Fukuichi Ohsawa, Miki Yamauchi, Hiroshi Nagaso, Shoichi Murakami, Jun
Baba and Aiko Sawa
Drug Discovery, Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd.,
760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan
Abstract: 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 microM 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.
Keywords: Phosphodiesterase, Rolipram, Inhibition, Purification, Brain