Albuquerque F C, Smith E H, Kellum J M
Department of Surgery, Medical College of Virginia/VCU, Richmond, Virginia, 23298-0161, USA.
J Surg Res. 1998 Jul 1;77(2):137-40. doi: 10.1006/jsre.1998.5361.
Previous studies demonstrate that both 5-hydroxytryptamine (5-HT) and cyclic AMP (cAMP) induce chloride efflux from crypt colonocytes in the rat distal colon; antagonist studies suggest that the 5-HT response is mediated primarily by the 5-HT4 receptor. Since this receptor is known to be positively coupled to adenylate cyclase, we postulated that 5-HT should induce generation of cAMP, which should be inhibited by 5-HT4 antagonists. Method. Mucosal cells from rat distal colon were taken by a sequential calcium chelation technique for enrichment of crypt cells. Cytokeratin stains demonstrated that >99% of cells were colonocytes. [3H]Thymidine uptake studies demonstrate a fivefold increased incorporation in this cell preparation compared to earlier fractions. 3-Isobutyl-l-methylxanthine (IBMX, 100 microM) was added to all cell suspensions in order to prevent cAMP metabolism. Cell suspensions were incubated for 2 min at 37 degreesC with different concentrations of 5-HT (n = 7). cAMP was measured by enzyme immunoassay. In another series of experiments, 5-HT (0.3 microM) stimulation of cAMP was similarly measured in the presence and absence of 5-HT receptor antagonists: 10 microM 5-HTP-DP (5-HT1P; n = 4), 0.1 microM ketanserin (5-HT2A; n = 4), 0.3 microM ondansetron (5-HT3; n = 4), 3 microM tropisetron (5-HT3 and 5-HT4; n = 4), and 10 nM GR-113808 (5-HT4; n = 5). Results. 5-HT produced a dose-dependent increase in cAMP. The increase was significant at concentrations >/=0.3 microM when compared to cells incubated with IBMX alone. In the second series of experiment, 5-HT-induced generation of cAMP at a dose of 0.3 microM was significantly inhibited in the presence of GR-113808 and tropisetron. Conclusion. 5-HT acts at a 5-HT4 receptor to induce production of cAMP in rat distal crypt colonocytes.
先前的研究表明，5-羟色胺（5-HT）和环磷酸腺苷（cAMP）均可诱导大鼠远端结肠隐窝结肠细胞中的氯离子外流；拮抗剂研究表明，5-HT反应主要由5-HT4受体介导。由于已知该受体与腺苷酸环化酶呈正偶联，我们推测5-HT应诱导cAMP的生成，而5-HT4拮抗剂应能抑制这一过程。方法。采用连续钙螯合技术获取大鼠远端结肠的黏膜细胞，以富集隐窝细胞。细胞角蛋白染色显示，>99%的细胞为结肠细胞。[3H]胸腺嘧啶摄取研究表明，与早期组分相比，该细胞制剂中的掺入量增加了五倍。向所有细胞悬液中加入3-异丁基-1-甲基黄嘌呤（IBMX，100 microM）以防止cAMP代谢。细胞悬液在37℃下与不同浓度的5-HT（n = 7）孵育2分钟。通过酶免疫测定法测量cAMP。在另一系列实验中，在存在和不存在5-HT受体拮抗剂的情况下，同样测量了5-HT（0.3 microM）对cAMP的刺激作用：10 microM 5-HTP-DP（5-HT1P；n = 4）、0.1 microM酮色林（5-HT2A；n = 4）、0.3 microM昂丹司琼（5-HT3；n = 4）、3 microM托烷司琼（5-HT3和5-HT4；n = 4）以及10 nM GR-113808（5-HT4；n = 5）。结果。5-HT使cAMP呈剂量依赖性增加。与仅用IBMX孵育的细胞相比，浓度≥0.3 microM时增加显著。在第二系列实验中，在存在GR-113808和托烷司琼的情况下，0.3 microM剂量的5-HT诱导的cAMP生成受到显著抑制。结论。5-HT作用于5-HT4受体以诱导大鼠远端隐窝结肠细胞中cAMP的产生。
