Intracellular endothelin type B receptor-driven Ca2+ signal elicits nitric oxide production in endothelial cells

Abstract

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.

FIGURE 1.

Control experiments. A, averaged Ca²⁺ responses to intracellular administration of endothelin-1 (10⁻¹⁰ m) in untransfected (black) or GFP-transfected (gray) U2OS cells. B, comparison of the amplitudes of ET-1 effects on [Ca²⁺]_i in untransfected and GFP-transfected U2OS control cells. C, averaged Ca²⁺ responses to BQ-123 (ET_A antagonist, 10⁻⁷ m, black) or BQ-788 (ET_B antagonist, 10⁻⁷ m, gray) microinjection in ET_B-GFP-transfected U2OS cells. D, comparison of the Ca²⁺ responses of ET_B-GFP-U2OS cells to intracellular administration of BQ-123 or BQ-788. E, averaged Ca²⁺ responses to BQ-123 (10⁻⁷ m, black) or BQ-788 (10⁻⁷ m, gray) microinjection in rat endothelial cells, endogenously expressing ET_B. F, comparison of the Ca²⁺ responses of endothelial cells to intracellular administration of BQ-123 or BQ-788.

FIGURE 2.

Intracellular administration of endothelin-1 elevates [Ca²⁺]_i in U2OS cells expressing ET_B receptors. A, averaged traces (n = 6) illustrating the Ca²⁺ responses to ET-1 (10⁻¹¹, 10⁻¹⁰, 10⁻⁹ m) and to ET-1 co-injected with the ET_A antagonist BQ-123 (10⁻⁷ m) or with the ET_B antagonist BQ-788 (10⁻⁷ m); arrows indicate the time point of injection. B, comparison of the increases in [Ca²⁺]_i produced by various concentrations of ET-1 in absence and presence of ET_A or ET_B antagonist; p < 0.05 compared with control (*) or to ET-1 10⁻¹⁰ m (#) microinjection. C–F, representative images showing ET_B-GFP fluorescence (first panels) and Fura-2 AM fluorescence ratio (F₃₄₀/F_{380 nm}) before, during and 6 min after microinjection (inj; second, third, and fourth panels, respectively) of control buffer (C), ET-1 (10⁻¹⁰ m) alone (D), or in presence of BQ-123 (E), or BQ-788 (F); arrows indicate the injected cells; the fluorescence scale (0–3) is illustrated in each panel and magnified in the second panel of C.

FIGURE 3.

Endothelin-1 microinjection increases [Ca²⁺]_i in RPMVEC via ET_B receptor activation. A, averaged traces (n = 6) illustrating concentration-dependent effect of ET-1 (10⁻¹¹–10⁻⁹ m) on [Ca²⁺]_i and the Ca²⁺ responses of ET-1 (10⁻¹⁰ m) co-injected with the ET_A antagonist BQ-123 (10⁻⁷ m) or with the ET_B antagonist BQ-788 (10⁻⁷ m); ET_B blockade abolished ET-1 effect. B, comparison of the amplitude of [Ca²⁺]_i elevations produced by various concentrations of ET-1 in absence and presence of ET_A or ET_B antagonist; p < 0.05 compared with control (*) or to ET-1 10⁻¹⁰ m (#) microinjection. C–F, typical fluorescence images of Fura-2 AM-loaded endothelial cells before (left), during (middle), and 6 min after (right) intracellular administration of control buffer (C), ET-1 (10⁻¹⁰ m) alone (D), or in presence of BQ-123 (E) or BQ-788 (F); arrows indicate the injected cells; the fluorescence scale (0–3) is illustrated in each panel and magnified in the first panel of C. inj, injection.

FIGURE 4.

Lysosomal localization of functional ET_B receptors in RPMVEC. A, the Ca²⁺ response to endothelin-1 is prevented by 1-h pretreatment with bafilomycin A1 (Baf, 1 μm) or rapamycin (Rap, 30 μm), but not brefeldin A (Bref, 10 μm); averaged traces from six experiments are shown. B, comparison of the increases in [Ca²⁺]_i produced by microinjected ET-1 in absence or presence of brefeldin A, bafilomycin A1, or rapamycin; *, p < 0.05 compared with ET-1 alone. C–F, representative images depicting Fura-2 AM fluorescence ratio (F₃₄₀/F_{380 nm}) of endothelial cells before (left), during (middle), and 6 min after (right) ET-1 microinjection in absence (C) or presence of brefeldin A (D), bafilomycin A1 (E), or rapamycin (F) pretreatment. inj, injection.

FIGURE 5.

Immunocytochemical localization of ET_B receptors to lysosomes in RPMVEC. A, identification of ET_B-GFP fluorescence, acidic organelles/lysosomes labeled with LysoTracker Red, and the nuclei labeled with DAPI in RPMVEC transiently transfected with GFP-labeled ET_B. In the overlay image, the colocalization of intracellular ET_B receptors with lysosomes is seen as orange fluorescence. B, pretreatment with bafilomycin A1 (Baf, 1 μm) markedly decreased both ET_B-GFP and LysoTracker Red fluorescence. C, disruption of lysosomes by pretreatment with glycyl-l-phenylalanine 2-naphthylamide (GPN) (100 μm) also markedly reduced the ET_B-GFP and LysoTracker Red fluorescence.

FIGURE 6.

Endothelin-1 mobilizes Ca²⁺ from the IP₃-dependent stores. A, averaged Ca²⁺ responses (n = 6) to ET-1 microinjection in RPMVEC incubated in Ca²⁺-free saline with inhibitors of Ca²⁺ release from the lysosomes (Ned-19) and from the endoplasmic reticulum/lysosomal IP₃R blockers xestospongin C (XeC) and heparin; endoplasmic reticulum ryanodine (Ry) receptor blocker ryanodine; or with the phospholipase C inhibitor U-73122. B, comparison of the [Ca²⁺]_i increases in response to intracellular administration of ET-1 in Ca²⁺-containing and Ca²⁺-free saline in the absence and presence of the indicated antagonists; *, p < 0.05 compared with ET-1 (10⁻¹⁰ m). ET-1 effects were blocked by inhibition of phospholipase C or of IP₃ receptors.

FIGURE 7.

Intracellular administration of ET-1 elevates endothelial NO levels. A, averaged traces of DAF-FM fluorescence indicating increases in NO level in response to microinjection of control buffer or ET-1 (upper left panel), in cells incubated with either NO synthase inhibitor l-NAME (upper right panel) or microautophagy blocker rapamycin (lower right panel), or in response to ET-1 co-injected with ET_A or ET_B antagonists BQ-123 and BQ-788, respectively (lower left panel). B, quantification of the ΔDAF (F/F_o)-FM fluorescence increases in RPMVEC in each of the conditions listed in A; p < 0.05 compared control (*) or to ET-1 (10⁻¹⁰ m) (#). Rap, rapamycin.

FIGURE 8.

Proposed signaling of endothelin-1 via endolysosomal ET_B receptor activation. Cytosolic endothelin-1 (ET-1), transferred to the endolysosomal lumen (Endo-Lys) via microautophagy, stimulates endolysosomal ET_B receptors, which in turn activate phospholipase C (PLC) located in the membrane of endolysosomal Ca²⁺ stores. Thus, IP₃ is released from membrane phosphoinositides and activates IP₃ receptors from the endoplasmic reticulum (ER) or endolysosomes. The subsequent IP₃-induced increase in cytosolic Ca²⁺ activates endothelial NO synthase (eNOS) to produce NO. The NO released from the endothelial cells leads to relaxation of the vascular smooth muscle.