Abstract

BackgroundPhosphodiesterases (PDEs) cleave phosphodiester bonds in cyclic nucleotides and play diverse roles in cell biology. PDE5A is a cytoplasmic phosphodiesterase which specifically degrades cyclic guanosine monophosphate (cGMP), a cell signaling molecule that plays important roles in neuronal signaling and vascular smooth muscle contraction. Inhibition of PDE5A induces headache resembling migraine headaches.AimTo test the hypothesis that Ser102 and Ser104 in PDE5A and/or their phosphoserine derivatives 1) regulate the intracellular localization and/or activity of PDE5A, and 2) modulate the interaction between PDE5A and pharmaceutical reagents in clinical or pre-clinical use for migraine headaches and other types of vascular dysfunction.MethodsWild type PDE5A or PDE5A with substitution mutations (Ser102Ala, Ser104Ala or Ser102Ala/Ser104Ala) were overexpressed in SK-N-AS neuroblastoma cells as C-terminal fusions with green fluorescent protein. Transfected cells were treated with sildenafil, cilostazol, glyceryl trinitrate, calcitonin gene-related peptide (CGRP) or sumatriptan. PDE5A-GFP fusion proteins were localized in fixed cells by immunofluorescence and PDE activity was quantified in cell extracts by standard in vitro assay using [3H] cGMP.ResultsThe intracellular distribution of wild-type, single and double mutant PDE5A was similar and was not altered by exposure to sildenafil, cilostazol, glyceryl trinitrate, calcitonin gene-related peptide (CGRP) or sumatriptan. PDE5 activity was similar for wild type, Ser102Ala and Ser104Ala PDE5A, but activity of the Ser102Ala/Ser104Ala mutant was approximately two-fold higher than wild type. Double mutant Ser102Ala/Ser104Ala migrated as a single band on a native acrylamide gel, while wild-type and single mutant PDE5A migrated as a doublet.InterpretationSer102 and Ser104 may influence the conformational flexibility of PDE5A, which may in turn influence phosphorylation status, allosteric regulation by cGMP or other as yet unknown regulatory mechanisms for PDE5A. PDE5A activation could be important in reversal of migraine-like headache.

Highlights

  • Headache and migraine-like pathophysiology can be induced in migraine patients by at least two endogenous signalling molecules, nitric oxide (NO) and calcitonin gene-related peptide (CGRP) [1,2,3]

  • Interpretation: Ser102 and Ser104 may influence the conformational flexibility of PDE5A, which may in turn influence phosphorylation status, allosteric regulation by cyclic guanosine monophosphate (cGMP) or other as yet unknown regulatory mechanisms for PDE5A

  • Sildenafil induces headache in healthy subjects and migraine-like headache in migraine patients, without significant concomitant effect on cerebral artery hemodynamics [10]. The latter observation suggests that PDE5A and its downstream effects on cGMP- and NO-mediated signalling play critical roles in pain signalling during migraine-like attacks [8]; the exact details of its influence on pain signalling during headache and migraine events are not fully understood

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Summary

Introduction

Headache and migraine-like pathophysiology can be induced in migraine patients by at least two endogenous signalling molecules, nitric oxide (NO) and calcitonin gene-related peptide (CGRP) [1,2,3]. CGRP initiates an intracellular signalling cascade predominantly involving cAMP [4], and NO increases intracellular levels of cGMP [5] The levels of both cAMP and cGMP are modulated by phosphodiesterases (PDE). Sildenafil induces headache in healthy subjects and migraine-like headache in migraine patients, without significant concomitant effect on cerebral artery hemodynamics [10]. The latter observation suggests that PDE5A and its downstream effects on cGMP- and NO-mediated signalling play critical roles in pain signalling during migraine-like attacks [8]; the exact details of its influence on pain signalling during headache and migraine events are not fully understood.

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