Angiotensin II Receptor Subtypes Research Articles

Angiotensin II receptor expression and inhibition in the chronically hypoxic rat lung.

1. Angiotensin II (AII) binding density and the effect of chronic AII receptor blockade were examined in the rat model of hypoxia-induced pulmonary hypertension. 2. [125I]-[Sar1,Ile2]AII binding capacity was increased in lung membranes from rats exposed to hypoxia (10% fractional inspired O2) for 7 days compared to normal rats (Bmax 108 +/- 12 vs 77 +/- 3 fmol mg-1 protein; P < 0.05), with no significant change in dissociation constant. Competition with specific AII receptor subtype antagonists demonstrated that AT1 is the predominant subtype in both normal and hypoxic lung. 3. Rats treated intravenously with the AT1 antagonist, GR138950C, 1 mg kg-1 day-1 rather than saline alone during 7 days of exposure to hypoxia developed less pulmonary hypertension (pulmonary arterial pressure: 21.3 +/- 1.7 vs 28.3 +/- 1.1 mmHg; P < 0.05), right ventricular hypertrophy (right/left ventricle weight ratio: 0.35 +/- 0.01 vs 0.45 +/- 0.01; P < 0.05) and pulmonary artery remodelling (abundance of thick-walled pulmonary vessels: 9.6 +/- 1.4% vs 20.1 +/- 0.9%; P < 0.05). 4. The reduction in cardiac hypertrophy and pulmonary remodelling with the AT1 antagonist was greater than that achieved by a dose of sodium nitroprusside (SNP) that produced a comparable attenuation of the rise in pulmonary arterial pressure during hypoxia. 5. The data suggest that AII, via the AT1 receptor, has a role in the early pathogenesis of hypoxia-induced pulmonary hypertension in the rat.

Identification and Characterization of AngiotensinIV Binding Sites in Rat Neurone and Astrocyte Cell Cultures

This study demonstrates the existence of the putative receptor for the hexapeptide (3-8) fragment of angiotensin II (AngIV) on rat astrocytes and neurons grown in cell culture. Binding of 125I-AngIV was saturable and distinct from that of the AngII receptor subtypes. Equilibrium binding was attained in 15 min in astrocytes and 75 min in neurons at 22 degrees C. The bound peptide was confirmed by HPLC to be intact AngIV while the bound peptide was substantially degraded, even in the presence of peptidase inhibitors. Scatchard analysis of equilibrium binding was consistent with a two binding site model, revealing a high affinity and a low affinity binding site in both cell types. In neurons, the respective association constants (Ka) were 2.72 +/- 0.23 nM-1 and 727 +/- 354 nM-1, with associated receptor densities of 109.30 +/- 58.87 and 1723 +/- 1167 fmol/mg protein. Similar analyses in astrocytes gave Kas of 5.71 +/- 2.85 nM-1 and 277 +/- 205 nM-1, and respective densities of 191.1 +/- 90.1 and 1425 +/- 1250 fmol/mg protein. However, the quantitative reliability of these binding isotherms may be influenced by the degration of unbound peptide. Competitive binding analysis was used to determine the specificity of the receptor site, with the relative order of affinities being AngIV > AngIII > AngII(4-8), and no displacement by AngII, Iosartan and PD123319 in either neurons or astrocytes. Autoradiography with 125I-AngIV performed on neuronal cultures demonstrated that binding was confined to a subpopulation of the total cells. These data support the existence of a specific binding site for AngIV in both neurons and astrocytes, consistent with the properties of binding reported previously in the brain, and distinguish this site from the AngII receptor subtypes.

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: ANGIOTENSIN II AND THE ADRENAL

1. Angiotensin II (AngII) evokes a variety of physiological responses in the adrenal gland. It is the major regulator of aldosterone secretion, in the medulla it enhances catecholamine release and it exerts trophic effects in the adrenal and stimulates growth factor secretion. 2. Angiotensin II acts via binding to specific receptors, located on the plasma membrane. Two pharmacologically distinct AngII receptor subtypes, type 1 (AT(1)) and type 2 (AT(2)) receptors, have been identified using the non-peptide antagonists Dup753 and PD 123177, respectively, and cDNA encoding each type have been identified. 3. In the adrenal, the AT(1) receptor modulates all the known biological effects of AngII. The expression of the AT(1) receptor is modulated at the mRNA and protein levels by many factors: conditions that increase levels of AngII (low sodium diet, renovascular hypertension, AngII infusion) up-regulate AT(1) receptor mRNA levels and binding and increase aldosterone secretion. 4. A tissue renin-angiotensin system has been found in the adrenal, suggesting an important paracrine role for AngII in aldosterone regulation. 5. The possible involvement of AT(1) receptors in human disease has been investigated by examining the role of AngII receptors in adrenal tumours. Binding and gene expression studies have shown that AngII receptors are abundantly expressed in aldosterone-producing adenoma (APA). 6. Densitometric analysis of AT(1) expression in APA showed no significant differences compared with normal and nontumorous adrenal. In addition, no mutations in the coding sequence of the AT(1) receptor have been found to date in adrenal tumours.

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: PRESENCE OF ANGIOTENSIN II AT2 RECEPTOR BINDING SITES IN THE ADVENTITIA OF HUMAN KIDNEY VASCULATURE

SUMMARY Angiotensin II (AngII) receptor subtypes in adult human kidney were pharmacologically characterized by in vitro autoradiography using the AngII receptor subtype‐selective antagonists, losartan and PD 123319, and the sensitivity to the reducing agent, dithiothreitol. High densities of AngII AT1 receptor binding occur in the glomeruli and the inner stripe of the outer medulla, while a moderate AT1 receptor binding is localized in the proximal convoluted tubules. AT2 receptor binding is observed predominantly in the intrarenal large blood vessels, including the arcuate, inter‐ and intra‐lobular arteries, and in the renal capsule. In the major renal artery, AT1 receptor binding is abundant in the media and adventitia, while AT2 receptor binding is observed mainly in the adventitia. At the light microscopic level using emulsion autoradiography, AT1 receptors are localized in the glomeruli and juxtaglomerular apparatus, as expected. However, in larger renal blood vessels, including the arcuate arteries, inter‐ and intra‐lobular arteries, intense AT2 receptor labelling occurs primarily in the adventitia, while the endothelium and vascular smooth muscle layers contain only low levels of AngII receptor binding. These results indicate that the adult human kidney displays two pharmacologically distinct AngII receptor subtypes, with AT1 predominating in the glomeruli, juxtaglomerular apparatus, proximal tubules and the inner stripe of the outer medulla, while AT2 predominates in the adventitia of the arcuate and interlobular arteries and the renal capsule. The functional significance of AT2 receptor binding sites in the adventitia of adult human kidney vessels remains to be elucidated.

Angiotensin II-receptor subtypes in human atria and evidence for alterations in patients with cardiac dysfunction

Angiotensin II (AII) has been implicated as an important factor in the pathophysiology of heart diseases. Following the recent identification of two subtypes of the AII receptor in cardiac tissue of animals, we investigated the possible occurrence of these, or similar, subtypes in human atrial tissue. In right-atrial tissue from patients undergoing heart surgery, we determined the AII-receptor profile in receptor binding studies, using [125I]-angiotensin as radioligand and subtypes to identify and quantify AII-receptor subpopulations. In 35 patients (23 requiring coronary bypasses, 10 valvular surgery and two combined coronary and valvular surgery), the left-ventricular ejection fraction was determined in the preoperative phase, and right- and left-atrial pressure during surgery. In membranes of human right atria, AII receptors are present in high density (median: Bmax = 294 fmol.mg-1 protein, range: 111-2073) and two different subtypes can be distinguished. Type-1 receptors (AT1) accounted for 33 +/- 10% of the population whereas type-2 receptors (AT2) made up 67 +/- 10% of the population. There was no correlation between any of the measured cardiac functions and total AII-receptor density or receptor affinity. However, the percentage of AT1 receptors was higher in the atria of patients with normal right-atrial pressure; left-ventricular ejection fraction was positively and right-atrial pressure inversely correlated with the percentage of AT1 receptors (r = 0.740 and -0.901, respectively; P < 0.001, for both). Moreover, the percentage of AT2 receptors was directly correlated with the levels of left-atrial pressure (r = 0.853; P < 0.001). It is concluded that the ratio of AT1 to AT2 receptors correlates well with right-atrial pressure and left-ventricular function. This is a first indication of a possible involvement of AII-receptor subtypes in the pathophysiology of cardiac dysfunctions.

Angiotensin II receptors and angiotensin II stimulation of ciliary activity in human fallopian tube.

Using an antibody (6313/G2) directed against a specific sequence in the extracellular domain of the type 1 angiotensin II receptor (AT1), we demonstrated the presence of angiotensin II (AII) receptors in human fallopian tube. Immunoperoxidase staining for AT1 receptor showed positive staining in the epithelium of the tubal mucosa. The intensity of staining varied depending upon the hormonal status at the time of salpingectomy, being strongest in the proliferative phase of the ovarian cycle and weakest after menopause. Ligand binding assay confirmed that the AII receptor concentration was highest in the mucosa of fallopian tubes from premenopausal women. Mucosa from the ampullary segment had higher concentrations of AII receptor than the fimbrial and isthmic segments in both premenopausal and postmenopausal women. Displacement studies using specific AII receptor subtype antagonists showed that approximately 60% of the total activity could be displaced by CGP42112B (type 2 specific) and 40% by losartan (AT1 specific). Immunoblotting confirmed that the antibody detected a protein of approximately 60 kDa. Functional studies showed that AII had a stimulatory action on tubal ciliary beat frequency, but had no significant effect on myosalpingseal activity. This effect was achieved at nanomolar concentrations of AII; further increases in the AII concentration were without additional effect. The stimulatory effect of AII was inhibited by the specific AT1 antagonist losartan, whereas the type 2 antagonist, CGP42112B, had no effect. The data demonstrate that AII may play an important role in ovum transport and fertility.

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.

Angiotensin II receptor subtypes in the duck subfornical organ: an electrophysiological and receptor autoradiographic investigation

The pharmacology of angiotensin II (AngII) receptors was investigated in the brain of ducks using receptor autoradiographic and electrophysiological methods. Using 125I[Val 5]AngII as a ligand, specific binding was observed in sections of the duck adrenal gland and in several brain areas involved in body fluid homeostasis. Displacement studies using the same antagonists as used for classifying mammalian AngII receptor subtypes revealed that the rank order of potencies in competition with AngII receptors in the adrenal gland and in the subfornical organ was: AngII > CGP-42112A > losartan > PD-123319. Electrophysiological recordings from spontaneously active neurons of duck SFO slices revealed that the majority of neurons could be excited by AngII (10 −7 M). The excitatory effect of AngII could be partially inhibited by CGP-42112A (10 −5 M), which proved to be more effective than equimolar losartan and far more effective than PD-123319. These data suggest that the neuronal AngII receptors in the SFO are pharmacologically distinct from the mammalian AT 1- and AT 2-receptors. Further, central AngII receptors of ducks share common pharmacological characteristics with AngII receptors in the duck adrenal gland and peripheral organs of other bird species.

Molecular cloning of the human AT2 receptor.

The peptide hormone angiotensin II (AII), the biologically active component of the renin-angiotensin system, exerts a wide variety of physiological effects on the cardiovascular, endocrine and central and peripheral nervous system (1). AII exerts its physiological effects in the target tissues through specific interactions with plasma membrane receptors (2). Given the diversity of AII-mediated events and the differential signal transduction mechanisms, it has been proposed that multiple AII receptor subtypes exist (3,4).

Human AT1 receptor gene regulation.

The peptide hormone angiotensin II (AII), the biologically active component of the renin-angiotensin system, regulates a variety of physiological responses including fluid homeostasis, aldosterone production, renal function and contraction of vascular smooth muscle (1). In addition, AII has actions in the central nervous system (2) and may play a role in development (3). AII binds to specific receptors that mediate intracellular Ca2+ mobilization through stimulation of phospholipase C and production of inositol trisphosphate (4), activation of Ca2+ channels (5), and inhibition of adenylate cyclase through a pertussis toxin-sensitive G-protein (6). Given the diversity of AII-mediated events and the differential signal transduction mechanisms, it has been proposed that multiple AII receptor subtypes exist (1,7).

Intracerebroventricular administration of angiotensin type 1 (AT 1) receptor antisense oligonucleotides attenuate thirst in the rat

The central actions of the peptide hormone angiotensin II (AngII) are importantly involved in body fluid homeostasis. Included amongst these actions is a potent dipsogenic response that has been implicated in the thirst that develops during many forms of extracellular dehydration. The use of highly selective receptor antagonists has revealed that the Type 1 (AT 1), and not the Type 2 (AT 2), AngII receptor subtype mediates this drinking response. More recently, antisense oligonucleotides specific for the AT 1 receptor have been developed and after intracerebroventricular (i.c.v.) administration, they significantly reduce the dipsogenic response elicited by a similar injection of AngII. In the present study AT 1 antisense oligonucleotides were used to further investigate their effect on experimentally induced thirst in the rat. In addition, immunohistochemical analysis of biotin-labeled oligonucleotides was performed in order to correlate the behavioral effects of the oligonucleotides with their distribution in the brain. The results demonstrated that the antidipsogenic effects of the oligonucleotides were dose and time-dependent and were limited to those thirst challenges that involve activation of the renin-angiotensin system. Collectively, these results demonstrate the efficacy and behavioral specificity of these oligonucleotides, as well as their utility in investigating the physiological role of cerebral AngII receptor subpopulations in various models of thirst.

Angiotensin II Activates the Na+/HCO3- Symport through a Phosphoinositide-independent Mechanism in Cardiac Cells

Angiotensin II (AngII) is a hormone that alters contractility as well as myocyte growth in heart. Since many hormones that regulate cardiac contractility have also been found to modulate intracellular pH (pHi) the goal of this study was to determine if AngII altered pHi in cultured neonatal rat ventricular myocytes. Changes in pHi were monitored in single cells using the fluorescent pH indicator carboxy-seminaphthorhodafluor-1. Application of 100 nM AngII resulted in a rapid, receptor-mediated alkalinization of 0.08 +/- 0.02 pH unit. The Na+/H+ exchanger was not involved since the response was HCO3(-)-dependent and amiloride-insensitive. Ammonia rebound experiments showed AngII increased the initial rate of recovery from an imposed acid load by 3.15-fold and showed that the hormone led to the selective activation of the Na+/HCO3- symport. In contrast, phorbol ester activation of protein kinase C led to the selective activation of Na+/H+ antiport in these cells. Pharmacological studies showed that the alkalinization was independent of the AngII receptor subtype 1 (AT1) phosphoinositide signaling path. In contrast, AngII activation of the symport was blocked by nanomolar AT2 receptor antagonist PD 123319. Superfusion of the myocytes with exogenous arachidonic acid (5 microM) mimicked the AngII-mediated alkalinization, further suggesting that the AT2 signaling pathway underlies the response. In summary, while most of the known actions of AngII in heart are mediated through AT1 receptors, activation of the Na+/HCO3- symport occurs through a distinct alternative path that is likely related to fatty acid production.

Nitric oxide inhibits angiotensin II-induced migration of rat aortic smooth muscle cell. Role of cyclic-nucleotides and angiotensin1 receptors.

Nitric oxide (NO) and angiotensin II (AII) can effect vascular smooth muscle cell (SMC) proliferation. However, the effects of such agents on SMC migration, an equally important phenomenon with regard to vascular pathophysiology, have received little attention. The objectives of the present study were: (a) to determine whether NO inhibits AII-induced migration of vascular SMCs; (b) to investigate the mechanism of the interaction of NO and AII on SMC migration; and (c) to evaluate the AII receptor subtype that mediates AII-induced SMC migration. Migration of rat SMCs was evaluated using a modified Boydens Chamber (transwell inserts with gelatin-coated polycarbonate membranes, 8 microns pore size). AII stimulated SMC migration in a concentration-dependent manner, and this effect was inhibited by sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP). In the presence of L-arginine, but not D-arginine, IL-1 beta, an inducer of inducible NO synthase, also inhibited AII-induced SMC migration, and this effect was prevented by the NO-synthase inhibitor, N-nitro-L-arginine methyl ester. The effects of NO donors on AII-induced SMC migration were mimicked by 8-bromo-cGMP. Also, the antimigratory effects of SNAP were partially inhibited by LY83583 (an inhibitor of soluble guanylyl cyclase) and by KT5823 (an inhibitor of cGMP-dependent protein kinase). Although 8-bromo-cAMP (cAMP) also mimicked the antimigratory effects of NO donors, the antimigratory effects of SNAP were not altered by 2',5'-dideoxyadenosine (an inhibitor of adenyl cyclase) or by (R)-p-adenosine-3',5'-cyclic phosphorothioate (an inhibitor of the cAMP-dependent protein kinase). Low concentrations of the subtype AT1-receptor antagonist CGP 48933, but not the subtype AT2-receptor antagonist CGP 42112, blocked AII-induced SMC migration. These findings indicate that (a) NO inhibits AII-induced migration of vascular SMCs; (b) the antimigratory effect of NO is mediated in part via a cGMP-dependent mechanism; and (c) AII stimulates SMC migration via an AT1 receptor.

Influence of tissue freezing on the binding of 125I-sarcosine 1, isoleucine 8 angiotensin II to angiotensin II receptor subtypes in the rat

Evaluation of angiotensin II (AII) receptor binding often necessitates freezing of the tissue of interest prior to assay of radioligand binding. This study evaluated the effects of freezing of various rat tissues at different rates on 125I-sarcosine 1, isoleucine 8 angiotensin II ( 125I-SI AII) binding to AII receptor subtypes. Slow freezing in a −20°C compartment significantly reduced 125I-SI AII binding to AT 1 receptors in the adrenal (51%), epididymis (34%), and liver (22%). Binding of 125I-SI AII to the AT 1 receptor in the brain was not significantly reduced. In the adrenal, both the Bmax and affinity of AT 1 receptors were decreased by freezing. But in the epididymis, only the affinity of AT 1 receptors was decreased. Binding of 125I-SI AII to AT 2 receptors in the adrenal, epididymis, and brain was also not significantly reduced by freezing. Further evaluation of the mechanism of the reduction in 125I-SI AII binding to AT 1 receptors in the adrenal indicated that both the receptor density and affinity for 125I-SI AII were decreased by freezing. Rapid freezing in a dry-ice bath caused even greater reductions in 125I-SI AII binding to AT 1 receptors in the adrenal. Snap freezing in liquid nitrogen decreased 125I-SI AII binding in adrenals to a similar extent as did slow freezing. These results suggest that studies of AII receptor subtypes that involve freezing of the tissues underestimate the density and affinity of the AT 1 receptor subtype.

Dissociation of increases in intracellular calcium and aldosterone production induced by angiotensin II (AII): evidence for regulation by distinct AII receptor subtypes or isomorphs.

In mammalian zona glomerulosa cells, angiotensin II (AII)-induced increases in intracellular Ca2+ ([Ca2+]i) and AII-induced aldosterone production seem to be inextricably linked. However, in avian adrenal steroidogenic (adrenocortical) cells studied thus far, inducible aldosterone production seems to be insensitive to alterations in the mobilization of cellular Ca2+. This raises the hypothesis that alternative signal transduction pathways are implemented to induce aldosterone production in avian adrenocortical cells. In the present study, this hypothesis was investigated by using isolated turkey (Meleagris gallopavo) adrenocortical cells that are known to be three times more sensitive to AII than to ACTH for aldosterone production. In isolated turkey adrenocortical cells, the mammalian AII receptor antagonist, [Sar1,Ile8]AII, was as efficacious as [Ile5]AII in stimulating aldosterone production, albeit it had about 1/150 the potency of [Ile5]AII. The actions of both analogs required extracellular K+, suggesting a voltage-sensitive event. However, a maximal aldosteronogenic concentration of [Sar1,Ile8]AII not only failed to increase [Ca2+]i but also completely blocked maximal (10(-8) M)[Ile5]AII-induced increases in [Ca2+]i when added before [Ile5]AII and partially dampened (approximately 50%) maximal [Ile5]AII-induced increases in [Ca2+]i when added after (3 min) [Ile5]AII. This blockade in [Ca2+]i elevation was surmounted by high concentrations of [Ile5]AII (> 10(-6) M). By contrast, [Sar1,Ile8]AII did not alter maximal aldosterone production induced by [Ile5]AII and vice versa, thus suggesting that the action of both analogs converged on the same aldosteronogenic pathway, and that AII-induced aldosterone production was not coupled to elevations in [Ca2+]i. Detailed homologous-heterologous ligand-binding analyses supported the presence of two AII-binding sites that were discriminated by [Sar1,Ile8]AII (dissociation constants, 4.2 +/- 1.4 and 21.9 +/- 2.2 nM; concentration distribution, approximately 40% and approximately 60%, respectively; mean +/- SE, n = 4) but not by [Ile5]AII (dissociation constant, 2.1 +/- 0.1 nM for both sites). In addition, [Sar1,Ile8]AII- and [Ile5]AII-binding sites exhibited different physicochemical and pharmacological properties. The sensitivity of [Sar1,Ile8]AII-binding sites was about twice that of [Ile5]AII-binding sites to dithiothreitol. In addition, whereas both the high- and low-affinity sites detected by [Sar1,Ile8] AII exhibited equivalent competitive sensitivities to the type-1 receptor, the nonpeptidic antagonist, losartan (DuP 753), the sensitivity of the low-affinity site was 2.7 times that of the high-affinity site to the type-2 receptor, nonpeptidic antagonist, PD123319. Taken collectively, the data suggest that in turkey adrenocortical cells, elevations in [Ca2+]i and aldosterone production are dissociable events regulated by distinct AII receptor subtypes or isomorphs.

Dissociation of increases in intracellular calcium and aldosterone production induced by angiotensin II (AII): evidence for regulation by distinct AII receptor subtypes or isomorphs

Dissociation of increases in intracellular calcium and aldosterone production induced by angiotensin II (AII): evidence for regulation by distinct AII receptor subtypes or isomorphs

Regulation of gene transcription of angiotensin II receptor subtypes in myocardial infarction.

Increasing evidence suggests that angiotensin II (AngII) acts as a modulator for ventricular remodeling after myocardial infarction. Using competitive reverse-transcriptase polymerase chain reaction, nuclear runoff, and binding assays, we examined the regulation of AngII type 1a and 1b (AT1a-R and AT1b-R) and type 2 receptor (AT2-R) expression in the infarcted rat heart as well as the effects of AngII receptor antagonists. AT1a-R mRNA levels were increased in the infarcted (4.2-fold) and noninfarcted portions (2.2-fold) of the myocardium 7 d after myocardial infarction as compared with those in sham-operated controls, whereas AT1b-R mRNA levels were unchanged. The amount of detectable AT2-R mRNA increased in infarcted (3.1-fold) and noninfarcted (1.9-fold) portions relative to that in the control. The transcription rates for AT1a-R and AT2-R genes, determined by means of a nuclear runoff assay, were significantly increased in the infarcted heart. The AngII receptor numbers were elevated (from 12 to 35 fmol/mg protein) in the infarcted myocardium in which the increases in AT1-R and AT2-R were 3.2- and 2.3-fold, respectively, while the receptor affinity was unchanged. Therapy with AT1-R antagonist for 7 d reduced the increase in AT1-R and AT2-R expressions in the infarcted heart together with a decrease in blood pressure, whereas therapy with an AT2-R antagonist did not affect mRNA levels and blood pressure. Neither AT1-R nor AT2-R antagonists affected the infarct sizes. These results demonstrated that myocardial infarction causes an increase in the gene transcription and protein expression of cardiac AT1a-R and AT2-R, whereas the AT1b-R gene is unaffected, and that therapy with an AT1-R antagonist, but not with an AT2-R antagonist, is effective in reducing the increased expression of AngII receptor subtypes induced by myocardial infarction.

Stable expression of a truncated AT1A receptor in CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling or desensitization.

Phosphorylation of serine and threonine residues in the carboxyl-terminal region of many G-protein-coupled receptors directs the rapid uncoupling from signal transduction pathways. In Chinese hamster ovary cells, we have stably expressed a truncated mutant of the angiotensin II (AT1A) receptor devoid of the carboxyl-terminal 45 amino acids, encompassing 13 serine/threonine residues. One clone, designated TL314 to indicate truncation after leucine 314, expressed a single class of angiotensin II receptors with a dissociation constant of 1.08 nM and a receptor density of 560 fmol/mg of protein (approximately 75,000 receptors/cell). A nonhydrolyzable analog of GTP accelerated the angiotensin II-induced dissociation of [125I]angiotensin II from TL314 plasma membranes 3.6-fold, indicating G-protein coupling. In TL314 cells, angiotensin II stimulated the release of intracellular calcium and the induction of mitogen-activated protein kinase activity, the level of which were comparable with the full-length AT1A receptor. The AII-stimulated calcium response was rapidly desensitized in both full-length and truncated AT1A receptors. Interestingly, angiotensin II-induced endocytosis of the truncated receptor was almost completely inhibited, suggesting that a recognition motif within the carboxyl-terminal 45 amino acids of the AT1A receptor promotes sequestration. Thus, truncation of the AT1A receptor after leucine 314 inhibits agonist-induced internalization without affecting the capacity of the expressed protein to adopt the correct conformation necessary for high affinity binding of angiotensin II, coupling to G-proteins, and activation of signal transduction pathways. The rapid desensitization and refractoriness of the angiotensin II-induced calcium transient in the TL314 cell line, in which putative carboxyl-terminal phosphorylation sites are absent, suggests that the mechanism of AT1A receptor desensitization differs from that of other prototypical G-protein-coupled receptors.

AT1 receptors and angiotensin actions in the brain and neuronal cultures of normotensive and hypertensive rats.

It is now widely accepted that a functional renin-angiotensin system is intrinsic to the brain and is of great physiological and pathophysiological importance. Over the past twenty-five years evidence has been mounting that demonstrates the profound effects of angiotensin II (AII) on fluid balance and cardiovascular regulation. Activation by AII of specific neuronal receptors localized on the cardioregulatory-relevant areas of the brain, is the key step in a cascade of cellular events that ultimately lead to the increase in blood pressure (BP) and modulation of baroreceptor function. With this review we will discuss: 1) the physiological evidence supporting the role of brain AII in BP control; 2) localization of AII receptor subtypes in the brain; 3) evidence demonstrating the usefulness of an in vitro neuronal cell culture system to study the cellular, molecular and genetic basis of the brain AII dependent-hypertensive state; and 4) our current understandings of the cellular mechanism of AII actions in the brain with the use of this in vitro neuronal culture system.

Angiotensin receptors in cardiovascular diseases.

1. Angiotensin II (AII) plays a major role in cardiovascular function via direct actions on the vasculature, kidney, adrenal, heart, brain and sympathetic nerves. The cellular effects of AII are extensive and encompass hypertrophy, hyperplasia and the deposition of extracellular matrix. 2. The actions of AII are mediated by the AT1 and AT2 membrane receptor subtypes, and additional forms of each subtype. Evidence is emerging that selective changes in AII receptor subtypes occur in cardiovascular diseases. 3. Thyroid dysfunction increased cardiac, liver and kidney AII receptor density but decreased adrenal gland receptor density. In the heart, there was a selective increase in AT2 receptor density. 4. Diabetes increased cardiac, liver and adrenal gland AII receptor densities but decreased kidney receptor density. 5. Hypertension increased AII receptor density in the heart and kidney. A corresponding increase in receptor mRNA was prevented by selective AT1 receptor antagonists. 6. The human heart contained AII receptors in all chambers; right atrial receptor density was increased in coronary artery bypass graft patients. 7. The presence of AII receptor changes in these models of cardiac hypertrophy and hypertension raises the possibility of using orally active, subtype-selective agonists and antagonists to treat particular forms of cardiovascular diseases.