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

COPII vesicles are formed at specific subdomains of the ER, termed ER exit sites (ERESs). Depending on the cell type, ERESs number from a few to several hundred per cell. However, whether these ERESs are functionally and compositionally identical at the cellular level remains unclear. Our live cell-imaging analysis in Saccharomyces cerevisiae revealed that the isoforms of cargo-adaptor subunits are unequally distributed to each ERES at steady state, whereas this distribution is altered in response to UPR activation. These results suggest that in S. cerevisiae cargo loading to ERES is dynamically controlled in response to environmental changes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]