title: Agonist-induced internalization and recycling of the human A(3) adenosine receptors: role in receptor desensitization and resensitization.
abstract: A(3) adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen-dependent nature of the therapeutic effects probably related to receptor desensitization and down-regulation. Here we studied the agonist-induced internalization of human A(3) adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine-5'-N-methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A(3) adenosine receptor showed a profile typical of these receptors in other cell lines (K:(D) = 1.3+/-0.08 nM; B(max) = 400+/-28 fmol/mg of proteins). The iodinated agonist, bound at 4 degrees C to whole transfected cells, was internalized by increasing the temperature to 37 degrees C with a rate constant of 0.04+/-0.034 min(-1). Agonist-induced internalization of A(3) adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short-term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02+/-0.0017 min(-1). Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.
authors.affiliation: Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Pisa, Italia.
authors.forename: M L
authors.initials: ML
authors.lastname: Trincavelli
authors.forename: D
authors.initials: D
authors.lastname: Tuscano
authors.forename: P
authors.initials: P
authors.lastname: Cecchetti
authors.forename: A
authors.initials: A
authors.lastname: Falleni
authors.forename: L
authors.initials: L
authors.lastname: Benzi
authors.forename: K N
authors.initials: KN
authors.lastname: Klotz
authors.forename: V
authors.initials: V
authors.lastname: Gremigni
authors.forename: F
authors.initials: F
authors.lastname: Cattabeni
authors.forename: A
authors.initials: A
authors.lastname: Lucacchini
authors.forename: C
authors.initials: C
authors.lastname: Martini
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D000345:Affinity Labels / Q000493:pharmacokinetics; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D003208:Concanavalin A / Q000494:pharmacology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006982:Hypertonic Solutions / Q000494:pharmacology; D007150:Immunohistochemistry; D007457:Iodine Radioisotopes / Q000032:analysis; D008024:Ligands; D011188:Potassium / Q000378:metabolism; D058906:Purinergic P1 Receptor Agonists; D011869:Radioligand Assay; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013696:Temperature; D014162:Transfection
chemical_list: D000345:Affinity Labels; D006982:Hypertonic Solutions; D007457:Iodine Radioisotopes; D008024:Ligands; D058906:Purinergic P1 Receptor Agonists; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1; D003208:Concanavalin A; C089722:N(6)-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide; D000241:Adenosine; D011188:Potassium