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.     

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC⁰-AngI and TOAC¹-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe⁸-His⁹ peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr⁴ fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.     

Conformational studies of TOAC-labeled bradykinin analogues in model membranes

Summary Spin-labeled analogues of bradykinin (BK) were synthesized containing the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) either before Arg¹ (TOAC⁰-BK) or replacing Pro³ (TOAC³-BK). Whereas the latter is inactive, the former retains about 70% of BK's activity in isolated rat uterus. A combined electron paramagnetic resonance (EPR)-circular dichroism (CD) approach was used to examine the conformational properties of the peptides in the presence of membrane-mimetic systems (negatively charged sodium dodecyl sulfate, SDS, and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, HPS). While the peptides bind to both monomeric and micellar SDS, no interaction occurs with HPS, evincing the contribution of electrostatic interactions. TOAC³-BK's EPR spectral lineshapes are broader than those of TOAC⁰-BK, indicating a more restricted degree of motion at position 3. Moreover, the motional freedom of both peptides decreased upon binding to SDS. BK and TOAC⁰-BK solution CD spectra indicate highly flexible conformations (possibly an equilibrium between rapidly interconverting forms), while TOAC³-BK's spectra correspond to a more ordered structure. SDS binding induces drastic changes in BK and TOAC⁰-BK spectra, indicating stabilization of similar folds. The micelle interface promotes a higher degree of secondary structure by favoring intramolecular hydrogen bonds. In contrast. TOAC³-BK spectra remain essentially unchanged. These results are interpreted as due to TOAC's ring imposing a more constrained conformation. This rigidity is very likely responsible for the inability of TOAC³-BK to acquire the correct receptor-bound conformation leading to loss of biological activity, On the other hand, the greater flexibility of TOAC⁰-BK and the similarity between its conformational behavior and that of the native hormone are probably related to their similar biological activity.     

Conformational studies of TOAC-labeled bradykinin analogues in model membranes

Spin-labeled analogues of bradykinin (BK) were synthesized containing the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) either before Arg¹ (TOAC⁰-BK) or replacing Pro³ (TOAC³-BK). Whereas the latter is inactive, the former retains about 70% of BK's activity in isolated rat uterus. A combined electron paramagnetic resonance (EPR)-circular dichroism(CD) approach was used to examine the conformational propertiesof the peptides in the presence of membrane-mimetic systems (negatively charged sodium dodecyl sulfate, SDS, and zwitterionicN-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, HPS). While the peptides bind to both monomeric and micellar SDS, no interaction occurs with HPS, evincing the contribution of electrostatic interactions. TOAC³-BK's EPR spectral lineshapes are broader than those of TOAC⁰-BK, indicating amore restricted degree of motion at position 3. Moreover, the motional freedom of both peptides decreased upon binding to SDS. BK and TOAC⁰-BK solution CD spectra indicate highly flexible conformations (possibly an equilibrium between rapidlyinterconverting forms), while TOAC³-BK's spectra correspondto a more ordered structure. SDS binding induces drastic changesin BK and TOAC⁰-BK spectra, indicating stabilization of similar folds. The micelle interface promotes a higher degree of secondary structure by favoring intramolecular hydrogen bonds. Incontrast, TOAC³-BK spectra remain essentially unchanged. These results are interpreted as due to TOAC's ring imposing a more constrained conformation. This rigidity is very likely responsible for the inability of TOAC³-BK to acquire the correct receptor-bound conformation, leading to loss of biological activity. On the other hand, the greater flexibility of TOAC⁰-BK and the similarity between its conformational behavior and that of the native hormone are probably related to their similar biological activity.     

CD-n.m.r. study of the solution conformation of bradykinin analogs containing alpha-aminoisobutyric acid

The conformation in aqueous solution of several alpha-aminoisobutyric acid (AIB)-containing analogs of bradykinin (BK) has been probed by complementary CD and 1H n.m.r. measurements. The conclusion reached is that substitution of AIB for Pro2 and/or Pro3 in BK stabilizes a degree of beta-turn conformation in the N-terminal tetrapeptide moiety of the resulting analogs. Changing the solvent from water to DMSO or TFE further enhances the contribution of particular hydrogen bonded structures to the time-averaged conformation of these peptides. Bradykinin and [AIB7]-BK adopt similar hydrogen bonded conformations in TFE, apparently with a contribution from a beta-turn involving their common Arg1-Pro2-Pro3-Gly4 moiety. The contrasting biological activities of BK and its AIB-analogs are considered in terms of the conformational analogy between the AIB-residue and cis' Pro and the propensity for a beta-turn at the N-terminus of the peptide.     

l-3,4-Dehydroproline analogs of bradykinin: Synthesis,biological activity,and solution conformation

Three analogs of bradykinin (BK) containing l-3,4-dehydroproline (l-Δ³Pro) at positions 2, 3, or 7 of the BK sequence have been synthesized by the solid-phase technique and assayed for their effects on isolated smooth muscle tissues and on the systemic arterial blood pressure of rats. In these assays, (l-Δ³Pro²)-BK and (l-Δ³Pro³)-BK are as potent as BK itself, and each appears to be more resistant to enzymic degradation than BK itself during passage through the pulmonary circulation. (l-Δ³Pro⁷)-BK has approximately 25% of the potency of BK and appears to be more susceptible to pulmonary degradation than is BK. The circular dichroism (CD) spectra for the three analogs in water are similar in profile between 250 and 200 nm, although with different intensities, and more closely parallel the CD spectrum of BK in trifluoroethanol (TFE) than in water. The CD spectra in TFE show changes for all three analogs from the aqueous spectra. The TFE CD spectra of (l-Δ³Pro³)-BK and (l-Δ³Pro⁷)-BK are similar in profile to the spectrum of BK in TFE, whereas the spectrum of (l-Δ³Pro²)-BK is considerably different from that of BK. The spectroscopic changes can be interpreted in terms of a change in polyproline-like character or β-fold formation, presumably imposed by the greater rigidity of the dehydroproline ring.     

Electron spin resonance of TOAC labeled peptides: folding transitions and high frequency spectroscopy

The unnatural, conformationally constrained nitroxide amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) stabilizes helical structure and provides a means for studying rigidly spin labeled peptides by electron spin resonance (ESR). Two new directions in TOAC research are described. The first investigates intermediates formed during alpha-helix unfolding. Double TOAC labeled alpha-helical peptides were unfolded at low temperature in aqueous solution with increasing concentrations of guanidine hydrochloride. Comparison of ESR spectra from two doubly labeled peptides suggests that 3(10)-helix emerges as an intermediate. The second research direction involves the use of high frequency ESR (140 GHz) at low temperature to assess dipolar couplings and, hence, distances between TOAC pairs in a series of 3(10)-helical peptides. Preliminary simulations suggest that high frequency ESR is able to extract correct distances between 6 and 11 A. In addition, the spectra appear to be very sensitive to the relative orientation of the TOAC labels.     

Gamma Ray Irradiation of the Vasoactive Peptide Bradykinin Reveals a Residue- and Position-Dependent Structural Modification

In this work the effect of radical species generated by gamma ray irradiation of aqueous solution upon structure of vasoactive peptide bradykinin (BK, RPPGFSPFR) was investigated. Increasing doses of 1–15 kGy Co⁶⁰ gamma radiation were applied to BK solutions and a progressive degradation of its structure in a non-linear mode was observed. Two main peptide derivatives generated by these treatments were isolated and characterized through a combined amino acid analysis and daughter ion scanning mass spectrometry approach. Notably, it was observed that only the Phe residue located at position 8 and not 5 of BK was oxidized by reactive hydroxyl radical species given rise to Tyr⁸-BK and m-Tyr⁸-BK analogues. Comparative circular dichroism (CD) experiments of these peptides revealed that BK presents greater conformational similarity to Tyr⁸-BK than to m-Tyr⁸-BK. These results are in agreement with the biological potencies of these compounds measured in rat uterus and guinea pig ileum muscle contractile experiments. In summary, gamma irradiation of BK solutions revealed a residue- and surprisingly, position-structural modification effect of reactive radicals even in small peptides. Also of value for peptide chemistry field, the approach of applying controlled strong electromagnetic radiation in solution seems to be an alternative and unique strategy for generating, in some cases, peptides derivatives with uncommon structures and valuable for their further therapeutic potential evaluations.     

Conformations of hydrophobic peptides in trifluoroethanol, water and in solid state: a circular dichroism and Fourier Transform Infrared study

The conformations of peptides corresponding to KLLIALVLCFLPLAALG have been examined in trifluoroethanol (TFE), aqueous medium by circular dichroism spectroscopy and in the solid state by Fourier Transform Infra Red Spectroscopy (FTIR). The 17-residue parent peptide and peptides corresponding to shorter segments LVLCFLPLAALG and CFLPLAALG showed preference for helical conformation in TFE. Even the shorter hydrophobic peptides corresponding to KLLIA and LVL showed propensity for beta-turn conformations in TFE. However, peptides corresponding to the relatively polar segment FLPLAALG were unordered in TFE. In water, peptides that showed ordered conformation in TFE preferred beta-conformation. In solid-state, FTIR spectra indicated that the hydrophobic peptides adopt beta-structures with extensive hydrogen bonded network in the solid-state. The hydrophobic core segment thus appears to dictate the conformational propensity of the peptide.     

Paramagnetic bradykinin analogues as substrates for angiotensin I-converting enzyme: Pharmacological and conformation studies

This study uses EPR, CD, and fluorescence spectroscopy to examine the structure of bradykinin (BK) analogues attaching the paramagnetic amino acid-type Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 3, 7, and 9. The data were correlated with the potencies in muscle contractile experiments and the substrate properties towards the angiotensin I-converting enzyme (ACE). A study of the biological activities in guinea pig ileum and rat uterus indicated that only Toac⁰-BK partially maintained its native biological potency among the tested peptides. This and its counterpart, Toac³-BK, maintained the ability to act as ACE substrates. These results indicate that peptides bearing Toac probe far from the ACE cleavage sites were more susceptible to hydrolysis by ACE. The results also emphasize the existence of a finer control for BK-receptor interaction than for BK binding at the catalytic site of this metallodipetidase. The kinetic kcat/Km values decreased from 202.7 to 38.9 μM⁻¹ min⁻¹ for BK and Toac³-BK, respectively. EPR, CD, and fluorescence experiments reveal a direct relationship between the structure and activity of these paramagnetic peptides. In contrast to the turn-folded structures of the Toac-internally labeled peptides, more extended conformations were displayed by N- or C-terminally Toac-labeled analogues. Lastly, this work supports the feasibility of monitoring the progress of the ACE-hydrolytic process of Toac-attached peptides by examining time-dependent EPR spectral variations.     

Short peptide constructs mimic agonist sites of AT1R and BK receptors

Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT₁R and BKRB₁ or BKRB₂ G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S–S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac¹-AngII and Toac⁰-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac³-AngII and Toac³-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT₁R and BKRB cyclic constructs based on the structure of the CXCR₄, an α-chemokine GPCR-type receptor.     

Combined effect of the DeltaPhe or DeltaAla residue and the p-nitroanilide group on a didehydropeptides conformation

Two series of dehydropeptides of the general formulae Boc-Gly-X-Phe-p-NA, Boc-Gly-Gly-X-Phe-p-NA, Gly-X-Gly-Phe-p-NA.TFA, and Boc-Gly-X-Gly-Phe-p-NA, with X = Delta(Z)Phe and DeltaAla, were studied with NMR in DMSO and CDCl(3)-DMSO, and with CD in MeOH, MeCN, and TFE. The NMR spectra measured in DMSO suggest that peptides with the DeltaPhe residue next to Phe are folded whereas peptides with Gly between DeltaPhe and Phe are less ordered. NMR spectra of DeltaAla-containing peptides indicate that these peptides are flexible and their conformational equilibria are populated by many different conformations. The CD spectra show that conformational properties of the peptides studied are distinctly influenced by a mutual position of the dehydroamino acid residue and the p-NA group. They indicate that all dehydropeptides with the DeltaPhe residue, Boc-Gly-DeltaAla-Phe-p-NA, and Boc-Gly-Gly-DeltaAla-Phe-p-NA adopt ordered conformations in all solvents studied, presumably of the beta-turn type. The last two peptides exhibit surprising chiroptical properties. Their spectra show exciton coupling-like couplets in the region of the p-NA group absorption. This shape of CD spectra suggests a rigid, chiral conformation with a fixed disposition of the p-NA group. The CD spectra indicate that Boc-Gly-DeltaAla-Gly-Phe-p-NA and Gly-DeltaAla-Gly-Phe-p-NA.TFA are unordered, independently of the solvent.     

N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes

Peptide agonists and antagonists of both bradykinin (BK) B(1) and B(2) receptors (B(1)R, B(2)R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ε-aminocaproyl (ε-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B(1)R: B-10376: CF-ε-ACA-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; B(2)R: B-10380: CF-ε-ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B(2)R agonists CF-ε-ACA-BK, AF350-ε-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B(1)R agonist B-10378 (CF-ε-ACA-Lys-des-Arg(9)-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ε-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors.     

A CD and an nmr study of multiple bradykinin conformations in aqueous trifluoroethanol solutions

CD and nmr studies have been carried out on aqueous trifluoroethanol (TFE) solutions of bradykinin (BK) and a bradykinin antagonist. The CD results exhibit a striking effect of TFE on the spectra of BK, with sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and the BK antagonist, with sequence D -Arg-Arg-Pro-Hyp-Gly-Thi-D -Ser-D -Cpg-Cpg-Arg [where Hyp is 4-hydroxy-L -proline; Thi refers to β-(2-thienyl)-L -alanine and Cpg refers to α-cyclopentylglycine]. The effect of increasing concentration of TFE in water on the difference ellipticity at 222 nm was examined and showed that BK may be a mixture of at least two different conformers, one of which largely forms when the TFE concentration is increased beyond 80%. The linear extrapolation of 100% of the difference ellipticity of BK at low TFE concentrations yields a value in agreement with that shown by the BK antagonist, indicating that the conformation of BK at the lower TFE concentrations is similar to that of the BK antagonist. The conformational analysis was carried out using both one-dimensional and two-dimensional ¹H-nmr techniques. The total correlation spectroscopy (TOCSY) spectrum of BK in a 60/40% (v/v) TFE/H₂O solution at 10°C and a nuclear Overhauser effect spectroscopy (NOESY) spectrum that shows only sequential H_α(i) – NH(i + 1) or the H_α(i) – H_δδ′(i + 1) NOEs indicate that the majority of the molecules adopt an all-trans extended conformation. The TOCSY for BK in the 95/5% (v/v) TFE/H₂O solution shows that there are two major conformations in the solution with about equal population. The NOESY experiment shows two new important cross peaks for one conformation, namely Pro²(α)-Pro³ (α) and the Pro²(α)-Gly⁴(NH), indicating a cis Pro²-Pro³ bond and a type VI β-turn between residues Arg¹ and Gly⁴ involving cis proline at position 3, respectively. The low temperature coefficient of Gly⁴ for this conformation suggests the presence of an intramolecular hydrogen bond, therefore a type VIa β-turn is present. The other conformation is all trans and extended. The BK antafonist shows difference CD spectra in TFE solutions referred to H₂O that are superficially indicative of a β-bend. However, nmr speaks against this possibility, as only one set of peaks were observed in the TOCSY and NOESY experiments, indicating an all-trans extended confirmation over the range of TFE concentrations. The BK-antagonist CD data suggest that solvent perturbation of the CD of an extended confirmation perturbation of the optical activity of the thienyl moiety of the peptide since the CD spectrum of N-acetyl-β-thienyl-L -alanine N-methylamide is strongly perturbed by TFE. The present results again demonstrate the complementary relationship between CD and nmr. © 1994 John Wiley & Sons, Inc.     

Low-ultraviolet circular dichroism spectroscopy of oligopeptides 1-95 and 96-168 derived from myelin basic protein of rabbit

Myelin basic protein (MBP) is a major protein constituent of the myelin sheath of the central nervous system, where it is believed to have functional alpha-helical segments. One element of the function of the protein might be "conformational adaptability" of specific regions of its amino acid sequence, since the purified protein appears to be largely devoid of ordered structure. To pursue this question, low-ultraviolet circular dichroism (CD) spectroscopy was conducted on the sequential thrombic peptides 1-95 and 96-168 of the protein in the presence of 0-92% trifluoroethanol (TFE), a solvent known to promote stable secondary structures in polypeptides. The series of CD spectra of the oligopeptides were subjected to a computerized best-fit analysis of four peptide conformations, the alpha-helix, beta-structure, beta-turn, and nonordered form. Agreement between experimental and best-fit composite spectra was achieved when standard CD curves of peptide conformations were derived from known theoretical spectra and experimental spectra of polypeptides. In dilute buffer alone, oligopeptides 1-95 and 96-168 evidence no alpha-helix but significant beta-structure (18% and 23%, respectively), as well as a predominant, extended nonordered conformation. However, the two parts of the protein differed in conformational adaptability. From 0% to 30% TFE, 96-168 exhibited concomitant transitions to 10% helix and 32% beta-structure from the nonordered form. In contrast, in 10-30% TFE, 1-95 underwent a transition to approximately 21% helix with partial loss of beta-structure as well as nonordered form; higher concentrations of TFE (40-75%) promoted additional transitions to both helix and beta-structure (totaling 33% and 25%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow Cytometric Analysis of Internal Calcium Mobilization via a B2-Bradykinin Receptor on a Subclone of PC-12 Cells

Single cell Ca2+ mobilization was studied by nonparametric, quantitative flow cytometry using a sort-selected subclone of PC-12 cells. The response of the parent PC-12 population to bradykinin (BK) was very heterogeneous and of a relatively low magnitude. Cells that exhibited maximal Ca2+ mobilization were singly sorted by flow cytometry, cultured, and reanalyzed. In one subclone, referred to as BK1, BK or the B2-BK receptor agonists Lys-BK and Met-Lys-BK (10 pM-1 microM) induced robust Ca2+ transients in 80% of the cells. All three peptides produced the same maximal responses. The B1-BK receptor agonist Des-Arg9-BK (1 nM-1 microM) failed to elicit Ca2+ mobilization in these cells. The responses to BK (10 and 100 nM) were inhibited by preincubation with the B2-receptor antagonists D-Arg0-Hyp3-thienyl5,8-D-Phe7-BK and D-Arg0-Hyp3-D-Phe7 (0.1 nM-10 microM) in a concentration-dependent manner. Des-Arg9-Leu8-BK, a B1-receptor antagonist, failed to block the BK responses at 0.1-10 microM. The agonist/antagonist profile of the BK responses indicated that the B2-BK receptor mediated the Ca2+ response in the BK1 subclone. Thus, flow cytometric analysis of a receptor-mediated Ca2+ response can be employed to select a homogeneously responsive subclone from a heterogeneous, clonal population that can improve the resolution of receptor-mediated second messenger generation at the single cell level.     

How bradykinin alters the lipid membrane structure: a spin label comparative study with bradykinin fragments and other cations

Electron spin resonance spectroscopy of several different spin labels was used to comparatively study the interaction of the cationic peptide hormone bradykinin (BK; Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), and some BK fragments (des-Arg(9)-BK, des-Arg(1)-BK, and Arg-Pro-Pro-Gly-Phe or BK(1-5)), with anionic vesicles of dimyristoyl phosphatidylglycerol (DMPG). For temperatures above the lipid gel-liquid crystal thermal transition (T(m) approximately 20 degrees C), membrane-incorporated spin labels indicated that all peptides (total concentration of 10 mol % relative to lipid) interact with the bilayer, turning the membrane less fluid, both at its surface and center, suggesting a partial penetration of the peptides into the membrane core. However, in the lipid gel phase (t < T(m)), BK was found to display a much stronger interaction with the membrane, decreasing the bilayer fluidity. At temperatures around 15 degrees C the BK-DMPG system was found to present a hysteresis, evinced by the different electron spin resonance spectra yielded upon cooling and heating the sample. System reversibility was found at all other temperatures (0-45 degrees C). That effect could not be assigned to the BK higher concentration at the membrane surface, due to its higher net charge (2(+)) compared to the fragments (1(+)), because ten times more des-Arg(9)-BK (100 mol %) yielded opposite result. Further, that was found to be a result rather different from those elicited by the other cations tested: the monovalent Na(+), the divalent Zn(2+), and the peptide pentalysine. The data presented here are discussed in the light of the different BK and BK fragments biological activities.     

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.     

Bradykinin and its receptors in non-mammalian vertebrates

The generation of bradykinin (BK) in blood by the action of the kallikrein-kinin system has been studied intensively in mammals but the system has received relatively little attention in non-mammalian vertebrates. The plasma of crocodilians and Testudines (turtles and tortoises) contains all the components of the kallikrein-kinin system found in mammals (prekallikrein activator, prekallikrein, kininogen, and kininases) and activation results in generation of [Thr6]-BK. Plasma of birds and snakes probably lacks a prekallikrein activator analogous to mammalian Factor XII but treatment with exogenous proteases (pig pancreatic kallikrein and/or trypsin) generates [Thr6, Leu8]-BK (chicken), [Ala1, Thr6]-BK (python) and [Val1, Thr6]-BK (colubrid snakes). The skins of certain frogs, particularly of the genus Rana, contain very high concentrations of BK-related peptides but their pathway of biosynthesis involves the action of cellular endoproteinase(s) cleaving at the site of single arginyl residues rather than by the action of the kallikrein-kinin system. Evidence for a prekallikrein activator in fish plasma is lacking but treatment with exogenous proteases generates [Arg0, Trp5, Leu8]-BK (trout and cod), [Trp5]-BK (bowfin and gar), [Met1, Met5]-BK (sturgeon). The cardiovascular actions and effects upon gastrointestinal smooth muscle of these peptides in their species of origin differ markedly. For example, intra-arterial injections of the native BK peptides into unanesthetized fish produce transient hypertension in the cod, complex depressor and pressor responses in the trout and bowfin and hypotension in the sturgeon. Pharmacological studies in snakes and fish and with the recombinantally expressed chicken BK receptor have demonstrated that the BK receptors in the tissues of non-mammalian vertebrates have appreciably different ligand binding properties from the well-characterized mammalian B1 and B2 receptors.