Pharmacology and Therapeutics - Research Publications

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    The absence of autonomic perivascular nerves in human colorectal liver metastases
    Ashraf, S ; Crowe, R ; Loizidou, MC ; Turmaine, M ; Taylor, I ; Burnstock, G (NATURE PUBLISHING GROUP, 1996-02-01)
    The peptidergic/aminergic innervation of normal liver and tumour blood vessels was investigated in order to determine vascular control with a view to improving the efficacy of hepatic arterial cytotoxic infusion in the treatment of colorectal liver metastases. Selected areas of liver metastases and macroscopically normal liver from resection specimens (n = 13) were studied using light microscope immunohistochemistry for the presence of protein gene product 9.5 (PGP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), substance P (SP) and tyrosine hydroxylase (TH). The ultrastructure of blood vessels supplying liver metastases and their perivascular innervation were also examined by transmission electron microscopy. In the normal liver, perivascular immunoreactive nerve fibres containing PGP, NPY and TH were observed around the interlobular blood vessels and along the sinusoids and the central vein of the hepatic lobule. The greatest density of immunoreactive nerve fibres was seen for PGP, followed (in decreasing order) by NPY and TH. VIP, SP and CGRP immunoreactivity was observed only in nerve bundles associated with the large interlobular blood vessels. In contrast, no perivascular immunoreactive nerves were observed in colorectal liver metastases. Electron microscopy confirmed the absence of perivascular nerves in liver metastases. In addition, it showed that the walls of these blood vessels were composed of a layer of endothelial cells surrounded by an incomplete or, very rarely in the periphery of the tumour, a complete, layer of synthetic phenotype of smooth muscle-like cells. These results imply that the blood vessels supplying liver metastases are bereft of normal neuronal regulation; whether there is a role for endothelial cell control of blood flow in these vessels is not yet known.
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    The generation of nitrite (NO(2) (-)) was used as an index of the production of nitric oxide by human and rat polymorphonuclear leukocytes (PMN) and rat peritoneal macrophages. Human peripheral blood PMN did not produce significant levels of NO(2) (-). Attempts to induce NO(2) (-) generation in human PMN by incubation with GM-CSF (1 nM), TNFalpha (0.3 nM), endotoxin (1 mug/ml) or formyl-Met-Leu-Phe (100 nM) for up to 16 h were not successful. Addition of human PMN primed by GM-CSF (1 nM) to rabbit aortic ring preparations precontracted with phenylephrine had no effect on tone. In contrast to these observations, PMN, isolated from the peritoneum of oyster glycogen treated rats, generated NO(2) (-) via a pathway sensitive to inhibition by the nitric oxide synthase inhibitor, N(G)-monomethyl L-arginine. However, peripheral blood rat PMN obtained from the same animals did not produce NO(2) (-), even during prolonged incubation for periods of up to 16 h. It is suggested that detectable NO production by PMN requires NO synthase activity to be induced either by the process of PMN migration or by exposure to certain cytokines produced locally at the site of inflammation.
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    Induction of integral membrane PAM expression in AtT-20 cells alters the storage and trafficking of POMC and PC1.
    Ciccotosto, GD ; Schiller, MR ; Eipper, BA ; Mains, RE (Rockefeller University Press, 1999-02-08)
    Peptidylglycine alpha-amidating monooxygenase (PAM) is an essential enzyme that catalyzes the COOH-terminal amidation of many neuroendocrine peptides. The bifunctional PAM protein contains an NH2-terminal monooxygenase (PHM) domain followed by a lyase (PAL) domain and a transmembrane domain. The cytosolic tail of PAM interacts with proteins that can affect cytoskeletal organization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PAM-1 expression. Upon induction, cells displayed a time- and dose-dependent increase in enzyme activity, PAM mRNA, and protein. Induction of increased PAM-1 expression produced graded changes in PAM-1 metabolism. Increased expression of PAM-1 also caused decreased immunofluorescent staining for ACTH, a product of proopiomelanocortin (POMC), and prohormone convertase 1 (PC1) in granules at the tips of processes. Expression of PAM-1 resulted in decreased ACTH and PHM secretion in response to secretagogue stimulation, and decreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using the inducible cell line model, we show that expression of integral membrane PAM alters the organization of the actin cytoskeleton. Altered cytoskeletal organization may then influence the trafficking and cleavage of lumenal proteins and eliminate the ability of AtT-20 cells to secrete ACTH in response to a secretagogue.