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

味蕾细胞的亚群对味觉刺激产生动作电位。II型味觉细胞具有许多味觉传导分子，但缺乏精细的突触，这导致了这些细胞中动作电位的功能意义的问题。我们研究了三磷酸腺苷（ATP）递质释放的依赖性味觉细胞动作电位。为了鉴定TT型味觉细胞，我们使用了表达来自gustducin启动子的绿色荧光蛋白（GFP）转基因的小鼠。动作电位记录的电极基底侧连接到一个。单个GFP阳性味觉细胞。通过在味素刺激的动作电位后立即收集电极溶液并使用a.荧光素酶测定以定量ATP。用a.顶端膜上的甜味、苦味或鲜味化合物增加了电极溶液中的ATP水平; ATP的量取决于放电速率。增加自发放电率也诱导ATP从表达味蛋白的味觉细胞释放。河豚毒素抑制表达味细胞的ATP释放，甘珀酸抑制低于检测限。我们的数据支持这一假设，即在II型味觉细胞的动作电位增强ATP释放通过半通道。

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.