Lenard M. Lichtenberger

Structural and hormonal alterations in the gastrointestinal tract of parenterally fed rats

This study examines the effect of prolonged absence of oral food intake on structural parameters of the gastrointestinal tract in rats maintained nutritionally by intravenous feeding for up to 3 weeks. During this time, their body weights increased by 22%. Controls fed a nearly isocaloric oral diet were sham operated and harnessed in the same manner as their parenterally fed counterparts. Parenteral feeding resulted in a significant decrease in the weights (per 100 g body weight) of the oxyntic gland area of the stomach, small intestine, and pancreas. The weights of the spleen, testes, kidneys, and antral region of the stomach were unaltered. In the small intestine there was a significant loss of DNA and a near doubling of the RNA:DNA ratio in the parenterally fed animals. In the absence of an oral diet antral gastrin levels decreased to one-thirtieth of the control level. The following conclusions are suggested by these results. First, the oral intake and/or physical presence of food within the gastrointestinal tract are necessary for structural maintenance of some tissues of that tract. Second, the disproportionate decrease in weight that occurs in certain tissues is apparently unrelated to the absence of nutrients which might normally be utilized directly from the lumen. Third, maintenance of normal tissue stores of the hormone, gastrin, is dependent on stimuli provided by oral ingestion and the presence of food in the gastrointestinal tract.

Action of Gastrin on Gastrointestinal Structure and Function

In previous communications we have reported using the rat fed by total parenteral nutrition to examine the effects of the absence of food from the gut on functional and structural parameters of the gastrointestinal tract. In the current study three groups of animals were fed parenterally; one received a continuous infusion of pentagastrin equal to about one-half the D50 for acid secretion, another received a comparable infusion of histamine, and a third group was given only the liquid diet. These animals were compared to orally fed sham operated controls. The parenterally fed animals had significantly lower levels of antral and serum gastrin. When compared to whole body weight, the weights of the oxyntic gland area of the stomach, the pancreas, and the small intestine were significantly lower. In addition, the total and specific activities of the disaccharidase enzymes were significantly reduced. Pentagastrin prevented both the decreases in weights of the gastrointestinal tissues and the decreases in dissaccharidase activity. Histamine was without effect. We conclude that pentagastrin prevents the changes in gastrointestinal structure and function caused by the absence of food from the gut and that the trophic action of gastrin is necessary for the maintenance of the functional and structural integrity of the gastrointestinal tract.

Effect of 16,16-Dimethyl Prostaglandin E2 on the Surface Hydrophobicity of Aspirin-Treated Canine Gastric Mucosa

The canine gastric mucosa has a uniquely hydrophobic or nonwettable surface that is rapidly disrupted by damaging agents such as aspirin. In this study we investigated the effects of acidified aspirin on the wettability of the luminal surface of gastric mucosae mounted in Ussing chambers in the presence of varying concentrations of 16,16-dimethyl prostaglandin E2. It was determined that surface hydrophobicity of the stomach, as measured by contact angle measurements, could be reduced by 50% with an aspirin concentration of 5 mM in the mucosal bath and that this change could be completely and significantly reversed by the addition of 16,16-dimethyl prostaglandin E2 (1 microgram/ml) to the nutrient compartment. 16,16-Dimethyl prostaglandin E2 at this dose was less effective in restoring the surface hydrophobicity in response to a higher concentration of aspirin (20 mM) that abolished the nonwettable property of the tissue. The reduced surface hydrophobicity in the presence of 5 mM aspirin could be increased in a dose response relationship to the nutrient 16,16-dimethyl prostaglandin E2 concentration, with an effect being seen at doses as low as 1 ng/ml. These results support the concept that prostaglandins may protect the stomach by the maintenance of a nonwettable hydrophobic lining between damaging agents in the lumen and the gastric epithelium.

Luminal surface hydrophobicity of canine gastric mucosa is dependent on a surface mucous gel

The contribution of the surface mucous gel to the stomachs hydrophobic luminal properties and how these properties are affected by both damaging and cytoprotective agents were studied. Canine gastric mucosa, determined to be hydrophobic in nature by contact angle analysis, had an adherent periodic acid-Schiff-reactive mucous gel layer over 85% of its luminal surface, as observed under light microscopy. Extracellular structures reactive with the phospholipid-selective stain, iodoplatinate, were observed ultrastructurally in these tissues, within and at the luminal interface of the surface mucous gel. Incubating the luminal surface of gastric mucosa mounted in Ussing chambers in acidified aspirin promoted the exfoliation of surface epithelium and markedly reduced surface hydrophobicity, surface periodic acid-Schiff reactivity, and transmucosal potential difference. Addition of 16,16-dimethyl prostaglandin E2 to the nutrient compartment of these chambers maintained surface hydrophobicity at control levels but did not prevent aspirin-induced reductions in potential difference or cellular damage to the surface epithelium. However, prostaglandin did attenuate exfoliation of aspirin-damaged surface mucous cells and preserved the surface mucous gel. These results indicate that the stomachs hydrophobic lining is closely associated with the presence of a surface mucous gel layer, is not an effective barrier to the penetration of lipid-soluble damaging agents such as acidified aspirin, and is maintained by exogenous prostaglandin as is the mucous gel layer, even in the presence of luminal aspirin. The ability of prostaglandin to maintain a hydrophobic mucous gel layer over compromised tissue may, in part, explain its ability to limit aspirin-induced injury and promote the recovery and restitution of the surface epithelium.

Phospholipid association reduces the gastric mucosal toxicity of aspirin in human subjects

OBJECTIVES:In previous studies on rats, we have shown that aspirin (ASA)-induced injury to the gastric mucosa is markedly reduced or completely abolished if ASA is chemically associated with the phospholipid, phosphatidylcholine (PC). We have also shown that the protective effect of PC does not influence the ability of ASA to inhibit mucosal cyclooxygenase (COX) activity in the stomach and other tissues. We therefore sought to assess the effect of PC-associated ASA (ASA/PC) on the gastric mucosa of normal volunteers and to compare the results with the use of ASA alone.METHODS:Sixteen normal healthy subjects were administered ASA or ASA/PC in a randomized, double-blind, crossover study. The subjects received ASA in a dose of 650 mg three times a day for 3 days or an equivalent dose of ASA chemically associated with PC. Endoscopy was performed at baseline and again on the morning of day 4, after the subjects had taken the final dose of the test drug. On both occasions, antral biopsy specimens were obtained for the assessment of mucosal COX activity and prostaglandin concentration.RESULTS:The number (mean ± SD) of gastric erosions seen with the ASA/PC formulation was significantly less than when ASA was used alone (8.7 ± 10.7 vs 2.9 ± 4.3; p < 0.025). A similar trend was seen in the duodenum but the difference was statistically not significant. The antral mucosal COX activity, as well as the level of prostaglandin 6-keto PGF1α, were reduced significantly (80–88%) and to a similar extent by both ASA and ASA/PC.CONCLUSIONS:The present study shows that acute aspirin-induced damage to the gastric mucosa can be reduced by chemically associating ASA with PC. The mechanism of mucosal protection provided by this compound is not related to any alteration in the ability of ASA to inhibit mucosal COX activity. We believe this protection is attributable to the maintenance of the defensive hydrophobic barrier of the gastric mucosa.

NSAID injury to the gastrointestinal tract: evidence that NSAIDs interact with phospholipids to weaken the hydrophobic surface barrier and induce the formation of unstable pores in membranes

In this review, we have discussed our current understanding of the barrier properties that are in place to protect the upper gastrointestinal mucosa from luminal acid, and the pathogenic mechanism by which nonsteroidal anti‐inflammatory drugs (NSAIDs) induce injury to the gastrointestinal tract. The changes in our view of the importance of NSAID‐induced cyclo‐oxygenase (COX) inhibition on the pathogenesis and prevention of NSAID‐induced gastrointestinal injury is presented. The focus of this paper has been placed on the effects of NSAIDs on the mucosal surface, and specifically the effect of these powerful drugs in inducing changes in the hydrophobicity, fluidity, biomechanical and permeability properties of extracellular and membrane phospholipids. Lastly, recent evidence is presented that salicylic acid and related NSAIDs may alter the stability of membranes, inducing the formation of unstable pores that may lead to back‐diffusion of luminal acid and membrane rupture. This understanding of the interaction of NSAIDs with membrane phos‐pholipids may prove valuable in the design of novel NSAID formulations with reduced gastrointestinal side‐effects.

Interaction of Indomethacin and Naproxen with Gastric Surface-active Phospholipids: A Possible Mechanism for the Gastric Toxicity of Nonsteroidal Anti-Inflammatory Drugs (NSAIDs)

The possibility that the molecular mechanism underlying the topical gastric irritancy of nonsteroidal anti-inflammatory drugs (NSAIDs) may involve alterations in the surface-active properties of gastric phospholipids was investigated. Indomethacin and naproxen were intragastrically administered to rats and the hydrophobicity of the luminal surface of the stomach wall was assessed by contact angle analysis. Both NSAIDs have the ability to attenuate the phospholipid-related hydrophobic properties of the gastric mucosa by more than 80-85% in a dose-dependent fashion. Potential molecular interactions between both NSAIDs and surface-active phospholipids were analyzed using fluorescent probes. Indomethacin has the ability to displace, in a dose-dependent manner, ANS (1-anilino-8-naphthalene sulphonate), a fluorescent anionic probe previously bound to the head group of phosphatidylcholine molecules. Estimations of the resonance fluorescence transfer between naproxen and the surface probe ANS or the hydrophobic probe, pyrene, bound to dipalmitoylphosphatidylcholine (DPPC) vesicles revealed that naproxen diffuses within the phospholipid bilayers. The dynamic of the gastric lipid material extracted from the surface scraping material (SSM) of the mucosa was altered by the NSAID as shown by the increase in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) (at 25 degrees, rSSM = 0.106+/-0.006, rssM + indomethacin = 0.137+/-0.005, and rSSM + naproxen = 0.133+/-0.007, P < 0.001). The thermodynamic behavior of a model bilayer containing DPPC was also perturbed by the NSAIDs tested. These results provide evidence that NSAIDs may reduce the ability of gastric surface-active phospholipids to form a hydrophobic protective layer.

Depression of Antral and Serum Gastrin Concentration by Food Deprivation in The Rat

In rats fasted 4 days, immunoreactive gastrin concentrations decreased to one-third of the fed levels in antral tissue and to one-eight of the fed levels in serum. The number of antral cells that reacted with fluorescent antigastrin antiserum was also correspondingly decreased. After refeeding, serum gastrin returned to normal levels in 6 days, whereas antral gastrin concentration recovered after 9 days. Normal gastrin levels were maintained in rats fed a nutritious liquid diet over a 6-day period, whereas tissue and serum hormone concentrations decreased to low levels in rats placed on a high bulk non-nutritive diet over the same period. These results suggest that food in the gastrointestinal tract is necessary for the maintenance of normal serum and antral gastrin concentration in rats. The effect of food is most likely attributable to chemical constituents and not distention by bulk.

INSIGHT INTO NSAID-INDUCED MEMBRANE ALTERATIONS, PATHOGENESIS AND THERAPEUTICS: CHARACTERIZATION OF INTERACTION OF NSAIDS WITH PHOSPHATIDYLCHOLINE

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most widely consumed pharmaceuticals, yet both the mechanisms involved in their therapeutic actions and side-effects, notably gastrointestinal (GI) ulceration/bleeding, have not been clearly defined. In this study, we have used a number of biochemical, structural, computational and biological systems including; Fourier Transform InfraRed (FTIR). Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) spectroscopy, and cell culture using a specific fluorescent membrane probe, to demonstrate that NSAIDs have a strong affinity to form ionic and hydrophobic associations with zwitterionic phospholipids, and specifically phosphatidylcholine (PC), that are reversible and non-covalent in nature. We propose that the pH-dependent partition of these potent anti-inflammatory drugs into the phospholipid bilayer, and possibly extracellular mono/multilayers present on the luminal interface of the mucus gel layer, may result in profound changes in the hydrophobicity, fluidity, permeability, biomechanical properties and stability of these membranes and barriers. These changes may not only provide an explanation of how NSAIDs induce surface injury to the GI mucosa as a component in the pathogenic mechanism leading to peptic ulceration and bleeding, but potentially an explanation for a number of (COX-independent) biological actions of this family of pharmaceuticals. This insight also has proven useful in the design and development of a novel class of PC-associated NSAIDs that have reduced GI toxicity while maintaining their essential therapeutic efficacy to inhibit pain and inflammation.

Phospholipase activity of Helicobacter pylori and its inhibition by bismuth salts - Biochemical and biophysical studies

In this study we measured phospholipase A (PLA) and C (PLC) activity of media filtrates and French Press lysates of the gastritis-inducing bacteriaHelicobacter pylori. We report here that bothH. pylori lysates and filtrates contain PLA1, PLA2, and C enzymes, which readily hydrolyze a radiolabeled dipalmitoylphosphatidylcholine (DPPC) and phosphorylcholine substrates, respectively. The specific activity of both PLA and C enzymes were greatest in the 6.5–7.0 and 8.4–8.8 pH ranges, respectively. Colloidal bismuth subcitrate (CBS) induced a dose-dependent inhibition of PLA2 and C activity of bothH. pylori lysates and filtrates. This inhibitory effect of CBS on PLA2 was antagonized in a dose-dependent fashion by the addition of CaCl2 to the incubation mixture, suggesting that calcium and bismuth may be competing for the same site on the enzyme. In contrast, the ability of bismuth salts to inhibit PLC activity ofH. pylori lysates was not antagonized by CaCl2. Employing a biophysical assay system for surface wettability, it was determined thatH. pylori lysates had the capacity to remove a synthetic phospholipid monolayer off a glass in a dose-dependent fashion. This ability of the bacterial lysates to catalyze the transformation of a hydrophobic surface to a wettable state was significantly attenuated in the presence of bismuth salts. Our experimental results are, therefore, consistent with the possibility thatH. pylori colonization compromises the stomachs barrier to acid by eroding a phospholipid lining, possibly a monolayer, on the surface of the gastric mucus gel and that this process is blocked in response to bismuth therapy.In this study we measured phospholipase A (PLA) and C (PLC) activity of media filtrates and French Press lysates of the gastritis-inducing bacteria Helicobacter pylori. We report here that both H. pylori lysates and filtrates contain PLA1, PLA2, and C enzymes, which readily hydrolyze a radiolabeled dipalmitoylphosphatidylcholine (DPPC) and phosphorylcholine substrates, respectively. The specific activity of both PLA and C enzymes were greatest in the 6.5-7.0 and 8.4-8.8 pH ranges, respectively. Colloidal bismuth subcitrate (CBS) induced a dose-dependent inhibition of PLA2 and C activity of both H. pylori lysates and filtrates. This inhibitory effect of CBS on PLA2 was antagonized in a dose-dependent fashion by the addition of CaCl2 to the incubation mixture, suggesting that calcium and bismuth may be competing for the same site on the enzyme. In contrast, the ability of bismuth salts to inhibit PLC activity of H. pylori lysates was not antagonized by CaCl2. Employing a biophysical assay system for surface wettability, it was determined that H. pylori lysates had the capacity to remove a synthetic phospholipid monolayer off a glass in a dose-dependent fashion. This ability of the bacterial lysates to catalyze the transformation of a hydrophobic surface to a wettable state was significantly attenuated in the presence of bismuth salts. Our experimental results are, therefore, consistent with the possibility that H. pylori colonization compromises the stomachs barrier to acid by eroding a phospholipid lining, possibly a monolayer, on the surface of the gastric mucus gel and that this process is blocked in response to bismuth therapy.