Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut

Abstract

The midgut of mosquito larvae maintains a specific lumen alkalinization profile with large longitudinal gradients (pH Ϸ 3 units⅐mm ؊1 ) in which an extremely alkaline (pH Ϸ 11) anterior midgut lies between near-neutral posterior midgut and gastric cecum (pH 7-8). A plasma membrane H ؉ V-ATPase energizes this alkalinization but the ion carriers involved are unknown. Capillary zone electrophoresis of body samples with outlet conductivity detection showed a specific transepithelial distribution of chloride and bicarbonate͞carbonate ions, with high concentrations of both anions in the midgut tissue: 68.3 ؎ 5.64 and 50.8 ؎ 4.21 mM, respectively. Chloride was higher in the hemolymph, 57.6 ؎ 7.84, than in the lumen, 3.51 ؎ 2.58, whereas bicarbonate was higher in the lumen, 58.1 ؎ 7.34, than the hemolymph, 3.96 ؎ 2.89. Timelapse video assays of pH profiles in vivo revealed that ingestion of the carbonic anhydrase inhibitor acetazolamide and the ion exchange inhibitor DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid), at 10 ؊4 M eliminates lumen alkalinization. Basal application of these inhibitors in situ also reduced gradients recorded with self-referencing pH-sensitive microelectrodes near the basal membrane by Ϸ65% and 85% respectively. Self-referencing chlorideselective microelectrodes revealed a specific spatial profile of transepithelial chloride transport with an efflux maximum in anterior midgut. Both acetazolamide and DIDS reduced chloride effluxes. These data suggest that an H ؉ V-ATPase-energized anion exchange occurs across the apical membrane of the epithelial cells and implicate an electrophoretic Cl ؊ ͞HCO 3 ؊ exchanger and carbonic anhydrase as crucial components of the steady-state alkalinization in anterior midgut of mosquito larvae.T he human stomach works at an acid pH near 1, whereas the larval mosquito midgut works at an alkaline pH near 11. The acid pH of mammalian stomachs has been thoroughly investigated and its mechanisms are understood in great detail. The alkaline pH of larval mosquito midguts has been little studied and remains largely a mystery. Recently developed techniques for direct analysis of the intact midgut in vivo and in semi-intact larvae, with a resolution that allows analytical assay of small patches of tissue, even of individual cells, have led to insights into the alkalinization mechanism.The principal function of the larval mosquito midgut is to digest and absorb nutrients. High pH values have been recorded in anterior midgut of several mosquito larvae, including Aedes aegypti (1), as well as in some lepidopteran larvae with high tannin diet (2). The alkaline environment of the anterior midgut lumen enhances dissociation of tannin-protein complexes and, therefore, would be advantageous for the nutrition of herbivorous and detritus-feeding larvae (3). The tannin-free protein could then be digested and absorbed at the near neutral pH of posterior midgut lumen (3, 4). This pattern of nutrient processing has led to the large longitudinal gradients of pH and ionic concentrat...

Bioanalytical profile of the l-arginine/nitric oxide pathway and its evaluation by capillary electrophoresis

“…UV absorbance detection was first used with CZE to detect simultaneously nitrate and nitrite in biological samples [244,245] at about the same time as HPLC technique was used for similar analyses [246]. Minor detection interference with other anions was observed by using various electrophoretic and separation modalities and buffers, except for chloride which may interfere with electrokinetic sample loading and nitrite detection in biological liquid and tissue samples [247][248][249]. A range of 210-220 nm for UV absorbance detection with apparent efficiency maximum at 214 nm is commonly used for the detection of nitrite and nitrate.…”

“…Conductivity-based assays were also optimized in a number of practical works relevant to the analysis of the L-arginine/NO pathway [101,179,[247][248][249][313][314][315][316][317]. The major challenge of CD/CCD in the determination of nitrite and nitrate in biological samples is the high concentration of Cl − .…”

“…The type of combination of CZE and ITP sample treatment can be especially useful for planar chip CE technologies such as the one introduced by Kaniansky and colleagues [318]. Our group has optimized a CZE/CD technique to measure nitrite and nitrate concentrations in hemolymph, tissues, and individual neurons samples from model mollusks [101,315], subepithelial and epithelial tissue microsamples from the midgut of mosquito larvae [249], and submicroliter tissue samples from mammalian neuronal tissues [247]. To reduce chloride interference and improve NO 2 − detection, we use sample cleanup with a home-made solid-phase microextraction (SPME) cartridge filled with Dionex OnGuard-Ag cationic exchange resin.…”

“…The first application of the technique for microanalysis of lung airway surface fluid (ASF) was with HPCE and CD, and was published in 1997 by Govindaraju et al [400]. Another example of nitrite/nitrate detection in epithelial samples investigated the profile of these anions in the midgut epithelia of the tropical disease vector mosquito larvae, Aedes aegypti [249]. Similar to neuronal model molluscs, this simple arthropod organism offers remarkable advantages in the study of epithelial functions at the level of individual cells.…”

“…Similar to neuronal model molluscs, this simple arthropod organism offers remarkable advantages in the study of epithelial functions at the level of individual cells. Submicroliter fragments of epithelial tissue (~100 cells) as well as tiny apical and basal liquid samples were analyzed using CZE, SPE and ITP staking injection [249]. In addition to major biological anions present in the samples, nitrite and nitrate were detected.…”