Intracellular Endothelin Type B Receptor-driven Ca2+ Signal Elicits Nitric Oxide Production in Endothelial Cells*

Background: The intracrine nature of endothelin-1 is largely correlated with nuclear signaling events. Results: In endothelial cells, endothelin-1 acting on endolysosomal ETB receptors increases cytosolic Ca2+ and nitric oxide. Conclusion: Endolysosomal ETB receptors are functional. Significance: We identify a new pathway for ET-1-induced intracrine signaling and provide the first evidence that intracellular G protein-coupled receptors are involved in redox signaling. Endothelin-1 exerts its actions via activation of ETA and ETB Gq/11 protein-coupled receptors, located in the plasmalemma, cytoplasm, and nucleus. Although the autocrine/paracrine nature of endothelin-1 signaling has been extensively studied, its intracrine role has been largely attributed to interaction with receptors located on nuclear membranes and the nucleoplasm. Because ETB receptors have been shown to be targeted to endolysosomes, we used intracellular microinjection and concurrent imaging methods to test their involvement in Ca2+ signaling and subsequential NO production. We provide evidence that microinjected endothelin-1 produces a dose-dependent elevation in cytosolic calcium concentration in ETB-transfected cells and endothelial cells; this response is sensitive to ETB but not ETA receptor blockade. In endothelial cells, the endothelin-1-induced Ca2+ response is abolished upon endolysosomal but not Golgi disruption. Moreover, the effect is prevented by inhibition of microautophagy and is sensitive to inhibitors of the phospholipase C and inositol 1,4,5-trisphosphate receptor. Furthermore, intracellular endothelin-1 increases nitric oxide via an ETB-dependent mechanism. Our results indicate for the first time that intracellular endothelin-1 activates endolysosomal ETB receptors and increase cytosolic Ca2+ and nitric oxide production. Endothelin-1 acts in an intracrine fashion on endolysosomal ETB to induce nitric oxide formation, thus modulating endothelial function.


Endothelin-1 exerts its actions via activation of ET A and ET B
G q/11 protein-coupled receptors, located in the plasmalemma, cytoplasm, and nucleus. Although the autocrine/paracrine nature of endothelin-1 signaling has been extensively studied, its intracrine role has been largely attributed to interaction with receptors located on nuclear membranes and the nucleoplasm. Because ET B receptors have been shown to be targeted to endolysosomes, we used intracellular microinjection and concurrent imaging methods to test their involvement in Ca 2؉ signaling and subsequential NO production. We provide evidence that microinjected endothelin-1 produces a dose-dependent elevation in cytosolic calcium concentration in ET B -transfected cells and endothelial cells; this response is sensitive to ET B but not ET A receptor blockade. In endothelial cells, the endothelin-1-induced Ca 2؉ response is abolished upon endolysosomal but not Golgi disruption. Moreover, the effect is prevented by inhibition of microautophagy and is sensitive to inhibitors of the phospholipase C and inositol 1,4,5-trisphosphate receptor. Furthermore, intracellular endothelin-1 increases nitric oxide via an ET B -dependent mechanism. Our results indicate for the first time that intracellular endothelin-1 activates endolysosomal ET B receptors and increase cytosolic Ca 2؉ and nitric oxide production. Endothelin-1 acts in an intracrine fashion on endolysosomal ET B to induce nitric oxide formation, thus modulating endothelial function.
Compelling evidence indicates that in addition to the classical localization and signaling at the plasma membrane, a wide variety of G protein-coupled receptors (GPCRs) 3 are expressed and fully functional intracellularly (1)(2)(3). Endothelin-1 (ET-1) acts on ET A and ET B receptors, G q/11 -coupled GPCRs, exerting opposing effects on vascular functions. In the cardiovascular human and rodent cells, ET A and ET B are localized to the plasma membrane, cytoplasm, nuclear membranes, and nucleoplasm (4 -6). ET-1 elevates both cytosolic and nuclear Ca 2ϩ concentrations; the rise in cysotolic Ca 2ϩ concentration, [Ca 2ϩ ] i , in response to ET-1 is seen in both intact and membrane-perforated cells (5). Moreover, ET-1 is involved in nitric oxide (NO) and reactive oxygen species generation both in the cytosol and in the nucleus (5).
Although the stimulation of nuclear membrane receptors elicits events largely restricted to the nucleus (3,5), receptors located on the membrane of other organelles trigger signaling cascades within the cytoplasm, resulting in rapid cell responses. Because ET B receptors were identified in the endolysosomal system (7)(8)(9), in this study, we examined whether or not endolysosomal ET B receptors are involved in Ca 2ϩ signaling and NO production in rat pulmonary microvascular endothelial cells (RPMVEC), which express ET B receptors (10 -12).
Calcium Imaging-Measurements of [Ca 2ϩ ] i were performed as described previously (13,14). Cells were incubated with 5 M Fura-2 AM (Invitrogen) in Hanks' balanced salt solution at room temperature for 45 min, in the dark, washed three times with dye-free Hanks' balanced salt solution, and then incubated for another 45 min to allow for complete de-esterification 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 (Nikon, Inc., Melville, NY). The microscope is equipped with a Perfect Focus System and a Photometrics CoolSnap HQ2 CCD camera (Photometrics, Tucson, AZ). 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 software (version 3.1, Nikon, Inc.). The ratio of the fluorescence signals (340/380 nm) was converted to Ca 2ϩ concentrations (15).
Intracellular Microinjection-Injections were performed using Femtotips II, InjectMan NI2, and FemtoJet systems (Eppendorf) as reported previously (16 -19). Pipettes were back filled with an intracellular solution composed of 110 mM KCl, 10 mM NaCl, and 20 mM HEPES (pH 7.2) (20) or the specific chemicals. The injection time was 0.4 s at 60 hectoPascal with a compensation pressure of 20 hPa to maintain the microinjected volume to Ͻ1% of cell volume, as measured by microinjection of a fluorescent compound (Fura-2-free acid) (20). The intracellular concentration of chemicals was determined based on the concentration in the pipette and the volume of injection. The cellular volume was estimated to 1000 m 3 (21).
Measurement of NO Levels-Intracellular NO was monitored with DAF-FM (4-amino-5-methylamino-2Ј,7Ј-difluorofluorescein, Invitrogen), a pH-insensitive fluorescent dye, as described previously (22). Cells were incubated at room temperature for 45 min in Hanks' balanced salt solution containing a low concentration (0.5 M) of DAF-FM. This condition significantly reduced the background autofluorescence and improved the signal-to-noise ratio of NO detection in single cells. After load-ing, cells were rinsed three times with saline. NO fluorescence was measured at a rate of 0.1 Hz using excitation/emission wavelengths of 488 nm/540 nm.

DISCUSSION
ET-1, first identified as an endothelium-derived vasoconstrictor peptide, is an autocrine and paracrine signaling factor with extensive modulatory effects on vascular function (29). ET-1 acts also as an intracrine, activating intracellular cognate receptors (2). Stimulation of ET-1 receptors at the plasma or nuclear membrane induces Ca 2ϩ release, NO, and reactive oxygen species formation in the cytosol or nucleus (5,30). Cardiovascular disease pathogenesis involves oxidative stress and further alteration of ET-1 and NO signaling pathways (30, 31). ET-1 immunoreactivity is expressed within the cytoplasm on the membranes of the endoplasmic reticulum (ER), mitochondria, and cytosolic vesicles of endothelial cells from human (4) and various rodent species, including rats (6). Moreover, the components responsible for ET-1 generation are present intracellularly in endothelial cells, as well as in other cardiovascular cells (6,32,33). These findings suggest that ET-1 is available within the cytoplasm to activate its intracellular receptors.
We and others (17, 34 -36) have previously reported that stimulation of intracellular GPCRs such as angiotensin II AT 1 receptors, CB 1 , and CB 2 cannabinoid receptors (16,37,38), or estrogen receptor GPER/GPR30 (18,19,39) leads to [Ca 2ϩ ] i elevation. Using a similar approach in this study, we tested the hypothesis that ET-1 may act in an intracrine fashion on intra-cellular ET B receptors to modulate endothelial functions. Using imaging methods and concurrent intracellular injection of ET-1, we provide the first evidence of functionality of intracellular ET B receptors in cells transiently transfected with the receptor. Because ET-1 is an endothelium-derived peptide, and ET B is the predominant type of endothelin receptor in endothelial cells, the next series of experiments were designed to evaluate whether intracellular ET B receptors are functional in RPMVEC. Similar to ET B -transfected cells, RPMVEC responded to ET-1 microinjection with a dose-dependent increase in [Ca 2ϩ ] i . Blocking intracellular ET B receptors abolished the effect of ET-1, whereas ET A antagonism did not affect it.
Interestingly, a previous study demonstrating cytoplasmic distribution of both ET A and ET B in aortic human vascular  endothelial cells also showed that the responses initiated by ET-1 at the plasma membrane are not dependent on plasmalemmal ET B but on ET A (4). According to the present study, the reverse is true in RPMVEC where the effects of microinjected ET-1 are mediated through intracellular ET B receptors. Our findings are particularly relevant considering the ability of endothelial cells to synthesize ET-1 (40 -42) and make it available within the cytoplasm (32,33,40) to activate its intracellular targets. Importantly, a majority of the responses elicited by ET-1 in the endothelium, including the release of vasorelaxant factors such as NO, prostacyclin, and endothelium-derived hyperpolarizing factor are ET B receptor-dependent (11).
We further examined the intracellular location of functional ET B receptors in endothelial cells. Previous studies have identified that ET B , similar to other GPCRs, may be targeted to the endolysosomes (7-9) or Golgi apparatus (43). Disruption of the Golgi apparatus did not affect the response of endothelial cells to intracellular ET-1, whereas inhibition of lysosomal acidification (25) completely abolished it. Our results indicate that degradation is not the only fate of endolysosomal ET B ; they are also functional and involved in ET-1 signaling. This may correlate with the remarkable stability of ET B receptors in these organelles (44).
In addition to playing a role in cellular degradation, increasing evidence supports lysosomes as key regulators of cell home-  ostasis (45) and as platforms for continued receptor-mediated signaling (1). Similar to the plasma membrane, the membrane of the endocytic vesicles is organized into specialized domains, working as a platform for the assembly of specific signaling complexes; these features allow the endolysosomal targeted receptors to initiate signaling from this intracellular compartment (46). Accordingly, various types of receptors, including GPCRs, have been reported to trigger signal transduction pathways upon their endolysosomal activation (1,47). However, the ET-1 binding pocket on the ET B receptor is located on the N-terminal side, thus within the lysosomal lumen (11). We have previously demonstrated that angiotensin II is transferred inside the endolysosomal vesicles via microautophagy (17), a process in which soluble cytosolic molecules are engulfed (48). Thus, we further tested whether a similar mechanism was applicable to endothelin. Indeed, rapamycin, an inhibitor of the final uptake reaction in the microautophagic process (26), prevented the cellular responses to microinjected endothelin-1. Two major events may be delimited in the microautophagic process: lysosomal membrane invagination/formation of autophagic tubes and vesicle scission (48). Although the former may occur with a 30-min lag, the latter is very rapid, occurring in seconds (26). Given that microautophagy is an ongoing process, important in housekeeping and in the maintenance of cytoplasmic mass (48), membrane invaginations are formed continuously and cytosolic components may be rapidly uptaken into the endolysosomal lumen. Indeed, activation of lysosomal ET B receptors occurred readily in response to microinjected ET-1.
Next, we defined the Ca 2ϩ pools mobilized by ET-1 in endothelial cells. The ET-1-induced rise in [Ca 2ϩ ] i was not modified in Ca 2ϩ -free saline, suggesting that Ca 2ϩ release, but not Ca 2ϩ influx, is the main mechanism underlying the ET-1-induced effect. The response to ET-1 was not affected by preventing lysosomal Ca 2ϩ release with the two-pore channel blocker Ned-19 (28,49); thus, despite their localization, activation of ET B receptors does not mobilize Ca 2ϩ via two-pore channel activation. Conversely, the effect of intracellular ET-1 was sensitive to IP 3 R but not ryanodine receptor blockade, pointing to the IP 3 -responsive stores as the major Ca 2ϩ pool mobilized upon activation of lysosomal ET B . It is largely accepted that the IP 3 Rs are located on the endoplasmic reticulum. However, it has been shown that IP 3 R subtypes 2 and 3 are located on acidfilled Ca 2ϩ stores (50). Based on these findings, we cannot exclude the participation of both types of Ca 2ϩ pools. Moreover, we found that the phospholipase C inhibitor U-73122 abolished the effect of ET-1; this is not surprising, given that ET B receptors couple to G q/11 (11) and that phospholipase C is present in the lysosomal membrane (51). Immunocytochemical experiments confirmed the endolysosomal location of ET B receptors.
To further probe for the physiological relevance of the Ca 2ϩ release in response to activation of endolysosomal ET B receptors, we examined the effect of microinjected endothelin on NO production. In endothelial cells, an elevation of [Ca 2ϩ ] i results in activation of endothelial NO synthase via Ca 2ϩ /calmodulin binding (52,53) and endothelial NO synthase phosphorylation (54), which leads to the release of the vasorelaxant mediator NO. Indeed, intracellular administration of ET-1 resulted in NO release, an effect that was completely contingent on endothelial NO synthase. Our results also indicate that microautophagy and ET B activation are critical steps for NO production in response to intracellular ET-1.
Thus, we propose a new pathway for ET-1-induced intracrine signaling: intracellular ET-1 is transferred to the endolysosomal lumen via microautophagy to trigger ET B -phospholipase C-dependent IP 3 generation. IP 3 further activates specific Ca 2ϩ release channels (IP 3 R) from the endoplasmic reticulum or endolysosomes; [Ca 2ϩ ] i elevation stimulates endothelial NO synthase, resulting in endothelial NO accumulation (Fig. 8). This mode of action of ET-1 is canonical, in that it is associated with its receptor, ET B , is second messenger-dependent, and occurs at a membranous compartment (55,56).
ET-1 released in the circulation is a very potent vasoconstrictor that has been implicated in the pathophysiology of systemic and pulmonary hypertension and in atherosclerosis (57,58). Although ET-1 acts on the vascular smooth muscle to produce vasoconstriction, activation of endothelial ET B receptors promotes vasorelaxation via NO release (59). ET-1 involvement in atherosclerosis is also dual. ET-1 promotes vasoprotection through ET B receptor-dependent generation of NO and reac-  NOVEMBER 30, 2012 • VOLUME 288 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 41029 tive oxygen species in endothelial cells (58). However, ET-1 stimulates vascular smooth muscle cell proliferation, inducing vascular hypertrophy and endothelial dysfunction, effects that are sensitive to ET A receptor blockade (58). Specific trapping of ET-1 to vascular endothelial cells may produce selective activation of lysosomal ET B , Ca 2ϩ mobilization and NO generation; this may shift its overall effects toward vasorelaxation/vasoprotection and prevent its potentially detrimental, vasoconstrictor/proliferative activity on adjacent vascular smooth muscle cells. Endothelial cell-specific ET B knock-out mice exhibit decreased endogenous release of NO and increased plasma endothelin-1 (60). Innovative approaches previously employed to demonstrate that another intracrine, angiotensin II, increases blood pressure upon intracellular trapping at the kidney level (55,61), may also prove useful in the case of ET-1. Should our findings be supported by in vivo data, selective targeting of ET B agonists to the endothelial cytosol may prove therapeutically beneficial in cardiovascular disorders associated with dysfunction of ET-1/NO pathway.

Endolysosomal ET B Receptors Signal through NO
To summarize, this study provides the first evidence that activation of endolysosomal ET B receptors increases cytosolic Ca 2ϩ concentration and nitric oxide production. Furthermore, we show here for the first time that intracellular receptors (namely, ET B ) may be involved in redox signaling. Our study suggests a new mechanism for ET B -mediated endothelium-dependent vasorelaxation and extends the current knowledge on intracrine signaling.