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 (AT₁) and type 2 (AT₂) 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 AT₁ receptor modulates all the known biological effects of AngII. The expression of the AT₁ 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 AT₁ 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 AT₁ 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 AT₁ expression in APA showed no significant differences compared with normal and nontumorous adrenal. In addition, no mutations in the coding sequence of the AT₁ receptor have been found to date in adrenal tumours.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: MOLECULAR MECHANISMS OF ANGIOTENSIN II (AT1a) RECEPTOR ENDOCYTOSIS

• 1 Angiotensin II (AngII) initiates a variety of cellular responses through activation of type 1 (AT₁; with subtypes AT_1a and AT_1b) and type 2 (AT₂) cell surface angiotensin receptors. Both AT₁ and AT₂ receptors couple to heterotrimeric guanyl nucleotide binding proteins (G-proteins) and generate intracellular signals following recognition of extracellular AngII, but only AT₁ is targeted for the rapid ligand-stimulated endocytosis (internalization) typical of many plasma membrane receptors.
• 2 AT₁ endocytosis proceeds through clathrin-coated pits and is independent of G-protein coupling which predicts that the AngII-AT₁ receptor complex attains a conformation necessary for interaction with the endocytotic machinery, but separate from receptor signalling activation.
• 3 The function of AT₁ endocytosis and the reason for the disparity between AT₁ and AT₂ endocytosis is not fully appreciated, but the latter probably reflects differences in the primary amino acid sequence of these two receptor types.
• 4 For many receptors that undergo internalization, it has been established that internalization motifs (2–6 amino acids, often incorporating crucial tyrosine and hydrophobic amino acids) within the cytoplasmic regions of the receptor mediate the selective recruitment of activated receptors into clathrin-coated pits and vesicles.
• 5 Mutagenesis studies on the AT_1a receptor, aimed at identifying such motifs, reveal that sites within the third cytoplasmic loop and the cytoplasmic carboxyl terminal region are important for AngII-stimulated AT_1a receptor endocytosis.

     

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

• 1 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.
• 2 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.
• 3 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.
• 4 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.
• 5 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.
• 6 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.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: ANGIOTENSIN RECEPTORS AND DEVELOPMENT: THE KIDNEY

• 1 This brief review examines the evidence that angiotensin II (AngII) is essential for kidney development.
• 2 Several components of the renin-angiotensin system (RAS) are detected in the foetal kidney early in development.
• 3 Angiotensin II is essential for normal foetal and neonatal renal function.
• 4 Angiotensin II receptors transduce important signals leading to growth and development.
• 5 Angiotensin receptor subtypes show spatial and temporal specificity of localization throughout renal development.
• 6 Angiotensin converting enzyme (ACE) inhibition or AngII receptor blockade (specifically AT₁ subtype blockade) results in functional and structural abnormalities of the developing kidney in both experimental and clinical situations.
• 7 While chronic postnatal RAS blockade in rats is associated with structural damage to tubules and blood vessels of the kidney, reports differ on whether treatment also affects glomerular induction and growth.
• 8 In metanephric organ culture, glomerular induction proceeds despite AngII receptor blockade.
• 9 In summary, the evidence suggests that AngII is not essential for nephron induction and glomerular development in the rat kidney. However, AngII is essential for normal growth and development of renal tubules and vasculature.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: ENDOGENOUS ANGIOTENSIN II LEVELS AND THE MECHANISM OF ACTION OF ANGIOTENSIN-CONVERTING ENZYME INHIBITORS AND ANGIOTENSIN RECEPTOR TYPE 1 ANTAGONISTS

• 1 Modification of endogenous angiotensin II (AngII)-mediated processes by inhibitors of the angiotensin-converting enzyme (ACE) and antagonists of the angiotensin type 1 (AT₁) receptor is dependent upon both the levels of each agent in the plasma and tissues and on the concomitant changes in plasma and tissue AngII levels.
• 2 Both ACE inhibitors and AT₁ receptor antagonists increase renin secretion and angiotensin peptide formation in plasma and extrarenal tissues. Clinical doses of ACE inhibitors produce incomplete inhibition of ACE and the increased AngI levels act to restore AngII towards basal levels. Clinical doses of AT₁ receptor antagonists produce incomplete blockade of AT₁ receptors and the increased AngII levels in plasma and extrarenal tissues counteract (to an unknown degree) the effects of the antagonist.
• 3 The effects of ACE inhibitors and AT₁ receptor antagonists on AngII levels show tissue specificity. Angiotensin II-mediated processes in the kidney are most sensitive to inhibition by these agents. ACE inhibitors reduce renal AngII levels at doses much less than those required to reduce AngII levels in plasma and other tissues. Moreover, in contrast to increased AngII levels in plasma and extrarenal tissues, renal AngII levels do not increase in response to AT₁ receptor antagonists. The inhibition of AngII-mediated processes in the kidney may, therefore, play a primary role in mediating the effects of ACE inhibitors and AT₁ receptor antagonists on blood pressure and other aspects of cardiovascular function and structure.
• 4 Combination of an ACE inhibitor with an AT₁ receptor antagonist prevents the rise in plasma AngII levels that occurs with AT₁ receptor antagonism alone. This combination would, therefore, be predicted to produce more effective inhibition of endogenous AngII-mediated processes than either agent alone. We must await further studies to determine whether the combination of ACE inhibition and AT₁ receptor antagonism results in superior clinical outcomes.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: PHYSIOLOGICAL ACTIONS OF ANGIOTENSIN II MEDIATED BY AT1 AND AT2 RECEPTORS IN THE BRAIN

• 1 Autoradiographic binding studies have shown that the AT₁ receptor is the predominant angiotensin II (AngII) receptor subtype in the central nervous system (CNS). Major sites of AT₁ receptors are the lamina terminalis, hypothalamic paraventricular nucleus, the lateral parabrachial nucleus, rostral and caudal ventrolateral medulla, nucleus of the solitary tract and the intermediolateral cell column of the thoraco-lumbar spinal cord.
• 2 While there are differences between species, AT₂ receptors are found mainly in the cerebellum, inferior olive and locus coeruleus of the rat.
• 3 Circulating AngII acts on AT₁ receptors in the subfornical organ and organum vasculosum of the lamina terminalis (OVLT) to stimulate neurons that may have a role in initiating water drinking.
• 4 Centrally administered AngII may act on AT₁ receptors in the median preoptic nucleus and elsewhere to induce drinking, sodium appetite, a sympathetic vasoconstrictor response and vasopressin secretion.
• 5 Recent evidence shows that centrally administered AT₁ antagonists inhibit dipsogenic, natriuretic, pressor and vasopressin secretory responses to intracerebroventricular infusion of hypertonic saline. This suggests that an angiotensinergic neural pathway has a role in osmoregulatory responses.
• 6 Central angiotensinergic pathways which include neural inputs to the rostral ventrolateral medulla may use AT₁ receptors and play a role in the function of sympathetic pathways maintaining arterial pressure.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: MOLECULAR DETERMINANTS OF PEPTIDE AND NON-PEPTIDE BINDING TO THE AT1 RECEPTOR

• 1 Several residues critically involved in AT₁ receptor ligand-binding and activation have now been identified based on mutational and biochemical studies.
• 2 Asp²⁸¹ and Lys¹⁹⁹ of the rat AT₁ receptor ion-pair with Arg² and the Phe³ α-COOH of angiotensin II (AngII), respectively, and the Asp²⁸¹/Arg² interaction is critical for full agonist activity.
• 3 Agonist activity of AngII also requires an interaction of the Phe² side chain with His²⁵⁶, which is achieved by docking of the α-COOH with Lys¹⁹⁹. Non-peptide agonists interact with Lys¹⁹⁹ and His²⁵⁶ in a similar fashion.
• 4 The crucial acid pharmacophores of AngII and the non-peptide antagonist, Iosartan, appear to occupy the same space within the receptor pocket. Binding of the tetrazole anion moiety of losartan involves multiple contacts, such as Lys¹⁹⁹ and His²⁵⁶. However, this interaction does not involve a conventional salt bridge, but rather an unusual lysine-aromatic interaction.
• 5 Asp¹ of AngII forms an ion-pair with His¹⁸³, which stabilizes the receptor-bound conformation of AngII but is not critical for receptor activation.
• 6 These interactions and the involvement of other residues in stabilizing the wild-type receptor conformation or in receptor/G-protein coupling are considered here.
• 7 Despite these insights, considerable effort is still needed to elucidate how ligand binding induces receptor activation, what determines the specificity of AT₁ receptor coupling to multiple G-proteins and the in vivo role of receptor down-regulation.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: EXPERIENCE WITH ANGIOTENSIN II ANTAGONISTS IN HYPERTENSIVE PATIENTS

• 1 The availability of orally active specific angiotensin receptor antagonists (AT₁ antagonists) has opened new therapeutic choices and provided probes to test the specific role of the renin-angiotensin system in the pathogenesis of cardiovascular disease.
• 2 The data available so far suggest that the antihypertensive efficacy of angiotensin receptor antagonists is comparable to that of angiotensin-converting enzyme (ACE) inhibitors. This provides further evidence that this latter class of drugs exerts its effect mainly through blockade of the renin-angiotensin enzymatic cascade. As expected, the association of a diuretic exerts an equally strong additive effect to the antihypertensive efficacy of both classes of drugs.
• 3 The most common side effect of ACE inhibitors, dry cough, does not occur with AT₁ antagonists, which confirms the long-held view that this untoward effect of the ACE inhibitors is due to renin-angiotensin-independent mechanisms.
• 4 Long-term studies with morbidity/mortality outcome results are needed, before a definite position can be assigned to this newcomer in the orchestra of modern antihypertensive drugs. Notwithstanding, this new class of agents already represents an exciting new addition to our therapeutic armamentarium.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: REGULATION OF RENAL TUBULAR SODIUM TRANSPORT BY ANGIOTENSIN II AND ATRIAL NATRIURETIC FACTOR

• 1 The effects of angiotensin II (AngII) on water and electrolyte transport are biphasic and dose-dependent, such that low concentrations (10^?12 to 10^?9 mol/L) stimulate reabsorption and high concentrations (10^?7 to 10^?6 mol/L) inhibit reabsorption. Similar dose-response relationships have been obtained for luminal and peritubular addition of AngII.
• 2 The cellular responses to AngII are mediated via AT₁ receptors coupled via G-regulatory proteins to several possible signal transduction pathways. These include the inhibition of adenylyl cyclase, activation of phospholipases A₂, C or D and Ca²⁺ release in response to inositol-1,4,5,-triphosphate or following Ca²⁺ channel opening induced by the arachidonic acid metabolite 5,6,-epoxy-eicosatrienoic acid. In the brush border membrane, transduction of the AngII signal involves phospholipase A₂, but does not require second messengers.
• 3 Angiotensin II affects transepithelial sodium transport by modulation of Na⁺/H⁺ exchange at the luminal membrane and Na⁺/HCO₃ cotransport, Na⁺/K⁺-ATPase activity and K⁺ conductance at the basolateral membrane.
• 4 Atrial natriuretic factor (ANF) does not appear to affect proximal tubular sodium transport directly, but acts via specific receptors on the basolateral and brush border membranes to raise intracellular cGMP levels and inhibit AngII-stimulated transport.
• 5 It is concluded that there is a receptor-mediated action of ANF on proximal tubule reabsorption acting via elevation of cGMP to inhibit AngII-stimulated sodium transport. This effect is exerted by peptides delivered at both luminal and peritubular sides of the epithelium and provides a basis for the modulation by ANF of proximal glomerulotubular balance. The evidence reviewed supports the concept that in the proximal tubule, AngII and ANF act antagonistically in their roles as regulators of extracellular fluid volume.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: CYTOKINES AND CARDIAC HYPERTROPHY: ROLES OF ANGIOTENSIN II AND BASIC FIBROBLAST GROWTH FACTOR

• 1 While the haemodynamic influences that cause cardiac hypertrophy are well known, the cellular and molecular mechanisms by which a mechanical stimulus is translated into a growth response by cardiac muscle have remained uncertain.
• 2 Current evidence suggests that a number of trophic factors may be released by cellular constituents of the heart, acting in an autocrine or paracrine manner to influence the growth response and phenotype of neighbouring cells.
• 3 Angiotensin II, acting via the AT₁ receptor subtype, and both basic fibroblast growth factor and heparin-binding epidermal growth factor have been shown to exert hypertrophic actions in vivo and in vitro. Studies also indicate that cardiac myocytes themselves are capable of releasing all of these cytokines in response to increased mechanical load.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: ROLE OF AT1 RECEPTORS IN THE CENTRAL CONTROL OF SYMPATHETIC VASOMOTOR FUNCTION

• 1 In a number of species, high concentrations of angiotensin II (AngII) receptors have been found in the rostral ventrolateral medulla (RVLM) in the hindbrain, which is an important region involved in the modulation of sympathetic vasomotor tone. The present review describes studies in which the contribution of angiotensin receptors in the brainstem to cardiovascular regulation, in particular sympathetic vasomotor reflexes, has been examined in conscious and anaesthetized rabbits.
• 2 In conscious rabbits, fourth ventricular infusions of AngII produced dose-dependent pressor responses as doses 400 times less than equipressor intravenous doses. Chronic baroreceptor denervation increased the sensitivity to AngII by 1000-fold. Administration of prazosin i.v. blocked the pressor response, suggesting that the mechanism involved sympathetic vasoconstriction.
• 3 The pattern of haemodynamic changes in response to AngII injected into the fourth ventricle (4V) involved decreased total peripheral conductance and mesenteric conductance, but a rise in hindlimb conductance. Sinoaortic denervation changed the hindlimb fall in conductance to an increase, suggesting that muscle vasomotor pathways were particularly inhibited by baroreceptor feedback mechanisms.
• 4 In anaesthetized rabbits, infusion of AngII into the RVLM increased blood pressure and transiently increased resting renal sympathetic nerve activity. The renal sympathetic baroreflex curves were shifted to the right and the upper plateau of the sympathetic reflex increase was markedly increased.
• 5 The pressor actions of 4V AngII were blocked by administration of a peptide antagonist injected into the RVLM or by the angiotensin AT₁ antagonist losartan injected into the 4V. These results suggest that mainly AT₁ receptors are involved and that the RVLM is a likely candidate site for the modulation of the renal sympathetic baroreflex.
• 6 Losartan administration into the 4V in conscious rabbits increased resting renal sympathetic tone and enhanced renal sympathetic baroreflex and chemoreflexes.
• 7 Our studies suggest that there are sympathoexcitatory AT₁ receptors in the RVLM accessible to AngII from the cerebrospinal fluid. In addition, an AT₁ receptor pathway normally inhibits the sympathoexcitation produced by baroreceptor unloading or chemoreceptor activation. The effect of losartan suggests that there is greater tonic activity within the sympathoinhibitory pathways. These two actions suggest that angiotensin receptors in the brainstem modulate sympathetic responses to specific afferent inputs, thus forming part of a potentially important mechanism for the integration of characteristic autonomic response patterns.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: FUNCTIONS OF ANGIOTENSIN PEPTIDES IN THE ROSTRAL VENTROLATERAL MEDULLA

• 1 It was first shown several years ago that the rostral part of the ventrolateral medulla (VLM) contains a high density of receptor binding sites for angiotensin II (AngII). In the present paper we briefly review recent studies aimed at determining the actions of both exogenous and endogenous angiotensin peptides in the rostral VLM, as well as their specific sites of action.
• 2 The results of these studies have shown that angiotensin peptides can excite pressor and sympathoexcitatory neurons in the rostral VLM, but do not appear to affect non-cardiovascular neurons in this region.
• 3 It is known that pressor neurons in the rostral VLM include both catecholamine and non-catecholamine neurons. There is evidence that, at least in conscious rabbits, both of these types of neurons are activated by AngII. The specific endogenous angiotensin peptide or peptides that affect pressor neurons in the rostral VLM have not yet been definitively identified.
• 4 It is also possible that different angiotensin peptides may have different effects on pressor neurons in the rostral VLM, mediated by different receptors. Further studies will be needed to define these different functions as well as the specific receptors and cellular mechanisms that subserve them.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: STRUCTURAL CHANGES IN THE RENAL VASCULATURE IN THE SPONTANEOUSLY HYPERTENSIVE RAT: NO EFFECT OF ANGIOTENSIN II BLOCKADE

• 1 There is strong evidence for a renal basis to the development of hypertension in the spontaneously hypertensive rat (SHR). Alterations of the SHR renal vasculature, including the glomerulus, may be involved in the initiation and maintenance of hypertension in this animal model.
• 2 The arterial walls of pre-glomerular vessels of the SHR are hypertrophied compared with WKY vessels. Unlike other vascular beds in the SHR, this hypertrophy is independent of angiotensin II (AngII).
• 3 Glomerular number and volume are similar between SHR and the normotensive Wistar-Kyoto (WKY) rats. These results provide no support for the theory that a reduced filtration surface area within the kidneys of the SHR contributes to the elevated blood pressure in these animals.
• 4 Intrarenal hypertrophy may have similar haemodynamic consequences to clipping of the main renal artery, as in Goldblatt hypertension. Further analysis of the role of pre-glomerular arterial hypertrophy is warranted to determine its involvement in the initiation and maintenance of hypertension in the SHR.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: TRANSGENIC RATS: TOOLS TO STUDY THE FUNCTION OF THE RENIN-ANGIOTENSIN SYSTEM

• 1 The development of the transgenic technology for the rat allowed the evaluation of gene functions in the cardiovascular system in vivo. New insights have been gained particularly in the functions of the renin-angiotensin system (RAS), as most transgenic rat models established so far carry genes of this system.
• 2 TGR(mREN2)27 is a rat harbouring the mouse Ren-2 gene and exhibiting fulminant hypertension. The plasma RAS in this animal is down-regulated; however, the tissue-specific production of angiotensin II is activated (e.g. in the adrenal gland, the brain and the vessel wall). The physiological consequences of this activation, which finally leads to hypertension, can be studied in TGR(mREN2)27, rendering it a valuable tool in the functional analysis of tissue RAS.
• 3 TGR(hREN) and TGR(hAOGEN) carry the human genes for renin and angiotensinogen, respectively. In these animals the species-specific interaction of the two proteins and the expression pattern of the genes can be studied. Furthermore, these animals can be used to test renin-inhibitory drugs for use in antihypertensive therapy.
• 4 Further refinement of transgenic methodology (e.g. by the development of gene targeting in rats), should enhance our understanding of the functions of the RAS in cardiovascular regulation.