Effects Of Arecolinehydrobromide On Intracellular Free Ca～(2+) Of Colonic Smooth Muscle Cells

Objective: 1.To establish an efficient culture method of smooth muscle cells (SMCs) from the rat colon in vitro. 2.To observe the effects of arecolinehydrobromide (Ah) on contraction of smooth muscle cells (SMCs) of rat colon and on intrcellular concentration of free Ca~2+.Methods: 1.Freshly isolated SMC from the muscle layer of the distal colon were prepared by collagenase digestion , and cultured in DMEM supplemented with 10% fetal bovine serum. The cells were subcultured and identified by immunocytochemical staining. 2.SMCs of rat colon were cultured and divided into four groups and loaded with specific fluorescent Ca~2+ indicator Fluo-3/AM. Laser scanning confocal microscope (LSCM) was used to detect intrcellular concentration of free Ca~2+ and cellular contract percentage.Results: 1.Freshly isolated SMCs were spindle-shaped with centrally located nuclei. In primary culture, SMCs attached to the culture vessels by 24 h, proliferated by 3-5 d, and reached confluency by 14d with a "hill-and-valley" pattern. Cultured cells were identified by intensely positive immunocytochemical staining to smooth muscle actin-specific. 2. In Ah stimulation group, a rapid elevation of intracellular [Ca~2+]i occurred shortly after adding Ah , which was depicted as a pulse wave. Then there was a gradually slow increase inintracellular [Ca~2+]i which reached its peak at 484.0172+47.55s. The peak was followed by a quite long plateau and afterward returned to resting level at 1400S. The cellular contraction percentage was 20.70 ± 0.072%. No spontaneous cellular contraction took place in normal control group in which there was a tendency of decrease in FI because of attenuation of indicator. In Ach stimulation group, there was a same tendency as which happened in Ah stimulation group. In Atropine group, the contractive effect was completely inhibited even after adding Ah in cells pretreated by atropine.Conclusion: 1.The procedure mentioned above is efficient and reproducible for isolation, culture and identification of rat colon smooth muscle cells. 2.Ah can cause contraction of rat colon SMCs and elevation in concentration of intracellular Ca .