VOLUME-SENSITIVE CHLORIDE CONDUCTANCE IN BOVINE CHROMAFFIN CELL-MEMBRANE

1. Bovine chromaffin cells were inflated by pressure applied through a pipette or swollen during intracellular perfusion with hypertonic solutions. Effects of such procedures on electrical properties of the membrane were studied by a combination of the tight-seal whole-cell patch-clamp technique and Fura-2 fluorescence measurements of free intracellular calcium concentration ([Ca2+]i). 2. Application of air pressure (about +5 cmH2O or 490 Pa) through the patch pipette caused an increase in the cell volume and concomitant development of an inwardly directed transient current at the holding potential of -60 mV. The current gradually increased to a peak value and subsequently decayed almost to its initial level within 5-10 min. A short pulse of pressure (5-10 s) was sufficient to elicit the whole sequence of events. 3. Intracellular free Ca2+ ion concentration, [Ca2+]i, steeply increased at the beginning of the pressure pulse to about 0.2-mu-M and either stayed at this level or decayed back to the more usual value of approximately 0.1-mu-M. 4. Similar changes in the transmembrane current and [Ca2+]i were observed during intracellular perfusion of cells with hypertonic solutions (30-50 mosM difference relative to the bath solution) or during extracellular application of hypotonic solution. 5. Swelling of non-perfused cells by extracellular application of hyposmotic solution caused the appearance of inward currents in cell-attached membrane patches held at a fixed potential -30 mV relative to the cell's resting potential. The kinetics of the current resembled those of the whole-cell current. 6. Intracellular introduction of guanosine triphosphate (GTP, 300-mu-M) significantly prolonged the duration (from 62 +/- 10 s, n = 5, to 98 +/- 8 s, n = 4, when measured at the level of half-amplitude), while introduction of the non-hydrolysable analogue of guanosine diphosphate (GDP), guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S, 300-mu-M), decreased the maximal rate of increase (from 11.4 +/- 2.6 pA/s, n = 6, to 3.2 +/- 2.1 pA/s, n = 10) of the current activated by pressure. 7. Lowering of the intracellular free Ca2+ ion concentration by introduction of 10 mM-EGTA did not significantly affect the current amplitude or time course. However, a rapid increase in the [Ca2+]i to micromolar levels (by activation of the voltage-operated calcium channels during membrane depolarization) could terminate development of the current activated by pressure and cause its fast decay to zero-current level. 8. The current activated by pressure or by osmotic stress was indistinguishable in many of its properties from a current recently described in the same preparation which is activated by intracellular perfusion of GTP-gamma-S. The similarities include activation time course, ionic selectivity, single-channel conductance, block by stilbene derivatives, independence of activity from membrane potential, inhibition by intracellular administration of neomycin, and inhibition by incubation in the presence of nordihydroguaiaretic acid (NDGA). 9. Based on the delayed time course, and on the influence of guanosine nucleotides, calcium, neomycin and NDGA, it is suggested that Cl- current activation is not a direct consequence of membrane stress, but rather that of activating intracellular signal pathways involving a G protein and, possibly, arachidonic acid metabolites.