Early third-instar larvae of wild-type Drosophila feed actively. The epithelium lining the gut is a single layer of cells, and is therefore an attractive preparation for study of nutrient and fluid and electrolyte transport. As there is considerable variation in the dimensions, cell types and transport functions along the distinct segments of the gut, we have developed an in vitro method for microperfusion of individual gut segments that enables independent control of solute composition of luminal and hemolymph compartments with simultaneous measurement of transepithelial potential (V t ) and resistance (Rt) and basolateral intracellular potential (V b ) and ionic activities with ion-selective microelectrodes. Midgut segments of 1 to 1.5mm length, lumen diameter 170 µm and outer diameter 200 µm, were dissected and perfused with isotonic insect Ringer at 23 o C. Details of the basic perfusion technique with glass micropipettes and measurement of electrical parameters have been described earlier (Tripathi & Boulpaep, 1988). Data are presented as means + SEM and compared by Student’s t-test using paired data. Cannulation of one end only yielded a lumen negative Vt of -17.8+0.4 mV (n=94). Simultaneous cannulation of both ends increased V t to -56.8+1.0 mV (n=94). R t measured by terminated cable analysis was 719+34 Ω.cm2 (n=26); the epithelium is therefore tighter than most tubular epithelia e.g. proximal renal tubules. Vb was -48.8+1.8 mV (n=58) and indicates that the apical membrane potential is of the order of 8 mV. Bilateral substitution of Cl with Gluconate changed Vt from -46.5+3.1 to -54.5+3.7 mV (n=8; p<0.005) and increased Rt from 822+68 Ω.cm2 to 2452+340 Ω.cm2 (n=8; p<0.0001. Bilateral substitution of Na with N-methyl-D-glucamine increased Rt from 641+71 Ω.cm>2 to 2313+535 Ω.cm2 (n=4; p<0.05). Bilateral replacement of K with Na, or HCO3 with HEPES were without significant effects. Bafilomycin A1 (a V-H+-ATPase inhibitor) added to the bath to a concentration of 750 nM reduced Vt from -27.4+1.2 to -21+1.6 mV (n= 3; p<0.005) without a significant increase in Rt. The inhibitory effect of bafilomycin was dose dependent and became significant at a concentration of 1µM. At 7.5µM transepithelial current was inhibited ~65%(control 59+8 µA.cm-2 to 21+2 µA.cm<-2 (n=5; p<0.001). We conclude that the secretion of base into the lumen by this segment is accomplished by an electrogenic V-ATPase extruding H+ to the hemolymph and is the primary driving force for electrolyte transport. The high Cl and Na permeability permits net solute transport which would also result in net water flow across the epithelium.
Supported by programme 9P-821.
TripathiS & Boulpaep EL (1988). Amer. J. Physiol.255, F188-203.