Revisiting the cellular mechanisms of strong luminal alkalinization in the anterior midgut of larval mosquitoes

Abstract

Here we critically review two recent hypotheses about the mechanism of strong alkalinization by the anterior midgut of mosquito larvae and our tests of these hypotheses. We present experimental evidence against the major components of transport models proposed in these hypotheses. Measurements of the transapical and transbasal proton electrochemical gradients provide an indication of driving forces faced by and generated by the transport mechanisms of the tissue. These measurements confirmed that basal V-ATPase energizes alkalinization. Serotonin stimulates the V-ATPase, as indicated by the ensuing increase in proton-motive force across the basal membrane. Moreover, the neurohormone resulted in a surprisingly large increase in the intracellular pH. The results of inhibitor studies indicate that, contrary to previous proposals, carbonic anhydrase is apparently not involved in supplying acid-base equivalents to the respective transporters. Furthermore, any apical processes proposed to be involved in alkali secretion or acid absorption must be Cl(-) independent and insensitive to DIDS, amiloride, Zn(2+) and ouabain. These results argue against the involvement of putative apical Cl(-)/HCO (-)(3) exchangers, apical H(+) channels, apical cation/proton exchangers and the importance of the apical Na(+)/K(+) pump. The studies analyzed here thus provide both a limitation and direction for further studies of the mechanism of strong alkalinization in this system.

Fig. 1.

Transepithelial profiles of the proton-motive force (in mV) calculated from the average membrane voltages and transmembrane pH gradients for (A) a control condition with mosquito saline of pH 7 on both sides of the tissue, (B) after stimulation with serotonin (pH=7 on both sides; lumen 100 mmol l^–1 NaCl) and (C) after increasing the pH of the luminal perfusate to 10. HL, hemolymph; L, lumen.

Fig. 2.

Model of hypothetical transport mechanisms involved in strong alkalinization based on earlier proposals (Boudko et al., 2001; Onken et al., 2004), focusing on anionic pathways in the apical membrane. CA, carbonic anhydrase.

Fig. 3.

Mean lumen negative transepithelial voltages (V_te; –s.e.m.) of six anterior midguts stimulated with serotonin (0.2 μmol l^–1) in the presence (gray bar) and absence (white bar) of luminal methazolamide (200 μmol l^–1), and photographs of a representative preparation of the anterior midgut of larval (fourth instar) Aedes aegypti at identical times after perfusion stop in the presence (right) and absence (left) of luminal methazolamide (200 μmol l^–1). [Figure reproduced from Onken and colleagues (Onken et al., 2008).]

Fig. 4.

Model of hypothetical transport mechanisms involved in strong alkalinization and amino acid absorption based on earlier proposals (Okech et al., 2008; Patrick et al., 2006), focusing on cationic pathways in the apical membrane.

Fig. 5.

Mean lumen negative transepithelial voltages (V_te; –s.e.m.) of five anterior midguts stimulated with serotonin (0.2 μmol l^–1) in the presence (gray bar) and absence (white bar) of luminal amiloride (200 μmol l^–1), and photographs of a representative preparation of the anterior midgut of larval (fourth instar) Aedes aegypti at identical times after perfusion stop in the presence (right) and absence (left) of luminal amiloride (200 μmol l^–1). [Figure reproduced from Onken and colleagues (Onken et al., 2008).]

Fig. 6.

Representative time-course of the lumen negative transepithelial voltage (V_te) of the anterior midgut of larval (fourth instar) Aedes aegypti in the presence of hemolymph-side mosquito saline and luminal 100 mmol l^–1 NaCl. After mounting of the tissue, V_te declines but successively recovers after addition of serotonin (0.2 μmol l^–1) to the hemolymph-side bath and glutamine (10 mmol l^–1) to the luminal perfusate. Washout of serotonin in the presence of luminal glutamine indicates that the effect of luminal glutamine is stimulated by serotonin.