Lysosome-Sarcoplasmic Reticulum Junctions
2004; Elsevier BV; Volume: 279; Issue: 52 Linguagem: Inglês
10.1074/jbc.m406132200
ISSN1083-351X
AutoresNicholas P. Kinnear, François‐Xavier Boittin, Justyn M. Thomas, Antony Galione, Allan M. Evans,
Tópico(s)Hearing, Cochlea, Tinnitus, Genetics
ResumoPrevious studies on pulmonary arterial smooth muscle cells have shown that nicotinic acid adenine dinucleotide phosphate (NAADP) evokes highly localized intracellular Ca2+ signals by mobilizing thapsigargin-insensitive stores. Such localized Ca2+ signals may initiate global Ca2+ waves and contraction of the myocytes through the recruitment of ryanodine receptors on the sarcoplasmic reticulum via Ca2+-induced Ca2+ release. Here we show that NAADP evokes localized Ca2+ signals by mobilizing a bafilomycin A1-sensitive, lysosome-related Ca2+ store. These lysosomal stores facilitate this process by co-localizing with a portion of the sarcoplasmic reticulum expressing ryanodine receptors to comprise a highly specialized trigger zone for NAADP-dependent Ca2+ signaling by the vasoconstrictor hormone, endothelin-1. These findings further advance our understanding of how the spatial organization of discrete, organellar Ca2+ stores may underpin the generation of differential Ca2+ signaling patterns by different Ca2+-mobilizing messengers. Previous studies on pulmonary arterial smooth muscle cells have shown that nicotinic acid adenine dinucleotide phosphate (NAADP) evokes highly localized intracellular Ca2+ signals by mobilizing thapsigargin-insensitive stores. Such localized Ca2+ signals may initiate global Ca2+ waves and contraction of the myocytes through the recruitment of ryanodine receptors on the sarcoplasmic reticulum via Ca2+-induced Ca2+ release. Here we show that NAADP evokes localized Ca2+ signals by mobilizing a bafilomycin A1-sensitive, lysosome-related Ca2+ store. These lysosomal stores facilitate this process by co-localizing with a portion of the sarcoplasmic reticulum expressing ryanodine receptors to comprise a highly specialized trigger zone for NAADP-dependent Ca2+ signaling by the vasoconstrictor hormone, endothelin-1. These findings further advance our understanding of how the spatial organization of discrete, organellar Ca2+ stores may underpin the generation of differential Ca2+ signaling patterns by different Ca2+-mobilizing messengers. Consideration of the wide variety of processes regulated by changes in intracellular Ca2+ concentration, from fertilization and gene expression to muscle contraction and cell death, can leave us in no doubt of the need for a versatile Ca2+ signaling system (1Berridge M. Lipp P. Bootman M. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4372) Google Scholar). Consequently, not all stimuli that initiate a given cell response, such as muscle contraction, do so by eliciting Ca2+ signals with a common spatiotemporal pattern (1Berridge M. Lipp P. Bootman M. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4372) Google Scholar). Conversely, not all stimuli that increase intracellular Ca2+ concentration initiate a common cell response. Thus, Prentki et al. (2Prentki M. Glennon M. Thomas A. Morris R. Matschinsky F. Corkey B. J. Biol. Chem. 1988; 263: 11044-11047Abstract Full Text PDF PubMed Google Scholar) proposed that "Ca2+ fingerprints" may be generated in an agonist-specific manner. However, the precise mechanisms that underpin stimulus-specific Ca2+ signaling remain obscure.We do know, however, that agonist specificity is determined, in part, by the release of Ca2+ from intracellular stores in a manner dependent on second messengers and their associated Ca2+ release channels. During pharmaco-mechanical coupling in smooth muscle, it has long been accepted that many receptors induce the production of inositol 1,4,5-trisphosphate (IP3), 1The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; SR, sarcoplasmic reticulum; NAADP, nicotinic acid adenine dinucleotide phosphate; RyR, ryanodine receptor; CICR, Ca2+-induced Ca2+ release; ER, endoplasmic reticulum; ET-1, endothelin-1; PSS, physiological salt solution; GPN, glycyl-l-phenylalanine 2-naphthylamide; ML-9, 1-(5-chloronapthalene-1-sulfonyl)-1H-hexahydro-1,4,diazepine hydrochloride; PGF2α, prostaglandin-F2α. 1The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; SR, sarcoplasmic reticulum; NAADP, nicotinic acid adenine dinucleotide phosphate; RyR, ryanodine receptor; CICR, Ca2+-induced Ca2+ release; ER, endoplasmic reticulum; ET-1, endothelin-1; PSS, physiological salt solution; GPN, glycyl-l-phenylalanine 2-naphthylamide; ML-9, 1-(5-chloronapthalene-1-sulfonyl)-1H-hexahydro-1,4,diazepine hydrochloride; PGF2α, prostaglandin-F2α. which leads to the activation of one or more of the known IP3 receptor (IP3R) subtypes on the SR and release of Ca2+ from this store (1Berridge M. Lipp P. Bootman M. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4372) Google Scholar, 3Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-2365Crossref PubMed Scopus (1724) Google Scholar). However, there is a growing body of evidence to support a role for Ca2+-mobilizing pyridine nucleotides in the regulation of intracellular Ca2+ signaling in a number of cell types, including smooth muscle (4Li P.L. Lee H.C. Nelson M.T. Meininger G.A. Van Breemen C. Acta Physiol. Scand. 2003; 179: 339-352Crossref PubMed Scopus (14) Google Scholar). Consistent with this proposal, the enzymes for the synthesis and metabolism of NAADP (5Wilson H.L. Evans A.M. Galione A. Br. J. Pharmacol. 1998; 124: 30PCrossref Scopus (29) Google Scholar, 6Yusufi A.N. Cheng J. Thompson M.A. Burnett J.C. Grande J.P. Exp. Biol. Med. (Maywood). 2002; 227: 36-44Crossref PubMed Scopus (38) Google Scholar) and cyclic ADP-ribose (7Wilson H.L. Dipp M. Thomas J.M. Lad C. Galione A. Evans A.M. J. Biol. Chem. 2001; 276: 11180-11188Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) have been shown to be associated with smooth muscle in addition to other cell types. In smooth muscle, cyclic ADP-ribose may mobilize Ca2+ from the SR by activating RyRs (8Kuemmerle J.F. Makhlouf G.M. J. Biol. Chem. 1995; 270: 25488-25494Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 9Kannan M.S. Fenton A.M. Prakash Y.S. Sieck G.C. Am. J. Physiol. 1996; 270: H801-H806PubMed Google Scholar, 10Tang W.X. Chen Y.F. Zou A.P. Campbell W.B. Li P.L. Am. J. Physiol. 2002; 282: H1304-H1310Crossref PubMed Scopus (90) Google Scholar, 11Boittin F-X. Dipp M. Kinnear N.P. Galione A. Evans A.M. J. Biol. Chem. 2003; 278: 9602-9608Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 12Wang Y.X. Zheng Y.M. Mei Q.B. Wang Q.S. Collier M.L. Fleischer S. Xin H.B. Kotlikoff M. Am. J. Physiol. 2004; 286: C538-C546Crossref PubMed Scopus (101) Google Scholar), whereas our studies on arterial smooth muscle (13Boittin F-X. Galione A. Evans A.M. Circ. Res. 2002; 91: 1168-1175Crossref PubMed Scopus (102) Google Scholar) are consistent with the idea that NAADP mobilizes Ca2+ from an undefined store that may be coupled to the SR (14Patel S. Churchill G. Galione A. Trends Biochem. Sci. 2001; 26: 482-489Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 15Lee H. Aarhus R. J. Cell Sci. 2000; 113: 4413-4420Crossref PubMed Google Scholar, 16Churchill G. Galione A. EMBO J. 2001; 20: 2666-2671Crossref PubMed Scopus (136) Google Scholar). Briefly, we have demonstrated that NAADP triggers spatially restricted Ca2+ bursts in arterial smooth muscle, which subsequently elicit a global Ca2+ wave and muscle contraction by CICR via RyRs (13Boittin F-X. Galione A. Evans A.M. Circ. Res. 2002; 91: 1168-1175Crossref PubMed Scopus (102) Google Scholar).The aforementioned findings raised the possibility that the spatiotemporal pattern of Ca2+ signals may also be determined via the selection of different intracellular Ca2+ stores in a manner dependent on the nature of the Ca2+-mobilizing messenger(s) recruited by a given stimulus (15Lee H. Aarhus R. J. Cell Sci. 2000; 113: 4413-4420Crossref PubMed Google Scholar, 17Churchill G. Okada Y. Thomas J. Genazzani A. Patel S. Galione A. Cell. 2002; 111: 703-708Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 18Mitchell K.J. Lai F.A. Rutter G.A. J. Biol. Chem. 2003; 278: 11057-11064Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 19Yamasaki M. Masgrau R. Morgan A.J. Churchill G.C. Patel S. Ashcroft S.J. Galione A. J. Biol. Chem. 2004; 279: 7234-7240Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 20Hohenegger M. Suko J. Gscheidlinger R. Drobny H. Zidar A. Biochem. J. 2002; 367: 423-431Crossref PubMed Scopus (107) Google Scholar, 21Masgrau R. Churchill G.C. Morgan A.J. Ashcroft S.J.H. Galione A. Curr. Biol. 2003; 13: 247-251Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Further support for this view has recently been provided by the proposal that NAADP triggers Ca2+ release from reserve granules in sea urchin eggs (17Churchill G. Okada Y. Thomas J. Genazzani A. Patel S. Galione A. Cell. 2002; 111: 703-708Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), the functional equivalent of lysosomes in mammalian cells. However, others have suggested that direct activation of RyRs on the SR/ER may underlie Ca2+ signaling by NAADP (20Hohenegger M. Suko J. Gscheidlinger R. Drobny H. Zidar A. Biochem. J. 2002; 367: 423-431Crossref PubMed Scopus (107) Google Scholar, 22Gerasimenko J.V. Maruyama Y. Yano K. Dolman N.J. Tepikin A.V. Petersen O.H. Gerasimenko O.V. J. Cell Biol. 2003; 163: 271-282Crossref PubMed Scopus (193) Google Scholar). The findings of the present investigation are consistent with the idea that a lysosome-related Ca2+ store is the point of origin of NAADP-mediated Ca2+ bursts and suggest that lysosomes may co-localize with RyRs in the SR of arterial smooth muscle cells to form a "trigger zone" for NAADP-dependent Ca2+ signaling. Our data also lend weight to the argument that NAADP-dependent Ca2+ signals may be evoked in a stimulus-specific manner (19Yamasaki M. Masgrau R. Morgan A.J. Churchill G.C. Patel S. Ashcroft S.J. Galione A. J. Biol. Chem. 2004; 279: 7234-7240Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), by the potent vasoconstrictor ET-1.EXPERIMENTAL PROCEDURESCell Isolation—Adult male Wistar rats (150–300 g) were sacrificed by cervical dislocation. The heart and lungs were removed en bloc and placed in physiological salt solution (PSS) of the following composition (mm): 130 NaCl, 5.2 KCl, 1 MgCl2, 1.7 CaCl2, 10 glucose, and 10 Hepes, pH 7.45. Single arterial smooth muscle cells were isolated from second order branches of the pulmonary artery. Briefly, an artery was dissected out and placed in a low Ca2+ solution of the following composition (mm): 124 NaCl, 5 KCl, 1 MgCl2, 0.5 NaH2PO4, 0.5 KH2PO4, 15 NaHCO3, 0.16 CaCl2, 0.5 EDTA, 10 glucose, 10 taurine, and 10 Hepes, pH 7.4. After 10 min, the artery was placed in the same solution containing 0.5 mg/ml papain (Fluka) and 1 mg/ml bovine serum albumin (Sigma), and kept at 4 °C overnight. The following day 0.2 mm 1,4-dithio-dl-threitol (Sigma) was added to the solution, to activate the protease, and the preparation was incubated for 1 h at room temperature. The tissue was then washed three times in fresh low Ca2+ solution without enzymes, and single smooth muscle cells were isolated by gentle trituration with a fire-polished Pasteur pipette. Cells were stored in suspension at 4 °C until required.Ca2+ Imaging—Cells were incubated for 30 min with 5 μm Fura-2-AM in Ca2+ free PSS in an experimental chamber on a Leica DMIRBE inverted microscope and then superfused with Fura-2-free PSS for at least 30 min prior to experimentation. Fura-2 fluorescence was measured using excitation wavelengths of 340 nm (F340) and 380 nm (F380), respectively, and an emission wavelength of 510 nm. Emitted fluorescence was monitored using a Hamamatsu 4880 image-intensifying charge-coupled device camera through a Zeiss Fluar 40×, 1.3-numerical aperture oil immersion lens and recorded and analyzed using Openlab imaging software (Improvision, UK) on an Apple Macintosh G4 personal computer. Fluorescence intensity was measured at ≤0.3 Hz, with background subtraction being carried out on-line. Changes in Fura-2 fluorescence are reported as the F340/F380 ratio.Intracellular Dialysis of NAADP and IP3—Ca2+-mobilizing second messengers were applied intracellularly in the whole cell configuration of the patch clamp technique, and in current clamp mode (I = 0) as described previously (13Boittin F-X. Galione A. Evans A.M. Circ. Res. 2002; 91: 1168-1175Crossref PubMed Scopus (102) Google Scholar). The pipette solution contained (mm): 140 KCl, 10 Hepes, 1 MgCl2, and 5 μm Fura-2, pH 7.4. The seal resistance, as measured using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), was ≥3 GΩ throughout each experiment. Series resistance was ≤10 mΩ, and pipette resistance was ≤3 mΩ. All experiments were carried out at room temperature (22 °C).Extracellular Application of Pharmacological Agents—For experiments on the effects of intracellular dialysis of NAADP, pharmacological agents (e.g. ryanodine) were applied directly to the bath solution. During investigations into the effects of ET-1, PGF2α, and bafilomycin A1, however, these and other pharmacological agents were applied extracellularly by using a microsuperfusion system via a flow pipe positioned close the cell under investigation, as described previously (23Langton P.D. J. Physiol. 1993; 471: 1-11Crossref PubMed Scopus (121) Google Scholar).Fluorescent Imaging of Subcellular Labeling by LysoTracker Red and BODIPY-ryanodine—Cells were incubated with 1 μm BODIPY FL-X ryanodine for 60 min, and 0.5–2 nm LysoTracker Red for 30 min. Images were acquired using a Deltavision microscope system (Applied Precision), consisting of an Olympus IX70 inverted microscope with an Olympus PlanApo 60×, 1.40-numerical aperture oil immersion objective and Photometric CH300 charge-coupled device camera. Single Z sections were taken at what was determined to be the center of the cell, or 40-× 0.2-μm sections were taken through the cell. Images were deconvolved and analyzed off-line using Softworx acquisition and analysis software (Applied Precision). In experiments involving glycyl-l-phenylalanine 2-naphthylamide (GPN), cells were pre-treated for 15 min with 10 μm ML-9 to prevent contraction.Measurement of NAADP Levels—Second and third order branches of the pulmonary arterial tree were removed as described above. The arteries were cut open longitudinally, and the endothelium was removed by gently rubbing the lumen with a cotton bud. After 30-min equilibration in PSS at 37 °C, arteries were transferred to either PSS (control), or PSS containing either 1 μm ET-1 or 2 μm PGF2α (test). Removing the buffer and adding ice cold 1.5 m HClO4 terminated the reaction. Tissue was immediately sonicated and left on ice for 20 min. Samples were then centrifuged at 13,000 rpm for 10 min, and the supernatant was removed. The pellet was retained to determine protein content. The supernatant was then neutralized with 2 m KHCO3 and left for a further 20 min on ice before being centrifuged as before to remove the KClO4 precipitate. Samples were stored at –80 °C.NAADP levels were subsequently assayed via a radioreceptor assay as previously described (21Masgrau R. Churchill G.C. Morgan A.J. Ashcroft S.J.H. Galione A. Curr. Biol. 2003; 13: 247-251Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Briefly, enzymatically synthesized 32P-labeled NAADP, 1.25% sea urchin (Lytechinus pictus) egg homogenate, and samples versus known concentrations of NAADP were incubated for 15 min at room temperature in intracellular-like buffer of the following composition (mm): 250 N-methylglucamine, 250 potassium gluconate, 20 Hepes, 1 MgCl2, pH 7.2. The binding reaction was terminated by rapid filtration (Brandell cell harvester) through GF/B filters (Whatman) with ice-cold buffer of the following composition (mm): 10 Hepes, 1 EDTA, pH 7.2. Bound NAADP was determined by using Cerenkov spectrometry.Data Analysis—Data are expressed as mean ± S.E. for n experiments. Differences were detected by use of a Student's unpaired t test.Drugs and Chemicals—Ryanodine, thapsigargin, IP3, NAADP, bafilomycin A1, GPN, ET-1, PGF2α, and ML-9 were obtained from Sigma. Fura-2-AM, BODIPY FL-X ryanodine, and LysoTracker Red were obtained from Molecular Probes. NAD (32P-radiolabeled) was obtained from Amersham Biosciences. Stock solutions of ryanodine, thapsigargin, ML-9, and GPN were made with Me2SO as the solvent. The minimum dilution of Me2SO was 1:1000 in PSS, at which concentration Me2SO alone was without effect on the responsiveness of the preparation. All other stock solutions were made in distilled H2O before further dilution in PSS.RESULTSNAADP Induces "Ca2+ Bursts" from a Bafilomycin-insensitive, Lysosome-related Ca2+ Store—Clearly, further investigations on NAADP-dependent Ca2+ signaling in arterial smooth muscle were required to advance our understanding of the fundamental mechanisms that control blood pressure, and cell function in general. To this end NAADP was applied intracellularly into isolated pulmonary arterial smooth muscle cells from a patch pipette, in the whole cell configuration and under current clamp conditions (I = 0). Changes in intracellular Ca2+ were reported by the fluorescence ratio (F340/F380) of the Ca2+ indicator, Fura-2. At a concentration of 10 nm, NAADP induced a global increase in the Fura-2 fluorescence ratio, from 0.7 ± 0.1 to 1.9 ± 0.1 (n = 17) in isolated pulmonary arterial smooth muscle cells. These effects occurred within 180 s of entering the whole-cell configuration (Fig. 1A). In paired cells, intracellular dialysis of NAADP-free pipette solution had little or no effect (n = 17; not shown). Our findings are consistent with the view that the events leading up to the initiation of global Ca2+ waves by NAADP are complex, in that there is a highly localized release of Ca2+, which precedes the global Ca2+ waves and the associated contractile response (13Boittin F-X. Galione A. Evans A.M. Circ. Res. 2002; 91: 1168-1175Crossref PubMed Scopus (102) Google Scholar). In general, this initial localized Ca2+ release event appears as 1) a uniform increase in Fura-2 fluorescence ratio around the entire perimeter of the cell (Fig. 2A, panel i) and 2) a spatially restricted "focal" Ca2+ signal covering an area of between 2 and 10 μm in diameter (Figs. 1A and 2A, panel ii). These localized Ca2+ signals initiated by NAADP have been named "Ca2+ bursts" (13Boittin F-X. Galione A. Evans A.M. Circ. Res. 2002; 91: 1168-1175Crossref PubMed Scopus (102) Google Scholar) and either decline back to basal levels or trigger a global Ca2+ wave (Fig. 1A). Previously, we have shown that NAADP induces Ca2+ bursts from a thapsigargin-insensitive store other than the SR and that these local Ca2+ signals are then amplified by subsequent CICR from the SR via RyRs (13Boittin F-X. Galione A. Evans A.M. Circ. Res. 2002; 91: 1168-1175Crossref PubMed Scopus (102) Google Scholar). We therefore investigated the possibility that the NAADP-sensitive Ca2+ store in vascular smooth muscle is an acidic, lysosome-related store as previously suggested by investigations on other cell types (17Churchill G. Okada Y. Thomas J. Genazzani A. Patel S. Galione A. Cell. 2002; 111: 703-708Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 19Yamasaki M. Masgrau R. Morgan A.J. Churchill G.C. Patel S. Ashcroft S.J. Galione A. J. Biol. Chem. 2004; 279: 7234-7240Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar).Fig. 2Lysosomes cluster to form spatially restricted units in isolated pulmonary arterial smooth muscle cells. A, pseudocolor images of the Fura-2 fluorescence ratio recorded in isolated pulmonary arterial smooth muscle cells showing the two forms of Ca2+ burst elicited by intracellular dialysis of 10 nm NAADP: i, a uniform increase in Ca2+ around the entire perimeter of the cell; ii, a spatially localized focal Ca2+ release event. In B: panel i, bright field image of an isolated pulmonary arterial smooth muscle cell. ii, deconvolved Z section through the same cell as in panel i shows lysosomes labeled with LysoTracker Red; note, lysosomes cluster to form a ring around the perimeter of the cell consistent with the form of Ca2+ burst in (A, panel i). Panels iii–v, effect on labeling of organelles by 0.5 nm LysoTracker Red5(panel iii), 10 (panel iv), and 15 (panel v) min after addition of 200 μm glycylphenylalanine 2-naphthylamide (GPN); same cell as shown in panel i. In C: panel i shows a bright field image from a different cell; panel ii, the corresponding deconvolved LysoTracker Red fluorescence image from a single Z section through the cell; panel iii, the corresponding three-dimensional reconstruction of deconvolved Z sections; note, lysosomes form a spatially restricted cluster ∼6 μm across as indicated by the white rectangle, consistent with the form of Ca2+ burst in (A, panel ii). In C: panel iv shows a schematic diagram of the volume occupied by the largest lysosomal cluster, indicated by the white rectangle in the cell shown in C panel iii. In C: panels v–viii, three-dimensional reconstruction of the largest lysosomal cluster in the cell shown in panel iii and indicated by the white rectangle, measured for volume through, 0° (v), 90° (vi), 180° (vii), and 270° (viii) of rotation; white lines indicate the distance measured for volume calculations (μm). In A (panels i and ii), B, and C, respectively, images were from different cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It has been demonstrated that Ca2+ uptake into lysosome-related organelles is driven by proton gradients maintained by vacuolar proton pumps (V-H+ ATPase (24Haller T. Dietl P. Deetjen P. Volkl H. Cell Calcium. 1996; 19: 157-165Crossref PubMed Scopus (103) Google Scholar, 25Christensen K.A. Myers J.T. Swanson J.A. J. Cell Sci. 2002; 115: 599-607Crossref PubMed Google Scholar)). Thus, we investigated the effect of the V-H+ ATPase inhibitor bafilomycin A1 on NAADP-induced Ca2+ signals. Extracellular application of bafilomycin A1 (100–300 nm) induced a global increase in Fura-2 fluorescence ratio from 0.63 ± 0.05 to 1.04 ± 0.07, which declined back to baseline within 45 min (n = 20; Figs. 1B and 4F). We therefore proceeded to study the effect on NAADP-induced Ca2+ signals of bafilomycin A1. Preincubation (45 min) of pulmonary arterial smooth muscle cells with 100 nm bafilomycin A1 blocked Ca2+ signals induced by the intracellular application of NAADP (10 nm, n = 7; Fig. 1C). In stark contrast, extracellular application of caffeine (2 mm) induced an increase in the Fura-2 fluorescence ratio from 0.64 ± 0.05 to 2.58 ± 0.02 (n = 4) in the absence of bafilomycin A1 and from 0.71 ± 0.02 to 2.18 ± 0.06 (n = 4) in the presence of bafilomycin A1. Similarly, intracellular dialysis of IP3 (1 μm) increased the Fura-2 fluorescence ratio from 0.61 ± 0.04 to 1.83 ± 0.38 (n = 4) in the absence of bafilomycin A1 and from 0.68 ± 0.06 to 2.22 ± 0.19 (n = 4) in the presence of bafilomycin A1 (Fig. 1D). These data suggest that bafilomycin A1 blocks Ca2+ mobilization by NAADP without affecting Ca2+ release from the SR through either RyRs or IP3Rs.Fig. 4ET-1 triggers Ca2+ release from lysosome-related organelles via NAADP. A, effect on the Fura-2 fluorescence ratio in an isolated pulmonary arterial smooth muscle cell of extracellular application of 2 μm PGF2α before and after incubation (50 min) of cells with 100 nm bafilomycin A1. B, shows the reproducible nature of the Ca2+ signal induced by extracellular application of 100 nm ET-1. C, extracellular application of 100 nm ET-1 before and after incubation (50 min) with bafilomycin A1; D, extracellular application of 100 nm ET-1 before and after incubation (15 min) of cells with 1 μm thapsigargin; E, extracellular application of 100 nm ET-1 before and after incubation (15 min) of cells with 20 μm ryanodine. F, bar chart shows the percent change (mean ± S.E.) in Fura-2 fluorescence ratio in pulmonary arterial smooth muscle cells induced by: intracellular dialysis of 10 nm NAADP before and after incubation with 100 nm bafilomycin A1, 1 μm thapsigargin, and 20 μm ryanodine, respectively; extracellular application of 100 nm ET-1 before and after incubation with 100 nm bafilomycin A1, 1 μm thapsigargin, and 20 μm ryanodine, respectively; extracellular application of 100–300 nm bafilomycin A1 before and after incubation with 1 μm thapsigargin and 20 μm ryanodine, respectively. G, bar chart shows the effect of 1 μm ET-1 and 2 μm PGF2α (30-s incubation), respectively, on the tissue levels of NAADP in second and third order branches of the pulmonary arterial tree without endothelium. Asterisk denotes p = <0.005 when compared with control. Abbreviations: baf., bafilomycin; thap., thapsigargin; ryan., ryanodine.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next we compared the pharmacology of global Ca2+ signals initiated by bafilomycin A1 and NAADP. When cells were incubated (15 min) with thapsigargin (1 μm), both bafilomycin A1 (100–300 nm) and NAADP (10 nm) failed to induce global Ca2+ signals and contraction. However, upon intracellular dialysis of NAADP, small amplitude, spatially restricted Ca2+ bursts were observed (Fig. 1E), as previously reported (13Boittin F-X. Galione A. Evans A.M. Circ. Res. 2002; 91: 1168-1175Crossref PubMed Scopus (102) Google Scholar). Similar results were observed by extracellular application of bafilomycin A1, in that small spatially restricted and asynchronous Ca2+ transients were observed in 8 out of 11 cells (Fig. 1F). The localized thapsigargin-insensitive Ca2+ bursts evoked by NAADP (10 nm) and spatially restricted Ca2+ release observed with bafilomycin A1 (100–300 nm) represented a local increase in the F340/F380 ratio within a given region of interest of 27 ± 3% (n = 8; see Fig. 4F below) and 25 ± 4% (n = 11; Fig. 4F), respectively. Similar results to those obtained in thapsigargin-treated cells were obtained by treating cells with ryanodine (20 μm). Following incubation (15 min) with ryanodine, NAADP (100 nm) and bafilomycin A1 (100 nm) again failed to induce global Ca2+ signals, and no cell contraction was observed. Once more, however, low amplitude, spatially restricted Ca2+ bursts were triggered by NAADP (Fig. 1G; n = 8), while bafilomycin A1 triggered spatially restricted Ca2+ signals in 5 out of 8 cells (Fig. 1H). The localized Ca2+ signals induced by NAADP (10 nm) and bafilomycin A1 (100 nm) in ryanodine-treated cells represented increases in the F340/F380 ratio of 33 ± 7% (n = 9; Fig. 4F) and 28 ± 3% (n = 8; Fig. 4F), respectively. These findings are consistent with the view that bafilomycin A1 and NAADP mobilize Ca2+ from a common thapsigargin- and ryanodine-insensitive acidic Ca2+ store.Lysosomes Form Tight Clusters in Pulmonary Arterial Smooth Muscle Cells—We next investigated the distribution of acidic stores/lysosomes within arterial smooth muscle cells using LysoTracker Red, a fluorescent weak base that accumulates in acidic compartments. Deconvolved images of Z sections (0.2 μm depth) through single isolated pulmonary arterial smooth muscle cells labeled with LysoTracker Red fluorescence (excitation 568 nm, emission 590 nm) revealed dense regions of labeling. As mentioned previously, upon analysis of pseudocolor images of the change in Fura-2 fluorescence ratio with time, two forms of initial Ca2+ burst were observed in response to intracellular application of NAADP: 1) a relatively uniform increase in Ca2+ around the entire perimeter of the cell (Fig. 2A, panel i; n = 4) or 2) a spatially restricted "focal" Ca2+ signal covering an area of between 2 and 10 μm in diameter (Fig. 2A, panel ii; n = 6 (13Boittin F-X. Galione A. Evans A.M. Circ. Res. 2002; 91: 1168-1175Crossref PubMed Scopus (102) Google Scholar)). Consistent with the spatial distribution of these two forms of local Ca2+ signal, the dense regions of LysoTracker Red labeling observed in pulmonary arterial smooth muscle cells appeared as either: 1) a relatively uniform ring around the perimeter of the cell that was ∼2 μm across (Fig. 2B, panels i and ii; n = 5) or 2) a tight, spatially restricted unit ∼2–6 μm across (Fig. 2C, panels i and ii; n = 6). To determine whether or not the labeled organelles were lysosome-related, cells were incubated with GPN, a substrate of the lysosomal exopeptidase cathepsin C that selectively disrupts lysosomes by osmotic lysis (26Berg T. Stromhaug E. Lovdal T. Seglen O. Berg T. Biochem. J. 1994; 300: 229-236Crossref PubMed Scopus (83) Google Scholar). To achieve this goal, cells were first incubated for 15 min with the selective inhibitor of myosin light-chain kinase 1-(5-chloronapthalene-1-sulfonyl)-1H-hexahydro-1,4,diazepine hydrochloride (ML-9 (27Ito M. Tanabe F. Sato A. Ishida E. Takami Y. Shigeta S. Int. J. Immunopharmacol. 1989; 11: 185-190Crossref PubMed Scopus (10) Google Scholar)) to prevent disruption of the spatial orientation of cells due to contraction by GPN. Under these conditions, GPN (200 μm) eliminated LysoTracker Red labeling in a time-dependent manner (Fig. 2B, panels i–v) consistent with these organelles being lysosome-related. Tight lysosomal clusters were seen to occupy a volume of 27.5 ± 10 μm3 within the cells from which volume measurements were taken (n = 3, Fig. 2C). These results suggest that lysosome-related organelles form dense clusters that may provide a Ca2+ store with a high degree of spatial organization in arterial smoot
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