Inhibitory effects of rolipram on partially purified phosphodiesterase 4 from rat brains.
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.
Ohsawa
F
F
Drug Discovery, Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., Yokohama, Japan.
Yamauchi
M
M
Nagaso
H
H
Murakami
S
S
Baba
J
J
Sawa
A
A
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008297:Male; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011760:Pyrrolidinones / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D020889:Rolipram; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000737:chemistry / Q000201:enzymology; D013379:Substrate Specificity
D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020889:Rolipram