The dermorphin peptide family

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Gen. Pharmac. Vol. 27, No. 7, pp. 1099-1107, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. ELSEVIER

REVIEW

The Dermorphin Peptide Family P. Melchiorri* and L. Negri INSTITUTE OF MEDICALPHARMACOLOGY,UNIVERSITYOF ROME, "LA SAPIENZA,"ROME, ITALY ABSTRACT. 1. In 1980, the skin of certain frogs belonging to the genus Phyllomedusinae was found to contain two new peptides that proved to be selective ~-opioid agonists. Given the name dermorphins, these were the first members of a peptide family that in the past 15 years has grown to reach a total of seven naturally occurring peptides and nearly 30 synthetic analogs. 2. Dermorphin peptides are potent analgesics in rodents and primates, including man. Some dermorphins can enter the blood-brain barrier and produce central antinociception after peripheral administration. 3. The dermorphin family also includes ~-opioid receptor selective agonists that produce intense opioid analgesia, but stimulate pulmonary ventilation. 4. Experiments in rats and mice chronically exposed to dermorphins have shown that not only do they have higher antinociceptive efficacy and potency than morphine, but they are also less likely than morphine to produce tolerance, dependence and opiate side effects. Copyright © 1996 Elsevier Science Inc. tEN PHAeaaAC 27;7:1099--1107, 1996. KEY WORDS. Demorphins, peptides, analgesics, morphine INTRODUCTION Amphibian skin contains a wide variety of peptides that are often homologous or even identical to the gastrointestinal hormones and neurotransmitters of the Mammalia (Erspamer, 1994). The discovely of enkephalins in the mammalian brain raised the question of whether or not opioid peptides existed in the amphibian skin. Research started in 1980 led, one year later, to the discovery of dermorphin in the skin of the frog PhyUomedusa sauvagei (Montecucchi et al., 1981a; Broccardo et al., 1981) and of a similar peptide, containing hydroxyproline instead of proline, in the skin of Phyllomedusa rohdei and PhyUomedusa burmeisteri (Montecucchi et al., 1981b). Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 dermorphin Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser-NH2 [Hyp6]dermorphin The very high dermorphin content in the skin of these Phyllomedusinae, about 50-80 ~g per g of fresh skin, made it easier to purify and sequence the peptides. More recently, screening of a cDNA library from the skin of Phyllomedusa bicolor (Richter et al., 1990) predicted the amino acid sequence of the following three additional dermorphin-analogs: Tyr-D-Ala-Phe-Gly-Tyr-Pro-Lys-OH [Lys7-OH]dermorphin Tyr-D-Ala-Phe-Trp-Tyr-Pro-Asn -OH [Trp4, AsnLOH]dermorphin Tyr-D-Ala-Phe-Trp-Am-OH [Trp4, AsnLOH]dermorphin 1-5 The two heptapeptides were later isolated from methanol extracts of the skin of the frog and their amino acid sequences were confirmed. The total amount of these peptides present in the skin of Phyllomedusa bicolor approaches that found in other Phyllomedusinae (about 25 p~gof [LysLOH]dermorphin and 68 ~g of [Trp 4, AsnL OH]dermorphin per g of tissue) (Mignogna et al., 1992). Common to the primary structure of all naturally occurring dermorphin-like peptides is the amino-terminal sequence Tyr-D-Ala* To whom correspondence should be addressed. Received 3 November 1995; accepted 1 December 1995.

Phe containing a D-amino acid isomer located between two aromatic rings. The D-Ala2 isomer is essential for the biological activity because its replacement by the L-isomer leads to practically inactive peptides (Erspamer, 1992). BIOSYNTHESIS OF DERMORPHINS Though dermorphin-like peptides and their precursors isolated from the amphibian skin all contain D-AIa in the same position (Erspamer, 1992), the triplet codon for alanine in the dermorphin gene apparently codes for the L-isomer (Richter et al., 1987, 1980) Expression of the RNA encoding the dermorphin precursor in transinfected HeLa or AtT-20 cells yields a polypeptide containing L-AIa at the relevant position (Seethaler et al., 1991 ). Thus L-Ala must be converted to D-Ala through an unusual posttranslational reaction that presumably takes place in the precursor itself, and that appears to be a unique feature of the amphibian skin. Because [L-Ala2]-containing peptides have never been found in amphibian skin extracts, the mechanism of epimerization is probably a quantitative inversion of the chirality of the c~-carbon of alanine, rather than a raeemization, which would result in an equimolar mixture of L-and D-isomers (Kreil, 1994). An alternative, but less specious, explanation may be that the D-amino acid is selectively incorporated into the nascent protein chain by an unknown mechanism that differs from that of the L-isomer (Lazarus and Attila, 1993). STRUCTURE-ACTIVITY RELATIONSHIP Extensive in vitro and in vivo studies have demonstrated that dermorphins are the most potent and selective I*-opioid receptor agonists among the naturally occurring opioids. The receptor affinity, selectivity and potency of dermorphins have been measured by binding assays on crude or synaptosomal preparations of brain membranes and by bioassays on two isolated organ preparations, electrically stimulated guinea pig ileum (GPI) and mouse vas deferens (MVD), that are rich in Ix- and ~-opioid receptors (Table 1). These studies

1100

P. Melchiorri and L. Negri

TABLE 1. Affinity and selectivity for I* and 8 opioid receptors, and biological activities on guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations of natural dermorphins (amidated and deamidated forms)

K/, nM (mean + SE) Peptide Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-OH Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser-NH2 Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser-OH Tyr-D-Ala-Phe-Gly-Tyr-Pro-Lys-NH2 high affinity site low affinity site Tyr-D-Ala-Phe-Gly-Tyr-Pro-Lys-OH Tyr-D-Ala-Phe-Trp-Asn-NH2 Tyr-D-Ala-Phe-Trp-Asn-OH Tyr-D-Ala-Phe-Trp-Tyr-Pro-Asn-NH2 Tyr-D-Ala-Phe-Trp-Tyr-Pro-Asn-OH DAMGO Morphine

ICs0, nM (mean -+ SE)

It system

8 system

I*/~

0.54 -+ 0.021 -0.65 -+ 0.03 -0.09 -+ 0.008 0.007 -+ 0.001 0.25 -+ 0.013 5.7 -+ 0.51 0.9 -+ 0.052 4.44 -+ 0.39 0.32 -+ 0.026 2.9 _+ 0.21 1.1 _+ 0.2 11.0 _+ 1.5

929 + 41 -1200 _+ 131 -1105 _+ 185

5.8 × l0 4 -5.4 × 10 -4 -8.1 x 10 5

1150 480 715 690 865 430 500

4.9 1.9 6.2 4.6 3.3 2.6 2.2

+ 172 + 45 -+ 73 + 57 + 69 -+ 58 _+ 48

× × × × X X ×

10 -3 10-3 10 -3 10 4 10 -4 10 -z 10 -2

GPI

MVD

GPI/MVD

1.29 4.5 1.6 4.9 1.15

+ 0.11 -+ 0.32 _+ 0.12 + 0.23 -+ 0.13

16.5 28.1 18.1 33.0 13.6

-+ 1.3 -+ 2.4 + 2.9 _+ 1.9 _+ 1.5

7.8 1.6 8.8 1.5 8.4

× × × × x

10 2 10< 10 -2 10 1 10 -2

3.82 5.00 13.10 0.58 1.24 7.1 150

-+ 0.45 -+ 0.52 -+ 2.12 -+ 0.06 -+ 0.09 -+ 0.9 -+ 18.5

56.3 73.7 205.0 6.6 10.4 115 1215

-+ 7.8 -+ 9.1 -+ 35.4 -+ 0.9 -+ 1.1 _+ 21 + 115

6.8 6.8 6.4 8.8 1.2 6.1 1.2

× × X × × × ×

10 _2 10 2 10 2 10 -2 10 -1 10 2 10 I

Ki, equilibrium dissociation constant of the competing ligand; bu-opioidreceptors have been labelled with pH]DAMGO, 0.5 nM; 8-opioid receptors have

been labellled with [3H]deltorphinI, 0.3 nM; 1*/8 represents selectivity for the ~-receptors. have revealed that the two dermorphin-related peptides from the skin of P. bicolor, [LysT]dermorphin and [Wrp4, AsnT]dermorphin, are able to distinguish between two Ix-opioid receptor subtypes (Negri et al., 1992). [LysT]dermorphin shows an affinity and selectivity for Ix-opioid receptors of rat brain membranes 10 times higher than that of DAMGO and dermorphin, and 100 times higher than that of morphine. However, the potency of [Lysr]dermorphin in inhibiting GPI contractions is slightly lower than that of dermorphin and DAMGO. In contrast, the apparent affinity of [Trp4, AsnT]dermor phin for brain Ix-opioid receptors is about ten times less than that of [Lys7]dermorphin. Nevertheless, on the guinea pig ileum, [Trp4, Asn7]dermorphin is more potent than dermorphin and [LysT]dermorphin in inhibiting electrically-stimulatedcontractions. Labeling Ix-opioid receptors of rat brain membranes with the Ix-selective ligand [H3]DAMGO, obtained displacement curves of [H3]DAMGO binding with graded concentrations of [Lysr]dermorphin that revealed 2 Ix-binding sites, one of high and the other of low affinity. The density of the low affinity site was progressively reduced by adding graded concentrations (0.1 to 0.5 nM) of [Trp4, Asn7]dermorphin to the binding medium, and the high affinity site was practically abolished by pretreatment of rats with the non-equilibrium Ixl-opioid receptor antagonist naloxonazine. These results suggest that the two binding sites represent two distinct Ix-opioid receptor subtypes. A likely explanation for the apparent conflict between affinity to central receptors and potency on peripheral organs of these two peptides may be that guinea pig ileum Ix-receptors more closely resemble the brain low-affinity type receptor that preferentially binds [Trp4, AsnT]dermorphin. Among the naturally occurring dermorphins, those with a free terminal carboxyl group have a Ix-affinity 30-100 times lower than that of their amidated analogs and are far less active on isolated organ preparations. The reason for the higher Ix-affinity of C-terminal amides may be suppression of the negative charge of the terminal carboxyl group. The binding epitope on the w-receptor protein contains negatively charged amino acids that can repulse negatively charged ligands (see later). The superior potency of the peptide amides on isolated organ preparations may depend on enhanced receptor affinity, as well as on the protection afforded by C-terminal amidation against carboxypeptidase cleavage. A large number of synthetic dermorphin analogs and homologues have been synthe-

sized and tested in extensive studies on structure-activity relationship (DeCastiglione et al., 1981; Melchiorri et al., 1982; 1991; Darlaket al., 1987; Mosberg et al., 1990; Sivanandaiah et al., 1989). Some shorter homologs of dermorphin appear of particular interest because they represent fragments produced in the metabolic breakdown of the peptide upon peripheral and central administration (Negri and Improta, 1984). In activating opioid receptors of guinea pig ileum, dermorphin 1-5 retains 50%, and dermorphin 1-4 retains 5% of the dermorphin potency, whereas dermorphin 1-3 is inactive (Table 2).In the rat brain, dermorphin 1-4 is the main endproduct of enzymatic degradation (Negri and Improta, 1984). Because alternative cleavage sites in the sequence of dermorphin precursors can generate C-terminal elongated dermorphins, Ix-affinity and bioactivity of synthetic analogs extended at the C-terminus have been also studied (Table 2). In binding assays employing rat brain membranes, elongated peptides carrying a net positive charge, for example dermorphin-Gly-Glu-Ala-Lys-Lys-Ile-Lys-Arg-NH2, show a Ix-affinity higher than that of dermorphin. But, in GPI assay, these peptides exhibit potencies comparable to or slightly lower than that of dermorphin (Ambo et al., 1994). In contrast, all positively charged dermorphins produce potent and long-lasting antinociception in rats and mice (Negri, personal communication). Elucidation of the sequence of rodent and human gene encoding brain Ix-opioid receptors by cDNA cloning (Wang et al., 1993) indicates that these Ix-receptor proteins contain negatively charged amino acid residues (Asp u4, Asp 147) within the putative transmembrane domains II and III that contribute to ligand binding sites (Surratt et al., 1994). For example, substitution of Ala for each Asp residue in the transmembrane domains reduces the binding of Ix-selective agonists to less than 5% of wild-type receptor values. These results may explain the high receptor affinity of the positively charged Ix-agonists and the increased potency produced by amidation of the terminal carboxyl group. They also indicate a difference between central (brain) and peripheral (GPI) I~-opioid receptors, because the peripheral receptor is not preferentially activated by positively charged agonists. The functional role of the D-amino acid located between two aromatic rings in the dermorphin sequence has been tested with numerous substitutions of the D-Ala2 residue. The L-AlaZisomer substitution produced a 100-fold decrease in Fu-receptor affinity and

The Dermorphin Peptide Family

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TABLE 2. Affinity for ~ and 8 opioid receptors, and biological activities on guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations of dermorphin homologues and analogues ICs0, nM (mean -+ SE) K/, nM (mean -+ SE) Peptide Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 Tyr-D-Ala-Phe-Gly-Tyr-Pro-NH2 Tyr-D-Ala-Phe-Gly-Tyr-NH2 Tyr-D-Ala-Phe-Gly-NH, Tyr-D-Ala-Phe-GIy-OH Tyr-D-Ala-Phe-NH2 DER-Gly-Glu-Ala-Lys-Lys-Ile-NH2 DER-Gly-Glu-Ala-Lys-Lys-Ile-Lys-Arg-NH2 Tyr-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 Tyr-D-Met-Phe-Gly-Tyr-Pro-Ser-NH2 Tyr-D-Pro-Phe-Gly-Tyr-Pro-Ser-NH2 Tyr-D-Arg-Phe-Gly-Tyr-Pro-Ser-NH2 Tyr-D-Arg-Phe-Gly-NH2 Tyr-D-Arg-Phe-Gly-OH Tyr-D-Arg-Phe-Lys-NH2 (DALDA) Tyr-D-Arg-Phe-Sar-OH (TAPS)

Ix system 0.540 1.6 0.98 11.9 138

8 system

-+ 0.021 +- 0.11 -+ 011 + 1.2 +_ 20 _ 0.149 _+ 0.01 0.002 _+ 0.0008 2171 _+ 151 92.4 + 6.3 2400 _+ 861 3.9 +_ 0.5 1.7 _+ 0.3 5.25 _+ 0.8 1.69 +_ 0.25 1.1 _+ 0.2

929 320 88 1800

_+ 41 -+ 19 +-- 8.6 -+ 180 --130 + 60 7.2 -+ 0.4 >40000 1340 _+ 162 1890 _+ 423 --->20000 --

GPI 1.29 -+ 0.11 2.73 + 0.35 2.88 -+ 0.37 34.5 -+ 4.71 676.8 _+ 75 262 _+ 30 1.53 _+ 0.47 1.37 + 0.39 >4000 47.0 -+ 5.1 >50000 92 +_ 10 10.8 _+ 3 23 _+ 4 254 + 27 6.6 + 0.8

MVD 16.5 43.2 23.8 263

-+ 1.3 -+ 39 +- 3.6 -+ 65 -4050 +_ 211 -->15000 249 _+ 25 >50000 ---781 + 146 --

Ki, equilibrium dissociation constant of the competing ligand; I~-opioidreceptors have been labelled with pH]DAMGO, 0.3 raM; 8-opioid receptors have

been labelled with pH]DPDPE; DER, dermorphin.

GPI activity (Table 2). Substitution of D-AIa2 with D-Met 2 or D-Lysz also reduced binding to the Ix-receptor and inhibitory activity on GPI electrically stimulated contractions, whereas substitution with D-Arg2 decreased GPI activity, yet preserved or enhanced ix-receptor affinity and analgesic potency. Typical examples are DALDA (Tyr-D-Arg-Phe-Lys-NH2) (Schiller et al., 1989) and TAPS (Tyr-D-Arg-Phe-Sar-OH) (Sato et al., 1987), two dermorphin-derived tetrapeptides that have high affinity for brain Ix-receptors and are more potent in inducing antinociception than in inhibiting GPI contractions.

PHARMACOKINETIC S Like many other peptides, dermorphin does not readily enter the blood-brain barrier. Nevertheless, dermorphin analogs containing basic amino acid residues appear to penetrate more easily into the brain after peripheral administration. Although, by ICV injection, D-Arg2 substituted tri- and tetra-peptides (Tyr-D-Arg-Phe-NHCH3, Tyr-D-Arg-Phe-N(CH3)2, Tyr-D-Arg-Phe-Gly-NH2, Tyr-D-ArgPhe-Gly-OH, Tyr-D-Arg-Phe-Sar-OH) are weaker analgesics than dermorphin, their antinociceptive potency is 2-10 times greater by SC administration (Sato et al., 1987; Chaki et al., 1988b; Sakurada et al., 1992; Paakkari et al., 1993b). Among naturally occurring dermorphins, [LysY]dermorphin has a relatively high rate of blood-tobrain influx and displays a saturable brain efflux (Negri et al., 1995). This high rate of brain entry may be due, at least in part, to a bloodbrain transport system similar to that described for other peptides (Barrera et al, 1989; Banks and Kastin, 1990). After IV injection in rats, dermorphin rapidly leaves the blood (tl/~= 1.3 min) and accumulates in liver and kidney. Although carcass and skin have a somewhat lower level of dermorphin, because of their mass they account for the major fraction of the drug in the body. Five minutes after the injection, the rat blood still contains about 7% of the injected peptide, 34% of which accounted for intact dermorphin and 60% by its break-down products (di-, tri- and tetra-N-terminal fragments). In vivo and in vitro experiments have demonstrated that the peptide is mainly destroyed by the liver and the kidney and partially excreted in the bile (Negri and Improta,

1984). Scalia et al. (1986) showed that dermorphin was much more resistant than Leu-enkephalin to human and rat plasma proteolitic enzymes (dermorphin tl/2=180 rain; Leu-enkephalin t1/2=2.1-4.5 min) and that, in the brain, dermorphin was cleared with a tt/2 of 29 min whereas Leu-enkephalin disappeared with a t~/2 of only 1 min. Experiments comparing enzymatic degradation of four natural dermorphins have unexpectedly shown that, within few minutes of incubation with brain homogenates, the biological activity of[Trp4] dermorphin and [Trp4, AsnV]dermorphin is completely destroyed whereas that of dermorphin, Tyr-D-Ala-Phe-Trp-Asn-NH2 and [LysT]dermorphin disappears slowly. The rapid cleavage of Trp 4 containing peptides probably occurs between Trp 4 and Tyr 5, an unusual site of endopeptidase activity. This type of peptidase activity in brain homogenates is resistant to conventional mixtures of protease inhibitors (Negri et al., 1992). Peptides containing in their sequence a basic amino acid as C-terminal amide (i.e. [Lys7] or [ArgT]dermorphin) showed the slowest degradation rate by brain enzymes and a prolonged duration of the antinociceptive response after ICV injection. Substitution of the second amino acid with another D-isomer does not modify the sites of degradation by brain enzymes, but may increase the resistance to peptidases. Sasaki et al. (1985) found that dermorphin and [D-Arg2]dermorphin underwent similar degradation pathway in the brain to produce the N-terminal tetrapeptide, but the Tyr-D-Arg bond was more stable than the Tyr-D-Ala bond to cleavage by aminopeptidase (Sato et al., 1992). This may explain why D-Arg substituted analogs induce relatively longer-lasting antinociception than D-Ala-containing peptides.

PHARMACOLOGICAL PROPERTIES Dermorphins are highly selective Ix-opioid agonists with no appreciable effects on K, ~ and putative ~ receptors. Central nervous system

ANTINOCICEPTIVE AND BEHAVIORALEFFECTS. Like Ix-opioid agonists, dermorphins produce antinociception, catalepsy, rigidity and sedation. When injected ICV naturally occurring dermorphins are

1102 potent analgesics, [Lys7]dermorphin being the analgesic with the highest potency and efficacy. The ICV, the IV and the SC antinociceptive EDs0 values of [LysT]dermorphin are 290, 30 and 20 times lower than those of morphine. By peripheral administration, the peptide is still 6 times more active than dermorphin. Other positively charged analogs are also potent antinociceptive peptides. [DArg 2, Sar4]dermorphin (1-4) administered ICV in mice is 255 times more potent than dermorphin (1-4) (Sato et al., 1987) and the [DArgqdermorphin tripeptide analogs Tyr-D-Arg-Phe-NHCH3 and Tyr-D-Arg-Phe-N(CH3)2 are more potent and longer-lasting antinociceptive agents than morphine (Sakurada et al., 1992 ). These data confirm the hypothesis that positively charged peptides are more active on central than on peripheral I*-opioid receptors and that the "analgesic receptor" represents a distinct b~-receptor subtype. This conclusion is corroborated by evidence that the antinociception produced by Arg- or Lys-containing peptides is extremely sensitive to the antagonism of naloxonazine, a hr,-preferring ligand (Paakari et al., 1993b; Negri et al., 1995). Dermorphin produces antinociception at supraspinal and at spinal levels. Peripherally injected dermorphin (1.9 txmol/kg, IP) blocked the high firing frequency evoked by a noxious stimulus (strong pressure, radiant heat) in the nucleus lateralis anterior and ventrobasal complex of the rat thalamus without affecting basal spontaneous firing (Braga et al., 1984). At the spinal level, dermorphin exerts profound effects on nociceptive processing in the rat dorsal horn. In laminectomized rats, dermorphin applied directly onto the exposed spinal cord produces a dose-dependent, selective inhibition of the C fibre-evoked response in dorsal horn nociceptive neurons (test stimulus being electrical pulses on the rat paw). The peptide inhibits both superficial and deep dorsal horn cells, leaving fibre-evoked activity relatively unaffected (Sullivan and Dickenson, 1988). In man, given by IT route, dermorphin has an analgesic potency 100 times greater than that of morphine (Basso et al., 1985). By IV infusion, at a rate of 5.5 Izg/kg/min for 30 min, the peptide induces a marked and long-lasting increase in the threshold of the nociceptive reflex both in healthy subjects and in spinal cordlesioned patients, indicating that nociceptive transmission is depressed mainly at spinal level (Basso et al., 1985; Sandrini et al., 1986). In addition to antinociception, ICV injection of dermorphin-like peptides modify locomotor behavior. Paakkari et al., (1990) observed increased motor activity in rats given an ICV injection of low doses of dermorphin, whereas high doses caused catatonia. Comparison of their antinociceptive potency (ADs0) and catatonic potency (CDs0) shows that some dermorphin analogs require relatively high dosage to produce catatonia. The ratio ADs0/CDs0 varies widely among the different agonists. [LysT]dermorphin, [DArg 2, Sarqdermorphin 1-4, Tyr-D-Arg-Phe-NHCH3 and Tyr-DArg-Phe-N(CH3)2 show the lowest ratio (0.07 to 0.12), dermorphin has an intermediate value (0.32) and [Trp4, AsnT-NH2]dermorphin has the highest ADs0/CD50 ratio, about 500 times higher than that of [LysT]dermorphin (Negri et al., 1992). Dermorphin-induced catatonia is characterized by catalepsy (inability to initiate movements from abnormal position), akinesia (loss of spontaneous locomotor activity) and intense rigidity of the skeletal muscles of trunk and tail (Broccardo et al, 1981). With [LysT]dermorphin, hypokinesia and akinesia prevail, rigidity appearing only at peptide doses close to the lethal dose. At ICV doses that leave the reaction time in the tailflick test unchanged, [Trip4, AsnT]dermorphin induces long lasting catalepsy. The cataleptic response to low doses of [Trp4, AsnT]dermorphin is accompanied with flaccidity of the trunk skeletal muscles and tonic contractions of finger extensor muscles (Negri et al., to be published). Neither the akinesia produced by [LysT]dermor-

P. Melchiorri and L. Negri phin nor the catalepsy produced by[Trp4, AsnT]dermorphin are prevented by naloxonazine pretreatment (Negri et al., 1995). In contrast, dermorphin rigidity is strongly reduced by naloxonazine pretreatment. This is a rather surprising finding, considering that morphine catatonia is significantly attenuated by naloxonazine, as is TAPS catatonia (Paakkari et al., 1993b). It is noteworthy that naloxonazine effectively reduces opioid rigidity but not opioid akinesia, and that rigidity is the prevailing component of morphine and TAPS catatonia. Dermorphin catalepsy is most evident after peptide injection into the nucleus accumbens. Bilateral electrolytic lesions of the caudate nucleus potentiate the cataleptic response but prevent the muscular rigidity provoked by dermorphin (Improta and Guglietta, 1985). Like morphine, some dermorphin analogs have a biphasic effect on rat locomotor activity. For example, the tetrapeptide Tyr-D-ArgPhe-Lys-NH2 (DALDA) (ICV, from 0.1 to 1.0 txg/rat) resulted in inhibition followed by activation of horizontal movement, rearing and stereotypes (Meyer and Mclaurin, 1995). Low ICV doses (50-100 pmol/rat) of dermorphin in rats cause rather sustained "grand mal" EEG seizures associated with twitches and slight tremors, whereas high doses (>200 pmol/rat) produce tonic contractions of trunk muscles. During the postconvulsive phase induced by low doses, animals become hyperactive, whereas high doses led to exophthalmus, mydriasis, rigidity and catatonia (Aloisi et al., 1982). Microinjections of dermorphin into various areas of the rat brain have shown that epileptic discharges are triggered only in the ventral hippocampus (CA1 and CA3 areas), amygdala and entorhinal cortex. The epileptic threshold is the lowest in the CA1 area of the ventral hippocampus, suggesting that ICV injections of the agonist trigger the epileptic activity in the ventral hippocampus (Greco et al., 1994). In rat hippocampal slices, low concentrations of dermorphin (50-100 nM) markedly enhanced the CA1 and CA3 population responses to threshold levels of stimulation from both the Schaffer collaterals and messy fibres, whereas they leave responses to higher stimulus levels almost unchanged. However, at 500 nM dermorphin, all responses become epileptiform and, in some slices, spontaneous bursting erupted (Jones et al., 1994), These data provide evidence that excessive I*-opioid receptor activation can be proconvulsant in the hippocampus, but that, within the normal threshold range, the receptors may function as facilitatory modulators of responses. Dermorphin (0.05-5 pmol) applied unilaterally or bilaterally into the locus ceruleus of the rat produces behavioural and electrocorticographic (ECoG) soporific effects (Desarro et al., 1992). In rabbits, dermorphin induces sedative effects. ICV injection of 1.2 nmol/rabbit produces an EEG pattern resembling that occurring during physiological drowsiness. By IV injection, dermorphin (500 nmol/kg) produces a strong increase in quiet immobility and a concomitant reduction in voluntary activities, lasting 20 min. Neither pattern nor frequency of hippocampal electric activity were affected (Fontani et al., 1993). In birds, dermorphin does not influence sensitivity to painful stimuli. When injected into the third ventricle of the chick, the peptide elicits a dose-dependent behavioural and electrocortical sedation or sleep and a decrease in body temperature with no analgesia. In producing sleep, dermorphin is 24 times as potent as [3-endorphin (Nistic6 et al., 1981). The tetrapeptide analog TAPA (Tyr-DArg-Pheq3-Ala-NH2) (0.3-3 ng, ICV) impairs passive avoidance learning in mice, resulting in a dysfunction of memory consolidation, without affecting other behavioral responses (Ukai et al, 1993). In squirrel monkeys, the behavioural effects of dermorphin (1.2-

The Dermorphin Peptide Family 37.3 nmol/kg, ICV) depend on the manner in which responding is controlled by its consequence (Nader and Barret, 1989). EFFECTSON MICTURITIONAND RENALFUNCTION. Dermorphin produces a dose-related (1-10 pmol/rat) marked inhibition of reflex mieturition in urethane-anaesthetized rats when injected by the IT route, but had little or no effect by the IV route (up to 0.1 ~mol/ kg), suggesting that the peptide acts at the spinal cord without involving the periphery (Maggi et al., 1989). ICV (0.1 nmol/kg), but not IV, injection of dermorphin, in conscious normal and renaldenervated rats produces an increase in urine flow rate and a sustained decrease in urinary sodium excretion. It does not alter the glomerular filtration rate or the effective renal plasma flow. This suggests that tx-opioid receptor agonists may exert an influence on tubular reabsorption of sodium via a ~-opioid receptor-mediated mechanism independent of intact renal innervation (Kapusta et al., 1993). EFFECTS ON PULMONARYVENTILATION. At low doses, both by ICV and peripheral administration, dermorphin and its analogs stimulate ventilatory minute volume. Paakkari et al. (1993b) demonstrated that up to a dose of 100 pmol/rat ICV that induces a naloxonazine-sensible catalepsy and full antinociception, TAPS, a putative I~l-agonist, stimulates ventilation. Respiratory stimulation is also antagonized by naloxonazine. At doses up to 1.2 I~mol/kg by SC route, [LysT]dermorphin increases respiratory frequency and minute volume, but not tidal volume of air breathing rats. (Negri et al, to be published). Only very high doses of dermorphin (1-30 nmol) and TAPS (1-10 nmol) injected ICV to conscious, unrestrained rats decrease ventilation rate and minute volume dose-dependently (Paakkari et al., 1993a, 1993b). Subcutanous doses of [LysT]dermorphin that reduce ventilatory frequency, but increase tidal volume, of air-breathing rats are 40 times higher than those inducing antinociception. However, antinociceptive doses of all dermorphins are able to reduce the hypercapnic response to inhaled CO2 (Negri et al, to be published). In pentobarbital anaesthetized rats, dermorphin (1.2 Ixmol/kg IV) significantly depresses the slope of the CO2 response curve, compared with the control, but has no significant effect on respiratory rate (Eager et al., 1994). In conscious unrestrained rats breathing air, the ventilatory depression induced by 1-30 nmol ICV of dermorphin is antagonized by naloxone and by the benzodiazepine antagonist, flumazenil. The benzodiazepine, alprazolam, potentiates the respiratory inhibition induced by 1 nmol of dermorphin but antagonizes that induced by a higher dose (3 nmol/rat), suggesting that the benzodiazepine/ GABA receptor complex modulates respiratory depression induced by central w-receptor stimulation in the rat (Paakkari et al., 1993a). In rabbits, dermorphin depresses pulmonary ventilation in a dosedependent manner (Marchioni et al., 1992). BODYTEMPERATURE. In many species, opioids alter the equilibrium point of the hypothalamic heat-regulatory mechanisms. Dermorphin, at a dose that induces catatonia (0.12 nmol, ICV), produces hypothermia ( - 3 ° C to -4°C) in rats kept at low temperature ( - 1 0 ° C or +4°C) and hyperthermia (+3°C) in rats mantained at room or higher ( + 34°C) temperature. These responses last over 3 hr and are similar in fed and fasted rats. (Broccardo, 1987a). In rabbits mantained at room temperature, dermorphin produces hypothermia (Marchioni et al., 1992). DRINKING BEHAVIOUR. l e g dermorphin produces a dosedependent (0.0012-0.0062 nmol/rat) inhibition of angiotensin II-

1103 induced drinking (31%-70%), probably acting at sub-fornical organ, as shown by the high sensitivity of this organ to direct injections of the peptide (0.5 ng=0.0006 nmol). The inhibition of water deprivation-induced drinking requires higher doses, making questionable the specificity of the effect (Perfumi et al., 1986). PITUITARY HORMONE RELEASE. Acute administration of dermorphin, in rats, enhances basal and stress-induced (acute cold swimming stress) plasma levels of corticosterone and [~-endorphinlike immunoreactivity. These effects are antagonized by pretreatment with naloxone. Long-term administration of dermorphin does not alter resting plasma levels of corticosterone and [3-endorphinlike immunoreactivity, but significantly reduced stress-induced increase in these hormones (Degli Uberti et al., 1995). In rats both SC (0.4-6.2 Ixmol/kg) and ICV (1.2 nmol/kg) injection of dermorphin enhance the release of prolactin frora two- to fifteen-fold, dose-dependently. The observation that, in isolated and dispersed rat pituitary cells, dermorphin has no prolactin-releasing effect, suggests that the peptide acts not on the pituitary, but on the hypothalamus (Giudici et al., 1984; Rossi et al., 1983). Administration of dermorphin in rats has variable and poor effects on LH, FSH, TRH, TSH and thyroid hormone release (Erspamer and Melchiorri, 1983). The increase of plasma TRH and TSH levels in response to cold exposure is inhibited by the peptide (Mitsuma et al., 1985). Single or repeated doses of 1.2 nmol dermorphin, into the third cerebral ventricle, produces maximal crop stimulation and also stimulation of the pituitary lactotrophs in the pigeon (Rotiroti et al., 1984). In normal human subjects, dermorphin infusion (5.5 Izg/kg/min per 30 min) greatly increases plasma prolactin and growth hormone concentrations ( Degli Uberti et al., 1983a, b). It also significantly increases plasma renin activity and decreases plasma cortisol levels, but poorly affected aldosterone and ACTH levels (Degli Uberti et al., 1983c). In fertile women and in post-menopausal women treated with estrogens and progestins, 1V infusion of dermorphin decreases plasma LH but not FSH levels.

Gastrointestinal tract Like other opioids, dermorphin inhibits gastric acid secretion, delays gastric emptying and slows intestinal and colonic propulsion. Dermorphin injected ICV (10-120 pmol) into pylorus-ligated rats inhibited (up to 90%) gastric acid secretion induced by water distension of the stomach. Gastric secretion is reduced, but H t concentration remains ahnost unchanged. The peptide also moderately inhibits insulin-stimulated gastric secretion but has no effect on histamine-stimulated secretion. Among the shorter analogs of dermorphin, the N-terminal tetrapeptide amide is the most active, whereas the N-terminal hexapeptide amide is inactive (hnprota et al., 1982; Guglietta et al., 1987). In dogs with gastric fistula and Heidenhain pouch, dermorphin causes a significant increase in basal acid secretion and in acid secretion stimulated by 2-deoxy-D-glucose, bethanechot or pentagastrin. Measurements of plasma gastrin suggest that the opioid-induced changes in acid secretion are not due to changes in gastrin release (Intorre et al. 1993). In humans, a SC dose of 2 mg dermorphin reduces gastrin-stimulated gastric acid secretion in normal volunteers and in subjects submitted to truncal vagotomy (Lezoche et al., 1983). When it was injected by the ICV route, dermorphin caused a dose-related (26-620 pmol) delay (7%-86%) of rat gastric emptying (Broccardo et al., 1982). Some evidence suggests a pituitary-adrenal mediation of this effect (Broccardo, 1987b). Tolerance to the gastric emptying inhibitory effect develops after

1104

P. Melchiorri and L. Negri

10 days of repeated SC administration of dermorphin, or after 2 days of ICV infiasion (Broccardo, 1985). Compared with dermorphin, [LysT]dermorphin is slightly (3-5 times) more potent, both in reducing acid gastric secretion and gastric emptying. By contrast, [Trp4, AsnT]dermorphin is 20-50 times less potent than dermorphin in inhibiting gastric acid secretion, but is 100-1000 times less potent in delaying gastric erupting. (Improta and Broccardo, 1994). Dermorphin inhibits colonic propulsion in the rat in a dose-related manner (75-750 pmol/rat, ICV). The N-terminal dermorphin tetrapeptide is 9% as active as dermorphin. Naloxone blunts, but does not abolish, the effect of dermorphin (Broccardo et al. 1982; Rossi et al.,1983). Ferr~ et al. (1986) observed in fed conscious rats a clearcut inhibition of duodenal and colonic myoelectric activity that was approximately of the same intensity (35% in duodenum and 80% in colon) regardless of the route of dermorphin administration, suggesting multiple sites of action (supraspinal, spinal and peripheral). Local application of dermorphin on guinea-pig ileum (intra-arterial infusion in peristaltic reflex preparations, or in isolated organ bath) potently abolishes peristaltic reflex or cooling-induced contractions (Erspamer, 1992). Basal and caerulein-stimulated rat pancreatic secretion is unaffected by IV injection of dermorphin (0.15 Izmol/kg), although the peptide sharply reduces 2-deoxyglucose-stimulated protein output and protein concentration (Baldieri et al., 1983). Cardiovascular

effects

In the conscious dog, I and 10 Ixg/kg of dermorphin IV causes a fugaceous increase (5 min) in blood pressure (30%) and heart rate (50%) (Sander and Giles, 1982). In rats, analgesic doses of dermorphin have no major effect on blood pressure or cardiac rate. ICV administration of supramaximal analgesic doses (0.01-1 nmol/rat) increases the systolic and diastolic blood pressure and the circulating levels of adrenaline and noradrenaline. Doses of 6-12 nmol/rat caused a small fall in arterial pressure and doses higher than 50 nmol/rat caused a consistent pressure fall accompanied by severe bradycardia (Feuerstein and Faden, 1983; Feuerstein and Zukowska, 1987; Portolano et al., 1991a). By the IV route, dermorphin causes a transitory 40 to 50% fall in blood pressure associated with moderate bradycardia both in normotensive (0.12-0.62 txmol/kg) and in spontaneously hypertensive rats (0.0012 txmol/kg). The blood pressure fall caused by IV dermorphin is partially antagonized by an antiserum to the atrial natriuretic factor, indicating that release of this factor may be involved in the cardiovascular effects of dermorphin (Portolano et al., 1991b). Erspamer (1994) reported that, in urethane-anaesthetized rabbits, the moderate decrease in blood pressure induced by dermorphin underwent evident tachyphylaxis upon repeated injection of the peptide. Using dermorphin and its analog TAPS, a putative ~t-receptor agonist, Paakkari et al. (1992) suggested that txt-opioid receptors mediate tachycardic responses, whereas t&-receptors mediate bradycardic responses to dermorphins. On the isolated guinea-pig left atrium, all natural dermorphins exert a moderate stimulating action (EDs0=5-10 nM) (Erspamer, 1994). TOLERANCE AND DEPENDENCE In rats and mice, central or peripheral administration of the dermorphin-like peptides induces a significantly slower development of tolerance to the antinociceptive effect than does morphine. Tolerance to the cataleptic response and to muscular rigidity appears earlier than to the antinociceptive effect (Broccardo et al., 1985; Negri et al., 1995). In mice, tolerance to morphine antinociception reaches a

maximum after 4-day continous SC infusion of the alkaloid, whereas tolerance to [Lys7]dermorphinantinociception progressively increases over 7-day infusion of the peptide. An evaluation of the antinociceptive effects of morphine in 7-day morphine-tolerant mice indicates a decrease in the maximum achievable antinociceptive response, as shown by the reduced slope of the dose-response curve. In contrast to the results seen with morphine, an even higher degree of tolerance did not alter the ability of [LysT]dermorphin to produce a maximal antinociceptive effect. These results indicate that [Lys7]dermorphin is a compound with greater efficacy than morphine or that, in tolerant animals, the peptide acts as a full agonist whereas morphine behaves as a partial agonist. Acute administration of morphine in dermorphin-tolerant rats results in a significantly decreased analgesic and cataleptic respose to the alkaloid, showing the onset of cross-tolerance. However, dermorphin cataleptic response is unaffected in morphine-tolerant rats, but analgesic potency is sharply reduced (Broccardo and Improta, 1985; Stevens and Yaksh, 1986). In mice, SC [Lys7]dermorphin produces a full cross-tolerance with morphine (Negri et al., 1995). A marked tolerance to [D-Arg2, Sar4]dermorphin (1-4) and morphine has also been seen in rats made tolerant to morphine. However, the antinociceptive activity of morphine in [D-Arg2, Sar4]dermorphin (1-4) -tolerant rats did not decline, suggesting that, compared with morphine, the peptide may have a more limited site of action in the nociceptive pathway. Interestingly [D-Arg2, Sar4]dermorphin (1-4) failed to substitute for morphine in morphine-dependent rats (Sakurada et al., 1993). Withdrawal symptoms precipitated by naloxone injection were considerably less severe in rats chronically exposed to dermorphin than in animals receiving morphine (Broccardo et al, 1985). Abrupt withdrawal of [Lys7]dermorphin in tolerant mice produces mild symptoms and no loss of body weight (Negri et al., 1995). Upon naloxone precipitation, [Lys7]dermorphin-dependent mice make fewer jumps and lose less urine and feces than morphine-dependent animals. Withdrawal hyperalgesia does not develop in [Lys7]dermorphin-dependent mice (Negri et al., 1995). Studies conducted with other dermorphin analogs have confirmed that withdrawal symptoms precipitated by naloxone are less intense in peptide-dependent than in morphine-dependent rats (Nakata et al., 1986; Chaki et al., 1988a). C O N C L U D I N G REMARKS The discovery of dermorphins in 1980 has provided exciting news on the opioid system, but it has also raised many problems. The efforts to demonstrate the existence of dermorphin-like peptides in mammalian tissues did not yet provide inconfutable evidence. Approaching the problem with the usual techniques, such as radioimmunoassay and HPLC purification, one has to fight against the very low amount eventually present and the possibility that owing to differences in their amino acid sequence, mammalian dermorphins do not cross-react with antisera raised against amphibian dermorphin. Neverthless, using a highly specific antidermorphin serum (Negri et al., 1981), Buffa et al. (1982) obtained a clear-cut immunostaining of nerve cells and fibres in nucleus arcuatus and other nervous stuctures of rat brain only after colchicine pretreatment. Moret al. (1989) have indentified in rat small intestine a peptide (2 ng/g tissue) behaving like authentic dermorphin by HPLC and immunoassay. The detection of immunoreactive material of high molecular weight in guinea pig and rat stomach (Tseu et al., 1985) and in rat brain and adrenals (Moret al., 1991), stomach and intestine (Mor e t al., 1990), argues in favour of the existence of endogenous precur-

The Dermorphin Peptide Family sors of dermorphin-like peptides in mammalian tissues. Immunocytochemical studies have revealed immunostained elements in the rat brain, including regions involved in pain control and motricity. The immunoreactivity was localized both in neuronal and nonneuronal cells (ependymal cells along the ventricles) and was often associated with brain capillaries; thus, suggesting that dermorphins may act not only as a neurotransmitter or neuromodulator, but also as a neuroendocrine agent. In the stratum pigmentis retinae of the rat, a very intense band of dermorphin-like immunofluorescence could be demonstrated (Erspamer, 1992). Studies on the biosynthesis of dermorphins have revealed a new posttranslational reaction catalyzed by an, as yet unknown, enzyme system that quantitatively converts the L-Ala residue located between two aromatic rings of the precursor into the D isomer. Studies of structure-activity relationships in the dermorphin peptide family have provided evidence of two distinct Ix-opioid receptor subtypes and of differential conformational requirements for activation of central and peripheral I*-opioid receptors. Dermorphins containing C-terminal amides of basic amino acids (Lys or Arg) can enter the blood-brain barrier more easily than other analogs and concentrate themselves in the SNC through a still poorly defined peptide transport system. Experiments on chronic exposure to dermorphins demonstrated that these opioids act prevalently at spinal level and are less likely than morphine to produce tolerance, dependence and opiate side effects but have higher antinociceptive efficacy and potency. New dermorphin analogs, selective for spinal opioid receptors and active by peripheral administration, offer great promise of providing novel potent analgesics with enhanced medical benefit and reduced toxicity. References Aloisi F., Passarelli F., Scotti A., De Carolis A. and Longo V. G. (1982) Central effects of dermorphin. Ann. Istituto Sup. Sanita 18, 1-6. Ambo A., Sasaky Y. and K. Suzuki (1994) Synthesis of carboxy-terminal extension analogs of dermorphin and evaluation of their opioid receptorbinding and opioid activities. Chem. Pharmac. Bull. 42, 888-891. Baldieri Linari M., R. Castellacci and G. Linari (1983) The effect of dermorphin on exocrine pancreatic secretion of the rat. hal. J. Gastroenterol. 15, 177-180. Banks W. A. and Kastin A. J. (1990) Peptide transport systems for opiates across the blood-brain barrier. Am. ]. Physiol. 259, El-E10. Barrera C. M., Banks W. A. and Kastin A. J. (1989) Passage of Tyr-MIF-1 from blood to brain. Brain Res. Bull. 23, 439-442. Basso N., Marcelli M., Ginaldi A. and DeMarco M. (1985) Intrathecal dermorphin in post-operative analgesia. Peptides 6, 177-179. Braga P. G., Tienco M., Biella G., Dall'Oglio G. and Franceschini F. (1984) Dermorphin, a new peptide from amphibian skin, inhibits the nociceptire thalamic neurons firing rate evoked by noxious stimuli. Neurosci. Lett. 52, 165, 169. Broccardo M. (1985) Development of tolerance to the effect of dermorphin on gastric emptying in the rat. Pharmac. Res. Commun. 17,345-350. Broccardo M. (1987a) Effect of dermorphin on body temperature in rats. Pharmac. Res. Commun. 19, 713-721. Broccardo M. (1987b) Pituitary-adrenal mediation of dermorphin-induced inhibition of gastric emptying in rats. Eur. J. Pharmac. 142, 151-154. Broccar&) M., Erspamer V., Falconieri Erspamer G., Improta G., Linari G., Melchiorri P. and Montecucchi P. C. (1981) Pharmacological data on dermorphins, a new class of potent opioid peptides from amphibian skin. Br. J. Pharmac. 73,625-631. Broccardo M., hnprota G. (1985) Cross-tolerance between dermorphin and morphine to analgesia and catalepsy in rats. Peptides 6, 165-169. Broccardo M., Improta G., Nargi M. and Melchiorri P. (1982) Effects of dermorphin on gastrointestinal transit in rats. Regul. Peptides 4, 91-96. Broccardo, M., hnprota G., Negri L. and Melchiorri P. (1985) Tolerance and physical dependence induced by dermorphin in rats. Eur. J. Pharmac. 110, 55-61. Buffa R., Solcia E., Magnoni E., Rinaldi G., Negri L. and Melchiorri P. (1982) hnmunohistochenrical demonstration of a dermorphin-like peptide in rat brain. Histochemistry 76, 273-276.

1105 Chaki K., Kawamura S., Kisara K., Sakurada S., Sasaki Y., Sato T. and Suzuki K. (1988a) Antinociception and physical dependence produced by [D-Atg2]dermorphin tetrapeptide analogs and morphine in rats. Br. J. Pharmac. 95, 15-22. Chaki K., Sakurada S., Sakurada T., Sato T., Kawamura S., Kisara K., Watanabe H. and Suzuki K. (1988b) Comparison of the antinociceptive effects of new [D-Arg2]dermorphin tetrapeptide analogs and morphine in mice. Pharmac. Biochem. Behav. 31,439-444. Darlak K., Grzonka Z., Salvadori S. and Tomatis R. (1987) Synthesis and biological activity of some cyclic dermorphins. Polish J. Chem. 62, 445450. De Castiglione R., Faoro F., Perseo G., Piani S., Santangelo F. and Melchiorri P. (1981) Synthetic peptides related to dermorphins. I. Synthesis and biological activities of the shorter homologues and of analogues of the heptapeptides. Peptides 2, 265-269. Degli Uberti E. C., Petraglia F., Bondanelli M., Guo A. L., Valentini A., Salvadori S., Criscuolo M., Nappi R.E. and Genazzani A.R. (1995) Involvement of ix-opioid receptors in the modulation of pituitary-adrenal axis in normal and stressed rats. J. Endocr. Invest. 18, l-7. Degli Uberti, E. C,, Trasforini G., Salvadori S., Margutti A., Tomatis R., Bianconi M., Rotola C. and Pansini R. (1983a) Prolactin-releasing activity of dermorphin, a new synthetic potent opiate-like peptide in man. J. Clin. Endocrinol. Metab. 56, 1032-1034. Degli Uberti E. C., Trasforini G., Salvadori S., Margutti A., Tomatis R., Rotola C., Bianconi M. and Pansini R. (1983b) Stimulatory effects ofdermorphin, a new synthetic opiate-like peptide, on human growth factor secretion. Endocrinology 37, 280-283. Degli Uberti E. C., Trasforini G., Salvadori S., Margutti A., Tomatis R., Bianconi M., Rotola C. and Pansini R. (1983c) Responses of plasma renin activity, a[dosterone, adrenocorticotropin, and cortisol to dermorphin, a new synthetic opiate-like peptide, in man.J. Clin. Endocrinol. Metab. 57, 1179-1185. Desarro G. B., Spagnolo C., Audino M. G., Marra R., Nistic6 G. (1992) Comparative behavioural and ECoG spectrum effects of dermorphin and Ala-deltorphin given into some areas of the rat brain. Res. Comm. Sub. Abuse 13, 147-165. Eager K. R., Robinson B. J., Galletly D. C., Miller J. H. (1994) Endogenous opioid modulation of hypercapnic-stimulated respiration in the rat. Resp. Physiol. 96, 13-24. Erspamer V. (1992) The opioid peptides of the amphibian skin. Int. J. Devel. Neurosc. 10, 3-30. Erspamer V. (1994) Bioactive secretions of the Amphibian integmnent. In Amphibian Biology, (Edited by Heatwole H.) p. 178-350. Surrey Beatty & Sons Publ. Erspamer V. and Metchiorri P. (1983) Actions of Amphibian skin peptides on the central nervous system and the anterior pituitary. In Neuroendocrine Perspectives, Vol. 2, (Edited by Muller E. E. and McLeod R. M.) pp. 37-106. Elsevier Sci. Publ., Amsterdam. Erspamer V., Melchiorri P., Falconieri Erspamer G., Negri L., Corsi R., Severini C., Barra D., Simmaco M. and Kreil G. (1989) Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for opioid binding sites. Proc. Natl. Acad. Sci. USA 86, 5188-5192. Ferr~ J. P., Du Ch., Soldani G. and Ruckenbush V. (1986) Peripheral versus central components of the effects of dermorphin on intestinal motility in fed rats. Regul. Peptides 13, 109-117. Feurestein G. and Faden A. 1. (1983) Central autonomic effect of dermorphin in conscious rats. J. Pharmac. Exp. Ther. 226, 13 18. Feurestein G. and Zukowska G. Z. (1987) Effect of dermorphin and morphine on the sympathetic and cardiovascular system in pithed rats. Neuropeptides 9, 139-150. Fontani G., Vergani L., Salvadori S., Voglino N., Aloisi A. M., Portatuppi F. and Degli Uberti E. C. (1993) Effect of dermorphin on behavior and hippocampal electrical-activity in rabbits. Life Sci. 52, 323-328. Giudici D., D'Urso R., Falaschi P., Negri L., Melchiorri P. and Motta M. (1984) Dermorphin stimulates prolactin secretion in the rat. Neuroendocrinol. 39, 236-244. Greco B., Prevost J. and Gioanni Y. (1994) Intracerebroventricular injection of dermorphin: search fi)r the epileptic induction thresholds. Neurorepcrrt 5, 2169-2172. Guglietta A., Irons B. J., Lazarus L. H. and Melchiorri P. (1987) Structure activity relationships of dermorphins on gastric secretion. Endocrinology_ 120, 2137-2143. lmprota G. and Broccardo M. (1994) Effect of selective mul, mu2 and delta2 opioid receptor agonists on gastric functions in the rat. Neuropharmacology 33,977-981. lmprota G, Broccardo M. , Lisi A. and Mdchiorri P. (1982) Neural regula-

1106 tion of gastric acid secretion in rats: influence of dermorphin. Regul. Pept/des 3, 251-256. Improta G. and Guglietta A. (1985) The role of caudate nucleus in dermorphin-induced catalepsy in rats. Pept/des 6, 161-164. Intorre L., Mengozzi G., Vanni E., Grassi F. and Soldani G. (1993) The role of peripheral opioid receptor subtypes in the modulation of gastric acid secretion and plasma ga~trin in dogs. Eur. J. Pharmac. 243, 265-272. Jones L. S., Grooms S. Y., Salvadori S. and Lazarus L. H. (1994) Dermorphin-induced hyperexcitability in hippocampal CA3 and CA1 "in vitro." Eur.J. Pharmac. 264, 39-48. Kapusta D. R., Obih J. C. and Dibona G. F. (1993) Central b~-opioid receptor mediated changes in renal-function in conscious rats. J. Pharmac. Exp. Ther. 265, 134-143. Kisara K., Sakurada T., Sakurada T., Sasaki T., Sato T., Suzuki K. and Watanabe H. (1986) Dermorphin analogue containing D-kyororphin: structure antinociceptive relationship in mice. Br. J. Pharmac. 87, 183-189. Kreil G. (1994). Peptides containing a D-amino acid from frogs and molluscs. J. Biol. Chem. 269, 10967-10970. Lazarus L. H. and Attila M. (1993) The toad, ugly and venomous, wears yet a precious jewel in his skin. Progress Neurobiol. 41,473-507. Lezoche E., DePasquale G., Carlini F., Procacciante F., Mariani F., Nigri R., Luminari N. and Speranza V. (1983) Effect of dermorphin, a new opiatelike peptide, on gastric secretion, exocrine pancreatic secretion and gall bladder motility in man. Regul. Peptides, Suppl 2, S139-S140. Maggi C. A., Giuliani S. and Meli A. (1989) Dermorphin inhibits micturition reflex in rats at a central site of action. J. Auton. Nerv. System 26, 11-15. Marchioni E., Maurelli M. and Tartara A. (1992) Quantitative EEG and autonomic patterns of synthetic peptides related to dermorphin. Neuropsychobiology 26, 81-88. Melchiorri P., Falconieri Erspamer G., Erspamer V., Guglietta A., DeCastiglione R., Faoro F., Perseo G., Piani S. and Santangelo F. (1982) Synthetic peptides related to the dermorphins. II. Synthesis and biological activities of new analogues. Peptides 3, 745-748. Melchiorri P., Negri L., Falconieri Erspamer G., Severini C., Corsi R., Soaje M., Erspamer V. and Barra D. (1991) Structure-activity relationships of the delta-opioid selective agonists, deltorphins. Eur. J. Pharmac. 195, 201-207. Meyer M.E. and Mclaurin B.I. (1995) DALDA (H-Tyr-D-Arg-Phe-LysNH2), a potent I*-opioid peptide agonist, affects various patterns of locomotor activities. Pharmac. Biochem. Behav. 61, 149-151. Mignogna G., Severini C., Simmaco M., Negri L., Falconieri Erspamer G., Kreil G. and Barra D. (1992) Identification and characterization of two dermorphins from skin extracts of the Amazonian frog Phyllomedusa bicolor. FEBS 302, 151-154. Mitsuma T., Noginori T. and Chaya A. (1985) Dermorphin inhibits basal and cold-induced thyrotropin secretion in rats. Endocrin. Exper. 19, 8390. Montecucchi P. C., de Castiglione R. and Erspamer V. (1981b) Identification of dermorphin and Hyp-dermorphin in skin extract of the Brazilian frog Phyllomedusa rohdei. Int. J. Pept/de Prot. Res. 17, 316-321. Montecucchi P. C., de Castiglione R., Piani S., Gozzini L., and Erspamer V. (1981a) Amino acid composition and sequence of dermorphin, a novel opiate-like peptide from the skin extracts of Phyllomedusa sauvagei. Int. J. Peptide Prot. Res. 17, 275-283. Mor A., Delfour A., Amiche M., Sagan S., Nicolas P., Grassi J. and Pradelles Ph. (1989) Dermorphin and related peptides in rat tissues. Neuropeptides 13, 51-57. Mor A., Delfour A. and Nicolas P. (1991) Identification of a D-Ala-containing polypeptide precursor for the peptide opioid, dermorphin. J. Biol. Chem. 266, 6264-6270. Mor A., Pradelles Ph., Delfour A., Montagne J. J., Quintere F. L., Conrath M. and Nicolas P. (1990) Evidence for pro-dermorphin processing products in rat tissues. Biochem. Biophys. Res. Commun. 170, 30-38. Mosberg H. I., Heyl D. L., Haaseth R. C, Omnaas J. R., Medzkihrasky F. and Smith C. B. (1990) Dermorphin-like tetrapeptides with 8-opioid receptor activity. 3. Effect of residue 3 modidfication on "in vitro" opioid activity. Mol. Pharmac. 38, 924-928. Nader W. and Barret J. (1989) Effects of corticotropin-releasing factor, tuftsin and dermorphin on behavior of squirrel monkeys maintained by different events. Peptides 10, 1199-1204. Nakata N., Sakurada S., Sakurada T., Kisara K., Sasaski Y. and Suzuki K. (1986) Physical dependence of a dermorphin tetrapeptide analogue, [DArgZ,Sar4]dermorphin (I-4) in the rat. Pharmac. Biochem, Behav. 24, 27-31. Negri L., Falconieri Erspamer G., Severini C., Melchiorri P. and Erspamer

P. Melchiorri and L. Negri V. (1992) Dermorphin related peptides from the skin ofPhyllomedusa bicolor and their amidated analogs activate two I*-opioid receptor subtypes which modulate antinociception and catalepsy, in the rat. Proc. Natl. Acad. Sci. USA 89, 7203-7207. Negri L. and Improta G. (1984) Distribution and metabolism of dermorphin in rats. Pharmac. Res. Commun. 16, 1183-1194. Negri L., Lattanzi R. and Melchiorri P. (1995) Production of antinociception by peripheral administration of [LysV]dermorphin, a naturally occurring peptide with high affinity for I*-opioid receptors. Br. J. Pharmac. 114, 57-66. Negri L., Melchiorri P., Falconieri Erspamer G. and Erspamer V. (1981) Radioimmunoassay of dermorphin-like peptides in mammalian and nonmammalian tissues. Peptide 2, 45-49. Nistic6 G., Desarro G. B., Rotiroti D., Melchiorri P. and Erspamer V. ( 1981 ) Comparative effcts of 13-endorphin and dermorphin on behaviour, electrocortical activity and spectrum power in chicks after intraventricular administration. Res. Commun. Physiol. Psychiat. Behaviour 6, 351-363. Paakkari P., Paakkari I., Feuerstein G., Siren A. L. (1992) Evidence for differential opioid mu~-receptor and mu2-receptor-mediated regulation of heart-rate in the conscious rat. Neuropharmacology 31, 777-782. Paakkari P., Paakkari I., Landes P., Siren A, L., Feuerstein G. (1993a) Respiratory mu-opioid and benzodiazepine interactions in the unrestrained rat. Neuropharmacology 32, 323-329. Paakkari P., Paakkari l., Siren A. L. and Feuerstein G. (1990) Respiratory and locomotor stimulation by low doses of dermorphin, a mu~ receptormediated effect. J. Pharmac. Exp. Ther, 252, 235-252. Paakkari P., Paakkari I., Vonhof S., Feuerstein G., Siren A. L. (1993b) Dermorphin analog Tyr-D-Arg2-Phe-Sarcosine induced opioid analgesia and respiratory stimulation - - The role of mu~-receptors. J. Pharmac. Exp. Ther. 266, 544-550. Perfumi M., DeCaro G., Massi M. and Venturi F. (1986) Inhibition of angiotensin I1 induced drinking by dermorphin given into the SFO or into the lateral ventricle of intact or SFP-lesioned rats. In The Physiology of Thirst and Sodium Appetite (Edited by Decaro G., Epstein A. N. and Massi M.) pp 257-263. Plenum Publish. Corp. Portolano F., Filippelli A., Marrazzo R., Susanna V., Russo S., Stella L., Losasso C., Molinaro L., Agrisani M., Falzarano C. and Marmo E. (1991a) Cardiovascular and respiratory effects of dermorphin in rats. Res. Commun. Clin. Pathol. Pharmac. 71, 131-152. Portolano F., Marrazzo R., Matera S., De Novellis V., Marmo M. and Rossi F. (1991b) Opiate peptidergic neurotransmission and cardiovascular and respiratory apparatus: Experimental research with endorphin and dermorphin on normotensive and hypertensive rats. Curr. Ther. Res. 50, 625-634. Puglisi Allegra S., Castellano G., Fillibeck U., Oliverio A. and Melchiorri P. (1982) Behavioural data of dermorphin in mice. Eur. J.Pharmac. 82, 223-227. Richter K., Egger R. and Kreil G. (1987) D-Alanine in the frog skin peptide dermorphin is derived from L-Alanine in the precursors. Science 238, 200-202. Richter K., Egger R., Negri L., Corsi R., Severini C. and Kreil G. (1990) cDNAs encoding [D-Ala2]deltorphin precursors from skin of Phyllomedusa bicolor also contain genetic information for three dermorphin-related opioid peptides. Proc. Natl. Acad. Sci. USA 87, 48364839. Rossi A., Di Salle E., Briatico G., Arcari G., DeCastiglione R. and Perseo G. (1983) Antinociceptive, prolactin-releasing and intestinal motility inhibiting activities of dermorphin and analogues after subcutaneous administration in the rat. Peptides 4, 477-580. Rotiroti D., German~ G., Nistic6 G., Melchiorri P. and Erspamer V. (1984) Stimulation of crop sac and pituitary lactotrophs after intraventricular administration of dermorphin in pigeons. Gen. Comp. Endocrinol. 56, 24-31. Sakurada S., Chaki K., Watanabe H., Nakata N., Sakurada T., Kisara K. and Suzuki K. (1992) Antinociceptive mechanism of [D-Arg2]dermorphin tripeptide analogs. J. Pharmac. Exp. Ther. 263, 793-799. Sakurada S., N. Nakata, Sakurada T., Chaki K., Kawamura S., Kisara K. and Suzuki K. (1993) Tolerance and cross-tolerance to the antinociceptive effects of [D-Arg2]dermorphin tripeptide analogs and morphine. Neuropharmacology 32, 689-693. Sander G. E. and Giles T. D. (1982) Enkephalin analogs and dermorphin in the conscious dog: structure-activity relationships. Peptides 3, 10171021. Sandrini G., Degli Uberti E., Salvadori S., Margutti A., Trasforini G., Tomatis R., Nappi G. and Pansini R. (1986) Dermorphin inhibits spinal nociceptive flexion reflex in humans. Brain Res. 371,364-367. Sasaki Y., Hosono M.,Matsui M. , Fujita N., Suzuki K., Sh. Sakurada, Sakur-

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