Induction of the angiotensin AT2 receptor subtype expression by differentiation of the neuroblastoma × glioma hybrid,NG-108-15

In vitro differentiation of the mouse neuroblastoma-rat glioma hybrid cell line, NG-108-15, with dimethyl sulphoxide (1.5%) and low serum (0.5%), produced a marked increase in the number of angiotensin II receptors, from a level at the limit of sensitivity using labelled angiotensin II with a high specific activity ([¹²⁵I]angiotensin II), in undifferentiated cells, to a B_max of 1077 (1070–1268) fmol/mg in 5-day-differentiated cells. The affinity (K_d) of radiolabelled angiotensin II for the receptors in differentiated cells was 8.1 (7.5–10) nM. The recently available selective non-peptide antagonists. DuP 753 and PD 123177 and the peptide analogues of angiotensin II. CGP 42112A and p-aminophenylalanine⁶ angiotensin II, were used to characterize the angiotensin II receptors by competing for ¹²⁵I-[Sar¹-Ile⁸]angiotensin II binding to membranes prepared from undifferentiated and differentiated cells. The predominant angiotensin II receptor subtype expressed by undifferentiated cells was AT₁ and after differentiation AT₂. This change in receptor expression was evident 2 days after initiation of differentiation, was maximal at 4–5 days and was stable for at least 8 days. Administration of angiotensin II induced intracellular Ca²⁺ mobilization in both undifferentiated and differentiated cells. This was antagonised by the selective AT₁ antagonist. DuP 753, indicating an action at the AT₁ receptor subtype in both undifferentiated and differentiated cells. The selective AT₂ antagonist, PD 123177 was without effect on the angiotensin II induced increase intracellular Ca²⁺. This effect of DuP 753 on Ca²⁺ was specific for angiotensin II since the drug had no effect on bradykinin induced increases in intracellular Ca²⁺. Differentiation of NG-108-15 cells causes an increased expression of the AT₂ receptor. The function of this subtype of receptor remains to be determined.     

Characterization of the angiotensin II receptor expressed by the human hepatoma cell line, PLC-PRF-5.

Radioligand binding studies were undertaken to establish the expression of angiotensin II (AII) receptors on the human hepatoma cell line, PLC-PRF-5. Cell membranes were shown to express a large number of AII receptors with high and low affinity binding sites having Bmax values of 1269 +/- 365 and 4190 +/- 1055 fmol/mg protein and affinities (Kd) of 2.0 +/- 0.3 nM and 8.7 +/- 0.4 nM, respectively. In intact cells a single class of AII binding site was seen with an affinity (Kd) of 6.7 +/- 1 nM and a Bmax value of 315 +/- 32 fmol/mg. In both membranes and intact cells AII, AIII and the selective angiotensin AT1 receptor antagonist, DuP 753, all had a high affinity for the receptor (Ki values in the nanomolar range), but the selective angiotensin AT2 ligands, PD 123177 and p-aminophenylalanine6 AII, had low affinity (Ki values in the micromolar range). These results indicate that the PLC-PRF-5 cells express the angiotensin AT1 receptor subtype. This was further supported by the demonstration of the sensitivity of the receptor to dithiothreitol (DTT). Pretreatment of membranes with DTT reduced [3H]AII binding in a concentration-dependent manner with an IC50 of 4.2 +/- 0.9 mM. The coupling of the AT1 receptor to signal transduction pathways was investigated. In intact cells AII (100 nM) evoked an increase in intracellular calcium ([Ca2+]i). This increase in [Ca2+]i was unaffected by PD 123177 (100 microM) but was abolished by DuP 753 (100 microM). Furthermore, AII (100 nM) did not inhibit forskolin (0.1-10 microM) stimulated cyclic AMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin II exerts its effect on aldosterone production and potassium permeability through receptor subtype AT1 in rat adrenal glomerulosa cells.

The stimulatory effect of angiotensin II (AT) on the accumulation of inositol phosphates and on aldosterone production is abolished by the AT1 selective receptor antagonist DuP753 while PD123177, an AT2 antagonist, is ineffective. Similarly, a depolarizing effect of AT (inhibition of K+/86Rb efflux) is prevented by DuP753. While mediators derived from phospholipase C activation have a central role in the stimulation of aldosterone production by AT, the effect of AT on K+ permeability is mimicked neither by elevation of cytoplasmic [Ca2+] by ionomycin nor by kinase C activation with phorbol ester. Our results suggest that AT stimulates phospholipase C and the subsequent steroid production by glomerulosa cells through AT1 receptors. In addition some events induced by the activation of AT1 receptors may not be mediated by the activation of phospholipase C.     

Angiotensin II AT1 receptors in rat superior cervical ganglia: characterization and stimulation of phosphoinositide hydrolysis.

Angiotensin II receptor number was higher in superior cervical ganglia of 2-week-old when compared to 8-week-old rats. In both young and adult rats, specific binding of [125I][Sar1]angiotensin II was displaced competitively by the AT1-receptor antagonist DuP 753 but not by the AT2-receptor competitor PD 123177. In ganglia from adult rats, DuP 753 competed with an IC50 of 113 nM. The stable guanine nucleotide GTP gamma S inhibited binding of [125I][Sar1]angiotensin II in young and adult rats by approximately 50% with IC50 values of 105 and 120 nM, respectively, suggesting that the angiotensin receptor is G-protein linked. Angiotensin II at a dose of 1 microM stimulated inositol phosphate formation 58% over control values in superior cervical ganglia from 8-week-old rats. This effect was totally blocked by 10 microM DuP 753 but not by 10 microM PD 123177. Our findings demonstrate that rat superior cervical ganglia contain AT1-type angiotensin receptors that are probably G-protein linked, and their stimulation results in increased inositol phospholipid metabolism.     

DuP 753 can antagonize the effects of angiotensin II in rat liver

Results obtained with the use of nonpeptide angiotensin II receptor antagonists have suggested the presence of multiple subtypes of angiotensin II receptors in rat adrenal gland. However, the effects of nonpeptide antagonists on second messenger production by angiotensin II have not been investigated. In rat liver, angiotensin II can both activate phospholipase C, generating inositol polyphosphates and raising internal calcium, and inhibit adenylate cyclase. DuP 753 and PD123177, two nonpeptide angiotensin II antagonists, were used to characterize the receptor population in rat liver and to investigate the possibility that different angiotensin II receptor subtypes couple to different second messenger pathways. DuP 753 could completely antagonize the binding of angiotensin II in rat liver membranes, with a K1 of 9.3 x 10(-9) M. PD123177 had no effect on the binding of angiotensin II in rat liver at concentrations between 1 x 10(-9) M and 3 x 10(-5) M, in contrast to its ability to inhibit angiotensin II binding in rat adrenal. At a concentration of 10(-5) M, DuP 753 could inhibit increases in internal free calcium, could prevent production of inositol polyphosphates, and could attenuate inhibition of adenylate cyclase produced by angiotensin II. PD123177 at concentrations between 1 x 10(-9) M and 3 x 10(-5) M was ineffective in all of these assays. The results indicate that DuP 753 can displace the binding of angiotensin II at all receptor sites in rat liver and that this drug can attenuate both of the second messenger events produced by the angiotensin II receptor.     

Nonpeptide angiotensin II receptor antagonists. Studies with DuP 753 and EXP3174 in dogs.

DuP 753 (or EXP3174) and PD123177 are nonpeptide angiotensin (AII)-specific ligands, which show high affinities for two AII receptor subtypes, i.e. AT1 and AT2 sites, respectively. In furosemide-treated conscious dogs with high renin, DuP 753 and EXP3714, but not PD123177, were as effective as captopril in lowering blood pressure. Both DuP 753 and EXP3174 exhibited selective vascular antagonism of AII. In conscious dogs with normal renin, DuP 753, but not captopril or EXP3174, caused a dose-dependent but transient decrease in blood pressure. In anesthetized dogs, DuP 753 and captopril caused similar renal vasodilatation and natriuresis. The renal hemodynamic effects of DuP 753 and captopril were more pronounced in dogs with sodium depletion. These results suggest that the AT1 receptor mediates the pressor and renal effects of AII in dogs. The acute transient hypotensive effect of DuP 753 in normal-renin conscious dogs is probably unrelated to AII antagonism.     

Homologous and heterologous phosphorylation of the AT(2) angiotensin receptor by protein kinase C

The angiotensin AT(2) receptor is an atypical seven transmembrane domain receptor that is coupled to activation of tyrosine phosphatase and inhibition of MAP kinase, and does not undergo agonist-induced internalization. An investigation of the occurrence and nature of AT(2) receptor phosphorylation revealed that phorbol ester-induced activation of protein kinase C (PKC) in HA-AT(2) receptor-expressing COS-7 cells caused rapid and specific phosphorylation of a single residue (Ser(354)) located in the cytoplasmic tail of the receptor. Agonist activation of AT(2) receptors by angiotensin II (Ang II) also caused rapid PKC-dependent phosphorylation of Ser(354) that was prevented by the AT(2) antagonist, PD123177, and by inhibitors of PKC. In cells coexpressing AT(1) and AT(2) receptors, Ang II-induced phosphorylation of the AT(2) receptor was reduced by either PD123177 or the AT(1) receptor antagonist, DuP753, and was abolished by treatment with both antagonists or with PKC inhibitors. These findings indicate that the AT(2) receptor is rapidly phosphorylated via PKC during homologous activation by Ang II, and also undergoes heterologous PKC-dependent phosphorylation during activation of the AT(1) receptor. The latter process may regulate the counteracting effects of AT(2) receptors on growth responses to AT(1) receptor activation.     

A newly found angiotensin II receptor subtype mediates cyclic GMP formation in differentiated Neuro-2A cells.

In search of the functional role of the newly found angiotensin II (Ang II) binding site which is expressed in differentiated Neuro-2A cells, we found that Ang II causes a marked stimulation of cGMP formation dose-dependently. The stimulation was blocked by the nonselective Ang II receptor antagonist [Sar1,Ile8]Ang II but not by the AT1 antagonist DuP 753 or the AT2 antagonist PD 123319. These results suggest that Ang II increased cGMP level via a new Ang II receptor subtype in differentiated Neuro-2A cells.     

Monoclonal antibodies to the nonpeptide angiotensin II receptor antagonist, losartan.

Two murine monoclonal antibodies were produced to losartan (DuP 753), a nonpeptide angiotensin II receptor antagonist. Using a solid phase competitive enzyme-linked immunosorbent assay (ELISA), each antibody was examined for its ability to bind to a set of losartan analogs that differ structurally in varying degrees. Both antibodies distinguished fine structural changes in the analogs, particularly at the R5 position of the imidazole ring. No cross-reactivity towards either antibody was observed with the natural ligand angiotensin II, the peptide antagonist saralasin, or the AT2 selective nonpeptide antagonist PD123177.     

Angiotensin II-elicited signal transduction via AT1 receptors in endothelial cells.

1. Angiotensin II (AII) actions are mediated by two distinct types of receptors: AT1, which includes two subtypes, AT1A and AT1B, and AT2. AII produces vasoconstriction on the vascular wall acting directly on smooth muscle cells via AT1 receptors. AII receptors have recently been demonstrated on endothelial cells. But the pharmacological characteristics of these receptors and the intracellular signal pathways coupled to them remain unclear. 2. The aim of this work was to characterize the AII receptor subtypes in rat aortic endothelial cells (RAEC) in primary culture and to evaluate the signal pathways coupled to these receptors by measuring the activation of phospholipase C (PLC) and phospholipase A2 (PLA2). 3. Labelled AII bound to RAEC in a specific, saturable manner. Scatchard analysis showed a Kd of 1.87 +/- 0.49 nM and a Bmax of 50.2 +/- 10.9 x 10(3) sites per cell. AII was displaced by the AT1-specific antagonist, DuP753 with a Ki of 17.37 +/- 1.49 nM, but not by the AT2 receptor analogues CGP42771B or PD123177. These data were confirmed by the finding of AT1 mRNA in endothelial cells. Analysis of RNA expression by RT-PCR showed the presence of both subtypes, AT1A and AT1B in endothelial cells, whereas smooth muscle cells express only AT1A. 4. The activation of PLC and PLA2 in response to AII was evaluated by measuring inositol phosphate production and arachidonic acid release, respectively. Both were enhanced by AII in a dose-dependent manner, and inhibited by DuP753, but not by PD123177. 5. We conclude that AT1 receptors are expressed by endothelial cells in primary culture and that phospholipase C and phospholipase A2 activated via this receptor.     

Enhancement of losartan (DuP 753)-induced angiotensin II receptor antagonism by PD123177 in rats

Losartan (DuP 753) and PD123177 are angiotensin II type 1 (AT₁) and type 2 (AT₂) receptor selective ligands, respectively. In rats, PD123177 did not exhibit angiotensin II antagonism or hypotensive activity but enhanced these activities of a submaximal dose of losartan. As PD123177 displaced losartan from its rat plasma protein binding sites and thus increased the free concentration of losartan, this may account for its enhancement of the in vivo activities of losartan in rats.     

Novel angiotensin II antagonists distinguish amphibian from mammalian angiotensin II receptors expressed in Xenopus laevis oocytes

Angiotensin II (AII) stimulates rapid increases in cytosolic Ca2+ concentrations in Xenopus laevis oocytes after binding to specific receptors located in the surrounding follicular cells. In follicular oocytes, the peptide AII receptor antagonists saralasin (IC50 = 25 nM) and CGP 42112A (IC50 = 400 nM) were orders of magnitude more potent than the non-peptide antagonists DuP 753 and PD-123177 (IC50 greater than 10 microM) as inhibitors of AII-induced Ca2+ mobilization. The relative potencies of the AII antagonists at the Xenopus AII receptor were completely different from their activities at the two known mammalian AII receptor subtypes. These results indicate that the ligand-binding domain of the amphibian AII receptor has a unique conformation that distinguishes with high specificity between peptide and non-peptide AII antagonists. The amphibian AII receptor is pharmacologically distinct from the AT1 receptor subtype, which mediates phosphoinositide hydrolysis and Ca2+ mobilization in mammalian adrenal cells.     

Angiotensin II receptor subtypes in the rat brain

The non-peptide angiotensin II (AII) receptor subtype selective antagonist, DuP 753, was used to characterize AII receptor binding sites in the rat brain. DuP 753 competed for specific 125I-[Sar1,Ile8]AII (125I-SIAII) binding in many brain nuclei (IC50 = 20-30 nM), but was a weak competitor at remaining sites (IC50 greater than 10(-4) M). DuP 753 sensitive binding sites (designated AII alpha subtype) correspond with areas where binding is inhibited by sulfhydryl reducing agents, whereas DuP 753 insensitive sites (AII beta) correspond with areas where binding is not inhibited by sulfhydryl reducing agents.     

Characterization of rat intestinal angiotensin II receptors.

In rat ileum and duodenum 125I-sarcosine1,isoleucine8-angiotensin II labels a single population of binding sites with comparable receptor densities of 98 and 94 fmol/mg protein, respectively. Radioligand binding was dose dependently antagonized by angiotensin II (AII) and related peptides. DuP 753, a selective antagonist for the angiotensin AT1 receptor subtype, potently inhibited radioligand binding in both tissues (Ki: 12.7 and 11.8 nM), while AT2-selective ligands like PD 123.177 or p-amino-phenylalanine6-AII were inactive in concentrations lower than 1 microM. The contractile response to AII (1 microM) in ileal longitudinal and circular smooth muscle preparations amounted to 96 and 16%, respectively, of the response to 100 microM methacholine. The contractile response to AII was inhibited by DuP 753 (pA2 7.53) but unaffected by PD 123.177 (pA2 less than 5). The AII effect in longitudinal duodenal preparations amounted to only 24% of the methacholine response and was totally abolished in the presence of 1 microM DuP 753. No contraction due to AII was observed in duodenal circular smooth muscle preparations. The results obtained demonstrate the existence of functional AT1 receptors in the rat ileum and duodenum. In the ileum these receptors are mainly located on the longitudinal smooth muscle and coupled to contraction. In duodenal smooth muscle AII receptors may be either less effectively coupled to contractile elements or involved in another, additional function.     

Access of peripherally administered DuP 753 to rat brain angiotensin II receptors.

The in vivo access of the nonpeptide angiotensin II (Ang II) antagonist, DuP 753 (10 mg kg-1, i.v.), to Ang II receptors of rat brain was investigated by in vitro autoradiography with [125I]-[Sar1, Ile8] Ang II as a ligand. DuP 753 markedly inhibited the binding to sites which contain exclusively AT1 receptors both outside and within the blood brain barrier, such as the circumventricular organs, paraventricular hypothalamic nucleus, median preoptic nucleus and nucleus of the solitary tract. However, binding to other nuclei containing AT2 receptors was not significantly inhibited. These results demonstrate that DuP 753 and/or its active metabolite readily cross the blood brain barrier in vivo and selectively inhibit binding to AT1 receptors in specific brain nuclei.     

Ability of angiotensin II to modulate striatal dopamine release via the AT1 receptor in vitro and in vivo.

1. The ability of angiotensin II to modulate dopamine release from rat striatal slices in vitro and in the intact rat striatum in vivo was assessed by the microdialysis technique. 2. In slices of rat striatum, angiotensin II (0.1-1.0 microM) induced a concentration-related increase in endogenous dopamine release which was maximal (approximately 250% above basal levels) within the first 2-4 min of agonist application and subsequently declined to near basal values. The angiotensin II-induced increase in dopamine release was Ca(2+)-dependent and was completely antagonized by the selective AT1 receptor antagonist, losartan (1.0 microM). In contrast, the AT2 receptor antagonist, PD123177 (1.0 microM) failed to modify the angiotensin II-induced response. Neither antagonist alone modified basal dopamine release from striatal slices. 3. In freely moving rats, angiotensin II (1.0-10 microM; administered via the microdialysis probe) induced a concentration-related increase in extracellular levels of dopamine which was maximal (approximately 150% above basal levels) within 20-40 min of agonist application and subsequently declined. The angiotensin II (10 microM)-induced increase in extracellular levels of dopamine was completely antagonized by the AT1 receptor antagonist, losartan (0.1-1.0 microM; administered via the microdialysis probe) but not by the AT2 receptor antagonist, PD123177 (1.0 microM; administered via the microdialysis probe). Neither antagonist alone modified basal extracellular levels of dopamine. 4. Homogenate radioligand binding studies with [125I]-angiotensin II (0.1 nm) identified relatively low levels of specific binding sites in rat striatal homogenates compared to homogenates of pyriform cortex (51.3 +/- 9.2 and 651.3 +/- 55.1 fmol g-1 wet weight, respectively, mean +/- s.e.mean, n = 3; non-specific binding defined by unlabelled angiotensin II). The majority of the specific [125I]-angiotensin II (0.1 nM) binding in the striatal and pyriform cortex homogenates was sensitive to the selective AT1 receptor antagonist, losartan (1.0 microM). 5. In conclusions the present study provides direct evidence that angiotensin II acting via the AT1 receptor subtype facilitates the release of dopamine in the rat striatum in vitro and in vivo. This receptor-mediated response may account for the modulation of dopamine-mediated behavioural responses by antagonists of the AT1 receptor and inhibitors of angiotensin converting enzyme.     

Angiotensin II receptors in the rat lung are of the AII-1 subtype.

In the rat lung [3H]angiotensin II ([3H]AII) recognizes a single class of binding sites with an equilibrium constant (KD) of 2.8 +/- 0.9 nM. The corresponding receptor density (Bmax) was 704 +/- 60 fmol/mg protein. Equilibrium of [3H]AII binding was reached after 60 min. The dissociation of the radioligand was strongly influenced by mono- and divalent cations and the non-hydrolyzable GTP analog 5'-guanylyl-imido-diphosphate (Gpp[NH]p), indicating the interaction with a G protein. Specific binding of [3H]AII to rat lung preparations was inhibited by different agonists and antagonists and sensitive to DuP 753 (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)bi phe nyl-4-yl) methyl]imidazol, potassium salt) but insensitive to PD 123177 (1-(4-amino-3-methyl-benzyl)-5-diphenylacetyl-4,5,6,7-tetrahydro-i midazo [4,5-c]pyridine-6-carboxylic acid) two non-peptide AII antagonists selective for either the AII-1 or AII-2 subtype. Furthermore, specific [3H]AII binding was inhibited by dithiothreitol. It is concluded that in the rat lung [3H]AII binds to receptors of the AII-1 subtype which are coupled to a G protein. These receptors may be involved in the regulation of pulmonary perfusion and resistance.     

Angiotensin II receptor subtypes and biological responses in the adrenal cortex and medulla.

Angiotensin II (AII) receptor subtypes and their potential coupling mechanisms were studied using recently developed peptide and nonpeptide antagonists in rat and bovine adrenal zona glomerulosa cells, as well as in membranes prepared from rat and bovine adrenal cortex and medulla. Comparison of the potencies of these novel antagonists to displace 125I-[Sar1,Ile8]AII from its binding sites revealed two distinct AII binding sites in membranes prepared from rat adrenal capsules (zona glomerulosa) and from rat adrenal inner zones containing the medulla. About 85% of the binding sites of the glomerulosa zone and 30% of those of the inner zones were of the AT1 subtype, with relative affinities for the nonpeptide antagonists Dup 753 and PD 123177 and the peptide antagonist CGP 42112A in the order of Dup 753 much greater than CGP 42112A greater than PD 123177. In contrast, the relative binding potencies for the other (AT2) population of binding sites were CGP 42112A greater than PD 123177 much greater than Dup 753. Neither AII nor its peptide antagonist [Sar1,Ile8]AII could distinguish between the two sets of binding sites. The effects of the new antagonists on functional responses of rat adrenal glomerulosa cells demonstrated that both AII-stimulated aldosterone production and the AII-induced inhibition of adrenocorticotropic hormone-stimulated cAMP formation were mediated by the AT1 receptor subtype. In bovine adrenals, only AT1 receptors were detected in membranes prepared from the cortex and the medulla, as well as in cultured glomerulosa cells. The relative inhibitory potency of Dup 753 was lower by an order of magnitude at bovine than at rat AT1 receptors. The inhibition of AII-induced aldosterone production by the various antagonists was closely correlated with their inhibitory potencies on 125I-[Sar1,Ile8]AII binding to bovine glomerulosa cells. These data suggest that the known effects of AII in adrenal glomerulosa cells are mediated through the AT1 receptor subtype and that the distribution and/or specificity of the AT2 receptors shows marked species variations.     

Inhibition of vascular smooth muscle cell growth by angiotensin type 2 receptor stimulation for in vitro organ culture model

Recent data suggest that AT2 receptors in smooth muscle cells (VSMCs) of adult vessels may counterbalance AT1 receptor activity by inhibiting cell growth. The role of AT2 receptors in VSMC proliferation was investigated by using an in vitro organ culture model in which the sprouting of tubular structures was assessed. In preparations with endothelium, tubular growth was unaffected by angiotensin II (Ang II) but was inhibited by 50 +/- 9% by losartan and was increased by 110 +/- 15% by the AT2 antagonist PD123177. In endothelium-deprived preparations, growth inhibition (-49.1 +/- 0.5%) was observed when angiotensin II was added together with losartan 1 microM, whereas stimulation (59.8 +/- 14%) was induced by angiotensin II with 1 microM PD123177. In cultured VSMCs angiotensin II slightly promoted growth that was inhibited by losartan but was unaffected by PD123177. AT1a, AT1b, and AT2 mRNA expression was demonstrated in cells isolated from tubular structures grown from intact and endothelium-deprived rings, but only AT1a and AT1b mRNA was detected in cultured VSMCs. In conclusion, this paper proposes an in vitro organ culture model in which the expression of AT2 receptors in VSMCs is preserved and demonstrates AT2 receptor-mediated inhibition of VSMC proliferation in adult vessels.     

Evidence for the AT1 subtype of the angiotensin II receptor in the rat submandibular gland

We have characterized angiotensin II (Ang II) receptor subtypes on rat submandibular gland membranes using a radioligand binding assay. [3H]Ang II binding to the membrane fractions exhibited both high (Kd =0.08 nm, Bmax =2.19 fmol/mg protein) and low (Kd =4.19 nm, Bmax = 13.7 fmol/mg protein) affinity. Ang 11, Ang III and saralasin completely displaced the [3H]Ang II binding, whereas CV-11974, an AT1 receptor antagonist and PD123319, an AT2 receptor antagonist maximally displaced up to approximately 87 and 13% of the total binding, respectively. [3H]DuP753 binding to the membrane fractions exhibited a single population of binding site with a Kd of 4.22 nM and Bmax of 3.77 pmol/mg protein. Ang II, Ang III and CV-11974 completely displaced the [3H]DuP753 binding with slope factors near unity, but PD123319 did not. These findings suggest that rat submandibular gland membranes contain predominantly the AT1 receptor subtype.