Protein kinase C-mediated inhibition of renal Ca2+ ATPase by physiological concentrations of angiotensin II is reversed by AT1- and AT2-receptor antagonists

Angiotensin II (Ang II) increases the cytosolic Ca2+ concentration in different cell types. In this study, we investigate the effect of Ang II on the Ca2+ ATPase of purified basolateral membranes of kidney proximal tubules. This enzyme pumps Ca2+ out of the cytosol in a reaction coupled to ATP hydrolysis, and it is responsible for the fine-tuned regulation of cytosolic Ca2+ activity. Ca2+-ATPase activity is inhibited by picomolar concentrations of Ang II, with maximal inhibition being attained at approximately 50% of the control values. The presence of raising concentrations (10(-11) to 10(-7) M) of losartan (an AT1-receptor antagonist) or PD123319 (an AT2-receptor antagonist) gradually reverts inhibition by Ang II. Both the phospholipase C (PLC) inhibitor U-73122 (10(-6) M) and the inhibitor of protein kinase C (PKC) staurosporine (10(-7) M) prevent inhibition of the Ca2+ pump by Ang II. Incubation of the previously isolated membranes with a PKC activator-the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (10(-8) M)-mimics the inhibition found with Ang II, and the effects of the compounds are not additive. Taken as a whole, these results indicate the Ang II inhibits Ca2+-ATPase by activation of a PKC system present in primed state in these membranes after binding of the hormone to losartan- and PD123319-sensitive receptors coupled to a PLC. Therefore, inhibition of the basolateral membrane Ca2+-ATPase by kinase-mediated phosphorylation appears to be one of the pathways by which Ang II promotes an increase in the cytosolic Ca2+ concentration of proximal tubule cells.     

Angiotensin II and its receptors in human endocardial endothelial cells: role in modulating intracellular calcium

The aims of the present study are to investigate the presence and distribution of angiotensin II (Ang II), as well as AT1 and AT2 receptors, in endocardial endothelial cells (EECs) and to determine if the effect of Ang II on intracellular calcium in these cells is mediated via the AT1 or the AT2 receptor. Immunofluorescence and 3D confocal microscopy techniques were used on 20-week-old fetal human EECs. Our results showed that Ang II and its receptors, the AT1 and the AT2 types, are present and exhibit a different distribution in human EECs. Ang II labelling is found throughout the cell with a fluorescence signal higher in the cytosol when compared with the nucleus. Like Ang II, the AT1 receptor fluorescence signal is also homogeneously distributed in human EECs but with a preferential labelling at the level of the nucleus, while the AT2 receptor labelling is solely present in the nucleus. Using fluo-3 and 3D confocal microscopy technique, superfusion of human EECs with increasing concentration of Ang II induced a dose-dependent sustained increase in free cytosolic and nuclear Ca2+ levels. This effect of Ang II on human EEC's intracellular Ca2+ ([Ca2+]) was completely prevented by losartan, an AT1 receptor antagonist. Our results suggest that Ang II, as well as AT1 and AT2 receptors, is present but differentially distributed in EECs of 20-week-old fetal human hearts, and that the AT1 receptor mediates the effects of Ang II on [Ca2+]i in these cells.     

Angiotensin II-induced increase of T-type Ca2+ current and decrease of L-type Ca2+ current in heart cells

The effect of angiotensin II (Ang II) on the T- and L-type calcium currents (I(Ca)) in single ventricular heart cells of 18-week-old fetal human and 10-day-old chick embryos was studied using the whole-cell voltage clamp technique. Our results showed that in both, human and chick cardiomyocytes, Ang II (10(-7)M) increased the T-type calcium current and decreased the L-type I(Ca). The effect of Ang II on both types of currents was blocked by the AT1 peptidic antagonist, [Sar1, Ala8] Ang II (2 x 10(-7)M). Protein kinase C activator, phorbol 12,13-dibutyrate, mimicked the effect of Ang II on the T- and L-type calcium currents. These results demonstrate that in fetal human and chick embryo cardiomyocytes Ang II affects the T- and L-type Ca2+ currents differently, and this effect seems to be mediated by the PKC pathway.     

Angiotensin II modulates the activity of Na+,K+-ATPase in cultured rat astrocytes via the AT1 receptor and protein kinase C-delta activation

In astrocytes the activity of the Na+,K(+)-ATPase pump maintains an inwardly directed electrochemical sodium gradient used by the Na+-dependent transporters and regulates the extracellular K+ concentration essential for neuronal excitability. We show here that incubation of cultured rat astrocytes with angiotensin II (Ang II) modulates Na+,K(+)-ATPase activity, in a dose- and time-dependent manner. Na+,K(+)-ATPase activation was mediated by binding of Ang II to AT1 receptors as it was completely blocked by DuP 753, a specific AT1 receptor subtype antagonist. Stimulation of Na+,K(+)-ATPase activity by Ang II was dependent on protein kinase C (PKC) activation because PKC antagonists abolished the inducing effect of Ang II and the PKC activator phorbol 12-myristate 13-acetate enhanced transporter activity. Ang II stimulated translocation of PKC-delta but not that of other PKC isoforms from the cytosol to the plasma membrane. These results indicate that the activity of Na+,K(+)-ATPase in astrocytes is increased by physiological concentrations of Ang II and that the AT1 receptor subtype mediates the Na+,K(+)-ATPase response to Ang II via PKC-delta activation.     

Angiotensin II inhibits inward rectifier K+ channels in rabbit coronary arterial smooth muscle cells through protein kinase Calpha

We investigated the effects of the vasoconstrictor angiotensin (Ang) II on the whole cell inward rectifier K(+) (Kir) current enzymatically isolated from small-diameter (<100 microm) coronary arterial smooth muscle cells (CASMCs). Ang II inhibited the Kir current in a dose-dependent manner (half inhibition value: 154 nM). Pretreatment with phospholipase C inhibitor and protein kinase C (PKC) inhibitors prevented the Ang II-induced inhibition of the Kir current. The PKC activator reduced the Kir currents. The inhibitory effect of Ang II was reduced by intracellular and extracellular Ca(2+) free condition and by G?6976, which inhibits Ca(2+)-dependent PKC isoforms alpha and beta. However, the inhibitory effect of Ang II was unaffected by a peptide that selectively inhibits the translocation of the epsilon isoform of PKC. Western blot analysis confirmed that PKCalpha, and not PKCbeta, was expressed in small-diameter CASMCs. The Ang II type 1 (AT(1))-receptor antagonist CV-11974 prevented the Ang II-induced inhibition of the Kir current. From these results, we conclude that Ang II inhibits Kir channels through AT(1) receptors by the activation of PKCalpha.     

Angiotensin II AT1 receptor internalization,translocation and de novo synthesis modulate cytosolic and nuclear calcium in human vascular smooth muscle cells

The present study was designed to verify if human (h) Angiotensin II (Ang II) type-1 receptor (hAT1R) undergoes internalization, nuclear translocation, and de novo synthesis in primary culture of human aortic vascular smooth muscle cells (hVSMCs) and if overexpression of this receptor modulates sustained free cytosolic ([Ca]c) and nuclear ([Ca]n) calcium. 3-dimensional (3-D) confocal microscopy was used to monitor free intracellular Ca2+ and hAT1R-green fluorescence protein (GFP) fusion protein in cultured hVSMCs. Immunofluorescence studies showed the presence of hAT1R and the absence of hAT2R in normal hVSMCs. Using 3-D imaging technique, hAT1 receptors were localized at the sarcolemma and in the cytosolic and nuclear compartments. In native as well as in normal hAT1R or hAT1R-GFP overexpressing hVSMCs, Ang II (10(-9) and 10(-4) M) induced internalization and nuclear translocation of this type of receptor. The internalization of hAT1Rs is mediated via clathrin-coated pits and vesicles pathway. This phenomenon of trancellular trafficking of receptors was associated with an increase of hAT1R. The Ang II induced increase of hAT1R density was prevented by the protein synthesis inhibitor cycloheximide. Overexpression of hAT1R and hAT1R-GFP decreased both basal cytosolic and nuclear Ca2+. In normal hVSMCs and low hAT1R-GFP overexpressing hVSMCs, Ang II (10(-15) to 10(-4) M) induced a dose-dependent sustained increase of [Ca]c and [Ca]n with an EC50 near 5 x 10(-11) and 5 x 10(-9) M, respectively. Our results suggest that hAT1Rs are the predominant type of Ang II receptors in aortic hVSMCs and are present in the sarcolemma, the cytosolic and the nuclear compartments. Ang II rapidly induces hAT1R internalization, nuclear translocation, as well as nuclear de novo synthesis of this receptor. The hAT1R overexpression in hVSMCs modulates sustained [Ca]c and [Ca]n.     

Angiotensin II mediates catecholamine and neuropeptide Y secretion in human adrenal chromaffin cells through the AT1 receptor

The aim of the present work was to study the effect of angiotensin II (Ang II) on catecholamines and neuropeptide Y (NPY) release in primary cultures of human adrenal chromaffin cells. Ang II stimulates norepinephrine (NE), epinephrine (EP) and NPY release from perifused chromaffin cells by 3-, 2- and 12-fold, respectively. The NPY release is more sustained than that of catecholamines. We found that the receptor-AT(2) agonist, T(2)-(Ang II 4-8)(2) has no effect on NE, EP and NPY release from chromaffin cells. We further showed that Ang II increases intracellular Ca(2+) concentration ([Ca(2+)](i)). The selective AT(1)-receptor antagonist Candesartan blocked [Ca(2+)](i) increase by Ang II, while T(2)-(Ang II 4-8)(2) was ineffective. These findings demonstrate that AT(1) stimulation induces catecholamine secretion from human adrenal chromaffin cells probably by raising cytosolic calcium.     

AT1 angiotensin II receptor mediates intracellular calcium mobilization in normal and cancerous breast cells in primary culture

Angiotensin II (Ang II) increases intracellular calcium concentration ([Ca2+]i) in both normal and cancerous human breast cells in primary culture. Maximal [Ca2+]i increase is obtained using 100nM Ang II in both cell types; in cancerous breast cells, [Ca2+]i increase (delta[Ca2+]i) is 135+/-10nM, while in normal breast cells it reaches 65+/-5 nM (P<0.0001). In both cell types, Ang II evokes a Ca2+ transient peak mediated by thapsigargin (TG) sensitive stores; neither Ca2+ entry through L-type membrane channels or capacitative Ca2+ entry are involved. Type I Ang II receptor subtype (AT1) mediates Ang II-dependent [Ca2+]i increase, since losartan, an AT1 inhibitor, blunted [Ca2+]i increase induced by Ang II in a dose-dependent manner, while CGP 4221A, an AT2 inhibitor, does not. Phospholipase C (PLC) is involved in this signaling mechanism, as U73122, a PLC inhibitor, decreases Ang II-dependent [Ca2+]i transient peak in a dose-dependent mode.Thus, the present study provides new information about Ca2+ signaling pathways mediated through AT1 in breast cells in which no data were yet available.     

Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.     

Effects of angiotensin II microinjected into medial amygdala on male sexual behavior in rats

Research was undertaken to study the role of central angiotensin in the modulation of male sexual behavior, testing the effect of angiotensin II (Ang II) injections into the medial amygdaloid nucleus (MeA). The sexual behavior of adult male Wistar rats was evaluated, 15 min after bilateral intra-amygdaloid microinjection (0.3 microl) of saline and 5 doses of Ang II: 10; 25; 50; 100, and 150 fmol. The effects of the Ang II receptor blockade were also studied. We tested the effect of coinjection of Ang II (50 fmol) with the AT1 antagonist, losartan (20 pmol) and the AT2 antagonist, CGP 42112 (1 pmol). Ang II inhibited sexual behavior and this inhibition was prevented by the coinjection of AT1 antagonist, losartan, or the AT2 antagonist, CGP 42112. Results show that Ang II has a powerful effect on male sexual behavior, which may be mediated by both AT1 and AT2 receptors.     

Neuropeptide Y induced increase of cytosolic and nuclear Ca2+ in heart and vascular smooth muscle cells

It was reported that neuropeptide Y (NPY) affects cardiac and vascular smooth muscle (VSM) function probably by increasing intracellular Ca2+. In this study, using fura-2 microfluorometry and fluo-3 confocal microscopy techniques for intracellular Ca2+ measurement, we attempted to verify whether the action of NPY receptor's stimulation in heart and VSM cells modulates intracellular Ca2+ and whether this effect is mediated via the Y1 receptor type. Using spontaneously contracting single ventricular heart cells of 10-day-old embryonic chicks and the fluo-3 confocal microscopy Ca2+ measurement technique to localize cytosolic ([Ca]c) and nuclear ([Ca]n) free Ca2+ level and distribution, 10-10 M of human (h) NPY significantly (P < 0.05) increased the frequency of cytosolic and nuclear Ca2+ transients during spontaneous contraction. Increasing the concentration of hNPY (10(-9) M) did not further increase the frequency of Ca2+ transients. The L-type Ca2+ channel blocker, nifedipine (10(-5) M), significantly (P < 0.001) blocked the spontaneous rise of intracellular Ca2+ in the absence and presence of hNPY (10(-10) and 10(-9) M). However, the selective Y1 receptor antagonist, BIBP3226 (10(-6) M), significantly decreased the hNPY-induced (10(-10) and 10(-9) M) increase in the frequency of Ca2+ transients back to near the control level (P < 0.05). In resting nonworking heart and human aortic VSM cells, hNPY induced a dose-dependent sustained increase of basal resting intracellular Ca2+ with an EC50 near 10(-9) M. This sustained increase was cytosolic and nuclear and was completely blocked by the Ca2+ chelator EGTA, and was significantly decreased by the Y1 receptor antagonist BIBP3226 in both heart (P < 0.05) and VSM (P < 0.01) cells. These results strongly suggest that NPY stimulates the resting basal steady-state Ca2+ influx through the sarcolemma and induces sustained increases of cytosolic and nuclear calcium, in good part, via the activation of the sarcolemma membrane Y1 receptor type in both resting heart and VSM cells. In addition, NPY also increased the frequency of Ca2+ transients during spontaneous contraction of heart cells mainly via the activation of the Y1 receptor type, which may explain in part the active cardiovascular action of this peptide.     

Comparison of angiotensin II (Ang II) effects in the internal anal sphincter (IAS) and lower esophageal sphincter smooth muscles

Studies were performed to compare the actions of Ang II in the internal anal sphincter (IAS) vs. lower esophageal sphincter (LES) smooth muscles in vitro, in opossum and rabbit. Studies also were carried out in isolated smooth muscle cells. In opossum, Ang II produced no discernible effects in the IAS, but did produce a concentration-dependent contraction in the LES. Conversely, in the rabbit, while Ang II caused a modest response in the LES, it caused a significant contraction in the IAS. The contractile responses of Ang II in the opossum LES were mostly resistant to different neurohumoral antagonists but were antagonized by AT1 antagonist losartan. AT2 antagonist PD 123,319, rather than inhibiting, prolonged the contractile action of Ang II. The contractile actions of Ang II in the opossum LES were not modified by the tyrosine kinase inhibitors (genistein and tyrphostin 1 x 10(-6) M) but were partially attenuated by the PKC inhibitor H-7 (1 x 10(-6) M), Ca2+ channel blocker nicardipine (1 x 10(-5) M), Rho kinase inhibitor HA-1077 (1 x 10(-7) M) or p(44/42) MAP kinase inhibitor PD 98059 (5 x 10(-5) M). The combination of HA-1077 and H-7 did not cause an additive attenuation of Ang II responses. Western blot analyses revealed the presence of both AT1 and AT2 receptors. We conclude that Ang lI-induced contraction of sphincteric smooth muscle occurs primarily by the activation of AT1 receptors at the smooth muscle cells and involves multiple pathways, influx of Ca2+, and PKC, Rho kinase and p(44/42) MAP kinase.     

Excitatory regulation of angiotensin II on gastric motility and its mechanism in guinea pig

In the present study, we investigated the effect of Ang II on gastric smooth muscle motility and its mechanism using intracellular recording and whole-cell patch clamp techniques. Ang II dose-dependently increased the tonic contraction and the frequency of spontaneous contraction in the gastric antral circular smooth muscles of guinea pig. ZD7155, an Ang II type 1 receptor (AT(1)R) blocker, completely blocked the effect of Ang II on the spontaneous contraction of gastric smooth muscle. In contrast, TTX, a sodium channel blocker, failed to block the effect. Furthermore, nicardipine, a voltage-gated Ca(2+)-channel antagonist, did not block the effect of Ang II on the tonic contraction of gastric smooth muscle, but external free-calcium almost completely blocked this effect. Both ryanodine, an inhibitor of calcium-induced Ca(2+) release (CICR) from ryanodine-sensitive calcium stores, and thapsigargin, which depletes calcium in calcium stores, almost completely blocked the effect of Ang II on tonic contraction. However, 2-APB, an inositol trisphosphate (IP(3)) receptor blocker, significantly, but not completely, blocked the Ang II effect on tonic contraction. We also determined that Ang II depolarized membrane potential and increased slow wave frequency in a dose-dependent manner. It also inhibited delayed rectifying potassium currents in a dose-dependent manner, but did not affect L-type calcium currents or calcium-activated potassium currents. These results suggest that Ang II plays an excitatory regulation in gastric motility via AT(1)R-IP(3) and the CICR signaling pathway. The Ang II-induced inhibition of delayed rectifying potassium currents that depolarize membrane potential is also involved in the potentiation of tonic contraction and the frequency of spontaneous contraction in the gastric smooth muscle of guinea pig.     

125I]EXP985: a highly potent and specific nonpeptide radioligand antagonist for the AT1 angiotensin receptor.

[125I]EXP985 is the first nonpeptide radioligand with high specific activity for the AT1 angiotensin receptor. The biochemical and pharmacological profiles of this ligand were determined using either ligand-receptor binding techniques in rat adrenal cortical microsomes or cellular Ca2+ mobilization in rat smooth muscle cells. Specific binding with 0.1 nM [125I]EXP985 increased slowly with time reaching an equilibrium at 60 min of incubation (22 degrees C). Scatchard analysis of the inhibition/binding data revealed a single class of binding sites having a Kd of 1.49 +/- 0.06 nM and a Bmax of 3.6 +/- 0.1 pmol/mg protein. These sites were saturable and the ligand-receptor complex dissociated with a t1/2 of 58 min. The binding was inhibited by Ang peptides with the following order of potency and IC50 (nM): Ang II (3.7) > Ang III (69) > Ang I (3650), and by the nonpeptide AT1 receptor antagonist, losartan, with an IC50 of 3.2 nM. PD123177, an AT2 selective antagonist, showed minimal inhibitory effect. Specific binding of [125I]EXP985 was found on rat aortic smooth cells. Ang II-induced Ca2+ mobilization in these cells was blocked by EXP985 in a noncompetitive manner. These data show that [125I]EXP985 (or its unlabeled) is a potent and highly specific radioligand or noncompetitive antagonist which represents a novel tool to further our understanding of the biochemistry of AT1 receptors.     

Angiotensin II type 1 receptor activation modulates L- and T-type calcium channel activity through distinct mechanisms in bovine adrenal glomerulosa cells

In adrenal zona glomerulosa cells, calcium entry is crucial for aldosterone production and secretion. This influx is stimulated by increases of extracellular potassium in the physiological range of concentrations and by angiotensin II (Ang II). The high threshold voltage-activated (L-type) calcium channels have been shown to be the major mediators for the rise in cytosolic free calcium concentration, [Ca2+]c, observed in response to a depolarisation by physiological potassium concentrations. Paradoxically, both T- and L-type calcium channels have been shown to be negatively modulated by Ang II after activation by a sustained depolarisation. While the modulation of T-type channels involves protein kinase C (PKC) activation, L-type channel inhibition requires a pertussis toxin-sensitive G protein. In order to investigate the possibility of additional modulatory mechanisms elicited by Ang II on L-type channels, we have studied the effect of PKC activation or tyrosine kinase inhibition. Neither genistein or MDHC, two strong inhibitors of tyrosine kinases, nor the phorbol ester PMA, a specific activator of PKC, affected the Ang II effect on the [Ca2+]c response and on the Ba2+ currents elicited by cell depolarisation with the patch-clamp method. We propose a model describing the mechanisms of the [Ca2+]c modulation by Ang II and potassium in bovine adrenal glomerulosa cells.     

Inositol(1,4,5)trisphosphate signal triggers a receptor-mediated ATP release

Intracellular signal transduction pathways involved in ATP release evoked by angiotensin II (Ang II) were investigated in cultured guinea pig Taenia coli smooth muscle cells. Ang II (0.3-1 microM) elicited substantial release of ATP from the cells, but not from a human fibroblast cell line. However, Ang II even at 10 microM failed to cause a leakage of lactate dehydrogenase (LDH) from the smooth muscle cells. The release of ATP by Ang II was suppressed by 10 microM SC52458, an AT1 receptor antagonist, not by 10 microM PD123319, an AT2 receptor antagonist. The evoked release of ATP was almost completely inhibited in the presence of 10 microM U73122, a phospholipase C inhibitor, and 0.5 microM thapsigargin, a Ca2+-ATPase inhibitor. Furthermore, the release was hampered by 50 microM BAPTA/AM, an intracellular Ca2+ chelator, but not by 0.1 microM nifedipine, a voltage gated Ca2+ channel inhibitor. The basal release of ATP was increased by BAPTA/AM, but was reduced by U-73122. Ang II enhanced instantaneously inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) accumulation in the cells. The enhancing effect was perfectly antagonized by SC52458. These findings suggest that intracellular Ca2+ signals activated via stimulation of Ins(1,4,5)P3 receptor are involved in the release of ATP evoked by Ang II.     

Sarcosine1,glycine8 angiotensin II is an AT1 angiotensin II receptor subtype selective antagonist

Studies predating the discovery of the two major subtypes of angiotensin II (Ang II) receptors, AT1 and AT2, revealed anomalous characteristics of sarcosine1,glycine8 Ang II (Sar1,Gly8 Ang II). It competed poorly for 125I-Ang II binding in bovine brain but potently antagonized dipsogenic responses to intracerebroventricularly administered Ang II. Subsequent recognition that bovine brain contains AT(2) receptors, while dipsogenic responses to Ang II are mediated by AT1 receptors, suggests that Sar1,Gly(8) Ang II is AT1 selective. Sar1,Gly8 Ang II competed for 125I-sarcosine1,isoleucine8 Ang II binding to AT1 receptors in pituitary, liver and adrenal (the latter with the AT2 selective antagonist PD 123,319) with Ki's of 0.66, 1.40 and 1.36 nM, respectively. In contrast, the Ki of Sar1,Gly8 Ang II for AT2 receptors in rat adrenal (with the selective AT1 antagonist losartan) was 52 nM. 125I-Sar1,Gly8 Ang II (0.5-3 nM) bound to AT1 receptors in pituitary, liver, heart, adrenal, and hypothalamic membranes with high affinity (Kd=0.43, 1.6, 2.3, 0.96 and 1.8 nM, respectively), but showed no saturable binding to the adrenal AT2 receptor. 125I-Sar1,Gly8 Ang II selectively labeled AT1 receptors in sections of adrenal using receptor autoradiography. Thus, binding studies reveal Sar1,Gly8 Ang II to be the first angiotensin peptide analog to show AT1 receptor selectivity. 125I-Sar1,Gly8 Ang II offers a new means to selectively radiolabel AT1 receptors and may help to characterize ligand docking sites and agonist switches for AT1 versus AT2 receptors.     

Angiotensin II stimulates intercellular adhesion molecule-1 via an AT1 receptor/nuclear factor-kappaB pathway in brain microvascular endothelial cells

Microvascular changes in the brain are significant causes of cerebral edema and ischemia injury. A number of studies suggest that angiotensin (Ang) II may be involved in the initiation and regulation of processes occurring in brain ischemia. We recently reported that Ang II injures brain microvascular endothelial cells (BMEC) partially via stimulating intercellular adhesion molecule-1 (ICAM-1) expression. However, the signaling cascade leading to Ang II-induced ICAM-1 expression in BMEC was unclear. The present study tested the hypothesis that Ang II induces ICAM-1 expression via an AT1 receptor/nuclear factor-kappaB (NF-kappaB) pathway in BMEC. Ang II directly stimulated the expression of ICAM-1 mRNA and protein in primary cultured BMEC. Ang II treatment also resulted in the degradation of IkappaBalpha and increase of NF-kappaB p65 subunit in the nucleus as well as the DNA binding activity of nuclear NF-kappaB. These effects were abolished by pretreatment with the selective AT1 receptor antagonists, losartan and compound EXP-2528, or losartan plus the AT2 receptor antagonist PD123319, but not by PD123319 alone. Moreover, there were no significant differences between the losartan and losartan plus PD123319 groups. These findings indicate that Ang II-induced ICAM-1 upregulation in brain microvascular endothelial cells may be mediated via an AT1 receptor/NF-kappaB pathway.