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Abstract
The extent to which cAMP-dependent protein kinase (PKA) mediates the inhibitory effects
of cAMP-elevating drugs on tumour necrosis factor (TNF) alpha release from lipopolysaccharide
(LPS)-stimulated human monocytes is equivocal. Here, we have investigated the role
of this kinase by exploiting the ability of certain novel cAMP analogues to inhibit
or activate PKA and the recently described cAMP-guanine nucleotide-exchange factors
(GEFs). Pre-treatment of monocytes with Rp-8-Br-cAMPS, a selective inhibitor of Type
I PKA that has no effect on basal or stimulated Rap1 (a downstream effector of cAMP-GEFs)
activity, potentiated LPS-induced TNFalpha output but had little or no effect on the
suppression of this cytokine effected by rolipram (a PDE4 inhibitor), prostaglandin
(PG) E2 and salbutamol (a beta2-adrenoceptor agonist). In contrast, Rp-8-pCPT-cAMPS,
which selectively blocks Type II PKA with only weak activity against Rap1, significantly
antagonised or abolished the inhibitory effect of these cAMP-elevating agents. Pre-treatment
of monocytes with 8-pCPT-2'-O-Me-cAMPS, a potent activator of cAMP-GEFs, failed to
suppress TNFalpha output at concentrations known to profoundly activate Rap1. Collectively,
these results indicate that cAMP-elevating drugs suppress TNFalpha release from LPS-stimulated
human monocytes by activating PKA independently of cAMP-GEFs. Furthermore, by using
phosphorothioate cAMP analogue PKA inhibitors we provide evidence that the Type II
PKA isoenzyme is functionally the most important.