SummaryThere is a resurgence of interest in the Drosophila midgut on account of its potential value in understanding the structure, development and function of digestive organs and related epithelia. The recent identification of regenerative or stem cells in the adult gut of Drosophila has opened up new avenues for understanding development and turnover of cells in insect and mammalian gastrointestinal tracts. Conversely, the physiology of the Drosophila gut is less well understood as it is a difficult epithelial preparation to study under controlled conditions. Recent progress in microperfusion of individual segments of the Drosophila midgut, in both larval and adult forms, has enabled ultrastructural and electrophysiological study and preliminary characterization of cellular transport processes in the epithelium. As larvae are more active feeders, the transport rates are higher than in adults. The larval midgut has at least three segments: an anterior neutral zone, a short and narrow acid-secreting middle segment and a long and wider posterior segment (which is the best studied) that secretes base (probably

SUMMARY1. Water flows generated by osmotic and hydrostatic pressure and electrical currents were measured in sheets of isolated rabbit ileum at 20 'C. Flows across the mucosal and serosal surfaces were monitored continuously by simultaneous measurement of tissue volume change (with an optical lever) and net water flows across one surface of the tissue (with a capacitance transducer).2. Osmotic gradients were imposed across the mucosal and serosal surfaces of the tissue separately, using probe molecules of various sizes from ethanediol (68 Da) to dextrans (161000 Da). Flows across each surface were elicited with very short delay. The magnitudes of the flows were proportional to the osmotic gradient and related to the size of the probe molecule. Osmotic flow across the mucosal surface was associated with streaming potentials which were due to electro-osmotic water flow.3. The mucosal surface is a heteroporous barrier with narrow (0 7 nm radius, Lp (hydraulic conductivity) = (7-6±+ 16) x 10-9 cm s-1 cmH20-1) cation-selective channels in parallel with wide neutral pores (ca. 6-5 nm radius, Lp = (2-3 + 0 2) x 10-7 cm s-1 cmH20-1) which admit large pressure-driven backflows from the submucosa to the lumen. There is additional evidence for a further set of narrow electroneutral pores < 0 4 nm radius with Lp < 7 x 10-9 cm s-1 cmH20-1.The serosal surface has neutral pores of uniform radius (ca. 6-5 nm), Lp = (7-6± 16) x 10-8 cm s-1 cmH20-'.4. Hypertonic serosal solutions (100 mM-sucrose) cause osmotic transfer of fluid from isotonic mucosal solutions into the submucosa, expand it, and elevate the tissue pressure to 19-6 + 3-2 cmH20 (n = 4). Conversely, hypertonic mucosal solutions (100 mM-sucrose) draw fluid out of the submucosa in the presence of isotonic serosal solutions, collapse the submucosa, and lower the tissue pressure to -87-7 + 4-6 cmH20 (n= 5).5. Water flows coupled to cation movement could be generated across the mucosal surface in both directions by brief direct current pulses. The short latency of onset and cessation of flow (< 2 s), absence of polarization potentials, and high electroosmotic coefficients (range 50-520 mol water F-1), together with the presence of streaming potentials during osmotically generated water flows indicate electro-* Present address:

A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by approximately 17-fold and approximately 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (V(t)) of 38 to 45 mV and high resistance (R(t)) of 800 to 1,400 Omega x cm(2) measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10(-4) M, ouabain 10(-4) M, bumetanide 10(-4) M), K(+) -free solutions, or organic solutes such as D-glucose 10 mM or DL-alanine 0.5 mM failed to affect V(t) or R(t). Bafilomycin-A(1) (3 to 5 microM) decreased V(t) by approximately 40% and short-circuit current (I(sc)) by approximately 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H(+) -ATPase located in the basal membrane. Gradients of H(+) were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO(3)(-)), driven by the basal V-H(+) -ATPase.

An electrophysiological approach is used to analyze the possible routes of osmotically driven water flow across the isolated perfused Ambystoma proximal tubule. The minimum hydraulic conductivities (Lp) of the cell membranes were estimated from the initial rate of change of intracellular activities of Na+ and K+ in response to a step gradient of 50 or 100 mosmol/kg sucrose. The Lp of the apical membrane is 1.30 X 10(-4) cm.s-1.osM-1 referred to the luminal epithelial surface and 2.45 X 10(-6) cm.s-1.osM-1 when corrected for amplification of the brush border (n = 8). The Lp of the basolateral membrane is 1.42 X 10(-4) cm.s-1.osM-1 referred to the basement membrane surface and 6.39 X 10(-6) cm.s-1.osM-1 when corrected for the amplification of the basal and lateral membranes (n = 5). Transepithelial water flows were generated in either direction by a unilateral step increase of osmolality with 100 mosmol sucrose. Bath-to-lumen flow increased paracellular transepithelial resistance (R3) by 48%; lumen-to-bath flow decreased R3 by only 3%. A bilateral increase in the osmolality of both solutions by 50 mosM had no significant effect on R3. Streaming potentials were observed during trans-epithelial water flow induced by unilateral gradients of sucrose; their polarity, magnitude, site of generation, and insensitivity to change of paracellular resistance are all indicative of water flow through paracellular structures, especially the lateral intercellular spaces. Contrary to earlier suggestions (J. M. Diamond, J. Membr. Biol. 51: 195-216, 1979), these potentials are not primarily diffusion potentials across anion-selective tight junctions resulting from solute polarization in the unstirred layers. Instead, a true electrokinetic basis for these streaming potentials is indicated by their continued presence after deletion of all Cl-. Thus water moves through both cellular and paracellular pathways in this epithelium.

(Lp) of the mucosal boundary of the lateral intercellular space is approximately 1 x 10-8 cm S-1 cmH20-1. This Lp is too low to sustain isotonicity ofthe flow emerging from the lateral intercellular space at the observed rates. Hypertonic fluid emerging from the lateral intercellular space is diluted by transcellular water flow generated by the hypertonicity of the submucosa and back-diffusion of solute via mucosal shunt