Caffeine-induced Ca²⁺ Oscillations In Horizontal Cells Of Carp Retina And The Contribution Of The Store-operated Ca²⁺ Entry Pathway

Ca²⁺ is a ubiquitous intracellular messenger that regulates numerous cellular processes. As the primary intracellular reservoir of Ca²⁺ and a major source of [Ca²⁺]i elevation, the endoplasmic reticulum（ER） is involved in a wide range of neuronal Ca²⁺-dependent processes, such as synaptic transmission and plasticity. Retinal horizontal cells（HCs） receive excitatory inputs from photoreceptors, and their feedback to photoreceptors underlies the formation of center-surround antagonistic receptive field of bipolar cells and ganglion cells. It was reported that ryanodine receptors（Ry Rs）, a type of ER Ca²⁺ release channel which can be activated by Ca²⁺, are expressed in HCs of the teleost retina. Ca²⁺ release from the ER is involved in many HCs cellular functions, including the modulation of synaptic strength between HCs and photoreceptors, GABA transporter currents, as well as voltage-gated Ca²⁺ channels. However, the detailed mechanisms of ER Ca²⁺ signaling are unclear, and this study is important for understanding phenomena, like synaptic plasticity of HCs, and their physiological functions.To investigate the mechanisms of release, depletion, and refilling of ER Ca²⁺, [Ca²⁺]i changes initiated by the activation of Ry Rs were studied in type I horizontal cells（H1 cells） of the carp retina using a Fluo-3-based Ca²⁺ imaging technique. Experimental results showed that exogenous application of caffeine, a Ry R agonist, induced oscillatory intracellular free Ca²⁺ concentration([Ca²⁺]i) responses in a duration- and concentration-dependent manner. In Ca²⁺-free Ringer’s solution, [Ca²⁺]i transients could also be induced by a brief caffeine application, whereas subsequent caffeine application induced no [Ca²⁺]i increase, which implied that extracellular Ca²⁺ was required for ER refilling, confirming the necessity of Ca²⁺ influx pathways for ER refilling. Depletion of ER Ca²⁺ stores by thapsigargin triggered a Ca²⁺ influx which could be blocked by the store-operated channel（SOC） inhibitor 2-aminoethoxydiphenyl borate（2-APB）, which proved the existence of store-operated Ca²⁺ entry pathways. Taken together, these results suggested that after being depleted by caffeine, the ER was replenished by Ca²⁺ influx via SOCs.[Ca²⁺]i oscillations result from a complex interplay of nonlinear processes, so in the second part, a mathematical model was constructed to simulate caffeine-induced [Ca²⁺]i oscillations in H1 cells of the carp retina, and we also studied the effect of caffeine concentration on [Ca²⁺]i oscillations and the possible mechanism underlying inhibition of [Ca²⁺]i oscillations by 2-APB. Model results showed that the caffeine-induced [Ca²⁺]i oscillations involve a number of cytoplasmic and endoplasmic Ca²⁺ processes that interact with each other. The model results also suggested that SOC-mediated Ca²⁺ entry is the main source of ER refilling, and the maintenance of caffeine-induced [Ca²⁺]i oscillations depends on the activation of SOCsThese results reveal the fine modulation of ER Ca²⁺ signaling, and the activation of the store-operated Ca²⁺ entry pathway guarantees the replenishment of the ER so that the cell can be ready for response to the subsequent stimulus.