“cAMP-Specific” Phosphodiesterase Contributes to cGMP Degradation in Cerebellar Cells Exposed to Nitric Oxide
- The Wolfson Institute for Biomedical Research, University College London, The Cruciform Building, Gower Street, London, United Kingdom
Abstract
Nitric oxide (NO) functions as a diffusible messenger in the central nervous system and elsewhere, exerting many of it physiological effects by activating soluble guanylyl cyclase, so increasing cellular cGMP levels. Hydrolysis of cyclic nucleotides is achieved by phosphodiesterases (PDEs) but the enzyme isoforms responsible for degrading cGMP in most cells have not been identified. We have devised a method for quantitatively monitoring the rate of breakdown of cGMP within intact cells and have applied it to rat cerebellar cell suspensions previously stimulated with NO. In contrast to previous findings in cultured cerebellar cells, there was no evidence from the use of selective inhibitors that PDE 1 participated importantly in cGMP hydrolysis. Moreover, procedures expected to increase PDE 1 activity by raising cytosolic Ca2+ concentrations (neurotransmitter agonists, Ca2+ ionophore) failed to influence cGMP breakdown. Instead, through the use of inhibitors selective for different PDE families, two isoforms were implicated: a “cGMP-specific” PDE (PDE 5), inhibited by sildenafil and zaprinast, and a “cAMP-specific” PDE (PDE 4), inhibited by low concentrations of rolipram and Ro-20–1724 and by milrinone. An explanation is offered for a participation of PDE 4 based on the high estimated intracellular cGMP concentration (∼800 μM) and the low affinity of the enzyme for cGMP. In accordance with predictions, recombinant PDE 4 was shown to hydrolyze high cGMP concentrations in a rolipram-sensitive manner. The widespread use of rolipram to test for a specific involvement of cAMP in cellular phenomena must therefore be questioned.
Footnotes
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Send reprint requests to: Professor John Garthwaite, Wolfson Institute for Biomedical Research, University College London, The Cruciform Building, Gower Street, London, WC1E 6BT (E-mail:john.garthwaite{at}ucl.ac.uk)
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↵1 This calculation assumes that, in the cerebellar cell suspension, the initial rate of cGMP degradation is 0.1 pmol/106 cells/s (see Fig. 1), that a third of this rate is rolipram-sensitive (see Fig. 3), and that astrocytes make up 6% of the cells (see text) and contain 20 μg cytosolic protein/106cells (based on authors' unpublished observation that 106cells in the cell suspension contain about 50 μg of total protein, of which 40% is cytosolic).
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This work was supported by The Wellcome Trust and a Medical Research Council Studentship (to T.C.B.).
- Abbreviations:
- NO
- nitric oxide
- sGC
- soluble guanylyl cyclase
- PDE
- phosphodiesterase
- nNOS
- neuronal nitric-oxide synthase
- DEA
- diethylamine
- Hb
- hemoglobin
- ODQ
- 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-l-one
- (1S,3R)-ACPD
- (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid
- EHNA
- erythro-9-(2-hydroxy-3-nonyl)adenine
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- Received March 28, 2000.
- Accepted September 27, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
