Multiple receptors for calcitonin gene-related peptide and amylin on guinea-pig ileum and vas deferens

1. The responses of the electrically stimulated guinea-pig ileum and vas deferens to human and rat calcitonin gene-related peptide (CGRP) and amylin were investigated. 2. The inhibition of contraction of the ileum produced by human alpha CGRP was antagonized by human alpha CGRP8-37 (apparent pA2 estimated at 7.15 +/- 0.23) > human alpha CGRP19-37 (apparent pA2 estimated as 6.67 +/- 0.33) > [Tyr0]-human alpha CGRP28-37. The amylin antagonist, AC187, was three fold less potent than CGRP8-37 in antagonizing human alpha CGRP. 3. Both human beta- and rat alpha CGRP inhibited contractions of the ileum, but this was less sensitive to inhibition by CGRP8-37 than the effect of human alpha CGRP. However, CGRP19-37 was twenty times more effective in inhibiting the response to rat alpha CGRP (apparent pA2 estimated as 8.0 +/- 0.1) compared to human alpha CGRP. 4. Rat amylin inhibited contractions in about 10% of ileal preparations; this effect was not antagonized by any CGRP fragment. Human amylin had no action on this preparation. 5. Both human and rat alpha CGRP inhibited electrically stimulated contractions of the vas deferens, which were not antagonized by 3 microM CGRP8-37 or 10 microM AC187. 6. Rat amylin inhibited the stimulated contractions of the vas deferens (EC50 = 77 +/- 9 nM); human amylin was less potent (EC50 = 213 +/- 22 nM). The response to rat amylin was antagonized by 10 microM CGRP8-37 (EC50 = 242 +/- 25 nM) and 10 microM AC187 (EC50 = 610 +/- 22 nM). 7. It is concluded that human alpha CGRP relaxes the guinea-pig ileum via CGRP1-like receptors, but that human beta CGRP and rat alpha CGRP may use additional receptors. These are distinct CGRP2-like and amylin receptors on guinea-pig vas deferens.

Nitric oxide in the rat gastrointestinal tract

This study concerns the nature of nitric oxide synthase (NOS) and the role of nitric oxide (NO) in the rat gastrointestinal tract. The major objectives were (i) to characterise NOS isoforms in the gastric glandular mucosa, (ii) to localise NOS isoforms in the rat gastric glandular mucosa, (iii) to investigate the role of NO in carbachol-stimulated gastric mucus secretion, (iv) to investigate the nature of NOS and small intestine. Immunoblotting was performed using polyclonal antisera raised against two peptides found in the rat brain NOS sequence and commercial monoclonal antibodies directed against neuronal and endothelial isoforms of NOS. A160kDa band was detected in brain and gastric mucosal samples with antibodies and antisera directed against neuronal NOS sequences, and a 140kDa band was detected in gastric mucosal samples using an anti-endothelial NOS antibody. An intense 160kDa neuronal NOS band was detected in a high-density fraction of gastric mucosal cells separated on a Percoll gradient. Detection of neuronal NOS by a carboxyl-terminal antiserum in samples of brain, but not of gastric mucosa, could be blocked by the peptide (20g/ml) against which the antibody was raised. After affinity purification, recognition of gastric mucosal NOS was blocked by peptide. Particulate neuronal NOS was found in the brain by immunoblotting while 94% of gastric mucosal enzyme was soluble. Gastric mucosal endothelial NOS was 95% particulate. 95% of NOS activity in the gastric mucosa was due to neuronal NOS. Paraformaldehyde- and acetone-fixed gastric mucosal sections were subject to immunocytochemistry using the above antibodies. Neuronal NOS was localised to the surface mucosal epithelial cells while endothelial NOS was associated with microvessels at the base of the mucosa and to larger vessels in the submucosa. Intragastric administration of carbachol or 16, 16-dimethyl prostaglandin E2 increased the thickness of the rat gastric mucus layer. The NOS inhibitor NG-nitro-L-arginine methyl ester dose-dependently, and selectively, prevented the stimulatory effect of carbachol. Ca2+-independent NOS activity in rat ileal, jejunal and colonic muscle was increased after LPS induction. Ca2+-dependent activity was not affected. Distribution of inducible NOS protein paralleled Ca2+ -independent activity. LPS treatment did not affect the content of neuronal NOS in colonic muscle.

Development of a novel in vitro system for nasal drug delivery development

There is currently, no ideal system for studying nasal drug delivery in vitro. The existing techniques such as the Ussing chamber and cell culture all have major disadvantages. Most importantly, none of the existing techniques accurately represent the interior of the nasal cavity, with its airflow and humidity; neither do they allow the investigation of solid dosage forms.The work in this thesis represents the development of an in vitro model system in which the interior characteristics of the nasal cavity are closely represented, and solid or minimal volume dosage forms can be investigated. The complete nasal chamber consists of two sections: a lower tissue, viability chamber and an upper nasal chamber. The lower tissue viability chamber has been shown, using existing tissue viability monitoring techniques, to maintain the viability of a number of epithelial tissues, including porcine and rabbit nasal tissue, and rat ileal and Payers' patch tissue. The complete chamber including the upper nasal chamber has been shown to provide tissue viability for porcine and rabbit nasal tissue above that available using the existing Ussing chamber techniques. Adaptation of the complete system, and the development of the necessary experimental protocols that allow aerosol particle-sizing, together with videography, has shown that the new factors investigated, humidity and airflow, have a measurable effect on the delivered dose from a typical nasal pump. Similarly, adaptation of the chamber to fit under a confocal microscope, and the development of the necessary protocols has shown the effect of surface and size on the penetration of microparticulate materials into nasal epithelial tissues. The system developed in this thesis has been shown to be flexible, in allowing the development of the confocal and particle-sizing systems. For future nasal drug delivery studies, the ability to measure such factors as the size of the delivered system in the nasal cavity, the depth of penetration of the formulation into the tissue are essential. Additionally, to have access to other data such as that obtained from drug transport in the same system, and to have the tissue available for histological examination represents a significant advance in the usefulness of such an in vitro technique for nasal delivery.

The targeting of corticosteroids to inflamed tissues

In recent years, much interest has focused on the beneficial effects of administering potentially harmful therapeutic agents in drug carriers so as to reduce their toxic side effects. Rheumatoid arthritis is a chronic systemic disease with progressive destruction of the Joints and long term patient disability, Corticosteroids have been shown to retard the progression of Joint destruction but are limited in their use due to adverse side effects,This project, following the line of investigation started by other workers, was designed to study the use of microspheres to deliver corticosteroids to inflamed tissues by both the oral and intravenous routes. Hydrocortisone (HC)-loaded albumin microspheres were prepared by three different methods, by direct incorporation of HC within the particles, by indirect incorporation of HC by the enzymatic conversion of hydrocortisone-21-phosphate (H-21-P) to HC within the particles, and by the adsorption of HC onto the surface. HC was also loaded with PLA microspheres. The level of corticosteriod loading and in vitro release from microspheres was determined by HPLC analysis. A reversed-phase, ion-pairing HPLC method was developed to simultaneously measure both HC and H-21-P. The highest level of corticosteroid loading was achieved using the incorporation of H-21-P with enzymatic conversion to HC method. However, HPLC analysis showed only 5% of the incorporated steroid was HC. In vitro release rates of steroid from albumin microspheres showed >95% of incorporated steroid was released within 2 hours of dissolution. Increasing the protein:steroid ratio, and the temperature and duration of microsphere stabilization, had little effect on prolonging drug release. In vivo studies, using the carrageenan-induced rat hind-paw model of inflammation, indicated steroid-incorporated microspheres administered both orally and intraperitoneally were not therapeutically advantageous when compared to equivalent free steroid doses. The ability of orally and intravenously dosed [125I]~albumin microspheres (2.67 μm mean diameter) to accumulate in acutely and chronically inflamed tissues was investigated, The subcutaneous air-pouch was the model of inflammation used, with carrageenan as the inflammatory stimulus. Acute and chronic inflammation was shown to be consistently formed  in pouch tissues in terms of cell infiltration and fluid exudate formation in the pouch cavity. Albumin microspheres were shown to accumulate in the inflamed tissues and pouch fluids after both oral and intravenous administration. Preliminary, confirmatory studies using latex microspheres and quantitation by GPC analysis, also indicated microsphere accumulation in both acutely and chronically inflamed air-pouch tissues. tntl lUr"'poucbtis,sues; The results indicate the uptake and transfer of microspheres across the gastrointestinal tract into the circulation and their migration through disrupted endothelium and basement membranes at the inflamed sites. , .

Aspects of signal transduction in the gastrointestinal tract

This study was undertaken to increase knowledge of the mechanisms of inter- and intracellular signalling in the gastrointestinal tract. Specific aims were: to use cell lines to elucidate factors affecting growth of gastric cells, to investigate the distribution and aspects of function of isoforms of protein kinase C in a gastric cell line and in the rat gastrointestinal tract and to determine the presence and regulation of nitric oxide synthase in gastrointestinal tissues from the rat and in cell lines. The gastric cancer cell line HGT-1 was used to investigate control of growth. Increases in cell number were found to be dependent on the seeding density of the cells. In cells plated at low density insulin, epidermal growth factor and gastrin all increased cell number. Gastrin produced a bell-shaped dose response curve with a maximum activity at 5nM. No effect of gastrin was apparent in cells plated at high density. α and β isoforms of protein kinase C were found, by immunoblotting procedures, to be widespread in the gastrointestinal tract of the rat, but protein kinase Cε was confined to the gastric mucosa and gastrointestinal smooth muscle. HGT-1 cells contained protein kinase C α and ε but β or γ were not detected. Preincubation of HGT-1 cells for 24h with 1μM phorbol-12,13-dibutyrate down-regulated protein kinase C α but not ε. The inhibition by the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) of the histamine-stimulated increase in cAMP in HGT-1 cells was down regulated by phorbol-12,13-dibutyrate. Inhibition of histamine-stimulation of adenylate cyclase by TPA was Ca2+-dependent and inhibited by the addition of an antibody to protein kinase C α. A role for protein kinase C α in modulating the effect of histamine on adenylate cyclase in HGT-1 cells is suggested. No nitric oxide synthase activity was detected in the gastrointestinal cell lines HGT-l, MKN-45 or CaCo-2. Ca2+-dependent nitric oxide synthase activity was observed in the gastric mucosa and the gastrointestinal smooth muscle from stomach to colon. The gastric: mucosal enzyme was soluble and showed half-maximal activity at 400nM Ca2+. Pretreatment of rats with endotoxin (3mg/kg body weight) induced nitric oxide synthase activity in both jejunal, ileal and colonic mucosa and muscle. A major portion of the induced activity in ileal and colonic mucosa was Ca2+-independent. Nitric oxide synthase activity in a high-density fraction of gastric mucosal cells was inhibited in a dose-dependent fashion by L-nitroarginine, NG-monomethyl-L-arginine, trifluoperazine and L-canavanine (in descending order of potency). Preincubation with okadaic acid and addition of ATPlMg2+ to the homogenisation buffer inhibited enzyme activity, which implies that phosphorylation inhibits gastric mucosal nitric oxide synthase.

Drug targeting with phagocytic polymorphonuclear leucocytes

1. Phagocytic polymorphonuclear leucocytes (PMNLs) or neutrophils have a marked avidity for the uptake of particulate material and are the first cell type to respond to inflammatory stimuli in vivo. 2. By harnessing these pathophysiological characteristics the inherent targeting capacity of the PMNL could be exploited to carry drug loaded particles to these sites. 3. In vitro chemotaxis of PMNLs was studied in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in the Blindwell chamber assay. 4. After phagocytosis of 1.1m polystyrene latex (PSL) beads at a range of incubation concentrations (5,10,20, and 30 beads/cell) the migration of the PMNL population was not significantly different from control, without beads. 5. The distribution of the beads within the filter showed that a disproportionately large number of PSL (50%) were associated with the cells on the surface of the filter that had not penetrated the filter. Eighty per cent of the PMNL population migrated and despite containing less PSL beads/cell, 50% of the dose was carried into the filter. Between 5 and 10% of these PSL were carried beyond 60m in the assay. 6. These results suggested heterogeneity of the PMNL population and to achieve efficient targeting with these cells preferential selection of the migratory sub-population would be needed. 7. The air-pouch model was then developed to study the focal accumulation of PMNLs in vivo. The PMNL isolated did not survive long enough in the circulation due to the trauma of the isolation procedure used; an alternative method will have to be employed.

Clonal composition of human adamantinomatous craniopharyngiomas and somatic mutation analyses of the patched (PTCH), Gsα and Gi2α genes

Craniopharyngioma is the most common childhood tumor and thought to arise from embryonic remnants of Rathke's pouch. The paucity of published data on the molecular basis of these tumors prompted us to examine 22 adamantinomatous craniopharyngiomas looking for genetic abnormalities. Using the X-linked polymorphic androgen receptor gene as a tool for X-chromosome inactivating analysis, we found that a subset of craniopharyngiomas are monoclonal and therefore are probably due to acquired somatic genetic defects. Thus, we investigated these tumours for mutations within three candidate genes, Gsα, Gi2α and patched (PTCH). Using single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis and direct sequencing, the presence of somatic mutations in these genes could not be demonstrated in any tumor. Our data indicate that a subset of craniopharyngiomas are monoclonal and the mutations in the PTCH, Gsα, and Gi2α contribute little if any to cranipharyngioma development.

Comparative genomic hybridization analysis of craniopharyngiomas

Object. Craniopharyngioma is the most common childhood brain tumor and is thought to arise from embryonic remnants of the Rathke pouch. Some craniopharyngiomas are monoclonal in origin and hence presumably harbor somatic genetic alterations, although the precise molecular mechanisms involved in craniopharyngioma development are unknown. The goal of this study was to identify genetic alterations in craniopharyngiomas. Methods. To gain insight into the molecular mechanisms involved in development of these tumors, the authors analyzed nine adamantinomatous craniopharyngiomas by using comparative genomic hybridization. Six tumors (67%) displayed at least one genomic alteration, and three had six or more alterations. Only two tumors displayed a decrease in DNA copy number, and in all others an increase in DNA copy number was noted. Conclusions. The authors conclude that a subset of craniopharyngiomas consists of monoclonal tumors arising from activation of oncogenes located at specific chromosomal loci.

Effect of nitric oxide on apoptotic activity in the rat gastrointestinal tract

The effect of nitric oxide (NO) on apoptosis in the gastrointestinal mucosa was investigated. Experiments involved long-term exposure of rat gastric mucosal cells in vitro to exogenous NO delivered from the NO, donor S-nitroso-N-acetyl-penicillamine, and the effect of intravenous administration of lipopolysaccharide in vivo, in the presence and absence of the selective inhibitor of inducible NO synthase N-(3-(aminomethyl)benzyl) acetamidine (1400 W). S-nitroso-N-acetyl-penicillamine produced a dose-related inhibition of caspase 3-like activity and DNA fragmentation in isolated gastric mucosal cells. Caspase 3-like activity and DNA fragmentation in gastric, ileal and colonic mucosa were increased both 5 and 24 h after injection of lipopolysaccharide (3 mg/kg, i.v.) to rats in vivo. Administration of 1400 W (5 mg/kg, i.v.) immediately after lipopolysaccharide enhanced caspase 3-like activity and DNA fragmentation above that found with lipopolysaccharide alone. In conclusion, data obtained both in vitro and in vivo suggest that NO exerts an anti-apoptotic effect on rat gastrointestinal mucosal cells. © 2001 Elsevier Science B.V.

Using an Infra-red sensor to measure the dynamic behaviour of N2O gas escaping through different sized holes

An anastomosis is a surgical procedure that consists of the connection of two parts of an organ and is commonly required in cases of colorectal cancer. About 80% of the patients diagnosed with this problem require surgery. The malignant tissue located on the gastrointestinal track must be resected and the most common procedure adopted is the anastomosis. Therefore, an anastomotic leak represents a significant problem and increases the duration of hospital stay, which is associated with remedial treatment and recovery, causing, as a result, a negative financial impact. A number of techniques to treat, prevent and even detect an anastomotic leakage are under investigation. However, studies show that these techniques are not always able to prevent an anastomotic leak from occurring. This paper discusses the monitoring of leakage through differently sized and differently positioned leak holes in phantom colons, using physical experiments and a Computational Fluid Dynamics package called FloWorks. © 2011 Taylor & Francis Group.

Using an infra-red sensor to measure the dynamic behaviour of N2O gas escaping through different sized holes

An anastomosis is a surgical procedure that consists of the re-connection of two parts of an organ and is commonly required in cases of colorectal cancer. Approximately 80% of the patients diagnosed with this problem require surgery. The malignant tissue located on the gastrointestinal track must be resected and the most common procedure adopted is the anastomosis. Studies made with 2,980 patients that had this procedure, show that the leakage through the anastomosis was 5.1%. This paper discusses the dynamic behavior of N2O gas through different sized leakages as detected by an Infra-Red gas sensor and how the sensors response time changes depending on the leakage size. Different sized holes were made in the rigid tube to simulate an anastomostic leakage. N2O gas was injected into the tube through a pipe and the leakage rate measured by the infra-red gas sensor. Tests were also made experimentally also using a CFD (Computational Fluid Dynamics) package called FloWorks. The results will be compared and discussed in this paper.