Sakai R, Shen J B, Pappano A J
Department of Pharmacology, University of Connecticut Health Center, Farmington 06030, USA.
J Cardiovasc Pharmacol. 1999 Aug;34(2):304-15. doi: 10.1097/00005344-199908000-00017.
We investigated the effect of carbachol (CCh) on L-type Ca2+ current (ICa(L)) enhanced by dialyzed adenosine 3',5'-cyclic monophosphate (cAMP) and/or bath-applied 3-isobutyl-1-methylxanthine (IBMX) in guinea pig isolated ventricular myocytes. At pipette concentrations ([cAMP]pip) from 30 microM to 1 mM, cAMP increased ICa(L) to 25.8 +/- 0.9 microA/cm2 (682 +/- 24.8% increase above control). CCh (100 microM) did not inhibit ICa(L) at any [cAMP]pip. IBMX, a nonselective phosphodiesterase (PDE) inhibitor, increased ICa(L) maximally at 300 microM IBMX (17.9 +/- 0.7 microA/cm2; 449 +/- 20% increase). CCh (100 microM) inhibited ICa(L) by 92 +/- 9.5% at 30 microM IBMX and 78 +/- 4.6% at 100 microM IBMX; this effect was reduced or absent at higher IBMX concentrations (300 and 1,000 microM). Coadministration of cAMP and IBMX also progressively suppressed inhibition by CCh. CCh had a negligible effect on ICa(L) at 750 microM IBMX in the absence of pipette cAMP and at 50 microM IBMX in the presence of 100 microM [cAMP]pip. ACh-activated K+ current (IK(ACh)) was unchanged in atrial myocytes dialyzed with 100 microM cAMP; this excludes a phosphorylation-dependent desensitization of the muscarinic receptor (mAChR) or Gi by cAMP. LY83583 (100 microM), an inhibitor of cyclic guanosine monophosphate (cGMP) production, attenuated inhibition of ICa(L) by CCh in the presence of IBMX. 8-Bromo-cGMP (8-Br-cGMP), an activator of cGMP-dependent protein kinase (PKG), mimicked CCh in its actions on ICa(L) raised by both cAMP (no significant change) and IBMX (49 +/- 5.1% inhibition). Okadaic acid, an inhibitor of type 1 and 2A phosphatases, blocked inhibition of IBMX-stimulated ICa(L) by either CCh or 8-Br-cGMP. Thus the ability of CCh to inhibit ICa(L) appears caused by cGMP/PKG activation of an okadaic acid-sensitive protein phosphatase, and elevated levels of cAMP protect against this action.
我们研究了卡巴胆碱（CCh）对豚鼠离体心室肌细胞中由透析的腺苷3',5'-环磷酸（cAMP）和/或浴加3-异丁基-1-甲基黄嘌呤（IBMX）增强的L型Ca2+电流（ICa(L)）的影响。在移液管浓度（[cAMP]pip）从30 microM至1 mM时，cAMP将ICa(L)增加至25.8±0.9微安/平方厘米（比对照增加682±24.8%）。在任何[cAMP]pip下，CCh（100 microM）均不抑制ICa(L)。IBMX是一种非选择性磷酸二酯酶（PDE）抑制剂，在300 microM IBMX时最大程度地增加ICa(L)（17.9±0.7微安/平方厘米；增加449±20%）。在30 microM IBMX时，CCh（100 microM）将ICa(L)抑制92±9.5%，在100 microM IBMX时抑制78±4.6%；在较高的IBMX浓度（300和1000 microM）时，这种作用减弱或消失。cAMP和IBMX共同给药也逐渐抑制CCh的抑制作用。在不存在移液管cAMP时，CCh对750 microM IBMX下的ICa(L)影响可忽略不计，在存在100 microM [cAMP]pip时，对50 microM IBMX下的ICa(L)影响也可忽略不计。在用100 microM cAMP透析的心房肌细胞中，乙酰胆碱激活的K+电流（IK(ACh)）未改变；这排除了cAMP对毒蕈碱受体（mAChR）或Gi的磷酸化依赖性脱敏作用。LY83583（100 microM）是一种环鸟苷单磷酸（cGMP）产生的抑制剂，在存在IBMX时减弱CCh对ICa(L)的抑制作用。8-溴-cGMP（8-Br-cGMP）是一种cGMP依赖性蛋白激酶（PKG）的激活剂，在对由cAMP（无显著变化）和IBMX（抑制49±5.1%）升高的ICa(L)的作用方面模拟CCh。冈田酸是1型和2A磷酸酶的抑制剂，可阻断CCh或8-Br-cGMP对IBMX刺激的ICa(L)的抑制作用。因此，CCh抑制ICa(L)的能力似乎是由cGMP/PKG激活一种对冈田酸敏感的蛋白磷酸酶引起的，而升高的cAMP水平可防止这种作用。
