Hypoxia increases intracellular calcium in glutamate-activated horizontal cells of goldfish retina via mitochondrial KATP channels and intracellular stores.

Abstract

Central neurons of the common goldfish (Carassius auratus) are exceptional in their capacity to survive Ca²⁺-induced excitotoxicity and cell death during hypoxia. Horizontal cells (HCs) are inhibitory interneurons of the retina that are tonically depolarized by the neurotransmitter, glutamate, yet preserve intracellular Ca²⁺ homeostasis. In HCs isolated from goldfish, and in the absence of glutamatergic input, intracellular Ca²⁺ concentration ([Ca²⁺]_i) is protected from prolonged exposure to hypoxia by mitochondrial ATP-dependent K⁺ (mK_ATP) channel activity. In the present study, we investigated the effects of hypoxia upon [Ca²⁺]_i in isolated HCs during tonic activation by glutamate to better predict the effects of hypoxia in the active retina. Dynamic changes in [Ca²⁺]_i were measured using the ratiometric Ca²⁺ indicator, Fura-2. Application of 100 μM glutamate during hypoxia (P_O2 = 25 mmHg) produced a 1.3-fold greater rise in [Ca²⁺]_i compared to the same glutamate stimulus during normoxia. The hypoxia-dependent increase in [Ca²⁺]_i was abolished by application of 5-hydroxydecanoic acid, which renders mK_ATP channels inactive. Extracellular Ca²⁺ did not contribute to the elevated [Ca²⁺]_i observed during hypoxia, as the effect persisted in Ca²⁺-free solution and during application of verapamil, an L-type Ca²⁺ channel blocker. By contrast, inhibition of the mitochondrial Ca²⁺ uniporter or ryanodine receptors (with ruthenium red or ryanodine, respectively) abolished the hypoxia-dependent rise in [Ca²⁺]_i. This study reports an mK_ATP-dependent rise in [Ca²⁺]_i during hypoxia in HCs activated by glutamate, and suggests roles for the mitochondria and intracellular Ca²⁺ stores in regulating this mechanism.