Faculty Bibliography

The aim of this investigation was to purify aminopeptidase M (APM) from the muscle layer of the small intestine, to compare it with APM of the mucosa and kidney, and to examine the degradation of gastrointestinal neural and hormonal peptides by muscle APM. APM was purified from the muscle and mucosa of the pig small intestine by DEAE-Sepharose and immuno-affinity chromatography. The specific activity of APM from muscle, mucosa, and kidney was 3900, 3000, and 3800 nmol/min per mg protein, respectively (substrate [Leu5]enkephalin). Muscle and mucosa APM contained four protein bands with apparent molecular weights of 150, 110, 73, and 52 kDa. Kidney APM contained three protein bands of 150, 110, and 56 kDa. The 150, 110, and 52/56 kDa bands cross-rected with an APM antiserum. APM from each tissue degraded [Leu5]enkephalin and [Met5]enkephalin, but not cholecystokinin-8, gastrin releasing peptide-10, somatostatin-14, substance P, and vasoactive intestinal peptide. The enzymes were identically inhibited by APM antiserum, amastatin, bestatin, actinonin, and 1, 10 phenanthroline. Non-mucosal APM may degrade enkephalins and terminate their biological actions.

In the digestive system, substance P is an excitatory transmitter to muscle, a putative excitatory neuro-neuronal transmitter, a vasodilator, and a mediator in inflammatory processes. Many of the biological effects of substance P are mediated by a high-affinity interaction with the tachykinin receptor neurokinin-1. The aim of the present study was to identify the sites of expression of this receptor in the rat stomach and intestine by immunohistochemistry with a polyclonal antiserum raised to the intracellular C-terminal portion of the rat neurokinin-1 receptor. Neurokinin-1 receptor immunoreactivity is present in a large population of enteric neurons. The relative density of these neurons along the gut is colon > ileum > stomach. In the intestine, stained neurons have a smooth cell body with processes that can be followed within and between plexuses, and make close approaches to other neuronal cells, but do not appear to project outside the plexuses, suggesting that they are interneurons. In the stomach, neurokinin-1 receptor-immunoreactive neurons are infrequent and have a poorly defined and irregular shape. Neurokinin-1 receptor immunoreactivity is also localized to numerous non-neuronal cells in the inner portion of the circular muscle layer of the small intestine, which have the appearance of small dark smooth muscle cells or interstitial cells of Cajal. These cells are postulated to form a "stretch-sensitive" system with the deep muscular plexus and thus constitute an important site of regulation of muscle activity. Double labeling immunofluorescence was used to simultaneously localize neurokinin-1 receptor and substance P/tachykinin immunoreactivities. These experiments demonstrate that in the enteric plexuses, substance P/tachykinin-immunoreactive varicose fibers encircle the cell bodies of most neurokinin-1 receptor-containing neurons, and in the inner portion of the circular muscle layer of the small intestine they lie close to neurokinin-1 receptor-immunoreactive non-neuronal cells. In addition, some enteric neurons express both neurokinin-1 receptor and substance P/tachykinin immunoreactivities. The present study provides strong evidence that the neurokinin-1 receptor is the tachykinin receptor mediating the actions of substance P on enteric neurons and smooth muscle.

T cell homing into extravascular sites requires penetration across the subendothelial basal lamina, a specialized nonfibrillar connective tissue structure that anchors endothelial cells to parenchymal surfaces. Herein, we show that normal human T cells express gelatinases A and B, two matrix metalloproteinases active against the major basal lamina constituents, collagen types IV and V. Expression is confirmed at both the mRNA and protein levels. Gelatinase B is expressed constitutively, whereas gelatinases A and B expression is induced by T cell activation. In vitro migration of resting T cells across a basal lamina equivalent is mediated by gelatinase B, because it is specifically blocked by GM6001, a hydroxamic acid inhibitor of matrix metalloproteinases. Inhibition of T cell homing by interference with gelatinase function may represent a useful approach to the treatment of T cell-mediated autoimmune diseases.

Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.

Endocytosis of the gastrin releasing peptide receptor (GRP-R) may regulate cellular responses to GRP. We observed endocytosis in transfected epithelial cells by confocal microscopy using cyanine 3-GRP (cyanine 3.18-labeled gastrin releasing peptide) and GRP-R antibodies. At 4 degrees C, cy3-GRP and GRP-R were confined to the plasma membrane. After 5 min at 37 degrees C, ligand and receptor were internalized into early endosomes with fluorescein isothiocyanate-transferrin. After 10 min, cy3-GRP and GRP-R were in perinuclear vesicles, and at 60 min cy3-GRP was in large, central vesicles, while GRP-R was at the cell surface. We quantified surface GRP-R using an antibody to an extracellular epitope and an 125I-labeled secondary antibody. After exposure to GRP, there was a loss and subsequent recovery of surface GRP-R. Recovery was unaffected by cycloheximide, and thus independent of new protein synthesis, but was attenuated by acidotropic agents, and therefore required endosomal acidification. Internalization of 125I-GRP, assessed using an acid wash, was maximal after 10-20 min, and was clathrin-mediated since it was inhibited by hyperosmolar sucrose and phenylarsine oxide. Thus, GRP and its receptor are rapidly internalized into early endosomes and then dissociate in an acidified compartment. GRP is probably degraded whereas the GRP-R recycles.

Substance P (SP) degradation by the cell-surface enzyme neutral endopeptidase (NEP), and endocytosis of the NK-1 receptor (NK-1R) are potential mechanisms that limit the responsiveness of cells to SP. These mechanisms were studied in epithelial cells (KNRK) transfected with cDNA encoding an epitope labeled Flag-NK-1R or NEP. In cells expressing NK-1R alone, specific saturable ¹²⁵I-Sp binding at 37°C was 28% ± 3% of total counts. Co-expression of NEP with the NK-1R reduced binding to 4 ± 0.3% of total counts. Addition of the NEP inhibitor thiorphan restored binding to control levels. In cells expressing NK-1R alone, SP (1 nM) stimulated an increase in [Ca²⁺](i) of 163 ± 16 nM. Go-expression of NEP reduced the [Ca²⁺](i) response to 52 ± 13 nM, and this response was restored to control levels by thiorphan. Antibodies to the NK-1R and rhodamine labeled SP were used to observe endocytosis in KNRK cells expressing the NK-1R alone. When cells were incubated with SP for 60 min at 4°C, NK-1R and rhodamine SP were confined to the plasma membrane. After 3 and 10 min at 37°C, there was a reduction in cell-surface staining and NK-1R and rhodamine-SP were localized in intracellular vesicles. Binding experiments with ¹²⁵I-Sp confirmed the rapid internalization of peptide. Therefore, the NK-1R and SP are internalized within minutes of binding. Thus, ligand degradation and receptor endocytosis limit the cellular response to SP.

We labeled substance P (SP), neurokinin A (NKA), and [Lys0]gastrin-releasing peptide-27 (GRP) with cyanine 3.18 (cy3). Cy3-peptides purified by HPLC were fully active, determined by [Ca2+]i mobilization. Binding was specific because it was abolished by unlabeled peptides and receptor antagonists. Transfected cells yielded a log-fold greater cy3 intensity than control cells by FACS. Confocal microscopy of transfected cells and cultured enteric neurons showed that cy3-SP bound to surface receptors and was internalized. Internalization was observed in living cells by capture of sequential images. Recovery of surface binding sites was monitored by flow cytometry using cy3-SP. Thus, cy3 neuropeptides can be used to isolate and study receptor-bearing cells.

Internalization of the NK1 receptor (NK1R) and substance P was observed in cells transfected with cDNA encoding the rat NK1R by using anti-receptor antibodies and cyanine 3-labelled substance P (cy3-substance P). After incubation at 4 degrees C, NK1R immunoreactivity and cy3-substance P were confined to the plasma membrane. Within 3 min of incubation at 37 degrees C, NK1R immunoreactivity and cy3-substance P were internalized into small intracellular vesicles located beneath the plasma membrane. Fluorescein isothiocyanate-labelled transferrin and cy3-substance P were internalized into the same vesicles, identifying them as early endosomes. After 60 min at 37 degrees C, NK1R immunoreactivity was detected in larger, perinuclear vesicles. Internalization of 125I-labelled substance P was studied by using an acid wash to dissociate cell-surface label from that which has been internalized. Binding reached equilibrium after incubation for 60 min at 4 degrees C with no detectable internalization. After 10 min incubation at 37 degrees C, 83.5 +/- 1.0% of specifically bound counts were internalized. Hyperosmolar sucrose and phenylarsine oxide, which are inhibitors of endocytosis, prevented internalization of 125I-labelled substance P and accumulation of NK1R immunoreactivity into endosomes. Acidotropic agents caused retention of 125I-labelled substance P within the cell and inhibited degradation of the internalized peptide. Continuous incubation of cells with substance P at 37 degrees C reduced 125I-substance P binding at the cell surface. Therefore, substance P and its receptor are internalized into early endosomes within minutes of binding, and internalized substance P is degraded. Internalization depletes NK1Rs from the cell surface and may down-regulate the response of a cell to substance P.

Substance P (SP) can cause plasma leakage at sites of inflammation by binding to neurokinin type 1 (NK1) receptors on the surface of endothelial cells. Internalization after ligand binding could reduce the number of NK1 receptors on the cell surface and thus participate in the desensitization and resensitization of the inflammatory response to SP. By using an antibody to the receptor, we directly observed SP-induced internalization of NK1 receptors into endosomes in endothelial cells of postcapillary venules in the rat tracheal mucosa. In the absence of SP, an average of 15 immunoreactive endosomes were present per endothelial cell. After an intravenous injection of SP, the number of immunoreactive endosomes peaked at 107 per cell at 3 min and gradually returned to the baseline by 120 min. In parallel experiments we observed that when cultured cells transfected with the NK1 receptor were exposed to rhodamine-SP and an antibody to an extracellular Flag epitope of the NK1 receptor, the SP was internalized with the receptor antibody. Both in the cultured cells and in the endothelial cells of intact animals, the prompt SP-induced internalization was accompanied by rapid, long-lasting desensitization to SP. These studies suggest that internalization of NK1 receptors by endothelial cells may be one of the mechanisms that limit the amount of plasma leakage at sites of inflammation.

Restitution, the rapid re-establishment of mucosal integrity following damage, involves cell migration and can be monitored by measuring transmucosal potential difference of tissue mounted in an Ussing chamber. The involvement of extracellular matrix proteins and matrix receptors was examined in the restitution of rat gastric mucosa. Undamaged mucosa maintained a potential difference of -32.7 +/- 2.2 mV for several hours. Mucosal exposure to 0.6 M NaCl for 1 min reduced this to -3.3 +/- 1.4 mV in 2-3 min. Thereafter, the potential difference returned in 60 min to plateau at -28.9 +/- 1.3 mV (88.5 +/- 3.6% of pre-exposure). Tissues mucosally treated with 1:100 anti-laminin antiserum maximally recovered following damage to 65.6 +/- 6.6% of pre-exposure potential difference (PD), while those treated with 1:100 anti-collagen IV or anti-fibronectin antisera recovered to 88.8 +/- 9.7% and 86.3 +/- 3.2%, respectively. Only the anti-laminin result was significantly different from controls. The anti-laminin effect was abolished by pre-incubation of the anti-laminin antiserum with purified rat laminin, suggesting that the effect was laminin specific. In experiments involving matrix protein receptors, tissues treated with alpha-lactalbumin, a protein altering the substrate specificity of cell surface laminin receptor/enzyme beta-1,4-galactosyltransferase, maximally recovered following damage to only 49.3 +/- 7.7% of pre-exposure PD, which was significantly different from controls, while those treated with anti-beta 1 integrin recovered to 85.0 +/- 9.7%. Our data suggest that laminin is involved in mediation of gastric mucosal restitution, possibly via beta-1,4-galactosyltransferase.