Angiotensin II and IV stimulate expression and release of plasminogen activator inhibitor-1 in cultured human coronary artery endothelial cells

There is increasing evidence that angiotensin II influences thrombogenesis by regulating the expression of plasminogen activator inhibitor-1 (PAI-1). In this study, the effects of angiotensin II and its receptors on the expression and release of PAI-1 and tissue-type plasminogen activator (t-PA) were examined in human coronary artery endothelial cells (HCAECs). As control, cells were treated with angiotensin IV. HCAECs incubated with angiotensin II increased the expression of PAI-1 mRNA in a concentration (10-9-10-5 M)- and time (6-24 h)-dependent manner. PAI-1 protein release was also increased in the culture medium of HCAECs treated with angiotensin II. The effects of angiotensin II (10-6 M) were blocked completely by the AT1 receptor blocker losartan (10-6 M) but not by the AT2 receptor blocker PD123319 (10-6 M). Angiotensin II pretreatment also slightly, but significantly, increased t-PA mRNA expression. This effect of angiotensin II on t-PA mRNA was blocked by losartan but not by PD123319. HCAECS treated with angiotensin II revealed large amounts of the lipid peroxidation product, malonaldehyde (MDA). The effects of angiotensin II on PAI-1 expression and MDA release were blocked by pretreatment of cells with alpha-tocopherol (10-5 M). In control experiments, treatment of HCAECs with angiotensin IV markedly increased PAI-1 mRNA expression and protein release. This effect of angiotensin IV was blocked by the AT4 receptor blocker divalinal (10-6 M). These observations indicate that AT1 receptor activation plays an important role in the stimulation of PAI-1 expression and release in response to angiotensin II. Upregulation of t-PA gene may reflect autoregulation in response to PAI-1 release. Angiotensin II-mediated activation of oxidation pathways may relate to uupregulation of PAI-1. This study also confirms that angiotensin IV upregulates PAI-1 expression in HCAECs.     

Prejunctional angiotensin receptors involved in the facilitation of noradrenaline release in mouse tissues.

The effect of angiotensin II, angiotensin III, angiotensin IV and angiotensin-(1-7) on the electrically induced release of noradrenaline was studied in preparations of mouse atria, spleen, hippocampus, occipito-parietal cortex and hypothalamus preincubated with [3H]-noradrenaline. The prejunctional angiotensin receptor type was investigated using the non-selective receptor antagonist saralasin (AT1/AT2) and the AT1 and AT2 selective receptor antagonists losartan and PD 123319, respectively. In atrial and splenic preparations, angiotensin II (0.01 nM-0.1 microM) and angiotensin III (0.01 and 0.1 nM-1 microM) increased the stimulation-induced overflow of tritium in a concentration-dependent manner. Angiotensin IV, only at high concentrations (1 and 10 pM), enhanced tritium overflow in the atria, while angiotensin-(1-7) (0.1 nM-10 microM) was without effect in both preparations. In preparations of hippocampus, occipito-parietal cortex and hypothalamus, none of the angiotensin peptides altered the evoked overflow of tritium. In atrial and splenic preparations, saralasin (0.1 microM) and losartan (0.1 and 1 microM), but not PD 123319 (0.1 microM), shifted the concentration-response curves of angiotensin II and angiotensin III to the right. In conclusion, in mouse atria and spleen, angiotensin II and angiotensin III facilitate the action potential induced release of noradrenaline via a prejunctional AT1 receptor. Only high concentrations of angiotensin IV are effective in the atria and angiotensin-(1-7) is without effect in both preparations. In mouse brain areas, angiotensin II, angiotensin III, angiotensin IV and angiotensin-(1-7) do not modulate the release of noradrenaline.     

Effects of angiotensin II and its metabolites in the rat coronary vascular bed: is angiotensin III the preferred ligand of the angiotensin AT2 receptor?

Aminopeptidases metabolize angiotensin II to angiotensin-(2-8) (=angiotensin III) and angiotensin-(3-8) (=angiotensin IV), and carboxypeptidases generate angiotensin-(1-7) from angiotensin I and II. Angiotensin-converting enzyme (ACE) inhibitors and/or angiotensin II type 1 (AT1) receptor blockers affect the concentrations of these metabolites, and they may thus contribute to the beneficial effects of these drugs, possibly through stimulation of non-classical angiotensin AT receptors. Here, we investigated the effects of angiotensin II, angiotensin III, angiotensin IV and angiotensin-(1-7) in the rat coronary vascular bed, with or without angiotensin AT1 - or angiotensin II type 2 (AT2) receptor blockade. Results were compared to those in rat iliac arteries and abdominal aortas. Angiotensin II, angiotensin III and angiotensin IV constricted coronary arteries via angiotensin AT1 receptor stimulation, angiotensin III and angiotensin IV being approximately 20- and approximately 8000-fold less potent than angiotensin II. The angiotensin AT2 receptor antagonist PD123319 greatly enhanced the constrictor effects of angiotensin III, starting at angiotensin III concentrations in the low nanomolar range. PD123319 enhanced the angiotensin II-induced constriction at submicromolar angiotensin II concentrations only. Angiotensin-(1-7) exerted no effects in the coronary circulation, although, at micromolar concentrations, it blocked angiotensin AT1 receptor-induced constriction. Angiotensin AT2 receptor-mediated relaxation did not occur in iliac arteries and abdominal aortas, and the constrictor effects of the angiotensin metabolites in these vessels were identical to those in the coronary vascular bed. In conclusion, angiotensin AT2 receptor activation in the rat coronary vascular bed results in vasodilation, and angiotensin III rather than angiotensin II is the preferred endogenous agonist of these receptors. Angiotensin II, angiotensin III, angiotensin IV and angiotensin-(1-7) do not exert effects through non-classical angiotensin AT receptors in the rat coronary vascular bed, iliac artery or aorta.     

Evidence for the involvement of different receptor subtypes in the pre- and postjunctional actions of angiotensin II at rat sympathetic neuroeffector sites.

1. The effects of the nonpeptide angiotensin II receptor (AT) antagonists losartan and PD 123319 on actions of angiotensin II in the rat caudal artery and rat vas deferens preparations were investigated. 2. Angiotensin II (1.0 microM) increased perfusion pressure in isolated segments of the rat caudal artery. This increase in perfusion pressure was prevented by the AT1-antagonist, losartan (0.1 microM) but was not affected by the AT2-antagonist, PD 123319 (0.1 microM). 3. Angiotensin II (0.1-3.0 microM) produced a concentration-dependent enhancement of the stimulation-induced (S-I) efflux of [3H]-noradrenaline from isolated segments of rat caudal artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline. The maximum enhancement of S-I efflux was approximately 60% with 1.0 microM angiotensin II. 4. Losartan (0.01 and 0.1 microM) reduced the enhancement of S-I efflux produced by 1.0 microM angiotensin II in the caudal artery. 5. PD 123319 (0.01 microM) did not affect the enhancement of S-I efflux produced by angiotensin II (1.0 microM) in the caudal artery. However, in a higher concentration (0.1 microM), PD 123319 reduced the enhancement of S-I efflux produced by 1.0 microM angiotensin II. 6. Angiotensin II produced concentration-dependent enhancement of the purinergic twitch responses (1 pulse/60 s) in the rat vas deferens. 7. Losartan (0.03 microM) and PD 123319 (0.03 microM) each reduced the angiotensin II-induced enhancement of the twitch responses in the rat vas deferens. 8. These findings indicate that the enhancement of sympathetic neuroeffector transmission in both the caudal artery and vas deferens of the rat involves angiotensin receptor subtype(s) sensitive to both losartan and PD 123319. In contrast, the direct vasoconstrictor effect of angiotensin II in the rat caudal artery involves activation of a receptor subtype sensitive only to losartan.     

Intracellular Angiotensin II and cell growth of vascular smooth muscle cells

1. We recently demonstrated that intracellular application of Angiotensin II (Angiotensin II(intr)) induces rat aorta contraction independent of plasma membrane Angiotensin II receptors. In this study we investigated the effects of Angiotensin II(intr) on cell growth in A7r5 smooth muscle cells. 2. DNA-synthesis was increased dose-dependently by liposomes filled with Angiotensin II as measured by [(3)H]-thymidine incorporation at high (EC(50)=27+/-6 pM) and low (EC(50)=14+/-5 nM) affinity binding sites with increases in E(max) of 58+/-4 and 37+/-4% above quiescent cells, respectively. Cell growth was corroborated by an increase in cell number. 3. Extracellular Angiotensin II (10 pM - 10 microM) did not modify [(3)H]-thymidine incorporation. 4. Growth effects of Angiotensin II(intr) mediated via high affinity sites were inhibited by liposomes filled with 1 microM of the non-peptidergic antagonists losartan (AT(1)-receptor) or PD123319 (AT(2)-receptor) or with the peptidergic agonist CGP42112A (AT(2)-receptor). E(max) values were decreased to 30+/-3, 29+/-4 and 4+/-2%, respectively, without changes in EC(50). The Angiotensin II(intr) effect via low affinity sites was only antagonized by CGP42112A (E(max)=11+/-3%), while losartan and PD123319 increased E(max) to 69+/-4%. Intracellular applications were ineffective in the absence of Angiotensin II(intr). 5. Neither intracellular nor extracellular Angiotensin I (1 microM) were effective. 6. The Angiotensin II(intr) induced growth response was blocked by selective inhibition of phosphatidyl inositol 3-kinase (PI-3K) by wortmannin (1 microM) and of the mitogen-activated protein kinase (MAPK/ERK) pathway by PD98059 (1 microM) to 61+/-14 and 4+/-8% of control, respectively. 7. These data demonstrate that Angiotensin II(intr) induces cell growth through atypical AT-receptors via a PI-3K and MAPK/ERK -sensitive pathway.     

Angiotensin II receptors involved in the enhancement of noradrenergic transmission in the caudal artery of the spontaneously hypertensive rat.

1. The effects of the AT1 receptor antagonist losartan and the AT2 receptor antagonist PD 123319, on actions of angiotensin II in isolated caudal arteries of spontaneously hypertensive (SH) and age-matched normotensive (Wistar-Kyoto) rats were compared. 2. Angiotensin II (0.1-3 microM) produced concentration-dependent increases in perfusion pressure in artery preparations from both SH and Wistar-Kyoto (WKY) rats, the maximal increase in the SH rat being significantly greater than the increase in WKY rats. The increase in perfusion pressure in preparations from both strains of rats was prevented by losartan (0.1 microM) and unaffected by PD 123319 (0.1 microM), indicating that the vasoconstrictor action of angiotensin II is subserved by AT1 receptors. 3. Angiotensin II (0.1-3 microM) produced concentration-dependent enhancement of both stimulation-induced (S-I) efflux of [3H]-noradrenaline and stimulation-evoked vasoconstrictor responses in isolated preparations of caudal artery from both SH and WKY rats, in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline. The maximum enhancement of S-I efflux produced by angiotensin II (1 microM) was significantly greater in artery preparations from WKY rats than in preparations from SH rats, whereas the maximum enhancement of stimulation-evoked vasoconstrictor responses was greater in preparations from SH rats than in those from WKY rats. 4. In artery preparations from both WKY and SH rats, the AT1 angiotensin II receptor antagonist, losartan (0.01 and 0.1 microM), reduced or abolished the enhancement of both S-I efflux and vasoconstrictor responses by 1 microM angiotensin II. 5. The combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced both the S-I efflux and stimulation-evoked vasoconstrictor response in caudal artery preparations from WKY rats, whereas 0.1 microM angiotensin alone was ineffective. The AT2 receptor antagonist PD 123319 (0.01 and 0.1 microM) prevented the enhancement of both S-I efflux and stimulation-evoked vasoconstrictor responses by the combination of angiotensin II and losartan. 6. In contrast to findings in WKY preparations and those previously obtained for arteries from another normotensive strain (Sprague-Dawley), in artery preparations from SH rats there was no synergistic interaction between losartan and angiotensin II. Rather, combinations of 0.1 microM angiotensin II and PD 123319 (both 0.01 and 0.1 microM) enhanced S-I [3H]-noradrenaline efflux, whereas 0.1 microM angiotensin II alone was without effect. Moreover, losartan (0.1 microM) prevented the enhancement of S-I efflux by the combination of angiotensin II and PD 123319. 7. The present findings indicate that in the caudal artery of WKY and SH rats, and as previously found in Sprague-Dawley preparations, angiotensin II receptors similar to the AT1B subtype subserve enhancement of transmitter noradrenaline release. 8. As previously suggested for Sprague-Dawley caudal artery preparations, the synergistic prejunctional interaction of losartan and 0.1 microM angiotensin II in caudal artery preparations from WKY rats may be due to either the unmasking by losartan of a latent population of angiotensin II receptors subserving facilitation of transmitter noradrenaline release, or blockade by losartan of an inhibitory action of angiotensin II on transmitter release. 9. The synergistic interaction of PD 123319 and 0.1 microM angiotensin II in caudal arteries of SH rats may also be explained by either of the mechanisms proposed for the normotensive strains, but the involvement of different receptor subtypes would need to be postulated for each of the proposed mechanisms.     

17beta-estradiol inhibits angiotensin II-induced collagen synthesis of cultured rat cardiac fibroblasts via modulating angiotensin II receptors

Circulating endogenous estrogen is considered to be cardiovascular protective, but the underlying mechanisms remain obscure. The cardiac fibroblasts, the most abundant cell type present in the heart, are responsible for the deposition of extracellular matrix. Angiotensin II has been known to stimulate cardiac collagen gene expression. The present study was designed to investigate the effect of 17beta-estradiol on the angiotensin II-induced proliferation and collagen synthesis in cultured cardiac fibroblasts by using real-time polymerase chain reaction (PCR), Western blot and 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide proliferation assay. Angiotensin II increased the cell proliferation and synthesis of collagen types I and III. Angiotensin II up-regulated the gene expression of the angiotensin AT(1) receptor and down-regulated the gene expression of the angiotensin AT(2) receptor in cardiac fibroblasts. The effects of angiotensin II was abolished by the angiotensin AT(1) receptor antagonist, losartan, but not by the angiotensin AT(2) receptor antagonist, PD 123319. 17beta-estradiol prevented increases in proliferation and attenuated the collagen synthesis in response to angiotensin II. The increased AT(1) receptor mRNA levels and decreased AT(2) receptor mRNA levels were partially reversed by 17beta-estradiol treatment. In conclusion, the down-regulation of angiotensin AT(1) receptor expression and function is likely to be an important mechanism accounting for the inhibitory effect of 17beta-estradiol on angiotensin II-stimulated proliferation and collagen synthesis in cardiac fibroblasts. This effect may confer at least in part the cardiac protective action of 17beta-estradiol under pathological conditions with increased activity of the rennin-angiotensin system.     

Interactions of ligands at angiotensin II-receptors and imidazoline receptors

Ligands for angiotensin II-(AT)-receptors and imidazoline receptors have structural similarities and influence blood pressure via various mechanisms. The goal of this study was to study the specificity of various ligands by displacement experiments. Antazoline, cimetidine, clonidine, efaroxan, guanabenz, guanethidine, idazoxan, moxonidine and rilmenidine up to a concentration of 100 microM failed to displace the specific binding of [125I]Sar1,Ile8 angiotensin II at the AT1-receptor characterized by losartan (IC50 = 26 +/- 12 nM) in liver homogenate. The same substances up to 100 microM produced no reduction of specific [125I]Sar1,Ile8 angiotensin II binding to the AT2-receptor of phaeochromocytoma cell membranes characterized by PD123319 (IC50 = 20 +/- 5 nM). Displacement experiments at the imidazoline I1-receptors were performed on bovine adrenal medulla membranes using [3H]clonidine after characterization by the I1-ligand clonidine (IC50 = 459 +/- 13 nM) and the I2-ligand idazoxan (IC50 = 3.29 +/- 0.88 microM). The investigated AT-receptor ligands angiotensin II, losartan, EXP 3174 and PD123319 revealed no displacement of [3H]clonidine up to a concentration of 100 microM. The I2-receptor in liver homogenate was characterized by displacement of [3H]idazoxan by cold idazoxan and clonidine (IC50 = 0.37 +/- 0.17 and 68 +/- 31 microM, respectively). The investigated AT-receptor ligands angiotensin II, losartan and PD123319 failed to displace [3H]idazoxan specifically up to 100 microM. Hence, the tested substances showed no cross-reactivity at the corresponding AT- and I-receptors up to 100 microM, a concentration markedly higher than the plasma concentrations achieved after therapeutic application.     

Cross-talk between angiotensin II and glucagon receptor signaling mediates phosphorylation of mitogen-activated protein kinases ERK 1/2 in rat glomerular mesangial cells

We have recently shown that the pancreatic hormone glucagon-induced phosphorylation of mitogen-activated protein (MAP) kinase ERK 1/2 as well as growth and proliferation of rat glomerular mesangial cells (MCs) via activation of cAMP-dependent protein kinase A (PKA)- and phospholipase C (PLC)/Ca2+-mediated signaling pathways. Since circulating glucagon and tissue angiotensin II (Ang II) levels are inappropriately elevated in type 2 diabetes, we tested the hypothesis that glucagon induces phosphorylation of ERK 1/2 in MCs by interacting with Ang II receptor signaling. Stimulation of MCs by glucagon (10 nM) induced a marked increase in intracellular [Ca2+]i that was abolished by [Des-His1, Glu9]-glucagon (1 microM), a selective glucagon receptor antagonist. Both glucagon and Ang II-induced ERK 1/2 phosphorylation (glucagon: 214+/-14%; Ang II: 174+/-16%; p<0.001 versus control), and these responses were inhibited by the AT1 receptor blocker losartan (glucagon + losartan: 77+/-14%; Ang II + losartan: 84+/-18%; p<0.01 versus glucagon or Ang II) and the AT2 receptor blocker PD 123319 (glucagon + PD: 78+/-7%; Ang II + PD: 87+/-7%; p<0.01 versus glucagon or Ang II). Inhibition of cAMP-dependent PKA with H89 (1 microM) or PLC with U73122 (1 microM) also markedly attenuated the phosphorylation of ERK 1/2 induced by glucagon (glucagon + U73122: 109+/-15%; glucagon + H89: 113+/-16%; p<0.01 versus glucagon) or Ang II (Ang II + U73122: 111+/-13%; Ang II + H89: 86+/-10%; p<0.01 versus Ang II). Wortmannin (1 microM), a selective PI 3-kinase inhibitor, also blocked glucagon- or Ang II-induced ERK 1/2 phosphorylation. These results suggest that AT1 receptor-activated cAMP-dependent PKA, PLC and PI 3-kinase signaling is involved in glucagon-induced MAP kinase ERK 1/2 phosphorylation in MCs. The inhibitory effect of PD 123319 on glucagon-induced ERK 1/2 phosphorylation further suggests that AT2 receptors also play a similar role in this response.     

Analysis of the vasorelaxant action of angiotensin II in the isolated rat renal artery

Taking into consideration that mechanisms involved in the vasodilatator actions of angiotensin II have not yet been completely elucidated, the present study was undertaken in order to examine the mechanisms underlying the angiotensin II-induced relaxation of rat renal artery (RRA). Angiotensin II produced concentration-dependent and endothelium-independent relaxation of isolated RRA. Angiotensin II-induced relaxation was partially reduced by inhibitors of nitric oxide synthase and guanylyl cyclase. The remaining dilatation was inhibited by a potassium channel blocker, charybdotoxin. Precontraction of RRA with high concentration of K(+) partially reduced angiotensin II-evoked relaxation, while indomethacin, glibenclamide, apamin and barium did not alter the angiotensin II concentration-response curve. Losartan had no effect on angiotensin II effect. Oppositely, HOE 140 and PD123319, separately or in combination, partially antagonized vasorelaxation induced by angiotensin II. Complete blockade of RRA response was obtained after simultaneous incubation of all three receptor antagonists HOE-140, PD123319, and losartan; L-NOARG plus HOE-140; or PD123319 plus charybdotoxin. These results indicate that angiotensin II produces endothelium-independent relaxation of RRA, which is most probably mediated by the interaction of the NO-cGMP pathway and K(+) channels. Moreover, we can assume that AT(1), AT(2), and B(2) receptors are involved in the vasorelaxant effect of angiotensin II.     

Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis

1. We have recently demonstrated that chronic infusion of Angiotensin II into apoE-/- mice promotes the development of abdominal aortic aneurysms. To determine the involvement of specific Angiotensin II receptors in this response, we co-infused Angiotensin II (1000 ng kg(-1) min(-1) for 28 days) with losartan (30 mg kg(-1) day(-1)) or PD123319 (3 mg kg(-1) day(-1)) to antagonize AT1 and AT2 receptors, respectively. 2. Infusion of Angiotensin II promoted the development of abdominal aortic aneurysms in 70% of mature female apoE-/- mice. The formation of aortic aneurysms was totally inhibited by co-infusion of Angiotensin II with losartan (30 mg kg(-1) day(-1); P=0.003). In contrast, the co-infusion of Angiotensin II with PD123319 resulted in a marked increase in the incidence and severity of aortic aneurysms. 3. To determine whether AT2 antagonism also promoted Angiotensin II-induced atherosclerosis, Angiotensin II was infused into young female apoE-/- mice that had little spontaneous atherosclerosis. In these mice, co-infusion of PD123319 led to a dramatic increase in the extent of atherosclerosis. This increase was associated with no change in plasma lipid concentrations and only transient and modest increases in blood pressure during co-infusion with PD123319. 4. While antagonism of AT1 receptors totally prevented the formation of aneurysms, antagonism of AT2 receptors promoted a large increase in the severity of Angiotensin II-induced vascular pathology.     

A pharmacological differentiation between postjunctional (AT<Subscript>1A</Subscript>) and prejunctional (AT<Subscript>1B</Subscript>) angiotensin II receptors in the rabbit aorta

The effects of angiotensin II and angiotensin III were compared at prejunctional and postjunctional AT₁ receptors of the rabbit thoracic aorta. Furthermore, the influence of PD123319, losartan and eprosartan on these effects was also compared. To study prejunctional effects, the tissues were preincubated with (³H)-noradrenaline, superfused and electrically stimulated (1 Hz, 2 ms, 50 mA, 5 min). To study postjunctional effects, non-cumulative concentration–response curves were determined. Both angiotensin II and angiotensin III were more potent prejunctionally than postjunctionally. In the case of angiotensin II, the EC₅₀ was 12 times lower at the prejunctional than at the postjunctional level, while that of angiotensin III was 30 times lower prejunctionally. Furthermore, whereas angiotensin II was about 33 times more potent than angiotensin III postjunctionally, it was only 12 times more potent than angiotensin III prejunctionally. Eprosartan did not differentiate between prejunctional and postjunctional effects of both angiotensins. In contrast, PD123319 and losartan did differentiate; however, whereas PD123319 concentration-dependently antagonised the facilitation of tritium release caused by angiotensin II and angiotensin III and had no influence on the contraction of the aortic rings elicited by the peptides, losartan did the opposite: it concentration-dependently antagonised the contractions caused by the peptides on the aortic rings and exerted no influence on the facilitatory effect of angiotensin II and angiotensin III. These results show that prejunctional and postjunctional receptors for angiotensin II and angiotensin III are different and underline the hypothesis that postjunctional AT₁ receptors belong to the AT_1A subtype, while prejunctional AT₁ receptors belong to the AT_1B subtype.     

Chronic hypotensive effects of losartan are not dependent on the actions of angiotensin II at AT 2 receptors.

We have previously demonstrated the chronic hypotensive effects of the AT1 antagonist, losartan, in normotensive, salt-replete rats. One explanation for this response is a reduction in vascular resistance due to blockade of AT1 receptors. Another explanation is that increases in angiotensin II levels during losartan administration can bind to AT2 receptors. Studies suggest that binding of angiotensin II at AT2 receptors lowers arterial pressure by vasodilation. We hypothesized that the chronic effects of losartan are mediated by activation of angiotensin II effects at AT2 receptors. We tested this hypothesis by infusing the AT2 receptor antagonist, PD123319 (74 mg/kg/day), in conjunction with losartan (10 mg/kg/day) for 10 days in rats and compared the hypotensive response in rats treated with losartan alone. After 6 days of treatment, arterial pressure decreased similarly in losartan (-21 +/- 2 mm Hg) and losartan+PD123319 (-23 +/- 2 mm Hg) treated rats. However, by day 10 of the infusion, arterial pressure had decreased to a greater extent (p < 0.05) in rats treated with losartan and PD123319 (-31 +/- 2 mm Hg) compared with rats treated with losartan alone (-22 +/- 2 mm Hg). We conclude that the hypotensive effects of losartan are not dependent on the actions of angiotensin II at AT 2 receptors in normotensive, salt-replete rats.     

ATP release by angiotensin II from segments and cultured smooth muscle cells of guinea-pig taenia coil

Effects of angiotensin II on ATP release were evaluated in segments and cultured smooth muscle cells from the guinea-pig taenia coli. In the segments, angiotensin II (0.3–3 M) elicited release of ATP which was blocked by losartan and SC-52458, non-peptide angiotensin II type-1 receptor (AT₁)-antagonists, but not by PD-123319, an AT₂-antagonist. In superfused cultured cells, 10M angiotensin II likewise elicited release of ATP Again the response was blocked by losartan and SC-52458 but not by PD-123319. These findings suggest that angiotensin II releases ATP from the smooth muscles by activation of angiotensin II-, presumably ATE₁-, receptors.     

Lack of heterologous receptor desensitization induced by angiotensin II type 1 receptor activation in isolated normal rat thoracic aorta

We tested whether heterologous receptor desensitization induced by activation of AT1 receptors may explain the purported relaxation produced by angiotensin II in normal rat aorta. Also, the role for AT2 receptors in the promotion of vasodilation was studied. In endothelium-intact and endothelium-denuded aortic rings, angiotensin II elicited biphasic contractions, which were significantly depressed when repeated in each tissue. Angiotensin II produced biphasic responses on phenylephrine preconstricted endothelium-intact and endothelium-denuded tissues, without reducing precontractile tone. These responses were abolished in the presence of the AT1 receptor antagonist losartan, but no relaxing responses to angiotensin II were uncovered. PD123319 did not influence angiotensin II responses in endothelium-intact tissues precontracted with phenylephrine; thus, under AT2 receptors blockade the contractile effects of angiotensin II were not overexposed. In conclusion, angiotensin II-induced biphasic responses can be attributed to AT1 receptors activation and rapid desensitization with time. Desensitization proved to be homologous in nature, since precontractile tone induced by phenylephrine was not depressed by angiotensin II (i.e., angiotensin II did not induce heterologous α1-adrenergic receptors desensitization). We found no functional evidence of the participation of AT2 receptors in angiotensin II elicited biphasic contractions. Angiotensin II does not exert relaxant effects in normal rat aorta.     

Multiple prejunctional actions of angiotensin II on noradrenergic transmission in the caudal artery of the rat.

1. Angiotensin II produced concentration-dependent enhancement of both stimulation-induced (S-I) efflux of [3H]-noradrenaline and stimulation-evoked vasoconstrictor responses in isolated preparations of rat caudal artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline. The threshold concentrations of angiotensin II for enhancement of S-I efflux (between 0.03 and 0.1 microM) and of the stimulation-evoked vasoconstrictor responses (about 0.3 microM) were 10-1000 times higher than those that have been found for several other vascular preparations. 2. The AT1 angiotensin II receptor antagonist losartan (0.01 and 0.1 microM), reduced or abolished the enhancement of S-I efflux by 1 and 3 microM angiotensin II and the enhancement of vasoconstrictor responses by 1 microM angiotensin II. Surprisingly, the combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced S-I efflux to a much greater extent than did 0.1 microM angiotensin II alone. Moreover, the combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced stimulation-evoked vasoconstrictor responses, in contrast to the lack of effect of 0.1 microM angiotensin II alone. 3. In a concentration of 0.01 microM, the angiotensin II AT2 receptor antagonist PD 123319 did not affect the enhancement of either S-I efflux or vasoconstrictor responses by angiotensin II. However, in a higher concentration (0.1 microM), PD 123319 antagonized the enhancement of both the S-I efflux and vasoconstrictor responses by angiotensin II. 4. In concentrations of 0.01 and 0.1 microM, PD 123319 prevented the marked enhancement of both S-I efflux and stimulation-evoked vasoconstrictor responses produced by the combination of 0.1 microM angiotensin II and 0.01 microM losartan. 5. The potentiation by losartan (0.01 microM) of the facilitatory effect of 0.1 microM angiotensin II on S-I efflux and on stimulation-evoked vasoconstriction was still observed in the presence of either the cyclooxygenase inhibitor indomethacin (3 microM), or the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM). 6. The findings confirm our previous suggestion that, in the rat caudal artery, angiotensin II receptors similar to the AT1B subtype subserve enhancement of transmitter noradrenaline release. 7. The synergistic prejunctional interaction of 0.01 microM losartan and 0.1 microM angiotensin II may be due to either the unmasking by losartan of a latent population of angiotensin II receptors also subserving facilitation of transmitter noradrenaline release, or alternatively, losartan may block an inhibitory action of angiotensin II on transmitter noradrenaline release which normally opposes its facilitatory effect.     

Downregulation of cardiac AT1-receptor expression and angiotensin II concentrations after long-term blockade of the renin-angiotensin system in cardiomyopathic hamsters.

We monitored cardiac angiotensin II concentration and AT1-receptor density after long-term blockade of the renin-angiotensin system in inbred control hamsters treated with placebo or losartan (100 mg/kg/day) and cardiomyopathic hamsters treated with placebo, low-(30 mg/kg/day), or high-dose (100 mg/kg/day) losartan or quinapril (100 mg/kg/day). All treatments were started at age 50 days. Angiotensin II-receptor density and affinity were measured by radioligand-binding assays, and ventricular angiotensin II concentration was determined by radioimmunoassay. After 125 and 275 days of treatment, both doses of losartan significantly reduced AT1-receptor density, whereas quinapril had no effect. The administration of both drugs resulted in significant reductions in ventricular angiotensin II concentration. The prolonged administration of losartan was associated with an increase in cardiac hypertrophy, suggesting that angiotensin II signaling is not directly involved or at least does not play a major role in the remodeling process observed in cardiomyopathic hamsters.     

Angiotensin II stimulates arachidonic acid release from bone marrow stromal cells.

INTRODUCTION: Angiotensin II (Ang II) is recognised as a regulator of haematopoiesis, but its actions within the bone marrow are not fully understood. Support of haematopoiesis by bone marrow stromal cells (MSC) is dependent on factors that include arachidonic acid and macrophage colony stimulating factor (MCSF), both of which are increased by Ang II stimulation in other tissues. To further elucidate the mechanisms of Ang II-regulated haematopoiesis, we determined whether Ang II-stimulation alters arachidonic acid release and MCSF secretion from MSC. METHODS: Cynomolgus monkey MSC isolated from bone marrow aspirates and the human HS-5 stromal cell line were studied for Ang II-mediated arachidonic acid (AA) release, while secretion of MCSF in response to Ang II was studied in HS-5 cells. Cells were labelled overnight with 3H-AA and the release of 3H-AA was measured in culture medium following 20 minutes stimulation with Ang II, alone or in combination with the AT1- or AT2-receptor antagonists, losartan and PD 123319, respectively. MCSF secretion into culture medium was measured using an enzyme immunoassay following 24 hours of treatment with Ang II alone or in combination with losartan or PD 123319. Phorbol-myristate-acetate, known to stimulate release of AA and MCSF, was used as a positive control in both experiments. RESULTS: In response to Ang II, release of 3H-AA from monkey and human MSC was increased (p<0.05) to 147+/-4% and 124+/-3% of control, respectively. The AT1- and AT2-receptor antagonists, losartan and PD 123319, individually reduced Ang II-stimulated 3H-AA release. In contrast, Ang II had no effect on secretion of MCSF from HS-5 cells. CONCLUSIONS: These results provide mechanistic evidence for Ang II-mediated haematopoiesis through AA release that may, in part, explain Ang II-facilitated recovery of haematopoiesis in experimental myelosuppression and the anaemias associated with Ang II receptor blockade.     

Identification and characterisation of angiotensin II receptor subtypes in human brain

Autoradiographic and homogenate binding studies using the radioligand, [¹²⁵I]angiotensin II, identified a heterogeneous distribution of specific binding sites (defined by angiotensin II, 1.0 μM) throughout the human forebrain. Highest AT receptor densities were detected in the paraventricular nucleus, median eminence, substantia nigra, putamen and caudate nucleus (2.4, 1.2, 1.0, 0.30 and 0.24 fmol/mg tissue equivalent, respectively). The AT₁ receptor antagonist, losartan (1.0 μM) competed for the majority of the specific binding. [¹²⁵I]Angiotensin II-specific binding (although not consistently above non-specific binding levels) was also detected in various other brain regions (e.g. amygdala, entorhinal cortex, hippocampus, inferior colliculus, nucleus accumbens, parietal cortex, periaquaductal grey, superior colliculus, striate cortex, temporal cortex, thalamus). In the presence of losartan (1.0 μM), angiotensin II, saralasin, losartan and PD123177 competed for [¹²⁵I]angiotensin II binding to membranes prepared from the cerebellum or substantia nigra with a rank order of affinity; angiotensin II = saralasin > PD123177 > losartan. In the presence of PD123177 (1.0 μM), the rank order of affinity of losartan and PD123177 was reversed. These studies indicate the presence of both AT₁ and AT₂ receptor subtypes within various regions of the human forebrain.     

Inhibition of Angiotensin II receptors during pregnancy induces malformations in developing rat kidney

Evidence suggests that Angiotensin II plays an important role in the complex process of renal organogenesis. Rat kidney organogenesis starts between E13-14 and lasts up to 2 weeks after birth. The present study demonstrates histologic modifications and changes in receptor localisation in animals born from mothers treated with Angiotensin II, Losartan or PD123319 (1.0 mg/kg/day) during late pregnancy. Angiotensin II-treated animals exhibited very well developed tubules in the renal medulla in coincidence with higher AT(1) binding. Control animals exhibited angiotensin AT(2) binding in the outer stripe of the outer medulla, while in the Angiotensin II-treated animals binding was observed to the inner stripe. In Angiotensin II-treated 1-week-old animals, the nephrogenic zone contained fewer immature structures, and more developed collecting tubules than control animals. Treatment with Losartan resulted in severe renal abnormalities. For newborn and 1-week-old animals, glomeruli exhibited altered shape and enlarged Bowman spaces, in concordance with a loss of [(125)I]Angiotensin II binding in the cortex. Blockade with PD123319 led to an enlarged nephrogenic zone with increased number of immature glomeruli, and less glomeruli in the juxtamedullary area. Autoradiography showed a considerable loss of AT(1) binding in the kidney cortex of PD123319-treated animals at both ages. The present results show for the first time histomorphological and receptor localisation alterations following treatment with low doses of Losartan and PD123319 during pregnancy. These observations confirm previous assumptions that in the developing kidney Angiotensin II exerts stimulatory effects through AT(1) receptors that might be counterbalanced by angiotensin AT(2) receptors.