Inhibition of substance P activity prevents stress-induced bladder damage

Substance P is a neuropeptide involved in inflammation, immune regulation and stress response. Stress may induce bladder damage by stimulating inflammatory response such as mast cell activation. We here examined the role substance P during stress-induced mast cell degranulation and urothelial injury in rat bladder. Adult Sprague-Dawley rats (200-270 g) were either exposed to cold-immobilization stress or substance P (SP) intracerebroventricularly. Different doses of substance P receptor (NK1R) antagonist CP 99994 were administered peripherally or centrally before the stress exposure. From each group, samples of the bladder were examined with light and electron microscope. Stress- and SP-injected centrally, increased the number of both granulated and degranulated mast cells. Ultrastructurally, urothelial degeneration with vacuolization in the cytoplasm and dilated intercellular spaces were seen. Both central and peripheral injection of CP 99994 prevented stress-induced urothelial degeneration as well as stress-induced mast cell degranulation. In conclusion, centrally and peripherally released substance P is involved in stress-induced bladder damage. Inhibition of NK1R prevents stress-induced pathological changes of urinary bladder and NK1R antagonist can be considered for the treatment of inflammatory bladder diseases.     

Remodeling of neural, glial, and tracheal cell populations in the developing Manduca wing

In the rat larynx, plasma exudation and edema formation were studied by light and electron microscopy after i.v. injections of the mast cell activator compound 48/80, substance P, and capsaicin. The morphological effects of substance P and capsaicin on connective tissue mast cells in vivo were also examined. Of the drugs tested, only compound 48/80 degranulated the connective tissue mast cells. All drugs induced a subepithelial plasma exudation in the subglottic region, with edema in the lamina propria and widened intraepithelial intercellular spaces, though the tight junction regions seemed intact. In the epiglottis, 10 min after compound 48/80 injection, there was edema in the lamina propria on the lingual side, with an intact and tight epithelial lining. No morphological sign of edema was found in the epiglottis after injection of substance P or capsaicin. The pronounced effect found in the epiglottic region after compound 48/80 injection was due to the release of mediators such as histamine and 5-hydroxytryptamine from the connective tissue mast cells. This study supports the belief that substance P in vivo mediates an increased vascular permeability by a direct effect on the blood vessels – a mechanism distinct from mast cell degranulation.     

Proliferative potential of murine peritoneal mast cells after degranulation induced by compound 48/80, substance P, tetradecanoylphorbol acetate, or calcium ionophore A23187.

Proliferative potential of degranulated mast cells was investigated. Mast cells were collected from the peritoneal cavity of mice, and degranulation was induced by compound 48/80, substance P, 12-O-tetradecanoylphorbol 13-acetate (TPA), or calcium ionophore A23187. The potentiality of colony formation in methylcellulose was not reduced by treatment of various concentrations of compound 48/80, substance P and TPA. When degranulation was induced by compound 48/80, substance P or TPA, proportion of highly degranulated mast cells containing less than five granules was rather small. In contrast, considerable proportion of highly degranulated mast cells was obtained after the treatment with the low concentration (0.1 microgram/ml) of A23187. These highly degranulated mast cells, which were individually picked up by the micromanipulator, proliferated not only in methylcellulose but also in the skin of mast cell-deficient WBB6F1-W/Wv mice. Inasmuch as we have already shown the proliferation of IgE-sensitized and Ag-stimulated mast cells, degranulated mast cells appear to retain the proliferative potential in general.     

In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular Ca(2+) concentration ([Ca(2+)](i)). The degranulatory EC(50) for the mast cell secretagogue compound 48/80 (C48/80; 10 microg/ml) and the neuropeptides CGRP (2.10(-8) M) and substance P (SP; 3.10(-8) M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 microg/ml) and CGRP and SP (both 10(-7) M) to Fluo-4-loaded MMC induced a transient rise in [Ca(2+)](i) after a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 microg/ml), indicating involvement of G(i) proteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [Ca(2+)](i), indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo.     

Substance P induces granulocyte infiltration through degranulation of mast cells

Substance P, a potent vasodilatory neuropeptide, is released from peripheral nerve endings of sensory neurons by various stimuli. Although in vitro incubation of rat and human mast cells with substance P causes their degranulation, it is not known whether inflammatory changes induced by substance P are mediated by degranulation of mast cells. We investigated this point by using genetically mast cell-deficient WBB6F1-W/Wv and WCB6F1-Sl/Sld mice. The s.c. injection of substance P induced degranulation of mast cells in the skin of WBB6F1-+/+ mice, and then a marked eosinophil infiltration around the degranulated mast cells. However, WBB6F1-W/Wv and WCB6F1-Sl/Sld mice showed little or no eosinophil infiltration in the skin after the injection of substance P. When the mast cell deficiency of WBB6F1-W/Wv mice was rescued either systemically by bone marrow transplantation or locally by injection of cultured mast cells, injection of substance P induced the infiltration of eosinophils, suggesting that substance P-induced eosinophil infiltration was mediated through degranulation of mast cells.     

Migrating mast cells in the gallbladder epithelium of cattle and sheep

Summary This paper reports the existence of mast cells in an epithelial location in the gallbladders of both cattle and sheep. The histochemical studies performed on these cells showed that their cytoplasmic granules contain heparin and biogenic amines in both species. Optical- and electron microscopic observations demonstrated that, in both species, mast cells from the connective tissue of the gallbladder diapedese across the basal lamina and migrate through the epithelium all the way to the luminal surface, and that a degranulation process takes place during this migration. The biochemical results showed a correlation between the number of mast cells present in the epithelium and the amount of heparin detected in the different regions of the gallbladders of the species studied. Unusually high contents of heparin were found in both cattle and sheep gallbladders, suggesting that they should be studied as possible commercial sources of this polimer.     

The effects of unilateral and bilateral neurectomy of inferior alveolar nerves on the frequency of PCNA-positive keratinocytes in basal layer of gingival epithelium in Lewis rats

The aim of our study was to establish the role of sensory denervation on gingival epithelium proliferation. We investigated the effect of unilateral (right side) and bilateral neurectomy of inferior alveolar nerve on PCNA-positive (PCNA+) cell frequences in the basal layer of gingival epithelium in male Lewis rats. The samples were taken in each group both from left and right side of gingiva, and the PCNA+ cells were counted separately in the epithelium from both sides in all groups. The results of our study indicate that the proliferation of gingival epithelium, expressed as percentage of PCNA+ nuclei of basal layer keratinocytes of gingival squamous epithelium, is better correlated with the trauma than with denervation.     

Response of cardiac mast cells to atrial natriuretic peptide

Previously, our laboratory demonstrated that cardiac mast cell degranulation induces adverse ventricular remodeling in response to chronic volume overload. The purpose of this study was to investigate whether atrial natriuretic peptide (ANP), which is known to be elevated in chronic volume overload, causes cardiac mast cell degranulation. Relative to control, ANP induced significant histamine release from peritoneal mast cells, whereas isolated cardiac mast cells were not responsive. Infusion of ANP (225 pg/ml) into blood-perfused isolated rat hearts produced minimal activation of cardiac mast cells, similar to that seen in the control group. ANP also did not increase matrix metalloproteinase-2 activity, reduce collagen volume fraction, or alter diastolic or systolic cardiac function compared with saline-treated controls. In a subsequent study to evaluate the effects of natriuretic peptide receptor antagonism on volume overload-induced ventricular remodeling, anantin was administered to rats with an aortocaval fistula. Comparable increases of myocardial MMP-2 activity in treated and untreated rats with an aortocaval fistula were associated with equivalent decreases in ventricular collagen (P < 0.05 vs. sham-operated controls). Cardiac functional parameters and left ventricular hypertrophy were unaffected by anantin. We conclude that ANP is not a cardiac mast cell secretagogue and is not responsible for the cardiac mast cell-mediated adverse ventricular remodeling in response to volume overload.     

Single modified cilia displayed by cells of human internal stratified epithelia (oral cavity,vagina)

Summary Serial sections of human vaginal and keratinized oral-gingival epithelia were investigated for ciliary structures. Most melanocytes of the gingival epithelium lacked cilia, whereas almost all basal keratinocytes of the deeper portion of the epithelial ridges possessed one cilium each. In the suprabasal layers of the ridges only a few keratinocytes exhibited a single cilium. In the basal layer, at the top of the connective tissue papillae, approximately every second keratinocyte displayed a single cilium. In the suprabasal layers above the ridges no ciliated keratinocytes were observed. The basal cells of the vaginal epithelium were endowed with cilia, while cilia were absent from the suprabasal cells. In the human forearm epidermis most melanocytes and keratinocytes are supplied with a single cilium; it has been suggested that they may play a role in light reception. However, the widespread occurrence of 9 + 0 cilia in epithelial cells of internal epithelia and their coincidence with the sites of renewal of keratinocytes suggests that a relationship may exist between solitary cilia and mitotic activity.     

Mitogenic action of substance P on the epithelial cells of the tongue

Substance P (10 and 100 ng/ml) stimulates the proliferation of basal cells from rat's tongue epithelium in primary cultures with 2.5% fetal calf serum. In serum-free medium substance P have no effect on the epithelial cell growth. This neuropeptide secreted by afferent nerve fibers of the tongue epithelium is suggested to have a neurotrophic influence on epithelial cells controlling their proliferation.     

Mast cell distribution and density in Lewis rat gingiva after bilateral neurectomy of inferior alveolar nerves

The effect of uni- and bilateral neurectomy of inferior alveolar nerve on mast cell (MC) density and topography were studied in Lewis rat gingival mucosa. The results were compared with unilateral sham operation effects. The samples were taken in each group both form left and right side of gingiva, MC were revealed by pinacyanol erythrosinate and counted in connective tissue adjacent to the basal membrane of gingival epithelium. MC were insensitive to sham operation and the differences were noted only in MC density after unilateral neurectomy between both sides of gingiva, and in distribution between the control and left side after bilateral neurectomy. The results suggests that neurectomy and sham operation trauma after 3 weeks have limited effect on mast cell density and distribution.     

Heparin modulates the growth and adherence and augments the growth-inhibitory action of TNF-alpha on cultured human keratinocytes

Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02-200 microg/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 microg/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-alpha is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2-20 microg/ml augmented or even potentiated this growth-inhibitory effect of TNF-alpha. The association of TNF-alpha with heparin was shown by demonstrating that TNF-alpha bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-alpha-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 microg/ml inhibited significantly by 12-13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 microg/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-alpha.     

Intracellular localization of serotonin in mast cells of the colon in normal and colitis rats

Intracellular localization of serotonin (5-HT) in the mast cells of two phenotypes in normal rat colon and dextran sodium sulphate-induced colitis was studied by immunoelectron microscopy with a quantitative analysis of the distribution of immunogold labelling. Mucosal mast cells in normal rats contained round shape secretory granules with varying electron density. Immunogold labelling for 5-HT was concentrated over the secretory granules. In mucosal mast cells from colitis rats, vacuolated granules without 5-HT labelling were frequently observed and immunogold labelling over the secretory granules was significantly increased compared to controls. On the other hand, connective tissue mast cells in normal rats contained oval shape secretory granules with homogeneous electron density. Their immunogold labelling was diffusely scattered over the secretory granules as well as over the cytoplasm. In connective tissue mast cells from colitis rats, secretory granules with high electron density were increased and the immunogold labelling over the secretory granules was much higher than that in controls. The present results suggest that intracellular localization of 5-HT is different in two phenotypes of mast cells and they may release 5-HT in a different manner. Mucosal mast cells may release 5-HT by a degranulation or exocytosis, while connective tissue mast cells may release 5-HT by a diacrine manner of secretion.     

Selective adhesion of mast cells to tracheal epithelial cells in vitro

In allergic and nonallergic lung diseases, if intraluminal mast cells adhere to airway epithelium, inflammatory mediators released from activated mast cells may reach high local concentrations and thus greatly affect airway function. To determine whether mast cells adhere to airway epithelial cells, radiolabeled or unlabeled dog mastocytoma cells were incubated with cultured dog tracheal epithelial cells, with extracellular matrix substrates, and with cryostat-cut sections of dog trachea. Mast cells adhered well to cultured epithelial cells (35 +/- 13% adhesion, mean +/- 1 SD, n = 23) but adhered poorly to types I and IV collagen or to fibronectin (less than 7.5% mean adhesion in all cases). Similarly, in tracheal tissue sections, mast cells adhered preferentially to epithelial cells in surface epithelium or in submucosal glands but not to basal membrane or connective tissue. Adhesion to cultured epithelial cells was a characteristics of a subpopulation of mast cells, could persist for more than 48 h, did not require energy or the presence of divalent cations, and was not mediated by a known family of leukocyte-associated adhesion glycoproteins. Adhesion was completely abolished by pretreatment of mast cells with pronase E or proteinase K but not with trypsin (up to 10 micrograms/ml at 37 degrees C for 20 min each). In contrast, pretreatment of cultured epithelial cells with any of these proteinases had no effect on adhesion. It is concluded that dog mastocytoma mast cells adhere to dog tracheal epithelial cells and do so selectively. It is suggested that mast cell adhesion to airway epithelium may play a role in the effectiveness of mast cell-epithelial cell interactions, and thus, in certain lung diseases, airway function may be affected by intraluminal mast cells more than is currently appreciated.     

Group II phospholipase A2 inhibitors suppressed lysophosphatidylserine-dependent degranulation of rat peritoneal mast cells.

Rat peritoneal mast cells were sensitized with IgE and challenged with the specific antigen in the presence of lysophosphatidylserine (lysoPS), an essential co-factor for rodent connective tissue mast cell degranulation, and the effects of phospholipase A2 inhibitors were examined. Mepacrine, a known inhibitor of phospholipase A2, at concentrations below 10(-5) M and anti-rat 14-kDa group II phospholipase A2 antibody inhibited histamine release, while they did not affect the prostaglandin generation. Like histamine release, prostaglandin generation in IgE- and antigen- challenged rat peritoneal mast cells was dependent on the presence of lysoPS. These results indicate that 14-kDa group II phospholipase A2 may play an essential role in IgE-, antigen-, and lysoPS-dependent degranulation process of rat peritoneal mast cells and that the mechanism whereby it participates may not be due to the production of lysoPS from PS in mast cell membranes.     

Degranulation of eosinophilic granule cells induced by capsaicin and substance P in the intestine of the rainbow trout (Oncorhynchus mykiss Walbaum)

Summary Adult rainbow trout (Oncorhynchus mykiss) were injected intraperitoneally with capsaicin, substance P, serotonin, or a control of saline vehicle or bovine serum albumin (0.5 g/g body weight). Fish were sacrificed 30 min and 1,2 and 4 h post-injection, the gut was dissected out, and a small section of the upper intestine was processed for electron microscopy. A significant proportion of eosinophilic granule cells (EGCs) of the intestine were in close association with non-myelinated neuronal bundles in all fish (4 fish per treatment and time period), but there was no significant difference between treatment or time, suggesting that the association was unaffected by these factors. Close examination of EGC ultrastructure showed that fish treated with capsaicin and substance P exhibited limited degranulation of the EGCs in the stratum compactum and extensive crinophagic-like degranulation in the lamina propria. Cells of the lamina propria contained characteristic multivesicular-like bodies. The degranulation was reminiscent of both mast cell degranulation and endocrine cell crinophagy. EGCs of fish treated with serotonin or a control were unaffected, suggesting that the serotoninergic neurons, believed to be involved in gut motility, were not responsible for degranulation. It is apparent that EGCs of the trout intestine may be under nervous control, as has been demonstrated previously for mammalian mast cells.     

The mast cell response to substance P: effects of neuraminidase, limulin, and some novel synthetic peptide antagonists

We have investigated certain aspects of the mechanism whereby substance P triggers secretion of 5-hydroxytryptamine (5-HT) from rat peritoneal mast cells in vitro. Substance P-induced release of 5-HT was inhibited following pretreatment of rat peritoneal cells with 0.01-1.0 units/ml neuraminidase; secretion induced by anti-IgE antibody was inhibited by pretreatment with 1.0 units/ml but not by lower concentrations of enzyme. Addition of the sialic acid-rich substances N-acetyl-neuraminlactose (up to 1.0 mM) and mucin (up to 1.0 mg/ml) to substance P in free solution failed to block the activity of the neuropeptide. Limulin, a sialic acid-specific lectin, failed to block substance P-induced secretion of 5-HT, but was found to possess intrinsic non-lytic secretory activity (at 5-20 micrograms/ml). Release of 5-HT induced by limulin was independent of that induced by substance P. A range of octapeptides incorporating the C-terminal sequence Gly-Ser-Phe-Phe, but differing in degree of cationicity and positioning of cationic residues in the four N-terminal positions, were tested for their capacity to antagonise the mast cell-triggering activity of substance P. A peptide incorporating two lysine residues at the N-terminus was found to have partial substance P antagonist activity; no effects on IgE-mediated secretion were observed.     

The role of mast cells in vascular permeability disorders in rats subjected to immobilization stress]

Disturbances of vascular permeability were studied by the "vascular labeling" technique in the mesentry during the 24-hour immobilization of rats. Administration of dimebolin (an antihistaminic preparation) decreased the number of labeled vessels and labeling intensity. This effect was expressed in the presence of mast cells only and was accompanied by the mast cell degranulation. The authors suppose that the mast cells contain a substance preventing the disturbance of vascular permeability and released during degranulation. Such substance might be heparin. Experiments showed that small doses of heparin failed to produce such effect. These results allowed one to conclude that mast cells played a double role in the mechanisms of disturbance of vascular permeability during immobilization--the damaging (by the action of histamine and serotonine) and the protective (by the released heparin) action.     

Purification and characterization of human skin mast cells. Evidence for human mast cell heterogeneity

Our previous studies of human lung and intestinal mast cells failed to show the heterogeneity found among mast cells in murine species. Recently, we and others have developed techniques for the enzymatic dispersion of human neonatal skin mast cells. In addition, we are now able to make single cell suspensions of mast cells from adult skin and to purify these cells to near homogeneity. Comparative studies of mast cells from these several sources have uncovered several major differences among them. Adult and neonatal skin mast cells themselves differ in that the former are 10-fold less sensitive to goat anti-human IgE, with maximal release occurring at 3.0 and 0.3 microgram/ml, respectively. Skin mast cells also differ in optimal temperature for release: adult mast cells respond maximally at 23 to 30 degrees C and neonatal cells at 37 degrees C. Skin mast cells from both sources are dramatically different from lung and intestinal mast cells in two aspects. First, skin mast cells are quite responsive to several stimuli--morphine sulfate (10(-4) to 10(-6) M), substance P (10(-5) to 10(-7) M), compound 48/80 (10 to 0.1 microgram/ml), f-Met peptide (10(-6) M), and C5a (10(-8) M)--to which the other mast cells fail to respond. Second, although stimulated skin mast cells produce prostaglandin D2, little leikotriene C4, if any, is generated, unlike lung or intestinal mast cells. These differences in inflammatory potential among human mast cells from various sites have important implications for the management of allergic and inflammatory responses.     

Role of gastric mast cells in the regulation of central TRH analog-induced hyperemia in rats

RX 77368 (RX) increases gastric mucosal blood flow by a vagal cholinergic mechanism. The relative roles of mucosal and connective tissue mast cells (MMC and CTMC) were investigated in RX-injected rats. Blood flow and mast cell degranulation were measured after intracisternal RX. RX significantly increased gastric mucosal blood flow, and sequentially degranulated CTMC and MMC. Ketotifen or doxantrazole inhibited the hyperemic response. Ondansetron, RS-039604-90, or famotidine, but not ketanserin or pyrilamine, reduced hyperemia. Mast cells mediate RX-induced gastric hyperemia via 5-HT3, 5-HT4, and H2 receptors; initial increase depends upon CTMC whereas MMC contributes to the later response.