22Na+ and 45Ca2+ fluxes were studied in the clonal nerve cell line PC12. Three distinct types of ion channels were found: (a) voltage-dependent Na+ channels, (b) voltage-dependent Ca2+ channels, and (c) acetylcholine-activated channels permeable to both ions. 2. 22Na+ uptake through voltage-dependent Na+ channels is induced by veratridine and scorpion venom, and is inhibited 50% by 5 X 10(-7) M-tetrodotoxin and greater than 98% by 5 X 10(-6) M-tetrodotoxin. 3. 45Ca2+ uptake through voltage-dependent Ca2+ channels is induced by depolarizing the cells in 50 mM-KCl. This flux is not dependent on the presence of Na+ in the medium and is insensitive to 5 X 10(-6) M-tetrodotoxin. However, 1 mM-Mn2+ causes a 95% inhibition of K+-induced 45Ca2+ uptake. 4. Veratridine and scorpion venom also induce voltage-dependent 45Ca2+ uptake which can be blocked by 1mM-Mn2+. In contrast to KCl-induced 45Ca2+ uptake, this flux is completely blocked by 5 X 10(-6) M-tetrodotoxin and is abolished by removal of Na+ from the medium. Thus the depolarizing stimulus for Ca2+ uptake in this case is Na+ from the medium. Thus the depolarizing stimulus for Ca2+ uptake in this case is Na+ influx through voltage-dependent Na+ channels. 5. Carbamylcholine induces both 22Na+ and 45Ca2+ fluxes which are blocked by nicotinic cholinergic antagonists with the exception of alpha-bungarotoxin. The 22Na+ flux occurs exclusively via acetylcholine receptor channels, as evidenced by the lack of effect of 5 X 10(-6) M-tetrodotoxin. In the presence of Na+, almost all of the 45Ca2+ uptake can be blocked by 1 mM-Mn2+ and thus occurs via voltage-dependent Ca2+ channels which are activated by the depolarizing Na+ influx. 6--8% of the total 45Ca2+ flux, however, is insensitive to 1 mM-Mn2+, suggesting that this portion of the uptake occurs via the acetylcholine receptor channels. In Na+-free medium, the Mn2+-resistant 45Ca2+ component increases to 40% of the total uptake, apparently due to lack of competition from Na+ for the acetylcholine receptor channels. This receptor-linked flux still causes sufficient depolarization to induce the additional 60% of the Ca2+ flux through voltage-dependent, Mn2+ sensitive Ca2+ channels. 6. Mn2+ inhibits Ca2+ flux through voltage-dependent Ca2+ channels by competing for entry through these channels. 50 mM-KCl induces 54Mn2+ fluxes in PC12 cells that are comparable in magnitude to 45Ca2+ fluxes. 7. In normal saline 45Ca2+ efflux from PC12 cells is several times more rapid than in Na+-free medium, indicating the presence of a Ca2+-Na+ exchange mechanism.
摘要
在克隆神经细胞系PC12中研究了(^{22}Na^+)和(^{45}Ca^{2 +})通量。发现了三种不同类型的离子通道：(a) 电压依赖性(Na^+)通道，(b) 电压依赖性(Ca^{2 +})通道，以及 (c) 对两种离子均通透的乙酰胆碱激活通道。2. 藜芦碱和蝎毒可诱导通过电压依赖性(Na^+)通道的(^{22}Na^+)摄取，5×10⁻⁷ M - 河豚毒素可抑制50%，5×10⁻⁶ M - 河豚毒素可抑制超过98%。3. 通过使细胞在50 mM - KCl中去极化来诱导通过电压依赖性(Ca^{2 +})通道的(^{45}Ca^{2 +})摄取。这种通量不依赖于培养基中(Na^+)的存在，并且对5×10⁻⁶ M - 河豚毒素不敏感。然而，1 mM - (Mn^{2 +})可使K⁺诱导的(^{45}Ca^{2 +})摄取抑制95%。4. 藜芦碱和蝎毒也可诱导电压依赖性(^{45}Ca^{2 +})摄取，其可被1 mM - (Mn^{2 +})阻断。与KCl诱导的(^{45}Ca^{2 +})摄取相反，这种通量可被5×10⁻⁶ M - 河豚毒素完全阻断，并且通过从培养基中去除(Na^+)而消除。因此在这种情况下(Ca^{2 +})摄取的去极化刺激是来自培养基的(Na^+)。因此在这种情况下(Ca^{2 +})摄取的去极化刺激是通过电压依赖性(Na^+)通道的(Na^+)内流。5. 氨甲酰胆碱可诱导(^{22}Na^+)和(^{45}Ca^{2 +})通量，除α - 银环蛇毒素外，这些通量可被烟碱胆碱能拮抗剂阻断。(^{22}Na^+)通量仅通过乙酰胆碱受体通道发生，5×10⁻⁶ M - 河豚毒素无作用证明了这一点。在有(Na^+)存在的情况下，几乎所有的(^{45}Ca^{2 +})摄取可被1 mM - (Mn^{2 +})阻断，因此通过由去极化的(Na^+)内流激活的电压依赖性(Ca^{2 +})通道发生。然而，总(^{45}Ca^{2 +})通量的6 - 8%对1 mM - (Mn^{2 +})不敏感，表明这部分摄取通过乙酰胆碱受体通道发生。在无(Na^+)培养基中，对(Mn^{2 +})有抗性的(^{45}Ca^{2 +})成分增加到总摄取的40%，显然是由于(Na^+)对乙酰胆碱受体通道缺乏竞争。这种受体连接的通量仍会引起足够的去极化，以诱导通过电压依赖性、对(Mn^{2 +})敏感的(Ca^{2 +})通道的另外60%的(Ca^{2 +})通量。6. (Mn^{2 +})通过竞争通过这些通道进入而抑制通过电压依赖性(Ca^{2 +})通道的(Ca^{2 +})通量。50 mM - KCl在PC12细胞中诱导的(^{54}Mn^{2 +})通量在大小上与(^{45}Ca^{2 +})通量相当。7. 在正常盐溶液中，PC12细胞的(^{45}Ca^{2 +})外流比在无(Na^+)培养基中快几倍，表明存在(Ca^{2 +}-Na^+)交换机制。
