Shirayama T, Pappano A J
Department of Pharmacology, University of Connecticut Health Center, Farmington, USA.
J Pharmacol Exp Ther. 1996 Dec;279(3):1274-81.
The effects of intracellular cyclic guanosine monophosphate (cGMP) on L-type calcium current (lCa) and contraction of ventricular myocytes enzymatically isolated from guinea pig hearts were investigated to test the hypothesis that cGMP increases contractions along with ICa in these cells. ICa and contractions, elicited every 15 sec, were recorded simultaneously with a whole-cell voltage-clamp method and a video edge-detector, respectively. Cells were superfused with Tyrode's solution (22 degrees C); the pipette solution contained 120 mM potassium aspartate, 30 mM KCl, 4 mM ATP, 5 mM N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid), 0.01 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and various concentrations of cGMP, which entered the cell interior through the patch electrode. In the presence of 3 nM isoproterenol (ISO) in the bath, ICa was increased 3.2-fold. ICa was further increased by 20% with 30 microM cGMP; cell contractions were also increased by 32%. When ICa was maximal in the presence of 30 nM ISO, cGMP no longer increased ICa or contractions, an indication that the effects of cGMP and ISO were additive. When ICa was increased maximally (4.3-fold) by 100 microM isobutylmethylxanthine, a nonselective phosphodiesterase inhibitor, application of 100 microM cGMP in the pipette decreased ICa by 53% and cell shortening by 64%. Cyclic GMP changed contraction in parallel with ICa in the presence of either ISO or isobutylmethylxanthine. 5'-GMP had no significant effect on ICa or contraction in the presence of ISO or isobutyl-methylxanthine. Cyclic GMP alone, at 30 microM, increased ICa by 25%; this effect on basal ICa was reversed by removal of cGMP from the pipette solution. We conclude that intracellular cGMP had two effects on ICa and contraction, namely, 1) an increase caused by an action on cGMP-inhibited phosphodiesterase and 2) a decrease attributed to activation of cGMP-dependent protein kinase.
研究了细胞内环磷酸鸟苷（cGMP）对从豚鼠心脏酶解分离的心室肌细胞L型钙电流（lCa）和收缩的影响，以验证cGMP在这些细胞中与lCa一起增加收缩的假说。分别用全细胞膜片钳法和视频边缘检测器同时记录每隔15秒诱发的lCa和收缩。细胞用台氏液（22℃）灌流；电极内液含有120 mM天冬氨酸钾、30 mM KCl、4 mM ATP、5 mM N-（2-羟乙基）哌嗪-N-（2-乙磺酸）、0.01 mM乙二醇双（β-氨基乙醚）-N，N，N'，N'-四乙酸和不同浓度的cGMP，cGMP通过膜片电极进入细胞内部。在浴液中存在3 nM异丙肾上腺素（ISO）时，lCa增加3.2倍。30 μM cGMP使lCa进一步增加20%；细胞收缩也增加32%。当在30 nM ISO存在下lCa达到最大值时，cGMP不再增加lCa或收缩，这表明cGMP和ISO的作用是相加的。当用100 μM异丁基甲基黄嘌呤（一种非选择性磷酸二酯酶抑制剂）使lCa最大增加（4.3倍）时，电极内液中加入100 μM cGMP使lCa降低53%，细胞缩短降低64%。在ISO或异丁基甲基黄嘌呤存在下，cGMP与lCa平行改变收缩。在ISO或异丁基甲基黄嘌呤存在下，5'-GMP对lCa或收缩无显著影响。单独30 μM的cGMP使lCa增加25%；从电极内液中去除cGMP可逆转这种对基础lCa的作用。我们得出结论，细胞内cGMP对lCa和收缩有两种作用，即1）通过作用于cGMP抑制的磷酸二酯酶引起增加，2）归因于cGMP依赖性蛋白激酶激活的降低。
