Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut

The midgut of mosquito larvae maintains a specific lumen alkalinization profile with large longitudinal gradients (pH ≈ 3 units⋅mm(−1)) in which an extremely alkaline (pH ≈ 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 bicarbonate/carbonate 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. Time-lapse 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 ≈65% and 85% respectively. Self-referencing chloride-selective 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 [Formula: see text] exchanger and carbonic anhydrase as crucial components of the steady-state alkalinization in anterior midgut of mosquito larvae.

蚊子幼虫的中肠具有独特的管腔碱化特征，存在显著的纵向pH梯度（约3个pH单位·毫米⁻¹），其中呈强碱性（pH≈11）的前中肠位于近中性的后中肠与胃盲囊（pH 7~8）之间。质膜H⁺V-ATP酶（plasma membrane H(+) V-ATPase）为该碱化过程提供能量，但所涉及的离子载体迄今尚未明确。采用出口电导检测的毛细管区带电泳（capillary zone electrophoresis）对虫体样本进行分析，结果显示氯离子与碳酸氢根/碳酸根离子呈现特异性的跨上皮分布：中肠组织内两种阴离子的浓度分别为68.3±5.64 mM与50.8±4.21 mM，含量均较高。血淋巴中的氯离子浓度（57.6±7.84 mM）高于管腔（3.51±2.58 mM）；而碳酸氢根则在管腔中浓度更高（58.1±7.34 mM），显著高于血淋巴（3.96±2.89 mM）。体内pH分布特征的延时视频检测实验显示，以10⁻⁴ M浓度投喂碳酸酐酶抑制剂乙酰唑胺（acetazolamide）与离子交换抑制剂DIDS（4,4′-二异硫氰酸二苯乙烯-2,2′-二磺酸，4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid），可完全消除管腔的碱化现象。在原位条件下施加上述抑制剂，也可使基底膜附近经自参比pH敏感微电极检测到的pH梯度分别降低约65%与85%。自参比氯离子选择性微电极检测结果显示，跨上皮氯离子转运具有特异性空间分布特征，在前中肠区域氯离子外流达到峰值。乙酰唑胺与DIDS均可抑制氯离子外流。上述数据表明，上皮细胞顶膜存在由H⁺V-ATP酶供能的阴离子交换过程，并提示电驱动型Cl⁻/HCO₃⁻交换体与碳酸酐酶是蚊子幼虫前中肠稳态碱化过程的关键组成成分。