Jurevicius J, Fischmeister R
Laboratoire de Cardiologie Cellulaire et Moléculaire, Institut National de la Santé et de la Recherche Médicale, Université de Paris-Sud, Faculté de Pharmacie, Châtenay-Malabry, France.
Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):295-9. doi: 10.1073/pnas.93.1.295.
The role of cAMP subcellular compartmentation in the progress of beta-adrenergic stimulation of cardiac L-type calcium current (ICa) was investigated by using a method based on the use of whole-cell patch-clamp recording and a double capillary for extracellular microperfusion. Frog ventricular cells were sealed at both ends to two patch-clamp pipettes and positioned approximately halfway between the mouths of two capillaries that were separated by a 5-micron thin wall. ICa could be inhibited in one half or the other by omitting Ca2+ from one solution or the other. Exposing half of the cell to a saturating concentration of isoprenaline (ISO, 1 microM) produced a nonmaximal increase in ICa (347 +/- 70%; n = 4) since a subsequent application of ISO to the other part induced an additional effect of nearly similar amplitude to reach a 673 +/- 130% increase. However, half-cell exposure to forskolin (FSK, 30 microM) induced a maximal stimulation of ICa (561 +/- 55%; n = 4). This effect was not the result of adenylyl cyclase activation due to FSK diffusion in the nonexposed part of the cell. To determine the distant effects of ISO and FSK on ICa, the drugs were applied in a zero-Ca solution. Adding Ca2+ to the drug-containing solutions allowed us to record the local effect of the drugs. Dose-response curves for the local and distant effects of ISO and FSK on ICa were used as an index of cAMP concentration changes near the sarcolemma. We found that ISO induced a 40-fold, but FSK induced only a 4-fold, higher cAMP concentration close to the Ca2+ channels, in the part of the cell exposed to the drugs, than it did in the rest of the cell. cAMP compartmentation was greatly reduced after inhibition of phosphodiesterase activity with 3-isobutyl-methylxanthine, suggesting the colocalization of enzymes involved in the cAMP cascade. We conclude that beta-adrenergic receptors are functionally coupled to nearby Ca2+ channels via local elevations of cAMP.
通过使用基于全细胞膜片钳记录和双毛细管细胞外微灌流的方法，研究了环磷酸腺苷（cAMP）亚细胞区室化在心脏L型钙电流（ICa）β-肾上腺素能刺激过程中的作用。将青蛙心室细胞的两端密封到两个膜片钳微电极上，并放置在由5微米薄壁隔开的两个毛细管管口之间大约中间的位置。通过从一种溶液或另一种溶液中去除Ca2+，可以抑制细胞的一半或另一半的ICa。将细胞的一半暴露于饱和浓度的异丙肾上腺素（ISO，1 microM）会使ICa产生非最大增加（347 +/- 70%；n = 4），因为随后将ISO应用于另一部分会诱导几乎相似幅度的额外效应，使增加幅度达到673 +/- 130%。然而，将细胞的一半暴露于福斯可林（FSK，30 microM）会诱导ICa的最大刺激（561 +/- 55%；n = 4）。这种效应不是由于FSK在细胞未暴露部分的扩散导致腺苷酸环化酶激活的结果。为了确定ISO和FSK对ICa的远距离效应，在零钙溶液中应用药物。向含药溶液中添加Ca2+使我们能够记录药物的局部效应。ISO和FSK对ICa的局部和远距离效应的剂量反应曲线被用作肌膜附近cAMP浓度变化的指标。我们发现，在暴露于药物的细胞部分，ISO在靠近Ca2+通道处诱导的cAMP浓度比细胞其余部分高40倍，但FSK仅诱导高4倍。在用3-异丁基-甲基黄嘌呤抑制磷酸二酯酶活性后，cAMP区室化大大降低，这表明参与cAMP级联反应的酶共定位。我们得出结论，β-肾上腺素能受体通过cAMP的局部升高与附近的Ca2+通道功能偶联。
