vasoactive-intestinal-peptide and Colonic-Neoplasms

Glucagon-like peptide-2 (GLP-2) is a peptide hormone with multiple beneficial effects on the intestine, including expansion of the mucosal surface area through stimulation of crypt cell proliferation, as well as enhancement of nutrient digestion and absorption. Recent advances in clinical trials involving GLP-2 necessitate elucidation of the exact signaling pathways by which GLP-2 acts. In particular, the GLP-2 receptor has been localized to several intestinal cell types that do not include the proliferating crypt cells, and the actions of GLP-2 have thus been linked to a complex network of indirect mediators that induce diverse signaling pathways. The intestinotropic actions of GLP-2 on the colon have been shown to be mediated through the actions of keratinocyte growth factor and insulin-like growth factor (IGF)-2, whereas small intestinal growth has been linked to IGF-1, IGF-2, and ErbB ligands, as well as the IGF-1 receptor and ErbB. The cellular source of these mediators remains unclear, but it likely includes the intestinal subepithelial myofibroblasts. Conversely, the anti-inflammatory and blood flow effects of GLP-2 are dependent on vasoactive intestinal polypeptide released from submucosal enteric neurons and nitric oxide, respectively. Finally, recent studies have suggested that GLP-2 not only modulates intestinal stem cell behavior but may also promote carcinogenesis in models of sporadic colon cancer. Further consideration of the molecular cross-talk and downstream signaling pathways mediating the intestinotropic effects of GLP-2 is clearly warranted.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Proliferation; Cell Transformation, Neoplastic; Colonic Neoplasms; Fibroblast Growth Factor 7; Glucagon-Like Peptide 2; Glucagon-Like Peptide-2 Receptor; Humans; Intestinal Mucosa; Intestines; Mice; Nitric Oxide; Rats; Receptors, Glucagon; Signal Transduction; Somatomedins; Stem Cells; Vasoactive Intestinal Peptide

HT 29, a cell line derived from a human colonic adenocarcinoma, is highly responsive to the vasoactive intestinal peptide (VIP) as shown by a more than 100-fold intracellular cAMP increase (Ka = 0.3 nM), the stimulations of protein kinase A (Ka = 0.1 nM) and the low-Km cAMP phosphodiesterase (Ka = 40 nM). Remarkably, adenylate cyclase, cAMP-dependent kinase and cAMP-specific phosphodiesterase are activated in a sequential manner. Binding studies with [125I]-labeled VIP indicate a high affinity site with a Kd value (0.5 nM) close to the activation constant value (Ka) of the three enzymes. The molecular structure of the VIP receptor was studied by immunological and chemical approaches. A monoclonal antibody (mAb 109-10-16) which partially decreased the binding of VIP to its receptor allowed the characterization of Mr = 53,000 and Mr = 48-49,000 polypeptides. More precise identification of protein components of the VIP receptor resulted from covalent cross-linking on intact HT 29 cells by four bifunctional reagents: dithiobis-(succinimidyl propionate) and its non-cleavable analog disuccinimidyl suberate, the photoactivable azido phenyl glyoxal and dimethylpimelimidate. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated a major band of Mr = 67,000 regardless of which cross-linker was used. The same band and an Mr = 49,000 species were found in experiments using a crude membrane fraction of HT 29 cells. Assuming one molecule of VIP (Mr = 3326) linked per polypeptide, these observations suggest that an Mr = 64,000 species belongs to the VIP specific plasma membrane receptor. This protein contains an Mr = 20,000 N-linked sialic acid rich oligosaccharidic moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenocarcinoma; Adenylyl Cyclases; Colonic Neoplasms; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Humans; Immunoassay; Membrane Lipids; Protein Kinases; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Topics: Animals; Colonic Neoplasms; Macrophages; Mice; Mice, SCID; RAW 264.7 Cells; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Peptide, Type II; Signal Transduction; Vasoactive Intestinal Peptide

Topics: Adenocarcinoma; Carcinoma, Neuroendocrine; Cell Differentiation; Colonic Neoplasms; Fatal Outcome; Humans; Leukemia, Hairy Cell; Liver Neoplasms; Male; Middle Aged; Mutation; Proto-Oncogene Proteins B-raf; Vasoactive Intestinal Peptide; Vipoma

Topics: Colonic Neoplasms; Ganglioneuroblastoma; Humans; Infant; Male; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a neurotransmitter that primarily functions as a vasodilator. VIP plays its role through binding to its receptors known as VIP/pituitary adenylate cyclase-activating peptide receptors (VPACs). In this study, we examined the expression of VPAC1 in human colon cancer tissues, analyzed the relationship between VPAC1 expression and cancer malignancy, and explored the possible mechanisms using immunohistochemistry and immunofluorescence double staining. The results showed that (1) poorly differentiated colon cancers have significantly higher VPAC1 expression than well-differentiated colon cancers do (p < 0.01); (2) phospho-epithelial growth factor receptor (EGFR) overexpression/activation in the cytoplasm of cancer cells is related to VPAC1 overexpression; (3) blood vessels surrounding colon cancer have significantly more VPAC1-positive than normal colon mucosa does; (4) tumor-associated macrophages (TAMs) of colon cancer have a higher level of VPAC1 expression than macrophages in normal colon mucosa do. These data suggest that VPAC1 overexpression is associated with poorer differentiation of colon cancer, which is likely caused by subsequent EGFR activation in cancer cells. In addition, VPAC1 overexpression in both blood vessels and macrophages in tumors may also play an important role in the development of aggressive cancer.

Topics: Adult; Cell Differentiation; Colonic Neoplasms; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Receptors, Vasoactive Intestinal Polypeptide, Type I; RNA, Messenger; Vasoactive Intestinal Peptide

As large amount of vasoactive intestinal peptide (VIP) receptors are expressed in various tumors and VIP-related diseases, radiolabeled VIP provides a potential PET imaging agent for VIP receptor. However, structural modification of VIP is required before being radiolabeled and used for VIP receptor imaging due to its poor in vivo stability. As a VIP analogue, [R(8, 15, 21), L(17)]-VIP exhibited improved stability and receptor specificity in preliminary studies. In this study, F-18 labeled [R(8,15,21), L(17)]-VIP was produced with the radiochemical yield being as high as 33.6% ± 3% (decay-for-corrected, n = 5) achieved within 100 min, a specific activity of 255 GBq/ μmol, and a radiochemical purity as high as 99% as characterized by radioactive HPLC, TLC, and SDS-Page radioautography. A biodistribution study in normal mice also demonstrated fast elimination of F-18 labeled [R(8,15,21), L(17)]-VIP in the blood, liver, and gastrointestinal tracts. A further micro-PET imaging study in C26 colon carcinoma bearing mice confirmed the high tumor specificity, with the tumor/muscle radioactivity uptake ratio being as high as 3.03 at 60 min following injection, and no apparent radioactivity concentration in the intestinal tracts. In addition, blocking experiment and Western Blot test further confirmed its potential in PET imaging of VIP receptor-positive tumor.

Topics: Animals; Carcinoma; Colonic Neoplasms; Diagnostic Imaging; Estradiol; Humans; Mice; Mice, Nude; Positron-Emission Tomography; Radiography; Vasoactive Intestinal Peptide

Diffuse ganglioneuromatosis of the digestive tract is a rare condition, especially in children. It is frequently associated with multiple endocrine neoplasia type 2b and less commonly with neurofibromatosis type 1 (NF1). We report the case of an 8-month-old baby presenting with vasoactive intestinal polypeptide (VIP)-secreting diffuse ganglioneuromatosis affecting the small intestine and the colon and responsible for severe hydric diarrhea. Postoperatively the infant's symptoms resolved and the serum VIP level was normal. NF1 was clinically suspected and then confirmed through genetic testing. Two years later, the child developed an optic pathway glioma, another tumor frequently associated with NF1.

Topics: Cecal Neoplasms; Colonic Neoplasms; Ganglioneuroma; Humans; Ileal Neoplasms; Infant; Male; Neoplasms, Multiple Primary; Neurofibromatosis 1; Vasoactive Intestinal Peptide

Neuroinflammation driven by the vanilloid-type ion channel receptor transient receptor potential vanilloid type 1 (TRPV-1) is suspected to play a role in the pathophysiology of inflammatory bowel disease. Because inflammatory bowel disease is known to elevate the risk of colon cancer, we examined postulated roles for TRPV-1-driven neuroinflammation in promoting colitis-associated and spontaneous colon cancer development. Using a well-established model of colitis-associated cancer (CAC), we found that mice genetically deficient in TRPV-1 showed a higher incidence and number of tumors in the distal colon. In like manner, genetic deficiency of TRPV-1 in the APC(Min/+) model of spontaneous colon cancer accentuated the number of colonic adenomas formed. Mechanistic analyses in the CAC model revealed an increased infiltration of inflammatory cells into the tumors along with elevated expression of interleukin (IL)-6 and IL-11 and activation of the STAT3 and NF-κB signaling pathways. Notably, TPRV-1-deficient mice exhibited a defect in expression of the anti-inflammatory neuropeptides, vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating peptide (PACAP) which contributed to the generation of a local proinflammatory environment. Together, our findings argue that by limiting neuroinflammatory processes, TRPV-1 exerts a protective role that restricts the initiation and progression of colon cancer.

Topics: Animals; Colitis; Colonic Neoplasms; Cytokines; Genes, APC; Mice; Mice, Inbred C57BL; Mutation; Neurogenic Inflammation; NF-kappa B; Pituitary Adenylate Cyclase-Activating Polypeptide; RNA, Messenger; STAT3 Transcription Factor; TRPV Cation Channels; Vasoactive Intestinal Peptide

Colon cancer is the third most common cancer and one of the leading causes of cancer-related death in the world. Therefore, identification of biomarkers with potential in recognizing the biological characteristics is a key problem for early diagnosis of colon cancer patients. In this study, we used a random forest approach to discover biomarkers based on a set of oligonucleotide microarray data of colon cancer. Real-time PCR was used to validate the related expression levels of biomarkers selected by our approach. Furthermore, ROC curves were used to analyze the sensitivity and specificity of each biomarker in both training and test sample sets. Finally, we analyzed the clinical significance of each biomarker based on their differential expression. A single classifier consisting of 4 genes (IL8, WDR77, MYL9 and VIP) was selected by random forests with an average sensitivity and specificity of 83.75 and 76.15%. The differential expression levels of each biomarker was validated by real-time PCR in 48 test colon cancer samples compared to the matched normal tissues. Patients with high expression of IL8 and WDR77, and low expression of MYL9 and VIP had a significantly reduced median survival rate compared to colon cancer patients. The results indicate that our approach can be employed for biomarker identification based on microarray data. These 4 genes identified by our approach have the potential to act as clinical biomarkers for the early diagnosis of colon cancer.

Topics: Algorithms; Area Under Curve; Biomarkers, Tumor; Cluster Analysis; Colonic Neoplasms; Data Interpretation, Statistical; Female; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Male; Middle Aged; Myosin Light Chains; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; ROC Curve; Transcription Factors; Vasoactive Intestinal Peptide

Vasoactive intestinal polypeptide (VIP) is known to cause the watery diarrhea, hypokalemia, and achlorhydria syndrome. A 14-year-old girl was admitted with a 4-year history of persistent uncontrollable diarrhea and hypokalemia. Computed tomographic evaluation of the neck, chest, and abdomen were normal. Numerous polyps covering the entire colon and rectum were noted on colonoscopy. The serum VIP level was 143 pg/mL. The patient underwent a total proctocolectomy with an ileal-J-pouch. The pathologic examination revealed ganglioneuromatosis. Postoperatively, the symptoms resolved, and the serum VIP level fell to lower than 5 pg/mL. This is an unusual case of the watery diarrhea, hypokalemia, and achlorhydria syndrome caused by ganglioneuromatosis of the entire colon and rectum.

Topics: Adolescent; Colonic Neoplasms; Colonic Pouches; Female; Ganglioneuroma; Humans; Proctocolectomy, Restorative; Rectal Neoplasms; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) receptors are expressed abundantly on many types of tumors and, hence, radiolabeled VIP analogues are being explored for tumor imaging and therapy. Here, we report synthesis of three VIP analogues and their radiolabeling with (99m)Tc via a novel tricarbonyl synthon. The radiolabeled product could be prepared in high yields (>95%) and stability. In vitro studies showed significant uptake of (99m)Tc(CO)((3))-VP05 in human colon carcinoma cells. Biodistribution studies in animal tumor model showed 0.4-1%ID/g tumor uptake.

Topics: Amino Acid Sequence; Animals; Colonic Neoplasms; Humans; Isotope Labeling; Mice; Organotechnetium Compounds; Radionuclide Imaging; Vasoactive Intestinal Peptide

A 15-mer phosphorothioate antisense oligonucleotide (ASON) complementary to the translation start region of the C-myc oncogene mRNA was labeled with 131I or 125I and the labelled compound was linked to the vasoactive intestinal peptide (VIP) to be bound covalently to a polylysine chain so as to deliver oligonucleotide into tumor cells. The effect of the VIP as carrier on cell uptake of ASON in tissue culture was evaluated in a human colon adenocarcinoma HT29 cell line. The efficacy of VIP-131-ASON on cell growth was evaluated using the MTT assay. Expression of c-myc-encoded protein was measured by flow cytometry. Sense and nosense control Oligonucleotides with VIP carrier were used as control. The results showed that VIP competed effectively with VIP-125I-ASON to bind the HT29 cells. Cell uptake was increased 3-4 fold using the VIP carrier compared to the same dosage of naked DNA. HT29 cells treated with VIP-131I-ASON complexes exhibited 4-fold lower proliferation than those treated with 13I-ASON and 6-fold lower proliferation than those treated with radioiodinated Sense and nosense DNA. Cancer protein expression of HT29 cells treated with VIP-131I-ASON was decreased 2-fold compare with that in 131I-ASON treated cell. The use of a VIP carrier greatly increased 131I-ASON cellular uptake and inhibition of cell proliferation and C-myc cancer protein expressing in HT29 cell by radioiodinated antisense Oligonucleotides.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Drug Carriers; Humans; Iodine Radioisotopes; Isotope Labeling; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-myc; Vasoactive Intestinal Peptide

A 15-mer phosphorothioate antisense oligonuclide (ASON) complementary to the translation start region of the C-myc oncogene mRNA was radioiodinated to enhance its antitumor activity, and vasoactive intestinal peptide bound covalently polylysine (VIP-polylysine) was used as a carrier to deliver the oligonucleotide into VIP receptor-positive tumor cells. The antitumor activity of radioiodinated ASON conjugated to VIP-polylysine(VIP-131I-ASON) was investigated in athymic mice bearing HT29 tumor xenografts in comparison with unconjugated radioiodinated ASON(131I-ASON), unlabelled ASON (VIP-ASON) and scrambled oligonucleotide (VIP-131I-MON) conjugated to VIP-polylysine. Conjugation 125I-ASON to VIP-polylysine resulted in a 5.6-fold decrease in the plasma clearance and a 3.4-fold increase in tumor uptake of the radiopharmaceutical. Athymic mice bearing HT29 tumor xenografts were treated with 4 weekly doses of VIP-131I-ASON and the antitumor effects were assessed by use of the slope of the tumor growth curve. VIP-131I-ASON exhibited strong antitumor effects against HT29 xenografts, decreasing tumor growth rate 9.67-, 7.90-fold more effectively than 131I-ASON and VIP-ASON at equivalent doses of ASON. Conversely, 131I-ASON, VIP-ASON or VIP-131I-MON caused no significant effect compared with the normal saline. These data indicated that use of a VIP-polylysine carrier greatly increased HT29 tumor uptake of ASON and treatment with the VIP-131I-ASON complexes resulted in tumor growth delay in human colon cancer xenograft.

Topics: Animals; Colonic Neoplasms; Genes, myc; Genetic Therapy; Iodine Radioisotopes; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Oligonucleotides, Antisense; Polylysine; Radiopharmaceuticals; Thionucleotides; Transplantation, Heterologous; Vasoactive Intestinal Peptide

To prepare VIP-125I-ASON and investigate the possibility of using it as an agent for diagnostic imaging and therapy of colon carcinoma.. The iodination of a 15-base single-stranded antisense oligonucleotide (ASON) complementary to C-myc oncogene mRNA was carried out in the presence of TICl3. The radiolabeled oligonucleotide was complexed with a VIP-polylysine conjugate under certain condition. 3-5 microCi VIP-125I-ASON was injected into the tail vein of the BALB/c nude mice bearing transplanted HT29 colon carcinoma; the nude mice were killed at specific intervals after injection, and the biodistrbution of VIP-125I-ASON in the organs were calculated.. The biodistributed experiment showed that the 125I-ASON was excreted by kidney mostly and by liver and spleen in part. The results of studies after the injection of VIP-125I-ASON differed from those of unconjugated 125I-ASON. The conjugation of VIP to the ASON resulted in a decrease in the plasma clearance of the radiopharmaceutical, which may be due to the reduction in the renal clearance of the ASON. The highest uptake of tumor tissue (5.89% ID/g at 2 h) was significantly higher than that in nude mice given unconjugated ASON (P < 0.05). Tumor to blood ratios and tumor to muscle ratios were optimal at 4 h.. VIP-125I-ASON has desirable stability and higher uptake in tumor. It may provide a new sensitive mean for diagnostic antisense imaging and radiotherapy of tumor in the future.

Topics: Animals; Colonic Neoplasms; Iodine Radioisotopes; Mice; Mice, Inbred BALB C; Mice, Nude; Oligonucleotides, Antisense; Radionuclide Imaging; Radiopharmaceuticals; Tissue Distribution; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is secreted from many cancer lines and VIP binding was observed in many tumors. We have shown before that VIP antagonists are potent inhibitors of neoplastic growth of neuroblastoma, lung and breast cancer cells in vitro. Here, the cultured colon cancer cell line HCT-15 that exhibited VIP receptor expression was treated with the VIP hybrid antagonist neurotensin(6-11)VIP(7-28). The antineoplastic activity was assessed by thymidine incorporation. Neurotensin(6-11)VIP(7-28) efficiently inhibited cancer growth with a maximal effect at nanomolar concentrations. Once the inhibitory properties of the VIP antagonist on colon cancer cells were established, the in vivo curative effects were analyzed. Sprague-Dawley rats were injected with azoxymethane (AOM) (15 mg/kg/week) for 2 weeks, providing artificial induction of colon tumors. The rats were then allocated into four experimental groups: (1) receiving no treatment; (2) receiving treatment with saline; (3, 4) receiving treatment with 10 or 20 microg of neurotensin(6-11)VIP(7-28), respectively. After 10 weeks of daily injections, rats were sacrificed and tumors assessed for stage, volume, location, differentiation and lymphocytic infiltrate. Embedded mucosa was assessed for dysplastic crypts. Results showed that the antagonist treatment reduced the tumor volume, staging, lymphocyte infiltrate and the number of dysplastic crypts. Thus, neurotensin(6-11)VIP(7-28) could serve as an effective cancer treatment and a preventing agent.

Topics: Animals; Cell Differentiation; Cell Division; Colonic Neoplasms; Dose-Response Relationship, Drug; Humans; Neoplasm Staging; Neurotensin; Peptide Fragments; Rats; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Polypeptide, Type I; RNA, Messenger; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The neuropeptides substance P (SP) and vasoactive intestinal peptide (VIP) mediate physiologic activities in the intestine, not least in relation to motility and inflammatory processes. Neuropeptides are up-regulated and play particular importance during tissue stress. This paper aims to quantify mucosal and smooth muscle SP, VIP and total innervation in human colon in short- and long-term perspectives after abdominal irradiation.. Colon specimens from 23 irradiated or non-irradiated patients were investigated with immunohistochemistry and computerized image analysis. Plasma levels of SP and VIP in 15 additional patients receiving radiotherapy were analyzed.. At 4-7 days after irradiation (5 x 5 Gy), the overall innervation, and also VIP and SP nerve fiber densities, were increased in both mucosa and circular muscle layer. In contrast, 5-6 weeks as well as several years after irradiation, the VIP and SP nerve fiber densities were decreased. No peptide changes were revealed in plasma.. The degree of VIP and SP intestinal innervation was increased after radiotherapy in the short-term perspective but it decreased in the long-term. In the short-term, SP may have pro-inflammatory and VIP anti-inflammatory effects and the peptides may have trophic effects and be related to the occurrence of motor changes. It cannot be excluded that the decrease in VIP and SP neuronal supply seen in the long-term may contribute to intestinal malfunction.

Topics: Abdomen; Aged; Carcinoma; Cell Count; Colon; Colonic Neoplasms; Female; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Intestinal Mucosa; Male; Middle Aged; Muscle, Smooth; Nerve Fibers; Neurons; Pelvis; Radiation Dosage; Radioimmunoassay; Rectal Neoplasms; Substance P; Thiolester Hydrolases; Time; Ubiquitin Thiolesterase; Vasoactive Intestinal Peptide

The enteric nerve plexus in the colon was investigated in rats with chemically induced colonic adenocarcinoma. Tissue specimens from the colons of four group rats, namely controls, treated animals without development of colonic macro- or microscopic changes, rats with dysplasia and lymphoid hyperplasia, and rats with colonic adenocarcinoma were studied using immunocytochemistry, and quantified by computerized image analysis. No morphometeric changes were found in the treated rats regarding the myenteric and submucosal ganglia, with the exception of nitric oxide synthase (NOS), where the number of nerve cell bodies/ganglia was reduced in the myenteric ganglia in rats with both lymphoid hyperplasia and dysplasia, and carcinoma. The relative volume density of protein gene product (PGP) 9.5-immunoreactive (IR) nerve fibres was higher in the muscularis propria in rats with lymphoid hyperplasia and dysplasia, and carcinoma. However the relative volume density of PGP 9.5-IR nerve fibres was higher in the submucosa in rats with carcinoma only. The relative volume density of substance P- and VIP-IR nerve fibres was significantly higher in the muscularis propria in rats with colonic carcinoma. The relative volume density of NOS-IR nerve fibres was significantly decreased in both muscularis propria and submucosa in rats with lymphoid hyperplasia and dysplasia, and carcinoma. These findings imply that regulatory signals of the enteric innervation may be involved in the pathogenesis of colorectal cancer.

Topics: Adenocarcinoma; Animals; Colon; Colonic Neoplasms; Enteric Nervous System; Immunohistochemistry; Male; Nerve Fibers; Nerve Tissue Proteins; Nitric Oxide; Precancerous Conditions; Rats; Rats, Sprague-Dawley; Substance P; Thiolester Hydrolases; Ubiquitin Thiolesterase; Vasoactive Intestinal Peptide

1. Three human adenocarcinoma cell lines, Colony-24 (Col-24), Col-6 and Col-1 have been studied as confluent epithelial layers able to transport ions vectorially in response to basolateral vasoactive intestinal polypeptide (VIP) and pancreatic polypeptides (PP). 2. Different species PP stimulated responses in Col-24 with Y(4)-like pharmacology. Bovine (b)PP, human (h)PP and porcine (p)PP were equipotent (EC(50) values 3.0--5.0 nM) while rat (r)PP, avian (a)PP and [Leu(31), Pro(34)]PYY (Pro(34)PYY) were significantly less potent. PYY was inactive. The PP pharmacology in Col-1 was comparable with Col-24. However, Col-6 cells were different; pPP had an EC(50) intermediate (22.0 nM) between that of bPP (3.0 nM) and hPP (173.2 nM), with aPP and rPP being at least a further fold less potent. 3. Deamidation of Tyr(36) in bPP (by O-methylation or hydroxylation) or removal of the residue resulted in significant loss of activity in Col-24. 4. GR231118 (1 microM) had no PP-like effects. In Col-24 and Col-1, GR231118 significantly attenuated bPP (30 nM) or hPP (100 nM) responses, but it did not alter bPP responses in Col-6. BIBP3226 and GR231118 both inhibited Y(1)-mediated responses which were only present in Col-6. 5. RT--PCR analysis confirmed the presence of hY(4) receptor mRNA in Col-24 and Col-1 epithelia but a barely visible hY(4) product was observed in Col-6 and we suggest that an atypical Y(4) receptor is expressed in this cell line.

Topics: Adenocarcinoma; Arginine; Colonic Neoplasms; Humans; Neuropeptide Y; Pancreatic Polypeptide; Peptides, Cyclic; Receptors, Neuropeptide Y; Receptors, Somatostatin; Reverse Transcriptase Polymerase Chain Reaction; RNA; Somatostatin; Structure-Activity Relationship; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

This study was aimed at the preparation of 131I-vasoactive intestinal peptide (VIP) and its preliminary application in clinical imaging. VIP was labeled with Na 131I using chloramine-T method, then isolated by Sephadex G-10 column chromatography and examined by silica 60F254 thin layer chromatography. The bacteria and pyrogen were examined and the safety test was carried out. One control and two patients suffering from abdomen tumor were investigated. The results showed that the labeling rate of 131I was 80% and the specific activity of 131I-VIP was 36 TBq/mmol. The radiochemical purity of 131I-VIP was over 98%, and it decreased to 95% after six hours' storage at 4 degrees C. It was proved that the 131I-VIP eluate had no bacteria, no pyrogen and no poison. The injected 131I-VIP was distributed into the lungs immediately and was eliminated through kidneys. The primary tumor could be visualized about half an hour to 3 hours after injection. This study demonstrates that 131I-VIP is suitable for in vivo imaging and may be used as an effective tracer to identify the tumor site in patients with VIP receptor positive carcinoma.

Topics: Animals; Colonic Neoplasms; Female; Humans; Iodine Radioisotopes; Male; Mice; Middle Aged; Peritoneal Neoplasms; Radionuclide Imaging; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is known to modulate inflammatory reactions, to have trophic effects, and to contribute to diarrhea and has been implicated as an important factor in several inflammatory conditions in the human gut. The aim of the present study was to investigate the effects of irradiation on the expression of VIP in the colon of patients operated on for adenocarcinoma. Some of the patients had received preoperative irradiation (25 Gy) within one week before the operation. Specimens of sigmoideum, 10 cm cranial to the margin of the cancer, were examined, by using antiserum against VIP and immunohistochemistry. There were numerous nerve fibers showing VIP-like immunoreactivity in the damaged mucosa, including the regions showing ulcerations. There was a higher degree of expression of VIP in the ganglion cells in the submucous plexuses in irradiated than nonirradiated patients. The study shows that there is a marked immunohistochemical expression of VIP concomitant with the occurrence of inflammatory and repair processes in the irradiation-damaged human colonic mucosa.

Topics: Adenocarcinoma; Colon; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Male; Vasoactive Intestinal Peptide

We investigated the effect of VIP on the liver metastases and angiogenesis by Colon 26-L5 carcinoma cells in mice. Daily systemic administration of VIP, beginning 3 days after tumor inoculation into a portal vein of mice, inhibited significantly the development of their liver metastases. Immunohistochemical staining for factor VIII-related antigen in the sections of liver metastases showed that the systemic administration of VIP caused significant prevention of angiogenesis within tumor masses. VIP (10-(10) to 10(-6) M) inhibited the invasion of reconstituted basement membrane (Matrigel) by hepatic sinusoidal endothelial (HSE) cells in a concentration-dependent manner in a Transwell chamber assay in vitro and achieved approximately 50% reduction of control at 10(-6) M. VIP (10(-6) M) also significantly suppressed the haptotactic migration of HSE cells to fibronectin, laminin or type I collagen substrates with a similar inhibition rate to the invasion assay. Exposure of VIP to HSE cells induced accumulation of intracellular cAMP in a concentration-dependent manner. The inhibitory effect of VIP (10(-6) M) on HSE cell migration was significantly abrogated in the presence of 3 x 10(-6) M H-89, a cAMP-dependent protein kinase inhibitor. VIP (10(-6) M) inhibited the morphogenesis of HSE cells into capillary-like structures on Matrigel-coated wells. VIP did not affect the proliferation of HSE cells and the production of gelatinases in HSE cells in vitro at the concentrations used in the invasion assay. These observations suggest that the anti-metastatic effect of VIP on liver metastases by Colon 26-L5 carcinoma cells in mice is partly due to the prevention of tumor angiogenesis probably through suppression of the motility of endothelial cells.

Topics: Animals; Collagen; Colonic Neoplasms; Cyclic AMP; Drug Combinations; Endopeptidases; Extracellular Matrix; Laminin; Liver; Liver Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Proteoglycans; Vasoactive Intestinal Peptide

Human colonic adenocarcinoma cell lines have conserved several features of the native tissue. Among these is the expression of cell surface receptors for hormones and neurotransmitters that may be involved in the regulation of proliferation and differentiation processes in these cancer cells. Here, we confirm that high-affinity binding sites for the Vasoactive Intestinal Polypeptide (VIP) and for the VIP analogue Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP), were expressed in 4 human colonic adenocarcinoma cell lines, HT29, SW403, DLD-1 and Caco-2, that spontaneously displayed variable phenotypic properties in culture. We demonstrated that after long-term treatments, VIP and PACAP significantly reduced cell proliferation in the 4 cell lines and modulated intracellular cAMP and cGMP levels. Furthermore, conspicuous differences were observed from one cell type to another concerning expression of the receptor subsets or the effects of the neuropeptides on cell growth and on cyclic nucleotides production.

Topics: Adenocarcinoma; Caco-2 Cells; Cell Division; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Growth Inhibitors; HT29 Cells; Humans; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Recent studies have shown that various gastrointestinal tumours express substantial amounts of vasoactive intestinal peptide (VIP) receptors. Based on these observations, we have developed a receptor scintigraphy using [123I]VIP as a radioligand. An initial series performed at our institution showed promising potential for visualization of various gastrointestinal adenocarcinomas by means of [123I]VIP. In this article, we now report the results obtained in 80 consecutive patients with colorectal adenocarcinoma. Eighty consecutive patients with histologically verified colorectal cancer underwent scanning by means of [123I]VIP (1 microg, approximately 150 MBq). Thirteen patients were free of tumour after complete resection of Dukes' C cancer, eight patients presented with primary and 14 with locally recurrent tumours but were free of metastases. Ten patients had locally recurrent disease and liver, lung or lymph node metastases. Disease confined to organ metastases (i.e. liver, lung or lymph nodes) was present in 35 patients. The size of the primary or recurrent tumours ranged between 3 and 6 cm, and the size of metastases was between 1 and 13 cm in diameter. Scan results were evaluated independently by two nuclear medicine physicians in a blinded way, and results were then compared with computerized tomography (CT)scans not older than 4 weeks. Seven out of eight primary (87%) and 21 out of 24 (82%) locally relapsing cancers were imaged with [123I]VIP. Negative VIP scans were obtained in all 13 patients in whom the cancers had been curatively resected. All patients with lymph node metastases showed positive VIP scans (four out of four), and positive scans were obtained in 25 out of 28 (89%) patients with liver metastases and in two out of three cases with lung metastases. In four patients with relapsing cancer, the VIP scan indicated the presence of disease before CT, and in two patients the diagnosis of scar tissue instead of a local recurrence of rectal cancer as suggested by CT could be established. We conclude that [123I]VIP receptor scanning is a sensitive method for radioimaging of colorectal cancer with the potential to provide valuable additional information to conventional radiological methods.

Topics: Adult; Aged; Colonic Neoplasms; Female; Humans; Iodine Radioisotopes; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Rectal Neoplasms; Tomography, Emission-Computed, Single-Photon; Vasoactive Intestinal Peptide

We previously reported that vasoactive intestinal polypeptide (VIP) significantly inhibited Matrigel invasion and haptotactic migration of murine colon 26-L5 carcinoma in vitro. To extend our study, we investigated the inhibitory mechanisms of VIP on Matrigel invasion of colon 26-L5 carcinoma, and the effect on metastatic properties of the tumor cells. VIP inhibited the invasion of the tumor cells in a concentration-dependent manner without affecting their growth, and achieved approximately 50% reduction at 10(-6) M. VIP also suppressed cell motility with a similar inhibition rate to the invasion assay. Time course study revealed that the motility was reduced by 40% when the tumor cells were preincubated with 10(-6) M VIP for 3 h. In contrast, 6-h pretreatment with 10(-6) M VIP caused the increased ability of the adhesion to both fibronectin and laminin with a 50% enhancement. A large amount of VIP1 receptor transcripts was expressed in the cells, whereas VIP2 receptor was undetectable, by RT-PCR and subsequent Southern blot hybridization. A specific antagonist for VIP1 receptor reversed the suppressed motility induced by VIP. Cryostat sections showed that the 3-h pretreatment of tumor cells with VIP caused the reduction of the arrest in the livers at 6 h after the tumor inoculation into a portal vein of mice. VIP could prevent the experimental liver metastasis of the tumor cells in a dose-dependent manner. The cells pretreated with 10(-6) M VIP for 3 h also showed the reduced ability of the liver metastasis. These results suggest that VIP could block the invasion and the metastasis of colon 26-L5 carcinoma through suppression of their motility.

Topics: Animals; Blotting, Southern; Cell Adhesion; Cell Movement; Colonic Neoplasms; Dose-Response Relationship, Drug; Fibronectins; Laminin; Liver Neoplasms; Mice; Mice, Inbred BALB C; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The effects of a number of agonists which inhibit intestinal chloride secretion were investigated in Colony-1 (Col-1) cells, a subpopulation derived from the HCA-7 human adenocarcinoma cell line. Neither peptide YY (PYY) or somatostatin 14-28 (SRIF) reduced short-circuit current (SCC) in Col-1 epithelial layers stimulated with vasoactive intestinal polypeptide (VIP), suggesting that their respective receptors are either absent in this cell line, or are not functionally coupled. A second member of the neuropeptide Y family, pancreatic polypeptide (PP), decreased VIP-elevated SCC with an EC50 of 25.6 nM. Maximal PP responses were unaffected by prior addition of PYY, indicating that Col-1 cells may express a PP specific, Y4-like receptor. The alpha 2-adrenoceptor agonist clonidine also attenuated VIP-stimulated SCC (EC50342 nM) through the alpha 2A receptor subtype, since clonidine responses were inhibited by yohimbine and rauwolscine but not altered by previous addition of prazosin. Col-1 cells responded to both apical and basolateral addition of VIP or clonidine; to an extent, this lack of sidedness reflects the ability of drugs to permeate through the Col-1 epithelial layers. Both PP and clonidine also inhibited SCC in unstimulated Col-1 cells or those pretreated with 3-isobutyl-1-methylaxanthine (IBMX) or a submaximal concentration of forskolin, agents which both directly elevate intracellular cAMP. After a maximal concentration of forskolin (10 microM), which increased SCC to a significantly greater extent than either VIP or IBMX, the effects of both agonists were negligible. The absence of PP and clonidine responses under these conditions may have implications for the mechanisms by which these agonists inhibit, chloride secretion in Col-1 epithelia. In addition carbachol reduced SCC stimulated by 10 microM forskolin, in contrast to control carbachol responses which consisted of a rapid decrease followed by a transient elevation in SCC; this observation suggests that Col-1 cells may also be a useful model for studying the interactions between Ca(2+)- and cAMP-dependent mechanisms involved in epithelial ion transport.

Topics: Adenocarcinoma; Adrenergic alpha-Agonists; Chlorides; Clonidine; Colonic Neoplasms; Cyclic AMP; Depression, Chemical; Epithelium; Humans; Pancreatic Polypeptide; Patch-Clamp Techniques; Receptors, Adrenergic, alpha-2; Receptors, Gastrointestinal Hormone; Somatostatin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

We investigated the effect of neuropeptides, which are vasoactive intestinal polypeptide (VIP), substance P, (SP), neuropeptide Y (NPY), neurokinin A (NKA), somatostatin (SOM), calcitonin gene-related peptide (CGRP), and leucine-enkephalin (L-ENK), on the invasion of murine Colon 26-L5 adenocarcinoma cells through a reconstituted basement membrane (Matrigel) using a Transwell cell culture chamber assay. VIP, SP, NPY, and L-ENK reduced invasive potential of tumor cells in a concentration-dependent manner, whereas SOM, CGRP, and NKA had no effect. Especially, VIP showed the most effective in inhibiting tumor invasion, and achieved 50% reduction at 10(-6) M. A similar effect by VIP was also observed in cell migration to fibronectin. VIP had no effect on the growth of tumor cells at the concentrations ranging from 10(-10) to 10(-6) M. The suppressed ability of the tumor cell motility by VIP (10(-6) M) was practically recovered by co-treatment with 2',5'-dideoxyadenosine, an adenylate cyclase inhibitor. These results indicate that VIP, among the neuropeptides used, could inhibit Matrigel invasion of Colon 26-L5 carcinoma cells through partial suppression of their motility, and the reduction was associated with an intracellular cAMP-mediated pathway.

Topics: Adenocarcinoma; Animals; Biocompatible Materials; Calcitonin Gene-Related Peptide; Cell Division; Cell Movement; Colforsin; Collagen; Colonic Neoplasms; Dideoxyadenosine; Dose-Response Relationship, Drug; Drug Combinations; Enkephalins; Laminin; Leucine; Mice; Neoplasm Invasiveness; Neurokinin A; Neuropeptide Y; Neuropeptides; Proteoglycans; Somatostatin; Substance P; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The neuropeptides substance P, vasoactive intestinal polypeptide, and the recently discovered peptide secretoneurin are neurotransmitters of the intrinsic nervous system of the gut and effect gut motility. The aim of this study was to investigate whether these neuropeptides are involved in the pathophysiology of large bowel ileus. Five patients underwent colonic resections for obstructive cancer of the colon. Full-thickness specimens of the resected colon were taken 10 cm proximal and 10 cm distal to the site of tumor obstruction. Substance P-, vasoactive intestinal polypeptide-, and secretoneurin-like immunoreactivities were measured in the specimens by radioimmunoassay. In addition immunocytochemistry was performed. Tissue levels of substance P, vasoactive intestinal polypeptide, and secretoneurin were lower in the prestenotic than in the poststenotic bowel segment. In accordance, immunocytochemistry revealed a denser staining of ganglion cells and fibers for all three neuropeptides in the poststenotic bowel. The decreased tissue levels of substance P, vasoactive intestinal polypeptide, and secretoneurin in the prestenotic bowel segment may contribute to the final decompensation of obstructive ileus.

Topics: Aged; Colon; Colonic Diseases; Colonic Neoplasms; Enteric Nervous System; Female; Humans; Intestinal Obstruction; Neuropeptides; Secretogranin II; Substance P; Vasoactive Intestinal Peptide

We have studied the resection specimens from 5 patients with idiopathic megarectum and megacolon and 10 control subjects with non-obstructing colonic cancer. Histological staining with haematoxylin and eosin, and immunocytochemical staining for protein gene product 9.5 (PGP9.5), S100 protein, vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP), and histochemical localization of NADPH diaphorase was performed. The amount of VIP and CGRP present in samples was measured using an enzyme-linked immunosorbent assay. Patients with idiopathic megarectum and megacolon showed hypertrophy of the muscularis mucosae and muscularis externa. The architecture of the innervation as assessed by immunoreactivity for PGP9.5 and S100 protein appeared normal. There was a decrease in the density of innervation of the longitudinal muscle in rectal tissue from patients with idiopathic megarectum, with fewer VIP- and NADPH-diaphorase-containing nerves. In the muscularis mucosae and lamina propria of the rectal samples of patients with idiopathic megarectum, VIP immunoreactivity was higher and more NADPH-diaphorase-containing nerves were seen. CGRP-immunoreactive nerve fibres were only seen in the myenteric plexus. No CGRP-immunoreactive cell bodies were seen. In summary, there is an increase in VIP and nitric oxide containing fibres in the muscularis mucosae and lamina propria and a decrease in the longitudinal muscle in rectal tissue of patients with idiopathic megarectum. Both are NANC (nonadrenergic noncholinergic) inhibitory transmitters in the gut and the possible relationship of the changes in their density with gut function is discussed.

Topics: Adolescent; Adult; Calcitonin Gene-Related Peptide; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Intestine, Large; Male; Megacolon; NADPH Dehydrogenase; Nerve Tissue Proteins; Rectal Diseases; S100 Proteins; Thiolester Hydrolases; Ubiquitin Thiolesterase; Vasoactive Intestinal Peptide

In order to evaluate the importance of cAMP and cAMP-dependent protein kinase (cAMPdPK) in the regulation of chloride efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, Caco-2, human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of regulatory subunit of cAMPdPK under control of the mouse metallothionein 1 promoter. Four stable transformants were isolated that expressed the mutant subunit in a Zn(2+)-inducible manner and exhibited Zn(2+)-inducible inhibition of cAMPdPK activity. The parental and transformed Caco-2 cells were examined for their abilities to regulate chloride efflux in response to various secretagogues using a radioactive iodide-efflux assay. In the transformants, induction of the protein kinase mutation with ZnSO4 markedly decreased chloride efflux in response to forskolin, the 8-(4-chlorophenylthio) analog of cAMP, vasoactive intestinal polypeptide, prostaglandin E2 and isoproterenol, whereas Zn(2+)-treated parental cells remained responsive to these secretagogues. Treatment with carbachol, calcium ionophores or phorbol ester did not acutely affect chloride efflux. Together, these studies indicate that cAMP and cAMPdPK are essential components of secretagogue-regulated chloride channel activity in the Caco-2 cell line. In whole cell patch clamp recordings, induction of the cAMPdPK mutation inhibited anionic conductances indicative of the CFTR chloride channel, whereas purified catalytic subunit of cAMPdPK, added intracellularly, reversed the inhibition. These latter results demonstrate that the CFTR chloride channels in the protein kinase-defective transformants are normal and that the protein kinase mutation specifically affects their regulation, presumably by direct phosphorylation.

Topics: Carbachol; Chloride Channels; Colforsin; Colonic Neoplasms; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dinoprostone; Enzyme Activation; Humans; Isoproterenol; Membrane Proteins; Mutation; Second Messenger Systems; Transformation, Genetic; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Zinc

The purpose of this study was to investigate the peculiarities of hormonal regulation of adenylate cyclase (AC) of blood lymphocytes in colorectal cancer patients and to compare these peculiarities with hormone sensitivity of AC of colorectal tumors and normal colonic mucosa. Basal and stimulated lymphocyte AC activity was studied in 51 healthy persons and 52 cancer patients (14 with colon cancer, 21 with rectal cancer and 17 with stomach cancer) aged 20-75 years. In 31 of 35 patients with colorectal cancer the AC activity was studied simultaneously in lymphocytes, tumor tissue and normal colonic mucosa. To evaluate basal and stimulated AC activity the measurement of c-AMP (Amersham kits) formed in the presence of ATP regenerating system was used. Basal and by VIP, pentagastrin and sodium fluoride stimulated AC activity in lymphocytes of gastrointestinal cancer patients was lower than in lymphocytes of healthy subjects of similar age. Stage dependence of the parameters under study was not found. There was a tendency for higher basal and stimulated lymphocyte AC activity in colon cancer patients as compared to stomach and rectal cancer patients. In colorectal cancer patients the peculiarities of lymphocyte AC reactions to stimulation were closer to those in tumor tissue but not to those in normal colonic mucosa. The reaction of lymphocyte AC to VIP and glucagon coincided more frequently with tumor AC reactions to the same hormones in case of hormone nonsensitive tumors. Thus, basal and stimulated lymphocyte AC activity in colorectal cancer patients was modified to some degree by tumor factors. Lymphocyte AC reactions to VIP and glucagon may be considered as indirect markers of hormone sensitivity of colonic tumors. Moreover, the probability of discovery of hormone nonsensitive tumors by this way is more reliable than hormone sensitive ones.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Adult; Aged; Calcitonin; Colonic Neoplasms; Epinephrine; Glucagon; Humans; Intestinal Mucosa; Lymphocytes; Middle Aged; Pentagastrin; Rectal Neoplasms; Sodium Fluoride; Stomach Neoplasms; Vasoactive Intestinal Peptide

The effects of combined administration of vasoactive intestinal peptide (VIP) and the ornithine decarboxylase (ODC) inhibitor, 1,3-diaminopropane (DAP), on development of colon tumors induced by azoxymethane (AOM), on ODC activity of the colon wall, and on the labelling index of colon epithelial cells were investigated in inbred Wistar rats. Rats received weekly subcutaneous injections of AOM for 10 weeks and subcutaneous injections of VIP every other day and drinking water containing DAP (2.5 milligrams) ad libitum until the end of the experiment at week 45. Administration of VIP significantly increased the incidence of colon tumors at week 45. It also resulted in significant increases in colon ODC activity and in the labelling index during administration of AOM, but not after its cessation. Administration of both DAP and VIP significantly reduced the enhanced colon carcinogenesis by VIP. The DAP significantly attenuated the VIP enhancement of colon ODC activity and of the labelling index during AOM administration. These findings indicate that ODC inhibition attenuated enhancement of colon carcinogenesis, and suggest that enhancement of colon carcinogenesis by VIP may be mediated through its polyamine biosynthesis.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Adenoma; Animals; Azoxymethane; Colonic Neoplasms; Diamines; Male; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Random Allocation; Rats; Rats, Wistar; Vasoactive Intestinal Peptide

1. Confluent epithelial layers of a human adenocarcinoma cell line called Colony-6 have been shown to respond to nanomolar concentrations of vasoactive intestinal polypeptide (VIP), peptide YY (PYY), neuropeptide Y (NPY) and somatostatin (Som). 2. The VIP-induced increase in basal short-circuit current (SCC) was attenuated by basolateral application of Som, PYY or NPY, and also by the Y1-receptor agonist [Leu31,Pro34]NPY, as well as pancreatic polypeptide (PP). High concentrations (0.1-3.0 microM) of NPY(2-36) were effective but the C-terminal fragment NPY(13-36) (0.1-1.0 microM) and desamidoNPY (0.6 microM) were not active. A rank order of agonist EC50 values was: PYY > NPY > [Leu31,Pro34]NPY > PP > NPY(2-36) >> NPY (13-36). 3. Receptors for all these peptides were preferentially located within the basolateral domain. Apical addition of PP (1 microM) and Som (100 nM) had no effect upon basal SCC while apical VIP (10 nM) responses were 18%, and apical PYY (100 nM) were 27% the size of respective basolateral controls (100%). 4. Cross-desensitization was observed between [Leu31,Pro34]NPY (1 microM) and both PYY (100 nM) and PP (1 microM) and between PYY and NPY(2-36) (1 microM), but was not significant between PYY (100 nM) and PP (1 microM). We suggest that either these cells express a single new Y-receptor with an unusual phenotype or that two Y-receptor populations exist in Colony-6 cells.

Topics: Adenocarcinoma; Colon; Colonic Neoplasms; Epithelium; Humans; Neuropeptide Y; Pancreatic Polypeptide; Peptide YY; Peptides; Receptors, Gastrointestinal Hormone; Receptors, Neuropeptide Y; Secretory Rate; Sensitivity and Specificity; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Our goal was to define the incidence of neuroendocrine carcinomas of the colon and rectum, the patterns of neuroendocrine expression, and the cellular subtype within neuroendocrine tumors. We attempted to determine whether differences in neuroendocrine expression or specific cell type influenced survival.. Over a ten-year period, 988 patients had resections for colorectal cancer. Using immunohistochemical staining methods specific for neuroendocrine markers, 39 (3.9 percent) neuroendocrine cancers were identified retrospectively. Tumors were also stained with monoclonal antibody A-80 which is specific for exocrine differentiation. In this way we were able to determine the extent of neuroendocrine differentiation such as pure neuroendocrine, predominant neuroendocrine, and equal neuroendocrine-exocrine expression.. Average patient age was 65.5 (range, 28-89) years; there were 25 males and 14 females. Nineteen tumors were located in the right colon, 11 in the left, and 9 were in the rectum. Three histopathologic patterns were identified: pure neuroendocrine (n = 11), predominantly neuroendocrine (n = 17), and cancers with equal exocrine and neuroendocrine differentiation (n = 7). Three cellular subtypes were seen: small-cell (n = 15), intermediate-cell (n = 15), and well-differentiated neuroendocrine cancers (n = 5). There was one Dukes A cancer, 7 Dukes B, 16 Dukes C, and 15 patients had metastases to distant sites at the time of diagnosis. As a group, neuroendocrine tumors have a poor prognosis: six-month survival was 58 percent, three-year survival was 15 percent, and five-year survival was 6 percent. Survival statistically correlated with tumor stage (P = 0.01) but not with age, sex, tumor location, histopathologic pattern, or neuroendocrine subtypes. Median survival for pure neuroendocrine carcinomas was seven months and for predominantly neuroendocrine carcinomas was five months. Tumors with equal neuroendocrine and exocrine differentiation had a median survival of 22 months (P = 0.3). Small-cell neuroendocrine carcinomas had a median survival of five months, intermediate-cell had 11 months, and well-differentiated had a median survival of 22 months (P = 0.1).. Neuroendocrine differentiation is found in at least 3.9 percent of colon and rectal cancers. Many of these tumors were initially diagnosed as "carcinoids," the diagnosis was changed to "neuroendocrine carcinoma" after immunohistochemical staining. Overall survival is poor especially for small-cell and pure neuroendocrine carcinomas.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoma, Neuroendocrine; Carcinoma, Small Cell; Colonic Neoplasms; Female; Follow-Up Studies; Humans; Incidence; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Phosphopyruvate Hydratase; Rectal Neoplasms; Serotonin; Survival Rate; Time Factors; Vasoactive Intestinal Peptide

This study has examined the involvement of the Ca(2+)-signalling pathway in the regulation of agonist-stimulated cAMP responses in the human colonic adenocarcinoma cell line, HT29-cl.19A. The muscarinic agonist, carbachol (CCh) stimualted rapid increases in cellular IP3 and cytosolic Ca2+, [Ca2+]i in HT29-cl.19A cells. These were accompanied by a small but significant increase in basal cAMP levels and a marked (3-4-fold) potentiation of both forskolin- (FSK) and VIP-stimulated cAMP generation. Similar effects were observed with two other Ca(2+)-mobilising agonists, neurotensin and ATP. The failure of CCh to elicit potentiation of adenylate cyclase in broken cell preparations indicated an indirect action. Potentiation could be mimicked by the calcium ionophore, ionomycin, and thapsigargin and inhibited 70-90% by depleting intracellular Ca2+ stores suggesting that a rise in [Ca2+]i is the primary mediator of this response. In contrast, increasing [Ca2+]i levels to > 500 nM caused a significant inhibition of FSK-stimulated cAMP generation. The involvement of protein kinase C (PKC) was also assessed. PKC activators phorbol 12,13 dibutyrate (PDB) and 1-oleoyl-2-acetyl glycerol (OAG) potentiated FSK-stimulated cAMP production by 50-70% though PDB markedly inhibited the cAMP response to the receptor-mediated cAMP agonist, VIP. Neither effect could be elicited by the inactive phorbol ester, 4 alpha-phorbol, 12,13 didecanoate (PDD). PKC inhibitors staurosporine and H7 reduced by approximately 25% the CCh-induced potentiation of FSK-stimulated cAMP generation. In conclusion, these results suggest that stimulation of the phosphoinositidase C pathway in HT29-cl.19A colonocytes induces a 'sensitisation' of the adenylate cyclase system resulting in a dramatic amplification of agonist-stimulated cAMP generation. Increases in [Ca2+]i appear to be an important mediator of potentiation though activation of PKC may also play a significant role.

Topics: Adenocarcinoma; Calcium; Calmodulin; Carbachol; Colforsin; Colonic Neoplasms; Cyclic AMP; Drug Synergism; Humans; Phosphoric Diester Hydrolases; Protein Kinase C; Signal Transduction; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Xanthines

Hydrogen peroxide (H2O2) is a reactive oxygen species that can be produced in the digestive tract by inflammatory cells or during reperfusion following ischemia. To evaluate a possible direct effect of H2O2 on epithelial secretory cells, well-differentiated colonic T84 cells were grown to confluence on permeable membranes and studied in Ussing chambers. In this model, where the measured short-circuit current (Isc) reflects electrogenic secretion, we observed that H2O2 stimulated a concentration-dependent and transient secretory response: 5.5 mM H2O2 produced a peak Isc of 12.4 microA/cm2 after 4 min, 2.2 mM H2O2 a peak Isc of 7.9 microA/cm2 after 4 min, and 1.1 mM H2O2 a peak Isc of 5.5 microA/cm2 after 16 min (N = 5). When 97 experiments using 5.5 mM H2O2 were reviewed, the mean peak Isc response was 8.9 +/- 0.5 microA/cm2. A similar secretory response was elicited whether H2O2 was added to the serosal, to the mucosal, or simultaneously to both sides of the T84 cell monolayer. This secretory response reflected transcellular chloride secretion because it was inhibited by the depletion of chloride in the medium and by the suppression of the Na+,K+,2Cl- co-transporter activity necessary for the chloride gradient driving chloride secretion. When T84 cell monolayer resistance was studied, 5.5 mM H2O2 produced a transient decrease in resistance, reflecting transcellular chloride secretion, and a gradual decline in resistance (75% of the initial value after 55 min). The secretory response to H2O2 was increased 2-fold in T84 cells maximally stimulated with 10 nM vasoactive intestinal peptide (VIP), a neuropeptide which acts via cAMP, demonstrating synergism between the two agents. In contrast, the secretory responses produced by H2O2 and carbachol, which acts through the Ca2+ pathway, were additive. A late inhibitory effect of H2O2 was also observed: in cells previously treated with 5.5 mM H2O2, the subsequent secretory responses to either VIP or carbachol were partially inhibited. These secretory effects were specific for the oxidant properties of H2O2 because they were inhibited by 450 U/mL catalase and by 5 mM dithiothreitol, but were unaffected by 50 microM deferoxamine B or Fe3+. H2O2 may be a potential modulator of intestinal or colonic secretion in certain pathologic conditions such as inflammation or ischemia-reperfusion.

Topics: Adenocarcinoma; Carbachol; Catalase; Chlorides; Colonic Neoplasms; Deferoxamine; Dithiothreitol; Electrochemistry; Humans; Hydrogen Peroxide; Iron; L-Lactate Dehydrogenase; Trypan Blue; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

A non-transformed small-intestinal cell line from the rat (IEC-6) and a human colon cancer cell line (HT 29) were examined for their trophic response to sensory neuropeptides. Substance P, neurokinin A (NKA), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), and peptide YY (PYY) were tested. Epidermal growth factor (EGF), insulin, and somatostatin-14 were also used. Interaction studies were performed on IEC-6 cells by combining EGF or insulin with somatostatin-14. The sensory neuropeptides had no effect either on IEC-6 cell growth and DNA synthesis or on HT29 cell growth. EGF and insulin stimulated cell growth and DNA synthesis in IEC-6 cells and cell growth in HT 29 cells in a dose-dependent fashion. Somatostatin-14 had no effect either alone or in combination with EGF or insulin on IEC-6 cell growth and DNA synthesis. HT 29 cell growth was inhibited by somatostatin-14 only in the presence of serum with a maximal and significant response at 10(-7) M. Our observations suggest that the sensory neuropeptides do not exert a direct growth-regulatory effect either on IEC-6 cells or on HT 29 cells. Somatostatin, however, inhibits serum-induced HT 29 cell growth but does not interfere directly with the proliferative effect of serum, EGF, or insulin on IEC-6 cells in this model.

Topics: Animals; Calcitonin Gene-Related Peptide; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Colonic Neoplasms; DNA, Neoplasm; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelium; Gastrointestinal Hormones; Humans; Insulin; Intestinal Mucosa; Intestines; Neurokinin A; Neurons, Afferent; Neuropeptides; Peptide YY; Peptides; Rats; Somatostatin; Substance P; Transforming Growth Factor beta; Vasoactive Intestinal Peptide

Cl- secretion in T84 cells evoked by a stimulus that activates protein kinase C, carbachol, was associated with elevated levels of 32P-labeled phosphatidic acid (PA). PA's role in the regulation of Cl- secretion was explored by examining the effect of exogenous PA (10(-4) M) on Cl- secretion and intracellular Ca2+ levels ([Ca2+]i) in monolayers. PA potentiated the effect of carbachol on [Ca2+]i and Cl- secretion, although it did not stimulate Cl- secretion by itself. PA had divergent effects on cyclic nucleotide-dependent Cl- secretion. It delayed Cl- secretion induced by vasoactive intestinal polypeptide [VIP, adenosine 3',5'-cyclic monophosphate (cAMP) dependent] but potentiated that induced by the heat-stable enterotoxin of Escherichia coli (STa; guanosine 3',5'-cyclic monophosphate dependent). PA did not alter AMP or GMP levels, suggesting that PA acts at a site distal to the generation of these second messengers. PA caused a slight increase in phosphorylation of protein kinase C substrates but not of cAMP-dependent protein kinase substrates. However, PA is probably not acting through a classical protein kinase C pathway, because we have previously shown that phorbol esters inhibit carbachol's actions, and the protein kinase C inhibitor staurosporine failed to block the effect of PA on VIP- or STa-stimulated Cl- secretion. Thus PA differentially regulates stimulated Cl- secretion in T84 cells, depending on the nature of the agonist.

Topics: Calcium; Carbachol; Chlorides; Chromatography, Thin Layer; Colonic Neoplasms; Cyclic AMP; Cytosol; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Humans; Inositol Phosphates; Kinetics; Phosphatidic Acids; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Phosphoproteins; Phosphorylation; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Studies demonstrate that some colon cancers possess receptors for various gastrointestinal hormones or neurotransmitters, the occupation of which can affect growth. These results are limited because frequently only a small number of tumors are studied, only 1 or 2 receptors are sought, and the effect on cell function is not investigated. In the present study, 10 recently characterized human colon cancer cell lines were studied to determine whether they possess receptors for any of 12 different gastrointestinal hormones or neurotransmitters and to determine whether these receptors mediate changes in cellular function. Each of the cell lines exhibited receptors for at least one radioligand. Receptors for vasoactive intestinal peptide (VIP) and muscarinic cholinergic agents occurred on 60%, bombesin and gastrin on 30%, beta-adrenergic agents and gastrin-releasing peptide (GRP) on 20%, and somatostatin, opiates, neuromedin B, and substance P on 10%. Analysis of [3H]N-methylscopolamine binding revealed a Kd of 0.2 nM for N-methylscopolamine with a binding capacity of 2500 sites/cell. With the agonist carbamylcholine, the receptor exhibited 2 classes of binding sites: one of high affinity (Kd 55 microM) representing 75% of the binding sites and one of low affinity (Kd 0.3 mM) representing 25% of the binding sites. Analysis of 125I-[Tyr4]bombesin binding revealed a receptor of high affinity (Kd 2.1 microM) with a binding capacity of 3300 sites/cell. Inhibition of binding by agonists revealed relative potencies of 125I-[Tyr4]bombesin greater than GRP much greater than neuromedin B, and two recently described antagonists were similar in potency to GRP. Analysis of 125I-VIP binding revealed a receptor having 2 classes of binding sites: one of high affinity (Kd 3.6 nM) and one of low affinity (Kd 1.7 microM) which represented the majority of the 5.5 x 10(6) binding sites/cell. The relative potencies of agonists were VIP greater than helodermin greater than peptide histidine methionine greater than secretin. Evaluation of biological activity mediated by the muscarinic cholinergic and bombesin receptors revealed an increase of intracellular calcium and of inositol triphosphate by specific receptor agonists. The presence or absence of receptors detected by binding correlated closely with the ability of selective receptor agonists to alter cell function. These results demonstrate the presence of several different receptors for gastrointestinal hormones or neurotransmitt

Topics: Bombesin; Calcium; Carbachol; Colonic Neoplasms; Humans; Inositol 1,4,5-Trisphosphate; N-Methylscopolamine; Receptors, Bombesin; Receptors, Gastrointestinal Hormone; Receptors, Neurotransmitter; Scopolamine Derivatives; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The stably differentiated human intestinal goblet cell line Cl.16E was used to study the effects of two structurally related regulatory peptides, neurotensin (NT) and neuromedin N (NN), on mucus secretion. NT and NN stimulated rapid release of mucins from filter-grown Cl.16E cells, this effect being dose related with a mean effective dose of 36 nM for NT and 422 nM for NN. The order of potency of NT, three NT fragments corresponding to the NH2-terminal part [NT-(1-11)] or to the COOH-terminal part [NT-(8-13) and NT-(9-13)], and NN in promoting mucin release and in inhibiting 125I-labeled NT binding to Cl.16E cell membranes was identical with NT greater than or equal to NT-(8-13) greater than NN greater than NT-(9-13) much greater than NT-(1-11) supporting the hypothesis that NT and NN stimulate mucin output through interaction with a common NT-preferring receptor. Scatchard analysis of equilibrium binding data showed one population of NT binding sites in Cl.16E cell membranes with the following characteristics: binding capacity (Bmax) was 141 fmol/mg of protein and dissociation constant (Kd) was 1.00 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Binding, Competitive; Calcium; Carbachol; Colon; Colonic Neoplasms; Cyclic AMP; Humans; Kinetics; Molecular Sequence Data; Mucins; Neurotensin; Peptide Fragments; Receptors, Neurotensin; Receptors, Neurotransmitter; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a widely distributed neuropeptide that has been considered a potential regulator of cell growth and differentiation in various tissues, including the gut. To examine this idea, we used a human colon carcinoma cell line (LoVo) as a model system and measured ornithine decarboxylase (ODC), because this is the rate-limiting enzyme for the formation of polyamines, which are thought to be key factors in regulating cell growth. LoVo cells, grown to about 80% confluence in F-12 medium containing 10% fetal bovine serum, were preincubated for 5 h in low serum medium (1% fetal bovine serum in F-12), and ODC activity was determined by measuring 14CO2 liberated from 14C-labeled ornithine. VIP caused a dose-related biphasic change in ODC, with activity increased at 10 pM, maximal (5-fold increase) at 10 nM, and decreased toward basal at 100 nM to 1 microM. Incubation of cells for 6 days with VIP in low serum medium showed similar changes in cell numbers, with growth being increased by doses in the 1 pM to 100 nM range and decreased at higher doses (greater than or equal to 100 nM). Exposure of cells to 5 mM alpha-difluoromethylornithine blocked both the VIP-induced increase in cell number and the VIP-induced increase in ODC activity. Increased ODC mRNA was detected after 2 h of exposure to VIP, a time at which ODC activity peaked after treatment, and the increase in ODC mRNA caused by VIP was dose-dependent. In related experiments LoVo cells were found to have high affinity VIP receptors (Kd = 0.4 nM), as assessed by examination of [125I]VIP binding in the presence of varying concentrations of unlabeled VIP. Studies of intracellular cAMP revealed a dose-related increase in cAMP in response to VIP (ED50 = 11 pM), and the adenylate cyclase activator forskolin increased both ODC activity and ODC mRNA. The findings support the idea that LoVo cells have VIP receptors linked to cAMP which can stimulate cell growth at least in part by increasing ODC synthesis and activity, thereby altering the production of polyamines. The decreased growth and ODC activity observed with high doses of VIP may involve a second messenger other than cAMP.

Topics: Adenocarcinoma; Blotting, Northern; Cell Division; Cell Line; Colonic Neoplasms; Cyclic AMP; Dose-Response Relationship, Drug; Eflornithine; Humans; Kinetics; Ornithine Decarboxylase; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; RNA, Messenger; RNA, Neoplasm; Vasoactive Intestinal Peptide

Although several lines of evidence implicate cAMP in the regulation of intestinal cell proliferation, the precise role of this second messenger in the control of the human colon cancer cell cycle is still unclear. In order to investigate the role of cAMP in HT29 cell proliferation, we have tested the effect of vasoactive intestinal peptide (VIP) and forskolin on DNA synthesis and cell number, focusing on the time-dependent efficacy of the treatment. The cells were arrested in G0/G1 phase by incubation for 24 h in serum-free medium and proliferation was re-initiated by addition of either 85 nM insulin or 0.5% fetal calf serum. In the presence of fetal calf serum, G1/S transition was found to occur earlier than with insulin. Exposure of the HT29 cells to 10(-5) M forskolin in the early stages of growth induction (within 12 h from FCS addition or within 14 h from insulin treatment) resulted in a significant inhibition of DNA synthesis and a delayed entry in the S phase. By contrast, VIP (10(-7) M) was inhibitory only when added within a narrow window (10 to 12 h or 12 to 14 h following FCS or insulin addition, respectively). The difference in efficiency of forskolin and VIP to inhibit cell proliferation may be correlated with their own potency to promote long-lasting cAMP accumulation. The combination of VIP plus forskolin had synergistic effects on both cAMP accumulation and cell-growth inhibition. Taken together, our data indicate that cAMP may act at a step in the late G1 or G1/S transition.

Topics: 1-Methyl-3-isobutylxanthine; Adenocarcinoma; Blood; Cell Division; Colforsin; Colonic Neoplasms; Cyclic AMP; Humans; Insulin; Interphase; Kinetics; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

In past studies we observed that the chloride channel blocker, diphenylamine-2-carboxylate (DPC) and chemically related drugs (Hoechst compounds 131, 143, 144) inhibited cAMP formation in mouse pituitary tumor cells. The object of this study was to determine whether these drugs inhibited chloride transport in human T-84 colonic carcinoma cells through an effect on cAMP metabolism. Chloride secretion (measured as 125I efflux from isotope-preloaded cells) was stimulated in a concentration-dependent manner by vasoactive intestinal polypeptide (VIP) (EC50 = 1.5 x 10(-10) M) which similarly increased cAMP synthesis (EC50 = 1.6 x 10(-8) M). The cAMP response to VIP was inhibited 17, 52, 55, and 78% maximally by DPC and compounds 144, 143, and 131, respectively. In untreated T-84 cells, 125I secretion fell by 66% after 3 min; VIP (10(-7) M) increased secretion about fivefold over the same period. Both basal and VIP-stimulated 125I secretion were inhibited up to 60% by compound 131. Pretreatment of cells with pertussis toxin did not attenuate the inhibitory effect of channel blockers on either VIP-stimulated cAMP synthesis or 125I secretion. The cationophore, A-23187, which had no effect on cAMP formation, and 8-Br-cAMP both stimulated 125I secretion from T-84 cells. These secretory responses were inhibited by compound 131. The mechanism by which phenylanthranilic acids antagonize cAMP synthesis and its significance is not known; however, the data suggest that this family of drugs may inhibit chloride transport by both cAMP-dependent and independent mechanisms.

Topics: Chloride Channels; Chlorides; Colonic Neoplasms; Cyclic AMP; Epithelium; Humans; Iodine; Iodine Radioisotopes; Membrane Proteins; ortho-Aminobenzoates; Pertussis Toxin; Stimulation, Chemical; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

A panel of rat colon adenocarcinoma cell lines (the Per series) were used to investigate the phenotype and karyotype changes induced by in vivo passage in the subcutis of athymic nude mice. One poorly and one well-differentiated tumor cell line were serially passaged through the athymic nude mouse and then back to the syngeneic rat host. Each of the primary and xenograft cell lines expressed fetal crypt cell ("CaCo") antigens. The well differentiated primary and xenograft lines (Per305, Per305N1, and Per305N2a) were different in each of their growth factor responsiveness in vitro [i.e. epidermal growth factor (EGF), bombesin, vasoactive intestinal peptide], their EGF receptor expression, their secretion of transforming growth factor-alpha, and their exhibition of anchorage independent (A-I) growth capabilities. The poorly differentiated primary and xenograft cell lines were also different but were all capable of A-I growth; their responsiveness to exogenous growth factor stimulation decreased with progressive in vivo passage, as did their basal unstimulated proliferation rate. Cytogenetic alterations detected were those associated with clinical specimens from various stages of malignancy, i. e. aneuploidy, structural aberrations, and marker chromosomes. Genetic and mitogenic individuality of each line demonstrated the diversity of the growth control mechanisms in neoplasms at different stages of progression.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Bombesin; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Karyotyping; Male; Mice; Mice, Nude; Neoplasm Transplantation; Phenotype; Receptors, Bombesin; Receptors, Neurotransmitter; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a neuroendocrine mediator found in the central and peripheral nervous system. Distinct subsets of neural, respiratory, gastrointestinal, and immune cells bear specific high-affinity receptors for VIP, which are associated with a guanine nucleotide-binding (G) protein capable of activating adenylate cyclase. A cDNA clone (GPRN1) encoding the human VIP receptor was identified in libraries prepared from the Nalm 6 line of leukemic pre-B lymphoblasts and the HT-29 line of colon carcinoma cells. The deduced 362-amino acid polypeptide sequence encoded by GPRN1 shares a seven-transmembrane-segment hydropathicity profile with other G protein-coupled receptors. Northern blot analyses identified a 2.7-kilobase transcript of the VIP receptor in Nalm 6 and HT-29 cells as well as in tissues from rat brain, colon, heart, lung, kidney, spleen, and small intestine. COS-6 cells transfected with GPRN1 bound 125I-labeled VIP specifically with a dissociation constant (Kd) of 2.5 nM. VIP--and less effectively secretin, peptide histidine isoleucine (PHI), and glucagon competitively displaced bound 125I-VIP from transfected COS-6 cells, with potencies in the order VIP greater than secretin = PHI much greater than glucagon. VIP stimulated adenylate cyclase activity in stably transfected Chinese hamster ovary K1 cells, inducing a 3-fold increase in the intracellular level of cAMP. When the antisense orientation of the VIP receptor clone was introduced into HT-29 cells, there was a 50% suppression of the specific binding of 125I-VIP and of the VIP-induced increase in cAMP level, relative to untransfected cells. The VIP receptor cloned exhibits less than or equal to 24% homology with other receptors in the same superfamily and thus represents a subset of G protein-coupled receptors for peptide ligands.

Topics: Amino Acid Sequence; Animals; Blotting, Northern; Cell Line; Cloning, Molecular; Colonic Neoplasms; Gene Library; Humans; Kinetics; Leukemia; Molecular Sequence Data; Poly A; Protein Conformation; Rats; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Recombinant Proteins; RNA, Messenger; Sequence Homology, Nucleic Acid; Transcription, Genetic; Transfection; Vasoactive Intestinal Peptide

Involvement of ornithine decarboxylase (ODC) in proliferation of the HT29 cell line and its control by either fetal calf serum (FCS) or vasoactive intestinal peptide (VIP) as an external signal increasing cAMP level were investigated. Activation of the polyamine-producing system appears to be a necessary step in the proliferative response of HT29 cells since cell growth is arrested by addition of difluoromethylornithine (DFMO, an inhibitor of ODC), then restored by further addition of putrescine into the culture medium. FCS deprivation results in decreased activity of ODC and arrest of cell growth. Addition of FCS induces reactivation of ODC peaking at 9 hr and re-initiates proliferation but does not affect cAMP level. VIP strongly and rapidly stimulated cAMP accumulation, which resulted in significant activation of ODC. When VIP-induced cAMP formation was hindered by the alpha 2-adrenergic agonist UK14304, activation of ODC was no longer observed. The dose-response curve for ODC activation by VIP indicates an EC50 value of 0.078 nM which falls within the range of physiological concentrations for this peptide. However, VIP fails to stimulate proliferation when cells are cultured either in an FCS-free medium or in the presence of a growth-limiting concentration of FCS. We conclude that the mechanisms of ODC activation by either FCS or VIP are different, the latter involving cAMP formation. Activation of ODC to produce polyamines is necessary to support the proliferative process in our model but the VIP-induced activation of the enzyme alone is not sufficient to promote cell growth.

Topics: Cell Division; Colonic Neoplasms; Cyclic AMP; Eflornithine; Fetal Blood; Humans; Ornithine Decarboxylase; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

In the present study the effect of vasoactive intestinal peptide (VIP), peptide histidine-methionine (PHM), and secretin on spontaneous cell mediated cytotoxicity of peripheral blood mononuclear cells against tumour target cells was evaluated. VIP stimulated cytotoxicity against CaCo-2 human colon cancer cells, whereas less effect was seen against K-562 erythroleukemia cells. Depletion of CD16+ natural killer cells almost completely abolished cytotoxicity and subsequent VIP incubation did not change residual activity. In contrast to PHM, which hardly influenced cytotoxicity, secretin was found to be more effective especially against K-562 target cells. These observations suggest a modulating role for the neuropeptide VIP in the cellular immune response against tumour cells, especially from the colon, resulting in increased activity of CD16+ natural killer cells. Secretin, seems to be less potent in modulating cellular cytotoxicity. These findings support the concept that gastrointestinal peptides can play a role in the regulation of cellular cytotoxicity against tumor cells.

Topics: Colonic Neoplasms; Cytotoxicity, Immunologic; Humans; Killer Cells, Natural; Leukemia, Erythroblastic, Acute; Leukocytes, Mononuclear; Neuroimmunomodulation; Peptide PHI; Secretin; Stimulation, Chemical; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive Intestinal Peptide (VIP) is able at the concentration 10 to 100 nM to induce in HT-29 cells 2'5' oligoadenylate (2'5' A) synthetase activity. The kinetics of this induction show that the maximal effect is attained after 24 hrs. VIP induces 2'5' A synthetase parallel to inhibition of vesicular stomatitis virus growth. The levels of these two induced activities after VIP treatment are comparable to those induced by the poly (I).poly (C), an inducer of IFN beta/alpha in mammalian cells. Moreover the anti-IFN beta/alpha antibodies abolish the VIP-induced 2'5' A synthetase whereas anti-IFN gamma antibodies are ineffective. The fact that VIP establishes an antiviral state in HT-29 cells potentiates new pharmaceutical applications for this neuropeptide.

Topics: 2',5'-Oligoadenylate Synthetase; Animals; Cell Line; Colonic Neoplasms; Dose-Response Relationship, Drug; Enzyme Induction; Humans; Interferons; Mice; Vasoactive Intestinal Peptide; Vesicular stomatitis Indiana virus; Virus Replication

Topics: Colonic Neoplasms; Humans; Intestinal Mucosa; Mucins; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The factors which influence the exocytosis of mucins are not well characterized. Since the physical properties of mucins may be affected significantly by the co-secretion of electrolytes and water, we studied the relationship between ion movement and mucin secretion in T84 cells, a human colonic adenocarcinoma cell line which has been well characterized with respect to apical chloride secretion. Secretion of mucin was assessed by immunoassay of mucin appearing in the medium within 30 min of stimulation. Cells were grown on plastic in DMEM/Ham's F12 medium and experiments were carried out at 70% confluence. Mucin secretion was stimulated by the calcium ionophore A23187, or A23187 plus vasoactive intestinal polypeptide. Stimulated mucin secretion was not affected by loop diuretics (furosemide (1 x 10(-3) M) or bumetanide (1 x 10(-4) M)), with or without the addition of ouabain (5 x 10(-5) M) and amiloride (1 x 10(-5) M), making it unlikely that transcellular chloride movements in necessary for mucin secretion. However, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; (1 x 10(-5) and 5 x 10(-5) M) and three potassium channel blockers BaCl2 (1 x 10(-3) and 5 x 10(-3) M), tetraethylammonium chloride (1 x 10(-2) M) and quinine (5 x 10(-4) M) inhibited mucin secretion. A DIDS-sensitive chloride channel or chloride/bicarbonate exchanger and a Ca2(+)-dependent potassium channel may play important roles in mucin secretion. Since plasma membranes are sparingly permeable to DIDS, the DIDS-sensitive site is likely to be on the apical plasma membrane, perhaps at an initiation locus for exocytosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenocarcinoma; Amiloride; Barium; Barium Compounds; Calcimycin; Cell Line; Chlorides; Colonic Neoplasms; Furosemide; Humans; Kinetics; Mucins; Ouabain; Potassium Channels; Quinine; Stilbenes; Tetraethylammonium; Tetraethylammonium Compounds; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.

Topics: Adenocarcinoma; Calcimycin; Carbachol; Cell Line; Cholera Toxin; Colonic Neoplasms; Cystic Fibrosis; Histamine; Humans; Immunodiffusion; Immunoenzyme Techniques; Intestine, Small; Microscopy, Electron; Mucins; Prostaglandins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The clonal cell line HT29-D4 was able to grow in a completely defined medium containing EGF, selenous acid, and transferrin in the presence of the anti-helminthic drug suramin. In the absence of suramin, the kinetics of cell growth and the cell density obtained were dependent on the external EGF concentration. In the presence of suramin, cell density reached a plateau independent of EGF concentration above 50 ng/ml. At the morphological level, suramin allowed hemicyst formation in the cell monolayer. The cells were polarized with a well-ordered brush border facing the culture medium and mature junctional complexes that divided the cell membrane in two distinct domains. The carcinoembryonic antigen was found to be restricted to the apical membrane domain while the major histocompatibility molecules HLA-ABC were segregated within the basolateral domain. The electrical parameters of suramin-treated cells grown on permeable filters were measured and demonstrated that the cell monolayer was electrically active. These properties were never found in the absence of the drug. Moreover, the vasoactive intestinal polypeptide (VIP) was able to induce a dramatic increase in cAMP only when it was added, in agreement with the localization of the VIP receptor, in the lower compartment of the culture chamber. In conclusion we described for the first time conditions allowing the growth of functionally differentiated human colic cell monolayers in chemically defined medium. This model will contribute to a better understanding of suramin action and of the mechanisms involved in cell polarization.

Topics: Adenocarcinoma; Carcinoembryonic Antigen; Cell Count; Cell Differentiation; Cell Division; Clone Cells; Colonic Neoplasms; Culture Media; Cyclic AMP; Epidermal Growth Factor; HLA Antigens; Humans; Suramin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Metabolic pathways of glucose utilization have been investigated in a human colon adenocarcinoma cell line (HT29) using carbon-13 Nuclear Magnetic Resonance spectroscopy. HT29 cells were adapted to grow on a polystyrene beaded microcarrier and were perfused when attached to the beads in a specially designed NMR cell. Abnormalities in carbohydrate metabolism already observed in several cancer cells were studied in HT29 cells fed with (1-13C)-enriched glucose. The cells were first perfused with a glucose-free medium for 2 h in order to deplete the intracellular store of glycogen, and they were subsequently perfused with a medium containing enriched glucose at an initial concentration of 5.5 mM. Sequential 13C-NMR spectra, recorded at 100.5 MHz (5 min accumulation), show that HT29 cells were able to utilize glucose through the glycolytic pathway while storing glucose as glycogen (glucose was utilized at a rate of 3.9 mumol/mg protein/hr). The glycolytic activity determined by the amount of lactic acid produced was 4.6 microns/mg protein/hr, corresponding to the formation of 1.2 lactic acid per glucose molecule. Glycogen accumulation corresponded to 16 micrograms/mg of protein. Treatment of HT29 with 10 nM vasoactive intestinal peptide (VIP) induced a transient decrease in the level of labelled glycogen to 50% of the initial value. Control level was recovered 12 min after VIP loading.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Glycogen; Glycolysis; Humans; Lactates; Magnetic Resonance Spectroscopy; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Regulation of active K+ influx and Na(+)-K(+)-Cl- cotransport activity in HT-29 cells by vasoactive intestinal peptide (VIP) was investigated. Both active K+ influx, defined as the ouabain-sensitive component, and Na(+)-K(+)-Cl- cotransport, defined as the ouabain-resistant bumetanide-sensitive component, of total K+ uptake were increased by VIP. VIP increased the maximum velocity (Vmax) values for both components with no change in apparent Michaelis constant (Km) values. Three lines of evidence support the role of adenosine 3',5'-cyclic monophosphate (cAMP) as a mediator of the VIP effects. 1) The rank order potencies of VIP and peptide histidineisoleucineamide (PHI) in binding and cAMP production (J. T. Turner, S. B. Jones, and D. B. Bylund, Peptides Fayetteville 7: 849, 1986) and K+ uptake were consistent; 2) alpha 2-adrenergic agonists inhibited both VIP-stimulated cAMP production (J. T. Turner, C. Ray-Prenger, and D. B. Bylund, Mol. Pharmacol. 28: 422, 1985) and K+ uptake; and 3) forskolin, but not dideoxyforskolin, mimicked the effects of VIP on K+ uptake. Because amiloride blocked the VIP-stimulated active K+ component, the VIP effects on active K+ influx may be secondary to a Na(+)-H+ antiporter-mediated increase in cellular Na+ content. Additional experiments indicated that pretreatment of cells with a protein kinase C activator, previously shown to decrease basal Na(+)-K(+)-Cl- cotransport activity and the apparent number of cotransporters in HT-29 cells (C. C. Franklin, J. T. Turner, and H. D. Kim, J. Biol. Chem. 264: 6667, 1989), did not change the magnitude of response of the remaining cotransporters after adenylate cyclase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenocarcinoma; Amiloride; Biological Transport; Bumetanide; Carrier Proteins; Cell Line; Chlorides; Colonic Neoplasms; Humans; Isoquinolines; Kinetics; Norepinephrine; Ouabain; Piperazines; Potassium; Protein Kinase C; Sodium-Potassium-Chloride Symporters; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Yohimbine

Transferrin (Tf) and vasoactive intestinal peptide (VIP) were labeled with horseradish peroxidase (HRP) and 125I, respectively. To determine whether two simultaneously incubated ligands are conveyed by the same population of endosomal vesicles in human colon carcinoma cells (HT-29), we used an analysis system derived from the cross-fire method for quantitation of autoradiographic data. This system permitted the collection of data and the statistical calculations required by the double labeling of the cells. HRP-labeled Tf organelles were chosen as reference structures of the endosomal apparatus and taken as the conventional source of the radiolabeling. Our data established from the co-localization hypothesis strongly suggest that after a 30-sec (T 1/2) and a 10-min (T10) internalization at 37 degrees C, VIP and Tf share in major part the same endocytic pathway and even the recycling route to the cell surface. At T10, most of the radiolabeling was located inside the tubulovesicular network, and we also detected slight radiolabeling inside the vesicles recycling Tf. The number of double-labeled endosomes involved in ligand traffic were advantageously observed with our computer-assisted analysis system.

Topics: Cell Line; Colonic Neoplasms; Histocytochemistry; Horseradish Peroxidase; Humans; Image Processing, Computer-Assisted; Microscopy, Electron; Organelles; Transferrin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The effects of vasoactive intestinal polypeptide (VIP), 16,16-dimethyl prostaglandin E2 (DMPGE2) and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) on protein phosphorylation were studied in relation to stimulation of chloride transport in cell suspensions of the human colon epithelial cell line Caco-2. In 36Cl-loaded cells, VIP and DMPGE2 within 1 min decreased cellular chloride content 35-40%, with half-maximal effects being elicited at 1.0 and 85 nM concentration, respectively. A similar effect on chloride content occurred after 10 min of treatment with 0.5 mM DBcAMP. For all three secretagogues, decreases in cellular chloride content were associated with increases in membrane permeability to chloride. DMPGE2 and VIP within 1 min, and DBcAMP within 10 min, increased the phosphorylation of an unidentified soluble protein of Mr = 42,000 and pI = 6.1, and of a protein of Mr = 20,200 and pI = 4.9 identified as myosin regulatory light chain. Between 10 and 30 min of stimulation, however, phosphorylation of the Mr = 42,000 protein and chloride transport activity remained elevated in DMPGE2- and DBcAMP-treated cells, whereas light chain phosphorylation returned to control level. No effect of secretagogues on phosphorylation was detected in the total particulate fraction or an integral membrane protein fraction. It is concluded that increased membrane permeability to chloride induced by cAMP-mediated secretagogues in Caco-2 is temporally associated with the increased phosphorylation of a Mr = 42,000 soluble protein.

Topics: 16,16-Dimethylprostaglandin E2; Biological Transport; Bucladesine; Cell Membrane Permeability; Chlorides; Chlorine; Colonic Neoplasms; Cyclic AMP; Furosemide; Humans; Isoelectric Point; Molecular Weight; Myosins; Phosphoproteins; Phosphorylation; Prostaglandins E, Synthetic; Radioisotopes; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The effects of a novel polypeptide, pancreatic spasmolytic polypeptide (PSP) on a colon carcinoma cell line (HCT 116) were examined. PSP stimulated the incorporation of [3H]thymidine into HCT 116 cells as well as cell proliferation in a dose-dependent manner. Maximal increase in [3H]thymidine incorporation of 50-60% occurred at 3-300 microM PSP. The VIP-mediated-increase in cAMP levels was reduced by PSP at greater than 1 microM concentrations. PSP is highly homologous to the estrogen-induced pS2 protein in MCF-7 breast cancer cells. We find that PSP also enhanced [3H]thymidine incorporation in MCF-7 cells. These findings indicate for the first time that PSP has growth stimulatory properties.

Topics: Animals; Breast Neoplasms; Cell Division; Colonic Neoplasms; Cyclic AMP; DNA; Humans; Intercellular Signaling Peptides and Proteins; Mucins; Muscle Proteins; Neuropeptides; Parasympatholytics; Peptides; Swine; Trefoil Factor-2; Trefoil Factor-3; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The effects of vasoactive intestinal polypeptide (VIP) and dibutyryl cyclic adenosine 3':5'monophosphate (dbcAMP) on two human colon carcinoma cell lines, HCT 116 and GEO, were investigated. VIP and dbcAMP inhibited the growth of both cell lines in monolayer culture in a dose-dependent manner. Within 6 h of treatment with 1 mM dbcAMP or 0.3 microM VIP, numerous mucin-like droplets were secreted by GEO cells. VIP and dbcAMP also increased carcinoembryonic antigen (CEA) secretion. In both cell lines, a 9-fold increase in conditioned medium CEA levels was observed at 1 mM dbcAMP and a 2.6-fold increase at 1.5 microM VIP. Time- and concentration-dependent evaluation in cAMP levels were elicited by VIP in the two cell lines. Immunocytochemical studies for cell-surface glycoprotein detection in GEO cells showed that VIP induced a morphological and functional organization of mucin-secreting cells. These results indicate that VIP and dbcAMP have antiproliferative and strong differentiation-promoting effects in colon cancer cells. This is the first report of VIP-induced mucin secretion in colon tumor cells.

Topics: Carcinoembryonic Antigen; Cell Differentiation; Cell Division; Colforsin; Colonic Neoplasms; Cyclic AMP; Dibutyryl Cyclic GMP; Dose-Response Relationship, Drug; Humans; Mucins; Theophylline; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

At the maximally effective concentration of 10 nM, VIP induced a marked (12.5-fold stimulation above basal), and sustained increase in short circuit current in the human intestinal epithelial cell line Cl.19A grown on permeable filters and placed in Ussing chambers. Half-maximal increase of Isc was observed for 0.1 nM VIP. This was well correlated with the VIP-stimulated adenylate cyclase activity (ED50:0.07 nM). Binding studies using 125I-VIP indicated that Cl.19A cells express a peptide-specific VIP receptor with a dissociation constant of 0.07 nM. Covalent labeling of receptors followed by SDS-PAGE analysis of membrane proteins resulted in the identification of a 63,000 dalton binding protein in Cl.19A cells.

Topics: Adenocarcinoma; Adenylyl Cyclases; Binding, Competitive; Colonic Neoplasms; Electric Conductivity; Epithelium; Humans; Intestines; Molecular Weight; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) induces phosphorylation of a basic 38,000 mol. wt protein in a human lymphoblastic cell line (Molt 4b) and a human colon carcinoma cell line (HT29). In both cell types, VIP interacts with specific high affinity receptors to activate adenylate cyclase and cAMP-dependent protein kinase. The two cell types appear to express homologous receptors with similar affinity and specificity for VIP, but the colonic epithelial cells express a greater number of receptors. HT29 colonic cells also exhibit a greater stimulation of adenylate cyclase and a higher phosphorylation index for the 38,000 mol. wt protein in response to VIP. This 38,000 mol. wt protein, which is phosphorylated in the presence of VIP, appears to be identical in both cell lines; it is phosphorylated in both lymphoblasts and colonic epithelial cells in the presence of forskolin, but not in the presence of phorbol 12-myristate 13-acetate. Phosphorylation of this 38,000 mol. wt protein may be an important step in VIP regulation of water and electrolyte secretion from colonic epithelial cells, and in VIP regulation of immunoglobulin and lymphokine secretion from lymphocytes.

Topics: Adenylyl Cyclases; Cell Line; Colforsin; Colon; Colonic Neoplasms; Epithelium; Humans; Lymphocytes; Molecular Weight; Phosphorylation; Proteins; Vasoactive Intestinal Peptide

Desensitization of human carcinoma colonic cells in culture (HT-29) to vasoactive intestinal peptide (VIP) has been reported previously (C. Boissard, J. C. Marie, G. Hejblum, C. Gespach, and G. Rosselin, Cancer Res., 46: 4406-4413, 1986). In the present study, we have determined the ultrastructural localization of VIP and its receptor after exposure of HT-29 cells to VIP monoiodinated on tyrosyl residue 10 together with the molecular forms and the activity of the internalized VIP receptor. Quantitative electron microscope autoradiography showed that after binding at the cell surface, VIP is internalized in heterogeneous endosomes. Cross-linking experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis were performed in different experimental conditions allowing us to selectively obtain cell surface-associated, internalized, or recycled receptors. No detectable alteration of the labeled VIP-receptor complex occurred during the internalization and recycling processes. Furthermore, a loss of the forskolin potentiation of the VIP-induced stimulation of adenylate cyclase was observed after VIP exposure. This feature was time and temperature dependent as was the VIP-induced loss of cell surface receptors, indicating that the internalized VIP receptor is dissociated from the adenylate cyclase.

Topics: Adenylyl Cyclases; Ammonium Chloride; Carcinoma; Colonic Neoplasms; Cyclic AMP; Endocytosis; Humans; Microscopy, Electron; Molecular Weight; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Preincubation with an alpha 2-adrenergic agonist sensitized subsequent forskolin- and vasoactive intestinal peptide-stimulated cyclic AMP production in HT29 cells, a human colonic adenocarcinoma cell line. Preincubation with somatostatin, another agonist negatively coupled to adenylate cyclase, sensitized forskolin-stimulated cyclic AMP production to a lesser extent. alpha 2-Adrenergic agonist preincubation also resulted in desensitization as indicated by a shift to the right in the dose-response curve of a subsequent challenge by an alpha 2-adrenergic agonist. In an effort to elucidate the mechanism for sensitization, we examined protein kinase C and the Na+/H+ antiporter. Whereas these components had marked effects on forskolin stimulation, there was no effect on sensitization. Changes in the concentration of extra-cellular Ca2+ or Mg2+ had no effect on either forskolin stimulation or sensitization. Pertussis toxin pretreatment caused a time-dependent decrease in sensitization, an attenuation of inhibition of cyclic AMP production, and a decrease in subsequent [32P]ADP-ribosylation by pertussis toxin. The time course for these three events was similar, implicating the inhibitory guanine nucleotide regulatory protein in the mechanism for alpha 2-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production. In addition, pertussis toxin dramatically decreased forskolin-stimulated cyclic AMP production, although with a different time course. These results suggest that the mechanism of sensitization is via an as yet undefined sequence of biochemical events that includes the inhibitory guanine nucleotide regulatory protein, but does not include inhibition of adenylate cyclase nor activation of the Na+/H+ antiporter.

Topics: Adenocarcinoma; Adenylate Cyclase Toxin; Blood Platelets; Carrier Proteins; Cell Line; Colforsin; Colonic Neoplasms; Cyclic AMP; Cycloheximide; Enzyme Activation; Humans; Kinetics; Norepinephrine; Pertussis Toxin; Protein Kinase C; Receptors, Adrenergic, alpha; Sodium; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

HT29-D4, a clone of the human colonic adenocarcinoma cell line (HT29), possesses at its cell surface specific binding sites for the vasoactive intestinal peptide (VIP) (KD = 0.5 nM). Their molecular weight was previously estimated to 117 kDa and 64 kDa. This clone underwent functional and structural differentiation when grown in a glucose-free galactose-containing medium. The [125I]VIP binding capacity of cells grown in this medium gradually declined while the cell density increased and reached a value close to zero when cell monolayer was able to form hemicysts. At this time, cells presented numerous tight junctions and desmosomes and a well organized brush border. Binding capacity could be recovered when the post-confluent monolayers were previously disaggregated with EDTA. Neither the affinity for VIP nor the molecular weight of the [125I]VIP cross-linked polypeptides were modified in these cells compared to cells grown in glucose-containing medium. However, surface receptor number of differentiated cells was twice that of undifferentiated cells. Leakproof differentiated cell monolayers grown on permeable substratum produced cAMP in response to VIP only when the peptide was present in the lower chamber of the culture wells. Taking these data altogether, we conclude that the localization of functional VIP receptors is restricted to the basolateral domain in differentiated post-confluent HT29-D4 cells.

Topics: Adenocarcinoma; Adenylyl Cyclases; Cell Differentiation; Cell Line; Colonic Neoplasms; Humans; Microscopy, Electron; Molecular Weight; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Detergents; Humans; Kinetics; Molecular Weight; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Solubility; Vasoactive Intestinal Peptide

This report describes a 7-year-old boy presenting with watery diarrhea, hypokalemia, and hypochlohydria associated with vasoactive intestinal polypeptide (VIP)-secreting ganglioneuromatosis involving the entire colon and rectum. The child's symptoms resolved following proctocolectomy, and the VIP levels returned to normal. Although 55 previous children have been reported with VIP-secreting tumors, this case is the first involving the entire colon and rectum.

Topics: Adenoma, Islet Cell; Child; Colonic Neoplasms; Ganglioneuroma; Humans; Male; Rectal Neoplasms; Vasoactive Intestinal Peptide; Vipoma

Vasoactive intestinal peptide (VIP) is a neuropeptide with broad tissue distribution. Although its precise function is unknown, it is thought to exert its effect, at least in part, by interacting with cell surface receptors. Nuclear receptors for VIP have now been identified by specific binding of 125I-labeled VIP to nuclei of a human colonic adenocarcinoma cell line (HT29) and by cross-linking of 125I-labeled VIP to its receptor on intact nuclei. In contrast, 125I-labeled transferrin shows only background binding to nuclei but significant binding to intact cells. Purity of the isolated nuclei was further substantiated by electron microscopy. The apparent molecular sizes of the VIP--cross-linked nuclear and cell surface receptors are similar but not identical.

Topics: Adenocarcinoma; Cell Line; Cell Nucleus; Colonic Neoplasms; Humans; Kinetics; Microscopy, Electron; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Treatment of HT29 cells with the tumor promoting phorbol ester PMA resulted in an attenuation of VIP-stimulated cAMP production in intact cells and VIP-stimulated adenylate cyclase activity in cell membranes. PMA did not decrease the ability of cholera toxin and forskolin to elevate cAMP levels in intact cells. Fluoride-stimulated adenylate cyclase activity in HT29 cells homogenates was not affected by PMA. The maximal VIP binding capacity of homogenates prepared from HT29 cells treated with PMA was decreased by 50%. It is concluded that protein kinase C regulates VIP receptor function possibly through phosphorylation of the VIP receptor.

Topics: Adenocarcinoma; Adenylyl Cyclases; Binding Sites; Cell Line; Cell Membrane; Colonic Neoplasms; Cyclic AMP; Humans; Phorbol Esters; Tetradecanoylphorbol Acetate; Vasoactive Intestinal Peptide

We have previously shown that the mono [125I]iodinated vasoactive intestinal peptide (125I-VIP) could be covalently cross-linked on intact colonic adenocarcinoma cells (HT29). A major Mr 67,000 and a minor Mr 120,000 cross-linked polypeptides have been characterized [Muller, Luis, Fantini, Abadie, Giannellini, Marvaldi & Pichon (1985) Eur. J. Biochem. 151, 411-417]. The glycoprotein nature of these species was investigated using endo-beta-acetylglucosaminidase F (Endo F) treatment, enzymic and chemical desialylation and wheat germ agglutinin (WGA)-Sepharose affinity chromatography. Affinity-labelled VIP-binding proteins solubilized by Nonidet P-40 bound to WGA-Sepharose and could be eluted specifically with N-acetyl-D-glucosamine. Treatment with Endo F resulted in an increased electrophoretic mobility of both polypeptides. The major and the minor VIP-binding proteins were converted respectively into Mr 47,000 and 100,000 species, indicating removal of 20 kDa of N-linked oligosaccharides. Deglycosylation with trifluoromethanesulphonic acid also led to a 20 kDa loss in mass of the Mr 67,000 component, indicating the absence of additional O-linked sugars on this polypeptide. The presence of sialic acid on the major VIP-binding protein was demonstrated after treatment of intact cells with neuraminidase or by chemical desialylation with hydrochloric acid. We conclude from this study that the VIP receptor from intact HT29-D4 cells is a glycoprotein with N-linked oligosaccharide side chains containing sialic acid.

Topics: Acetylglucosaminidase; Adenocarcinoma; Carrier Proteins; Cell Line; Chromatography, Affinity; Colonic Neoplasms; Glycoproteins; Humans; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Sialic Acids; Vasoactive Intestinal Peptide

Incubation of monolayers of HT29-D4 cells (a clone of the human colonic adenocarcinoma cell line HT29) in the presence of 17.5 microM cycloheximide resulted in an increase in the number of vasoactive intestinal peptide (VIP) binding sites at the cell surface without any change in the affinity of receptor for its ligand. The increase in 125I-VIP-binding capacity was dose-dependent between 0.35 microM and 17.5 microM cycloheximide and was correlated with the inhibition of protein biosynthesis. At higher concentrations of drug (17.5-100 microM) a plateau corresponding to a twofold increase in VIP-binding capacity was reached independently of the extent of protein synthesis inhibition. We found that VIP receptors of HT29-D4 cells with such an enhanced binding capacity behaved like those of control cells with respect to receptor internalization and recycling (i.e. the cycle of occupied receptors was insensitive to cycloheximide). After inactivation of 90% of cell-surface VIP receptors by alpha-chymotrypsin, we observed a biphasic kinetic of reappearance of VIP-binding sites. 40% of VIP-binding sites reappeared very quickly (less than 5 min) and 100% within 17 h. The fast recovery of VIP receptors was probably due to the deployment of new binding sites from an intracellular pool. The rate and extent of recovery of these receptors were similar in control cells and in cycloheximide-treated cells. However, the slow recovery was inhibited in cycloheximide-treated cells probably because a pool of immature receptors was depleted by the drug before the alpha-chymotrypsin treatment. Our data are consistent with the existence of two different intracellular pathways of occupied and unoccupied VIP receptors.

Topics: Adenocarcinoma; Binding Sites; Cell Line; Chymotrypsin; Colonic Neoplasms; Cycloheximide; Dose-Response Relationship, Drug; Humans; Kinetics; Methionine; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

The effects of vasoactive intestinal peptide (VIP) on the incidence and histology of colonic tumors induced by azoxymethane (AOM) were investigated in Wistar rats. Rats were given 20 micrograms/kg body weight of VIP every other day for 12 weeks and from experimental week 3, were given 10 weekly injections of 7.4 mg/kg body weight of AOM. The administration of VIP before and during AOM treatment resulted in a significant increase in the incidence of colonic tumors in week 40. Furthermore, it caused a significant increase in the labeling index of the colonic mucosa during AOM treatment. These findings indicate that VIP enhanced the development of colonic tumors. This effect may have been related to its effect in increasing proliferation of cells in the colonic mucosa during administration of the carcinogen.

Topics: Animals; Azoxymethane; Cell Division; Colon; Colonic Neoplasms; Intestinal Mucosa; Male; Rats; Rats, Inbred Strains; Vasoactive Intestinal Peptide

The disappearance of vasoactive-intestinal-peptide (VIP) binding sites at the cell surface of a cultured target cell, originating from a human colonic adenocarcinoma (HT 29 cell line), was studied, after preexposition of the cell to the peptide, as a function of time, VIP concentration and temperature. Maximum effect (60-80% loss of binding capacity) was obtained after a 5-10 min exposure of the cells at 37 degrees C with a VIP concentration of 100 nM. The t1/2 of maximum disappearance was less than 2 min and the concentration of native VIP giving half-maximum decrease in 125I-VIP binding was 6 nM. The affinity of remaining binding sites for VIP was not affected compared to that of control cells (Kd = 0.3 nM). Disappearance of VIP binding sites was specific since, with the same conditions of preincubation, the specific binding of 125I-labeled epidermal growth factor to HT 29 cells was not modified. The phenomenon was reversible and 90% of binding capacity could be restored in less than 60 min by incubating cells in VIP-free medium. Correlatively we showed, by two independent experimental procedures, that 125I-VIP, initially bound to HT 29 cells, was maximally internalized after 10 min of incubation at 37 degrees C. All the data strongly suggest that: internalization of VIP is receptor-mediated; upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Humans; Hydrogen-Ion Concentration; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Temperature; Time Factors; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) receptors are widely distributed in different tissues or carcinoma cells originating from entoderm and have been shown to regulate the growth of colonic adenocarcinoma cells through the action of cyclic AMP (cAMP). After exposure of cultured HT-29 human colonic carcinoma cells to 10(-8) M VIP, the cAMP-mediated signals in response to a new challenge with this neuropeptide were strongly attenuated as a function of time (half-life, less than 3 min) and VIP concentrations (half-maximal desensitization, 4 X 10(-9) M VIP). Desensitization is receptor mediated as indicated by: (a) the pharmacological specificity of the desensitization (VIP greater than secretin); (b) the considerable decrease of the potentiative action of VIP on forskolin-induced cAMP generation; and (c) the close temporal relationship between VIP receptor desensitization and the disappearance of the VIP binding sites from the cell surface. Desensitization is reversible upon the removal of VIP. Recovery of functional VIP receptors is insensitive to cycloheximide treatment, is critically dependent upon temperature, and in optimal conditions (37 degrees C) does not exceed 75 and 55% of the binding of 125I-VIP monoiodinated on tyrosine residue and VIP-induced cAMP production, respectively. The characteristics of the desensitization and internalization/recycling of the VIP receptors in carcinoma cells in culture are consistent with the transient action of this neurotransmitter and underline the biological significance of these processes. The study of drugs and natural agents interfering with membrane regulation of VIP receptor density and activity may be of considerable importance in intestinal cell tumor biology.

Topics: Carcinoma; Cell Compartmentation; Cells, Cultured; Colforsin; Colonic Neoplasms; Cyclic AMP; Cycloheximide; Dose-Response Relationship, Drug; Humans; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Secretin; Temperature; Time Factors; Vasoactive Intestinal Peptide

In undifferentiated clonal HT29-18 cells, VIP produced an important (17-fold) and persistent increase in cyclic AMP generation for 10 min. This activation measured at 37 degrees C in the absence of IBMX was transient for 2 min and was considerably reduced (4-fold increase) in enterocyte-like cells. Retrodifferentiation of HT29-18 cells (after substitution of galactose for glucose) resulted in a partial reversion of VIP receptor activity. Cyclic AMP levels reached a peak value at 1 min but remained elevated over basal values for 10 min. Similarly, maturation of intestinal mucosae in 19-day-old rat fetuses and 5 day-old rats was accompanied by a remarkable reduction of VIP receptor activity. Cyclic AMP phosphodiesterase, adenylate cyclase activities and VIP receptor desensitization might explain this phenomenon. In contrast, the potency of VIP and the pharmacological properties of the VIP receptor are unchanged. We therefore assume that VIP receptor activity is an indicator of intestinal cell differentiation and we suggest that this neuropeptide may be a regulator of the normal and tumoral development of the intestinal mucosae in man and rat.

Topics: Adenylyl Cyclases; Animals; Cell Differentiation; Clone Cells; Colonic Neoplasms; Cyclic AMP; Female; Fetus; Humans; Intestinal Mucosa; Kinetics; Male; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

The initial event of VIP action is its interaction with a specific receptor at the surface of a target cell. The understanding of the fine mechanism of action of VIP requires the characterization and of course the purification of the receptor. The understanding of the mechanism which regulates the number of receptor sites at the cell surface, if it occurs, is also a way to characterize the properties of VIP receptor. In this paper we report a number of data which represent several attempts to characterize VIP receptor in a human colonic adenocarcinoma cell (HT 29 cells). We have characterized a monoclonal antibody which partially inhibits 125I-VIP binding to HT 29 cells. We have specifically cross-linked, on intact HT 29 cells, a major polypeptide of Mr-64,000 with 125I-VIP using DTSP or DSS as cross-linking reagents. This polypeptide behaves like a high affinity binding site for VIP. We have demonstrated that VIP is rapidly internalized in HT 29 cells (in less than 10 minutes) and that simultaneously VIP receptors were no more detectable on the cell surface by cross-linking experiments. This suggests that VIP is internalized together with its receptor.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Binding Sites; Cell Line; Colonic Neoplasms; Cross-Linking Reagents; Humans; Immunochemistry; Molecular Weight; Peptides; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) was found to cause a dose-dependent decrease in fructose 2,6-bisphosphatase concomitant with an increase in cyclic AMP in cultured HT29 cancer cells from human colon. The maximum effect was a 41% decrease obtained with 10 nM-VIP, and half-maximum effect was obtained with 0.75 nM-VIP. The effect of 2.5 nM-VIP was almost totally counteracted (i.e. fructose 2,6-bisphosphate concentration was restored) by either adrenaline (1 microM) or the alpha 2-adrenergic agonist UK-14304 (1 microM); the alpha 2-agonist clonidine (1 microM) was less efficient, since the VIP effect was decreased by 72% only. The adrenaline effect was totally antagonized by 1 microM-yohimbine. It is concluded that, in the HT29 cancer cells, the fructose 2,6-bisphosphate-producing system is sensitive to variations of cyclic AMP concentration and is under the dual control of VIP and alpha 2-adrenergic receptors.

Topics: Adenocarcinoma; Brimonidine Tartrate; Cell Line; Clonidine; Colonic Neoplasms; Cyclic AMP; Epinephrine; Fructosediphosphates; Hexosediphosphates; Humans; Quinoxalines; Vasoactive Intestinal Peptide; Yohimbine

The kinetics of VIP processing in different compartments (medium, plasma membrane and intracellular) by HT-29 cells was studied using direct biochemical methods and a homogeneous monoiodinated VIP. The compartmental radioactivity was characterized by HPLC fractionation and specific receptor binding. VIP once bound to the cell surface remains intact and is rapidly and maximally internalized (less than 10 min) at 37 degrees C. Then two different processes occur: (1) release of degradation products 125I and monoiodinated tyrosine in the medium; (2) VIP remains intact in the cells representing 67.2 +/- 4.7% of total radioactivity up to 90 min. The overall processing of VIP is time- and temperature-dependent and maximal internalization of VIP with minimal medium release is observed at 20 degrees C. Our results demonstrate a receptor mediated internalization of VIP and that at least two intracellular pathways may exist in the cycle of VIP. One is associated with a complete degradation of VIP detected in the extracellular medium and is optimal at 37 degrees C. The other results in the presence of intact intracellular VIP and is optimal at 20 degrees C.

Topics: Cell Compartmentation; Chromatography, High Pressure Liquid; Clone Cells; Colon; Colonic Neoplasms; Epithelium; Humans; Kinetics; Temperature; Vasoactive Intestinal Peptide

We have characterized the alpha 2-adrenergic receptor in membranes from the human colonic adenocarcinoma cell line, HT29, using the recently introduced alpha 2-agonist 5-bromo-6-[2-imidazolin-2-yl-amino]quinoxaline [( 3H]UK-14,304), two other radioligands, and a series of adrenergic agonists and antagonists. We also investigated alpha 2-agonist inhibition of HT29 cell adenylate cyclase and reversal of inhibition by alpha-adrenergic antagonists. [3H] Yohimbine saturation experiments indicated a single class of sites with a KD of 0.61 nM which agreed with the kinetically determined KD of 0.62 nM. Computer analysis of kinetic and saturation experiments with [3H]UK-14,304 revealed two classes of sites. From the saturation data, one site had high affinity for the radioligand (0.14 nM) and comprised 33% of the total number of sites, whereas the other site had lower affinity (6.1 nM). The total number of sites labeled by [3H]UK-14,304 (360 fmol/mg of protein) was approximately equal to the number of sites labeled by [3H]yohimbine (330 fmol/mg), whereas [3H]para-aminoclonidine labeled fewer sites of a single class. Rank order potencies of adrenergic agonists and antagonists obtained from competition binding assays indicated that: the same receptors were labeled by the three radioligands, and the receptors were of the alpha 2 subtype. UK-14,304 and epinephrine inhibited forskolin- and vasoactive intestinal peptide-stimulated adenylate cyclase in a dose-dependent manner up to 32%. Inhibition of the enzyme was reversed by yohimbine and, less potently, by phentolamine and prazosin in a dose-dependent manner. The HT29 cell line appears to be a useful model system for the investigation of the regulation and mechanism of action of alpha 2-adrenergic receptors in human tissues.

Topics: Adenocarcinoma; Adenylyl Cyclase Inhibitors; Adrenergic alpha-Agonists; Brimonidine Tartrate; Cell Line; Clonidine; Colforsin; Colonic Neoplasms; Dose-Response Relationship, Drug; Guanosine Triphosphate; Humans; Kinetics; Magnesium; Quinoxalines; Radioligand Assay; Receptors, Adrenergic, alpha; Vasoactive Intestinal Peptide; Yohimbine

In the present work, [3H]clonidine was used to characterize alpha 2-adrenoceptors on the human adenocarcinoma cell line HT 29. The effects of alpha 2-adrenergic stimulation on cellular cyclic AMP levels were also investigated. The binding of [3H]clonidine on HT 29 cell membrane preparations was rapid and reversible. Scatchard analysis of the saturation curves indicated the existence of a single class of non-interacting sites with a KD of 1.29 +/- 0.07 nM and a Bmax of 114 +/- 7 fmol/mg of cell membrane protein. The binding sites for [3H]clonidine showed the required specificity for alpha 2-adrenoceptors. The potencies of alpha-adrenergic compounds to displace [3H]clonidine binding ranked as follows: yohimbine greater than phentolamine much greater than prazosin for antagonists and clonidine greater than epinephrine greater than norepinephrine greater than phenylephrine much greater than amidephrine for agonists. When tested on intact cells, epinephrine, norepinephrine and clonidine were found to counteract, in a dose-dependent manner, the increase of cyclic AMP triggered by vasoactive intestinal peptide (VIP). Such inhibitory effects were abolished by the addition of yohimbine but not of prazosin. The physiological amines were the most efficient agonists: both epinephrine and norepinephrine inhibited VIP-induced cyclic AMP accumulation by 50-55% with KD values of 50 nM and 300 nM respectively. Clonidine was a partial agonist only, provoking a weak (25-30%) inhibition of VIP-induced cyclic AMP accumulation even at high concentrations. These results indicate that, like normal colocytes, human colon adenocarcinoma cells HT 29 possess alpha 2-adrenoceptors, the stimulation of which is associated with an inhibition of cyclic AMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenocarcinoma; Cell Line; Clonidine; Colonic Neoplasms; Cyclic AMP; Humans; Kinetics; Receptors, Adrenergic, alpha; Tritium; Vasoactive Intestinal Peptide

The human colon adenocarcinoma cell-line HT 29 has been shown to possess functional alpha 2-adrenergic receptors. Here, [3H] clonidine was used as radioligand to study the evolution of alpha 2-adrenergic receptivity during the time course of HT 29 cell culture. Scatchard analysis of the saturation curves indicates that the number of [3H]clonidine binding sites increases throughout the 17 day culture period. The maximal number of alpha 2-adrenoceptors is found during the stationary phase of growth, when cell density is high and mitotic rate low. Moreover, the use of adrenaline and clonidine as alpha 2-adrenergic agonists reveals a relationship between the number of receptors and the intensity of the biological effect associated with their stimulation (inhibition of the VIP-induced cyclic AMP accumulation).

Topics: Adenocarcinoma; Cell Line; Cell Membrane; Clonidine; Colonic Neoplasms; Cyclic AMP; Humans; Receptors, Adrenergic, alpha; Tritium; Vasoactive Intestinal Peptide

Human colonic carcinoma Caco-2 cells grown in vitro form epithelial layers of highly polarized cells. Unlike colonic adsorptive cells they possess a mucosal membrane with very limited ionic conductance, even after exposure to aldosterone. When grown on filters, Caco-2 cells were sensitive to various secretagogues; these included 10(-5) M dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and 10(-10) M vasoactive intestinal peptide, both of which, added serosally, enhanced the short-circuit current. The same applied to mucosal forskolin. Caco-2 cell sensitivity to serosal epinephrine was lower. Ion substitutions and 22Na-36Cl flux measurements indicated the possibility of secretagogue-dependent chloride secretion. Measurements on cells grown on Petri dishes and exposed to 1 mM DBcAMP for 1 h enabled detection of more profound modifications. Sustained 20-mV cell depolarization and a large reduction in the relative electrical resistance of the mucosal membrane were concomitant with a sizable decrease in 36Cl accumulation. These results suggest that Caco-2 cells, which to some extent resemble colonic crypt cells, possess the cAMP-dependent mucosal chloride conductance characteristic of secretory cells.

Topics: Bucladesine; Cell Line; Chlorides; Colforsin; Colonic Neoplasms; Diterpenes; Epithelium; Humans; Mucous Membrane; Sodium; Vasoactive Intestinal Peptide

[125I]Monoiodinated vasoactive intestinal peptide (125I-VIP) was cross-linked with human colonic adenocarcinoma cells (HT29 cells) grown as a monolayer using dithiobis(succinimidylpropionate) as cross-linking reagent. The cross-linked polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A major polypeptide of Mr = 67 000 was characterized and it behaved like a high-affinity binding site for VIP according to the following data. The concentration of native VIP (0.5 nM) giving half-maximum inhibition of 125I-VIP covalent cross-linking with this polypeptide was very similar to that giving half-maximum displacement of 125I-VIP on HT 29 cells (0.6 nM). Glucagon or insulin was unable to inhibit the labelling of the Mr-67 000 component. In our experimental conditions neither specific 125I-VIP binding nor covalent labelling was observed with monolayers of Madin Darby canine kidney epithelial cells (MDCK cells) or African green monkey kidney fibroblasts (Vero cells) while the Mr-67 000 polypeptide was also characterized with human rectal adenocarcinoma cells (HRT 18 cells), known to possess the VIP receptor. Preincubation of HT 29 cells with native VIP at 37 degrees C, before 125I-VIP binding and subsequent cross-linking reaction, decreased the labelling of the Mr-67 000 polypeptide up to 80%. Assuming one molecule of 125I-VIP cross-linked per polypeptide, we have characterized, for the first time, a major polypeptide of Mr = 64 000, which belongs to the high-affinity VIP binding site of an intestinal human cell line.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Humans; Iodine Radioisotopes; Isotope Labeling; Protein Binding; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Commitment of HT29-18 cells to enterocyte-like differentiation by glucose removal is related to a decreased capacity to generate cAMP after treatment with vasoactive intestinal peptide (VIP), forskolin or sodium fluoride. In contrast, the potency of VIP (EC50 = 1.1 - 1.3 X 10(-10) M) and the pharmacological specificity of the VIP receptor (VIP greater than rh GRF 1-43 greater than PHI greater than secretin) are unchanged during differentiation and retrodifferentiation. These results indicate that disturbances in VIP receptor-post-receptor activity, involving cell surface VIP receptors, membrane and intracellular transducers of hormonal information, occur during enterocyte-like differentiation of the HT29-18 subclone.

Topics: Adenylyl Cyclases; Cell Differentiation; Clone Cells; Colforsin; Colonic Neoplasms; Cyclic AMP; Diterpenes; Humans; Intestinal Mucosa; Intestines; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Sodium Fluoride; Vasoactive Intestinal Peptide

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.

Topics: Adenosine Triphosphate; Anions; Barium; Biological Transport; Bucladesine; Bumetanide; Calcimycin; Calcium; Cell Line; Cell Membrane; Colonic Neoplasms; Cyclic AMP; Epithelium; Humans; Hydrogen-Ion Concentration; Kinetics; Ouabain; Potassium; Quinidine; Radioisotopes; Rubidium; Vasoactive Intestinal Peptide

The human colon adenocarcinoma cell line HT-29 in culture exhibits a cyclic AMP production system highly sensitive to vasoactive intestinal peptide (VIP), making HT-29 cells a unique cultured cell system for studying the mechanism of VIP action [Laburthe, Rousset, Boissard, Chevalier, Zweibaum & Rosselin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2772-2775]. The quantitative characteristics of VIP receptors in HT-29 cells and their structural requirement and molecular size were studied. 125I-labeled VIP bound in a time-dependent manner to HT-29 cell homogenates. At equilibrium (60 min incubation at 30 degrees C), unlabelled VIP in the 0.01-10 nM concentration range competed with 125I-VIP for binding to cell homogenates. Scatchard analysis of binding data gave a straight line, indicating that VIP bound to a single population of sites with a KD of 0.12 +/- 0.02 nM and a capacity of 120 +/- 9 fmol/mg of protein. The structural requirement of these receptors was studied with peptides structurally related to VIP, either natural or synthetic. Several peptides inhibited 125I-VIP binding to HT-29 cell homogenates with the following order of potency, which is typical of the human VIP receptor: VIP (IC50 = 0.1 nM) greater than VIP-(2-28)-peptide (IC50 = 13 nM) greater than human growth hormone releasing factor (IC50 = 56 nM) greater than peptide histidine isoleucine amide (IC50 = 80 nM) greater than secretin (IC50 greater than 10 000 nM). To characterize the molecular component(s) of the VIP receptor in HT-29 cells, 125I-VIP was covalently bound to cell homogenates by using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulphate/polyacrylamide-gel autoradiographic studies of affinity-labelled cell homogenates revealed two major bands, corresponding to 125I-VIP-protein complexes of Mr 66 000 and 16 000. The labelling of the Mr-66 000 component was specific, since it was abolished by native VIP, whereas that of the Mr-16 000 component was not. Densitometric scanning of autoradiographs indicated that the labelling of the Mr-66 000 complex was inhibited by low VIP concentrations in the 0.1-10 nM range (IC50 = 0.6 nM), but was unaffected by 1 microM-glucagon or octapeptide of cholecystokinin. It was also decreased by VIP-(2-28)-peptide with a potency 1% that of VIP. Assuming that one molecule of 125I-VIP bound per molecule of protein, one protein of Mr 63 000 was identified as a component of the VIP receptor in HT-29 cells.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Growth Hormone-Releasing Hormone; Humans; Kinetics; Peptide PHI; Peptides; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Secretin; Vasoactive Intestinal Peptide

Three major forms of monoiodinated VIP (M125I-VIP) were isolated after chloramine-T iodination and HPLC purification. The iodinated tyrosine residue was located in each form of M125I-VIP using arginase C and trypsin digestion for obtaining defined fragments containing only one tyrosine residue. The HPLC isolated iodinated fragments thus obtained were used for HPLC comigration studies with iodinated synthetic C and N terminal VIP fragments and for amino acid analysis. The first two eluting peaks 1 and 2 are (M125I-Tyr10-VIP); peak 1 has an oxidized methionine; peak 3 is a (M125I-Tyr22-VIP) which also has an oxidized methionine. A reduced counterpart of peak 3 named peak 4 was isolated by further HPLC analysis. The ability of the different species of M125I-VIP to stimulate adenosine cyclic 3',5'-phosphate (cAMP) production in transformed colonic cells in culture (HT-29) was compared to that of native VIP. The mean potencies of the M125I-VIP species expressed as a percentage relative to the potency of native VIP were, peak (1): 0.98; (2): 0.84; (3): 1.38; (4): 1.48, in the range of concentrations tested (2-60 pM). The M125I-Tyr22-VIP are significantly more active than native VIP (P less than 0.01). Oxidation of methionine or iodination of tyrosine 10 does not significantly modify the biological activity of VIP. We conclude that iodination of Tyr-22 located in the apolar helical COOH-terminal of VIP increases the effectiveness of VIP interaction with its receptors. Thus the tyrosyl residue and the localized hydrophobic features of VIP are critically involved in the function of this neurotransmitter.

Topics: Adenocarcinoma; Arginase; Cell Line; Chromatography, High Pressure Liquid; Colonic Neoplasms; Cyclic AMP; Humans; Iodine Radioisotopes; Peptide Fragments; Radioisotope Dilution Technique; Structure-Activity Relationship; Trypsin; Vasoactive Intestinal Peptide

We used liposomes made with phospholipids of fatty acid chain length ranging from C12:0 to C16:0 to modify the cAMP dependent protein kinase (PK) activity of HT 29 cells induced by VIP or forskolin. Both VIP and forskolin effects were inhibited in dilauroylphosphatidylcholine (DLPC) treated cells. PK activity was slightly lowered when cells were treated by dimyristoylphosphatidylcholine (DMPC) liposomes. However neither VIP nor forskolin-induced PK activities were affected with dipalmitoylphosphatidylcholine (DPPC) liposomes. Furthermore, the binding of [125I]VIP to DLPC treated cells was drastically lowered whereas no change was observed when cells were incubated with DMPC or DPPC liposomes. On the other hand, the interaction of HT 29 cells with DLPC vesicles provoked a decrease in membrane cholesterol content with subsequent increase in membrane fluidity. These findings provide evidence that, in HT 29 cells, the mechanisms of VIP-receptor interaction and of adenylate cyclase activation is lipid dependent and is regulated by membrane fluidity.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Humans; Kinetics; Liposomes; Lysophosphatidylcholines; Membrane Fluidity; Phosphatidic Acids; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Topics: Adenocarcinoma; Casein Kinases; Cell Line; Colonic Neoplasms; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Gastrointestinal Hormones; Humans; Phosvitin; Protamine Kinase; Protein Kinases; Rectal Neoplasms; Subcellular Fractions; Vasoactive Intestinal Peptide

The mechanisms by which bacterial enterotoxins cause secretory diarrheas have been well defined, and the definitions of such mechanisms have been important in developing a consistently successful therapeutic approach. The less common secretory diarrheas, caused by the interaction of hormones of tumor origin with the gut small intestinal mucosa have also been clearly defined, and their pathogenetic mechanisms are similar to those by which the cholera and E. coli enterotoxins cause secretory diarrhea. The mechanisms by which histamine and gastrin of tumor origin cause gastric hypersecretion are less clearly delineated; secretory diarrhea caused by both of these agents can be stopped by total gastrectomy without removal of the responsible tumor. The secretory diarrhea caused by villous adenomas of the colon, which does not appear to be related to a distally produced humoral agent, results in the same picture of hypokalemic acidosis that is characteristic of the nonbacterial secretory diarrheas originating in the small intestine and is cured by resection of the responsible tumor.

Topics: Adenoma; Antigens, Bacterial; Bacterial Toxins; Cholera; Cholera Toxin; Colonic Neoplasms; Diarrhea; Enterotoxins; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Fimbriae Proteins; Hormones, Ectopic; Humans; Hydrogen-Ion Concentration; Intestinal Mucosa; Neoplasms, Hormone-Dependent; Prostaglandins; Vasoactive Intestinal Peptide; Water-Electrolyte Balance; Zollinger-Ellison Syndrome

Receptors, i.e. specific binding sites for vasoactive intestinal peptide (VIP), have been characterized in human colonic epithelial cells isolated by EDTA treatment using 125I-labeled porcine VIP. The binding was time and temperature dependent. Conditions of apparent equilibrium were obtained at 15 C after 45 min of incubation in the presence of 2.1-7.4 micrograms cell DNA-ml; these conditions minimized the degradation of the peptide and the binding sites. Native VIP competitively inhibited the binding of [125I]VIP in the range of 3 x 10(-11)-10(-7) M, and half-maximal inhibition was observed at 2 x 10(-9) M VIP. Scatchard analysis of these data was consistent with the existence of two classes of binding sites: 7.8 x 10(-9) high affinity sites/microgram DNA with a dissociation constant (Kd) of 1.4 x 10(-9) M, and 12.0 x 10(10) low affinity sites/microgram DNA with a Kd of 46 x 10(-9) M. Among the natural hormones structurally related to VIP, gastric inhibitory polypeptide (GIP) and glucagon had no effect on the binding of labeled porcine VIP. Porcine secretin inhibited [125I]VIP binding, but at doses 1000 times higher than those of porcine VIP. Studies of the coupling between the binding of VIP and the stimulation of cAMP formation indicated a nonlinear relationship between the two processes, with full activation of the cAMP-producing system with occupancy of only a limited number of the binding sites. The presence of binding sites with high affinity for VIP coupled with the cAMP production in human colonic epithelial cells support the concept that this peptide may contribute to the physiological regulation of the functions of the human colonic epithelium in normal and pathological conditions.

Topics: Binding, Competitive; Colon; Colonic Neoplasms; Cyclic AMP; Epithelium; Gastrointestinal Hormones; Humans; Hydrogen-Ion Concentration; Kinetics; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E1 (PGE1) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10(-9) M VIP promotes a rapid and specific activation of the lower Km cyclic AMP phosphodiesterase (1.7-fold); at 25 degrees C the effect is maintained for more than 15 min, while at 37 degrees C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer (Ka equals 4 x 10(-10) M) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 x 10(-7) M secretin, 10(-5) M isoproterenol and 10(-5) M PGE1; PGE2 and epinephrine are without effect. In HRT 18 cells VIP is less active (Ka equals 2 x 10(-9) M) whereas 10(-6) M PGE1, 10(-6) M PGE2 and 10(-5) M epinephrine are potent inducers of th phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenocarcinoma; Animals; Bucladesine; Catecholamines; Cells, Cultured; Colonic Neoplasms; Cyclic AMP; Enzyme Activation; Gastrointestinal Hormones; Prostaglandins E; Protein Kinases; Rectal Neoplasms; Secretin; Vasoactive Intestinal Peptide

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Cyclic AMP; Gastrointestinal Hormones; Glycogen; Humans; In Vitro Techniques; Phosphorylase a; Vasoactive Intestinal Peptide

This study was undertaken to assess the role of vasoactive intestinal peptide (VIP) in the control of cyclic adenosine 3':5'-monophosphate production in colonic tumor cells. Seven human colorectal adenocarcinoma cell lines in culture were investigated (HT-29, HRT-18, SW-480, Caco-2, CO-115, CO-125, and HCT-8R). These cell-lines had a cyclic adenosine 3':5'-monophosphate production system which was very sensitive to VIP but less so to prostaglandin E1 and/or isoproterenol. Nonintestinal human malignant epithelial cells, such as HeLa (cervix) and Caki-1 and Caki-2 (kidney), by contrast, did not respond to VIP. The dose-response relationships of malignant colorectal cells were compared to those obtained with epithelial cells of normal human colon and showed that: (a) maximal responses were observed with 0.1 micro M VIP in both malignant and normal cells; (b) half-maximal responses were elicited by VIP concentrations in the 0.3 to 2 nM range in malignant cells (1.2 nM in normal cells), thus indicating the high apparent affinity of the cells to VIP; and (c) the magnitudes of the responses (stimulated:basal ratios) were highly variable in malignant cells, ranging from 225 in HT-29 cells to 3.5 in Caco-2 cells, but were more constant, in the order of 25, in normal cells. Secretin, a VIP agonist in intestinal tissue, stimulated cyclic adenosine 3':5'-monophosphate accumulation in all colorectal cells, but with a 1000- to 5000-fold lower potency than did VIP. These results show that the VIP-sensitive adenylate cyclase system operates in malignant as well as in normal colon epithelial cells.

Topics: Adenocarcinoma; Adenylyl Cyclases; Animals; Cell Line; Colonic Neoplasms; Cyclic AMP; Gastrointestinal Hormones; HeLa Cells; Humans; Isoproterenol; Mice; Neoplasms, Experimental; Prostaglandins E; Rectal Neoplasms; Vasoactive Intestinal Peptide

Topics: Child, Preschool; Colon; Colonic Neoplasms; Ganglioneuroma; Gastrointestinal Hormones; Gastrointestinal Motility; Humans; Infant; Infant, Newborn; Intestinal Obstruction; Megacolon; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide, secretin, catecholamines and prostaglandin E1 stimulate the accumulation of cyclic AMP in HT 29 cells (see Laburthe, M. et al. (1978) Proc. Natl. Acad. Sci. U.S. 75, 2772-2775). In the present work maximal activation of protein kinases has been obtained at similar or even lower concentrations of the effectors. Maximal stimulation also requires a phosphodiesterase inhibitor. Type I and type II cyclic AMP-dependent protein kinases from basal and stimulated cells have been characterized by DEAE-Sepharose chromatography. Further identidication of the kinase has been carried out by gel electrophoresis and assay of the enzymes in the gel slabs. Comparison of the radioautography patterns of high speed supernatant lysate from basal and stimulated cells shows: First, that one type I and two type II cyclic AMP-dependent protein kinases plus one or two major and two minor cyclic AMP-independent protein kinases are present in HT 29 cells. Second, that all three holoenzymes are fully dissociated upon maximal stimulation, while the activity of the independent kinases appears unchanged.

Topics: Adenocarcinoma; Cells, Cultured; Chromatography, Gel; Colonic Neoplasms; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Epinephrine; Glucagon; Humans; Isoproterenol; Prostaglandins E; Protein Kinases; Secretin; Stimulation, Chemical; Theophylline; Vasoactive Intestinal Peptide

Human colonic adenocarcinoma plasma membranes exhibit specific receptors for VIP. Adenylate cyclase activity is stimulated by a VIP concentration as low as 10(-10) mol/1.

Topics: Adenocarcinoma; Adenylyl Cyclases; Cell Membrane; Colonic Neoplasms; Gastrointestinal Hormones; Humans; Receptors, Cell Surface; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a potent and efficient stimulator of adenosine 3':5'-cyclic monophosphate (cAMP) accumulation in a human colon carcinoma cell line, HT 29. cAMP accumulation is sensitive to a concentration of VIP as low as 3x10(-12) M. Maximum VIP-induced cAMP levels were observed with 10(-9) M VIP and are about 200 times above the basal levels. Half-maximum cAMP production was obtained at 3x10(-10) M VIP. (125)I-Labeled VIP was found to bind to HT 29 cells; this binding was competitively inhibited by concentrations of unlabeled VIP between 10(-10) and 10(-7) M. Half-maximum inhibition of binding was observed with 2x10(-9) M VIP. Secretin also stimulated cAMP accumulation in HT 29 cells, but its effectiveness was 1/1000 that of VIP. The other peptides tested at 10(-7) M, such as insulin, glucagon, bovine pancreatic polypeptide, somatostatin, octapeptide of cholecystokinin, neurotensin, and substance P, did not stimulate cAMP accumulation. Prostaglandin E(1) and catecholamines stimulated cAMP production but were 1/2.3 and 1/5.5 as efficient as VIP, respectively. Another malignant cell line from the gut, the human rectal tumor cell line HRT 18, is also sensitive to VIP. In HRT 18 cells, VIP stimulated cAMP accumulation with a maximal effect at 10(-8) M; half-maximum stimulation was observed at about 10(-9) M. These results demonstrate the presence of VIP receptors in two malignant human intestinal cell lines (HT 29 and HRT 18) in culture and provide a model for studying the action of VIP on cell proliferation.

Topics: Adenocarcinoma; Catecholamines; Cell Line; Colonic Neoplasms; Cyclic AMP; Dose-Response Relationship, Drug; Gastrointestinal Hormones; Hormones; Prostaglandins; Receptors, Cell Surface; Secretin; Vasoactive Intestinal Peptide