Rolipram

Manufactured by Cayman Chemical

Sourced in United States

Rolipram is a phosphodiesterase 4 (PDE4) inhibitor. It is a laboratory compound used in research to study the effects of PDE4 inhibition.

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Rolipram [RP] (2 citations)

20 protocols using rolipram

Long-term Potentiation Induction and Regulation

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Transverse hippocampal slices (400 μm) were made using a standard mechanical tissue chopper and allowed to recover in oxygenated (95% O₂/5% CO₂) artificial cerebrospinal fluid (ACSF) at 32 °C for 1 h. cLTP was induced using 200 nM N-methyl-d-aspartate (NMDA; Cayman Chemical, Ann Arbor, MI, USA) in 0 Mg²⁺ ACSF for 10 min followed by 0.1 μM rolipram + 50 μM forskolin (Cayman Chemical, Ann Arbor, MI, USA) in 0 Mg²⁺ ACSF for 15 min [29 (link),30 (link)]. ACSF lacking Mg²⁺ was used as NMDA receptors are normally blocked by such ions.
After 1 h of recovery, slices undergoing the cLTP induction protocol were incubated in ACSF with 25 μM β-lactone for 30 min or the following specific kinase inhibitors for 1 h; 20 μM U0126 (Cayman Chemical, Ann Arbor, MI, USA), 5 μM KT5720 (Cayman Chemical, Ann Arbor, MI, USA), or 5 μM KT5823 (Cayman Chemical, Ann Arbor, MI, USA); or the neddylation inhibitor MLN4924 (2 μM) (Cayman Chemical, Ann Arbor, MI, USA) followed by cLTP induction. Slices were then collected at different timepoints and processed for immunohistochemistry.

Smith S.G., Haynes K.A, & Hegde A.N. (2020). Degradation of Transcriptional Repressor ATF4 during Long-Term Synaptic Plasticity. International Journal of Molecular Sciences, 21(22), 8543.

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Biochemical Compound Acquisition Protocol

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Silymarin and silibin were obtained from Sigma Aldrich, St. Louis, MO, USA; milrinone, rolipram, and IBMX were obtained from Cayman, Ann Arbor, MI, USA.

Meng R., Mahadevan J., Oseid E., Vallerie S, & Robertson R.P. (2016). Silymarin Activates c-AMP Phosphodiesterase and Stimulates Insulin Secretion in a Glucose-Dependent Manner in HIT-T15 Cells. Antioxidants, 5(4), 47.

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Preparation of Compound Stock Solutions

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100 mM stock solutions of all screened compounds were prepared in individual, sterile amber vials. All stock solutions were dissolved in sterile DMSO, except L-arginine, which was solubilized using dH₂O, due to solubility constraints. Sterile cell-culture grade DMSO, tamsulosin hydrochloride, nifedipine, isoproterenol, butoxamine, adenosine and L-arginine were obtained from Sigma Aldrich. PGE1, PGE2, rolipram, L-arginine, ondansetron, celecoxib, diclofenac, mirabegron, sildenafil, atropine, vardenafil and tiotropium were obtained from Cayman Chemical. Y-27632 dihydrochloride was obtained from R&D Systems. Stock solutions when unused were stored at −20 °C

Lee C.X., Cheah J.H., Soule C.K., Ding H., Whittaker C.A., Karhohs K., Burds A.A., Subramanyam K.S., Carpenter A.E., Eisner B.H, & Cima M.J. (2019). Identification and local delivery of vasodilators for the reduction of ureteral contractions. Nature biomedical engineering, 4(1), 28-39.

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Pharmacological Inhibition of PDE Signaling

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Piclamilast/RP73401^{29 (link), 30 (link)}, Rolipram^{31 (link)}, Roflumilast^{32 (link)}, Cilostamide^{33 (link)}, Chlorcyclizine³⁴, Spiperone³⁵, and Pimozide³⁶ were from Cayman Chemical (Ann Arbor, MI, USA). YM976^{37 (link)} was obtained from Tocris/Bio-Techne (Minneapolis, MN, USA), Naloxone³⁸ from MP Biomedicals (Irvine, CA), Domperidone³⁹ from Alfa Aesar (Haverhill, MA, USA), Ketanserin³⁵ from TCI America (Portland, OR, USA), Propranolol^{40 , 41} from Millipore Sigma (St. Louis, MO, USA), and RS25344^{42 (link)} from Santa Cruz Biotech (Santa Cruz, CA, USA). All drugs were initially dissolved in DMSO, subsequently diluted into phosphate-buffered saline (PBS), pH 7.4, containing final concentrations of 5% DMSO and 5% Cremophor EL (Millipore Sigma, St. Louis, MO, USA), and were applied by intraperitoneal injection (100 μl per 20 g body weight).

McDonough W., Rich J., Aragon I.V., Saleh L.A., Boyd A., Richter A., Koloteva A, & Richter W. (2020). Inhibition of Type 4 cAMP-phosphodiesterases (PDE4s) in mice induces hypothermia via effects on behavioral and central autonomous thermoregulation. Biochemical pharmacology, 180, 114158.

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Regulations of Inflammatory Cytokine Secretion

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RAW264.7 cells and HEK293 cells are obtained from ATCC, and maintained in DMEM (Dulbecco′s modified Eagle′s medium) supplemented with 10% fetal bovine serum (FBS) from Gibico. BSA (bovine serum albumin) and anti-BSA antibody (α-BSA) are obtained from Invitrogen and MP Biomedicals, respectively. Dimethyl sulfoxide (DMSO) is obtained from Sigma-Aldrich. Rolipram and Roflumilast are obtained from Cayman Chemical and APExBIO, respectively. 6-Bnz-cAMP (PKA agonist) and 8-pCPT-2′-O-Me-cAMP (Epac agonist) are obtained from Biolog Life Science Institute. PKA inhibitor H-89 is obtained from Beyotime Biotechnology. ELISA kits for measuring cAMP, TNF-α, MIP-1α, MIP-1β, MIP-2 and KC are all obtained from R&D Systems. ELISA kit for measuring mouse albumin is obtained from Bethyl Laboratories.

Yan C., Chen J., Tang H., Deng C., Zhang Q, & Wang X. (2023). IgG immune complex-induced acute lung injury is ameliorated by cAMP via down-regulation of C/EBP- and AP-1-mediated transcriptions. Journal of Inflammation (London, England), 20, 34.

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Pharmacological Modulation of PDE Inhibitors

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Rolipram [35 (link)] (4-(3-cyclopentyloxy-4-methoxyphenyl)pyrrolidin-2-one), Roflumilast [36 ,37 (link)] (3-(cyclopropylmethoxy)-N-(3,5-dichloropyridin-4-yl)-4-(difluoromethoxy)benzamide), and Yohimbine (methyl (1S,15R,18S,19R,20S)-18-hydroxy-1,3,11,12,14,15,16,17,19,19,20,21-dodecahydroyohimban-19-carboxylate) were from Cayman Chemical (Ann Arbor, MI, USA). YM976 [38 ,39 (link)] (4-(3-chlorophenyl)-1,7-diethylpyrido [2,3-d] pyrimidin-2-one) was from Tocris/Bio-Techne (Minneapolis, MN, USA). Isoprenaline/Isoproterenol (4-[1-hydroxy-2-(propan-2-ylamino)ethyl]benzene-1,2-diol) and Propranolol (1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol) were from Millipore Sigma (St. Louis, MO, USA), and RS25344 (1-(3-nitrophenyl)-3-(pyridin-4-ylmethyl) pyrido [2,3-d] pyrimidine-2,4-dione) was obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). All drugs were initially dissolved in DMSO, subsequently diluted into phosphate-buffered saline (PBS), pH 7.4, containing final concentrations of 5% DMSO (dimethyl sulfoxide) and 5% Cremophor EL (MilliporeSigma, St. Louis, MO, USA), and were applied by intraperitoneal (i.p.) injection (100 μL per 20 g body weight).

Boyd A., Lochmaier P., Irelan D., Fiedler E., Lee J.Y., Fouty B., Abou Saleh L, & Richter W. (2022). Non-Selective PDE4 Inhibition Induces a Rapid and Transient Decrease of Serum Potassium in Mice. Biology, 11(11), 1582.

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Culturing HBEC6-KT and Calu-3 Airway Cells

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The human bronchial epithelial cell (HBEC6-KT) line was cultured in Keratinocyte Serum-Free Medium 1X (Gibco) according to the manufacturers directions and supplemented with P/S (VWR) at 1X^{70 (link)}. Human airway epithelial cell line Calu-3 (ATCC HTB-55) were cultured in Minimum Essential Medium Alpha Medium (Corning) and supplemented with Premium Grade FBS (VWR) at 10%, HEPES (Corning) at 1X, and P/S (VWR) at 1X. The following chemical reagents were dissolved in DMSO, cAMP elevating agents Forskolin (Cayman Chemical) and Isoproterenol (Cayman Chemical), ABCC4 inhibitors MK-571 (Cayman Chemical) and Ceefourin 1 (Abcam), PDE inhibitors Roflumilast (Cayman Chemical) and Rolipram (Cayman Chemical), and CFTR Inhibitor (Selleck Chemicals).

Nguyen J.P., Bianca M., Huff R.D., Tiessen N., Inman M.D, & Hirota J.A. (2021). Modulation of cAMP metabolism for CFTR potentiation in human airway epithelial cells. Scientific Reports, 11, 904.

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Purification and Analysis of Prostaglandin Analogs

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Proton NMR spectra were recorded
in solvent in CDCl₃ on a Varian Inova 400 (400 MHz) instrument.
Thin layer chromatography was performed on precoated, aluminum-backed
plates (silica gel 60 F₂₅₄, 0.25 mm thickness) from EM
Science and was visualized by UV lamp. Column chromatography was performed
with silica gel cartridges on Teledyne-ISCO machine. An Agilent LCMS
instrument was used to measure purity of the products. Elemental analyses
were performed by Atlantic Microlab Inc. (Norcross, GA). Chemicals
and drugs PGE₂, BW245C, iloprost, and rolipram were purchased
from Cayman Chemical.

Ganesh T., Jiang J., Yang M.S, & Dingledine R. (2014). Lead Optimization Studies of Cinnamic Amide EP2 Antagonists. Journal of Medicinal Chemistry, 57(10), 4173-4184.

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Investigating IGF-1 Signaling Pathways

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Chemicals and other materials were obtained from the following sources: H₂O₂, forskolin, Rp-cAMP, and H89 from Sigma; Rolipram from Cayman Chemical; Recombinant Rat IGF-1 from Prospec; DMEM/F12 (1:1), MEM+GlutaMax, 100 × penicillin–streptomycin (Pen/Strep) and fetal bovine serum from Gibco; Superscript II reverse transcriptase from Invitrogen; RNase inhibitor, dNTP mixture and oligo (dT) from Promega; PCR master mix, protein size marker, SuperSignal West Pico and Femto chemiluminescent substrates from Thermo scientific; Amersham Hybond-P (PVDF membrane) and ECL Prime Western Blotting Detection Reagent from GE Healthcare; Lab-Tek Chambered cover glass from Nunc; CE3F4 from TOCRIS.

Shim M.S., Kim K.Y., Bu J.H., Nam H.S., Jeong S.W., Park T.L., Ellisman M.H., Weinreb R.N, & Ju W.K. (2018). Elevated intracellular cAMP exacerbates vulnerability to oxidative stress in optic nerve head astrocytes. Cell Death & Disease, 9(3), 285.

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Evaluating T Cell Inhibition Compounds

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Following cell isolation, CD4+ and CD8+ T cells were plated out in a 96-well plate (minimum of 5×10⁴ cells/well) in RPMI (GIBCO) containing 10% fetal calf serum and 1% penicillin/streptomycin. For inhibition studies, cells were incubated with the following compounds for 1 hour in standard culture conditions (5%CO₂, 37°C): RORγt inhibitor 10 µM25 (Pfizer), MTX, 5 mg/mL (Cayman Chemical), rolipram (a PDE4 inhibitor) 10 µM (Cayman Chemical) or dimethyl sulfoxide (DMSO) control (0.1%). All drugs were diluted to maintain a final concentration of 0.1% DMSO. Cells were stimulated using anti-CD3/CD28 (GIBCO) for 48 hours. To test the potential of compounds to affect cell viability, flow cytometry was used to assess CD45+, CD3+, CD4/CD8+ and live dead viability by aqua zombie.

Watad A., Rowe H., Russell T., Zhou Q., Anderson L.K., Khan A., Dunsmuir R., Loughenbury P., Borse V., Rao A., Millner P.A., Bragazzi N.L., Amital H., Cuhtbert R., Wittmann M., Sharif K., Kenna T., Brown M.A., Newton D., Bridgewood C, & McGonagle D.G. (2020). Normal human enthesis harbours conventional CD4+ and CD8+ T cells with regulatory features and inducible IL-17A and TNF expression. Annals of the Rheumatic Diseases, 79(8), 1044-1054.

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