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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.

Tachyphylaxis to both angiotensin II (ANG II) and Sar1-ANG II is observed in normal rabbit aorta rings, but helical strips show tachyphylaxis only to Sar1-ANG II, whereas everted rings are not tachyphylactic to either analogue. In normal rings, a good correlation was observed between intraluminal pH and degree of tachyphylaxis to both analogues, suggesting that rate-limiting access of the agonists to their site of action may enhance tachyphylaxis in this preparation. Membrane potential and intracellular Na+ activity measurements, as well as the relaxation by K+ of norepinephrine-contracted preparations in K(+)-free medium, indicated that helical strips are more depolarized than everted rings due to Na+ leakage into the smooth muscle cells. These results suggest that the differences in the degree of tachyphylaxis induced by angiotensin in different rabbit aorta preparations are due to a less accessible site of action in normal rings and to the higher intracellular Na+ and more depolarized state of helical strips relative to everted rings.

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.

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.

Angiotensin tachyphylaxis was studied in the isolated guinea pig ileum under isometric conditions. 2. In tachyphylactic organs, the initial phasic response to angiotensin was abolished, while the tonic component of the response became faster in its onset and in its reversal (relaxation). 3. The relaxation of non-tachyphylactic organs after angiotensin washout was significantly slower than with bradykinin, des-amino-angiotensin and hexanoyl-angiotensin. This difference did not occur in tachyphylactic organs. 4. There was no correlation between production of tachyphylaxis or rate of relaxation after washout and the partition coefficient of the four peptides in an organic:aqueous solvent system. 5. It is suggested that tachyphylaxis depends on a slowly reversible alteration of a calcium translocation step in the stimulus-response coupling.

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.

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.