Acute Stress in Lesser-Spotted Catshark (Scyliorhinus canicula Linnaeus, 1758) Promotes Amino Acid Catabolism and Osmoregulatory Imbalances

Abstract

Acute-stress situations in vertebrates induce a series of physiological responses to cope with the event. While common secondary stress responses include increased catabolism and osmoregulatory imbalances, specific processes depend on the taxa. In this sense, these processes are still largely unknown in ancient vertebrates such as marine elasmobranchs. Thus, we challenged the lesser spotted catshark (Scyliorhinus canicula) to 18 min of air exposure, and monitored their recovery after 0, 5, and 24 h. This study describes amino acid turnover in the liver, white muscle, gills, and rectal gland, and plasma parameters related to energy metabolism and osmoregulatory imbalances. Catsharks rely on white muscle amino acid catabolism to face the energy demand imposed by the stressor, producing NH₄⁺. While some plasma ions (K⁺, Cl^- and Ca²⁺) increased in concentration after 18 min of air exposure, returning to basal values after 5 h of recovery, Na⁺ increased after just 5 h of recovery, coinciding with a decrease in plasma NH₄⁺. These changes were accompanied by increased activity of a branchial amiloride-sensitive ATPase. Therefore, we hypothesize that this enzyme may be a Na⁺/H⁺ exchanger (NHE) related to NH₄⁺ excretion. The action of an omeprazole-sensitive ATPase, putatively associated to a H⁺/K⁺-ATPase (HKA), is also affected by these allostatic processes. Some complementary experiments were carried out to delve a little deeper into the possible branchial enzymes sensitive to amiloride, including in vivo and ex vivo approaches, and partial sequencing of a nhe1 in the gills. This study describes the possible presence of an HKA enzyme in the rectal gland, as well as a NHE in the gills, highlighting the importance of understanding the relationship between acute stress and osmoregulation in elasmobranchs.

Keywords: NHE; Scyliorhinus canicula; air exposure; amiloride; amino acid consumption; elasmobranchs; osmoregulation; stress.

Figure 1

Amino acid consumption and NH₄⁺ production in S. canicula after air exposure. Muscle free amino acids (in µmol g⁻¹ wet weight; (A)), and plasma free amino acids (in mmol L⁻¹; (B)) and NH₄⁺ (in mmol L⁻¹; (C)) in S. canicula after air exposure and recovery. Data are expressed as mean ± s.e.m. Asterisks (*) indicate significant differences between both groups at each time (p < 0.05, n = 7–8).

Figure 2

Plasma sodium (A), chloride (B), potassium (C), and calcium (D) in S. canicula after air exposure and recovery. Data are expressed as mean ± s.e.m. Asterisks (*) indicate significant differences between both groups at each time (p < 0.05, n = 7–8).

Figure 3

Gill Na⁺/K⁺-ATPase (NKA); (A), amiloride-sensitive ATPase (possibly a Na⁺/H⁺-exchanger from the NHE family); (B), and rectal gland omeprazole-sensitive ATPase (possibly a H⁺/K⁺-ATPase from the family HKA); and (C), activities in S. canicula after air exposure and recovery. Data are expressed as mean ± s.e.m. Asterisks (*) indicate significant differences between both groups at each time (p < 0.05, n = 7–8).

Figure 4

Biochemical inhibition of an ouabain/bafilomycin A1-insensitive ATPase enzyme in gills of S. canicula by amiloride. Dose-dependent inhibition of this ATPase activity by amiloride, and half-maximal inhibitory concentration (IC50), calculated with the best-fit equation was 9.9 µmol L⁻¹. The data-points show mean ± s.e.m. from five independent assays in each of which amiloride-sensitive ATPase activity was measured in triplicate.

Figure 5

Phylogenetic and tree sequence analyses of slc9a1 (NHE1) from S. canicula. (A) Phylogenetic tree of the partial protein sequences of the identified small-spotted catshark NHE1 (indicated by black arrow) and other members of NHE family identified in other vertebrates. The phylogenetic tree was inferred using maximum likelihood method. The percentage of replicates trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Scale bar refers to a phylogenetic distance of 0.10 amino acid substitutions per site. Accession numbers are: human Homo sapiens, NM_003047.5 slc9a1, XM_047445572.1, slc9a2, NM_001284351.3 slc9a3; house mouse Mus musculus, NP_058677.1 slc9a1, NP_001028461.2 slc9a2, NP_001074529.1 slc9a3; starry ray Amblyraja radiata, XP_032900998.1 slc9a1, XP_032878489.1 slc9a2, XP_032872420.1 slc9a3; whale shark Rhincodon typus, XM_020514167.1 slc9a1; Australian ghostshark Callorhinchus milli, XM_007894944.1 scl9a1, spotted gar Lepisosteus oculatus, XM_015349113.1 slc9a1; white weakfish Atractoscion nobilis MW962258.1 slc9a1a, MW962257.1 slc9a1b, MW962261.1 slc9a2, MW962259.1 slc9a3; obscure pufferfish Takifugu obscurus, AB200332.1 slc9a1; Japanese seabass Lateolabrax maculatus, MF481092.1 slc9a1; zebrafish Danio rerio NM_001113480.1 slc9a1b, EF591983.1 slc9a2, EF591984.1 slc9a3; Japanse rice fish Oryzias melastigma, XM_024286873.2 slc9a2; greater amberjack Seriola dumerili, XP_022596365.1 slc9a2; and common carp Cyprinus carpio, XM_042730904.1 slc9a2; (B) Multiple amino acid sequence alignments of slc9a1 from different vertebrate species. The transmembrane regions are shown in bold and underlined.

Figure 6

Plasma NH₄⁺ (A), pH (B), Na⁺ (C), and urea (D) in S. canicula exposed to air and in vivo recovered in 100 µM amiloride for 5 h. The experiment includes an undisturbed-control group named “Sham”, a group sampled immediately after 18 min air exposure “Air(0h)”, and 5 h after recovery in control water “Air(5h)” or containing 100 µM amiloride “Air(5h)+Amiloride”. Data are expressed as mean ± s.e.m. Different letters indicate significantly different groups (p < 0.05, n = 12–14).

Figure 7

Amiloride-sensitive ATPase activity, putatively associated to a Na⁺/H⁺(NH₄⁺)-exchanger (NHE) (expressed as control %), in ex vivo gill explants of S. canicula incubated at different pH (A), or NH₄⁺ (B) and Na⁺ (C) concentrations. Data are expressed as mean ± s.e.m. Different letters indicate significantly different groups (p < 0.05, n = 8).