Extracellular H⁺ induces Ca²⁺ signals in respiratory chemoreceptors of zebrafish

Abstract

Neuroepithelial cells (NECs) of the fish gill are respiratory chemoreceptors that detect changes in O₂ and CO₂/H⁺ and are homologous to type I cells of the mammalian carotid body. In zebrafish (Danio rerio), stimulation of NECs by hypoxia or hypercapnia initiates inhibition of K⁺ channels and subsequent membrane depolarisation. The goal of the present study was to further elucidate, in zebrafish NECs, the signalling pathways that underlie CO₂/H⁺ sensing and generate intracellular Ca²⁺ ([Ca²⁺]_i) signals. Breathing frequency was elevated maximally in fish exposed to 5 % CO₂ (~37.5 mmHg). Measurement of [Ca²⁺]_i in isolated NECs using Fura-2 imaging indicated that [Ca²⁺]_i increased in response to acidic hypercapnia (5 % CO₂, pH 6.6) and isocapnic acidosis (normocapnia, pH 6.6), but not to isohydric hypercapnia (5 % CO₂, pH 7.6). Measurement of intracellular pH (pH_i) using BCECF demonstrated a rapid decrease in pH_i in response to acidic and isohydric hypercapnia, while isocapnic acidosis produced a smaller change in pH_i. Intracellular acidification was reduced by the carbonic anhydrase inhibitor, acetazolamide, without affecting [Ca²⁺]_i responses. Moreover, intracellular acidification using acetate (at constant extracellular pH) was without effect on [Ca²⁺]_i. The acid-induced increase in [Ca²⁺]_i persisted in the absence of extracellular Ca²⁺ and was unaffected by Ca²⁺ channel blockers (Cd²⁺, Ni²⁺ or nifedipine). The results of this study demonstrate that, unlike type I cells, extracellular H⁺ is critical to the hypercapnia-induced increase in [Ca²⁺]_i in NECs. The increase in [Ca²⁺]_i occurs independently of pH_i and appears to originate primarily from Ca²⁺ derived from intracellular stores.