The structures of four bombesins and their cloned precursor-encoding cDNAs from acid-solvated skin secretion of the European yellow-bellied toad, Bombina variegata

Peptides 36 (2012) 221–229

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The structures of four bombesins and their cloned precursor-encoding cDNAs from acid-solvated skin secretion of the European yellow-bellied toad, Bombina variegata夽 Bing Bai a,b,1 , Hui Wang a,b,1 , Yilu Xue b , Youjia Wu b , Mei Zhou b , Minjie Wei a , Tianbao Chen b,∗ , Lei Wang a,b,∗∗ , Chris Shaw b a b

School of Pharmaceutical Sciences, China Medical University, Shenyang 110001, Liaoning, China Natural Drug Discovery Group, School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK

a r t i c l e

i n f o

Article history: Received 18 May 2012 Received in revised form 31 May 2012 Accepted 31 May 2012 Available online 9 June 2012 Keywords: Amphibian Skin Peptide Bombesin Smooth muscle Cloning

a b s t r a c t Four different bombesins (bombesin, His6 -bombesin, Phe13 -bombesin and Asp2 -, Phe4 -SAP-bombesin) have been identified by a systematic sequencing study of peptides in reverse phase HPLC fractions of the skin secretion of the European yellow-bellied toad, Bombina variegata, that had been solvated in 0.1% (v/v) aqueous trifluoroacetic acid (TFA) and stored frozen at −20 ◦ C for 12 years. By using a 3 - and 5 -RACE PCR strategy, the corresponding biosynthetic precursor-encoding cDNAs of all four peptides were cloned from a cDNA library made from the same long-term frozen, acid-solvated skin secretion sample following thawing and lyophilization. Canonical bombesin and His6 -bombesin are classical bombesin sub-family members, whereas Phe13 -bombesin and Asp2 -, Phe4 -SAP-bombesin, belong to the litorin/ranatensin subfamily of bombesin-like peptides (BLPs). Assignment of these peptides to respective sub-families, was based upon both their primary structural similarities and their comparative pharmacological activities. An interesting observation in this study, was that the nucleotide sequences of the open-reading frames of cloned cDNAs encoding bombesin and its His6 -substituted analog, were identical except for a single base that was responsible for the change observed at the position 6 residue in the mature peptide from Asn to His. In contrast, the precursor cDNA nucleotide sequences encoding the Phe13 -bombesins, exhibited 53 base differences. The pharmacological activities of synthetic replicates of each bombesin were compared using two different mammalian smooth muscle preparations and all four peptides were found to be active. However, there were significant differences in their relative potencies. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Bombesin, a tetradecapeptide that terminates in a C-terminal methioninamide, was originally isolated from the skin of the European fire-bellied toad, Bombina bombina, as an agent that was capable of inducing smooth muscle contraction [1]. Subsequently, two bombesin-like peptides, gastrin-releasing peptide

夽 The nucleotide sequences have been deposited in EMBL Nucleotide Sequence Database under the accession codes, HE608246 (bombesin), HE608247 (His6 -bombesin), HE794026 (Phe13 -bombesin) and HE794027 (Asp2 -, Phe4 -SAPbombesin). ∗ Corresponding author. Tel.: +44 0 2890 972200; fax: +44 0 2890 247794. ∗∗ Corresponding author at: School of Pharmaceutical Sciences, China Medical University, Shenyang 110001, Liaoning, China; Natural Drug Discovery Group, School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK. Tel.: +44 0 2890 972200; fax: +44 0 2890 247794. E-mail addresses: [email protected] (T. Chen), [email protected] (L. Wang). 1 These authors contributed equally to the work. 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.05.020

(GRP) and neuromedin B (NMB), were identified in extracts of mammalian gastric tissues and spinal cord, respectively [12,13]. Many bombesins and bombesin-like peptides have been isolated from amphibian skins, especially those from frogs and toads [6,16]. Such is the number of known structures of these peptides, which they have been divided into three sub-families based on discrete structural relationships and to a lesser extent, on pharmacological actions. These are the bombesin subfamily, the litorin/ranatensin subfamily and the phyllolitorin subfamily [5]. GRP and neuromedin B (NMB) are the counterparts of bombesins and litorin/ranatensinlike peptides in mammalian tissues and although they both occur in different molecular forms, they represent the only mammalian bombesin-like peptides known [16]. Together, these peptides possibly possess the broadest spectrum of biological activities of any group of endogenous mammalian neuro-endocrine peptides and they mediate such through classical G-protein-coupled receptors that include the NMB-preferring receptor (BB1 ), the GRP-preferring receptor (BB2 ) and the bombesin receptor-subtype-3 (BB3 ). These G-protein coupled receptors are expressed in many mammalian

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tissues such as the brain, but especially in the gastrointestinal (GI) tract. However, the BB3 -receptor is an exception as it is only expressed within the central nervous system [8,9,16]. Many studies on mammalian bombesin-like peptides have revealed that GRP and NMB are widely distributed in the central nervous system and peripheral tissues, particularly in the gastrointestinal tract [8,9,16]. GRP is found predominantly in neurons of the submucosal and myenteric plexuses. NMB is also found in the gastrointestinal tract in significantly lower amounts than GRP. Bombesin and bombesin-like peptides have a variety of biological functions, including smooth muscle contraction, stimulation of the growth of carcinoma cell lines, stimulation of secretion of other gastrointestinal hormones and regulation of appetite, feeding behavior and thermoregulation [8,9,11,20,21]. Here, we report the identification and characterization of four bombesins, including canonical bombesin, His6 -bombesin, Phe13 -bombesin and Asp2 -, Phe4 -SAP-bombesin, from European yellow-bellied toad (Bombina variegata) skin secretion with subsequent molecular cloning of their respective biosynthetic precursor-encoding cDNAs. All peptides were present in reverse phase HPLC fractions of skin secretion and respective precursorencoding cDNAs were cloned from a cDNA library constructed from skin secretion material that had been stored acid-solvated and kept frozen for 12 years. The pharmacological activities of synthetic replicates of each bombesin were compared using both rat urinary bladder and rat uterus smooth muscle preparations. 2. Materials and methods 2.1. Collection and storage of B. variegata skin secretion Specimens of B. variegata (n = 12) were obtained from a commercial source as captive-bred metamorphs and were raised to maturity in vivaria for a period of 18 months. The skin secretions were obtained from the toads by gentle electrical stimulation (4 ms pulse width, 50 Hz, 5 V), using platinum electrodes rubbed over the moistened dorsal skin surface for 10 s intervals [18]. Secretions were washed from the skin using deionized water, snap frozen in liquid nitrogen and lyophilized. The sample used in the present study represented 15 mg dry weight of lyophilized skin secretion that had been dissolved in 5 ml of trifluoroacetic acid (TFA)/water (0.1:99.9, v/v), clarified by centrifugation and the decanted supernatant frozen at −20 ◦ C and stored in this state for 12 years. A sample (0.5 ml) was originally removed for reverse phase HPLC analysis prior to freezing of the remainder. The frozen sample was removed from the freezer, thawed at room temperature, then snapfrozen in liquid nitrogen prior to lyophilization. Approximately 12.5 mg dry weight of skin secretion residue was recovered following this procedure. 2.2. Reverse phase HPLC fractionation of skin secretion Five milligrams of the lyophilized skin secretion residue were dissolved in 0.5 ml of 0.05/99.95 (v/v) trifluoroacetic acid (TFA)/water and re-clarified by centrifugation. The supernatant was decanted and directly subjected to reverse-phase HPLC fractionation using a Cecil CE4200 Adept (Cambridge, UK) gradient reverse phase HPLC system fitted with an analytical column (Phenomenex C-5, 0.46 cm × 25 cm). The linear elution gradient employed was formed from 0.05/99.5 (v/v) TFA/water to 0.05/19.95/80.0 (v/v/v) TFA/water/acetonitrile in 240 min at a flow rate of 1 ml/min. Samples of 100 ␮l from each chromatographic fraction were removed, lyophilized and stored at −20 ◦ C prior to analysis of myoactivity using rat urinary bladder and uterus smooth muscle bioassays.

2.3. Identification and structural characterization of the four bombesins Reverse phase HPLC fraction #s 129, 130, 134 and 139 were found to possess contractile activity on the rat urinary bladder smooth muscle strips (for details of assays see Section 2.6) and the major peptide present in samples of each fraction was subjected to primary structural analysis by MS/MS fragmentation sequencing using a LCQ-Fleet mass spectrometer. 2.4. Molecular cloning of respective bombesin precursor-encoding cDNAs Polyadenylated mRNA was isolated from 5 mg of lyophilized, acid-stored skin secretion dissolved in stabilization buffer, using magnetic oligo-dT beads as described by the manufacturer (Dynal Biotech, UK), and was subsequently reverse-transcribed. The resultant cDNA library was subjected to 3 -rapid amplification of cDNA ends (RACE) procedures to obtain full-length skin bombesin precursor-encoding nucleic acid sequence data using a SMARTRACE kit essentially as described by the manufacturer (Clontech, UK). Briefly, the 3 -RACE reactions employed an UPM primer (supplied with the kit) and degenerate sense primers (S1: 5 CARCARMGIYTIGGIMAYC-3 , S2: 5 -CARCARMGIYTIGGIAAYCA-3 and S3: 5 -CARGAYWSITTYGGIAAYCA-3 ) that were complementary to the N-terminal amino acid sequence, Q-Q-R-L-G-N/H-Q-, of bombesin/H6 -bombesin, QQRLGNQ – of F13 -bombesin and QDSFGNQ – of Asp2 -, Phe4 -SAP-bombesin. PCR products from the 3 -RACE reactions, were gel purified and cloned using a pGEM-T vector system (Promega Corporation) and sequenced using an ABI 3100 automated sequencer. The sequence data obtained from the 3 -RACE product was used to design specific antisense primers; AS1: 5 -GTCCCATGTCTCAGGCACAAATATA3 , AS2: 5 -CCCATGTCTCAGGCACAAATATATT-3 and AS3: 5 GTTGTTGGGGTGGAGAGCGAAAT-3 , to conserved sites within the 3 non-translated regions of bombesin/H6 -bombesin, F13 bombesin and Asp2 -, Phe4 -SAP-bombesin precursor-encoding cDNAs, respectively. 5 -RACE reactions were carried out using these primers in conjunction with the NUP primer and resultant products were purified, cloned and sequenced. 2.5. Solid-phase peptide synthesis of the four bombesins Once unequivocal primary structures and post-translational modifications had been established for the four bombesins, replicates of each peptide were synthesized using solid-phase Fmoc chemistry by means of a PS3 automated solid-phase peptide synthesizer (Protein Technologies, Inc., AZ, USA). Following cleavage from the resin and deprotection, each synthetic peptide was analyzed by both reverse phase HPLC and MALDI-TOF mass spectrometry to establish degree of purity and authenticity. For pharmacological experiments, standardization of the synthetic peptide was achieved by acid hydrolysis of a known gravimetric quantity of lyophilizate followed by amino acid analysis using an Applied Biosystems PTH-amino acid analyzer. 2.6. Rat urinary bladder and uterus smooth muscle pharmacology Male Wistar rats (250–300 g) were euthanized by carbon dioxide asphyxiation followed by cervical dislocation under appropriate UK Government animal licences. The rats were placed dorsal surface down and the abdomen was opened by means of an incision along the mid ventral line and subcutaneous fat was carefully dissected. The exposed urinary bladder was removed from each rat, emptied of urine and placed in ice-cold Kreb’s solution (118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3 , 1.15 mM NaH2 PO4 , 2.5 mM

B. Bai et al. / Peptides 36 (2012) 221–229 Table 1 Alignment of amino acid sequences of the four bombesins isolated from the skin secretion of Bombina variegata. Sites of substituted amino acids are in bold typeface. Bombesin His6 -bombesin Phe13 -bombesin Asp2 -, Phe4 -SAP-bombesin

pQQRLGNQWAVGHLMa pQQRLGHQWAVGHLMa pQQRLGNQWAVGHFMa pQDSFGNQWARGHFMa

CaCl2 , 1.1 mM MgCl2 and 5.6 mM glucose), equilibrated with 95% O2 , 5%CO2 . Muscle strips, 2 mm × 10 mm, were dissected from the bladder under a dissection microscope. These were tied at each end with a fine silk thread (0.2 mm diameter) with one end subsequently attached to a fixed pin and the other to a transducer in a 2 ml organ bath containing Kreb’s solution at 37 ◦ C flowing at 2 ml/min with constant bubbling of 95% O2 , 5%CO2 . After a 20 min equilibration period, muscle strips were tested for viability using 60 mM KCl. Following this, viable preparations were used to screen samples of reverse phase HPLC fractions of B. variegata skin secretion for myoactivity. Subsequently, once the active peptides had been identified, structurally characterized and chemically synthesized, muscle strips were used to analyze dose–response relationships with synthetic replicates of each. Solutions of each synthetic peptide, ranging in concentration from 10−11 to 10−5 M, were made in Kreb’s solution and were used to construct dose–response curves. Solutions of each peptide, in increasing concentration, were added to the bladder muscle strips, which were put under 0.5 g of tension. Between each peptide treatment, strips were subjected to 5 min washes and 5 min equilibration periods. Each concentration of each peptide was applied to a minimum of six muscle strips. Changes in tension of the bladder muscle strips were recorded and amplified through pressure transducers connected to a PowerLab System (AD Instruments Pty Ltd.). Data were analyzed to obtain the mean and standard error of responses by Student’s t-test and dose–response curves were constructed using a “best-fit” algorithm through the data analysis package provided. Responses were plotted as percentages of maximal contraction (60 mM KCl) against final molar concentrations of each peptide present in the organ bath. For uterus smooth muscle preparations, female Wistar rats were used and treated as described previously for males. Uterine horns were removed in their entirety and placed in ice-cold Kreb’s solution which was vigorously aerated with mixture gas (95% CO2 ; 5% O2 ). Each uterine horn was halved and individual strips were mounted in a 2 ml organ bath, perfused with Krebs’ solution at 37 ◦ C 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, in contrast to bladder muscle strips, whose tension changes were recorded, changes in spontaneous contraction frequencies were recorded for these preparations. 3. Results 3.1. Isolation and characterization of bombesin peptides Screening of the reverse phase HPLC fractions (n = 200) of B. variegata skin secretion using smooth muscle bioassays, detected activity in fraction #s 129, 130, 134 and 139 (Fig. 1A). Major peptides in these fractions, whose singly and doubly charged ions were detected following LC/MS analysis, possessed individual parent molecular masses of 1621 Da, 1643 Da, 1653 Da and 1663 Da, respectively (Fig. 1B–E). The primary structures of each peptide were established by MS/MS sequencing (Tables 1 and 2) (later

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confirmed following cloning of respective precursor-encoding cDNAs) as: (1) (2) (3) (4)

pGlu-QRLGNQWAVGHLM-NH2 – bombesin – 1621 Da. pGlu-QRLGHQWAVGHLM-NH2 – His6 -bombesin – 1643 Da. pGlu-QRLGNQWAVGHFM-NH2 – Phe13 -bombesin – 1653 Da. pGlu-DSFGNQWARGHFM-NH2 – Asp2 -, Phe4 -SAP-bombesin – 1663 Da.

The relative abundance of each bombesin, as determined by integrated peak height following reverse phase HPLC fractionation, was His6 -bombesin > Phe13 -bombesin > bombesin > Asp2 -, Phe4 SAP-bombesin. Note: SAP-bombesin was originally reported from the skin of the oriental fire-bellied toad, Bombina orientalis [15], a species that is closely related to B. variegata despite widely disparate global distributions (Central/Southern Europe vs. North-East Asia). The prefix, SAP-, refers to site-substitution of amino acids compared to canonical bombesin with S representing serine at position 3 (substituting for arginine, R), A representing arginine at position 10 (substituting for valine, V) and P representing phenylalanine at position 13 (substituting for leucine, L). This is non-standard nomenclature. Using standardized nomenclature, with arginine represented as R and phenylalanine as F, the peptide should be called SRF-bombesin. However, to avoid confusion, the original nomenclature has been used here. 3.2. Molecular cloning of skin bombesin biosynthetic precursor-encoding cDNAs Four bombesin precursor-encoding cDNAs were consistently cloned from the acid-stored skin secretion-derived libraries of B. variegata (Fig. 2A–D). Two of these cDNAs contained an identical putative signal peptide (MSAIPLNRILPLGFLLIFSFISLSSC-) and each encoded a single copy of a different bombesin peptide. The first encoded canonical bombesin and the second encoded a novel bombesin analog, named (His) H6 -bombesin. The open-reading frames of canonical bombesin and H6 -bombesin precursor-encoding cDNAs consisted of 84 amino acid residues (Fig. 3). The other bombesin precursor-encoding cDNAs were different from these but each encoded a single copy of the peptides, (Phe) F13 -bombesin and Asp2 -, Phe4 -SAP-bombesin. These cDNAs contained open-reading frames consisting of 119 and 120 amino acid residues, respectively (Fig. 4). Alignment of H6 -bombesin and bombesin precursor nucleotide sequences (Fig. 3A) and openreading frame amino acid sequences (Fig. 3B), using the AlignX programme of the Vector NTI Bioinformatics suite (Informax), revealed a very high degree of primary structural similarity of both nucleic acid and amino acid sequence. Fig. 4A and B compares the nucleotide sequences and open-reading frame amino acid sequences of cDNAs encoding the biosynthetic precursors of F13 bombesin and Asp2 -, Phe4 -SAP-bombesin. From this alignment, it can be clearly seen that both of these bombesin precursor-encoding cDNAs display many more differences in both nucleotide sequence and resultant primary structures than those encoding bombesin and its His6 -substituted analog. Also, the common C-terminal tetrapeptide amide, GHFMa, present in F13 -bombesin and Asp2 -, Phe4 -SAP-bombesin, shows unequivocally that both of these peptides are members of the litorin/ranatensin subfamily of bombesin peptides. 3.3. Smooth muscle pharmacology Synthetic replicates of all four bombesins produced dosedependent contractions of urinary bladder smooth muscle and increased spontaneous contractions in uterus smooth muscle

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Fig. 1. (A) Region of reverse phase HPLC chromatogram of Bombina variegata skin secretion indicating elution positions/retention times of smooth muscle-contracting fractions (arrows). (B) Mass spectrum of fraction #129 showing bombesin ((M+2H)2+ = m/z 811.05). (C) Mass spectrum of fraction #134 showing His6 -bombesin ((M + 2H)2+ = m/z 822.24). (D) Mass spectrum of fraction #130 showing Phe13 -bombesin ((M + 2H)+2 = m/z 827.82). (E) Mass spectrum of fraction # 139 showing Asp2 -, Phe4 -SAP-bombesin ((M + 2H)+2 = m/z 832.46).

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Table 2 Electrospray ion-trap MS/MS fragmentation datasets derived from ions corresponding in molecular mass to (A) bombesin, (B) His6 -bombesin, (C) Phe13 -bombesin and (D) Asp2 -, Phe4 -SAP-bombesin. Singly and doubly charged b-ion and y-ion fragment m/z ratios for each bombesin were predicted using the MS-Product program available on-line through Protein Prospector. Observed fragment ions are indicated in bold type-face and are underlined. #1

b(1+)

b(2+)

Seq.

y(1+)

y(2+)

#2

A 1 2 3 4 5 6 7 8 9 10 11 12 13 14

113.02333 241.08191 397.18303 510.26710 567.28857 681.33150 809.39008 995.46940 1066.50652 1165.57494 1222.59641 1359.65532 1472.73939

57.01530 121.04459 199.09515 255.63719 284.14792 341.16939 405.19868 498.23834 533.75690 583.29111 611.80184 680.33130 736.87333

E-Gln→pyro-Glu Q R L G N Q W A V G H L M-amidated

1508.79039 1380.73181 1224.63069 1111.54662 1054.52515 940.48222 812.42364 626.34432 555.30720 456.23878 399.21731 262.15840 149.07433

754.89883 690.86954 612.81898 556.27695 527.76621 470.74475 406.71546 313.67580 278.15724 228.62303 200.11229 131.58284 75.04080

14 13 12 11 10 9 8 7 6 5 4 3 2 1

B 1 2 3 4 5 6 7 8 9 10 11 12 13 14

113.02333 241.08191 397.18303 510.26710 567.28857 704.34748 332.40606 1018.48538 1089.52250 1138.59092 1245.61239 1382.67130 1495.75537

57.01530 121.04459 199.09515 255.63719 284.14792 352.67738 416.70667 509.74633 545.26489 594.79910 623.30983 691.83929 748.38132

E-Gln→pyro-Glu Q R L G H Q W A V G H L M-amidated

1531.80637 1403.74779 1247.64667 1134.56260 1077.54113 940.48222 812.42364 626.34432 555.30720 456.23878 399.21731 262.15840 149.07433

766.40682 702.37753 624.32697 567.78494 539.27420 470.74475 406.71546 313.67580 278.15724 228.62303 200.11229 131.58284 75.04080

14 13 12 11 10 9 8 7 6 5 4 3 2 1

C 1 2 3 4 5 6 7 8 9 10 11 12 13 14

113.02333 241.08191 397.18303 510.26710 567.28857 631.33150 809.39008 995.46940 1066.50652 1165.57494 1222.59641 1359.65532 1506.72374

57.01530 121.04459 199.09515 255.63719 284.14792 341.16939 405.19868 498.23834 533.75690 583.29111 611.80184 680.33130 753.86551

E-Gln→pyro-Glu Q R L G N Q W A V G H F M-amidated

1542.77474 1414.71616 1258.61504 1145.53097 1088.50950 974.46657 846.40799 660.32867 589.29155 490.22313 433.20166 296.14275 149.07433

771.89101 707.86172 629.81116 573.26912 544.75839 487.73692 423.70763 330.66797 295.14941 245.61520 217.10447 148.57501 75.04080

14 13 12 11 10 9 8 7 6 5 4 3 2 1

D 1 2 3 4 5 6 7 8 9 10 11 12 13 14

113.02333 228.05028 315.08231 462.15073 519.17220 633.21513 761.27371 947.35303 1018.39015 1174.49127 1231.51274 1368.57165 1515.64007

57.01530 114.52878 158.04479 231.57900 260.08974 317.11120 381.14049 474.18015 509.69871 587.74927 616.26001 684.78946 758.32367

E-Gln→pyro-Glu D S F G N Q W A R G H F M-amidated

1551.69107 1436.66412 1349.63209 1202.56367 1145.54220 1031.49927 903.44069 717.36137 646,32425 490.22313 433.20166 296.14275 149.07433

776.34917 718.83570 675.31968 601.78547 573.27474 516.25327 452.22398 359.18432 323.66576 245.61520 217.10447 148.57501 75.04080

14 13 12 11 10 9 8 7 6 5 4 3 2 1

(Fig. 5A–D). H6 -bombesin was found to be more potent in bladder tissue than canonical bombesin, which was a surprising finding for this single site-substituted analog (Fig. 5A). In the uterus smooth muscle preparation, both peptides were essentially equipotent in terms of increasing the frequency of spontaneous contractions (Fig. 5B). F13 -bombesin displayed essentially the same effect as Asp2 -, Phe13 -SAP-bombesin on the urinary bladder preparation,

however, the maximum tension change observed for Asp2 -, Phe4 SAP-bombesin was around 1.3 g which was greater than the value observed for F13 -bombesin of around 1.0 g (Fig. 5C). F13 -bombesin and Asp2 -, Phe4 -SAP-bombesin exhibited almost equipotent effects in increasing the frequency of spontaneous contractions in the uterus smooth muscle (Fig. 5D). All EC50 values for each peptide on each preparation were in the range of 10−8 –10−7 M.

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A) 1 51 101 151 201 251 301 351

C) 1 51 101 151 201 251 301 351

B) M S A I P L N R I L P L G F L L I ATGTCTGCGA TTCCTCTGAA CAGGATCCTG CCTCTAGGGT TCCTGCTGAT TACAGACGCT AAGGAGACTT GTCCTAGGAC GGAGATCCCA AGGACGACTA · F S F I S L S S C M E F V E D P N TTTCTCCTTC ATCTCTCTGT CCAGCTGCAT GGAGTTCGTT GAAGATCCTA AAAGAGGAAG TAGAGAGACA GGTCGACGTA CCTCAAGCAA CTTCTAGGAT · N Q G G L G L Q Q R L G N Q W A ACAATCAGGG TGGTCTCGGC CTGCAGCAGA GGCTGGGGAA TCAGTGGGCA TGTTAGTCCC ACCAGAGCCG GACGTCGTCT CCGACCCCTT AGTCACCCGT V G H L M G K K S L Q D T D F E E GTGGGTCACT TGATGGGTAA GAAGAGCCTG CAGGACACAG ACTTTGAAGA CACCCAGTGA ACTACCCATT CTTCTCGGAC GTCCTGTGTC TGAAACTTCT · M E S F A K R N V E N M R A A L L GATGGAAAGT TTTGCTAAAC GTAACGTTGA GAACATGAGA GCTGCCCTCC CTACCTTTCA AAACGATTTG CATTGCAACT CTTGTACTCT CGACGGGAGG Q E Q N R A E S E R E L R H A Q TGCAGGAGCA GAACAGAGCA GAATCAGAAA GAGAGCTGCG GCATGCACAG ACGTCCTCGT CTTGTCTCGT CTTAGTCTTT CTCTCGACGC CGTACGTGTC L V V R N I L E Q Y L K N M Q N * TTGGTAGTAA GGAACATCTT GGAGCAGTAT CTGAAGAATA TGCAGAATTA AACCATCATT CCTTGTAGAA CCTCGTCATA GACTTCTTAT ACGTCTTAAT * G C P L N R I L P L G F L F H M S A I ATGTCTGCGA TTCCTCTGAA CAGGATCCTG CCTCTAGGGT TCCTATTTCA TACAGACGCT AAGGAGACTT GTCCTAGGAC GGAGATCCCA AGGATAAAGT · L L I F S F I S L S S C M E F V E CCTGCTGATT TTCTCCTTCA TCTCCCTGTC CAGCTGCATG GAGTTCGTTG GGACGACTAA AAGAGGAAGT AGAGGGACAG GTCGACGTAC CTCAAGCAAC · D P N N R G R I S L Q Q R L G N AAGATCCCAA CAATCGGGGC AGAATCAGCC TGCAGCAGAG GCTGGGGAAT TTCTAGGGTT GTTAGCCCCG TCTTAGTCGG ACGTCGTCTC CGACCCCTTA Q W A V G H F M G K K S L Q D T D CAGTGGGCAG TGGGTCACTT CATGGGTAAG AAGAGCCTAC AGGACACAGA GTCACCCGTC ACCCAGTGAA GTACCCATTC TTCTCGGATG TCCTGTGTCT · F E E M E S F A K R N V E N M R A CTTTGAAGAG ATGGAAAGTT TTGCTAAACG TAACGTTGAG AACATGAGAG GAAACTTCTC TACCTTTCAA AACGATTTGC ATTGCAACTC TTGTACTCTC · A L L Q E Q N R A G S E R E L R CTGCCCTCCT GCAGGAGCAG AACAGAGCAG GATCAGAAAG AGAGCTGCGG GACGGGAGGA CGTCCTCGTC TTGTCTCGTC CTAGTCTTTC TCTCGACGCC H A Q L V V R N I L E Q Y L K N M CATGCACAGT TGGTAGTAAG GAACATCTTG GAGCAGTATC TGAAGAATAT GTACGTGTCA ACCATCATTC CTTGTAGAAC CTCGTCATAG ACTTCTTATA · Q N * GCAGAATTAG CGTCTTAATC

1 51 101 151 201 251 301 351

D) 1

51 101 151 201 251 301 351

M S A I P L N R I L P L G F L L I ATGTCTGCGA TTCCTCTGAA CAGGATCCTG CCTCTAGGGT TCCTGCTGAT TACAGACGCT AAGGAGACTT GTCCTAGGAC GGAGATCCCA AGGACGACTA · F S F I S L S S C M E F V E D P N TTTCTCCTTC ATCTCTCTGT CCAGCTGCAT GGAGTTCGTT GAAGATCCTA AAAGAGGAAG TAGAGAGACA GGTCGACGTA CCTCAAGCAA CTTCTAGGAT · N Q G G L G L Q Q R L G H Q W A ACAATCAGGG CGGTCTTGGC CTGCAGCAGA GGCTGGGGCA TCAGTGGGCA TGTTAGTCCC GCCAGAACCG GACGTCGTCT CCGACCCCGT AGTCACCCGT V G H L M G K K S L Q D T D F E E GTGGGTCACT TGATGGGTAA GAAGAGCCTG CAGGACACAG ACTTTGAAGA CACCCAGTGA ACTACCCATT CTTCTCGGAC GTCCTGTGTC TGAAACTTCT · M E S F A K R N V E N M R A A L L GATGGAAAGT TTTGCTAAAC GTAACGTTGA GAACATGAGA GCTGCTCTCC CTACCTTTCA AAACGATTTG CATTGCAACT CTTGTACTCT CGACGAGAGG · Q E Q N R A E S E R E L R H A Q TGCAGGAGCA GAACAGAGCA GAATCAGAAA GAGAGCTGCG GCATGCACAG ACGTCCTCGT CTTGTCTCGT CTTAGTCTTT CTCTCGACGC CGTACGTGTC L V V R N I L E Q Y L K N M Q N * TTGGTAGTAA GGAACATCTT GGAGCAGTAT CTGAAGAATA TGCAGAATTA AACCATCATT CCTTGTAGAA CCTCGTCATA GACTTCTTAT ACGTCTTAAT G C M S A I P L N R I L P L G F L L I ATGTCTGCGA TTCCTCTGAA CAGGATCCTG CCTCTAGGGT TCCTGCTGAT TACAGACGCT AAGGAGACTT GTCCTAGGAC GGAGATCCCA AGGACGACTA · F S S L S L S S C M E F V E D P N TTTCTCCTCT CTCTCTCTGT CCAGCTGCAT GGAGTTCGTT GAAGATCCTA AAAGAGGAGA GAGAGAGACA GGTCGACGTA CCTCAAGCAA CTTCTAGGAT · N Q G G L N L Q D S F G N Q W A ACAATCAGGG CGGTCTCAAC CTGCAGGACA GCTTCGGGAA TCAGTGGGCA TGTTAGTCCC GCCAGAGTTG GACGTCCTGT CGAAGCCCTT AGTCACCCGT R G H F M G K K S L Q D T D F E E AGGGGTCACT TCATGGGTAA GAAGAGCCTA CAGGACACAG ACTTTGAAGA TCCCCAGTGA AGTACCCATT CTTCTCGGAT GTCCTGTGTC TGAAACTTCT · M E S F D K R N V E N M R A A L L GATGGAAAGT TTTGATAAAC GTAACGTTGA GAACATGAGA GCTGCCCTCC CTACCTTTCA AAACTATTTG CATTGCAACT CTTGTACTCT CGACGGGAGG · Q E Q N R A E S E R E L R H A Q TGCAGGAGCA GAACAGAGCA GAATCAGAAA GAGAGCTGCG GCATGCACAG ACGTCCTCGT CTTGTCTCGT CTTAGTCTTT CTCTCGACGC CGTACGTGTC L V V R N L I E E Y L K T I R N K TTGGTAGTAA GGAACCTCAT TGAAGAGTAC CTGAAGACAA TAAGGAATAA AACCATCATT CCTTGGAGTA ACTTCTCATG GACTTCTGTT ATTCCTTATT · N R L * GAACCGCCTT TAA CTTGGCGGAA ATT

Fig. 2. Nucleotide and translated amino acid sequences of cloned cDNAs encoding the biosynthetic precursors of (A) bombesin, (B) His6 -bombesin, (C) Phe13 -bombesin and (D) Asp2 -, Phe4 -SAP-bombesin. Putative signal peptides are double-underlined, mature peptides are single-underlined and stop codons are indicated by asterisks.

4. Discussion In a previous report, we described that the transcriptomes of the granular glands of many frogs and toads which secrete bioactive peptide-based defensive skin secretions, can be recovered from lyophilized secretions, permitting construction of robust cDNA libraries from which selected peptide precursor-encoding cDNAs can be successfully cloned by PCR procedures [3]. Here, we have examined skin secretion samples that were solvated in TFA/water (0.1%; v/v) and stored frozen at −20 ◦ C for 12 years and have demonstrated that polyadenylated mRNA templates, which are an essential factor for such studies, persist in a format that permits capture, reverse transcription and subsequent PCR-based cloning procedures. A secretion sample from the European yellowbellied toad, B. variegata, was used as a model, as we had samples stored acid-solvated and frozen for many years and this is a species from which a number of skin peptides had both been isolated and cloned [2]. As the sample contained 0.1% TFA/water, in order to make it entirely compatible with the reagents used in cDNA library construction, it was thawed, then immediately snap-frozen in liquid nitrogen and lyophilized. Samples of lyophilized residue were subsequently removed for reverse phase HPLC fractionation of peptides and for polyadenylated mRNA capture and cDNA library construction as a prelude to PCR amplification and molecular cloning of specific peptide-encoding transcripts. The canonical bombinid toad skin peptide, bombesin, was chosen as a model peptide for this study for several reasons. Although representing one of the original peptides found in bombinid toad

skin [1], and subsequently found to occur in several molecular forms in some species, there has been no systematic study performed on B. variegata to date. Bombesins usually possess a Cterminal methioninamide (Ma) residue and an internal sequence tryptophanyl (W) residue [5,6,17], both of which are among the most acid-labile of amino acids and are also subject to oxidation. Chemical modification (such as oxidation) can readily be detected by use of mass spectrometry and can often result in significant lowering if not elimination of bioactivity for peptides such as bombesins [14,19]. Bombesin, originally isolated through its potent activity on a variety of smooth muscle preparations, was targeted through employing smooth muscle screens of reverse phase HPLC fractions of the stored skin secretion. Several myoactive fractions were identified using this approach and through a combination of techniques including MS/MS fragmentation sequencing and molecular cloning of biosynthetic precursor-encoding cDNAs, the primary structures of four different bombesins were unequivocally established. Two of these bombesins, the canonical peptide and its Phe13 -substituted analog, had been described before in B. variegata skin secretion [1] and the others, His6 -bombesin and Asp2 -, Phe4 -SAP-bombesin, were novel. One of these novel peptides, His6 bombesin, belongs to the bombesin subfamily and contains the canonical C-terminal tetrapeptide amide, -GHLM-NH2 . However, the results of smooth muscle bioassays revealed that while both this peptide and canonical bombesin were active in causing contraction of both preparations, His6 -bombesin was more potent in urinary bladder. This result indicates that just a single amino acid

B. Bai et al. / Peptides 36 (2012) 221–229

A)

Bombesin H6-Bombesin Bombesin H -Bombesin 6

Bombesin H6-Bombesin Bombesin H6-Bombesin Bombesin H6-Bombesin Bombesin H6-Bombesin Bombesin H6-Bombesin Bombesin H6-Bombesin Bombesin H6-Bombesin Bombesin H6-Bombesin Bombesin H6-Bombesin Bombesin H6-Bombesin Bombesin H6-Bombesin

227

1 50 (1) ATGTCTGCGATTCCTCTGAACAGGATCCTGCCTCTAGGGTTCCTGCTGAT (1) ATGTCTGCGATTCCTCTGAACAGGATCCTGCCTCTAGGGTTCCTGCTGAT 51 100 (51) TTTCTCCTTCATCTCTCTGTCCAGCTGCATGGAGTTCGTTGAAGATCCTA (51) TTTCTCCTTCATCTCTCTGTCCAGCTGCATGGAGTTCGTTGAAGATCCTA 101 150 (101) ACAATCAGGGTGGTCTCGGCCTGCAGCAGAGGCTGGGGAATCAGTGGGCA (101) ACAATCAGGGCGGTCTTGGCCTGCAGCAGAGGCTGGGGCATCAGTGGGCA 151 200 (151) GTGGGTCACTTGATGGGTAAGAAGAGCCTGCAGGACACAGACTTTGAAGA (151) GTGGGTCACTTGATGGGTAAGAAGAGCCTGCAGGACACAGACTTTGAAGA 201 250 (201) GATGGAAAGTTTTGCTAAACGTAACGTTGAGAACATGAGAGCTGCCCTCC (201) GATGGAAAGTTTTGCTAAACGTAACGTTGAGAACATGAGAGCTGCTCTCC 251 300 (251) TGCAGGAGCAGAACAGAGCAGAATCAGAAAGAGAGCTGCGGCATGCACAG (251) TGCAGGAGCAGAACAGAGCAGAATCAGAAAGAGAGCTGCGGCATGCACAG 301 350 (301) TTGGTAGTAAGGAACATCTTGGAGCAGTATCTGAAGAATATGCAGAATTA (301) TTGGTAGTAAGGAACATCTTGGAGCAGTATCTGAAGAATATGCAGAATTA 351 400 (351) GCAAAGAAATGTGTCTTCCTGTACATACAGAAATATATTTGTGCCTGAGA (351) GCAAAGAAATGTGTCTTCCTGTACATACAGAAATATATTTGTGCCTGAGA 401 450 (401) CATGGGACTTATTTTAAACATTCCAAAGTTTATTGTTTACAAAAAATCCT (401) CATGGGACTTATTTTAAACATTCCAAAGTTTATTGTTTACAAAAAATCCT 451 500 (451) GAATTCTAAAGACAATAAGAATTTTTCATTTATAATTTTAATTTAAGATC (451) GAATTCTAAAGACAATAAGAATTTTTCATTTATAATTTTAATTTAAGATC 501 550 (501) CATTTTCTAAATTTAAAGTATAAAAACAACTCCTCTTCAGAG-TATGTAC (501) CATTTTCTAAATTTAAAGTAAAAAAACAACTCCTCTTCAGAGATATGTAC 551 600 (550) GGAATATTTTTTCTGACATTTTATGCAGTGTTCTAACTAAAACCTGTGAA (551) GGAATATTTTTTCTGACATTTTATGCAGTGTTCTAACTAAAACCTGTGAA 601 650 (600) TAAAAGTCATTCTTTGCTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (601) TAAAAGTCATTCCTTGCACACAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

1 50 (1) MSAIPLNRILPLGFLLIFSFISLSSCMEFVEDPNNQGGLGLQQRLGNQWA (1) MSAIPLNRILPLGFLLIFSFISLSSCMEFVEDPNNQGGLGLQQRLGHQWA 51 100 Bombesin (51) VGHLMGKKSLQDTDFEEMESFAKRNVENMRAALLQEQNRAESERELRHAQ H6-Bombesin (51) VGHLMGKKSLQDTDFEEMESFAKRNVENMRAALLQEQNRAESERELRHAQ 101 116 Bombesin (101) LVVRNILEQYLKNMQN H6-Bombesin (101) LVVRNILEQYLKNMQN B) Bombesin H6-Bombesin

Fig. 3. Alignment of nucleotide sequences of bombesin and His6 -bombesin precursors from Bombina variegata (A). Alignment of translated open-reading frame amino acid sequences of bombesin and H6 -bombesin precursors from Bombina variegata (B). Identical residues are shaded in black.

228

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A)

F13-Bombesin 2 4 D -, F -SAP-Bombesin F13-Bombesin D2-, F4-SAP-Bombesin F13-Bombesin D -, F -SAP-Bombesin 2

4

F13-Bombesin D -, F -SAP-Bombesin 2

4

F13-Bombesin D -, F -SAP-Bombesin 2

4

F13-Bombesin D2-, F4-SAP-Bombesin F13-Bombesin D -, F -SAP-Bombesin 2

4

F13-Bombesin D -, F -SAP-Bombesin 2

4

F13-Bombesin D2-, F4-SAP-Bombesin F13-Bombesin D2-, F4-SAP-Bombesin F13-Bombesin D -, F -SAP-Bombesin 2

4

F13-Bombesin D -, F -SAP-Bombesin 2

4

F13-Bombesin D2-, F4-SAP-Bombesin B) F13-Bombesin 2 4 D -, F -SAP-Bombesin F13-Bombesin D -, F -SAP-Bombesin 2

4

F13-Bombesin D2-, F4-SAP-Bombesin

1 50 (1) ATGTCTGCGATTCCTCTGAACAGGATCCTGCCTCTAGGGTTCCTATTTCA (1) ATGTCTGCGATTCCTCTGAACAGGATCCTGCCTCTAGGGTT--------51 100 (51) CCTGCTGATTTTCTCCTTCATCTCCCTGTCCAGCTGCATGGAGTTCGTTG (42) CCTGCTGATTTTCTCCTCTCTCTCTCTGTCCAGCTGCATGGAGTTCGTTG 101 150 (101) AAGATCCCAACAATCGGGGCAGAATCAGCCTGCAGCAGAGGCTGGGGAAT (92) AAGATCCTAACAATCAGGGCGGTCTCAACCTGCAGGACAGCTTCGGGAAT 151 200 (151) CAGTGGGCAGTGGGTCACTTCATGGGTAAGAAGAGCCTACAGGACACAGA (142) CAGTGGGCAAGGGGTCACTTCATGGGTAAGAAGAGCCTACAGGACACAGA 201 250 (201) CTTTGAAGAGATGGAAAGTTTTGCTAAACGTAACGTTGAGAACATGAGAG (192) CTTTGAAGAGATGGAAAGTTTTGATAAACGTAACGTTGAGAACATGAGAG 251 300 (251) CTGCCCTCCTGCAGGAGCAGAACAGAGCAGGATCAGAAAGAGAGCTGCGG (242) CTGCCCTCCTGCAGGAGCAGAACAGAGCAGAATCAGAAAGAGAGCTGCGG 301 350 (301) CATGCACAGTTGGTAGTAAGGAACATCTTGGAGCAGTATCTGAAGAATAT (292) CATGCACAGTTGGTAGTAAGGAACCTCATTGAAGAGTACCTGAAGACAAT 351 400 (351) GCAGAATTAGCAAAGAAATGTGTCTTCCTGTACATACAGAAATATATTTG (342) AAGGAAT----A-AGAA------CCGCCTTTA-------ATATTGACTGG 401 450 (401) TGCCTGAGACATGGGACTTATTTTAAACATTCCAAAGTTTATTGTTTACT (374) GCCCTGGGCAA----ACATTCCCCTAACCCCCCCCATTCAATTTCGCTCT 451 500 (451) CCTGAATTCTAAAGACAATAAGAACTTTTCATTTATAATTTTAATTCAAG (420) CC---ACCCCAACAACCCAAAAAACAACTAA---ATCAATTTATTT---501 550 (501) ATCCATTTTCTAAATTTAAAGTAAAAAAACAACTCCTCTTCAGAGATATC (460) -TATATATTATTATTTTAAATGATCA--TCCACTGTT-----GAGTTA-551 600 (551) GTACGAATAATTTTTTCTGACATTTTATGCAGTGTGTAACTAAAACGTGT (500) ------ATCATTTT-------------------------CAAAAAAATAT 601 650 (601) GAATAAAAGT-CATTCCTTGCACACAAAAAAAAAAAAAAAAAAAAAAAAA (519) GAAATAAATTGCAATATGATCACAAAAAAAAAAAAAAAAAAAAAAAAAAA

1 50 (1) MSAIPLNRILPLGFLFHLLIFSFISLSSCMEFVEDPNNRGRISLQQRLGN (1) MSAIPLNRILPLG---FLLIFSSLSLSSCMEFVEDPNNQGGLNLQDSFGN 51 100 (51) QWAVGHFMGKKSLQDTDFEEMESFAKRNVENMRAALLQEQNRAGSERELR (48) QWARGHFMGKKSLQDTDFEEMESFDKRNVENMRAALLQEQNRAESERELR 101 123 (101) HAQLVVRNILEQYLKNMQN---(98) HAQLVVRNLIEEYLKTIRNKNRL

Fig. 4. Alignment of nucleotide sequences of Phe13 -bombesin and Asp2 -, Phe4 -SAP-bombesin precursors from Bombina variegata (A). Alignment of translated open-reading frame amino acid sequences of Phe13 -bombesin and Asp2 -, Phe4 -SAP-bombesin precursors from Bombina variegata (B). Identical residues are shaded in black.

substitution can have profound effects on bioactivity. Phe13 bombesin and Asp2 -, Phe4 -SAP-bombesin both belong to the litorin/ranatensin subfamily having the canonical C-terminal tetrapeptide amide, -GHLM-NH2 , which directs such peptides to a different subtype of bombesin receptor in mammalian tissues. There are four amino acid sequence differences between these peptides at positions 2–4 and 10. Comparing the bioactivities of all four bombesin peptides, there were few significant differences in potencies observed in both smooth muscle preparations. These data taken together, would suggest the presence of both major

bombesin receptor subtypes (BB1 (NMB-preferring) and BB2 (GRPpreferring)) on each smooth muscle preparation. The structural diversity of bombesin peptides in the skin secretions of amphibians, demonstrated here in B. variegata and in previous studies of other species [5,6,17], increases the likelihood that in the event of a predator encounter, some components of the defensive secretion will find an appropriate target receptor in the tissues of the predator and cause an effect that is of a lifepreserving benefit to the amphibian. Although mammalian bladder and uterus smooth muscle has been employed here in a model

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Fig. 5. (A) Dose response curves of bombesin and His6 -bombesin using rat urinary bladder smooth muscle preparations. (B) Dose response curves of bombesin and His6 bombesin using rat uterus smooth muscle preparations. (C) Dose response curves of Phe13 -bombesin and Asp2 -, Phe4 -SAP-bombesin using rat urinary bladder smooth muscle preparations. (D) Dose response curves of Phe13 -bombesin and Asp2 -, Phe4 -SAP-bombesin using rat uterus smooth muscle preparations. Each data point represents the mean and standard error of five replicates.

bioassay, GRP/NMB receptors have a much wider tissue distribution in mammals [7] and presumably also in sub-mammalian vertebrates such as birds, reptiles, other amphibians and fishes, that also prey upon frogs/toads. Little however is known about receptor types and their distribution in these taxa. In keeping with this, the identification of natural ligand for the bombesin-like receptor sub-type 3 (BB3 ) remains an elusive goal [10]. The range of bombesin-like peptides present in defensive skin secretions of frogs and toads may parallel the structural diversity established for bradykinin-like peptides from this source [4,17]. It is now well-accepted that the structural diversity of these peptides probably reflects the spectrum of predators encountered by individual species as evidence has accumulated with respect to both endogenous ligands and receptor structure/activity requirements in representative species of these taxa [4,17]. While the endogenous bombesin-like peptides in mammalian tissues mediate a wide range of biological functions [8,9,16,21], further exploration of the even greater and more diverse library of such structural analogs in amphibian skin secretions, may provide some valuable drug leads for future human therapeutics. The demonstration of the long-term stability of both peptidome and transcriptome in samples of skin secretion stored in the manner described in this study, may aid in the acquisition of data from archived material once considered inappropriate for this purpose. References [1] Anastasi A, Erspamer V, Bucci M. Isolation and amino acid sequences of alytesin and bombesin, two analogous active tetradecapeptides from the skin of European discoglossid frogs. Arch Biochem Biophys 1972;148:443–6. [2] Bai B, Zhang Y, Wang H, Zhou M, Yu Y, Ding S, Chen T, Wang L, Shaw C. Parallel peptidome and transcriptome analyses of amphibian skin secretions using archived frozen acid-solvated samples. Mol Biotechnol 2012, http://dx.doi.org/10.1007/s12033-012-9551-6. [3] Chen T, Farragher S, Bjourson AJ, Orr DF, Rao P, Shaw C. Granular gland transcriptomes in stimulated amphibian skin secretions. Biochem J 2003;371:125–30.

[4] Conlon JM. Bradykinin and its receptors in nonmammalian vertebrates. Regul Pept 1999;79:71–81. [5] Erspamer V. Discovery, isolation, and characterization of bombesin-like peptides. Ann NY Acad Sci 1988;547:3–9. [6] Erspamer V. Bioactive Secretions of the Integument. In: Heatwole H, Barthalmus GT, editors. Amphibian Biology, vol. 1. The Integument. Chipping Norton: Surrey Beatty & Sons; 1994. p. 179–350. [7] Falconieri EG, Severini C, Erspamer V, Melchiorri P, Delle FG, Nakajima T. Parallel bioassay of 27 bombesin-like peptides on 9 smooth muscle preparations. Structure–activity relationships and bombesin receptor subtypes. Regul Pept 1988;21:1–11. [8] Gonzalez N, Moody TW, Igarashi H, Ito T, Jensen RT. Bombesin-related peptides and their receptors: recent advances in their role in physiology and disease states. Curr Opin Endocrinol Diabetes Obes 2008;15:58–64. [9] Majumdar ID, Weber HC. Biology of mammalian bombesin-like peptides and their receptors. Curr Opin Endocrinol Diabetes Obes 2011;18:68–74. [10] Majumdar ID, Weber HC. Biology and pharmacology of bombesin receptor subtype-3. Curr Opin Endocrinol Diabetes Obes 2012;19:3–7. [11] McCoy JG, Avery DD, Bombesin:. potential integrative peptide for feeding, and satiety. Peptides 1990;11:595–607. [12] McDonald TJ, Jörnvall H, Nilsson G, Vagne M, Ghatei M, Bloom SR, Mutt V. Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochem Biophys Res Commun 1979;90:227–33. [13] Minamino N, Kangawa K, Matsuo H, Neuromedin B. a novel bombesin-like peptide identified in porcine spinal cord. Biochem Biophys Res Commun 1983;114:541–8. [14] Mignogna G, Severini C, Erspamer GF, Siciliano R, Kreil G, Barra D. Tachykinins and other biologically active peptides from the skin of the Costa Rican phyllomedusid frog Agalychnis callidryas. Peptides 1997;18:367–72. [15] Nagalla SR, Barry BJ, Falick AM, Gibson BW, Taylor JE, Dong JZ, Spindel ER. There are three distinct forms of bombesin. Identification of [Leu13 ]bombesin, [Phe13 ]bombesin, and [Ser3 ,Arg10 ,Phe13 ]bombesin in the frog Bombina orientalis. J Biol Chem 1996;271:7731–7. [16] Ohki-Hamazaki H, Iwabuchi M, Maekawa F. Development and function of bombesin-like peptides and their receptors. Int J Dev Biol 2005;49:293–300. [17] Spindel ER. Amphibian bombesin-like peptides. In: Kastin AJ, editor. Handbook of Biologically Active Peptides. Academic Press; 2006. p. 277–89. [18] Tyler MJ, Stone DJM, Bowie JH. A novel method for the release and collection of dermal, glandular secretions from the skin of frogs. J Pharmacol Toxicol Methods 1992;28:199–200. [19] Vetter I, Davis JL, Rash LD, Anangi R, Mobli M, Alewood PF, Lewis RJ, King GF. Venomics: a new paradigm for natural products-based drug discovery. Amino Acids 2011;40:15–28. [20] Yamada K, Wada E, Santo-Yamada Y, Wada K. Bombesin and its family of peptides: prospects for the treatment of obesity. Eur J Pharmacol 2002;440:281–90. [21] Ye˘gen BC. Bombesin-like peptides: candidates as diagnostic and therapeutic tools. Curr Pharm Des 2003;9:1013–22.