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.

Effect of tyrosine ionization upon biological activities of angiotensin II and two new peptide analogues.

The role of the tyrosine side-chain in the smooth muscle contracting activity of angiotensin III was investigated by determining intrinsic activities and ED50 values of [4-(3-chlorotyrosine)]angiotensin II and [4-(3-benzyltyrosine)]angiotensin II in the isolated guinea-pig ileum and rat uterus. [4-(3-chlorotyrosine)]angiotensin II activity was compared with that of angiotensin II at different pH values, in which the ratio of their degrees of phenolic ionization varied. The results indicated that deprotonation of the phenolic group hinders binding to smooth muscle cell receptors, but not triggering of the response by the hormone-receptor complex. Steric hindrance by the benzyl substituent in [4-(3-benzyltyrosine)]angiotensin II reduced both receptor-binding and triggering of the response.

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.

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.

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.

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.

Central nervous system kinin receptors and the hypertensive response mediated by bradykinin.

1. Bradykinin (Bk) administered intracerebroventricularly to the rat causes an increase in arterial pressure. 2. Analogues of Bk with agonist and antagonist activity were injected, over a wide dose-range, into the posterior region of the fourth ventricle of unanaesthetized rats implanted with permanent ventricular canullae, and blood pressure was measured directly from the abdominal aorta. 3. The analogues Ile-Ser-Bk (T-kinin) and Lys-Lys-Bk, which interact with both B1 and B2 Bk receptors, produced pressor effects similar to those of Bk, although of greater duration, whereas des-Arg9-Bk, a B1-receptor agonist, had no effect. 4. The B1-antagonist des-Arg9-[Leu8]-Bk did not alter the Bk pressor response, but D-Arg-[Hyp3, Thi5,8,D-Phe7]-Bk, which interacts both with B1- and B2-receptors blocked the responses to Bk, T-kinin and Lys-Lys-Bk and caused parallel shifts to the right of the Bk dose-response curves. Neither antagonist, by itself, had any effect on blood pressure. 5. It is concluded that the central pressor response to Bk is mediated by receptors of the B2 subtype.

Kinin receptors of the central nervous system of spontaneously hypertensive rats related to the pressor response to bradykinin.

1. Kinin analogues bradykinin (BK), T-kinin, Met-Lys-BK, Lys-Lys-BK, Des-Arg9-BK with agonist activity and D-Arg0-Hyp3-Thi5,8-D-Phe7-BK (DAHTDBK) and Arg9-Leu8-BK with antagonist activity were injected into the posterior portion of the fourth cerebral ventricle of unanaesthetized rats implanted with permanent cannulae and arterial pressure was measured directly from the abdominal aorta. 2. The spontaneously hypertensive rats (SHR) were more sensitive than normotensive Wistar rats (NWR) to the pressor effect of BK and other kinin analogues. The SHR did not differ in sensitivity of the pressor response to centrally administered angiotensin II or endothelin-1. 3. Experiments with selective kinin agonists and antagonists revealed that in the SHR, as in the NWR, the receptors which mediated the central pressor response are of the BK2 subtype. 4. Measurements of the pressor activity of kinins with different degrees of susceptibility to degradation, as well as experiments with kininase inhibitors, enalaprilat and CPP-Ala-Ala-Phe-pAB, suggest that the kininase activity in the central nervous system of SHR is reduced in comparison to that of NWR. 5. The SHR also showed increased sensitivity to BK and Lys-Lys-BK, compared with the NWR, when the kinins were injected following the administration of a mixture of the kininase inhibitors, suggesting that mechanisms other than kininase activity may play a role in the increased sensitivity of the SHR to the central pressor action of kinins. 6. An in vivo characterization of the kinin receptors which mediate the central pressor response showed that the interaction with DAHTDBK was reversible and of competitive nature. The pA2 in vivo estimated for the kinin receptors of the SHR was 0.7 logarithm units larger than that obtained in the NWR. 7. The kinin receptors which mediate the central BK pressor effect in the SHR are of the BK2 subtype and are similar to receptors in the NWR. The increased sensitivity to kinins in the SHR may be explained, at least in part, by their decreased kininase activity. At present it is impossible to conclude whether the difference observed in the pA2 represents an increased affinity of the kinin receptors or can be attributed to differences amongst strains in the enzymatic degradation of the antagonist.

The role of calcium in the response of rabbit aorta to angiotensin.

The role of Ca++ in the stimulus-contraction coupling of the response of the isolated rabbit aorta to angiotensin II was investigated. Angiotensin was found to have lower intrinsic activity than epinephrine and to be more sensitive to acute exposure of the organ to Ca++-free medium. Two minutes after removal of Ca++, the maximal responses to angiotensin and epinephrine were reduced by 40% +/- 8% and 7% +/- 5%, respectively. Further loss of response for the two agonists followed parallel time courses. In another series of experiments, angiotensin tachyphylaxis was obtained in the rabbit aorta by administration of either [1-sarcosine]angiotensin or betainyl-angiotensin. The intrinsic activity of [1-sarcosine]angiotensin was lower than that of angiotensin and was not affected by removal of Ca++. It is concluded that the low intrinsic activity and the tachyphylaxis may be dependent on a strong binding of the molecule's positively charged N-terminus to sites responsible for release of Ca++ into the cell.

Low frequency of anti-Plasmodium falciparum circumsporozoite repeat antibodies and rate of high malaria transmission in endemic areas of Rondonia State in northwestern Brazil.

In areas studied in the Rondonia State of Brazil, a high rate of malaria transmission and a low prevalence of anti-(NANP)4 antibodies are reported. The entomologic data are comparable to those observed in some malaria-endemic areas of Africa and Asia. However, the frequency of individuals with antibodies to the immunodominant epitope of the circumsporozoite protein of Plasmodium falciparum recorded in the four localities of Rondonia state was very low when compared with the frequencies recorded in other African and Asian endemic areas. Most of the studies performed in Africa and Asia concerned the native population of hyperendemic areas, whereas we studied a migrant population who were mostly from malaria-free areas of Brazil and living in Rondonia State for 2-4 years. In positive individuals the antibody production was influenced by previous malaria experience, suggesting that infective bites must occur in cumulative numbers before anti-(NANP)4 antibodies are detected. Therefore, it is possible that the individuals described in this report have not been exposed long enough to malaria infection to develop detectable levels of anti-(NANP)4 antibodies.

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.

Changes in residues 1 and 4 of angiotensin II affect differently its agonist and tachyphylactic properties.

Two series of angiotensin II analogues with modifications at positions 1 or 4 of the peptide chain were studied with respect to their tachyphylactic properties and to the kinetics of relaxation of the guinea-pig ileum after a contractile response to maximally effective concentrations. Tachyphylaxis was measured by the decrease in response amplitude after three successive treatments ("tachyphylactic index") and the relaxation rate was evaluated by the time taken for the tonus to reach half of its value at the moment of agonist washout ("half relaxation time"). A correlation between tachyphylactic index and half relaxation time was found for the series of position 1 analogues, but not for the position 4 analogues. For the two series, the half relaxation times of the tachyphylactic analogues decreased from the first to the third of a series of successive treatments. Bulky substituents at position 1, which did not greatly affect the agonist activity, suppressed the tachyphylactic property. The results provide evidence that the agonist and tachyphylactic properties of angiotensin II are due to its interaction, respectively, with an agonist site and a "tachyphylaxis" site on the receptor and that the structural requirements for binding to the two sites are different.

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.

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 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.

Inhibition of renin by conformationally restricted analogues of angiotensinogen.

[Cys(5),Cys(10)]Angiotensinogen-(5-14)-peptide analogues and homologues were synthesized, in which a disulphide bond between residues 5 and 10 stabilized a 9-->6 beta-turn proposed for the substrate. These compounds were competitive inhibitors of human and pig renins with K(i) values of the order of 10(-6)-10(-5)m, indicating that the conformation proposed for the substrate is important for the interaction with the enzyme.

Effect of lipophilic character on the biological activity of synthetic angiotensin peptides.

[Ile5]angiotensin II (angiotensin) derivatives bearing acetyl, propionyl, butyryl, pentanoyl or hexanoyl moieties at the N-terminal amino group were synthesized. The myotropic effects in vitro (on guinea-pig ileum and rat uterus) of desamino-angiotensin and of the above compounds did not correlate with their partition coefficients in butan-1-ol/acetic acid/water. The pressor effects in vivo in rats showed a negative correlation with the partition coefficients, discouraging further attempts to raise the pressor potency of angiotensin analogues by increasing their hydrophobicity. The half-times for onset and reversal of the biological responses also did not correlate with partition coefficients, but reversal was retarded by the presence of a free amino group. It is concluded that partition between aqueous medium and the lipophilic receptor environment is not a limiting factor for angiotensin activity.