Foskett J K, Gunter-Smith P J, Melvin J E, Turner R J
Physiology Department, Armed Forces Radiobiology Research Institute, Bethesda, MD 20814.
Proc Natl Acad Sci U S A. 1989 Jan;86(1):167-71. doi: 10.1073/pnas.86.1.167.
Muscarinic stimulation of fluid secretion by mammalian salivary acinar cells is associated with a rise in the level of intracellular free calcium ([Ca2+]i) and activation of a calcium-sensitive potassium (K+) conductance in the basolateral membrane. To test in the intact cell whether the rise of [Ca2+]i precedes activation of the K+ conductance (as expected if Ca2+ is the intracellular messenger mediating this response), [Ca2+]i and membrane voltage were measured simultaneously in carbachol-stimulated rat parotid acinar cells by using fura-2 and an intracellular microelectrode. Unexpectedly, the cells hyperpolarize (indicating activation of the K+ conductance) before fura-2 detectable [Ca2+]i begins to rise. This occurs even in Ca2+-depleted medium where intracellular stores are the only source of mobilized Ca2+. Nevertheless, when the increase in [Ca2+]i was eliminated by loading cells with the Ca2+ chelator bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetate (Me2BAPTA) and stimulating in Ca2+-depleted medium, membrane hyperpolarization was also eliminated, indicating that a rise of [Ca2+] is required for the agonist-induced voltage response. Stimulation of Me2BAPTA-loaded cells in Ca2+-containing medium dramatically accentuates the temporal dissociation between the activation of the K+ conductance and the rise of [Ca2+]i. The data are consistent with the hypothesis that muscarinic stimulation results in a rapid localized increase in [Ca2+]i at the acinar basolateral membrane followed by a somewhat delayed increase in total [Ca2+]i. The localized increase cannot be detected by fura-2 but is sufficient to open the Ca2+-sensitive K+ channels located in the basolateral membrane. We concluded that a receptor-mobilized intracellular store of Ca2+ is localized at or near the basolateral membrane.
毒蕈碱对哺乳动物唾液腺泡细胞分泌液体的刺激作用与细胞内游离钙（[Ca2+]i）水平的升高以及基底外侧膜中钙敏感钾（K+）电导的激活有关。为了在完整细胞中测试[Ca2+]i的升高是否先于K+电导的激活（如果Ca2+是介导此反应的细胞内信使，则预期如此），通过使用fura-2和细胞内微电极，在卡巴胆碱刺激的大鼠腮腺腺泡细胞中同时测量[Ca2+]i和膜电压。出乎意料的是，在fura-2可检测到的[Ca2+]i开始升高之前，细胞就发生了超极化（表明K+电导被激活）。即使在Ca2+耗尽的培养基中，细胞内储存是动员的Ca2+的唯一来源时，这种情况也会发生。然而，当用Ca2+螯合剂双（2-氨基-5-甲基苯氧基）乙烷-N，N，N'，N'-四乙酸（Me2BAPTA）加载细胞并在Ca2+耗尽的培养基中刺激时，消除了[Ca2+]i的增加，膜超极化也被消除，表明[Ca2+]的升高是激动剂诱导的电压反应所必需的。在含Ca2+的培养基中刺激加载了Me2BAPTA的细胞，显著加剧了K+电导激活与[Ca2+]i升高之间的时间分离。这些数据与以下假设一致，即毒蕈碱刺激导致腺泡基底外侧膜处[Ca2+]i迅速局部增加，随后总[Ca2+]i有所延迟增加。这种局部增加不能被fura-2检测到，但足以打开位于基底外侧膜中的Ca2+敏感K+通道。我们得出结论，受体动员的细胞内Ca2+储存位于基底外侧膜或其附近。
