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1. The effects of balloon injury on the reactivity of ipsilateral and contralateral carotid arteries were compared to those observed in arteries from intact animals (control arteries). 2. Carotid arteries were obtained from Wistar rats 2, 4, 7, 15, 30 or 45 days after injury and mounted in an isolated organ bath. Reactivity to angiotensin II (Ang II), phenylephrine (Phe) and bradykinin (BK) was studied. Curves were constructed in the absence or presence of endothelium or after incubation with 10 microm indomethacin, 500 microm valeryl salicylate or 0.1 microm celecoxib. 3. Phe, Ang II and BK maximum effects (Emax) were decreased in ipsilateral arteries when compared to control arteries. No differences were observed among pD2 or Hill coefficient. 4. Emax to Phe (4 and 7 days) and to Ang II (15 and 30 days) increased in the contralateral artery. In addition, Phe or Ang II reactivity was not significantly different in aorta rings from control or carotid-injured animals. 5. The increased responsiveness of contralateral artery was not due to changes in carotid blood flow or resting membrane potential. The endothelium-dependent inhibitory component is not present in the contraction of contralateral arteries and it is not related to superoxide anion production. 6. Indomethacin decreased contralateral artery responsiveness to Phe and Ang II. Valeryl salicylate reduced the Ang II response in contralateral and control arteries. Celecoxib decreased the Phe Emax of contralateral artery. 7. In conclusion, decreased endothelium-derived factors and increased prostanoids appear to be responsible for the increased reactivity of contralateral arteries after injury.

The involvement of AT1 and AT2 receptor subtypes in the response of the isolated rat uterus to angiotensin II (AngII) was studied throughout the estrous cycle. The AngII potency varied during the different estrous cycle phases, as indicated by significantly different pD2 values. No significant differences were observed in AngII metabolism among different estrous phases. Morphological analysis indicated that external and internal myometrium layers were thicker during estrus. In addition, the highest resting membrane potential was also observed during this phase, when compared with the proestrus and diestrus phases. The AngII-induced uterine contractions were blocked by losartan. Different losartan pD2 values were observed. PD123319 had no effect on the contractile response to AngII. The results also indicate that estrous cycle-dependent changes in AngII potency are correlated with uterine morphological and/or membrane potential changes., (Copyright 2002 S. Karger AG, Basel)

Ca2+ pathways activated by angiotensin II and carbachol were evaluated in the circular muscle of the guinea-pig ileum by recording mechanical and electrical activities. Transient contractions induced by angiotensin II were greatly reduced by Ca2+ removal from the medium whereas carbachol-induced responses were not significantly altered. Nifedipine had no effect on the responses to both agonists. A high concentration of tetrodotoxin (0.1 microM) inhibited angiotensin II-induced contractile responses without affecting the depolarization, whereas 1 mM Ni2+ inhibited the mechanical and electrical effects. Neither tetrodotoxin nor Ni2+ affected carbachol-induced effects. These results indicate that angiotensin II-induced phasic contractions depend on extracellular Ca2+ but not on voltage-dependent L-type Ca2+ channels. It is suggested that angiotensin II activates Ni2+-sensitive Na+ and non-specific cationic channels, whereas the responses to carbachol are dependent on receptor-activated Ca2+ release. Furthermore the different response of the longitudinal and circular muscles to the inhibitory effects of tetrodotoxin and Ni2+ on the angiotensin II- and carbachol-induced contractions indicates that these agonists exert their own myogenic effects on each layer and are able to trigger different Ca2+ mobilization pathways.

Angiotensin II (AII) tachyphylaxis occurs in the guinea pig ileum, but is not induced by analogs lacking the N-terminal amino group or the Arg2 guanidino group. Both AII and Lys2AII increased cell inositol trisphoshate content in cultured intestinal smooth muscle cells. Protein kinase C inhibition by staurosporine or downregulation by prolonged incubation with phorbol reverted tachyphylaxis of the inositol trisphoshate response, but not that of the Na+ uptake response, indicating that the uncoupling of the phosphoinositide signal system by protein kinase C did not involve all processes distal to receptor activation. Tachyphylaxis of the Na+ uptake response was prevented when receptor internalization was blocked by reduction of the temperature (4 degrees C) or by pretreatment of the cells with phenylarsine oxide. Acid washings, which prevented tachyphylaxis of the 24Na+ influx response, also prevented tachyphylaxis of the contractile response of the guinea pig ileum to AII. Although these findings suggest that sequestration or internalization of the AII receptor might be involved in AII tachyphylaxis, binding of [125I]AII and of [125I]Lys2AII to the cells was equally unaffected by repeated administrations of the peptides. The results suggest that conformational change of the AII-receptor complex within the plasma membrane, but not internalization, is the most important factor responsible for tachyphylaxis.

Simultaneous recordings of the tension and intracellular Ca2+ concentration of guinea-pig ileum longitudinal smooth muscle strips, as well as 24Na+ and 45Ca2+ influx measurements in cultured myocytes from the same tissue, were used to investigate the mechanisms underlying angiotensin-induced desensitization and tachyphylaxis. Angiotensin II and [2-lysine]-angiotensin II (Lys2All), incubated for prolonged periods (10 min) with muscle strips, induced fading of the contractile response (desensitization) and reappearance of the intracellular Ca2+ concentration oscillations, which were inhibited during the initial increase in cytosolic Ca2+. The desensitization was paralleled, in cultured myocytes, by inhibition of the 45Ca2+ but not of the 24Na+ influxes which were initially stimulated by the peptides. On the other hand, repeated administrations of angiotensin II (but not of Lys2All) caused gradual reduction of the contractile response and of the 24Na+ influx stimulation evoked by the agonist (tachyphylaxis). Treatment with phorbol 12-13 dibutyrate accelerated the desensitization induced by both angiotensin II and by Lys2All and aggravated the tachyphylaxis to angiotensin II. The results support the hypothesis that activation of protein kinase C is responsible for the desensitization and that tachyphylaxis is due to the slow dissociation of angiotensin II from a postulated Na(+)-dependent regulatory site on the receptor.

The effects of angiotensin II (ANG) on Na+ and Ca++ fluxes in cultured intestinal smooth muscle cells from the guinea pig ileum were studied and correlated with the contraction and desensitization observed in whole muscles. The effects of ANG were compared with those of acetylcholine (ACh), an agonist that acts at muscarinic receptors in the intestinal smooth muscle and which does not induce desensitization. Both ANG and ACh stimulated 24Na+ influx upon addition to the cells, and this stimulation persisted for at least 30 min. Both agonists also stimulated 45Ca++ uptake but ANG's effect was transient, whereas that of ACh was persistent. Short-term (30 min) treatment with PMA (phorbol-12-myristate-13-acetate) caused a fade of the tonic response of the whole muscle to ANG, and also blocked this hormone's stimulating effect on 45Ca++, but not on 24Na+ influx. Long-term (7 hr) treatment with PMA, which suppresses protein kinase C activity, restored ANG's ability to stimulate 45Ca++ influx. The stimulating effects of ACh on 24Na+ and 45Ca++ influxes were not affected by short- or long-term treatment of the cells with PMA. Our results suggest that ANG desensitization involves protein kinase C inhibition of a step in the stimulus-response chain that is subsequent to phospholipase C-activation.

The belief that the smooth muscle contracting activity of angiotensin II (angiotensin) in the guinea pig ileum is partly mediated by release of acetylcholine was reexamined, with the following results. 1. Atropine did not reduce the maximum contraction produced by angiotensin, although it caused a shift to the right of the log dose-response curve (dose ratio = 2.2). A similar shift was observed with histamine, bradykinin and BaCl2. 2. A moderate potentiation of angiotensin by eserine was also observed, which was similarly found for the other agonists. 3. A previous report that atropine blocks the fast (phasic) component of the isometric response of the ileum to angiotensin was not confirmed. The disappearance of the phasic component was found to be due to a tachyphylactic change in the response. 4. Depolarization by high doses of nicotine, and inhibition of acetylcholine synthesis by hemicholinium, did not affect the response to angiotensin. 5. Ilei in which the intramural ganglia had been destroyed by incubation at 4 degrees 48-56 h responed maximally to angiotensin, without loss of the phasic component of the response. It is concluded that the available evidence does not support the participation of a cholinergic mechanism in the effect of angiotensin upon the guinea pig ileum.

The ability of angiotensin II (AII) and of seven N-terminally modified peptide analogs to induce tachyphylaxis in isolated preparations of the rat uterus and the guinea-pig ileum was found to correlate well with the degree of amino group protonation. No correlation was found between tachyphylaxis and the affinity for the rat uterus receptors, as measured by the reciprocals of ED50 values. Dissociation between the tachyphylactic property and receptor affinity in the rat uterus was also evident by lowering the calcium concentration in the medium, which resulted in increased tachyphylaxis without significant changes in ED50 values. In the guinea-pig ileum, tachyphylaxis correlated with the reciprocal of ED50, but there was a better correlation with amino group protonation. [6-Pyrazolylalanine] AII was approximately as tachyphylactic as AII in both smooth muscles studied, indicating that protonation of the histidine side-chain may not be important for the manifestation of tachyphylaxis. The recovery of the guinea-pig ileum for AII tachyphylaxis was not affected by repeated treatments with BaCl2, but it was partially impaired by [Suc1]AII, des-Asp-AII, bradykinin and histamine.

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.

The effect of modifications of the N-terminal end of the angiotensin II (AII) molecule on its ability to induce tachyphylaxis in the isolated rabbit aorta was studied by analyzing the effects of several analogs of AII. No tachyphylaxis to AII was observed, but (1-sarcosine)-AII, (1-guanido-acetic)-AII and (1-glycine)-AII induced marked tachyphylaxis. (1-Betaine)-AII, (2-lysine)-AII, (2-ornithine)-AII and (1-sarcosine,2-lysine)-AII were unable to induce tachyphylaxis in the rabbit aorta. A positively charged N terminus and the guanidino group of the Arg2 side chain appear to be needed for the manifestation of the tachyphylactic property, while the Asp1 side chain is responsible for the absence of tachyphylaxis to AII. It is proposed that the analogs that are able to induce tachyphylaxis, after binding to the receptor to produce the agonistic response, induce a slow conversion of the hormone-receptor complex into a tachyphylactic state, and that this conversion is promoted by the interaction of the N terminus and the guanidino group of the analog with their complementary sites.

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. Prostaglandin E2 (prostaglandin) potentiated the responses of the guinea-pig ileum to angiotensin, bradykinin, histamine and acetylcholine without significant differences in the effects observed. 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. No cross-tachyphylaxis between angiotensin and adrenaline was observed in the rabbit mesenteric and coeliac arteries. 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. The results with the rabbit isolated arteries indicate that tachyphylaxis to angiotensin in these organs is not caused by prostaglandin release.

We have examined the responsiveness of the guinea-pig ileum and taenia coli to angiotensin (ANG) analogues that were previously shown to be either able ([1-sarcosine]-ANG, Sar1-ANG) or unable ([2-lysine]-ANG, Lys2-ANG) to induce tachyphylaxis in the ileum. The taenia coli, in which tachyphylaxis had not been previously shown to occur, was strongly tachyphylactic to Sar1-ANG, but not to Lys2-ANG. The contractile responses of the ileum, as well as the contractile and electrical (sucrose-gap) events in the taenia coli, in response to the two ANG analogues, were used to investigate the tachyphylactic phenomenon and the role of Na+ in the manifestation. Relaxation of the ileum and repolarizations of the taenia coli were faster after treatment with Lys2-ANG than after Sar1-ANG. In the tachyphylactic state, relaxation and repolarization after Sar1-ANG became as fast as after Lys2-ANG. In "low-Na+" (80 mmol/l) medium, as well as in ouabain-treated preparations, the responses of the ileum to ANG analogues were similar to those of tissues in the tachyphylactic state. Addition of Ca2+ to taenia coli preparations previously treated with the two ANG analogues in Ca2+-free medium, caused contractile and electrical responses only in the case of Sar1-ANG. It is proposed that ANG tachyphylaxis is due to changes at the level of the receptor causing increased Na+ permeability which leads to a decreased Na+ gradient across the cell membrane.

The development of tachyphylaxis to [1-sarcosine]-angiotensin II was studied in helical strips and everted rings of rabbit aorta. Strips, but not everted rings, developed marked (greater than 50%) tachyphylaxis to the peptide, when challenged repeatedly at 1-h intervals. Measurements of the membrane potentials showed no difference between the two preparations, but strips were more sensitive to KCl than everted rings. These results suggest that the strips are more depolarized than the everted rings due to lesions caused by the spiral cutting. This partial depolarization may underlie the tachyphylactic phenomenon.

Tachyphylaxis to [Ile5]angiotensin II (angiotensin) in the isolated guinea pig ileum was found to be more severe when the Ca2+ concentration or the temperature of the medium were lowered, or when glucose was absent. Incubation with indomethacin or prostaglandin E2 did not affect the onset of tachyphylaxis or recovery from the tachyphylactic state. The angiotensin dose-response curves of tachyphylactic organs were shifted to the right, and the maximum responses were depressed in proportion to the conditioning doses of the hormone. The recovery from tachyphylaxis followed zero-order kinetics and was not affected by Ca2+ concentration or pH. The temperature dependence of the rate of recovery yielded a value of 14.6 kcal/mol for the activation energy in the physiological temperature range. It is concluded that tachyphylaxis results from the tight binding of angiotensin to superficial calcium-binding sites in the smooth muscle cell membrane. Recovery from tachyphylaxis appears to involve displacement of angiotensin by calcium in a process that is dependent on active transport.

The structural requirements for the production of angiotensin tachyphylaxis in the guinea-pig ileum were studied by analyzing the tachyphylactic properties of the following synthetic analogues of angiotensin II (AII): [1-sarcosine]AII, [1-betaine]AII; [1-guanidinoacetic]AII; betainyl-AII; [2-lysine]AII; [2-ornithine]AII. In the non-atropinized ileum, no tachyphylaxis was observed with any of the following analogues: [2-lysine]AII, [2-ornithine]AII, [2-ornithine]AII, [1-betaine]AII and betainyl-AII. [1-Guanidinoacetic]AII induced tachyphylaxis, but to a smaller degree than AII, while [1-sarcosine]AII was significantly more tachyphylactic than AII. Similar results were obtained in the atropinized ileum, except that moderate tachyphylaxis was also observed with betainyl-AII and [1-betaine]AII. The analogues with lysine or ornithine residues in position 2 did not induce tachyphylaxis under any of the conditions studied. It is concluded that, besides the protonated N-terminal amino group, the guanidino group of the Arg2 side-chain is essential for the manifestation of angiotensin tachyphylaxis in the guinea-pig ileum.

Octanoyl[8-leucyl]angiotensin II (oct-LAT) was synthesized with the aim of obtaining a longer acting angiotensin (AT) inhibitor. The new compound, with a partition coefficient K=7.00 in n-BuOH-HOAc-H2O, was compared with [Leu8]angiotensin II (LAT, K=0.18) as an AT antagonist in two isolated smooth muscle preparations and in the rat blood pressure assay. The two compounds were equally potent in the rat uterus, but LAT was more effective in the guinea pig ileum and the in vivo assay. LAT's effect was longer lasting in the smooth muscles, but the duration of in vivo inhibition was the same for the two compounds. It is concluded that partitioning between external medium and biophase is not a limiting factor for antagonistic potency and permanence of effects of 8-substituted AT derivatives.

The development of tachyphylaxis to angiotensin II and to [1-sarcosine]angiotensin II ([Sar1]angiotensin II) was studied in rabbit aorta helical strips, normal rings and everted rings, with and without a functional endothelium. Normal rings developed tachyphylaxis to both angiotensin II and [Sar1]angiotensin II, helical strips only to [Sar1]angiotensin II and everted rings did not show tachyphylaxis. The presence or absence of a functional endothelium had no effect on the development of tachyphylaxis in any of the preparations. Measurement of the intraluminal pH of the normal rings with an antimony minielectrode showed that under steady state conditions there was a pH gradient between the intima side of the vessel wall and the outside bath medium. This could be responsible for the greater susceptibility of the normal rings to tachyphylaxis.

In order to evaluate the importance of some structural features of asparagyl(1)-Valyl(5)-angiotensin II (angiotensinamide) for its pharmacological actions, the relative potencies of angiotensinamide and five peptide analogues were studied on the blood pressure of the rat, the isolated rat uterus and the isolated guinea-pig ileum. All the modifications of the angiotensinamide structure that were studied led to a decrease of potency which, however, was not the same on all three preparations. The importance of the guanido group, the phenolic group and the length of the peptide chain for the pharmacological activities of these peptides is discussed.