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Biomedical subjects

T J Layden

Publications and source records attributed to T J Layden.

At least 55 records · Page 3Linked to original sources
PubMed1993

Rabbit esophageal cells show regulatory volume decrease: ionic basis and effect of pH.

BACKGROUND: Regulatory volume decrease (RVD) after osmotic cellular swelling has been shown in several gastrointestinal epithelia but not in esophageal cells. In acid reflux disease, esophageal injury may be related in part to loss of RVD. METHODS: Isolated basal esophageal cells were exposed to an external hyposmolar solution, and changes in relative cell size were assessed using a Coulter counter (Hilaleah, FL) in the presence of K+ and Cl- transport inhibitors and at varying extracellular pH (pHo). RESULTS: At pHo 7.4, a 30% hyposmotic dilution of the external solution caused an initial peak cell swelling (1.15 +/- 0.05-fold) followed by a return to starting cell size by 5 minutes (RVD). RVD was inhibited by Ba2+ (4 mmol/L), quinine (1 mmol/L), or increasing the [K+]o > or = 10 mmol/L. RVD was also inhibited by depleting [Cl-]i or in the presence of 0.5 mmol/L 4,4'-diisothiocyanastostilbene-2,2'-disulfonic acid disodium salt (DIDS) or 50 mumol/L diphenylamine-2-carboxylate, a Cl- conductance inhibitor. To test the effect of pH on RVD, cells in solutions at pHo 7.4, 7.0, or 6.8 were subjected to hyposmotic stress; RVD was significantly inhibited at pHo 6.8. This pH-dependent inhibition of RVD was reversed in the presence of valinomycin, a K+ ionophore. CONCLUSIONS: These studies show that isolated esophageal cells possess RVD mechanisms that are mediated by Cl(-)- and pH-dependent K+ effluxes. RVD appears to be inhibited by a decrease in pHo, suggesting the possibility that acid-induced esophageal injury results from inhibition of normal volume regulatory mechanisms.

Animals↗
PubMed1993

Rabbit esophageal cells possess K+ channels: effect of hyposmotic stress on channel activity.

BACKGROUND: In many cell types, basolateral K+ channels are important in maintaining transepithelial Na+ absorption and regulatory volume decrease (RVD) after hyposmolar stress. However, in the esophagus the effect of K+ transport in maintaining baseline short-circuit current (SCC) (Na+ absorption) and RVD is unknown. METHODS: Ussing chambers were used to evaluate changes in SCC of rabbit esophageal mucosa in response to serosal Ba2+ (4 mmol/L), quinine (1 mmol/L), and increasing serosal [K+]. To determine whether K+ channel(s) are activated in RVD, changes in SCC in response to serosal hyposmolarity (156 mOsm) were assessed in the presence or absence of serosal quinine. RESULTS: Serosal Ba2+, quinine, or increased serosal [K+] caused a decline in baseline SCC. Serosal hyposmolarity caused an increase in SCC that was not blocked by mucosal application of amiloride (10(-4) mmol/L). In contrast, serosal quinine completely blocked the hyposmolar-induced increase in SCC. CONCLUSIONS: These studies suggest that rabbit esophageal cells possess Ba(2+)- and quinine-sensitive basolateral K+ channel(s) that are active under baseline conditions. Potassium conductance(s) also appear to be activated by external serosal hyposmolarity and may be involved in the process of RVD.

Amiloride↗
PubMed1993

Gallstones: choosing the right therapy despite vague clinical clues.

Therapeutic decisions are quite clear-cut for asymptomatic gallstone disease and acute cholecystitis. However, the appropriate therapeutic course for older patients with chronic cholecystitis may be less obvious. Watchful waiting may be reasonable for patients with mild and infrequent symptoms. For healthy patients, cholecystectomy is recommended if symptoms are becoming more frequent and severe. Laparoscopy may reduce the complication rate and be safely performed even in those with underlying medical illness. Oral dissolution therapy can be attempted for qualifying symptomatic patients who are at poor surgical risk or who refuse surgery. Shock wave lithotripsy and contact dissolution therapy show some promise but are currently experimental.

Acute Disease↗
PubMed1992

Rabbit esophageal cell cytoplasmic pH regulation: role of-antiport and-dependenttransport systems.

Regulation of cytoplasmic pH (pHi) of esophageal cells assumes importance as these cells can be exposed to mucosally absorbed acid during gastroesophageal reflux episodes. In this study, we examined whether esophageal cells possess pHi transport systems. Esophageal cells were harvested utilizing a gentle trypsin technique that yieldedcells per esophagus. Cells were attached to a glass cover slip that had been pretreated with rat-tail collagen, and pHi was measured continuously in a spectrofluorometer utilizing 2',7'-bis(2-carboxyethyl)-5(-6)- carboxyfluoroscein acetoxymethyl ester as a pH-sensitive fluorescent probe.The basal pHi of cells exposed to a-containing solution averaged 7.52 ± 0.20 (n = 6). The pHi declined slightly but not significantly to 7.46 ± 0.12 with the addition of 5%and 28 mMWhen H2 4,4'-diisothiocyanatostilbene- 2,2'-disulfonic acid (DIDS; 0.5 mM) was added, pHi was unchanged. However, addition ofM amiloride caused pHi to decrease to 7.29 ± 0.18 (P less than 0.01). When cells were acidified (pHi 6.3-7.0) using a(20 mM) pulse technique, pHi was rapidly restored toward neutrality in the presence of a-free externalconcentration ([]o)-containing solution (pH units/min = 0.26 ± 0.12; n = 8). Alkalinization was completely blocked withM amiloride. In the presence ofM amiloride, 28 mM, and 5%, acidified cells also alkalinized, although at a slower rate (0.11 ± 0.04 pH units/min; n = 16).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Epidemiology considerations in peptic ulcer disease.

Peptic ulcer disease is a common clinical problem. The lifetime risk of peptic ulcer disease is at least 10%. Millions of Americans are affected each year, imposing a major economic burden on the health care system. The overall hospitalization and mortality rates for peptic ulcer disease seem to have decreased substantially over the past few decades. There is much to suggest, however, that these changes were occurring even before effective medical therapy became available. In addition, other influences, such as increasing age of the population at risk, changes in smoking prevalence, and increasing use of nonsteroidal anti-inflammatory drugs, have impacted on the changing epidemiology of peptic ulcer disease. Hospitalization rates for patients with complications of peptic ulcer disease have remained relatively stable or, especially in the case of elderly women with gastric ulcers, increased significantly in recent years. Understanding the epidemiology of peptic ulcer disease will allow improved assessment of the effects of medical and surgical therapy and, hopefully, provide better clues to the etiology of this diverse group of diseases.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1991

Isolation, characterization, and attachment of rabbit distal colon epithelial cells.

The authors investigated various enzymatic digestion procedures for isolating epithelial cells from the distal colon of New Zealand White male rabbits. Rabbit mucosa was washed, diced, and digested for 90 minutes in one of five different solutions, including a new combination consisting of 0.03% collagenase IV and 0.1% pronase (solution V). Solution I (0.3% dispase) yielded 14.2 +/- 8.2 x 10(6) colonocytes/g mucosa, solution II (0.15% dispase and 0.03% collagenase) yielded 7.7 +/- 2.8 x 10(6) colonocytes/g mucosa, and solution III (0.03% collagenase IV) yielded 15.4 +/- 10(6) cells/g mucosa. Solutions I-III have previously been described for the isolation of colonocytes. Solution IV (0.1% pronase and 325 U/mL DNAase) was originally described for the isolation of nasal epithelial cells but yielded only 2.5 +/- 1.2 x 10(6) cells/g mucosa when applied to the isolation of colonocytes. The new combination of pronase and collagenase, solution V, yielded significantly more colonocytes, 34.5 +/- 3.0 x 10(6) cells/g mucosa, than previously described methods (P less than 0.01). Inclusion of 5 mmol/L ethylenediaminetetraacetic acid in any of the solutions enhanced neither viability nor yield. The digestion product of solution V could be enriched for crypts by serial low-speed centrifugations. The epithelial origin of the colonocytes was confirmed by immunofluorescent staining for cytokeratins. Functional viability was tested by determining the presence of a Na+/H+ exchanger, using the pH fluorescent dye bis(carboxymethyl)-5(6)-carboxyfluorescein acetoxymethyl ester to measure intracellular pH. The authors document that sodium-dependent restoration of intracellular pH in colonocytes acid-loaded to a pH of 6.30 occurred at a rate of 0.19 +/- 0.02 pH U/min. Amiloride at concentrations of 1 mmol/L completely inhibited operation of the exchanger, as did sodium substitution with choline or tetramethylammonium. Lineweaver-Burke analysis at this intracellular pH showed a Michaelis constant of 10.71 mmol/L Na+ and a maximum velocity of 0.12 pH U/min. Exposing the colonocytes to 100 nmol/L phorbol 12,13-dibutyrate increased antiporter activity by 62.0%. Finally, the authors describe the synthesis of a new biomatrix composed of the basement membrane of 3T3 NIH fibroblasts that permits significantly improved colonocyte attachment than to glass, plastic, collagen types I or IV, or matrigel.

Animals↗
PubMed1991

In vivo evidence of altered chloride but not potassium secretion in cystic fibrosis rectal mucosa.

In cystic fibrosis, cyclic adenosine monophosphate-mediated chloride secretion is abnormal in respiratory, small intestinal, and rectal mucosa. Calcium-mediated chloride secretion is also aberrant in CF small intestinal mucosa in cystic fibrosis, in contrast to the respiratory epithelia, where it appears to be normal. To determine whether this disparity between calcium- and cyclic adenosine monophosphate-mediated chloride secretion exists in cystic fibrosis rectal mucosa in vivo, transrectal potential difference was measured in age-matched adult cystic fibrosis subjects (n = 8) and control subjects (n = 9) in response to 10-minute luminal perfusions of bethanechol (1 mmol/L) or theophylline (5 mmol/L). In response to bethanechol, an initial (1-minute) negative change in potential difference (-1.4 +/- 1.1 mV; mean +/- SEM) was seen in control subjects, in contrast to a positive change in mean potential difference (+2.5 +/- 1.0 mV) in cystic fibrosis subjects (control vs. cystic fibrosis, P less than 0.05). After 1 minute, mean potential differences changes in both control and cystic fibrosis subjects were positive. Theophylline perfusion resulted in a significant (P less than 0.01) difference in potential difference response between groups; at 10 minutes, the potential difference became more negative (-3.6 +/- 1.4 mV) in control subjects and more positive in cystic fibrosis subjects (+3.9 +/- 1.4 mV). To determine whether second messenger-mediated potassium secretion contributed to the observed potential difference changes in response to bethanechol and theophylline, studies were repeated in the presence of barium chloride, a known blocker of potassium conductance. In the control group, barium chloride significantly enhanced the theophylline-induced negative potential difference change (P less than 0.05) and reduced the positive potential difference change seen with bethanechol alone. In subjects with cystic fibrosis, barium chloride completely abolished the previously seen positive potential difference change in response to either bethanechol or theophylline alone. These in vivo studies suggest that there is active potassium secretion in both control and cystic fibrosis rectal mucosa in response to cyclic adenosine monophosphate- and calcium-dependent secretagogues and that the magnitude of the potential difference changes attributable to barium-inhibitable potassium secretion is the same in cystic fibrosis and control subjects. In contrast, it appears that in cystic fibrosis rectal mucosa in vivo, calcium- as well as cyclic adenosine monophosphate-dependent chloride secretion is aberrant.

Adult↗
PubMed1991

Age-related effects of chronic ethanol intake on vitamin A status in Fisher 344 rats.

The present study was designed to investigate the interaction of age and ethanol on vitamin A status in rats. Rats aged 2 and 19 mo were fed a liquid diet containing 36% of total energy as ethanol or pair-fed a diet containing isoenergetic carbohydrate in place of ethanol. After 3 wk older rats had lower serum retinol (P = 0.04) and higher vitamin A concentrations in liver (P = 0.0001), esophagus (P = 0.0001) and the proximal (P = 0.03) and distal (P = 0.0001) colon than younger animals. Hepatic microsomal cytochrome P-450, retinyl ester hydrolase (REH) and cellular retinol-binding protein (cRBP) were significantly reduced; acyl coenzyme A: retinol acyltransferase (ARAT) was increased; and alcohol (retinol) dehydrogenase (ADH) activity was unchanged with age. Ethanol ingestion increased serum retinol as well as esophageal and colonic vitamin A levels in both age groups. Hepatic cRBP decreased further in the older rats with ethanol feeding, but no change was noted in the percentage of hepatic vitamin A as retinol or retinyl esters. Ethanol ingestion decreased REH (P = 0.0001) and ARAT activities (P = 0.02) and increased cytochrome P-450 (P = 0.04) but had no effect on the activity of ADH in either age group. These data indicate that, regardless of age, chronic ethanol ingestion significantly alters the tissue distribution of vitamin A; however, ethanol reduced cRBP levels only in older rats.

Acyltransferases↗
PubMed1991

Diarrhea associated with tube feeding: the importance of using objective criteria.

Since the published incidence of tube feeding-associated diarrhea varies widely between 2.3 and 68%, and because commonly used definitions of diarrhea are subjective and imprecise, we determined the actual fecal output of malnourished patients during the first 6 days of tube feeding. Nine patients on the same isoosmolar formula were evaluated for diarrhea. Patients were interviewed daily as to whether they thought diarrhea was present or absent. Frequency of defecation was recorded, and stool was collected daily for weight. When assessed subjectively, the incidence of diarrhea rose from 11% on day 1, to 25% on day 4, and to 43% by day 6. The group (n = 4) complaining of diarrhea averaged 3.0 bowel movements per day, whereas those denying diarrhea (n = 5) averaged fewer than 0.2 bowel movements per day (p less than 0.01). Stool weight, however, plateaued for all patients by day 6 at 52.7 +/- 35.7 g of stool per day. No patient produced in excess of 250 g of stool per day. We suggest that traditional subjective measures for assessing diarrhea may be flawed and should not be relied upon when evaluating and treating tube feeding-associated diarrhea.

Adult↗
PubMed1991

In vitro formation of retinoic acid from retinal in rat liver.

Enzymatic conversion of retinal to retinoic acid in rat liver cytosol was detected using a rapid and sensitive assay based on high pressure liquid chromatography (HPLC). This retinal oxidase assay system did not require extraction steps or any other manipulation of the sample mixture once the sample vial was sealed for incubation. The product (retinoic acid) and the reactant (retinal) were separated by HPLC in 14.0 min with a sensitivity of 15 and 40 pmol per injection for retinoic acid and retinal, respectively. Enzymatic activity was observed to be linear with protein concentration (0-2.4 mg/mL) and time (0-30 min) and displayed a broad pH maximum of 7.7-9.7. The enzyme exhibited Michaelis-Menten single-substrate kinetics with an apparent Km of 0.25 mM. The average specific activity in nine normal rats was 35.6 +/- 3.3 nmol retinoic acid formed/h per mg protein. Incubation of the enzyme with zinc did not affect the rate of retinoic acid synthesis. Dithiothreitol inhibited the reaction. Both NAD and NADH stimulated retinoic acid formation. Formation of retinol was also observed when these pyridine nucleotides were added to the reaction mixture, indicating the presence of retinal reductase activity. The results of kinetic studies suggest that NADH may act indirectly to stimulate retinoic acid formation.

Aldehyde Oxidoreductases↗
PubMed1990

Rabbit esophageal cells possess an Na+,H+ antiport.

The development of esophagitis is the result of hydrogen ion diffusion into the mucosa leading to cellular acidification and necrosis. In these studies, whether esophageal cells possess transport system(s) that can respond to cytoplasmic acidification was assessed; specifically, whether esophageal cells possess an Na+,H+ antiport was determined. Nucleated esophageal cells were isolated from rabbit esophagi using a trypsin-digestion technique that yielded 5-8 x 10(6) cells per esophagus, of which 74% +/- 3% were basal and 26% +/- 8% were squamous. Trypan blue was excluded by 95% +/- 2% of the cells. Cytoplasmic pH (pHi) was measured using the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6) carboxyfluorescein acetoxymethyl ester. Cells were acidified to the desired pHi by suspension in solutions with varying external pH (pHo) in the presence of nigericin. When cells acidified to pHi 6.3 were suspended in a choline chloride solution (pHo 7.4), cytoplasmic pHi did not increase. In contrast, Nao+ caused a concentration-dependent increase in the rate of cytoplasmic alkalinization with saturation occurring above 50 mmol/L Nao+. The transporter behaved according to first-order Michaelis-Menten type kinetics with respect to external Na+ and had an apparent Km for Nao+ of 38.4 mmol/L. In contrast, the transporter behaved with greater than first-order kinetics with respect to external Na+ and had an apparent Km for Nao+ of 38.4 mmol/L. In contrast, the transporter behaved with greater than first-order kinetics with respect to cytoplasmic hydrogen ion concentration. Amiloride (10(-4) mol/L) caused a reversible inhibition of Na(+)-dependent alkalinization. Amiloride-sensitive cytoplasmic alkalinization was not observed when either cholineo or Ko+ was substituted for Nao+, while Lio+ resulted in alkalinization that was 60% +/- 8% of that seen with equimolar concentrations of Nao+. The basal pHi of cells suspended in a bicarbonate-free 130 mmol/L NaCl solution (pHo 7.4) averaged 7.42 +/- 0.03 (n = 10); amiloride (10(-4) mmol/L caused the basal pHi to decrease to 7.26 +/- 0.05 (n = 10; P less than 0.0025). When cells were suspended in a choline chloride (pHo 7.4) solution, pHi averaged 7.29 +/- 0.06 (n = 10) (P less than 0.0025 compared with Nao+). These studies indicate that nucleated esophageal cells obtained from rabbits possess an amiloride-sensitive Na+,H+ antiport that functions to regulate basal pHi and responds to intracellular acidification.

Amiloride↗
PubMed1990

Esophageal mucosal resistance. A factor in esophagitis.

The development of esophageal damage depends on a number of factors. The components in the refluxate, including H+ ion, pepsin, bile salts, and pancreatic enzymes, are able to permeate the mucosa and cause injury. These agents may act individually or in combination. Balancing the effects of these damaging agents is the "esophageal mucosal barrier." This barrier is an integrated complex of anatomic and physiologic components that acts to maintain the integrity of the mucosa. Although the relative efficacy of the various components in developing an effective barrier is not understood completely, their physiologic and clinical importance in the face of "noxious" luminal contents remains critical. Understanding the interplay between the injurious agents in the refluxate and the esophageal mucosal barrier may allow for the development of new therapeutic measures in the treatment and prevention of gastroesophageal reflux disease.

Bile Acids and Salts↗
PubMed1989

In vitro stimulation of rat liver retinyl ester hydrolase by ethanol.

Retinyl ester hydrolase (REH), the enzyme which converts retinyl esters to retinol, was partially characterized from whole liver homogenates of rats using an HPLC method with quantitation of retinol product. Optimal results were obtained by incubation of 1 mg of whole homogenate protein with 900 microM all-trans-retinyl palmitate and 275 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in a 0.1 M Tris-maleate buffer, pH 7.0, for 1 h at 37 degrees C. The enzyme assay proved to be sensitive and reproducible, with an interanimal coefficient of variation of 13% (n = 7). Because ethanol has been shown to mobilize vitamin A from the liver, we tested its effect on REH activity at several concentrations. In concentrations ranging from 0.01 to 0.5 M, ethanol added in vitro caused a concentration related increase in REH activity (from 20 to 86% above baseline activity). This increase was specific to ethanol as acetaldehyde, 1-propanol, and t-butanol either did not change or significantly decreased REH activity over the range of concentrations tested. The range of concentrations of ethanol causing stimulation in our assays was within the range of concentrations seen in the blood of rats after acute ethanol ingestion. Stimulation of REH activity could explain, in part, the well-known effects of ethanol on mobilization of vitamin A from liver stores.

Acetaldehyde↗
PubMed1989

Effects of acute starvation on vitamin A status in rats.

Maintenance of vitamin A stores in the body is dependent on a number of basic metabolic processes. These processes, such as protein and carbohydrate metabolism, are disrupted in acute starvation, and, as a result, alterations in vitamin A status may result. We investigated this possibility in 8-week-old Sprague-Dawley male rats. The rats were starved for 24, 48, and 72 hr but had free access to water. At 24 hours of starvation, the plasma retinol concentration was depressed, but not significantly so. After 48 and 72 hours of starvation, however, the plasma retinol concentration decreased to less than half of the control values (61 +/- 4 vs 124 +/- 12 nmol/dl at 72 hours, mean +/- SEM, (p less than 0.005). The hepatic retinoid (retinyl esters + retinol) concentration (nmol/g liver) was increased at 24 and 48 hours of starvation compared to controls (p less than 0.05), and by 72 hours the concentration was 56% greater in starved rats than in fed controls (p less than 0.001). The total hepatic retinoid content (mumol/total liver) was decreased moderately at all periods of starvation compared to controls (p less than 0.05). In both starved and fed animals, the total hepatic content per 100 g body weight, a measure of total vitamin A reserves, was statistically the same. These results demonstrate that acute starvation in rats alters the vitamin A equilibrium between the plasma and hepatic stores without affecting the overall vitamin A reserves.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1988

Effect of zinc deficiency on hepatic enzymes regulating vitamin A status.

The elevation of hepatic vitamin A (VA) in zinc deficiency (ZD) is thought to be due, in part, to a reduced synthesis of plasma retinol-binding protein with a subsequent decrease in the release of retinol into the circulation. We hypothesized that the hepatic VA elevation may also be secondary to a change in the activity of enzymes that either regulate retinol degradation or affect the synthesis or hydrolysis of retinyl esters. To examine this question, 20 rats were divided into two groups and pair-fed for 3 wk. ZD rats received a ZD diet (zinc 2.3 mg/kg diet) and controls were fed a zinc-sufficient diet (zinc 50 mg/kg diet). The enzymes studied were retinyl ester hydrolase (REH) and microsomal acyl coenzyme A:retinol acyl transferase (ARAT), the principal enzymes regulating retinyl ester hydrolysis and synthesis. The activities of retinol (alcohol) dehydrogenase (ADH) and retinal oxidase (RO), enzymes regulating retinol degradation to polar metabolites, were also studied. ZD caused an increase in both total hepatic VA concentration and total hepatic VA content, but did not alter the ratio of retinol to retinyl esters. The specific activities of REH and ARAT were not affected by ZD. However, ZD caused a significant reduction in the activity of ADH, the enzyme that catalyzes the first step in retinol oxidation. In contrast, the activity of RO, the enzyme that regulates the irreversible oxidation of retinal to retinoic acid, was significantly increased in ZD rats. These findings indicate that the elevation in hepatic levels of VA in ZD rats may be, in part, secondary to decreased retinol degradation.

Acyl Coenzyme A↗
PubMed1988

Rectum has abnormal ion transport but normal cAMP-binding proteins in cystic fibrosis.

The luminal membranes of involved tissues in cystic fibrosis (CF) are relatively impermeable to Cl and the regulation of Cl transport by adenosine 3',5'-cyclic monophosphate (cAMP)-mediated hormones is abnormal. We investigated the human rectum as a putative model for CF. We compared in vivo transrectal potential difference (PD) in CF and in normal subjects in response to sequential perfusions with various test solutions. The base-line PD was different in normal (-35.5 +/- 4.0 mV; lumen negative; mean +/- SE; n = 9) and CF subjects (-23.4 +/- 3.1 mV; n = 6; P less than 0.025) and was eliminated by amiloride (10(-4) M) perfusion in both groups by 3 min. However, in response to a Cl-free solution with amiloride, all six CF subjects exhibit less of a change in PD (PD, -2.2 +/- 1.2 mV vs. -11.7 +/- 1.5 mV in 6 controls; P less than 0.01). Furthermore, normal subjects (n = 7) respond to a 5 mM theophylline + amiloride perfusion with an increase in lumen-negative PD, whereas, CF subjects (n = 6) show no increase in lumen-negative PD. Rectal biopsy specimens from four normal and four CF subjects exhibit similar (2- to 3-fold) increases in theophylline-induced cAMP content and have similar cAMP-binding proteins (CF, n = 3; control, n = 3). We conclude that the rectum is an involved epithelium in CF in which the aberration may lie at a point beyond the binding of cAMP to its protein kinase.

Adult↗