Modulation of AT1 receptor-mediated contraction of rat uterine artery by AT2 receptors

1. The aim of this study was to characterize the angiotensin II receptors in isolated uterine arteries from non pregnant and pregnant rats, since it has been reported from binding studies that ovine uterine arteries contain AT₂ receptors.
2. Uterine arterial segments were obtained from virgin, non-pregnant and late pregnant (18–21 days) Sprague-Dawley rats and mounted in small vessel myographs. Concentration-response curves were constructed to angiotensin II (1 nM–10 μM) in the absence and presence of various angiotensin II receptor subtype selective compounds. These included losartan (AT₁ antagonist; 1, 10 and 100 nM), PD 123319 (AT₂ antagonist; 1 μM) and CGP 42112 (AT₂ agonist; 1 μM). Responses to angiotensin II were measured as increases in force (mN) and expressed as a per cent of the response to a K⁺ depolarizing solution.
3. Losartan (1, 10 and 100 nM) caused significant concentration-dependent rightward shifts of the angiotensin II concentration-response curve in uterine arteries from non-pregnant and pregnant rats. The pA₂ values calculated from these data were 9.8 and 9.2, respectively, although the slope of the Schild plot in the non-pregnant group was less than unity.
4. PD 123319 (1 μM) caused significant 6- and 3 fold leftward shifts of the angiotensin II concentration-response curve in uterine arteries from non-pregnant and pregnant rats, respectively. In vessels from pregnant rats, PD 123319 also significantly increased the maximum response to angiotensin II.
5. CGP 42112 (1 μM) attenuated the response to angiotensin II of uterine arteries from non-pregnant rats. This was reflected by a 14 fold rightward shift of the angiotensin II concentration-response curve and a decrease in the maximum response. In uterine arteries from pregnant rats, CGP 42112 (1 μM) caused a 3 fold rightward shift of the angiotensin II concentration-response curve, but had no effect on the maximum response.
6. PD 123319 (1 μM) and CGP 42112 (1 μM) had no effect on the concentration-response curves to phenylephrine (PE) of uterine arteries from non-pregnant or pregnant rats. In addition, CGP 42112 (1 nM–1 mM) had no vasodilator effect on tissues precontracted with phenylephrine.
7. These results suggest that the contractile responses of the rat uterine artery are mediated by the AT₁ receptor. Furthermore, in this vascular preparation, the AT₂ receptor appears to inhibit the response mediated by the AT₁ receptor, although, this is not uniform between the non-pregnant and pregnant states.

     

Molecular characterisation of the interactions between olmesartan and telmisartan and the human angiotensin II AT1 receptor

BACKGROUND AND PURPOSE: Whereas some angiotensin II (Ang II) type 1 receptor blockers (ARBs) produce surmountable antagonism of AT(1) receptors, others such as olmesartan and telmisartan display varying degrees of insurmountability. This study compared the molecular interactions of olmesartan and telmisartan with the human AT(1) receptor, using well characterised in vitro methods and model systems. EXPERIMENTAL APPROACH: CHO-K1 cells that stably express human AT(1) receptors (CHO-hAT(1) cells) were used in several pharmacological studies of olmesartan and telmisartan, including direct radioligand binding and inhibition of Ang II-induced inositol phosphate (IP) accumulation. KEY RESULTS: Both ARBs were found to be competitive antagonists that displayed high affinity, slow dissociation, and a high degree of insurmountability for the AT(1) receptor (the latter greater with olmesartan). Their receptor interactions could be described by a two-step process with the initial formation of a loose complex (IR) and subsequent transformation into a tight binding complex (IR*). In washout experiments, [(3)H] telmisartan dissociated from the receptor with a half-life of 29 min and the Ang II-mediated IP accumulation response was 50% maximally restored within 24 min, whereas values for [(3)H] olmesartan were 72 min and 76 min, respectively. CONCLUSIONS AND IMPLICATIONS: The high degree of insurmountability, slow dissociation, and high affinity of olmesartan for its receptor may relate to its ability to stabilise IR* via the carboxyl group of its imidazole core. In comparison, telmisartan displays a less potent interaction with the receptor.     

Effect of saponin and filipin on antagonist binding to AT 1 receptors in intact cells

In the present study, [ 3H ]-candesartan binding experiments were performed on intact Chinese Hamster Ovary cells transfected with the human AT1 receptor (CHO-AT1 cells). Cells were pre-treated with 0.01mg/ml saponin or filipin. Both pre-treatments resulted in an increased dissociation rate and decreased affinity of the insurmountable non-peptide antagonist [3H ]-candesartan. A similar decrease in affinity was observed for the peptide antagonist Sar1-Ile8 angiotensin II and for other non-peptide antagonists, irrespectively of their degree of insurmountability. A similar discrepancy in [ 3H ]-candesartan binding was earlier observed when comparing intact CHO-AT1 cells and membrane preparations thereof. This similarity is further highlighted by the observations that saponin or filipin no longer affect [ 3H ]-candesartan binding to CHO-AT1 cell membranes and that both agents permeabilise the CHO-AT1 cells. This suggests that the intracellular composition and/or organisation of living cells play an active role with regard to antagonist-AT1 receptor interactions.     

Characterization of angiotensin II antagonism displayed by Ib, a novel nonpeptide angiotensin AT(1) receptor antagonist

The pharmacologic profile of Ib, 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one, a novel nonpeptide angiotensin AT(1) receptor antagonist, was investigated by receptor-binding studies, functional in vitro assays with rabbit and rat aorta, and in vivo experiments in rats. Ib inhibited [(125)I] angiotensin II binding to AT(1) receptors in rat liver membranes (K(i)=2.5+/-0.5 nM) and did not interact with AT(2) receptors in bovine cerebellar membranes. In functional studies with rat and rabbit aorta, Ib inhibited the contractile response to angiotensin II (pD(2)' value: 7.43 and 7.29, respectively) with a significant reduction in the maximum. In pithed rats, Ib inhibited the angiotensin II induced pressor response in a dose-related manner. After intravenous administration, Ib produced a dose-dependent antihypertensive effects in spontaneously hypertensive rats and renal hypertensive rats. These results suggest that Ib is a potent angiotensin AT(1) selective receptor antagonist with a mode of insurmountable antagonism.     

Chronic blockade of angiotensin AT1 receptors improves cardinal symptoms of metabolic syndrome in diet-induced obesity in rats

Background and Purpose

AT₁ receptor antagonists decrease body weight gain in models of murine obesity. However, fewer data are available concerning the anti-obesity effects of these antagonists, given as a treatment after obesity had been established.

Experimental Approach

In spontaneously hypertensive rats, obesity was established by cafeteria diet (CD) feeding for 19 weeks. Rats were then were treated with telmisartan (8 mg·kg⁻¹·d⁻¹) or amlodipine (10 mg·kg⁻¹·d⁻¹; serving as blood pressure control) or telmisartan + amlodipine (2 + 10 mg·kg⁻¹·d⁻¹; to control for dose-dependency) for 17 weeks. Rats receiving only chow (C_chow) or CD-fed rats treated with vehicle (C_CD) served as controls.

Key Results

The CD feeding induced obesity, hyperphagia, hyperlipidaemia, and leptin and insulin resistance. Telmisartan reduced the CD-induced increase in body weight and abdominal fat mass. Whereas energy intake was higher rather than lower, the respiratory ratio was lower. After telmisartan, leptin-induced energy intake was reduced and respiratory ratio was increased compared with C_CD rats. Telmisartan also decreased plasma levels of triglycerides, free fatty acids and low-density lipoprotein. Amlodipine alone or the combination telmisartan + amlodipine did not affect body weight and eating behaviour. Telmisartan, but not amlodipine and telmisartan + amlodipine, improved glucose utilization. The decrease in BP reduction was almost the same in all treatment groups.

Conclusions and Implications

Telmisartan exerted anti-obesity effects and restored leptin sensitivity, given as a treatment to rats with obesity. Such effects required high doses of telmisartan and were independent of the decrease in blood pressure.     

Endogenous angiotensin II and the reflex response to stimulation of cardiopulmonary serotonin 5HT3 receptors

1. Angiotensin (Ang) II modulates cardiovascular baroreflexes; whether or not the peptide influences chemosensitive cardiovascular reflexes is not known. We tested the hypothesis that Ang II modulates the reflex control of sympathetic nerve activity exerted by 5-hydroxytryptamine 3 (5HT₃) cardiopulmonary receptors.
2. The 5HT₃ receptor agonist phenylbiguanide (PBG), infused intravenously for 15 min, elicited a sustained reflex decrease of renal sympathetic nerve activity (RSNA) but only transient (<3 min) changes of arterial blood pressure (BP) and heart rate (HR) in methohexital-anaesthesized rats.
3. Infusion of Ang II at a dose that did not affect baseline BP, HR and RSNA enhanced the PBG-evoked reflex decrease of RSNA (−54±5% in Ang II treated versus −33±6% in control rats after 15 min PBG, P<0.05, n=6 each) in methohexital-anaesthetized rats.
4. The angiotensin converting enzyme (ACE) inhibitor lisinopril blunted the reflex responses to PBG in anaesthetized as well as conscious animals. The effect of the ACE inhibitor was abolished by concomitant infusion of Ang II.
5. The reflex response to stimulation of cardiopulmonary 5HT₃ afferents was also impaired by the Ang II type 1 receptor (AT₁) blocker ZD7155 but not by the type 2 (AT₂) blocker PD 123319.
6. Infusion of a volume load to stimulate cardiopulmonary baroreceptors induced a gradual decrease of RSNA which was impaired by exogenous Ang II (RSNA −26±6% in Ang II treated versus −47±6% in control rats after volume load, P<0.05, n=6 each) but unaffected by ACE inhibition.
7. The reflex control of RSNA by cardiopulmonary 5HT₃ receptors is enhanced by Ang II via AT₁ receptors. Thus, Ang II facilitates a chemosensitive cardiovascular reflex, in contrast to its inhibitory influences on mechanosensitive reflexes.

     

AT1 receptor blocker-insensitive mutant AT1A angiotensin receptors reveal the presence of G protein-independent signaling in C9 cells

Although mutant receptors are highly useful to dissect the signal transduction pathways of receptors, they are difficult to study in physiological target tissues, due to the presence of endogenous receptors. To study AT(1) angiotensin receptors in their physiological environment, we constructed a mutant receptor, which differs only from the AT(1A) receptor in its reduced affinity for candesartan, a biphenylimidazole antagonist. We have determined that the conserved S109Y substitution of the rat AT(1A) receptor eliminates its candesartan binding, without exerting any major effect on its angiotensin II and peptide angiotensin receptor antagonist binding, internalization kinetics, beta-arrestin binding, and potency or efficacy of the inositol phosphate response. To demonstrate the usefulness of this mutant receptor in signal transduction studies, we combined it with substitution of the highly conserved DRY sequence with AAY, which abolishes G protein activation. In rat C9 hepatocytes the S109Y receptor caused ERK activation with the same mechanism as the endogenous AT(1) receptor. After combination with the DRY/AAY mutation G protein-independent ERK activation was detected demonstrating that this approach can be used to study the angiotensin II-stimulated signaling pathways in cells endogenously expressing AT(1) receptors.     

The human angiotensin AT(1) receptor supports G protein-independent extracellular signal-regulated kinase 1/2 activation and cellular proliferation

The angiotensin AT(1) receptor is a key regulator of blood pressure and body fluid homeostasis, and it plays a key role in the pathophysiology of several cardiovascular diseases such as hypertension, cardiac hypertrophy, congestive heart failure, and arrhythmia. The importance of human angiotensin AT(1) receptor signalling is illustrated by the common use of angiotensin AT(1) receptor-inverse agonists in clinical practice. It is well established that rodent orthologues of the angiotensin AT(1) receptor can selectively signal through G protein-dependent and -independent mechanisms in recombinant expression systems, primary cells and in vivo. The in vivo work clearly demonstrates profoundly different cellular consequences of angiotensin AT(1) receptor signalling in the cardiovascular system, suggesting pharmacological potential for drugs which specifically affect a subset of angiotensin AT(1) receptor actions. However, it is currently unknown whether the human angiotensin AT(1) receptor can signal through G protein-independent mechanisms - and if so, what the physiological impact of such signalling is. We have performed a detailed pharmacological analysis of the human angiotensin AT(1) receptor using a battery of angiotensin analogues and registered drugs targeting this receptor. We show that the human angiotensin AT(1) receptor signals directly through G protein-independent pathways and supports NIH3T3 cellular proliferation. The realization of G protein-independent signalling by the human angiotensin AT(1) receptor has clear pharmacological implications for development of drugs with pathway-specific actions and defined biological outcomes.     

Role of angiotensin II in the response to endothelin-1 of goat cerebral arteries after ischemia–reperfusion

As angiotensin II may underlie the deleterious effects of some vascular diseases, we have examined the role of this peptide on the cerbrovascular endothelin-1 action after ischemia–reperfusion. In anesthetized goats, 1 hour-occlusion followed by 1 hour-reperfusion of the left middle cerebral artery (MCA) was induced, and then segments 3-mm in length from branches of the right MCA (control) and the left MCA (ischemic) were obtained for isometric tension recording. Endothelin-1 (10^− 11–10^− 7 M) produced a contraction that was higher in ischemic than in control arteries, and in control but not in ischemic arteries this contraction was potentiated by angiotensin II (10^− 7 M). Losartan (3 × 10^− 6 M), antagonist of AT₁ receptors, did not affect the response to endothelin-1 in control arteries, but reduced it both in ischemic arteries and angiotensin II-treated control arteries. PD123,319 (3 × 10^− 6 M), antagonist of AT₂ receptors, or the inhibitor of nitric oxide synthesis l-NAME (10^− 4 M) did not alter the arterial effects of endothelin-1. Therefore, angiotensin II may potentiate the constriction to endothelin-1 in normal cerebral arteries by activating AT₁ receptors. The observed cerebrovascular increased response to endothelin-1 after ischemia–reperfusion might be related in part to activation of AT₁ receptors under this condition.     

Down-regulation of aortic and cardiac AT1 receptor gene expression in transgenic (mRen-2) 27 rats

1. Transgenic(TG) (mRen-2) rats overexpressing the mouse renin gene develop fulminant hypertension and cardiac hypertrophy. Since the activation of AT₁ receptor by angiotensin II is involved in blood pressure regulation, cardiac performance and myocardial growth, we investigated the biological effects of angiotensin II and the regulation of the AT₁ receptor in the heart and aorta of TGR (mRen-2)27 rats in comparison to control animals.
2. Contraction studies on isolated cardiac muscle strips reveal that angiotensin II exerts no positive inotropic effect on the left ventricular myocardium of both, transgenic and control rats. In contrast, angiotensin II leads via AT₁ receptor activation in the left atrium of control rats to a significant contraction (130±5% of basal contraction) which is not detectable in left atrium preparations of the transgenic animals. Furthermore, AT₁ receptor activation causes a profound contraction of aortic rings isolated from control rats amounting to 1.39±0.2 mN mg⁻¹ wet weight, whereas aortic rings from TGR (mRen-2)27 rats contract only minimally upon angiotensin II stimulation (0.2±0.02 mN mg⁻¹ wet weight).
3. These altered physiological responses of angiotensin II in the transgenic rats are in part due to a marked down-regulation of the AT₁ receptor in atrial, ventricular and aortic tissue of these transgenic animals in comparison to control Sprague-Dawley rats, as shown by radioligand binding assays and quantitative polymerase chain reaction (PCR) experiments. The AT₁ receptor density B_max in the left atrium was 1.3±0.08 fmol mg⁻¹ protein in control rats (K_D 1.1±0.18 nmol l⁻¹) and 0.94±0.15 fmol mg⁻¹ protein (K_D 2.1±0.3 nmol l⁻¹. In the aorta B_max values were 15.1±0.5 fmol mg⁻¹ protein (K_D 1.9±0.27 nmol l⁻¹) for control rats and 11.3±0.76 fmol mg⁻¹ protein (K_D 1.9±0.27 nmol l⁻¹) for the TGR(mRen-2)27 rats AT₁ receptor mRNA was reduced in the transgenic animals to 46±3% in the left atrium, 50±11% in the left ventricle and 40±3% in the aorta, respectively.
4. Together, the AT₁ receptor is down-regulated in TGR (mRen-2)27 rats in comparison to wildtype Sprague Dawley rats leading to a profoundly decreased response of cardiac and aortic tissue upon stimulation with angiotensin II.

     

Stimulation of angiotensin AT2 receptors by the non-peptide agonist,Compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats

Background and purpose:

Angiotensin type 2 receptor (AT₂ receptor) stimulation evokes vasodilator effects in vitro and in vivo that oppose the vasoconstrictor effects of angiotensin type 1 receptors (AT₁ receptors). Recently, a novel non-peptide AT₂ receptor agonist, Compound 21, was described, which exhibited high AT₂ receptor selectivity.

Experimental approach:

Functional cardiovascular effects of the drug candidate Compound 21 were assessed, using mouse isolated aorta and rat mesenteric arteries in vitro and in conscious spontaneously hypertensive rats (SHR).

Key results:

Compound 21 evoked dose-dependent vasorelaxations in aortic and mesenteric vessels, abolished by the AT₂ receptor antagonist, PD123319. In vivo, Compound 21 administered alone, at doses ranging from 50 to 1000 ng·kg⁻¹·min⁻¹ over 4 h did not decrease blood pressure in conscious normotensive Wistar-Kyoto rats or SHR. However, when given in combination with the AT₁ receptor antagonist, candesartan, Compound 21 (300 ng·kg⁻¹·min⁻¹) lowered blood pressure in SHR only. Further analysis in separate groups of conscious SHR revealed that, at a sixfold lower dose, Compound 21 (50 ng·kg⁻¹·min⁻¹) still evoked a significant depressor response in adult SHR (∼30 mmHg) when combined with different doses of candesartan (0.01 or 0.1 mg·kg⁻¹). Moreover, the Compound 21-evoked depressor effect was abolished when co-infused (50 µg·kg⁻¹·min⁻¹ for 2 h) with the AT₂ receptor antagonist PD123319.

Conclusion and implications:

Collectively, our results indicate that acute administration of Compound 21 evoked blood pressure reductions via AT₂ receptor stimulation. Thus Compound 21 can be considered an excellent drug candidate for further study of AT₂ receptor function in cardiovascular disease.     

Positive inotropic action of angiotensin II in the pithed rat

Summary The cardiovascular effects of angiotensin II were examined in aortic blood pressure-controlled and-uncontrolled pithed rats. Angiotensin II induced a dose-dependent increase in diastolic blood pressure, left ventricular pressure (LVP), dP/dt (the first derivative of LVP) and heart rate in pithed rats. The maximal responses for these parameters were similar to those to noradrenaline, except for the rise in diastolic blood pressure, where noradrenaline caused a greater increase than angiotensin II. After treatment with propranolol, the positive chronotropic effect of angiotensin II was abolished. Angiotensin II produced a dose-dependent increase in diastolic blood pressure, which was similar to that of vasopressin, and an increase in dP/dt_max, which proved much greater than that of vasopressin. When aortic blood pressure was controlled and the -receptors were blocked by propranolol, angiotensin II caused a dose-dependent increase in dP/dt_max without affecting the left ventricular enddiastolic pressure. The same results were obtained after both - and -adrenoceptors were blocked by propranolol and phentolamine. Losartan but not PD 123177 caused parallel rightward shifts of the dose-response curve of angiotensin II for dP/dt_max in the aortic blood pressure controlled pithed rat without altering the maximal response. It is concluded that in the pithed rat angiotensin II produced an increase in myocardial contractile force which is not mediated by - or -adrenoceptors. The inotropic effect appears to be mediated by angiotensin receptors, of the AT₁-subtype.Correspondence to J. Zhang at the above address     

Structural determinants for the activation mechanism of the angiotensin II type 1 receptor differ for phosphoinositide hydrolysis and mitogen-activated protein kinase pathways

While the mechanism whereby the angiotensin II type 1 receptor (AT(1) receptor) activates its classical effector phospholipase C-beta (PLC-beta) has largely been elucidated, there is little consensus on how this receptor activates a more recently identified effector, the p42/44 mitogen-activated protein kinases (p42/44(MAPK)). Using transfected COS-1 cells, we investigated the activation of this signaling pathway at the receptor level itself. Previous mutational studies that relied on phosphoinositide turnover as an index of receptor activation have indicated that key residues in the second and seventh transmembrane domains participate in AT(1) receptor activation mechanisms. Thus, we introduced a variety of mutations-AT(1)[D74N], AT(1)[Y292F], AT(1)[N295S], and AT(1)[AT(2) TM7], which is composed of a chimeric substitution of the AT(1) seventh transmembrane domain with its AT(2) counterpart. These mutations that strongly diminished the receptor's ability to activate PLC-beta had little to no effect on its ability to activate p42/44(MAPK), which not only suggests that p42/44(MAPK) does not exclusively lie downstream of the G-protein G(q)/PLC-beta pathway but also indicates that more than one activation state may exist for the AT(1) receptor. The failure of a protein kinase C inhibitor to block AT(1) receptor activation of p42/44(MAPK) further corroborated evidence that the receptor's activation of p42/44(MAPK) is largely independent of the G(q)/PLC-beta/PKC pathway. Taken together, the experimental evidence strongly suggests that the mechanism whereby the AT(1) receptor activates p42/44(MAPK) is fundamentally different from that for PLC-beta, even at the level of the receptor itself.     

Conformational induction is the key process for activation of the AT1 receptor

It is currently unclear whether activation of the AT1 receptor by agonists involves conformational selection or induction. We evaluated the pharmacological properties of wild type and N111G CAM human AT1 receptors stably expressed in HEK293 cells. Although [Sar1]-Ang II and Ang IV were full agonists at both receptors, the potency of Ang IV was 280-fold lower at the wild type receptor. [Sar1, Ile8]-Ang II was only a full agonist at the N111G CAM AT1 receptor. [Sar1]-Ang II and [Sar1, Ile8]-Ang II displayed similar high affinity binding to both receptors. In contrast, Ang IV displayed low affinity binding to the wild type and high affinity binding to the N111G CAM AT1 receptor. Based on these observations we provide strong evidence that conformational induction is the key process for activation of the AT1 receptor. Only by the creation of CAMs can conformational selection be envisaged to take place.     

Antagonist interaction with endogenous AT(1) receptors in human cell lines

Using Chinese Hamster Ovary cells expressing human AT(1) receptors cells (CHO-hAT(1)), it was previously shown that insurmountable inhibition of the angiotensin II response by non-peptide antagonists is related to the duration of their receptor occupancy. In the present study it was shown that these antagonists displayed similar binding characteristics to endogenously expressed AT(1) receptors in human adrenal cortex cells (NCI-h295) and renal vascular smooth muscle cells (HVSMC). Competition binding studies with [(3)H]candesartan for NCI-h295 cells, with [(125)I]Sar(1)-Ile(8) angiotensin II for HVSMC and with both radioligands for CHO-hAT(1) cells displayed the same potency order for unlabelled antagonists: candesartan>EXP3174>irbesartan>losartan. The AT(2) receptor antagonist PD123319 displayed low potency in all instances. The apparent half-lives of the antagonist-AT(1) receptor complexes in NCI-h295 cells and HVSMC were comparable to those obtained under identical conditions with CHO-hAT(1) cells. Angiotensin II increased the inositol phosphate accumulation dose dependently with half-maximal response at 17.4+/-1.6nM for NCI-h295 cells and 4.5+/-0.8nM for HVSMC. Pre-incubation of the cells with losartan only produced concentration-dependent rightward shifts of the angiotensin II concentration-response curve. The maximal response was decreased by 85-92% with candesartan, 70-88% with EXP3174 and 60% with irbesartan. The similar binding and inhibitory properties of these antagonists among the investigated cell types validates the use of CHO-hAT(1) cells for investigating pharmacological properties of human AT(1) receptors.     

Influence of Fimasartan (a Novel AT1 Receptor Blocker) on Catecholamine Release in the Adrenal Medulla of Spontaneously Hypertensive Rats

The aim of this study was to determine whether fimasartan, a newly developed AT₁ receptor blocker, can affect the CA release in the isolated perfused model of the adrenal medulla of spontaneously hypertensive rats (SHRs). Fimasartan (5~50 µM) perfused into an adrenal vein for 90 min produced dose- and time-dependently inhibited the CA secretory responses evoked by ACh (5.32 mM), high K⁺ (56 mM, a direct membrane depolarizer), DMPP (100 µM) and McN-A-343 (100 µM). Fimasartan failed to affect basal CA output. Furthermore, in adrenal glands loaded with fimasartan (15 µM), the CA secretory responses evoked by Bay-K-8644 (10 µM, an activator of L-type Ca²⁺ channels), cyclopiazonic acid (10 µM, an inhibitor of cytoplasmic Ca²⁺-ATPase), and veratridine (100 µM, an activator of Na⁺ channels) as well as by angiotensin II (Ang II, 100 nM), were markedly inhibited. In simultaneous presence of fimasartan (15 µM) and L-NAME (30 µM, an inhibitor of NO synthase), the CA secretory responses evoked by ACh, high K⁺, DMPP, Ang II, Bay-K-8644, and veratridine was not affected in comparison of data obtained from treatment with fimasartan (15 µM) alone. Also there was no difference in NO release between before and after treatment with fimasartan (15 µM). Collectively, these experimental results suggest that fimasartan inhibits the CA secretion evoked by Ang II, and cholinergic stimulation (both nicotininc and muscarinic receptors) as well as by membrane depolarization from the rat adrenal medulla. It seems that this inhibitory effect of fimasartan may be mediated by blocking the influx of both Na⁺ and Ca²⁺ through their ion channels into the rat adrenomedullary chromaffin cells as well as by inhibiting the Ca²⁺ release from the cytoplasmic calcium store, which is relevant to AT₁ receptor blockade without NO release.     

Role of H(2)O(2) in hypertension, renin-angiotensin system activation and renal medullary disfunction caused by angiotensin II

BACKGROUND AND PURPOSE

Activation of the intrarenal renin-angiotensin system (RAS) and increased renal medullary hydrogen peroxide (H₂O₂) contribute to hypertension. We examined whether H₂O₂ mediated hypertension and intrarenal RAS activation induced by angiotensin II (Ang II).

EXPERIMENTAL APPROACH

Ang II (200 ng·kg⁻¹·min⁻¹) or saline were infused in Sprague Dawley rats from day 0 to day 14. Polyethylene glycol (PEG)-catalase (10 000 U·kg⁻¹·day⁻¹) was given to Ang II-treated rats, from day 7 to day 14. Systolic blood pressure was measured throughout the study. H₂O₂, angiotensin AT₁ receptor and Nox4 expression and nuclear factor-κB (NF-κB) activation were evaluated in the kidney. Plasma and urinary H₂O₂ and angiotensinogen were also measured.

KEY RESULTS

Ang II increased H₂O₂, AT₁ receptor and Nox4 expression and NF-κB activation in the renal medulla, but not in the cortex. Ang II raised plasma and urinary H₂O₂ levels, increased urinary angiotensinogen but reduced plasma angiotensinogen. PEG-catalase had a short-term antihypertensive effect and transiently suppressed urinary angiotensinogen. PEG-catalase decreased renal medullary expression of AT₁ receptors and Nox4 in Ang II-infused rats. Renal medullary NF-κB activation was correlated with local H₂O₂ levels and urinary angiotensinogen excretion. Loss of antihypertensive efficacy was associated with an eightfold increase of plasma angiotensinogen.

CONCLUSIONS AND IMPLICATIONS

The renal medulla is a major target for Ang II-induced redox dysfunction. H₂O₂ appears to be the key mediator enhancing intrarenal RAS activation and decreasing systemic RAS activity. The specific control of renal medullary H₂O₂ levels may provide future grounds for the treatment of hypertension.     

Blood pressure response to angiotensin II is enhanced in obese Zucker rats and is attributed to an aldosterone-dependent mechanism

BACKGROUND AND PURPOSE

Plasma aldosterone levels correlate positively with obesity, suggesting a link between the hypertension associated with obesity and increased mineralocorticoid levels. We tested the hypothesis that aldosterone is involved in the BP response to angiotensin II (AngII) in obese rats.

EXPERIMENTAL APPROACH

Lean (LZR) and obese (OZR) Zucker rats were treated with AngII (9 µg·h⁻¹; 4 weeks), and BP and plasma AngII and aldosterone were determined.

KEY RESULTS

Chronic AngII increased the BP in OZR markedly more so than in LZR. Plasma AngII levels in LZR and OZR were similar after AngII treatment. The AngII stimulated a rise in plasma aldosterone that was sixfold more in OZR than in LZR. The thickness of the zona glomerulosa of the adrenal glands was selectively increased by AngII in OZR. Adrenal mRNA levels of CYP11B2 aldosterone synthase and the AT_1B receptor were selectively increased in AngII-treated OZR. The BP response to chronic AngII stimulation was diminished in OZR after adrenalectomy when plasma aldosterone was absent. Acute bolus injections of AngII did not increase the BP response or aldosterone release in OZR.

CONCLUSIONS AND IMPLICATIONS

The AngII-induced BP response is enhanced in obesity and this is associated with a specific increase in circulating aldosterone. Due to the AngII-induced growth of the zona glomerulosa in OZR, the AT_1B receptors and aldosterone synthase may be selectively enhanced in obesity under concomitant AngII stimulation, increasing the adrenal synthesis of aldosterone. Our results confirm functionally that aldosterone plays a major role in obesity-related hypertension.     

In vitro effect of sanguinarine alkaloid on binding of [3H]candesartan to the human angiotensin AT1 receptor

The type of interaction of 5-methyl-2,3,7,8-bis(methylenedioxy)benzo[c]phenanthridinium (sanguinarine), an alkaloid isolated from the root of Bocconia frutescens L., with the human angiotensin AT(1) receptor was evaluated in both intact cells and membrane binding of [3H](2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H-benzimidazoline-7-carboxylic acid) ([3H]candesartan). The results indicate that the inhibition of [3H]candesartan binding by sanguinarine is independent of cell viability, since the alkaloid inhibited at a similar extent radioligand binding on both intact Chinese hamster ovary (CHO) cells transfected with the human angiotensin AT(1) receptor (hAT(1)) and their cell membranes (K(i)=0.14 and 1.10 microM, respectively). The unsuccessful recovery of [3H]candesartan binding after washing sanguinarine off the cells suggested a nearly irreversible or slow reversible interaction. Saturation binding studies showed a substantial reduction of the B(max) without affecting the K(d). In addition, the presence of 2-n-butyl-4chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole (losartan) could not prevent sanguinarine inhibition of [3H]candesartan binding neither. The present findings indicate that sanguinarine interacts with the receptor in a slow, nearly irreversible and noncompetitive manner.     

Elevated plasma endothelin-1 level in streptozotocin-induced diabetic rats and responsiveness of the mesenteric arterial bed to endothelin-1

1. Both the plasma endothelin-1 (ET-1) levels and the plasma glucose levels were markedly elevated in streptozotocin (STZ)-induced diabetic rats.
2. The maximum contractile response of the mesenteric arterial bed to ET-1 was significantly reduced, and the vasodilatation induced by the ET_B-receptor agonist IRL-1620 in the mesenteric arterial bed was significantly reduced in STZ-induced diabetic rats.
3. ET-1 (10⁻⁸ M) caused a transient vasodilatation followed by a marked vasoconstriction in methoxamine-preconstricted mesenteric arterial beds. The ET-1-induced vasodilatation was significantly larger in beds from diabetic rats than in those from age-matched controls. By contrast, the ET-1-induced vasoconstriction was significantly smaller in STZ-induced diabetic rats than in the controls.
4. Both removal of the endothelium with Triton X-100 and preincubation with BQ-788 (10⁻⁶ M) (ET_B-receptor antagonist) abolished the ET-1-induced vasodilatation. Preincubation with BQ-485 (10⁻⁶ M) or BQ-123 (3×10⁻⁶) (ET_A-receptor antagonist) significantly augmented the ET-1-induced vasodilatation in control mesenteric arterial beds, but not that in beds from diabetic rats.
5. These results demonstrate that marked increases not only in plasma glucose, but also in plasma ET-1 occur in STZ-induced diabetic rats. We suggest that the decreased contractile response and the increased vasodilator response of the mesenteric arterial bed to ET-1 may both be due to desensitization of ET_A receptors, though ET_B receptors may also be desensitized. This desensitization may result from the elevation of the plasma ET-1 levels seen in STZ-induced diabetic rats.