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Hydrolysis of cyclic GMP in rat peritoneal macrophages
Hanna Witwicka1, Marcin Kobiałka, Wojciech A Gorczyca
1Laboratory of Signaling Proteins, L Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.
Abstract:
Intact rat peritoneal macrophages (rPM) treated with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases (PDEs), accumulated more cGMP than untreated cells. A PDE activity toward [(3)H]cGMP was detected in the soluble and particulate fractions of rPM. The hydrolysis of cGMP was Ca(2+)/calmodulin-independent but increased in the presence of cGMP excess. Similar results were obtained when [(3)H]cAMP was used as a substrate. The hydrolytic activity towards both nucleotides was inhibited in the presence of IBMX. Therefore, the PDEs of families 2, 5, 10 and 11 are potential candidates for cGMP hydrolysis in the rPM. They may not only regulate the cGMP level in a feedback-controlled way but also link cGMP-dependent pathways with those regulated by cAMP.
Insights
Rat peritoneal macrophages treated with IBMX accumulated more cGMP. Phosphodiesterase (PDE) activity hydrolyzes both cGMP and cAMP, suggesting PDEs regulate these pathways and link them.
Area of Science:
- Cellular biology
- Biochemistry
- Pharmacology
Background:
- Rat peritoneal macrophages (rPM) play crucial roles in immune responses.
- Cyclic guanosine monophosphate (cGMP) is a key second messenger involved in various cellular processes.
- Phosphodiesterases (PDEs) regulate intracellular levels of cyclic nucleotides like cGMP and cyclic adenosine monophosphate (cAMP).
Purpose of the Study:
- To investigate the presence and characteristics of PDE activity in rat peritoneal macrophages.
- To identify potential PDE families involved in cGMP hydrolysis within rPM.
- To understand how PDEs may regulate cGMP and cAMP signaling pathways in macrophages.
Main Methods:
- Treatment of intact rat peritoneal macrophages (rPM) with 3-isobutyl-1-methylxanthine (IBMX).
- Assay of phosphodiesterase (PDE) activity using radiolabeled cGMP and cAMP as substrates.
- Analysis of PDE activity in soluble and particulate fractions of rPM.
- Investigation of Ca(2+)/calmodulin independence and substrate excess effects on PDE activity.
Main Results:
- IBMX treatment led to increased cGMP accumulation in rPM.
- PDE activity hydrolyzing both [(3)H]cGMP and [(3)H]cAMP was detected in rPM.
- cGMP hydrolysis was Ca(2+)/calmodulin-independent and enhanced by cGMP excess.
- The hydrolytic activity towards both nucleotides was inhibited by IBMX.
Conclusions:
- Rat peritoneal macrophages possess PDE activity capable of hydrolyzing both cGMP and cAMP.
- PDEs from families 2, 5, 10, and 11 are potential candidates for cGMP hydrolysis in rPM.
- These PDEs may regulate cGMP levels via feedback mechanisms and link cGMP and cAMP signaling pathways.