The concentration of cytosolic free calcium in vertebrate rod outer segments measured with fura-2

Abstract

The use of fluorescent indicators such as fura-2 (Grynkiewicz et al., 1985) to measure the cytosolic free calcium activity in retinal rods is complicated by the rods' sensitivity both to the fluorescence and to the light that excites it. By stimulating fluorescence from large numbers of rods in whole, loaded retinas and averaging repeated measurements, however, we have been able to monitor changes in free [Ca2+]i during exposure to nonsaturating lights under physiological conditions. Retinas, isolated from the bullfrog Rana catesbeiana, were loaded with fura-2 by incubation and mounted, receptor-side up, in a perfusion chamber placed on the stage of a specially designed apparatus. A step of light delivered from above, whose wavelength alternated between 340 and 380 nm every 110 msec, excited fluorescence from 24 mm2 of retina and evoked a light response (the aspartate-isolated pIII component of the electroretinogram--ERG). By comparing the fluorescence intensities excited by the 2 wavelengths (corrected for background and dark-noise), the free [Ca2+]i of the rod outer segment was determined. In darkness, the [Ca2+]i of the outer segment was found to be approximately 220 nM. A bright light caused it to fall exponentially to approximately 140 nM, with a time constant of approximately 1.6 sec. The value of [Ca2+]i at the onset of illumination was independent of stimulus intensity over a 2 log-unit range, and in all cases the fall was monotonic. After terminating the illumination, [Ca2+]i rose again to its time-zero value.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of Cytotoxicity of Mononucleoside Phosphotriester Derivatives Bearing Biolabile Phosphate Protecting Groups in Normal Human Bone Marrow Progenitor Cells

“…Among the byproducts, formaldehyde is undoubtedly the most worrying with regards to biological toxicity. Thus, applied to isolated cells or such tissues as the retina, the toxicity of acyloxymethyl esters must be prevented by antidotes such as ascorbate and pyruvate (Ratto et al, 1988;Tsien and …”

Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-dependent adenylyl cyclase.

“…Since Ca2+ levels in ROSs reflect recent light signaling, and neuronal adenylyl cyclase is commonly regulated by Ca2+/calmodulin, the possibility that ROS adenylyl cyclase might be regulated by Ca2+ was tested. In bovine ROS preparations in which soluble components are retained, adenylyl cyclase activity exhibited a striking dependence on free [Ca2+] over its physiological range (7,(23)(24)(25) in ROS cytosol (Fig. 2).…”

Oxygen consumption in the isolated toad retina

“…Inhibiting the Na+/Ca 2+ transporter is known to elevate intracellular [Ca 2+] in photoreceptors Nakatani, 1984, 1985 ;Miller and Korenbrot, 1987) (Goldstein, 1979). The [Ca 2÷] in the subretinal space was probably somewhat higher, but within the physiological range for intracellular [Ca 2+] in photoreceptors (Ratto et al, 1988;Korenbrot and Miller, 1989). The superfusing solution was bubbled with 100% 02 in a reservoir that was gravity fed into the open chamber, where the PO2 was about 400 mmHg.…”