Plasma membrane-bound cyclic AMP phosphodiesterase activity in 3T3-L1 adipocytes

Abstract

Plasma membranes were isolated from 3T3-L1 adipocytes. Plasma membrane phosphodiesterase (PM-PDE) was measured in the presence of 5 μM cilostamide. Time course and cAMP dose response ranging from 0 to 2 μM were measured. PM-PDE remained linear up to 20 min. Non-linear curve fitting analysis showed that the low K m cAMP dose data fit a two component curve significantly better than a one component curve, indicating that there are two iso-forms of PDE in the plasma membrane of 3T3-L1 adipocytes, similar to swine adipocytes. The K m and V max values for this two component curve were K m1=0.12 μM, V max1=3.08 pmol min −1 mg −1 protein, and K m2=3.67 μM, V max2=83.8 pmol min −1 mg −1 protein. Inhibitors of PDE1, PDE2 and PDE5 failed to inhibit PM-PDE, as observed in swine adipocyte plasma membranes. However, PDE4 inhibitors were three-fold more effective at inhibiting PDE in 3T3-L1 PM compared to swine adipocyte PM. One mM 1, 3-dipropyl-8- p-sulfophenylxanthine (DPSPX) inhibited PM-PDE by approximately 75% in both preparations. These data demonstrate that PM-PDE is distinct from microsomal membrane PDE and may be responsible for extracellular cAMP metabolism to AMP in 3T3-L1 adipocytes.