High affinity 3H-phenylalanine uptake by brush border membrane vesicles from whole larvae of Aedes aegypti (AaBBMVw)
Graphical abstract
Highlights
► AaBBMVw are valid for studying characteristics of nutrient amino acid transporters. ► The Phe concentration in the lumen of 4th instar Anopheles gambiae midgut is 0.1 μM. ► The [H+], the K0.5 for Phe±, Na+ and K+ and for 3H-Phe uptake are all ⩽1 μM. ► Concentrations can be set in AaBBMVw that are in their native lipid backgrounds. ► AaBBMVw can be prepared from >300,000 larvae, frozen and shipped anywhere.
Introduction
Fresh water mosquito larvae use Na+ for nerve action potentials and for symport (i.e. co-transport) of nutrient amino acids from the alimentary canal lumen into the cells. Preparation of nutrients for digestion occurs in the anterior midgut where Na+ carbonate is the principal component of the highly alkaline lumen (Boudko et al., 2001, Smith et al., 2007). In larvae of Anopheles gambiae, amino acids are the principal energy substrates, predominant osmolytes and protein building blocks. The uptake of Phe, an essential amino acid, is coupled to Na+ or K+ by a nutrient amino acid transporter, AgNAT8 (Meleshkevitch et al., 2006). The affinities of AgNAT8 for Phe and Na+ heterologously expressed in oocytes were: and (Meleshkevitch et al., 2006). Yet the Na+ concentration in fresh water is ∼0.1 mM and in the lumen of Aedes aegypti midgut is ∼0.1 μM as deduced from its affinity during Phe uptake. The concentration of Phe in the midgut lumen is ∼0.1 μM (Fig. 1) during feeding stages and drops to near zero when feeding ceases during larval molts. If AgNAT8 is like AaNAT8, then how can transporters with millimolar affinities for Na+ and Phe carry out symport at the micromolar concentrations of Phe and Na+ in the midgut lumen?
The unexpectedly low Phe concentration in the lumen was matched by an equally unexpected high affinity for Phe during measurements of 3H Phe-uptake by AaBBMVw reported below. It appears that Ae. aegypti has evolved with a high affinity Na+-coupled Phe transporter. A promising candidate for such a transporter is AaNAT8 which is 73% identical to its Anopheline orthologue, AgNAT8 (Meleshkevitch et al., 2006) which has been immuno-localized to the apical plasma membranes of epithelial cells in gastric caeca and posterior midgut of An. gambiae larvae (Okech et al., 2008a, Okech et al., 2008b). AgNAT8 has been expressed heterologously and localized to the plasma membrane of Xenopus laevis oocytes (Meleshkevitch et al., 2006). Key components of its amino acid uptake spectrum reported there were Phe ⩾ Tyr > > Trp > > 5-HTP > Leu (Fig. 2). These data are subject to the limitation that mosquito transporters which are expressed in frog oocytes are surrounded by foreign “boundary lipids” (Hidalgo, 1987) that are likely to alter their properties. Moreover, the ∼0.1 molar Na+ concentration in the classical oocyte bathing solution is a million times greater than the ∼0.1 μM Na+ concentration in larval mosquito midgut (Fig. 1).
In an attempt to understand this discrepancy we studied tritium-labeled phenylalanine (3H-Phe) uptake into brush border membrane vesicles that were isolated from whole larvae of Ae. aegypti (Abdul-Rauf and Ellar, 1999). These vesicles are derived from apical microvilli of gastric caeca, posterior midgut and Malpighian tubules (Harvey et al., 2010). The apical membranes in epithelial cells of these regions in An. gambiae have been shown by immuno-histochemistry to contain not only AgNAT8 (Okech et al., 2008a) but also the H+ V-ATPase and the Na+/H+ antiporter, AgNHA1 (Fig. 3). BBMVw can be viewed as tiny bits of isolated midgut epithelial cell apical membranes. In this study, the properties of 3H-Phe uptake in native brush border membrane vesicles from whole Ae. aegypti larvae are compared with the published properties of Phe uptake deduced from electrical measurements on Xenopus oocyte membranes that were heterologously over-expressing AgNHA8 (Meleshkevitch et al., 2006).
Section snippets
Why study vesicles from Ae. aegypti rather than An. gambiae?
The apical membrane in gastric caeca, anterior midgut, posterior midgut and Malpighian tubules of An. gambiae is folded outwardly as projections that are called microvilli in electron micrographs but brush borders in light micrographs (because they resemble a brushy horizon). Microvilli on the apical membrane in anterior midgut of An. gambiae are short projections each enclosing a mitochondrion (Fig. 4A). By contrast in posterior midgut they are long and devoid of mitochondria (Fig. 4B).
Concentration of Phe in midgut lumen
The results of measurements of Phe in the lumen by HPLC were unexpected. The concentration of Phe in the lumen was 0.1 μM (Fig. 1) which is more than a thousand times less than the widely assumed values.
Evidence that the vesicles are from microvilli
Comparison of the ultrastructure of Ae. aegypti microvilli in vivo (Fig. 4C) with that of AaBBMVw (Fig. 4D) make it clear that the vesicles come from the microvilli. The presence of dense glycocalyx bordering the microvilli in vivo (Fig. 4C) and the isolated vesicles in vitro (Fig. 4D) provides
Amino acids, pH and voltage in mosquito larvae
Amino acids are the principal energy substrates for insects and are the principal osmolytes in insect blood. They are also substrates for protein synthesis during the ∼1000-fold increase in mass as larvae grow from the 1st to 4th instar. Unlike mammals with acidic stomachs and mildly alkaline intestines, mosquitoes have a longitudinal pH gradient that starts at ∼6 in the foregut, increases to ∼8.5 just outside gastric caeca, increases further to a maximum of ∼11 in anterior midgut, then drops
Conclusions
5.1. All components of alkali metal ion-coupled 3H-Phe uptake operate in the sub-micromolar range: 3H-Phe uptake is maximal when the concentration of H+ is 0.1 μM (pH 7); during 3H-Phe uptake, the K0.5 for Phe is 0.5 μM, for Na+ is 0.4 μM and for K+ is 0.1 μM. The concentration of Phe in the lumen of larval midguts is 0.1 μM.
5.2. Brush border membrane vesicles from whole Ae. aegypti larvae are further validated as preparations for membrane transport studies.
5.3. Unlimited quantities of AaBBMVw can
Acknowledgements
This research was supported in part by NIH Research Grants AI-52436, AI-30464 (WRH) and AI-45098 (PJL), American Heart Association Scientist Development Grant 10SDG3830004 (MAX), by funds from The Emerging Pathogens Institute and The Whitney Laboratory for Marine Bioscience, University of Florida and by a gift from Mrs. Barbara Mayer of Storrs, CT, We thank Dr. Michael J. Adang from the University of Georgia for the antibody to APN2 and Ms. M. Lynn Milstead for the artwork and assistance with
References (30)
- et al.
Isolation and characterization of brush border membrane vesicles from whole Aedes aegypti larvae
Journal of Invertebrate Pathology
(1999) - et al.
Efficiency of amino acid transport into preserved brush border membrane vesicles from lepidopteran larval midgut
Comparative Biochemistry and Physiology
(1992) - et al.
K+-dependent phenylalanine uptake in membrane vesicels isolated from the midgut of Philosamia cynthia larvae
Biochimica et Biophysica Acta
(1980) - et al.
H+V-ATPase-energized transporters in brush border membrane vesicles from whole larvae of Aedes aegypti
Journal of Insect Physiology
(2010) - et al.
Neutral amino acid symport in larval Manduca sexta midgut brush border membrane vesicles deduced from cation-dependent uptake of leucine, alanine, and phenylalanine
Biochimica et Biophysica Acta
(1993) Ion currents of Xenopus laevis oocytes: state of the art
Biochimica et Biophysica Acta
(1999)- et al.
A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane
Journal of Biological Chemistry
(1991) - et al.
Leucine transport across plasmamembranes from the scud Echinogammarus stammeri amphipoda: gammaridae
Annales de Limnologie
(2006) - et al.
Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut
Proceedings of the National Academy of Sciences of the United States of America
(2001) Amino acid transport across mammalian intestinal and renal epithelia
Physiological Reviews
(2008)