Determination of peptide contact points in the human angiotensin II type I receptor (AT1) with photosensitive analogs of angiotensin II

To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-L-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the [Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166-199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of [Sar1, Bpa8]AngII interacts with the second extracellular loop of the AT1 receptor.     

Comparative studies of angiotensin II and its analog [Sar1Ala8]angiotensin II on the density of cellular monolayer of cell cultures from green monkey kidney

The effects of the octapeptide angiotensin II (AT II) and its analog [Sar1Ala8]AT II on the cell density in cell culture from green monkey kidney (GMK) were studied. AT II and [Sar1Ala8]AT II provoked a decrease of the number of living cells depending on the concentration (0.001, 0.01 and 0.1 nM) and time of incubation (24, 48 and 72 hours), both peptides having a very similar activity. These data indicate that AT II and [Sar1Ala8]AT II may act on the same class of angiotensin receptors in GMK cells.     

Photoaffinity labeling of the angiotensin II receptor; pharmacology of the labeling peptides in the dark.

The biological activities of photoaffinity labeling analogs of angiotensin II (ATII) and their precursors were measured in rabbit aorta strips in the dark. Most of the analogs behave as reversible, specific agonists, one as a competitive inhibitor. The activities are discussed in line with the current view of structural requirements. The modifications consisted of substitutions on the aromatic nuclei of Tyr4 and Phe8 in [Sar1]ATII with (4'-NO2) Phe, (4'-NH2)Phe, (4'N3)Phe, (4'-N2 +)Phe, and (4'-NH2-3', 5'-I2)Phe. It is shown that the affinity of the ATII analogs modified in position 4 depends on the electronegativity and not on space-filling properties of the aromatic residue; rising electronegativity lowers the affinity, i.e. [sar1, (4'-NO2)Phe4]ATII has no more measurable activity. Substituting the aromatic side chain in position 8 of [Sar1]ATII gives well-binding analogs with intrinsic activities from 0 to 100% and activity seems to depend only on stereochemical requirements. Agonists and partial agonists bear rather small groups like -NH2, -N3, -NO2, and -N2 +. The only antagonist [Sar1, (4'-NH2-3',5'-I2)Phe8]ATII resembles the antagonist E1Sar1, Leu8]ATII in competitivity and binding.     

Angiotensin II receptors in the human placenta are type AT1

Membrane angiotensin II receptors were measured in human placenta by means of 125I [Sar1 Ile8] All (angiotensin II antagonist) and characterized by using 2 other antagonists of angiotensin II: Dup 753 and CGP 42112A. These are specific and selective ligands which enable identification of AT1 and AT2 receptor subtypes respectively. The [Sar1 Ile8] All affinity is similar (Kd approximately 1 nmol.l-1) in the 3 different placental structures examined. However, the Bmax of villous tissues is approximately 9 times higher than that observed in chorionic plate but remains near that found in basal plate. In the central area of the placenta, mean values of 125I [Sar1 Ile8] All binding observed at a single concentration of 0.15 nmol.l-1 are 242 +/- 31 fmol/mg proteins in basal plate, 300 +/- 35 in villous tissues and 36 +/- 8 in chorionic plate. The umbilical vein and arteries respectively have 8.8 +/- 4.8 and 4.0 +/- 1.7 fmol/mg protein. The subtype analysis shows that only AT1 receptor is present in placental tissues. The Bmax values as well as those obtained by the relative measurement performed at a fixed 125I [Sar1 Ile8] All concentration of 0.15 nmol.l-1 indicate that the highest concentrations of angiotensin II receptors are found in placental villous tissues.     

Analysis of the adrenal angiotensin II receptor with the photoaffinity labeling method

The angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, AT) receptor of bovine adrenocortex has been investigated with photosensitive analogues of AT. In a first series of experiments, we have shown that isolated cortical cells secrete aldosterone in a permanent and specific manner if they have been photolyzed in the presence of the photolabel [Sar1,(4'-N3)Phe8]AT. This permanent stimulation is in contrast to the smooth muscle assays where under similar conditions a permanent and specific block was always observed. It is assumed that the irreversible occupation of the AT receptor produces this effect. In a second type of experiment, we have shown that the AT binding site on adrenocortical membranes can be specifically and irreversibly occupied under similar conditions and that this occupation can be prevented in a competitive manner by the presence of nonphotosensitive hormone. Using a radioactive label, [Sar1,(3'-125I)Tyr4,(4'-N3)Phe8]AT, we have identified the AT receptor as a 300-kDa protein by means of gel filtration under nonreducing and nondenaturating conditions. Under reducing and denaturing conditions, a subunit of 60 kDs was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The AT receptor is proposed to be a 300-kDa protein with one binding subunit of 60 kDa.     

Reduction of superior mesenteric hemodynamic responsiveness to [Sar1, Thr8]-angiotensin II and bradykinin, but not sodium nitroprusside, in the presence of homocysteine infusion

Hyperhomocysteinemia (HHcy) has been shown to be an independent risk factor for cardiovascular diseases, superior mesenteric thrombosis and inflammatory bowel disease. Superior mesenteric artery (SMA) supplies the intestine and reduced SMA blood flow results in intestinal ischemia. Although in vitro studies have shown that endothelium-dependent vasorelaxation of SMA is reduced in the presence of homocysteine incubation, it is not confirmed with in vivo studies. In this work, we evaluated responsiveness of SMA to endothelium-dependent or -independent vasodilators and a vasoconstrictor in the absence and presence of acute HHcy in vivo to clarify effect of HHcy on superior mesenteric vascular function. Sodium nitroprusside (SNP), bradykinin (BK), and [Sar1,Thr8]angiotensin II ([Sar1,Thr8]-ANG II) were intravenously administrated in sequence in male Sprague-Dawley rats with or without D,L-homocysteine infusion (6 mg/kg/min) through femoral vein. Agonists-induced changes in carotid artery blood pressure, superior mesenteric blood flow and vascular resistance were measured in the present study. We found that acute HHcy infusion had little effects on SNP-induced hemodynamic changes; however, BK-induced changes in blood pressure, blood flow and vascular resistance were significantly reduced in the presence of HHcy infusion. Additionally, HHcy also markedly decreased [Sar1,Thr8]-ANG II-induced superior mesenteric hemodynamic changes. These results demonstrated that responsiveness of SMA to vasoconstrictor, endothelium-dependent, but not endothelium-independent vasodilator, was inhibited in the presence of Hcy infusion. This HHcy-associated vascular hyporesponsiveness to vasoconstrictors and endothelium-dependent vasodilators may partially contribute to circulatory dysfunctions.     

Design,synthesis and biological evaluation of cyclic angiotensin II analogues with 3,5 side-chain bridges. Role of C-terminal aromatic residue and ring cluster for activity and implications in the drug design of AT1 non-peptide antagonists

The novel amide linked angiotensin II (ANG II) cyclic analogues: gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Ile(8)] ANG II (I) and gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Phe(8)] ANG II (II) have been designed, synthesized and bioassayed in anesthetized rabbits in order to unravel structural ring cluster characteristics important for receptor activation. Analogue I with Ile at position 8 was an inhibitor of Angiotensin II while analogue II with Phe at position 8 was found to be an agonist. Similar results were reported for cyclic compounds that have reversed the linking between positions 3 and 5. The overall results show that positions 3 and 5 do not govern the biological activity of the synthetic analogues. It also appears that the aromatic ring cluster (Tyr-His-Phe) in agonist peptides is an essential stereo-electronic feature for Angiotensin II to exert its biological activity. A non-peptide mimetic of ANG II, 1-[2'-[(N-benzyl)tetrazol-5-yl]biphenyl-4-yl]methyl]-2-hydroxymethylbenzimidazole (BZI8) has been designed and synthesized. This molecule is more rigid and much less active than AT(1) non-peptide mimetic losartan probably because it lacks to mimic the orientation of tetrazole and the pharmacophore segments of butyl chain and imidazole ring.     

Synthesis and study of a cyclic angiotensin II antagonist analogue reveals the role of pi*--pi* interactions in the C-terminal aromatic residue for agonist activity and its structure resemblance with AT(1) non-peptide antagonists.

The novel amide linked Angiotensin II (ANG II) cyclic analogue cyclo(3, 5) -[Sar(1)-Lys(3)-Glu(5)-Ile(8)] ANG II (18) has been designed, synthesized and bioassayed in anesthetized rabbits. The constrained cyclic analogue with a lactam amide bridge linking a Lys-Glu pair at positions 3 and 5 and possessing Ile at position 8, was synthesized by solution procedure using the maximum protection strategy. This analogue was found to be inhibitor of Angiotensin II. NMR spectroscopy coupled with computational analysis showed clustering between the side chains of the key aminoacids Tyr(4)-His(6)-Ile(8) similar to that observed with ANG II. The obtained data show that only pi*--pi* interactions observed in ANG II or its superagonist Sar(1) [ANG II] are missing. Therefore, it can be concluded that these interactions are essential for agonist activity. Conformational analysis comparisons between AT(1) antagonists losartan, eprosartan and irbesartan with C-terminal segment of cyclic compound 18 revealed structural similarities.     

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.     

Brain angiotensin: critical role in the ongoing regulation of body fluid homeostasis and cardiovascular function

The existence of an endogenous brain-angiotensin system and its association with cardiovascular and body water regulation has been recognized for over a decade. Nevertheless, the importance of the brain-angiotensin system to the instantaneous regulation of these processes has not been firmly established. A 5-minute intracerebroventricular (ICV) infusion of the angiotensin antagonist, [Sar1,Thr8]-AII, effectively lowered the blood pressure in normotensive rats. Additionally, application of the selective aminopeptidase inhibitor, bestatin, given alone, resulted in a dramatic increase in blood pressure and a robust drinking response. Both effects were 100% blockable by [Sar1,Thr8]-AII pretreatment. Predictably, an aminopeptidase inhibitor, bestatin, greatly elongated the half-lives of AII and AIII in the cerebroventricles. Since neither of these treatments included the introduction of exogenous angiotensins, we have concluded that perturbations of the endogenous brain-angiotensin system are effective at rapidly influencing both cardiovascular and body fluid homeostasis, thus highlighting the paramount role played by brain angiotensin in their ongoing regulation.     

Nephrectomy and a newly identified binding site for angiotensin II in the rat adrenal cortex.

Angiotensin II (Ang II) binding sites in adrenal glands of nephrectomized rats were investigated by in vitro autoradiography using 125I-[Sar1,Ile8]Ang II as ligands. Ang II binding site was increased to 161% in the cortex and decreased to 67% in the medulla 48 h after nephrectomy. In the medulla, the AT2 antagonist (PD123177, 5 microM) inhibited specific binding by 90% whereas the AT1 antagonist (DuP753, 5 microM) inhibited by only 10%. In contrast, in the cortex, neither DuP753 (5 microM) nor PD123177 (5 microM) substantially inhibited the binding. Binding in the presence of either the AT1 or AT2 antagonist was abolished by the simultaneous presence of both antagonists. These results suggest the presence of a new Ang II binding site with unique pharmacological properties and differing from currently known subtypes of Ang II receptors, in the adrenal cortex after nephrectomy.     

Distribution of angiotensin II receptors in the brain of nonhuman primates

Angiotensin II binding sites were demonstrated at discrete nuclei in the brain of three nonhuman primate species by autoradiography, using the agonist ligand, [Sar1]AII. Although there were some differences in location of the binding sites, all three species exhibited a characteristic pattern of distribution in areas related to water intake, vasopressin secretion, and blood pressure regulation through modulation of sympathetic activity. Studies in the cynomolgus monkey with the antagonist ligand, [Sar1,Ile8]AII, which localizes in pathways as well as nuclei, revealed novel regions of binding including the habenular-interpeduncular pathway, ventral bundle, and XII nerve, in addition to the X nerve. These data indicated that AII, as in other species, has a role in the central homeostatic control mechanisms in the primate.     

125I[Sar(1)Ile(8)] angiotensin II has a different affinity for AT(1) and AT(2) receptor subtypes in ovine tissues

Iodinated angiotensin II (Ang II) and its analogues are often assumed to have equal affinities for AT(1) and AT(2) receptor subtypes. However, using saturation and competition binding assays in several tissues from pregnant, nonpregnant, and fetal sheep, we found the affinity of 125I[Sar(1)Ile(8)] Ang II for Ang II receptors was different (P<0.05) between tissue types. The dissociation constants (Kd) and half maximal displacements of [Sar(1)Ile(8)] Ang II (Sar IC(50)) were directly related (P<0.05) to proportions of AT(1) receptors, and inversely related (P<0.05) to proportions of AT(2) receptors in tissues from all groups combined, in tissues from individual groups (pregnant, nonpregnant or fetal), and in some individual tissues (uterine arteries and aortae). This suggests that 125I[Sar(1)Ile(8)] Ang II has a different affinity for AT(1) and AT(2) receptors in ovine tissues. The Kds of 125I[Sar(1)Ile(8)] Ang II for "pure" populations of AT(1) and AT(2) receptors were 1.2 and 0.3 nM, respectively, i.e. affinity was four-fold higher for AT(2) receptors. We corrected the measured proportions of the receptor subtypes using their fractional occupancies. In tissues which contained at least 10% of each receptor subtype, the corrected proportions were significantly altered (P<0.05), even in some tissues, to the extent of being reversed.     

Identification of angiotensin II receptor and determination of its subtypes in rabbit and guinea pig fetal tissue]

Membrane angiotensin II receptors were measured in trophoblastic tissues using a 2-step procedure. The first step consisted of the relative measurement performed at a fixed 125I[Sar1 Ile8]AII concentration of 0.15 nM in order to determine which tissues had a sufficient number of binding sites for studying the competition curves. The second consisted of determining the maximal binding (Bmax) and the dissociation constant (Kd) for [Sar1 Ile8] AII and the receptor subtypes in these tissues. The relative binding measurement revealed a significant number of occupied sites in rabbit fetal placenta and chorion (159 +/- 17 and 51 +/- 10 fmol/mg proteins) and in guinea pig chorion (132 +/- 12). The mean values of the other trophoblastic tissues were 3-10-fold lower in the 2 species. The competition curves obtained from tissues with high angiotensin II binding receptors showed the predominance of the AT2 subtype in rabbit fetal placenta (AT1/AT2 = 25/75) and of the AT1 receptor in guinea pig chorion (97/3) and in rabbit chorion (90/10). The [SAR1 Ile8] AII affinity (Kd) obtained from Scatchard plot analysis was 1.2 +/- 0.2 nM (n = 5) in fetal placenta and 1.2 (n = 1) in rabbit chorion and 0.5 +/- 0.1 (n = 3) in guinea pig chorion. In these tissues, the respective Bmax values were 1,281 +/- 115 (n = 5), 263 (n = 1) and 1,188 +/- 134 fmol/mg proteins (n = 3). These findings indicate that rabbit fetal placenta and chorion and guinea pig chorion are the most important sites of action for the renin-angiotensin system present in trophoblastic tissues.     

Role of N-glycosylation in the expression and functional properties of human AT1 receptor.

The role of N-glycosylation in the pharmacological properties and cell surface expression of AT1 receptor was evaluated. Using site-directed mutagenesis, we substituted both separately and simultaneously the asparagine residues in all three putative N-linked glycosylation consensus sequences (N-X-S/T) of AT1 receptor (positions 4, 176, and 188) with aspartic acid. Expression of these mutant receptors in COS-7 cells followed by photolabeling with [125I]-[p-benzoyl-Phe8]AngII and SDS-PAGE revealed ligand-receptor complexes of four different molecular sizes, indicating that the three N-glycosylation sites are actually occupied by oligosaccharides. Binding studies showed that the affinity of each mutant receptor for [Sar1,Ile8]Ang II was not significantly different from that of wild-type AT1 receptor. Moreover, the functional properties of all mutant receptors were unaffected as evaluated by inositol phosphate production. However, the expression levels of the aglycosylated mutant were 5-fold lower than that of the wild-type AT1 receptor. Use of green fluorescent protein-AT1 receptor fusion proteins in studying the cellular location of the aglycosylated mutant demonstrated that it was distributed at a much higher density to the ER-Golgi complex than to the plasma membrane in HEK 293 cells. Together, these results suggest an important role of N-glycosylation in the proper trafficking of AT1 receptor to the plasma membrane.     

Insertion of an electronegative sulfur atom in the side chain of position 5 of angiotensin II: changes in the tachyphylactic properties of the peptide.

Angiotensin II (AII) analogs bearing n-Leu, Met or S-substituted groups for cysteine at position 5 were studied regarding their agonistic and tachyphylactic properties. It was shown that these analogs lowered the relative affinity towards the AT1 receptor as determined by contractile responses, which could be due to the removal of the beta-branching residue at position 5. Insertion of a sulfur atom in a different position away from the attached backbone carbon atom presented no significant difference in EC50 values for these analogs. Interestingly, the S-bearing analogs at position 5 were full agonists but the tachyphylactic property was lost, in contrast to [n-Leu5]AII, which still induced reduction of the contractile responses. Nevertheless after replacing the Asp with Sar in position 1 (Sar1) tachyphylaxis was again established. It is concluded that the insertion of Met or an S-substituted cysteine into the side chain at position 5 of AII may promote interactions with its receptor due to the slight electronegative character of the sulfur atom and changes in the restricted conformational freedom of the Ile5 residue in the AII molecule. This was overcome by Sar1, probably through interactions due to its fully protonated N-terminal amino group and favoring the conformation responsible for the tachyphylaxis phenomenon.     

Agonistic activities of isoleucine8-angiotensin II in man

In order to clarify the importance of C-terminal phenylalanine in angiotensin II (ANG II) molecule, agonistic activities of a C-terminal substituted peptide, isoleucine8-angiotensin II (Ile8-ANG II), were studied in comparison with those of sarcosine1-, isoleucine8-angiotensin II (Sar1-, Ile8-ANG II) and isoleucine5-angiotensin II (Ile5-ANG II) in 5 normal men. When infused iv at a rate of 600 pmol/kg X min for 30 min, Ile8-ANG II and Sar1-, Ile8-ANG II raised the blood pressure to the same extent (15/15 mmHg on the average), while the average blood pressure increase was 21/21 mmHg after an iv infusion of Ile5-ANG II at a rate of 5 pmol/kg X min for 30 min. Duration of the pressor action after the cessation of each infusion was 50-90, 90-120 and 10-25 min, respectively. In each case plasma renin activity (PRA) decreased and plasma aldosterone (PA) increased. When infused iv at a rate of 10 pmol/kg X min (maximum non-pressor dose) for 120 min, both Ile8-ANG II and Sar1-, Ile8-ANG II lowered PRA and increased PA gradually, but 100 mg oral captopril given immediately before these infusions caused no significant increase in PRA or no significant decrease in PA but again a decrease in PRA and an increase in PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective down-regulation of AT2 receptors in uterine arteries from pregnant ewes given 24-h intravenous infusions of angiotensin II

Previously, we showed that uterine arteries from late gestation pregnant ewes infused intravenously with angiotensin II (Ang II) for 24 h, displayed heightened responsiveness to Ang II in vitro. Furthermore, we found that a small population of ewes with a "preeclampsia-like" disorder also displayed this. Therefore, we have investigated the density and affinity of Ang II receptor subtypes in the uterine arteries from these groups. Ang II receptor binding was measured using 125I [Sar1Ile8] Ang II. Proportions of AT1 and AT2 receptors were determined by inhibiting 125I [Sar1Ile8] Ang II with losartan (AT1 antagonist) or PD 123319 (AT2 antagonist). Uterine arteries from 24-h Ang II-infused ewes had a lower proportion of AT2 receptors (56.2+/-2.3%) than control (saline-infused) ewes (84.1+/-1.0%; P<0.05). The density of AT2 receptors was reduced (P<0.05) while the density of AT1 receptors was not different. Thus, 24-h infusions of Ang II selectively down-regulated AT2 receptors in the uterine artery, resulting in heightened Ang II reactivity. By contrast, the binding properties of Ang II receptor subtypes in uterine arteries from ewes with the "preeclampsia-like" disorder were not different from control ewes.     

Short term memory in the calcium messenger system. Evidence for a sustained activation of protein kinase C in adrenal glomerulosa cells.

If adrenal glomerulosa cells are treated with angiotensin II for a period of 20-30 min, their subsequent response to either a rechallenge with the same concentration of angiotensin II or treatment with BAY K 8644, a calcium channel agonist, differs from the responses of control cells. Perifusion of control cells with 10 nM-angiotensin II leads to an increase in aldosterone secretory rate from 44 +/- 7 to 166 +/- 9 pg/min per 10(6) cells, but perifusion of cells pretreated for a 20 min period with angiotensin II leads to an increase in secretory rate from 51 +/- 9 to 209 +/- 18 pg/min per 10(6) cells. Likewise, treatment of control cells with 10 nM-BAY K 8644 leads to no significant increase in aldosterone secretory rate, but treatment of previously exposed cells to angiotensin II leads to an increase in rate from 51 +/- 9 to 130 +/- 11 pg/min per 10(6) cells. This memory effect is time-dependent in two ways: cells must be exposed to angiotensin II for 20 min or more before it is apparent; the longer the time between removal of angiotensin II and the rechallenge, the less effect these agents have on aldosterone secretory rate. When cells are exposed to angiotensin II for 20 min and then treated with [Sar1,Ala8]angiotensin II, a competitive antagonist of angiotensin II action, the aldosterone secretory rate falls to basal with a half time of 5-7 min. If BAY K 8644 is added simultaneously with [Sar1,Ala8]angiotensin II, the secretory rate falls with a halftime of 35-60 min. BAY K 8644 increases Ca2+ influx rate to the same extent in the presence or absence of [Sar1,Ala8]angiotensin II, and does not alter the effect of either angiotensin II or [Sar1,Ala8]angiotensin II on the production of inositol tris-, bis-, or mono-phosphate. In cells treated with 10 nM-angiotensin II for either 20, 30 or 45 min, the extent of phosphorylation of four cellular proteins is increased. If cells treated for 20 min with angiotensin II are then treated with [Sar1,Ala8]angiotensin II, and examined 15 min later (35 min), there is no longer an increase in the extent of phosphorylation of any of the four proteins. If such cells are then treated with 10 nM-BAY K 8644 and re-examined 5 min later (40 min), all four patients show an increase in the extent of phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of angiotensin-(1-7) from angiotensin II by the venom of Conus geographus

The binding of [3H]angiotensin II to AT(1) receptors on Chinese Hamster Ovary cells expressing the human AT(1) receptor (CHO-AT(1) cells) is potently inhibited by venoms of the marine snails Conus geographus and C. betulinus. On the other hand, the binding of the nonpeptide AT(1) receptor-selective antagonist [3H]candesartan is not affected but competition binding curves of angiotensin II and the peptide antagonist [Sar(1),Ile(8)]angiotensin II (sarile) are shifted to the right. These effects resulted from the breakdown of angiotensin II into smaller fragments that do not bind to the AT(1) receptor. In this context, angiotensin-(1-7) is the most prominent fragment and angiotensin-(1-4) and angiotensin-(1-5) are also formed but to a lesser extent. The molecular weight of the involved peptidases exceeds 50 kDa, as determined by gel chromatography and ultrafitration.