Evidence that cyclic AMP phosphodiesterase inhibitors suppress TNFα generation from human monocytes by interacting with a ‘low‐affinity’ phosphodiesterase 4 conformer
Authors: Souness John E. , Griffin Miriam , Maslen Christopher , Ebsworth Karen , Scott Lisa C. , Pollock Kenneth , Palfreyman Malcolm N. , Karlsson Jan‐Anders 
Published: July 2012

We have investigated the inhibitory effects of RP 73401 (piclamilast) and rolipram against human monocyte cyclic AMP‐specific phosphodiesterase (PDE4) in relation to their effects on prostaglandin (PG)E2‐induced cyclic AMP accumulation and lipopolysaccharide (LPS)‐induced TNFα production and TNFα mRNA expression.
PDE4 was found to be the predominant PDE isoenzyme in the cytosolic fraction of human monocytes. Cyclic GMP‐inhibited PDE (PDE3) was also detected in the cytosolic and particulate fractions. Reverse transcription polymerase chain reaction (RT‐PCR) of human monocyte poly (A+) mRNA revealed amplified products corresponding to PDE4 subtypes A and B of which the former was most highly expressed. A faint band corresponding in size to PDE4D was also observed.
RP 73401 was a potent inhibitor of cytosolic PDE4 (IC50: 1.5 ± 0.6 nM, n = 3). (±)‐Rolipram (IC50: 313 ± 6.7 nM, n = 3) was at least 200 fold less potent than RP 73401. R‐(−)−rolipram was approximately 3 fold more potent than S‐(+)‐rolipram against cytosolic PDE4.
RP 73401 (IC50: 9.2 ± 2.1 nM, n = 6) was over 50 fold more potent than (±)‐rolipram (IC50: 503 ± 134 nM, n = 6)) in potentiating PGE2‐induced cyclic AMP accumulation. R‐(−)−rolipram (IC50: 289 ± 121 nM, n = 5) was 4.7 fold more potent than its S‐(+)‐enantiomer (IC50: 1356 ± 314 nM, n = 5). A strong and highly‐significant, linear correlation (r = 0.95, P < 0.01, n = 13) was observed between the inhibitory potencies of a range of structurally distinct PDE4 inhibitors against monocyte PDE4 and their ED50 values in enhancing monocyte cyclic AMP accumulation. A poorer, though still significant, linear correlation (r = 0.67, P < 0.01, n = 13) was observed between the potencies of the same compounds in potentiating PGE2‐induced monocyte cyclic AMP accumulation and their abilities to displace [3H]‐rolipram binding to brain membranes.
RP 73401 (IC50: 6.9 ± 3.3 nM, n = 5) was 71 fold more potent than (±)‐rolipram (IC50: 490 ± 260 nM, n = 4) in inhibiting LPS‐induced TNFα release from monocytes. R‐(−)−rolipram (IC50: 397 ± 178 nM, n = 3) was 5.2‐fold more potent than its S‐(+)‐ enantiomer (IC50: 2067 ± 659 nM, n = 3). As with cyclic AMP, accumulation a closer, linear correlation existed between the potency of structurally distinct compounds in suppressing TNFα with PDE4 inhibition (r = 0.93, P < 0.01, n = 13) than with displacement of [3H]‐rolipram binding (r = 0.65, P < 0.01, n = 13).
RP 73401 (IC50: 2 nM) was 180 fold more potent than rolipram (IC50: 360 nM) in suppressing LPS (10 ng ml−1)‐induced TNFα mRNA.
The results demonstrate that RP 73401 is a very potent inhibitor of TNFα release from human monocytes suggesting that it may have therapeutic potential in the many pathological conditions associated with over‐production of this pro‐inflammatory cytokine. Furthermore, PDE inhibitor actions on functional responses are better correlated with inhibition of PDE4 catalytic activity than displacement of [3H]‐rolipram from its high‐affinity binding site, suggesting that the native PDE4 in human monocytes exists predominantly in a ‘low‐affinity’ state.