Figure 8.

Effect of TTX, low [Ca²⁺]_o, Gd³⁺, flufenamic acid, or SKF-96365 on glycine release evoked by the application of a hypotonic solution. A, TTX application did not significantly change the amplitude of the sniffer currents (105.8 ± 9.8% of control; n = 5) evoked by the application of a hypotonic solution, indicating that the glycine release evoked by membrane stretch caused by cell swelling is independent of Na⁺ action potential firing. B, To determine whether glycine release evoked by hypotonic solution is dependent on external Ca²⁺, we analyzed the effect of a low Ca²⁺ external solution (0 mm [Ca²⁺]_o and 1 mm EGTA). The amplitude of the evoked sniffer current was significantly (Wilcoxon's test, p < 0.01) and reversibly decreased by 56.9 ± 11% (n = 4) in the presence of a Ca²⁺-free solution, indicating that glycine release in response to membrane stretch necessitates Ca²⁺ influx. C, Application of 100 μm gadolinium (Gd³⁺) reversibly inhibited the sniffer response to hypotonic shock by 61.2 ± 7.4% (n = 4). D, E, A single application (2–4 min) of 100 μm flufenamic acid or 100 μm SKF-96365 evoked an inhibition of the sniffer response by 89.1 ± 2.6% (n = 5) and 81.6 ± 5.4% (n = 8), respectively. The sniffer responses induced by hypotonic solution were still decreased 10 min after the end of the application of these inhibitors. This was not attributable to direct inhibition of glycine-evoked responses (supplemental Fig. 7, available at www.jneurosci.org as supplemental material). Ten minutes after the end of the application of flufenamic acid and SKF-96365, sniffer response were decreased by 81.4 ± 7.3 and 62.4 ± 4.6%, respectively. The inhibition evoked by flufenamic acid still remains significantly higher than Gd³⁺-evoked inhibition (Mann–Whitney test, p < 0.05). In two sniffer patches, we found that inhibition of the sniffer responses still occurred 20 min after washing out SKF-96365 or flufenamic acid (data not shown).