Intracellular organelles and calcium homeostasis in rods and cones

Abstract

The role of intracellular organelles in Ca2+ homeostasis was studied in salamander rod and cone photoreceptors under conditions that simulate photoreceptor activation by darkness and light. Sustained depolarization evoked a Ca2+ gradient between the cell body and ellipsoid regions of the inner segment (IS). The standing pattern of calcium fluxes was created by interactions between the plasma membrane, endoplasmic reticulum (ER), and mitochondria. Pharmacological experiments suggested that mitochondria modulate both baseline [Ca2+]i in hyperpolarized cells as well as kinetics of Ca2+ entry via L type Ca2+ channels in cell bodies and ellipsoids of depolarized rods and cones. Inhibition of mitochondrial Ca2+ sequestration by antimycin/oligomycin caused a three-fold reduction in the amount of Ca2+ accumulated into intracellular organelles in both cell bodies and ellipsoids. A further 50% decrease in intracellular Ca2+ content within cell bodies, but not ellipsoids, was observed after suppression of SERCA-mediated Ca2+ uptake into the ER. Inhibition of Ca2+ sequestration into the endoplasmic reticulum by thapsigargin or cyclopiazonic acid decreased the magnitude and kinetics of depolarization-evoked Ca2+ signals in cell bodies of rods and cones and decreased the amount of Ca2+ accumulated into internal stores. These results suggest that steady-state [Ca2+]i in photoreceptors is regulated in a region-specific manner, with the ER contribution predominant in the cell body and mitochondrial buffering [Ca2+] the ellipsoid. Local [Ca2+]i levels are set by interactions between the plasma membrane Ca2+ channels and transporters, ER and mitochondria. Mitochondria are likely to play an essential role in temporal and spatial buffering of photoreceptor Ca2+.

Tauroursodeoxycholic Acid Prevents Retinal Degeneration in Transgenic P23H Rats

“…In this context, ellipsoids of photoreceptors exhibit a high density of mitochondria that provide energy for phototransduction and the maintenance of Ca 2ϩ homeostasis. 52 The energy required for synaptic vesicular trafficking and regulation of the cytosolic Ca 2ϩ levels in the presynaptic terminals of photoreceptors is provided by a single giant mitochondrion located in each rod spherule and by an average of five medium-sized mitochondria in each cone pedicle. 27 Dysfunctional mitochondria cause an energy deficit, leading to an increase of reactive oxygen species (ROS) levels, the activation of mitochondriadependent apoptotic pathways, 53,54 and an abnormal elevation of cytosolic Ca 2ϩ .…”

Intracellular pH modulates inner segment calcium homeostasis in vertebrate photoreceptors

“…3, C and D). Unlike voltage-dependent and store-dependent Ca 2ϩ mechanisms, which were shown to be distributed in a highly polarized fashion across the inner segment (33,(63)(64)(65)(66)69), the pH-sensitive mechanism appears to be distributed uniformly across the cell body and ellipsoid region. Alkalinization strongly reduced [Ca 2ϩ ] i within the perikaryal region.…”

“…Instead, we used high and low concentrations of potassium to depolarize or hyperpolarize rod and cone inner segments loaded with fura-2 or Fluo-4 indicator dyes. The 20 mM KCl depolarized dissociated rod photoreceptors by ϳ10 mV, causing an elevation of inner segment [Ca 2ϩ ] i due to activation of L-type voltageoperated Ca 2ϩ channels and Ca 2ϩ release from internal stores (3,(63)(64)69). [Ca 2ϩ ] i elevations induced by high K ϩ typically consisted of an initial transient followed by a sustained plateau; unless noted otherwise, the experimental manipulations were performed during the plateau phase.…”

“…After being washed, cells were triturated in L-15 medium, plated onto sterilized clean glass coverslips coated with concanavalin A (1 mg/ml; Sigma-Aldrich), and allowed to settle for 40 min. Dissociated rod and cone inner segments were unambiguously identified by the morphology of the respective cell bodies and ellipsoids (e.g., 39,48,55,64). Most extracellular solutions were HCO 3 Ϫ / CO 2-free to minimize activation of HCO 3 Ϫ /Cl Ϫ exchange mechanisms and contained (in mM): 97 NaCl, 2 KCl, 2 CaCl 2, 2 MgCl2, 10 HEPES, 20 glucose, 1 pyruvic acid, 2 lactic acid, and 0.3 ascorbic acid at 240 mOsm.…”

“…After sequential image acquisition of cells' fluorescence at 495/440 nm or 340/380 nm, the backgrounds were subtracted using commercial software (Metafluor; Universal Imaging, West Chester, PA and NIS Elements, Melville, NY). Calibration of [Ca 2ϩ ]i and pHi was carried out as described previously (63)(64)(65)(66). Intracellular pH was calibrated from 495-to-440 ratios using the nigericin/high extracellular K ϩ method (10 M nigericin/90 mM KCl).…”