Adenosine A₁ Receptor-Mediated Inhibition of Protein Kinase A-Induced Calcitonin Gene-Related Peptide Release from Rat Trigeminal Neurons

Abstract

Calcitonin gene-related peptide (CGRP), a potent vasodilator, has been implicated in the pathogenesis of migraine. Its release from adult rat trigeminal neurons in culture was shown to be markedly increased by the activation of adenylate cyclase with forskolin. Modulation of this secretion was investigated by a number of agents with known inhibitory effects on cAMP generation mediated via receptor coupling to G(i/o) proteins. Significantly, forskolin-stimulated CGRP release could be closely correlated with the phosphorylation of the protein kinase A (PKA) substrate cyclic AMP response element-binding protein (CREB). Forskolin-stimulated CGRP release could be potently and effectively inhibited by the adenosine A(1) receptor-selective agonist GR79236X (pIC(50) = 7.7 +/- 0.1, maximal inhibition 65 +/- 2.5% at 300 nM), whereas the A(2A) (CGS21680) and the A(3) (2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide) receptor-selective agonists were without effect. GR79236X-mediated inhibition was abolished by the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Immunocytochemical studies and Western analysis revealed the presence of adenosine A(1) receptors on trigeminal neurons. However, despite the additional detection of 5-hydroxytryptamine (5-HT)(1B) receptors on these cells, the clinically effective antimigraine 5-HT(1B/1D) agonist sumatriptan did not inhibit forskolin-stimulated CGRP release nor did it show any effect on the concomitant CREB phosphorylation. In contrast, the mu-opioid agonist fentanyl elicited a 74 +/- 4% reduction in CGRP levels. Forskolin-stimulated CGRP release and CREB phosphorylation could be mimicked by incubation of the cells with chlorophenylthio-cAMP and blocked by pretreatment with the PKA inhibitor myrPKI(14-22). Taken together, the present data confirm the PKA-dependence of forskolin-stimulated CGRP release and suggest that A(1) adenosine agonists may warrant further investigation in models of migraine and neurogenic inflammation.

Current and prospective pharmacological targets in relation to antimigraine action

“…In the rat, the adenosine A 1 receptor antagonist DPCPX prevented the inhibitory effect of GR79236 on neurogenic vasodilatation, which was primarily mediated by prejunctional A 1 -receptors (Honey et al 2002). GR79236 has no effect on resting meningeal artery diameter in rats (Honey et al 2002) and inhibits forskolin-stimulated CGRP release in rats (Carruthers et al 2001). Likewise, in cats, GR79236 has been shown to significantly reduce the CGRP levels in the external jugular vein ( Fig.…”

Sustained morphine treatment augments basal CGRP release from cultured primary sensory neurons in a Raf-1 dependent manner

“…In addition, a direct AC activator, forskolin, was reported to stimulate CGRP release from trigeminal sensory neurons. Forskolin-stimulated CGRP release from the sensory neurons was blocked by a selective PKA inhibitor myr-PKI 14-22 (Carruthers et al, 2001), suggesting a crucial role for cAMP-dependent protein kinase (PKA) in the regulation of CGRP release from cultured sensory neurons. On the other hand, it was found earlier that sustained opioid agonist treatment upregulates the cAMP signal transduction cascade (AC superactivation) in primary sensory DRG neurons (Crain et al, 1998;Terwilliger et al, 1991).…”

“…Activation of the cAMP transduction pathway is known to modulate presynaptic neurotransmitter synthesis and release (Carruthers et al, 2001, Supowit et al, 1995, Leenders and Sheng, 2005. Thus, it was shown earlier that treatment with a non-hydrolyzable cAMP analogue (dibutyryl cAMP) upregulates CGRP mRNA and peptide concentrations in cultured rat DRG neurons (Supowit et al, 1995).…”

“…However, cAMPdependent protein kinase (PKA) is known to play a major role in the regulation of presynaptic neurotransmitter synthesis and release (Carruthers et al, 2001;Supowit et al, 1995;Leenders and Shen, 2005;Rathee et al, 2002). On the other hand, sustained opioid agonist treatment was shown to upregulate the cAMP signal transduction cascade (adenylyl cyclase (AC) superactivation) in multiple regions of the nervous system (Nestler and Aghajanian, 1997), including the primary sensory dorsal root ganglion (DRG) neurons (Crain and Shen, 1998;Terwilliger et al, 1991).…”

Sensitization of Calcitonin Gene-Related Peptide Receptors by Receptor Activity-Modifying Protein-1 in the Trigeminal Ganglion

“…However, increased CGRP secretion from cultured trigeminal ganglia has been reported after forskolin stimulation, which mimics cAMP production by the CGRP receptor (Carruthers et al, 2001). We replicated this finding under our conditions (2.3-fold increase) (data not shown).…”