Rasgado-Flores H, Santiago E M, Blaustein M P
Department of Physiology, School of Medicine, University of Maryland, Baltimore 21201.
J Gen Physiol. 1989 Jun;93(6):1219-41. doi: 10.1085/jgp.93.6.1219.
Coupled Na+ exit/Ca2+ entry (Na/Ca exchange operating in the Ca2+ influx mode) was studied in giant barnacle muscle cells by measuring 22Na+ efflux and 45Ca2+ influx in internally perfused, ATP-fueled cells in which the Na+ pump was poisoned by 0.1 mM ouabain. Internal free Ca2+, [Ca2+]i, was controlled with a Ca-EGTA buffering system containing 8 mM EGTA and varying amounts of Ca2+. Ca2+ sequestration in internal stores was inhibited with caffeine and a mitochondrial uncoupler (FCCP). To maximize conditions for Ca2+ influx mode Na/Ca exchange, and to eliminate tracer Na/Na exchange, all of the external Na+ in the standard Na+ sea water (NaSW) was replaced by Tris or Li+ (Tris-SW or LiSW, respectively). In both Na-free solutions an external Ca2+ (Cao)-dependent Na+ efflux was observed when [Ca2+]i was increased above 10(-8) M; this efflux was half-maximally activated by [Ca2+]i = 0.3 microM (LiSW) to 0.7 microM (Tris-SW). The Cao-dependent Na+ efflux was half-maximally activated by [Ca2+]o = 2.0 mM in LiSW and 7.2 mM in Tris-SW; at saturating [Ca2+]o, [Ca2+]i, and [Na+]i the maximal (calculated) Cao-dependent Na+ efflux was approximately 75 pmol#cm2.s. This efflux was inhibited by external Na+ and La3+ with IC50's of approximately 125 and 0.4 mM, respectively. A Nai-dependent Ca2+ influx was also observed in Tris-SW. This Ca2+ influx also required [Ca2+]i greater than 10(-8) M. Internal Ca2+ activated a Nai-independent Ca2+ influx from LiSW (tracer Ca/Ca exchange), but in Tris-SW virtually all of the Cai-activated Ca2+ influx was Nai-dependent (Na/Ca exchange). Half-maximal activation was observed with [Na+]i = 30 mM. The fact that internal Ca2+ activates both a Cao-dependent Na+ efflux and a Nai-dependent Ca2+ influx in Tris-SW implies that these two fluxes are coupled; the activating (intracellular) Ca2+ does not appear to be transported by the exchanger. The maximal (calculated) Nai-dependent Ca2+ influx was -25 pmol/cm2.s. At various [Na+]i between 6 and 106 mM, the ratio of the Cao-dependent Na+ efflux to the Nai-dependent Ca2+ influx was 2.8-3.2:1 (mean = 3.1:1); this directly demonstrates that the stoichiometry (coupling ratio) of the Na/Ca exchange is 3:1. These observations on the coupling ratio and kinetics of the Na/Ca exchanger imply that in resting cells the exchanger turns over at a low rate because of the low [Ca2+]i; much of the Ca2+ extrusion at rest (approximately 1 pmol/cm2.s) is thus mediated by an ATP-driven Ca2+ pump.(ABSTRACT TRUNCATED AT 400 WORDS)
通过测量内部灌注且以ATP供能、Na⁺泵被0.1 mM哇巴因抑制的巨藤壶肌细胞中的²²Na⁺外流和⁴⁵Ca²⁺内流，研究了耦合的Na⁺外流/Ca²⁺内流（以Ca²⁺内流模式运行的Na/Ca交换）。内部游离Ca²⁺（[Ca²⁺]i）通过含有8 mM乙二醇双（2-氨基乙基醚）四乙酸（EGTA）和不同量Ca²⁺的Ca-EGTA缓冲系统进行控制。用咖啡因和线粒体解偶联剂（羰基氰化物-4-(三氟甲氧基)苯腙，FCCP）抑制内部储存库中的Ca²⁺螯合。为了使Ca²⁺内流模式的Na/Ca交换条件最大化，并消除示踪剂Na/Na交换，标准Na⁺海水（NaSW）中的所有外部Na⁺都被Tris或Li⁺取代（分别为Tris-SW或LiSW）。在两种无Na⁺溶液中，当[Ca²⁺]i增加到10⁻⁸ M以上时，观察到一种外部Ca²⁺（Cao）依赖性的Na⁺外流；这种外流在[Ca²⁺]i = 0.3 μM（LiSW）至0.7 μM（Tris-SW）时达到半最大激活。在LiSW中，Cao依赖性Na⁺外流在[Ca²⁺]o = 2.0 mM时达到半最大激活，在Tris-SW中为7.2 mM；在饱和的[Ca²⁺]o、[Ca²⁺]i和[Na⁺]i时，最大（计算得出）的Cao依赖性Na⁺外流约为75 pmol·cm²·s。这种外流被外部Na⁺和La³⁺抑制，其半数抑制浓度（IC50）分别约为125 mM和0.4 mM。在Tris-SW中也观察到一种Na⁺i依赖性的Ca²⁺内流。这种Ca²⁺内流也需要[Ca²⁺]i大于10⁻⁸ M。内部Ca²⁺激活了来自LiSW的Na⁺i非依赖性Ca²⁺内流（示踪剂Ca/Ca交换），但在Tris-SW中，几乎所有的Ca²⁺i激活的Ca²⁺内流都是Na⁺i依赖性（Na/Ca交换）。在[Na⁺]i = 30 mM时观察到半最大激活。内部Ca²⁺在Tris-SW中激活Cao依赖性Na⁺外流和Na⁺i依赖性Ca²⁺内流这一事实意味着这两种通量是耦合的；激活（细胞内）的Ca²⁺似乎不是由交换体转运的。最大（计算得出）的Na⁺i依赖性Ca²⁺内流为-25 pmol/cm²·s。在6至106 mM之间的各种[Na⁺]i下，Cao依赖性Na⁺外流与Na⁺i依赖性Ca²⁺内流的比率为2.8 - 3.2:1（平均值 = 3.1:1）；这直接证明了Na/Ca交换的化学计量比（耦合比）为3:1。这些关于Na/Ca交换体耦合比和动力学的观察结果表明，在静息细胞中，由于[Ca²⁺]i较低，交换体的周转率较低；因此，静息时大部分Ca²⁺外流（约1 pmol/cm²·s）是由ATP驱动的Ca²⁺泵介导的。（摘要截断于400字）
