Biochemical and functional assessment of equine lymphocyte phosphodiesterases and protein kinase C.
Lymphocytes play an important role in allergic inflammation and have been implicated in the pathogenesis of equine allergic skin and respiratory disease. Targeting intracellular signalling pathways in human lymphocytes has demonstrated a role for both phosphodiesterase and protein kinase C in cell activation. The aim of this study was to measure total cyclic nucleotide hydrolysing phosphodiesterase activity and to identify the phosphodiesterase and protein kinase C isoenzymes present in equine lymphocytes. The functional significance of these isoenzymes was then investigated by examining their role in peripheral blood mononuclear cell proliferation using isoenzyme selective inhibitors. Total cyclic adenosine monophosphate hydrolysing phosphodiesterase activity was double that of cyclic guanosine monophosphate (30+/-2 pmol/min mg versus 16+/-3 pmol/min mg for cyclic adenosine and cyclic guanosine monophosphate phosphodiesterase activity, respectively). Evidence for the presence of PDE1, 3, 4 and 5 was obtained and PKCalpha, beta, delta, eta, iota, theta and zeta were identified. Selective inhibitors of PDE4, PKCdelta and conventional PKCs alpha and beta caused significant inhibition of mitogen-induced peripheral blood mononuclear cell proliferation. This study demonstrates a functional role for specific signalling isoenzymes and suggests that, in the context of allergic inflammation, targeting inflammatory cells involved in disease pathogenesis with relevant isoenzyme inhibitors may have therapeutic potential.
Rickards
K J
KJ
Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Campus, North Mymms, Hertfordshire AL9 7TA, UK. karen.rickards@kcl.ac.uk
Page
C P
CP
Hamblin
A S
AS
Goode
N T
NT
Cunningham
F M
FM
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000494:pharmacology; D002455:Cell Division / Q000276:immunology; D000242:Cyclic AMP / Q000276:immunology; D006152:Cyclic GMP / Q000276:immunology; D006736:Horses / Q000097:blood / Q000276:immunology*; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000276:immunology* / Q000378:metabolism; D011493:Protein Kinase C / Q000276:immunology* / Q000378:metabolism
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; C015482:protein kinase modulator; D000242:Cyclic AMP; D011493:Protein Kinase C; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP