Sarmesin is a partial agonist of angiotensin-II receptors in rabbit, but not in rat, aortic rings

Sarmesin, [Sar1, Tyr(Me)4]angiotensinII], has been reported to be a competitive angiotensin II (AII) receptor antagonist in rat smooth muscle preparations (Scanlon et al., (1984), Life Science 34, 317-321). In the present study, sarmesin displaced AII from its binding sites in rat aortic smooth muscle cells and in a rabbit aorta membrane preparation (IC50 5 and 6 nM resp.; Ki 4.1 and 5.3 resp.) In rabbit aortic rings, sarmesin (0.003-3 microM) produced concentration-dependent contractions (ED50 89 nM) and this effect was inhibited by saralasin. No contraction was observed in the rat aorta up to 100 microM. In rabbit aortic rings, sarmesin, at the same concentrations that produced contraction, inhibited contractions induced by AII in a competitive manner (pA2 7, 26). These results indicate that, in rabbit aortic rings sarmesin is a partial agonist of AII receptors.     

Angiotensin II binding sites in frog kidney and adrenal.

Angiotensin II (AII) binding sites were localized and quantified in kidney and adrenal of the frog Rana temporaria by quantitative in vitro autoradiography. AII binding was present in kidney glomeruli and in interrenal tissue of the outer zone of the adrenal gland. Saturation experiments showed that [125I]-[Val5]AII binds to a single class of binding sites with a dissociation constant (Kd) of 548 +/- 125 pM in glomeruli and 593 +/- 185 pM in interrenal tissue (n = 8). The corresponding maximal binding capacities (Bmax) were 2.48 +/- 0.71 and 3.05 +/- 1.02 fmol/mm2, respectively. AII binding was displaced by unlabeled angiotensin analogues in the rank order: [Sar1]AII greater than human AII greater than [125I]-[Val5]AII = [Val5]AII = human AIII much greater than human AI. The AII binding sites in glomeruli and interrenal tissue suggest an influence of AII on glomerular filtration rate and adrenal steroid secretion to take part in osmomineral regulation of the frog.     

Purification and identification of endogenous anti-opioid substances from bovine brain

Two fractions with high potency for reversing the inhibitory effect of Met-enkephalin on the electrically induced contractility of guinea-pig ileum have been purified from bovine brain extract. Unexpectedly, one isolated peptide was identified as [Val5]-angiotensin I and the other fraction was [Val5]-angiotensin II, as judged by chromatographic comparisons on HPLC and amino acid analysis. Since angiotensins did not affect opioid binding to brain membrane, we consider that angiotensins may act as physiological antagonists to the opioid system in the brain, as well as in the guinea-pig ileum.     

Evidence for oxytocin receptors in the urinary bladder of the rabbit

Experiments were performed on isolated detrusor smooth muscle from New Zealand White rabbits. Oxytocin was shown to exhibit high intrinsic contractile activity on isolated strips of detrusor muscle, where the maximum contractile amplitude was 12% greater than control responses to 1 microM carbachol. Repeated applications of 1 microM oxytocin were associated with tachyphylaxis representing a 49% decrease in the amplitude which became reproducible after several applications without further decay of contractile strength. Dose-response experiments indicated that threshold contractions to oxytocin occur at 3 nM and were maximum at 10 microM with mean effective concentration of 125 nM. The contractile responses to 1 microM oxytocin were not antagonized by phentolamine, atropine, methysergide, saralasin, or naloxone, but were partially inhibited by 1 microM of indomethacin. Ligand binding studies on partially purified membrane preparations from detrusor smooth muscle were performed over a range of 78 pM to 10 nM with 125I-labelled oxytocin. Scatchard analysis of specific bound receptors indicated a KD of 2.5 nM and Bmax of 187 fmol/mg protein and a second compartment that was unsaturable at the concentrations of ligand employed. Nonspecific binding ranged from 36 to 77% of the total binding.     

Des-aspartate-angiotensin I and angiotensin AT1 receptors in rat cardiac ventricles

The binding of 125I-[Sar1,Ile8]angiotensin II and 125I-angiotensin II to ventricular membranes of rat heart was studied. Displacement of bound 125I-[Sar1,Ile8]angiotensin II by its cold equivalents, angiotensin I, angiotensin II, angiotensin III, des-aspartate-angiotensin I, losartan, PD123319 and CGP42112B supports the presence of the AT1 and the near absence of the AT2 angiotensin receptor in adult rat ventricle. The presence of binding sites for des-aspartate-angiotensin I could account for its reported cardioprotective actions. Binding of 125I-angiotensin II but not that of 125I-[Sar1,Ile8]angiotensin II was partially displaced by GppNHp suggesting that a portion of the receptor population was in the active state with dissociated G-protein. Saturation experiments carried out in the absence and presence of 1 mM GppNHp showed similar magnitude of decrease in the number of receptors (Bmax from 26.2+/-1.3 to 15.7+/-1.1 fmol/mg protein) in [125I]-angiotensin II binding. However, the guanine nucleotide had no effect on the binding of 125I-[Sar1,Ile8]angiotensin II as has also been reported elsewhere, and may suggest that Sar1-Ile8-angiotensin II, being a partial agonist, binds to both the G-protein coupled and uncoupled states of the angiotensin receptors. The present study demonstrates that des-aspartate-angiotensin I binds to angiotensin receptors in the heart, and provides further evidence for its involvement in the pathophysiology of the organ.     

Potentiation of pressor response to angiotensin II at the preoptic area in spontaneously hypertensive rat

Cardiovascular responses to angiotensin II(AII) at the preoptic area (POA) were compared between normotensive Wistar Kyoto rat (WKY) and spontaneously hypertensive rat(SHR) by measuring blood pressure and heart rate under unrestrained, conscious state via a catheter implanted chronically into the abdominal aorta and by injection of drugs into POA through a chronic guide cannula. AII injected into POA at doses of 0.3 ng and 1 ng produced a dose-dependent pressor response, accompanied with a slight decrease of heart rate, in both WKY and SHR. However, in SHR, the pressor response to AII was more than 2 times greater than that in WKY and was quick in onset and lasted about 30 min. When AII in combination with [Sar1, Ile8]-angiotensin II (0.5 microgram), an AII receptor antagonist, were simultaneously administered to POA, the pressor response to AII was strongly inhibited in both WKY and SHR. The results suggest that the pressor response to AII due to its receptor stimulation at POA is markedly potentiated in SHR.     

Antagonists of angiotensin II containing N-methyl-L-alanine and DL-nipecotic acid in position 7.

[1-sarcosine, 7-N-methyl-L-alanine, 8-isoleucine]-Angiotensin II and [1-sarcosine, 7-DL-nipecotic acid, 8-isoleucine]-angiotensin II were synthesized by the solid-phase method and purified by cation-exchange chromatography and high-pressure liquid chromatography. In the isolated rat uterus these analogs and less than 0.1% of the myotropic activity of angiotensin II and inhibited angiotensin II with pA2 values of 8.2 and 7.8, respectively. In the rat pressor assay (vagotomized ganglion blocked rat) these analogs had 0.9 and 2.8%, respectively, of the pressor activity of angiotensin II. The results show that the proline residue in position 7 of [Sar1,Ile8]-angiotensin II may be replaced by other secondary amino acids without disrupting interactions at angiotensin II receptors.     

Agonist-induced down-regulation of the angiotensin II receptor in primary cultures of rat hepatocytes

The ability of angiotensin II to down-regulate its receptor was tested on rat hepatocytes in primary culture for 4 h. Angiotensin II treatment decreased [3H]angiotensin II specific binding in a concentration- and time-dependent manner. The effect was maximum with 1 microM angiotensin II and after 2 h. There was a decrease in the maximum number of binding sites (56% of control) with no significant effect on the apparent dissociation constant. The down-regulation was blocked by the angiotensin II antagonist [Val4,Ile7]angiotensin III and was not induced by other hormones (e.g. vasopressin, norepinephrine, or glucagon) or by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate or A23187 ionophore. The decrease in angiotensin II receptors resulted in correlated decreases in the potency of angiotensin II to activate phosphorylase or lower glucagon-induced cAMP accumulation. However, high concentrations of the agonist were still able to elicit maximal responses in both parameters. Down-regulation of the receptor was not dependent upon active Gi, since it was still observed after ADP-ribosylation and inactivation of Gi by pertussis toxin. The above results indicate that the down-regulation of the hepatic angiotensin II receptor induced by its agonist is homologous and does not involve Gi, Ca2+, or protein kinase C. The correlation of receptor loss with decreases in the potency of angiotensin to activate phosphorylase and inhibit glucagon-induced cAMP accumulation is consistent with the idea that a single receptor population regulates two different messengers, i.e. calcium and cAMP.     

Different pharmacological anatomy in the paraventricular hypothalamic nucleus, supraoptic nucleus, and suprachiasmatic nucleus of rats: quantitative autoradiography on angiotensin II receptor binding sites

Angiotensin II (AII) and vasopressin (VP) play important roles in cardiovascular function. Using 125I-[Sar1,Ile8]-angiotensin II (125I-SI-AII), a potent AII antagonist, AII receptor binding sites were autoradiographically localized in three VP-producing areas of the hypothalamus and compared in hypertensive and normotensive rats. Within three major VP-producing areas, AII receptor binding was highest in the paraventricular hypothalamic nucleus and lowest in the supraoptic nucleus, suggesting that a differential AII regulation of separate VP systems exists in the brainstem. No statistical difference in 125I-SI-AII receptor binding was found between WKY and SHR rats in each of the three major VP-producing nuclei studied. These results are consistent with a role of AII receptors in a subtle and complicated regulation of VP in cardiovascular function.     

Specific, high-affinity binding sites for angiotensin II on Mycoplasma hyorhinis.

Mycoplasmataceae are known to express various proteins that are similar to those present in mammals. We report a strain of Mycoplasma hyorhinis isolated from opossum kidney cells with specific, high-affinity binding sites for human angiotensin II (Kd = 5.1 +/- 1.9 nM). In contrast, two strains of M. hominis revealed no specific binding. These binding sites resembled mammalian angiotensin II receptors by their high affinity and by their sensitivity to dithiothreitol. However, they are different from mammalian angiotensin II receptors in that they bind angiotensin I with high affinity (Kd = 1.6 +/- 0.29 nM) but not angiotensin III (Kd approximately 330,000 nM). [125I]-angiotensin II binding was not inhibited by angiotensin receptor subtype antagonists DuP 753 and CGP 42112A but it was sensitive to bacitracin and aprotinin. Positions Asp1, Ile5, His6 and Pro7 were essential for binding to M. hyorhinis as deletion of these residues led to a more than 10,000-fold decrease in affinity.     

Activation of angiotensin II receptors in brain potentiates the stimulating effect of endogenous opioid neurons on central sympathetic outflow

Endogenous opioid peptides appear to have neurotransmitter or neuromodulator functions in brain mediating a wide variety of effects. We have reported that intracisternal administration of synthetic human beta-endorphin increases plasma concentration of catecholamines, apparently by acting at unknown brain sites to increase sympathetic outflow to the adrenal medulla and sympathetic nerves. In the present study we examined the possibility that angiotensin II, acting in brain, modulates endorphin-induced catecholamine secretion. Simultaneous intracisternal administration of angiotensin II 1.0 nmol together with synthetic human beta-endorphin 1.45 nmol potentiated the plasma epinephrine, norepinephrine and dopamine responses to intracisternal beta-endorphin. In contrast, simultaneous intracisternal administration of the angiotensin II antagonist, [Sar1, Val5, Ala8]-angiotensin II (saralasin), 1.1 nmol together with beta-endorphin, blunted the plasma epinephrine, norepinephrine and dopamine responses to beta-endorphin. These data are consistent with the hypothesis that activation of angiotensin II receptors in brain potentiates the endorphin-induced stimulation of central sympathetic outflow. It remains to be demonstrated whether angiotensin II acting in brain to modulate activity of opioid neurons is synthesized in brain or is derived peripherally.     

Isolation and purification of angiotensin II using affinity and high-pressure liquid chromatography

Peptides have been found in a variety of tissues including brain. To purify the peptide angiotensin II, a three-step method for the isolation and purification has been developed using extraction, affinity chromatography, and high-pressure liquid chromatography. Angiotensin II antiserum purified by affinity chromatography was covalently coupled to Affi-gel 10 (Affi-gel 10-AB). The efficiency and usefulness of this column for the purification of angiotensin II from biological sources were tested with 125I- and 3H-labeled (Ile5)-angiotensin II added to rat brains prior to extraction. After extraction, the recoveries for both peptides were 74 and 75%, respectively. Recovery after the purification on Affi-gel 10-AB was 84 and 82%. Thirty-two percent of the radioactivity was not retained and 50% of the radioactivity could be eluted with 0.1 M Na citrate buffer containing 1 M NaCl using a stepwise pH gradient. Characterization by HPLC of the unretained radioactivity from the Affi-gel 10-AB column showed one peak for [125I]angiotensin II, coeluting with the [125I]angiotensin II standard and two minor peaks. Only 30% of unretained [3H]angiotensin II could be identified as intact [3H]angiotensin II on HPLC. Both [125I]angiotensin II and [3H]angiotensin II elutable at pH 5.0 and 4.0 on Affi-gel 10-AB could be demonstrated as highly purified [125I]angiotensin II and [3H]angiotensin II on HPLC with a purity of more than 90%. On HPLC, the recovery was 81% for [125I]angiotensin II and 99% for [3H]angiotensin II. The recovery for the entire three-step procedure was about 60%. The loading capacity of the Affi-gel 10-AB column for (Ile5)-angiotensin II was 550 ng.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inability of [125I]Sar1, Ile8-Angiotensin II to Move Between the Blood and Cerebrospinal Fluid Compartments

The angiotensin II competitive antagonist [125I]-Sar1, Ile8-angiotensin II was not transported from the vascular space to the cerebroventricular space in either intact or nephrectomized rats. In addition [125I]Sar1, Ile8-angiotensin II lacked the capacity to move in the opposite direction over a 20-min collection period following cerebroventricular infusion. These data suggest that angiotensins lack the capacity to move freely between the blood and cerebrospinal fluid compartments and are consistent with the notion that blood-borne and cerebroventricular angiotensins access different receptor populations.     

Relative biological activities of Asn1-,Val5-angiotensin II, Ile5-angiotensin II and Sar1-angiotensin II in man

Biological activities of asn1-,val5-angiotensin II (Hypertensin, Ciba, Asn1-,Val5-ANG II), ile5-angiotensin II (human angiotensin II, Ile5-ANG II) and sar1-angiotensin II (Sar1-ANG II) were compared in man. In 7 normal men 5 pmol/kg X min each of Asn1-,Val5-ANG II, Ile5-ANG II and Sar1-ANG II was infused iv from 0900 h to 0930 h at 1-week intervals. Average increments of blood pressure at the end of the infusions were 11/12, 23/20 and 36/30 mmHg, respectively (significant differences among the 3: P less than 0.001), average decrements of plasma renin activity were 0.30, 0.32 and 0.27 ng/ml X H, respectively (no significant difference among the 3), average increments of plasma aldosterone were 1.1, 2.3 and 4.4 ng/100 ml, respectively (significant difference between the former 2: P les than 0.001, between the latter 2: P less than 0.02), and durations of blood pressure rise after the cessation of these infusions (T) were 2-5 (average 5) min, 10-25 (average 20) min and 35-60 (average 40) min, respectively (significant difference between the former 2:less than P 0.01, between the latter 2: P less than 0.001). From these results it is evident that the pressor and steroidogenic actions of Ile5-ANG II are significantly stronger than those of Asn1-,Val5-ANG II and that the duration of pressor action of the former is much longer than that of the latter. Therefore, when the activities of angiotensin II (ANG II) derivatives are compared with those of ANG II in man, Ile5-ANG II--natural human ANG II--should always be used instead of Asn1-,Val5-ANG II. The pressor and steroidogenic actions and T of Sar1-ANG II are significantly stronger or longer than those of Ile5-ANG II. The reason for this is thought to be that Sar1-ANG II is bound tightly to the vascular and adrenal ANG II receptors and is not readily metabolized.     

Molecular characterization of angiotensin II type II receptors in rat pheochromocytoma cells.

The binding sites and biochemical effects of angiotensin (A) II were investigated in rat pheochromocytoma (PC12W) cells. Sarcosine1, [125I]-tyrosine4, isoleucine8-AII ([125I]-SI-AII) bound to a saturable population of sites on membranes with an equilibrium dissociation constant (Kd) of 0.4 nM and a binding site maximum of 254 fmol/mg protein. Competitive displacement of [125I]-SI-AII by agonists and antagonists elucidated a rank order of potency of AIII greater than or equal to AII greater than PD 123177 greater than AI greater than [des-Phe]AII [AII(1-7)] much greater than DuP 753. The stable guanine nucleotide analog 5'-guanylyl imidodiphosphate did not alter the binding affinity or slope of the inhibition curves for AI, AII, AIII, or AII(1-7). Treatment of PC12W cells with AII or AIII did not affect the free intracellular calcium concentration, phosphoinositide metabolism, arachidonate release, cyclic GMP, or cyclic AMP concentrations. [125I]-AII binding sites remained on the cell surface and were not internalized after 2 h at 37 degrees C. Angiotensin II did not stimulate tyrosine, serine, or threonine phosphorylation. Northern analysis of PC12W mRNA with an AT1 receptor gene probe failed to produce an RNA:DNA hybrid at low stringency. These data indicate that PC12W cells express a homogeneous population of AT2 binding sites which differ significantly from AT1 receptors in signal transduction and molecular structure. AT2 sites may act via potentially novel, biochemical pathways or, alternatively, be vestigial receptors.     

Quantitative autoradiography of angiotensin II receptors in the SHR brain

Several lines of evidence indicate brain angiotensin II is associated with the elevation of blood pressure seen in the spontaneously hypertensive rat (SHR). These include an increased pressor response to intracerebroventricularly administered angiotensin II and a reduction of blood pressure in response to centrally administered angiotensin II receptor antagonists. Using quantitative receptor autoradiography, we have detected greater angiotensin II receptor binding in a number of discrete brain nuclei of the 6-week-old SHR when compared to age-matched Wistar-Kyoto controls. Tissue sections from various brain regions were labeled with [¹²⁵I]-angiotensin II according to a previously described method. Autoradiograms were generated by apposing the labeled tissue sections to LKB Ultrofilm along with brain paste standards which contained known amounts of [¹²⁵I]. Quantitation of the binding, utilizing computer-assisted microdensitometry, indicated greater [¹²⁵I]-angiotensin II binding in several brain areas implicated in cardiovascular control including the subfornical organ, nucleus of the solitary tract, dorsal motor nucleus of the vagus, locus coeruleus, supraoptic nucleus and the organum vasculosum of the lamina terminalis. Scatchard analysis of the binding in the nucleus of the solitary tract indicated an increased receptor number (B_max) was responsible for the change while binding in two forebrain structures, the subfornical organ and supraoptic nucleus, showed alterations in receptor number and affinity (K_d). Several other brain regions, unrelated to cardiovascular control, exhibited no change in [¹²⁵I]-angiotensin II binding. Since the increased receptor binding was present primarily in brain regions related to cardiovascular control, we conclude that an increased angiotensin II receptor affinity and density is indicated as a factor in the etiology of the high blood pressure seen in the SHR.     

Nociceptin receptor activation inhibits tachykinergic non adrenergic non cholinergic contraction of guinea pig isolated bronchus

We studied the action of nociceptin (NC) on the atropine-resistant contractions of the guinea pig isolated bronchus evoked by the electrical field stimulation (EFS), an effect that is mediated by the activation of excitatory non adrenergic-non cholinergic (eNANC) nerves and the subsequent release of tachykinins. The functional site by which NC acts in this preparation was investigated using few different NC receptor agonists and the newly discovered NC receptor antagonist, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 ([F/G]NC(1-13)NH2). NC inhibited in a concentration dependent manner (pEC50 7.14; Em - 87 +/- 3% of control values) EFS induced contractions. NC effect was mimicked by the NC analogues, NCNH2 and NC(1-13)NH2, but not by NC(1-9)NH2. NC (1 microM) did not affect the contractile effects of exogenously applied neurokinin A (1 microM). [F/G]NC(1-13)NH2 (10 microM) completely prevented the inhibition induced by NC (1 microM), whereas naloxone (1 microM) was found inactive. Both naloxone and ([F/G]NC(1-13)NH2 were per se inactive on basal resting tone as well as on the electrically induced contractions. The present findings show that NC inhibits the atropine-resistant EFS-induced contraction in the guinea pig bronchus by inhibiting eNANC nerves, and suggest the presence of NC receptors, distinct from opioid receptors, on the nerves of the guinea pig bronchus.     

Cerebroventricular and Intravascular Metabolism of [125I]Angiotensins in Rat

This study compared the metabolism of [125I]angiotensin II (AII), [125I]angiotensin III (AIII), and [125I]Sar1,Ile8-AII (SI-AII) in the vascular and cerebroventricular compartments. Using HPLC methods to monitor degradation the following t1/2 values were established in the vascular compartment: AII, 12.7 +/- 1.4 s; AIII, 16.3 +/- 0.7 s; and SI-AII, 100.7 +/- 7.3 s. HPLC analysis also revealed that [125I]AII is converted in an obligatory manner to [125I]AIII during its degradation sequence. Cerebrospinal fluid contained no degradative capacity for [125I]AII but exhibited a significant capacity to degrade [125I]AIII. A technique that combined the intra-cerebroventricular injection of [125I]angiotensins followed by focused microwave fixation to stop all peptidase activity was used to determine the half-life of [125I]angiotensins in the ventricular space. Results indicated very rapid metabolism of angiotensins with the following t1/2 values: AII, 23.0 s; and AIII, 7.7 s. This extremely rapid, differential, and sequential metabolism of AII and AIII in two relevant body fluid compartments underscores the need for caution when interpreting data derived from intravascular and intracerebroventricular application of angiotensins. In addition the faster metabolism of AIII than AII in the ventricular space indicates that the actual potency of AIII at central angiotensin receptors is being underestimated.     

Differential prejunctional effect of captopril and saralasin on neurogenic vasoconstriction in pithed normotensive rats

The present study describes a differential inhibitory effect of captopril and [Sar1 Ala8]angiotensin II (saralasin) on the neurogenic vasoconstriction in pithed normotensive rats. In pithed normotensive rats with intact kidneys captopril more profoundly inhibited the vasopressor response to spinal stimulation than observed for saralasin. Bilateral nephrectomy also diminished the hypertensive response to spinal stimulation. After bilateral nephrectomy, 1 h previously, captopril but not saralasin diminished the hypertensive response to spinal stimulation. After bilateral nephrectomy, 18-24 h previously, captopril did not produce an additional reduction of the vasopressor response to spinal stimulation. In contrast, saralasin significantly potentiated the neurogenic vasoconstriction. The results suggest that both captopril and saralasin diminish the hypertensive response to spinal stimulation by producing dilatation of vascular smooth muscle in pithed normotensive rats. Apart from this common mechanism, a differential effect of captopril and saralasin on the neurogenic vasoconstriction can be observed. In contrast to saralasin, captopril may depress the neurogenic vasoconstriction in pithed normotensive rats by blocking the sympathofacilitatory action induced by subpressor levels of angiotensin II (AII). In pithed normotensive rats, saralasin may mimic the sympathofacilitatory action of subpressor AII.     

Importance of the N-terminal domain of the type I angiotension II antagonist [Sar1,Ile8]ANG II for receptor blockade

Analogues of the Type I angiotensin (ANG) antagonist, [Sar1,Ile8]ANG II, in which the N-terminal dipeptide was modified were synthesized by the solid phase method and purified by reversed-phase HPLC. Antagonist potencies (pA2) of the peptides were determined on the rat isolated uterus using ANG II as the agonist. Substitution of the Arg residue occupying position 2 of [Sar1,Ile8]ANG II (pA2 8.1) by Gly, Ala, Nle, Phe, Pro or Sar reduced the antagonist potency to pA2 = 7.0, 6.8, 6.7, 6.8, 5.8 and 5.3, respectively. Deletion of the N-terminal Sar residue in these same peptides gave pA2 = 6.8, 5.7, 5.5, 5.9, 6.1 and 7.5, respectively. The characteristically long duration of action of [Sar1,Ile8] was absent for all of these analogues including (des1, Sar2, Ile8]ANG II. These findings demonstrate that the antagonist potencies of Type I angiotensin antagonists for smooth muscle receptors, and also the long duration of action, are dependent on the location of positive charges within the peptide and on the conformation of the molecule in determining favorable electrostatic interactions with the receptor. A model is proposed in which the two positively charged loci on the angiotensin molecule (N-terminus and Arg) interact with two corresponding anionic binding sites on the smooth muscle receptor. The possibility that the prolonged duration of action of [Sar1, Ile8]ANG II results from binding to a different site on the angiotensin receptor from that occupied by ANG II is discussed in relation to the present findings.