Importance of Tyr409 and Tyr414 in constructing the substrate pocket of human aminopeptidase B.

Aminopeptidase B (APB, EC 3.4.11.6) preferentially hydrolyzes basic amino acids of synthetic substrates and requires a physiological concentration of chloride anions for optimal activity. Several amino acid residues of APB responsible for its enzymatic activity have been elucidated. In this study, we further searched for residues critical to its enzymatic activity, especially toward peptide substrates. APB residues Tyr409 (Y409) and Tyr414 (Y414), both of which were critical to its hydrolytic activity toward synthetic substrates, were predicted by molecular modeling to be involved in cleaving peptide substrates via its interaction with amino acids in the P1' cleavage site. Using site-directed mutagenesis, several mutant APBs were prepared. In contrast to synthetic substrates, wild-type and Y409F/Y414F double mutant enzymes showed P1'-dependent cleavage of peptide substrates, indicating that both tyrosine residues were not indispensable for hydrolytic activity toward peptide substrates. Moreover, the Y409F/Y414F double mutant enzyme cleaved peptides with a Pro residue at the P1' site, which is uncommon among the M1 family of aminopeptidases. These results suggested that Tyr409 and Tyr414 of APB play important roles in enzymatic function and characteristic properties of APB via proper formation of the S1' site.

Crosslinked Network with Rotatable Binding Sites Based on Monocarboxylated α-Cyclodextrin [2]Rotaxane Capable of Angiotensin†III Recognition.

Synthetic receptors selective for target peptides or proteins have received attention because of their potential applications in the separation of biomolecules and biomedical diagnostics. Herein, a [2]rotaxane-based functional monomer containing monocarboxylated α-cyclodextrin (α-CD) was synthesized, and its crosslinked polymers were evaluated to determine their binding ability to a model peptide, angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe), containing an arginine (Arg) residue. The binding ability of the resulting polymers toward angiotensin III, angiotensin IV (Val-Tyr-Ile-His-Pro-Phe), and FMRF-amide (Phe-Met-Arg-Phe) was examined by the batch-binding assay and compared with that of control polymers, in which maleic acid-introduced α-CD was chemically crosslinked. The results suggest that the [2]rotaxane-based functional monomer in the crosslinked polymer contributes to the high affinity toward angiotensin III. The α-CD motion and rotation within the [2]rotaxane-based crosslinked polymer may be applicable for designing molecular recognition materials.

On-plate self-desalting and matrix-free LDI MS analysis of peptides with a surface patterned sample support.

A hydrophobic-hydrophilic-hydrophobic pattern has been produced on the surface of a silicon substrate for selective enrichment, self-desalting, and matrix-free analysis of peptides in a single step. Upon sample application, the sample solution is first confined in a small area by a hydrophobic F-SAM outer area, after which salt contaminants and peptides are selectively enriched in the hydrophilic and hydrophobic areas, respectively. Simultaneously, matrix background noise is significantly reduced or eliminated because of immobilization of matrix molecules. As a result, the detection sensitivity is enhanced 20-fold compared with that obtained using the usual MALDI plate, and interference-free detection is achieved in the low m/z range. In addition, peptide ions can be identified unambiguously in the presence of NH4HCO3 (100 mM), urea (1 M), and NaCl (1 M). When the device was applied to the analysis of BSA digests, the peptide recovery and protein identification confidence were greatly improved.

Differential mechanisms of activation of the Ang peptide receptors AT1, AT2, and MAS: using in silico techniques to differentiate the three receptors.

The renin-angiotensin system is involved in multiple conditions ranging from cardiovascular disorders to cancer. Components of the pathway, including ACE, renin and angiotensin receptors are targets for disease treatment. This study addresses three receptors of the pathway: AT1, AT2, and MAS and how the receptors are similar and differ in activation by angiotensin peptides. Combining biochemical and amino acid variation data with multiple species sequence alignments, structural models, and docking site predictions allows for visualization of how angiotensin peptides may bind and activate the receptors; allowing identification of conserved and variant mechanisms in the receptors. MAS differs from AT1 favoring Ang-(1-7) and not Ang II binding, while AT2 recently has been suggested to preferentially bind Ang III. A new model of Ang peptide binding to AT1 and AT2 is proposed that correlates data from site directed mutagenesis and photolabled experiments that were previously considered conflicting. Ang II binds AT1 and AT2 through a conserved initial binding mode involving amino acids 111 (consensus 325) of AT1 (Asn) interacting with Tyr (4) of Ang II and 199 and 256 (consensus 512 and 621, a Lys and His respectively) interacting with Phe (8) of Ang II. In MAS these sites are not conserved, leading to differential binding and activation by Ang-(1-7). In both AT1 and AT2, the Ang II peptide may internalize through Phe (8) of Ang II propagating through the receptors' conserved aromatic amino acids to the final photolabled positioning relative to either AT1 (amino acid 294, Asn, consensus 725) or AT2 (138, Leu, consensus 336). Understanding receptor activation provides valuable information for drug design and identification of other receptors that can potentially bind Ang peptides.

Mass spectrometry for the molecular imaging of angiotensin metabolism in kidney.

To better understand the tissue distribution and activity of enzymes involved in angiotensin II (Ang II) processing, we developed a novel molecular imaging method using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Mouse kidney sections (12 µm) were incubated with 10-1,000 µmol/l Ang II for 5-15 min at 37°C. The formed peptides Ang III and Ang-(1-7) were identified by MALDI-TOF/TOF. A third metabolite, Ang-(1-4), was generated from further degradation of Ang-(1-7). Enzymatic processing of Ang II was dose and time dependent and absent in heat-treated kidney sections. Distinct spatial distribution patterns (pseudocolor images) were observed for the peptides. Ang III was localized in renal medulla, whereas Ang-(1-7)/Ang-(1-4) was present in cortex. Regional specific peptide formation was confirmed using microdissected cortical and medullary biopsies. In vitro studies with recombinant enzymes confirmed activity of peptidases known to generate Ang III or Ang-(1-7) from Ang II: aminopeptidase A (APA), Ang-converting enzyme 2 (ACE2), prolyl carboxypeptidase (PCP), and prolyl endopeptidase (PEP). Renal medullary Ang III generation was blocked by APA inhibitor glutamate phosphonate. The ACE2 inhibitor MLN-4760 and PCP/PEP inhibitor Z-pro-prolinal reduced cortical Ang-(1-7) formation. Our results establish the power of MALDI imaging as a highly specific and information-rich analytical technique that will further aid our understanding of the role and site of Ang II processing in cardiovascular and renal pathologies.

TOF-SIMS with argon gas cluster ion beams: a comparison with C60+.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an established technique for the characterization of solid sample surfaces. The introduction of polyatomic ion beams, such as C(60), has provided the associated ability to perform molecular depth-profiling and 3D molecular imaging. However, not all samples perform equally under C(60) bombardment, and it is probably naïve to think that there will be an ion beam that will be applicable in all situations. It is therefore important to explore the potential of other candidates. A systematic study of the suitability of argon gas cluster ion beams (Ar-GCIBs) of general composition Ar(n)(+), where n = 60-3000, as primary particles in TOF-SIMS analysis has been performed. We have assessed the potential of the Ar-GCIBs for molecular depth-profiling in terms of damage accumulation and sputter rate and also as analysis beams where spectral quality and secondary ion yields are considered. We present results with direct comparison with C(60) ions on the same sample in the same instrument on polymer, polymer additive, and biomolecular samples, including lipids and small peptides. Large argon clusters show reduced damage accumulation compared with C(60) with an approximately constant sputter rate as a function of Ar cluster size. Further, on some samples, large argon clusters produce changes in the mass spectra indicative of a more gentle ejection mechanism. However, there also appears to be a reduction in the ionization of secondary species as the size of the Ar cluster increases.

Peptide-phospholipid complex formation at liquid-liquid interfaces.

Two peptides known to interact with receptors embedded in cell membranes, angiotensin III (Ang III) and Leu-enkephalin (LeuEnk), were studied electrochemically at the interface formed between two immiscible electrolyte solutions modified by an adsorbed monolayer of dipalmitoylphosphatidylcholine (DPPC). The results indicate that cationic angiotensin III transfer can be facilitated by the interfacial formation of a complex with DPPC. The complexation constant was determined by voltammetry and found to be equal to 5.2 x 10(4) M(-1). For neutral Leu-enkephalin, a current only observable in the presence of the lipidic monolayer results from the formation of a complex between the lithium cation, LeuEnk or LeuEnk dimer and the phospholipid. For both peptides, the peptide-lipid complexes were identified by biphasic electrospray ionization mass spectrometry using a setup consisting of a dual-channel microchip, which puts in contact two immiscible phases at the Taylor cone and makes possible the study of interfacial complexes. The stability of the 1:1 complexes between lysine, diphenylalanine, Ang III, and LeuEnk and DPPC were evaluated by varying the temperature of the heated capillary of the mass spectrometer. Finally, from the complementary use of voltammetry and mass spectrometry, a mechanism for the interaction between these two biologically relevant peptides and DPPC monolayers is formulated.

The effect of the secondary structure on dissociation of peptide radical cations: fragmentation of angiotensin III and its analogues.

Fragmentation of protonated RVYIHPF and RVYIHPF-OMe and the corresponding radical cations was studied using time- and collision energy-resolved surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Peptide radical cations were produced by gas-phase fragmentation of Co (III)(salen)-peptide complexes. Both the energetics and the mechanisms of dissociation of even-electron and odd-electron angiotensin III ions are quite different. Protonated molecules are much more stable toward fragmentation than the corresponding radical cations. RRKM modeling of the experimental data suggests that this stability is largely attributed to differences in threshold energies for dissociation, while activation entropies are very similar. Detailed analysis of the experimental data obtained for radical cations demonstrated the presence of two distinct structures separated by a high free-energy barrier. The two families of structures were ascribed to the canonical and zwitterionic forms of the radical cations produced in our experiments.

Hydrolysis of various bioactive peptides by goat brain dipeptidylpeptidase-III homologue.

DPP-III from goat brain was purified to apparent electrophoretic homogeneity which showed several characteristics similar to other reported DPP-IIIs although it possesses dissimilar molecular weight and different inhibition behavior. Thin layer chromatographic studies with goat brain DPP-III revealed that it hydrolyses Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) at the Gly-Gly bond producing Tyr-Gly and Gly-Phe-Leu with no further degradation of liberated tripeptide. (Ala)(4) is hydrolyzed to dialanine whereas trialanine is not cleaved. ACTH, angiotensin II and III were also hydrolyzed whereas angiotensin I was not. It was concluded that the enzyme requires at least a tetrapeptide to act and that it removes a dipeptidyl moiety from the NH(2)-terminus of the studied peptides. Goat brain DPP-III may be involved in the metabolism of very important bioactive peptides such as enkephalins and angiotensins.

Lack of angiotensin II conversion to angiotensin III increases water but not alcohol consumption in aminopeptidase A-deficient mice.

Elevated central concentrations of the vasopressor octapeptide angiotensin (Ang) II increase the water intake in mammals. Recently, we showed that central AngII is also crucial in alcohol-consuming behavior. Since the heptapeptide AngIII, an AngII metabolite, is discussed to mediate AngII-related effects, we investigated water and alcohol consumption in mice, genetically deficient in aminopeptidase A (APA), a peptidase responsible for AngII conversion to AngIII. Sixteen male APA-deficient mice and their age matched wild-type controls were monitored on their water intake under basal conditions and total fluid and alcohol intake before and after social stress in a two-bottle free-choice paradigm. Alterations were connected to the regulation in activity of Ang-related peptidases (APA, ACE; ACE2) in brain regions involved in alcohol intake and peripheral organs. In comparison to their wild-type controls, APA-deficient mice drank significantly more water but not more alcohol at all investigated time points. A reduction in water intake, as observed in wild-type animals after social stress, did not occur in knockout mice. However, the reduction in alcohol consumption after social stress was significantly reduced in both strains. Alcohol consumption upregulated all three peptidases in the kidney, but not in lung. Notable, renal ACE2 activity was significantly higher in APA-deficient mice under basal condition. While the inhibition of AngII metabolism to AngIII does not influence the alcohol intake, water consumption in mice deficient for APA was significantly elevated. These differences induced by an altered AngII/AngIII ratio oppose the hypothesis that central AngII and AngIII act in a congruent pattern.

Formation of anionic peptide radicals in vacuo.

In this paper, we demonstrate for the first time the formation of radical anionic peptides [M - 2H]*- through a one-electron transfer mechanism upon low-energy collision-induced dissociation (CID) of gas-phase singly charged [Mn(III)(salen)(M - 2H)]*- complex ions [where salen is N,N'-ethylenebis(salicylideneiminato) and M is an angiotensin III derivative]. The types of fragment ions formed from [M - 2H]*- share some similarities with those from the cationic radical peptides M*+ and [M + H]*2+, but differ significantly from those of the corresponding deprotonated peptides [M - H]-. Fragmentation of [M - 2H]*- radical anionic angiotensin III derivatives leads preferentially to product ions of side-chain cleavage of amino acid residues, z-type and minor x-type fragment ions, most of which are types rarely observed in low-energy CID spectra of deprotonated analogs. The degree of competitive dissociation of the complexes is highly dependent on the nature of the substituted salen derivatives. The yields of anionic peptide radicals were enhanced to the greatest extent when electron withdrawing groups were positioned at the 5 and 5' positions, but the effect was rather modest when such groups resided at the 3 and 3' positions. Substituting a cyclohexyl unit of a salen with phenyl or naphthyl moieties at the 8 and 8' positions also facilitated electron-transfer pathways.

Isotope ratio monitoring of small molecules and macromolecules by liquid chromatography coupled to isotope ratio mass spectrometry.

In the field of isotope ratio mass spectrometry, the introduction of an interface allowing the connection of liquid chromatography (LC) and isotope ratio mass spectrometry (IRMS) has opened a range of new perspectives. The LC interface is based on a chemical oxidation, producing CO2 from organic molecules. While first results were obtained from the analysis of low molecular weight compounds, the application of compound-specific isotope analysis by irm-LC/MS to other molecules, in particular biomolecules, is presented here. The influence of the LC flow rate on the CO2 signal and on the observed delta13C values is demonstrated. The limits of quantification for angiotensin III and for leucine were 100 and 38 pmol, respectively, with a standard deviation of the delta13C values better than 0.4 per thousand. Also, accuracy and precision of delta13C values for elemental analyser-IRMS and flow injection analysis-IRMS (FIA-LC/MS) were compared. For compounds with molecular weights ranging from 131 to 66,390 Da, precision was better than 0.3 per thousand, and accuracy varied from 0.1 to 0.7 per thousand. In a second part of the work, a two-dimensional (2D)-LC method for the separation of 15 underivatised amino acids is demonstrated; the precision of delta13C values for several amino acids by irm-LC/MS was better than 0.3 per thousand at natural abundance. For labelled mixtures, the coefficient of variation was between 1% at 0.07 atom % excess (APE) for threonine and alanine, and around 10% at 0.03 APE for valine and phenylalanine. The application of irm-LC/MS to the determination of the isotopic enrichment of 13C-threonine in an extract of rat colon mucosa demonstrated a precision of 0.5 per thousand, or 0.001 atom %.

A mechanistic investigation of the enhanced cleavage at histidine in the gas-phase dissociation of protonated peptides.

Enhanced gas-phase cleavage of peptides adjacent to histidine was investigated. The peptides examined were angiotensins III (RVYIHPF) and IV (VYIHPF) as well as synthetic peptide analogues with altered key residues ((R)VYI-X-Z-F; X = F or H and Z = A, P, or Sar) or a fixed charge M3P(+)CH(2)C(O)-VYIHPF. While all singly protonated peptide ions containing both histidine and arginine fragment nonselectively, the doubly protonated peptide ions with arginine and histidine, and the singly protonated peptides containing histidine but not arginine, cleave in a selective manner. In particular, dominant complementary b+/y+ product ions resulting from cleavage between the HP amide bond are observed. For the fixed-charge derivative, selective cleavage occurs only if a proton is added to produce a doubly charged precursor. The results are consistent with involvement of a protonated histidine in the selective cleavage. The ratio of b+/y+ is determined by the identity of the residue C-terminal to histidine and by the ability of protonated histidine to transfer a proton to the C-terminal leaving fragment. This was probed further by systematically changing the residue C-terminal to histidine and by alkylating histidine. The results indicate that while b+/y+ complementary ion pairs dominate in doubly protonated RVYIHPF, b5(2+) and b6(2+) product ions dominate the spectra of doubly protonated RVYIHAF. Also, dominant b5(2+) product ions are observed when the histidine side chain is alkylated (H) in doubly protonated RVYIHPF. Based on all of the results, a selective fragmentation mechanism for enhanced cleavage at histidine involving an atypical b ion structure is proposed.

Primary and secondary locations of charge sites in angiotensin II (M + 2H)2+ ions formed by electrospray ionization.

High-energy tandem mass spectrometry and molecular dynamics calculations are used to determine the locations of charge in metastably decomposing (M + 2H)2+ ions of human angiotensin II. Charge-separation reactions provide critical information regarding charge sites in multiple charged ions. The most probable kinetic energy released (Tm.p.) from these decompositions are obtained using kinetic energy release distributions (KERDs) in conjunction with MS/MS (MS2), MS/MS/MS (MS3), and MS/MS/MS/MS (MS4) experiments. The most abundant singly and doubly charged product ions arise from precursor ion structures in which one proton is located on the arginine (Arg) side chain and the other proton is located on a distal peptide backbone carbonyl oxygen. The MS3 KERD experiments show unequivocally that neither the N-terminal amine nor the aspartic acid (Asp) side chain are sites of protonation. In the gas phase, protonation of the less basic peptide backbone instead of the more proximal and basic histidine (His) side chain is favored as a result of reduced coulomb repulsion between the two charge sites. The singly and doubly charged product ions of lesser abundance arise from precursor ion structures in which one proton is located on the Arg side chain and the other on the His side chain. This is demonstrated in the MS3 and MS4 mass-analyzed ion kinetic energy spectrometry experiments. Interestingly, (b7" + OH)2+ product ions, like the (M + 2H)2+ ions of angiotensin II, are observed to have at least two different decomposing structures in which charge sites have a primary and secondary location.

Combined NMR and molecular modeling study of an iduronic acid-containing trisaccharide related to antithrombotic heparin fragments.

An iduronic acid-containing trisaccharide, methyl-O-(4-O-methyl-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1-->4)-O- (2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-->4)-O-2,6-di-O-sulfo-alpha-D-glucopyranoside, related to antithrombotic heparin fragments has been subjected to a combined NMR and molecular modeling investigation. The conformational behavior of the two constituting disaccharide segments was investigated using a systematic grid search approach with the MM3 force field along with the proper parameters for the sulfate ester group. The exploration of the potential energy surfaces of the trisaccharide was performed through the use of the CICADA methods interfaced with the MM3 force field. In all cases, the 2-O-sulfo-alpha-L-iduronate moiety was given the three favored ring conformations (1)C4, (4)C1, and (2)S0. Conformations were clustered into families, four of which are likely to exhibit significant occupancy in solution. The different low-energy conformational families display different orientations at the glycosidic linkages and/or different ring shapes for the iduronate ring. The (2)S0 conformation is the major one for the 2-O-sulfo-alpha-L-iduronate but is still in equilibrium with the (1)C4 ring shape. The occurrence of such a conformational equilibrium in solution was probed via high-resolution NMR spectroscopy through measurements of coupling constants and NOE build-up. These results are in keeping with the observation that 2-O-sulfated pentasaccharides display a similar affinity for antithrombin III as their 2-N-sulfated counterparts.

Determination of endogenous peptides with in vitro ACE inhibitory activity in normotensive human plasma by the fluorometric HPLC method.

An in vitro degradation test of angiotensin (ANG) II or III in normotensive supine human plasma from 9 healthy male subjects confirmed the production of smaller ANG metabolites with angiotensin I-converting enzyme inhibitory activity. These metabolites were identified as ANG (3-8), ANG (5-8), and ANG (3-4), whose respective peptide concentrations were determined by our proposed naphthalene-2,3-dialdehyde (NDA)-HPLC method to be 64 +/- 9, 39 +/- 5, 176 +/- 22, and 197 +/- 35 fmol/ml of plasma.

Conformational analysis of neuropeptide Y-[18-36] analogs in hydrophobic environments.

The interactive and conformational behavior of a series of neuropeptide Y-[18-36] (NPY-[18-36]) analogs in hydrophobic environments have been investigated using reversed-phase high-performance liquid chromatography (RP-HPLC) and circular dichroism (CD) spectroscopy. The peptides studied comprised a series of 16 analogs of NPY-[18-36], each containing a single D-amino acid substitution. The influence of these single L-->D substitutions on the alpha-helical conformation of the NPY-[18-36] analogs in different solvent environments was determined by CD spectroscopy. Retention parameters related to the hydrophobic contact area and the affinity of interaction were determined with an n-octadecyl (C18) adsorbent. Structural transitions for all peptides were manifested as significant changes in the hydrophobic binding domain and surface affinity between 4 degrees C and 37 degrees C. The results indicated that the central region of NPY-[18-36] (residues 23-33) is important for maintenance of the alpha-helical conformation. Moreover, L-->D amino acid residue substitutions within the N- and C-terminal regions, as well as Asn29 and Leu30, do not appear to affect the secondary structure of the peptide. These studies demonstrate that RP-HPLC provides a powerful adjunct for investigations into the induction of stabilized secondary structure in peptides upon their interaction with hydrophobic surfaces.

Effect of the position of a basic amino acid on C-terminal rearrangement of protonated peptides upon collision-induced dissociation.

Internal rearrangement involving the loss of the C-terminal amino acid residue upon collision-induced dissociation (CID) or metastable decomposition was studied for protonated peptides. To investigate the structural characteristics of peptides responsible for this rearrangement, a series of synthetic peptides were prepared and subjected to B/E-linked scan or tandem mass spectrometric analyses using a four-sector instrument. The results showed that the position of a basic amino acid in the peptide sequence and its basicity have a significant influence on the rearrangement. Arginine (Arg) located at the n-1 position facilitates the rearrangement with about twice as many rearrangement ions as is observed for the other Arg-containing peptides. This can be attributed to the interaction of a positively charged guanidino group of Arg with its own carbonyl group via a salt bridge which is tightly formed in vacuo between a guanidino and carboxylate groups, the mechanism of which is analogous to that previously proposed for the formation of similar rearrangement ions observed in the spectra of metal-cationized peptides. This association would result in the facile attack of the C-terminal hydroxyl group on the penultimate carbonyl group, leading to the rearrangement. In addition, the rearrangement ion was observed both in metastable decomposition and high-energy CID spectra obtained by B/E-linked scan analyses without or with gas, respectively, but in a sequence dependent manner.

Sequence-specific fragmentation generated by matrix-assisted laser desorption/ionization in a quadrupole ion trap/reflectron time-of-flight device.

Sequence-specific fragmentation for structural analysis has been generated by activation of ions via matrix-assisted laser desorption/ionization (MALDI) in an ion trap storage/reflectron time-of-flight device (IT/reTOF). The key to this work is that ion decay can be induced by MALDI activation but requires an extended period of time to occur in large peptides. This extended decay period, which may be in excess of 20 ms, is provided in these experiments using the long storage times of the ion trap device. The ions are stored until decay is complete and then rapidly pulsed into a reflectron TOF for analysis. Since the ions decay within the trap, they ultimately appear as stable ions in the reTOF rather than as metastable decay peaks. The ion fragmentation was found to depend strongly on laser power and the rf voltage placed on the ring electrode of the trap. The fragmentation obtained was shown to be similar to but different from that observed in FAB-low-energy CAD. In particular, enhanced fragmentation was obtained in the lower mass range and large species could be more easily fragmented than with FAB-low-energy CAD. The types of fragmentation for several target peptides are discussed.

Angiotensin-I- and -III-mediated cardiovascular responses in the freshwater North American eel, Anguilla rostrata: effect of Phe8 deletion.

Cardiovascular responses to synthetic eel [Asn1, Val5, Gly9]-ANG-I, ANG-III (2-8), and ANG-I (1-7) were measured in conscious and resting freshwater North American eels. Indwelling Doppler flow probes were placed on the ventral and dorsal aortas, a pressure catheter in the lienomesenteric artery, and a peptide delivery catheter in the caudal vein. Twenty-five and 150 ng.kg-1 ANG-III increased baseline cardiac output (CO) (15.23 +/- 0.31 ml.min-1.kg-1; n = 5) by 23 and 47%, respectively, by increasing stroke volume (SV) but not heart rate (HR). ANG-I (150 ng.kg-1) also elevated CO (62%) by increasing both SV (44%) and HR (14%). Estimated branchial shunting (BS) was increased by 150 ng.kg-1 ANG-I and -III suggesting that more blood perfused the arteriovenous pathway in the gills. Dorsal aortic blood pressure (PDA) (3.08 +/- 0.12 kPa) was elevated by 150 ng.kg-1 ANG-I (67%) and -III (52%). Pressor responses temporally preceded the blood flow increases and there was a significant increase in systemic vascular resistance (RSYS) at the peak pressor responses. At the peak flow responses, increased CO was solely responsible for the increase in PDA; RSYS had returned to baseline values. Pressor responses to ANG-III decayed more rapidly (18.6 min) compared with those of ANG-I and -II (36 min). ANG-I (1-7) had no measurable effect on cardiovascular function indicating that the carboxyl-terminal 8-phenylalanine is an absolute requirement for the hormonal activity of angiotensin in fishes.