Measurement of cytoplasmic calcium concentration in the rods of wild‐type and transducin knock‐out mice

Abstract

A 10 μm spot of argon laser light was focused onto the outer segments of intact mouse rods loaded with fluo‐3, fluo‐4 or fluo‐5F, to estimate dark, resting free Ca2+ concentration ([Ca2+]i) and changes in [Ca2+]i upon illumination. Dye concentration was adjusted to preserve the normal physiology of the rod, and the laser intensity was selected to minimise bleaching of the fluorescent dye. Wild‐type mouse rods illuminated continuously with laser light showed a progressive decrease in fluorescence well fitted by two exponentials with mean time constants of 154 and 540 ms. Rods from transducin α‐subunit knock‐out (Trα–/–) animals showed no light‐dependent decline in fluorescence but exhibited an initial rapid component of fluorescence increase which could be fitted with a single exponential (τ∼1–4 ms). This fluorescence increase was triggered by rhodopsin bleaching, since its amplitude was reduced by pre‐exposure to bright bleaching light and its time constant decreased with increasing laser intensity. The rapid component was however unaffected by incorporation of the calcium chelator BAPTA and seemed therefore not to reflect an actual increase in [Ca2+]i. A similar rapid increase in fluorescence was also seen in the rods of wild‐type mice just preceding the fall in fluorescence produced by the light‐dependent decrease in [Ca2+]i. Dissociation constants were measured in vitro for fluo‐3, fluo‐4 and fluo‐5F with and without 1 mm Mg2+ from 20 to 37 °C. All three dyes showed a strong temperature dependence, with the dissociation constant changing by a factor of 3–4 over this range. Values at 37 °C were used to estimate absolute levels of rod [Ca2+]i. All three dyes gave similar values for [Ca2+]i in wild‐type rods of 250 ± 20 nm in darkness and 23 ± 2 nm after exposure to saturating light. There was no significant difference in dark [Ca2+]i between wild‐type and Trα–/– animals.

Why photoreceptors die (and why they don't)

“…2). Such a decrease in free Ca 2þ has actually been measured both from normal mouse rods exposed to light (50) and from rods of Rpe65 À/À mice even in darkness, as a consequence of the equivalent background. (34,46) Although apoptosis triggered by a decrease in Ca 2þ might seem a novel mechanism, there are several lines of evidence that give it credence.…”

“…Even when all the channels are closed, recent measurements indicate that the free Ca 2þ probably never descends below about 10-20 nM. (50,74) The reason for this may have been discovered by Paul Schnetkamp and his collaborators: (75) the activity of the exchanger is apparently inhibited when the intracellular Ca 2þ concentration becomes very low. The slowing down of the exchanger appears to stop the efflux of Ca 2þ and prevent the Ca 2þ concentration from reaching a dangerously low level.…”

Cardiac function in an endothermic fish: cellular mechanisms for overcoming acute thermal challenges during diving

“…Background fluorescence (F 0 ) was measured in each cell in a region that did not have localized or transient fluorescent elevation. The Kd of Fluo-4 was adjusted for in vivo temperature dependence [44]: 829 nM at 288C, 1594 at 148C and 1922 nM at 88C. Kinetic analyses of Ca 2þ transients are described in detail in [41] and in the electronic supplementary material.…”

Workflow and Methods of High-Content Time-Lapse Analysis for Quantifying Intracellular Calcium Signals

“…The [Ca 2+ ] i inside cells are represented by the average of fluorescence intensity of the cells as: (4) where K d is the ion dissociation constant (K d =345nM), F min is the fluorescence intensity in the absence of calcium, F max is the fluorescence intensity in the saturation of calcium, and F is the average fluorescence intensity of the cells (Woodruff et al 2002).…”