sq-23377 and Colonic-Neoplasms

Endocannabinoids and related N-acylethanolamines (NAEs) are involved in regulation of gut function, but relatively little is known as to whether inflammatory cytokines such as IFNγ affect their levels. We have investigated this in vitro using cultures of T84 colon cancer cells.. T84 cells, when cultured in monolayers, differentiate to form adult colonic crypt-like cells with excellent permeability barrier properties. The integrity of the permeability barrier in these monolayers was measured using transepithelial electrical resistance (TEER). NAE levels were determined by ultra-performance liquid chromatography-tandem mass spectrometric analysis. Expression of the enzymes involved in NAE and 2-arachidonoylglycerol (2-AG) turnover were assessed with qPCR.. IFNγ treatment for 8 or 24 h increased levels of both endocannabinoids (anandamide and 2-AG) and the related NAEs. The treatment did not affect the rate of hydrolysis of either anandamide or palmitoylethanolamide by intact cells, and in both cases, fatty acid amide hydrolase (FAAH) rather than NAE-hydrolysing acid amidase (NAAA) was mainly responsible for the hydrolysis of these NAEs. IFNγ treatment reduced the TEER of the cells in a manner that was not prevented by inhibition of either FAAH or NAAA but was partially reversed by apical administration of the NAE palmitoylethanolamide.. IFNγ treatment mobilized endocannabinoid and related NAE levels in T84 cells. However, blockade of anandamide or NAE hydrolysis was insufficient to negate the deleterious effects of this cytokine upon the permeability barrier of the cell monolayers.

Topics: Amides; Arachidonic Acids; Cell Culture Techniques; Cell Line, Tumor; Chromatography, High Pressure Liquid; Colonic Neoplasms; Endocannabinoids; Ethanolamines; Glycerides; Humans; Interferon-gamma; Ionomycin; Palmitic Acids; Polyunsaturated Alkamides

Calcyclin Binding Protein/(Siah-1 interacting protein) (CacyBP/SIP) acts as an oncogene in colorectal cancer. The nuclear accumulation of CacyBP/SIP has been linked to the proliferation of cancer cells. It has been reported that intracellular Ca2+ induces the nuclear translocation of CacyBP/SIP. However, the molecular mechanism of CacyBP/SIP nuclear translocation has yet to be elucidated. The purpose of this study was to test whether the Ca2+-dependent binding partner S100 protein is involved in CacyBP/SIP nuclear translocation in colon cancer SW480 cells.. The subcellular localization of endogenous CacyBP/SIP was observed following the stimulation of ionomycin or BAPTA/AM by immunofluorescence staining in SW480 cells. S100A6 small interfering RNAs (siRNA) were transfected into SW480 cells. Immunoprecipitation assays detected whether S100 protein is relevant to the nuclear translocation of CacyBP/SIP in response to changes in [Ca2+]i.. We observed that endogenous CacyBP/SIP is translocated from the cytosol to the nucleus following the elevation of [Ca2+]i by ionomycin in SW480 cells. Co-immunoprecipitation experiments showed that the interaction between S100A6 and CacyBP/SIP was increased simultaneously with elevated Ca2+. Knockdown of S100A6 abolished the Ca2+ effect on the subcellular translocation of CacyBP/SIP.. Thus, we demonstrated that S100A6 is required for the Ca2+-dependent nuclear translocation of CacyBP/SIP in colon cancer SW480 cells.

Topics: Active Transport, Cell Nucleus; Calcium; Calcium Ionophores; Calcium-Binding Proteins; Cell Line, Tumor; Cell Nucleus; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Ionomycin; RNA Interference; S100 Calcium Binding Protein A6

Functional activation of beta-catenin/Tcf signaling plays an important role in the early events in colorectal carcinogenesis. We examined the effect of ionomycin against beta-catenin/Tcf signaling in colon cancer cells. Reporter gene assay showed that ionomycin inhibited beta-catenin/Tcf signaling efficiently. In addition, the inhibition of beta-catenin/Tcf signaling by ionomycin in HEK293 cells transiently transfected with a constitutively mutant beta-catenin gene, whose product is not phosphorylated by GSK3beta, indicates that its inhibitory mechanism is related to beta-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunoprecipitation analysis, western blot and electrophoretic mobility shift assay. As a result, our data reveal that the association of beta-catenin and Tcf-4 is disrupted and the amount of beta-catenin product in the nucleus is decreased by ionomycin in a concentration-dependent manner. Moreover, ionomycin strongly suppressed the binding of the Tcf complexes to its specific DNA-binding sites. The significance of the current work is that ionomycin is a negative regulator of beta-catenin/Tcf signaling in colon cancer cells and its inhibitory mechanism is related to the decreased nuclear beta-catenin products and to the suppressed binding of Tcf complexes to consensus DNA.

Topics: beta Catenin; Binding Sites; Blotting, Western; Cell Nucleus; Cells, Cultured; Colonic Neoplasms; DNA; Down-Regulation; Electrophoretic Mobility Shift Assay; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunoprecipitation; Ionomycin; Kidney; Luciferases; Mutation; Phosphorylation; Promoter Regions, Genetic; Response Elements; Signal Transduction; TCF Transcription Factors; Trans-Activators; Transfection

To investigate the potential role of cytokines in promoting Fas ligand (FasL)-expressing colon cancer cells.. Immunohistochemical SABC method was used to observe the expression of Fas receptor and ligand in SW620 colon cancer cell line and Jurkat T cells in order to provide the morphological evidence for the functions of Fas receptor and ligand. To examine the cytotoxicity of effector cells, CytoTox96(r) non-radioactive cytotoxicity assay was adopted to measure the lactate dehydrogenase-releasing value after SW620 cells were co-cultured with Jurkat T lymphocytes.. The FasL of colon cancer SW620 cells was positive. The positive substances were distributed in the cell membrane and cytoplasm. The Fas receptor of colon cancer SW620 cells was negative. The Fas receptor and ligand of Jurkat T lymphocytes turned out to be positive. The positive substances were distributed in the cell membrane. After phytohemagglutinin (PHA)-stimulated Jurkat T lymp-hocytes were co-cultured with phorbol 12-myristate 13-acetate (PMA)-plus-ionomycin-stimulated (for 48 h) SW620 cells or tumor necrosis factor-alpha (TNF-alpha)-stimulated (for 48 h) SW620 cells or unstimulated SW620 cells for 4 h, the cytotoxicity of SW620 cells to PHA-stimulated Jurkat cells at effector-to-target ratios of 10:1, 5:1, 2.5:1, and 1.25:1 was 74.6%, 40.8%, 32.4%, and 10.9% (F = 8.19, P<0.05); or 54.9%, 35.3%, 22.0%, and 10.3% (F = 11.12, P<0.05); or 14.9%, 10.5%, 6.9%, and 5.8% (F = 3.45, P<0.05). After PHA-stimulated Jurkat T lymphocytes were co-cultured with unstimulated SW620 cells for 8 h, the cytotoxicity of SW620 cells to PHA-stimulated Jurkat cells at effector-to-target ratios of 5:1, 2.5:1, and 1.25:1 from the experiment was 83.9%, 74.1%, and 28.5% (F = 137.04, P<0.05) respectively. Non-radioactive cytotoxicity assay showed that the apoptotic rate of Jurkat cells remarkably increased with the increase of planting concentration of SW620 cells and co-culture time after the SW620 cells were co-cultured with the Jurkat T lymphocytes. The cytotoxicity was significantly enhanced by PMA+ionomycin or TNF-alpha.. The FasL expressed in human colon cancer cells may be regulated by endogenous factors in the microenvironment of the host and facilitate the escape of tumor cells from the host immune system.

Topics: Cell Communication; Cell Line, Tumor; Coculture Techniques; Colonic Neoplasms; Cytokines; Cytotoxicity, Immunologic; Fas Ligand Protein; fas Receptor; Glioma; Humans; Immunohistochemistry; Ionomycin; Ionophores; Jurkat Cells; Membrane Glycoproteins; Phytohemagglutinins; T-Lymphocytes; T-Lymphocytes, Regulatory; Tetradecanoylphorbol Acetate

Neuropeptide Y is known to exert inhibitory effects on ion secretion in the intestine by reducing the activity of adenylyl cyclase. In the human intestinal epithelial cell line HT29cl.19A, it has been previously shown that neuropeptide Y inhibits the electrophysiological phenomena related to Cl(-) secretion, when induced by elevation of cAMP via forskolin. Moreover, the secretion induced via elevation of intracellular calcium levels via muscarinic activation can be inhibited by neuropeptide Y. Part of these inhibitions appeared to be due to lowered calcium activity in the epithelial cells, thereby reducing the basolateral K(+) conductance. The phorbol ester 4-phorbol-12,13-dibutyrate (PDB) can induce secretion in this cell line via activation of protein kinase C as well; however, the effect of neuropeptide Y on this pathway has not yet been studied. In the present experiments, it is shown that neuropeptide Y inhibits the PDB-induced secretion at two sides: one located in the apical membrane and another in the basolateral membrane. It is shown that the latter effect can, at least partially, be explained via a direct effect of neuropeptide Y on the K(+) conductance. This was concluded from the observation that neuropeptide Y could also reduce basolateral K(+) conductance when intracellular calcium was dramatically increased by ionomycin. The observed inhibitory effects suggest that neuropeptide Y is a very powerful antisecretory peptide in human intestinal epithelial cells.

Topics: Calcium; Chlorides; Colforsin; Colonic Neoplasms; Cyclic AMP; Electric Conductivity; HT29 Cells; Humans; Ionomycin; Membrane Potentials; Neuropeptide Y; Phorbol 12,13-Dibutyrate; Potassium; Protein Kinase C

Fas ligand (FasL) belongs to the TNF superfamily. It is induced in activated lymphocytes and eliminates Fas-positive lymphocytes, resulting in the down-regulation of immune responses. FasL has also been detected in tissues other than lymphoid cells. We investigated the expression and function of FasL on human colon cancer cells. FasL mRNA was detected by RT-PCR in all six colon cancer cell lines tested and was not found on fibroblasts. FasL protein was detected in DLD-1, LoVo, HCT-116 and RPMI 4788 cells by immunohistochemical staining. DLD-1, LoVo and WiDr were cytotoxic against mouse T lymphoma cells which were transfected with human Fas receptor cDNA. The cytotoxicity was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) and ionomycin. Our data suggest that the FasL expressed in human colon cancer cells may be regulated by endogenous factors in the microenvironment of the host and facilitates the escape from the host immune system.

Topics: Apoptosis; Coculture Techniques; Colonic Neoplasms; Fas Ligand Protein; Gene Expression; Humans; Ionomycin; Lymphocyte Activation; Lymphocytes; Membrane Glycoproteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

HGF/SF (0.5-100 ng/ml) induced rapid membrane ruffling, formation of microspikes, and increased motility of HT115 cells within 5 min of addition. These effects were not accompanied by any change in cytosolic free Ca2+ concentration. However, ATP (0.5-10 mM) induced a transient rise in cytosolic free Ca2+ concentration in HT115 cells from a resting concentration of 100 nM to a peak of 400 nM before returning to baseline within 3 min. The addition of ATP to cells treated with HGF/SF inhibited both membrane ruffling and cell movement. The effect of ATP was attributed to the transient rise in cytosolic free Ca2+ concentration, because cytosolic BAPTA, which prevented the rise in cytosolic free Ca2+ concentration, also abolished the inhibitory effect of ATP. Raising cytosolic free Ca2+ concentration with ionomycin and ADP also inhibited membrane ruffling. It was thus concluded that transiently raised cytosolic free Ca2+ concentration inhibited HGF/SF-induced membrane ruffling of HT115 cells.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Calcium; Cell Line; Cell Membrane; Cell Movement; Chelating Agents; Colonic Neoplasms; Cytosol; Egtazic Acid; Hepatocyte Growth Factor; Humans; Ionomycin; Kinetics; Microscopy, Electron, Scanning; Time Factors; Tumor Cells, Cultured; Video Recording

Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C1 cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C1 was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10 microM) increased furosemide-insensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanide-insensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloride-depleted cells. When cells with normal chloride content were depolarized (135 mM medium potassium + 10 microM valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl- approximately Br- > NO3- > I-. The electrogenic transport pathway was inhibited in chloride-depleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.

Topics: Anions; Biological Transport; Bumetanide; Carrier Proteins; Cations; Cell Membrane Permeability; Cell Polarity; Cell Size; Chloride Channels; Chlorides; Colforsin; Colonic Neoplasms; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophysiology; Furosemide; Gluconates; Humans; Intestinal Mucosa; Intracellular Fluid; Ionomycin; Nitrates; Organ Specificity; Quinolinium Compounds; Sodium-Potassium-Chloride Symporters; Tumor Cells, Cultured; Valinomycin

Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na >> Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single KBIC channels, the carbachol-stimulated Isc was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the KBIC channel mediates basolateral membrane K conductance in T84 cell monolayers during stimulation by cholinergic secretagogues.

Topics: 4-Aminopyridine; Calcimycin; Carbachol; Cell Membrane; Charybdotoxin; Colonic Neoplasms; Cyclic AMP; Epithelium; Fluorescence; Humans; Ionomycin; Membrane Potentials; Patch-Clamp Techniques; Potassium Channels; Quinidine; Scorpion Venoms; Temperature; Tetraethylammonium; Tetraethylammonium Compounds; Time Factors; Tumor Cells, Cultured

The patch-clamp technique was combined with camera-based intracellular Ca2+ concentration ([Ca2+]i) imaging to identify the single-channel basis of the Ca(2+)-dependent Cl- conductance in human colonic adenocarcinoma cells (HT-29). Cl- channels were activated when membrane patches were excised into solutions containing high (1 microM) Ca2+ concentrations. Their single-channel conductance, measured by amplitude histogram analysis, averaged 13 pS at -90 mV and 16 pS at +90 mV membrane potential (MP). In multiple channel patches, Cl- currents showed properties similar to Ca(2+)-activated whole cell currents: outward rectification and time-dependent activation at depolarizing MP. Channel activity disappeared shortly after patch excision from the cell. In cell-attached patches, Cl- channel opening was infrequent at resting [Ca2+]i values (96 +/- 18 nM), but when [Ca2+]i was increased by the Ca2+ ionophore ionomycin (1 microM), Cl- channels were activated with a time course that paralleled the [Ca2+]i rise. Repetitive ionophore exposure produced equivalent rises in [Ca2+]i, but the corresponding Cl- channel activity became progressively reduced. The Ca(2+)-mediated agonist neurotensin (50 nM) elicited a transient Cl- channel activation that preceded the generalized cellular [Ca2+]i rise. Channel activation with neurotensin occurred in the absence of pipette Ca2+ but was abolished by preloading cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus, in response to the Ca(2+)-mediated agonist neurotensin, Cl- channel activation results from Ca2+ mobilization from intracellular pools localized within the vicinity of the plasma membrane. The Ca2+ dependency, voltage sensitivity, and kinetics of this 15-pS Cl- channel indicate that it is the basis of the whole cell Ca(2+)-activated Cl- current.

Topics: Adenocarcinoma; Calcium; Cell Differentiation; Cell Membrane; Chloride Channels; Chlorides; Colonic Neoplasms; Egtazic Acid; Humans; Ion Channel Gating; Ion Channels; Ionomycin; Meglumine; Membrane Potentials; Membrane Proteins; Neurotensin; Tumor Cells, Cultured

The regulation of 15-pS Cl- channels by Ca(2+)-mobilizing agonists was investigated by simultaneous cell-attached patch and intracellular Ca2+ concentration ([Ca2+]i) measurements. Cells were loaded with a synthetic peptide made from the calmodulin binding domain of Ca2+/calmodulin-dependent protein kinase II. This caused inhibition of Cl- channel activity without any corresponding effect on either agonist-induced [Ca2+]i mobilization or K+ channel activation. Calmodulin therefore confers Ca2+ sensitivity to the 15-pS channel. When patches were excised from the cell, Cl- channel activity ran down. Channel rundown was not reversed by ATP or calmodulin. When recording from cell-attached patches of detergent-treated cells, similar phenomenology was observed. Therefore, other factors that are lost upon plasma membrane permeabilization are required for the functioning of Ca(2+)-dependent Cl- channels. After rundown of these channels, a large-conductance, multistate, Ca(2+)-insensitive Cl- channel was seen. The smallest subconductance state of this channel was of similar magnitude to that of the Ca(2+)-dependent Cl- channel. Furthermore, its voltage and halide sensitivities were similar to those reported for the 15-pS Cl- channel and Ca(2+)-dependent whole cell Cl- currents. Because this channel is not observed in the intact cell, this may be a remnant conductance of the Ca(2+)-sensitive 15-pS Cl- channel.

Topics: Adenocarcinoma; Calcium; Calmodulin; Cell Differentiation; Cell Membrane; Chloride Channels; Colonic Neoplasms; Digitonin; Egtazic Acid; Homeostasis; Humans; Ion Channels; Ionomycin; Kinetics; Meglumine; Membrane Potentials; Membrane Proteins; Neurotensin; Tumor Cells, Cultured

We have investigated the regulation of the intestinal mucin gene MUC2 in HT29 cells. Surprisingly, sodium butyrate, an effective inducer of aspects of colonic cell differentiation in HT29 cells, fails to induce MUC2 during short-term exposure, despite the fact that it has been used to select stably differentiated clones of HT29 that resemble goblet cells and produce mucin. However, 12-O-tetradecanoylphorbol-13-acetate and forskolin, which trigger the protein kinase C- and A-dependent signal transduction pathways, respectively, are potent inducers of MUC2 gene expression. 12-O-Tetradecanoylphorbol-13-acetate and forskolin operate through distinct mechanisms, with the former requiring de novo protein synthesis and the latter not. Experiments using specific protein kinase inhibitors suggest that both inducers operate by triggering their respective signal transduction pathways. Nuclear runoff analyses suggest that post-transcriptional (rather than transcriptional) mechanisms are important in the accumulation of MUC2 mRNA. Finally, we show that in several cell lines from human mucinous tumors, characterized by elevated levels of mucin production, MUC2 expression is very high and constitutive compared to forskolin-treated HT29 cells. Thus, the different regulation of MUC2 in HT29 cells and in mucinous tumor cell lines may reflect molecular pathways that characterize colon carcinomas of different histology and pathology.

Topics: 1-Methyl-3-isobutylxanthine; Adenocarcinoma; Bucladesine; Calcimycin; Colforsin; Colonic Neoplasms; DNA Probes; Ethers, Cyclic; Gene Expression Regulation, Neoplastic; Humans; Intestinal Mucosa; Ionomycin; Ionophores; Kinetics; Mucins; Okadaic Acid; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured

The whole-cell mode of the patch-clamp technique has been used to monitor ionic currents in T84 colonic carcinoma cells. The cells were stimulated by either a cAMP cocktail, ionomycin or hypotonicity. Sizeable currents with distinct kinetics were observed after the stimulation with the different agonists. These kinetically distinct Cl- currents also presented a differential sensitivity to the anti-oestrogen Tamoxifen and to the halide I-. Tamoxifen only inhibits the volume activated Cl- current without affecting the other two. Substitution of extracellular Cl- by I- shifted the reversal potential towards more negative values both in the hypotonicity and ionomycin activated Cl- currents. The cAMP activated current responded to the Cl- substitution by I- with a blockade of both outward and inward currents, in addition to the displacement of the zero current level towards positive values. Thus, the use of these two simple tools, I- and tamoxifen, allows the distinction of Cl- channels in epithelial cells.

Topics: Carcinoma; Chlorides; Colonic Neoplasms; Cyclic AMP; Electric Conductivity; Hypotonic Solutions; Iodides; Ionomycin; Tamoxifen; Tumor Cells, Cultured

The differentiated clone 19A of the HT-29 human colon carcinoma cell line was used as a model to study the intracellular electrophysiological effects of interaction of the cAMP, the protein kinase C (PKC) and the Ca2+ pathways. (a) A synergistic effect between ionomycin and forskolin was observed. From intracellular responses it was concluded that the synergistic effect is caused by activation of an apical Cl- conductance by protein kinase A and a basolateral K+ conductance by Ca2+. (b) A transient synergistic effect of ionomycin and the phorbol ester phorbol dibutyrate (PDB) was found. The decrease of the response appeared to be due to PKC-dependent inactivation of the basolateral K+ conductance. The synergism is caused by PKC-dependent increase of the apical Cl- conductance and Ca(2+)-dependent increase of the basolateral K+ conductance. (c) The effects of carbachol and PDB were not fully additive presumably because of their convergence on PKC activation. (d) Forskolin and PDB, when added in this order, had a less than additive effect. Results of cell-attached patch-clamp studies, presented in the accompanying paper, showed a synergistic effect of forskolin and PDB on non-rectifying small-conductance Cl- channels. Assuming that these channels are involved in the transepithelial responses it is suggested that forskolin and PDB induce a modulatory, synergistic increase of the apical Cl- conductance when both pathways are activated simultaneously. (e) The HT-29cl.19A cells differ from T84 cells in that the latter did n ot respond with an increase of the short-circuit current to addition of phorbol ester. this may be due to a very low expression of PKA alpha.

Topics: Biological Transport; Calcium; Carbachol; Chloride Channels; Colforsin; Colonic Neoplasms; Cyclic AMP; Drug Synergism; Electric Conductivity; Humans; Ionomycin; Phorbol 12,13-Dibutyrate; Potassium Channels; Protein Kinase C; Tumor Cells, Cultured

Increases of cytosolic Ca2+, as occur with agonists such as ATP, neurotensin (NT), hypotonic cell swelling and ionomycin, enhance the membrane conductance (GM) and hence the input conductance (GI) of HT29 cells. In the present study we have examined whether these increases in GM are paralleled by exocytosis. To this end the membrane capacitance (CM) of HT29 cells was measured by patch clamp techniques. Two methods to monitor CM were used: a direct method (DM) and a phase tracking method (PTM). With the DM the following results were obtained. NT (10(-8) mol/l, n = 9) increased GM and CM significantly from 2.4 +/- 0.3 nS and 23.5 +/- 3 pF to 32 +/- 8 nS and 27.3 +/- 3.1 pF respectively. ATP (10(-4) mol/l, n = 29) had a very similar effect. GM and CM were increased from 5.7 +/- 1 nS and 36 +/- 4.4 pF to 111 +/- 21 nS and 44 +/- 5.4 pF respectively. Hypotonic cell swelling (160 mosmol/l, n = 18) had a comparable effect: GM and CM were increased from 4.9 +/- 1 nS and 30 +/- 4.1 pF to 46 +/- 10 nS and 37 +/- 4.9 pF respectively. Ionomycin (10(-7) mol/l, n = 4) gave similar results. With the PTM it was possible to monitor the rapid changes in GM and CM, as they were induced by ATP (n = 42) and NT (n = 29), with high time resolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Calcium; Chloride Channels; Colonic Neoplasms; Cytosol; Exocytosis; Humans; Hypotonic Solutions; Ionomycin; Membrane Potentials; Neurotensin; Tumor Cells, Cultured

We have examined the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the phosphoinositol signal transduction pathway in the human colon cancer-derived cell line CaCo-2 and have studied the regulation of intracellular calcium ([Ca2+]i) and pH (pHi) by this secosteroid. CaCo-2 cells were prelabeled with [3H]myoinositol and treated with 10(-8) M 1,25-(OH)2D3 or vehicle for 90 sec. 1,25-(OH)2D3 caused a decrease in labeled phosphatidylinositol-4-5-bis-phosphate and an increase in labeled inositol 1,4,5-trisphosphate. Treatment with 10(-8) M 1,25-(OH)2D3 for 90 sec also raised the cellular content of diacylglycerol. In a dose-dependent manner, 1,25-(OH)2D3 caused the translocation of protein kinase-C activity from the cytosolic to the membrane fraction, which occurred after as little as 15 sec of exposure to the secosteroid, peaked at about 1-5 min, and then returned toward baseline values. In these CaCo-2 cells, baseline [Ca2+]i was 258 +/- 2 nM (mean +/- SE), as assessed using the fluorescent dye fura-2. After exposure to 10(-8) M 1,25-(OH)2D3, [Ca2+]i rapidly increased to 392 +/- 14 nM after 100 sec, fell, and then subsequently rose to a plateau of 350 +/- 3 nM after 400 sec. In Ca(2+)-free buffer, 1,25-(OH)2D3 caused only a transient rise in [Ca2+]i, indicating that 1,25-(OH)2D3 stimulated both the release of intracellular calcium stores and calcium influx. 1,25-(OH)2D3 caused a dose-dependent decrease in pHi in CaCo-2 cells, as assessed by the fluorescent dye BCECF, which was not observed in cells suspended in Na(+)-free buffer or pretreated with amiloride, indicating that the secosteroid inhibited Na(+)-H+ exchange. No effect of 1,25-(OH)2D3 on pHi was observed in cells in a Ca(2+)-free buffer or pretreated with the phospholipase-C inhibitor U-73,122, which also blocked the rise in [Ca2+]i, or in cells pretreated with the Ca2+/calmodulin inhibitor calmidazolium. Taken together, these studies indicate that 1,25-(OH)2D3 rapidly stimulates membrane phosphoinositide breakdown in CaCo-2 cells, generating the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, causing translocation of protein kinase-C to the membrane, and increasing [Ca2+]i by both releasing calcium stores and promoting calcium influx. Secondary to the rise in [Ca2+]i, Na(+)-H+ exchange is inhibited by a calcium/calmodulin-dependent pathway.

Topics: Amiloride; Calcitriol; Carrier Proteins; Cell Line; Cell Membrane; Colonic Neoplasms; Cytosol; Diglycerides; Humans; Hydrogen-Ion Concentration; Inositol; Inositol Phosphates; Ionomycin; Kinetics; Membrane Lipids; Phosphatidylinositols; Protein Kinase C; Sodium; Sodium-Hydrogen Exchangers

The gene responsible for cystic fibrosis (CF) has recently been cloned and sequenced. When transfected into CF epithelial cells, normal transcripts of this gene correct the underlying defect in CF, i.e. cAMP-dependent Cl- secretion is restored. Thus, the protein encoded by this gene, designated "cystic fibrosis transmembrane conductance regulator" (CFTR), somehow participates in the Cl- secretory response. In this paper we have correlated CFTR gene expression with cAMP and Ca(2+)-dependent Cl- secretion in unpolarized (parental) and polarized (Cl.19A) clones of the human colonic adenocarcinoma cell line HT-29. These cell lines were found to express equally high levels of CFTR mRNA at 4 days post-passage. In addition, protein expression (determined by immunoprecipitation) was also identical. The cAMP-generating agonist forskolin had little effect on 125I efflux from the unpolarized cells. In contrast, this agonist increased 125I efflux 3-fold in polarized cells. The lack of response in the unpolarized cells was not due to the inability of forskolin to raise cAMP levels. Neurotensin, a Ca(2+)-mobilizing agonist, stimulated 125I efflux from both cell lines. In the polarized cells, the magnitude of this response was attenuated at 8 days post-seeding. At this time, the undifferentiated line attained some cAMP responsiveness. This latter effect was paralleled by the appearance of monolayers within areas of the multicell layer. Cell-attached patch-clamp recording from apical membrane patches of polarized cells revealed the presence of a forskolin-stimulated 8-pS Cl- channel; no channel activity was observed in forskolin-stimulated unpolarized cells. Ca(2+)-activated Cl- channels were found in both cell lines. In agreement with the 125I efflux data, the single-channel activation response to [Ca2+]i was smaller in the polarized cell line. From these studies, we can conclude that CFTR expression, measured both at the mRNA and protein level, does not correlate with the colonocyte's ability to secrete chloride ions in response to a cAMP-generating agonist. Cyclic AMP-dependent Cl- secretion requires cellular polarization; specifically, the delineation of an apical membrane. Differences in the cellular location of CFTR during differentiation are likely to explain our results. In contrast, Ca(2+)-stimulated Cl- secretion occurred independently of cellular polarization but was reduced when the cells formed tight junctions.

Topics: Adenocarcinoma; Calcium; Cell Differentiation; Cell Line; Chloride Channels; Chlorides; Colforsin; Colonic Neoplasms; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Epithelium; Humans; Ion Channels; Ionomycin; Kinetics; Membrane Potentials; Membrane Proteins; Microscopy, Electron; Neurotensin; Poly A; RNA, Messenger

Muscarinic agonists stimulate Cl- secretion across monolayers of the colon tumor epithelial cell line, T84. The muscarinic receptor has been characterized in T84 cell homogenates by radioligand binding using [3H]N-methylscopolamine ([3H]NMS). [3H]NMS bound to a single population of sites at 25 degrees C in 100 mM NaCl, 20 mM HEPES, 10 mM MgCl2, pH 7.4 buffer, with calculated Kd = 278 (+/- 44) pM and Bmax = 40 (+/- 6) fmol/mg protein (n = 4). Binding was reversible (diss. t1/2 = 18 +/- 3 min) and stereoselective (dexetimide Ki = 0.3 nM) much greater than levetimide (Ki = 8300 nM). Antagonists exhibited the following rank order of potencies and Ki values (nM): atropine (0.54) greater than 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) (0.84) greater than dicyclomine (14) = hexahydrosiladifenidol (18) greater than pirenzepine (136) greater than AF-DX 116 (3610). The same sequence was observed for inhibition of carbachol-induced 125I efflux from T84 monolayers. This is indicative of an M3 'glandular' muscarinic receptor. Coupling to second messenger systems was examined by labelling monolayers with [14C]arachidonic acid (AA) or [3H]inositol. Carbachol (0.3 mM) did not release [14C]AA from labelled lipids, but ionomycin produced a dose-dependent increase in media [14C]AA. Carbachol (0.3 mM) elevated inositol monophosphate 14-fold. The results suggest that muscarinic agonists stimulate Cl- secretion by interacting with an M3 receptor coupled to inositide lipid hydrolysis.

Topics: Arachidonic Acid; Atropine; Carbachol; Chloride Channels; Chlorides; Colonic Neoplasms; Humans; Ion Channels; Ionomycin; Membrane Proteins; Parasympatholytics; Phosphatidylinositols; Receptors, Muscarinic; Signal Transduction; Tumor Cells, Cultured

The present study demonstrates the activation of Cl- channels in HT29 cells by agonist (ATP, neurotensin, carbachol) increasing cytosolic Ca2+, by hypotonic cell swelling and by cGMP. Cell-attached nystatin patch-clamp (CAN) as well as slow and fast whole-cell recordings were used. The cell membrane potential was depolarized in a dose-dependent manner with half-maximal effects at 0.4 mumol/l for ATP, 60 pmol/l for neurotensin and 0.8 mumol/l for carbachol. The depolarization, which was caused by Cl- conductances increases, occurred within 1 s and was accompanied by a simultaneous and reversible increase of the input conductance of the cell-attached membrane from 295 +/- 32 pS to 1180 +/- 271 pS (ATP; 10 mumol/l, n = 21) and 192 +/- 37 pS to 443 +/- 128 pS (neurotensin; 1 nmol/l, n = 8). The effects of the agonists could be mimicked by ionomycin (0.2 mumol/l), suggesting that an increase in intracellular Ca2+ was responsible for the activation of Cl- channels. The depolarization was followed by a secondary hyperpolarization. Hypotonic cell swelling also depolarized the cells and induced an increase in the membrane conductance. With 120 mmol/l NaCl the depolarization was 10 +/- 0.8 mV and the cell-attached conductance increased from 228 +/- 29 pS to 410 +/- 65 (n = 26) pS. NaCl at 90 mmol/l and 72.5 mmol/l had even stronger effects. Comparable conductance increases were also obtained when the different agonists or hypotonic cell swelling were examined in whole cell experiments. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mumol/l) did not prevent the effects of Ca(2+)-increasing hormones and of hypotonic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Calcium; Carbachol; Carcinoma; Chlorides; Colonic Neoplasms; Culture Media; Cyclic GMP; Cytosol; Hypotonic Solutions; Ion Channels; Ionomycin; Membrane Potentials; Neurotensin; Nitrobenzoates; Osmolar Concentration; Tumor Cells, Cultured

The regulation of chloride conductance was investigated in the T84 human colon carcinoma cell line by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium. The permeable cAMP analog 8-Br-cAMP (100 microM) and the calcium ionophore ionomycin (1 microM) activate a chloride conductance. A prolonged (4 h) preincubation of cells with phorbol 12-myristate 13-acetate (100 nM) or with the diacylglycerol analog 1-oleoyl-2-acetyl-glycerol (100 microM): (i) down-modulates to almost zero the protein kinase C activity in the membranes; (ii) inhibits the activation of the chloride conductance mediated by 8-Br-cAMP but not by calcium; (iii) reduces the mRNA without changing the expression of the protein product of the cystic fibrosis gene. The data suggest that PKC is essential for the activation of the cAMP-dependent chloride conductance in T84 cells.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Biological Transport; Chloride Channels; Chlorides; Colonic Neoplasms; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diglycerides; Down-Regulation; Electric Conductivity; Fluorescence; Fluorescent Dyes; Humans; Ionomycin; Membrane Proteins; Protein Kinase C; Protein Kinases; Quinolinium Compounds; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Neurotensin receptors have been shown previously to be coupled to polyphosphoinositide turnover and intracellular Ca2+ ([Ca2+]i) mobilization in HT-29 colonic epithelial cells (Bozou et al. Biochem. J. 264: 871, 1989; Turner et al. J. Pharmacol. Exp. Ther. 253: 1049, 1990). In this study, neurotensin was found to enhance dramatically the Ba2(+)- and tetraethylammonium chloride-sensitive K(+)-efflux rate (measured with 86Rb+) in the presence of ouabain and bumetanide, with basal efflux increasing 4.5 +/- 0.5-fold with 10 nM neurotensin. The K(+)-efflux rate, which was partially dependent on the extracellular Ca2+ concentration, was also increased by carbachol and ATP, two other [Ca2+]i-mobilizing agonists in HT-29 cells, as well as by the Ca2+ ionophores ionomycin and A23187, suggesting that the efflux was through Ca2(+)-activated K+ channels. Pretreatment of cells with neurotensin, carbachol, or ATP desensitized subsequent neurotensin-stimulated efflux by 82, 57, and 63%, respectively, confirming our previous results which indicated homologous and heterologous desensitization of the neurotensin receptor-signal transduction pathway. Pretreatment of cells with the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and mezerein did not affect [Ca2+]i mobilization or K+ efflux directly but desensitized neurotensin-stimulated efflux by greater than 80%. Pretreatment (2 h) with PMA also decreased K+ efflux in response to ionomycin by 59%, although ionomycin-induced [Ca2+]i mobilization was not inhibited. Downregulation of protein kinase C by overnight pretreatment with PMA resulted in recovery of ionomycin-stimulated efflux. These results suggest that agonist-stimulated Ca2(+)-activated K+ channels in HT-29 cells are regulated at multiple steps in the signal transduction pathway.

Topics: Adenocarcinoma; Adenosine Triphosphate; Barium; Calcium; Carbachol; Cell Line; Colonic Neoplasms; Humans; Ionomycin; Kinetics; Neurotensin; Potassium; Potassium Channels; Rubidium; Tetradecanoylphorbol Acetate; Tetraethylammonium; Tetraethylammonium Compounds

We developed a convenient flux assay that permits simultaneous measurement of Cl and K conductance pathways in Cl-secreting epithelial cells. Monolayers of the colonic tumor cell line T84 were preloaded with 125I and 86Rb, and isotope effluxes were monitored by a sample-replace procedure. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agonists forskolin and prostaglandin E2 increased I efflux with little effect on Rb efflux, whereas the Ca-mediated agonists ionomycin, A23187, and carbachol increased both I and Rb effluxes. Simultaneous determinations of I and Cl or Rb and K effluxes indicated that I and Rb provide good measures of the effluxes of Cl and K, respectively. Forskolin- and ionomycin-stimulated I effluxes were inhibited by the Cl-channel blockers diphenylamine-2-dicarboxylate (DPC), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), and 2-[cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H- inden-5-yl)oxy]acetic acid (IAA-94) and by high external K. The Rb efflux evoked by ionomycin was inhibited by the K-channel blockers Ba and charybdotoxin. These findings suggest that I and Rb effluxes provide qualitative estimates of agonist-stimulated Cl and K conductance pathways. Thus this method can provide a simple and relatively inexpensive screening assay for Cl and K conductances in cultured cells to assess the effects of agonist, blockers, or genetic manipulations.

Topics: Barium; Bumetanide; Carbachol; Cell Line; Charybdotoxin; Chloride Channels; Chlorides; Colforsin; Colonic Neoplasms; Dinoprostone; Epithelium; Humans; Iodides; Iodine Radioisotopes; Ion Channels; Ionomycin; Kinetics; Mannitol; Membrane Proteins; ortho-Aminobenzoates; Potassium Channels; Radioisotope Dilution Technique; Rubidium; Rubidium Radioisotopes; Scorpion Venoms; Sodium; Tumor Cells, Cultured

To explore the role of calcium in mediating the action of carbachol in chloride-secreting epithelia, we simultaneously measured intracellular free [Ca] ([Ca]i) and the potassium conductance (gK) of the basolateral membrane in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value ([Ca]'i) normalized to control. To assess changes in basolateral gK, we measured the short circuit current (Isc) in the presence of luminal amphotericin and a transepithelial mucosa-to-serosa K+ gradient (Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson. 1986. J. Gen. Physiol. 88:237-251). Treatment of the monolayers with carbachol resulted in a parallel increase and then decrease in [Ca]'i and gK. The carbachol-induced changes in gK appeared to be dependent on the increase in [Ca]i because stimulation of gK was significantly diminished when the hormone-induced increase in [Ca]'i was blunted, either by loading the cells with BAPTA or by reducing the extracellular [Ca]. The carbachol-stimulated increase in gK appeared to be the direct result of the increase in steady-state [Ca]'i. The changes in gK and [Ca]'i after stimulation with carbachol were correlated and ionomycin also increased gK and [Ca]'i in a parallel manner. The carbachol-induced delta gK per delta[Ca]'i, however, was greater than that after ionomycin. Because ionomycin and carbachol appear to open the same channel, a conclusion based on inhibitor and selectivity experiments, carbachol may have a second action that amplifies the effect of calcium on gK.

Topics: Amphotericin B; Calcium; Carbachol; Cell Membrane; Colonic Neoplasms; Egtazic Acid; Electric Conductivity; Humans; Ionomycin; Microscopy, Fluorescence; Potassium; Tumor Cells, Cultured

Activation of M3 muscarinic receptors in HT-29 cells by carbachol rapidly increases polyphosphoinositide breakdown. Pretreatment of these cells with carbachol (0.1 mM) for 5 h completely inhibits the subsequent ability of carbachol to increase [3H]inositol monophosphate ([3H]InsP) accumulation, paralleled by a total loss of muscarinic binding sites. In contrast, protein kinase C (PK-C)-mediated desensitization by incubation with phorbol esters [PMA (phorbol 12-myristate 13-acetate)], leading to a time- and dose-dependent inhibition of cholinergically stimulated InsP release (95% inhibition after 4 h with 0.1 microM-PMA), is accompanied by only a 40% decrease in muscarinic receptor binding, which suggests an additional mechanism of negative-feedback control. Neither carbachol nor PMA pretreatment had any effect on receptor affinity. Incubation with carbachol for 15 min caused a small increase of membrane-associated PK-C activity (15% increase, P less than 0.05) as compared with the potency of phorbol esters (PMA) (3-4-fold increase, P less than 0.01). Long-term incubation (4-24 h) with PMA resulted in a complete down-regulation of cytosolic and particulate PK-C activity. Stimulation of InsP release by NaF (20 mM) was not affected after a pretreatment with phorbol esters or carbachol, demonstrating an intact function of G-protein and phospholipase-C (PL-C) at the effector side. Determination of PL-C activity in a liposomal system with [3H]PtdInsP2 as substrate, showed no change in PL-C activity after carbachol (13 h) and short-term PMA (2.5 h) pretreatment, whereas long-term preincubation with phorbol esters (13 h) caused a small but significant decrease in PL-C activity (19%, P less than 0.05). Our results indicate that agonist-induced desensitization of phosphoinositide turnover occurs predominantly at the receptor level, with a rapid loss of muscarinic receptors. Exogenous activation of PK-C by phorbol esters seems to dissociate the interaction between receptor and G-protein/PL-C, without major effects on total cellular PL-C activity.

Topics: Carbachol; Colonic Neoplasms; Enzyme Activation; GTP-Binding Proteins; Humans; Hydrolysis; Inositol; Ionomycin; N-Methylscopolamine; Phosphatidylinositols; Phosphorylation; Protein Kinase C; Receptors, Muscarinic; Scopolamine Derivatives; Sodium Fluoride; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Type C Phospholipases

Free cytosolic Ca2+ [( Ca2+]i) has been implicated as a second messenger mediating the ion transport effects of carbachol, histamine, taurodeoxycholate, ionomycin, and 4-bromo-A23187 (4-BrA23187) in T84-cells. In this study, we correlated short-circuit current (Isc, reflective of Cl- secretion) and [Ca2+]i responses in T84-cell monolayers stimulated by these agents to evaluate the role of [Ca2+]i in Cl- secretory responses. Time-course studies showed that the duration of [Ca2+]i and Isc responses did not correlate with one another. Isc responses were more prolonged than [Ca2+]i responses with carbachol and histamine (both derived [Ca2+]i partly from intracellular sources), less prolonged than [Ca2+]i with taurodeoxycholate, and continued to increase after [Ca2+]i stabilized with ionomycin and 4-BrA23187. Isc and [Ca2+]i responses to histamine and carbachol were additive. A comparison of the magnitude of [Ca2+]i and Isc responses in cells stimulated by different agonists showed that the change in [Ca2+]i accompanying equivalent Isc responses varied greatly, suggesting that secretagogues vary in their dependency on [Ca2+]i. These findings suggest the existence of multiple [Ca2+]i-mediated effector mechanisms or the existence of multiple mediators that augment or attenuate the action of [Ca2+]i.

Topics: Calcium; Carbachol; Cell Line; Chloride Channels; Chlorides; Colonic Neoplasms; Cytosol; Egtazic Acid; Ethers; Histamine; Humans; Ion Channels; Ionomycin; Kinetics; Membrane Proteins; Second Messenger Systems

To examine the role of calcium in mediating carbachol's action in secretory epithelia, we simultaneously measured intracellular free [Ca] [( Ca]i) and transepithelial chloride transport in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value [( Ca]'i) normalized to control. Chloride transport was measured as the short-circuit current (Isc) with a voltage clamp. Monolayers were pretreated with cyclic AMP to augment the response of Isc to carbachol, a procedure that did not qualitatively change the response of the monolayer to carbachol. The carbachol-induced changes in Isc appeared to be dependent on the increase in [Ca]i. First, carbachol caused both Isc and [Ca]'i to increase in parallel. Isc increased from 32 +/- 5 to 70 +/- 9 microA and then declined to 57 +/- 16 microA while [Ca]'i increased from 72 +/- 14 to 156 +/- 22 nM and then declined to 133 +/- 45 nM. Second, the carbachol-induced increases in Isc and [Ca]'i were correlated. The greater the hormone-stimulated rise in [Ca]'i, the higher the increase in Isc. Third, carbachol's stimulation of Isc was blunted by preventing the calcium spike with the cellular calcium buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Although the carbachol-induced increase in [Ca]'i appeared necessary for the increase in Isc, it was not clear if carbachol's action was solely the result of an increase in [Ca]'i. Increasing [Ca]'i with ionomycin, although causing Isc and [Ca]'i to increase in parallel, failed to increase Isc to the levels observed with carbachol. These experiments suggest that although the carbachol-induced increase in Isc is dependent on the increase in [Ca]i, the hormone may activate a second process that increases the sensitivity of the calcium-activated transport process to changes in [Ca]i.

Topics: Calcium; Carbachol; Chelating Agents; Chlorides; Colonic Neoplasms; Cyclic AMP; Egtazic Acid; Electrophysiology; Homeostasis; Humans; Ionomycin; Osmolar Concentration; Tumor Cells, Cultured

It has previously been shown that neurotensin binds to high-affinity receptors in the adenocarcinoma HT29 cell line, and that receptor occupancy leads to inositol phosphate formation. The present study was designed to investigate further the effects of neurotensin on calcium mobilization and protein kinase C (PKC) activation in HT29 cells, and to assess the role of GTP-binding proteins (G-proteins) in the neurotensin response. Direct measurements of cytosolic Ca2+ variations using the fluorescent indicator quin 2 showed that neurotensin (0.1-1 microM) elicited Ca2+ transients in HT29 cells. These transients occurred after the neurotensin-stimulated formation of Ins(1,4,5)P3, as measured by means of a specific radioreceptor assay. In addition, the peptide induced a decrease in the 45Ca2+ content of cells previously equilibrated with this isotope. The peptide effect was rapid, long-lasting and concentration-dependent, with an EC50 of 2 nM. Phorbol 12-myristate 13-acetate (PMA) inhibited by 50% the neurotensin effects on both intracellular Ca2+ and inositol phosphate levels. The inhibition by PMA was abolished in PKC-depleted cells. Pertussis toxin had no effect on either the Ca2+ or inositol phosphate responses to neurotensin. Epidermal growth factor (EGF) receptors which are present in HT29 cells have been shown to be down-regulated through phosphorylation by PKC in a variety of systems. Here, PMA markedly (70-80%) inhibited EGF binding to HT29 cells. Scatchard analysis revealed that PMA abolished the high-affinity component of EGF binding, an effect that was totally reversed in PKC-depleted cells. In contrast, neurotensin slightly (10-20%) inhibited EGF binding to HT29 cells, and its effect was only partly reversed by PKC depletion. Neurotensin had no detectable effect on sn-1,2-diacylglycerol levels in HT29 cells, as measured by a specific and sensitive enzymic assay. In membranes prepared from HT29 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors with a dissociation constant of 0.27 nM. Sodium and GTP inhibited neurotensin binding in a concentration-dependent manner. Maximal inhibition reached 80% with Na+ and 35% with GTP.IC50 values were 20 mM and 0.2 microM for Na+ and GTP respectively. Li+ and K+ were less effective than Na+ and the effects of GTP were shared by GDP and guanosine-5'-[beta gamma- imido]triphosphate but not by ATP. Scatchard analysis of binding data indicated that Na+ and GTP converted the high-affinity

Topics: Adenocarcinoma; Aminoquinolines; Calcium; Cell Line; Cell Membrane; Colonic Neoplasms; Diglycerides; Enzyme Activation; Fluorescent Dyes; Humans; Inositol 1,4,5-Trisphosphate; Ionomycin; Kinetics; Neurotensin; Protein Kinase C; Receptors, Neurotensin; Receptors, Neurotransmitter; Ribonucleotides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

To investigate the mechanism of control of intestinal apolipoprotein B (apoB) secretion, we studied the effects of fatty acids and calcium ionophores on the human intestinal model cell line Caco-2. Although treatment with various fatty acids (18:1w9, 18:2w6, and 20:5w3) complexed to bovine serum albumin resulted in a dramatic redistribution of apoB-100 from the low density and high density lipoproteins to the very low density lipoprotein fraction, there was no effect of any of the fatty acids on the overall rate of total apoB (apoB-100 and apoB-48) secretion. Treatment of differentiated monolayers with calcium ionophores A23187 or ionomycin caused dose-specific increases (125% at 1 microM) in the accumulation of total apoB, but not apoA-I, in conditioned medium as measured by specific immunoassays. Incubation studies with 35S-labeled Caco-2 apoB,E-containing low density lipoprotein particles revealed that treatment with ionomycin over a broad concentration range had no effect on the reuptake of secreted apoB-100. The effect on A23187 on total apoB secretion was blocked by prior chelation of medium calcium and was significantly enhanced by the addition of calcium (up to 50 mM) to the medium. The effect of A23187 was significantly blunted by treatment with the calmodulin antagonist trifluoperazine (10 microM). The time course of A23187 action on Caco-2 apoB secretion required at least 6 h to occur. In contrast to the concentration of apoB in the medium, cellular apoB content was not influenced by treatment with ionophore. Pulse-chase experiments demonstrated a significant reduction in the synthesis-secretion interval for apoB-100 and apoB-48 after 24 h of exposure to ionomycin. Neither fatty acid treatment nor stimulation with ionophore affected the ratio of apoB-100 to apoB-48 produced by the cells. These findings with calcium ionophores implicate the involvement of calcium ion in the mechanism of intestinal apoB secretion. A role for calcium-dependent processes in apoB production raises the possibility that, rather than fatty acid flux, calcium-evoked or calcium-dependent hormones may be important regulators of apoB secretion.

Topics: Adenocarcinoma; Apolipoprotein A-I; Apolipoproteins A; Apolipoproteins B; Calcimycin; Calcium; Calmodulin; Centrifugation, Density Gradient; Colonic Neoplasms; Ethers; Fatty Acids; Humans; Immunoassay; Ionomycin; Kinetics; Trifluoperazine; Tumor Cells, Cultured