Isolation of the V-ATPase A and c subunit cDNAs from mosquito midgut and malpighian tubules

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164

174

SUMMARY This study describes the expression patterns of P-type Na+/K+-ATPase and V-type H+-ATPase in the larval and adult forms of the mosquito Aedes aegypti and provides insight into their relative importance in ion transport function of key osmoregulatory organs. RT-PCR assays indicate that, at the level of the gene,both ATPases are expressed in all of the osmoregulatory tissues of larvae(midgut, Malpighian tubules, rectum and anal papillae) and adults (stomach,Malpighian tubules, anterior hindgut and rectum). Immunohistochemical studies determined that both ATPases are present in high levels in all the relevant organs, with the exception of the larval rectum (P-type Na+/K+-ATPase only). In larval gastric caeca, ATPase location corresponds to the secretory (basal P-type Na+/K+-ATPase, apical V-type H+-ATPase) and ion-transporting (V-type H+-ATPase on both membranes) regions as previously described. The two ATPases switch membrane location along the length of the larval midgut, indicating three possible regionalizations,whereas the adult stomach has uniform expression of basolateral P-type Na+/K+-ATPase and apical V-type H+-ATPase in each cell. In both larval and adult Malpighian tubules, the distal principal cells exhibit high expression levels of V-type H+-ATPase (apically and cytoplasmically) whereas P-type Na+/K+-ATPase is highly expressed in stellate cells found only in the distal two-thirds of each tubule. By contrast, the proximal principal cells express both P-type Na+/K+-ATPase (basal) and V-type H+-ATPase(apical). These results suggest a functional segregation along the length of the Malpighian tubules based on cell type and region. P-type Na+/K+-ATPase is the only pump apparent in the larval rectum whereas in the larval anal papillae and the adult hindgut (including the anterior hindgut and rectum with rectal pads), P-type Na+/K+-ATPase and V-type H+-ATPase localize to the basal and apical membranes, respectively. We discuss our findings in light of previous physiological and morphological studies and re-examine our current models of ion transport in these two developmental stages of mosquitoes that cope with disparate osmoregulatory challenges.

Section: Midgutmentioning

confidence: 93%

P-type Na+/K+-ATPase and V-type H+-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquitoAedes aegypti

Patrick

Aimanova

Sanders

et al. 2006

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164

174

“…Degenerate PCR primers were designed on the basis of sequences from selected animals (Pan et al, 1991;Graf et al, 1992;Sander et al, 1992;Hille et al, 1993;Hernando et al, 1995;Gill et al, 1998) to obtain a partial cDNA fragment of killifish V-ATPase A-subunit, sense: (Fig. 4).…”

Section: Molecular Identification Of the A-subunit Of V-atpasementioning

confidence: 99%

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

Katoh

Hyodo

Kaneko

2003

127

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.

“…Gill et al (1998) isolated cDNA clones of two V-ATPase subunits from the stomach of Aedes aegypti larvae, and a high level of expression of one of these subunits was observed in the anterior stomach (Filippova et al, 1998). Zhuang et al (1999) showed that V-ATPases are localized on the basal membranes of the anterior stomach, whereas the ATPase is found in the apical membrane of the caeca and posterior stomach.…”

mentioning

confidence: 99%

The transepithelial voltage of the isolated anterior stomach of mosquito larvae (Aedes aegypti): pharmacological characterization of the serotonin-stimulated cells

Onken

Moffett

2004

SUMMARYThe lumen-negative transepithelial voltage (Vte) of the isolated and perfused anterior stomach of mosquito larvae (Aedes aegypti) was studied with a `semi-open' preparation in which one end of the gut was ligated onto a perfusion pipette and the other end remained open to the bath. All experiments were performed with serotonin-stimulated preparations. Vte was abolished after addition of 2.5 mmol l-1 dinitrophenol and depended on the presence of Cl-. Na+ substitution experiments showed that a major part of Vte depended on the presence of this cation in the hemolymph side of the epithelium. Addition of 10 μmol l-1concanamycin (78±6% inhibition) or 2.5 mmol l-1 ouabain(15±2% inhibition) to the bath partially inhibited Vte. DPC (0.5 mmol l-1) or DIDS (0.1 mmol l-1) reduced Vte when applied to the hemolymph side of the epithelium (to 49±8% or 78±3% of the control,respectively). When present on both sides of the epithelium, these inhibitors caused further Vte reductions (to 23±4% or 35±4% of the control, respectively). Hemolymph-side furosemide (0.1 mmol l-1) or BaCl2 (5 mmol l-1) reduced Vte by 13±3% or 23±4% of the control,respectively. When applied to the hemolymph side of the epithelium, amiloride(0.2 mmol l-1) significantly decreased Vte by 35±6% of the control, whereas the drug caused no further effect when it was subsequently also applied to the luminal side of the epithelium. The above results are the basis for an extended model for the cellular mechanisms of NaHCO3 secretion/HCl absorption involved in alkalization of the anterior stomach of mosquito larvae.