Activation of the cAMP-protein kinase A pathway facilitates Na+ translocation by the Na+-K+ pump in guinea-pig ventricular myocytes
Activation of the cAMP-protein kinase A pathway facilitates Na+ translocation by the Na+-K+ pump in guinea-pig ventricular myocytes
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DOI：
10.1111/j.1469-7793.2000.t01-2-00561.x
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发表时间：
2000-03-15
影响因子：
5.5
通讯作者：
Glitsch, HG
中科院分区：
文献类型：
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作者：
Kockskämper, J;Erlenkamp, S;Glitsch, HG
1.在30℃全细胞电压钳上观察腺苷环化酶激动剂Forskolin对豚鼠心室肌细胞Na~+-K~+泵产生的稳态电流和瞬时电流的影响。在含有144 mM Na+(Na-o(+))和10 mM K+(K-o(+))的外液中，由5 mM移液管Na+(Na-pip(+))激活的稳态Na+-K+泵电流(i-p)在-20 mV时被Forsklin(4mM)可逆地增大到对照电流的133+/-4%(n=15)。Forsklin类似物1，9-二脱氧Forsklin(10 MU M)不激活腺苷酸环化酶，不增加i-p(n=2)。蛋白激酶A(PKA)抑制剂H-89(10 MU M)在Forsklin持续存在的情况下，逆转Forsklin诱导的i-p升高(n=3)。在50 mM Na-pip(+)的存在下，Forsklin对i-p的影响持续存在，这确保了Na+-K+泵的内部Na+结合部位接近饱和。在此条件下，当吸管内游离钙浓度为0.013 nM(n=5)时，药物使i-p增加到对照i-p的142nM/-3%(n=5)；当游离[Ca~(2+)](Pip)为15 nM(n=9)时，药物使i-p增加到对照i-p的138%/-4%。在无Na+外液中，经50 mM Na-pip(+)和1.5 mM K-o(+)激活的i-p同样可被Forsklin升高，但幅度小于在含Na+介质中(116+/-3%，n=10)。为了专门研究泵浦周期中Na+分支的部分反应，研究了在电中性Na+-Na+交换条件下的瞬时泵浦电流。由电压跳跃引起的暂态泵浦电流呈现出一个初始峰值，然后呈单指数衰减。移动电荷(Q)和电流衰减速率常数随膜电位(V)变化。Q-V关系符合Boltzmann分布，其特征为中点电压(V-0.5)和最大可动电荷量(Q(Max))。Forskolin(2-10muM)使V-0.5向负值偏移，而Delta Q(Max)不受影响(n=11)。用8-溴-cAMP(500 mU M；n=2)模拟Forsklin对瞬时泵电流的影响，并用PRA的多肽抑制剂(PKI，10 mU M；n=5)消除该效应。我们得出结论，豚鼠心室肌细胞cAMP-PKA通路的激活至少部分是通过调节Na+泵循环中的部分反应来增加Na+-K+泵电流的。在生理条件下，对心脏Na+-K+泵的刺激可能有助于缩短动作电位时程，并抵消交感神经刺激时增加的被动肌膜Na+和K+流量。
1. The effects of the adenylyl cyclase activator forskolin on steady-state and transient currents generated by the Na+-K+ pump were studied in guinea-pig ventricular myocytes by means of whole-cell voltage clamp at 30 degrees C.2. In external solution containing 144 mM Na+ (Na-o(+)) and 10 mM K+ (K-o(+)), steady-state Na+-K+ pump current (I-p) activated by 5 mM pipette Na+ (Na-pip(+)) at -20 mV was reversibly augmented by forskolin (4 mu M) to 133 +/- 4% of the control current (n = 15). The forskolin analogue 1,9-dideoxyforskolin (10 mu M), which does not activate adenylyl cyclases, did not increase I-p (n = 2). Application of the protein kinase a (PKA) inhibitor H-89 (10 mu M) in the continued presence of forskolin reversed the forskolin-induced elevation of I-p (n = 3).3. The forskolin effect on I-p persisted in the presence of 50 mM Na-pip(+) which ensured that the internal Na+-binding sites of the Na+-K+ pump were nearly saturated. Under these conditions, the drug increased I-p to 142 +/- 3% of the control I-p when the pipette free Ca2+ concentration ([Ca2+](pip)) was 0.013 nM (n = 5) and to 138 +/- 4% of the control I-p when free [Ca2+](pip) was 15 nM (n = 9).4. In Na+-free external solution, I-p activated by 50 mM Na-pip(+) and 1.5 mM K-o(+) was likewise increased by forskolin but to a lesser extent than in Na+-containing medium (116 +/- 3% of control, (n = 10).5. In order to investigate exclusively partial reactions in the Na+ limb of the pump cycle, transient pump currents under conditions of electroneutral Na+-Na+ exchange were studied. Transient pump currents elicited by voltage jumps displayed an initial peak and then decayed monoexponentially. Moved charge (Q) and the rate constant of current decay varied with membrane potential (V). The Q- V relationship followed a Boltzmann distribution characterized by the midpoint voltage (V-0.5) and the maximum amount of movable charge (Delta Q(max)). Forskolin (2-10 mu M) shifted V-0.5 to more negative values while Delta Q(max) was not affected (n = 11). The effects of forskolin on transient pump currents were mimicked by 8-bromo-cAMP (500 mu M; n = 2) and abolished by a peptide inhibitor of PRA (PKI, 10 mu M; n = 5).6. We conclude that activation of the cAMP-PKA pathway in guinea-pig ventricular myocytes increases Na+-K+ pump current at least in part by modulating partial reactions in the Na+ limb of the pump cycle. Under physiological conditions, the observed stimulation of the cardiac Na+-K+ pump may serve to shorten the action potential duration and to counteract the increased passive sarcolemmal Na+ and K+ fluxes during sympathetic stimulation of the heart.