Measurement of cytosolic Ca2+ concentration in Limulus ventral photoreceptors using fluorescent dyes.
Several Ca-sensitive fluorescent dyes (fura-2, mag-fura-2 and Calcium Green-5N) were used to measure intracellular calcium ion concentration, Cai, accompanying light-induced excitation of Limulus ventral nerve photoreceptors. A ratiometric procedure was developed for quantification of Calcium Green-5N fluorescence. A mixture of Calcium Green-5N and a Ca-insensitive dye, ANTS, was injected in the cell and the fluorescence intensities of both dyes were used to calculate the spatial average of Cai within the light-sensitive R lobe of the photoreceptor. In dark-adapted photoreceptors, the initial Cai was 0.40 +/- 0.22 microM (SD, n = 7) as measured with fura-2. Cai peaked in the light-sensitive R lobe at 700-900 ms after the onset of an intense measuring light step, when the spatial average of Cai within the R lobe reached 68 +/- 14 and 62 +/- 37 microM (SD, n = 5) as measured with mag-fura-2 and Calcium Green-5N, respectively. The rate of Cai rise was calculated to be approximately 350 microM/s under the measuring conditions. The resting level of Mg2+ was estimated to be 1.9 +/- 0.9 mM, calculated from mag-fura-2 measurements. To investigate the effect of adapting light on the initial Cai level in the R lobe, a 1-min step of 420 nm background light was applied before each measurement. The first significant (P < 0.05) change in the initial level of Cai occurred even at the lowest adapting light intensity, which delivered approximately 3 x 10(3) effective photons/s. The relative sensitivity of the light-adapted photoreceptors was linearly related to the relative Cai on a double log plot with slope between -4.3 and -5.3. We were unable to detect a Cai rise preceding the light-activated receptor potential. The Cai rise, measured with Calcium Green-5N, lagged 14 +/- 5 ms (SD, n = 32) behind the onset of the receptor potential at room temperature in normal ASW. In the absence of extracellular Ca2+ and at 10 degrees C, this lag increased to 44 +/- 12 ms (SD, n = 17).
Ukhanov
K Y
KY
Department of Zoology, University of Maryland, College Park 20742, USA.
Flores
T M
TM
Hsiao
H S
HS
Mohapatra
P
P
Pitts
C H
CH
Payne
R
R
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D003623:Dark Adaptation / Q000502:physiology; D004594:Electrophysiology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D006737:Horseshoe Crabs / Q000378:metabolism*; D066298:In Vitro Techniques; D008086:Lissamine Green Dyes; D008274:Magnesium / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009900:Optic Nerve / Q000166:cytology / Q000502:physiology; D010775:Photic Stimulation; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism*
D005456:Fluorescent Dyes; D008086:Lissamine Green Dyes; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; C024537:green 5; D008274:Magnesium; D002118:Calcium; D016257:Fura-2