1 In the present study, the contractile effects of angiotensin III (AIII) and angiotensin IV (AIV) compared with those of angiotensin II (AII) were determined in rat aortic ring preparations. 2 All three peptides caused concentration-dependent contractions with similar maximal responses. AIII proved approximately 4 times less potent than AII, whereas AIV was about 1000 times less active than AIL.3 The selective AT,-receptor antagonist, losartan (10-300 nM) caused parallel rightward shifts of the concentration-response curves (CRC) for all three peptides. The Schild plot slopes for the effect of losartan on AIII curves were significantly lower than unity (P<0.05). The selective AT2-receptor antagonist, PD123177 did not influence the CRCs for AII and AIV. However, the AIII curves were moderately shifted leftward in the presence of PD123177 (0.1 and 1 JiM).4 Destruction of the endothelium or incubation with the NO-synthesis inhibitor N0-monomethyl-Larginine acetate (L-NMMA) (0.1 mM) significantly enhanced the contractile responses to all three peptides. 5Tachyphylaxis was investigated by constructing a second CRC for all three peptides, after an interval of 1 h. The presence of endothelium significantly enhanced the development of tachyphylaxis to all three peptides. However, in endothelium-denuded preparations, the E,,,,,, value of the second curve elicted by AII was about 50%, compared with the first one, whereas for AIII and AIV E,,,, values were as high as 90% and 100%, respectively. 6 Our results indicate that both AIII and AIV are less potent but similarly efficacious vasoconstrictor agents compared with AII. Their contractile effects are also mediated by AT,-receptors and probably modulated by endothelium. Tachyphylaxis induced by AIII and AIV proved weaker than that for AII. Tachyphylaxis appears to be enhanced by the presence of an intact endothelium.

1 [3H]-clonidine binding was investigated in membranes isolated from the ventral medulla oblongata of the rabbit where clonidine produced a hypotensive effect which was not mediated by adrenoceptors.[3H]-clonidine specific binding, as defined by the difference between the binding of [3H]-clonidine in the presence and in the absence of 10 M cirazoline, occurred at two sites: a high affinity site with a KD = 2.9 ± 0.7 nM and a Bma, of 40 ± 8 fmol mg-' protein and a low affinity site with a KD = 18.2 ± 0.4 nM and a B,_ of 66 + 14 fmol mg-' protein.2 The high affinity sites being catecholamine-sensitive were identified as a2-adrenoceptors. The low affinity binding of [3H]-clonidine was insensitive to catecholamines, as well as to other M2-adrenoceptor specific probes, and could be inhibited with high affinity only by compounds which lowered blood pressure when directly injected in the nucleus reticularis lateralis of the ventral brainstem, or by antagonists.3 It was concluded that in the ventral medulla of the rabbit, [3H]-clonidine labelled M2-adrenoceptors and imidazoline receptors (IRs). Only the latter were related to the hypotensive effects of.clonidine and rilmenidine directly injected into the rostroventrolateral medulla oblongata (RVLM) of the rabbit. The methodological problems regarding the study of IRs with [3H]-clonidine are discussed.

The influence of vascular endothelium on angiotensin II-induced contraction and the underlying mechanisms in the rabbit renal artery were investigated. In endothelium-intact preparations, angiotensin II (3-100 nM) caused a concentration-dependent increase in tension by maximally (Emax) 0.74 +/- 0.05 g. Removal of the endothelium significantly enhanced the angiotensin II-induced contractions (Emax: 3.91 +/- 0.19 g). Indomethacin (10 microM) did not influence the angiotensin II-induced contractions. Methylene blue (10 microM) and NG-methyl-l-arginine (L-NMMA, 5 microM) significantly enhanced angiotensin II-induced contractions by 418 +/- 29% and 200 +/- 14%, respectively, in endothelium intact preparations, but not in those devoid of endothelium. L-arginine (1 mM), but not D-arginine, reversed the L-NMMA-induced enhancement of the angiotensin II-induced contraction. The present results suggest that angiotensin II-induced contractions in rabbit renal artery are largely subject to the influence of the endothelium. The endothelium-derived relaxant factor (EDRF), rather than cyclo-oxygenase products, appears to be involved in mediating the inhibitory effects of the endothelium. Nitric oxide (NO) derived from endothelium may play a major role in inhibiting angiotensin II-induced contractions in this preparation.

1 The direct positive chronotropic effects of angiotensin II (All) and its degradation products angiotensin III (AIII) and angiotensin IV (AIV) were established in pithed rats and in rat spontaneously beating right atria. 2 In pithed rats, All, AIII and AIV caused dose-dependent tachycardia with similar maximal responses (110 beats min'-). The /3-adrenoceptor antagonist propranolol (3.37 x 10-6 mol kg-') but not the aIadrenoceptor antagonist prazosin (2.38 x 10-7 mol kg-') significantly reduced these effects (P<0.05; n = 7-8), but 20-25% of the responses could not be blocked by propranolol. 3 In isolated atria, All, AIII and AIV caused concentration-dependent increases in beating rate with similar maximal responses to All and AIII (34.3 + 0.4 and 34.7 + 0.4 beats min'-; n = 9-10), and a lower maximal response to AIV (26.8 +0.6 beats min'-; P<0.05; n = 8). AIII was about 9 times less potent than All, whereas AIV proved approximately 3800 times less potent than All. Neither propranolol (1 yM) nor prazosin (1 gM) could influence the effects of the angiotensin peptides. 4 In isolated atria, the selective AT,-receptor antagonist, losartan (10, 100 and 300 nM) caused parallel rightward shifts of the concentration-response curves for All and AIII, whereas the selective AT2-receptor antagonist PD123177 (1 gM) did not influence the effects of All and AIII. The aminopeptidase-A and -M inhibitor amastatin (10 tM), significantly steepened the slope of the AIII curves and increased the potency of AIII about 6 fold. Amastatin did not influence the responses to All. 5 Our results indicate that both in vivo and in vitro, exogenous All and AIII induced a direct dosedependent chronotropic effect, which is independent of the adrenergic system. This chronotropic effect is mediated by AT,-subtype receptors.

The cardiovascular effects of angiotensin II were examined in aortic blood pressure-controlled and -uncontrolled pithed rats. Angiotensin II induced a dose-dependent increase in diastolic blood pressure, left ventricular pressure (LVP), dP/dt (the first derivative of LVP) and heart rate in pithed rats. The maximal responses for these parameters were similar to those to noradrenaline, except for the rise in diastolic blood pressure, where noradrenaline caused a greater increase than angiotensin II. After treatment with propranolol, the positive chronotropic effect of angiotensin II was abolished. Angiotensin II produced a dose-dependent increase in diastolic blood pressure, which was similar to that of vasopressin, and an increase in dP/dtmax, which proved much greater than that of vasopressin. When aortic blood pressure was controlled and the beta-receptors were blocked by propranolol, angiotensin II caused a dose-dependent increase in dP/dtmax without affecting the left ventricular enddiastolic pressure. The same results were obtained after both beta- and alpha-adrenoceptors were blocked by propranolol and phentolamine. Losartan but not PD123177 caused parallel rightward shifts of the dose-response curve of angiotensin II for dP/dtmax in the aortic blood pressure controlled pithed rat without altering the maximal response. It is concluded that in the pithed rat angiotensin II produced an increase in myocardial contractile force which is not mediated by beta- or alpha-adrenoceptors. The inotropic effect appears to be mediated by angiotensin receptors, of the AT1-subtype.