Grönroos E, Thodeti C K, Sjölander A
Department of Laboratory Medicine, Lund University, Malmö, Sweden.
Cell Calcium. 1998 Jul;24(1):9-16. doi: 10.1016/s0143-4160(98)90084-7.
Although the LTD4-induced Ca2+ influx in human epithelial cells has been shown to be regulated by a pertussis toxin-sensitive heterotrimeric G-protein, most likely a G alpha i3 protein [Adolfsson J.L.P., Ohd J.F., Sjölander A. Leukotriene D4-induced activation and translocation of the G-protein alpha i3-subunit in human epithelial cells. Biochem Biophys Res Commun 1996; 226: 413-419], the signalling pathway further downstream is still unclear. In the present study, we investigated the possible involvement of cAMP and protein kinase A activity in the LTD4-induced Ca2+ influx in the epithelial cell line Int 407. Stimulation with LTD4, but not with the calcium ionophore ionomycin, triggered a rapid increase (peak at 7 s) in the cellular cAMP level, an effect that was totally abolished by pertussis toxin. Furthermore, the LTD4-induced Ca2+ signal was reduced by 60% when cells that had been pre-incubated with the protein kinase A inhibitor Rp-cAMPS (50 microM for 30 min) were stimulated in a calcium containing medium. In contrast, Rp-cAMPS had no apparent effect on the LTD4-induced Ca2+ signal when the cells were stimulated in a calcium-depleted medium. The immediate LTD4-induced protein tyrosine phosphorylation (15 s), previously shown to be necessary for the subsequent Ca2+ influx, was abolished not only by pretreatment with pertussis toxin but also by exposure to Rp-cAMPS. Furthermore, direct activation of the cellular adenylyl cyclase activity by treatment with forskolin alone induced a prompt Ca2+ signal in the presence, but not in the absence, of extracellular Ca2+, identical results were obtained with the cell permeable cAMP analogue 8-bromo-cAMP. In addition, forskolin induced protein tyrosine phosphorylation similar to that seen with LTD4. These results suggest that protein kinase A activity participates in the regulation of the LTD4-induced Ca2+ influx at a site that is downstream of the activation of the pertussis toxin-sensitive G-protein but upstream of a LTD4-stimulated tyrosine kinase(s).
尽管已表明白三烯D4（LTD4）诱导的人上皮细胞Ca2+内流受百日咳毒素敏感的异源三聚体G蛋白调节，很可能是Gαi3蛋白[阿道夫松J.L.P.，奥德J.F.，舍兰德A.白三烯D4诱导人上皮细胞中G蛋白αi3亚基的激活和转位。生物化学与生物物理研究通讯1996；226：413 - 419]，但其下游的信号通路仍不清楚。在本研究中，我们调查了环磷酸腺苷（cAMP）和蛋白激酶A活性在LTD4诱导的上皮细胞系Int 407中Ca2+内流过程中可能的参与情况。用LTD4刺激而非钙离子载体离子霉素刺激，引发细胞内cAMP水平迅速升高（7秒时达到峰值），该效应被百日咳毒素完全消除。此外，当用蛋白激酶A抑制剂Rp - cAMPS（50微摩尔，处理30分钟）预孵育的细胞在含钙培养基中受到刺激时，LTD4诱导的Ca2+信号降低了60%。相反，当细胞在无钙培养基中受到刺激时，Rp - cAMPS对LTD4诱导的Ca2+信号没有明显影响。先前已表明LTD4诱导的即刻蛋白酪氨酸磷酸化（15秒）是随后Ca2+内流所必需的，不仅用百日咳毒素预处理可消除该磷酸化，用Rp - cAMPS处理也可消除。此外，单独用福司可林处理直接激活细胞腺苷酸环化酶活性，在有细胞外钙存在时可迅速诱导Ca2+信号，但在无细胞外钙时则不能，细胞可渗透的cAMP类似物8 - 溴 - cAMP也得到了相同结果。此外，福司可林诱导的蛋白酪氨酸磷酸化与LTD4诱导的相似。这些结果表明，蛋白激酶A活性在百日咳毒素敏感的G蛋白激活下游但LTD4刺激的酪氨酸激酶上游的位点参与LTD4诱导的Ca2+内流的调节。
