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HORMONES AND SIGNALING

Alteration of intracellular histamine H₂ receptor cycling precedes antagonist-induced upregulation

¹First Department of Medicine and ²Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan

Submitted 2 December 2004 ; accepted in final form 10 June 2005

Long-term administration of a histamine H₂ receptor (H₂R) antagonist (inverse agonist) induces upregulation of H₂R in parietal cells, which may be relevant to the rebound hypersecretion of gastric acid that occurs after withdrawal of treatment. The mechanisms underlying this effect are unknown. We hypothesized that the H₂R upregulation could be related to receptor trafficking and used H₂R-green fluorescent protein (H₂R-GFP) to test the hypothesis. Human H₂R-GFP was generated and functionally expressed in HEK-293 cells. Binding of the H₂R antagonist [³H]tiotidine was performed to quantify H₂R expression, and H₂R-GFP was imaged in living cells by confocal and evanescent wave microscopy. The binding affinity of [³H]tiotidine was not significantly different between H₂R-GFP- and wild-type H₂R-expressing HEK-293 cells, both of which had constitutive activity of adenylate cyclase. Visualization of H₂R-GFP revealed that the agonist-induced H₂R internalization and the antagonist-induced recycling of the internalized H₂R from the recycling endosome within 2 h. Long exposure to the antagonist increased GFP fluorescence in the plasma membrane and also induced upregulation of H₂R-GFP estimated by the binding assay, whereas long exposure to the agonist enhanced degradative trafficking of H₂R-GFP. We examined whether the upregulation reflected an increase in receptor synthesis. Treatment with antagonist did not augment H₂R mRNA, and subsequent inhibition of protein synthesis by cycloheximide had no effect on H₂R upregulation. These findings suggested that upon exposure to an antagonist (inverse agonist), the equilibrium between receptor endocytosis and recycling is altered before H₂R upregulation, probably via suppressing H₂R degradation.

endocytosis; recycling; inverse agonist; internalization; constitutive activity