Soluble Vascular Adhesion Protein-1 Mediates Spermine Oxidation as Semicarbazide-Sensitive Amine Oxidase: Possible Role in Proliferative Diabetic Retinopathy.

Purpose/Aim of the study: To explore the possible role of vascular adhesion protein-1 (VAP-1) via its enzymatic function as a semicarbazide-sensitive amine oxidase (SSAO) in the pathogenesis of proliferative diabetic retinopathy (PDR). MATERIALS AND METHODS: The levels of soluble VAP-1/SSAO and the unsaturated aldehyde acrolein (ACR)-conjugated protein, Nε-(3-formyl-3, 4-dehydropiperidino) lysine adduct (FDP-Lys), were measured in vitreous fluid samples of PDR and non-diabetic patients using ELISA. Recombinant human VAP-1/SSAO (rhVAP-1/SSAO) was incubated with spermine, with or without semicarbazide or RTU-1096 (a specific inhibitor for VAP-1/SSAO). Immunofluorescence assays were performed to assess the localization of VAP-1/SSAO and FDP-Lys in fibrovascular tissues from patients with PDR. The impact of ACR on cultured retinal capillary endothelial cells was assessed using a cell viability assay and total glutathione (GSH) measurements. RESULTS: The levels of sVAP-1/SSAO and FDP-Lys were elevated in the vitreous fluid of patients with PDR. Incubation of rhVAP-1 with spermine resulted in the generation of hydrogen peroxide and FDP-Lys and the production was inhibited by semicarbazide and RTU-1096. In fibrovascular tissues, FDP-Lys and VAP-1/SSAO were present in endothelial cells. ACR stimulation reduced GSH levels in the cultured endothelial cells in a dose-dependent manner and caused cellular toxicity. CONCLUSIONS: Our results indicate the pathological role of sVAP-1/SSAO to generate hydrogen peroxide and toxic aldehyde ACR, both of which are associated with oxidative stress, as a consequence of spermine oxidation in eyes with PDR.

Implication for functions of the ectopic adipocyte copper amine oxidase (AOC3) from purified enzyme and cell-based kinetic studies.

AOC3 is highly expressed in adipocytes and smooth muscle cells, but its function in these cells is currently unknown. The in vivo substrate(s) of AOC3 is/are also unknown, but could provide an invaluable clue to the enzyme's function. Expression of untagged, soluble human AOC3 in insect cells provides a relatively simple means of obtaining pure enzyme. Characterization of enzyme indicates a 6% titer for the active site 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor and corrected k(cat) values as high as 7 s(-1). Substrate kinetic profiling shows that the enzyme accepts a variety of primary amines with different chemical features, including nonphysiological branched-chain and aliphatic amines, with measured k(cat)/K(m) values between 10(2) and 10(4) M(-1) s(-1). K(m)(O(2)) approximates the partial pressure of oxygen found in the interstitial space. Comparison of the properties of purified murine to human enzyme indicates k(cat)/K(m) values that are within 3 to 4-fold, with the exception of methylamine and aminoacetone that are ca. 10-fold more active with human AOC3. With drug development efforts investigating AOC3 as an anti-inflammatory target, these studies suggest that caution is called for when screening the efficacy of inhibitors designed against human enzymes in non-transgenic mouse models. Differentiated murine 3T3-L1 adipocytes show a uniform distribution of AOC3 on the cell surface and whole cell K(m) values that are reasonably close to values measured using purified enzymes. The latter studies support a relevance of the kinetic parameters measured with isolated AOC3 variants to adipocyte function. From our studies, a number of possible substrates with relatively high k(cat)/K(m) have been discovered, including dopamine and cysteamine, which may implicate a role for adipocyte AOC3 in insulin-signaling and fatty acid metabolism, respectively. Finally, the demonstrated AOC3 turnover of primary amines that are non-native to human tissue suggests possible roles for the adipocyte enzyme in subcutaneous bacterial infiltration and obesity.

Preliminary studies of the effects of vascular adhesion protein-1 inhibitors on experimental corneal neovascularization.

Vascular adhesion protein-1 (VAP-1) controls the adhesion of lymphocytes to endothelial cells and is upregulated at sites of inflammation. Moreover, it expresses amine oxidase activity, due to the sequence identity with semicarbazide-sensitive amine oxidase. Recent studies indicate a significant role for VAP-1 in neovascularization, besides its contribution to inflammation. Pathological blood vessel development in severe ocular diseases (such as diabetes, age-related macula degeneration, trauma and infections) might lead to decreased visual acuity and finally to blindness, yet there is no clear consensus as to its appropriate treatment. In the present case study, the effects of two VAP-1 inhibitors on experimentally induced corneal neovascularization in rabbits were compared with the effects of a known inhibitor of angiogenesis, bevacizumab, an anti-vascular endothelial growth factor antibody. In accordance with recent literature data, the results of the preliminary study reported here indicate that the administration of VAP-1 inhibitors is a potentially valuable therapeutic option in the treatment of corneal neovascularization.

Estrogen regulates amiloride-binding protein 1 through CCAAT/enhancer-binding protein-beta in mouse uterus during embryo implantation and decidualization.

Embryo implantation is an intricate interaction between receptive uterus and active blastocyst. The mechanism underlying embryo implantation is still unknown. Although histamine and putrescine are important for embryo implantation and decidualization, excess amount of histamine and putrescine is harmful. Amiloride binding protein 1 (Abp1) is a membrane-associated amine oxidase and mainly metabolizes histamine and putrescine. In this study, we first showed that Abp1 is strongly expressed in the decidua on d 5-8 of pregnancy. Abp1 expression is not detected during pseudopregnancy and under delayed implantation but is detected after estrogen activation. Because Abp1 is mainly localized in the decidua and also strongly expressed during in vitro decidualization, Abp1 might play a role during mouse decidualization. The regulation of estrogen on Abp1 is mediated by transcription factor CCAAT/enhancer-binding protein-ß. Abp1 expression is also regulated by cAMP, bone morphogenetic protein 2, and ERK1/2. Abp1 may be essential for mouse embryo implantation and decidualization.

Vascular adhesion protein-1 enhances tumor growth by supporting recruitment of Gr-1+CD11b+ myeloid cells into tumors.

Cancer growth is regulated by several nonmalignant cell types, such as leukocytes and endothelial cells, which reside in the stroma of the tumor. Vascular adhesion protein-1 (VAP-1) is an amine oxidase enzyme that is expressed on the surface of endothelial cells. It supports leukocyte traffic into inflamed tissues, but nothing is known about its possible role in cancer biology in vivo. Here, we report that B16 melanoma and EL-4 lymphoma remain smaller in VAP-1-deficient mice than in wild-type controls. We found an unexpected defect in tumor angiogenesis in the absence of VAP-1. VAP-1 also selectively enhanced the recruitment of Gr-1+CD11b+ myeloid cells into the tumors. Generation of mice expressing enzymatically inactive VAP-1 showed that the oxidase activity of VAP-1 was necessary to support neoangiogenesis, myeloid cell recruitment, and tumor growth in vivo. These data describe VAP-1 as the first adhesion molecule known to be involved in the recruitment of Gr-1+CD11b+ myeloid cells into tumors. They also suggest that VAP-1 is a potential new tool for immunotherapy of tumors that could be exploited to reduce tumor burden by controlling the traffic of Gr-1+CD11b+ myeloid cells.

Vascular adhesion protein-1 blockade suppresses choroidal neovascularization.

Vascular adhesion protein-1 (VAP-1) is an endothelial cell adhesion molecule involved in leukocyte recruitment. Leukocytes and, in particular, macrophages play an important role in the development of choroidal neovascularization (CNV), an integral component of age-related macular degeneration (AMD). Previously, we showed a role for VAP-1 in ocular inflammation. Here, we investigate the expression of VAP-1 in the choroid and its role in CNV development. VAP-1 was expressed in the choroid, exclusively in the vessels, and colocalized in the vessels of the CNV lesions. VAP-1 blockade with a novel and specific inhibitor significantly decreased CNV size, fluorescent angiographic leakage, and the accumulation of macrophages in the CNV lesions. Furthermore, VAP-1 blockade significantly reduced the expression of inflammation-associated molecules such as tumor necrosis factor (TNF) -alpha, monocyte chemoattractant protein (MCP) -1, and intercellular adhesion molecule (ICAM) -1. This work provides evidence for an important role of VAP-1 in the recruitment of macrophages to CNV lesions, establishing a novel link between VAP-1 and angiogenesis. Inhibition of VAP-1 may become a new therapeutic strategy in the treatment of AMD.

Anticancer drugs and hyperthermia enhance cytotoxicity induced by polyamine enzymatic oxidation products.

A correlation between regulation of cell proliferation and polyamine metabolism is described. The latter can enter protein synthesis through the modification of eukaryotic initiation factor 5A (eIF5A) and the formation of the peculiar amino acid hypusine. Specific inhibitors of hypusine formation induce apoptosis that can be potentiated by the combination with cytokines such as interferonalpha (IFNalpha) that itself decreases hypusine synthesis. We have also demonstrated that the concomitant treatment of cancer cells with IFNalpha and the protein synthesis inhibitor fusion protein TGFalpha/Pseudomonas Aeruginosa toxin synergize in inducing cancer cell growth inhibition. Another way used by polyamines to induce apoptosis is the generation of intracellular oxidative stress through the interaction with bovine serum amine oxidase (BSAO). This enzyme used simultaneously to spermine induces apoptosis, necrosis, inhibition of cell proliferation and inhibition of DNA and protein synthesis in several cell types. The enzymatic oxidation products of polyamine, H2O2 and aldehyde(s) cause these effects. We have recently found that the cytotoxicity of anti-cancer agents, either etoposide or docetaxel, in cancer cells is potentiated in the presence of BSAO/Spermine. In conclusion, polyamine metabolites could be useful in the design of new therapeutic strategies.

Involvement of semicarbazide-sensitive amine oxidase-mediated deamination in lipopolysaccharide-induced pulmonary inflammation.

Semicarbazide-sensitive amine oxidase (SSAO) resides on the vascular endothelium and smooth muscle cell surface and is capable of deaminating short chain aliphatic amines and producing toxic aldehydes and hydrogen peroxide. The enzyme, also known as a vascular adhesion protein-1, is involved in the inflammation process. This intriguing protein with dual functions is increased in the serum of diabetic and heart failure patients. In the present study we assessed the involvement of SSAO in a lipopolysaccharide-induced pulmonary inflammation model using transgenic mice that overexpress human vascular adhesion protein-1. Overexpression of SSAO activity increased the formation of protein-formaldehyde deposits in tissues. Lysine residues of proteins were the primary targets for cross-linkage with formaldehyde derived from deamination of methylamine. Lipo-polysaccharide-induced increases in inflammatory cells in the bronchoalveolar lavage (BAL) fluid were significantly higher in the transgenic than in the nontransgenic mice. BAL cell counts were also higher in the untreated transgenic than in nontransgenic mice. Blocking SSAO activity with a selective inhibitor significantly reduced the number of neutrophils as well as levels of macrophage inflammatory protein-1alpha, granulocyte colony-stimulating factor, tumor necrosis factor-alpha, and interleukin-6 in the BAL fluid. Inhalation of methylamine also increased BAL neutrophil counts. Together, these results suggest a role for SSAO-mediated deamination in pulmonary inflammation.

Vascular adhesion protein-1 plays an important role in postischemic inflammation and neuropathology in diabetic, estrogen-treated ovariectomized female rats subjected to transient forebrain ischemia.

Endothelial vascular adhesion protein-1 (VAP-1) facilitates leukocyte adhesion and infiltration. This relates partly to the function of VAP-1 as a semicarbazide-sensitive amine oxidase (SSAO). We examined the effects of VAP-1/SSAO inhibition [via LJP-1207 (N'-(2-phenyl-allyl)-hydrazine hydrochloride)] on pial venular leukocyte adhesion and infiltration (at 2-10 h of reperfusion) and neuropathology (at 72 h of reperfusion) after transient forebrain ischemia (TFI). A model associated with increased postischemic inflammation was used-i.e., diabetic ovariectomized (OVX) female rats given chronic estrogen replacement therapy (ERT). We compared rats treated, either at the onset or at 6 h of reperfusion, with saline or LJP-1207. Additional rats, rendered neutropenic 24 h before TFI, were studied. In saline-treated controls, intravascular accumulation of adherent leukocytes gradually increased, reaching 15 to 20% of the venular area, at which point neutrophil infiltration commenced (at approximately 6 h). In the rats given LJP-1207 at the onset of reperfusion, limited neutrophil adhesion ( approximately 5% maximum) and no infiltration were observed. These results generally paralleled those in neutropenic rats. In rats treated at 6 h of reperfusion, the pattern of neutrophil adhesion was similar to that of the saline-treated group up to 6 h, but further infiltration was essentially prevented. Neurologic outcomes and histopathology were similar to one another in the LJP-1207-treated and neutropenic groups and significantly improved over those in saline-treated controls. Thus, VAP-1-mediated post-TFI leukocyte adhesion/infiltration in diabetic OVX females given chronic ERT contributes substantially to neuropathology. One implication is that specifically preventing leukocyte infiltration provides a substantial measure of neuroprotection. This could explain the finding of LJP-1207 having at least a 6-h therapeutic window in this model.

Carbohydrates located on the top of the "cap" contribute to the adhesive and enzymatic functions of vascular adhesion protein-1.

Vascular adhesion protein 1 (VAP-1) is an endothelial adhesion molecule with an enzymatic activity. It deaminates biogenic amines, resulting in the formation of aldehydes and hydrogen peroxide. During the enzymatic reaction a transient Schiff base is formed between endothelial VAP-1 and its leukocytic ligand, and this interaction is important for lymphocyte adhesion. VAP-1 monomer has six potential N-linked, and three putative O-linked glycosylation sites and an SSSS sequence potentially forming an attachment site for an adjacent O-linked site. In this work we modeled the carbohydrate decorations on a structural model of VAP-1, and studied which of those potential glycosylation sites are utilized, and whether those decorations accessible to a lymphocyte ligand are important in lymphocyte adhesion and enzymatic activity of VAP-1. We show that, unlike the O-linked attachment sites, all six N-linked glycosylation sites are in use. Furthermore, mutation of the N-linked attachment sites strategically located on the top of the molecule reduces lymphocyte adhesion in non-static conditions, and enhances the catalytic activity of membrane-bound human VAP-1 in static conditions, suggesting that glycosylation regulates the functional properties of VAP-1.

Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells.

Vascular adhesion protein-1 (VAP-1) is an amine oxidase and adhesion receptor that is expressed by endothelium in the human liver. The hepatic sinusoids are perfused by blood at low flow rates, and sinusoidal endothelium lacks selectin expression and has low levels of CD31, suggesting that VAP-1 may play a specific role in lymphocyte recruitment to the liver. In support of this we now report the constitutive expression of VAP-1 on human hepatic sinusoidal endothelial cells (HSEC) in vitro and demonstrate that VAP-1 supports adhesion and transmigration of lymphocytes across these cells under physiological shear stress. These are the first studies to report the function of VAP-1 on primary human endothelial cells. Under static conditions lymphocyte adhesion to unstimulated HSEC was dependent on VAP-1 and ICAM-2, whereas adhesion to TNF-alpha-stimulated HSEC was dependent on ICAM-1, VCAM-1, and VAP-1. Under conditions of flow, blocking VAP-1 reduced lymphocyte adhesion to TNF-alpha-treated HSEC by 50% and significantly reduced the proportion of adherent lymphocytes that transmigrated across cytokine or LPS-activated endothelium. In addition, inhibition of the amine oxidase activity of VAP-1 reduced both adhesion and transmigration of lymphocytes to a level similar to that seen with VAP-1 Ab. Thus, VAP-1 can support transendothelial migration as well as adhesion, and both functions are dependent on its enzymatic activity. In the absence of selectins and CD31, VAP-1 may play a specific role in lymphocyte recruitment via hepatic sinusoidal endothelium. Moreover, since VAP-1 is induced on nonhepatic endothelium in response to inflammation, its ability to support lymphocyte transendothelial migration may be an important systemic function of VAP-1.

Vascular adhesion protein 1 mediates binding of immunotherapeutic effector cells to tumor endothelium.

Tumor-infiltrating lymphocytes (TIL) can be used as an immunotherapeutic tool to treat cancer. Success of this therapy depends on the homing and killing capacity of in vitro-activated and -expanded TIL. Vascular adhesion protein 1 (VAP-1) is an endothelial molecule that mediates binding of lymphocytes to vessels of inflamed tissue. Here, we studied whether VAP-1 is involved in binding of TIL, lymphokine-activated killer (LAK) cells, and NK cells to vasculature of the cancer tissue. We demonstrated that VAP-1 is expressed on the endothelium of cancer vasculature. The intensity and number of positive vessels varied greatly between the individual specimens, but it did not correlate with the histological grade of the cancer. Using an in vitro adhesion assay we showed that VAP-1 mediates adhesion of TIL, LAK, and NK cells to cancer vasculature. Treatment of the tumor sections with anti-VAP-1 Abs diminished the number of adhesive cells by 60%. When binding of different effector cell types was compared, it was evident that different cancer tissues supported the adhesion of TIL to a variable extent and LAK cells were more adhesive than TIL and NK cells to tumor vasculature. These data suggest that VAP-1 is an important interplayer in the antitumor response. Thus, by up-regulating the expression of VAP-1 in tumor vasculature, it can be possible to improve the effectiveness of TIL therapy.

Human leukocyte subpopulations from inflamed gut bind to joint vasculature using distinct sets of adhesion molecules.

Reactive arthritis can be triggered by inflammatory bowel diseases. We hypothesized that migration of mucosal immune cells from inflamed gut to joints could contribute to the development of reactive arthritis. Here we isolated gut-derived leukocytes from patients with Crohn's disease and ulcerative colitis. Using function-blocking mAbs and in vitro frozen section adhesion assays we studied whether these cells bind to synovial vessels and which molecules mediate the interaction. The results showed that mucosal leukocytes from inflammatory bowel diseased gut bind well to venules in synovial membrane. Small intestinal lymphocytes adhered to synovial vessels using multiple homing receptors and their corresponding endothelial ligands (CD18-ICAM-1, alpha(4)beta(7)/alpha(4)beta(1)-integrin-VCAM-1, L-selectin-peripheral lymph node addressins, and CD44). Of these, only ICAM-1 significantly supported binding of immunoblasts. In contrast, P-selectin glycoprotein ligand-1-P-selectin interaction accounted for practically all synovial adherence of mucosal macrophages. In addition, blocking of vascular adhesion protein-1 significantly inhibited binding of all these leukocyte subsets to joint vessels. We conclude that different leukocyte populations derived from inflamed gut bind avidly to synovial vessels using distinct repertoire of adhesion molecules, suggesting that their recirculation may contribute to the development of reactive arthritis in inflammatory bowel diseases.

Human vascular adhesion protein-1 in smooth muscle cells.

Human vascular adhesion protein-1 (VAP-1) is a dual-function molecule with adhesive and enzymatic properties. In addition to synthesis in endothelial cells, where it mediates lymphocyte binding, VAP-1 is expressed in smooth muscle cells. Here we studied the expression, biochemical structure, and function of VAP-1 in muscle cells and compared it to those in endothelial cells. VAP-1 is expressed on the plasma membrane of all types of smooth muscle cells, but it is completely absent from cardiac and skeletal muscle cells. In tumors, VAP-1 is retained on all leiomyoma cells, whereas it is lost in half of leiomyosarcoma samples. In smooth muscle VAP-1 predominantly exists as a approximately 165-kd homodimeric glycoprotein, but a trimeric (approximately 250 kd) form of VAP-1 is also found. It contains N-linked oligosaccharide side chains and abundant sialic acid decorations. In comparison, in endothelial cells dimeric VAP-1 is larger, no trimeric forms are found, and VAP-1 does not have N-glycanase-sensitive oligosaccharides. Unlike endothelial VAP-1, VAP-1 localized on smooth muscle cells does not support binding of lymphocytes. Instead, it deaminates exogenous and endogenous primary amines. In conclusion, VAP-1 in smooth muscle cells is structurally and functionally distinct from VAP-1 present on endothelial cells.

Circulating form of human vascular adhesion protein-1 (VAP-1): increased serum levels in inflammatory liver diseases.

Vascular adhesion protein-1 (VAP-1) is a dimeric 170-kDa endothelial transmembrane molecule that under normal conditions is most strongly expressed on the high endothelial venules of peripheral lymph nodes and on hepatic endothelia. It is a glycoprotein that mediates tissue-selective lymphocyte adhesion in a sialic acid-dependent manner. In this study, we report the detection of a soluble form of VAP-1 in circulation. We developed a quantitative sandwich ELISA using novel anti-VAP-1 mAbs and used it to determine the levels of soluble VAP-1 (sVAP-1) in the serum of healthy individuals and in patients with inflammatory diseases. In healthy persons, circulating sVAP-1 concentrations were 49 to 138 ng/ml. Immunoblotting studies revealed that the apparent molecular mass of dimeric sVAP-1 is slightly (approximately 10 kDa) higher than that of transmembrane VAP-1 under nonreducing conditions. In contrast, the electrophoretic mobilities of monomeric sVAP-1 and transmembrane VAP-1 were similar after reduction and boiling. Adhesion assays showed that the circulating sVAP-1 modulates lymphocyte binding to endothelial cells. Inflammation can cause an elevation of serum sVAP-1 levels, because sVAP-1 concentrations in patients with certain liver diseases were two- to fourfold higher than those in normal individuals. In contrast, rheumatoid arthritis and inflammatory bowel diseases were not associated with elevated levels of sVAP-1. These findings indicate that there is a functionally active, soluble form of VAP-1 in circulation and suggest that the serum level of sVAP-1 might be a useful marker of disease activity in inflammatory liver diseases.

Role of polyamines in the early adaptive response to jejunectomy in the rat: effect of DFMO on the ileal villus:crypt axis.

To study the role of the polyamines putrescine, spermidine and spermine and of the enzymes controlling their synthesis (ornithine decarboxylase; ODC) and degradation (diamine oxidase; DAO) along the villus:crypt axis at the crucial early stage of the ileal adaptive response to jejunectomy, we measured polyamine concentrations and the activities of ODC, DAO and alkaline phosphatase (a marker of enterocyte maturity) in epithelial cells isolated by the Weiser technique from villus tips, mid villi, lower villi and crypts 4 days after surgery in transected control (TRC) and jejunectomised rats untreated or given the specific ODC blocker, alpha-difluoromethyl ornithine (DFMO, 2% in drinking water beginning 3 days before surgery). In the TRCs, there was a diminishing villus tip-to-crypt gradient not only in alkaline phosphatase but also in ODC and DAO activities. After jejunectomy, there were up to 93% increases in mean enterocyte ODC activity when compared with the corresponding cell fractions from the TRCs, but in both the control and jejunectomised rats, DFMO treatment markedly inhibited ODC activity (p less than 0.05-0.01) and reduced spermidine and particularly putrescine concentrations (p less than 0.005-0.001) in all four cell fractions. Only 4 days post-operation, jejunectomy stimulated a significant increase in ileal wet weight but DFMO treatment completely prevented this adaptive response and significantly reduced segmental intestinal weight (mg/cm) in the TRCs. These results (i) extend our knowledge of polyamines and related enzymes along the villus:crypt gradient in the normal intestine, (ii) provides the first data on these variables after resection, and (iii) lend further support to the hypothesis that changes in enterocyte ODC activity and in putrescine and spermidine concentrations play an important role in initiating the ileal adaptive response to proximal small bowel resection in the rat.

Histamine-mediated hyposmotic stimulation of gastric acid secretion.

Gastric glands incubated in hyposmotic medium (200 mOsm) accumulated aminopyrine, a measure of acid secretion, to the same extent as that of paired glands in isomotic medium containing histamine (10(-4) M). These maximal responses to hyposmolality and histamine were not additive. The hyposmotic response peaked earlier than the histamine response. Hyposmotic stimulation was nearly abolished by preincubation of the glands with metiamide and cimetidine, H-2 histamine antagonists. In the presence of histaminase, no hyposmotic stimulation occurred. The response to forskolin, a stimulant of adenylate cyclase, was equivalent in hyposmotic and isosmotic media. These results indicate that hyposmolality releases histamine from a paracrine cell in the gastric gland and that histamine binds to H-2 receptors on the parietal cell to initiate a cyclic AMP-mediated stimulation of acid secretion.

Hormones and polyamines in intestinal and pancreatic adaptation.

The evidence for and against an enteropancreatic trophic axis is reviewed. Luminal nutrition is essential for the maintenance of normal intestinal mucosal, and exocrine pancreatic, structure and function. Exclusion of luminal nutrition leads to mucosal hypoplasia and hypofunction with similar changes in the pancreas. The trophic effect of luminal nutrition may be mediated through the release of regulatory peptides with endocrine or paracrine effects. Enteroglucagon is the strongest candidate for the role of 'enterotrophin' while cholecystokinin (CCK) markedly influences pancreatic growth. Thus, CCK not only stimulates exocrine pancreatic secretion but makes acinar cells divide and the pancreas grow. The cellular mechanisms whereby trophic peptides influence normal and adaptive growth are also discussed with emphasis on polyamines (putrescine, spermidine and spermine) and the key enzymes controlling their synthesis (ornithine decarboxylase; ODC) and degradation (diamine oxidase; DAO). When polyamine synthesis is blocked with the ODC inhibitor, difluoromethyl ornithine (DFMO), the adaptive intestinal hyperplasia of pancreatico-biliary diversion is either inhibited or completely prevented. A proposed sequence of events might be as follows: luminal nutrients, particularly long-chain fats, reach the ileum and colon and stimulate increased enteroglucagon release. Enteroglucagon binds to cell receptors and triggers an intracellular cascade involving ODC and the polyamines, which, in turn, stimulate RNA polymerase, DNA, RNA and protein synthesis, cell division, and adaptive tissue growth.