The role of hydrogen sulphide in the control of breathing in hypoxic zebrafish (Danio rerio)

Abstract

Key pointsr Hydrogen sulphide (H 2 S), a gaseous neurotransmitter, is involved in oxygen sensing in glomus cells, which are oxygen-sensing cells found in the mammalian carotid body.r Neuroepithelial cells (NECs) are oxygen-sensing cells of fish and are thought to be phylogenetic precursors of mammalian glomus cells; however, the oxygen-sensing mechanisms of these cells remain largely unknown.r Both adult and larval zebrafish responded to exogenous H 2 S by increasing ventilation in a dose-dependent manner; H 2 S increased intracellular [Ca 2+ ] in NECs.r Inhibiting endogenous H 2 S production decreased or abolished the ventilatory response to hypoxia in both adult and larval zebrafish.r The results demonstrate an important role for H 2 S in oxygen sensing in zebrafish.Abstract The current study investigated the role of hydrogen sulphide (H 2 S) in oxygen sensing, intracellular signalling and promotion of ventilatory responses to hypoxia in adult and larval zebrafish (Danio rerio). Both larval and adult zebrafish exhibited a dose-dependent increase in ventilation to sodium sulphide (Na 2 S), an H 2 S donor. In vertebrates, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are enzymes that catalyse the endogenous production of H 2 S. In adult zebrafish, inhibition of both CBS and CSE with aminooxyacetate (AOA) and propargyl glycine (PPG) blunted or abolished the hypoxic hyperventilation, and the addition of Na 2 S to the water partially rescued the effects of inhibiting endogenous H 2 S production. In zebrafish larvae (4 days post-fertilization), gene knockdown of either CBS or CSE using morpholinos attenuated the hypoxic ventilatory response. Furthermore, the intracellular calcium concentration of isolated neuroepithelial cells (NECs), which are putative oxygen chemoreceptors, increased significantly when these cells were exposed to 50 μM Na 2 S, supporting a role for H 2 S in Ca 2+ -evoked neurotransmitter release in these cells. Finally, immunohistochemical labelling showed that NECs dissociated from adult gill contained CBS and CSE, whereas cutaneous NECs in larval zebrafish expressed only CSE. Taken together, these data show that H 2 S can be produced in the putative oxygen-sensing cells of zebrafish, the NECs, in which it appears to play a pivotal role in promoting the hypoxic ventilatory response.

Hydrogen sulfide promotes calcium uptake in larval zebrafish

“…This smaller size possibly reflected posttranslational cleavage of CBS as reported in mammalian systems (9). Consistent with previous observations, the reduced cbsb mRNA expression by morpholino knockdown [Kumai et al (13); Porteus et al (33)] also resulted in a reduction in its protein expression (Fig. 8E).…”

“…As reported previously [Kumai et al (13); Porteus et al (33)], the CBSb morpholino was originally designed to splice out exon 3, but it appeared to reduce total CBSb mRNA levels, likely by premature mRNA degradation. In the present study, we further confirmed the effectiveness of the CBSb knockdown by Western blot using a specific antibody against the zebrafish CBSb.…”

“…Although it has been reported that H 2 S concentration in the plasma or blood is typically in the micromolar range (measured as total sulfide) in mammals and fish (26), critical measurements of intracellular H 2 S are lacking. Previous studies suggested that H 2 S is involved in cardiorespiratory control, oxygen sensing, and Na ϩ transport in mammals and fish (1,13,25,27,31,33). In mammalian systems, it was also demonstrated that H 2 S exposure increased cytosolic Ca 2ϩ levels (15,22,24).…”

“…The CSE morpholino (5=-CGC ACA AGA GTG AAC AGC TCT CTG T-3=) was designed to splice out exon 1 (57-219 bp of NC_007117.5), and the CBSb morpholino (5=-TTG TCC TGT GAG AAA AAG CTG CAT T-3=) was designed to splice out exon 3 (385-491 bp of NM_001014345.2). The effectiveness of these morpholinos was confirmed previously (13,33). The morpholinos were diluted in Danieau buffer [58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO 4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES (pH 7.6)] plus 0.05% phenol red, and 4 ng were injected into one-cell stage embryos.…”

A role for nitric oxide in the control of breathing in zebrafish (Danio rerio)

“…Although there is an increasing awareness of the physiological significance of gasotransmitters in adult lower vertebrates (Fago et al, 2012;Olson et al, 2012), as in mammals, there are few data concerning their role during early development. To our knowledge, only two studies (Hedrick et al, 2005;Porteus et al, 2014) have systematically compared the interactive effects of gasotransmitters and developmental age and only one of these assessed the O 2 chemosensory response (Porteus et al, 2014). In that study, it was shown that endogenously produced H 2 S augmented the hyperventilatory response to hypoxia in both adult and larval (4 days post-fertilization, dpf) zebrafish, Danio rerio (Porteus et al, 2014).…”