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Bleached pigment produces a maintained decrease in outer segment Ca2+ in salamander rods.

Sampath AP, Matthews HR, Cornwall MC, Fain GL - J. Gen. Physiol. (1998)

Bottom Line: The resting level of Ca2+i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 +/- 5% (SEM, n = 9) of its value in the dark-adapted rod.Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+i.These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science, University of California, Los Angeles, Los Angeles, California 90095, USA.

ABSTRACT
A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca2+i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca2+i estimates of 670 +/- 250 nM in a dark-adapted rod and 30 +/- 10 nM during response saturation after exposure to bright light (mean +/- SD). The resting level of Ca2+i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 +/- 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

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Changes in circulating current, sensitivity, and fluo-3  fluorescence during the photopigment cycle. (A) Circulating current recorded by the suction pipette measured as the current suppressed by the laser exposure or by a saturating flash of light. (B)  Response sensitivity measured using trains of dim flashes delivering 0.74 (dark-adapted, before the first laser exposure), 11  (bleached), and 2.6 (regenerated) photon μm−2 at 570 nm. (C)  Fluo-3 fluorescence in response to trains of laser pulses presented  to the rod when dark adapted, after bleaching, and after regeneration. Under each condition, two successive trains of laser pulses  were presented in rapid succession; the values plotted represent  the maximum and minimum fluorescence levels in darkness and  after complete suppression of the circulating current. In each case,  the column representing the second fluorescence measurement  has been displaced slightly to the right for clarity. Top traces denote the delivery of bright flashes (Light), trains of laser pulses (Laser), and superfusion with phospholipid vesicles containing 11-cis- retinal (11-cis).
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Figure 7: Changes in circulating current, sensitivity, and fluo-3 fluorescence during the photopigment cycle. (A) Circulating current recorded by the suction pipette measured as the current suppressed by the laser exposure or by a saturating flash of light. (B) Response sensitivity measured using trains of dim flashes delivering 0.74 (dark-adapted, before the first laser exposure), 11 (bleached), and 2.6 (regenerated) photon μm−2 at 570 nm. (C) Fluo-3 fluorescence in response to trains of laser pulses presented to the rod when dark adapted, after bleaching, and after regeneration. Under each condition, two successive trains of laser pulses were presented in rapid succession; the values plotted represent the maximum and minimum fluorescence levels in darkness and after complete suppression of the circulating current. In each case, the column representing the second fluorescence measurement has been displaced slightly to the right for clarity. Top traces denote the delivery of bright flashes (Light), trains of laser pulses (Laser), and superfusion with phospholipid vesicles containing 11-cis- retinal (11-cis).

Mentions: The principal objective of this study was to investigate how Ca2+i changes in the rod outer segment after photopigment bleaching. This was achieved by making repeated measurements of fluo-3 fluorescence from a single salamander rod when dark adapted, after exposure to laser light sufficiently intense to bleach a substantial fraction of the photopigment, and after regeneration with 11-cis-retinal, as illustrated in Figs. 6 and 7. First, the sensitivity of the dark-adapted rod was measured with dim flashes. Then laser pulses were delivered to investigate the decline of Ca2+i from its dark-adapted level (Fig. 6 A). The corresponding fluorescence intensity evoked by the first laser pulse has been plotted in Fig. 7 C, together with the level to which it declined after the bleach-induced suppression of the dark current. These values reflect relative Ca2+i levels before and immediately after the bleaching laser exposure. These two sequences of laser illumination, each consisting of four 20-ms laser pulses, are calculated to have bleached >99% of the photopigment within the area of the laser spot (see methods), while scattered laser light is likely to have caused significant bleaching within the remainder of the outer segment.


Bleached pigment produces a maintained decrease in outer segment Ca2+ in salamander rods.

Sampath AP, Matthews HR, Cornwall MC, Fain GL - J. Gen. Physiol. (1998)

Changes in circulating current, sensitivity, and fluo-3  fluorescence during the photopigment cycle. (A) Circulating current recorded by the suction pipette measured as the current suppressed by the laser exposure or by a saturating flash of light. (B)  Response sensitivity measured using trains of dim flashes delivering 0.74 (dark-adapted, before the first laser exposure), 11  (bleached), and 2.6 (regenerated) photon μm−2 at 570 nm. (C)  Fluo-3 fluorescence in response to trains of laser pulses presented  to the rod when dark adapted, after bleaching, and after regeneration. Under each condition, two successive trains of laser pulses  were presented in rapid succession; the values plotted represent  the maximum and minimum fluorescence levels in darkness and  after complete suppression of the circulating current. In each case,  the column representing the second fluorescence measurement  has been displaced slightly to the right for clarity. Top traces denote the delivery of bright flashes (Light), trains of laser pulses (Laser), and superfusion with phospholipid vesicles containing 11-cis- retinal (11-cis).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1887770&req=5

Figure 7: Changes in circulating current, sensitivity, and fluo-3 fluorescence during the photopigment cycle. (A) Circulating current recorded by the suction pipette measured as the current suppressed by the laser exposure or by a saturating flash of light. (B) Response sensitivity measured using trains of dim flashes delivering 0.74 (dark-adapted, before the first laser exposure), 11 (bleached), and 2.6 (regenerated) photon μm−2 at 570 nm. (C) Fluo-3 fluorescence in response to trains of laser pulses presented to the rod when dark adapted, after bleaching, and after regeneration. Under each condition, two successive trains of laser pulses were presented in rapid succession; the values plotted represent the maximum and minimum fluorescence levels in darkness and after complete suppression of the circulating current. In each case, the column representing the second fluorescence measurement has been displaced slightly to the right for clarity. Top traces denote the delivery of bright flashes (Light), trains of laser pulses (Laser), and superfusion with phospholipid vesicles containing 11-cis- retinal (11-cis).
Mentions: The principal objective of this study was to investigate how Ca2+i changes in the rod outer segment after photopigment bleaching. This was achieved by making repeated measurements of fluo-3 fluorescence from a single salamander rod when dark adapted, after exposure to laser light sufficiently intense to bleach a substantial fraction of the photopigment, and after regeneration with 11-cis-retinal, as illustrated in Figs. 6 and 7. First, the sensitivity of the dark-adapted rod was measured with dim flashes. Then laser pulses were delivered to investigate the decline of Ca2+i from its dark-adapted level (Fig. 6 A). The corresponding fluorescence intensity evoked by the first laser pulse has been plotted in Fig. 7 C, together with the level to which it declined after the bleach-induced suppression of the dark current. These values reflect relative Ca2+i levels before and immediately after the bleaching laser exposure. These two sequences of laser illumination, each consisting of four 20-ms laser pulses, are calculated to have bleached >99% of the photopigment within the area of the laser spot (see methods), while scattered laser light is likely to have caused significant bleaching within the remainder of the outer segment.

Bottom Line: The resting level of Ca2+i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 +/- 5% (SEM, n = 9) of its value in the dark-adapted rod.Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+i.These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science, University of California, Los Angeles, Los Angeles, California 90095, USA.

ABSTRACT
A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca2+i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca2+i estimates of 670 +/- 250 nM in a dark-adapted rod and 30 +/- 10 nM during response saturation after exposure to bright light (mean +/- SD). The resting level of Ca2+i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 +/- 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

Show MeSH
Related in: MedlinePlus