Intracellular Ca 2+ measurements in living cells

Transcription

1 Equilibrium Res Vol.(2) Intracellular Ca 2+ measurements in living cells Narinobu Harada Harada ENT Clinic Intracellular Ca 2+ acts as the second messenger in a variety of cells. Many cellular functions are tightly regulated by the intracellular Ca 2+ concentrations ([Ca 2+ ]i). Therefore, measurement of the [Ca 2+ ]i is of critical importance. Calcium-sensitive dual excitations, indicators, such as fura-2, are now widely used to measure the [Ca 2+ ]i in living cells. The development of techniques allowing the measurement of [Ca 2+ ]i has contributed noticeably to our understanding of many cellular functions. Digital video microscopy, confocal laser scanning microscopy, and multiphoton microscopy allow accurate spatial analysis of [Ca 2+ ]i at the subcellular level. Fura-2 has been used commonlytomeasurethe[ca 2+ ]i because of the sensitivity and specificity of the method. It can be loaded into living cells with little disruption of the cellular functions. Fura-2 acetoxymethyl ester (AM) is a lipid-soluble derivative that is often used extracellularly because of its ability to pass through cell membranes, whereas fura-2 by itself cannot be introduced into the cells. For fura-2 measurements, measurements of the fluorescence intensities at two excitation wavelengths can be used to obtain an estimate of the [Ca 2+ ]i, independent of the dye concentration and the thickness of the cell membrane?. In this review, I summarize the advantages and pitfalls of using fura-2 and other standard Ca 2+ indicators, methods used to measure the [Ca 2+ ]i, including simultaneous measurements of cell movements and the changes in the [Ca 2+ ]i, and procedures used for measuring the cellular and/or subcellular Ca 2+ concentrations in living cells from the cochlea, such as the outer and inner hair cells. Key words: intracellular Ca 2+, fura-2, cochlea, simultaneous measurement, calcium imaging Ca 2+ Fura-2 [Ca 2+ ]i (81)

2 Equilibrium Res Vol.(2) Ca 2+ Ca 2+ [Ca 2+ ]i 100 nm 10 7 M Ca M Ca 2+ [Ca 2+ ]i Tsien quin 2 fura-2 indo-1 Ca 2+ Ca 2+ CCD Ca 2+ [Ca 2+ ]i fura-2 Ca 2+ [Ca 2+ ]i Ca 2+ Fura-2 Ca 2+ Ca 2+ Fura-2 AM AM Ca 2+ AM fura-2/am fura-2 Ca 2+ [Ca 2+ ]i 100 nm 10 μm Fura nm 380 nm 500 nm Fura-2 Ca nm 500 nm 380 nm 500 nm Indo nm 420 nm 470 nm [Ca 2+ ]i fluo nm 530 nm NADH NADP 480 nm fura-2 fura-2/am fura-2 Ca 2+ mmnacl 150; KCl 5; MgCl2 1; D-Glucose 5; HEPES 10; CaCl2 2 ph mosm fura-2/am Pluoronic 127 DMSO fura-2/am fura-2 5 μm fura-2/am (82)

3 Equilibrium Res Vol.(2) fura-2/am 5 μm fura-2/am fura-2/am fura -2/AM fura-2/am fura-2 /AM fura-2/am fura-2 fura-2/am fura-2 fura-2/am fura-2/am 340 nm 380 nm ARGUS-100/CA fura nm 380 nmdm nm fura-2 CCD ARGUS 100 fura-2/am fura-2 fura-2 Fura nm 380 nm (83)

4 Equilibrium Res Vol.(2) 380 nm 510 nm 340 nm 380 nm ratio ATP fura-2 [Ca 2+ ]i [Ca 2+ ]i [Ca 2+ ]i 340 nm 380 nm 340 nm/380 nm [Ca 2+ ]i 340 nm 380 nm Fura-2 fura nm fura nm ratio ATP [Ca 2+ ]i [Ca 2+ ]i DM nm 340 nm 380 nm 500 nm 510 nm CCD ARGUS 100 fura nm [Ca 2+ ]i photo bleaching 380 nm 340 nm 340 nm/380 nm 340 nm 380 nm 340 nm/380 nm 300 μl 600 μl microperpex pump LKB 2132 ATP (84)

5 Equilibrium Res Vol.(2) ) 90 A 60 Fluorescence intensity * Ratio(f340/f380nm) min Ratio(f340/f380nm) B ATP 10 sec Ionomycin [Ca 2+ ]i ionomycin 340 nm 380 nm ionomycin 380 nm 340 nm ionomycin inonomycin ATP ATP P2 [Ca 2+ ]i [Ca 2+ ]i Ca 2+ Ca 2+ fura-2fura- Ratio(f340/f380nm) ATP 60 sec ATP [Ca 2+ ]i ATP [Ca 2+ ]i 2/AM fura-2 Ca 2+ Calcium Calibration Buffer Kit, Molecular Probe Inc fura-2 Ca 2+ Ca 2+ (85)

6 Equilibrium Res Vol.(2) Ratio (f340/f380nm) )62 IA? Ratio (f340/f380nm) )62 Ratio (f340/f380nm) )62 P2 ATP [Ca 2+ ]i [Ca 2+ ]i ATP [Ca 2+ ]i [Ca 2+ ]i [Ca 2+ ]i ratio [Ca 2+ ]i [Ca 2+ ]i ratio [Ca 2+ ]i reviewer ratio [Ca 2+ ]i in vitro in vivo Ca 2+ fura-2 [Ca 2+ ]i [Ca 2+ ]i [Ca 2+ ]i 610 nm 600 nm fura-2 CCD Ca 2+ [Ca 2+ ]i K + PBFI fura nm 380 nm (86)

7 Equilibrium Res Vol.(2) 500 nm K + fluo-3 NO DAF nm 530 nm 510 nm nm 530 nm fluo-3 DAF nm 530 nm fura-2 AQUACOSMO 340 nm 380 nm nm nm [Ca 2+ ]i NO fura-2 NO Ca 2+ fura-2 Ca 2+ GFP cameleon cameleon FRET Ca 2+ Tsien, RY: New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19: , 1980 Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca 2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: , 1985 Minta A, Kao JP, Tsien RY: Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem 264: , 1989 Harada N, Ernst A, Zenner HP: Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea. Brain Res 614: , 1993 Harada N, Ernst A, Zenner HP: Volume regulation in guinea pig outer hair cells and the role of intracellular calcium. Acta Otolaryngol (Stockh) 500: 39 41, 1993 Han DY, Yamashita T, Harada N, et al: Calcium mobilization in isolated cochlear spiral ganglion cells of the guinea pig. Acta Otolaryngol (Stockh) 506: 26 29, 1993 Han DY, Harada N, Tomoda K et al: Characterization of the calcium influx induced by depolarization of guinea pig cochlear spiral ganglion cells. ORL-J-Otorhinolaryngol-Relat- Spec 56: , 1994 Harada N, Han DY, Komeda M, et al : Glutamate-induced intracellular Ca 2+ elevation in isolated spiral ganglion cells of the guinea pig cochlea. Acta Otolaryngol (Stockh) 114: , 1994 (87)

8 Equilibrium Res Vol.(2) Harada N, Ernst A, Zenner HP: Intracellular calcium changes by hyposmotic activation of cochlear outer hair cells in the guinea pig. Acta Otolaryngol (Stockh) 114: , 1994 Harada N, Cho H, Ikeda H, et al: Extracellular ATP elevated intracellular Ca 2+ in cochlear spiral ganglion cells of the guinea pig. Proc. Sendai Symposium 5: , 1995 Cho H, Harada N, Yamashita T. Extracellular ATP-induced Ca 2+ mobilization of type I spiral ganglion cells from the guinea pig cochlea. Acta Otolaryngol (Stockh) 117: , 1997 Chen L, Harada N, Yamashita T: Thimerosalinduced Ca 2+ mobilization in isolated guinea pig cochlear outer hair cells. Acta Otolaryngol (Stockh) 539: 28 33, 1998 Nagata M, Harada N, Chen L, et al: Extracellular ATP-induced calcium signaling in isolated vestibular ganglion cells of the guinea pig. Acta Otolaryngol (Stockh) 120: , 2000 Shen J, Harada N, Yamashita T: Nitric oxide inhibits adenosine 5 -triphosphate-induced Ca 2+ response in inner hair cells of the guinea pig cochlea. Neurosci Lett 337: , 2003 Yukawa H, Shen J, Harada N, et al: Acute effects of glucocorticoids on ATP-inudced Ca 2+ mobilization and nitric oxide production in cochlear spiral ganglion neurons. Neuroscience 130: , 2005 Shen J, Harada N, Nakazawa H, et al: Involvement of the nitric oxide/ cyclic GMP pathway and neuronal nitric oxide synthase in ATP-induced Ca 2+ signalling in cochlear inner hair cells. Eur. J. Neurosci. 21: , 2005 Shen J, Harada N, Kubo N, et al: Functional expression of transient receptor potential vanilloid 4 in the mouse cochlea. Neuroreport 17: , 2006 Shen J, Harada N, Nakazawa H, et al; Role of nitric oxide on ATP-induced Ca 2+ signaling in outer hair cells of the guinea pig cochlea. Brain Res. 1081: , 2006 Takeda-Nakazawa H, Harada N, Shen J, et al: Hyposmotic stimulation-induced nitric oxide production in outer hair cells of the guinea pig cochlea. Hear Res. 230: , 2007 Konishi M, Olson A, Hollingworth S, et al: Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements. Biophys J 54: , 1988 Baylor SM, Hollingworth S: Measurements and interpretation of cytoplasmic [Ca 2+ ]i signals from calcium-indicator dyes. News in Physiological Sciences 15: 19 26, 2000 Brette F, Calaghan SC, Lappin S, et al: Biphasic effects of hyposmotic challenge on excitation-contraction coupling in rat ventricular myocytes. Am J Physiol 279: H 1963 H 1971, 2000 GFP hrd@wood.odn.ne.jp (88)