Characterization of gastric ATPase vesicle transport

The microsomal fraction of both dog and hog gastric mucosa contains a K⁺ activated ATPase. ATP phosphorylates a peptide of c̅ 100,000 Mᵣ in both species, and dephosphorylation is stimulated by K⁺. By a combination of differential and zonal density gradient centrifugation a membrane fraction is produced containing almost exclusively this peptide region. These vesicles, upon the addition of ATP, take up H⁺ and extrude K⁺. The action of ionophores such as nigericin or valinomycin and the uptake of lipid permeable anions such as thiocyanate or anilino-naphthosulfonic acid indicate the lack of a potential difference during transport. Reconstitution of this material into a planar bilayer indicates that ATP activates a K⁺ conductance and hence, in the presence of K⁺ also a low potential difference is observed. These data suggest that this H⁺ pump is non-electrogenic as prepared in the vesicular form. Using an antibody obtained from rabbits immunized with the highly purified membrane fractions, it was demonstrated that this membrane was derived uniquely from gastric parietal cells. Hence, based on the ability of this ATPase to actively transport H⁺, its cellular origin and on the well known K⁺ requirement for acid secretion in amphibia and mammals, it is concluded that this ATPase is a component of the HCl secretory mechanism of gastric mucosa.Paper I Secretion by in Vitro Amphibian Gastric Mucosa. IN: Physiology of Gastric Secretion: NATO Institute, Myren, J., ed, p. 186 -202, Oslo Press, 1968. • Paper II Frog Gastric Mucosal ATPase. Proc. Soc. Exptl. Biol. Med. 119: 1023 -1027, 1965. • Paper III Action of SCN on Rat Liver Mitochondria. Proc. Soc. Exptl. Biol. Med. 133: 456 -459, 1970. • Paper IV Action of Thiocyanate on Gastric Mucosa in Vitro. Biochim. Biophys. Acta 173: 509 -517, 1969. • Paper V Role of ATP and ATPase in Gastric Acid Secretion. IN: Gastric Secretion, (Sachs, G., Heinz, E., Ullrich, K.J., eds), Academic Press, New York, pp. 321 -343, 1972. • Paper VI Properties of ATPase of Gastric Mucosa. V. Preparation of membranes and mitochondria by zonal centrifugation. Biochim. Biophys. Acta 311: 545 -564, 1973. • Paper VII Characterization of Gastric Mucosal Membranes. VI. The presence of channel- forming substances. Biochim. Biophys. Acta 332: 233 -247, 1974. • Paper VIII Characterization of Gastric Mucosal Membranes. Composition of gastric cell membranes and poly - peptide fractionation using ionic and nonionic detergents. Arch. Biochem. Biophys. 161: 456 -471, 1974. • Paper IX Pronase Method for Isolation of Viable Cells From Necturus Gastric Mucosa. Gastroenterology 61: 189 -200, 1971. • Paper X Specific Effect of Acetylsalicylic Acid on Cation Transport of Isolated Gastric Mucosal Cells. Am. J. Physiol. 235: E16 -E21, 1978. • Paper XI Studies on Adenyl Cyclase in Necturus Gastric Mucosa. Arch. Biochem. Biophys. 143: 123 -126, 1971. • Paper XII Adenyl and Guanyl Cyclase in Rabbit Gastric Mucosa. Am. J. Physiol. 225: 1359 -1363, 1973. • Paper XIII Action of Cholinergic Drugs on Necturus Gastric Mucosa. Am. J. Physiol. 210: 1056 -1060, 1970. • Paper XIV Action of Burimamide, a Histamine Antagonist, on Acid Secretion in Vitro. Am. J. Physiol. 226: 898- 902, 1974. • Paper XV Effects of Sodium Removal on Acid Secretion by the Frog Gastric Mucosa. Proc. Soc. Exptl. Biol. Med. 123: 47 -52, 1966. • Paper XVI Ion Transport by Amphibian Antrum in Vitro. I. General Characteristics. Am. J. Physiol. 228: 1188 -1198, 1975. • Paper XVII Quantitation of Conductance Pathways in Antral Gastric Mucosa. J. Gen. Physiology 65: 645 -662, 1975. Paper XVIII Properties of Gastric Antrum. III. Selectivity and modification of shunt conductance. Gastroenterology 72: 72 -77, 1977. • Paper XIX A Molecular Approach to Epithelial Conductance: Gastric Mucosa. Aired Benzon Symp. V. Transport Mechanisms in Epithelia, Munksgaard, Copenhagen, pp. 257 -274, 1973. • Paper XX Conductance Pathways in Epithelial Tissues. Exp. Eye Res. 16: 241 -249, 1973. • Paper XXI Electrical Properties of Isolated Cells of Necturus Gastric Mucosa. Biochim. Biophys. Acta 241: 261 -272, 1971. • Paper XXII Microelectrode Studies of Gastric Mucosa and Isolated Gastric Cells. Symp. Med. Hoechst. IN: Electrophysiology of Epithelial Cells, p. 257 -279, 1971. • Paper XXIII Microelectrode Studies of Fundic Gastric Mucosa: Cellular Coupling and Shunt Conductance. J. Membr. Biol. 19: 105 -128, 1974, • Paper XXIV The Action of Amytal on Frog Gastric Mucosa. Biochim. Biophys. Acta 143: 522 -531, 1967. Paper XXV Metabolism of Dog Gastric Mucosa. I. Nucleotide Levels in Parietal Cells. J. Biol. Chem. 250: 8321 -8329, 1975. • Paper XXVI Metabolism of Dog Gastric Mucosa. Levels of glycolytic citric acid cycle and other intermediates. J. Biol. Chem. 252: 8572 -8581, 1977. • Paper XXVII Redox Involvement in Acid Secretion in the Amphibian Gastric Mucosa. J. Membr. Biol. 35: 189 -204, 1977. • Paper XXVIII REVIEW: H⁺ Transport by a Non-electrogenic Gastric ATPase as a Model for Acid Secretion. Rev. Physiol. Biochem. Pharmac. 79: 133 -167, 1977. • Paper XXIX Characterization of Gastric Mucosal Membranes. VIII. Localization of peptides by iodination and phosphorylation. J. Biol. Chem. 250: 4802 -2809, 1975. • Paper XXX Characterization of Gastric Mucosal Membranes. IX. Fractionation and Purification of K⁺- ATPase containing vesicles by zonal centrifugation and free flow electrophoresis technique. Biochim. Biophys. Acta. 465: 311 -330, 1977. • Paper XXXI A Non -electrogenic H⁺ Pump in Plasma Membranes of Hog Stomach. J. Biol. Chem. 251: 7690 -7698, 1976. • Paper XXXII Proton Transport by Gastric Membrane Vesicles. Biochim. Biophys. Acta 464: 313 -327, 1977. • Paper XXXIII Cation Transport by Gastric H⁺ + K⁺ ATPase. J. Membr. Biol. 32: 361 -381, 1977. • Paper XXXIV Use of l-anilino-8-naphthalene sulfonate as a Probe of Gastric Vesicle Transport. J. Membr. Biol. 32: 301 -318, 1977. • Paper XXXV Reconstitution of a Proton Pump from Gastric Mucosa. J. Membr. Biol. 35: 285 -301, 1977. • Paper XXXVI Metabolic and Membrane Aspects of Gastric H⁺ Transport. Gastroenterology 73: 931 -940, 1977. • Paper XXXVII Tissue and Cell Localization of Hog Gastric Plasma Membrane by Antibody Technique. Proc. Symp. Gastric. Ion Transport, Special Suppl. Acta Physiol. Scand (Obrink, K.J. and Flemstrom,-G., eds) p. 293 -305, 1978. • Paper XXXVIII Transport Parameters of Gastric Vesicles. Proc. Symp. Gastric Ion Transport, Special Suppl. Acta Physiol. Scand. (Obrink, K.J. and Flemstrom, G., eds) p. 409 -426, 1978. • Paper XL Enzymic Modification of Gastric Transport ATPase. IN: Frontiers of Biological Energetics (Dutton, P.L., Leigh, J., Scarpa, A., eds) Academic Press, New York, Vol. 1, p. 545 -554, 1978. • Paper XLI Transport Characteristics of Frog Gastric Membranes Biochim. Biophys. Acta 551: 432 -447, 1979. • Paper XLII Quantitation of Hydrogen Ion and Potential Gradients in Gastric Plasma Membrane Vesicles. Biochemistry 17: 3345 -3353, 1978

Laser doppler assessment of gastric mucosal blood flow in normals and its relationship to the systemic activity of growth peptides in healing and non healing gastric ulcers.

Thesis (M.Med.Sc.)-University of Natal, Durban, 1999.The pattern of mucosal blood flow in normal human stomachs, and benign gastric ulcers was assesed with laser Doppler flowmetry and the relationship between a single determination of ulcer blood flow and the systemic level of growth factors was investigated. A significant ascending gradient in mucosal blood flow from the antrum to fundus was demonstrated. Different levels of cellular activity in the regions of the stomach may explain this gradient. In the gastric ulcers that healed on standard medical therapy mucosal blood flow was significantly increased in comparison to normal stomachs. In the ulcers that were refractory to standard medical therapy mucosal blood flow was significantly lower than in normal stomachs and healing ulcers. Higher systemic levels of the growth factor bFGF were demonstrated in healing ulcers compared to non-healing ulcers. Gastric mucosal blood flow can increase in response to the increased metabolic demands of healing, however impairment of this response may be an important factor preventing healing of benign gastric ulcers. It would appear that non-healing of gastric ulcers can be predicted at initial diagnosis by reduced peri-ulcer gastric mucosal blood flow and low blood levels of bFGF

Understanding intestinal and pancreatic hormone secretion in health and type 2 diabetes: (pre-)clinical studies and technical innovations

The gastrointestinal (GI) tract and pancreatic islets are key components of the endocrine system, responsible for the release of an array of peptide hormones, which orchestrate metabolic homeostasis through regulation of energy intake, nutrient digestion, absorption and metabolism. Of numerous hormones released from the gut, the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), govern the secretion of both insulin and glucagon from pancreatic islets. Together, these hormones play a critical role in maintaining glucose homeostasis. Disrupted secretion and/or action of the incretins and pancreatic hormones underpins the development of type 2 diabetes (T2D) - a global epidemic characterised by elevated blood glucose concentrations and associated with devastating micro- and macro-vascular complications. Accordingly, an improved understanding of the physiology and pathophysiology of GI and pancreatic hormones in health and T2D is of major relevance to the development of effective strategies to both prevent and better manage T2D. This thesis comprises a series of clinical and preclinical evaluations that provide novel insights into the determinants of GI and islet hormone secretion (Chapters 3-6). In addition, it details the cross-disciplinary collaborative development of two ‘organ-on-a-chip’ platforms for dissecting the secretory function of both intestinal tissues and pancreatic islets (Chapters 7-8). Chapter 1 provides an overview of the secretion and action of GI hormones arising from the complex interaction between luminal nutrients/bioactive compounds and the gut mucosa, and details conventional and innovative research tools/platforms that are indispensable for the investigation of GI hormone secretion. Chapter 2 summarises the molecular mechanisms underlying insulin secretion from pancreatic islets, with a focus on the role of Ca2+ signalling, and systematically reviews the development of diverse research platforms that are fundamental to progressing islet research. Given the substantial sex-related differences in glucose metabolism and risk of T2D, the study described in Chapter 3 explores the sex disparity in incretin hormone secretion, and compares the incretin and glycaemic responses to standardised intraduodenal glucose infusions within the physiological range of gastric emptying between healthy young men and women. While insulin resistance and consequently a relative deficiency in insulin secretion are recognised as key metabolic derangements in T2D, there is accumulating evidence indicating that excessive glucagon secretion also underpins the development of dysglycaemia during both the fasting and postprandial phases. In the liver, insulin and glucagon signalling pose counter-regulatory effects on hepatic glucose production. Alterations in hepatic function have the potential to disrupt hepatic insulin and glucagon signalling, leading to pathological changes in insulin and glucagon secretion. The study reported in Chapter 4 evaluates the relationships of blood glucose, plasma insulin, C-peptide and glucagon, both during fasting and after 75g oral glucose, with serum liver enzymes in healthy and T2D subjects, and in T2D subjects before and after a mixed meal. Given the major role of the rate of gastric emptying (GE) in determining nutrient digestion and absorption, GE may influence the glucagon and glycaemic responses in T2D. Therefore, Chapter 5 further examines the relationships of plasma glucagon and blood glucose with the rate of gastric emptying (GE) of a standardised mashed potato meal in individuals with well-controlled T2D. Strategies that are effective for modulating GI and pancreatic hormone secretion have the potential to improve glycaemic control in T2D. The recent recognition that the GI tract can detect a range of physiological and pharmacological bitter substances via a family of type 2 monomeric G-protein-coupled receptors, namely bitter taste receptors (BTRs), to release GI hormones has led to growing interest in the administration of bitter tastants to stimulate GI hormone secretion for the management of metabolic disorders, including T2D. However, the effects of bitter substances beyond the GI tract have received little attention. Chapter 6 reports the effect of a bitter substance, denatonium benzoate (DB), on insulin secretion in a series of in vitro and ex vivo experiments using a rodent pancreatic β-cell line, INS-1 832/13 cells, and isolated mouse pancreatic islets. In the latter, the effects of DB on the secretion of other islet hormones, including glucagon, GLP-1 and somatostatin, were also characterised. While the currently available cell/tissue models and in vivo tools have substantially advanced the knowledge on the physiology and pathophysiology of incretins and islet hormones, there is rising demand for sophisticated biomimetic platforms to address the increasingly complicated biological challenges and improve the translational success from benchtop to bedside. To this end, the development of a gut-on-a-chip (GOC) system is described in Chapter 7 which facilitates continuous monitoring of dynamic GLP-1 secretion from primary mouse intestinal tissue. Similarly, the development and customisation of a microfluidic sensing platform is described in Chapter 8, allowing quantification of the dynamic changes of Ca2+ and insulin concurrently, enabling investigation of the secretory function of isolated islets.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 202

Gastrointestinal injury following cardiopulmonary bypass

The gastrointestinal (GI) tract may be the source of a number of bacterial and non-bacterial mediators, which may contribute to the development of morbidity and mortality following episodes of gut hypoperfusion/ ischaemia. The aim of this thesis has been to identify the changes in gut blood flow, oxygenation and function following cardiopulmonary bypass (CPB) and their relationship to the development of post-CPB morbidity. The findings are summarised below: The retrospective study identified age (>65 yr) and CPB time as risk factors for the development of post-CPB intra-abdominal complications . Tonometrically determined values for intramucosal pH (pHi) need temperature correction to avoid calculation of erroneously high values during hypothermic CPB. Considerable hypoperfusion occurs during hypothermic CPB, with laser Doppler flowmetry (LDF) falling to approximately 45% of pre-CPB values. The gastric and colonic pHi becomes acidotic (<7.35) during the re-warming and immediate post-CPB period. Intramucosal acidosis occurs at a time when mucosal LDF blood flow is normal or supranormal. CPB increases gut permeability and reduces the absorption of the monosaccharides, 3-O-m-D-glucose, D-xylose & L-rhamnose. Post-CPB gut permeability has a temporal relationship with the CPB time. Pulsatile flow attenuates the increase in post-CPB gut permeability. Endotoxaemia occurs during CPB but is not associated with the production of TNFα; pulsatile flow attenuates this endotoxaemia. When examining perfusion and patient factors, the best predictor for a protracted ventilation & ICU stay for patients was a low gastric pHi (<7.35). A canine model of CPB supported the clinical findings, but also found that: (a) changes in large vessel blood flow do not indicate more dynamic alterations in small vessel blood flow (b) blood flow is prioritised to the mucosa at the expense of the serosal aspects of the bowel wall (c) in the re-warming phase of hypothermic CPB & the immediate post-CPB period, when intramucosal acidosis occurs, there is a disparity between gut oxygen consumption & delivery (b) increased expression of vasoactive intestinal peptide was found in the neural plexus of the submucosa post-CPB, which may indicate a role in preserving mucosal blood flow during periods of hypoperfusion

Teratology Studies on Lewisite and Sulfur Mustard Agents: Effects of Sulfur Mustard in Rats and Rabbits

Sulfur mustard (HD) was administered to rats and rabbits by intragastric intubation. Rats were dosed daily from 6 through 15 days of gestation (dg) with 0. 0.5, 1.0 or 2.0 mg of HD/kg; rabbits were dosed with 0, 0.4, 0.6 or 0.8 mg/kg on 6 through 19 dg. Maternal animals were weighed periodically and, at necropsy, were examined for gross lesions of major organs and reproductive performance; live fetuses were weighed and examined for external, internal and skeletal defects. In rats, reductions in body weights were observed in maternal animals and their female fetuses at the lowest administered dose (0.5 mg/kg), but the incidence of fetal malformations was not increased. In rabbits the highest administered dose (0.8 mg/kg) induced maternal mortality and depressed body weight measures but did not affect fetal development. These results suggest that orally administered HD is not teratogenic in rats and rabbits since fetal effects were observed only at dose levels that induced frank maternal toxicity. Estimations of dose ranges for "no observable effects levels" in rats and rabbits, respectively, were: 0.8 mg/kg in their fetuses

The development of a polymer patch for the treatment of oesophageal leaks and perforations

Oesophageal leaks and perforations carry mortality and morbidity. Management options include surgery, stents, drains, and negative pressure therapy; all current treatment options, however, have significant associated morbidity. Here we investigate an alternative approach using a temporary polymer patch to adhere to and seal the oesophageal defect whilst supporting healing and function without damaging local tissue. This approach could offer a timely, cost effective and minimally invasive approach, especially for environments where specialised and complex surgery may not be available such as resource limited military and humanitarian healthcare settings. To quantitatively compare perforation management options (including the novel patch) a novel ex vivo model of oesophageal perforation repair was developed. The patch had a degradable poly(ε-caprolactone urea) urethane backbone with a polyhedral oligomeric silsesquioxane (POSS PCLU) component. The study successfully developed an ex vivo porcine oesophagus bench top model for testing the strength of oesophageal repair techniques. The model allowed a maximum pressure of 100kPa and was capable of quantitatively assessing perforation repair approaches. A major consideration in the patch approach to perforation repair is the adhesion of the patch to the oesophagus. The study demonstrates that the surface chemistry of the patch can be modified to improve adhesion and increase attachment strength. Here we found that both modifications of the tissue adhesion site in vivo (by removal of the outer epithelial layer of the oesophagus) and by modifying the surface chemistry of the patch (by plasma treatment) it was possible to enhance fibrin patch adhesion to the oesophagus. The optimisation of the patch surface chemistry and host tissue site for fibrin-based adhesion could have an impact in the medical use of polymer patches throughout the body

Immunological studies in diabetes mellitus

The studies presented here were all undertaken in the labor- atories of Dr. W. James Irvine, Reader in Medicine, University of Edinburgh. Dr. Irvine kindly permitted me access to his technical facilities and arranged for help and tuition in laboratory techniques from his permanent staff. He suggested avenues along which new research should be directed and gave active help and support in submitting applications for research grants. Above all, he gave me the benefit of his own immense expertise in the field of medical immunology, in which he is an international authority. It was a privilege to work with him and I owe him an enormous debt of gratitude.Professor K.W. Donald, University of Edinburgh, was good enough to allow me to spend a prolonged period in laboratory -based research during my tenure of a senior registrarship in his department. More- over he actively encouraged the work and lent the weight of his support to applications for research grants. I could not have engaged in work of this nature without his assistance and advice and I am grateful to him for all his kindness.Substantial finance assistance for many of the studies presented here was obtained from research grants made (jointly or separately) to Dr. Irvine and myself by the Medical Research Council, the Scottish Home and Health Department and the South- Eastern Regional Hospital Board. These grants were quite invaluable in allowing the purchase of expensive equipment and reagents and in providing finance for trained technical help. To all these bodies I extend my thanks and my gratitude for their support.e studies of immunology in diabetes mellitus which form the basis of this thesis were performed upon patients attending the Diabetic Department, Edinburgh Royal Infirmary, under the care of Dr. L.J.P. Duncan and Dr. B.F. Clarke. Drs. Duncan and Clarke spent many years in teaching me the rudiments of clinical diabetes mellitus and were good enough to allow me an unlimited facility to study the patients under their care. Meaningful studies of immuno- logy in diabetes could not have been undertaken without access to their patients and I am extremely grateful for their help and support as well as their tuitionMany other friends and colleagues collaborated in some of the studies presented here. Their contributions are acknowledged individually in the relevant chapters but I would extend particular thanks to Dr. W.J. Penhale, Senior MRC Research Worker, who taught me a great deal of basic immunology, initiated and directed the development of one particular assay system used in the work and was an authoritative source of information on difficult technical questions

Volume 10, issue 1

The mission of CJS is to contribute to the effective continuing medical education of Canadian surgical specialists, using innovative techniques when feasible, and to provide surgeons with an effective vehicle for the dissemination of observations in the areas of clinical and basic science research. Visit the journal website at http://canjsurg.ca/ for more.https://ir.lib.uwo.ca/cjs/1083/thumbnail.jp

Absorption and bioavailability of prednisolone in the dog

Incomplete gastrointestinal absorption of a drug from a formulation has long been recognised, as the 19th century quotation above shows. Only in the past 10 to 15 years has attention been directed to studying this problem and determining the factors necessary for efficient absorption from the gastrointestinal tract and at devising methods for the study and routine testing of individual batches of formulation intended for oral administration