THE EFFECT OF Na⁺ AND Cl^- REMOVAL AND OF LOOP DIURETICS ON ACETYLCHOLINE-EVOKED MEMBRANE POTENTIAL CHANGES IN MOUSE LACRIMAL ACINAR CELLS

¹ Medical Research Council, Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, P.O. Box 147, Liverpool L69 3BX

Intracellular micro-electrode recordings were made from acinar units in mouse lacrimal gland segments superfused with physiological saline solutions. Two micro-electrodes were used: one for recording the membrane potential and the other for current injection. A third extracellular micropipette was used for local ionophoretic acetylcholine (ACh) application. The ACh-evoked membrane potential change was recorded at the spontaneous resting potential and after the resting potential had been increased or decreased by direct current injection. The membrane potential at which ACh did not evoke any change in potential (null potential, reversal potential) (E_ACh) was determined. E_ACh was about -53 mV under normal ionic conditions. When all Cl^- in the superfusion fluid was replaced by NO₃^- or all Na+ replaced by N-methyl-D-glucamine⁺ (NMDG⁺) or Tris⁺, E_ACh was shifted to more negative values. During exposure to Cl^--free NO₃^- solution or Na⁺-free NMDG⁺ solution E_ACh was about -72 mV. These effects on E_ACh were fully reversible. Inclusion of the loop diuretics piretanide (2 x 10^-4 M) or furosemide (l0^-3 M) also shifted E_ACh towards more negative values. In these cases E_ACh was the same as during exposure to Na⁺-free Tris⁺ solution with a value of about -62 mV. Replacement of extracellular Cl^- by Br^- had no effect on E_ACh whereas replacement of Na⁺ by Li⁺ shifted E_ACh towards less negative values. The results can most easily be understood in terms of a cellular transport model in which Ca²⁺-activated Cl^- channels are present in the luminal membrane whereas the basolateral membrane contains three transport proteins: the Na⁺-K⁺-pump, the Na⁺-K⁺-2Cl^- co-transporter and the Ca²⁺-activated K⁺ channel. These three transport proteins function together as a Cl^- pump. ACh acts to open both Cl^- and K⁺ channels via an increase in the free [Ca²⁺]_i. When the Na⁺-K⁺-2Cl^- co-transporter is blocked by Na⁺ removal or by the loop diuretics, [Cl^-]_i drops and E_Cl (the Cl^- equilibrium potential) becomes more negative. The permeability of the Cl^- pathway may also decrease.