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• W1969914617 abstract "Increases in the concentration of free ATP within the islet β-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+(KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary β-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar (∼1 mm). Elevations in extracellular glucose concentration (3–30 mm) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration. Increases in the concentration of free ATP within the islet β-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+(KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary β-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar (∼1 mm). Elevations in extracellular glucose concentration (3–30 mm) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration. Increases in extracellular glucose concentration stimulate the exocytosis of insulin from islet β-cells. This is probably achieved by an increase in glycolysis and flux through the citrate cycle (1Ashcroft F.M. Kakei M. J. Physiol. 1989; 416: 349-367Crossref PubMed Scopus (122) Google Scholar), leading to elevated intracellular levels of likely coupling factors (2Prentki M. Tornheim K. Corkey B.E. Diabetologia. 1997; 40: S32-S41Crossref PubMed Scopus (152) Google Scholar), including ATP. Closure of ATP-sensitive K+ channels (3Ashcroft F.M. Harrison D.E. Ashcroft S.JH. Nature. 1984; 312: 446-448Crossref PubMed Scopus (876) Google Scholar, 4Cook D.L. Hales C.N. Nature. 1984; 311: 271-273Crossref PubMed Scopus (969) Google Scholar, 5Rorsman P. Diabetologia. 1997; 40: 487-495Crossref PubMed Scopus (249) Google Scholar) then leads to plasma membrane depolarization and the influx of Ca2+ through voltage gated Ca2+ channels. Increases in the total intracellular concentration of ATP have been measured in isolated islets (6Meglasson M.D. Nelson J. Nelson D. Erecinska M. Metabolism. 1989; 38: 1188-1195Abstract Full Text PDF PubMed Scopus (45) Google Scholar) and cell lines (7Civelek V.N. Deeney J.T. Kubik K. Schultz V. Tornheim K. Corkey B.E. Biochem. J. 1996; 315: 1015-1019Crossref PubMed Scopus (58) Google Scholar) exposed to increases in extracellular glucose concentration. However, the measured changes are generally small and difficult to interpret because of the large depot of intragranular ATP, and the presence of non-β-cells (8Detimary P. Jonas J.C. Henquin J.C. J. Clin. Invest. 1995; 96: 1738-1745Crossref PubMed Scopus (107) Google Scholar). Furthermore, such measurements give no indication of the concentration of unbound ATP. Unfortunately, measurements of free [ATP] in living cells, for example by 31P NMR (9Scholz T.D. Laughlin M.R. Balaban R.S. Kupriyanov V.V. Heineman F.W. Am. J. Physiol. 1995; 268: H82-H91PubMed Google Scholar), cannot easily be extended to the islet micro-organ and do not provide sufficient sensitivity to detect changes at the cellular or subcellular level. This is an important question because differences in [ATP] at different intracellular sites have been predicted. In particular, locally high ATP consumption by the plasma membrane Na+-K+ and Ca2+-ATPase, may mean that [ATP] is lower in this domain than in the bulk of the cell cytosol (1Ashcroft F.M. Kakei M. J. Physiol. 1989; 416: 349-367Crossref PubMed Scopus (122) Google Scholar). Similarly, the electrogenic nature of the mitochondrial ATP/ADP translocase (10Klingenberg M. Trends Biochem. Sci. 1979; 4: 249-252Abstract Full Text PDF Scopus (76) Google Scholar) is predicted to create differences in ATP/ADP ratio across the inner mitochondrial membrane (cytosolic high). The role of changes in free Ca2+ ion concentration ([Ca2+]) in regulating β-cell metabolism and ATP concentration is controversial. Increases in [Ca2+] following plasma membrane depolarization act both to stimulate ATP requiring processes (i.e. secretory granule movement and exocytosis) (11Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2001) Google Scholar) and possibly to enhance mitochondrial oxidative metabolism (12Denton R.M. McCormack J.G. FEBS Lett. 1980; 119: 1-8Crossref PubMed Scopus (249) Google Scholar, 13McCormack J.G. Halestrap A.P. Denton R.M. Physiol. Rev. 1990; 70: 391-425Crossref PubMed Scopus (1149) Google Scholar). Recent measurements of total ATP content of whole islets have suggested that the former may dominate and that Ca2+ influx may diminish glucose-induced increases in ATP/ADP ratio (14Detimary P. Gilon P. Henquin J.-C. Biochem. J. 1998; 333: 269-274Crossref PubMed Scopus (147) Google Scholar). The use of firefly luciferase, targeted to discrete intracellular domains, should provide an extremely sensitive method of monitoring free [ATP] dynamically and at the subcellular level. In previous studies, we have shown that photon counting imaging of total luciferase activity in single cells provides a convenient means to measure changes in gene expression in single cells (15Rutter G.A. White M.R.H. Tavare J.M. Curr. Biol. 1995; 5: 890-899Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16Kennedy H.J. Viollet B. Rafiq I. Kahn A. Rutter G.A. J. Biol. Chem. 1997; 272: 20636-20640Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Luciferase has previously been employed to measure intracellular ATP concentration in single cardiac myocytes (17Bowers K.C. Allshire A.P. Cobbold P.H. J. Mol. Cell Cardiol. 1992; 24: 213-218Abstract Full Text PDF PubMed Scopus (64) Google Scholar) and hepatocytes (18Koop A. Cobbold P.H. Biochem. J. 1993; 295: 165-170Crossref PubMed Scopus (40) Google Scholar), but only after microinjection of the purified protein. In recent reports, Maechleret al. (19Maechler P. Wang H. Wollheim C.B. FEBS Lett. 1998; 422: 328-332Crossref PubMed Scopus (82) Google Scholar, 20Maechler P. Kennedy E.D. Wang H. Wollheim C.B. J. Biol. Chem. 1998; 273: 20770-20778Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) have shown that expression of recombinant luciferase provides a means of monitoring cytosolic ATP concentration in large populations (>10,000) of INS-1 β-cells. Unfortunately, such measurements fail to take account of the likely heterogeneity in the behavior of individual β-cells (21Pipeleers D. Kiekens R. Ling Z. Wilikens A. Schuit F. Diabetologia. 1994; 37: S57-S64Crossref PubMed Scopus (133) Google Scholar). Here, we use photon counting to image ATP concentrations dynamically and at the subcellular level in single living primary β-cells and derived MIN6 cells. We further extend this technique to allow the imaging of [ATP] in two cellular subdomains, the mitochondrial matrix and the subplasmallemal region, by the molecular targeting of luciferase. We demonstrate that exposure to elevated glucose concentrations causes increases in [ATP] in each of the compartments analyzed, coincident with an increase in mitochondrial membrane potential. Comparison of the kinetics of [ATP] changes, and dependence on increases in mitochondrial free [Ca2+] ([Ca2+]m), 1The abbreviation used is: [Ca2+]m, [Ca2+]c, and [Ca2+]pm, free Ca2+concentration in the mitochondrial matrix, cytosol, and plasma membrane regions, respectively; cLuc, cytosolic firefly luciferase; mLuc, mitochondrial matrix firefly luciferase; pmLuc, plasma membrane firefly luciferase; ΔΨ, mitochondrial membrane potential, TMREE, tetramethylrhodamine ethyl ester.1The abbreviation used is: [Ca2+]m, [Ca2+]c, and [Ca2+]pm, free Ca2+concentration in the mitochondrial matrix, cytosol, and plasma membrane regions, respectively; cLuc, cytosolic firefly luciferase; mLuc, mitochondrial matrix firefly luciferase; pmLuc, plasma membrane firefly luciferase; ΔΨ, mitochondrial membrane potential, TMREE, tetramethylrhodamine ethyl ester. also suggests that activation of strategically located mitochondria may preferentially enhance [ATP] immediately beneath the plasma membrane. Cytosolic (untargeted) firefly luciferase in plasmid pGL3 basic (Promega) was placed under cytomegalovirus immediate gene control by subcloning the cytomegalovirus promoter element from plasmid pcDNA3 (Invitrogen) as an 876-nucleotide BglII-HindIII fragment into the upstream multiple cloning site of pGL3, generating plasmid cLuc. Plasma-membrane targeted luciferase was prepared by polymerase chain reaction amplification of the 617-nucleotide translated region (minus stop codon) of synaptosome-associated protein of 25 kDa cDNA, with primers 5′ T.TTT.GAC.GAG.ACC.ATG.GCC.GAG.GAC.GCA and 5′TT.TTC.CAT.GGT.ACC.ACT.TCC.CAG.CAT.CTT (NcoI sites underlined) and the 629-nucleotide polymerase chain reaction fragment digested and subcloned into the plasmid pGL3-control (Promega) under SV40 immediate early gene promoter control, generating plasmid pmLuc. Correct orientation of the insert was verified by restriction mapping with SmaI and SalI, and confirmed by automated DNA sequencing. To allow higher levels of expression of this construct under cytomegalovirus promoter control, theHindIII-BamHI fragment of pmLuc was subcloned into plasmid pcDNA3; essentially identical data were obtained with either plasmid. For the preparation of mLuc, DNA sequences encoding a mitochondrial presequence and the hemagglutinin HA1 tag were added to the luciferase cDNA as follows. A fragment of wild-type luciferase cDNA was first amplified from plasmid pGL2 (Promega) using the following primer: 5′AAAG.CTT.AAT.GGA.AGA.CGC.CAA.AAA.CAT.AAA.GAA.A (corresponding to the sequence encoding amino acids 1–9 of luciferase;HindIII site underlined) and GAA.GAT.GTT.GGG.GTG.TTG.TAA.CAA.T (downstream of the endogenousClaI site of the luciferase cDNA and encoding amino acids 456–465). The polymerase chain reaction product was digested with the enzymes HindIII and ClaI and fused in frame to the ClaI/HindIII fragment encoding the HA1 tag (22Bastianutto C. Clementi E. Codazzi F. Podini P. De Giorgi F. Rizzuto R. Meldolesi J. Pozzan T. J. Cell Biol. 1995; 130: 847-855Crossref PubMed Scopus (168) Google Scholar). A ClaI fragment was thus generated that, in an appropriately prepared pBSK+ plasmid, could be fused in frame with theEcoRI/HindIII fragment encoding the amino-terminal 33 amino acids of cytochrome oxidase subunit 8 (COX8) (25 amino acids of the cleavable presequence plus 8 amino acids of the mature polypeptide) (23Rizzuto R. Nakase H. Darras B. Francke U. Fabrizi G.M. Mengel T. Walsh F. Kadenbach B. DiMauro S. Schon E.A. J. Biol. Chem. 1989; 264: 10595-10600Abstract Full Text PDF PubMed Google Scholar) and the ClaI/SalI fragment encoding the carboxyl-terminal portion of luciferase (amino acids 457–556). The whole final construct (shown schematically in Fig. 1) was excised via PstI/SalI digestion and cloned into the expression vectors VR1012 (Vical Research Inc., San Diego, CA) under modified cytomegalovirus promoter control. Primary rat islet β-cells were isolated by collagenase (PanPlus, Serva) digestion and purified on a discontinuous bovine serum albumin gradient (16Kennedy H.J. Viollet B. Rafiq I. Kahn A. Rutter G.A. J. Biol. Chem. 1997; 272: 20636-20640Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Cells were dissociated with trypsin before 24 h culture on Cell-TakTM-treated glass coverslips. Plasmids (0.2–0.4 mg·ml−1 in 10 mm Tris, 0.2 mmEDTA) were microinjected using glass borosilicate capillaries and an Eppendorf 5171 transjector/micromanipulator, as described (16Kennedy H.J. Viollet B. Rafiq I. Kahn A. Rutter G.A. J. Biol. Chem. 1997; 272: 20636-20640Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 24Rutter G.A. Burnett P. Rizzuto R. Brini M. Murgia M. Pozzan T. Tavare J.M. Denton R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5489-5494Crossref PubMed Scopus (220) Google Scholar). MIN6 cells were cultured on poly-l-lysine-treated coverslips (25Pouli A.E. Karagenc N. Bright N. Arden S. Schofield G.S. Wasmeier C. Hutton J.C. Rutter G.A. Biochem. J. 1998; 330: 1399-1404Crossref PubMed Scopus (35) Google Scholar). Injected primary β-cells cells were cultured 16–24 h in an atmosphere 5% CO2, and at 3 mmglucose. For all experiments in which the effects of elevated glucose or K+ concentrations were tested, MIN6 cells were cultured at 3 mm glucose for 24 h before imaging. Such cells responded robustly to elevated glucose concentrations with enhanced insulin secretion (4–10-fold above basal levels) (26Zhao C. Rutter G.A. FEBS Lett. 1998; 430: 213-216Crossref PubMed Scopus (57) Google Scholar). Cells were imaged in modified Krebs-Ringer bicarbonate medium (0.2 ml) comprising 125 mm NaCl; 3.5 mm KCl; 1.5 mmCaCl2; 0.5 mm MgSO4; 0.5 mm KH2PO4; 2.5 mmNaHCO3, 10 mm Hepes-Na+, pH 7.4, containing the indicated glucose concentration and equilibrated with 95:5 O2:CO2. Cells were maintained on the temperature-controlled (37 °C) stage of an Olympus IX-70 microscope (UPlanApo × 10, 0.4 numerical aperture air objective), located in a sealed dark housing. Medium was rapidly (<2 s) changed by the addition of an equal volume through a remotely located syringe. For calibration of signals, cells were lysed in “intracellular medium” comprising 20 mm Hepes, 140 mm KCl, 5 mm NaCl, 10.2 mm EGTA, 6.67 mmCaCl2, 1 mm luciferin, 20 μg·ml−1 digitonin plus additions of ATP, MgSO4, and CoA as indicated. Data were captured with an intensified charge-coupled device camera comprising a low-noise S-20 multi-alkali photocathode and three in-series microchannel plates (Photek ICCD216; Photek Ltd., St. Leonards-on-Sea, East Sussex, United Kingdom), maintained at 4 °C. Single photon events were captured at 25-ms intervals by time-resolved imaging, which allowed the spatial and temporal coordinates of each photon event to be held in matrix format. In this way, luminescence changes of any individual cell or group of cells could be analyzed for the entire time course of an individual experiment. When required, images corresponding to the selected area of interest within the image field were generated retrospectively over the desired integration period. Aequorin imaging was performed in cells expressing mitochondrially targeted aequorin (27Rizzuto R. Simpson A.W.M. Brini M. Pozzan T. Nature. 1992; 358: 325-327Crossref PubMed Scopus (782) Google Scholar) as described previously (24Rutter G.A. Burnett P. Rizzuto R. Brini M. Murgia M. Pozzan T. Tavare J.M. Denton R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5489-5494Crossref PubMed Scopus (220) Google Scholar). Cells were maintained at 37 °C for 2 h in serum-free Dulbecco's modified Eagle's medium containing 3 mm glucose, and then loaded with TMREE (10 nm) for 60 min. Confocal imaging was performed in KREBS, initially containing 3 mm glucose, using a Leica TCS-NT inverted confocal microscope, fitted with a × 40 oil immersion objective, with illumination at 568 nm from a krypton/argon laser. Fluorescence from groups of 4–7 single cells was analyzed off-line; this compensated for the considerable movement of individual mitochondria in and out of the confocal plane. Cells were fixed and permeabilized 24 h after microinjection using 4% (v/v) paraformaldehyde plus 0.2% Triton X-100. Primary polyclonal rabbit anti-luciferase antibody (Promega) was revealed with tetra-methyl-rhodamine-conjugated anti-rabbit immunoglobulin G (Sigma). Confocal images were obtained using a Leica TCS 4D/DM IRBE laser scanning confocal microscope equipped with a krypton/argon laser (568 nm excitation line) and analyzed off-line using a Silicon Graphics workstation. MIN6 cells were transiently transfected with the lipoamine Tfx-50TM(Promega) as per the manufacturer's instructions. Cells were extracted into buffer comprising 20 mm Hepes, 0.1% Triton X-100 (pH 7.2) and assayed in intracellular medium (see above) using an LB-9501 luminometer (EG & Berthold, Bad Wildbad, Germany). Data are presented as means ± S.E. for the number of observations given. ForK m measurement of the different luciferases, statistical significance was determined by Fischer test for the improvement in fitting individual K m values compared with fitting a common value (28Woodward R.H. Davies O.L. Goldsmith P.L. Statistical Methods in Research and Production. Longman Group Ltd., London1972: 178-236Google Scholar). ATP content of MIN6 cell populations was determined in extracts using firefly lantern extracts (29Stanley P.E. Williams S.G. Anal. Biochem. 1969; 29: 381-392Crossref PubMed Scopus (663) Google Scholar). Intracellular pH was measured by monitoring fluorescence changes at the single cell level using 2′7′-bis(carboxyethyl) 5(6)carboxyfluorescein as an intracellular pH indicator. Briefly, cells grown on glass coverslips and preloaded with 2′7′-bis(carboxyethyl) 5(6)carboxyfluorescein for 20 min were perifused continuously with Krebs-Ringer bicarbonate medium at a flow rate of 2 ml min−1 on the stage of a Nikon Diaphot microscope equipped with a × 40 oil immersion objective. The ratio of the emitted light at two excitation wave lengths (440/490 nm) was used to monitor intracellular pH, using commercially available software (Cairn Instruments, Faversham, Kent, United Kingdom) for data acquisition. Luminescence imaging of firefly luciferase provides adequate resolution at the single cell level but is not readily amenable to confocal or deconvolution methods necessary to achieve subcellular resolution (24Rutter G.A. Burnett P. Rizzuto R. Brini M. Murgia M. Pozzan T. Tavare J.M. Denton R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5489-5494Crossref PubMed Scopus (220) Google Scholar). We therefore used the molecular approach of targeting luciferase to distinct subcellular domains by fusion with specific peptide sequences. Fig. 1 shows the constructs used in this study. Mitochondrially targeted luciferase (mLuc) was based on wild-type Photinus pyralis firefly luciferase (M r 65 kDa), extended at the amino terminus via the 26-amino acid amino-terminal signal peptide of cytochrome c oxidase subunit VIII (23Rizzuto R. Nakase H. Darras B. Francke U. Fabrizi G.M. Mengel T. Walsh F. Kadenbach B. DiMauro S. Schon E.A. J. Biol. Chem. 1989; 264: 10595-10600Abstract Full Text PDF PubMed Google Scholar). This presequence is cleaved soon after mitochondrial import of the parental protein (30Pfanner N. Meijer M. Curr. Biol. 1997; 7: R100-R103Abstract Full Text Full Text PDF PubMed Google Scholar). pmLuc was generated by fusion with synaptosome-associated protein of 25 kDa, a neuoronal v-SNARE (11Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2001) Google Scholar) targeted to the plasma membrane and neurite extensions after palmitoylation at two amino-terminal cysteine residues (31Hess D.T. Slater T.M. Wilson M.C. Skene J.H. J. Neurosci. 1992; 12: 4634-4641Crossref PubMed Google Scholar). These constructs targeted luciferase to the mitochondrial matrix and the plasma membrane respectively (Fig. 1), as predicted from the behavior of aequorin and green fluorescent protein targeted by identical strategies (32Rizzuto R. Brini M. Pizzo P. Murgia M. Pozzan T. Curr. Biol. 1995; 5: 635-642Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 33Marsault R. Murgia M. Pozzan T. Rizzuto R. EMBO J. 1997; 16: 1575-1581Crossref PubMed Scopus (161) Google Scholar). Limited localization (< 10% of total) to a vesicular, intracellular compartment was also observed after expression of pmLuc and may represent association with secretory vesicles (34Tagaya M. Genma T. Yamamoto A. Kozaki S. Mizushima S. FEBS Lett. 1996; 394: 83-86Crossref PubMed Scopus (33) Google Scholar). The sensitivity of recombinant expressed luciferase to [ATP] was first determined in cell extracts. These assays were performed in the presence of likely intracellular concentrations of CoA (0.01 mm) (35Liang Y. Matschinsky F.M. Diabetes. 1991; 40: 327-333Crossref PubMed Google Scholar), a known regulator of enzyme activity (36Ford S.R. Buck L.M. Leach F.R. Biochim. Biophys. Acta. 1995; 1252: 180-184Crossref PubMed Scopus (31) Google Scholar). The presence of CoA greatly reduces the complex “flash” kinetics of the enzyme, believed to result either from the formation of the luciferyl·AMP intermediate, or the accumulation of the reaction product, oxyluciferin (37DeLuca M. Wannlund J. McElroy W.D. Anal. Biochem. 1979; 95: 194-198Crossref PubMed Scopus (61) Google Scholar), probably by enhancing the breakdown of the less stable enzyme·luciferyl·CoA intermediate (36Ford S.R. Buck L.M. Leach F.R. Biochim. Biophys. Acta. 1995; 1252: 180-184Crossref PubMed Scopus (31) Google Scholar, 38Sherf B.A. Wood K.V. Promega Notes. 1994; 49: 14-21Google Scholar). Calibration of the responses of each expressed and extracted luciferase to [ATP] indicated a similar K m for [ATP] of each construct, close to 1 mm (Fig. 2). We next demonstrated that luminescence from the expressed recombinant chimeras could be imaged in single living cells over the likely time frame of changes in intracellular [ATP]. Photon production was imaged in single luciferase-expressing MIN6 cells using an intensified and cooled charge-coupled device camera (15Rutter G.A. White M.R.H. Tavare J.M. Curr. Biol. 1995; 5: 890-899Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 39Kennedy H.J. Viollet B. Kahn A. Rutter G.A. Diabetic Med. 1997; 14: S8Google Scholar) attached to an inverted optics microscope equipped with a × 10 objective lens. In the presence of 1 mm luciferin, this technique allowed 2–5-s resolution in MIN6 cells, in which high levels of luciferase expression could be achieved, and 5-s resolution in primary β-cells (see below, Fig. 8). As observed previously (40Craig F.F. Simmonds A.C. Watmore D. McCapra F. White M.R.H. Biochem. J. 1991; 276: 637-641Crossref PubMed Scopus (57) Google Scholar) this concentration of luciferin was close to saturating for photon production by single cells, with little further increase in luminescence observed in the presence of 2 mm luciferin or above (data not shown). Ouabain (100 μm) caused a small increase in luminescence from cells expressing cLuc (5 ± 1%, n = 16 cells) but a more substantial increase in luminescence from cells expressing pmLuc (14 ± 1.5%, n = 15 cells) and mLuc (11.5 ± 1.5%, n = 16), as expected by the relief of ATP consumption by the plasma membrane Na+/K+-ATPase. It should be noted that these and smaller changes could readily be detected. Indeed, changes in luminescence in populations of 8–12 cells of as little as 3% were found to be statistically significant when integrated over a 40-s interval (results not shown). To determine the value of [ATP] in each subcompartment, we monitored the luminescence of single cells before and after permeabilization in the presence of ATP (Fig. 2). Single MIN6 cells were permeabilized with digitonin at the likely subdomain pH (7.2 for the cytosol and sub-plasma membrane region, or pH 7.8 for the mitochondrial matrix), intracellular free CoA (0.01 mm) (35Liang Y. Matschinsky F.M. Diabetes. 1991; 40: 327-333Crossref PubMed Google Scholar) and Mg2+(0.5 mm) (41Corkey B.E. Duszynski J. Rich T.L. Matschinsky B. Williamson J.R. J. Biol. Chem. 1986; 261: 2567-2574Abstract Full Text PDF PubMed Google Scholar, 42Rutter G.A. Osbaldeston N.J. McCormack J.G. Denton R.M. Biochem. J. 1990; 271: 627-634Crossref PubMed Scopus (60) Google Scholar). Permeabilization caused a time-dependent decrease in luminescence from each construct, presumably reflecting loss of ATP from the cell cytosol or conversion of intramitochondrial ATP to ADP (for mLuc). Re-addition of ATP caused the re-appearance of luminescence after an initial small burst, as observed in extracts (Fig. 2). Comparison of the steady-state luminescence level before and after permeabilization with the obtained standard curves indicated resting [ATP] values in the low mm range in each compartment (Fig. 2). To confirm that any stimulus-induced luminescence change was due, principally, to changes in intracellular [ATP] and was unlikely to be the result of changes in the concentration of other luciferase substrates or effectors, we monitored the effects of changes in these parameters on firefly luciferase activity in vitro and in living cells. Because luciferase activity was increased in vitro by increases in pH (activity at 1 mm ATP: 1.0, 1.2, and 1.4 arbitrary units at pH 6.8, 7.2, and 7.6, respectively), we first investigated whether pH changes may contribute to any observed luminescence change in response to cell stimulation. Transient intracellular alkalinization of cells with 10 mmNH4Cl increased the luminescence output from cells expressing each construct by 15–20%, whereas acidification with 10 mm Na+ acetate caused a decrease in light output by about the same amount. Under the conditions used in these studies, exposure of MIN6 cells to 30 mm glucose caused little or no change in intracellular pH (26Zhao C. Rutter G.A. FEBS Lett. 1998; 430: 213-216Crossref PubMed Scopus (57) Google Scholar). However, 70 mm K+ caused a small (3–5%) decrease in 2′7′-bis(carboxyethyl) 5(6)carboxyfluorescein fluorescence ratio, which could be mimicked by treatment with 4 mmNa+-acetate (data not shown), likely to correspond to a pH decrease of < 0.05 pH units (43Wang X. Levi A.J. Halestrap A.P. Am. J. Physiol. 1996; 270: H476-H484PubMed Google Scholar). We next tested the effects of increasing CoA concentration on extracted luciferase. In islets, Liang and Matschinsky (35Liang Y. Matschinsky F.M. Diabetes. 1991; 40: 327-333Crossref PubMed Google Scholar) have reported that increasing extracellular glucose from 2.5 to 25 mm raised islet CoA content by about 6% during a 30-min perfusion, from 6.8 pmol/μg DNA (equivalent to 6.8 μmol/liter, assuming 10 ngDNA·islet−1, and a cell volume of 2 pl) to 7.2 pmol/μg DNA. In our hands, a 500% increase in [CoA], from 10 to 50 μm, was required to increase luciferase luminescence in cell extracts by 28%. It should be noted that an increase in intracellular concentration of O2, another key luciferase substrate, is unlikely in response to elevated extracellular glucose concentrations, because these usually provoke increases in O2 consumption (44Panten U. Klein H. Endocrinology. 1982; 111: 1595-1600Crossref PubMed Scopus (34) Google Scholar) and an increased intracellular NAD(P)H/NAD(P)+ ratio (see below). Finally, no changes in the luminescence were apparent in cells transfected with the non-ATP-utilizing luciferase from the sea pansy, Renilla reniformis (16Kennedy H.J. Viollet B. Rafiq I. Kahn A. Rutter G.A. J. Biol. Chem. 1997; 272: 20636-20640Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) (data not shown). As a functional assay of the correct targeting of mitochondrial luciferase in living cells, we determined whether [ATP)c and [ATP]m could be altered independently in the presence of atractyloside, a potent inhibitor of the mitochondrial adenine nucleotide translocase (10Klingenberg M. Trends Biochem. Sci. 1979; 4: 249-252Abstract Full Text PDF Scopus (76) Google Scholar). As shown in Fig. 3, exposure to atractyloside caused a time-dependent decrease in luminescence from cells microinjected with cytosolic luciferase (Fig. 3). By contrast, in cells microinjected with cDNA encoding mitochondrial luciferase, an anti-parallel increase in the luminescence of mitochondrial luciferase was apparent. Exposure to 30 mm glucose of MIN6 cells expressing mLuc provoked a rapid, stable (for at least 10 min) increase in luminescence (Fig. 4, a and b). Blockade of Ca2+ influx slowed the apparent glucose-induced [ATP]m increase but had little or no effect on the final extent of the [ATP]m change (Fig. 4b; TableI). Furthermore, the effect of glucose could be mimicked, in part, by an increase in intracellular [Ca2+], provoked by exposure to high [K+] (Fig. 5, a and b). Unlike the stable luminescence i" @default.
• W1969914617 created "2016-06-24" @default.
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• W1969914617 creator A5036647095 @default.
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• W1969914617 date "1999-05-01" @default.
• W1969914617 modified "2023-10-14" @default.
• W1969914617 title "Glucose Generates Sub-plasma Membrane ATP Microdomains in Single Islet β-Cells" @default.
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