indicated, 0.1222,with the n singular = 5). In addition, application the of combined HOE 140 (prep =‐ 0.7931,treatmentn = of 5) (FigureHOE 1408). and These R715 data exhibited indicated, no as significant expected, as expected, that the RAP‐L1, T6‐BK ‐induced relaxation of the rat tail arterial smooth muscle was differencethat the RAP-L1, with the T6-BK singular -induced application relaxation of HOE of the 140 rat (p tail = 0.7931, arterial n smooth= 5) (Figure muscle 8). These was most data likely indicated, to be most likely to be mediated by BK B2 receptors. asmediated expected, by that BK B2 the receptors. RAP‐L1, T6‐BK ‐induced relaxation of the rat tail arterial smooth muscle was most likely to be mediated by BK B2 receptors.

Figure 8. Rat tail artery smooth muscle tissues were pre‐treated with R175 (10−6 M) or HOE140 (10−6 M) −5 orFigureFigure their 8.combination 8. RatRat tail tail artery artery as smoothindicated smooth muscle followed muscle tissues tissuesby administrationwere were pre‐treated pre-treated of with 10 MR175 with RAP (10 R175‐L1,−6 M) (10T6 or‐−BK. 6HOE140M) (*** or p < HOE140 (10 0.001).−6 M) Allor(10 their −data6 M) combinationpoints or their represent combination as indicated the mean as followed indicated± SEM of by five followed administration applications. by administration of 10−5 M RAP of‐L1, 10− T65 M‐BK. RAP-L1, (*** p < 0.001). T6-BK. All(*** datap < 0.001). points All represent data points the mean represent ± SEM the of mean five applications.± SEM of ﬁve applications. The analogue RAP‐L1, T6, L8‐BK showed no effect on rat tail artery smooth muscle preparations −11 −5 from The1 × 10analogue to 1 × RAP 10 ‐ ML1, (Figure T6, L8‐ BKS1). showed However, no effectwhen onrat rat tail tail artery artery was smooth pre‐treated muscle with preparations RAP‐L1, The analogue RAP-L1, T6, L8-BK−11 showed no−6 effect on rat tail artery smooth muscle preparations T6, L8‐BK dose−11 range −from5 1 × 10 M to 1 × 10 M followed by administration of BK at maximal fromfrom 1 1 ×× 1010− to11 1to × 110× M10 −(Figure5 M (Figure S1). However, S1). However, when rat when tail artery rat tail was artery pre‐ wastreated pre-treated with RAP with‐L1, effective concentration 10−6 M as described previously [9], maximum inhibition of BK‐induced T6, L8‐BK dose range from 1 × 10−11 M to× 1 × −1011−6 M followed× −by6 administration of BK at maximal RAP-L1, T6, L8-BK dose range−6 from 1 −105 M to 1 10 M followed−6 by administration of relaxation was observed at−6 10 M and 10 M. Hence, we selected 10 M as the most effective effectiveBK at maximal concentration effective 10 concentration M as described 10−6 M previously as described [9], previously maximum [ 9inhibition], maximum of inhibitionBK‐induced of inhibitoryrelaxation concentrationwas observed onat 10rat−6 tailM andartery 10− 5smooth M. Hence, muscles we selected (Figure 109).−6 InM theas the second most serieseffective of −6 experiments,inhibitory concentration when the rat ontail ratartery tail smooth artery musclesmooth preparations muscles (Figure were treated9). In thewith second 10 M seriesRAP‐L1, of experiments, when the rat tail artery smooth muscle preparations were treated with 10−6 M RAP‐L1,

Toxins 2016, 8, 283 8 of 15

BK-induced relaxation was observed at 10−6 M and 10−5 M. Hence, we selected 10−6 M as the most Toxinseffective 2016 inhibitory, 8, 283 concentration on rat tail artery smooth muscles (Figure9). In the second series8 of of 15 Toxinsexperiments, 2016, 8, 283 when the rat tail artery smooth muscle preparations were treated with 10−6 M RAP-L1,8 of T6, 15 T6, L8‐BK followed by the application of BK concentration range from 10−11 M to 10−5 M, the potency L8-BK followed by the application of BK concentration range from 10−11 M to 10−5 M, the potency of −11 −9 −5 −5 ofT6, BKL8‐inducedBK followed maximum by the applicationrelaxation of BKrat concentrationtail artery smooth range frommuscle−9 10 at M 10 −to5 10M toM, 10the potencyM was BK-induced maximum relaxation of rat tail artery smooth muscle at 10 M to 10 −9 M was dramatically−5 dramaticallyof BK‐induced decreased maximum (nearly relaxation 40%–60%), of rat although tail artery the IC smooth50 values muscle were 1.20 at ±10 0.08 M nM to vs. 10 0.50 M ± 0.56was decreased (nearly 40%–60%), although the IC50 values were 1.20 ± 0.08 nM vs. 0.50 ± 0.56 nM. Similar nM.dramatically Similar to decreased RAP‐L1, (nearlyT6, L8‐BK, 40%–60%), HOE140 although pre‐treatment the IC largely50 values antagonize were 1.20 ±BK 0.08‐induced nM vs. relaxation, 0.50 ± 0.56 to RAP-L1, T6, L8-BK, HOE140 pre-treatment largely antagonize BK-induced relaxation, whereas this whereasnM. Similar this to effect RAP was‐L1, notT6, L8detected‐BK, HOE140 under R715pre‐treatment pre‐treatment largely (Figure antagonize 10). BK‐induced relaxation, effect was not detected under R715 pre-treatment (Figure 10). whereas this effect was not detected under R715 pre‐treatment (Figure 10).

Figure 9. RAP‐L1, T6, L8‐BK dose‐dependent inhibition response observed in the rat artery smooth muscleFigure 9.preparations RAP-L1,RAP‐L1, T6, followed L8-BKL8‐BK dose-dependentdose by ‐presencedependent of inhibition maximal bradykinin response observed effective in concentration the rat artery (10 smooth−6 M). −6 Eachmuscle point preparations represents followed the mean by and presence standard ofof error maximalmaximal of six bradykininbradykinin replicates of effectiveeffective three rats. concentrationconcentration (10(10−6 M).M). Each point represents the mean and standard errorerror ofof sixsix replicatesreplicates ofof threethree rats.rats.

Figure 10. 10. BKBK dose–response dose–response curves curves using using rat arterial rat arterial smooth smooth muscle muscle in the absence in the absence (black) and (black) presence and −6 ofpresenceFigure RAP 10.‐L1, of BK T6, RAP-L1, dose–response L8‐BK T6, (red), L8-BK HOE curves (red), 140 using (violet) HOE rat 140 arterialor (violet)R715 smooth (green) or R715 muscleat a (green) single in the dose at absence a single of 10− 6(black) dose M. of and 10 presenceM. of RAP‐L1, T6, L8‐BK (red), HOE 140 (violet) or R715 (green) at a single dose of 10−6 M. To further assess the receptors that might be involvedinvolved in thethe antagonism,antagonism, co-administrationco‐administration of HOE140To further or R715 assess with the 10− 66receptors MM RAP RAP-L1,‐L1, that T6, T6, might L8 L8-BK‐BK be was wasinvolved employed employed in the prior prior antagonism, to to induction induction co‐administration of of rat rat tail artery of relaxationHOE140 or by by R715 10 10−6− withM6 M BK. BK.10 Consistent−6 M Consistent RAP‐ L1,with withT6, the L8 theprevious‐BK previous was data, employed data, R715 R715 hadprior a had trendto induction a trendto attenuate to of attenuate rat BK’s tail effect,artery BK’s butrelaxationeffect, no but statistic noby 10 statistic −difference6 M BK. difference Consistent was wasdetermined with determined the previous(p = ( p0.0773,= 0.0773,data, n R715 =n 5),= 5),had RAP RAP-L1, a ‐trendL1, T6, to T6, attenuateL8 L8-BK‐BK and and BK’s HOE140 effect, significantlybutsigniﬁcantly no statistic attenuatedattenuated difference rat rat tailwas tail artery determined artery dilation dilation induced(p = induced0.0773, by BK n by (=p = 5),BK 0.0028 RAP (p and‐=L1, 0.0028 0.0006,T6, L8andn‐BK= 5,0.0006, and respectively). HOE140 n = 5, respectively).significantlyCo-administration attenuatedCo‐administration of R715 rat with tail RAP-L1, ofartery R715 T6, dilationwith L8-BK RAP induced did‐L1, not T6, furtherby L8 ‐BKBK enhance (didp = not0.0028 thefurther BK and attenuation enhance 0.0006, then effect, = BK 5, whileattenuationrespectively). co-administration effect, Co‐administration while ofco‐ HOE140administration of withR715 RAP-L1, with of HOE140 RAP T6,‐L1, L8-BK with T6, RAP exhibitedL8‐‐BKL1, didT6, a L8 synergeticnot‐BK further exhibited inhibition enhance a synergetic activitythe BK inhibitionattenuationto almost blockactivity effect, BK-induced whileto almost co‐administration block rat tail BK artery‐induced relaxation of HOE140 rat tail (p arterywith< 0.0001, RAP relaxation‐nL1,= 5)T6, (Figure ( L8p <‐ BK0.0001, 11exhibited). n = 5) a(Figure synergetic 11). inhibition activity to almost block BK‐induced rat tail artery relaxation (p < 0.0001, n = 5) (Figure 11).

Toxins 2016, 8, 283 9 of 15 Toxins 2016, 8, 283 9 of 15

FigureFigure 11. Rat 11. tailRat tail artery artery smooth smooth muscle muscle tissues tissues were were treated treated by BK (10 (10−6− M),6 M), R175 R175 (10 (10−6 M),−6 HOE140M), HOE140 (10−6(10M)−6 singleM) single dose dose or or their their combination combination as indicated (* (* p p< <0.05; 0.05; ** **p

3. Discussion 3. Discussion Frog skin secretions contain large number of bioactive components, including antimicrobial, Frogprotease skin inhibitory, secretions and contain pharmacological large number relevant of peptides. bioactive As we components, stated in the includingintroduction, antimicrobial, discrete proteaseBRP inhibitory,may have unique and pharmacological ligand‐receptor relevantspecificity peptides. and potency, As we which stated is vital in the to introduction,provide valuable discrete BRPstructure may have‐interaction unique ligand-receptorrelationship information specificity for agonistic and potency, and antagonistic which is research. vital to Therefore, provide valuable we structure-interactiondecided to focus on relationship “shotgun cloning” information novel BRP for agonisticby using BK and‐derived antagonistic degenerate research. primer in Therefore, this we decidedproject. In to addition, focus on BRPs “shotgun from amphibian cloning” secretions novel BRP and venoms by using are BK-derivedconsidered to degeneratebe homologous primer in thiscounterparts project. Inof addition,endogenous BRPs mammalian from amphibian polypeptide secretions hormones. and Therefore, venoms are study considered on their to be physiological and pathological relevance is essential for us to understand metabolic homeostasis in homologous counterparts of endogenous mammalian polypeptide hormones. Therefore, study on vivo, which could provide us novel insight into cardiac, vascular, renal and inflammatory associated theirdisorders physiological [10–13]. and pathological relevance is essential for us to understand metabolic homeostasis in vivo, whichIn this could study, provide we describe us novel the structural insight into and cardiac, functional vascular, characterizations renal and inflammatory of a novel BRP associated first disordersidentified [10– 13in ].the skin secretions of Chinese Large frog, Odorrana livida, through “shotgun” cloning of Ina biosynthetic this study, precursor we describe‐encoding the cDNA structural from a and lyophilised functional skin characterizationssecretion‐derived cDNA of a library novel BRP first(Figure identified 1). Both in the the skinopen‐ secretionsreading frame of nucleotide Chinese Large sequences frog, andOdorrana amino acid livida sequences, through encoding “shotgun” cloningthe ofprecursors a biosynthetic of BRPs precursor-encoding from Amolops and cDNA Odorrana from (Figure a lyophilised 4) elicited skin unparalleled secretion-derived similarity cDNA library(identity: (Figure 64%–90%),1). Both which the open-reading suggests a possible frame evolutionary nucleotide connection sequences between and amino these two acid genera sequences of encodingfrogs the[14]. precursors The mature of novel BRPs BRP, from RAPAmolops‐L1, T6‐andBK, hasOdorrana a three (Figureresidue 4N)‐terminal elicited unparalleledextension RAP, similarity and two site‐substitutions L1, T6‐BK compared with canonical BK. In addition, the T6‐BK was found (identity: 64%–90%), which suggests a possible evolutionary connection between these two genera among several species of reptiles such as crocodilians, chelonians, and varanid lizards which contain of frogswhole [14 components]. The mature of the novel kallikrein BRP,‐kinin RAP-L1, system T6-BK, in mammals has a [6]. three Threonine residue 6 Nis -terminalone of the extensionmost RAP,common and two site site-substitutions‐substitutions of amphibian L1, T6-BK skin compared secretion with‐derived canonical BRPs, which BK. Inreflects addition, their evolutional the T6-BK was foundadaptations among several to particular species predators of reptiles [15]. such It is believed as crocodilians, that the replacement chelonians, of and Threonine varanid at position lizards which6 containof BK whole is associated components with enhanced of the kallikrein-kinin effect of contracting system rat ileum in mammals with equivalent [6]. Threonine potent to relax 6 is rat one of the mosttail artery common compared site-substitutions with BK [16,17]. of amphibian Meanwhile, skin N‐terminal secretion-derived or C‐terminal BRPs, extended which BRPs reflects are their evolutionalcommonly adaptations identified to from particular amphibian predators secretions [15]. and It is venoms. believed This that study the replacement reported the of ‐ ThreonineRAP‐ at positionextension 6 at of N BK‐terminal is associated end BRP from with Odorrana enhanced livida effect. The ofBRPs contracting Amolopkinin rat‐W1/W2 ileum isolated with equivalent from potentAmolops to relax wuyiensis rat tail exhibited artery comparedhigh degree withof sequence BK [16 similarity,17]. Meanwhile, with RAP‐L1,N-terminal T6‐BK, with or theC-terminal N‐ terminal extension motif structure ‐RAA‐ and ‐RVA‐, respectively. Both the Amolopkinin‐W1 and extended BRPs are commonly identified from amphibian secretions and venoms. This study reported Amolopkinin‐W2 exhibited inhibitory effect of BK‐induced intestinal smooth muscle contraction, while the -RAP-no direct extension effects on at bladder N-terminal and uterine end BRP smooth from muscleOdorrana preparations livida. Thewere BRPs detected Amolopkinin-W1/W2 [14]. Despite the isolated from Amolops wuyiensis exhibited high degree of sequence similarity with RAP-L1, T6-BK, with the N-terminal extension motif structure -RAA- and -RVA-, respectively. Both the Amolopkinin-W1 and Amolopkinin-W2 exhibited inhibitory effect of BK-induced intestinal smooth muscle contraction, while no direct effects on bladder and uterine smooth muscle preparations were detected [14]. Despite Toxins 2016, 8, 283 10 of 15 the fact that the precise functions of diverse N- or C-terminal extension of BRPs still remains elusive, they are predicted to be involved in prolonging the interaction time of these peptides as ligands with target receptors, which could be adopted as a defensive strategy by host to survival [18,19]. In terms of artery relaxation, considering the fact that a lot of vasodilators were involved in the release of nitric oxide (NO) through a common downstream signalling, and NO is widely accepted as the final substance that induces smooth muscle relaxation and inflammatory status [20]. The eNOS (NO releaser) inhibitor, L-NIO, was pre-treated in arterial smooth muscle to test whether the well-established vasodilation via BK induction was influenced. Since the result did not show any significant difference, it implied that NO is not possible to be associated with this ex vivo rat tail artery smooth muscle model. The myotropic potency of this novel identified RAP-L1, T6-BK peptide is relatively weak compared to BK, not only in contraction of rat bladder, ileum and uterus, but also in dilating vessels. Whilst, the novel peptide seemed to exhibited higher efficacy for the receptors compared to BK, which could be deduced from the Figure7d that at the concentration of 10 −7 M, RAP-L1, T6-BK have similar vasodilate capacity with BK, and when the concentration raised to 10−6 M and 10−5 M, RAP-L1, T6-BK dramatically enhanced the vasodilation on rat tail artery smooth muscle compared to BK, which indicated that the threshold concentration for RAP-L1, T6-BK and BK to induce a significant rat tail artery relax is differentiated, BK is more potency at lower concentration (10−9 M–10−7 M), but RAP-L1, T6-BK is more effective at higher concentration (10−6 M–10−5 M). In consideration of this, the primary structure similarity between them proposed us the hypothesis that RAP-L1, T6-BK mediates these effects through the cognate G protein-coupled receptors, and thus, we examined the arterial smooth muscle relaxing property modulated by RAP-L1, T6-BK in the presence of HOE140, a well-established BK B2 antagonist and R715, a selective BK B1 antagonist. Surprisingly, we observed a significant attenuation (nearly 60%) in HOE140 treatment condition, while R715 barely antagonize RAP-L1, T6-BK, which suggests a BK B2 receptors-related mechanism. Further experiments are still needed to address this issue thoroughly. Additionally, stimulation of neurokinin receptors in rat bladder, muscarinic receptors in ileal and endothelin receptors in uterine smooth muscle, which leads to intracellular calcium concentration, are reported to be involved in the contractions of these tissues [21–25], the relationship between RAP-L1, T6-BK and these receptors are worthy to be investigated in the future. The analogue peptide RAP-L1, T6, L8-BK completely abrogated those bioactivities. Of interest is that only the leucine residue substitution at position 8 of BK would cause such an astonishing function loss. Leucine 1 is a common substitution occurred in BRPs, compared with valine substation at position 1, it normally exhibits moderate agonistic effect of BK [26]. As the fact that Leu8-BK was reported as a BK receptor antagonist [27], we performed further experiments to examine whether RAP-L1, T6-BK and its analogue RAP-L1, T6, L8-BK could inhibit BK-induced relax on rat tail artery smooth muscle, the results revealed that, as expected, RAP-L1, T6-BK did not show BK inhibition property (Figure S1), but RAP-L1, T6, L8-BK significantly reduced BK relaxation activity on rat tail artery nearly 50% in the concentration range 10−8 M–10−5 M. These data suggested that although this novel BRP has a three residues -RAP- extension and leucine 1, threonine 6 substitutions, the leucine 8 still plays the essential and dominant role in reversing the BK’s effect. To investigate the precise manner how RAP-L1, T6, L8-BK exert BK antagonistic effect, again, we pre-treated rat tail artery smooth muscle with specific BK B1 receptors and/or B2 receptors inhibitors. When the tissues pre-treated with R157, BK-induced artery relaxation has a trend to be attenuated, but no statistical differences were observed. In contrast, when the tissues were pre-treated with HOE140, BK-induced artery relaxation was largely impaired. This probably due to that B1 receptors are rare expressed in rat tail artery or B2 receptor-mediated signalling is mainly responsible for BK-induced relaxation in rat tail artery, further examination by pre-treating the tissues with RAP-L1, T6, L8-BK along or together with R157 showed similar BK inhibition potency, which implies that the peptide might exert the effect in a B2 receptors-dependent manner. While RAP-L1, T6, L8-BK pre-treatment along with HOE140 almost abolished BK-induced relaxation suggested a novel synergistic inhibition mechanism via the Toxins 2016, 8, 283 11 of 15

BK B2 receptors might be involved compared with HOE140. In terms of this additive effect, others receptors, rather than BK receptors, activated by RAP-L1, T6, L8-BK is also a feasible option. Therefore, the primary structure initiated ligands-receptor interaction study of BRPs could provide researchers valuable information for developing novel molecular therapeutics. Taken together, due to the inherent characteristics of the physiopathological signiﬁcance of diverse BRPs for their potential to treat multiple vascular, cardiac diseases, they have been focused on for many years. In this project, we reported a novel BRP and its analogue isolated from Chinese large frog Odorrana livida, which presented distinct bio-functionalities, have unique structure/activity relationship, and continuously provides virtues for drug design.

4. Materials and Methods

4.1. Specimen Preparation and Secretion Harvesting Adult Chinese Large Odorous frogs, Odorrana livida (n = 3, the length from snout to vent was 8–10 cm) were obtained from the mountain areas of Fujian province, People’s Republic of China. The gentle electrical stimulation (5 V, 50 Hz, 4 ms plus width) (C.F. Palmer, London, UK) was performed to induce the secretion from the glands of the frog dorsal surface. After secretion harvesting, the frogs were released. This method was non-lethal to frogs. The distilled deionized water was used to rinse the frog skin surface for secretions collection. The secretions were then subjected to snap frozen with liquid nitrogen, lyophilized and stored at −20 ◦C before use. Sampling of skin secretion was performed by Mei Zhou under UK Animal (Scientiﬁc Procedures) Act 1986, project license PPL 2694, issued by the Department of Health, Social Services and Public Safety, Northern Ireland. Procedures had been vetted by the IACUC of Queen’s University Belfast, and approved on 1 March 2011.

4.2. “Shotgun” Cloning of a Novel BRP Encoded cDNA Library Five milligrams of lyophilized Odorrana livida skin secretion was dissolved in lysis/Binding Buffer. The extraction of polyadenylated mRNA was performed by using Dynabeads® mRNA DIRECT™ Kit (Dynal Biotech, Liverpool, UK). The cDNA library was obtained in reverse transcription reaction. Both 30-RACE and 50-RACE PCRs were employed to obtain the novel full-length prepro-BRP nucleic acid sequence by using SMART-RACE kit (Clontech, Oxford, UK). Briefly, the 30-RACE PCR reaction employed a specific sense primer (50-CCRVCNGGGTTYASSCCWTTY-30) (R = A/G; V = A/C/G; N = A/C/T/G; Y = C/T; S = C/G; W = A/T) that was complementary to the nucleic acid sequence encoding the common -PPGFSPF- and -PPGFTPF- internal amino acid sequences of ranid frog skin BRPs and a UPM primer (supplied with the kit). The 30-RACE reactions were purified and cloned into pGEM®-T Easy Vector (Promega Corporation, Southampton, UK) and sequenced using an ABI 3100 automated capillary sequencer. The 50-RACE PCR employed an antisense primer (AS: 50-CCGTATGTCATTTAGCTAAATGATGA-30), which was designed to a highly conserved domain located within the 30 non-translated region of the obtained 30-RACE transcripts, and a UPM primer. In addition, the 50-RACE products were gel-purified, cloned, and sequenced.

4.3. Identification and Structure Characterization of a Novel BRP from the Odorrana livida Skin Secretion An aliquoted sample of five milligrams lyophilized Odorrana livida skin secretion was dissolved in 1 mL trifluoroacetic acid (TFA)/water (0.05/99.95, v/v), followed by centrifugation to clarify the sample solution. The clear supernatant was decanted and pumped into an analytical reverse phase HPLC column (pheomenex C-18, 250 × 10 mm) for fractionation by using a Cecil Adept 4200 HPLC system (Amersham Biosciences, Buckinghamshire, UK). The linear gradient elute method was used from TFA/water (0.05/99.95, v/v) to TFA/water/Acetonitrile (0.05/19.95/80.00, v/v/v) over 240 min at a flow rate of 1 mL/min. The fractions were collected automatically at one-minute intervals; the effluent absorbance was continuously monitored at 214 nm. Fractions were lyophilized and stored at −20 ◦C prior to being subjected to the smooth muscle assay. Meanwhile, the molecular Toxins 2016, 8, 283 12 of 15 masses of polypeptides in the collected fractions were analysed through matrix-assisted, laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) on a linear time-of-flight Voyager DE mass spectrometer (PerSeptive Biosystems, Framingham, MA, USA). The sample was analysed in positive detection mode using α-cyano-4-hydroxycinnamic acid as the matrix. Internal instrument calibration was performed by using standard peptides of established molecular masses, which provides the determined accuracy of ±0.1%. Fractions with the masses coincidence with those deduced mature peptides from the molecular cloning were subjected to MS/MS fragmentation sequencing analysis by using a Thermoquest gradient reversed phase HPLC system (Thermo Fisher Scientific, San Francisco, CA, USA), fitted with an analytical column (C-18), and interfaced with a Thermoquest LCQ™ Deca electrospray ion-trap mass spectrometer (Thermo Fisher Scientific, San Francisco, CA, USA).

4.4. Chemical Synthesis of Novel BRP (RAP-L1, T6-BK) and Its Modified Analogue RAP-L1, T6, L8-BK Once the unequivocal primary structure of the novel bradykinin-related peptide had been determined through MS/MS fragmentation sequencing and molecular cloning of its biosynthetic precursor-encoding cDNA, Fmoc chemistry based solid phase peptide synthesis was performed to produced RAP-L1, T6-BK peptide replicates by using Tribute® Peptide Synthesizer (Protein Technologies, Inc., Tucson, AZ, USA). The analogue RAP-L1, T6, L8-BK was also synthesized with the same method. All synthetic peptides were purified with reverse phase HPLC. The authenticity of their primary structures and purity were confirmed via MALDI-TOF MS and LCQ MS/MS fragmentation sequencing.

4.5. Preliminary Study of Inherent Cytotoxicity of the Novel BRPs Prior to the smooth muscle assay for evaluating the pharmacological actions of BRPs, the MTT cell viability assay was performed. Cultured human microvessel endothelial cells (HMECs) were used to assess the possible inﬂuence of inherent cytotoxicity caused by the synthetic novel BRPs. In detail, the HMECs were seeded in 96-well plates with the density 5000 cells/well. After overnight incubation, HMECs were subjected to 6 h starvation prior to being treated with synthetic BRPs (RAP-L1, T6-BK and RAP-L1, T6, L8-BK) ranging from 10−11 M to 10−5 M for 24 h. Cell viabilities were assessed by MTT assay, as described previously [28].

4.6. Ex Vivo Rat Bladder, Ileum, Uterus and Tail Artery Smooth Muscle Pharmacology Wistar rats (200–250 g) were euthanized by asphyxiation with carbon dioxide in accordance with institutional animal experimentation ethics and UK animal research guidelines. The rats were placed on their dorsal surfaces, an incision was made along the mid ventral line towards the bottom of the abdomen and subcutaneous fat was carefully removed. The smooth muscle tissues of bladder, ileum, uterus, and tail artery were dissected gently followed by being placed in ice-cold Krebs’ solution immediately (118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.15 mM NaH2PO4, 2.5 mM CaCl2, 1.1 mM MgCl2 and 5.6 mM glucose), and equilibrated with a mixed gas (95% CO2 and 5% O2). The tissues were dissected and mounted as shown in the table to obtain maximum viability and to achieve the best response (Table1).

Table 1. The cutting size, mounting way and basal tension of rat smooth muscle tissues.

Smooth Muscle Tissues Cutting Size Mounting Way Basal Tension (g) 10 mm × 2 mm Bladder Hooked longitudinally (length × width) Uterus Entire Uterine horns Mounted horizontally 0.5–1.0 Ileum 0.5 cm length ring Mounted horizontally Proximal rat tail artery 2 mm width ring Mounted horizontally Toxins 2016, 8, 283 13 of 15

After dissection, small strips of particular tissues were connected to a triangular hook. Then, the removed tissues were mounted to the force transducers in a 2 mL organ bath which were perfused with Krebs’ solution (118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.15 mM NaH2PO4, 2.5 mM CaCl2, ◦ 1.1 mM MgCl2 and 5.6 mM glucose) at 37 C and constantly bubbling 95% O2 and 5% CO2 gas mixture. The prepared tissues were attached to the force transducer in order to detect the tension changes. The responses of tension change were recorded and ampliﬁed through pressure transducers connected to a PowerLab System (AD Instruments Pty Ltd., Oxford, UK). The peptide solutions of RAP-L1, T6-BK and RAP-L1, T6, L8-BK were prepared in the range of 10−11 M to 10−5 M with fresh Krebs’ solutions to construct the dose–response curves. Each concentration of individual peptide was applied to a minimum of six muscle strips. For the rat tail artery preparations, endothelial linings were essentially removed from the dissected smooth muscle, a basal tension of 0.5 g was applied to the organ bath incubated tail artery before adding phenylephrine (1 × 10−5 M) for 10 min to achieve constriction plateaux. To further exclude the possible vasorelaxant effect caused by endothelial-derived nitric oxide (NO), tail artery smooth muscles were pre-treated with/without 0.5 µM eNOS (endothelium nitric oxide synthase) inhibitor N5-(1-Iminoethyl)-L-ornithine dihydrochloride (L-NIO, Tocris Biosciences, San Diego, CA, USA) for 20 min, prior to performing a BK sequential concentration (10−11 M to 10−5 M) -induced vasodilation. For urinary bladder and ileum smooth muscle preparations, the bladder and ileum strips were gradually exposed to increasing tension until 0.5 g was reached and maintained. The preparations were then exposed to peptides as described above and relative changes in tension were recorded. For uterus smooth muscle preparations, female Wistar rats were euthanized by carbon dioxide. The dissected uterine horns were mounted in each organ bath, incubated with 37 ◦C Krebs’ solution for 10 min with no tension. The uterus strips were gradually exposed to increasing tension until 0.5 g was reached and maintained. The uterus smooth muscle preparations were exposed to peptides as described previously and, changes in spontaneous contraction frequencies, instead of tension change, were recorded. To examine what type of BK receptors in rat tail artery smooth muscle were targeted by BRPs, a single 10−6 M dose of BK B1 receptor antagonist R715 and/or the BK B2 receptor antagonist, HOE140 (Sigma Aldrich, Irvine, UK) were applied to artery smooth muscle preparations prior to peptide treatment. Data from this study were analysed by one-way ANOVA with Bonferroni’s post-test and two-way ANOVA using GraphPad Prism software (version 5.01, San Diego, CA, USA). A p value less than 0.05 was considered signiﬁcant.

Supplementary Materials: The following are available online at www.mdpi.com/2072-6651/8/10/283/s1, Figure S1: Dose–response curves of administration of BK and BRPs using rat arterial smooth muscle. Figure S2: MALDI-TOF (Perceptive Biosystem, Bedford, MA, USA) mass spectrum of synthetic peptide RAP-L1, T6, L8-BK. Author Contributions: Conceived of and designed the experiments: Tianbao Chen, Yuxin Wu, and Lei Wang. Performed the experiments: Jie Xiang, and Chengbang Ma. Analysed the data: Jie Xiang, Yuxin Wu, and Lei Wang. Contributed reagents/materials/analysis tools: Hui Wang, Shaodong Guo, and Mei Zhou. Wrote the paper: Yuxin Wu and Jie Xiang. Edited the paper: Tianbao Chen, Yuxin Wu, and Chris Shaw. Conﬂicts of Interest: The authors declare no conﬂict of interest. Data Deposition Footnote: The nucleotide sequence of the cDNA encoding the novel bradykinin-related peptide RAP-L1, T6-BK precursor from the skin secretion of Odorrana livida, has been deposited in the EMBL Nucleotide Sequence Database under the accession code: LT576450.

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