Purification to apparent homogeneity of the somatostatin receptor of the human cell line HGT-1 of gastric origin

This communication reports the isolation and the purification of the gastric somatostatin receptor from the human cell line HGT-1. The receptor has been extracted from the cell membrane by Triton X 100, and a monoclonal antibody to this was prepared. A series of affinity chromatographies (Sepharose-antibody and Sepharose-somatostatin-14) and a final purification by steric exclusion on high performance liquid chromatography columns (HPLC) allowed us to obtain a fraction enriched 20,000 fold in 125I-Tyrll-somatostatin-14 specific binding (apparent dissociation constant: 7.6 x 10(-8) M). This fraction corresponded to a molecular mass of about 90 kDa (in presence of detergent) and to a maximal binding capacity of more than 10,000 pmol/protein. It therefore has a theoretical homogeneity close to 100%.     

Desensitization by histamine of H2 receptor-mediated adenylate cyclase activation in the human gastric cancer cell line HGT-1

Short-term treatment of cultured HGT-1 cells with histamine produced a time-dependent (half-life: 20 min) and homologous desensitization of histamine H2 receptor activity mediating cAMP generation in HGT-1 cells and gastric acid secretion in normal gastric mucosa. Histamine treatment resulted in loss of response of the adenylate cyclase to histamine in purified plasma membranes, but had no effect on basal, vasoactive intestinal peptide (VIP)- or NaF-stimulated enzyme activities. We propose that the desensitization of gastric histamine H2 receptor by histamine evidenced in cellular or subcellular preparations from HGT-1 cells could be involved in the physiological regulation and pharmacological control of gastric cell function in man.     

Solubilization and immunopurification of a somatostatin receptor from the human gastric tumoral cell line HGT-1

The human gastric tumoral cell line HGT-1 was previously shown to contain a membrane somatostatin receptor negatively coupled to adenylate cyclase through a pertussis toxin-sensitive inhibitory GTP-binding regulatory protein (Gi) (Reyl-Desmars, F., Laboisse, C., and Lewin, M. J. M. (1986) Regul. Pept. 16, 207-215). In this study, we have solubilized this receptor in a free unoccupied form using Triton X-100 as detergent and [125I-Tyr11]somatostatin-14 to monitor specific binding. Furthermore, we have prepared a monoclonal antibody against a chromatographically enriched soluble receptor fraction and used this antibody (30F3) to immunopurify the receptor in conjunction with Sepharose-somatostatin-14 immunopurification and steric exclusion high pressure liquid chromatography (HPLC). The purified fraction showed 18,600-fold enrichment in terms of specific binding (i.e. from 0.6 +/- 0.05 to 11,300 +/- 830 pmol/mg of protein) and a single dissociation constant (kappa D) of 76 +/- 8 nM. On HPLC, it migrated as a single and symmetric 90-kDa peak. Moreover, after 125I-protein labeling, it gave a single 90-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. On the other hand, the 30F3 monoclonal antibody immunoblotted with a single 90-kDa band contained in the HGT-1 cell membrane. We therefore suggest that this antibody is specific to the HGT-1 membrane somatostatin receptor, that this receptor has a molecular mass of 90 kDa, and that we have obtained a homogeneous preparation of nondenatured receptor suitable for further cloning studies.     

Molecular cloning of the human histamine H2 receptor.

We utilized the technique of polymerase chain reaction with oligonucleotide primers based upon the nucleotide sequence of the canine H2 histamine receptor gene which we recently isolated to clone its human homologue. Transfection of a construct of this gene in Colo-320 DM cells led to the expression of a receptor that bound to [methyl-3H] tiotidine and was linked to 3',5'cyclic adenosine monophosphate (cAMP) generation in response to histamine. Both cAMP generation and [methyl-3H] tiotidine binding were inhibited with the H2 histamine receptor selective antagonist cimetidine but not diphenhydramine or thioperamide which are, respectively, H1 and H3 histamine receptor antagonists. These data confirm that we have successfully cloned a novel gene encoding the human H2 histamine receptor.     

Characterization of the subtype of muscarinic receptor coupled to the stimulation of phosphoinositide hydrolysis in 132-1N1 human astrocytoma cells.

Stimulation of muscarinic receptors increases phosphoinositide (PI) hydrolysis in 132-1N1 human astrocytoma cells. To evaluate the subtype of receptors which mediate PI hydrolysis in 132-1N1 cells, the effects of: a) the nonselective M1 agonist, carbachol; b) the selective M1 agonist, 4-hydroxy-2-butynyl-trimethylammonium chloride-m-chlorocarbinilate (McN-343); c) the nonselective antagonists, atropine and scopolamine; d) the relatively selective M1 antagonist, pirenzepine; e) the relatively selective M2 antagonists, AF-DX 116 (11-2-diethylaminomethyl-1-piperidinylacetyl-5, 11-dihydro-6H-pyrido-2,3-b-1,4-benzodiazepine-6-one) and methoctramine and f) the relatively selective M3 antagonist, hexahydrosila-difenidol (HHSiD) on PI hydrolysis in 132-1N1 cells were studied. The cell pools of inositol-phospholipids were prelabelled by incubating 132-1N1 cells in a low inositol containing medium (CMRL-1066) supplemented with [3H]inositol (2 microCi/ml) for 20-24 hours at 37 degrees C. The cells were washed and resuspended in a physiological salt solution, and PI hydrolysis was measured by accumulation of [3H]inositol-1-phosphate (IP) in the presence of 10 mM LiCl. Carbachol produced time and concentration dependent PI hydrolysis (EC50, 37 microM). McN-A343 did not cause significant hydrolysis of PI in 132-1N1 cells indicating that the receptor was not of M1 type. All the above muscarinic antagonists caused a concentration dependent decrease in the level of IP in response to carbachol (100 microM). The rank order of their affinities (pA2 values) was: atropine (8.8) > HHSiD (7.6) > pirenzepine (6.8) > methoctramine (6.0) > AF-DX 116 (5.8). This rank order supports the concept that M3 (other names, M2 beta, glandular M2) receptors are linked to PI hydrolysis in 132-1N1 cells. HHSiD, which is selective for M3 receptors of the smooth muscle has higher affinity for muscarinic receptors in 132-1N1 cells than AF-DX 116 which is selective for M2 receptors in cardiac tissue. If the receptor in 132-1N1 cells had been M2, part of the rank order for affinities would have been methoctramine > AF-DX 116 > HHSiD > pirenzepine. From all of these observations, the muscarinic receptor for PI hydrolysis in 132-1N1 cells is tentatively characterized as of M3 type.     

Identification of a dual histamine H1/H3 receptor ligand based on the H1 antagonist chlorpheniramine

Combining the first generation H(1) antihistamine chlorpheniramine (1) with H(3) ligands of the alkylamine type has led to the identification of compound 9d, a dual ligand of both the H(1) and H(3) receptors.     

Sorting of the FGF receptor 1 in a human glioma cell line

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase promoting tumor growth in a variety of cancers, including glioblastoma. Binding of FGFs triggers the intracellular Ras/Raf/ERK signaling pathway leading to cell proliferation. Down-regulation of FGFR1 and, consequently, inactivation of its signaling pathways represent novel treatment strategies for glioblastoma. In this study, we investigated the internalization and endocytic trafficking of FGFR1 in the human glioma cell line U373. Stimulation with FGF-2 induced cell rounding accompanied by increased BrdU and pERK labeling. The overexpression of FGFR1 (without FGF treatment) resulted in enhanced phosphorylated FGFR1 suggesting receptor autoactivation. Labeled ligand (FGF-2-Cy5.5) was endocytosed in a clathrin- and caveolin-dependent manner. About 25 % of vesicles carrying fluorescently tagged FGFR1 represented early endosomes, 15 % transferrin-positive recycling endosomes and 40 % Lamp1-positive late endosomal/lysosomal vesicles. Stimulation with FGF-2 increased the colocalization rate in each of these vesicle populations. The treatment with the lysosomal inhibitor leupeptin resulted in FGFR1 accumulation in lysosomes, but did not enhance receptor recycling as observed in neurons. Analysis of vesicle distributions revealed an accumulation of recycling endosomes in the perinuclear region. In conclusion, the shuttling of receptor tyrosine kinases can be directly visualized by overexpression of fluorescently tagged receptors which respond to ligand stimulation and follow the recycling and degradation pathways similarly to their endogenous counterparts.     

Characterization of functional histamine H1 receptors on a cultured smooth muscle cell line

Cultured cells of the smooth muscle line DDT1MF-2, which was derived from a hamster vas deferens tumor, expressed histamine H1-type receptors and responded biochemically and functionally to H1-specific stimulation. The H1-receptor antagonist [3H]-pyrilamine bound specifically to 9.7 x 10(6) sites/DDT1MF-2 cell with a dissociation constant (Kd) of 219 nM. The addition of histamine to suspensions of fura-2-loaded DDT1MF-2 cells elicited a rapid, transient, and stimulus concentration-dependent increase in the intracellular concentration of Ca2+ with an EC50 of 3 x 10(-5) M, which demonstrated H1 receptor specificity. Moreover, in order to evaluate in vitro contractile response of individual DDT1MF-2 cells, the degree of intracellular actin polymerization was quantified by a DNase inhibition assay. The percentage of nonpolymerized or G-actin in DDT1MF-2 cells was reduced in a histamine concentration-dependent manner with an EC50 of 1 x 10(-5) M and H1 receptor specificity. Histamine-induced actin polymerization was accompanied by changes in cell shape that were consistent with cellular contraction, as assessed by flow cytometry. The H1-type receptors of cultured DDT1MF-2 cells thus couple histamine stimulation to a variety of functional responses of smooth muscle cells.     

Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC3H1 cell line

We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC3H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, 2H, 13C, 15N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher mass (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme (s = 7.40 +/- 0.04 S, n = 36). Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme (i.e. Km for ATP and EC50 for Mn2+ or Mg2+) did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control (cells in light medium). After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value indicating that the heavy isotope-labeled enzyme replaced the pre-existing light form of the molecule. These observations show that the rapid decrease in adenylate cyclase activity and the synthesis of heavy adenylate cyclase molecules are two separate events. The relative amounts of heavy and light components of forskolin-stimulated adenylate cyclase obtained in sucrose gradient differential sedimentation were determined as a function of time beginning 24 h after the transfer into the heavy medium. The decrease of the pre-existing light form could be represented by simple first order kinetics with a half-time of 40 h. This result suggests that the metabolic renewal of forskolin-stimulated adenylate cyclase is comparable to that of most plasma membrane proteins.     

Characterization of JOK-1, a human gastric epithelial cell line

Summary Human gastric epithelial cells were isolated from samples of human gastric lining and immortalized with simian virus 40 (SV40) to generate the stable human gastric epithelial cell line “JOK-1” These cells express conventional, epithelial markers (vimentin, cytokeratin-18, occludin, N-and E-cadherins, β-catenin, ZO-1, ZO-2, mucin, epithelial specific antigen) as well as SV40 large T-antigen. These cells rapidly externalized E-cadherin in response to acidic medium, and exhibited epitheliallike barrier properties that are also regulated by media pH. In contrast, the kidney epithelial cell line “MDCK” also expresses serveral epithelial markers (vimentin, cytokeratin-18, occludin, N-and E-cadherin, β-catenin, ZO-1, ZO-2, epithelial specific antigen), but does not express mucin, or large T-antigen. However, MDCK rapidly internalize their E-cadherin from the cell surface and increase the solute flux in an acidic medium. These data suggest that the JOK-1 cell line is a potentially useful cell line for developing models of gastric epithelial function, development, and disease.     

Autoregulation of enterochromaffin-like cell histamine secretion via the histamine 3 receptor subtype.

INTRODUCTION: The neuroendocrine histamine-secreting cell of the gastric fundus, the enferochromaffin-like cell, is the principal regulator of parietal cell acid secretion. We have proposed that histamine may regulate its own synthesis and release via an autocrine mechanism. The purpose of this study was to evaluate the role of the histamine receptor subtypes H1, H2 and H3 in the regulation of this phenomenon. METHODS: Purified ECL cells were isolated by pronase digestion and EDTA exposure of the rat stomach, followed by particle size and density separation using counterflow elutriation and Nycodenz gradient centrifugation, 24-hr cultured cells were pretreated for 30 min with the agents; H1 receptor agonist (2-[(3-trimethyl)-diphenyl] histamine) (TMPH), H1 receptor antagonist (terfenadine); H2 receptor agonist (dimaprit) or antagonist (cimetidine or loxitidine); or H3 receptor agonist (imetit) or antagonist (thioperamide) (all tested, 10(-10)-10(-6) M). Gastrin was then used to stimulate histamine secretion. Histamine secretion was quantified by specific enzyme-immunoassay. RESULTS: Basal histamine secretion was 2.7 +/- 0.14 nmol/10(3) cells. Gastrin-stimulated (10 nM) levels were 4.6 +/- 0.4 nmol/10(3) cells (p < .01). TMPH inhibited both basal and gastrin driven histamine secretion with a maximal effect (34 percent) (1.78 +/- 0.08 nmol/10(3) cells) and an IC50 of > 5 x 10(-7) M. H1 receptor antagonism did not alter histamine secretion alone or in combination with gastrin. Neither H2 receptor stimulation nor antagonism had any effect on histamine secretion alone or in combination with gastrin. Gastrin-induced histamine secretion was dose-dependently inhibited by imetit (H3 agonist) with a maximal effect (2.4 +/- 0.6 nmol/10(3) cells) (p < .05) and an IC50 of 10(-9) M. Conversely, Thioperamide (H3 antagonist) dose-dependently augmented gastrin-stimulated histamine secretion with a maximum effect (5.7 +/- 0.5 nmol/10(3) cells) (p < .05) at 10(-8) M and an EC50 of 7 x 10(-10) M. CONCLUSION: These data are consistent with the presence of an H3 receptor on the ECL cell which modulates gastrin-stimulated histamine secretion. Our observations support the proposal that a histamine-mediated short-loop autocrine regulatory mechanism of ECL cell secretion exists.     

Presence of histamine H2-receptors on human gastric carcinoma cell line MKN-45 and their increase by retinoic acid treatment.

Histamine dose-dependently stimulated cyclic AMP production in human gastric carcinoma cell line MKN-45, and this effect was inhibited by cimetidine but not by pyrilamine. Moreover, not only histamine but also cimetidine displaced the specific binding of [3H]tiotidine to these cells, whereas pyrilamine had no effect. On the other hand, pretreatment of MKN-45 cells with retinoic acid (RA) significantly enhanced histamine-induced increase of cyclic AMP production, although the cyclic AMP response to either forskolin or NaF was not affected. Finally, RA treatment increased the number of histamine receptor without altering its affinity. Thus, it appears that histamine H2-receptors are present on MKN-45 cells, and that RA treatment enhances the action of histamine on these cells by increasing the number of H2-receptors.     

Bimodal regulation of the human H1 histamine receptor by G protein-coupled receptor kinase 2

The H1 histamine receptor (H1HR) is a member of the G protein-coupled receptor superfamily and regulates numerous cellular functions through its activation of the G(q/11) subfamily of heterotrimeric G proteins. Although the H1HR has been shown to undergo desensitization in multiple cell types, the mechanisms underlying the regulation of H1HR signaling are poorly defined. To address this issue, we examined the effects of wild type and mutant G protein-coupled receptor kinases (GRKs) on the phosphorylation and signaling of human H1HR in HEK293 cells. Overexpression of GRK2 promoted H1HR phosphorylation in intact HEK293 cells and completely inhibited inositol phosphate production stimulated by H1HR, whereas GRK5 and GRK6 had lesser effects on H1HR phosphorylation and signaling. Interestingly, catalytically inactive GRK2 (GRK2-K220R) also significantly attenuated H1HR-mediated inositol phosphate production, as did an N-terminal fragment of GRK2 previously characterized as a regulator of G protein signaling (RGS) protein for Galpha(q/11). Disruption of this RGS function in holo-GRK2 by mutation (GRK2-D110A) partially reversed the quenching effect of GRK2, whereas deletion of both the kinase activity and RGS function (GRK2-D110A/K220R) effectively relieved the inhibition of inositol phosphate generation. To evaluate the role of endogenous GRKs on H1HR regulation, we used small interfering RNAs to selectively target GRK2 and GRK5, two of the primary GRKs expressed in HEK293 cells. A GRK2-specific small interfering RNA effectively reduced GRK2 expression and resulted in a significant increase in histamine-promoted calcium flux. In contrast, knockdown of GRK5 expression was without effect on H1HR signaling. These findings demonstrate that GRK2 is the principal kinase mediating H1 histamine receptor desensitization in HEK293 cells and suggest that rapid termination of H1HR signaling is mediated by both the kinase activity and RGS function of GRK2.     

Phorbol ester-mediated desensitization of histamine H1 receptors on a cultured smooth muscle cell line

The present study was undertaken in order to examine the effect of protein kinase C (PKC) on histamine H1 receptors (H1R) present on the smooth muscle cell line, DDT1MF-2. [3H]-pyrilamine binding revealed that specific [3H]-pyrilamine binding sites were reduced by pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, but not the Kd. The TPA analogue, 4 alpha phorbol 12,13-didecanoate, which does not activate PKC, failed to induce down-regulation of H1R. TPA-induced down-regulation of H1R was inhibited by pretreatment with 1-(5-Isoquinilinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, in a dose dependent manner. The H-7 analogue, H-8, which is a less potent inhibitor of PKC, but a potent inhibitor of cyclic nucleotide dependent protein kinase, had no effect on H1R. Moreover, treatment with TPA inhibited histamine-induced increases in [Ca2+]i in cells loaded with the fluorescent indicator, indo-1. These data suggest that H1R in DDT1MF-2 cells are functionally regulated by PKC.     

Solubilization, purification and molecular characterization of H2 histamine receptor from human tumoral gastric cells HGT-1

This communication reports the solubilization, the purification and the molecular characterization of the H2-histamine receptor from the cell line HGT-1 derived from a human gastric cancer. The receptor has been solubilized by Triton X100 and purified by gel filtration onto Sephacryl, affinity-chromatography (Sepharose-famotidine) and high performance liquid chromatography (HPLC). The purified receptor specifically bound the H2 selective ligand 3H-methyltiotidine with a kD of 160 nM (vs 50 nM for the intact HGT-1 cell) and a maximal binding capacity of 14,000 pmol/mg protein which represents a 12,170-fold enrichment and a degree of purity of 98%. It is a glycoprotein of 70 kDa molecular mass containing N-acetylglucosamine residues.     

CLIC4 interacts with histamine H3 receptor and enhances the receptor cell surface expression

Histamine H3 receptor (H3R), one of G protein-coupled receptors (GPCRs), has been known to regulate neurotransmitter release negatively in central and peripheral nervous systems. Recently, a variety of intracellular proteins have been identified to interact with carboxy (C)-termini of GPCRs, and control their intracellular trafficking and signal transduction efficiencies. Screening for such proteins that interact with the C-terminus of H3R resulted in identification of one of the chloride intracellular channel (CLIC) proteins, CLIC4. The association of CLIC4 with H3R was confirmed in in vitro pull-down assays, coimmunoprecipitation from rat brain lysate, and immunofluorescence microscopy of rat cerebellar neurons. The data from flowcytometric analysis, radioligand receptor binding assay, and cell-based ELISA indicated that CLIC4 enhanced cell surface expression of wild-type H3R, but not a mutant form of the receptor that failed to interact with CLIC4. These results indicate that, by binding to the C-terminus of H3R, CLIC4 plays a critical role in regulation of the receptor cell surface expression.     

Re-examination of [3H]mepyramine binding assay for histamine H1 receptor using quinine.

[3H]Mepyramine, a potent antagonist of the histamine H1 receptor, has been widely used as a radioligand binding assay for the H1 receptor. Previously, we purified a mepyramine binding protein (MBP) from rat liver, but found that its partial amino acid sequences were very similar to those of debrisoquine 4-hydroxylase isozymes (P450 db1 and db2), which are members of the superfamily of cytochrome P450. Using cloned histamine H1 receptor cDNA, we found that [3H]mepyramine could bind only the H1 receptor and did not bind MBP in the presence of 10(-5) M quinine, an inhibitor of debrisoquine 4-hydroxylase isozymes. We developed a method to determine the contents of the H1 receptor and MBP separately using [3H]mepyramine and quinine and found that MBP is abundant in certain areas of bovine brain.