Demonstration of different contractile mechanisms for angiotensin II and des-Asp 1-angiotensin II in rabbit aortic strips ( angiotensin III / [ Sarl ] angiotensin II / calcium ion / smooth muscle )

Evidence of selective inhibition, differences in dose-response relationships, and cross-tachyphylaxis studies suggest that separate receptors and/or mechanisms may be involved in responses to angiotensin (Ang), [Sar']Ang II, and Ang III (= des-Aspl-Ang II). The extracellular Ca2+ requirement for contractile responses induced by angiotensin peptides and norepinephrine was determined in rabbit aortic strips. Responses to K+ and [Sar'JAng II were attenuated markedly by treatment with SKF425A, verapamil, or Ca2+-free buffer. The response to Ang II was not impaired by verapamil, was blocked partially by SKF-525A, and was reduced markedly in Ca2+-free medium. Norepinephrineand Ang III-induced contractions were not dependent on extracellular Ca2+. K+, Ang II, and [Sar'JAng II required extracellular Ca2+ to induce contraction of the rabbit aorta. The data indicate that Ang III may have a mechanism of action that differs from that of [Sarl]Ang II and Ang II. Several lines of evidence indicate that effector organ responses to the octapeptides [1-sarcosinejangiotensin II ([Sar']Ang II) and angiotensin II (Ang II), and to the heptapeptide angiotensin III (Ang III, which is des-Aspl-angiotensin II) may be mediated by different receptors or mechanisms of action. While these structural analogs of angiotensin, which are full agonists, may produce parallel dose-response curves of comparable maximal response, exceptions to this behavior have been found in various tissues from several species. In the isolated rat uterus, Ang II and III displayed nonparallel dose-response curves (1, 2), and the maximal inotropic response to Ang III was lower than that to Ang II in both rabbit atria (3) and cat papillary muscle (4). These results could be interpreted to indicate that the heptapeptide might act preferentially on a population of receptors or by a mechanism of action different from Ang II. Other studies have indicated a greater angiotensin receptor affinity for heptapeptide than for octapeptide analogs. Angiotensininduced aldosterone biosynthesis in both rat and dog adrenal zona glomerulosa is blocked more easily by the competitive antagonist des-Asp'-[Ile8]Ang II than by octapeptide antagonists (5, 6). Similar results recently were reported by Taub et al. regarding the antagonism of angiotensin-mediated hemodynamic changes in the kidney (7); these potent inhibitory actions of the heptapeptide analog are in marked contrast to its poor antagonism manifest in other peripheral vascular beds. Many smooth muscles develop tachyphylaxis to Ang II. Two recent studies present evidence of the failure to produce cross-tachyphylaxis to Ang II and III and of difficulty in obtaining tachyphylaxis to Ang III in rat uterus and aortic strips. Indeed, Ang II tachyphylaxis in rat aorta could be reversed by treatment with indomethacin while that to Ang III was unaffected by such treatment (1, 2). In preparations that become tachyphylactic, responses to Ang II are blocked totally by lowering the extracellular Ca2+ concentration and by treatment with verapamil, SKF-525A, and tetracaine (8). This is in contrast to the rabbit aorta (no Ang II tachyphylaxis) for which intracellular Ca2+ seems paramount in the mediation of the response to Ang II. However, [Sarl]Ang II does produce tachyphylaxis in the rabbit aorta (2), and it seems reasonable to assume that the contractile response to this analog may be blocked by pretreatment with verapamil. The present study was undertaken to determine the contractile activity in rabbit aortae of [Sarl]Ang II, Ang II, and Ang III in Ca2+-free buffer and in the presence of agents that inhibit Ca2+ influx or release of loosely bound Ca2+. MATERIALS AND METHODS Aortic strips were obtained from male New Zealand white rabbits (2-3 kg). The rabbits were killed by cervical dislocation and the aorta was rapidly removed, cleaned, and placed in oxygenated physiological salt solution (PSS) of the following composition (millimolar): NaCI, 111; NaHCO3, 25; KCI, 5; NaH2PO4, 1; MgCI2, 0.5; glucose, 11; CaCl2, 1.4. Four helically cut rabbit aortic strips (0.25 X 2 cm) were prepared from one length of thoracic aorta according to the method of Furchgott and Bhadrakom (9). Each strip was mounted in a 10-ml organ bath containing physiological salt solution at 370 and gassed with 95% 02/5% CO2. The strip was placed under an initial 2 g of passive tension and allowed to equilibrate for about 2 hr. During this equilibration period the buffer was changed every 10 min. The final resting tension was adjusted to 1 g and the isometric contractile responses of the aortic strips to angiotensins were recorded using Grass force-displacement transducers (FT-03C) coupled to a Brush Mark 220 recorder or a Grass model 7 polygraph. Dose-response curves were determined from the data obtained for [Sarl]Ang II, Ang II, Ang III and norepinephrine. In studies that evaluated the role of extracellular Ca2+ in the generation of responses, verapamil or SKF-525A was administered 30 min prior to the agonists or at the peak of the maximal response to a given agonist. The concentrations of SKF-525A and verapamil used in these experiments inhibited the K+induced contractile response by 90%. Between exposures to an agonist, the preparation was washed repeatedly and allowed to return to basal resting tension. To substantiate data obtained with organic Ca2+ antagonists, tissues were incubated in Ca2+_free buffer for 5 min prior to exposure to agonists and maintained in Ca2+_free buffer while dose-response relationships were determined as in the control situation. Materials: [Sar']Ang II was generously supplied by Richard Freer, Medical College of Virginia, Richmond, VA. Ang II and Abbreviation: Ang, angiotensin. 5725 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5726 Medical Sciences: Ackerly et al.