Felbel J, Trockur B, Ecker T, Landgraf W, Hofmann F
Institut für Physiologische Chemie, Medizinische Fakultät, Universität des Saarlandes, Homburg-Saar, Germany.
J Biol Chem. 1988 Nov 15;263(32):16764-71.
The regulation of the cytosolic calcium concentration was investigated in freshly isolated adult bovine tracheal smooth muscle cells using fura 2. These cells contain 1.1 and 1.8 pmol of cGMP kinase and cAMP kinase per mg protein, respectively. Carbachol, histamine, serotonin, isoproterenol, and salbutamol increased the cytosolic calcium in a dose-dependent manner from 79 nM to about 650 nM. Preincubation of these cells for 20 min with isoproterenol, forskolin, 8-Br-cAMP and 8-(4-Cl-phenyl)thio-cAMP did not lower carbachol-induced increases in cytosolic calcium concentration, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the atrionatriuretic factor, isobutylmethylxanthine, and 8-Br-cGMP lowered cytosolic calcium. The active fragment of cGMP kinase, but not the catalytic subunit of cAMP kinase lowered carbachol-induced calcium levels. Carbachol released calcium from intracellular stores and increased calcium influx from the extracellular space. The influx was inhibited by preincubation with the calcium channel blockers nitrendipine or gallopamil. Both carbachol-stimulated pathways were suppressed by 8-Br-cGMP. Isoproterenol increased only the influx of calcium from the outside by a channel which was blocked by calcium channel blockers or 8-Br-cGMP. Forskolin and 8-Br-cAMP lowered carbachol- and isoproterenol-stimulated increases in calcium when added shortly before or after the addition of the agonist. In addition, isoproterenol decreased carbachol-stimulated calcium levels when added 10 s after carbachol. The calcium stimulatory effect of isoproterenol was abolished by preincubation of the cells with pertussis toxin or cholera toxin. These results show (a) that the beta 2-adrenoceptor couples in isolated tracheal smooth muscle cells to a dihydropyridine- and pertussis toxin-sensitive calcium channel; (b) that the same channel is opened by carbachol; (c) that cGMP kinase is very effective in decreasing elevated cytosolic calcium concentrations, whereas cAMP-dependent protein kinase has a variable effect on stimulated cytosolic calcium levels.
利用fura 2对新鲜分离的成年牛气管平滑肌细胞中胞质钙浓度的调节进行了研究。这些细胞每毫克蛋白质分别含有1.1皮摩尔和1.8皮摩尔的cGMP激酶和cAMP激酶。卡巴胆碱、组胺、5-羟色胺、异丙肾上腺素和沙丁胺醇以剂量依赖方式使胞质钙从79纳摩尔增加至约650纳摩尔。用异丙肾上腺素、福斯可林、8-溴-cAMP和8-(4-氯苯基)硫代-cAMP对这些细胞预孵育20分钟，并未降低卡巴胆碱诱导的胞质钙浓度升高，而佛波酯12-O-十四烷酰佛波醇13-乙酸酯、心钠素、异丁基甲基黄嘌呤和8-溴-cGMP降低了胞质钙。cGMP激酶的活性片段而非cAMP激酶的催化亚基降低了卡巴胆碱诱导的钙水平。卡巴胆碱从细胞内储存库释放钙并增加钙从细胞外空间的内流。该内流被用钙通道阻滞剂尼群地平或加洛帕米预孵育所抑制。两种卡巴胆碱刺激的途径均被8-溴-cGMP抑制。异丙肾上腺素仅通过一个通道增加钙从外部的内流，该通道被钙通道阻滞剂或8-溴-cGMP阻断。在加入激动剂之前或之后不久加入福斯可林和8-溴-cAMP，可降低卡巴胆碱和异丙肾上腺素刺激引起的钙增加。此外，在卡巴胆碱加入10秒后加入异丙肾上腺素，可降低卡巴胆碱刺激的钙水平。用百日咳毒素或霍乱毒素对细胞预孵育可消除异丙肾上腺素的钙刺激作用。这些结果表明：(a) 在分离的气管平滑肌细胞中，β2-肾上腺素能受体与对二氢吡啶和百日咳毒素敏感的钙通道偶联；(b) 同一通道被卡巴胆碱打开；(c) cGMP激酶在降低升高的胞质钙浓度方面非常有效，而cAMP依赖性蛋白激酶对刺激的胞质钙水平具有可变作用。
