Giron Maria Cecilia, Bin Anna, Brun Paola, Etteri Sabrina, Bolego Chiara, Florio Chiara, Gaion Rosa Maria
Department of Pharmacology and Anesthesiology, University of Padova, Padova, Italy.
Gastroenterology. 2008 Apr;134(4):1116-26. doi: 10.1053/j.gastro.2008.01.030. Epub 2008 Jan 17.
BACKGROUND & AIMS: Extracellular adenosine plays a relevant role in regulating intestinal motility and preventing inflammatory processes. Adenosine 3',5'-cyclic monophosphate (cAMP) extruded from cells may be converted to adenosine monophosphate and then to adenosine by ecto-phosphodiesterase and CD73/ecto-5'nucleotidase, respectively, thus representing a source of adenosine. Our purpose was to assess the existence of a functional extracellular cAMP-adenosine pathway in intestinal tissue, obtaining evidence for CD73 expression and evaluating the effect of cAMP on ileum motility.
The formation of cAMP metabolites in rat ileum strips incubated with exogenous cAMP or [(3)H]cAMP was monitored by high-performance liquid chromatography. CD73 was detected by immunoprecipitation and immunofluorescence. The functional activity of exogenous cAMP on ileum strips was recorded by measuring tension changes.
In ileum strips, the generation of cAMP-derived adenosine monophosphate, adenosine, and inosine was time and concentration dependent and was blocked by phosphodiesterase or CD73 inhibitors in a manner consistent with exogenous cAMP being processed through the extracellular cAMP-adenosine pathway. Accordingly, [(3)H]cAMP uptake in ileum strips was negligible. Immunofluorescence revealed CD73 surface expression on intestinal smooth muscle cells and intact smooth muscle. Exogenous cAMP concentration-dependently increased ileum muscle tension partially inhibited by adenosine inactivation or receptor blockade. Forskolin-stimulated endogenous cAMP induced concentration-dependent ileum relaxations.
A functioning extracellular cAMP-adenosine pathway featuring CD73 expression is present in rat ileum and affects intestinal motility. Extracellular cAMP may therefore act on intestinal muscle both directly by binding to specific smooth muscle cell membrane sites and indirectly through its degradation products.
细胞外腺苷在调节肠道蠕动和预防炎症过程中发挥着重要作用。从细胞中挤出的3',5'-环磷酸腺苷（cAMP）可分别通过胞外磷酸二酯酶和CD73/胞外5'-核苷酸酶转化为单磷酸腺苷，然后再转化为腺苷，从而成为腺苷的一个来源。我们的目的是评估肠道组织中功能性细胞外cAMP-腺苷途径的存在，获取CD73表达的证据，并评估cAMP对回肠蠕动的影响。
通过高效液相色谱法监测与外源性cAMP或[³H]cAMP孵育的大鼠回肠条中cAMP代谢产物的形成。通过免疫沉淀和免疫荧光检测CD73。通过测量张力变化记录外源性cAMP对回肠条的功能活性。
在回肠条中，cAMP衍生的单磷酸腺苷、腺苷和肌苷的生成具有时间和浓度依赖性，并被磷酸二酯酶或CD73抑制剂阻断，其方式与外源性cAMP通过细胞外cAMP-腺苷途径进行处理一致。因此，回肠条中[³H]cAMP的摄取可忽略不计。免疫荧光显示肠道平滑肌细胞和完整平滑肌表面表达CD73。外源性cAMP浓度依赖性地增加回肠肌肉张力，腺苷失活或受体阻断可部分抑制该作用。福司可林刺激的内源性cAMP诱导浓度依赖性的回肠松弛。
大鼠回肠中存在以CD73表达为特征的功能性细胞外cAMP-腺苷途径，并影响肠道蠕动。因此，细胞外cAMP可能通过与特定平滑肌细胞膜位点结合直接作用于肠道肌肉，并通过其降解产物间接发挥作用。
