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N Bunnett

Publications and source records attributed to N Bunnett.

9 recordsLinked to original sources
PubMed2003

Protease-activated receptor 2: activation, signalling and function.

PARs (protease-activated receptors) are a family of four G-protein-coupled receptors for proteases from the circulation, inflammatory cells and epithelial tissues. This report focuses on PAR(2), which plays an important role in inflammation and pain. Pancreatic (trypsin I and II) and extrapancreatic (trypsin IV) trypsins, mast cell tryptase and coagulation factors VIIa and Xa cleave and activate PAR(2). Proteases cleave PAR(2) to expose a tethered ligand that binds to the cleaved receptor. Despite this irreversible activation, PAR(2) signalling is attenuated by beta-arrestin-mediated desensitization and endocytosis, and by lysosomal targeting and degradation, which requires ubiquitination of PAR(2). beta-Arrestins also act as scaffolds for the assembly of multi-protein signalling complexes that determine the location and function of activated mitogen-activated protein kinases. Observations of PAR(2)-deficient mice support a role for PAR(2) in inflammation, and many of the effects of PAR(2) activators promote inflammation. Inflammation is mediated in part by activation of PAR(2) in the peripheral nervous system, which results in neurogenic inflammation and hyperalgesia.

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PubMed2001

Apical and basolateral EGF receptors regulate gastric mucosal paracellular permeability.

Previous studies found that monolayers formed from canine oxyntic epithelial cells in primary culture displayed remarkable resistance to apical acidification and both mitogenic and migratory responses to epidermal growth factor (EGF) treatment. In our present studies, we found that EGF increased transepithelial resistance (TER) but not short-circuit current in these monolayers. Parallel effects of EGF on decreasing mannitol flux and increasing TER implicate direct regulation of paracellular permeability. EGF acting at either apical and basolateral receptors rapidly increased TER, but the apical response was sustained whereas the basolateral response was transient. (125)I-labeled EGF binding revealed specific apical binding, but receptor numbers were 25-fold lower than on the basolateral surface. Both apical and basolateral EGF activated tyrosine phosphorylation of EGF receptors (EGFR), beta-catenin, and cellular substrate as evident on confocal microscopy. Although apical EGF activated a lesser degree of receptor autophosphorylation than basolateral EGF, phosphorylation of beta-catenin was equally prominent with apical and basolateral receptor activation. Together, these findings indicate that functional apical and basolateral EGFR exist on primary canine gastric epithelial cells and that these receptors regulate paracellular permeability. The sustained effect of apical EGFR activation and prominent phosphorylation of beta-catenin suggest that apical EGFR may play a key role in this regulation.

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PubMed1997

Mechanisms attenuating cellular responses to neuropeptides: extracellular degradation of ligands and desensitization of receptors.

Neuropeptides make up one of the largest and functionally most diverse groups of signaling molecules. They exert their effects by interacting with members of the large family of G-protein-coupled receptors, which transmit information about the extracellular environment to the interior of the cell by interacting with the heterotrimeric G-proteins. Cellular responses to neuropeptides are usually rapidly attenuated. Mechanisms of signal attenuation include removal of peptides from the extracellular fluid and receptor desensitization. Peptides are removed from the extracellular fluid principally by enzymatic degradation by cell surface enzymes, exemplified by neutral endopeptidase. Receptor desensitization is mediated by receptor phosphorylation by G-protein receptor kinases and second messenger kinases, interaction of receptors with arrestins, and consequent receptor uncoupling from G-proteins. Peptides also induce endocytosis of their receptors, which may contribute to desensitization by depleting the cell surface of high-affinity receptors. Recycling and processing of internalized receptors, which include dissociation of receptors from their ligands and receptor dephosphorylation, contribute to resensitization of cellular responses. These regulatory mechanisms are important for they determine the ability of cells to respond to agonists, and defects may result in uncontrolled stimulation of cells, which could cause disease. A greater understanding of the processes that modulate signaling by neuropeptides may lead to the development of novel receptor antagonists and agonists and help to explain the mechanism of drug tolerance.

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PubMed1997

The control of microvascular permeability and blood pressure by neutral endopeptidase.

Plasma extravasation from postcapillary venules is one of the earliest steps of inflammation. Substance P (SP) and bradykinin (BK) mediate extravasation and cause hypotension. The cell-surface enzyme neutral endopeptidase (NEP) inactivates both peptides. Thus, absence of NEP may predispose development of inflammation and hypotension. We examined these possibilities in mice in which the NEP gene was deleted by homologous recombination. There was widespread basal plasma extravasation in postcapillary venular endothelia in NEP-/- mice, which was reversed by recombinant NEP and antagonists of SP (NK1) and BK (B2) receptors. Mean arterial blood pressure was 20% lower in NEP-/- animals, but this was unaffected by reintroduction of recombinant NEP and the kinin receptor antagonists. The hypotension was also independent of nitric oxide (NO), because NEP-/- mice treated with a NO synthase inhibitor remained hypotensive relative to the wild type. Thus, NEP has important roles in regulating basal microvascular permeability by degrading SP and BK, and may regulate blood pressure set point through a mechanism that is independent of SP, BK and NO. The use of NEP antagonists as candidate drugs in cardiovascular disease is suggested by the blood pressure data reported herein.

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PubMed1997

Substance P and bradykinin stimulate plasma extravasation in the mouse gastrointestinal tract and pancreas.

Neurogenic inflammation is mediated by release of tachykinins from sensory nerves, which stimulate plasma extravasation from postcapillary venules. Because there are conflicting results regarding the importance of neurogenic inflammation in the gastrointestinal tract, we quantified plasma extravasation using Evans blue and identified sites of the leak using Monastral blue in the mouse. Substance P and bradykinin stimulated extravasation from postcapillary venules in the stomach, small and large intestine, pancreas, urinary bladder, trachea, and skin by two- to sevenfold by interacting with NK1 and B2 receptors, respectively. Stimulation of sensory nerves with capsaicin also induced extravasation. Capsaicin- and bradykinin-stimulated extravasation was attenuated by an NK1-receptor antagonist and is thus mediated by release of tachykinins and activation of the NK1 receptor. We conclude that 1) substance P stimulates extravasation in the gastrointestinal tract and pancreas of mice by interacting with the NK1 receptors, and 2) capsaicin and bradykinin induce plasma extravasation by stimulating tachykinin release from sensory nerves. Thus neurogenic mechanisms mediate inflammation in the gastrointestinal tract and pancreas of the mouse.

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PubMed1987

Degradation of endogenous heptadecapeptide gastrin by endopeptidase 24.11 in the pig.

Hydrolysis of heptadecapeptide gastrin (G-17) by endopeptidase 24.11 (EC 3.4.24.11) was studied in vivo and in vitro in the pig. Ion exchange chromatography and radioimmunoassay with three region-specific antisera were used to identify the products of porcine G-17 degradation. Incubation of antral extracts with pure endopeptidase 24.11 resulted in a substantial loss of intact G-17: 80% C-terminal immunoreactivity was lost in 60 min. This hydrolysis was completely inhibited by phosphoramidon, which is a specific inhibitor of endopeptidase 24.11. In antral extracts G-17 accounted for greater than 95% of total C-terminal immunoreactivity, compared with less than 60% C-terminal immunoreactivity in the gastric venous outflow; shorter C-terminal forms comprised the major part of the remaining immunoreactivity. After infusion of phosphoramidon, the concentration of intact G-17 was increased, and there was a corresponding reduction in the concentration of other C-terminal immunoreactive fragments. We conclude that endopeptidase 24.11 degrades G-17 in vitro and in vivo and may be responsible for the generation of C-terminal fragments from G-17 after secretion from the porcine antral mucosa.

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PubMed1986

Chromatographic and immunochemical studies on postsecretory processing of gastrin in the pig.

Immunoreactive forms of gastrin in antral mucosal extracts and in gastric venous plasma of the pig were compared using ion-exchange and gel-filtration chromatography and radioimmunoassay using three region-specific antiserums. In antral mucosal extracts, gastrin heptadecapeptide (G-17) accounted for over 90% of the total C-terminal immunoreactivity, but in gastric venous plasma it accounted for only 47% of total C-terminal immunoreactivity. The remaining C-terminal immunoreactivity was accounted for by shorter C-terminal forms. Unsulfated and sulfated G-17 contributed 44.1 and 49.2%, respectively, of C-terminal immunoreactivity in antral mucosa. In contrast, they contributed 14 and 30%, respectively, to total C-terminal immunoreactivity in gastric venous plasma. Incubation of antral extracts with plasma in vitro resulted in a slow loss of intact G-17 (32.3% in 60 min) that could not account for the production of C-terminal fragments in vivo. Moreover, when antral extracts were infused into the gastroepiploic artery, over 90% of the gastrin present in the antral venous outflow corresponded to G-17. These observations suggest that it is unlikely that enzymes involved in the generation of the C-terminal forms are located either in blood or on the luminal side of the endothelial membrane. It is proposed, then, that antral gastrin is converted into shorter C-terminal fragments at or before the time it enters the circulation and that the major storage forms of gastrin in tissue account for less than 50% of the material in the gastric venous outflow.

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