AT Receptor Binding Research Articles

Adapter proteins and promoter regulation of the angiotensin AT2 receptor — implications for cardiac pathophysiology

The angiotensin AT( 2) receptor (AT(2)R) represents an important component of the renin-angiotensin system since it is involved in the (patho) physiology of different cardiovascular and neuronal diseases. Furthermore, AT(2) receptors can partly mediate beneficial effects of angiotensin AT( 1) receptor (AT(1)R) blockers, and direct pharmacological AT( 2) receptor agonism emerges as a novel therapeutic strategy. This review discusses the constitutive and ligand-mediated activity as well as the signal transduction of the AT(2) receptor, focusing on adapter proteins which directly bind to this receptor. Direct protein-protein interaction partners of the AT(2) receptor described so far include the transcription factor promyelocytic zinc finger protein, AT(2) receptor binding protein and the AT(1) receptor. In addition, the putative crosstalk of the AT(2) receptor with the renin/ prorenin receptor (RER) via the promyelocytic zinc finger protein (PLZF) and the role of oestrogens on the regulation of the AT(2) receptor are presented. Conceiving the coupling of the AT(2) receptor to different adapter proteins with distinct and partly opposing cellular effects and the implications of its constitutive activity might help to overcome the current controversies on the (patho)physiological role of the AT(2) receptor.

High blood pressure in aged Fischer 344 X Brown Norway F1 rats is associated with increased oxidative stress, NFkB activation and exaggerated renal AT1R response to Ang II

Aging is characterized by a progressive decline in physiological functions. Although the underlying mechanisms are not well understood, increasing number of evidence points towards oxidative stress as one of the primary determinants of aging. In this study, we compared 3 month‐ (adult) and 21 month‐ (aged) old Fischer 344 X Brown Norway F1 rats for age‐related changes in kidney function. We measured blood pressure, sodium potassium ATPase activity in proximal tubules, AT1 receptor binding in proximal tubular membranes, NFkB transcription factor in nuclear fractions and oxidative stress markers such as protein carbonyls and urinary 8‐isoprostane. Our results showed an increase in systolic, diastolic and mean arterial blood pressure in aged animals. In response to Ang II, there was increased stimulation of sodium potassium ATPase in aged animals accompanied by increased AT1R binding. Protein carbonyls and 8‐isoprostane were also increased in aged animals. We also found increased NFkB in nuclear fractions from aged animals. While Ang II activates NFkB through AT1 receptors, NFkB has putative response elements on AT1 receptor promoter. Therefore, it appears that age‐related increase in oxidative stress increased renal AT1R function via activating NFkB. This may have caused increased sodium retention and hypertension in aged animals (NIH/NIA AG25056, AG29904).

Design, synthesis, and docking studies of telmisartan analogs for the treatment of metabolic syndrome

In our early studies, telmisartan was found to be a moderate peroxisome proliferator-activated receptor (PPAR) gamma activator in the human PPARγ-GAL-4 cell-based transactivation assay. Thus, novel analogs of telmisartan were designed, synthesized, and evaluated in the AT1 receptor binding assay and PPAR gamma transactivation assay. A total of 11 compounds were designed based on docking in both AT1 receptor model and PPAR gamma active pocket and synthesized. Introduction of an additional acidic group at the para position of the distal phenyl ring of telmisartan decreased affinity towards AT1 receptor and PPARγ activity. In the present study, the molecule with best results was MT003 with weak PPARγ activity (8% of maximum PPARγ activation achieved by full agonist rosiglitazone at 10 μM) and good binding affinity (Ki = 650 ± 139 nM) towards the AT1 receptor. Docking of MT003 into AT1 receptor model and PPAR gamma showed very similar interactions with the receptors as AT1 antagonist telmisartan and PPAR gamma agonist rosiglitazone.

Variety of Angiotensin Receptors in 3T3-L1 Preadipose Cells and Differentiated Adipocytes

A local paracrine acting angiotensin (ANG) system of preadipocytes and mature adipocytes is involved in metabolic effects and tissue differentiation. The present study reports on the investigation of binding affinities for various angiotensin receptors including their relevance in 3T3-L1 adipocytes and preadipocytes and 3T3-442A preadipocytes. Competitive binding studies using both 125I-ANG II and its more stable analogue 125I-SARILE for investigating AT1/AT2 binding sites in 3T3-L1 preadipocytes reveal a biphasic competition curve with KDs at a low and high nanomolar range. By using the AT2 receptor selective ligand 125I-CGP4112A the presence of high affinity AT2 binding sites in preadipocytes was observed. High nonspecific binding and a low receptor number is characteristic for all these experiments. An AT4 binding site (binding site for ANG IV) exists in 3T3-L1 and F442A preadipocytes and adipocytes with a high nanomolar KD. This low binding affinity was confirmed by a biological assay, the IRAP assay (=insulin regulated aminopeptidase assay). IRAP is associated with the AT4 receptor, which is a binding site at the luminal part of membrane bound IRAP. The curves for competition binding and for inhibition of IRAP activity are superimposable with respect to angiotensin IV. In conclusion, AT1 and AT2 binding sites are present in preadipocytes. AT2 receptor binding affinities are shown in preadipocytes for the first time. The description of a non-AT1/AT2 binding site with low affinity remains speculative albeit of high interest because antidiabetic and obesity related effects of angiotensin peptides and sartanes as antagonists are observed at these high concentrations. Local concentrations of ANG II and their degradation products may be extremely high. The low amounts of AT1 and AT2 binding sites emphasize the relevance of other binding sites in adipose tissue development and metabolic effects. The AT4 binding site seems to be one of the predominant receptors in adipose cells. Other degraded, but still bioactive peptides like ANG III, IV and ANG(1-7), activating receptors not influenced by ANG II, could be of importance.

Angiotensin II AT1 receptor blockade selectively enhances brain AT2 receptor expression, and abolishes the cold-restraint stress-induced increase in tyrosine hydroxylase mRNA in the locus coeruleus of spontaneously hypertensive rats

Spontaneously hypertensive rats, a stress-sensitive strain, were pretreated orally for 14 days with the AT1 receptor antagonist candesartan before submission to 2 h of cold-restraint stress. In non-treated rats, stress decreased AT1 receptor binding in the median eminence and basolateral amygdala, increased AT2 receptor binding in the medial subnucleus of the inferior olive, decreased AT2 binding in the ventrolateral thalamic nucleus and increased tyrosine hydroxylase mRNA level in the locus coeruleus. In non-stressed rats, AT1 receptor blockade reduced AT1 receptor binding in all areas studied and enhanced AT2 receptor binding in the medial subnucleus of the inferior olive. Candesartan pretreatment produced a similar decrease in brain AT1 binding after stress, and prevented the stress-induced AT2 receptor binding decrease in the ventrolateral thalamic nucleus. In the locus coeruleus and adrenal medulla, AT1 blockade abolished the stress-induced increase in tyrosine hydroxylase mRNA level. Our results demonstrate that oral administration of candesartan effectively blocked brain AT1 receptors, selectively increased central AT2 receptor expression and prevented the stress-induced central stimulation of tyrosine hydroxylase transcription. The present results support a role of brain AT1 and AT2 receptors in the regulation of the stress response, and the hypothesis that AT1 receptor antagonists may be considered as potential therapeutic compounds in stress related disorders in addition to their anti-hypertensive properties.

Angiotensin II Inhibits the ROMK-like Small Conductance K Channel in Renal Cortical Collecting Duct during Dietary Potassium Restriction

Base-line urinary potassium secretion in the distal nephron is mediated by small conductance rat outer medullary K (ROMK)-like channels. We used the patch clamp technique applied to split-open cortical collecting ducts (CCDs) isolated from rats fed a normal potassium (NK) or low potassium (LK) diet to test the hypothesis that AngII directly inhibits ROMK channel activity. We found that AngII inhibited ROMK channel activity in LK but not NK rats in a dose-dependent manner. The AngII-induced reduction in channel activity was mediated by AT1 receptor (AT1R) binding, because pretreatment of CCDs with losartan but not PD123319 AT1 and AT2 receptor antagonists, respectively, blocked the response. Pretreatment of CCDs with U73122 and calphostin C, inhibitors of phospholipase C (PLC) and protein kinase C (PKC), respectively, abolished the AngII-induced decrease in ROMK channel activity, confirming a role of the PLC-PKC pathway in this response. Studies by others suggest that AngII stimulates an Src family protein-tyrosine kinase (PTK) via PKC-NADPH oxidase. PTK has been shown to regulate the ROMK channel. Inhibition of NADPH oxidase with diphenyliodonium abolished the inhibitory effect of AngII or the PKC activator phorbol 12-myristate 13-acetate on ROMK channels. Suppression of PTK by herbimycin A significantly attenuated the inhibitory effect of AngII on ROMK channel activity. We conclude that AngII inhibits ROMK channel activity through PKC-, NADPH oxidase-, and PTK-dependent pathways under conditions of dietary potassium restriction.

Activation of brain renin-angiotensin-aldosterone system by central sodium in Wistar rats

Functional studies indicate that the sympathoexcitatory and pressor responses to an increase in cerebrospinal fluid (CSF) [Na+] by central infusion of Na+-rich artificial cerebrospinal fluid (aCSF) in Wistar rats are mediated in the brain by mineralocorticoid receptor (MR) activation, ouabain-like compounds (OLC), and AT1-receptor stimulation. In the present study, we examined whether increasing CSF [Na+] by intracerebroventricular infusion of Na+-rich aCSF activates MR and thereby increases OLC and components of the renin-angiotensin system in the brain. Male Wistar rats received via osmotic minipump an intracerebroventricular infusion of aCSF or Na+-rich aCSF, in some groups combined with intracerebroventricular infusion of spironolactone (100 ng/h), antibody Fab fragments (to bind OLC), or as control gamma-globulins. After 2 wk of infusion, resting blood pressure and heart rate were recorded, OLC and aldosterone content in the hypothalamus were assessed by a specific ELISA or radioimmunoassay, and angiotensin-converting enzyme (ACE) and AT1-receptor binding densities in various brain nuclei were measured by autoradiography using 125I-labeled 351 A and 125I-labeled ANG II. When compared with intracerebroventricular aCSF, intracerebroventricular Na+-rich aCSF increased CSF [Na+] by approximately 5 mmol/l, mean arterial pressure by approximately 20 mmHg, heart rate by approximately 65 beats/min, and hypothalamic content of OLC by 50% and of aldosterone by 33%. Intracerebroventricular spironolactone did not affect CSF [Na+] but blocked the Na+-rich aCSF-induced increases in blood pressure and heart rate and OLC content. Intracerebroventricular Na+-rich aCSF increased ACE and AT1-receptor-binding densities in several brain nuclei, and Fab fragments blocked these increases. These data indicate that in Wistar rats, a chronic increase in CSF [Na+] may increase hypothalamic aldosterone and activate CNS pathways involving MR, and OLC, leading to increases in AT1-receptor and ACE densities in brain areas involved in cardiovascular regulation and hypertension.

AT1, AT2 and mas Receptor Expression in the Uteroplacental Unit of Normotensive and Hypertensive Rats

Late pregnancy is associated with enhanced uterine and placental levels of angiotensin peptides but a marked decrease in Ang-(1–7) occurs during hypertensive pregnancy. We investigated AT1, AT2 and mas/AT1-7 receptors in the uteroplacental unit of normal (NP) and reduced uterine perfusion pressure (RUPP) pregnant rats at late gestation by receptor autoradiography using 125I-Sar1-Thr8-Ang II and reverse transcriptase real time PCR. RUPP rats had higher MAP (109±1 vs 121±1 mmHg, p<0.05). In myo- and endometrium of virgin uterus there was 80-90% competition with the AT1 receptor antagonist losartan and ~10–20% competition with AT2/mas receptor antagonists (PD123319 and D-Ala). AT1 binding density was higher in myo- vs. endometrium (142 ± 68 vs 50 ± 34, p<0.01). In NP and RUPP, AT1 binding predominates in the mesometrial triangle with little to no competition with PD or D-Ala. The AT1 density was 1.6 fold greater in RUPP vs. NP (p<0.05). AT1, AT2 and mas receptor mRNA was lower in uterus of NP (58%, 98% and 76%, p<0.05) and RUPP (55%, 97% and 81%) vs virgin. AT1 receptor binding and mRNA in fetal and fetal/maternal placenta respectively were similar between RUPP and NP. In the fetal placenta of NP, 23% competition occurred with PD (p=.0505) and in RUPP there was 17% competition with D-Ala. AT1 receptor accounts for the majority of binding in the uteroplacental unit of both NP and RUPP. During normal and hypertensive pregnancy expression of all receptors are reduced in the uteroplacental unit; however in the fetal placenta there is a shift from AT2 to mas/AT1-7 receptors in hypertensive pregnancy. Supported by NHLBI51952 and AHA 0565390U

Long-lasting angiotensin type 1 receptor binding and protection by candesartan: comparison with other biphenyl-tetrazole sartans

The ability of biphenyl-tetrazole angiotensin type 1 (AT1) receptor antagonists (BTsartans) to block angiotensin II (Ang II)-mediated responses has been extensively investigated in vascular tissues and, more recently, in cell lines expressing the human AT1-receptor. When pre-incubated, BTsartans acted surmountably (shifting the Ang II concentration-response curve to the right) or insurmountably (also decreasing the maximal response). It was shown that their insurmountable behaviour is due to the formation of tight, long-lasting complexes with the receptor. Partial insurmountable antagonism is due to the co-existence of tight and loose complexes. The proportion of insurmountable antagonism, the potency and the dissociation rate of the BTsartans decreases in the order: candesartan > EXP3174 (losartan's active metabolite) > valsartan > irbesartan >> losartan. It is of interest to explore how tight AT1-receptor binding of BTsartans such as candesartan might contribute to their long-lasting clinical effect. Computer-assisted simulations (COPASI program) were performed to follow the receptor-occupation and protection by different antagonists as a function of time. Free antagonist concentrations were allowed to decrease exponentially with time. The simulations suggest that slow dissociation does not tangibly prolong receptor occupancy if the free antagonist is eliminated at a slower pace (as is the case for BTsartans). Yet when surmountable and insurmountable antagonists occupy the same amount of receptors, insurmountable antagonists offer appreciably better protection against fluctuations in natural messenger concentration. Slow receptor dissociation and slow antagonist elimination are likely to act in synergy to produce long-lasting receptor protection.

Design and Synthesis of New Tetrazolyl- and Carboxy-biphenylylmethyl-quinazolin-4-one Derivatives as Angiotensin II AT1 Receptor Antagonists

A series of novel quinazolin-4-ones was designed and their molecular modeling simulation fitting to a new HipHop 3D pharmacophore model using CATALYST was examined. Several compounds showed significant high simulation fit values. The designed compounds were synthesized and eight of them were biologically evaluated in vitro using an AT1 receptor binding assay, where compound XX competed weakly against radiolabeled Sar1Ile8-angiotensin II (Ang II) binding, compounds XIV and XXII showed moderate competition, and compound XXV showed almost equal ability to displace radiolabeled Sar1Ile8-Ang II binding to AT1 receptors as losartan. In vivo biological evaluation study of compounds XIV, XXII, and XXV on both normotensive and hypertensive rats revealed that compound XXV demonstrated higher hypotensive and antihypertensive activity than the reference compound losartan. To obtain a highly active compound from a candidate set of only eight tested compounds illustrates the power and utility of our pharmacophore model.

Synthesis and AT2 receptor-binding properties of angiotensin II analogues.

The present study investigates the importance of the amino acid side chains in the octapeptide angiotensin II (Ang II) for binding to the AT2 receptor. A Gly scan was performed where each amino acid in Ang II was substituted one-by-one with glycine. The resulting set of peptides was tested for affinity to the AT2 receptor (porcine myometrial membranes). For a comparison, the peptides were also tested for affinity to the AT1 receptor (rat liver membranes). Only the substitution of Arg2 reduced affinity to the AT2 receptor considerably (92-fold when compared with Ang II). For the other Gly-substituted analogues the affinity to the AT2 receptor was only moderately affected. To further investigate the role of the Arg2 side chain for receptor binding, we synthesized some N-terminally modified Ang II analogues. According to these studies a positive charge in the N-terminal end of angiotensin III [Ang II (2-8)] is not required for high AT2 receptor affinity but seems to be more important in Ang II. With respect to the AT1 receptor, [Gly2]Ang II and [Gly8]Ang II lacked binding affinity (Ki > 10 microM). Replacement of the Val3 or Ile5 residues with Gly produced only a slight decrease in affinity. Interestingly, substitution of Tyr4 or His6, which are known to be very important for AT1 receptor binding, resulted in only 48 and 14 times reduction in affinity, respectively.

Hypersensitivity of the adrenal cortex to trophic and secretory effects of angiotensin II in Lyon genetically-hypertensive rats.

In Lyon hypertensive (LH) rats, a model of low-renin genetic hypertension, we investigated adrenal sensitivity to angiotensin II in terms of angiotensin II receptor (AT1 and AT2 receptors) regulation, morphological changes, and aldosterone and corticosterone secretion. Twelve-week-old LH rats, compared with normotensive LN and LL rats, were either untreated or treated for 4 weeks with AT1 receptor antagonist irbesartan (50 mg/kg/d), angiotensin-converting enzyme inhibitor perindopril (3 mg/kg/d), or perindopril (3 mg/kg/d) plus angiotensin II infusion (200 ng/kg/min). At 16 weeks, untreated LH rats had high systolic blood pressure (P<0.05), low aldosterone (P<0.05), and increased corticosterone (P<0.05) plasma levels. AT1-receptor binding density in the zona glomerulosa was similar in the three strains. In LH rats, angiotensin II infusion increased the relative adrenal weight from 10.5+/-0.3 to 16.7+/-0.7 mg/100g (P<0.05), whereas this change was very modest in normotensive rats. Zona glomerulosa enlarged and plasma aldosterone increased after angiotensin II infusion in the 3 strains, but more markedly in LH versus normotensive rats (2.4- versus 1.3- and 1.6-fold, respectively; 20- versus 10-fold in normotensive rats, P<0.05). Surprisingly, after angiotensin II infusion, despite the absence of angiotensin II receptors in the three strains, the zona fasciculata-reticularis enlarged 1.5-fold and plasma corticosterone increased 1.7-fold only in LH rats (P<0.05), suggesting an indirect control of this compartment by angiotensin II. The hypertrophy and hypersecretory activity of both zona glomerulosa and zona fasciculata-reticularis in LH rats in response to angiotensin II point to the adrenal cortex as a pivotal tissue in the pathophysiology of hypertension in LH rats.

Angiotensin II AT(1) and AT(2) receptors contribute to maintain basal adrenomedullary norepinephrine synthesis and tyrosine hydroxylase transcription.

Angiotensin II (Ang II) AT(1) receptors have been proposed to mediate the Ang II-dependent and the stress-stimulated adrenomedullary catecholamine synthesis and release. However, in this tissue, most of the Ang II receptors are of the AT(2) type. We asked the question whether AT(1) and AT(2) receptors regulate basal catecholamine synthesis. Long-term AT(1) receptor blockade decreased adrenomedullary AT(1) receptor binding, AT(2) receptor binding and AT(2) receptor protein, rat tyrosine hydroxylase (TH) mRNA, norepinephrine (NE) content, Fos-related antigen 2 (Fra-2) protein, phosphorylated cAMP response element binding protein (pCREB), and ERK2. Long-term AT(2) receptor blockade decreased AT(2) receptor binding, TH mRNA, NE content and Fra-2 protein, although not affecting AT(1) receptor binding or receptor protein, pCREB or ERK2. Angiotensin II colocalized with AT(1) and AT(2) receptors in ganglion cell bodies. AT(2) receptors were clearly localized to many, but not all, chromaffin cells. Our data support the hypothesis of an AT(1)/AT(2) receptor cross-talk in the adrenomedullary ganglion cells, and a role for both receptor types on the selective regulation of basal NE, but not epinephrine formation, and in the regulation of basal TH transcription. Whereas AT(1) and AT(2) receptors involve the Fos-related antigen Fra-2, AT(1) receptor transcriptional effects include pCREB and ERK2, indicating common as well as different regulatory mechanisms for each receptor type.

Up-regulation of components of the renin-angiotensin system in the bile duct–ligated rat liver

Background & Aims: Angiotensin II (ANG II) has profibrotic actions in the heart and kidney, whereas blockade of the renin angiotensin system (RAS) attenuates injury. This study examines whether the RAS is present in the liver and examines its regulation in the bile duct–ligation model of hepatic fibrogenesis. Methods: Sham-operated and bile duct–ligated (BDL) Sprague-Dawley rats were studied. Gene and protein expression of hepatic renin, angiotensinogen, angiotensin-converting enzyme (ACE), and the angiotensin receptors AT1 and AT2 were assessed using real-time reverse-transcription polymerase chain reaction, in vitro autoradiography, and immunohistochemistry. Results: Angiotensinogen and renin messenger RNA were detected in sham liver but were not increased following BDL. Angiotensinogen protein was widely distributed in hepatocytes in both normal and injured livers, but in BDL livers, it was also expressed within areas of active fibrogenesis. Both ACE and AT1 receptor genes were up-regulated following BDL. The low level of ACE activity in sham animals was significantly increased in areas of active fibrogenesis in BDL livers. The AT1 receptor was present in both normal and diseased liver parenchyma, with increased AT1 receptor binding seen in fibrotic areas in the diseased liver. The AT2 receptor gene was not detected in normal or diseased liver. Conclusions: Key elements of the RAS are present in normal liver tissue, and there is major up-regulation of the system in the bile duct-ligated liver. These findings are in keeping with recent experimental studies that have demonstrated antifibrotic effects of RAS blockade in the bile duct–ligated liver.GASTROENTEROLOGY 2002;123:1667-1676

Perindopril alters vascular angiotensin-converting enzyme, AT(1) receptor, and nitric oxide synthase expression in patients with coronary heart disease.

Angiotensin-converting enzyme inhibitors (ACEi) reduce cardiovascular morbidity and mortality by improving coronary perfusion, reducing ventricular hypertrophy and remodeling, and preventing progression of coronary atherosclerosis. However, the cellular mechanisms underlying the beneficial effects of ACEi are not fully understood. We studied the in vivo effects of ACE inhibition with perindopril on cellular expression of ACE, AT(1) receptors and 2 nitric oxide synthase (NOS) isoforms, endothelial (eNOS) and inducible NOS (iNOS), in human blood vessels using quantitative in vitro autoradiography and immunocytochemistry. Seven patients with ischemic heart disease were treated with perindopril (4 mg/d) for up to 5 weeks before elective coronary bypass surgery, whereas controls did not receive the ACEi (n=7). Perindopril decreased plasma ACE by 70% and the plasma angiotensin II to angiotensin I ratio by 57% and reduced vascular ACE to approximately 65% of control levels in both endothelium and adventitia. By contrast, AT(1) receptor binding in vascular smooth muscle cells was increased by 80% in patients treated with perindopril as confirmed by immunocytochemistry. eNOS was expressed primarily in endothelial cells, whereas little iNOS expression occurred in vascular smooth muscle cells of untreated patients. Both eNOS and iNOS expression seemed to increase during perindopril treatment. These results suggest that suppression of angiotensin II formation in the vascular wall and increased expression of eNOS and iNOS during ACE inhibition may be beneficial in reversing endothelial dysfunction in patients with cardiovascular disease. Because vascular AT(1) receptor expression is increased during chronic ACE inhibition, more clinical studies are required to determine whether it is necessary to combine ACE inhibitors and AT(1) receptor antagonists in clinical management of heart failure, coronary heart disease, and hypertension

Intrarenal AT(1) receptor and ACE binding in ANG II-induced hypertensive rats.

The intrarenal expression of angiotensin II (ANG II) type 1 (AT(1)) receptors and angiotensin-converting enzyme (ACE) was determined in ANG II-induced hypertensive rats (80 ng/min; 2 wk). Systolic blood pressure averaged 184 +/- 3 and 125 +/- 1 mmHg in ANG II-infused compared with Sham rats on day 12. Total kidney AT(1) receptor protein levels were not altered significantly. AT(1) receptor binding mapped by quantitative in vitro autoradiography was significantly decreased in glomeruli (172 +/- 25 vs. 275 +/- 34 disintegrations. min(-1). mm(-2)) and the inner stripe of the outer medulla (121 +/- 17 vs. 178 +/- 19 disintegrations. min(-1). mm(-2)), but not proximal convoluted tubules (48 +/- 9 vs. 58 +/- 6 disintegrations. min(-1). mm(-2)) of ANG II-infused compared with Sham rats. Proximal tubule ACE binding was significantly augmented (132 +/- 4 vs. 97 +/- 3 disintegrations. min(-1). mm(-2)) in ANG II-infused rats. In summary, during ANG II-induced hypertension, glomeruli and inner stripe of the outer medulla have reduced AT(1) receptor binding. Proximal convoluted tubules exhibit maintained AT(1) receptor density and increased ACE binding, which together with the elevated ANG II levels suggest that ANG II exerts a sustained influence on tubular reabsorption and consequently contributes to the development and maintenance of ANG II-dependent hypertension.

Renin-angiotensin blockade improves renal cGMP production via non-AT(2)-receptor mediated mechanisms in hypertension-induced by chronic NOS inhibition in rat.

To investigate the changes in the angiotensin II (Ang II) receptors and nitric oxide (NO)-cGMP pathway in the rat kidney after nitric oxide synthase (NOS)blockade. Captopril, an angiotensin-converting enzyme (ACE)inhibitor, 20 mg/100 ml; and/or L-158,809 (an Ang II AT1-receptor antagonist, 5 mg/100 ml) and L-NAME (NOS inhibitor, 50 mg/100 ml) were administered orally for 12 weeks. Blood pressure (BP),urinary albumin, urinary cGMP excretion, plasma ANP, and plasma renin activity were measured. In vitro autoradiography was used to locate the Ang II receptors in the kidney. Captopril and L- 158,809 treatments normalised BP and prevented the appearance of albuminuria in rats receiving L-NAME. Urinary cGMP excretion was significantly increased in L-158,809-treated rats compared with the non-treated group, suggesting that the dysfunctional NO system may be activated by the treatment. AT1-receptor binding in the kidney was inhibited to about 40% of the control value after administration of L- 158,809. The AT2-receptor binding was inhibited to less than 15% of the control value. NOS inhibition had no effect on receptor binding. Blockade of NOS causes hypertension and renal damage. Treatment with an ACE inhibitor and/or Ang II receptor antagonist prevented these changes equally effectively. The stimulatory effect of AT1-receptor antagonism on cGMP production was not mediated by AT2-receptor-dependent mechanisms, since renalAT2-receptor binding density was suppressed following treatment with L-158,809. AT1-receptor blockade per se favours activation of humoral pathways that stimulate cGMP production potentially contributing to renal and vascular protection in hypertension and chronic renal disease.

Increased angiotensin‐converting enzyme and type 1 angiotensin receptors in cortical vasculature and tubulointerstitium of chronically rejected human kidney allografts

SUMMARY: Chronic rejection of human renal allografts after transplantation is characterized by interstitial infiltration, arteriosclerosis and glomerulosclerosis in the grafts. Apart from tissue HLA compatibility, angiotensin II (AII) may be implicated in accelerating the processes of chronic rejection. In the present study, the cellular distribution of angiotensin‐converting enzyme (ACE) and type 1 AII (AT1) receptors was mapped and compared in non‐rejected human kidneys (n = 8) and chronically rejected renal allografts (n = 9) using complementary immunohistochemistry and quantitative in vitro autoradiography. Chronically rejected allografts showed typical histopathological characteristics of tissue rejection, including concentric intimal thickening of intrarenal arteries, extensive or focal glomerulosclerosis, tubular atrophy and severe tubulointerstitial fibrosis. In rejected allografts, total ACE binding in the cortex was decreased to 46% of that in non‐rejected kidneys (P &lt; 0.01), whereas AT1 receptor binding in the glomeruli and the inner stripe of the outer medulla was maintained. However, ACE and AT1 receptor binding were increased in the cortical tubulointerstitium of chronically rejected allografts. In non‐rejected kidneys, strong ACE immunostaining occurred in proximal tubules and vascular endothelium, whereas AT1 receptors occurred in vascular smooth muscle cells, as expected. In rejected allografts, intense ACE and AT1 receptor immunostaining were detected not only in the same sites as those non‐rejected kidneys, but also in cortical tubulointerstitium between atrophied tubules and surrounding the glomeruli. AT1 receptors were markedly up‐regulated in vascular smooth muscle cells of thickened atherosclerotic vessels. These results provide novel morphological evidence that increased expression and/or altered distribution of ACE and AT1 receptors in cortical tubulointerstitium and in the neointima of intrarenal arteries may play an important role in the progression of chronic rejection of human renal allografts after transplantation.

Peripheral Administration of an Angiotensin II AT1 Receptor Antagonist Decreases the Hypothalamic-Pituitary-Adrenal Response to Isolation Stress

Angiotensin II, which stimulates AT1 receptors, is a brain and peripheral stress hormone. We pretreated rats with the AT1 receptor antagonist candesartan for 13 d via sc-implanted osmotic minipumps, followed by 24-h isolation in individual metabolic cages. We measured angiotensin II receptor-type binding and mRNAs and tyrosine hydroxylase mRNA by quantitative autoradiography and in situ hybridization, catecholamines by HPLC, and hormones by RIA. Isolation increased AT1 receptor binding in hypothalamic paraventricular nucleus as well as anterior pituitary ACTH, and decreased posterior pituitary AVP. Isolation stress also increased AT1 receptor binding and AT1B mRNA in zona glomerulosa and AT2 binding in adrenal medulla, adrenal catecholamines, tyrosine hydroxylase mRNA, aldosterone, and corticosterone. Candesartan blocked AT1 binding in paraventricular nucleus and adrenal gland; prevented the isolation-induced alterations in pituitary ACTH and AVP and in adrenal corticosterone, aldosterone, and catecholamines; abolished the increase in AT2 binding in adrenal medulla; and substantially decreased urinary AVP, corticosterone, aldosterone, and catecholamines during isolation. Peripheral pretreatment with an AT1 receptor antagonist blocks brain and peripheral AT1 receptors and inhibits the hypothalamic-pituitary-adrenal response to stress, suggesting a physiological role for peripheral and brain AT1 receptors during stress and a possible beneficial effect of AT1 antagonism in stress-related disorders.

Candesartan decreases the sympatho-adrenal and hormonal response to isolation stress.

A change from group housing to isolation in unfamiliar metabolic cages represents, for rodents, a significant emotional stress. We studied the effect of candesartan, a peripheral and central angiotensin II AT1-receptor antagonist, on the hormonal and sympathetic response to acute isolation. We pretreated rats with 1 mg/kg/day candesartan for 13 days via subcutaneously implanted osmotic minipumps, followed by 24-hour isolation in individual metabolic cages. We measured brain, pituitary and adrenal angiotensin II (Ang II) receptor binding by quantitative autoradiography and adrenal hormones and catecholamines by RIA and HPLC. Isolation increased adrenal catecholamines, aldosterone and corticosterone, AT1-receptor binding in the zona glomerulosa and AT2-receptor binding in the adrenal medulla. Candesartan pretreatment decreased adrenal catecholamines, aldosterone and corticosterone, AT1-receptor binding in adrenal zona glomerulosa and medulla, pituitary gland and the hypothalamic paraventricular nucleus, and AT2-receptor binding in adrenal medulla, but increased AT2-receptor binding in zona glomerulosa. We conclude that peripheral and central AT1-receptor blockade with candesartan decreases the sympatho-adrenal and hormonal response to acute stress. Our results indicate that Ang II is an important stress hormone and suggest that blockade of the physiologically active AT 1-receptors could influence stress-related disorders.