Phosphodiesterase (PDE) assays

Determination of the total PDE3 and PDE4 activities was done using 1 μM cAMP as substrate and 10 μM of either the PDE3 selective inhibitor, Cilostimide, or the PDE4 selective inhibitor, Rolipram.

Cell lines

U937 cells

Preparation method

The solubility of this compound in DMSO is >13 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

0 ~ 10000 nM; 20 mins

Applications

In U937 cells, Rolipram increased protein kinase A (PKA)-mediated cAMP-response-element-binding protein (CREB) phosphorylation in a dose-dependent manner. In contrast, Rolipram dose-dependently inhibited p38 MAP kinase phosphorylation induced by interferon-γ, with an IC50 value of 294 ± 99 nM.

Animal models

SD rats

Dosage form

300 μg/100 g; i.v.

Applications

In SD rats, Rolipram significantly reduced mean arterial pressure. In SD rats treated with lipopolysaccharide and Rolipram, cardiac index significantly increased only at 3 hrs. In addition, Rolipram significantly decreased sinusoidal volumetric flow and increased sinusoidal diameter, which indicated protective effects of Rolipram on sinusoidal microhemodynamics in endotoxemia.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

Rolipram is a selective phosphodiesterases PDE4 inhibitor with IC50s of 3 nM, 130 nM and 240 nM for PDE4A, PDE4B, and PDE4D, respectively.

The PDE4 selective inhibitor, Rolipram, inhibits immunopurified PDE4B and PDE4D activities similarly, with IC50s of approx. 130 nM and 240 nM respectively. In contrast, Rolipram inhibits immunopurified PDE4A activity with a dramatically lower IC50 of around 3 nM. Rolipram increases phosphorylation of cAMP-response-element-binding protein (CREB) in U937 cells in a dose-dependent fashion, which implies the presence of both high affinity (IC50 approx. 1 nM) and low affinity (IC50 approx. 120 nM) components. Rolipram dose-dependently inhibits the IFN-gamma-stimulated phosphorylation of p38 MAPK in a simple monotonic fashion with an IC50 of approx. 290 nM[1]. Rolipram is a selective PDE4 inhibitor that inhibits all PDE4 isoforms A, B, C and D. Rolipram inhibits LPS-induced TNF production in a dose-dependent manner (IC50 25.9 nM), and maximal/submaximal inhibition is observed with 2 μM drug concentration in J774 cells[2].

TNF mRNA and protein expression is induced by LPS in peritoneal macrophages (PM) from WT mice, and that is clearly (by 74 and 63% for TNF mRNA and TNF protein, respectively) inhibited by Rolipram. LPS-induced TNF production is enhanced in PM from MKP-1(-/-) mice as compared to that in PM from WT mice, which is in line with the published results. Interestingly, the inhibition of TNF mRNA and protein expression by Rolipram is markedly attenuated in PM from MKP-1(-/-) mice and does not reach statistical significance[2]. Repeated administration of Rolipram (1.25 mg/kg, i.p.) reduces the number of escape failures in learned helplessness rats[3].

References:
[1]. MacKenzie SJ, et al. Action of rolipram on specific PDE4 cAMP phosphodiesterase isoforms and on the phosphorylation of cAMP-response-element-binding protein (CREB) and p38 mitogen-activated protein (MAP) kinase in U937 monocyticcells. Biochem J. 2000 Apr
[2]. Korhonen R, et al. Attenuation of TNF production and experimentally induced inflammation by PDE4 inhibitor rolipram is mediated by MAPK phosphatase-1. Br J Pharmacol. 2013 Aug;169(7):1525-36.
[3]. Shalaby AM, et al. Effect of rolipram, a phosphodiesterase enzyme type-4 inhibitor, on γ-amino butyric acid content of the frontal cortex in mice exposed to chronic mild stress. J Pharmacol Pharmacother. 2012 Apr;3(2):132-7.