Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, MA, USA.
Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, MA, USA; Department of Ophthalmology, Tufts University School of Medicine, Boston, MA, USA.
Exp Eye Res. 2023 Aug;233:109526. doi: 10.1016/j.exer.2023.109526. Epub 2023 Jun 7.
The aim of these studies was to investigate the involvement of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP) and its downstream effectors in oxytocin (OXT)-mediated lacrimal gland myoepithelial cell (MEC) contraction. Lacrimal gland MEC were isolated and propagated from alpha-smooth muscle actin (SMA)-GFP mice. RNA and protein samples were prepared to analyze G protein expression by RT-PCR and western blotting; respectively. Changes in intracellular cAMP concentration were measured using a competitive ELISA kit. To increase intracellular cAMP concentration, the following agents were used: forskolin (FKN, a direct activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of the phosphodiesterase that hydrolyzes cAMP), or a cell permeant cAMP analog, dibutyryl (db)-cAMP. In addition, inhibitors and selective agonists were used to investigate the role of cAMP effector molecules, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in OXT-induced MEC contraction. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. The adenylate cyclase coupling G proteins, Gαs, Gαo, and Gαi, are expressed in lacrimal gland MEC at both the mRNA and protein levels. OXT increased intracellular cAMP in a concentration-dependent manner. FKN, IBMX and db-cAMP significantly stimulated MEC contraction. Preincubation of cells with either Myr-PKI, a specific PKA inhibitor or ESI09, an EPAC inhibitor, resulted in almost complete inhibition of both FKN- as well as OXT-stimulated MEC contraction. Finally, direct activation of PKA or EPAC using selective agonists triggered MEC contraction. We conclude that cAMP agonists modulate lacrimal gland MEC contraction via PKA and EPAC activation which also play a major role in OXT induced MEC contraction.
这些研究的目的是探讨第二信使 3'，5'-环腺苷酸（cAMP）及其下游效应物在催产素（OXT）介导的泪腺肌上皮细胞（MEC）收缩中的作用。泪腺 MEC 是从 α-平滑肌肌动蛋白（SMA）-GFP 小鼠中分离和培养的。通过 RT-PCR 和 Western blot 分别分析 RNA 和蛋白质样品中的 G 蛋白表达；分别。使用竞争 ELISA 试剂盒测量细胞内 cAMP 浓度的变化。为了增加细胞内 cAMP 浓度，使用了以下试剂：forskolin（FKN，一种激活腺苷酸环化酶的直接激活剂）、3-异丁基-1-甲基黄嘌呤（IBMX，一种水解 cAMP 的磷酸二酯酶抑制剂）或细胞可渗透的 cAMP 类似物，二丁酰基（db）-cAMP。此外，还使用抑制剂和选择性激动剂来研究 cAMP 效应分子蛋白激酶 A（PKA）和 cAMP 激活的交换蛋白（EPAC）在 OXT 诱导的 MEC 收缩中的作用。实时监测 MEC 收缩，并用 ImageJ 软件量化细胞大小的变化。在 mRNA 和蛋白质水平上，泪腺 MEC 表达与腺苷酸环化酶偶联的 G 蛋白 Gαs、Gαo 和 Gαi。OXT 以浓度依赖的方式增加细胞内 cAMP。FKN、IBMX 和 db-cAMP 显著刺激 MEC 收缩。细胞预先孵育 Myr-PKI（一种特异性 PKA 抑制剂）或 ESI09（一种 EPAC 抑制剂）可导致 FKN 和 OXT 刺激的 MEC 收缩几乎完全抑制。最后，使用选择性激动剂直接激活 PKA 或 EPAC 可引发 MEC 收缩。我们得出结论，cAMP 激动剂通过 PKA 和 EPAC 激活来调节泪腺 MEC 收缩，PKA 和 EPAC 激活在 OXT 诱导的 MEC 收缩中也发挥主要作用。
