Reversible Alkalinization by Manduca Sexta Midgut

1. Midguts of the larva of the tobacco hornworm larva, Manduca sexta, were isolated, pinned out and double perfused on the stage of an inverted microscope. The pH gradients across the anterior, middle and posterior regions of the gut were measured with a double-barrelled pH microelectrode, simultaneously with electrical potentials and the transepithelial potential difference (TEP). 2. The microenvironment surrounding the apical surface is more alkaline, and that surrounding the basal surface more acid, than either the perfusion medium or the intracellular pH. Under double perfusion, a stable gradient of 1.4 pH units is observed across the middle midgut. A similar gradient is found across the anterior midgut, but no significant pH difference occurs across the posterior midgut. 3. The pH gradient across the middle midgut is reversibly and symmetrically collapsed by anoxia, implying that it is sustained by a process requiring oxidative phosphorylation. 4. The time course of decay and reconstitution of the pH gradient matches closely the activity of electrogenic K+ pumping, as measured by the TEP. 5. These results are consistent with a model for high pH generation which links electrogenic K+ transport into the goblet cavities with net alkalinization of the lumen of the anterior and middle midgut regions.