The effect of intracellular calcium ions on adrenaline-stimulated adenosine 3':5'-cyclic monophosphate concentrations in pigeon erythrocytes, studied by using the ionophore A23187.
The effect of intracellular calcium ions on adrenaline-stimulated adenosine 3':5'-cyclic monophosphate concentrations in pigeon erythrocytes, studied by using the ionophore A23187.
复制标题
使用离子载体 A23187 研究细胞内钙离子对鸽子红细胞中肾上腺素刺激的 3:5-环单磷酸腺苷浓度的影响。
作者：
A. Campbell;K. Siddle
1.用二价阳离子载体A23187增加鸽红细胞内Ca ~（2+）浓度，以探讨肾上腺素引起的cAMP含量增加是否受完整细胞内Ca ~（2+）变化的影响。2.孵育的细胞与肾上腺素，在浓度范围0.55- 55 μ M，导致在一段时间内的环AMP的浓度增加60分钟。肾上腺素的作用被抑制90%以上的离子载体A23187（1.9 μ M）在1 mM-Ca 2+的存在下。这种抑制作用可以通过降低离子载体的浓度或细胞外Ca ~（2+）的浓度来减弱，并且与肾上腺素的浓度无关。3.离子载体A23187的作用依赖于孵育时间。时程研究表明，只有当细胞与离子载体孵育至少15分钟，然后再加入肾上腺素时，才能观察到离子载体A23187的最大抑制作用。4.离子载体A23187的抑制作用依赖于细胞外Ca ~（2+）浓度。在不存在Mg 2+的情况下，离子载体A23187（1.9 μ M）抑制肾上腺素的作用约。30%不添加Ca 2+，约66%与10 μ M-Ca 2+和超过90%与添加的Ca 2+浓度大于30 μ M。然而，即使在EGTA [乙二氧基双（乙胺）四乙酸盐]（0.1- 10 mM）存在下，离子载体A23187也引起至少30%的环AMP反应抑制，这可能是由于细胞Mg 2+浓度的降低。5.在细胞与离子载体A23187孵育后加入EGTA导致肾上腺素抑制作用的部分逆转。6.列入Mg 2+（2 mM）在孵育介质中拮抗离子载体A23187的抑制作用。当在加入细胞之前将离子载体A23187加入到含有Mg 2+的培养基中时，这种效果最显著。7.细胞内Mg ~（2+）含量降低了约10%。在有Ca 2+（1 mM）但无Mg 2+存在下，与离子载体A23187（1.9 μ M）孵育20分钟后，50%。当培养基中还存在Mg 2+（2 mM）时，离子载体A23187引起约100%的增加。细胞Mg ~（2+）含量为80%。离子载体A23187对细胞K+含量无显著影响。8.离子载体A23187在一定条件下引起细胞ATP含量的降低。由于当ATP不显著降低时也能显示对环AMP含量的影响，因此看来细胞中ATP的降低不能解释离子载体A23187对环AMP的影响。9.离子载体A23187（1.9 μ M），与1 mM-Ca 2+，并没有提高环AMP降解完整的细胞，表明离子载体A23187对环AMP含量的影响是通过抑制腺苷酸环化酶介导的，而不是刺激环AMP磷酸二酯酶。10.得出结论，在完整的鸽子红细胞腺苷酸环化酶可能被抑制细胞内浓度的Ca 2+在1-10 μ M的范围内。
1. The bivalent cation ionophore A23187 was used to increase the intracellular concentration of Ca2+ in pigeon erythrocytes to investigate whether the increase in cyclic AMP content caused by adrenaline might be influenced by a change in intracellular Ca2+ in intact cells. 2. Incubation of cells with adrenaline, in the concentration range 0.55--55 muM, resulted in an increase in the concentration of cyclic AMP over a period of 60 min. The effect of adrenaline was inhibited by more than 90% with ionophore A23187 (1.9 muM) in the presence of 1 mM-Ca2+. This inhibition could be decreased by decreasing either the concentration of the ionophore or the concentration of extracellular Ca2+, and was independent of the concentration of adrenaline. 3. The effect of ionophore A23187 depended on the time of incubation. Time-course studies showed that maximum inhibition by ionophore A23187 was only observed when the cells were incubated with the ionophore for at least 15 min before the addition of adrenaline. 4. The inhibition by ionophore A23187 depended on the concentration of extracellular Ca2+. In the absence of Mg2+, ionophore A23187 (1.9 muM) inhibited the effect of adrenaline by approx. 30% without added Ca2+, by approx. 66% with 10 muM-Ca2+ and by more than 90% with concentrations of added Ca2+ greater than 30 muM. However, even in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate](0.1--10 mM), ionophore A23187 caused an inhibition of the cyclic AMP response of at least 30%, which may have been due to a decrease in cell Mg2+ concentration. 5. The addition of EGTA after incubation of cells with ionophore A23187 resulted in a partial reversal of the inhibition of the effect of adrenaline. 6. Inclusion of Mg2+ (2 mM) in the incubation medium antagonized the inhibitory action of ionophore A23187. This effect was most marked when the ionophore A23187 was added to medium containing Mg2+ before the addition of the cells. 7. The cellular content of Mg2+ was decreased by approx. 50% after 20 min incubation with ionophore A23187 (1.9 muM) in the presence of Ca2+ (1 mM) but no Mg2+. When Mg2+ (2 mM) was also present in the medium, ionophore A23187 caused an increase of approx. 80% in cell Mg2+ content. Ionophore A23187 had no significant effect on cell K+ content. 8. Ionophore A23187 caused a decrease in cell ATP content under some conditions. Since effects on cyclic AMP content could also be shown when ATP was not significanlty lowered, it appeared that a decrease in ATP in the cells could not explain the effect of ionophore A23187 on cyclic AMP. 9. Ionophore A23187 (1.9 muM), with 1 mM-Ca2+, did not enhance cyclic AMP degradation in intact cells, suggesting that the effect of ionophore A23187 on cyclic AMP content was mediated through an inhibition of adenylate cyclase rather than a stimulation of cyclic AMP phosphodiesterase. 10. It was concluded that in intact pigeon erythrocytes adenylate cyclase may be inhibited by intracellular concentrations of Ca2+ in the range 1-10 muM.