Intracellular angiotensin II activates rat myometrium

Abstract

Angiotensin II is a modulator of myometrial activity; both AT₁ and AT₂ receptors are expressed in myometrium. Since in other tissues angiotensin II has been reported to activate intracellular receptors, we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrium, using calcium imaging. Intracellular injection of angiotensin II increased cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT₁ receptor antagonist losartan but not by the AT₂ receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca²⁺]_i. Blockade of AT₁ receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca²⁺]_i produced by intracellular injection of angiotensin II; this indicates that microautophagy is a critical step in transporting the peptide into the endo-lysosomes lumenum. The response to angiotensin II was slightly reduced in Ca²⁺-free saline, indicating a major involvement of Ca²⁺ release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP₃) receptors with heparin and xestospongin C or inhibition of phospholipase C (PLC) with U-73122 abolished the response to angiotensin II, supporting the involvement of PLC-IP₃ pathway. Angiotensin II-induced increase in [Ca²⁺]_i was slightly reduced by antagonism of ryanodine receptors. Taken together, our results indicate for the first time that in myometrial cells, intracellular angiotensin II activates AT₁-like receptors on lysosomes and activates PLC-IP₃-dependent Ca²⁺ release from endoplasmic reticulum; the response is further augmented by a Ca²⁺-induced Ca²⁺ release mechanism via ryanodine receptors activation.

angiotensin ii is an eight amino acid peptide that exerts its actions by activation of G protein-coupled receptors, namely AT₁ and AT₂ receptors. Increasing evidence suggests that angiotensin II acts not only as an endocrine/paracrine factor, but also as an autacoid or intracrine peptide, with the ability to signal from within the cell, without stimulating plasma membrane receptors (21, 38). We previously reported that intracellular administration of angiotensin II via liposomes increases contractility of rat aorta via activation of intracellular receptors (7). Similarly, microinjection of angiotensin II increases cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in vascular smooth muscle cells (13, 16) or kidney proximal tubule cells (49). The intracellular angiotensin II can result either via uptake from the interstitium or by intracellular synthesis. In the latter case, intracellular renin converts angiotensinogen to angiotensin I, which is further converted to the active angiotensin II by chymase or by the angiotensin-converting enzyme, both of which are present intracellularly (21). The intracellular renin-angiotensin system has been identified in the heart (11), kidney (18, 19, 34, 48), and uterus (37).¹

Autoradiography and binding studies reveal that the uterus expressed high levels of angiotensin II receptors; both AT₁ and AT₂ receptors are present in the uterus. The prevalence of myometrial AT₁ versus AT₂ receptors varies in different species and physiological states (42, 44). Extracellular angiotensin II induces dose-dependent contractions of the myometrium (12, 32, 44). This effect is sensitive to the AT₁ blocker losartan and depends on the estrous cycle (1, 47). Since an increase in [Ca²⁺]_i is essential in uterine contractility, in this study we assessed the effects of intracellular injection of angiotensin II on [Ca²⁺]_i using calcium imaging.

MATERIALS AND METHODS

Primary culture of myometrial cells.

Primary myometrial cells were prepared from the rat uterus according to the procedure described by Krizsan-Agbas et al. (20) with some modifications. Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee. Adult (nonpregnant) female Sprague-Dawley rats (Ace Animals, Boyertown, PA) were anesthetized with urethane (1.2 g/kg ip); uterine horns were immediately excised and immersed in cold Hanks balanced salt solution (HBSS). The myometrium was rapidly separated from endometrium and stroma and then minced into fragments (about 1 mm³). Tissues were digested for 1.5 h at 37°C by collagenase type I (600 U/ml, Sigma-Aldrich, St. Louis, MO) in DMEM culture medium, followed by trituration with a sterile Pasteur pipette and filtration through a cell strainer with 70-μm pores. After collection, the cells were washed twice by centrifugation (1,000 RPM, 10 min) and resuspended in fresh DMEM medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂; the medium was changed every 2–3 days.

Calcium imaging.

Measurements of intracellular Ca²⁺ concentrations were performed as previously described (8). Briefly, cells were incubated with 5 μM fura-2 AM (Invitrogen, Carlsbad, CA) in HBSS at room temperature for 45 min, in the dark, washed three times with dye-free HBSS, and then incubated for another 45 min to allow for complete deesterification of the dye. Coverslips (25 mm diameter) were subsequently mounted in an open bath chamber (RP-40LP, Warner Instruments, Hamden, CT) on the stage of an inverted microscope Nikon Eclipse TiE (Optical Apparatus, Ardmore, PA). The microscope was equipped with a Perfect Focus System and a Photometrics CoolSnap HQ2 CCD camera (Roper Scientific, Optical Apparatus). During the experiments the Perfect Focus System was activated. Fura-2 AM fluorescence (emission = 510 nm), following alternate excitation at 340 and 380 nm, was acquired at a frequency of 0.25 Hz. Images were acquired and analyzed using NIS-Elements AR 3.1 software (Nikon/Optical Apparatus). The ratio of the fluorescence signals (340/380 nm) was converted to Ca²⁺ concentrations (14). In Ca²⁺-free experiments, CaCl₂ was omitted and 2.5 mM EGTA was added.

Intracellular microinjection.

Injections were performed using Femtotips II, InjectMan NI2, and FemtoJet systems (Eppendorf) as previously reported (8). Pipettes were back filled with an intracellular solution composed of 110 mM KCl, 10 mM NaCl, and 20 mM HEPES (pH 7.2) (14) or angiotensin II. The injection time was 0.4 s at 60 hPa with a compensation pressure of 20 hPa to have <0.1% of cell volume microinjected (14). Blockade of AT₁ receptors internalization was done by cells pretreatment for 5 h with phenylarsine oxide (PAO, 30 μM), an agent that is known to inhibit the AT₁ internalization in rat myometrium (24). Blockade of microautophagy was done by preincubating the cells for 1 h with 30 μM rapamycin (22).

Statistical analysis.

One-way ANOVA or two-way ANOVA were used; a P value of <0.05 was considered statistically significant.

RESULTS

Intracellular injection of angiotensin II increases [Ca²⁺]_i in myometrial cells.

The mean basal [Ca²⁺]_i of rat myometrial cells was 67 ± 1.5 nM (n = 154 cells). Intracellular microinjection of control buffer produced a small but not significant increase in [Ca²⁺]_i by 19 ± 3 nM (n = 6 cells) (Fig. 1, A, C, D). Microinjection of angiotensin II (10⁻⁹ M, 10⁻⁸ M, and 10⁻⁷ M final intracellular concentrations) produced a robust and transitory increase in [Ca²⁺]_i by 131 ± 12 nM, 382 ± 17 nM, and 865 ± 44 nM, respectively (n = 6 for each concentration tested) (Fig. 1, B–D).

Fig. 1.

Intracellular administration of ANG II produced a dose-dependent increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in rat myometrial cells. A and B: fluorescent images of fura-2 AM-loaded myometrial cells illustrating the levels of basal [Ca²⁺]_i (left) during injection of control buffer or ANG II (10⁻⁷ M, middle) and 6 min after injection (right). The fluorescence intensity scale (0–4) is illustrated on each panel and magnified in A (left). C: intracellular administration of ANG II (10⁻⁹, 10⁻⁸, and 10⁻⁷ M) produced a fast, robust and transitory increase in [Ca²⁺]_i; averaged traces from 6 experiments are shown. D: comparison of the increase in [Ca²⁺]_i produced by control injection and ANG II; ANG II (10⁻⁹, 10⁻⁸, and 10⁻⁷ M, intracellular concentrations) produced a dose-dependent increase in [Ca²⁺]_i. *P < 0.05 compared with control.

Angiotensin II activates intracellular AT₁ receptors in myometrial cells.

Since both AT₁ and AT₂ receptors are present in the myometrium, the next series of experiments was designed to identify which type of receptor mediates the Ca²⁺-mobilizing effects of angiotensin II. Pretreatment with and concomitant injection of losartan (1 μM, 15 min), an AT₁ receptor antagonist, abolished the effect of angiotensin II on [Ca²⁺]_i, (Fig. 2, B, E, F). Coinjection of saralasin (1 μM final concentration inside the cells) with angiotensin II largely reduced the angiotensin II-evoked response; in the presence of saralasin, angiotensin II increased [Ca²⁺]_i only by 127 ± 6 nM (Fig. 2, D–F). Pretreatment with and coinjection of PD-123319 (1 μM, 15 min), an AT₂ receptor antagonist, did not significantly affect the response to angiotensin II. In the presence of PD-123319, intracellular injection of angiotensin II increased [Ca²⁺]_i by 836 ± 61 nM versus 865 ± 44 nM, in the absence of PD-123319 (Fig. 2, C–E).

Fig. 2.

ANG II activates intracellular AT₁ receptors in myometrial cells. A–D: fluorescent images of fura-2 AM-loaded myometrial cells illustrating the levels of basal [Ca²⁺]_i (left) during microinjection of ANG II (10⁻⁷ M, middle), alone (A), or in the presence of losartan (B), PD-123319 (C), or saralasin (D), and 6 min after injection (right). The fluorescence intensity scale (0–4) is illustrated on each panel and magnified in A (left). E: averaged traces from 6 experiments indicating the increase in [Ca²⁺]_i produced by ANG II alone or in the presence of AT₁ receptor antagonist, losartan, AT₂ receptor antagonist (PD 123319), or of AT₁ and AT₂ receptors antagonist, saralasin; losartan and saralasin abolished the increase in [Ca²⁺]_i produced by injection of ANG II. F: comparison of the increase in [Ca²⁺]_i produced by ANG II in the absence and presence of losartan, PD-123319, and saralasin. *P < 0.05 compared with ANG II alone.

Intracellular AT₁ receptors are located on endo-lysosomes.

Pretreatment with bafilomycin A1 (1 μM, 1 h), a V-type ATPase inhibitor (6), abolished the Ca²⁺ response to angiotensin II (Fig. 3, B, F, G) (n = 6). In cells pretreated with brefeldin A (10 μM, 1 h), which disrupts Golgi apparatus, angiotensin II (10⁻⁷ M) increased [Ca²⁺]_i by 827 ± 76 nM (n = 6) (versus 865 ± 44 nM produced angiotensin II alone) (Fig. 3, C, F, G). Blockade of AT₁ receptor endocytosis with PAO (30 μM) had no significant effect on intracellular angiotensin II-evoked [Ca²⁺]_i increase (798 ± 28 vs. 865 ± 44 nM; Fig. 3, D, F, G). In contrast, the blockade of microautophagy with rapamycin (30 μM) abolished the elevation in [Ca²⁺]_i induced by intracellular injection of angiotensin II (Fig. 3, E–G).

Fig. 3.

Intracellular AT₁ receptors are located in lysosomes in rat myometrial cells. A–E: fluorescent images of fura-2 AM-loaded myometrial cells illustrating the levels of basal [Ca²⁺]_i (left) during microinjection of ANG II (10⁻⁷ M, middle) alone (A) or in cells pretreated with bafilomycin A1 (B), brefeldin A (C), phenylarsine oxide (PAO, D), or rapamycin (E) and 6 min after injection (right). The fluorescence intensity scale (0–4) is illustrated on each panel and magnified in A (left). F: pretreatment with bafilomycin A1 (1 μM, 1 h), or rapamycin (30 μM, 1 h), but not with brefeldin A (10 μM, 1 h), or PAO (30 μM, 5 h) prevented the ANG II-induced increase in [Ca²⁺]_i; averaged traces from 6 experiments for each treatment are shown. G: comparison of the increase in [Ca²⁺]_i produced by ANG II in the absence and presence of brefeldin A, bafilomycin A1, PAO, or rapamycin. *P < 0.05 compared with ANG II alone.

Angiotensin II mobilizes Ca²⁺ from endoplasmic reticulum pool.

In Ca²⁺-free EGTA (2.5 mM)-containing HBSS, intracellular injection of angiotensin II produced a fast and transitory increase in [Ca²⁺]_i by 609 ± 27 nM (n = 6). The response was insensitive to the blockade of NAADP-signaling by Ned-19 (5 μM, 15 min) (35) (Fig. 4A). The response to angiotensin II was markedly reduced by blocking IP₃ receptors with xestospongin C and heparin (48 ± 12 nM, n = 6, Fig. 4A) or by inhibiting PLC by pretreatment with U-73122; pretreatment with the inactive derivative U-73343 did not affect the response (Fig. 4A), supporting the PLC specificity of the response. Pretreatment with ryanodine (1 μM, 15 min) slightly reduced the response (438 ± 21 nM, n = 6, Fig. 4A). These results indicate the major involvement of IP₃-dependent Ca²⁺ pathways and a participation of Ca²⁺-induced Ca²⁺ release mechanism.

Fig. 4.

ANG II-induced Ca²⁺ mobilization involves release of Ca²⁺ from endoplasmic reticulum (ER). A: averaged responses (n = 6) to intracellular injection of ANG II in the presence of the mentioned antagonists of lysosomal and ER Ca²⁺ release; experiments were carried out in Ca²⁺-free saline (0 Ca²⁺). The response to ANG II was not affected by blocking NAADP signaling with Ned-19 or the inactive derivative of PLC inhibitor U-73343; markedly reduced by inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) antagonists (xestospongin C, XeC, and heparin) or phospholipase C (PLC) inhibitor U-73122, slightly reduced by pretreatment with the ryanodine receptor antagonist (ryanodine, Ry). B: comparison of the amplitude of the [Ca²⁺]_i increases produced by intracellular injection of ANG II in the presence of the mentioned antagonists. *P < 0.05 compared with ANG II (10⁻⁷ M); **P < 0.05 compared with ANG II and Ned-19 and ANG II and xestospongin C and heparin or ANG II and Ned-19.

DISCUSSION

Previous studies indicate that angiotensin II induces depolarization and stimulates the contractility of nonpregnant rat myometrium (31). Increase of [Ca²⁺]_i and stimulation of myosin light chain kinase via Ca²⁺-calmodulin is essential for myometrial contraction. Since in other tissues such as vasculature and kidney angiotensin II has been reported to activate intracellular receptors (7, 13, 16, 49), we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrial cells, using calcium imaging. The first important observation of our study is that intracellular application of angiotensin II (10⁻⁹ to 10⁻⁷ M) resulted in dose-dependent increments in [Ca²⁺]_i in rat myometrial cells. Intracellular AT₂ receptor blockade by PD-123319 did not affect the angiotensin II-induced increase in [Ca²⁺]_i, whereas losartan, an AT₁ receptor antagonist, or saralasin (nonspecific AT₁ and AT₂ antagonist) abolished it. These results indicate that the observed response is dependent on AT₁ receptors located inside the cell and support an intracrine role for angiotensin II in the myometrium. This finding is consistent with previous reports that describe [Ca²⁺]_i elevation upon intracellular stimulation of angiotensin II receptors in renal proximal tubule cells (49) or vascular smooth muscle cells (16).

It is not unusual that a G protein-coupled receptor (GPCR) signals from within the cell. Upon internalization, some GPCRs have been shown to direct a β-arrestin-mediated activation of the mitogen-activated protein kinase cascade, which appears to happen selectively on the endosomes (26). Intracellular GPCRs are either located at the Golgi, where they undergo modifications that will further allow insertion and functionality at the plasma membrane, or targeted at the endo-lysosomal compartment upon internalization. The AT_1A receptor is known to remain associated with β-arrestin upon endocytosis (2, 47), being further trafficked to cytosolic vesicular structures; however, it is neither dephosphorylated nor efficiently recycled back to the cell surface (2). In our experiments, angiotensin II-induced Ca²⁺ response was not affected by brefeldin A, a Golgi apparatus disruptor (4); however, pretreatment with bafilomycin A₁, a V-type ATPase inhibitor that prevents endo-lysosomal acidification (6), abolished the response. These results indicate that functional intracellular angiotensin II receptors are located at endo-lysosomal level. Endosomes have previously been implicated as important sites for receptor-initiated signaling, as they contain a wide range of signaling molecules in their membranes and are able to recruit scaffolding proteins and signaling mediators (40, 43, 46). Apparently this is an intriguing observation because in endo-lysosomal system the NH₂-terminal of receptors (binding site) is located in the lumenum and COOH-terminal is located in the cytoplasmic side. Moreover, saralasin, a nonspecific AT₁ and AT₂ blocker with peptidic structure largely reduced elevation of [Ca²⁺]_i produced by intracellular injection of angiotensin II. These results suggest that the NH₂-terminal of endo-lysosomal AT₁ receptors are translocated, and both NH₂- and COOH-terminal are on cytoplasmic side or both peptides (angiotensin II and saralasin) are transported into the endo-lysosomal lumenum. In our opinion, the translocation of NH₂-terminal to the cytoplasmic side was very unlikely, and next step was to identify how those peptides are transported inside the endo-lysosomes.

Microautophagy is a process that transfers cytoplasmic components such as old or unfolded proteins into endo-lysosomes by direct invagination of endo-lysosomal membranes followed by scission of the vesicle (for review see Ref. 9). The invaginations phase occurs with a lag period of 20–30 min (41); meanwhile, the second phase is a very fast process (22). Using a luciferase assay, Kunz et al. (22) reported that after the invaginations are formed, 40% of the uptake occurred and luciferase was transported into the lysosomes even after an incubation of only 2 min. Viewed in this context, the intracellular injected angiotensin II or saralasin could be rapidly transported into endo-lysosomes via ongoing microautophagy. The fact that rapamycin, which blocks microautophagy (22), abolished the angiotensin II-induced increase in [Ca²⁺]_i suggests that this process is a key step in transporting the peptides inside the endo-lysosomes.

Next, we characterized the calcium pathways and pools involved in the response to intracellular angiotensin II in the rat myometrium. In the absence of extracellular Ca²⁺, the response to angiotensin II is only slightly decreased indicating a major involvement of Ca²⁺ release from internal stores. Intracellular organelles reportedly involved in Ca²⁺ release include the endoplasmic reticulum (ER) and acidic Ca²⁺ stores such as lysosomes (3, 36). Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca²⁺ from lysosomes (25, 36). Pretreatement with Ned-19, an inhibitor of NAADP signaling (35) did not affect angiotensin II-induced Ca²⁺ response. In contrast, xestospongin C and heparin that prevent IP₃R-mediated Ca²⁺ release from the ER, significantly decreased, angiotensin II-induced response.

AT₁ receptors are known to couple with G_q/11 proteins that can further activate various signal transduction mediators, including phospholipase C (PLC) (45). PLC has been identified in lysosomal membranes (30, 39), thus we hypothesized it is activated following angiotensin II stimulation of AT₁ receptors at this site. Accordingly, we tested the effect PLC inhibitor U-73122 (5) or its negative control U-73343 on angiotensin II response. Pretreatment with U-73122, but not with U-73343, abolished angiotensin II-induced increase in [Ca²⁺]_i, thereby confirming that PLC activation is a required step for ER release of Ca²⁺ following intracellular AT₁ receptor activation.

Antagonism of ryanodine receptors, another type of Ca²⁺ release channels located on the ER, slightly reduced the response to angiotensin II; this indicates that Ca²⁺ release via Ca²⁺-induced Ca²⁺ release (CICR) mechanism may amplify the IP₃-dependent Ca²⁺ release. In summary, our findings indicate that intracellular angiotensin II transferred to lysosomes via microautophagy, activates AT₁-like receptors located on lysosomes, which in turn stimulates lysosomal PLC to hydrolyze phosphatidylinositol-(4,5)-bisphosphate (PIP₂) and form IP₃. IP₃ activates IP₃R to release Ca²⁺ from the ER; CICR may further increase [Ca²⁺]_i levels via ryanodine receptor activation (Fig. 5). A similar mechanism has been demonstrated in renal proximal tubule cells (12, 23, 32, 44, 49) and is consistent with the accepted contractile effect of angiotensin II on the myometrium, which involves AT₁ receptor activation and elevation of [Ca²⁺]_i (23, 28, 32).

Fig. 5.

Proposed mode of action of ANG II via activation of intracellular AT₁ receptors. Intracellular ANG II transferred into the endo-lysosomes (Lys) via microautophagy acts on AT₁ receptors situated on acid-filled Ca²⁺ stores and activates lysosomal PLC followed by production of IP₃ from phosphatidylinositol 4,5-bisphosphate (PIP₂). IP₃ activates IP₃R on ER, increasing [Ca²⁺]_i. The increase in [Ca²⁺]_i may activate ryanodine receptors (RyR) and further potentiates Ca²⁺ release from the ER via a Ca²⁺-induced Ca²⁺ release (CICR) mechanism.

Although the presence of a local renin-angiotensin system (RAS) in the uterus has been suggested, the significance of an uterine RAS is not clear. It appears that in pregnant women (29) and ewes (33) plasma angiotensin II levels present a fivefold increase compared with nonpregnant controls, suggesting a possibility that angiotensin II receptors may suffer internalization. Important differences in levels of expression of AT₁ and AT₂ receptors and reactivity to angiotensin II were reported in the myometrium of pregnant versus nonpregnant women (10). Whether or not the location of functional angiotensin II receptors plays a role in these differences it is not known. While most of evidence for functional intracellular angiotensin II receptors is provided by in vitro, or ex vivo studies, very recently, an elegant study using an innovative approach established the physiological in vivo effects of cytoplasmic angiotensin II-AT₁ interaction on proximal tubule epithelial function (27). The authors used adenoviral transfer of intracellular cyan fluorescent fusion of angiotensin II selectively in proximal tubules and provided evidence that trapping angiotensin II intracellulary increased the blood pressure in rats and mice (46). This novel approach can distinguish the intracrine actions of angiotensin II in the physiological responses in an intact animal model (17).

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant RO1HL-90804 (to E. Brailoiu).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).