pH is critical to the regulation of expression of the β_2-adrenergic receptor gene in hypoxia

摘要

Expression and function of the β2-adrenergic receptor (β2-AR), a critical modulator of motor function, is altered in ischemic tissues. However, the mechanism by which ischemia influences gene expression remains controversial, in part because of the conflicting results reported by numerous investigators. To determine the relative importance of hypoxia and acidosis on β2-AR expression and function, steady-state mRNA levels and receptor function were measured in DDT1MF-2 hamster smooth muscle cells grown in 10% serum and 3 nM epinephrine in 5% CO2 (pH 7.50) and then exposed for 48 h to either combined hypoxia with acidosis (through incubation in 2% O2, 10% CO2, mean pH 7.14 at 48 h), hypoxia alone (2% O2, 2.5% CO2, pH 7.36), normoxia-acidosis (21% O2, 10% CO2, pH 7.12) or continued normoxia (21% O2, 2.5% CO2, pH 7.49). Combined hypoxia-acidosis downregulated the β2-AR membrane density by 50% compared to hypoxia alone and normoxia alone at 48 h. β2-AR coupling in these cells, as measured by cellular cAMP production in response to 10?4 M isoproterenol, was decreased by hypoxia but increased by acidosis. The effect of hypoxia-acidosis on Bmax was abolished by inhibiting transcription with 1.0 μg/ml actinomycin D. A quantitative reverse transcriptase polymerase chain reaction assay demonstrated a decrease in steady-state mRNA concentration with hypoxia-acidosis. Our experiments demonstrate an important distinction between the effects of modeled hypoxia and ischemia on β2-AR gene expression.