Six novel tachykinin- and bombesin-related peptides from the skin of the Australian frog Pseudophryne güntheri

Peptides, Vol. 11, pp. 29%304. ©Pergamon Press plc, 1990. Printed in the U.S.A.

0196-9781/90 $3.00 + .00

Six Novel Tachykinin- and Bombesin-Related Peptides From the Skin of the Australian Frog Pseudophryne giintheri MAURIZIO SIMMACO, CINZIA SEVERINI,* DANIELA DE BIASE, D O N A T E L L A B A R R A , I F R A N C E S C O B O S S A , J O H N D. R O B E R T S , t PIETRO MELCHIORRI* AND VITTORIO ERSPAMER*

Dipartimento di Scienze Biochimiche and Centro di Biologia Molecolare del CNR *Istituto di Farmacologia, Universitgt di Roma La Sapienza, 00185 Roma, Italy fDepartment of Zoology, The University of Western Australia, Nedlands, Australia R e c e i v e d 19 O c t o b e r 1989

SIMMACO, M., C. SEVERINI, D. DE BIASE, D. BARRA, F. BOSSA, J. D. ROBERTS, P. MELCHIORRI AND V. ERSPAMER.

Six novel tachykinin- and bombesin-relatedpeptides from the skin of the Australianfrog Pseudophryne giJntheri. PEPTIDES 11(2) 299-304, 1990. --Six novel peptides belonging to the tachykinin and bombesin families were isolated and sequenced from extracts of the skin of the Australian myobatrachid frog Pseudophryne giintheri. One of these peptides (PG-L) was of the homhesin family and may be considered an N-elongation of the litorin/ranatensin molecule, with which it shares an identical spectrum of activity on isolated smooth muscle preparations. The other five peptides were of the tachykinin family with two of these peptides (PG-SPI and PG-SPII) related to substance P and three (PG-KI, PG-KII and PG-KIII) to kassinin. In contrast to the basic nature of substance P, the PG-SP peptides showed a clear acidic character and displayed a more potent and sustained action on isolated smooth muscle preparations and rat blood pressure than did substance P. Two of the three PG-K peptides were more potent than kassinin; PG-KIII was considerably less potent. PG-KI and PG-KII were also present in a deamidated, poorly active, form.

Pseudophryne giintheri skin Bombesin

Isolation

Litorin-like peptides Bioactivity

Substance P-like peptides

THE skin of the Australian myobatrachid frogs of the genus Pseudophryne, like that of other amphibians, represents a source of a variety of active compounds including biogenic amines, alkaloids and peptides (8, 11, 16, 19). It was shown in a previous paper (16) that the skin of Ps. gu'ntheri in particular contained unusually large concentrations (up to 500-2000 Ixg per g dry tissue) of 5-hydroxytryptamine (5-HT), as well as lesser amounts of other unknown indole compounds. Large amounts of a bombesin-like peptide (20-150 Ixg/g) and of several tachykinin-like peptides (50-200 ixg/g expressed as uperolein) were also present. It is well known that the large bombesin and tachykinin families are both comprised of numerous members. These are represented in amphibian skin and gut, in the central nervous system and gut of mammals and all other vertebrate species, as well as in invertebrate tissues. The bombesin peptide family has been divided, on the basis of chemical structure, of activity in parallel bioassays, and of receptor binding with radiolabeled agonists, into three subfamilies having their prototypes in bombesin, litorin/ranatensin and phyllolitorin, respectively. The tachy-

Kassinin-like peptides

Tachykinin

kinin family in turn may be divided into two subfamilies having their prototypes in substance P/physalaemin and kassinin/eledoisin, respectively. Both the bombesin- and the tachykinin-related peptides are now the focus of intense research, because of their probable involvement as neurotransmitters, neuromodulators and growth factors, in numerous physiological and pathological processes. Hence the great interest in enriching the list of natural members of these peptide families. Amphibian skin peptides often have been the springboard for the detection of analogous peptides in mammalian gut and nervous system (10). The purpose of this study was to isolate the different active peptides of Ps. gitntheri skin, determine their primary structure, and carry out a preliminary parallel bioassay in comparison with prototype peptides of the pertinent families or subfamilies. METHOD

Amphibian Material One hundred and fifty specimens of Ps. gu'ntheri were col-

XRequests for reprints should be addressed to Prof. Donatella Barra, Dipartimento di Scienze Biochimiche, Universit/~La Sapienza, Piazzale Aldo Moro 5, 00185 Roma, Italia.

299

SIMMACO ET AL.

300

lected by one of us (Dr, Roberts) in Westem Australia, near Perth, in May 1987. The skins were removed from the animals immediately after killing them, carefully spread out and dried in the shade. After arrival in the Rome laboratory, they were minced with scissors and immersed in twenty parts (w/v) of 80% aqueous methanol. The liquid was decanted after a week and the skins extracted for another week with fifteen parts of the same solvent. The methanol extracts were combined and filtered. Kept in dark bottles and refrigerated (2-3°C), they can be stored for months without appreciable loss of activity, as assessed by periodical bioassay against standard synthetic peptides. The total weight of the skins was 11.2 g (0.075 g/skin). The material extracted by methanol weighed 1.6 g, representing 14% of the dry weight of the skins. In addition to Ps. giintheri skins, we obtained from the same source twenty skins of Ps. occidentalis (total weight 1 g). They were processed as above, but not studied in detail. Only the generic tachykinin- and bombesin-like activity was determined.

Absorption on Alumina Column The extract obtained from 10 g dried skin of Ps. gu'ntheri was evaporated under reduced pressure at 45°C, and the residue (1.4 g) reconstituted in 10 ml distilled water and 190 ml 99% ethanol. The liquid was loaded onto a chromatographic column filled with 200 g of alkaline alumina (Merck, Darmstadt, FRG) equilibrated with 99% ethanol. The column was eluted stepwise by adding 400 ml portions of decreasing ethanol concentration (from 95 to 20%) as shown in Fig. 1. Fractions of 200 ml were collected. Each fraction was submitted to a preliminary bioassay in order to locate bombesins (rat uterus), tachykinins (guinea pig ileum) and caeruleins (guinea pig gall bladder). Alkaloids were visualized on paper or thin layer chromatograms by the Dragendorff reagent; 5-HT by the stable diazonium salt NNCD (2-chloro-4-nitrobenzenediazonium 2-naphthalenesulfonate).

Bioassay Smooth muscle preparations used in this study were as follows: a) rat uterus (Tyrode solution at 30°C) or, alternatively, rat urinary bladder (Krebs solution at 37-38°C) for bioassay of bombesin-like peptides; b) guinea pig ileum and rabbit large intestine (Tyrode solution at 37°C) for generic bioassay of tachykinins; rat duodenum, hamster urinary bladder (Tyrode or Krebs solution at 38°C) for the distinction between physalaemin-like (aromatic) tachykinins and kassinin-like (aliphatic) tachykinins; c) guinea pig gall bladder (Tyrode solution at 38°C) for detection and bioassay of caerulein. The organ bath had a capacity of 10 ml and was gassed with 5% CO2 + 95% 02. The mechanical activity of all the above smooth muscle preparations was recorded isometrically by a strain-gauge transducer (DY 2, force up to 10 g, Basile, Milan, Italy) and displayed on a recording microdynamometer (Unirecord, Basile, Milan, Italy). The rat blood pressure was recorded from a carotid artery of animals (300-400 g) anesthetized with ethylurethane (1-1.5 g, intraperitoneally) by means of a Trantee Pressure Transducer model 880 (Bentley, Irvine, CA) connected with the Unirecord. Injections were given via the jugular vein. In order to avoid blood coagulation, heparin (1000 units/kg) was administered IV before the experiment. Methods have been described in detail in preceding papers (2, 3, 9, 10), in which sensitivity and specificity of each preparation are also indicated. Peptide Purification Active material was further purified by reverse phase high

performance liquid chromatography (RP-HPLC) using a Beckman (Berkeley, CA) model 332 instrument, on an Aquapore RP-300 column (7 x 250 mm, Brownlee Labs, Applied Biosystems, Santa Clara, CA). Peptides were eluted on a linear gradient of 5 to 60% acetonitrile/2-propanol, 4:1 (v/v) in 0.1% trifluoroacetic acid, at a flow rate of 2.5 ml/min. Elution of the peptides was monitored on a Beckman 165 spectrophotometer at 220 and 280 nm. In correspondence of an absorbance peak the effluent was collected in a tube and lyophilized. A 2% aliquot of the material from each tube was then used for assay of biological activity.

Structure Determination N-terminal analyses were performed after derivatization of the peptides with dansyl-chloride [DNS-C1, (13)]. Amino acid analyses were performed with an LKB 2131 Alpha Plus instrument (Pharmacia Biochrom, Cambridge, UK) after hydrolysis of the peptides (1-2 nmol) in 6 N HCI for 24 hr in vacuo. Automated Edman degradation was performed on an Applied Biosystems (Foster City, CA) model 470A gas phase sequencer equipped with an Applied Biosystems model 120A PTH-analyzer for the on-line identification of amino acid derivatives. One to two nmol of a particular peptide were loaded on a precycled, polybrene-coated, trifluoroacetic acid-activated, glass fiber filter. The N-terminal pyroglutamate was removed by digestion of the peptide with pyroglutamate aminopeptidase (Boehringer, Mannheim, FRG), followed by HPLC isolation of the unblocked peptide and automated Edman degradation (15). Alternatively, this moiety was cleaved after treatment of the peptide with 1 N HC1 in methanol at room temperature (20-22°C) for 24 hr (17), tbllowed by direct automated sequence determination. The possible presence of amidated residues was determined by digesting aliquots of the peptides with carboxypeptidase Y (Boehringer, Mannheim, FRG) in 20 mM HEPES (Sigma, St. Louis, MO), pH 8. The released residues were identified by HPLC after derivatization with DNS-C1 (18). Where necessary, aliquots of the peptides were digested either with pepsin (Sigma) in 5% formic acid (enzyme to substrate ratio 1/50) at room temperature (20-22°C) for 5 min, or with Staphylococcus aureus V8 protease (Miles, Naperville, IL) in 0.1 M ammonium bicarbonate at 37°C for 3 hr (same E/S ratio), The fragments were purified by RP-HPLC on an analytical column (Aquapore RP-300, 4.6x250 mm, Brownlee Labs), under the conditions specified above.

Drugs and Reagents Physalaemin (mol.wt. 1265), kassinin (mol.wt. 1335), substance P (mol.wt. 1348), litorin (mol.wt. 1085), 5-hydroxytryptamine creatinine sulphate (5-HT) and heparin (sodium salt, grade II, 150 units/mg) were purchased from Sigma (St. Louis, MO); the NNCD reagent from Fluka (Buchs, Switzerland). Caerulein (mol. wt. 1352) was a gift from the Farmitalia Carlo Erba Research Laboratories (Milan, Italy). HPLC grade solvents were from Farmitalia Carlo Erba (Milan, Italy); gas phase sequencer chemicals were from Applied Biosystems (Foster City, CA). All other chemicals were of reagent grade. RESULTS The analysis of the crude Ps. gantheri methanol extract showed a bombesin-like activity on the rat uterus preparation equivalent to 50-60 Ixg litorin per g dry skin, and a tachykinin-like activity on the guinea pig ileum preparation ranging from 400 to 800 tzg/g physalaemin. Moreover, the occurrence of large amounts of 5-HT, together with that of remarkable amounts (as deduced from the

PSEUDOPHRYNE G(]NTHERI SKIN PEPTIDES

301

800-

EtOH 801

E t O H 70=

600-

_

TACHYKININS o r~

D.

4

0

o

~

E LU T I 0 It T I fl ( (el~) laG-Kill

951 952 901 90~ 801 80= 701 70= 601 60= 501 502 40 30 20 ETHANOL

ELUATE

FIG. 1. Biological activity of the eluate from alumina column loaded with a crude skin extract of Ps. giintheri and developed with descending concentration of ethanol. For each ethanol concentration either two 200-ml fractions or one 400-ml fraction were collected as indicated on the x-axis (e.g., 951 and 952 mean the first and the second 200-ml fraction collected after elution with 95% ethanol). Elution profiles of bombesins, expressed as litorin (dotted columns),and tachykinins, expressed as physalaemin (black columns), are reported. The chromatographic behavior of pseudophrynaminol (PSOL) and 5-hydroxytryptamine (5-HT) is also indicated.

:.t OH 60=

1,o

<~

0.0

0

3'* (LUTION TII~(

Dragendorff reaction) of a pseudophrynamine A-like alkaloid (possibly pseudophrynaminol), was confirmed (16). The latter compounds were not further investigated in the present research.

Peptide Purification

6 (~lN)

FIG. 2. Reverse phase HPLC of the peptide fractions eluted from the alumina column with 80, 70 and 60% ethanol concentration (see Fig. 1). Chromatographic conditions: column, Aquapore RP-300, 7 × 250 mm; solvent A, 0.2% trifluoroacetic acid; solvent B, 0.1% trifluoroacetic acid in acetonitrile/2-propanol, 4:1 (v/v); flow rate 2.6 ml/min. Linear gradients from 5 to 50% solvent B over 35, 25 and 55 min in 801 , 702 and 602 eluates, respectively. The active peptides are indicated by the corresponding abbreviation. KI-OH and KII-OH represent the deamidated forms of PG-KI and PG-KII, respectively.

The elution profile from the alumina column of amines, alkaloids and peptides from the methanol extract of Ps. giintheri skin is shown in Fig. 1. The phryniscamine A-like alkaloid emerged in the first 95% ethanol eluate and 5-HT chiefly in the 70 l eluate, with smaller amounts in 802 and 702 eluates. Bombesinlike activity (rat uterus preparation) emerged in the eluate 801, with some activity present in eluate 802. The tachykinin-like activity (guinea pig ileum and rabbit colon preparations) appeared in eluates 702 tO 40, with a distinct peak in eluate 602. No caerulein-like activity could be detected with the guinea pig gall bladder assay. Whereas values for bombesin activity (expressed in terms of litorin) are reliable, those for tachykinin activity (expressed as physalaemin) are only approximate, because eluates 702 to 40 contain a mixture of several tachykinin peptides of different relative activity. Eluates 801 , 702 and 602 were further processed for purification and isolation of the individual peptides. The elution profiles from the HPLC column are reported in Fig. 2. Each HPLC fraction was submitted to specific bioassays in order to locate the biological activities. In eluate 80= the bombesin-like activity was associated to the peak indicated as PG-L (Ps. giintheri litorin-like peptide) in Fig. 2. In eluates 702 and 602 the tachykinin-like activity was associated to peaks indicated as PG-SP (Ps. giintheri substance P-like peptides) or PG-K (Ps. gfintheri kassinin-like peptides) in Fig. 2. These abbreviations were introduced retrospectively according to the primary structure homologies.

purification is reported in Table 1. Direct N-terminal analysis gave no results and the presence of a pyroglutamyl residue at each N-terminus was suspected. To circumvent this problem, two procedures were attempted: 1) removal of the pyroglutamyl residue by incubation with pyroglutamyl aminopeptidase, followed by HPLC isolation of the unblocked peptide and automated sequence determination (15) and 2) treatment of the peptide with methanolic HC1, followed by direct automated sequence analysis (17). The former procedure allowed determination of the structure of PG-L, but failed in the case of the other peptides, possibly because of the presence of proline in the second position, as verified by the latter procedure. In the case of peptides PG-SPI, PG-KI and PG-KII, the structure was confirmed after digestion of an aliquot with S. aureus V8 protease or pepsin, followed by purification and analysis of the fragments. The amidation state of the C-terminal residue was ascertained by digestion with carboxypeptidase Y under conditions that favor the peptidase activity but inhibit the amidase activity of the enzyme (4). The sequence information, that allowed the reconstruction of the structure of the bioactive peptides, is summarized in Fig. 3.

Structure Determination

Parallel Bioassay of Isolated Natural Peptides

The amino acid composition of the active peptides after HPLC

Data presented in this paragraph are only preliminary to a more

SIMMACO ET AL.

302

TABLE 1

i' / ,

A M I N O ACID C O M P O S I T I O N O F PSEUDOPHRYNE G(flNTHERI SKIN PEPTIDES

Amino Acid Asp Glu Pro Gly Ala Val Met Leu Phe His Tip*

PG-L

2.0 1.0 3.7 0.8 0.6 0.6

(2) (1) (4) (1) (1) (1)

0.7 (1) 0.8 (1) + (1)

'

PG-KI

PG-KII

PG-KIII

PG-SPI

PG-SPII

0.9 1.7 1.6 0.9

(1) (2) (2) (1)

1.8 2.0 1.7 1.1

(2) (2) (2) (1)

1.0 2.0 1.8 1.1

(l) (2) (2) (1)

1.6 2.0 1.8 1.0

1,7 2.0 1.7 1.1

0.7 1.0 0.7 0.7 0.8

(1) (1) (i) (1) (1)

0.7 0.8 0.8 0.8

(1) (1) (1) (1)

0.7 0.9 1.O 0.9 0.8

(1) (1) (1) (1) (1)

1.0 (1) 0.9 (1) 1.7 (2)

(2) (2) (2) (1)

(2) (2) (2) (1)

pGlu-Gly-G1y-G1y-Pro-G1n-Trp-Ala-Val-G1y-His-Phe-Net-NH 2

pGI u-Pro-Hi s-Pro-Asp-Glu-Phe-Val -Gly-Leu-Net-NH 2 ,

PG-KII

~

-"g' i

--,,

~-/~

~

,

d pG1u-Pro-Asn-Pro-Asp-G1 u-Phe-VaI-Gly-Leu-Net -NH~

;

~

~--i.~---,---W--,---,~-i.._~___ ~dr_

PG-KIII

PG-SPI

pGlu-Pro-Ht s-Pro-Asn-Glu-Phe-Val -Gly-Leu-Met -Nit 2

pG1u-Pro-Asn-Pro-Asp-Glu-Phe-Phe-Gly-Leu-14et -NH. -p .-.._p ~ ,

PG-SPII

Sl

,~l

SZ

!---

i

i

,

r --

-

'

thorough pharmacological study, which presupposes the availability of larger amounts of synthetic peptides. However, they are sufficient to give a clear-cut indication on the differences in potency of the various peptides in a few typical test preparations. The potency of the peptides was determined at threshold dose levels and expressed as a percentage of that of the reference peptide. Efficacy, i.e., maximum obtainable response, was not determined in this study. The molar concentration of all natural peptides submitted to bioassay was established by quantitative amino acid analysis. Three to five experiments were carried out

PG-KI

lnlllil

0.8 (1) 0.8 (1) 1.7 (2)

The composition from sequence analysis of each peptide is indicated by the numbers in parentheses. *Presence of tryptophan was indicated by absorbance at 280 nm and Ehrlich reaction.

PG-L

i . y. . . .i . . .f. I / , ' I/,/ / . , - ; ; i

,

/_... pG1u-Pro-Asn-Pro-Asn-G1 u-Phe- Phe-Gly-Leu-Net- NH^

7

FIG. 4. Effect of graded concentrations (nM) of PG-SPI and substance P (SP) on isolated guinea pig ileum. In this experiment the Ps. giintheri peptide was approximately three times more potent than SP and elicited a contraction that reached its peak somewhat slower, but was more sustained than that provoked by SP. Concentration of the natural peptide PG-SPI was determined by amino acid analysis. *Wash.

with the individual peptides on each preparation. PG-litorin displayed a stimulant effect on the rat uterus and the rat urinary bladder which was indistinguishable, both qualitatively and quantitatively, from that caused by either litorin or ranatensin. On the guinea pig ileum, like all bombesins, it caused irregular spike-contractions with strong tachyphylaxis. Concerning the kassinin-like peptides, PG-KI and PG-KII were decidedly more potent, by 2 to 4 times, than kassinin on the guinea pig ileum, the rabbit colon and the hamster urinary bladder; PG-KII! presented, in the same preparations, 15-25% of the activity of kassinin, whereas deamidated PG-KI and PG-KII showed only 2-5% of the potency of kassinin. The PG-SP peptides, like substance P, elicited a powerful contraction of the guinea pig ileum and the rabbit colon, but were virtually inactive on the rat uterus preparation and the hamster urinary bladder. On the guinea pig ileum both PG-SPI and PG-SPII were 2-3 times more potent than substance P and, in turn, PG-SPI was approximately 30% more potent than PG-SPII. Response of the intestinal smooth muscle was considerably more sustained, as clearly shown in Fig. 4. In a few experiments on rat blood pressure hypotension caused by PG-SPI was more intense and, above all, more sustained than that elicited by equimolar doses of substance P. In addition to Ps. gf~ntheri and Ps. occidentalis, considered in this study, six other Pseudophryne species have been submitted for screening for active peptides in previous studies on Australian amphibians (16). A synopsis of present and previous results obtained on methanol extracts of dried skins is shown in Table 2. Tachykinin activity is expressed as i~g physalaemin equivalents, bombesin activity as ~g litorin equivalents, always per g dry skin. DISCUSSION

FIG. 3. Summary of sequence studies on Ps. gantheri peptides. The residues above the arrow ( 7 ) were identified by automated Edman degradation. The sequence determined by carboxypeptidase Y digestion is indicated by arrows ( ~ ) above the corresponding residues. Fragments obtained after digestion with S. aureus V8 protease (S) or pepsin (P) are indicated by solid lines.

The Pseudophryne giintheri skin represents a striking example of the variety and abundance of active compounds which may occur in the skin of a single amphibian species. It contains a wide array of peptides displaying bombesin- or tachykinin-like activities, as well as alkaloids and biogenic amines. The alkaloid

P S E U D O P H R Y N E G U N T H E R I SKIN PEPTIDES

303

TABLE 2 TACHYKININAND BOMBESINCONTENTSIN THE SKIN OF EIGHT PSEUDOPHRYNE SPECIES Tachykinins (Rabbit Colon) Ps. Ps. Ps. Ps. Ps. Ps. Ps. Ps.

giintheri occidentalis bibronii corroboree nichollsi semimarmorata australis coriacea

[150] [20] [37] [12] [12] [20] [2] [374]

400-800 100-150 2-4 1-2 <1 1-2 5-6 <1

(4) (3) (3) (3) (2) (3) (4) (2)

Bombesins (Rat Uterus) 50-60 10-15 10-20
(4) (3) (3) (2) (2) (2) (4) (4)

Tachykinin- and bombesin-like activities are expressed as la,g physalaemin and litorin, respectively, per g dry skin. In brackets is the number of skins; in parentheses the number of experiments.

alcohol (pseudophryniscaminol) probably derives from hydrolysis of pseudophrynamine A (8). The biogenic amine 5-HT is present in larger amounts than has been observed in the skin of most other amphibian species. The bioactive peptides have been purified and their primary structure elucidated. The bombesin-like activity was associated to one litorin-like peptide (PG-L); the tachykinin-like activity to three kassinin-like peptides (PG-KI, PG-KII and PG-KIII) and to two substance P-like peptides (PG-SPI and PG-SPII). In HPLC experiments, numerous other peaks were recorded on the chromatograms. Although these were inactive in the present biological screening procedure, they may be active in other bioassay systems. They may represent novel peptides unrelated to the tachykinins or bombesins, but part of distinct peptide families. We are persuaded that their study may prove highly rewarding. The tachykinins identified in the Ps. giintheri skin may be of critical importance in the investigation of the relationship between

structure and activity in the field of natural peptides. They may also indicate, especially in the case of substance P, the relative importance of the N- and the C-moiety of the molecule in eliciting both central and peripheral effects. Moreover, antisera raised against the novel peptides, for example against the N-moiety of PG-SPI, may be used in the search for PG-SPI analogs in mammalian tissues. The structures of the novel peptides are compared in Table 3 with representative sequences of established peptides from each family. PG-litorin, the only representative of the bombesin family, may be considered an N-elongation of litorin, since it contains three Gly residues inserted after the pGlu of litorin or ranatensin. It was indistinguishable in potency and in the shape of the contraction curve both from litorin and ranatensin in the rat uterus and rat urinary bladder preparations. Three kassinin-like undecapeptides were isolated from the Ps. gi~'ntheri skin. Two of these peptides (PG-KI and PG-KII) were also present in their deamidated form. All three amidated forms showed a potent stimulant action on the guinea pig ileum, the rabbit colon and the hamster urinary bladder. However, whereas the potency of PG-KI and PG-KII exceeded that of kassinin, that of PG-KIII was barely 20-25%. This suggests that the Asp residue, in position 7 from the C-terminus, which occurs in all other known natural kassinin-like peptides, is important for potency of action on this preparation. The deamidated forms of the PG-K peptides, like deamidated substance P (7), showed little activity in our smooth muscle preparations. It is not known whether deamidation occurs in the living skin or is a postmortem artifact. The PG-SP peptides were both amidated undecapeptides differing sharply from substance P in that the two Gin residues, in position 5 and 6, are substituted either with an Asp-Glu or an Asn-Glu pair and by the substitution of the Arg and Lys residues by a pGlu and an Asn residue, respectively. This gives to the PG-SP peptides a distinct acidic character, which is opposite to the basic character of substance P and its analog [Arg3]substance p, recently described in chicken intestine (6). These substantial

TABLE 3 STRUCTUREOF THE NOVELPS. G•NTHERI BOMBESIN (PG-L) AND TACHYKININS (PG-SP AND PG-K) AS COMPAREDTO THAT OF RELATEDNATURALPEPTIDES Bombesin-Like Peptides PG-L

pGlu_Gly.Gly_Gly_Pro_Gln_Trp_Ala_Val_Gly_His_Phe_Met_NH 2

Litorin

pGlu

Gln-Trp-Ala-Val-Gly-His-Phe-Met-NHz

Kassinin-Like Peptides PG-KI PG-KII PG-KIII Kassinin Neurokinin A (Subst. K) Neurokinin B

pGlu-Pro - His-Pro-Asp-Glu-Phe-Val-Gly-Leu_Met_NH2* pGlu-Pro-Asn-Pro-Asp-Glu-Phe-Val-Gly-Leu-Met-NH2* pGlu-Pro-His-Pro-Asn-Glu-Phe-Val-Gly-Leu_Met_NH 2 Asp-Val-Pro-Lys-Ser-Asp-Glu-Phe-Val-Gly-Leu_Met.NH 2 His-Lys-Thr- Asp-Ser-Phe-Val-Gly-Leu-Met_NH 2 Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met_NH 2 Substance P-Like Peptides

PG-SPI PG-SPII Substance P

pGlu_Pro_Asn_Pro_Asp_Glu_Phe.Phe.Gly_Leu_Met_NH 2 pGlu_Pro_Asn_Pro_Asn_Glu_Phe.Phe_Gly_Leu_Met_NH 2 Arg -Pro _Lys_Pro_Gln.Gln_Phe.Phe_Gly.Leu_Met_NH2

The structure of PG peptides was determined in the course of the present work, as summarized in Fig. 3. For other peptides, the sequence is taken from: (12), litorin; (1), kassinin; (14), neurokinin A and B; (5), substance P. *Also present in a deamidated form.

304

SIMMACO E T AL.

changes resulted in an increase in potency of action of the PG-SPs versus substance P and in a clear-cut prolongation of action. From inspection of Table 2 it may be seen that among the eight examined Pseudophryne species, only Ps. gu'ntheri and Ps. occidentalis from Western Australia contained high amounts of tachykinin peptides. Remarkable amounts of bombesin activity, on the contrary, were found not only in the skin of the above two species, but also in that of Ps. bibronii and Ps. australis. The existence of striking differences in the peptide content of the skin of various amphibian species belonging to the same genus was a frequent, nearly regular finding in our studies on amphibian skin peptides. The reason for this fact is at present completely obscure, although it may obviously have some taxonomical significance. The biological significance of peptides in amphibian skin is poorly understood, with the exception of that of peptides involved in the defense of the cutaneous tissue against microbial attack. We refer to the magainins and related peptides (21), and perhaps also to the mast cell degranulating and chemotactic peptides (20), possibly involved in immunological processes.

However, independently of their function in the skin, we have assigned for years a paramount importance to the study of amphibian skin peptides. As repeatedly stated, this study has substantially contributed to the expansion of our knowledge in the field of bioactive peptides, often heralding the discovery of new neuropeptides, strictly related to the amphibian molecules, in mammalian tissues, especially brain and gastrointestinal tract. Some of the new tachykinins described in this paper (especially the acidic SP-like peptides) may find a counterpart in mammalian tissues. ACKNOWLEDGEMENTS Collection in Australia of the Pseudophrine material studied in this paper was sponsored by the Fidia Research Laboratories, Abano Terme, Italy. This work was supported in part by the CNR special project "Chimica Fine II," contract No. 89.00804.72, by a CNR grant No. 89.02416.04, and by a grant from the Ministero della Pubblica Istruzione, Italy. The technical supervision and coordination of Mr. Marco Federici is gratefully acknowledged.

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