Halothane facilitates the translocation of GRK-2 and phosphorylation of beta 2-adrenergic receptor in rat synaptosomes

Purpose: To examine the effect of halothane on beta 2-adrenergic receptor p hosphorylation and on G-protein coupled receptor kinase (GRK), responsible for beta 2-receptor downregulation. Methods: Rat forebrain synaptosomes were incubated for 30 min with halothan e 1 or 2%. The cytosolic and membrane fractions were separated, and phospho rylation activity of recombinant beta 2-adrenergic receptor was quantified autoradiographically using P-32 labeled adenosine triphosphate. Phosphoryla tion activity of a specific GRK-2 substrate, was examined by measuring P-32 binding. Subcellular localization-of the enzyme was immunologically analyz ed by Western blotting. Results: Halothane 2% decreased the phosphorylation activity of the recombi nant receptor in the cytosol fraction, regardless of 1 mu M isoproterenol ( ISP) (P < 0.01), which activity in the membrane fraction was increased (P < 0.01). Phosphorylation activity of the synthetic peptide decreased in the cytosol obtained from synaptosomes exposed to halothane 2% (P < 0.05). In c ontrast, activity in the membrane increased by exposure to halothane 2% (P < 0.01). The concentration of GRK-2 decreased in the cytosol obtained from synaptosomes exposed to halothane 1% or 2% (decreases of 8.3 +/- 1.2% @ 1%, and 18.0 +/- 2.1% @ 2%, P < 0.05). In the membrane, exposure to halothane 1% or 2% increased the GRK-2 amount dose dependently (22.5 +/- 3.1% @ 1%, a nd by 45.7 +/- 6.1% @ 2%, P < 0.01). Conclusion: Halothane could facilitate translocation of GRK-2 and possibly promote the downregulation of O-2-adrenergic receptors in the synaptic memb rane. The anesthetic action and hemodynamic suppressive action of halothane may be related to this phenomenon.