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Timing of Ca(2+) release from intracellular stores and the electrical response of Limulus ventral photoreceptors to dim flashes.

Payne R, Demas J - J. Gen. Physiol. (2000)

Bottom Line: Ca(i) was also measured during steps of light delivering approximately 10(5) effective photons/s to photoreceptors that had been bleached with hydroxylamine so as to reduce their quantum efficiency.Elevations of Ca(i) were detected at the earliest times of the electrical response to the steps of light, when a significant receptor potential had yet to develop.Light-induced elevations of Ca(i) resulted from Ca(2+) release from intracellular stores, being abolished by cyclopiazonic acid (CPA), an inhibitor of endoplasmic reticulum Ca(2+) pumps, but not by removal of extracellular Ca(2+) ions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Maryland, College Park, MD 20742, USA. rp12@umail.umd.edu

ABSTRACT
Light-induced release of Ca(2+) from stores in Limulus ventral photoreceptors was studied using confocal fluorescence microscopy and the Ca(2+) indicator dyes, Oregon green-5N and fluo-4. Fluorescence was collected from a spot within 4 microm of the microvillar membrane. A dual-flash protocol was used to reconstruct transient elevations of intracellular free calcium ion concentration (Ca(i)) after flashes delivering between 10 and 5 x 10(5) effective photons. Peak Ca(i) increased with flash intensity to 138 +/- 76 microM after flashes delivering approximately 10(4) effective photons, while the latent period of the elevation of Ca(i) fell from approximately 140 to 21 ms. The onset of the light-induced elevation of Ca(i) was always highly correlated with that of the receptor potential. The time for Ca(i) to exceed 2 microM was approximately equal to that for the receptor potential to exceed 8 mV (mean difference; 2.2 +/- 6.4 ms). Ca(i) was also measured during steps of light delivering approximately 10(5) effective photons/s to photoreceptors that had been bleached with hydroxylamine so as to reduce their quantum efficiency. Elevations of Ca(i) were detected at the earliest times of the electrical response to the steps of light, when a significant receptor potential had yet to develop. Successive responses exhibited stochastic variation in their latency of up to 20 ms, but the elevation of Ca(i) and the receptor potential still rose at approximately the same time, indicating a shared process generating the latent period. Light-induced elevations of Ca(i) resulted from Ca(2+) release from intracellular stores, being abolished by cyclopiazonic acid (CPA), an inhibitor of endoplasmic reticulum Ca(2+) pumps, but not by removal of extracellular Ca(2+) ions. CPA also greatly diminished and slowed the receptor potential elicited by dim flashes. The results demonstrate a rapid release of Ca(2+) ions that appears necessary for a highly amplified electrical response to dim flashes.

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(A) Receptor potential (solid line) and reconstructed elevation of Cai (symbols) recorded after a flash that delivered ∼50 effective photons to a photoreceptor filled with fluo-4. The bar beneath the trace indicates the timing of the flash, 50-ms duration, 5 log10 U attenuation. (B) The rising edge of the response in A is shown on an expanded time scale, together with an estimate of Cai and a receptor potential recorded during a step of illumination by the unattenuated laser beam, delivering ∼108 effective photons/s. The bars below the traces indicate the onset and duration of the stimuli.
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Figure 6: (A) Receptor potential (solid line) and reconstructed elevation of Cai (symbols) recorded after a flash that delivered ∼50 effective photons to a photoreceptor filled with fluo-4. The bar beneath the trace indicates the timing of the flash, 50-ms duration, 5 log10 U attenuation. (B) The rising edge of the response in A is shown on an expanded time scale, together with an estimate of Cai and a receptor potential recorded during a step of illumination by the unattenuated laser beam, delivering ∼108 effective photons/s. The bars below the traces indicate the onset and duration of the stimuli.

Mentions: By varying the time between dim and bright flashes, the time course of the elevation of Cai that followed the dim flash could be reconstructed (Fig. 1), provided adequate time for dark adaptation was allowed between the paired flashes. To extend the time-window during which measurements of Cai could be made, the latent period of the response to the bright flash could be extended to 70–150 ms by lowering the quantum efficiency of the cell through chemical bleaching of rhodopsin. In comparing the waveforms of the reconstructed calcium signals and the receptor potential in Fig. 2, Fig. 4, Fig. 6, and Fig. 10, it is important to note that the receptor potential shown is a representative response to the dim flash alone, recorded between presentations of the dual flashes. As Fig. 1 illustrates, there was some variation in the latency of the receptor potential from flash to flash, which becomes significant for dim flashes delivering <1,000 effective photons. This variation reduces the accuracy with which the latency of the estimated elevations of Cai and that of the receptor potential can be compared. Stable recordings of dye fluorescence and membrane potential were generally obtained for up to 90 min after injection of the dye. With ∼10 min between dual flash presentations required for dark adaptation in unbleached photoreceptors, reconstructions of elevations of Cai were usually limited to a maximum of 9 or 10 dual flash presentations.


Timing of Ca(2+) release from intracellular stores and the electrical response of Limulus ventral photoreceptors to dim flashes.

Payne R, Demas J - J. Gen. Physiol. (2000)

(A) Receptor potential (solid line) and reconstructed elevation of Cai (symbols) recorded after a flash that delivered ∼50 effective photons to a photoreceptor filled with fluo-4. The bar beneath the trace indicates the timing of the flash, 50-ms duration, 5 log10 U attenuation. (B) The rising edge of the response in A is shown on an expanded time scale, together with an estimate of Cai and a receptor potential recorded during a step of illumination by the unattenuated laser beam, delivering ∼108 effective photons/s. The bars below the traces indicate the onset and duration of the stimuli.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2232888&req=5

Figure 6: (A) Receptor potential (solid line) and reconstructed elevation of Cai (symbols) recorded after a flash that delivered ∼50 effective photons to a photoreceptor filled with fluo-4. The bar beneath the trace indicates the timing of the flash, 50-ms duration, 5 log10 U attenuation. (B) The rising edge of the response in A is shown on an expanded time scale, together with an estimate of Cai and a receptor potential recorded during a step of illumination by the unattenuated laser beam, delivering ∼108 effective photons/s. The bars below the traces indicate the onset and duration of the stimuli.
Mentions: By varying the time between dim and bright flashes, the time course of the elevation of Cai that followed the dim flash could be reconstructed (Fig. 1), provided adequate time for dark adaptation was allowed between the paired flashes. To extend the time-window during which measurements of Cai could be made, the latent period of the response to the bright flash could be extended to 70–150 ms by lowering the quantum efficiency of the cell through chemical bleaching of rhodopsin. In comparing the waveforms of the reconstructed calcium signals and the receptor potential in Fig. 2, Fig. 4, Fig. 6, and Fig. 10, it is important to note that the receptor potential shown is a representative response to the dim flash alone, recorded between presentations of the dual flashes. As Fig. 1 illustrates, there was some variation in the latency of the receptor potential from flash to flash, which becomes significant for dim flashes delivering <1,000 effective photons. This variation reduces the accuracy with which the latency of the estimated elevations of Cai and that of the receptor potential can be compared. Stable recordings of dye fluorescence and membrane potential were generally obtained for up to 90 min after injection of the dye. With ∼10 min between dual flash presentations required for dark adaptation in unbleached photoreceptors, reconstructions of elevations of Cai were usually limited to a maximum of 9 or 10 dual flash presentations.

Bottom Line: Ca(i) was also measured during steps of light delivering approximately 10(5) effective photons/s to photoreceptors that had been bleached with hydroxylamine so as to reduce their quantum efficiency.Elevations of Ca(i) were detected at the earliest times of the electrical response to the steps of light, when a significant receptor potential had yet to develop.Light-induced elevations of Ca(i) resulted from Ca(2+) release from intracellular stores, being abolished by cyclopiazonic acid (CPA), an inhibitor of endoplasmic reticulum Ca(2+) pumps, but not by removal of extracellular Ca(2+) ions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Maryland, College Park, MD 20742, USA. rp12@umail.umd.edu

ABSTRACT
Light-induced release of Ca(2+) from stores in Limulus ventral photoreceptors was studied using confocal fluorescence microscopy and the Ca(2+) indicator dyes, Oregon green-5N and fluo-4. Fluorescence was collected from a spot within 4 microm of the microvillar membrane. A dual-flash protocol was used to reconstruct transient elevations of intracellular free calcium ion concentration (Ca(i)) after flashes delivering between 10 and 5 x 10(5) effective photons. Peak Ca(i) increased with flash intensity to 138 +/- 76 microM after flashes delivering approximately 10(4) effective photons, while the latent period of the elevation of Ca(i) fell from approximately 140 to 21 ms. The onset of the light-induced elevation of Ca(i) was always highly correlated with that of the receptor potential. The time for Ca(i) to exceed 2 microM was approximately equal to that for the receptor potential to exceed 8 mV (mean difference; 2.2 +/- 6.4 ms). Ca(i) was also measured during steps of light delivering approximately 10(5) effective photons/s to photoreceptors that had been bleached with hydroxylamine so as to reduce their quantum efficiency. Elevations of Ca(i) were detected at the earliest times of the electrical response to the steps of light, when a significant receptor potential had yet to develop. Successive responses exhibited stochastic variation in their latency of up to 20 ms, but the elevation of Ca(i) and the receptor potential still rose at approximately the same time, indicating a shared process generating the latent period. Light-induced elevations of Ca(i) resulted from Ca(2+) release from intracellular stores, being abolished by cyclopiazonic acid (CPA), an inhibitor of endoplasmic reticulum Ca(2+) pumps, but not by removal of extracellular Ca(2+) ions. CPA also greatly diminished and slowed the receptor potential elicited by dim flashes. The results demonstrate a rapid release of Ca(2+) ions that appears necessary for a highly amplified electrical response to dim flashes.

Show MeSH
Related in: MedlinePlus