DUAL REGULATION BY CAMP OF BETA-HEXOSAMINIDASE-INDUCED MITOGENESIS IN BOVINE TRACHEAL MYOCYTES

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 H-3-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 muM) by radioimmunoassay using I-125-cAMP as tracer. Exposure of quiescent cells to purified human placental hexosaminidase B (5 mug/ml, 50 nM) caused a significant transient increase in cAMP accumulation (49 to 107 fmol/mug 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 muM). The increase in H-3-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 muM) or forskolin (a direct activator of catalytic subunit of adenylyl cyclase, 24 muM) up to 6 h enhanced H-3-thymidine incorporation. In contrast, a prolonged exposure (18 to 30 h) to these agents inhibited H-3-thymidine incorporation. Cholera toxin (CT; 0.1 to 5.0 nM) inhibited hexosaminidase-induced increase in H-3-thymidine incorporation. Repeated exposure of cells to CT (1 nM), forskolin (24 muM), or cpt-cAMP (100 muM) in fresh medium at 6-h intervals inhibited H-3-thymidine incorporation in the absence of hexosaminidase. These data suggest that cAMP has a dual regulatory influence on BTM proliferation. An early transient increase in cAMP accumulation in quiescent cells is involved in the mitogenesis and contributes to hexosaminidase-induced mitogenesis, whereas a persistent elevation of cAMP acts as an antimitogen.