Vacuolar-type proton pump in the basolateral plasma membrane energizes ion uptake in branchial mitochondria-rich cells of killifishFundulus heteroclitus, adapted to a low ion environment

Abstract

SUMMARYWe examined the involvement of mitochondria-rich (MR) cells in ion uptake through gill epithelia in freshwater-adapted killifish Fundulus heteroclitus, by morphological observation of MR cells and molecular identification of the vacuolar-type proton pump (V-ATPase). MR cell morphology was compared in fish acclimated to defined freshwaters with different NaCl concentrations: low (0.1 mmol l-1)-, mid (1 mmol l-1)-and high (10 mmol l-1)-NaCl environments. MR cells, mostly located on the afferent-vascular side of the gill filaments, were larger in low- and mid-NaCl environments than in the high-NaCl environment. Electron-microscopic observation revealed that the apical membrane of well-developed MR cells in low- and mid-NaCl environments was flat or slightly projecting, and equipped with microvilli to expand the surface area exposed to these environments. On the other hand, in the high-NaCl environment, the apical membrane was invaginated to form a pit, and MR cells often formed multicellular complexes with accessory cells, although the NaCl concentration was much lower than that in plasma. We cloned and sequenced a cDNA encoding the A-subunit of killifish V-ATPase. The deduced amino acid sequence showed high identity with V-ATPase A-subunits from other vertebrate species. Light-microscopic immunocytochemistry, using a homologous antibody, revealed V-ATPase-immunoreactivity in Na+/K+-ATPase-immunoreactive MR cells in low-NaCl freshwater, whereas the immunoreactivity was much weaker in higher NaCl environments. Furthermore, immuno-electron microscopy revealed V-ATPase to be located in the basolateral membrane of MR cells. These findings indicate that MR cells are the site responsible for active ion uptake in freshwater-adapted killifish, and that basolaterally located V-ATPase is involved in the Na+ and/or Cl- absorbing mechanism of MR cells.

Novel and potential physiological roles of vacuolar-type H+-ATPase in marine organisms

“…For example, the rates of Cl − uptake in larval (Bayaa et al, 2009) and adult zebrafish (Boisen et al, 2003), catfish (Goss et al, 1992) and rainbow trout (reviewed in Goss et al, 1994) have been reported as 200-700 μmol kg −1 h −1 , and are therefore comparable to the rates of HCO 3 − secretion (and thus Cl − uptake) in post-fed dogfish sharks. Additionally, basolateral VHA has been reported in gill ionocytes from freshwater stingray (Piermarini and Evans, 2001;Piermarini et al, 2002), bull shark (Reilly et al, 2011), killifish (Katoh et al, 2003) and rainbow trout (Tresguerres et al, 2006a), as well as in skin ionocytes from medaka embryos (Lin et al, 2012). An early study found evidence for Cl − /HCO 3 − exchange in the osmoconforming hagfish, and proposed that the driving force for the evolution of ion uptake in freshwater fish was A/B regulation (Evans, 1984).…”

Ion uptake and acid secretion in zebrafish (Danio rerio)

“…In tilapia, type-II and type-III MR cells are analogous to zebrafish NCC cells and HR cells, respectively. However, localization of H + -ATPase (with an anti-bovine adrenal medulla V-ATPase subunit A antibody) in tilapia embryonic skin was found to be confined to the apical membrane of pavement cells in a previous study (Hiroi et al, 1998), and this needs to be confirmed because different results were obtained using an antikillifish V-ATPase A subunit antibody (Katoh et al, 2003) (T. Kaneko and J. Hiroi, personal communication).…”

Cloning and Expression of Vacuolar Proton-Pumping ATPase Subunits in the Follicular Epithelium of the Bullfrog Endolymphatic Sac

“…An ORF of 1,854 bp encoded a protein of 617 amino acids with a molecular mass calculated to be 68,168 Da. The bullfrog A-subunit showed the highest amino acid sequence similarity to the Xenopus laevis V-ATPase A-subunit (94%; AAH44025), followed by a high similarity to the rat (88%; XP-340988), mouse (88%; Laitala- Leinonen et al, 1996), human (88%; van Hille et al 1993), pig (87%; Sander et al 1992), and killifish A-subunits (87%; Katoh et al, 2003). Fig.…”