Biochemical and Biophysical Research Communications
Regular ArticleDesensitization of Canine Histamine H2 Receptor Expressed in Chinese Hamster Ovary Cells
Abstract
Canine histamine H2 receptor DNA was transfected into Chinese hamster ovary cells using an expression vector. Expression of H2 receptors was demonstrated by immunoblotting with specific antibodies and the binding of tiotidine, an H2 receptor antagonist. H2 receptor-specific cAMP production was observed only in the cells expressing canine H2 receptor with 10−9-10−4 M histamine in a dose-dependent manner. Preincubation of transfected cells with 10 μM histamine for 10 min or 60 min at 37°C decreased both the maximal response and the sensitivity of the subsequent histamine-stimulated cAMP production, showing desensitization. Under these circumstances, tiotidine binding was decreased by 25% in intact cells. A similar decrease in the tiotidine binding was observed also in the membrane without changes in the affinity, whereas no decrease in total H2 receptor number was observed. Thus, desensitization of histamine H2 receptor was associated with the sequestration of receptors.
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Shining light on the histamine H<inf>2</inf> receptor: Synthesis of carbamoylguanidine-type agonists as a pharmacological tool to study internalization
2021, Bioorganic and Medicinal Chemistry LettersSo far, only little is known about the internalization process of the histamine H2 receptor (H2R). One promising approach to study such dynamic processes is the use of agonistic fluorescent ligands. Therefore, a series of carbamoylguanidine-type H2R agonists containing various fluorophores, heterocycles, and linkers (28–40) was synthesized. The ligands were pharmacologically characterized in several binding and functional assays. These studies revealed a significantly biased efficacy (Emax) for some of the compounds, e.g. 32: whereas 32 acted as strong partial (Emax: 0.77, mini-Gs recruitment) or full agonist (Emax: 1.04, [35S]GTPγS binding) with respect to G protein activation, it was only a weak partial agonist regarding β-arrestin1/2 recruitment (Emax: 0.09–0.12) and failed to promote H2R internalization (confocal microscopy). On the other hand, H2R internalization was observed for compounds that exhibited moderate agonistic activity in the β-arrestin1/2 pathways (Emax ≥ 0.22). The presented differently-biased fluorescent ligands are versatile molecular tools for future H2R studies on receptor trafficking and internalization e.g. using fluorescence microscopy.
H-2 histamine receptor
2007, xPharm: The Comprehensive Pharmacology ReferenceThe histamine H2 receptor gene has been cloned from human, rat, mouse, dog and guinea pig. The receptor is present in gastric parietal cells, vascular smooth muscle and neutrophils as well as in the central nervous system. H2 receptor antagonists have been used for many years clinically for the treatment of peptic ulcers in the gastrointestinal tract and are among the most widely prescribed drugs across the western world.
Unique roles of G protein-coupled histamine H <inf>2</inf> and gastrin receptors in growth and differentiation of gastric mucosa
2004, European Journal of PharmacologyDisruption of histamine H2 receptor and gastrin receptor had different effects growth of gastric mucosa: hypertrophy and atrophy, respectively. To clarify the roles of gastrin and histamine H2 receptors in gastric mucosa, mice deficient in both (double-null mice) were generated and analyzed. Double-null mice exhibited atrophy of gastric mucosae, marked hypergastrinemia and higher gastric pH than gastrin receptor-null mice, which were unresponsive even to carbachol. Comparison of gastric mucosae from 10-week-old wild-type, histamine H2 receptor-null, gastrin receptor-null and double-null mice revealed unique roles of these receptors in gastric mucosal homeostasis. While small parietal cells and increases in the number and mucin contents of mucous neck cells were secondary to impaired acid production, the histamine H2 receptor was responsible for chief cell maturation in terms of pepsinogen expression and type III mucin. In double-null and gastrin receptor-null mice, despite gastric mucosal atrophy, surface mucous cells were significantly increased, in contrast to gastrin-null mice. Thus, it is conceivable that gastrin-gene product(s) other than gastrin-17, in the stimulated state, may exert proliferative actions on surface mucous cells independently of the histamine H2 receptor. These findings provide evidence that different G-protein coupled-receptors affect differentiation into different cell lineages derived from common stem cells in gastric mucosa.
Structural and functional characterization of gastric mucosa and central nervous system in histamine H<inf>2</inf> receptor-null mice
2003, European Journal of PharmacologyTo examine the physiological role of the histamine H2 receptor, histamine H2 receptor-null mice were generated by homologous recombination. Histamine H2 receptor-null mice, which developed normally and were fertile and healthy into adulthood, exhibited markedly enlarged stomachs and marked hypergastrinemia. The former was due to hyperplasia of gastric gland cells (small-sized parietal cells, enterochromaffin-like cells and mucous neck cells which were rich in mucin), but not of gastric surface mucous cells, which were not increased in number as compared with those in wild-type mice despite the marked hypergastrinemia. Basal gastric pH was slightly but significantly higher in histamine H2 receptor-null mice. Although carbachol but not gastrin induced in vivo gastric acid production in histamine H2 receptor-null mice, gastric pH was elevated by both muscarinic M3 and gastrin antagonists. Thus, both gastrin and muscarinic receptors appear to be directly involved in maintaining gastric pH in histamine H2 receptor-null mice. Interestingly, gastric glands from wild-type mice treated with an extremely high dose of subcutaneous lansoprazole (10 mg/kg body weight) for 3 months were very similar to those from histamine H2 receptor-null mice. Except for hyperplasia of gastric surface mucous cells, the findings for gastric glands from lansoprazole-treated wild-type mice were almost identical to those from gastric glands from histamine H2 receptor-null mice. Therefore, it is possible that the abnormal gastric glands in histamine H2 receptor-null mice are secondary to the severe impairment of gastric acid production, induced by the histamine H2 receptor disruption causing marked hypergastrinemia. Analyses of the central nervous system (CNS) of histamine H2 receptor-null mice revealed these mice to be different from wild-type mice in terms of spontaneous locomotor activity and higher thresholds for electrically induced convulsions. Taken together, these results suggest that (1) gastrin receptors are functional in parietal cells in histamine H2 receptor-null mice, (2) abnormal gastric glands in histamine H2 receptor-null mice may be secondary to severe impairment of gastric acid production and secretion and (3) histamine H2 receptors are functional in the central nervous system.
Regulation of phospholipase C activation by the number of H<inf>2</inf> receptors during Ca<sup>2+</sup>-induced differentiation of mouse keratinocytes
2002, Biochemical PharmacologyWe have reported previously that the histamine H2 receptor (H2R) can stimulate the phospholipase C (PLC) signaling pathway in mouse keratinocytes. In the present work, we examined the physiological mechanisms involved in this activation by studying histamine metabolism and H2R expression and coupling during mouse keratinocyte differentiation. Ca2+-induced differentiation decreased histidine decarboxylase (HDC) mRNA, the enzyme responsible for histamine synthesis, by 68.9±5.0%. Concomitantly, intracellular histamine content and its release into the extracellular medium were reduced significantly by 68.2±2.0 and 74.1±1.7%, respectively. Binding of []tiotidine to H2Rs present on the surface of whole cells was also decreased by cellular differentiation [(18.17±2.1)×104 vs. (6.27±0.87)×104 sites/cell, undifferentiated and differentiated cells, respectively], without affecting H2R affinity. Northern blot and reverse transcriptase–polymerase chain reaction (RT–PCR) analysis of the H2R mRNA showed that the expression was also down-regulated at the transcriptional level. Moreover, the inhibition of H2R expression strongly affected the ability of the receptor to induce PLC activation. Our findings suggest that H2R signaling through the PLC second messenger system is inhibited during keratinocyte differentiation by an autocrine loop involving down-regulation of H2R expression and inhibition of histamine metabolism.
Palmitoylation of the canine histamine H2 receptor occurs at Cys<sup>305</sup> and is important for cell surface targeting
2001, Biochimica et Biophysica Acta - Molecular Cell ResearchTo determine the presence and functional role of the histamine H2 receptor (H2R) palmitoylation, a receptor with a Cys305 to Ala (A305 receptor) mutation was generated. Wild-type (WT) and A305 receptors were tagged at their N-termini with a hemagglutinin (HA) epitope. WT, but not A305, receptors incorporated [3H]palmitate by metabolic labeling, indicating that the H2R is palmitoylated at Cys305. Immunocytochemistry of WT and A305 receptors expressed in COS7 cells revealed WT receptors to be distributed at the plasma membrane, while the majority of A305 receptors were localized intracellularly with only a small portion being at the plasma membrane. However, the affinity of the A305 receptor for tiotidine was comparable to that of the WT receptor. In addition, when the amounts of cell surface receptors as determined by anti-HA antibody binding were equivalent, A305 receptors mediated production of more cAMP than WT receptors. Preincubation of COS7 cells expressing each receptor with 10−5 M histamine for 30 min reduced subsequent cAMP production in response to histamine via the receptors to similar extents, indicating that palmitoylation is not necessary for desensitization. In addition, cell surface A305 receptors were capable of being internalized from the cell surface at a rate and extent similar to those of WT receptors. Finally, CHO cell lines stably expressing either WT or A305 receptors were incubated with 10−5 M histamine for 1, 6, 12 and 24 h. Total amounts of WT and A305 receptors, as determined by tiotidine binding, were reduced by incubation, indicating downregulation. Downregulation of the A305 receptor was more extensive than that of the WT receptor. Thus, palmitoylation of the H2R might be important for targeting to the cell surface and stability.