Mechanism of cellular uptake of genotoxic silica nanoparticles.

Mechanisms for cellular uptake of nanoparticles have important implications for nanoparticulate drug delivery and toxicity. We have explored the mechanism of uptake of amorphous silica nanoparticles of 14 nm diameter, which agglomerate in culture medium to hydrodynamic diameters around 500 nm. In HT29, HaCat and A549 cells, cytotoxicity was observed at nanoparticle concentrations ≥ 1 µg/ml, but DNA damage was evident at 0.1 µg/ml and above. Transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy confirmed entry of the silica particles into A549 cells exposed to 10 µg/ml of nanoparticles. The particles were observed in the cytoplasm but not within membrane bound vesicles or in the nucleus. TEM of cells exposed to nanoparticles at 4°C for 30 minutes showed particles enter cells when activity is low, suggesting a passive mode of entry. Plasma lipid membrane models identified physical interactions between the membrane and the silica NPs. Quartz crystal microbalance experiments on tethered bilayer lipid membrane systems show that the nanoparticles strongly bind to lipid membranes, forming an adherent monolayer on the membrane. Leakage assays on large unilamellar vesicles (400 nm diameter) indicate that binding of the silica NPs transiently disrupts the vesicles which rapidly self-seal. We suggest that an adhesive interaction between silica nanoparticles and lipid membranes could cause passive cellular uptake of the particles.

A highly selective fluorescent chemosensor for zinc ion and imaging application in living cells.

A new 2,6-bis(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-4-methylphenol (1) serves as a highly selective and sensitive fluorescent probe for Zn(2+) in a HEPES buffer (50 mM, DMSO:water = 1:9 (v/v), pH = 7.2) at 25 °C. The increase in fluorescence in the presence of Zn(2+) is accounted for by the formation of dinuclear Zn(2+) complex [Zn(2)(C(35)H(25)N(6)O)(OH)(NO(3))(2)(H(2)O)] (2), characterized by X-ray crystallography. The fluorescence quantum yield of the chemosensor 1 is only 0.019, and it increases more than 12-fold (0.237) in the presence of 2 equiv of the zinc ion. Interestingly, the introduction of other metal ions causes the fluorescence intensity to be either unchanged or weakened. By incubation of cultured living cells (A375 and HT-29) with the chemosensor 1, intracellular Zn(2+) concentrations could be monitored through selective fluorescence chemosensing.

Unlocking the ultrastructure of colorectal cancer cells in vitro using selective staining.

AIM: To characterise differences between three widely used colorectal cancer cell lines using ultrastructural selective staining for glycogen to determine variation in metastatic properties. METHODS: Transmission electron microscopy was used in this investigation to help identify intracellular structures and morphological features which are precursors of tumor invasion. In addition to morphological markers, we used selective staining of glycogen as a marker for neoplastic cellular proliferation and determined whether levels of glycogen change between the three different cell lines. RESULTS: Ultrastructural analysis revealed morphological differences between the cell lines, as well as differentiation into two sub-populations within each cell line. Caco-2 cells contained large glycogen deposits as well as showing the most obvious morphological changes between the two sub-populations. SW480 cells also contained large glycogen stores as well as deep cellular protrusions when grown on porous filter membranes. HT-29 cells had trace amounts of glycogen stores with few cellular projections into the filter pores and no tight junction formation. CONCLUSION: Morphology indicative of metastatic properties coincided with larger glycogen deposits, providing strong evidence for the use of selective staining to determine the neoplastic properties of cells.

Characterization of four clones derived from human adenocarcinoma cell line, HT29, and analysis of their response to sodium butyrate.

The differentiation of colorectal cancer cells is associated with the arrest of tumor growth and tumor regression. However, the mechanism of such tumor cell differentiation has not yet been elucidated. Several adenocarcinoma cell lines, including HT29 which differentiates only upon stimulation with a differentiation agent, have been used for the study of colorectal cells. Since we had previously obtained variable results during analyses of these cells, we selected several clones of this cell line. In this study, four clones of the parental HT29 cells, H8, G9, G10 and A3, were characterized. All of them differentiated upon treatment with sodium butyrate as the differentiation agent but they appeared different in their response regarding some of the markers of differentiation. As revealed by ultrastructural analysis, H8 and G10 clones formed numerous intercellular cysts with microvilli whereas these structures were found only occasionally in G9 and A3 clones. An elevated level of the indicator of cell differentiation, CEACAM 1, was found in H8 and G10 clones and the activity of alkaline phosphatase, another important marker of colorectal cell differentiation, was up-regulated and highly increased upon butyrate treatment in the H8 clone. Phosphorylation of p38 MAPK was increased in H8 and A3 butyrate-treated clones. According to the levels of cleaved PARP and activated caspase-3, the apoptotic response to butyrate appeared similar in all four clones, while electronoptic analysis revealed that clones G9 and A3 were more perceptive to butyrate-induced apoptosis. In conclusion, our data showed considerable heterogeneity in morphology and some enzymatic activity of the cloned cells. This fact may contribute to the evidence that many HT29 cells possess multipotent information similar to that of stem cells of the normal intestinal crypt.

Fibroblast growth factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death.

TWEAK, a TNF family member, is produced by IFN-gamma-stimulated monocytes and induces multiple pathways of cell death, including caspase-dependent apoptosis, cathepsin B-dependent necrosis, and endogenous TNF-alpha-mediated cell death, in a cell type-specific manner. However, the TWEAK receptor(s) that mediates these multiple death pathways remains to be identified. Recently, fibroblast growth factor-inducible 14 (Fn14) has been identified to be a TWEAK receptor, which was responsible for TWEAK-induced proliferation of endothelial cells and angiogenesis. Because Fn14 lacks the cytoplasmic death domain, it remains unclear whether Fn14 can also mediate the TWEAK-induced cell death. In this study, we demonstrated that TWEAK could induce apoptotic cell death in Fn14 transfectants. A pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, rather sensitized the Fn14 transfectants to TWEAK-induced cell death by necrosis via reactive oxygen intermediates and cathepsin B-dependent pathway. By using newly generated agonistic anti-Fn14 mAbs, we also observed that Fn14 is constitutively expressed on the cell surface of all TWEAK-sensitive tumor cell lines, and can transmit the multiple death signals. Moreover, an anti-Fn14 mAb that blocks TWEAK-Fn14 interaction could totally abrogate TWEAK binding and TWEAK-induced cell death in all TWEAK-sensitive tumor cell lines. These results revealed that the multiple pathways of TWEAK-induced cell death are solely mediated by Fn14.

Culture and co-culture of DU145 prostate carcinoma, osteoblasts and HT-29 colon carcinoma cells on a fabricated type III collagen matrix.

DU145 prostate carcinoma cells cultured on type III collagen possessed a highly migratory potential which was twice as much as HT-29 colon carcinoma cells. Prior to attachment to collagen, DU145 cells were highly reactive for fibronectin and after attachment clear zones between cells and collagen suggested protease activity. HT-29 cells attached to type III collagen forming dome-like polyps, however, tight and/or gap junctions were not observed. hFob osteoblasts were co-cultured with DU145 to establish a prostate cancer-collagen matrix barrier-bone cell metastasis model. Osteoblasts maintained their differentiated osteoblastic characteristics on one side of the collagen barrier, demonstrating high alkaline phosphatase, osteocalcin and insulin growth factor (IGF) activities. hFob cell growth was prominent adjacent to demineralized bone matrix particles (BMPs) embedded in type III collagen. The collagen matrix was deteriorated on the DU145 side of the collagen barrier. The DU145-collagen III-hFob model will allow an evaluation of the influence of the matrix on prostate cancer-bone cell interaction and regulation by growth factors.

Response of HT29 cells to butyrate depends on time of exposure and glucose deprivation.

The high level of alkaline phosphatase activity in HT29 cells induced after 2 or 5 days of butyrate treatment was decreased during their prolonged exposure (about 30 days) to this agent together with a decrease of sensitivity to apoptosis. However, an enormous additive effect on alkaline phosphatase activity was found after butyrate treatment of glucose-starved cells. In concert with this finding, the substructural analysis revealed a dense brush border, tendency to polarization and morphologically normal mitochondria. It can be concluded that prolonged butyrate treatment of HT29 cells attenuated their response to this agent. On the other hand, glucose deprivation, as another inductor of differentiation, was found to increase the sensitivity of HT29 cells to butyrate.

Adherence of yeast and filamentous forms of Candida albicans to cultured enterocytes.

OBJECTIVE: Systemic candidiasis is a major cause of complicating infections in intensive care units. Morbidity and mortality are high, even in those who receive appropriate antifungal therapy. Because the intestinal tract is considered a major portal of entry for systemic candidiasis, experiments were designed to clarify the ability of yeast and filamentous forms, as well as the INT1 gene product, to influence adherence of Candida albicans to the intestinal epithelium. DESIGN: Controlled. SETTING: University teaching hospital research laboratory. SUBJECTS: Mature Caco-2 and HT-29 cultured enterocytes. INTERVENTIONS: C. albicans INT1 mutant strains, defective in filament production, were used to observe the ultrastructural surface interactions of C. albicans with cultured intestinal epithelial cells, namely Caco-2 and HT-29 cells. These mutant strains also were used to quantify the effect of the INT1 gene product on C. albicans adherence (yeast and filamentous forms) to cultured enterocytes. Ultrastructural surface interactions of C. albicans with cultured enterocytes were observed with high resolution scanning electron microscopy. C. albicans adherence to cultured enterocytes was quantified by using a colorimetric enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: Both yeast and filamentous forms of C. albicans appeared tightly adherent to the apical surface of cultured enterocytes, and INT1 appeared to have little, if any, effect on these ultrastructural surface interactions. The distal ends of C. albicans filaments appeared to mediate adherence to enterocyte apical microvilli, and thigmotropism (contact guidance) appeared to play a role in C. albicans adherence. The absence of functional INT1 was associated with decreased adherence of C. albicans yeast forms to cultured enterocytes. CONCLUSIONS: Although functional INT1 appeared to facilitate adherence of C. albicans yeast forms to cultured enterocytes, the role of INT1 in adherence of filamentous forms was unclear, and both yeast and filamentous forms could adhere to, and perhaps invade, the apical surface of cultured enterocytes.

Transient vs. prolonged histone hyperacetylation: effects on colon cancer cell growth, differentiation, and apoptosis.

The role of histone hyperacetylation in regard to growth, differentiation, and apoptosis in colon cancer cells was assessed in an in vitro model system. HT-29 cells were grown in +/-10% fetal bovine serum with either 5 mM sodium butyrate or 0.3 microM trichostatin A [single dose (T) or 3 doses 8 h apart (TR)] for 24 h. Serum-starved HT-29 cells were further treated with epidermal growth factor or insulin-like growth factor I for an additional 24 h. Apoptosis was quantified with propidium iodide and characterized by electron microscopy. Northern blot analyses were performed with cDNA probes specific for intestinal alkaline phosphatase, Na-K-2Cl cotransporter, the cell cycle inhibitor p21, and the actin control. Flow cytometric analysis revealed a time-dependent growth suppression along with early induction of p21 mRNA in the butyrate, T, and TR groups. Histone hyperacetylation, assessed by acid-urea-triton gel electrophoresis, was transient in the T group but persisted for up to 24 h in the butyrate and TR groups. Induction of apoptosis, growth factor unresponsiveness, and differentiation occurred in the butyrate- and TR-treated cells but not those treated with a single dose of trichostatin A. Thus transient hyperacetylation of histones is sufficient to induce p21 expression and produce cellular growth arrest, but prolonged histone hyperacetylation is required for induction of the programs of differentiation, apoptosis, and growth factor unresponsiveness.

Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport.

PURPOSE: The objective of this study was a systematic characterization and evaluation of cell culture models based on mixtures of Caco-2/HT29-MTX co-cultures for their use in screening for drug absorption and intestinal permeability in comparison to the properties of the respective mono-cultures. METHODS: Co-cultures of Caco-2 cells (absorptive-type) and HT29-MTX cells (goblet-type) were set up. Three different co-cultures (initial seeding ratios Caco-2/HT29-MTX: 90/10, 70/30, and 50/50) were grown on permeable filter supports, and monolayers were used for permeability studies with model compounds for paracellular absorption (atenolol, furosemide, H334/75, mannitol, terbutaline), transcellular absorption (antipyrine, ketoprofen, metoprolol, piroxicam), carrier-mediated absorption (D-glucose, Gly-Pro, and L-phenylalanine) as well as substrates for carrier-mediated secretion via P-glycoprotein (cimetidine and talinolol). Electrophysiological and microscopic controls were performed to characterize the cell cultures. RESULTS: For compounds undergoing passive intestinal absorption permeabilities were generally higher in co-cultures than in Caco-2 monolayers, yielding highest values in pure HT29-MTX monolayers. This difference was most obvious for compounds transported via the paracellular pathway, where HT29-MTX cells may be up to 30 times more permeable than Caco-2 cells, whereas for lipophilic and highly permeable compounds, the difference in permeability values was less obvious. For drugs undergoing intestinal secretion mediated by P-glycoprotein, co-cultivation of Caco-2 cells with HT29-MTX cells led to increased apical to basolateral permeability which was decreased in the opposite direction, consistent with the fact that HT29-MTX cells do not express P-glycoprotein. When a carrier-mediated absorption mechanism is involved, the permeabilities observed were lower than the values reported for human small intestine and co-cultivation of HT29-MTX cells with Caco-2 cells resulted in even lower values as compared to the plain Caco-2 cultures. CONCLUSIONS: Co-cultures of HT29-MTX and Caco-2 cells offer the opportunity of modifying the permeability barrier of the cell monolayers both with respect to paracellular resistance and secretory transport via P-gp. Thus, in special cases, they allow more flexibility in adapting the in vitro system to the in vivo situation as compared to the monocultures. Another advantage is the obvious robustness of the method with respect to the reproducibility of the results. A problem remaining, however, is the quantitative expression of carriers involved in intestinal uptake of many nutrients and drugs.

Induced differentiation in HT29, a human colon adenocarcinoma cell line.

The human colon adenocarcinoma cell line HT29 displays an undifferentiated phenotype under standard growth conditions. When these cells were cultured for 21 days and then treated with forskolin, most of the cells formed brush borders on their apical surfaces. Brush border formation was inhibited by cytochalasin D but not by colchicine. Colchicine, nocodazole and taxol were found to induce differentiation and apoptosis in HT29 cells. Differentiation was characterized by flattening of the cells, formation of brush borders on apical surfaces and tight junctions between adjacent cells. Apoptosis was characterized by detachment of round cells from the cell layer, condensation of nuclear DNA and annexin V binding to cell surfaces. Treatment with colchicine or forskolin induced the association of E-cadherin to the cytoskeleton fraction of subconfluent HT29 cells. This effect was less prominent in post confluent cells. Our data indicate that microtubule-interfering agents may serve as an important tool in the study of differentiation and apoptosis in intestinal carcinoma.

Follicle-like structures formed by intestinal cell lines derived from the HT29-D4 adenocarcinoma cell line: morphological and functional characterization.

When cultured in high glucose containing medium, the human colon carcinoma cell line HT29-D4 and a clone derived by transfection with the MDR1 cDNA (MDR31) form multilayers of unorganized cells which are not polarized and are linked by desmosomes. Within these multilayers appear spontaneously large multicellular follicle-like-structures (FLS) where polarized cells linked by tight junctional complexes surround a lumen. Electron microscopy showed that some FLS display well developed brush borders with densely packed microvilli. Others have irregularly oriented microvilli of various lengths or are even completely devoid of apical differentiation. The lumen contains a variable amount of amorphous osmiophilic material. The apical surface of FLS forming cells express dipeptidylpeptidase IV, carcinoembryonic antigen, the mucin MUC1 and for the transfected cells the gp-170 protein. The organic anion fluorescein is transported from the cell to the lumen of FLS. Rhodamine 123, a substrate of the gp-170 ABC transporter is also concentrated in the lumen formed by MDR31 cells. Verapamil and cyclosporine A inhibited this last transport. Cyclic AMP stimulates the formation of these structures since treatment of post-confluent multilayers dramatically increased the number of FLS in HT29-D4 and MDR31 cell cultures within 24 h. The spontaneous formation of these morphologically and functionally polarized structures appeared at random and might respond to the coincidence of fluctuating parameters of the regulatory pathways (cAMP, Ca2+).

A specifically radiolabeled somatostatin analog with strong antitumor activity induces apoptosis and accumulates in the cytosol and the nucleus of HT29 human colon carcinoma cells.

The new heptapeptide somatostatin analog TT-232 decreases proliferation of HT-29 human colon carcinoma cells in vitro by reducing mitotic and increasing apoptotic activity. We have synthesized and characterized a specifically tritium labeled 3H-Tyr3-TT-232 (30 Ci/mmol) to investigate the effect and the fate of this antitumor peptide on human colon tumor cells. 3H-labeled TT-232 could be detected on the cell surface, on cytoplasmic membranes and also in the nucleus of HT-29 cells, 1-6 h after the administration of 0.5 and 50 microg/mL [3H]TT-232. Binding and internalization of TT-232 to human colon tumor cells at a relatively high dose provide further evidence for the existence of low-affinity somatostatin receptors in such cells, which might mediate the apoptosis-inducing effect. Our data suggest the possible use of TT-232 in the treatment of human colon tumors.

The isoflavone genistein inhibits internalization of enteric bacteria by cultured Caco-2 and HT-29 enterocytes.

The dietary isoflavone genistein is the focus of much research involving its role as a potential therapeutic agent in a variety of diseases, including cancer and heart disease. However, there is recent evidence that dietary genistein may also have an inhibitory effect on extraintestinal invasion of enteric bacteria. To study the effects of genistein on bacterial adherence and internalization by confluent enterocytes, Caco-2 and HT-29 enterocytes (cultivated for 15-18 d and 21-24 d, respectively) were pretreated for 1 h with 0, 30, 100, or 300 micromol/L genistein, followed by 1-h incubation with pure cultures of Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, or Escherichia coli. Pretreatment of Caco-2 and HT-29 enterocytes with genistein inhibited bacterial internalization in a dose-dependent manner (r = 0.60-0.79). Compared to untreated enterocytes, 1-h pretreatment with 300 micromol/L genistein was generally associated with decreased bacterial internalization (P < 0. 05) without a corresponding decrease in bacterial adherence. Using Caco-2 cell cultures, decreased bacterial internalization was associated with increased integrity of enterocyte tight junctions [measured by increased transepithelial electrical resistance (TEER)], with alterations in the distribution of enterocyte perijunctional actin filaments (visualized by fluorescein-labeled phalloidin), and with abrogation of the decreased TEER associated with S. typhimurium and E. coli incubation with the enterocytes (P < 0.01). Thus, genistein was associated with inhibition of enterocyte internalization of enteric bacteria by a mechanism that might be related to the integrity of the enterocyte tight junctions, suggesting that genistein might function as a barrier-sustaining agent, inhibiting extraintestinal invasion of enteric bacteria.

Resistance to apoptosis and cyclooxygenase-2 expression in a human adenocarcinoma cell line HT29 CL.19A.

BACKGROUND: COX-2 expression increases the tumorigenic potential of enterocytes. Tumorigenic effect is partially linked to an inhibition of programmed cell death which is one of the most important components in maintaining intestinal epithelium integrity. MATERIALS AND METHODS: We analyzed apoptosis in HT29 cl.19A cells cultured over 3 weeks, in the presence (10%) or in the absence of fetal bovine serum (FBS), by analysis of genomic DNA fragmentation after agarose gel electrophoresis, morphological measurement of apoptosis using DAPI chromatin staining, and transmission electron microscopy (TEM) to identify apoptotic cellular morphological changes. RESULTS: Regardless of the methods used, no apoptotic signs were observed for each culture condition, even if cells were cultured 3 weeks in the absence of FBS. CONCLUSION: Using HT29 cl.19A cells (untransfected cells), we found that intrinsic or constitutive COX-2 expression in adenocarcinoma cell line was associated with spontaneous resistance to apoptosis.

Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated.

Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degreesC, or in streptolysin O-permeabilized cells by manipulating the intracellular environment. In both cases, Cer transfer was not inhibited, as demonstrated by the biosynthesis of ceramide monohexosides and sphingomyelin (SM) de novo from metabolically (with [14C]serine) labelled Cer. This assay is based on the knowledge that Cer is synthesized, starting from serine and palmitoyl-CoA, at the ER, whereas glycosphingolipids and SM are synthesized in the (early) Golgi apparatus. Formation of [14C]glycosphingolipids and [14C]SM was observed under conditions that block vesicle-mediated vesicular stomatitis virus glycoprotein transport. These results indicate that [14C]Cer is transferred from ER to Golgi by a non-vesicular mechanism.

Change of calretinin expression in the human colon adenocarcinoma cell line HT29 after differentiation.

Calretinin is a Ca(2+)-binding protein of the EF-hand family which is expressed in colon adenocarcinomas and colon-derived tumor cell lines (e.g. WiDr), but is absent from normal human enterocytes. Its function has not as yet been elucidated, but some lines of evidence lead us to postulate its involvement in cell proliferation in these cells. In order to test whether calretinin is correlated with an undifferentiated, proliferating, or with a differentiated, state of cells, its expression was studied in the human colon adenocarcinoma clonal cell line HT29-18, which can be caused to differentiate into enterocyte-like cells by replacing glucose with galactose in the culture medium (glucose starvation differentiation). Treatment of HT29-18 cells with galactose led to a drop in the calretinin mRNA level and in protein expression as evidenced by immunocytochemical staining and Western blot analysis of cytosolic cell extracts. These results suggest that calretinin is present in HT29-18 cancer cells, mostly in those which are in the undifferentiated state. The possibility that calretinin is involved in maintaining the cells in an undifferentiated (cancerous) state is discussed.

HT29-MTX/Caco-2 cocultures as an in vitro model for the intestinal epithelium: in vitro-in vivo correlation with permeability data from rats and humans.

The diverse secretory and absorptive functions of the intestinal epithelium are conducted by a mixed population of absorptive cells and mucus-producing goblet cells as the major cell types. In order to approach the main characteristics in an in vitro model, a coculture system of absorptive Caco-2 cells and mucus-secreting HT29-MTX cells was developed and the permeability of a range of different drugs was tested. Variable goblet cell frequency can be achieved, preserving a significant barrier to drug transport and maintaining the differentiated features of both cell types. Absorption rates for actively transported drugs are rather underestimated in the cell culture model when compared to in vivo data. However, a good correlation with fraction absorbed in humans was attained separating the range of passively transported drugs into two groups of well-absorbable compounds with Peff > or = 10 x 10(-6) cm/s and drugs that are absorbed 40-70% with Peff = 0.1-1 x 10(-5) cm/s. A permeability of Peff < 0.1 x 10(-5) cm/s is suggested for low absorbable drugs.

The functional investigation of a human adenocarcinoma cell line, stably transfected with the neuropeptide Y Y1 receptor.

1. The human adenocarcinoma cell line, HT-29, has been stably transfected with the cDNA sequence for the rat neuropeptide Y (NPY) Y1 receptor, and three Y1 clones (Y1-4, Y1-7 and Y1-16) have been isolated which express high levels of specific [125I]-PYY binding. We have studied the functional responses or lack of responses to peptide YY (PYY) and its analogues in the three transfected clones and HT-29 wild type (wt) cells. 2. Vasoactive intestinal polypeptide (VIP) produced long-lasting increases in short-circuit current (SCC) in both HT-29 wt cells and the Y1 clones. VIP EC50 values were 8.4-11.7 nM in all four cases. The elevation in SCC after a maximal concentration of VIP (30 nM) was significantly greater in Y1-7 cells than in either HT-29 wt epithelia or the other Y1 cell lines. 3. PYY (100 nM) and human pancreatic polypeptide (hPP; 1 microM) were ineffective in HT-29 wt cells under either basal or stimulated conditions. In contrast, basolateral additions of PYY reduced both basal and VIP-stimulated SCC in all three Y1 clones. After VIP, the PYY EC50 values (in nM) were 18.6 in Y1-4, 8.0 in Y1-7 and 52.5 in Y1-16 hPP (1 microM) produced only small and transient responses in each transfected cell type. 4. The Y1 receptor agonist, [Leu31, Pro34] NPY (1 microM) was also effective in the three Y1 cell lines. In the Y1-7 clone the EC50 value for the effect of this peptide was 149 nM, 18.6 fold less potent than PYY. 5. PYY and the Y1-selective non-peptide antagonist, BIBP 3226 displaced [125I]-PYY binding from Y1-7 cell membranes with Ki values of 2.0 and 3.1 nM respectively. In the Y1-7 clone, BIBP 3226 fully inhibited the reductions in VIP-stimulated SCC induced by 30 nM PYY, with an IC50 of 27.2 nM and 30 nM BIBP 3226 caused a parallel rightward shift on the PYY concentration-response curve, with an approximate pKB of 8.0. 6. HT-29 clones stably expressing the Y1 receptor therefore show responses to PYY and its analogues that are characteristic of that subtype, and the Y1-7 clone in particular will be useful in the assessment of novel Y1-specific drugs. This approach will also allow the functional study of NPY Yi receptors with selected mutations.