Vol. 59, Issue 6, 1533-1541, June 2001

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

Alan M. Carruthers,¹ Lynda A. Sellers, David W. Jenkins, Emma M. Jarvie, Wasyl Feniuk, and Patrick P. A. Humphrey

Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom

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 A₁ receptor-selective agonist GR79236X (pIC₅₀ = 7.7 ± 0.1, maximal inhibition 65 ± 2.5% at 300 nM), whereas the A_2A (CGS21680) and the A₃ (2-chloro-N⁶-(3-iodobenzyl)-adenosine-5'-N-methyluronamide) receptor-selective agonists were without effect. GR79236X-mediated inhibition was abolished by the A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Immunocytochemical studies and Western analysis revealed the presence of adenosine A₁ 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 µ-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 A₁ adenosine agonists may warrant further investigation in models of migraine and neurogenic inflammation.