We previously reported that the activation of histamine H 3 receptors (H 3 Rs) selectively counteracts the facilitatory action of adenosine A 2A receptors (A 2A Rs) on GABA release from rat globus pallidus (GP) isolated nerve terminals (synaptosomes). In this work, we examined the mechanisms likely to underlie this functional interaction. Three possibilities were explored: (a) changes in receptor affinity for agonists induced by physical A 2A R/H 3 R interaction, (b) opposite actions of A 2A Rs and H 3 Rs on depolarization-induced Ca 2+ entry, and (c) an A 2A R/H 3 R interaction at the level of adenosine 3′,5′-cyclic monophosphate (cAMP) formation. In GP synaptosomal membranes, H 3 R activation with immepip reduced A 2A R affinity for the agonist 2-p-(2-carboxyethyl)phenethylamino-5′-Nethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680) (K i control 4.53 nM; + immepip 9.32 nM), whereas A 2A R activation increased H 3 R affinity for immepip (K i control 0.63 nM; + CGS-21680 0.26 nM). Neither A 2A R activation nor H 3 R stimulation modified calcium entry through voltage-gated calcium channels in GP synaptosomes, as evaluated by microfluorometry. A 2A Rmediated facilitation of depolarization-evoked [2,3-3 H]-γ-aminobutyric acid ([ 3 H]-GABA) release from GP synaptosomes (130.4 ± 3.6% of control values) was prevented by the PKA inhibitor H-89 and mimicked by the adenylyl cyclase activator forskolin or by 8-Bromo-cAMP, a membrane permeant cAMP analogue (169.5 ± 17.3 and 149.5 ± 14.5% of controls). H 3 R activation failed to reduce the facilitation of [ 3 H]-GABA release induced by 8-Bromo-cAMP. In GP slices, A 2A R activation stimulated cAMP accumulation (290% of basal) and this effect was reduced (− 75%) by H 3 R activation. These results indicate that in striato-pallidal nerve terminals, A 2A Rs and H 3 Rs interact at the level of cAMP formation to modulate PKA activity and thus GABA release.

No abstract

No abstract

Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 μM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.

No abstract