Role of metalloproteinases in the development and healing of acetic acid-induced gastric ulcer in rats

The three main components involved in thrombosis and haemostasis are thrombin, platelets, and plasmin. Almost all inhibitors of thrombosis are focused either on the inhibition of thrombin or on the inhibition of platelets. We designed a construct using the fibrinolytic activity of staphylokinase, fused via a cleavable linker to an antithrombotic peptide of 29 amino acids. The peptide was designed to include three inhibitory regions: (1) the Arg-Gly-Asp (RGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and cloned into an expression vector as a 3' fusion to the staphylokinase gene. Gene expression was induced in E. coli Top 10 cells and the fusion protein, designated PLATSAK, was purified using metal affinity chromatography. The purified fusion protein significantly lengthened the activated partial thromboplastin time and thrombin time and inhibited the amidolytic activity of thrombin. The fibrinolytic activity was almost equal to that of recombinant staphylokinase as measured with a thrombelastograph. Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside. Our results confirm that it is feasible to design and produce a hybrid multifunctional protein that targets various components of the haemostatic process.     

Proteinases of rat mast cells. Peritoneal but not intestinal mucosal mast cells express mast cell proteinase 5 and carboxypeptidase A

Six basic proteins of 26 to 38 kDa with isoelectric points (pI) > or = 8.5 were abundant in proteins separated by two-dimensional SDS-PAGE from adult rat peritoneal mast cells (MC). One was identified previously as rat mast cell proteinase (RMCP) 1, a chymase of 26 to 28 kDa, pI > 9.0. Microsequence analyses showed that two polypeptides of about 29 and 30 kDa had NH2 terminal amino acid sequences homologous to mouse MC proteinase 5 (MCP-5), whereas the amino terminals of the 33, 35, and 36 kDa proteins were homologous to MC carboxypeptidase A (MC-CPA). Rabbit Abs produced against synthetic peptides of the identified NH2 terminal sequences were used in immunoblot studies. At least three proteins reacted with Abs to MC-CPA, whereas Abs to MCP-5 detected three adjacent polypeptides, rather than just the two identified by using microsequence analysis. Removal of oligosaccharide side chains using peptide:N-glycosidase F reduced the heterogeneity of each set of three polypeptides (MCP-5 and MC-CPA) to a band of each protein of a lower M(r). The serine proteinase inhibitor [3H]diisopropylfluorophosphate ([3H]DFP) bound to a proteinase of 30 to 35 kDa, which is probably MC tryptase (pI < or = 6.0). Immunoblot analysis of proteins from intestinal mucosal mast cells showed RMCP-2, but not RMCP-1, MCP-5, or MC-CPA. This is the first report of MCP-5 in the rat and of clearly distinguishable glycosylated forms of MC CPA. These proteinases appear to be restricted in their distribution to selected MC populations, but little is known about their functions.     

Intraepithelial infiltration by mast cells with both connective tissue-type and mucosal-type characteristics in gut, trachea, and kidneys of IL-9 transgenic mice

IL-9 transgenic mice were analyzed for the presence of mast cells in different tissues. In these mice, increased mast cell infiltration was found in the gastric and intestinal epithelium as well as in the upper airways and kidney epithelium, but not in other organs, such as skin. IL-9 transgenic mast cells do not show signs of massive degranulation such as that found in IL-4 transgenic mice and are not involved in spontaneous pathologic changes. Gastric mast cells showed a phenotype related to connective-type mast cells, since they were stained by safranin, and strong expression of mouse mast cell protease-4 and -5 was found in this organ. However, they also expressed proteases related to the mucosal cell type, such as mouse mast cell protease-1 and -2. In vitro, although IL-9 by itself did not induce mast cell development from bone marrow progenitors, it strongly synergized with stem cell factor for the growth and differentiation of mast cells expressing the same protease pattern as that observed in IL-9 transgenic mice. Since constitutive stem cell factor expression was observed in vivo, and anti-c-Kit Abs inhibited IL-9 transgenic mastocytosis in the gut, this synergistic combination of factors is likely to be responsible for the mastocytosis observed in IL-9 transgenic mice. Taken together, these data demonstrate that IL-9 induces the in vivo amplification of a nonclassical mast cell subset with a mucosal localization but expressing proteases characteristic of both connective tissue-type and mucosal mast cells.     

Complement peptide C3a inhibits IgE-mediated triggering of rat mucosal mast cells

The relationship between mast cells' secretory response to stimulation via their type 1 Fc epsilon receptors (Fc epsilon RI) and that provided by the C3a fragment of the complement system was investigated in the rat mucosal-type mast cell line RBL-2H3. These cells are known to be unresponsive to the so-called 'peptidergic' stimulus provided by cationic agents, such as anaphylatoxins, neuropeptides or polyamines. We now observed that C3a effectively inhibits the Fc epsilon RI clustering induced secretion of RBL-2H3 cells. This inhibition is dose-dependent and takes place at a C3a concentration range of 0.4-12.5 nM, i.e. at least three orders of magnitude lower than those where this anaphylatoxin exerts its secretory stimulus to 'serosal' mast cells. In order to identify where C3a interferes in the Fc epsilon RI coupling cascade, we have studied its effect on the cells' protein phosphorylation pattern, hydrolysis of phosphatidyl inositides, transient rise in free cytosolic Ca2+ ion concentration and Ca2+ uptake. All these processes were found to be inhibited by a similar C3a concentration range.     

Malabsorption of zinc in rats with acetic acid-induced enteritis and colitis

Acute intestinal inflammation was established in rats by intraluminal administration of acetic acid into loops of distal ileum, proximal jejunum or ascending colon. The study included two control groups of intact (untreated) rats and sham-operated (saline-treated) rats for each intestinal segment. A third group of rats received acetic acid. Histological evaluation demonstrated that acetic acid treatment induced a mild inflammatory response. Two days after treatment, zinc absorption was measured using ligated 10-cm loops of each segment in which 65Zn was injected intraluminally. 65Zn absorption by the ileum, jejunum and colon was markedly reduced in those rats in which inflammation was induced by acetic acid. The liver showed the highest uptake of radioisotope, but the relative tissue distribution generally followed the amount of absorption. The surgical procedure itself seemed to reduce zinc absorption. No changes in [3H]leucine absorption were observed between sham-operated and acetic acid-treated controls. There was no significant serosal-->luminal secretion of intramuscularly injected 65Zn in any of the studied segments. Therefore, based upon the data obtained, we conclude that acetic acid-induced intestinal inflammation reduces absorption of zinc by the small and large intestine, and that a surgical procedure (laparotomy) also reduces zinc absorption. The mechanism of this inflammation is such that malabsorption shows some specificity.     

Effect of putative phospholipase A2 inhibitors on acetic acid-induced acute colitis in the rat

Phospholipase activation may play an important role in ulcerative colitis. This hypothesis was tested by evaluating the effect of two non-selective phospholipase (PL) A2 inhibitors, quinacrine and p-bromophenacyl-bromide (pBPB), on acetic acid-induced colitis in the rat. The calcium antagonist verapamil, which may also act as a PLA2 inhibitor, was also tested. Acute colitis was induced in an isolated colonic segment by instillation of 4 per cent acetic acid for 15 s; this induces a uniform colitis after 4 days. The severity of colitis was evaluated histologically, by measuring myeloperoxidase (MPO) activity and by determining plasma exudation into the lumen of the colon (permeability) with 125I-labelled albumin given intravenously. All three putative PLA2 inhibitors tested were found to prevent the development of colitis. Intravenous administration of quinacrine 10 mg kg-1 at 30 min before instillation of acetic acid resulted in a normal mucosal appearance, normal MPO activity and a significantly reduced increase in plasma exudation into the colon. A similar effect was achieved using verapamil. Intracolonic administration of either quinacrine or pBPB also prevented acetic acid-induced colitis. However, three doses, starting immediately after acetic acid administration and repeated on the first and second days, were needed to achieve this, whereas one dose produced only a partial effect. PLA2 may play an important role in acetic acid-induced colitis and inhibition of its activity may offer an alternative mode of treatment in ulcerative colitis.     

Quantitative and ultrastructural analysis of rectal mucosal mast cells in acute infectious diarrhea

The role of mast cells, potential mediators of mucosal immunity and inflammation, was studied morphologically in the rectal mucosa in two acute diarrheal diseases, cholera and shigellosis. Quantitation of mucosal mast cells showed that they were significantly higher in the deeper lamina propria where blood vessels and nerves were more abundant. There was no difference in mast cell counts or degranulation in the mucosa in both groups of patients and controls. Intraepithelial mast cells were decreased in the patients. The prevalence of lipid bodies was significantly higher in mast cells from patients with cholera and shigellosis (P < 0.01). These findings suggest that mast cell populations are more dense around blood vessels and nerves and that inflammatory mediators derived from arachidonic acid metabolites, as indicated by the lipid bodies, are the response of mast cells to the alterations in diarrhea, despite differences in the etiology of diarrhea.     

Role of endothelin-1 in repeated electrical stimulation-induced microcirculatory disturbance and mucosal damage in rat stomach

Ollier's disease is a chondromatosis of the long bones that occurs rarely but that is highly disabling because it causes severe dysmetria and deformity of the lower limbs. Surgical correction of these skeletal changes is obstructed by poor mechanical resistance of the bone tissue affected and by the amount of lengthening required to even the lower limbs. It is the purpose of this study to indicate the surgery of choice for the treatment of this disease, comparing the two most recent methods used: Wagner's technique and the Ilizarov method. The latter is more reliable in terms of mechanical hold and the possibility of correcting severe deformities, producing bone regenerate of excellent quality even in major lengthening procedures. These results were obtained by adapting the Ilizarov method to the features of the chondromatous bone, thanks to the extreme malleability of the circular external fixator.     

The role of mast cells in mucosal permeability changes during ischemia-reperfusion injury of the small intestine

The ipsilateral primary motor cortex (M1) plays a role in voluntary movement. In our studies, we used repetitive transcranial magnetic stimulation (rTMS) to study the effects of transient disruption of the ipsilateral M1 on the performance of finger sequences in right-handed normal subjects. Stimulation of the M1 ipsilateral to the movement induced timing errors in both simple and complex sequences performed with either hand, but with complex sequences, the effects were more pronounced with the left-sided stimulation. Recent studies in both animals and humans have confirmed the traditional view that ipsilateral projections from M1 to the upper limb are mainly directed to truncal and proximal muscles, with little evidence for direct connections to distal muscles. The ipsilateral motor pathway appears to be an important mechanism for functional recovery after focal brain injury during infancy, but its role in functional recovery for older children and adults has not yet been clearly demonstrated. There is increasing evidence from studies using different methodologies such as rTMS, functional imaging and movement-related cortical potentials, that M1 is involved in ipsilateral hand movements, with greater involvement in more complex tasks and the left hemisphere playing a greater role than the right.     

Degranulating activity in hybrid cells derived from rat peritoneal mast cells and ehrlich ascites

Fusion of rat mast cells and Ehrlich ascites tumor cells was mediated by HVJ. Compound 48/80-induced degranulation occurred in the fused cells formed from two mast cells and one tumor cell, but not in the fused cells from one mast cell and two or more tumor cells.     

Evidence for a neurotensin receptor in rat serosal mast cells

OBJECTIVE AND DESIGN: The ability of neurotensin (NT) at nmolar levels to stimulate exocytosis of the mast cell suggested that it could play a role in neuro-immune-endocrine interactions. The inhibition by a specific receptor antagonist of NT's mast cell stimulation suggested the presence of a specific mast cell NT receptor. We have here employed several probes to determine if a specific neurotensin receptor was present on rat serosal mast cells. MATERIAL: Serosal mast cells were isolated from the peritoneal and pleural cavities of male Sprague-Dawley rats. METHODS: Immunocytochemistry with an antibody raised against the C-terminal peptide of the neurotensin receptor was utilized. The same antibody was employed in immunoblotting following SDS gel electrophoresis of mast cell extracts. An RNA probe for ribonuclease protection assays (RPA) was prepared using the rat brain neurotensin receptor cDNA and polymerase chain reaction was carried out using primers based on the rat brain neurotensin receptor sequence. RESULTS: Mast cells showed specific staining with the anti-neurotensin receptor antibody and this same antibody revealed a protein on SDS gels migrating as a 70 kDa species. Ribonuclease protection assays revealed the predicted protected fragment at approximately 450 bp while PCR amplification gave a major product at 843 bp. CONCLUSIONS: These results indicate that a specific neurotensin receptor is present on the rat mast cell.     

Exocytotic proteins in enterochromaffin-like (ECL) cells of the rat stomach

Proteins participating in vesicular docking and fusion have been identified in the nervous system. Such proteins appear to be important for the molecular regulation of exocytosis also in non-neuronal cells. The enterochromaffin-like (ECL) cells of the gastric acid-secreting (oxyntic) mucosa secrete histamine and chromogranin A-derived peptides, such as pancreastatin. Using immunohistochemistry, we have examined whether the ECL cells of the rat stomach, identified with antibodies to histidine decarboxylase (HDC, the histamine-forming enzyme), express the same exocytotic proteins as neurons. The ECL cells displayed immunoreactivity for synaptophysin, synaptotagmin III, vesicle-associated membrane protein-2 (VAMP-2), cysteine string protein (CSP), vesicular monoamine transporter-2 (VMAT-2), synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin, and Munc-18, but not for synaptotagmin I/II and VAMP-1. Synaptophysin and VMAT-2 could be detected not only in the ECL cells, but also in a population of HDC-negative cells. The demonstration of synaptotagmin III in only a limited number of ECL cells suggests the existence of a subpopulation of ECL cells. The results show that several exocytotic proteins, previously identified in neurons, are present in rat stomach ECL cells. Hence, proteins engaged in vesicular docking and in the fusion of granule/vesicle membrane with plasma membrane seem to exist in both neurons and endocrine cells.     

Effect of frusemide on Cl- channel in rat peritoneal mast cells

Frusemide can be used as an antiasthma drug and appears to inhibit the release (conditioned by activation of Cl- channels) of mast cell proinflammatory mediators. We studied the cause of the effects of frusemide, checking its action on Cl- channels. The patch-clamp technique was used to study single-channel currents, and differences in electrical potential of the cellular membrane of rat peritoneal mast cells were measured. In inside-out configuration, outwardly-rectifying Cl- channels were identified whose conductance was 2.4/1.7 pS at positive and negative voltages. In cell-attached configuration, the open probability (Po) of the channel increased with depolarization or with the presence of cyclic adenosine monophosphate (cAMP) in the incubation medium. Po increased with a rise of cytoplasmic free calcium concentration [Ca2+] and was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and by 4-4'-diisothiocyanatoostilbene-2-2'-disulphonic acid (DIDS). These channels seem to be the main cause of mast cell Cl- conductance. Frusemide (10(-5) and 10(-3) M) did not affect Cl- channel activity when using excised patches. In cell-attached configuration experiments, the presence of frusemide (from 10(-5) to 10(-3) M) in the cell incubation medium, increasingly reduced Po (median inhibitory concentration (IC50) = 4.3 x 10(-7) M). In similar conditions, bumetanide also inhibited Po (IC50 = 5.7 x 10(-3) M). The results of this study suggest that frusemide can inhibit mast cell Cl- channels only via an indirect mechanisms, which probably involves an inhibition of a Na(+)-K(+)-2Cl- symport.     

Postoperative complications and mortality in peptic ulcer of the duodenum and stomach

During 19 years (from 1974 to 1992) 5205 operations for complicated ulcer disease of duodenum (4201 observation), stomach (915) and of double localization (89) were conducted. In 585 of 5205 patients complications occurred: of operative wound healing-in 17.4%, in the region of intervention--in 56%, dysfunction of other organs and systems--in 26.6%, 44 (0.84%) of them died. The mortality after conduction of stomach resection according to Bilroth-I was 0.8%, according to Bilroth-II--5.5%, after vagotomy--1%.     

Gastric mucosal EGF and PDGF receptor expression with ulcer healing by ebrotidine

OBJECTIVES: Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) play important roles in the process of mucosal repair and restitution, and their biological effects are mediated by receptors located on the target cell surfaces. The purpose of this study was to assess the effect of the antiulcer agent, ebrotidine, on the expression of EGF and PDGF receptors with chronic ulcer healing. METHODS: Chronic gastric ulcers were developed in the rat by acetic acid technique. The animal were divided into two groups and were treated twice daily for 14 consecutive days, either with ebrotidine at 100 mg/kg, or placebo. At different stages of treatment, the animals were sacrificed and used for the isolation and quantification of gastric mucosal EGF and PDGF receptors. RESULTS: The binding assays revealed that ulcer healing was accompanied by an increase in mucosal expression of both types of receptors. A 1.7-1.8-fold increase in PDGF and EGF receptors occurred by the 4th day after the development of ulcer and reached a maximum of 3-fold increase by the 14th day, when the ulcer was essentially healed. Treatment with ebrotidine caused accelerated ulcer healing (7 days) which was accompanied by a significant enhancement in receptor expression. Compared to the controls, a 1.5-fold increase in EGF and 1.7-fold increase in PDGF receptor expression occurred by the 7th day of ebrotidine treatment, and a 1.4- to 1.5-fold increase was still observed at the 14th day of treatment. CONCLUSIONS: The results suggest that ebrotidine is capable of enhancement of gastric mucosal proliferative activities associated with ulcer healing through the stimulation of EGF and PDGF receptor expression.