Wang Y X, Kotlikoff M I
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6046, USA.
Am J Physiol. 1997 Aug;273(2 Pt 1):C509-19. doi: 10.1152/ajpcell.1997.273.2.C509.
We investigated the muscarinic activation of Ca(2+)-activated Cl- currents [ICl(Ca)] in voltage-clamped equine tracheal myocytes. The threshold of cytosolic free Ca2+ concentration ([Ca2+]i) required for activation of ICl(Ca) was 202 +/- 22 nM, and full activation of the current occurred at 771 +/- 31 nM. Hexahydro-sila-difenidol (M3 antagonist) inhibited the methacholine-induced phasic [Ca2+]i increase and ICl(Ca) in a concentration-dependent manner, whereas methoctramine (M2 antagonist) only slightly attenuated the [Ca2+]i increase and ICl(Ca) (14.8 and 21.4%, respectively), consistent with incomplete selectivity. Dialysis of heparin (10 mg/ml) blocked methacholine-induced [Ca2+]i and ICl(Ca) but had no effect on the caffeine-induced Ca2+ release or ICl(Ca); inositol 1,4,5-trisphosphate (100 microM) induced ICl(Ca) and blocked the methacholine current. Conversely, ruthenium red (50 microM) prevented the caffeine-induced [Ca2+]i release and ICl(Ca) but had no effect on methacholine-induced [Ca2+]i or current. Intracellular dialysis of the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7, 500 microM) or the Ca2+/calmodulin-dependent protein kinase inhibitor KN93 (5 microM) had no effect on the [Ca2+]i increase or ICl(Ca). Pertussis toxin (0.5 mg/ml) did not affect the increase in [Ca2+]i or ICl(Ca). Dialysis with antibodies directed against the alpha-subunit of Gq/G11 (Gq alpha/ G alpha 11) blocked the methacholine-induced ICl(Ca) in a concentration-dependent manner, whereas anti-G alpha i-1/G alpha 1-2 antibodies (1:35) and anti-G alpha i-3/G(o) alpha antibodies (1:35) were without effect. The results indicate that stimulation of phospholipase C via M3/Gq proteins is the predominant signaling pathway for the activation of ICl(Ca); at high agonist concentrations, Ca(2+)-induced Ca2+ release does not appear to play a prominent role in muscarinic signaling.
我们研究了电压钳制的马气管肌细胞中，毒蕈碱对钙激活氯电流[ICl(Ca)]的激活作用。激活ICl(Ca)所需的胞质游离钙浓度([Ca2+]i)阈值为202±22 nM，电流完全激活发生在771±31 nM。六氢硅二苯哌啶（M3拮抗剂）以浓度依赖性方式抑制乙酰甲胆碱诱导的[Ca2+]i相位增加和ICl(Ca)，而甲氧基氨（M2拮抗剂）仅轻微减弱[Ca2+]i增加和ICl(Ca)（分别为14.8%和21.4%），这与不完全选择性一致。用肝素（10 mg/ml）透析可阻断乙酰甲胆碱诱导的[Ca2+]i和ICl(Ca)，但对咖啡因诱导的钙释放或ICl(Ca)无影响；肌醇1,4,5-三磷酸（100 microM）诱导ICl(Ca)并阻断乙酰甲胆碱电流。相反，钌红（50 microM）可阻止咖啡因诱导的[Ca2+]i释放和ICl(Ca)，但对乙酰甲胆碱诱导的[Ca2+]i或电流无影响。钙调蛋白拮抗剂N-(6-氨基己基)-1-萘磺酰胺（W-7，500 microM）或钙/钙调蛋白依赖性蛋白激酶抑制剂KN93（5 microM）的细胞内透析对[Ca2+]i增加或ICl(Ca)无影响。百日咳毒素（0.5 mg/ml）不影响[Ca2+]i或ICl(Ca)的增加。用针对Gq/G11α亚基（Gqα/Gα11）的抗体透析以浓度依赖性方式阻断乙酰甲胆碱诱导的ICl(Ca)，而抗Gαi-1/Gα1-2抗体（1:35）和抗Gαi-3/G(o)α抗体（1:35）则无作用。结果表明，通过M3/Gq蛋白刺激磷脂酶C是激活ICl(Ca)的主要信号通路；在高激动剂浓度下，钙诱导的钙释放似乎在毒蕈碱信号传导中不发挥突出作用。
