A.C.M. Paiva

Synthesis and properties of spin-labeled angiotensin derivatives

Abstract We have synthesized three derivatives of the peptide hormone angiotensin, containing as their N-terminal residue the spin-labeled amino acid 2,2,6,6-tetramethylpiperidine-N-oxide-4-amino-4-carboxylicacid. (TOAC). The angiotensin analogs displayed considerable biological activity, indicating that the spin label is not too great a perturbation for the hormone-receptor interaction. Studies of the effect of pH upon the electron paramagnetic resonance (EPR) spectra of the spin-labeled angiotensins indicated that deprotonation of the terminal amino group leads to changes in spectral parameters similar to those displayed by model compounds (TOAC and TOAC-glycine). In view of the slow exchange between the two forms at pH values where both the protonated and unprotonated forms coexist in considerable amounts, computer simulations demonstrate that the EPR spectra are superpositions of the spectra for each form. The EPR spectra of the spin-labeled hormone derivatives were shown to be indicative of a pH-dependent conformationai change, corroborating previous conclusions drawn from other studies. This study demonstrates the usefulness of spin-labeled analogs for the investigation of conformational properties of small peptides.

EFFECT OF INDOMETHACIN AND PROSTAGLANDIN ON THE SMOOTH MUSCLE CONTRACTING ACTIVITY OF ANGIOTENSIN AND OTHER AGONISTS

1 Indomethacin had an equal inhibitory effect on the response of the guinea‐pig isolated ileum to angiotensin II (angiotensin), bradykinin, histamine and acetylcholine. This effect did not seem to result from inhibition of prostaglandin synthesis, as it did not depend on the time of treatment with indomethacin. 2 Prostaglandin E2 (prostaglandin) potentiated the responses of the guinea‐pig ileum to angiotensin, bradykinin, histamine and acetylcholine without significant differences in the effects observed. 3 In the rabbit isolated mesenteric and coeliac arteries, indomethacin had an equal potentiating effect on the responses to angiotensin and to adrenaline. In these organs pre‐incubation with indomethacin was necessary for the effect to be observed, and this effect lasted for 2 h or more after that drug was removed from the medium. 4 No cross‐tachyphylaxis between angiotensin and adrenaline was observed in the rabbit mesenteric and coeliac arteries. 5 It is concluded that the effects of indomethacin and prostaglandin on the response of the guinea‐pig ileum to the four agonists result from an action on the smooth muscle contractile mechanism per se rather than from an inhibitory action on the release of endogenous prostaglandin produced by the four agonists. 6 The results with the rabbit isolated arteries indicate that tachyphylaxis to angiotensin in these organs is not caused by prostaglandin release.

Role of Ca+‐dependent K‐channels in the membrane potential and contractility of aorta from spontaneously hypertensive rats

1 Contractile responses to KCl and membrane potentials were determined in aortic rings from spontaneously hypertensive rats (SHR), normotensive Wistar rats (NWR) and Wistar Kyoto rats (WKY) both in the absence and in the presence of the Ca2+‐dependent K‐channel blockers, apamin and tetraethylammonium (TEA). 2 Compared to NWR, aortic rings from WKY and SHR were less reactive and their Ca2+ uptake after stimulation with K+ was decreased. 3 Smooth muscle cell membrane potentials were higher in aortae from SHR and WKY than in NWR aortae, whereas SHR had higher K+ and lower Na+ intracellular activities than WKY and NWR, suggesting overactivity of the Na+/K+ pump in the hypertensive animals. 4 Treatment with apamin caused depolarization of WKY and SHR aortae, and increased their contractile responses to the same level as those of the NWR. Treatment with TEA also caused depolarization of aortae from WKY and SHR, but in the SHR the depolarization induced by TEA was smaller than that produced by apamin and the contractile responses to KCl did not reach the level of those of aortae from NWR. 5 It is concluded that overactivity of Ca2+‐dependent K‐channels in aortae of WKY and SHR contributes to their higher membrane potentials and lower responsiveness to vasoconstrictor stimuli. In SHR, an overactive Na+/K+ pump is also present, and the contribution of apamin‐sensitive Ca2+‐dependent K‐channels to the membrane potential and reactivity appears to be more relevant than that of TEA‐sensitive channels.

Ionization constants and thermodynamic parameters of histidine and derivatives

Abstract The pKa values for the proton dissociation of carboxyl, imidazolium, and ammonium groups for histidine and ten of its derivatives were determined electrometrically at seven temperatures in the range 10–40°C. The ΔH and ΔS values were estimated from the temperature dependence of the dissociation constants of histidine and its derivatives. These results and the pKa values compared in terms of inductive effect suggest an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring. The charge and the solvation effects of the neighboring groups are the main factors that determine the imidazole group pKa in histidine and its studied derivatives. The Nτ-H tautomer is favored over the Nπ-H by 1.6 kcal/mol, indicating that the inductive substituent effect at position 4 of the imidazole ring is the major component in determining this tautomeric preference.

Sodium-dependence of the non-specific desensitization of the guinea-pig ileum induced by acetylcholine and histamine

1 The isometric maximal responses of the guinea‐pig ileum to acetylcholine and to histamine (but not those to prostaglandin E2 and to high K+) exhibited a secondary transient increase in tonus during the tonic component of the contraction. 2 After desensitizing treatment with acetylcholine or histamine, the isometric responses to either agonist showed decreased phasic and enhanced tonic components, whereas both components of the response to prostaglandin E2 were markedly depressed. 3 During the desensitizing treatment the degree of desensitization went through a maximum that coincided with the occurrence of the secondary tonic increment. 4 In low‐Na+ medium, or in ouabain‐treated tissues, the responses to the three agonists were similar to the respective responses in the desensitized state. 5 It is concluded that the non‐specific desensitization is due to changes in Na+ translocation and that the increased tonic component of the isometric response is due to a reduced Na+ gradient across the cell membrane and consequent increase in Ca2+ loading.

The importance of the N-terminal end of angiotensin II for its pressor and oxytocic activities☆

Abstract The importance of the N-terminal end of angiotensin II for its biological activity has been investigated by a comparative study of the pressor and oxytocic potencies of Val 5 angiotensin II and the following peptide analogues: (1) Asp(NH 2 ). Arg. Val. Tyr. Val. His. Pro. Phe; (2) Gly. Arg. Val. Tyr. Val. His. Pro. Phe; (3) Arg. Val. Tyr. Val. His. Pro. Phe; (4) Val. Tyr. Val. His. Pro. Phe. It was found that: (1) the absence of the N-terminal aspartyl residue of angiotensin II resulted in a parallel reduction of the pressor and oxytocic activities to 26 and 23 per cent, respectively; (2) the removal of the N-terminal aspartyl-arginyl portion of angiotensin II abolished the two activities; (3) the blocking of the free γ-carboxyl group of the aspartyl residue by an amide bond caused a greater reduction of oxytocic (73 per cent) than pressor activity (47 per cent).

Irreversible Inhibition of Angiotensin II Activity on the Rat Uterus by an Alkylating Angiotensin Derivative

An alkylating analogue of angiotensin II (AII) containing a nitrogen mustard group (p-[N, N-bis(2-chloroethyl) aminojphenylbutyryl at the N-terminus ([Chi1]-All) was shown to be an irreversible specific inhibitor of the action of AII on the isolated rat uterus. The inhibition was observed when the Ca++ concentration in the medium was 0.18 mM, and was more intense in calcium-free medium. It was not observed in the presence of 1 mM Ca++. It is concluded that a site near (but not on) the AII receptor is alkylated by [Chi1 ]-AII, and the properties of this site suggest that it is the same one involved in the phenomenon of tachyphylaxis.

Reactivity with p-nitrophenyl acetate and interaction between the amino and imidazole groups of histidine and related compounds

Abstract The study of the reaction of p-nitrophenyl acetate (PNPA) with histidine and certain derivatives showed that the species in which the amino group is unprotonated (R(NH2)Im) react with second-order rate constants ( k 2 am ) that are higher than predicted by a Bronsted relation for a series of neutral amino acids. The reason for this behavior was investigated through an analysis of the kinetics of the reaction of PNPA with these compounds in order to assess the reactivities of the amino and imidazole groups in the two species R(NH 3 + ) Im and R(NH 2 ) Im . The rate constant for the reaction with the imidazole group ( k 2 im ) of Nπ-methyl histidine agrees with the value predicted by a Bronsted relation obtained from a series of model imidazole compounds. Nτ-Methyl histidine, however, is unreactive, indicating that Nτ is the reactive nitrogen in the imidazole ring of histidine. The k 2 im values found for histidine, histidine methyl ester, and Nα-dimethyl histidine are lower than predicted by the Bronsted relation. This behavior was found to be due to low reactivity of the R(NH 3 + ) Im species, in contrast with the normal reactivity of R(NH 2 ) Im . The evidence presented suggests that the lower reactivity of R(NH 3 + ) Im is due to an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring, which displaces the tautomeric equilibrium toward the unreactive Nτ-H form. The higher reactivity of the imidazole group in the species R(NH2)Im, relative to that in R(NH 3 + ) Im , is responsible for the observed high k 2 am values for histidine, for histidine methyl ester, for Nτ-methyl histidine, and for Nα-dimethyl histidine, in contrast with the normal k 2 am value found for Nτ-methyl histidine. The conclusions from this study of histidine and its derivatives support the proposal of an interaction between the protonated N-terminal amino group and the imidazole ring of His6 in the octapeptide hormone angiotensin.