An improved chamber for the short-circuiting of epithelia.

Since the pioneering work of Ussing & Zerahn (1951), the design of short-circuit apparatus has been modified only slightly. Such chambers generally consist of two halves, between which the epithelium is stretched. The assembly is clamped together and filled with saline, which is circulated in each half chamber by gas lift pumps, usually connected to the main chamber with plastic pipes. There are two current electrodes, one at each end of the chamber, and usually two voltagesensing electrodes close to the tissue. The current passed through the chamber is adjusted until the p.d. between the sensing electrodes becomes zero. Under such conditions, only actively transported ions move across the tissue, and the shortcircuit current is a precise measure of the net flux of charged species. Shortcircuiting is thus a method of choice for investigating transport phenomena in epithelia. In this paper, we describe a chamber which differs significantly from the classical design, and illustrate its performance with specific reference to a tissue containing a potent electrogenic pump, the midgut of the lepidopteran larva, Manduca sexta (Harvey, Cioffi, Dow & Wolfersberger, 1983). The chamber (Fig. 1) consists of a short cylinder of Perspex, drilled axially with an 8-mm hole. Current electrodes of thin silver sheet are held against either end by Perspex endplates (Fig. 1), which are held to the main chamber with screws. The central bore is divided into two chamber 'halves' by a milled slot, into which the tissue-bearing aperture (Fig. 1) is inserted. Three voltage-sensing electrodes (Ag/AgCl wires, insulated to 1 mm below the tip, for normal use, or agar bridges to calomel electrodes for Ag-sensitive tissues) are inserted into the chamber, through rubber seals, on either side of the aperture (A-C in Fig. 1). The three electrode arrangement compensates for the resistivity of the saline, when this low resistivity tissue (10011cm) is short-circuited (Wood & Moreton, 1978). Circulation of fluid is accomplished with gas-lift pumps, which are integral within the main chamber block. Gas (oxygen or nitrogen) is supplied at constant pressure to the two gas inlets (F,G, Fig. 1).