Binding affinities and activation of Asp712Ala and Cys100Ser mutated kinin B1 receptor forms suggest a bimodal scheme for the molecule of bound-DABK.

Mutant forms of kinin B(1) receptor (B(1)R) and analogs of the full agonist des-Arg(9)-bradykinin (DABK) were investigated aiming to verify the importance of selected receptor residues and of each agonist-peptide residue in the specific binding and activation. Linked by a specific disulfide bond (Cys(100)-Cys(650)), the N-terminal (N(t)) and the EC3 loop C-terminal (C(t)) segments of angiotensin II (AngII) receptor 1 (AT(1)R) have been identified to form an extracellular site for binding the agonist N(t) segment (Asp(1) and Arg(2) residues). Asp(712) residue at the receptor EC3 loop binds the peptide Arg(2) residue. By homology, a similar site might be considered for DABK binding to B(1)R since this receptor contains the same structural elements for composing the site in AT(1)R, namely the disulfide bond and the EC3 loop Asp(712) residue. DABK, Ala(n)-DABK analogs (n=Ala(1)-, Ala(2)-, Ala(3)-, Ala(4)-, Ala(5)-, Ala(6)-, Ala(7)-, Ala(8)-DABK), and other analogs were selected to binding wild-type, Asp712Ala and Cys100Ser mutated B(1)R receptors. The results obtained suggested that the same bimodal scheme adopted for AngII-AT(1)R system may be applied to DABK binding to B(1)R. The most crucial similarity in the two cases is that the N(t) segments of peptides equally bind to the homologous Asp(712) residue of both AT(1)R and B(1)R extracellular sites. Confirming this preliminary supposition, mutation of residues located at the B(1)R extracellular site as EC3 loop Asp(712) and Cys(100) caused the same modifications in biological assays observed in AT(1)R submitted to homologous mutations, such as significant weakening of agonist binding and reduction of post-receptor-activation processes. These findings provided enough support for defining a site that determines the specific binding of DABK to B(1)R receptors.

Carbamazepine inhibits angiotensin I-converting enzyme, linking it to the pathogenesis of temporal lobe epilepsy.

We find that a common mutation that increases angiotensin I-converting enzyme activity occurs with higher frequency in male patients suffering from refractory temporal lobe epilepsy. However, in their brains, the activity of the enzyme is downregulated. As an explanation, we surprisingly find that carbamazepine, commonly used to treat epilepsy, is an inhibitor of the enzyme, thus providing a direct link between epilepsy and the renin-angiotensin and kallikrein-kinin systems.

Bradykinin-related peptides from Phyllomedusa hypochondrialis.

Bradykinin related peptides (BRPs) present in the water-soluble secretion and freshly dissected skin fragments of Phyllomedusa hypochondrialis were investigated by mass spectrometry techniques. Eighteen BRPs, along with their post-translational modifications, were characterized in the secretion by de novo MS/MS sequencing and direct MALDI imaging experiments of the frog skin. These molecules revealed strong sequence similarities to the main plasma kinin of some mammals and reptiles. Such a diversity of molecules, within the same peptide family, belonging to a single amphibian species may be related to functional specializations of these peptides and a variety of corresponding receptors that might be present in a number of different predators. Also, a novel analog, [Val]1,[Thr]6-bradykinyl-Gln,Ser had its biological activity positively detected in cell culture expressing the human bradykinin B2 receptor and in guinea pig ileum preparations.

Synthesis and immunological activity of a branched peptide carrying the T-cell epitope of gp43, the major exocellular antigen of Paracoccidioides brasiliensis.

The 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis (PCM), a prevalent fungal infection in South America. A 15-mer sequence from gp43, denominated P10, induced T-CD4+ T helper 1 cellular immune responses in mice of three different haplotypes and protected against intratracheal challenge by a virulent isolate of P. brasiliensis. In an attempt to improve delivery of P10, a promiscuous antigen also presented by human leucocyte antigen-DR alleles, aiming at immunotherapy, we synthesized a multiple antigen peptide with the protective T-cell epitope expressed in a tetravalent 13-mer analog of P10 (M10). M10 induced specific lymph node cell proliferation in mice preimmunized with peptides in complete Freund's adjuvant (CFA). In addition, M10 immunization without CFA significantly protected intratracheally infected mice. We conclude that M10 is a candidate for an anti-PCM vaccine. In this report we describe: (1) the synthesis of M10; (2) the induction of M10-elicited T-cell response and (3) in vivo protection of mice immunized with M10 and challenged by a virulent strain of P. brasiliensis.

Tachyphylactic properties of angiotensin II analogs with bulky and hydrophobic substituents at the N-terminus.

Tachyphylaxis, defined as the acute loss of response of some smooth muscles upon repeated stimulations with angiotensin II (Ang II), has been shown to be dependent mainly on the N-terminal region of the ligand. To further study the structural requirements for the induction of tachyphylaxis we have synthesized Ang II analogs containing the bulky and very lipophilic substituents 9-fluorenylmethyloxycarbonyl (Fmoc) and 9-fluorenylmethyl ester (OFm) at the alpha-amino (Nalpha-Fmoc-Ang II) or the beta-carboxyl ([Asp(OFm)1]-Ang II) groups of the Asp1 residue, respectively. In binding assays with Chinese hamster ovary cells transfected with the AT1 Ang II receptor, Nalpha-Fmoc-Ang II bound with high affinity, whereas [Asp(OFm)1]-Ang II showed lower affinity. In biological assays, these two analogs were full agonists and showed 30 and 3%, respectively, of the Ang II potency in contracting the guinea-pig ileum smooth muscle. The two analogs induced tachyphylaxis, in spite of the lack of a free amino group in Nalpha-Fmoc-Ang II. Thus, analogs with Fmoc- or OFm-type groups coupled to the Asp1 residue, whether at the amino or carboxyl functions, induce tachyphylaxis through an unreported mechanism. Based in these findings and those available from the literature, an alternate molecular interaction mode between Ang II N-terminal portion and the AT1 receptor is proposed to explain the tachyphylactic phenomenon.

Synthesis and pharmacological properties of TOAC-labeled angiotensin and bradykinin analogs.

Angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label were synthesized by solid phase methodology. Ammonium hydroxide (pH 10, 50 degrees C, l h) was the best means for reverting nitroxide protonation occurring during peptide cleavage. EPR spectra yielded rotational correlation times for internally labeled analogs that were nearly twice as large as those of N-terminally labeled analogs. Except for TOAC(1)-AngII and TOAC(0)-BK, which showed high intrinsic activities, other derivatives were inactive in smooth muscle preparations. These active paramagnetic analogs may be useful for conformational studies in solution and in the presence of model and biological membranes.

Evaluation of the trifluoromethanosulfonic acid/trifluoroacetic acid/thioanisole cleavage procedure for application in solid-phase peptide synthesis.

As an extension of our investigation of peptidyl-resin linkage stability towards different cleavage procedures used in the solid-phase peptide synthesis (SPPS) technique, the present paper evaluated the trifluoromethanesulfonic acid (TFMSA)/trifluoroacetic acid (TFA)/thioanisole method, varying the type of resin (benzhydrylamine-resin, BHAR; methylbenzhydrylamine-resin, MBHAR and 4-(oxymethyl)-phenylacetamidomethyl-resin, PAMR) and peptide resin-bound residue (Gly and Phe). The vasoactive angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue linked to those resins used routinely in tert-butyloxycarbonyl (Boc)-SPPS chemistry were submitted comparatively to a time course study towards TFMSA/TFA cleavage. At 0 degrees C, [Gly8]-AII was completely removed from all resins in less than 6 h, but the hydrophobic Phe8 moiety-containing AII sequence was only partially cleaved (not more than 15%) from BHAR or MBHAR in this period. At 25 degrees C, [Gly8]-AII cleavage time decreased to less than 2 h irrespective of the solid support, and quantitative removal of AII from PAMR and MBHAR occurred in less than 3 h. However, about 10-15 h seemed to be necessary for cleavage of AII from BHAR, and in this extended cleavage reaction a significant increase in peptide degradation rate was observed. Regardless of the cleavage temperature used, the decreasing order of acid stability measured for resins was BHAR>MBHAR>PAMR. Collectively, these findings demonstrated the feasibility of applying TFMSA/TFA solution as a substitute for anhydrous HF at the cleavage step in Boc-SPPS methodology. Care should be taken however, as the cleavage efficacy depends on multiple factors including the resin, peptide sequence, the time and temperature of reaction.

Fmoc-POAC: [(9-fluorenylmethyloxycarbonyl)-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid]: a novel protected spin labeled beta-amino acid for peptide and protein chemistry.

The stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) is the only spin labeled amino acid that has been used to date to successfully label peptide sequences for structural studies. However, severe difficulty in coupling the subsequent amino acid has been the most serious shortcoming of this paramagnetic marker. This problem stems from the low nucleophilicity of TOAC's amine group towards the acylation reaction during peptide chain elongation. The present report introduces the alternative beta-amino acid 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid (POAC), potentially useful in peptide and protein chemistry. Investigations aimed at addressing the stereochemistry of this cyclic molecule through X-ray diffraction measurements of crystalline and bulk samples revealed that it consists only of the trans conformer. The 9-fluorenylmethyloxycarbonyl group (Fmoc) was chosen for temporary protection of the POAC amine function, allowing insertion of the probe at any position in a peptide sequence. The vasoactive octapeptide angiotensin II (All, DRVYIHPF) was synthesized by replacing Pro7 with POAC. The reaction of Fmoc-POAC with the peptidyl-resin occurred smoothly, and the coupling of the subsequent amino acid showed a much faster reaction when compared with TOAC. POAC7-AII was obtained in good yield, demonstrating that, in addition to TOAC, POAC is a convenient amino acid for the synthesis of spin labeled peptide analogues. The present findings open the possibility of a wide range of chemical and biological applications for this novel beta-amino acid derivative, including structural investigations involving its differentiated bend-inducing characteristics.

Fluorescence study of conformational properties of melanotropins labeled with aminobenzoic acid.

The native hormone alpha-melanocyte-stimulating hormone (alpha-MSH) and its more potent analog [Nle(4),D-Phe(7)]alpha-MSH (NDP-alpha MSH), labeled at the amino terminal with the fluorescent aminobenzoic acid (Abz) isomers, were examined by fluorescence methods. We observed energy transfer between the tryptophan(9) residue acting as donor and Abz as acceptor, the transfer being more pronounced to the ortho-form of the acceptor. Within the hypothesis that different peptide conformations coexist in equilibrium during the fluorescence decay, we supposed that the intensity decay was modulated by an acceptor-donor distance distribution function f(r). From the time-resolved fluorescence experimental data, we recovered the distance distribution between Abz and Trp(9), using the CONTIN program, within the framework of the Förster resonance energy transfer model. The methodology proved to be useful to provide quantitative information about conformational dynamics of melanotropins and its dependency on the solvent. In aqueous medium, alpha-MSH has a broad Abz-Trp(9) distance distribution, reflecting the structural flexibility of the peptide. Three different distance populations could be identified in the labeled analog NDP-alpha MSH in water, indicating distinct conformational states for the synthetic peptide, compared with the native hormone. Measurements in trifluoroethanol resulted in the recovery of two Abz-Trp(9) distance populations, both for the native and the analog hormones, reflecting the decrease, induced by the solvent, of the conformational states available to the peptides.

Comparative EPR and fluorescence conformational studies of fully active spin-labeled melanotropic peptides.

Similar to melanocyte stimulating hormone (alpha-MSH), its potent and long-acting analogue, [Nle(4), D-Phe(7)]alpha-MSH, when labeled with the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac), maintains its full biological potency, thus validating any comparative structural investigations between the two labeled peptides. Correlation times, calculated from the electron paramagnetic resonance signal of Toac bound to the peptides, and Toac-Trp distances, estimated from the Toac fluorescence quenching of the Trp residue present in the peptides, indicate a more rigid and folded structure for the potent analogue as compared to the hormone, in aqueous medium.

Mapping the domain of troponin T responsible for the activation of actomyosin ATPase activity. Identification of residues involved in binding to actin.

The in vitro Ca(2+) regulation of the actomyosin Mg(2+)-ATPase at physiological ratios of actin, tropomyosin, and troponin occurs only in the presence of troponin T. We have previously demonstrated that a polypeptide corresponding to the first 191 amino acids of troponin T (TnT-(1-191)) activates the actomyosin Mg(2+)-ATPase in the presence of tropomyosin. In order to further characterize this activation domain, we constructed troponin T fragments corresponding to residues 1-157 (TnT-(1-157)), 1-76 (TnT-(1-76)), 77-157 (TnT-(77-157)), 77-191 (TnT-(77-191)), and 158-191 (TnT-(158-191)). Assays using these fragments demonstrated the following: (a) residues 1-76 do not bind to tropomyosin or actin; (b) residues 158-191 bind to actin cooperatively but not to tropomyosin; (c) the sequence 77-157 is necessary for troponin interaction with residue 263 of tropomyosin; (d) TnT-(77-191) on its own activates the actomyosin ATPase activity as described previously for TnT-(1-191). TnT-(1-157), TnT-(1-76), TnT-(77-157), TnT-(158-191), and combinations of TnT-(158-191) with TnT-(1-157) or TnT-(77-157) showed no effect on the ATPase activity. We conclude that the activation of actomyosin ATPase activity is mediated by a direct interaction between amino acids 77 and 191 of troponin T, tropomyosin, and actin.

First synthesis of a fully active spin-labeled peptide hormone.

For the first time in the electron spin resonance (ESR) and peptide synthesis fields, a fully active spin-labeled peptide hormone was reported. The ESR spectra of this alpha-melanocyte stimulating hormone (alpha-MSH) analogue (acetyl-Toac0-alpha-MSH) where Toac is the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, suggested a pH-independent conformation and a more restricted movement comparatively to the free Toac. Owing to its equivalent biological potency in a skin pigmentation assay as compared to the native alpha-MSH and its unique characteristic (paramagnetic, naturally fluorescent and fully active), this analogue is of great potential for investigation of relevant physiological roles reported for alpha-MSH.

Structure of two fragments of the third cytoplasmic loop of the rat angiotensin II AT1A receptor. Implications with respect to receptor activation and G-protein selection and coupling.

The structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT1A receptor one of the cytoplasmic domains responsible for G-protein coupling are still unknown. The three-dimensional structures of two overlapping peptides mapping the entire i3 loop and shown to differently interact with purified G-proteins have been obtained by simulated annealing calculations, using NMR-derived constraints collected in 70% water/30% trifluoroethanol solution. While the NH2-terminal half, Ni3, residues 213-231, adopts a stable amphipathic alpha-helix, extending over almost the entire peptide, a more flexible conformation is found for the COOH-terminal half, Ci3, residues 227-242. For this peptide, a cis-trans isomerization around the Lys6-Pro7 peptide bond generates two exchanging isomers adopting similar conformations, with an alpha-helix spanning from Asn9 to Ile15 and a poorly defined NH2 terminus. A quite distinct structural organization is found for the sequence EIQKN, common to Ni3 and Ci3. The data do suggest that the extension and orientation of the amphipathic alpha-helix, present in the proximal part of i3, may be modulated by the distal part of the loop itself through the Pro233 residue. A molecular model where this possibility is considered as a mechanism for G-protein selection and coupling is presented.

Structure of the C-terminal fragment 300-320 of the rat angiotensin II AT1A receptor and its relevance with respect to G-protein coupling.

Angiotensin II AT1A receptor is coupled to G-protein, and the molecular mechanism of signal transduction is still unclear. The solution conformation of a synthetic peptide corresponding to residues 300-320 of the rat AT1A receptor, located in the C-terminal cytoplasmic tail and indicated by mutagenesis work to be critical for the G-protein coupling, has been investigated by circular dichroism (CD), nuclear magnetic resonance (NMR) and restrained molecular dynamics calculations. The CD data indicate that, in acidic water, at concentration below 0.8 mM, the peptide exists in a predominantly coil structure while at higher concentration it can form helical aggregates; addition of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. Using NMR-derived constraints, an ensemble of conformers for the peptide has been determined by restrained molecular dynamics calculations. Analysis of the converged three-dimensional structures indicates that a significant population of them adopts an amphipathic alpha-helical conformation that, depending upon experimental conditions, presents a variable extension in the stretch Leu6-Tyr20. An equilibrium with nonhelical structured conformers is also observed. We suggest that the capability of the peptide to modulate its secondary structure as a function of the medium dielectric constant, as well as its ability to form helical aggregates by means of intermolecular hydrophobic interactions, can play a significant role for G-protein activation.

Comparative evaluation of the synthesis and purification of transmembrane peptide fragments. Rat bradykinin receptor fragment 64-97 as model.

The 34-residue peptide CTVAEIYLGNLAGADLILASGLPFWAITIANNFD (TM-34), corresponding to the 64-97 sequence of the rat bradykinin, receptor, was selected as a model of hydrophobic transmembrane peptide segment for systematic study of synthesis and purification strategies. Application of conventional Boc/Bzl chemistry resulted in very low yield of the synthesis (around 4%) when DMF was used as the solvent for coupling reactions. As shorter resin-bound fragments of TM-34 showed improved swelling in 80% NMP/DMSO, the synthesis was repeated in this mixed solvent and the yield increased to 12%. A comparative synthesis using optimized Fmoc chemistry and Fmoc-(FmocHmb) derivatives of Ala and Leu to prevent aggregation did not provide any detectable TM-34. Taken together, these results illustrate the synthetic problems associated with hydrophobic sequences, almost regardless of the chemistry used. As expected, the hydrophobicity of TM-34 and of most of its minor fragments made them scarcely soluble in common solvents. Purification could be achieved by loading the crude materials dissolved in 90% AcOH onto a C4 HPLC column and eluting with a TFA/MeCN linear gradient. CD studies of the TM-34 and of the shorter fragment with the 74-97 sequence (TM-24) showed a higher percentage of alpha-helix structure for the latter. This suggests that the shorter sequence may better represent the correct transmembrane region of the second helix of the rat bradykinin receptor.

Spin-labeled extracellular loop from a seven-transmembrane helix receptor: studies in solution and interaction with model membranes.

A spin-labeled pentadecapeptide was synthesized containing 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) as the N-terminal amino acid and residues 253-266 (EYWSTFGNLHHISL) of the mass oncogene receptor, a membrane-bound protein from the G-protein coupled receptors family. According to predictions, this protein folds into seven transmembrane helices connected by three extra- and three intracellular loops, and the peptide encompasses part of the third extracellular loop and part of the seventh helix. Electron paramagnetic resonance (EPR) spectra of the spin-labeled peptide (TOAC-14) were obtained in aqueous solution as a function of pH and temperature, in a secondary structure-inducing solvent [trifluoroethanol (TFE)], and in the presence of detergent micelles and phospholipid bilayers. The charged and uncharged amino groups of TOAC and TOAC-14 yielded spectra with different isotropic hyperfine splittings (aN). The slow exchange between protonated and unprotonated forms in the EPR time scale gave rise to composite spectra weighted by the Henderson-Hasselbalch equation. Plots of aN vs pH allowed the determination of the amino group pK values (8.4 and 4.5, for TOAC and TOAC-14, respectively). A small change in aN centered at pH 6.5 was ascribed to the titration of the histidines. Values of calculated rotational correlation times were indicative of a pH-induced conformational change. A conformational change was also observed in TFE. TOAC-14 bound to micelles irrespective of peptide and detergent head group charge. In contrast, the peptide bound to phospholipid bilayers only when both carried opposite charges. The slow exchange (in the EPR time scale) between membrane-bound and free TOAC-14 allowed the calculation of the peptide's partition coefficient. The spectral line shapes were affected by aggregate size and degree of packing of the constituent molecules. It is proposed that pH, polarity, and lipid environment can affect the conformation of water-exposed regions of membrane-bound receptors, thereby playing a role in the mechanism of signal transduction.

Conformational changes upon binding of a receptor loop to lipid structures: possible role in signal transduction.

The mas oncogene codes for a seven transmembrane helix protein. The amino acid sequence 253-266, from the third extracellular loop and beginning of helix 7, was synthesized either blocked or carrying an amino acid spin label at the N-terminus. Peptide binding to bilayers and micelles was monitored by ESR, fluorescence and circular dichroism. Binding induced tighter lipid packing, and caused an increase of peptide secondary structure. While binding to bilayers occurred only when peptide and phospholipid bore opposite charges, in micelles the interaction took place irrespective of charge. The results suggest that changes in lipid packing could modulate conformational changes in receptor loops related to the triggering of signal transduction.

Seasonal variation of anti-RESA/Pf155 Plasmodium falciparum antibodies in three localities from the state of Amapá, Brazil.

Anti-RESA/Pf155 antibodies were assayed in sera of individuals from three localities (Laranjal do Jari, Vila Padaria and Vila Paraíso) in the State of Amapá, Brazil, during the long-rains and short-rains seasons. All of these had negative blood smears for malaria. Most of the sera collected were positive in Indirect Fluorescent Antibody (IFA) with P. falciparum parasites, with no seasonal variation. A high percentage of these sera (62% to 100%) was RESA positive by Modified Indirect Fluorescent Antibody (MIFA), with a significant (p < 0.05) increase of geometric mean titers during the short-rains season, when the transmission of the disease is highest. ELISA with three repetitive RESA peptides (EENV)3 (4 x 3), (EENVEHDA)2 (8 x 2) and (DDEHVEEPTVA)2 (11 x 2) did not reveal statistically significant seasonal variations, although a small enhancement of positivity was observed in V. Padaria (15.3 to 38.8%) in the short-rains season with the 8 x 2 peptides, and with 4 x 3 and 8 x 2 peptides in V. Paraíso, with a decrease in 11 x 2. MIFA titers appeared to be correlated mainly to the peptide 4 x 3 and it was the immunodominant in the three localities.

Binding of peptide fragments from a seven helix membrane receptor to lipid bilayers and to micelles.

Membrane proteins influence the organizational and motional properties of lipids, while the conformation and function of these proteins (receptors, channels, enzymes, pumps) are affected by the lipid environment. Model systems consisting of peptides and lipids can provide information at a molecular level about the interactions between proteins and lipids in biological membranes. We have synthesized peptides (residues 253-266 (EYWSTFGNLHHISL) from the seven-helix receptor expressed by the mas oncogene), having free or blocked N- and C-terminals. An analog was obtained by linking a spin-labeled amino acid to the N-terminal via a peptide bond. Several spectroscopic techniques were employed to study the interaction between the peptides and lipophilic systems (zwitterionic and negatively charged phospholipid bilayers, and negatively charged, positively charged, zwitterionic and nonionic micelles). Peptide conformational changes were monitored by circular dichroism (CD). The peptides acquired an increased secondary structure upon binding to the lipid systems. Additional evidence for peptide incorporation into micelles came from fluorescence measurements which indicated a blue shift of the tryptophan's emission wavelength, and from ESR spectra of the spin-labeled analog. While narrow lines were obtained in the aqueous phase, line broadening indicative of slower motion was observed in the presence of the lipophilic aggregates. The slow exchange between the two media allowed the evaluation of partition coefficients. The spectra in aqueous solution were also sensitive to conformational changes as a function of pH, allowing the determination of the N-terminal pK. ESR spectra of lipid spin probes incorporated into phospholipid bilayers indicated that the lipids became more immobilized upon binding of the peptides.

Endothelin (16-21): biphasic effect and no desensitization on the guinea-pig isolated ileum.

1. In the guinea-pig ileum the C-terminal hexapeptide of the endothelins, endothelin (16-21), induced a biphasic effect (relaxation followed by contraction) qualitatively similar to that seen in the responses to endothelins 1 and 3. Both components of the response were concentration-dependent in the range studied (2-100 microM). 2. The response induced by endothelin (16-21) was inhibited in low-sodium (80 mM) medium. 3. Repeated administration of endothelin (16-21) induced no desensitization of the preparation, contrasting with the tachyphylaxis induced by endothelin-1 and endothelin-3 in the guinea-pig ileum. 4. Tissues rendered tachyphylatic to endothelin-1 or endothelin-3 responded normally to endothelin (16-21). 5. The results suggest that the C-terminal tail of the endothelins contains the message for the biphasic response, whereas the N-terminal domain may be responsible for the strong binding to the receptor and for the tachyphylactic properties of endothelin-1 and endothelin-3, in the guinea-pig isolated ileum. However, the possibility that endothelin (16-21) may be acting on a site other than the endothelin receptor cannot be ruled out.