Contractile Effects of Angiotensins Are Independent of Receptors Internalization in Rat Aorta (original) (raw)
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British Journal of Pharmacology, 1999
1 We studied the eect of intracellular angiotensin II (Ang II) and related peptides on rat aortic contraction, whether this eect is pharmacologically distinguishable from that induced by extracellular stimulation, and determined the Ca 2+ source involved. 2 Compounds were delivered into the cytoplasm of de-endothelized aorta rings using multilamellar liposomes. Contractions were normalized to the maximum obtained with phenylephrine (10 75 M). 3 Intracellular administration of Ang II (incorporation range: 0.01 ± 300 nmol mg 71 ) resulted in a dose-dependent contraction, insensitive to extracellular administration (10 76 M) of the AT 1 receptor antagonist CV11947, the AT 2 receptor antagonist PD 123319, or the non-selective AT receptor antagonist and partial agonist saralasin ([Sar 1 ,Val 5 ,Ala 8 ]-Ang II (P50.05). 4 Intracellular administration of CV11947 or PD 123319 right shifted the dose-response curve about 1000 fold or 20 fold, respectively. PD 123319 was only eective if less than 30 nmol mg 71 Ang II was incorporated. 5 Contraction was partially desensitized to a second intracellular Ang II addition after 45 min (P50.05). 6 Intracellular administration of Ang I and saralasin also induced contraction (P50.05). Both responses were sensitive to intracellular CV11947 (P50.05), but insensitive to PD 123319. The response to Ang I was independent of intracellular captopril. 7 Contraction induced by extracellular application of Ang II and of Ang I was abolished by extracellular pre-treatment with saralasin or CV11947 (P50.05), but not with PD 123319. Extracellular saralasin induced no contraction. 8 Intracellular Ang II induced contraction was not aected by pre-treatment with heparin ®lled liposomes, but completely abolished in Ca 2+ -free external medium. 9 These results support the existence of an intracellular binding site for Ang II in rat aorta. Intracellular stimulation induces contraction dependent on Ca 2+ -in¯ux but not on Ins(1,4,5)P 3 mediated release from intracellular Ca 2+ -stores. Intracellular Ang I and saralasin induce contraction, possibly via the same binding site. Pharmacological properties of this putative intracellular receptor are clearly dierent from extracellular stimulated AT 1 receptors or intracellular angiotensin receptors postulated in other tissue.
Differential β-arrestin binding of AT1and AT2angiotensin receptors
FEBS Letters, 2005
Agonist stimulation of G protein-coupled receptors causes receptor activation, phosphorylation, b-arrestin binding and receptor internalization. Angiotensin II (AngII) causes rapid internalization of the AT 1 receptors, whereas AngII-bound AT 2 receptors do not internalize. Although the activation of the rat AT 1A receptor with AngII causes translocation of b-arrestin2 to the receptor, no association of this molecule with the AT 2 receptor can be detected after AngII treatment with confocal microscopy or bioluminescence resonance energy transfer. These data demonstrate that the two subtypes of angiotensin receptors have different mechanisms of regulation.
European Journal of Pharmacology, 1992
Angiolcnsin-II (All) stimulates smooth muscle contraction hy activating a~rgiatcnain AT, rcccptor which induces intr;lccllular Ca' ' rclcasc and Ca' ' influx from cxtraccllular space. In this study, cffcct 01' cxlraccllular C$ ' concentration ([Ca' ' I,,) on angiotcnsin AT, rcccptor-mcdiutcd contraclilc rcsponsc IO All hiis hccn cxamincd in the ahscncc and prcscncc of [Sar'.AI$]AII in rahhit aorta. A dccrcasc in agonist potency and ian incrcnsc in antilgtki potency for dcprcssion wcrc ohscrvcd in low [CL,' ' I,,. Data wcrc: intcrprctcd hy applying an cxplanalory model dcvclopcd previously. The result indicates that [Gil' ' I,, is linked to thl: efl'icacy cxprcssion 01' A!! al angiotcnsin AT, receptor and this prompted speculation about the underlying mechanism. Angiotcnsin: Angiotcnsin AT, rcccptors; Ci\' ' ; Efficacy G proteins stimulating phospholipascs which lead to a dCCtTilSC iR Cyclic f-!MP, iRCrCil!XS in irlositol i,4.%trisphosphate, diacylglyccrol, intraccllulitr C;I' ' rclcasc and Cii'+ influx from cxtraccllular space (Akxandcr Cl ill., 19X5; Gricndling cl al., 1087; Muscha Stcckclings ct al., 1002). Rclc%C Ol' Gil" from intriKCllular slorcs, sarcoplasmic reticulum for cxamplc, provides iI tran-SiCRt CoRtraCtk9R. However, miliRtCRilRCC Of the CORlractilc rcsponsc requires Ca" influx through a rcccptor-linked 0.1~' channel (K;lrilki and Weiss. I%#). It has hccn shown that rcccptor-mcdiatcd cndocytosis and cxocytosis occur wilh irngiotcnsin AT, receptors, bound to All as well as its pcptidc ligands and. thcsc processes also involve Ca'+ mobilisation (Griendling ct aI., 1987; Ulliim and LiRilS, I~NO). Complex ilntilgOniS1 profilcs associated with pcptidr and nonpuplidc All antagonists ilt the angiotcnsin AT, receptor have been rcporlcd carlicr (Freer cl al., I9XO; Wang ct al.. 1990). BaScd OII iI scrics of examinations in it range of isolated smooth muscle assays using rcprcscntntivc compounds, this aRtilgc9RiSt cornplcxity charactcriscd hy unSaturirhlc rightward-shifts concurrent with saturahlc dcprussion of All concentration-cffcct (E/log[A]) CUIV~S, hits bCCR satisfactorily accounted for using iIn sxpitinatory modei ILiu, 1092; Liu cl al., 1002) dcvclopod from the operational model of iigc!nism (Black and Lcff, 19H.i). The irnportancc of iRtrilCC~hlklr CB'+ in stimulating smooth muscle CORtrilCt~l9R has IMXR well rccogniscd. Howcvcr.
Regulatory Peptides, 2004
h-Arrestins play a role in AT 1 endocytosis by binding the cytoplasmic, C-terminus region T332 -S338, the major site of angiotensin II (Ang II)-induced phosphorylation. However, the processes responsible for recruiting h-arrestin to the activated receptor are poorly defined. In this study, we used CHO-K1 and HEK 293 cells expressing wild-type or mutant AT 1 to investigate two possibilities: activated AT 1 induces global relocation of h-arrestins to the plasma membrane or the phosphorylated C-terminus acts as bait to attract h-arrestins. Results obtained using high osmolarity and dominant-negative h-arrestin confirmed that internalization of AT 1 in both CHO-K1 and HEK 293 cells is predominately via clathrin-mediated endocytosis involving h-arrestin, and substitution of T332, S335, T336 and S338 with alanine to preclude phosphorylation markedly attenuated AT 1 internalization. Confocal microscopy revealed that wild-type AT 1 induced a timedependent translocation of GFP-tagged h-arrestins 1 and 2 to the cell surface. In contrast, the TSTS/A mutant did not traffic h-arrestin 1 at all, and only trafficked h-arrestin 2 weakly. Results of rescue-type experiments were consistent with the idea that both h-arrestins are able to interact with the non-phosphorylated receptor, albeit with much lower affinity and h-arrestin 1 less so than h-arrestin 2. In conclusion, this study shows that the high affinity binding of h-arrestins to the phosphorylated C-terminus is the predominant mechanism of agonist-induced h-arrestin recruitment to the cell surface and AT 1 receptor. D
Effect of angiotensin receptor blockade in the rabbit aorta: influence of the endothelium
Canadian Journal of Physiology and Pharmacology, 1996
Contractile responsiveness of the rabbit aorta (endothelium intact and denuded) to angiotensin I, II, and III was compared. The effects of converting-enzyme inhibition with enalapril, the selective AT 1-receptor antagonist (losartan), and the AT 2-receptor antagonist (PD 123319) on these contractile profiles were examined. In all preparations, it was found that the angiotensins produced concentration-dependent increases in tension. Differences in sensitivity were encountered; in endothelium-intact preparations, the mean EC 50 values (nM with 95% confidence interval in parentheses) for angiotensin I, II, and III were 9 (95% CI 7-11), 40 (20-60), and 30 (10-40), respectively, and for denuded preparations they were 20 (11-29), 0.8 (0.7-0.9), and 30 (20-40), respectively. Enalapril decreased the maximal tension developed to angiotensin I and II, which was greater in endothelium-intact preparations. Losartan was a competitive antagonist against angiotensin I and angiotensin II in both intact and denuded preparations, with pA 2 values as follows: against angiotensin I, 9.0 and 9.3 for intact and denuded, respectively; against angiotensin II, 8.3 and 8.9 for intact and denuded, respectively. Losartan antagonized angiotensin III, but the slopes of the Schild analysis were significantly less than unity. In endothelium-intact preparations, PD 123319 failed to significantly antagonize responsiveness to angiotensin I. Against angiotensin II, PD 123319 was a competitive antagonist with a pA 2 of 8.3. The antagonism for PD 123319 against angiotensin III was insurmountable. In endothelium-denuded preparations, PD 123319 failed to antagonize angiotensin I and angiotensin III. Although PD 123319 appeared to inhibit the responsiveness of the rabbit aorta by angiotensin II, the slope of the Schild plot was significantly less than unity. These experiments provide evidence that angiotensin I possesses different actions from angiotensin II and III and that a functional endothelium modulates the underlying vascular response to angiotensin. In addition, the endothelium modulates the antagonism by losartan and PD 123319, supporting the notion that the endothelium possesses distinct angiotensin receptors.
Vascular Pharmacology, 2011
We tested whether heterologous receptor desensitization induced by activation of AT 1 receptors may explain the purported relaxation produced by angiotensin II in normal rat aorta. Also, the role for AT 2 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 AT 1 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 AT 2 receptors blockade the contractile effects of angiotensin II were not overexposed. In conclusion, angiotensin II-induced biphasic responses can be attributed to AT 1 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 α 1-adrenergic receptors desensitization). We found no functional evidence of the participation of AT 2 receptors in angiotensin II elicited biphasic contractions. Angiotensin II does not exert relaxant effects in normal rat aorta.
β-Arrestin- and Dynamin-Dependent Endocytosis of the AT1Angiotensin Receptor
Molecular Pharmacology, 2001
The major mechanism of agonist-induced internalization of G protein-coupled receptors (GPCRs) is -arrestinand dynamindependent endocytosis via clathrin-coated vesicles. However, recent reports have suggested that some GPCRs, exemplified by the AT 1 angiotensin receptor expressed in human embryonic kidney (HEK) 293 cells, are internalized by a -arrestinand dynamin-independent mechanism, and possibly via a clathrin-independent pathway. In this study, agonist-induced endocytosis of the rat AT 1A receptor expressed in Chinese hamster ovary (CHO) cells was abolished by clathrin depletion during treatment with hyperosmotic sucrose and was unaffected by inhibition of endocytosis via caveolae with filipin. In addition, internalized fluorescein-conjugated angiotensin II appeared in endosomes, as demonstrated by colocalization with transferrin. Overexpression of -arrestin1(V53D) and -arres-tin1(1-349) exerted dominant negative inhibitory effects on the endocytosis of radioiodinated angiotensin II in CHO cells. GTPase-deficient (K44A) mutant forms of dynamin-1 and dynamin-2, and a pleckstrin homology domain-mutant (K535A) dynamin-2 with impaired phosphoinositide binding, also inhibited the endocytosis of AT 1 receptors in CHO cells. Similar results were obtained in COS-7 and HEK 293 cells. Confocal microscopy using fluorescein-conjugated angiotensin II showed that overexpression of dynamin-1(K44A) and dynamin-2(K44A) isoforms likewise inhibited agonist-induced AT 1 receptor endocytosis in CHO cells. Studies on the angiotensin II concentration-dependence of AT 1 receptor endocytosis showed that at higher agonist concentrations its rate constant was reduced and the inhibitory effects of dominant negative dynamin constructs were abolished. These data demonstrate the importance of -arrestinand dynamin-dependent endocytosis of the AT 1 receptor via clathrin-coated vesicles at physiological angiotensin II concentrations. The pressor octapeptide hormone, angiotensin II (Ang II), exerts the majority of its physiological effects on cardiovascular regulation and saltwater balance by activating the G q-coupled AT 1 angiotensin receptor (De Gasparo et al., 2000). The AT 1 receptor also activates intracellular signaling pathways that stimulate cell growth including activation of tyrosine kinases and small GTP-binding proteins (Berk, 1999; De Gasparo et al., 2000), and is rapidly internalized after Ang II binding (Thomas, 1999; Hunyady et al., 2000). Agonist-induced endocytosis of G protein-coupled receptors (GPCRs) initiates a process by which desensitized receptors are resensitized and recycled to the plasma membrane (Krupnick and Benovic, 1998). Sequestration of the  2-adrenergic receptor has been shown to require the binding of -arrestin proteins to its cytoplasmic tail after agonist-induced activation and phosphorylation by G protein-coupled receptor kinases (Zhang et al., 1996; Ferguson et al., 1997; Krupnick and Benovic, 1998). -arrestins direct the phosphorylated receptors to clathrin-coated pits and induce the formation of clathrin-coated vesicles (Goodman et al., 1996). The role of -arrestins in receptor internalization has been demonstrated for several GPCRs (Bü nemann et al., 1999). Although -arrestins translocate to the plasma membrane upon agonist stimulation of many GPCRs (Zhang et al., 1999), it has been reported that the internalization of some of
Naunyn-schmiedebergs Archives of Pharmacology, 2004
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 AT1A subtype, while prejunctional AT1 receptors belong to the AT1B subtype.