Dual regulation by cAMP of beta-hexosaminidase-induced mitogenesis in bovine tracheal myocytes.
Dual regulation by cAMP of beta-hexosaminidase-induced mitogenesis in bovine tracheal myocytes.
复制标题
cAMP 对牛气管肌细胞 β-己糖胺酶诱导的有丝分裂的双重调节。
DOI：
10.1165/ajrcmb/7.6.614
复制
发表时间：
1992
影响因子：
6.4
通讯作者：
Malik,KU
中科院分区：
文献类型：
--
作者：
Lew,DB;Nebigil,C;Malik,KU
Materials and MethodsIsolation and Culture of Tracheal Smooth Muscle Cells Primary cultures of BTM were prepared by a modified method of Kotlikoffand co-workers (11), as described previously (1). Approximately 1 g of dissected bovine trachealis muscle was minced and digested by two successive 45-min incubations at 37 C in 10 ml fresh minimum essential medium containing 0.2 mM CaCb, collagenase (Sigma Type IV, 350 U/ml), protease (bovine pancreas, 0.3 mg/ml), trypsin inhibitor (soybean, 0.4 mg/ml), and globulin-free crystalline bovine serum albumin (2 mg/ml). Dissociated cells were washed by centrifugation and maintained in T-75 flasks in medium Ml99 containing 10% fetal bovine serum (FBS) and antimicrobial-antimycotic solution. Tracheal smooth muscle cells prepared by this method are recognized by monoclonal antibody raised against synthetic smooth muscle actin homogenously (1). Cells of passages 0 to 4 from 108 cow tracheas were used for experiments.Experimental Protocol Protocoll. The first series of experiments were performed to determine the time course of the effect of {j-hexosaminidase (Hex B) and mannose bovine serum albumin (ManBSA) on cAMP accumulation in BTM. We performed time-course experiments over 48 h according to the following protocol. Confluent tracheal smooth cells were detached bytrypsinization and seeded (100,000 cells/16-mm well) in Ml99 with 10% FBS. Approximately 72 h later, confluent cells were incubated with fresh medium containing 0.4% FBS. Fortyeight hours later, quiescent cells were washed twice with Ca2+-free and Mg2+-free Hanks' balanced salt solution. Cells were preincubated with M199 without serum for 5 min, supernatant was removed, and the procedure was repeated. Just prior to the experimental period, medium was changed with serum-free fresh M199. Isobutyl methylxanthine (IBMX; 100 t-tM) was added 15 min prior to each stopping time point. At the end of each experimental period, supernatant was removed and 5 mM sodium acetate buffer (pH 4.2, 1 mllwell) was added. Cell extracts were frozen and thawed, and acid-insoluble protein components were removed by 3 min of boiling and centrifugation at 1,500 x g for 15 min at 4 C. The supernatant was acetylated by adding 5 t-tl of triethylamine and acetic anhydride mixture (2: 1, vollvol) to 50-t-t1 aliquots of cAMP standards and samples. Acetylated samples were assayed for cAMP by radioimmunoassay using! 25I-cAMP as tracer (12). Protein content of the cell extract was measured by Lowry's protein assay (13). cAMP accumulation was expressed as fmollt-tg protein. The results were correlated with the previous results on time-course response on 3H-thymidineincorporation induced by Hex B (1).
