Angiotensin induces the urinary peristaltic machinery during the perinatal period.

The embryonic development of mammalian kidneys is completed during the perinatal period with a dramatic increase in urine production, as the burden of eliminating nitrogenous metabolic waste shifts from the placenta to the kidney. This urine is normally removed by peristaltic contraction of the renal pelvis, a smooth muscle structure unique to placental mammals. Mutant mice completely lacking angiotensin type 1 receptor genes do not develop a renal pelvis, resulting in the buildup of urine and progressive kidney damage. In mutants the ureteral smooth muscle layer is hypoplastic and lacks peristaltic movements. We show that angiotensin can induce the ureteral smooth muscles in organ cultures of wild-type, but not mutant, ureteral tissues and that, in wild-type mice, expression of both renal angiotensin and the receptor are transiently upregulated at the renal outlet at birth. These results reveal a new role for angiotensin in the unique cellular adaptations of the mammalian kidney to the physiological stresses of postnatal life.

Purification of the hepatic vasopressin receptor using a novel affinity column.

A vasopressin receptor was purified, using a novel affinity column, from rat liver plasma membranes treated with guanosine 5'-(3-O-thio)triphosphate and solubilized with 0.8% cholate. Incubation of the membranes with the GTP analogue resulted in a dissociation of the receptor-guanine nucleotide regulatory protein complex. This manipulation, although resulting in a low-affinity state of the receptor, facilitated purification. The solubilized receptor was assayed using a new reconstitution procedure in which the soluble extracts were inserted into lipid vesicles composed of phosphatidylcholine and phosphatidylinositol. The receptor was purified by sequential chromatography on Q-Sepharose and hydroxyapatite. The use of a novel affinity column, a V1-vasopressin antagonist-agarose, resulted in a near-homogeneous preparation of a protein which exhibited an Mr = 58,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of purified receptor, as well as crude membrane preparations cross-linked to [125I]arginine vasopressin, also revealed a protein band with an approximate Mr = 58,000. These findings indicate that V1-antagonist affinity chromatography should be useful for purifying adequate amounts of the receptor for studies of structure and function.

Agonist-induced phosphorylation of the endogenous AT1 angiotensin receptor in bovine adrenal glomerulosa cells.

A polyclonal antibody was raised in rabbits against a fusion protein immunogen consisting of bacterial maltose-binding protein coupled to a 92-amino acid C-terminal fragment of the rat AT1b angiotensin II (Ang II) receptor. The antibody immunoprecipitated the photoaffinity-labeled bovine AT1 receptor (AT1-R), but not the rat AT2 receptor, and specifically stained bovine adrenal glomerulosa cells and AT1a receptor-expressing Cos-7 cells, as well as the rat adrenal zona glomerulosa and renal glomeruli. The antibody was employed to analyze Ang II-induced phosphorylation of the endogenous AT1-R immunoprecipitated from cultured bovine adrenal glomerulosa cells. Receptor phosphorylation was rapid, sustained for up to 60 min, and enhanced by pretreatment of the cells with okadaic acid. Its magnitude was correlated with the degree of ligand occupancy of the receptor. Activation of protein kinase A and protein kinase C (PKC) also caused phosphorylation of the receptor, but to a lesser extent than Ang II. Inhibition of PKC by staurosporine augmented Ang II-stimulated AT1-R phosphorylation, suggesting a negative regulatory role of PKC on the putative G protein-coupled receptor kinase(s) that mediates the majority of AT1-R phosphorylation. The antibody should permit further analysis of endogenous AT1-R phosphorylation in Ang II target cells.

Physicochemical characterization of photoaffinity-labeled angiotensin II receptors.

Specific receptor sites for angiotensin II (AII) were analyzed in the adrenal cortex and other target tissues including liver, anterior pituitary gland, and smooth muscle, after covalent labeling with the radioactive photoaffinity analog 125I-[Sar1,(4-N3)Phe8]-AII. The photoreactive AII derivative retained high affinity for adrenal receptors and full steroidogenic activity in adrenal glomerulosa cells. In bovine adrenal cortex, covalent labeling of AII receptors by the photoreactive analog was specifically inhibited by [Sar1]AII with an IC50 of about 5 nM. The Mr of the covalent AII-receptor complex during polyacrylamide gel electrophoresis of the labeled protein under reducing conditions was 58,000. Under non-reducing conditions, a minor band with Mr of 105,000 was also observed. Two labeled species were also found during gel permeation chromatography of the detergent-solubilized complex, with Mrs of 64,000 and 86,000. The pl of the solubilized photolabeled complex was absorbed to wheat germ lectin Sepharose 6MB and could be eluted by N-acetylglucosamine. The Mrs of specific AII-binding sites in several target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed significant differences within and between species. The most striking differences were between rat adrenal cortex (79,000) and both rat liver (60,000) and bovine adrenal cortex (58,000). After enzymatic deglycosylation, the Mr of the major component present in the bovine and rat adrenal cortex decreased by 40% and 55% to 35,000 and 34,000, respectively, suggesting that variations in carbohydrate content contribute to the physical heterogeneity of AII receptors in individual target tissues.

Molecular size characterization of oxytocin receptors in rabbit amnion.

The binding of oxytocin (OT) to receptors in rabbit amnion cells stimulates PGE2 release. We previously studied the binding characteristics, changes in receptor concentration during pregnancy, and agonist specificity of OT action on amnion cells. In this study the molecular size of OT receptors in rabbit amnion was estimated by photoaffinity labeling, radiation inactivation, and gel filtration of solubilized receptor, using an iodinated OT antagonist, [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid, 2-O-methyltyrosine,4-threonine,8-ornithine,9-tyrosylamide]vasotoci n (OTA), or [3H]OT. Two electrophoretic bands, about 50 and 65 kilodaltons (kDa), were specifically covalently labeled with azidobenzoyl-[125I]OTA. Both sizes correspond to that determined by radiation inactivation, about 55 kDa, using [3H]OT binding to assess the fraction of receptor sites remaining. When we used [125I]OTA, the radiation inactivation size was about 30 kDa. These differences in radiation inactivation size suggest that the receptor binding site is comprised of more than one domain and that the binding of the antagonist involves fewer points of interaction than does OT. The molecular size of the receptor estimated from [125I]OTA binding by detergent-solubilized extracts of amnion membranes was about 350 kDa, as determined by gel filtration on columns of Sepharose 6B. Although the functional size of the receptor is about 65 kDa, it appears to be closely associated with other membrane proteins. The size estimates of amnion OT receptors agree with those in rabbit myometrium and rat mammary gland, both of which differ from amnion by contracting in response to OT. Despite different responses, OT receptors in different tissues appear to be very similar in size.

Determination of the functional size of oxytocin receptors in plasma membranes from mammary gland and uterine myometrium of the rat by radiation inactivation.

Gel filtration of detergent-solubilized oxytocin (OT) receptors in plasma membrane fractions from both regressed mammary gland and labor myometrium of the rat, showed that specific [3H]OT binding was associated with a heterogeneously sized population of macromolecules. As radiation inactivation is the only method available to measure the apparent molecular weights of membrane proteins in situ, we used this approach to define the functional sizes of OT receptors. The results indicate that both mammary and myometrial receptors are uniform in size and of similar molecular mass. Mammary and myometrial receptors were estimated to be 57.5 +/- 3.8 (SD) and 58.8 +/- 1.6 kilodaltons, respectively. Knowledge of the functional size of OT receptors will be useful in studies involving the purification and characterization of the receptor and associated membrane components.

Solubilization and properties of oxytocin receptors from rat mammary gland.

Oxytocin (OT)-binding activity was extracted with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfonate (CHAPSO) from rat involuted mammary glands with about 20% yield. The binding in detergent extracts was characterized and shown to be similar or identical to that of OT receptors on intact plasma membranes. Solubilized receptors had a high affinity site (Kd approximately 2 nM) and a lower affinity component, whereas the membrane receptor has only the high affinity site. Several synthetic OT analogs inhibited [3H]OT binding in the same rank order in both solubilized and intact membrane preparations. Both solubilized and membrane-associated receptor required Mn2+ for [3H]OT binding. The concentration of OT-binding sites in solubilized extracts of uterine myometrium from rats in late pregnancy was substantially greater in uteri from rats in labor than in that from rats 2 days before labor, as we have seen previously with receptors on intact membranes. The affinity of the solubilized myometrial receptor (Kd approximately 5 nM) was comparable to that of the membrane-associated receptor. Binding of [3H]OT to solubilized extracts of intestinal smooth muscle, which is not a target for OT, was negligible. Gel filtration analysis on columns of Sepharose 6B indicated that the solubilized [3H]OT-binding component from mammary gland was present in multiple mol wt forms, but the smallest major form eluted with an average apparent mol wt of about 40,000. These studies indicate that CHAPSO-solubilized binding sites for [3H]OT are the same as those in intact membranes and, therefore, are components of the OT receptor.

Angiotensin-II-binding sites on hepatocyte nuclei.

Angiotensin-II (Ang II) stimulates gene expression and cell growth in several cell types. Studies that have shown localization of Ang II to nuclei of myocytes and hepatic nuclear Ang II binding suggest that these actions may be mediated by nuclear receptors. We characterized Ang II binding to rat liver nuclei, which were free of plasma membrane based on enzyme analysis and electron microscopy. At 18 C, specific binding of 0.1-0.3 nM [125I]Ang II to nuclei and nuclear envelopes reached equilibrium by 2 h. Unlabeled Ang II inhibited [125I]Ang II binding to nuclei with an IC50 of 1.4 +/- 0.2 nM (+/- SE; n = 6). In half of the nuclear preparations, a lower affinity site (IC50, 50.4 +/- 23.6 nM), which accounted for 7-32% of specific Ang II binding, was detected by Scatchard analysis. Results similar to these were obtained with nuclear envelopes. Other Ang peptides competed for binding in the rank order: Ang III (IC50, 2.1 nM) greater than Ang I (IC50, 33) greater than [Des-Phe8]Ang II (IC50, 362) greater than [Des-Asp1-Des-Arg2]Ang II (IC50, 736). Losartan (DuP 753), an AT1 receptor antagonist, inhibited binding (IC50, 10.9 +/- 0.9 nM), whereas the AT2 receptor antagonist PD123177 did not. The pH optimum for binding to nuclear envelopes was 7, with binding more sensitive to low (5 and 6) than high (8 and 9) pH. Nonhydrolyzable GTP analogs accelerated displacement of bound [125I]Ang II by 10(-5) M Ang II. Differences were noted in pH sensitivity, time course, binding affinity for Ang I, II, and III, and rate of dissociation between nuclei or nuclear envelopes and plasma membrane Ang II binding. These results suggest that nuclear envelopes have a G-protein-coupled Ang II-binding site, which belongs to the AT1 class of Ang II receptors, with properties different from the plasma membrane receptor.

Solubilization of angiotensin II receptors in rat glomeruli.

125I-labelled angiotensin II (A II) specifically binds to solubilized receptors extracted from rat isolated glomeruli using CHAPS (3-[3-( cholamidopropyl ) dimethylammonio ]-1-propanesulfonate). The yield of solubilization of the binding sites was 3.3%. Equilibrium was reached after 15-20 min and specific binding represented 75% of total binding. Dissociation of the hormone-receptor complex after addition of an excess of A II was very slow in the presence of Ca2+ and Mg2+. [Sar1 Ala8] A II and [Sar1 Ile8] A II were more potent as competitive inhibitors of 125I-labelled A II than A II itself and its heptapeptide. These basic features of 125I-labelled A II binding to the extracted material were similar to those observed previously with untreated glomeruli.

Determination of the functional molecular size of vasopressin isoreceptors.

The molecular size of vasopressin receptors in the intact membrane-bound state was determined by radiation inactivation (target size analysis). For the V1 receptor in rat liver a molecular size of (76 +/- 8) kDa was determined. For the V2 receptor in rat kidney and bovine kidney molecular sizes of (95 +/- 4) and (108 +/- 11) kDa were found. Statistical analysis gave evidence for size differences between rat liver and rat kidney receptors or differences between rat liver and bovine kidney receptors, but not between kidney receptors from different species. The results suggest that V1 and V2 receptors can be distinguished by functional properties as well as by their size.

Angiotensin II receptor isoforms in the rat adrenal gland: studies with the selective subtype antagonists DuP 753 and CGP42112A.

The angiotensin II (Ang II)-binding sites in rat adrenal gland membranes were characterized using 125I-radiolabelled Ang II. While Scatchard analysis identified a single population of Ang II receptor sites, isoelectric focusing (IEF) on polyacrylamide gels revealed four peaks of specific Ang II binding which migrated to isoelectric points (pI values) 6.8, 6.7, 6.5 and 6.3. In binding assays in the presence of an excess of the Ang II receptor AT1 subtype antagonist DuP 753, a monophasic dose-dependent displacement of 125I-labelled Ang II binding by the Ang II receptor AT2 subtype antagonist CGP42112A was observed, and vice versa. In this system, reduction of disulphide bridges using 1 mmol dithiothreitol (DTT)/l markedly increased the number of binding sites in the adrenal zona glomerulosa without affecting receptor affinity. Using IEF, it was found that both DuP 753 and CGP42112A were able to reduce specific binding of each of the four peaks to some extent. However, the predominant effect of DuP 753 was to reduce the labelling of the isoform at pI 6.7 substantially, while CGP42112A significantly inhibited the specific 125I-labelled Ang II binding to the pI 6.3 isoform. When DuP 753 and CGP42112A were used together, specific binding of 125I-labelled Ang II to the isoforms of pI values 6.8, 6.7 and 6.3 was completely eliminated. These data suggest that the four peaks of specific binding found may be composed of different isoforms of both AT1 and AT2 receptor subtypes and that the Ang II receptor isoforms which migrated to pI 6.7 and pI 6.3 are predominantly composed of AT1 and AT2 receptor subtypes respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilized adrenal angiotensin II receptors: studies on the site of action of sodium and calcium ions, and on the role of disulfide bridges.

The zwitterionic detergent, 3[(3-cholamidopropyl)dimethylammonio)-1-propane sulfonate (CHAPS), was used to extract angiotensin receptors from the adrenal zona glomerulosa of two species, the rat and beef. The solubilized receptors retained the different properties displayed by particulate receptors in these two species, as well as a preserved affinity and specificity. Moreover, the adrenal receptors lost their ability to be affected by sodium and calcium ions, and by guanyl nucleotides, indicating that the site of action of these modulators is distinct from the receptor itself. Dithiothreitol treatment enhanced the binding of agonist ([125I]angiotensin II) and antagonist ([125I](Sar1,Ala8)-angiotensin II) to particulate and solubilized rat adrenal receptors, an effect attributable to inhibition of tracer degradation. In contrast, agonist and antagonist binding to particulate bovine receptors was decreased by dithiothreitol, but agonist binding to soluble receptors was increased. In addition to showing important species differences in angiotensin-degrading activity of adrenal receptor preparations, this work suggests that bovine receptors may contain essential sulfhydryl groups for binding and recognition of angiotensin peptides as agonists or antagonists.

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.

Vasopressin antisense peptide interactions with the V1 receptor.

The molecular recognition hypothesis, that peptide ligands and their receptor binding sites are encoded by complementary nucleotide sequences, was tested for arginine vasopressin (AVP) and its V1 receptor. Binding of [125I] [d(CH2)5,Sar7]AVP (a selective V1 vasopressin antagonist radioligand) or [3H]AVP to rat liver plasma membranes was inhibited by peptides known to bind to V1 receptors but not by the AVP complementary peptide (Ser-Ser-Trp-Ala-Val-Leu-Glu-Val-Ala) (PVA). Rabbit anti-PVA antibodies were nonimmunoreactive with any protein in rat liver membranes or in a partially purified preparation from rat liver containing reconstitutable vasopressin binding activity. Furthermore, there was no suppression of the AVP pressor effect by PVA in vivo using a rat blood pressure bioassay. These findings do not support the hypothesis that the V1 receptor binding site is encoded by the antisense DNA strand to AVP.

Angiotensin II receptor subtypes and contractile responses in portal vein smooth muscle.

The selective biphenylimidazole and tetrahydroimidazopyridine antagonists exemplified by losartan (DuP 753) and PD 123319 have been shown to bind selectively to angiotensin AT1 and AT2 receptor subtypes, respectively. To characterize which subtypes of angiotensin II receptors are expressed in mammalian portal vein smooth muscle, we performed, using both membrane and strip preparations, [3H]angiotensin II binding experiments and then contraction experiments to investigate the functional relevance of these binding sites. Specific binding of [3H]angiotensin II was of high affinity, saturable and reversible. Specific binding of [3H]angiotensin II was completely displaced by angiotensin II and the peptide antagonist [Sar1,Ile8]angiotensin II. The inhibition of [3H]angiotensin II binding by losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl)- methyl]imidazole, potassium salt) and DuP 532 (2-n-propyl-4-pentafluoroethyl-1-[(2'-(1H-tetrazol-5-yl)biph enyl-4-yl)- methyl]imidazole-5-carboxylic acid) was biphasic and LIGAND curve-fitting analysis revealed two populations of specific binding sites. One subpopulation represented 75% of the total binding and showed high affinity for angiotensin II, losartan and DuP 532, but low affinity for the peptide angiotensin AT2 receptor antagonist CGP 42112A (N-alpha-nicotinoyl-Tyr-Lys-[N-alpha-CBZ-Arg]-His-Pro-Ile-OH) and thus appeared identical to the cloned angiotensin AT1 receptor subtype. The remaining 25% of the sites showed nearly 1000-fold lower affinity for losartan, 6500-fold lower affinity for DuP 532 and high affinity for PD 123319 (S-1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-diphenylacetyl- 4,5,6,7-tetrahydro-1H-imidazo-[4,5-c] pyridine-6-carboxylic acid, difluoroacetate monohydrate) and CGP 42112A, with values of Ki in the same range (nM) as those found for losartan and DuP 532 at angiotensin AT1 binding sites. These sites appear to be angiotensin AT2 receptors. Only the angiotensin AT1 receptor subtype interacted with G-proteins, as indicated by the 80% inhibition of [3H]angiotensin II binding in the presence of guanosine 5'-O-(3-thiophosphate) or fluoroaluminates. Although the angiotensin II-induced contraction was completely inhibited by losartan with a pA2 value of 8.8, PD 123319 reduced the angiotensin II-induced contraction by 20-25%, indicating that both angiotensin AT1 and AT2 receptor subtypes are functional in portal vein smooth muscle.

Autoradiographic localization of angiotensin II receptors in the rat brainstem.

The microscopic localization of angiotensin II receptors in the rat brainstem has been accomplished utilizing in vitro receptor autoradiographic techniques. These receptors are highly localized to discrete nuclear regions of the brainstem. Significant densities of autoradiographic grains, indicating the presence of specifically bound [125I]-angiotensin II, were observed in regions of the film corresponding to the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, nucleus intercalatus, nucleus commissuralis, substantia gelatinosa of the trigeminal nerve and the inferior olivary nucleus. Previous immunohistochemical studies have indicated the presence of immunoreactive angiotensin II in several of these regions, especially those concerned with central cardiovascular regulatory mechanisms. These results provide additional evidence in support of the hypothesized role of angiotensin II as a central neuromodulator in the regulation of systemic blood pressure.

Solubilization of human platelet vasopressin receptors.

The human platelet membrane receptor for vasopressin (AVP) has been solubilized with the cholic acid derivative detergent 3-( [3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. Rapid and simple separation of free tritiated AVP ( [3H]AVP) from the solubilized receptor-hormone complex was done by filtration through polyethylenimine-treated filters. [3H]AVP binds to this soluble receptor with an equilibrium dissociation constant of 11.03 +/- 1.86 nM and a maximal number of binding sites = 288 +/- 66 fmol/mg protein while the corresponding values of the membrane-bound receptor are 1.62 +/- 0.21 nM and 237 +/- 38 fmol/mg of protein, respectively. The Ki value for native AVP derived from competition experiments is 11.02 +/- 2.05 nM for the soluble receptor. Competition experiments with specific vascular and renal antagonists confirm that the solubilized receptor belongs to the V1-vascular subtype.

Characterization of the functional angiotensin II-receptor complex isoform in rat liver plasma membrane.

In rat liver plasma membrane a single angiotensin II (Ang II) binding site (Kd of 3.71 +/- 0.33 nM and Bmax of 1143.7 +/- 83.9 fmol/mg protein) was identified using radioligand binding assay. Pharmacologically, this receptor match with the AT1 receptor subtypes in term of affinity for the selective antagonist Losartan, and probably with the AT1A receptor form in term of insensitivity for the antagonist PD123319. Nevertheless, using polyacrylamide gel isoelectric focusing, two 125I-Ang II binding sites migrating to pI 6.8 and 6.5 were found in these membrane preparations. Monophasic displacement of 125I-Ang II bound to isoform migrating at pI 6.8 clearly indicate that this isoform represents a functional Ang II-receptor complex. In contrast, the high concentrations of agonist and peptidic derivates of Ang necessary to displace 125I-Ang II bound to isoform migrating at pI 6.5 indicate that this atypical 125I-Ang II binding site represents a biologically nonfunctional Ang II binding molecule, presumably a nonspecific 125I-Ang II binding site.

Thoughts and studies on purification of the angiotensin II receptor.

The angiotensin receptor is the only macromolecular component of the renin-angiotensin system which has not yet been purified and characterized in the isolated state. A purified preparation could be useful for identifying the amino acid residues it preferentially recognizes in various positions of defined peptide ligands, and for elucidating the proximate molecular mechanism by which the binding event is transduced into a cellular response. Such knowledge should expedite the development of receptor antagonists which might be more physiologically specific than other inhibitors of the system. This paper elaborates on these thoughts, and describes some recent progress in purification of the rabbit hepatic receptor.