Faculty Bibliography

Proteinase-activated receptor 2 (PAR-2) is a recently characterized G-protein coupled receptor that is cleaved and activated by pancreatic trypsin. Trypsin is usually considered a digestive enzyme in the intestinal lumen. We examined the hypothesis that trypsin, at concentrations normally present in the lumen of the small intestine, is also a signaling molecule that specifically regulates enterocytes by activating PAR-2. PAR-2 mRNA was highly expressed in the mucosa of the small intestine and in an enterocyte cell line. Immunoreactive PAR-2 was detected at the apical membrane of enterocytes, where it could be cleaved by luminal trypsin. Physiological concentrations of pancreatic trypsin and a peptide corresponding to the tethered ligand of PAR-2, which is exposed by trypsin cleavage, stimulated generation of inositol 1,4,5-trisphosphate, arachidonic acid release, and secretion of prostaglandin E2 and F1alpha from enterocytes and a transfected cell line. Application of trypsin to the apical membrane of enterocytes and to the mucosal surface of everted sacs of jejunum also stimulated prostaglandin E2 secretion. Thus, luminal trypsin activates PAR-2 at the apical membrane of enterocytes to stimulate secretion of eicosanoids, which regulate multiple cell types in a paracrine and autocrine manner. We conclude that trypsin is a signaling molecule that specifically regulates enterocytes by triggering PAR-2.

There is increasing experimental evidence that the neurologic system can directly participate in cutaneous inflammation and wound healing. Recent studies indicate that neuropeptides released by cutaneous nerves such as c-fibers can activate a number of target cells including keratinocytes, Langerhans cells, mast cells, and endothelial cells. One such neuropeptide, substance P (SP), is able to specifically bind to murine and human keratinocytes and induce the release of cytokines such as interleukin 1 (IL-1). Other studies demonstrate that SP can also activate mast cells to produce the potent pro-inflammatory cytokine tumor necrosis factor alpha (TNF alpha). More recently, we examined the effect of cutaneous neuropeptides on human dermal microvascular endothelial cell (HDMEC) activities. Our studies indicate that the c-fiber-derived calcitonin gene-related peptide (CGRP) is capable of stimulating HDMEC to secrete the neutrophil chemotactic factor interleukin 8 (IL-8). In addition, SP is able to directly activate HDMEC to express high levels of the important cellular adhesion molecule vascular cellular adhesion molecule 1 (VCAM-1). Thus, these studies support the role that the neurologic system may play in mediating the biologic processes that occur during inflammation and wound healing in the skin.

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.

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.

Extracellular signaling molecules regulate intracellular events by way of complex transduction assemblies composed of several proteins: receptor, G protein, effector, inactivating enzyme. Much is known about the structure and function of these transducer proteins. A signaling molecule initiates transduction by binding to the receptor which then prompts the G protein to undergo a reaction cycle. This cycle involves guanine nucleotide binding and hydrolysis, G protein subunit dissociation, and interactions with an effector (e.g. adenylyl cyclase, phospholipase C), as well as with inactivating molecules. The result is altered generation of intracellular second messengers, protein transcription, or another profound cellular response. This signal transduction system also contains multiple mechanisms for turning off the signal such as phosphorylating, internalizing, or downregulating receptors, uncoupling the receptor-G protein complex, or cell-surface peptidases, and precipitating conformational changes in transducer elements. These aspects of signal transduction are examined in two well studied systems, namely the beta 2-adrenergic and the substance P transducers. Both complexes are important physiological neuroregulators in the gut and elsewhere. Pathophysiological mechanisms involving aberrent signal transduction have been implicated in various diseases including major common illnesses such as heart failure and gastrointestinal disorders such as cholera, other infectious diarrheas, and colitis.

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.

The large and functionally diverse group of G-protein-coupled receptors includes receptors for many different signalling molecules, including peptide and non-peptide hormones and neuro-transmitters, chemokines, prostanoids and proteinases. Their principal function is to transmit information about the extracellular environment to the interior of the cell by interacting with the heterotrimeric G-proteins, and they thereby participate in many aspects of regulation. Cellular responses to agonists of these receptors are usually rapidly attenuated. Mechanisms of signal attenuation include removal of agonists from the extracellular fluid, receptor desensitization, endocytosis and down-regulation. Agonists are removed by dilution, uptake by transporters and enzymic degradation. Receptor desensitization is mediated by receptor phosphorylation by G-protein receptor kinases and second-messenger kinases, interaction of phosphorylated receptors with arrestins and receptor uncoupling from G-proteins. Agonist-induced receptor endocytosis also contributes to desensitization by depleting the cell surface of high-affinity receptors, and recycling of internalized receptors contributes to resensitization of cellular responses. Receptor down-regulation is a form of desensitization that occurs during continuous, long-term exposure of cells to receptor agonists. Down-regulation, which may occur during the development of drug tolerance, is characterized by depletion of the cellular receptor content, and is probably mediated by alterations in the rates of receptor degradation and synthesis. These regulatory mechanisms are important, as they govern the ability of cells to respond to agonists. A greater understanding of the mechanisms that modulate signalling may lead to the development of new therapies and may help to explain the mechanism of drug tolerance.

Although agonist-induced endocytosis of G-protein-coupled receptors is critical for receptor desensitization and resensitization, receptor motifs that interact with the endocytic apparatus have not been adequately characterized. We examined the effects of mutating the rat neurokinin-1 receptor on endocytosis using 125I-substance P, fluorescent substance P, and receptor antibodies. Substance P induced rapid internalization of wild-type receptors that were targeted to perinuclear endosomes. Truncation of the C-tail at residues 324, 342, and 354 reduced internalization up to 60% and caused retention of receptors at the cell surface and in superficial endosomes. Mutation of Tyr-341 and Tyr-349 in potential tyrosine-containing endocytic motifs of the C-tail also impaired internalization. A Y305A mutant within the putative NPX2-3Y endocytic motif of the seventh transmembrane domain showed impaired signaling and was minimally expressed at the plasma membrane but was found in cytoplasmic vesicles. In contrast, a Y305F mutant signaled normally and was normally expressed at the plasma membrane but showed impaired internalization. Thus, endocytosis of the neurokinin 1 receptor relies on several tyrosine-containing sequences in the C-tail and seventh transmembrane domain.

The survival, differentiation, and maintenance of responsive neurons are regulated by nerve growth factor (NGF), which is secreted by the target and interacts with receptors on the axon tip. It is uncertain how the NGF signal is communicated retrogradely from distal axons to neuron cell bodies. Retrograde transport of activated receptors in endocytic vesicles could convey the signal. However, little is known about endocytosis of NGF receptors, and there is no evidence that NGF receptors continue to signal after endocytosis. We have examined early events in the membrane traffic of NGF and its receptor, gp140(TrkA) (TrkA), in PC12 cells. NGF induced rapid and extensive endocytosis of TrkA in these cells, and the receptor subsequently moved into small organelles located near the plasma membrane. Some of these organelles contained clathrin and alpha-adaptin, which implies that TrkA is internalized by clathrin-mediated endocytosis. Using mechanical permeabilization and fractionation, intracellular organelles derived from endocytosis were separated from the plasma membrane. After NGF treatment, NGF was bound to TrkA in endocytic organelles, and TrkA was tyrosine-phosphorylated and bound to PLC-gamma1, suggesting that these receptors were competent to initiate signal transduction. These studies raise the possibility that NGF induces formation of signaling endosomes containing activated TrkA. They are an important first step in elucidating the molecular mechanism of NGF retrograde signaling.

The distribution of neurokinin-2 (NK2) tachykinin receptors was investigated by immunohistochemistry in the guinea-pig oesophagus, stomach, small and large intestine. Receptor immunoreactivity occurred at the surfaces of smooth muscle cells throughout the digestive tract. Nerve fibre varicosities in enteric ganglia were also immunoreactive. In myenteric ganglia, these varicosities were most numerous in the ileum, frequent, but less dense, in the proximal colon and caecum, rare in the distal colon, extremely infrequent in the rectum and duodenum, and absent from the stomach and oesophagus. Reactive varicosities were rare in the submucous ganglia. Reactive nerve fibres in the mucosa were only found in the caecum and proximal colon. Strong NK2 receptor immunoreactivity was also found on the surfaces of enterocytes at the bases of mucosal glands in the proximal colon. Receptors were not detectable on the surfaces of nerve cells or on non-terminal axons. Reactivity did not occur on nerve fibres innervating the muscle. Denervation studies showed that the immunoreactive varicosities in the myenteric plexus of the ileum were the terminals of descending interneurons. Immunoreactivity for nitric oxide synthase was colocalised with NK2 receptor (NK-R) immunoreactivity in about 70% of the myenteric varicosities in the small intestine. Bombesin immunoreactivity occurred in about 30% of NK2-R immunoreactive varicosities in the small intestine.