Perianin A, Synderman R
Department of Pharmacological Sciences, Genentech, Inc., South San Francisco, California 94080.
J Biol Chem. 1989 Jan 15;264(2):1005-9.
The stimulation of polymorphonuclear leukocytes (PMNs) by chemoattractants triggers a rapid rise in cytosolic free calcium concentration(s) ([Ca2+]i), which quickly returns to base line, suggesting a role for calcium removal in the homeostasis of activated PMNs. To investigate cytosolic calcium homeostasis, PMNs were treated with a fluoroprobe and ionomycin to induce a sustained elevation of [Ca2+]i. The cells were then stimulated, and attenuation of the fluorescence signal was measured as an indication of calcium loss from the cytosol. The formyl peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and 1,2-dioctanoyl-sn-glycerol, but not the inactive phorbol ester 4 alpha-phorbol didecanoate, induced a dose-dependent decrease in [Ca2+]i in ionomycin-pretreated cells. However, the decline in [Ca2+]i caused by PMA was sustained and occurred following a lag time, whereas the response to fMLP was immediate, lasted approximately 2 min, and then was followed by a return of [Ca2+]i to its initial level. The restoration of [Ca2+]i required extracellular calcium. Varying the ionomycin concentration allowed studies at different initial [Ca2+]i, which in untreated PMNs was approximately 135 nM. In contrast to fMLP, PMA did not lower calcium at concentrations below 200 nM. The decline in [Ca2+]i induced by fMLP, but not PMA, was blocked by pertussis toxin. In contrast, the decrease in [Ca2+]i caused by PMA and 1,2-dioctanoyl-sn-glycerol, but not fMLP, was inhibited by the protein kinase C antagonists staurosporine, H-7, and sphingosine. These results suggest that formyl peptide chemoattractants transiently stimulate an activity which lowers [Ca2+]i to normal intracellular levels. Activation of this process appears to be independent of protein kinase C. An additional cytosolic calcium lowering activity, dependent on protein kinase C, operates at [Ca2+]i above 200 nM. Thus, activated PMNs can use at least two processes for attentuation of elevated cytosolic calcium levels.
趋化因子对多形核白细胞（PMNs）的刺激会引发胞质游离钙浓度（[Ca2+]i）迅速升高，随后又迅速恢复至基线水平，这表明钙的移除在活化的PMNs的内环境稳定中发挥作用。为了研究胞质钙稳态，用荧光探针和离子霉素处理PMNs以诱导[Ca2+]i持续升高。然后刺激细胞，并测量荧光信号的衰减，以此作为胞质钙流失的指标。甲酰肽趋化因子N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）、佛波酯肉豆蔻酸乙酸酯（PMA）和1,2-二辛酰-sn-甘油，但非无活性的佛波酯4α-佛波十二烷酸酯，在离子霉素预处理的细胞中诱导[Ca2+]i呈剂量依赖性降低。然而，PMA引起的[Ca2+]i下降是持续的，且有一段滞后时间，而对fMLP的反应是即时的，持续约2分钟，随后[Ca2+]i恢复到初始水平。[Ca2+]i的恢复需要细胞外钙。改变离子霉素浓度可在不同的初始[Ca2+]i水平下进行研究，未处理的PMNs中的初始[Ca2+]i约为135 nM。与fMLP不同，PMA在浓度低于200 nM时不会降低钙水平。fMLP诱导的[Ca2+]i下降，但PMA诱导的[Ca2+]i下降不受百日咳毒素阻断。相反，PMA和1,2-二辛酰-sn-甘油引起的[Ca2+]i下降，但fMLP引起的[Ca2+]i下降不受蛋白激酶C拮抗剂星形孢菌素、H-7和鞘氨醇抑制。这些结果表明，甲酰肽趋化因子短暂刺激一种活性，该活性将[Ca2+]i降低至正常细胞内水平。这一过程的激活似乎与蛋白激酶C无关。另一种依赖蛋白激酶C的胞质钙降低活性在[Ca2+]i高于200 nM时起作用。因此，活化的PMNs可至少利用两种过程来降低升高的胞质钙水平。
