Action potential-dependent ATP release from taste receptor cells : development of real-time micro determination for neurotransmitter release at single ceII level

味蕾细胞的亚群产生动作电位，以响应有味道的刺激。II型味觉细胞有许多味觉转导分子，但缺乏精细的突触，这导致了动作电位在这些细胞中的功能意义的问题。我们研究了味觉细胞释放三磷酸腺苷（ATP）递质对动作电位的依赖性。为了鉴定TT型味觉细胞，我们使用了表达gustducin启动子转基因绿色荧光蛋白（GFP）的小鼠。动作电位记录由电极基底侧连接到一个单一的gfp阳性味觉细胞。通过在味觉刺激动作电位后立即收集电极溶液，并使用荧光素酶测定法定量ATP，监测表达味觉素的味觉细胞释放ATP。用尖膜上的甜、苦或鲜味化合物刺激表达gustducin的味觉细胞可增加电极溶液中的ATP水平；ATP的数量取决于燃烧速率。自发放电率的增加也诱导了表达味觉素的味觉细胞释放ATP。河豚毒素抑制表达gustducin的味觉细胞释放ATP，卡贝诺洛酮抑制其释放低于检测限。我们的数据支持II型味觉细胞的动作电位通过半通道增强ATP释放的假设。

Subsets of taste bud cells generate action potentials in response to sapid stimuli. Type II taste cells have many taste transduction molecules but lack well elaborated synapses, which leads to the question in the functional significance of action potentials in these cells. We examined the dependence of adenosine triphosphate (ATP) transmitter release from taste cells upon action potentials. To identify Type TT taste cells we used mice expressing a green fluorescence protein (GFP) transgene from the gustducin promoter. Action potentials were recorded by an electrode basolaterally attached to a. single GFP-positive taste cell. ATP release was monitored from gustducin-expressing taste cells by collecting the electrode solution immediately after tastant-stimulated action potentials and using a. luciferase assay to quantify ATP. Stimulation of gustducin-expressing taste cells with a. sweet, bitter, or umami compound on the apical membrane increased ATP levels in the electrode solution; the amount of ATP depended on the firing rate. Increased spontaneous firing rates also induced ATP release from gustducin-expressing taste cells. ATP release from gustducin-expressing taste cells was depressed by tetrodotoxin and inhibited below the detection limit by carbenoxolone. Our data support the hypothesis that action potentials in Type II taste cells enhance ATP release through hemichannels.