ABSTRACT
Pharmacological characterization of adenosine A(1) and A(2A) receptors in human brain caudate nucleus membranes led to non-cooperative binding of radiolabelled ligands. In human caudate nucleus but not in cortex, the agonist binding to A(1) receptors was modulated by the agonist binding to A(2A) receptors indicating a functional negative cross-talk. Accordingly, the A(1) receptor-activation-mediated G(i)-dependent guanosine 5'-o-(3-[(35)S]thio-triphosphate) binding was modulated by agonist binding to A(2A) receptors. A(2A) receptors occupation led to a decrease in the potency of A(1) receptor agonists. These results indicate that A(1) but not A(2A) receptors activation, likely occurring at low adenosine concentrations, engages a G(i)-mediated signaling; however, when both receptors are occupied by adenosine, there is an A(2A) receptor-mediated impairment of G(i)-operated transducing units. These findings are relevant to get insight into the complex relationships derived from co-expression of multiple neurotransmitter/neuromodulator receptors subtypes that individually are coupled to different G proteins. A further finding was the demonstration that the A(2A) receptor agonist, CGS 21680, at high concentrations able to significantly bind to the A(1) receptor, behaved as a partial agonist of the later receptor. This fact might be taken into account when characterizing CGS 21680 actions in human cells expressing A(1) receptors when the compound is used at micromolar concentrations.
Subject(s)
Caudate Nucleus/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Multiprotein Complexes/metabolism , Receptor Cross-Talk/physiology , Receptor, Adenosine A1/metabolism , Receptor, Adenosine A2A/metabolism , Adenosine A1 Receptor Agonists , Adenosine A2 Receptor Agonists , Binding, Competitive/physiology , Caudate Nucleus/drug effects , Cell Membrane/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/agonists , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Humans , Multiprotein Complexes/agonists , Protein Binding/drug effects , Protein Binding/physiologyABSTRACT
Thaumatin-like proteins (TLPs) have been established as a new family of fruit and pollen allergens. The aim of this study was to develop a two-site ELISA for the quantification of the thaumatin-like kiwi allergen (Act d 2) in kiwifruit extracts and kiwifruit-containing food products. Genomic DNA (gDNA) of Act d 2 was amplified and the deduced amino acid sequence was determined to obtain a primary structure. Act d 2 purified from kiwifruit extract by HPLC was identified by Edman degradation and MS. Balb/c mice were immunized with Act d 2 for the production of mAbs by hybridoma technology. The optimized ELISA measured Act d 2 concentrations ranging from 0.2 to 9.0 ng/mL, with intra- and interassay coefficients of variation of 3.65 and 10.44%, respectively. The developed ELISA is a useful method for the quantification of the thaumatin-like kiwi allergen in kiwifruit extracts as well as the allergen level in kiwifruit-containing food products. It may be a helpful analytical tool for the evaluation of the stability (integrity) of fruit allergen extracts for in vitro diagnosis.
