Firestone Institute for Respiratory Health, St. Joseph’s Hospital and the Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
Am J Physiol Lung Cell Mol Physiol. 2012 Aug 15;303(4):L334-42. doi: 10.1152/ajplung.00103.2012. Epub 2012 Jun 8.
Caffeine is sometimes used in cell physiological studies to release internally stored Ca(2+). We obtained evidence that caffeine may also act through a different mechanism that has not been previously described and sought to examine this in greater detail. We ruled out a role for phosphodiesterase (PDE) inhibition, since the effect was 1) not reversed by inhibiting PKA or adenylate cyclase; 2) not exacerbated by inhibiting PDE4; and 3) not mimicked by submillimolar caffeine nor theophylline, both of which are sufficient to inhibit PDE. Although caffeine is an agonist of bitter taste receptors, which in turn mediate bronchodilation, its relaxant effect was not mimicked by quinine. After permeabilizing the membrane using β-escin and depleting the internal Ca(2+) store using A23187, we found that 10 mM caffeine reversed tone evoked by direct application of Ca(2+), suggesting it functionally antagonizes the contractile apparatus. Using a variety of molecular techniques, we found that caffeine did not affect phosphorylation of myosin light chain (MLC) by MLC kinase, actin-filament motility catalyzed by MLC kinase, phosphorylation of CPI-17 by either protein kinase C or RhoA kinase, nor the activity of MLC-phosphatase. However, we did obtain evidence that caffeine decreased actin filament binding to phosphorylated myosin heads and increased the ratio of globular to filamentous actin in precontracted tissues. We conclude that, in addition to its other non-RyR targets, caffeine also interferes with actin function (decreased binding by myosin, possibly with depolymerization), an effect that should be borne in mind in studies using caffeine to probe excitation-contraction coupling in smooth muscle.
咖啡因有时被用于细胞生理学研究以释放内部储存的 Ca(2+)。我们获得的证据表明，咖啡因也可能通过以前未描述的不同机制起作用，并试图更详细地研究这种机制。我们排除了磷酸二酯酶 (PDE) 抑制的作用，因为这种作用 1）不能通过抑制蛋白激酶 A 或腺苷酸环化酶逆转；2）不能通过抑制 PDE4 加剧；3）不能被亚毫摩尔浓度的咖啡因或茶碱模拟，这两者都足以抑制 PDE。尽管咖啡因是苦味受体的激动剂，而苦味受体反过来又介导支气管扩张，但奎宁并不能模拟其松弛作用。在用 β-七叶皂甙和 A23187 耗尽内部 Ca(2+)库使膜穿孔后，我们发现 10 mM 咖啡因逆转了直接应用 Ca(2+)引起的张力，表明它在功能上拮抗了收缩装置。使用各种分子技术，我们发现咖啡因不影响肌球蛋白轻链激酶 (MLCK)对肌球蛋白轻链 (MLC)的磷酸化、MLCK 催化的肌动蛋白丝运动、蛋白激酶 C 或 RhoA 激酶对 CPI-17 的磷酸化，也不影响肌球蛋白磷酸酶的活性。然而，我们确实有证据表明咖啡因降低了磷酸化肌球蛋白头部与肌动蛋白丝的结合，并增加了预收缩组织中球状肌动蛋白与丝状肌动蛋白的比例。我们的结论是，除了其对 RyR 的其他非靶点作用外，咖啡因还干扰了肌动蛋白的功能（肌球蛋白结合减少，可能伴有解聚），在使用咖啡因研究平滑肌兴奋-收缩偶联的研究中应牢记这一点。
