I. Introduction

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Blood pressure was measured for the first time in 1733 by Stephen Hales, in a dramatic experiment on a horse, by inserting a brass pipe into the carotid artery. The technique of modern blood pressure measurement was introduced in 1905 by Nicolai Korotkov using the stethoscope invented by Laennec in 1815 and the relatively recently devised wraparound inflatable rubber cuff. The latter was first described by Riva-Rocci in 1896 and was improved by von Recklinghausen in 1901 (Freis, 1995).

The first insight into the regulation of blood pressure came from the discovery of a pressor principle by Tigerstedt and Bergman in 1897. They called this factor "renin" because it was extracted from the kidney. This pioneering work led to the description of reno-vascular hypertension in animals and in humans (Goldblatt et al., 1934). However, it was not until 1940 (Braun-Menendez et al., 1940) that a vasoconstrictor substance was isolated from renal venous blood from the ischemic kidney of a Goldblatt hypertensive dog. A similar finding was made simultaneously and independently by Page and Helmer (1940) after the injection of renin into an intact animal. This group also isolated a so-called "renin activator" that later proved to be angiotensinogen. The pressor substance was named "hypertensin" in Argentina and "angiotonin" in the United States and was later isolated and shown to be an octapeptide (Skeggs et al., 1956; Bumpus et al., 1957; Elliott and Peart, 1957). There were differences between laboratories concerning interpretations and nomenclature but in fact hypertensin and angiotonin were the same substance. In 1958, Braun-Menéndez and Page agreed on the hybrid term angiotensin for the highly potent pressor octapeptide. This proved to be an appropriate choice, given the later recognition of angiotensin's numerous actions in addition to its hypertensive effects. The sequence of angiotensin II is Asp-Arg-Val-Tyr-Ile-His-Pro-Phe in the human, horse, and pig. In bovine angiotensin II, the isoleucine residue in position 5 is replaced by valine.

Following this major discovery, the various components of the cascade leading to the formation of angiotensin II were characterized, including angiotensinogen, angiotensin converting enzyme (ACE),² and angiotensins I, II, and III (Table 1). The synthesis of the peptide angiotensin II by Bumpus et al. (1957) and by Rittel et al. (1957) was followed by a continuing series of investigations into the structure-activity relationship of angiotensin analogs, mainly in the hope of finding a peptide antagonist.

In 1987, a committee of the International Society for Hypertension, The American Heart Association, and the World Health Organization proposed abbreviating angiotensin to Ang using the decapeptide angiotensin I as the reference for numbering the amino acids of all angiotensin peptides (Dzau et al., 1987).

Angiotensin II plays a key role in the regulation of cardiovascular homeostasis. Acting on both the "content" and the "container", Ang II regulates blood volume and vascular resistance. The wide spectrum of Ang II target tissues includes the adrenals, kidney, brain, pituitary gland, vascular smooth muscle, and the sympathetic nervous system. Angiotensin is not only a blood-borne hormone that is produced and acts in the circulation but is also formed in many tissues such as brain, kidney, heart, and blood vessels. This has led to the suggestion that Ang II may also function as a paracrine and autocrine hormone, which induces cell growth and proliferation and controls extracellular matrix formation (Dzau and Gibbons, 1987; Griffin et al., 1991; Weber et al., 1995a,b). Other angiotensin-derived metabolites such as angiotensin 2-8 (Ang III), angiotensin 1-7, or angiotensin 3-8 (Ang IV) have all been shown to have biological activities (Table 1) (Peach, 1977; Schiavone et al., 1990; Ferrario et al., 1991; Ferrario and Iyer, 1998; Wright et al., 1995).

As for other peptide hormones, Ang II was postulated to act on a receptor located on the plasma membrane of its target cells. This receptor should possess the dual functions of specific recognition of the ligand and stimulation of the characteristic cellular response. Comparison of changes in steroidogenesis in the adrenal cortex, adrenal catecholamine release, and developed tension in aortic strips in response to Ang I, Ang II, and Ang III clearly indicated different affinities of these target organs for the three peptides (Peach, 1977; Devynck and Meyer, 1978). These pharmacological experiments showed that effector organs responded to Ang I, II, and III with 2 to 3 log differences in potency from tissue to tissue. Based on these studies, Ang II receptor selectivity for the agonists was proposed to be structure-activity related. Comparison of Ang II and a large number of synthetic agonists and antagonists formed by substituting various amino acids of Ang II indicated marked dissimilarities between the analogs in each of the preparations, suggesting differences in the structure of the receptor sites (Khosla et al., 1974; Papadimitriou and Worcel, 1974; Peach and Levens, 1980).

Early binding studies detected sites with binding characteristics that differed between the various target tissues (Peach and Levens, 1980). Also, receptor density was up- or down-regulated in different tissues following either Ang II infusion or Na⁺ restriction (Aguilera and Catt, 1978). The characterization of receptor types in rat liver and kidney cortex (Gunther, 1984; Douglas, 1987; Bouscarel et al., 1988b) suggested further Ang II receptor heterogeneity. An early classification proposed for Ang II receptor types was based on studies in only a few tissues or species (Levens et al., 1980; Peach and Levens, 1980; Ferrario et al., 1991). It was not until the end of the 1980s that tools became available to demonstrate the existence of at least two receptor types in many tissues for which the conventional peptide analogs such as saralasin have high affinity but little or no selectivity. These included the nonpeptide antagonists losartan (or Ex89 or DuP 753) and PD123177, and a new generation of peptide ligands such as CGP42112 and p-aminophenylalanine Ang II (Chiu et al., 1989a; Whitebread et al., 1989; Speth and Kim, 1990). This new development was made simultaneously and independently in three different laboratories, and the initial nomenclature was confusing: the receptor sensitive to losartan was called 1, B, or , and that with no affinity for losartan was termed 2, A, or . The High Blood Pressure Research Council in 1990 and the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) in 1992 therefore appointed a subcommittee³ to address the problem, and a classification was proposed in 1991 and updated in 1995 (Bumpus et al., 1991; de Gasparo et al., 1995).

To obtain a "fingerprint" capable of identifying distinct receptors, three main criteria have been proposed: operational, transductional, and structural (Humphrey et al., 1994). The operational criteria include the drug-related characteristics of the receptor, such as ligand binding affinities, and selective agonists and antagonists. The receptor-effector coupling events constitute the transductional criteria, and the receptor sequence and gene cloning represent the structural criteria. It is clear that all of these criteria are not necessarily achieved simultaneously and at an early stage. The coupling mechanism may not have a major influence on receptor pharmacology but it helps in differentiating receptor types. Also, receptors with diverse structures may respond to the same endogenous ligands. Finally, receptors may be cloned without having a known pharmacology. The combination of the three criteria should clearly help in defining true receptor types.

Any such classification will essentially evolve as our knowledge increases. Nevertheless, there is a need for an official scheme that will help to avoid confusion among investigators. Two Ang II receptor types fulfill the three classification criteria, and are termed AT₁ and AT₂ receptors. According to the NC-IUPHAR recommendation, the AT₁ and AT₂ receptors have an IUPHAR Receptor Code of 2.1.Ang.01.000.00.00 and 2.1 Ang.02.000.00.00 (Humphrey and Barnard, 1998). The first two numbers indicated the structural class: they are seven transmembrane domain, G protein-coupled receptor (GPCR) member of the rhodopsin subclass (2.1). The receptor family is abbreviated Ang. The types indicated as 01 and 02 for AT₁ and AT₂. The following series of null are reserved for splice variants chronologically numbered according to identification within species.

Two other receptors (AT₃ and AT₄) have been proposed, based on operational criteria, but their transduction mechanisms are unknown and they have not yet been cloned. The name AT₃ was initially given to a binding site described in the Neuro-2a mouse neuroblastoma cell line that was not blocked by either AT₁-specific losartan, or AT₂-specific PD123319 and was not affected by GTP analogs (Chaki and Inagami, 1992). This AT₃ binding site, which has a low affinity for Ang III, should be called a non-AT₁-non-AT₂ site until more information about its nature has been obtained. The endogenous ligand for the AT₄ receptor is Ang 3-8 or Ang IV. Its binding properties and physiological characteristics, described in more detail in another section, are sufficiently different from those of the AT₁ and AT₂ receptor to warrant keeping the name AT₄ for this putative Ang IV-selective receptor until the binding protein is cloned and further characterized.

The strategy of expression cloning was successfully applied to the AT₁ receptors of rat smooth muscle and bovine adrenal gland, and subsequently the corresponding receptors of mouse, rabbit, human, pig, dog, turkey, and frog angiotensin receptors were cloned and sequenced. The nonmammalian receptors have 60% identity with the mammalian receptor and are pharmacologically distinct in their ligand binding properties. Hydropathy analysis indicated that both AT₁ and AT₂ receptors contain seven hydrophobic transmembrane segments forming  helices in the lipid bilayer of the cell membrane. The structural information for the AT₁ receptor is coded as follows: h 359 aa, P30556, chr.3. This indicates that the human AT₁ receptor contains 359 amino acids, with the sequence reported in the SwissProt file under the number 30556 and the gene coding for the receptor (abbreviated AGTR1) is located on chromosome 3 q. Similarly, the structural coding for rat and mouse AT₁ receptor is r 359 aa, P29089, P25095 and m 359 aa, P29754, P29755 as there are two subtypes A and B in rat and mouse located on chromosomes 17 and 2 and 13 and 3, respectively. The AT₂ receptor is only 34% identical with its AT₁ counterpart (Fig. 1). The structural information is coded h 363 aa, P50052, chr.X q22-q23 as the gene AGTR2 is located on human chromosome X with the cytogenetic location q23-q24. For rat and mouse, the respective information is r 363 aa, P35351 and m 363 aa, P35374. As in human, the AT₂ receptor in rodents is also located on chromosome X. 

View larger version (50K): [in this window] [in a new window]   Fig. 1.   Secondary structure and consensus sequence of the mammalian angiotensin AT1 receptor. The amino acid sequence shown is based on the derived sequences of five individual cloned mammalian AT1 receptors. The amino acid residues that are highly conserved among G protein-coupled receptors are indicated by bold letters. The positions of the three extracellular carbohydrate chains, and of the two extracellular disulfide bonds, are also indicated.

An evolutionary analysis based on the alignment of cloned AT₁ receptor sequences, using the CLUSTAL algorithm of PC/gene, has suggested that rat and mouse AT₁ receptors coevolved. (Sandberg, 1994). Amphibian and avian receptors diverged early during evolution. So far, gene duplication has been observed only in rats and mice (see following section). Two isoforms of the AT₁ receptor derived by alternative splicing of the same gene have been reported in man (Curnow et al., 1995). They have similar binding and functional properties. A receptor with as much as 97% identity to the AT₁ receptor has been cloned from human placenta (Konishi et al., 1994). It differs in its C-terminal amino acid sequence, tissue distribution, and pharmacological properties. The gene has not been cloned and it may well be a splice variant of the AT₁ receptor.

	II. The Type 1 (AT1) Angiotensin Receptor

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The angiotensin AT₁ receptor mediates virtually all of the known physiological actions of angiotensin II (Ang II) in cardiovascular, renal, neuronal, endocrine, hepatic, and other target cells. These actions include the regulation of arterial blood pressure, electrolyte and water balance, thirst, hormone secretion, and renal function. The AT₁ receptor belongs to the G protein-coupled receptor (GPCR) superfamily and is primarily coupled through pertussis toxin-insensitive G proteins to the activation of phospholipase C and calcium signaling. The AT₁ receptors of several species have been cloned and their amino acid sequences determined from the respective cDNAs. Ang II binding to the AT₁ receptor induces a conformational change in the receptor molecule that promotes its interaction with the G protein(s), which in turn mediate signal transduction via several plasma membrane effector systems. These include enzymes, such as phospholipase C, phospholipase D, phospholipase A₂, and adenylyl cyclase, and ion channels, such as L-type and T-type voltage-sensitive calcium channels. In addition to activating several intracellular signaling pathways that mediate agonist-induced phenotypic responses in a wide variety of Ang II target cells, the agonist-occupied AT₁ receptor undergoes desensitization and internalization in the same manner as many other GPCRs.

The cellular responses to AT₁ receptor signaling include smooth muscle contraction, adrenal steroidogenesis and aldosterone secretion, neuronal activation, neurosecretion, ion transport, and cell growth and proliferation. The AT₁ receptor is coupled not only to the well recognized G_q-mediated calcium and protein kinase C signaling pathways, but also to intracellular signaling cascades that extend into the nucleus. These pathways regulate gene transcription and the expression of proteins that control growth responses and cell proliferation in several Ang II target tissues. Some of the latter consequences of AT₁ receptor activation are counteracted by the structurally dissimilar AT₂ receptor, which antagonizes the effects of AT₁-mediated growth responses in several cell types, in particular endothelial cells, cardiomyocytes, and ovarian granulosa cells. These actions of the AT₂ receptor are described in more detail below. This account of the AT₁ receptor will address its gene expression, ligand binding, activation and signal transduction pathways, and physiological roles in the regulation of the activity and growth of its major target cells in cardiovascular, neuronal, and endocrine tissues.

The angiotensin receptor was identified as a functional entity by Lin and Goodfriend (1970), who first described the binding of radioiodinated Ang II to its receptor sites in the adrenal gland. These sites were subsequently shown to be located in the plasma membrane (Glossmann et al., 1974a), and the binding reaction was found to be influenced by the ambient Na⁺ concentration and guanyl nucleotides (Glossmann et al., 1974b,c). The G proteins had not been discovered at that time, but this finding indicated that the binding activity of a noncyclic AMP-coupled receptor is regulated by guanine nucleotides. Subsequent studies showed that the AT₁ receptor is coupled to both G_q and G_i proteins in the adrenal glomerulosa zone and several other tissues in the rat.

Many of the properties of the angiotensin II receptor were first identified in studies on the adrenal gland and liver, both of which are abundant sources of receptors that are coupled to well defined physiological responses (Saltman et al., 1975; Campanile et al., 1982). As in the rat adrenal gland, guanine nucleotides reduced agonist binding of ¹²⁵I-Ang II to hepatic receptors, largely by increasing its dissociation rate constant. Guanine nucleotides also decreased the number of high-affinity binding sites for Ang II, but not those for the peptide antagonist, [Sar¹,Ala⁸]Ang II. These changes were accompanied by inhibition of adenylyl cyclase activity in hepatic membranes, and of cyclic AMP production in intact hepatocytes (Crane et al., 1982). The high-affinity Ang II receptors in the liver were found to be inactivated by dithiothreitol, with a concomitant loss of Ang II-induced stimulation of glycogen phosphorylase in isolated hepatocytes (Gunther, 1984). These and related studies also presaged the existence of angiotensin II receptor types with distinct biochemical properties and intracellular mechanisms of action. Differential effects of guanine nucleotides on receptor binding of Ang II agonist and antagonist ligands were also observed in the bovine adrenal gland. This effect was evident for both membrane-bound and solubilized receptors. Concerning the latter, the association of the agonist-occupied receptor with a putative G protein was suggested by its larger size on steric exclusion HPLC (De Lean et al., 1984).

The ability of Ang II to inhibit glucagon-stimulated cyclic AMP production in hepatocytes, and adenylyl cyclase activity in hepatic membranes, was consistent with its coupling to an inhibitory G protein, now termed G_i. This was confirmed by the ability of pertussis toxin to prevent the inhibitory action of Ang II on adenylyl cyclase. The ability of GTPS to further reduce receptor binding affinity when all G_i molecules were ADP-ribosylated by the toxin indicated that Ang II receptors are also coupled to other G protein(s) that could mediate actions of Ang II on additional signaling pathways (Pobiner et al., 1985). Subsequent studies on cultured hepatocytes revealed a single population of Ang II binding sites and demonstrated that agonist and antagonist analogs had parallel actions on cytosolic calcium and phosphorylase activity, as did treatment with dithiothreitol to inactivate the receptors (Bouscarel et al., 1988a). Reconstitution studies in hepatocyte membranes showed that G_i3 is the major form of G_i in these cells and is responsible for coupling the Ang II receptor to agonist-induced inhibition of adenylyl cyclase (Pobiner et al., 1991). One of the few physiological actions of Ang II that is mediated by G_i, rather than G_q/11, is the AT₁ receptor-dependent stimulation of angiotensinogen production in the rat liver (Klett et al., 1993).

The relatively low abundance of the AT₁ receptor in most Ang II target tissues, and the instability of the solubilized receptor molecule, impeded efforts to isolate and sequence the receptor protein. For this reason, expression cloning from bovine adrenal and rat smooth muscle cells was necessary to isolate the cDNAs encoding the receptor proteins of these species (Sasaki et al., 1991; Murphy et al., 1991). Both AT₁ receptors were found to be typical seven transmembrane domain proteins, composed of 359 amino acids and with a molecular mass of about 41 kDa. The extracellular regions, composed of the N terminus and the three extracellular loops, contain three N-glycosylation sites and four cysteine residues (Fig. 1). Each of the consensus sites is glycosylated in the native AT₁ receptor (Jayadev et al., 1999), which has a molecular mass of about 65 kDa. In addition to the two conserved cysteines that form a disulfide bond between the first and second extracellular loops of all GPCRs, the AT₁ receptor contains an additional pair of extracellular cysteine residues. These are located in the N-terminal region and the third extracellular loop, and form a second disulfide bond that maintains the conformation of the AT₁ receptor protein (Ohyama et al., 1995). The latter disulfide bond, which is not present in the AT₂ receptor, renders the AT₁ receptor susceptible to inactivation by dithiothreitol and other reducing agents. The cytoplasmic region of the receptor, which is composed of the three intracellular loops and the C-terminal cytoplasmic tail, contains consensus sites for phosphorylation by several serine/threonine kinases, including protein kinase C (PKC) and GPCR kinases. Several of the specific residues that are phosphorylated during AT₁ receptor activation have been identified, but there are no confirmed reports of agonist-induced tyrosine phosphorylation of the AT₁ receptor or other GPCRs.

Similar structural features are present in several other cloned mammalian and nonmammalian AT receptors. The rat and mouse AT₁ receptors exist as two distinct subtypes, termed AT_1A and AT_1B, that are 95% identical in their amino acid sequences. The two subtypes are also similar in terms of their ligand binding and activation properties but differ in their tissue distribution, chromosomal localization, genomic structure, and transcriptional regulation. None of the other cloned mammalian AT₁ receptors, including those from cow (Sasaki et al., 1991), human (Bergsma et al., 1992; Curnow et al., 1992), pig (Itazaki et al., 1993), rabbit (Burns et al., 1993), and dog (Burns et al., 1994) appear to have subtypes. The two AT₁ subtypes in the rodent genome may be the consequence of a gene duplication event that occurred during evolution after the branching of rodents from the mammalian phylogenetic tree (Aiyar et al., 1994b).

The rat AT_1A receptor gene is 84 kb in length and contains three introns and four exons, the third of which (~2 kb) includes the entire 1077-base pair (bp) coding sequence of the receptor protein as well as 5' and 3' untranslated sequences (Langford et al., 1992; Murasawa et al., 1993; Takeuchi et al., 1993). The first two small exons encode alternatively spliced 5' untranslated sequences, and the fourth exon (1 kb) encodes an additional 3' untranslated sequence. A 2.3-kb transcript is found in all AT_1A-expressing tissues and contains exons 2 and 3. An additional 3.3-kb transcript containing exons 2, 3, and 4 is present in vascular smooth muscle cells and several other tissues but is not found in the brain. The transcription start site of the AT_1A receptor gene is located about 70 kb upstream from the exon that encodes the receptor protein. The rat AT_1B receptor gene is about 15 kb in length and contains two introns and three exons, the first two of which encode 5' untranslated sequences. The third exon contains the entire coding region of the receptor and the 3' untranslated sequence. The AT_1B receptor has 92 and 95% homology with the AT_1A at the nucleotide level and amino acid levels, respectively (Guo and Inagami, 1994) and is expressed in relatively few tissues as a 2.4-kb transcript. The rat AT_1A and AT_1B receptor genes are located on chromosomes 17q12 and 2q24, respectively (Tissir et al., 1995).

AT_1A and AT_1B receptors exhibit similar ligand binding and signal transduction properties but differ in their tissue distribution and transcriptional regulation. In the rat, AT_1A and AT_1B receptor mRNAs are expressed in numerous tissues, including adrenal, kidney, heart, aorta, lung, liver, testis, pituitary gland, and brain. AT_1A transcripts are predominantly expressed in all tissues except the adrenal and pituitary glands, where the AT_1B message is the major subtype. AT_1A receptors are abundantly expressed in vascular smooth muscle cells, in which their properties and regulation have been extensively investigated. In the adult mouse, AT_1A receptors are expressed in the kidney, liver, adrenal gland, ovary, brain, testis, lung, heart, and adipose tissue. In contrast, AT_1B receptors are confined to the adrenal gland, brain, and testis (Burson et al., 1994).

Studies on the tissue-specific expression of AT₁ receptor by in situ hybridization revealed that liver, heart, and lung contain solely AT_1A receptors, whereas the anterior pituitary gland contains only AT_1B receptors (Gasc et al., 1994). In the adrenal gland, the zona glomerulosa contains both AT_1A and AT_1B transcripts, the zona fasciculata contains little of either subtype, and only AT_1A mRNA is present in the medulla. In the kidney, AT_1A mRNA is present in mesangial and juxtaglomerular cells, proximal tubules, vasa recta, and interstitial cells, whereas AT_1B mRNA is found only in mesangial and juxtaglomerular cells, and in the renal pelvis. In male rats, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the relative abundance of AT_1A transcripts is 100% in liver, 85% in lung, 73% in kidney, 48% in adrenals, and 15% in the pituitary gland (Llorens-Cortes et al., 1994). In contrast to the adult animal, only AT_1A receptors are expressed in the pituitary gland during fetal and postnatal life.

The expression of the AT_1A receptor is stimulated by glucocorticoids, which act via one of three putative glucocorticoid responsive elements located in its promoter region (Guo et al., 1995). In the rat heart, where the AT_1A receptor is expressed in 10-fold excess over the AT_1B receptor, treatment with dexamethasone increased AT_1A and AT_1B mRNA levels by 100 and 300%, respectively (Della Bruna et al., 1995). Conversely, deoxycorticosterone acetate suppressed AT_1A mRNA levels by 70%, indicating that glucocorticoids and mineralocorticoids exert reciprocal actions on AT_1A receptor levels in the heart. In the heart and aorta, transcripts for both AT₁ subtypes were reduced by treatment with an AT₁ receptor antagonist. However, the AT_1B subtype was preferentially reduced, suggesting that the expression of AT_1B receptors in the adrenal is dependent on the activity of the renin-angiotensin system (Kitami et al., 1992).

Estrogens also influence the expression of AT₁ receptors, and exert divergent actions on subtype abundance in the pituitary gland and vascular smooth muscle. Estrogen treatment suppresses the expression of AT_1B but not AT_1A mRNA in the pituitary gland (Kakar et al., 1992). On the other hand, AT_1A receptor expression in vascular smooth muscle is elevated in ovariectomized rats and restored to normal by estrogen replacement (Nickenig et al., 1996). In cultured vascular smooth muscle cells, a high concentration of estradiol (1 µM) reduced AT_1A mRNA by about 30%. Whether estrogen deficiency leads to increased vascular AT₁ receptor expression in the human has yet to be determined.

Other forms of hormonal regulation of AT₁ receptor expression include the insulin-induced up-regulation of vascular AT₁ receptor expression, which has been attributed to a post-translational mechanism (Nickenig et al., 1998). In cultured vascular smooth muscle cells, insulin caused a doubling of AT₁ receptor density and a concomitant increase in the Ang II-induced intracellular Ca²⁺ response. This increase in receptor content, which was dependent on tyrosine phosphorylation and the intracellular Ca²⁺ response, was due to an increase in receptor mRNA stability rather than increased gene transcription. In rat astrocytes, growth hormone but not insulin-like growth factor 1 (IGF-1) also increased AT_1A receptor expression. This was associated with an increase in gene transcription and elevated mRNA levels. AT_1B receptors, which were much less abundant than the AT_1A subtype, were not affected by growth hormone treatment (Wyse and Sernia, 1997). On the other hand, nitric oxide (NO) caused a marked decrease in AT_1A gene expression in vascular smooth muscle cell (VSMC) that was independent of changes in cyclic GMP. This was accompanied by an inhibitory action of NO on the expression of a reporter gene containing 616 bp of the AT₁ receptor gene promoter, and reduced association with a DNA binding protein that interacts with this region (Ichiki et al., 1998).

The human AT₁ receptor contains 359 amino acids, and its deduced amino acid sequence is 95% identical with those of the rat and bovine AT₁ receptors (Curnow et al., 1992; Bergsma et al., 1992; Furuta et al., 1992). The receptor is derived from a single large gene that contains five exons ranging in size from 59 to 2014 bp (Guo et al., 1994). The open reading frame of the AT₁ receptor is located on exon 5. The other four exons participate to varying degrees in alternative splicing to produce mature RNAs that encode two receptor isoforms that are translated with different efficiencies (Curnow et al., 1995). The inclusion of exon 2 occurs in up to 50% of AT₁ mRNAs and inhibits the translation of the downstream AT₁ receptor sequence. In about one-third of AT₁ transcripts, the splicing of exon 3 to exon 5 yields a receptor with a 32 amino acid N-terminal extension. The ligand binding and signaling properties of this receptor are similar to those of the predominant shorter isoform of the AT₁ receptor.

The human AT₁ receptor gene is located on the q22 band of chromosome 3 (MEM number 106165) (Curnow et al., 1992; Davies et al., 1994). An additional human AT₁ receptor gene was suggested by the report of a human cDNA clone that differed from the known sequence in 10 of its 359 residues (Konishi et al., 1994), but subsequent studies have not confirmed the existence of a second gene (Curnow, 1996; Su et al., 1996). However, most human Ang II target tissues also express the slightly longer and functionally similar AT₁ receptor that results from alternative splicing of exons 3/5 as noted above. The longer isoform appears to be better expressed at the plasma membrane in cell transfection studies, but there is no evidence to suggest that it has a significant physiological role in AT₁ receptor function (Curnow, 1995).

Expression of the human AT₁ receptor is enhanced by epidermal growth factor in transfected COS-7 cells (Guo and Inagami, 1994b). Relatively little is known about the control of expression of the AT₁ receptor in most Ang II target tissues in the human. In the reproductive system, both Ang II and its AT₁ and AT₂ receptor types are present in the endometrium and exhibit cyclic changes during the menstrual cycle with a maximum in the early secretory phase (Ahmed et al., 1995). AT₁ receptors are expressed in the glands and the endometrial blood vessels and may participate in uterine vascular regulation and regeneration of the endometrium after menstruation. The human placenta expresses the AT₁ receptor and all other components of the renin-angiotensin system. The receptors are present throughout gestation in the syncytiotrophoblast and cytotrophoblast, and in the fetal vascular endothelial cells (Cooper et al., 1999). AT₁ receptor mRNA transcripts (2.4 kb) and receptor protein (83 kDa) increase progressively during pregnancy and reach their maximal level in the term placenta (Petit et al., 1996).

A local renin-angiotensin system is also present in human adipose tissue, with expression of angiotensinogen, ACE, and AT₁ receptor genes in omental and s.c. fat and cultured adipocytes (Engeli et al., 1999). The extent to which these components are related to the development of hypertension and obesity-related disorders has yet to be established. In the human kidney, AT₁ receptors are expressed in the renal vasculature, glomeruli, and the vasa recta bundles in the inner stripe of the outer medulla (Goldfarb et al., 1994). AT₁ receptors are diminished in the glomeruli of patients with chronic renal disease (Wagner et al., 1999). The AT₁ receptors expressed in cultured human mesangial cells mediate Ang II-induced hypertrophy and proliferative responses, implying that Ang II may be involved in the pathogenesis of glomerulosclerosis (Orth et al., 1995).

Similar effects of Ang II are mediated by AT₁ receptors in human pulmonary artery smooth muscle cells, in which Ang II stimulates DNA and protein synthesis. This response was associated with activation of mitogen-activated protein kinase (MAPK) and was prevented by losartan and by the MAPK inhibitor, PD-98059. These findings suggest that Ang II-induced activation of the AT₁ receptor initiates signaling pathways that participate in growth and remodeling of the human vascular system (Morrell et al., 1999).

In erythroid progenitor cells, which express both AT₁ and erythropoietin (EPO) receptors, Ang II enhances EPO-stimulated erythroid proliferation in vitro (Mrug et al., 1997). In vivo, the ₂-adrenergic receptor-induced production of EPO in normal subjects was inhibited by losartan treatment, implying that Ang II is a physiological regulator of EPO production in the human (Freudenthaler et al., 1999).

1. AT₁ Receptor Gene Polymorphisms and Cardiovascular Disease. The discovery of several polymorphisms in the human AT₁ receptor gene, one of which (A1166C) was more frequent in hypertensive subjects (Bonnardeaux et al., 1994), initiated a series of studies on the role of such mutations in the genesis of hypertension and other cardiovascular disorders. Subsequently, this polymorphism was reported to act synergistically with the angiotensin converting enzyme DD genotype on the risk of myocardial infarction (Tiret et al., 1994). However, the results of subsequent reports on this topic have not been consistent. In some studies, the A1166C polymorphism had no effect on ambulatory blood pressure, left ventricular mass, or carotid arterial wall thickness (Castellano et al., 1996; Schmidt et al., 1997). In other reports, the same AT₁ receptor gene polymorphism was associated with increased coronary arterial vasoconstriction in response to methylergonovine maleate (Amant et al., 1997), essential hypertension (Szombathy et al., 1998; Kainulamen et al., 1999), and increased left ventricular mass but not hypertension (Takami et al., 1998). An analysis of the role of this polymorphism in rats overexpressing the mutant human AT₁ receptor in the myocardium suggested that it is associated with increased responsiveness to Ang II. This may lead to cardiac hypertrophy under high-renin conditions or during pressure and volume overload (Van Geel et al., 1998).

In the Xenopus laevis oocyte, endogenous Ang II receptors were detected in the ovarian follicular cells that surround the oocyte. These receptors mediate Ang II-induced elevations of cytoplasmic Ca²⁺ in the oocyte via gap junctions between follicular cells and oocyte (Sandberg et al., 1990, 1992b) and are thus functionally identifiable as AT₁ receptors. However, the amphibian (xAT) receptor for Ang II did not recognize the nonpeptide antagonist, DuP753, that inhibits the binding and actions of Ang II at the mammalian AT₁ receptor (Sandberg et al., 1991). The xAT receptor cDNA was cloned from a Xenopus myocardial cDNA library to investigate the structural basis of this functional distinction in ligand binding. The xAT receptor is a 41-kDa protein containing 362 amino acids that has 60% amino acid identity and 65% nucleotide homology with the coding regions of known mammalian AT₁ receptors (Ji et al., 1993; Aiyar et al., 1994a). When expressed in Xenopus oocytes, xAT receptors mediate Ang II-induced Ca²⁺ mobilization and are pharmacologically distinct from mammalian AT₁ receptors. Receptor transcripts are present in Xenopus lung, liver, kidney, spleen, and heart, but not in adrenal, intestine, and smooth muscle. Mutational analyses of xAT and rat AT₁ receptors have largely elucidated the structural basis of their individual ligand binding properties, as described below.

Gene targeting experiments have provided several important insights into the physiological role of the renin-angiotensin system in cardiovascular regulation, fluid and electrolyte balance, and development. Deletion of the genes encoding angiotensinogen (Tanimoto et al., 1994; Kim et al., 1995) and ACE (Krege et al., 1995; Esther et al., 1996) revealed that the lack of Ang II in Agt/ or Ace/ mice was associated with hypotension, reduced survival, and marked abnormalities in renal development. Most of the Agt null animals died before weaning and most of the ACE null mice died within 12 months. The Agt and ACE null animals that survived until adult life had severe renal lesions. In both cases, the kidneys showed focal areas of cortical inflammation, thickened arterial walls, and medullary hypoplasia with a consequent deficit in urinary concentration. An additional feature of interest in the Ace/ animals was impaired fertility in the male animals, although histologically the sperm appeared to be normal and had normal motility (Krege et al., 1995; Esther et al., 1997). This was dependent on the loss of a testis-specific ACE isozyme that is expressed in round and elongating spermatids and was associated with defects in sperm transport in the oviduct and binding to the zona pellucida of the oocyte (Hagaman et al., 1998). In mice lacking both ACE isozymes, male fertility was selectively restored by sperm-specific expression of the testicular isoenzyme (Ramaraj et al., 1998).

The effects of disruption of the mouse gene encoding the AT₁ receptor types were in part predictable from the results of deleting the genes encoding angiotensinogen and ACE. Mice lacking a functional AT_1A receptor had a significant reduction of resting blood pressure (ca. 20 mm Hg) and lacked the pressor/depressor responses to infused Ang II that occur in normal animals. However, the Agtr1a(/) animals showed no marked impairment of development and survival and had no major abnormalities in the heart, vascular system, and kidney (Ito et al., 1995; Sugaya et al., 1995a,b; Chen et al., 1997). Closer examination of the Agtr1a(/) animals revealed a slight decrease in survival, marked hypertrophy of the renal juxtaglomerular cells, and a moderate degree of mesangial expansion (Oliviero et al., 1997). Also, the tubuloglomerular feedback loop that regulates sodium delivery to the distal tubule was not detectable in AT_1A knockout mice, indicating a specific role of AT₁ receptor in the operation of this homeostatic mechanism (Schnermann et al., 1997). The absence of the severe renal lesions observed in animals lacking angiotensinogen or ACE at first suggested that the AT_1A receptor is not a critical determinant of normal renal development and structure. However, the more severe effects of angiotensinogen and ACE deficiency, and the prominent inhibitory action of losartan treatment on renal structure and function in neonatal mice, indicated that Ang II action through the AT₁ receptor is essential for normal renal growth and development (Tufro-McReddie et al., 1995).

Subsequent studies revealed that Ang II infusions in Agtr1a(/) mice treated with enalapril to reduce endogenous Ang II production cause dose-related elevations in blood pressure that were prevented by AT₁ receptor antagonists. These pressor responses were much smaller than those observed in wild-type mice, but nevertheless demonstrated that AT_1B receptors participate in blood pressure regulation in the absence of the AT_1A receptor (Oliverio et al., 1997). This was confirmed by the finding that Ang II-induced calcium mobilization was similar in vascular smooth muscle cells from AT_1A-deficient and wild-type mice, and was blocked by losartan (Zhu et al., 1998b). Thus, the AT_1B receptor contributes substantially to Ang II action in the cardiovascular system in the absence of the AT_1A receptor, and presumably is subsidiary to the major AT_1A subtype in normal animals. This is supported by data obtained in mice with a double knockout of the AT_1A and AT_1B receptors, which have a more severe phenotype and lower blood pressure than mice lacking only the AT_1A receptor (Oliviero et al., 1998a; Tsuchida et al., 1998).

These conclusions were confirmed by the finding that mice lacking both AT_1A and AT_1B receptors showed impaired growth and marked abnormalities in renal structure and function. The renal abnormalities in the double knockout animals were similar to those seen in Agtr/ and Ace/ mice and were accompanied by comparable decreases in blood pressure and the complete absence of pressor responses to Ang II. In these animals, inhibition of converting enzyme by enalapril caused a paradoxical increase in blood pressure that could result from impairment of AT₂ receptor signaling and possibly an inhibitory effect on renal sodium excretion (Oliviero et al., 1998). These observations demonstrated that although the AT_1B receptor has a relatively minor role in normal animals, its contributions to growth, renal development, and cardiovascular regulation can compensate for much of the loss of the major regulatory actions of the AT_1A receptors in Agtr1a/ animals.

Studies on the role of the AT₁ receptors in sodium homeostasis revealed that Agtr1a/ mice have a further fall in blood pressure during sodium restriction and, unlike wild-type mice, develop negative sodium balance. However, these animals showed normal increases in plasma aldosterone levels during sodium depletion, consistent with the abundance of AT_1B receptors in the adrenal zona glomerulosa. These findings suggest that the hypotension observed in Agrt1a/ mice results from sodium deficiency and blood volume depletion and are consistent with the major role of AT_1A receptors in renal sodium resorption. The mechanisms by which Ang II regulates water homeostasis through its actions in the kidney and the brain were also clarified by observations in AT_1A receptor-deficient mice (Oliviero et al., 1998a,b). Agtr1a/ mice have a significant defect in urinary sodium concentration and develop marked serum hypotonicity during water deprivation. This does not result from impairment of vasopressin action on water permeability in the distal tubule but from the inability to maintain a maximal sodium gradient in the kidney. This change is not increased by losartan treatment and appears to be solely dependent on AT_1A receptor function. In contrast, the central action of Ang II on drinking responses appears to be mediated by AT_1B receptors, since it is largely retained in Agtr1a/ mice but is almost abolished in the mice lacking AT_1B receptor (Davisson et al., 1998).

In contrast to the hypertension and impaired vascular responses observed in AT₁-deficient mice, knockout of the AT₂ receptor leads to elevation of blood pressure and increased vascular sensitivity to Ang II (Hein et al., 1995a; Ichiki et al., 1995b). This has suggested that the AT₂ receptor may exert a protective action in blood pressure regulation by counteracting AT₁ receptor function. Such an action could be exerted in part by reduced expression of the AT₁ receptor, which is increased in the vascular smooth muscle of AT₂-deficient mice (Tanaka et al., 1999). However, the sustained hypersensitivity to Ang II in such animals is also attributable to loss of the counter-regulatory action of renal bradykinin and cyclic GMP formation (an index of NO production) (Siragy et al., 1999). The relative contributions of these two factors to AT₂-dependent vascular regulation, and the extent to which AT₂ receptor deficiency could contribute to sustained blood pressure elevation, as in human hypertension, have yet to be determined (see Section III, D).

1. Determinants of Ang II Bioactivity. The major features of the Ang II octapeptide (Asp¹-Arg-Val-Tyr-Ile/Val-His-Pro-Phe⁸) that determine its biological activity were identified in early studies on the in vivo and in vitro actions of structurally modified Ang II peptides (Khosla et al., 1974). All of the biological responses that were analyzed in these early reports were mediated by what is now defined as the AT₁ receptor. These findings were extended by the development of radioligand-receptor binding assays that used radioiodinated Ang II or its peptide analogs for in vitro analysis of the hormone-receptor interaction. More recent studies on the properties of cloned and mutant AT₁ receptors have led to the development of ligand-receptor models that incorporate the major features currently believed to be important in agonist binding to the AT₁ receptor. They have also provided further insights into the nature of the peptide binding site and the structural features that determine receptor activation, G protein coupling, and agonist-induced desensitization and internalization of the receptor.

2. Agonist Binding Site of the AT₁ Receptor. Amino acids in the AT₁ receptor that are essential for Ang II binding include the four cysteine residues that form the two external disulfide bonds and several other residues located in the exposed surface regions of the receptor (Fig. 2). In addition, polar or charged residues located within the hydrophobic transmembrane domains, including Lys¹⁰² at the top of TM helix III and Lys¹⁹⁹ near the top of TM helix V, participate in agonist binding. Some of the extracellular residues contribute to ligand interaction and stabilization of Ang II binding and others to the conformational change that causes receptor activation. The additional disulfide bridge between the amino terminal region and the third extracellular loop of the AT₁ receptor appears to stabilize the receptor and may be necessary to maintain the proximity of the extracellular amino acids that are involved in peptide binding. Cleavage of this disulfide bond probably accounts for the impairment of AT₁ receptor binding by reducing agents (Ohyama et al., 1995).

View larger version (56K): [in this window] [in a new window]   Fig. 2.   Amino acids that contribute to the agonist binding site of the AT1 receptor. Residues implicated in Ang II binding are shown as blue letters on pink background. The positions of the glycosylation sites, and of the conserved residues among GPCRs, are also shown.

3. Antagonist Binding of the AT₁ Receptor. Since Ang II is a major regulator of blood pressure, aldosterone secretion, and fluid homeostasis, and is also an important etiological factor in hypertension and other cardiovascular disorders, blockade of Ang II formation or action by ACE inhibitors or receptor antagonists is of major therapeutic importance. Early attempt to develop therapeutic agents able to block the Ang II receptor impeded by the peptidic nature of antagonists such as saralasin, which lacked oral activity and showed agonistic properties (Pals et al., 1979). More recently, based on imidazole derivatives first described by Furukawa et al. (1982), it became possible to develop specific nonpeptide Ang II receptor antagonists that specifically and selectively block the angiotensin AT₁ receptor (Timmermans et al., 1993; Goodfriend et al., 1996). The first of this series to reach the clinic, losartan, was followed by a large number of orally active AT₁ antagonists (Table 2).These can be classified in two groups depending on the presence of a biphenyltetrazole moiety, as in the prototype drug, losartan, in their structure. Receptor binding of nonpeptide Ang II antagonists is saturable and usually reversible and is independent of the pathway responsible for the synthesis of Ang II. This could be relevant to comparisons with ACE inhibitors, given the possible role of alternative Ang II-generating enzymes such as chymase, in human tissues (Urata et al., 1996).