Sunagawa M, Yokoshiki H, Seki T, Sperelakis N
Department of Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio, USA.
J Vasc Res. 1998 Sep-Oct;35(5):303-9. doi: 10.1159/000025599.
In the present study, we investigated the actions of calmodulin (CaM) and CaM-dependent protein kinase II (CaMK-II) on the L-type Ca2+ currents (ICa(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), using the whole-cell voltage clamp method. The peak IBa (Ca2+ channel using 5 mM Ba2+ as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of -60 mV. To test the effect of CaM on IBa, 1 microM calmidazolium (CMZ), an inhibitor of CaM, was added to the pipette solution (pCa of 6.5 or 300 nM [Ca]i). The amplitude of maximally activated IBa was -4.3 +/- 0.5 pA/pF (n = 13) for control and -8.1 +/- 0.9 pA/pF (n = 14) in the presence of CMZ. This difference was statistically significant (p = 0.016). The CMZ stimulation of IBa was not abolished when 5 microM KN-62, a specific inhibitor of CaMK-II, was included in the pipette (-9.5 +/- 1.1 pA/pF; n = 10). Introduction of CaMK-II itself intracellularly had no effect on the basal IBa. On the other hand, the CMZ stimulation of IBa was prevented by both H-7, a nonspecific protein kinase inhibitor, and H-89, a specific inhibitor of protein kinase A (PK-A). Since CMZ is a strong inhibitor of Ca2+/CaM-dependent phosphodiesterase (type I PDE), we studied the effect of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MIBMX), another specific inhibitor of the PDE. MIBMX, like CMZ, stimulated IBa: control, -4.6 +/- 0.4 pA/pF (n = 10); MIBMX, -9.6 +/- 1.2 (n = 8), and CMZ, -7.9 +/- 0.9 (n = 15). 0.1 mM 8Br-cAMP, a membrane permeable cAMP analogue, stimulated IBa by +42%: before, -3.7 +/- 0. 7 pA/pF; after, -5.2 +/- 1.0 (n = 6). In conclusion, Ca2+ channels of VSM cells might not be directly regulated by the CaM/CaMK-II pathway. Therefore, the CMZ stimulation of IBa might occur due to the increase in intracellular concentration of cAMP produced by inhibition of CaM-dependent PDE.
在本研究中，我们使用全细胞膜片钳方法，研究了钙调蛋白（CaM）和钙调蛋白依赖性蛋白激酶II（CaMK-II）对培养的血管平滑肌（VSM）细胞（A7r5细胞系）L型Ca2+电流（ICa(L)）的作用。从-60 mV的钳制电位施加至+10 mV的测试电位，每隔15 s诱发一次峰值IBa（使用5 mM Ba2+作为电荷载体的Ca2+通道）。为了测试CaM对IBa的影响，将1 μM钙调蛋白拮抗剂（CMZ），一种CaM抑制剂，添加到移液管溶液中（细胞内游离Ca2+浓度为6.5或300 nM）。对照情况下最大激活的IBa幅度为-4.3±0.5 pA/pF（n = 13），在存在CMZ时为-8.1±0.9 pA/pF（n = 14）。这种差异具有统计学意义（p = 0.016）。当移液管溶液中包含5 μM KN-62（一种CaMK-II特异性抑制剂）时，CMZ对IBa的刺激并未消除（-9.5±1.1 pA/pF；n = 10）。细胞内引入CaMK-II本身对基础IBa没有影响。另一方面，H-7（一种非特异性蛋白激酶抑制剂）和H-89（蛋白激酶A（PK-A）的特异性抑制剂）均可阻止CMZ对IBa的刺激。由于CMZ是Ca2+/CaM依赖性磷酸二酯酶（I型PDE）的强效抑制剂，我们研究了另一种PDE特异性抑制剂8-甲氧基甲基-3-异丁基-1-甲基黄嘌呤（MIBMX） 的作用。MIBMX与CMZ一样，刺激了IBa：对照，-4.6±0.4 pA/pF（n = 10）；MIBMX，-9.6±1.2（n = 8），CMZ，-7.9±0.9（n = 15）。0.1 mM 8-溴-cAMP（一种可透过细胞膜的cAMP类似物）使IBa增加了42%：之前，-3.7±0.7 pA/pF；之后,-5.2±1.0（n = 6）。总之，VSM细胞的Ca2+通道可能不受CaM/CaMK-II途径的直接调节。因此，CMZ对IBa的刺激可能是由于抑制CaM依赖性PDE导致细胞内cAMP浓度增加所致。
