P2X antagonists inhibit styryl dye entry into hair cells.

The styryl pyridinium dyes, FM1-43 and AM1-43, are fluorescent molecules that can permeate the mechanotransduction channels of hair cells, the sensory receptors of the inner ear. When these dyes are applied to hair cells, they enter the cytoplasm rapidly, resulting in a readily detectable intracellular fluorescence that is often used as a molecular indication of mechanotransduction channel activity. However, such dyes can also permeate the ATP receptor, P2X(2). Therefore, we explored the contribution of P2X receptors to the loading of hair cells with AM1-43. The chick inner ear was found to express P2X receptors and to release ATP, similar to the inner ear of mammals, allowing for the endogenous stimulation of P2X receptors. The involvement of these receptors was evaluated pharmacologically, by exposing the sensory epithelium of the chick inner ear to 5 microM AM1-43 under different experimental conditions and measuring the fluorescence in hair cells after fixation of the tissue. Pre-exposure of the tissue to 5 mM EGTA for 15 min, which should eliminate most of the gating "tip links" of the mechanotransduction channels, deceased fluorescence by only 44%. In contrast, P2X receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid [PPADS], suramin, 2',3'-O-(2,4,6-trinitrophenyl) ATP [TNP-ATP], and d-tubocurarine) had greater effects on dye loading. PPADS, suramin, and TNP-ATP all decreased intracellular AM1-43 fluorescence in hair cells by at least 69% when applied at a concentration of 100 microM. The difference between d-tubocurarine-treated and control fluorescence was statistically insignificant when d-tubocurarine was applied at a concentration that blocks the mechanotransduction channel (200 microM). At a concentration that also blocks P2X(2) receptors (2 mM), d-tubocurarine decreased dye loading by 72%. From these experiments, it appears that AM1-43 can enter hair cells through endogenously activated P2X receptors. Thus, the contribution of P2X receptors to dye entry should be considered when using styryl pyridinium dyes to detect hair cell mechanotransduction channel activity, especially in the absence of explicit mechanical stimulation of stereocilia.

Developmental changes in calcium current pharmacology and somatostatin inhibition in chick parasympathetic neurons.

Voltage-dependent calcium (Ca2+) currents were characterized and modulatory effects of somatostatin were measured in acutely dissociated chick ciliary ganglion neurons at embryonic stages 34, 37, and 40. This developmental time period coincides with the period of synapse formation between ciliary ganglion neurons and peripheral eye muscles. At all three developmental stages Ca2+ current could be blocked almost completely by combined application of omega-CgTX GVIA and nitrendipine. At young embryonic ages there was significant overlap in sensitivity, with approximately 75% of the current sensitive to either blocker applied independently. By stage 40, there was very little or no overlap in sensitivity, with approximately 75% of the current blocked by omega-CgTX GVIA (N-type) and 30% blocked by nitrendipine (L-type). These data are consistent with earlier findings that the pharmacology of acetylcholine release from ciliary ganglion nerve terminals changes during development from sensitivity to both dihydropyridines and omega-CgTX GVIA to selective sensitivity to omega-CgTX GVIA (Gray et al., 1992). Somatostatin reduced Ca2+ current by 50-60% at all three developmental stages. At early developmental stages somatostatin receptors coupled predominantly to the current that was sensitive to both omega-CgTX GVIA and nitrendipine. By stage 40, somatostatin primarily inhibited classically defined N-type current (selectively sensitive to omega-CgTX GVIA). Thus, somatostatin receptor coupling to Ca2+ channels persisted throughout development as Ca2+ current pharmacology changed.