Regional distribution of endothelin receptor in porcine cardiovascular tissues

To clarify effecting sites of endothelin (ET) in a circulation system, we have identified specific receptors for porcine ET(ET-1) and investigated the distribution in the porcine cardiovascular tissues. Scatchard analysis of 125I-porcine ET binding indicated the presence of a single class of high-affinity binding sites. The binding was highly specific for ET-1, because (1) none of the other various peptides or Ca2+-channel antagonists affected the binding, (2) the scission of disulfide bonds, the digestion of the C-terminal 6-amino acid residues, or nitrophenylsulfenylization of the C-terminal Trp21 of ET-1 markedly reduced the binding ability and, (3) ET-1 showed the highest affinity for the vascular receptor among three ET isopeptides. Cardiac atria possessed the highest density (2.7 pmol/mg protein) of ET receptors of all the tissues examined, including thoracic aorta, cardiac atria and cardiac ventriculi, basilar, renal, coronary and pulmonary branch arteries, coronary, renal and jugular veins, and small vessels of pia mater encephali. Small vessels, renal and coronary arteries also showed relatively high density (0.8-1.4 pmol/mg protein). Various veins examined also showed considerable density (0.45-0.74 pmol/mg protein). The apparent Kd of cardiac ET receptors (0.76 nM) was significantly greater than that of the receptors of the other tissue (0.06-0.14 nM). The extensive distribution and the local enrichment of ET receptor in a cardiovascular system strongly suggests that ET is one of the essential endogenous substances to control the tone of the vasculature.     

A potent and specific agonist, Suc-[Glu9,Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor.

A series of C-terminal linear peptides of endothelin (ET)-1 and their N alpha-succinyl (Suc) analogs were synthesized and their binding affinities for the two subtypes of ET receptor, ETA and ETB, in porcine lung membranes were examined. Among the synthetic analogs, Suc-[Glu9,Ala11,15]-ET-1(8-21), IRL 1620, was the most potent and specific ligand for the ETB receptor (KiETA/KiETB approximately equal to 120,000) as judged by the Ki values for ETA (1.9 microM) and ETB (16 pM) receptors. IRL 1620 was 60 times more selective for the ETB receptor than ET-3 (KiETA/KiETB approximately equal to 1,900). IRL 1620 (10(-9)-10(-7) M) induced contractions of the guinea pig trachea with a comparable potency to those of ET-1 or ET-3, suggesting that IRL 1620 is a potent ETB receptor agonist.     

Functional endothelin/sarafotoxin receptors in rat heart myocytes: structure-activity relationships and receptor subtypes

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) family were characterized in newborn rat heart myocytes using human and rat endothelins (ET-1 and ET-3, respectively), SRTX-b and SRTX-c. Binding studies in intact cells and homogenates revealed significantly higher affinities of ET-1 and SRTX-b than of ET-3 and SRTX-c towards these receptors. This binding profile of ET/SRTX peptides points to their interaction with the receptor subtype designated E-S alpha. All four peptides induced time- and dose-dependent phosphoinositide hydrolysis with the following rank order of potency: ET-1 greater than SRTX-b greater than SRTX-c greater than ET-3. Thus, ET-3 which possesses an intermediate affinity toward the receptor was the least effective with regard to this response. These results confirm and extend our earlier report that the ET/SRTX peptides interact with a newly characterized receptor(s) associated with phosphoinositide metabolism and Ca2+ mobilization. The initiation of inositol phosphate formation is largely independent of extracellular Ca2+, verapamil and nifedipine, indicating that the ET/SRTX peptides are not agonists for the voltage-dependent Ca2+-channels.     

Analysis of endothelin related peptides in culture supernatant of porcine aortic endothelial cells: evidence for biosynthetic pathway of endothelin-1

We investigated the molecular forms of endothelin (ET) related peptides in culture supernatant of porcine aortic endothelial cells by high performance liquid chromatography coupled with radioimmunoassays for ET related peptides. We isolated and sequenced a C-terminal peptide (big ET-1(22-39] of big ET-1(1-39) and its N-terminal truncated form (big ET-1(23-39] in addition to ET-1(1-21) and its oxidized form, [Met7 (0)]ET-1(1-21). The total contents of the two C-terminal peptides of big ET-1(1-39) are approximately equal to those of ET-1(1-21) and its oxidized form on a molar basis in the culture supernatant. Furthermore, we isolated big ET-1(1-39) although its content is approximately 2% of that of ET-1(1-21). These results strongly suggest that ET-1(1-21) and big ET-1(22-39) are generated from big ET-1(1-39) by specific processing between Trp21-Val22.     

Two distinct types of endothelin receptors are present on chick cardiac membranes

Competitive displacement experiments of 125I-endothelin (ET)-1, -2, or -3 binding to chick cardiac membranes were performed with unlabeled ET-1, -2, -3, and sarafotoxin S6b (STX) as competitors. 125I-ET-1 and -2 binding was competitively inhibited by increasing concentrations of these unlabeled peptides in the same order; i.e. ET-2 greater than or equal to ET-1 greater than ET-3 greater than STX. In contrast, the order of potency in displacing 125I-ET-3 binding was ET-3 greater than ET-2 greater than or equal to ET-1 greater than STX. Affinity labeling of the membranes by cross-linking with 125I-ET-1 and -2 via disuccinimidyl tartarate yielded one major specific band with an apparent Mr = 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. On the other hand, affinity labeling with 125I-ET-3 showed that two major and one minor bands of Mr = 34,000, 46,000, and 53,000, respectively, were specifically labeled. These results indicate the presence of two distinct types of ET receptors, one of which has higher affinity for ET-1 and -2 than ET-3 and the other is conversely ET-3-preferring.     

Receptor binding affinity and biological activity of C-terminal elongated forms of endothelin-1

Endothelin-1 (21 amino acids; ET-21) is considered to be derived from a precursor, proendothelin (38 amino acids; ET-38). In order to make the physiological significance of this conversion clear, we synthesized various C-terminal elongated derivatives of ET-21, such as ET-22, ET-23, ET-25, ET-31, ET-36 and ET-38 (each number implies the number of amino acid residues), and measured their receptor binding affinities and biological activities. When inhibition of [¹²⁵I]ET-21 binding to cultured rat smooth muscle cells (A10 cells) was measured, ET-21 inhibited with the highest affinity (IC₅₀ = 1.6 × 10⁻¹⁰ M) and the affinity of ET-38 was 30-fold less than that of ET-21. The binding affinities of the C-terminal elongated peptides were reduced with increasing number of amino acid residues, except for ET-22 whose affinity was lower than those of other peptides (IC₅₀ = 1.6 × 10⁻⁸ M). When contractions of rat aortic segments induced by these peptides were measured, ET-21 was the most potent (EC₅₀ = 2.8 × 10⁻¹⁰ M). All C-terminal elongated peptides, including ET-38, were more than 100-fold less active. It is noteworthy that ET-22 was the least potent peptide (EC₅₀ = 1.2 × 10⁻⁷ M). When bolus doses of C-terminal elongated peptides were administered to chemically denervated rats, the time-dependent change in blood pressure induced by each peptide was different from that induced by ET-21. Although ET-21 elicited a three phase depressor/pressor blood pressure response (an initial rapid hypotension, then a rapid transient hypertension followed by a slowly developing long-lasting hypertensive effect), the C-terminal elongated peptides, including ET-38, did not cause the initial transient hypotensive response. Very interestingly, the ability of the peptides to induce the rapid phase of hypertension in vivo does not seem to be correlated with the affinity of each peptide for the smooth muscle cell receptor, since the peptides with lower affinities for the smooth muscle receptor, such as ET-22, ET-23 and ET-25, showed more potent hypertensive effects. On the other hand, the slow and long-lasting hypertensive effect is likely to be related to the affinity of the compounds. The maximal hypertensive effects of cumulatively administered ET-21 derivatives were similar to those of ET-21. These results suggest that ET-21 is the most potent vasoconstrictor among the peptides and that the conversion from ET-38 to ET-21 may be important as an activation process.     

Importance of the phenotypic state of vascular smooth muscle cells on the binding and the mitogenic activity of endothelin

Smooth muscle cells of the rabbit aorta, when grown in vitro, express distinguishable forms of phenotypes (contractile and synthetic). On contractile cells, ET-1 specifically bound to a single class of high affinity (KD = 128 pM) and high capacity (Bmax = 66,000 sites/cell) binding sites. But, whereas affinity of [125I]-ET-1 was not significantly affected by phenotypic modulation, synthetic cells displayed a 10-fold lower [125I]-ET-1 binding capacity than contractile smooth muscle cells. Similarly, the mitogenic effect of ET-1 on smooth muscle cells was considerably lower for synthetic than for contractile cells. The ET-1 receptor on primary cells was recognized by sarafotoxin S6b and the different ET-related peptides with an order of potency [ET-1 greater than S6b greater than ET-3 greater than Big ET-1 much greater than ET(16-21)] identical to that inducing smooth muscle cell growth. Therefore, these data indicate that the binding and the mitogenic effects of ET-1 on smooth muscle cells might be of different magnitudes depending on the phenotypic state of these cells.     

Ala1,3,11,15]endothelin-1 analogs with ETB agonistic activity.

A linear peptide analog of endothelin (ET)-1, [Ala1,3,11,15]ET-1 (4AlaET-1), and its truncated peptide analogs were synthesized to study the structural requirements of ET-1 for the recognition of ETs-nonselective ETB receptors. ET-1 exhibited sub-nanomolar binding to two distinct ET receptor subtypes (ETA and ETB), but 4AlaET-1 bound to ETB with an affinity 1,700 times higher than that seen during binding to ETA. The truncated linear peptides 4AlaET-1(6-21), 4AlaET-1(8-21) and N-acetyl-4AlaET-1(10-21) still had high affinity for ETB, whereas 4AlaET-1(6-20) and 4AlaET-1(11-21) displayed remarkably reduced affinity for ETB. Therefore, ET-1 requires the Glu10-Trp21 sequence for ETB binding, but not the disulfide bridges. These ETB-binding peptides elicit endothelium-dependent vasorelaxation of porcine pulmonary arteries in parallel with the binding affinity for ETB, suggesting that they are ETB agonists.     

The effects of the endothelin family peptides on cultured osteoblastic cells from rat calvariae

The activities of three isoforms of the endothelin (ET) family peptides, ET-1, ET-2 and ET-3, were studied in cultured osteoblastic cells from neonatal rat calvariae. All three isoforms induce stimulation of DNA synthesis and reductions in cellular alkaline phosphatase activity in a dose-dependent manner with the rank order of potency: ET-1 congruent to ET-2 greater than ET-3. The 125I-labeled ET binding and affinity-cross linking experiments show the presence of a single class of the ET binding sites with a more than 10-fold higher affinity for ET-1 and ET-2 as compared to ET-3. The endothelins dose-dependently stimulate the production of inositol phosphates and induce mobilization of Ca2+ with the similar relative potency to that for the receptor binding. These results indicate that osteoblastic cells possess the endothelin receptor with a high affinity for ET-1 and ET-2 that is coupled to phospholipase C, and that the endothelins modulate cellular functions via this receptor.     

Structure-activity relationship of endothelin: importance of charged groups

Endothelin (ET)-related peptides including ET-1 (1-39) were synthesized, and their constricting activity in rat pulmonary artery rings and pressor activity in unanesthetized rat were measured to elucidate their structure-activity relationship. The vasoconstrictor activities of ET-2, ET-3 and sarafotoxin S6b were one-half, one-60th and one-third that of ET-1, respectively. Such differences in biological activities should mainly arise from sequence heterogeneity at the N-terminal portion, especially at positions 4 to 7. All of the blocked ETs at the amino or carboxyl termini showed greatly decreased activities. A monocyclic analog, in which Cys3 and Cys11 were replaced by Ala, showed one-third the activity of ET-1; however, its deamino dicarba analog was almost completely inactive. Significant activities were retained even with replacement of amino acids at positions Ser4, Ser5, Leu6, Met7, Lys9, Tyr13, and Trp21 by Ala, Ala, Gly, Met(0), Leu, Phe, and Tyr or Phe, respectively. On the other hand, replacement of Asp8, Glu10 and Phe14 by Asn, Gln and Ala, respectively, resulted in complete loss of the biological activity. These results indicated that two disulfide bonds in ET molecule were not essential for the expression of vasoconstricting activity. Both terminal amino and carboxyl groups, carboxyl groups of Asp8 and Glu10, and the aromatic group of Phe14 seemed to be contributing, more or less, to the expression of the biological activities.     

Hybrid peptides constructed from RES-701-1, an endothelin B receptor antagonist, and endothelin; binding selectivity for endothelin receptors and their pharmacological activity

Hybrid peptides were constructed from endothelin B receptor (ET_B) selective antagonist RES-701-1 (1) and endothelin (ET-1). They have N-terminal 10 amino acids derived from 1 and C-terminal 10 amino acids derived from ET-1. RES-701-1(1-10)-[Ala15]ET-1(12-21) and its analogues substituted or truncated at the residues derived from RES-701-1 had proved to possess high receptor binding activity selective for ET_B as well as 1. Substitutions at the residues derived from ET-1 had produced some analogues that possessed high affinity not only for ET_B but for ET_A. Although all analogues had antagonistic effects on ET_A, some analogues had proved to function as agonist on ET_B confirmed by the changes in intracellular calcium concentrations of ET receptor-transfected COS-7 cells. We have found four types of ET receptor-binding peptides: (1) ET_B-selective agonist with weak ET_A antagonism (3, KT7421); (2) ET_B-selective antagonist with weak ET_A antagonism (29, KT7539); (3) ET_B agonist with potent ET_A antagonism (27, KT7538); and (4) non-selective ET_A/ET_B antagonist (26, KT7540).     

Endothelin-1, -2 and -3 directly and big-endothelin-1 indirectly elicit an abdominal constriction response in mice.

When injected intraperitoneally into mice, endothelins ET-1, ET-2, ET-3 and big-endothelin-1[1-38] (big-ET-1[1-38]) produced a dose-related, robust and easily quantified abdominal constriction response within 20 min. The ED50 values for this response were 0.026, 0.005, 0.131, and 0.043 mg/kg, respectively. Hence, this test could provide a convenient in vivo endpoint for endothelin activity. The results also imply that ET-1, ET-2, ET 3 or big-ET-1[1-38] may be nociceptive under certain conditions. Morphine (4 mg/kg, s.c.) administered 30 min prior completely blocked the response produced by ET-1. Thus, in conjunction with other indicators, the test may also serve as an in vivo screen for agents useful in the treatment of abdominal or visceral pain. The effect of big-ET-1[1-38], but not ET-1, was blocked by pretreatment with the enzyme inhibitor phosphoramidon (10 mg/kg, s.c., 30 min prior), implying that the big-ET-1[1-38] must first be enzymatically cleaved, presumably to ET-1, in order to elicit the abdominal constriction response. This test might also serve as a discriminative antinociceptive screen, because the response to ET-1 was not blocked by acetaminophen (400 mg/kg, p.o.), ibuprofen (75 mg/kg, p.o.) or indomethacin (1.0 mg/kg, p.o.).     

Structural and dynamic characterization of the aromatic amino acids of the human immunodeficiency virus type I nucleocapsid protein zinc fingers and their involvement in heterologous tRNA(Phe) binding: a steady-state and time-resolved fluorescence study.

The steady-state and time-resolved fluorescence properties of two zinc-saturated 18-residue synthetic peptides with the amino acid sequence of the NH2-terminal (NCp7 13-30 F16W, where the naturally occurring Phe was replaced by a Trp residue) and the COOH-terminal (NCp7 34-51) zinc finger domains of human immunodeficiency virus type I nucleocapsid protein were investigated. Fluorescence intensity decay of both Trp 16 and Trp 37 residues suggested the existence of two fully solvent-exposed ground-state classes governed by a C = 2.2 equilibrium constant. The lifetimes of Trp 16 classes differed from those of Trp 37 essentially because of differences in nonradiative rate constants. Arrhenius plots of the temperature-dependent nonradiative rate constants suggested that the fluorescence quenchers involved in both classes and in both peptides were different and the collisional rate of these quenchers with the indole ring was very low, probably because of the highly constrained peptide chain conformation. The nature of the ground-state classes was discussed in relation to 1H nuclear magnetic resonance data. Using Trp fluorescence to monitor the interaction of both peptides with tRNA(Phe) we found that a stacking between the indole ring of both Trp residues and the bases of tRNA(Phe) occurred. This stacking constituted the main driving force of the interaction and modified the tRNA(Phe) conformation. Moreover, the binding of both fingers to tRNA(Phe) was noncooperative with similar site size (3 nucleotide residues/peptide), but the affinity of the NH2-terminal finger domain (K = 1.3 (+/- 0.2) 10(5) M-1) in low ionic strength buffer was one order of magnitude larger than the COOH-terminal one due to additional electrostatic interactions involving Lys 14 and/or Arg 29 residues.     

Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination

We investigated the molecular forms of endothelin (ET) related peptides in porcine spinal cord by high performance liquid chromatography coupled with radioimmunoassays using three antisera raised against ET-1 and C-terminal fragments of ET-1 and big ET-1. ET-1 and its oxidized form were isolated as major immunoreactive peptides and sequenced. Furthermore, immunoreactivities like ET-3 and big ET-1(22-39) (contents: less than 8% and less than 1% of ET-1, respectively) were detected based on their chromatographic retention times and characteristics of immunoreactivity to the antisera. Big ET-1 was only scarcely detected. Immunohistochemical study showed the presence of ET-1-like immunoreactivity in motoneurons, dorsal horn neurons and dot- and fiber-like structures in the dorsal horn of lumbar spinal cord. These results indicate that ET-1 is present not only in endothelial cells but also in spinal cord, and that big ET-1 is converted into ET-1 in spinal cord by specific processing between Trp21-Val22. The data also indicate that ET-1 may act as a neuropeptide in the central nervous system.     

Purification and characterization of recombinant human endothelin receptor type A

Human endothelin receptor type A (ET(A)) is a G-protein coupled receptor that mediates vasoconstriction of blood vessels. To determine the structural characteristics and signaling mechanism of ET(A), we have expressed recombinant ET(A) as a fusion protein with p9 envelope protein from phi6 bacteriophage. The His-tag-labeled p9-ET(A) fusion protein was highly expressed in the membrane fraction of Escherichia coli and purified to homogeneity by single affinity chromatography after solubilization with detergents. Purified p9-ET(A) appeared as an oligomer and presented mainly as an α-helical structure. The protein also showed specific binding to endothelin-1 (ET-1) and the alpha subunit of G(q) protein with apparent K(D) values of 17 and 20 nM, respectively. An antagonist of ET(A), bosentan, prevented the interaction between p9-ET(A) and ET-1 in a concentration-dependent manner. These results indicate that recombinant p9-ET(A) has a competent conformation for interactions with ET-1 and the alpha subunit of G(q) protein.     

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors.

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.     

Endothelin and structurally related analogs distinguish between endothelin receptor subtypes.

The endothelin (ET) analog ET-1[1,3,11,15-Ala] was compared with ET-1, ET-2, ET-3 and sarafotoxins (SRTX) S6b and S6c for receptor binding and function. All the peptides exhibited high affinity binding and contracted rabbit pulmonary artery with near equal potency. In rat aorta both ET-3 and ET-1 [1,3,11,15-Ala] bound with much lower affinity than ET-1 while ET-3 displayed weak contractile potency and ET-1 [1,3,11,15-Ala] and SRTX-c were inactive. In rat left atria, ET-1 [1,3,11,15-Ala] and SRTX-c were weak inhibitors of binding and were also functionally inactive, whereas ET-1, ET-2, ET-3, and SRTX-b were equipotent in producing contractile responses. The data support the idea of there being a predominance of ETA receptors in rat aorta and ETB receptors in rabbit pulmonary artery. In rat left atria, the ET receptor could not be readily classified into ETA or ETB and suggests the existence of a new receptor subtype.     

Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal D-Trp or D-Tpi.

In an attempt to produce more powerful (effective) bombesin/GRP receptor antagonists, the D forms of Trp or Trp analog (Tpi) were introduced at position 6 in two pseudononapeptides, Leu13 psi (CH2NH)Leu14-bombesin(6-14) and Leu13 psi(CH2NH)Phe14-bombesin (6-14). These antagonists were tested for their ability to inhibit basal and gastrin releasing peptide (GRP) (14-27)-induced amylase release from rat pancreatic acini in a superfusion assay. They were also assessed for the inhibition of 125I-Tyr4-bombesin binding to Swiss 3T3 and small cell lung carcinoma cell line H-345 and the mitogenic response of Swiss 3T3 cells induced by GRP(14-27). The peptides, when given alone, did not stimulate amylase secretion, but were able to inhibit gastrin releasing peptide (14-27)-induced amylase release. All of the antagonists showed strong binding affinities for Swiss 3T3 and H-345 cells and suppressed the GRP(14-27)-induced increase of [3H]thymidine incorporation into DNA of Swiss 3T3 cells at nanomolar concentrations. Antagonist D-Tpi6,Leu13 psi (CH2NH)Leu14-bombesin (6-14)(RC-3095) was slightly more potent in these assays than D-Trp6,Leu13 psi (CH2NH)Leu14-bombesin (6-14)(RC-3125). Nevertheless, D-Trp6,Leu13 psi (CH2NH)Phe14-bombesin (6-14) showed the highest binding affinity for Swiss 3T3 and H345 cells and it was the most potent inhibitor of GRP(14-27)-induced amylase secretion. This antagonist RC-3420 was particularly effective in inhibiting the growth of Swiss 3T3 cells, exhibiting an IC50 value less than 1 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design of chimeric peptide ligands to galanin receptors and substance P receptors.

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1) less than 0.1 nM and KD(2) approximately 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD approximately 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD approximately 1 nM) or its N-terminal fragment galanin-(1-13) (KD approximately 1 microM), which constitutes the N-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD = 0.4 microM), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors.(ABSTRACT TRUNCATED AT 250 WORDS)