Lew D B, Nebigil C, Malik K U
Department of Pediatrics and Pharmacology, College of Medicine, University of Tennessee, Memphis.
Am J Respir Cell Mol Biol. 1992 Dec;7(6):614-9. doi: 10.1165/ajrcmb/7.6.614.
beta-Hexosaminidases, potent mitogens in bovine tracheal myocytes (BTM), stimulate a rapid and transient increase in intracellular cyclic adenosine monophosphate (cAMP) accumulation. The objective of this study was to elucidate the contribution of cAMP in hexosaminidase-induced airway muscle proliferation. Rate of DNA synthesis was measured by 3H-thymidine incorporation in quiescent cells prepared by a low-serum treatment (0.4%) for 48 h after reaching confluency in microtiter wells. cAMP accumulation was measured in acetylated cell extracts in the presence of isobutyl methylxanthine (100 microM) by radioimmunoassay using 125I-cAMP as tracer. Exposure of quiescent cells to purified human placental hexosaminidase B (5 micrograms/ml, 50 nM) caused a significant transient increase in cAMP accumulation (49 to 107 fmol/micrograms protein, or a 20- to 70-fold increase from basal level). Maximum increase occurred at 15 min followed by a rapid decline in cAMP accumulation within 30 min after exposure to hexosaminidase. Similar results were obtained in cells treated with neoglycoprotein mannose bovine serum albumin (100 to 500 nM). The increase in cAMP accumulation was inhibited by mannan (mannose receptor blocker, 0.1 mg/ml), as well as phenylisopropyladenosine (PIA; A1 receptor agonist that inhibits adenylyl cyclase, 0.1 to 1.0 microM). The increase in 3H-thymidine incorporation induced by hexosaminidase B was also inhibited by mannan and PIA. Exposure to 8-(4-chlorophenylthio)-cAMP (cpt-cAMP; a cell-permeable analog of cAMP, 100 microM) or forskolin (a direct activator of catalytic subunit of adenylyl cyclase, 24 microM) up to 6 h enhanced 3H-thymidine incorporation. In contrast, a prolonged exposure (18 to 30 h) to these agents inhibited 3H-thymidine incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)
β-己糖胺酶是牛气管肌细胞（BTM）中的强效促有丝分裂原，可刺激细胞内环状腺苷单磷酸（cAMP）积累迅速且短暂增加。本研究的目的是阐明cAMP在己糖胺酶诱导的气道平滑肌增殖中的作用。DNA合成速率通过在微孔板中汇合后用低血清处理（0.4%）48小时制备的静止细胞中掺入³H-胸腺嘧啶核苷来测定。在存在异丁基甲基黄嘌呤（100μM）的情况下，通过使用¹²⁵I-cAMP作为示踪剂的放射免疫测定法，在乙酰化细胞提取物中测量cAMP积累。将静止细胞暴露于纯化的人胎盘己糖胺酶B（5μg/ml，50nM）会导致cAMP积累显著短暂增加（49至107fmol/μg蛋白质，或比基础水平增加20至70倍）。最大增加发生在15分钟，随后在暴露于己糖胺酶后30分钟内cAMP积累迅速下降。在用新糖蛋白甘露糖牛血清白蛋白（100至500nM）处理的细胞中获得了类似结果。cAMP积累的增加受到甘露聚糖（甘露糖受体阻滞剂，0.1mg/ml）以及苯异丙基腺苷（PIA；抑制腺苷酸环化酶的A1受体激动剂，0.1至1.0μM）的抑制。己糖胺酶B诱导的³H-胸腺嘧啶核苷掺入增加也受到甘露聚糖和PIA的抑制。暴露于8-（4-氯苯基硫代）-cAMP（cpt-cAMP；一种可渗透细胞的cAMP类似物，100μM）或福斯可林（腺苷酸环化酶催化亚基的直接激活剂，24μM）长达6小时可增强³H-胸腺嘧啶核苷掺入。相反，长时间暴露（18至30小时）于这些试剂会抑制³H-胸腺嘧啶核苷掺入。（摘要截断于250字）
