Alteration of intracellular histamine H₂receptor cycling precedes antagonist-induced upregulation

Abstract

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.

A nucleic acid-hydrolyzing antibody penetrates into cells via caveolae-mediated endocytosis, localizes in the cytosol and exhibits cytotoxicity

“…Next, we further examined whether the cytosolic release of 3D8 scFv from caveolae/caveosome re- quires the fusion with early endosomes and late endosomes, or lysosomes. As uptake by caveosomes is a slower process than that by clathrin-coated pits [30], HeLa cells were incubated with 3D8 scFv for 2 h or transferrin for 30 min before being fixed and then fluorescently labeled with Abs against 3D8 scFv and EEA-1 (early endosomal antigen-1) or lysotrackerred, agent accumulated in late endosomes/lysosomes [31]. The resulting overlays revealed that the signal of 3D8 scFv merged with only that of cav-1, but not with EEA-1 and lysotracker-red, whereas transferrin that is delivered to early and/or late endosomes before being recycled to plasma membrane was colocalized with EEA-1 and lysotracker-red, but not with cav-1 (Fig.…”

Antihistaminergics and inverse agonism: Potential therapeutic applications

“…Alternatively, inverse agonists may require chronic administration and therefore exert some undesirable effects. For instance, receptor up-regulation could occur as it was observed for the H 2 receptor after long-term treatment with cimetidine or ranitidine (Osawa et al, 2005;Smit et al, 1996), providing a plausible explanation for the tolerance to these inverse agonists after prolonged clinical use. In the same way, Milligan and colleagues showed that inverse agonists also up-regulate ȕ 2 -and Į 1B -ARs (Georgieva et al, 2008;Lee et al, 1997;MacEwan and Milligan, 1996).…”

Can H2‐receptor upregulation and raised histamine explain an anaphylactoid reaction on cessation of ranitidine in a 19‐year‐old female? A case report

“…They suggested that this observation reflected an increase in receptor number (or upregulation). Upregulation is believed to relate to suppression of H 2 receptor degradation and the recycling of H 2 receptors .…”