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Intracellular angiotensin II inhibits heterologous receptor stimulated Ca2+ entry
Life Sciences 70 (2001) 171–180
Intracellular angiotensin II inhibits heterologous receptor stimulated Ca21 entry Catalin M. Filipeanua, Eugen Brailoiua,1, Robert H. Henninga, Leo E. Deelmana, Dick de Zeeuwa, S. Adriaan Nelemans a,b,* a
Groningen University Institute for Drug Exploration (GUIDE), Department of Clinical Pharmacology, University of Groningen, A. Deusinglaan 1, 9713AV Groningen, The Netherlands b University Centre for Pharmacy, Department of Molecular Pharmacology University of Groningen, A. Deusinglaan 1, 9713AV Groningen, The Netherlands Received 1 February 2001; accepted 15 June 2001
Abstract Recent studies show that angiotensin II (AngII) can act from within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane AngII receptors. The role of this intracellular AngII (AngIIi) is unclear. Besides direct effects of AngIIi on cellular processes one could hypothesise a possible role of AngIIi in modulation of cellular responses induced after heterologous receptor stimulation. We therefore examined if AngIIi influences [Ca21]i in A7r5 smooth muscle cells after serotonin (5HT) or UTP receptor stimulation. Application of AngIIi using liposomes, markedly inhibited 45Ca21 influx after receptor stimulation with 5HT or UTP. This inhibition was reversible by intracellular administration of the AT1-antagonist losartan and not influenced by the AT2-antagonist PD123319. Similar results were obtained in single cell [Ca21]i measurements, showing that AngIIi predominantly influences Ca21 influx and not Ca21 release via AT1-like receptors. It is concluded that AngIIi modulates signal transduction activated by heterologous receptor stimulation. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Intracellular angiotensin II; Ca21 influx; Ca21 release; Serotonin; UTP; A7r5 cells
Introduction Cellular effects of angiotensin II (AngII) observed under physiological and patho-physiological conditions are attributed to the interaction with specific AT1- and AT2- type AngII receptors in the plasma membrane [1]. These receptors are coupled to different signal trans* Corresponding author. Tel.: 1131-50-363-3323; fax: 1131-50-363-6908. E-mail address: [email protected] (S.A. Nelemans) 1 Present address: Department of Pharmacology, James Q College of Medicine, East Tennessee State University, Johnson City, Tennessee, USA. 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )1 3 8 9 -3
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duction pathways. Stimulation of AT1-receptors activates phospholipase C, formation of Ins(1,4,5)P3, which subsequently discharges Ca21 from internal stores and activates MAP kinase, whereas AT2-receptors increase cGMP levels and inhibit cell growth [2, 3]. The AT1receptor subtype can rapidly desensitise after stimulation followed by receptor internalisation. During this process it can deliver AngII inside the cell [4]. Intracellular AngII binding proteins have been reported in liver [5], storage granules of cardiac myocytes [6], mesangial cells [7], and within the cytosolic fraction of placenta [8]. Recent studies show that AngII can act from within the cell, possibly via intracellular receptors, which differ in their pharmacology from typical plasma membrane AngII receptors. Although their physiological function is not yet clear, important cellular effects have been observed after stimulation of these putative receptors. Intracellular application of AngII (AngIIi) inhibited cell communication through gap-junctions in heart muscle [9], induced [Ca21]i increases in vascular smooth muscle cells [10], initiated tyrosine phosphorylation in myocytes [11], elicited contraction of rat aorta [12] and stimulated cell growth in vascular smooth muscle cells [13]. Under both physiological and patho-physiological conditions cells are often exposed to a multitude of hormones and other mediators of signal transduction. Crosstalk between different receptor systems is essential for physiological functioning, determining the resulting cellular response. Second messengers induced after receptor stimulation with different agents (i.e. ATP, adenosine, histamine, bradykinin) modulate other second messenger systems, either activated by themselves or by the other agents. Crosstalk have been described at the level of cAMP, Ins(1,4,5)P3, [Ca21]i and/or contraction after stimulation of heterologous receptors in smooth muscle [15–18]. With respect to AngII, only Ca21 dependent transactivation of EGF receptors was reported after stimulation of the plasma membrane AT1-receptor [19]. There are no data published on a possible role of intracellular AngII in the modulation of cellular responses induced after stimulation of different plasma membrane receptors (heterologous receptor stimulation). Therefore, we investigated the effects of intracellular application of AngII on [Ca21]i in A7r5 smooth muscle cells after serotonin (5HT) and UTP receptor stimulation. Methods Cell culture A7r5 vascular smooth muscle cells (kindly provided by Dr. H. De Smedt, K. U. Leuven, Belgium) were grown in 75cm2 flasks in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (50 mg ml21) and streptomycin (50 units ml21) at 378C in a humidified atmosphere (5 % CO2). The cells were subcultured at 95% confluency by trypsinization. 45Ca21 experiments were performed in 6 well plates (Costar, 9.6 cm2 well21) at a density of 105cells well21. For fluorescence experiments the cells were plated 24–72 hours before the start of the experiment in LabTek II (type 155382) chambers. Chemicals All culture media were obtained from Gibco BRL (U.S.A.). Inositol 1,4,5-trisphosphate sodium salt was obtained from Boehringer (Germany). Fura2-AM and AngII-fluorescein
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were obtained from Molecular Probes (U.S.A.). 45CaCl2 (specific activity: 19.3 Ci g21) and D-[2-3H]inositol 1.4,5-trisphosphate (specific activity: 3.3 Ci mmol21) were obtained from Dupont-NEN (U.S.A.). AngII was supplied by the Academic Hospital Pharmacy of the University of Groningen. Losartan (2-n- butyl-4-chloro-5-hydroxymethyl-1-[(29-(1H-tetrazol5-yl)biphenyl-4-yl)methyl]imidazole) was provided by Merck Sharp & Dohme (U.S.A.) and PD123319 ( (s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate) by Park Davis Company (U.S.A.). All other compounds were obtained from Sigma (U.S.A.). Liposomes preparation and intracellular application Liposomes were prepared as described previously [20, 21], using 10 mg phosphatidylcholine per ml of solution containing the substance to be incorporated. The number of lamellae was decreased by addition of diethyl ether in a ratio of 1/10 (v/v). AngII was dissolved at a concentration of 1026 M in 140 mM KCl solution (pH 7.0). Control liposomes contained only 140 mM KCl. In order to remove non-incorporated solutes, liposomes were subjected to dialysis, 2 times during 120 min, against buffer solution in a ratio of 1/600 v/v. (Sigma dialysis tubing, molecular weight cut-off: 12400 dalton). The buffer solution had the following composition (mM): 145 NaCl, 5 KCl, 0.5 MgSO4, 0.5 CaCl2, 10 glucose and 10 HEPES (pH adjusted to 7.4 with NaOH). The amount of Ang II delivered into the cells by this procedure was estimated by loading the cells with 1026 M fluorescein-AngII filled liposomes and subsequent cell permeabilization with saponin comparable to the method described for intracellular adenosine delivery [20]. Fluorescence was measured with excitation wavelength 470 nm, emission wavelength 520 nm, and a bandpass filter of 4 nm (Aminco Bowman LS Series 2). Liposome-incapsulated fluorescein-AngII amounted to 12.4 6 0.9 % (n54) of the initial amount in the aqueous phase. Angiotensin II incorporated into the cells with an efficiency of 2.3 6 0.4 % (n54). The actual [AngIIi] can be calculated if an estimation of the cell volume is established. The cellular volume of A7r5 cells was estimated by determination of the maximal radius (r) of spherical cells and calculation of the volume (V) according to V54/3*p*r3. Cells were slightly attached to the culture dish and the culture medium was sequentially diluted with an equal volume of water. The osmotic swelling process was followed under microscopy and the maximal diameter reached was estimated with a standard micrometer in view. The maximal diameter was 13.4 6 0.2 mm (n564), resulting in a volume of 1.26 6 0.02 pL cell21. Therefore, if the protocol is used to deliver liposomes filled with 1026 M AngII to the cells, this will result in an estimated intracellular [AngII] of 18 6 3 mmol/L. 45
Ca21 uptake by intact cells
These experiments were performed as described previously [21]. In brief, culture medium was replaced 1 hr before the start of the experiment with a buffer solution containing (mM): 145 NaCl, 5 KCl, 0.5 MgSO4, 0.5 CaCl2, 10 glucose and 10 HEPES (pH adjusted to 7.4 with NaOH). Uptake of 45Ca21 was measured at room temperature (22–24 8C) and started by removing the solution and replacing it by the same buffer (1 ml) supplemented with 10 mCi 45 Ca21 (specific activity 19.3 Ci g21) and indicated compounds. The liposomes were added at a ratio of 1/20 (v/v) in the buffer solution. Aspiration of the solution and addition of 1ml ice-
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cold buffer stopped uptake of 45Ca21 in the absence of CaCl2 after 5 min. Thereafter, cells were washed 3 times with buffer without CaCl2 and containing 2 mM EGTA. Cells were lysed in the presence of NaOH (1 ml, 1 M) and radioactivity was measured by liquid scintillation counting. Data were corrected for non-specific binding as determined by adding buffer with 45 Ca21 and immediately terminating uptake. [Ca21]i measurements Cells were loaded with 5 mM fura2-AM for 45 min at 378C. Calcium measurements were performed using a S100 Axiovert inverted microscope (Zeiss). The 340/380 ratio was acquired at room temperature with at a frequency by 1 Hz using a cooled CCD camera (SensiCam) and Imaging Workbench 2.2. software (Axon Instruments). Liposomes were added at a ratio of 1/20 vol/vol, 5 min before agonist addition. Ratio values were transformed in [Ca21]i at the end of the experiment [22]. Measurement of Ins (1,4,5)P3 Mass measurement of Ins (1,4,5)P3 was performed using a ligand binding assay as described earlier in detail [17]. Statistics All experiments were performed in series with n $ 4. The results are expressed as mean 6 S.D. Statistical differences were tested either by ANOVA analysis followed by Bonferroni post-test or by unpaired Student’s t-test considering p # 0.05 significantly different. Results A7r5 cells represent an attractive model to investigate the effects of intracellular AngII. Since these cells do not respond to extracellular addition of AngII (1026 M) in terms of [Ca21]i or Ins(1,4,5)P3 levels (Table 1) one can study the effects of AngIIi on signal transduction processes without interference of cellular signalling activated by plasma membrane An-gII receptors. We used liposomes to administer AngII intracellularly. Various compounds can be delivered by liposomes, while plasma membrane integrity is maintained (20, 21, 23). Cells are also not metabolically compromised by liposome treatment, since methylene blue is still excluded and incubation with control liposomes (24 hr) did not affect cell growth (data not
Table 1 Effects of extracellular AngII on [Ca21]i and Ins(1,4,5)P3 levels in A7r5 cells
Basal AngII
[Ca21]i (nM)
Ins(1,4,5)P3 (pmol/105 cells)
57 6 6 58 6 6
0.23 6 006 0.22 6 0.04
Peak [Ca21]i and Ins(1,4,5)P3 were measured 30 s after stimulation with extracellular AngII (1026 M). n $ 6 in each case.
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Fig. 1. Effects of intracellular AngII on 45Ca21 uptake induced by 5HT or UTP. Panel A: 45Ca21 uptake induced by extracellular 5HT (1025 M) for 5 min (first bar), in the presence of liposomes filled with 140 mM KCl (Lcon) or filled with either 1026 M Ang II(1LAngII) alone or together with 1026 M losartan (1LAngII 1Llos) or 1026 M PD 123319 (1LAngII 1LPD). Panel B: idem for extracellular stimulation with UTP (1023 M). Data are presented as mean 6 S.D. (n56) and expressed as % of uptake in the absence of extracellular 5HT or UTP amounting 85 dpm/ 105 cells. * P,0.05 vs control stimulation (5HT or UTP alone).
shown). Moreover, pre-treatment with control liposomes (filled with 140 mM KCl) did not affect Ca21 increases induced by either 5HT or UTP (Fig.1). Influx of 45Ca21 was increased about 2-fold after stimulation with 5HT or UTP for 5 min (Fig. 1). This increase was partially inhibited by liposomes containing 1026 M AngII, whereas control liposomes were ineffective. The effect of AngII filled liposomes was unaffected by the extracellular addition of the AT1-receptor antagonist losartan (1026 M) or the AT2-receptor antagonist PD123319 (1026 M, n56, data not shown). In contrast, inclusion of losartan into liposomes together with AngII reversed the inhibition of 45Ca21 influx by AngII. Liposomes containing both AngII and PD123319 (1026 M) still inhibited 45Ca21 influx (Fig. 1). The effect of AngIIi on 5HT and UTP induced changes in [Ca21]i homeostasis was confirmed by single cell [Ca21]i measurements using fura2 fluorescence. Pre-incubation with Ang II containing liposomes reduced the 5HT and UTP induced [Ca21]i increases similarly
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Fig. 2. Effects of intracellular AngII on [Ca21]i induced by 5HT or UTP. Upper panels: Changes in [Ca21]i as measured by single cell fura2 fluorescence induced by extracellular 5HT (A, 1025 M) or UTP (B, 1023 M) in the absence (a) or presence (b) of liposomes filled with 1026 M AngII. Lower panels: maximal [Ca21]i increases induced by 1025 M 5HT (left panel) or 1023 M UTP (right panel) in the absence (first bar) or the presence of liposomes (5 min pre-incubation) filled with 140 mM KCl (Lcon) or with 1026 M AngII (LAngII). Data are presented as mean 6 S.D. (n56). * P,0.05 vs control.
as in the 45Ca21 uptake experiments to approximately 30 % (Fig. 2). Control liposomes filled with 140 mM KCl were ineffective. The inhibitory effect was reversed by losartan containing liposomes, but not by PD123319 containing liposomes (Table 2). These results show that intracellular AngII exerts its effect through an AT1-like receptor, probably via inhibition of Ca21 influx. To clarify which part of Ca21 signalling is affected by intracellular AngII we further investigated this effect on 5HT induced changes in [Ca21]i. Using a Ca21 chelation / readmission protocol, Ca21 release and Ca21 influx could be discriminated. Stimulation with 5HT predominantly elevates [Ca21]i via Ca21 influx, which is largely mediated via voltage dependent Ca21 channel, as demonstrated by the almost complete inhibition by 1026 M verapamil (Fig. 3). Intracellular application of AngII inhibits this 5HT induced Ca21 influx to a similar extent as verapamil, possibly indicating that inhibition of this channel by AngIIi occurs, whereas it does not affect Ca21 release.
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Table 2 Inhibition of 5HT induced [Ca21]i increases by intracellular Ang II. Effects of antagonists [Ca21]i increases L control 1 5HT L AngII 1 5HT L(AngII 1 losartan) 1 5HT L(AngII 1 PD123319) 1 5HT
100 6 8 (%) 27 6 8 a 72 6 16 a, b 30 6 10 a
Increases in peak [Ca21]i were measured after stimulation with 5HT (1025 M) and liposomes filled with AngII (10 M) either in the absence or presence of losartan (1026 M) or PD123319 (1026 M). n $ 6 in each case. a) different from L control 1 5HT (P,0.05). b) different from L AngII 1 5HT (P,0.05). 26
Discussion Effects of AngIIi can be mediated via mechanisms atypical for plasma membrane AngII receptors in various cell types [9–13]. This implies that patho-physiological actions of AngII might occur intracellularly and therefore not accessible to classical pharmacological agents working at the plasma membrane. Many studies described crosstalk between components of different receptor classes and at different levels downstream receptor activation [23–25]. However, the novelty of the present work is to show interactions between an agonist (AngII) and heterologous receptor stimulation not initiated by AngII at the plasma membrane level, but by intracellular receptor stimulation. In this study we have used A7r5 cells, which do not have functional responses after stimulation extracellular AngII, as concluded from the results on Ins(1,4,5)P3 formation and [Ca21]i in this study and other experiments [13, 26]. Therefore interference of the results obtained by AngIIi on Ca21 homeostasis with possible effects induced by plasma membrane AT-receptors receptor is excluded. AngIIi potently reduced the Ca21 signal evoked by 5HT and UTP stimulation. In view of the experiments performed with the antagonist losartan and PD123319, it is suggested that this effect of AngIIi be mediated by an AT1-like receptor. The ineffectiveness of AngI indicates that it might be different from common plasma membrane AT1-receptors [25]. We have recently shown that AngIIi increase [Ca21]i in non-stimulated A7r5 cells [27]. These effects were insensitive to both losartan and PD123319, suggesting that multiple intracellular AngII receptor types be coupled to different signal transduction mechanisms in A7r5 cells. Reductions of Ca21 increases induced by AngIIi are the not the consequence of inhibition of Ca21 release from intracellular stores, but can be totally attributed to inhibition of Ca21 influx. The parallel effect of AngIIi and verapamil indicates that the L-type Ca21 channel might be the target for this effect. This novel finding of the AngIIi induced inhibition of heterologous receptor activated Ca21 influx can be supported by the observations made in rat cardiomyocytes showing reduction in amplitude of inward voltage-dependent Ca21 current after dialysing AngII into the cell [26]. Increases in [Ca21]i by AngIIi occurred through voltage independent Ca21 channels in non-stimulated A7r5 cells, indicating either the presence of a different receptor or a different coupling mechanism downstream receptor activation under these conditions. In analogy, we have recently shown that AngIIi stimulation of cell growth in
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Fig. 3. Differential effects of intracellular AngII on Ca21 release and Ca21 influx. Changes in intracellular Ca21 release and Ca21 influx were measured by single cell fura2 fluorescence after stimulation with extracellular 5HT (1025 M) under Ca21 free conditions and after subsequent restoration of the normal [Ca21] in the buffer solution. Left panel: tracing a) control, b) in the presence of verapamil (verap, 1026 M, 5 min pre-incubation), c) in the presence of liposomes (5 min pre-incubation) filled with 1026 M AngII (LAngII), Right panel: maximal Ca21 increase. Open bars: Ca21 release, hatched bars: Ca21 influx. Data are presented as mean 6 S.D. (n56). * P,0.05 vs control.
A7r5 cells is mediated by two receptor subtypes, one with high affinity for AngIIi, partially sensitive to losartan and PD123319 and a second one with low affinity for AngIIi and insensitive to both antagonists [13]. Inhibition of heterologous receptor activated Ca21 influx might be of physiological relevance in the process of fine-tuning of multiple receptor signaling pathways. Also over stimulation due to exposure to different mediators can be avoided by such a mechanism, particular beneficial under patho-physiological conditions in which Ca21 overload is implicated like e.g. ischemia. For such a negative protective feedback mechanism availability of AngIIi is essential. It is likely that AngIIi can fulfil this role, since intracellular pools of AngII were observed in cardiomyocytes [5], renal endosomes [28] and lymphoctes [29] under physiological conditions. It is not clear at present if AngII can be produced intracellularly, or if these depos-
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its are the result of AngII internalization together with the AT1-receptor [30, 31]. However, the observation that the amount of intracellular AngII changes under various conditions is an indication that AngIIi might play an important role in adaptive changes to patho-physiological situations. In conclusion, AngIIi inhibited Ca21 entry induced after heterologous receptor stimulation with either 5HT or UTP via an AT1-like receptor in A7r5 smooth muscle cells. Acknowledgments C.M. Filipeanu is recipient of an Ubbo Emmius fellowship from the Groningen University Institute for Drug Exploration (GUIDE). References 1. Griendling KK, Ushio-Fukai M, Lassegue B, Alexander RW. Angiotensin II signaling in vascular smooth muscle. New concepts. Hypertension 1997; 29: 366–73. 2. Hunyady L, Balla T, Catt KJ. The ligand binding site of the angiotensin AT1 receptor. Trends Pharmacol Sci 1996; 17: 135–140. 3. Horiuchi M, Akishita M, Dzau, V.J. Recent progress in angiotensin II type 2 receptor research in the cardiovascular system. Hypertension 1999; 33: 613–21. 4. Hunyady L, Catt KJ, Clark AJ, Gaborik Z. Mechanisms and functions of AT(1) angiotensin receptor internalization. Regul Pept 2000; 91: 29–44. 5. Kiron MA, Soffer RL. Purification and properties of a soluble angiotensin II-binding protein from rabbit liver. J Biol Chem 1989; 264: 4138–4142. 6. Sadoshima J, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell 1993; 75: 977–984. 7. Mercure C, Ramla D, Garcia R., Thibault G., Deschepper CF., Reudelhuber TL. Evidence for intracellular generation of angiotensin II in rat juxtaglomerular cells. FEBS Lett 1998; 422: 395–399. 8. Li X, Shams M, Zhu J, Khalig A, Wilkes M, Whittle M, Barnes N, Ahmed A. Cellular localization of AT1 receptor mRNA and protein in normal placenta and its reduced expression in intrauterine growth restriction. Angiotensin II stimulates the release of vasorelaxants. J Clin Invest 1998; 101: 442–454. 9. De Mello WC. Renin-angiotensin system and cell communication in the failing heart. Hypertension 1996; 27: 1267–1272. 10. Haller H, Lindschau C, Erdmann B, Quass P, Luft FC. Effects of intracellular angiotensin II in vascular smooth muscle cells. Circ Res 1996; 79: 765–772. 11. Haller H, Lindschau C, Quass P, Luft FC. Intracellular actions of angiotensin II in vascular smooth muscle cells. J Am Soc Nephrol 1999; Suppl 11:S75–83. 12. Brailoiu E, Filipeanu CM, Tica A, Toma CP, de Zeeuw D, Nelemans SA. Contractile effects by intracellular angiotensin II via receptors with a distinct pharmacological profile in rat aorta. Br J Pharmacol 1999; 126: 1133–1138. 13. Filipeanu CM, Henning RH, de Zeeuw D, Nelemans A. Intracellular Angiotensin II and cell growth of vascular smooth muscle cells. Br J Pharmacol 2001; 132:1590–1596. 14. Dickenson JM, Hill SJ. Intracellular cross-talk between receptors coupled to phospholipase C via pertussis toxin-sensitive and insensitive G-proteins in DDT1MF-2 cells. Br J Pharmacol 1993; 109: 719–24. 15. Manolopoulos VG, Pipili-Synetos E, Den Hertog A., Nelemans A. Inositol phosphates formed in rat aorta after alpha1-adrenoceptor stimulation are inhibited by forskolin. Eur J Pharmacol 1991; 207: 29–36. 16. Gerwins P, Fredholm BB. Stimulation of adenosine A1 receptors and bradykinin receptors, which act via different G proteins, synergistically raises inositol 1,4,5-trisphosphate and intracellular free calcium in DDT1 MF-2 smooth muscle cells. Proc Natl Acad Sci U S A 1992; 89: 7330–4.
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17. Sipma H, Duin M, Hoiting B, den Hertog A, Nelemans, A. Regulation of histamine- and UTP-induced increases in Ins(1,4,5)P3, Ins (1,3,4,5)P4 and Ca21 by cyclic AMP in DDT1 MF-2 cells Br J Pharmacol 1995; 114: 383–90. 18. Eguchi, S., Numaguchi, K., Iwasaki, H., Matsumoto, T., Yamakawa, T., Utsunomiya, H., Motley, E.D., Kawakatsu, H., Owada, K.M., Hirata, Y., Marumo, F. and Inagami, T. Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem 1998; 273: 8890–8896. 19. Brailoiu E, Serban DN, Slatineanu S, Filipeanu CM, Petrescu BC, Branisteanu DD. Effects of liposomeentrapped adenosine in the isolated rat aorta. Eur J Pharmacol 1993; 250: 489–492. 20. Filipeanu CM, Brailoiu E, Petrescu G, Nelemans SA. Extracellular and intracellular arachidonic acidinduced contractions in rat aorta. Eur J Pharmacol 1998; 349: 67–73. 21. Sipma H, van der Zee L, van den Akker J, den Hertog A, Nelemans A. The effect of the PKC inhibitor GF109203X on the release of Ca21 from internal stores and Ca21 entry in DDT1 MF-2 cells. Br J Pharmacol 1996; 119: 730–736. 20. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca21 indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440–3449. 21. Brailoiu E, van der Kloot WV. Bromoacetylcholine and acetylcholinesterase introduced via liposomes into motor nerve endings block increases in quantal size. Pfluegers Arch-Eur J Physiol 1996; 432: 413–418. 22. Luttrell LM, Daaka Y, Lefkowitz RJ. Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Current Opinion Cell Biol 1999; 11: 177–183. 23. Selbie LA, Hill SJ. G protein-coupled-receptor cross-talk: the fine-tuning of multiple receptor-signalling pathways. Trends Pharmacol Sci. 1998; 19: 87–93. 24. Bornfeldt KE, Krebs EG. Crosstalk between protein kinase A and growth factor receptor-signalling pathways in arterial smooth muscle. Cell Signal 1999; 11: 465–477. 25. Tschudi MR, Luscher TF. Age and hypertension differently affect coronary contractions to endothelin-1, serotonin, and angiotensins. Circulation 1995; 91: 2415–22. 26. De Mello WC. Intracellular angiotensin II regulates the inward calcium current in cardiac myocytes. Hypertension 1998; 32: 976–82. 27. Filipeanu CM, Brailoiu E, Kok JW, Henning RH, de Zeeuw D, Nelemans SA. Intracellular angiotensin II elicits Ca21 increases in A7r5 vascular smooth muscle cells. Eur. J. Pharmacol 2001; 420: 9–18. 28. Imig, JD, Navar, GL, Zou, LX, O’Reilly, KC, Allen, PL, Kaysen, JH, Hammond, TG, Navar, LG. Renal endosomes contain angiotensin peptides, converting enzyme, and AT(1A) receptors. Am J Physiol 1999; 277: F303–311. 29. Hermann K, Ring J. Association between the renin angiotensin system and anaphylaxis. Adv Exp Med Biol 1995; 377: 299–309. 30. van Kats JP, de Lannoy LM, Danser JAH, van Meegen JR, Verdouw PD, Schalekamp MA. Angiotensin II type 1 (AT1) receptor-mediated accumulation of angiotensin II in tissues and its intracellular half-life in vivo. Hypertension 1997; 301: 42–9. 31. De Mello WC, Danser AH. Angiotensin II and the heart: on the intracrine renin-angiotensin system. Hypertension 2000; 35: 1183–8.
Intracellular angiotensin II inhibits heterologous receptor stimulated Ca21 entry Catalin M. Filipeanua, Eugen Brailoiua,1, Robert H. Henninga, Leo E. Deelmana, Dick de Zeeuwa, S. Adriaan Nelemans a,b,* a
Groningen University Institute for Drug Exploration (GUIDE), Department of Clinical Pharmacology, University of Groningen, A. Deusinglaan 1, 9713AV Groningen, The Netherlands b University Centre for Pharmacy, Department of Molecular Pharmacology University of Groningen, A. Deusinglaan 1, 9713AV Groningen, The Netherlands Received 1 February 2001; accepted 15 June 2001
Abstract Recent studies show that angiotensin II (AngII) can act from within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane AngII receptors. The role of this intracellular AngII (AngIIi) is unclear. Besides direct effects of AngIIi on cellular processes one could hypothesise a possible role of AngIIi in modulation of cellular responses induced after heterologous receptor stimulation. We therefore examined if AngIIi influences [Ca21]i in A7r5 smooth muscle cells after serotonin (5HT) or UTP receptor stimulation. Application of AngIIi using liposomes, markedly inhibited 45Ca21 influx after receptor stimulation with 5HT or UTP. This inhibition was reversible by intracellular administration of the AT1-antagonist losartan and not influenced by the AT2-antagonist PD123319. Similar results were obtained in single cell [Ca21]i measurements, showing that AngIIi predominantly influences Ca21 influx and not Ca21 release via AT1-like receptors. It is concluded that AngIIi modulates signal transduction activated by heterologous receptor stimulation. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Intracellular angiotensin II; Ca21 influx; Ca21 release; Serotonin; UTP; A7r5 cells
Introduction Cellular effects of angiotensin II (AngII) observed under physiological and patho-physiological conditions are attributed to the interaction with specific AT1- and AT2- type AngII receptors in the plasma membrane [1]. These receptors are coupled to different signal trans* Corresponding author. Tel.: 1131-50-363-3323; fax: 1131-50-363-6908. E-mail address: [email protected] (S.A. Nelemans) 1 Present address: Department of Pharmacology, James Q College of Medicine, East Tennessee State University, Johnson City, Tennessee, USA. 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )1 3 8 9 -3
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duction pathways. Stimulation of AT1-receptors activates phospholipase C, formation of Ins(1,4,5)P3, which subsequently discharges Ca21 from internal stores and activates MAP kinase, whereas AT2-receptors increase cGMP levels and inhibit cell growth [2, 3]. The AT1receptor subtype can rapidly desensitise after stimulation followed by receptor internalisation. During this process it can deliver AngII inside the cell [4]. Intracellular AngII binding proteins have been reported in liver [5], storage granules of cardiac myocytes [6], mesangial cells [7], and within the cytosolic fraction of placenta [8]. Recent studies show that AngII can act from within the cell, possibly via intracellular receptors, which differ in their pharmacology from typical plasma membrane AngII receptors. Although their physiological function is not yet clear, important cellular effects have been observed after stimulation of these putative receptors. Intracellular application of AngII (AngIIi) inhibited cell communication through gap-junctions in heart muscle [9], induced [Ca21]i increases in vascular smooth muscle cells [10], initiated tyrosine phosphorylation in myocytes [11], elicited contraction of rat aorta [12] and stimulated cell growth in vascular smooth muscle cells [13]. Under both physiological and patho-physiological conditions cells are often exposed to a multitude of hormones and other mediators of signal transduction. Crosstalk between different receptor systems is essential for physiological functioning, determining the resulting cellular response. Second messengers induced after receptor stimulation with different agents (i.e. ATP, adenosine, histamine, bradykinin) modulate other second messenger systems, either activated by themselves or by the other agents. Crosstalk have been described at the level of cAMP, Ins(1,4,5)P3, [Ca21]i and/or contraction after stimulation of heterologous receptors in smooth muscle [15–18]. With respect to AngII, only Ca21 dependent transactivation of EGF receptors was reported after stimulation of the plasma membrane AT1-receptor [19]. There are no data published on a possible role of intracellular AngII in the modulation of cellular responses induced after stimulation of different plasma membrane receptors (heterologous receptor stimulation). Therefore, we investigated the effects of intracellular application of AngII on [Ca21]i in A7r5 smooth muscle cells after serotonin (5HT) and UTP receptor stimulation. Methods Cell culture A7r5 vascular smooth muscle cells (kindly provided by Dr. H. De Smedt, K. U. Leuven, Belgium) were grown in 75cm2 flasks in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (50 mg ml21) and streptomycin (50 units ml21) at 378C in a humidified atmosphere (5 % CO2). The cells were subcultured at 95% confluency by trypsinization. 45Ca21 experiments were performed in 6 well plates (Costar, 9.6 cm2 well21) at a density of 105cells well21. For fluorescence experiments the cells were plated 24–72 hours before the start of the experiment in LabTek II (type 155382) chambers. Chemicals All culture media were obtained from Gibco BRL (U.S.A.). Inositol 1,4,5-trisphosphate sodium salt was obtained from Boehringer (Germany). Fura2-AM and AngII-fluorescein
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were obtained from Molecular Probes (U.S.A.). 45CaCl2 (specific activity: 19.3 Ci g21) and D-[2-3H]inositol 1.4,5-trisphosphate (specific activity: 3.3 Ci mmol21) were obtained from Dupont-NEN (U.S.A.). AngII was supplied by the Academic Hospital Pharmacy of the University of Groningen. Losartan (2-n- butyl-4-chloro-5-hydroxymethyl-1-[(29-(1H-tetrazol5-yl)biphenyl-4-yl)methyl]imidazole) was provided by Merck Sharp & Dohme (U.S.A.) and PD123319 ( (s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate) by Park Davis Company (U.S.A.). All other compounds were obtained from Sigma (U.S.A.). Liposomes preparation and intracellular application Liposomes were prepared as described previously [20, 21], using 10 mg phosphatidylcholine per ml of solution containing the substance to be incorporated. The number of lamellae was decreased by addition of diethyl ether in a ratio of 1/10 (v/v). AngII was dissolved at a concentration of 1026 M in 140 mM KCl solution (pH 7.0). Control liposomes contained only 140 mM KCl. In order to remove non-incorporated solutes, liposomes were subjected to dialysis, 2 times during 120 min, against buffer solution in a ratio of 1/600 v/v. (Sigma dialysis tubing, molecular weight cut-off: 12400 dalton). The buffer solution had the following composition (mM): 145 NaCl, 5 KCl, 0.5 MgSO4, 0.5 CaCl2, 10 glucose and 10 HEPES (pH adjusted to 7.4 with NaOH). The amount of Ang II delivered into the cells by this procedure was estimated by loading the cells with 1026 M fluorescein-AngII filled liposomes and subsequent cell permeabilization with saponin comparable to the method described for intracellular adenosine delivery [20]. Fluorescence was measured with excitation wavelength 470 nm, emission wavelength 520 nm, and a bandpass filter of 4 nm (Aminco Bowman LS Series 2). Liposome-incapsulated fluorescein-AngII amounted to 12.4 6 0.9 % (n54) of the initial amount in the aqueous phase. Angiotensin II incorporated into the cells with an efficiency of 2.3 6 0.4 % (n54). The actual [AngIIi] can be calculated if an estimation of the cell volume is established. The cellular volume of A7r5 cells was estimated by determination of the maximal radius (r) of spherical cells and calculation of the volume (V) according to V54/3*p*r3. Cells were slightly attached to the culture dish and the culture medium was sequentially diluted with an equal volume of water. The osmotic swelling process was followed under microscopy and the maximal diameter reached was estimated with a standard micrometer in view. The maximal diameter was 13.4 6 0.2 mm (n564), resulting in a volume of 1.26 6 0.02 pL cell21. Therefore, if the protocol is used to deliver liposomes filled with 1026 M AngII to the cells, this will result in an estimated intracellular [AngII] of 18 6 3 mmol/L. 45
Ca21 uptake by intact cells
These experiments were performed as described previously [21]. In brief, culture medium was replaced 1 hr before the start of the experiment with a buffer solution containing (mM): 145 NaCl, 5 KCl, 0.5 MgSO4, 0.5 CaCl2, 10 glucose and 10 HEPES (pH adjusted to 7.4 with NaOH). Uptake of 45Ca21 was measured at room temperature (22–24 8C) and started by removing the solution and replacing it by the same buffer (1 ml) supplemented with 10 mCi 45 Ca21 (specific activity 19.3 Ci g21) and indicated compounds. The liposomes were added at a ratio of 1/20 (v/v) in the buffer solution. Aspiration of the solution and addition of 1ml ice-
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cold buffer stopped uptake of 45Ca21 in the absence of CaCl2 after 5 min. Thereafter, cells were washed 3 times with buffer without CaCl2 and containing 2 mM EGTA. Cells were lysed in the presence of NaOH (1 ml, 1 M) and radioactivity was measured by liquid scintillation counting. Data were corrected for non-specific binding as determined by adding buffer with 45 Ca21 and immediately terminating uptake. [Ca21]i measurements Cells were loaded with 5 mM fura2-AM for 45 min at 378C. Calcium measurements were performed using a S100 Axiovert inverted microscope (Zeiss). The 340/380 ratio was acquired at room temperature with at a frequency by 1 Hz using a cooled CCD camera (SensiCam) and Imaging Workbench 2.2. software (Axon Instruments). Liposomes were added at a ratio of 1/20 vol/vol, 5 min before agonist addition. Ratio values were transformed in [Ca21]i at the end of the experiment [22]. Measurement of Ins (1,4,5)P3 Mass measurement of Ins (1,4,5)P3 was performed using a ligand binding assay as described earlier in detail [17]. Statistics All experiments were performed in series with n $ 4. The results are expressed as mean 6 S.D. Statistical differences were tested either by ANOVA analysis followed by Bonferroni post-test or by unpaired Student’s t-test considering p # 0.05 significantly different. Results A7r5 cells represent an attractive model to investigate the effects of intracellular AngII. Since these cells do not respond to extracellular addition of AngII (1026 M) in terms of [Ca21]i or Ins(1,4,5)P3 levels (Table 1) one can study the effects of AngIIi on signal transduction processes without interference of cellular signalling activated by plasma membrane An-gII receptors. We used liposomes to administer AngII intracellularly. Various compounds can be delivered by liposomes, while plasma membrane integrity is maintained (20, 21, 23). Cells are also not metabolically compromised by liposome treatment, since methylene blue is still excluded and incubation with control liposomes (24 hr) did not affect cell growth (data not
Table 1 Effects of extracellular AngII on [Ca21]i and Ins(1,4,5)P3 levels in A7r5 cells
Basal AngII
[Ca21]i (nM)
Ins(1,4,5)P3 (pmol/105 cells)
57 6 6 58 6 6
0.23 6 006 0.22 6 0.04
Peak [Ca21]i and Ins(1,4,5)P3 were measured 30 s after stimulation with extracellular AngII (1026 M). n $ 6 in each case.
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Fig. 1. Effects of intracellular AngII on 45Ca21 uptake induced by 5HT or UTP. Panel A: 45Ca21 uptake induced by extracellular 5HT (1025 M) for 5 min (first bar), in the presence of liposomes filled with 140 mM KCl (Lcon) or filled with either 1026 M Ang II(1LAngII) alone or together with 1026 M losartan (1LAngII 1Llos) or 1026 M PD 123319 (1LAngII 1LPD). Panel B: idem for extracellular stimulation with UTP (1023 M). Data are presented as mean 6 S.D. (n56) and expressed as % of uptake in the absence of extracellular 5HT or UTP amounting 85 dpm/ 105 cells. * P,0.05 vs control stimulation (5HT or UTP alone).
shown). Moreover, pre-treatment with control liposomes (filled with 140 mM KCl) did not affect Ca21 increases induced by either 5HT or UTP (Fig.1). Influx of 45Ca21 was increased about 2-fold after stimulation with 5HT or UTP for 5 min (Fig. 1). This increase was partially inhibited by liposomes containing 1026 M AngII, whereas control liposomes were ineffective. The effect of AngII filled liposomes was unaffected by the extracellular addition of the AT1-receptor antagonist losartan (1026 M) or the AT2-receptor antagonist PD123319 (1026 M, n56, data not shown). In contrast, inclusion of losartan into liposomes together with AngII reversed the inhibition of 45Ca21 influx by AngII. Liposomes containing both AngII and PD123319 (1026 M) still inhibited 45Ca21 influx (Fig. 1). The effect of AngIIi on 5HT and UTP induced changes in [Ca21]i homeostasis was confirmed by single cell [Ca21]i measurements using fura2 fluorescence. Pre-incubation with Ang II containing liposomes reduced the 5HT and UTP induced [Ca21]i increases similarly
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Fig. 2. Effects of intracellular AngII on [Ca21]i induced by 5HT or UTP. Upper panels: Changes in [Ca21]i as measured by single cell fura2 fluorescence induced by extracellular 5HT (A, 1025 M) or UTP (B, 1023 M) in the absence (a) or presence (b) of liposomes filled with 1026 M AngII. Lower panels: maximal [Ca21]i increases induced by 1025 M 5HT (left panel) or 1023 M UTP (right panel) in the absence (first bar) or the presence of liposomes (5 min pre-incubation) filled with 140 mM KCl (Lcon) or with 1026 M AngII (LAngII). Data are presented as mean 6 S.D. (n56). * P,0.05 vs control.
as in the 45Ca21 uptake experiments to approximately 30 % (Fig. 2). Control liposomes filled with 140 mM KCl were ineffective. The inhibitory effect was reversed by losartan containing liposomes, but not by PD123319 containing liposomes (Table 2). These results show that intracellular AngII exerts its effect through an AT1-like receptor, probably via inhibition of Ca21 influx. To clarify which part of Ca21 signalling is affected by intracellular AngII we further investigated this effect on 5HT induced changes in [Ca21]i. Using a Ca21 chelation / readmission protocol, Ca21 release and Ca21 influx could be discriminated. Stimulation with 5HT predominantly elevates [Ca21]i via Ca21 influx, which is largely mediated via voltage dependent Ca21 channel, as demonstrated by the almost complete inhibition by 1026 M verapamil (Fig. 3). Intracellular application of AngII inhibits this 5HT induced Ca21 influx to a similar extent as verapamil, possibly indicating that inhibition of this channel by AngIIi occurs, whereas it does not affect Ca21 release.
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Table 2 Inhibition of 5HT induced [Ca21]i increases by intracellular Ang II. Effects of antagonists [Ca21]i increases L control 1 5HT L AngII 1 5HT L(AngII 1 losartan) 1 5HT L(AngII 1 PD123319) 1 5HT
100 6 8 (%) 27 6 8 a 72 6 16 a, b 30 6 10 a
Increases in peak [Ca21]i were measured after stimulation with 5HT (1025 M) and liposomes filled with AngII (10 M) either in the absence or presence of losartan (1026 M) or PD123319 (1026 M). n $ 6 in each case. a) different from L control 1 5HT (P,0.05). b) different from L AngII 1 5HT (P,0.05). 26
Discussion Effects of AngIIi can be mediated via mechanisms atypical for plasma membrane AngII receptors in various cell types [9–13]. This implies that patho-physiological actions of AngII might occur intracellularly and therefore not accessible to classical pharmacological agents working at the plasma membrane. Many studies described crosstalk between components of different receptor classes and at different levels downstream receptor activation [23–25]. However, the novelty of the present work is to show interactions between an agonist (AngII) and heterologous receptor stimulation not initiated by AngII at the plasma membrane level, but by intracellular receptor stimulation. In this study we have used A7r5 cells, which do not have functional responses after stimulation extracellular AngII, as concluded from the results on Ins(1,4,5)P3 formation and [Ca21]i in this study and other experiments [13, 26]. Therefore interference of the results obtained by AngIIi on Ca21 homeostasis with possible effects induced by plasma membrane AT-receptors receptor is excluded. AngIIi potently reduced the Ca21 signal evoked by 5HT and UTP stimulation. In view of the experiments performed with the antagonist losartan and PD123319, it is suggested that this effect of AngIIi be mediated by an AT1-like receptor. The ineffectiveness of AngI indicates that it might be different from common plasma membrane AT1-receptors [25]. We have recently shown that AngIIi increase [Ca21]i in non-stimulated A7r5 cells [27]. These effects were insensitive to both losartan and PD123319, suggesting that multiple intracellular AngII receptor types be coupled to different signal transduction mechanisms in A7r5 cells. Reductions of Ca21 increases induced by AngIIi are the not the consequence of inhibition of Ca21 release from intracellular stores, but can be totally attributed to inhibition of Ca21 influx. The parallel effect of AngIIi and verapamil indicates that the L-type Ca21 channel might be the target for this effect. This novel finding of the AngIIi induced inhibition of heterologous receptor activated Ca21 influx can be supported by the observations made in rat cardiomyocytes showing reduction in amplitude of inward voltage-dependent Ca21 current after dialysing AngII into the cell [26]. Increases in [Ca21]i by AngIIi occurred through voltage independent Ca21 channels in non-stimulated A7r5 cells, indicating either the presence of a different receptor or a different coupling mechanism downstream receptor activation under these conditions. In analogy, we have recently shown that AngIIi stimulation of cell growth in
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Fig. 3. Differential effects of intracellular AngII on Ca21 release and Ca21 influx. Changes in intracellular Ca21 release and Ca21 influx were measured by single cell fura2 fluorescence after stimulation with extracellular 5HT (1025 M) under Ca21 free conditions and after subsequent restoration of the normal [Ca21] in the buffer solution. Left panel: tracing a) control, b) in the presence of verapamil (verap, 1026 M, 5 min pre-incubation), c) in the presence of liposomes (5 min pre-incubation) filled with 1026 M AngII (LAngII), Right panel: maximal Ca21 increase. Open bars: Ca21 release, hatched bars: Ca21 influx. Data are presented as mean 6 S.D. (n56). * P,0.05 vs control.
A7r5 cells is mediated by two receptor subtypes, one with high affinity for AngIIi, partially sensitive to losartan and PD123319 and a second one with low affinity for AngIIi and insensitive to both antagonists [13]. Inhibition of heterologous receptor activated Ca21 influx might be of physiological relevance in the process of fine-tuning of multiple receptor signaling pathways. Also over stimulation due to exposure to different mediators can be avoided by such a mechanism, particular beneficial under patho-physiological conditions in which Ca21 overload is implicated like e.g. ischemia. For such a negative protective feedback mechanism availability of AngIIi is essential. It is likely that AngIIi can fulfil this role, since intracellular pools of AngII were observed in cardiomyocytes [5], renal endosomes [28] and lymphoctes [29] under physiological conditions. It is not clear at present if AngII can be produced intracellularly, or if these depos-
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