Salivary acini were isolated enzymatically from submandibular glands of adult male mice. The patch-clamp technique was employed to record K+ currents in cell-attached patches of basolateral membrane. Application of acetylcholine (10(-5) M) to the medium bathing the cells results in a pronounced and sustained activation of the K+ channels in the cell-attached patches, an effect mediated by an intracellular second messenger. In the present study we investigate the mechanism by which acetylcholine achieves activation of K+ channels. 2. The effects of acetylcholine on single-channel activity were shown to be dependent on extracellular Ca2+, i.e. due to Ca2+ influx. In cells bathed in Ca2+-free medium acetylcholine activation resulted in no increase in single-channel open probability. This blockade could be reversed by reintroduction of Ca2+ to the extracellular fluid in the continued presence of the agonist. The effects of acetylcholine in control medium (1.2 mM-Ca2+) were mimicked by the Ca2+ ionophore, A23187 (10(-8) M). 3. In K+-depolarized cells (bathed in a Na+-free, 145 mM-KCl solution) there was no evidence of any voltage-activated Ca2+ influx pathway. In the K+-depolarized cells acetylcholine application was no longer associated with any increase in the open probability of the K+ channels. K+ channels could be activated by adding A23187 (10(-8) M) to the high-K+ solution. 4. Cells bathed in another Na+-free (N-methyl-D-glucamine substituted for Na+) but non-depolarizing solution were also refractory to acetylcholine. K+ currents could, however, be activated in patches attached to these cells by application of A23187 (10(-8) M) or by the introduction of 20 mM-Na+ to the extracellular fluid in the presence of acetylcholine. The increased activity associated with the reintroduction of Na+ was totally reversed by atropine, i.e. it was receptor regulated. 5. The data presented above indicate that the cholinergic regulation of K+ channels is secondary to the receptor-regulated activation of a Ca2+ influx pathway. There is no evidence of voltage-activated Ca2+ influx in these cells. The cholinergic activation of Ca2+ influx is abolished in Na+-depleted cells. We conclude that the Na+ dependency indicates either that Na+ is involved in the gating of some voltage-independent Ca2+ channel or that Ca2+ entry is via a coupled Na+-Ca2+ co- or countertransport pathway.
摘要
从成年雄性小鼠的下颌下腺中酶法分离出唾液腺泡。采用膜片钳技术记录基底外侧膜细胞贴附式膜片中的钾离子电流。向浴有细胞的培养基中加入乙酰胆碱（10⁻⁵ M）会导致细胞贴附式膜片中钾离子通道显著且持续激活，该效应由细胞内第二信使介导。在本研究中，我们探究乙酰胆碱实现钾离子通道激活的机制。2. 乙酰胆碱对单通道活性的影响显示依赖于细胞外钙离子，即由于钙离子内流。在无钙离子培养基中培养的细胞，乙酰胆碱激活并未导致单通道开放概率增加。在激动剂持续存在的情况下，向细胞外液重新加入钙离子可逆转这种阻断作用。钙离子载体A23187（10⁻⁸ M）可模拟乙酰胆碱在对照培养基（1.2 mM - 钙离子）中的作用。3. 在钾离子去极化细胞（浴于无钠、145 mM - 氯化钾溶液中）中，没有任何电压激活的钙离子内流途径的证据。在钾离子去极化细胞中，加入乙酰胆碱不再与钾离子通道开放概率增加相关。向高钾溶液中加入A23187（10⁻⁸ M）可激活钾离子通道。4. 浴于另一种无钠（用N - 甲基 - D - 葡糖胺替代钠）但非去极化溶液中的细胞对乙酰胆碱也不敏感。然而，通过加入A23187（10⁻⁸ M）或在乙酰胆碱存在的情况下向细胞外液中加入20 mM - 钠，可激活贴附于这些细胞的膜片中的钾离子电流。与重新加入钠相关的活性增加可被阿托品完全逆转，即它是受受体调节的。5. 上述数据表明，钾离子通道的胆碱能调节继发于受体调节的钙离子内流途径的激活。在这些细胞中没有电压激活的钙离子内流的证据。在钠离子缺乏的细胞中，乙酰胆碱对钙离子内流的激活作用被消除。我们得出结论，钠离子依赖性表明要么钠离子参与了某些电压非依赖性钙离子通道的门控，要么钙离子进入是通过耦合的钠 - 钙同向或反向转运途径。
