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cUMP hydrolysis by PDE3B

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Abstract

Previous results indicate that the phosphodiesterase PDE3B hydrolyzes cUMP. Also, almost 50 years ago, cUMP-hydrolytic activity was observed in rat adipose tissue. We intended to characterize the enzyme kinetics of PDE3B-mediated cUMP hydrolysis, to determine the PDE3B binding mode of cUMP, and to analyze cUMP hydrolysis in adipocyte preparations. Educts (cNMPs) and products (NMPs) of the PDE reactions as well as intracellular cNMPs were quantitated by HPLC-coupled tandem mass spectrometry. PDE3B expression was determined by qPCR and Western blot. Docking studies were performed with the PDE3B crystal structure PDB ID 1SO2 (complex with a dihydropyridazine inhibitor). PDE3B hydrolyzed cUMP (Km ~ 550 μM, Vmax ~ 76 μmol/min/mg) and cAMP (Km ~ 0.7 μM, Vmax ~ 4.3 μmol/min/mg) in a milrinone (PDE3-selective inhibitor)-sensitive manner (Ki for inhibition of cUMP hydrolysis: 205 nM). cUMP forms one hydrogen bond with PDE3B (uracil 3-NH with side chain oxygen of Q988). Two hydrogen bonds stabilize cAMP binding. cCMP does not interact with PDE3B. Possibly, the cytosine base cannot form hydrogen bonds with PDE3B, and the 4-NH2 group clashes with L987 of the enzyme. Adipocyte differentiation of 3T3-L1 MBX cells increased mRNA of PDE3B, but not of PDE3A. Significant amounts of cUMP were detected in differentiated and undifferentiated 3T3-L1 MBX cells. 3T3-L1 MBX adipocyte lysates and rat epididymal adipose tissue membranes contained milrinone-sensitive cUMP-hydrolytic activity. PDE3B is a low-affinity and high-velocity phosphodiesterase for cUMP. The cUMP-hydrolyzing activity described almost 50 years ago for rat adipose tissue is caused by PDE3, probably by the isoform PDE3B.

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Abbreviations

3T3-L1 :

MBX cells

murine pre-adipocyte cell line (subclone of the 3T3-L1 cell line)

AMP :

adenosine 5′-monophosphate

BSA :

bovine serum albumin

cAMP :

adenosine 3′,5′-cyclic monophosphate

cCMP :

cytidine 3′,5′-cyclic monophosphate

C/EBPα :

CCAAT/enhancer-binding protein alpha

cGMP :

guanosine 3′,5′-cyclic monophosphate

CMP :

cytidine 5′-monophosphate

cNMP :

nucleoside 3′,5′-cyclic monophosphate

cUMP :

uridine 3′,5′-cyclic monophosphate

DMEM :

Dulbecco’s modified Eagle medium (cell culture medium)

DMSO :

dimethylsulfoxide

EDTA :

ethylenediaminetetraacetic acid

ExoY :

Exotoxin of Pseudomonas aeruginosa with nucleotidyl cyclase properties

FABP4 :

fatty acid binding protein 4 (also known as adipocyte protein 2 or aP2)

FCS :

fetal calf serum

GMP :

guanosine 5′-monophosphate

GST :

glutathione S-transferase (used as protein tag)

HCN :

hyperpolarization-activated cyclic nucleotide-gated channel

HPLC-MS/MS :

high-performance liquid chromatography-coupled tandem mass spectrometry

IBMX :

3-isobutyl-1-methylxanthine

K m :

Michaelis-Menten constant

MRP :

multidrug resistance-associated protein

NCS :

neonatal calf serum

NMP :

nucleoside 5′-monophosphate

PBS :

phosphate-buffered saline

PDE :

phosphodiesterase

PKA :

protein kinase A

PKG :

protein kinase G

PPARγ :

peroxisome proliferator-activated receptor gamma

Pref-1 :

preadipocyte factor 1

P/S + G :

penicillin/streptomycin + glutamine

qPC :

quantitative real-time PCR

sAC :

soluble adenylyl cyclase

SD :

standard deviation

SEM :

standard error of the mean

sGC :

soluble guanylyl cyclase

UMP :

uridine 5'-monophosphate

Vmax :

maximum velocity of an enzymatic reaction under saturating conditions

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Acknowledgements

We thank Prof. Dr. Harald Genth (Institute of Toxicology, MHH), Dr. Sabine Wolter (Institute of Pharmacology, MHH), and Dr. Thorsten Gnad (Institute of Pharmacology and Toxicology, University of Bonn) for excellent scientific discussions as well as Ms. Annette Garbe (Research Core Unit Metabolomics, MHH) and Ms. Solveig Kälble (Institute of Pharmacology, MHH) for outstanding technical support.

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Author notes

  1. Jessica Ostermeyer and Franziska Golly contributed equally to this work.

Authors and Affiliations

  1. Hannover Medical School, Institute of Pharmacology, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

    Jessica Ostermeyer, Franziska Golly, Roland Seifert & Erich H. Schneider

  2. Research Core Unit Metabolomics, Hannover Medical School, Institute of Pharmacology, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

    Volkhard Kaever

  3. Department of Medicinal Chemistry II, Institute of Pharmacy, University of Regensburg, 93040, Regensburg, Germany

    Stefan Dove

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  1. Jessica Ostermeyer
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Contributions

Enzyme kinetics data: 60% JO, 40% FG

Western blot data: 0% JO, 100% FG

cNMP content of 3T3-L1 cells: 0% JO, 100% FG

qPCR data: 60% JO, 40% FG

Enzyme activity in differentiated 3T3-L1 cells: 0% JO, 100% FG

Enzyme activity in undifferentiated 3T3-L1 cells: 100% JO, 0% FG

Enzyme activity in rat adipose tissue: 100% JO, 0% FG

Molecular modeling: 100% SD

HPLC-MS/MS measurements: 100% VK

Corresponding author

Correspondence to Erich H. Schneider.

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Ostermeyer, J., Golly, F., Kaever, V. et al. cUMP hydrolysis by PDE3B. Naunyn-Schmiedeberg's Arch Pharmacol 391, 891–905 (2018). https://doi.org/10.1007/s00210-018-1512-6

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  • DOI: https://doi.org/10.1007/s00210-018-1512-6

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