Several previous studies have demonstrated that the phosphodiesterase 4 selective inhibitor rolipram affects cellular function at a much lower concentration than the reported Ki value for phosphodiesterase 4 inhibition. In this study, we examined the inhibitory effect of rolipram on rat brain phosphodiesterase 4 to determine the heterogeneity of the enzyme activity. Partial purification of various phosphodiesterases from the rat brain was performed by anion-exchange chromatography. The eluant was pooled into four fractions, two of which manifested cAMP-selective phosphodiesterase activity that was blocked by 10 μM of rolipram, indicating the presence of phosphodiesterase 4 in these fractions. The IC50 of rolipram (racemate) of these two fractions was 492 and 79 nM, respectively. The R-(-)-enantiomer of rolipram inhibited the cAMP-phosphodiesterase activity in the latter fraction 10 times more than did S-(+)-rolipram, and the inhibition of the former fraction was less stereospecific. Dixon plot analysis revealed that the rolipram enantiomers inhibited the cAMP-phosphodiesterase in the latter fraction in a multiphasic manner, with two Ki values, one at the micromolar level and the other at the sub-micromolar level, respectively, for both of the enantiomers. These results suggest that there is a heterogeneity for phosphodiesterase 4 in the rat brain, and some of the phosphodiesterase forms are sensitive to rolipram.
The Japanese Pharmacological Society 1998