Seensalu R, Avedian D, Barbuti R, Song M, Slice L, Walsh J H
CURE: Digestive Diseases Research Center, Department of Medicine, UCLA, Los Angeles, California 90073, USA.
J Clin Invest. 1997 Sep 1;100(5):1037-46. doi: 10.1172/JCI119614.
Isolated canine G cells in primary culture have been used to study calcium, protein kinase C (PKC), and rho/cytoskeletal-dependent intracellular pathways involved in bombesin- stimulated gastrin release. A method to obtain highly purified G cells by culture (64% G cells) after flow cytometry on elutriated fractions of cells from digested canine gastric antral mucosa has been developed. Pretreatment of G cells with thapsigargin (10(-8)-10(-6) M) and release experiments in Ca2+-containing or -depleted media showed that influx of Ca2+ into the cells and not acute release from intracellular stores plays an important role in bombesin-stimulated gastrin release. Inhibition of PKC by the specific inhibitor GF 109 203X did not affect bombesin-stimulated release. Rho, a small GTP-binding protein that regulates the actin cytoskeleton, is specifically antagonized by Clostridium botulinum C3 exoenzyme. C3 (10 microg/ml) enhanced basal and bombesin-stimulated gastrin release by 315 and 266%, respectively. The importance of the cytoskeleton for regulation of gastrin release was emphasized by a more pronounced release of gastrin when the organization of the actin cytoskeleton was disrupted by cytochalasin D (5 x 10(-)7 and 10(-)6 M). Wortmannin, a potent inhibitor of phosphoinositide-3-kinase, did not alter bombesin-stimulated gastrin release. Thus, it is concluded that bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by PKC, and is enhanced by disruption of rho/cytoskeletal pathways.
原代培养的分离犬G细胞已被用于研究参与蛙皮素刺激胃泌素释放的钙、蛋白激酶C（PKC）和rho/细胞骨架依赖性细胞内途径。已开发出一种方法，通过对消化后的犬胃窦黏膜细胞的淘洗级分进行流式细胞术分选后培养（64%为G细胞）来获得高度纯化的G细胞。用毒胡萝卜素（10⁻⁸ - 10⁻⁶ M）预处理G细胞，并在含Ca²⁺或无Ca²⁺的培养基中进行释放实验，结果表明Ca²⁺流入细胞而非细胞内储存的急性释放，在蛙皮素刺激的胃泌素释放中起重要作用。特异性抑制剂GF 109 203X对PKC的抑制并不影响蛙皮素刺激的释放。Rho是一种调节肌动蛋白细胞骨架的小GTP结合蛋白，可被肉毒杆菌C3外毒素特异性拮抗。C3（10微克/毫升）分别使基础和蛙皮素刺激的胃泌素释放增加315%和266%。当肌动蛋白细胞骨架的组织被细胞松弛素D（5×10⁻⁷和10⁻⁶ M）破坏时，胃泌素释放更为明显，这强调了细胞骨架对胃泌素释放调节的重要性。渥曼青霉素是一种有效的磷脂酰肌醇-3-激酶抑制剂，它不会改变蛙皮素刺激的胃泌素释放。因此，得出结论：蛙皮素诱导犬G细胞释放胃泌素受Ca²⁺刺激而非PKC，并且rho/细胞骨架途径的破坏会增强这种释放。
