Relationship between light sensitivity and intracellular free Ca concentration in Limulus ventral photoreceptors. A quantitative study using Ca-selective microelectrodes.

Abstract

The possible role of Ca ions in mediating the drop insensitivity associated with light adaptation in Limulus ventral photoreceptors was assessed by simultaneously measuring the sensitivity to light and the intracellular free Ca concentration (Ca i) ; the latter was measured by using Ca-selective microelectrodes . In dark-adapted photoreceptors, the mean resting Cai was 3 .5 ± 2 .5,M SD (n = 31) . No correlation was found between resting Cai and absolute sensitivity from cell to cell . Typically, photoreceptors are not uniformly sensitive to light ; the Cai rise evoked by uniform illumination was 20-40 times larger and faster in the most sensitive region of the cell (the rhabdomeral lobe) than it was away from it . In response to a brief flash, the Cai rise was barely detectable when 10 2 photons were absorbed, and it was saturated when^-10 5 photons were absorbed . During maintained illumination, starting near the threshold of light adaptation, steady Cai increases were associated with steady desensitizations over several log units of light intensity : a 100-fold desensitization was associated with a 2 .5-fold increase in Ca ; . After a bright flash, sensitivity and Cai recovered with different time courses : the cell was still desensitized by -0 .5 log units when Ca i had already recovered to the prestimulus level, which suggests that under those conditions Cai is not the rate-limiting step of dark adaptation . Ionophoretic injection of EGTA markedly decreased the light-induced Cai rise and increased the time to peak of the light response, but did not alter the resting Ca;, which suggests that the time to peak is affected by a change in the capacity to bind Ca2' and not by resting Ca i . Lowering the extracellular Ca 21 concentration (Ca .) first decreased Cai and increased sensitivity. Longer exposure to low Ca. resulted in a further decrease of Ca ; but decreased rather than increased Address reprint requests to Dr.

A lingering elevation of Cai accompanies inhibition of inositol 1,4,5 trisphosphate-induced Ca release in Limulus ventral photoreceptors.

“…As a further assurance that the slow response of the electrodes did not greatly distort the measurement of the decay of Ca i transients, we compared Ca i transients recorded using singleand double-barreled, Ca-sensitive electrodes. Because their sensing area is generally larger for the same tip diameter, single-barreled Ca electrodes are usually faster than double-barreled electrodes (Levy and Fein, 1985). We measured the InsPs-and the light-induced Cai increase in two cells using single-barreled Ca electrodes and found their decay time to be in the same range as those measured with double-barreled, Ca-selective microelectrodes.…”

“…Ventral nerves from Limulus were pinned into a plexiglas chamber and superfused with artificial seawater (ASW) as described previously (Millecchia and Mauro, 1969;Levy and Fein, 1985). For measurements with Ca-selective electrodes, cells were stimulated with white light from a 45-W tungsten lamp that was brought through a shutter into the specimen plane using an optic fiber (3 mm diameter).…”

“…The method for measuring intracellular Ca 2+ using Ca-selective microelectrodes is the same as that described earlier (Levy and Fein, 1985), except that double-barreled, Ca-selective microelectrodes were used. Double-barreled, Ca-selective microelectrodes were pulled from O glass capillaries (style 1A; R & D Glass Co., Spencerville, MD) and then slightly beveled.…”

“…The A-and R-lobes can easily be identified if one strips the cells of their glia (Stern et al, 1982) or scans the photoreceptors with a microspot of light while recording the membrane potential (Levy and Fein, 1985). In previous studies it was found that light and InsP3 release Ca ~÷ predominantly from the R-lobe (Levy and Fein, 1985;Payne and Fein, 1987).…”

“…However, the low resting aequorin luminescence under these conditions, the nonlinear relationship between Cai and luminescence, and the localized nature of the InsP~-induced increase of Cai would make it difficult to quantify any elevation of Cai remaining after an injection of InsP3, even if lingering aequorin luminescence were detectable by an improved method. We have therefore measured InsP~-induced elevations in Cai using Ca-selective electrodes, which have a higher sensitivity to small elevations of Cai (Levy and Fein, 1985). After an injection of InsP3 we measured an elevation of Cai that lingered for tens of seconds.…”