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Extra-Gastric Expression of the Proton Pump H<Sup>+</Sup>/K<Sup>+</Sup>-Atpase in the Gills and Kidney O

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Extra-Gastric Expression of the Proton Pump H<Sup>+</Sup>/K<Sup>+</Sup>-Atpase in the Gills and Kidney O © 2020. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2020) 223, jeb214890. doi:10.1242/jeb.214890 RESEARCH ARTICLE Extra-gastric expression of the proton pump H+/K+-ATPase in the gills and kidney of the teleost Oreochromis niloticus Ebtesam Ali Barnawi1, Justine E. Doherty1,Patrıciá Gomes Ferreira1 and Jonathan M. Wilson1,2,* ABSTRACT drive direct and indirect ion fluxes are the basolateral Na+/K+- Potassium regulation is essential for the proper functioning of excitable ATPase that provides a sodium motive force and an apical vacuolar- tissues in vertebrates. The H+/K+-ATPase (HKA), which is composed type proton-ATPase that provides a proton motive force (Evans of the HKα1 (gene: atp4a)andHKβ (gene: atp4b) subunits, has an et al., 2005; Hwang et al., 2011). However, Choe et al. (2004) have + + established role in potassium and acid–base regulation in mammals provided some evidence of a role of the H /K -ATPase (HKA) in and is well known for its role in gastric acidification. However, the role of ion regulation in elasmobranch fish gills. HKA in extra-gastric organs such as the gill and kidney is less clear, The main site of HKA expression is the stomach in vertebrates especially in fishes. In the present study in Nile tilapia, Oreochromis (Shin et al., 2009; Wilson and Castro, 2010). HKA is composed of α β niloticus, uptake of the K+ surrogate flux marker rubidium (Rb+)was HK 1 (gene: ATP4A)andHK (ATP4B) subunits (Pedersen and β demonstrated in vivo; however, this uptake was not inhibited with Carafoli, 1987). The -subunit is unique to the gastric HKA and is omeprazole, a potent inhibitor of the gastric HKA. This contrasts with involved in stabilizing cellular targeting of the transporter, α gill and kidney ex vivo preparations, where tissue Rb+ uptake was while the larger -subunit catalyses ATP hydrolysis and ion α significantly inhibited by omeprazole and SCH28080, another gastric translocation. The gastric HKA HK 1 subunit belongs to the large HKA inhibitor. The cellular localization of this pump in both the gill and subgroup of the P-type ATPase IIc family that also includes the α + + α kidney was demonstrated using immunohistochemical techniques with non-gastric/colonic HKA (HK 2) and Na /K -ATPases (NK ) custom-made antibodies specific for Atp4a and Atp4b. Antibodies (Pedersen and Carafoli, 1987). – against the two subunits showed the same apical ionocyte distribution Most studies of ion and/or acid base regulation in FW and + − + pattern in the gill and collecting tubules/ducts in the kidney. Atp4a saltwater (SW) teleost studies have focused on Na ,Cl and Ca + antibody specificity was confirmed by western blotting. RT-PCT was ions (Evans et al., 2005). However, K is also an ion of interest, as used to confirm the expression of both subunits in the gill and kidney. it is an essential and natural element that plays a critical role in nerve, Taken together, these results indicate for the first time K+ (Rb+)uptake muscle and many other vital cell functions, such as metabolism, + in O. niloticus and that HKA is implicated, as shown through the ex vivo growth and repair (Talling, 2010). In addition, K affects the ’ uptake inhibition by omeprazole and SCH28080, verifying a role for kidney s ability to reabsorb bicarbonate as the main extracellular HKA in K+ absorption in the gill’s ionocytes and collecting tubule/duct buffer to metabolic acids. During periods of positive potassium segments of the kidney. balance, potassium efflux pathways in SW tilapia (Furukawa et al., 2012) and FW zebrafish (Abbas et al., 2011) and medaka (Horng KEY WORDS: Potassium regulation, Fresh water, Acid–base et al., 2017) have been studied showing that orthologues to the regulation mammalian renal outer medullary potassium channel (ROMK) are present apically in branchial and skin ionocytes. In FW larval medaka, INTRODUCTION Horng et al. (2017) have proposed a paracellular uptake pathway In the aquatic environment, fishes are exposed to many challenges between skin keratinocytes that requires a high transepithelial that require regulation of ions and acid–base balance (Evans et al., potential (TEP) to operate using a SIET (scanning ion-selective 2005; Marshall and Grosell, 2005). In fresh water (FW), fishes electrode technique) approach. Studies by Eddy (1985) and Gardaire compensate for the diffusive loss of ions by active uptake of ions, and colleagues (Gardaire and Isaia, 1992; Gardaire et al., 1991) driven by pumps (Evans et al., 2005). They also maintain water suggest the possibility of branchial uptake of K+ from the water in homeostasis by getting rid of osmotically gained water by excreting rainbow trout, although the mechanism remains unknown. According copious amounts of dilute urine (Marshall and Grosell, 2005). In to Caplan (1998), H+/K+-ATPase is certainly also involved in extra- FW teleost fishes, acid–base transport takes place across the gill gastric roles such as renal acid–base regulation in tetrapods including epithelium via exchange mechanisms located in ionocytes mammals. Welling (2013) and Gumz et al. (2010) additionally [mitochondrion-rich ‘chloride’ cells (CCs)] and possibly in summarized that kidney HKA has an established role in potassium pavement or respiratory cells (PVCs) (Evans et al., 2005; Wilson, and acid–base regulation, which controls potassium absorption, and 2011). The two main pumps that have been identified in fish gill to could potentially contribute to efficient renal potassium excretion. Proton pump inhibitors (PPIs) and acid pump antagonists (APAs) are drugs that are the mainstay treatments for all acid-related 1Department of Biology, Wilfrid Laurier University, Waterloo, ON, Canada, N2L 3C5. diseases by inhibiting acid secretion with varying efficiency 2Molecular Physiology, Centro Interdisciplinar de Investigação Marinha e Ambiental, 4450-208 Matosinhos, Portugal. (Codina and DuBose, 2006; Shin et al., 2009). Omeprazole was the first drug of the PPIs class to be introduced into clinical use (in *Author for correspondence ( [email protected]) 1989) that inhibits the gastric HKA by covalent binding; hence, the P.G.F., 0000-0002-6132-1356; J.M.W., 0000-0003-3681-1166 duration of its effect is longer than expected from its levels in the blood. Unlike PPIs such as omeprazole, APAs do not require acid Received 17 September 2019; Accepted 26 June 2020 activation. Journal of Experimental Biology 1 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214890. doi:10.1242/jeb.214890 Oreochromis niloticus is also a well-studied species for fish Rubdium uptake rate was determined by measuring rubidium biology and is widely used in aquaculture globally (Maclean et al., accumulation in muscle over time using terminal sampling. 2002). Therefore, it was chosen as a model species of significance as Oreochromis niloticus (n=16, 6–15 g) were captured randomly its genome has been sequenced and annotated (ensembl.org). The from the 180 l holding tanks using a hand net, then moved to one of O. niloticus genome only has the HKα1 gene atp4a (Castro et al., four 3 l tanks (n=4 per tank) on flow through with aeration. The water 2014) and lacks the non-gastric proton pump paralogue HKα2 gene was maintained at ∼pH 7.8. Fish were acclimated overnight, and the atp12a, which simplifies analysis of the results. The apparent HKα2 Rb+ flux was initiated by stopping flow to the tanks and adding a gene (atp12a) teleost orthologue (ENSHHUG00000034153) in specific final concentration of Rb+ (0.1, 0.5, 1 or 2.5 mmol l−1 RbCl) Hucho hucho has been incorrectly annotated in ensembl.org and to each tank. An initial water sample of 10 ml was taken to check the although at least two orthologues do exist in the ancient water [Rb+]. At 0, 3, 6 and 12 h, O. niloticus were killed with an Polypteriform ray-finned fish Erpetoichthys calabaricus overdose of MS222 (0.2 g l−1; Syndel Laboratories, Nanaimo, BC, (ENSECRG00000015423 and ENSECRG00000016690), none Canada) and epaxial muscle samples (usually 0.2–1 g) were excised are found in teleost genomes (J.M.W., unpublished observation). for measurement of [Rb+]. Sample processing is described in The potential significance of extra-gastric HKA expression in ‘Analytical techniques: Rb+ kinetics’,below. teleost fishes remains to be resolved. In this paper, we provide the first evidence for the expression of the gastric proton pump HKA (genes: In vivo pharmacological inhibition of the proton pump with atp4a and atp4b) in a teleost fish and demonstrate its potential omeprazole function in potassium regulation. The central objective was to Omeprazole was used as a proton pump inhibitor to evaluate the investigate the role of this pump in extra-gastric potassium regulation activity of HKA for this experiment by comparing sham-injected in the gill and kidney of the teleost fish O. niloticus.Rb+ wasusedasa controls and omeprazole-injected fish. Treatment fish were injected surrogate flux marker for K+ and two experimental approaches were with 5 mg kg−1 omeprazole dissolved in DMSO and 0.9% sodium taken: in vivo and ex vivo Rb+ uptake assays to analyse HKA activity chloride, 12 h before and at the start of the Rb uptake periods. The in the gill and kidney. To determine whether HKA was specifically control fish were injected with an equivalent volume of 0.9% NaCl involved in Rb+ uptake, we performed a pharmacological inhibition with 2% DMSO. For injection, fish were anaesthetized with 0.2 g l−1 assay of Rb+ uptake with the gastric HKA inhibitors omeprazole of MS222, weighed and the injection volume calculated. and SCH28080. In addition, a tissue viability assay confirmed the Oreochromis niloticus were moved to individual 500 ml tanks with stability of the ex vivo preparations. Specifically, the lactate aeration and flow to the tanks was stopped after the second injection.
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