Presence Of PD123319 Research Articles

ATR1 Angiotensin II Receptor Reduces Hemoglobin S Polymerization, Phosphatidylserine Exposure, and Increases Deformability of Sickle Cell Disease Erythrocytes.

Angiotensin II (Ang II) regulates blood volume and stimulates erythropoiesis through AT1 (ATR1) and AT2 (ATR2) receptors, found in multiple tissues, including erythrocytes. Sickle cell disease (SCD) patients present altered Ang II levels. Hemoglobin S polymerization, deformability and phosphatidylserine translocation are important features of mature erythrocytes, therefore, our hypothesis is Ang II affects these parameters and, if it does, what would be the influence of AT1R and AT2R on these effects. A polymerization assay (PA), deformability, and annexin V binding were performed in SCD erythrocytes samples adding Ang II, ATR1 antagonist (losartan or eprosartan), and ATR2 antagonist (PD123319). Through the PA test, we observed a dose-dependent polymerization inhibition effect when comparing Ang II to control. Losartan did not affect the level or the rate of Ang II inhibition, while PD123319 showed an increased level of protection against polymerization, and eprosartan brought levels back to control. Ang II was able to reduce the translocation of phosphatidylserine from the inner to the outer leaflet, a marker of eryptosis, in the presence of PD123319. Also, ATR1 showed a positive effect increasing deformability. Our data shows that ATR1 is important for maintenance of erythrocyte physiological function in SCD and for prolonging its life.

Endothelial AT1 and AT2 pathways in aortic responses to angiotensin II after stress and ethanol consumption in rats

Stress and ethanol are important cardiovascular risk factors. Their vascular and blood pressure (BP) effects were evaluated alone and in combination. Adult male Wistar rats (8–10 per group) were separated into control, ethanol (ethanol 20% in drinking water for 6 weeks), stress (restraint 1 h/d 5 d/week for 6 weeks), and ethanol/stress (in combination) groups. Systolic BP was evaluated weekly. Concentration–response curves for contractile responses to angiotensin II in the absence and the presence of losartan (AT1-blocker), PD123-319 (AT2-blocker), L-NAME (nitric oxide synthase inhibitor), or indomethacin (cyclooxygenase inhibitor) were obtained in isolated intact and endothelium-denuded aortas. Effective concentration 50% (EC50) and maximum response (MR) were compared among groups using MANOVA/Tukey tests. Stress and stress plus ethanol increased BP. Ethanol and stress, alone and in combination, did not alter angiotensin responses of intact aortas. PD123-319 decreased MR to angiotensin II in intact aortas from the ethanol and ethanol/stress groups relative to control in the presence of PD123-319. Losartan increased MR to angiotensin II in intact aortas from the stress and ethanol/stress groups relative to control in the presence of losartan. None of the protocols altered angiotensin responses of denuded aortas. Neither indomethacin nor L-NAME altered angiotensin responses of intact aortas from the experimental groups. Thus ethanol and ethanol plus stress may alter endothelial signaling via AT1-receptors, without changing systemic BP. Stress and stress plus ethanol may alter endothelial signaling via AT2-receptors, and thereby increase BP. Knowledge of such vascular changes induced by stress and/or ethanol may contribute to understanding adverse cardiovascular effects of stress and ethanol consumption in humans.

Abstract 307: Enhancement of Cerebrovascular Relaxation by Angiotensin II Type 2 Receptor Agonist, C21, is Lost in Type 2 Diabetes

Background: Angiotensin type 1 receptor (AT1R) blockers provide vascular protection and improve stroke outcomes in young otherwise healthy animals. These effects are believed to be mediated by the indirect stimulation of AT2R signaling. The AT2R agonist, compound 21 (C21), improves endothelial function in peripheral vascular beds but its effect on cerebral endothelial function remains unknown. It is important to determine the vascular effects of C21 in diabetes, a comorbid condition which is known to worsen stroke outcomes. Methods: Endothelium-dependent relaxation was assessed in male Wistar and Type 2 diabetic Goto-Kakizaki (GK) rats (n=3-6) by measuring acetylcholine (ACh, 1 nM - 5 μm) induced dilatory response in basilar arteries. In a subset of experiments C21 dose response curves were generated (0.1 nM - 1 μM) or vessels were pre-incubated with 100 nM C21 ± 1 μM PD123319 (AT2R blocker) for 30 min prior to Ach dose response curves. Area under the curve (AUC) and half maximal effective concentration (EC50 nM) were calculated as indices of total relaxation and receptor sensitivity, respectively. Angiotensin receptors expression was measured by immunoblotting of brain homogenates. Results: AT2R agonist C21 dose response curves showed no basilar reactivity in either control or diabetic animals. Pre-incubation with C21 enhanced relaxation to Ach in control animals (vehicle 146.7 ± 3.9 vs C21 pretreatment 229.6 ± 11.6), which was abolished by the blockade of AT2R (176.9 ± 25.5, p=0.007). Similarly, C21 improved sensitivity in control animals (vehicle 110.5 ± 32 vs C21 pretreatment 11.9 ± 2) which was abolished in the presence of PD123319 (93.8 ± 31, p=0.04). Basilar artery relaxation (AUC) was impaired in diabetic GK rats (93.9 ± 1.8%) as compared to controls (146.7 ± 3.9%) and C21 had no effect (98 ± 8.9%) indicating a disease and treatment interaction (p<0.001). Normalized AT1R expression levels were 1 ± 0.04 and 0.98 ± 0.05 in control vs diabetic rats and respective AT2R levels were 1 ± 0.1 and 0.84 ± 0.09. Conclusion: C21 improves vascular relaxation in control but not in diabetic rats in an AT2R-dependent manner. Underlying mechanisms blunting response to C21 need to be further investigated and may impact the subsequent development of C21 as a treatment for stroke.

Relationship between renal Ang (1‐7) induced sodium and water excretion and AT2 receptors (1088.7)

Ang (1‐7) causes a natriuresis, which is mediated by intrarenal (IR) Mas receptors but is also dependent upon functional IR AT1 receptors. This study investigated the role played by IR AT2 receptors in mediating the renal excretory actions of Ang (1‐7) normally and when the RAS was stimulated by a low sodium diet. Male Wistar rats (n= 5‐6) were placed on normal (NNa+, 0.2%) or low (LNa+, 0.03%) sodium diets for 2 weeks. Rats were anaesthetized (chloralose/urethane) and prepared for mean arterial pressure (MAP) and left renal function measurement and IR infusion of saline and drugs. Data ±SEM were subjected to a Students t‐test. Basal MAP was 93±4 and 91±2mmHg and basal fractional sodium excretion (FENa) was 1.4±0.3 and 0.7±0.2 % in NNa+ and in LNa+ rats, respectively. IR infusion of Ang1‐7 had no effect on MAP but increased FENa by 227% and 427% in NNa+ and LNa+ rats respectively (both P<0.05). In the presence of PD123319, Ang (1‐7) induced increases in FENa excretion were blunted at 75% and 34% in NNa+ and LNa+ rats respectively (both P<0.05). These data imply that functional IR AT2 receptors may have a permissive effect upon the natriuretic actions of Ang (1‐7) at a tubular level.Grant Funding Source: IRCSET

Angiotensin II type 2 receptor blockade inhibits fatty acid synthase production through activation of AMP-activated protein kinase in pancreatic cancer cells

The lipogenesis-promoting enzyme fatty acid synthase is highly expressed in pancreatic ductal adenocarcinoma. Angiotensin II, which is the principal hormone of the renin angiotensin system, is generated actively in the pancreas and has been shown to increase the expression of fatty acid synthase. The angiotensin II type 2 receptor has been proposed to play an important role in lipogenesis and fat deposition. In this study, we explored the potential role of the angiotensin II type 2 receptor in fatty acid synthase regulation in pancreatic ductal adenocarcinoma cells, and we evaluated the mechanisms involved. Fatty acid synthase messenger RNA and protein in pancreatic ductal adenocarcinoma cell lines treated with or without angiotensin II (10(-6) to 10(-8) mol/L) in the presence or absence of the angiotensin II type 2 receptor blocker PD123319 (10(-4) to 10(-6) mol/L) were analyzed by real-time polymerase chain reaction and Western blotting. The total-AMP-activated protein kinase and phospho-AMP-activated protein kinase, total-acetyl CoA carboxylase and phospho-acetyl CoA carboxylase, and LKB1/STK11 were analyzed by Western immunoblotting. The tissue localization of the angiotensin II type 2 receptor was examined by immunohistochemistry in invasive pancreatic ductal adenocarcinoma lesions and matching normal tissue. Angiotensin II type 2 receptor treatment increased fatty acid synthase expression and promoter activity in significantly pancreatic ductal adenocarcinoma cells; these effects were blocked significantly in the presence of PD123319. Interestingly, angiotensin II also induced angiotensin II type 2 receptor expression in pancreatic ductal adenocarcinoma cells. PD123319, C75, and AICAR decreased fatty acid synthase protein levels, but only PD123319 increased LKB1/STK11 levels. All 3 agents activated AMP-activated protein kinase differentially and inhibited acetyl CoA carboxylase. Angiotensin II type 2 receptor messenger RNA levels were upregulated significantly in 20 of the 25 neoplastic tissues examined (80%) when compared with matching controls. Angiotensin II type 2 receptor protein was localized in the malignant ducts and in the stromal cells. Our data demonstrate a previously unknown involvement of the angiotensin II type 2 receptor in pancreatic ductal adenocarcinoma cell fatty acid synthesis and suggest that its blockade has potential as a novel chemopreventive and antilipogenic mechanism for human pancreatic ductal adenocarcinoma through the activation of AMP-activated protein kinase, which could have detrimental effects on cancer cell survival.

Nerve stimulation induced overflow of neuropeptide Y and modulation by angiotensin II in spontaneously hypertensive rats

The sympathetic nervous system and renin-angiotensin system are both thought to contribute to the development and maintenance of hypertension in experimental models such as the spontaneously hypertensive rat (SHR). We demonstrated that periarterial nerve stimulation (NS) increased the perfusion pressure (PP) and neuropeptide Y (NPY) overflow from perfused mesenteric arterial beds of SHRs at 4-6, 10-12, and 18-20 wk of age, which correspond to prehypertensive, developing hypertensive, and maintained hypertensive stages, respectively, in the SHR. NS also increased PP and NPY overflow from mesenteric beds of Wistar-Kyoto (WKY) normotensive rats. NS-induced increases in PP and NPY were greater in vessels obtained from SHRs of all three ages compared with WKY rats. ANG II produced a greater increase in PP in preparations taken from SHRs than WKY rats. ANG II also resulted in a greater increase in basal NPY overflow from 10- to 12-wk-old and 18- to 20-wk-old SHRs than age-matched WKY rats. ANG II enhanced the NS-induced overflow of NPY from SHR preparations more than WKY controls at all ages studied. The enhancement of NS-induced NPY overflow by ANG II was blocked by the AT1 receptor antagonist EMD-66684 and the angiotensin type 2 receptor antagonist PD-123319. In contrast, ANG II greatly enhanced norepinephrine overflow in the presence of PD-123319. Both captopril and EMD-66684 decreased neurotransmitter overflow from SHR mesenteric beds; therefore, we conclude that an endogenous renin-angiotensin system is active in this preparation. It is concluded that the ANG II-induced enhancement of sympathetic nerve stimulation may contribute to the development and maintenance of hypertension in the SHR.

Angiotensin II‐induced contractions in human jejunal wall musculature in vitro

Angiotensin II is well known for its contractile effects on smooth muscle cells. This effect is also present in the gut previously shown in animal models. The aim of this study was to clarify expression and localization of angiotensin II receptors in the human small intestine and to explore the pharmacological profile of angiotensin II effects in vitro. Strips of jejunal muscle wall from 32 patients undergoing bariatric surgery were used to record isometric tension in vitro in response to angiotensin II (10(-10)-10(-5) M) alone and in the presence of PD123319 (10(-7) M), losartan (10(-7) M), PD123319 (10(-7) M) and losartan (10(-7) M) in combination, tetrodotoxin (TTX) (10(-6) M), atropine (10(-6) M) and guanethidine (3 x 10(-6) M). Western blot, immunohistochemistry and RT-PCR were performed on corresponding muscle samples to identify expression and localization of key components of the renin-angiotensin system. Angiotensin II elicited concentration-dependent contraction in both longitudinal and circular jejunal muscle wall strips; neither TTX, atropine nor guanethidine affected this action. Losartan alone and in combination with PD123319 shifted the concentration-response curve to the right. Transcription of angiotensinogen, ACE and angiotensin II types 1 and 2 receptor RNA was detected in all patients. Immunohistochemistry detected angiotensin II type 1 receptors in the musculature; both angiotensin II types 1 and type 2 receptors were found in the myenteric plexus. This pharmacological analysis indicates that the contractile action elicited by angiotensin II on jejunal wall musculature is primarily mediated through the angiotensin II type 1 receptor located on the musculature.

The effect of angiotensin 1–7 on tyrosine kinases activity in rat anterior pituitary

Angiotensin 1–7 (Ang 1–7) is a peptide originated from Ang II. It is known that in vessels Ang 1–7 shows opposite effects to Ang II. Ang 1–7 can modify processes of proliferation. However, Ang 1–7 action in pituitary gland cells was never studied. Moreover, the specific binding sites for Ang 1–7 are still unknown. The aim of this study was to examine the effects of Ang 1–7 on tyrosine kinases (PTKs) activity in the anterior pituitary. The reaction of phosphorylation was carrying out in presence of different concentration of Ang 1–7 and losartan (antagonist of AT1 receptor) and PD123319 (antagonist of AT2). Our results show that Ang 1–7 inhibited activity of PTK to 60% of basic activity. Losartan did not change the Ang 1–7-induced changes in PTKs activity. The presence of PD123319 together with Ang 1–7 caused stronger inhibition PTKs activity than Ang 1–7 alone. These observations suggest that Ang 1–7 binds to the novel, unknown, specific for this peptide receptor.

Evidence for a Functional Interaction of the Angiotensin-(1–7) Receptor Mas With AT 1 and AT 2 Receptors in the Mouse Heart

The aim of this study was to evaluate the angiotensin (Ang)-(1-7) effects in isolated mouse hearts. The hearts of male C57BL/6J and knockout mice for the Ang-(1-7) receptor Mas were perfused by the Langendorff method. After a basal period, the hearts were perfused for 20 minutes with Krebs-Ringer solution (KRS) alone (control) or KRS containing Ang-(1-7) (0.22 pmol/L), the Mas antagonist A-779 (115 nmol/L), the angiotensin type 1 receptor antagonist losartan (2.2 micromol/L), or the angiotensin type 2 receptor antagonist PD123319 (130 nmol/L). To evaluate the involvement of Ang receptors, prostaglandins, and nitric oxide in the Ang-(1-7) effects, the hearts were perfused for 20 to 30 minutes with KRS containing either A-779, losartan, PD123319, indomethacin, or NG-nitro-L-arginine methyl ester (L-NAME) alone or in association with subsequent Ang-(1-7) perfusion. In addition, hearts from Mas-knockout mice were perfused for 20 minutes with KRS containing Ang-(1-7) (0.22 pmol/L) and losartan. Ang-(1-7) alone did not change the perfusion pressure. Strikingly, in the presence of losartan, 0.22 pmol/L Ang-(1-7) induced a significant decrease in perfusion pressure, which was blocked by A-779, indomethacin, and L-NAME. Furthermore, this effect was not observed in Mas-knockout mice. In contrast, in the presence of PD123319, Ang-(1-7) produced a significant increase in perfusion pressure. This change was not modified by the addition of A-779. Losartan reduced but did not abolish this effect. Our results suggest that Ang-(1-7) produces complex vascular effects in isolated, perfused mouse hearts involving interaction of its receptor with angiotensin type 1- and type 2-related mechanisms, leading to the release of prostaglandins and nitric oxide.

Diminution of angiotensin II-induced contraction of the abdominal aorta isolated from Watanabe heritable hyperlipidemic rabbits

The purpose of this study was to investigate the changes in vasocontractile responses in atherosclerosis, using abdominal aortic strips isolated from Watanabe heritable hyperlipidemic (WHHL) rabbits and Japanese White (control) rabbits. The aortic strips from WHHL rabbits showed a significantly lower contractile response to angiotensin II than that in strips from control rabbits. The contractile responses to phenylephrine and 5-hydroxytryptamine were not different in WHHL and control groups. The contractile response to angiotensin II was higher in endothelium-denuded aortic strips than in endothelium-intact strips, but to a greater extent in the control group than in the WHHL group. The contractile response to angiotensin II in the absence of the endothelium was also lower in the WHHL group than in the control group. Pretreatment with N(G)-nitro-L-arginine significantly increased the contractile response to angiotensin II in the endothelium-intact aortic strips in both the WHHL and control groups, while pretreatment with diclofenac did not affects the aortic contractile response to angiotensin II. The contractile responses to angiotensin II in the presence of N(G)-nitro-L-arginine and diclofenac were lower in the WHHL group than in the control group. The contractile response to angiotensin II in the presence of PD123319 was also lower in the WHHL group than in the control group. Endothelium-dependent relaxation by acetylcholine occurred to the some extent in the WHHL and control groups. These results suggest that the WHHL rabbit abdominal aorta displays attenuated angiotensin II-induced contraction, mainly due to an abnormality in the angiotensin II-specific contractile pathway of the medial smooth muscle.

Angiotensin II type 2 receptor-mediated duodenal mucosal alkaline secretion in the rat.

The aims of this study were to elucidate the distribution of angiotensin receptors (AT(1) and AT(2)) in the duodenal wall and to investigate whether AT(2) receptors are involved in the regulation of duodenal mucosal alkaline secretion, which is of importance for the mucosal defense against gastric acid. Immunohistochemistry was used to locate AT(1) and AT(2) receptors in chloralose-anesthetized rats. Duodenal mucosal alkaline output was measured by use of in situ pH-stat titration. Immunohistochemistry demonstrated a distinct staining for both AT(1) and AT(2) receptors in the lamina propria of the villi and also for AT(1) receptors in the muscularis interna. When angiotensin II was infused in the presence of the AT(1) receptor antagonist losartan, mucosal alkaline secretion increased by ~50%. This response was inhibited by the AT(2) receptor antagonist PD-123319. The AT(2) receptor agonist CGP-42112A increased mucosal alkaline secretion by ~50%. This increase was absent in the presence of PD-123319 but not in the presence of losartan or the local anesthetic lidocaine. We conclude that angiotensin II stimulates duodenal mucosal alkaline secretion by activation of AT(2) receptors located in the duodenal mucosa/submucosa.

Characterisation of vasopressin V 1A, angiotensin AT 1 and AT 2 receptor distribution and density in normotensive and hypertensive rat brain stem and kidney: effects of restraint stress

In the present study, we have examined neurochemical correlates that may be involved in the differential cardiovascular responses observed in normotensive and hypertensive rats during stress. Using a restraint stress paradigm, both normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive rats (SHR) underwent acute (1 h restraint in a perspex tube), chronic (1 h restraint for ten consecutive days) or no restraint (control) stress. Following cessation of restraint, rats were processed by incubating sections of brain stem and kidney with [ 125I]-HO-LVA (0.03 nM) or [ 125I]Sar 1Ile 8-AngiotensinII (0.5 nM), in the presence of PD123319 (10 μM) or losartan (10 μM), to determine the distribution and density of vasopressin V 1A, angiotensin AT 1 and AT 2 receptors, respectively. Analysis of autoradiograms indicated changes in the density of radioligand binding in acutely and chronically-stressed rats, as compared to controls. For example, V 1A binding in the medial nucleus tractus solitarius (SolM) decreased in the WKY but increased in the SHR. AT 1 binding in SolM did not significantly change in the WKY but decreased in the SHR with repeated restraint. In kidney slices, AT 1 binding decreased with stress in the WKY (−17%) but increased in SHR (+10–15%). AT 2 binding in the kidney showed a pattern similar to that of AT 1 binding in SHR, but not WKY. Graded increases in V 1A binding were measured in kidney medulla and cortex of both strains (+50–60% with chronic restraint). These results suggest that physiological adaptation to restraint is associated with specific changes in V 1A, AT 1 and AT 2 receptor density within brain nuclei and kidney.

Endogenous nitric oxide and epoxyeicosatrienoic acids modulate angiotensin II-induced constriction in the rabbit afferent arteriole.

Nitric oxide (NO) and epoxyeicosatrienoic acids (EETs), cytochrome P450 epoxygenase metabolites of arachidonic acid, are released by the vascular endothelium and play important roles in the control of glomerular haemodynamics. We examined whether endogenous NO or EETs modulate angiotensin II- (AngII) induced constriction in isolated microperfused afferent arteriole (Af-Art) of the rabbit kidney. When Af-Arts were treated with NG-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthese; 10-4 mol L-1) or miconazole (an inhibitor of P450 epoxygenase; 10-6 mol L-1), basal diameter was decreased by 34.5 +/- 2.2 and 13.9 +/- 3.2%, respectively. AngII added to both the bath and lumen decreased the diameter of Af-Arts in a dose-dependent manner. Pretreatment with either L-NAME or miconazole also augmented the constrictor response to AngII. AngII at 10-8 mol L-1 decreased the diameter to 39.2 +/- 1.4, 32.9 +/- 3.6, and 12.7 +/- 4.6%, in control, L-NAME-, and miconazole-treated group, respectively. In order to study whether the AngII type2 (AT2) receptor modulates AngII action via NO or EETs, we repeated the experiments in the presence of PD123319 (an AT2 receptor antagonist; 10-7 mol L-1). In the presence of PD123319, L-NAME still augmented the constrictor response to AngII, however, miconazole had no effect. In the presence of PD123319, AngII at 10-8 mol L-1 decreased the diameter to 25.0 +/- 4.6, 9.4 +/- 4.0, and 26.0 +/- 3.3%, in control, L-NAME-, and miconazole-treated group, respectively. These results suggest that (1) tonic release of NO and EETs attenuates the vasoconstrictor response to AngII in Af-Arts and (2) AT2 receptor seems to be coupled to EETs rather than the NO pathway.

Expression of the AT2 receptor developmentally programs extracellular signal-regulated kinase activity and influences fetal vascular growth.

Angiotensin II type 2 (AT2) receptor is abundantly expressed in vascular smooth muscle cells (VSMC) of the fetal vasculature during late gestation (embryonic day 15-20), during which the blood vessels undergo remodeling. To examine directly the influence of AT2 receptor expression in the developmental biology of VSMC, we studied cultures of VSMC from fetal and postnatal wild-type (Agtr2(+)) and AT2 receptor null (Agtr2(-)) mice. Consistent with in vivo data, AT2 receptor binding in cultured Agtr2(+) VSMC increased by age, peaking at embryonic day 20, and decreased dramatically after birth. Angiotensin II-induced growth in Agtr2(+) VSMC (embryonic day 20) was increased by the AT2 receptor blocker PD123319, indicating that the AT2 receptors are functional and exert an antigrowth effect in Agtr2(+) VSMC. Growth of VSMC in response to serum decreased age dependently and was higher in Agtr2(-) than in Agtr2(+), inversely correlating with AT2 receptor expression. However, serum-induced growth in Agtr2(+) and Agtr2(-) VSMC and the exaggerated Agtr2(-) VSMC growth was maintained even in the presence of PD123319 or losartan, an AT1 receptor blocker. Moreover, Agtr2(-) VSMC showed greater growth responses to platelet-derived growth factor and basic fibroblast growth factor, indicating that Agtr2(-) cells exhibit a generalized exaggerated growth phenotype. We studied the mechanism responsible for this phenotype and observed that extracellular signal-regulated kinase (ERK) activity was higher in Agtr2(-) VSMC at baseline and also in response to serum. ERK kinase inhibitor PD98059 inhibited both growth and ERK phosphorylation dose-dependently, while the regression lines between growth and ERK phosphorylation were identical in Agtr2(+) and Agtr2(-) VSMC, suggesting that increased ERK activity in Agtr2(-) VSMC is pivotal in the growth enhancement. Furthermore, the difference in ERK phosphorylation between Agtr2(+) and Agtr2(-) was abolished by vanadate but not by okadaic acid, implicating tyrosine phosphatase in the difference in ERK activity. These results suggest that the AT2 receptor expression during the fetal vasculogenesis influences the growth phenotype of VSMC via the modulation of ERK cascade.

The receptor subtype mediating the action of angiotensin II on intracellular sodium in rat proximal tubules.

1. An investigation was undertaken to explore the subtype of receptor involved in mediating the actions of angiotensin II on intracellular sodium content in suspensions of isolated proximal tubules of the rat. 2. Intracellular sodium content of the proximal tubules was measured with 23Na n.m.r. spectroscopy and under these conditions basal sodium content of the tubular cells was 69.04+/-1.73 nmol mg(-1) dry weight and the ATP levels, at 8.3+/-0.9 nmol ATP mg(-1) protein, were consistent with active respiration by the tissue. 3. In the presence of 10(-4) M PD123319, a selective non-peptide AT2 receptor antagonist, intracellular sodium levels rose from steady state by 30% (P < 0.01; n = 7) within 10 min of exposure to angiotensin II 10(-11) M. Over the subsequent 30 min steady state levels were re-established. Administration of angiotensin II 10(-11) M, in the presence of the selective AT1 receptor antagonist, losartan at either 10(-6) M (n = 5) or 10(-4) M (n = 6), was without effect on intracellular sodium levels, which were significantly different (P < 0.001) from those observed when PD 123319 was present. 4. Angiotensin II 10(-5) M, administered to the tubular suspension in the presence of 10(-4) M PD123319, decreased (P < 0.01, n = 6) intracellular sodium content by 16% in the first 5 min, but in the following 25 min returned to steady state levels. However, in the presence of losartan 10(-4) M, angiotensin II 10(-5) M had no effect on intracellular sodium content which was markedly different (P < 0.001) from that obtained in the presence of PD123319. 5. These findings show that at both the high and low concentrations of angiotensin II, its modulation of intracellular sodium levels within the proximal tubule cells is mediated via the activation of AT1 receptors. The intracellular mechanism underlying this effect remain to be investigated.

Angiotensin II-mediated Growth and Antigrowth Effects in Cultured Neonatal Rat Cardiac Myocytes and Fibroblasts

Angiotensin II (Ang II) stimulates cardiovascular growth and remodeling via AT1receptors. Recent experiments have shown that Ang II may also exert antiproliferative effects via AT2receptors. We studied the effects of Ang II on protein and DNA content and synthesis rate in unstimulated and endothelin-1 (ET-1)-stimulated neonatal rat cardiomyocytes and fibroblasts, isolated from 1–3-day-old Wistar strain pups. Total protein and total DNA, as well as [3H]leucine and [3H]thymidine incorporation were measured following incubation with either vehicle, Ang II, ET-1 or Ang II+ET-1, both in the presence or absence of the AT1receptor blocker losartan or the AT2receptor blocker PD123319. In myocytes, ET-1 increased total protein (+38% relative to control) as well as [3H]leucine (+66%) and [3H]thymidine (+77%) incorporation. Ang II did not affect any of these parameters, nor did it influence the ET-1-induced responses. However, in the presence of PD123319 Ang II stimulated [3H]leucine (+24%) and [3H]thymidine (+30%) incorporation. In fibroblasts, ET-1 and Ang II did not significantly affect total DNA and [3H]thymidine incorporation. Ang II tended to increase total protein in these cells, an effect which was significant only in the presence of PD123319 (+17%). Ang II stimulated [3H]leucine incorporation (+24%) in fibroblasts. This effect was absent with losartan and enhanced in the presence of PD123319. These data demonstrate that AT1receptor-mediated proliferative effects of Ang II in neonatal cardiac cells may become apparent only when its AT2receptor-mediated antigrowth effects are blocked. The net growth effect of Ang II therefore depends on the cellular AT1/AT2receptor ratio. Ang II does not appear to interfere with ET-1-induced effects.