Biomedical subjects

W M Sun

Publications and source records attributed to W M Sun.

At least 91 records · Page 5Linked to original sources

PubMed1996

Alterations in glutathione homeostasis in mutant Eisai hyperbilirubinemic rats.

Eisai hyperbilirubinemic rats (EHBR) are mutant Sprague-Dawley rats that exhibit impaired biliary organic anion and reduced glutathione (GSH) secretion. In addition, liver GSH levels are twice that of age-matched controls. The mechanisms for the defect in biliary GSH secretion and the increase in cell GSH are not fully understood. We previously showed that canalicular membrane-enriched vesicles isolated from EHBR livers exhibited normal GSH transport. In the present study, we examined the steady-state rat canalicular reduced glutathione transporter (RcGshT) messenger RNA (mRNA) and protein levels, as well as the mechanisms for the increase in cell GSH. Both Northern and Western blot analyses of EHBR livers showed nearly identical RcGshT mRNA and polypeptide levels, respectively, as compared with controls. Treatment with phenobarbital, which increased steady-state RcGshT mRNA by five- to sixfold, RcGshT polypeptide, and biliary GSH secretion by onefold in controls, had a smaller effect on steady-state RcGshT-mRNA level in EHBR (by 1.5-fold) and did not increase RcGshT polypeptide or biliary GSH secretion. In examining possible mechanisms for increased liver GSH, both cysteine level and gamma-glutamylcysteine synthetase (GCS) activity were significantly higher than controls, while the activity of GSH synthetase was unchanged. Northern and Western blot analyses also showed increased steady-state GCS heavy subunit (GCS-HS) mRNA and polypeptide levels, respectively. In addition to liver, GSH levels in kidney, duodenal, jejunal, and ileal mucosa of EHBR were 200% to 300% of age-matched control rats. GCS activity was also increased in kidney cytosol of EHBR. Thus, the defect in biliary GSH secretion in EHBR most likely is either at the posttranslational level of RcGshT or in the inhibition exerted by retained endogenous organic anions. In addition, there is a widespread up-regulation of GSH synthesis capacity in the tissues of EHBR.

Analysis of Variance↗

PubMed1996

Changes in S-adenosylmethionine synthetase in human liver cancer: molecular characterization and significance.

S-adenosylmethionine synthetase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) and is essential to normal cell function. There are two forms of SAMS, liver-specific and nonliver-specific (often referred to as "kidney"), which are products of two different genes. SAMS isoenzymes differ greatly in kinetic parameters and sensitivity to inhibition by methionine analogs. The current work studied changes in SAMS and their significance in liver cancer. Northern blot analysis showed that while normal liver expresses only liver-specific SAMS, both HepG2 and HuH-7 cells express only nonliver-specific SAMS. Absence of liver-specific SAMS messenger RNA (mRNA) was not because of gene deletion or rearrangement but complete lack of gene transcription. Reverse-transcription polymerase chain reaction (RT-PCR) with liver- and kidney-specific SAMS primers showed that liver-specific SAMS mRNA was absent with only kidney SAMS mRNA present in HepG2, HuH-7, Hep3B, and HuH-1 cells, and four consecutive hepatocellular carcinoma (HCC) specimens. Normal liver tissues from the same patients express both forms of SAMS mRNA. As a result of the change in SAMS expression, SAMS activity was higher in HepG2 and HuH-7 cells at physiologically relevant methionine concentrations but lower at high (mmol/L) methionine concentrations than rat hepatocytes. Treatment with ethionine and seleno-D,L-ethionine, two inhibitors known to have I50 values 50 to 60 times lower against SAMS purified from Novikoff hepatoma cells as compared with SAMS purified from normal rat liver, resulted in increased cell lysis in HepG2 and HuH-7 cells but not cultured rat hepatocytes. These agents did not affect cellular adenosine triphosphate (ATP) levels but inhibited SAMS activity in HepG2 and HuH-7 cells when added to their protein extracts. In summary, expression of SAMS is altered in human liver cancer. This occurrence may provide a potentially exploitable target for cancer chemotherapy.

Animals↗

PubMed1996

Hyperglycaemia affects proximal gastric motor and sensory function in normal subjects.

OBJECTIVE: Hyperglycaemia delays gastric emptying in normal subjects and patients with diabetes mellitus by uncertain mechanisms and may affect the perception of somatic sensations. The effects of hyperglycaemia on the motor function of the proximal stomach and the perception of gastric distension were evaluated in normal subjects. DESIGN: Paired studies were performed in randomized order in 10 healthy volunteers on separate days during euglycaemia and hyperglycaemia (blood glucose approximately equal to 15 mmol/l). METHODS: With a barostat and a balloon positioned in the proximal stomach, tasting subjects underwent a stepwise gastric distension. Each 2 mmHg step was maintained at a constant pressure for 2 min. The volume of the barostat balloon was measured and perception of the sensations of fullness, desire to belch, nausea, abdominal discomfort and hunger was scored at each step. RESULTS: Hyperglycaemia was associated with an increase in proximal gastric compliance (P < 0.01) evident from 2 mmHg above basal intragastric pressure. Perception scores for the sensations of nausea and desire to belch were greater during hyperglycaemia than euglycaemia (P < 0.05) in relation to both pressure at each step and volume. Hyperglycaemia did not affect perception of the sensations of abdominal discomfort, fullness or hunger. CONCLUSIONS: Hyperglycaemia increases proximal gastric compliance, reflecting a reduction in gastric tone. This may contribute to the previously observed delay in gastric emptying associated with hyperglycaemia. Hyperglycaemia appears to increase the perception of some of the sensations induced by gastric distension.

Abdominal Pain↗

PubMed1996

Disturbances in anorectal function in patients with diabetes mellitus and faecal incontinence.

OBJECTIVE: The pathophysiology of faecal incontinence in diabetes mellitus is poorly understood. The study was designed to document the anorectal dysfunctions in diabetic patients with faecal incontinence. METHODS: Multiport anorectal manometry and electromyography were done in 11 diabetic patients with faecal incontinence and in 20 healthy controls. RESULTS: Basal and squeeze pressures were reduced (P < 0.05) in the diabetic patients compared with the control subjects. During basal recording six patients showed regular oscillations in anal electrical activity and pressure with an amplitude of 10-40 (median: 25) cmH2O and a frequency of 6-10 (median: 8) min-1. Nine patients also exhibited spontaneous transient anal relaxations with an amplitude of 15-50 (median: 40) cmH2O and a duration of 15-720 (median: 60)s, and in six of them leakage occurred as the anal pressure fell below the rectal pressure. None of the control subjects showed oscillation or spontaneous relaxations. In patients there was a greater tendency for repetitive rectal contractions in response to rectal distension and reduced rectal compliance (P < 0.01). During rectal distension four patients showed no anal relaxation, and in the remainder relaxation occurred at an abnormally high threshold. However, the residual pressures were lower (P < 0.05) than in control subjects and often fell below rectal pressure, whereupon leakage occurred. There was no significant difference in the distension thresholds for rectal sensation between patients and control subjects, but in 9/11 patients the perception of rectal sensation was delayed by more than 2s (P < 0.05). CONCLUSION: These results indicate that aetiology of faecal incontinence in diabetic patients is multifactorial and, suggest for the first time, that instability of the internal sphincter probably plays a major role.

Adult↗

PubMed1996

Effects of glyceryl trinitrate on the pyloric motor response to intraduodenal triglyceride infusion in humans.

The retardation of gastric emptying induced by infusion of triglyceride into the small intestine is associated with suppression of antral pressure waves and stimulation of basal pyloric tone in combination with phasic pressure waves localized to the pylorus. The role of nitric oxide (NO) mechanisms in the control of pyloric motility was evaluated in 12 healthy male subjects (21-43 years), using the NO donor glyceryl trinitrate (GTN). Antropyloric pressures were measured with a manometric assembly incorporating nine sideholes, spanning the antrum and proximal duodenum, and a pyloric sleeve sensor. On separate days, an intraduodenal triglyceride infusion (10% intralipid at 1 mL min-1) was started during antral phase I activity and continued for 60 min. On one of the days GTN (600 micrograms) was given sublingually 20 min after start of the triglyceride infusion. The tonic pyloric motor response to triglyceride [5.6 (SEM 0.8,) vs. 2.7 (1.3) mmHg, P < 0.001] and both the number 3.2 (0.2) vs. 2.2 (0.2) min-1, P < 0.05] and amplitude [40 (4) vs. 27 (5) mmHg, P < 0.05] of phasic isolated pyloric pressure waves were reduced by GTN. These observations suggest that NO mechanisms are involved in the regulation of pyloric motor activity in humans.

Adolescent↗

PubMed1996

Effects of cisapride on gastric emptying of oil and aqueous meal components, hunger, and fullness.

To evaluate the effects of cisapride on gastric emptying of extracellular fat and hunger and fullness 10 volunteers consumed a meal consisting of 60 ml technectium-99m (99mTc)-V-thiocyanate labelled olive oil and 290 ml indium-113m (113mIn) labelled soup after taking cisapride (10 mg four times daily orally) and placebo, each for four days, in randomised, double blind fashion. Gastric emptying was quantified scintigraphically. Hunger and fullness before and after the meal were evaluated using visual analogue scales. Cisapride accelerated gastric emptying of oil and aqueous components by reducing the lag phase mean (SEM) (20.3 (7.0) min v 40.7 (4.1) min (p < 0.05) for oil and 4.1 (2.5) min v 10.0 (3.1) min (p < 0.05) for aqueous). Cisapride had no effect on the post-lag emptying rate of oil. Treatment with cisapride was associated with reduced retention of oil in the proximal stomach (p < 0.05). Subjects were more hungry before ingestion of the meal while receiving cisapride (6.7 (0.9) v 3.9 (0.7), p < 0.001). The scores for hunger at 120 and 180 minutes were inversely related to gastric emptying of oil on both cisapride (r > -0.62, p < 0.05) and placebo (r > -0.86, p < 0.001). Fullness increased after the meal while receiving placebo (p < 0.01), but not cisapride and postprandial fullness was less with cisapride at (30 min; 0.4 (0.3) v 3.3 (1.0), p < 0.05). With placebo, but not cisapride, the score for fullness at 15 minutes was inversely related to emptying of the aqueous phase (r = 0.68, p < 0.05). These results show that in normal volunteers after ingestion of an oil/aqueous meal: (a) postprandial hunger is inversely related to gastric emptying of oil, while fullness is inversely related to gastric emptying of the aqueous phase, (b) cisapride affects the intragastric distribution and accelerates gastric emptying of both oil and aqueous meal components, and (c) cisapride increases preprandial hunger and reduces postprandial fullness.

Adolescent↗

PubMed1996

Appetite regulation by carbohydrate: role of blood glucose and gastrointestinal hormones.

To investigate the mechanisms by which intestinal carbohydrate affects eating behavior, seven fasted, healthy male volunteers received intraduodenal infusions of glucose or saline over a 90-min period while blood glucose levels were matched by use of intravenous glucose and saline infusions. A second study examined the effect of intraduodenal glucose on eating behavior when the gastrointestinal hormone response was inhibited by intravenous octreotide. Intravenous glucose infusion did not affect hunger or satiety. In contrast, intraduodenal infusion of glucose suppressed hunger, increased fullness and satiety ratings, reduced energy intake, and resulted in higher plasma insulin responses compared with the intravenous glucose infusion. Octreotide abolished the plasma insulin response to intraduodenal glucose and reversed the changes in ratings and eating behavior. This study has shown that the effects of intestinal glucose on appetite are not mediated via an increase in blood glucose but are likely to reflect small intestinal stimulation of release of either insulin or intestinal incretins.

Adult↗

PubMed1996

Hyperglycemia affects proximal gastric motor and sensory function during small intestinal triglyceride infusion.

Hyperglycemia slows gastric emptying in normal individuals and patients with diabetes mellitus and may affect both somatic and visceral sensation. The effects of hyperglycemia on proximal gastric motility and sensation during intraduodenal infusion of a triglyceride emulsion were evaluated using a barostat in six normal subjects during euglycemia and hyperglycemia (approximately 15 mmol/l). Isobaric distension induced greater bag volumes during hyperglycemia compared with euglycemia at 3 (452 +/- 26 vs. 343 +/- 12 ml, P < 0.05) and 4 mmHg (600 +/- 55 vs. 497 +/- 50 ml, P < 0.05) above basal pressure. During isovolumetric distension, intrabag pressure was less during hyperglycemia at 500 (2.5 +/- 0.3 vs. 3.5 +/- 0.5 mmHg above basal pressure, P < 0.05) and 600 ml (3.0 +/- 0.4 vs. 4.5 +/- 0.5 mmHg above basal pressure, P < 0.05). Perception of nausea (P < 0.05) and fullness (P < 0.05) was increased during hyperglycemia compared with euglycemia. We conclude that hyperglycemia 1) reduces proximal gastric tone during intraduodenal triglyceride infusion, an effect that may contribute to delayed gastric emptying, and 2) increases the intensity of nausea and fullness during intraduodenal triglyceride infusion and proximal gastric distension, indicative of an effect on visceral sensation.

Adult↗

PubMed1995

Anorectal function in patients with complete spinal transection before and after sacral posterior rhizotomy.

BACKGROUND/AIMS: The implantation of spinal stimulators to facilitate defecation in patients with complete spinal transection involves division of the posterior sacral nerve roots. The aim of this study was to investigate the role of spinal reflexes in anorectal function. METHODS: Anorectal manometry and electromyography were performed in 14 patients with supraconal spinal cord transection (C6-T12) before and after complete sacral posterior rhizotomy and in 30 normal controls. RESULTS: Patients with spinal transection lost conscious control of the external anal sphincter. Reflex responses to intra-abdominal pressure and to rectal distention were eliminated after rhizotomy, indicating that they are spinal reflexes. Rhizotomy also eliminated giant rectal contractions induced by rectal distention in these patients. In contrast, the exaggerated sphincter relaxation induced by rectal distention was not influenced by rhizotomy. Discriminant rectal sensation was lost, but patients with thoracic cord lesions perceived a dull pelvic sensation during rectal distention even after rhizotomy. CONCLUSIONS: The exaggerated anorectal smooth muscle responses and absent conscious control of the anorectum may explain why patients with complete spinal transection experience uncontrollable reflex defecation, and the persistence of external anal sphincter contraction during straining may impair fecal expulsion. The elimination of these responses after posterior rhizotomy prevented reflex defecation while facilitating manual evacuation.

Adult↗

PubMed1995

Relationship between fractional calcium absorption and gastric emptying.

The relationship between calcium absorption and gastric emptying and the precision of measurement of fractional calcium absorption using a single isotope technique were evaluated in 14 normal postmenopausal women (age range 61-72 years). On two occasions separated by between 5 and 15 days, each subject was given 250 mL water containing 0.2 MBq of 45Ca in 20 mg of calcium carrier as the chloride, 20 mg kg-1 paracetamol and 9 MBq of 99mTc sulphur colloid. Venous blood samples were taken at -2, 15, 30, 45, 60, 90, 120, 150 and 180 min after consumption of the drink, and gastric emptying (GE) was monitored with a gamma camera. Fractional calcium absorption in the first hour (alpha 6) was calculated from the blood samples obtained at 15, 30, 45, 60, 90 and 120 min. An absorption rate was also derived from the 60 min sample using only a calibration curve (alpha 1). There were close correlations between radiocalcium absorption on the two study days (r = 0.89, P < 0.001 for both alpha 1 and alpha 6) and between alpha 1 and alpha 6 (r = 0.93, P < 0.001). Plasma paracetamol concentrations at 15 min were directly related to the early phase of GE (r = 0.42, P < 0.05). In contrast, calcium absorption was inversely related to GE (r = 0.45, P < 0.05). We conclude that radiocalcium absorption is not greatly influenced by gastric emptying rate and that the single blood sample procedure has similar precision to the six-blood sample test.

Acetaminophen↗

PubMed1995

Postural changes in proximal gastric volume and pressure measured using a gastric barostat.

A barostat was used to examine the effect of changes in posture on the volume and pressure in a bag positioned in the proximal stomach of 14 normal volunteers. Volumes in the supine position were compared with those in the standing, left lateral and right lateral positions at a constant pressure 2 mmHg above basal intragastric pressure. A separate series of measurements was then used to evaluate the effects of the same postural changes on pressure within the bag whilst its volume was kept constant. Changing from the supine to the left lateral position decreased bag volume by 62% when pressure was controlled; pressure increased by 60% when volume was controlled. In contrast, movement from the supine to the right lateral position resulted in a 68% increase in bag volume and a 31% fall in pressure. Moving from supine to standing had inconsistent effects on bag volume and pressure. There was a negative correlation between the magnitudes of the changes in pressure and volume (r2 = 0.557). The observed effects of posture probably result from changes in the compression of the stomach by abdominal viscera and indicate that subject position must be specified and maintained constant in studies of proximal gastric motor function using a barostat.

Adult↗

PubMed1995

Effect of drink temperature on antropyloroduodenal motility and gastric electrical activity in humans.

There is little information on the motor mechanisms underlying the effects of meal temperature on gastric emptying. The effects on antropyloric pressures and the surface electrogastrogram of ingesting drinks at 4 degrees C, 37 degrees C, and 50 degrees C (350 ml normal saline and 50 ml low calorie (7 kj) orange cordial) given in randomised order were measured over 60 minutes in 12 normal volunteers (10 men and 2 women, aged 18-55 years). The warm and cold drinks suppressed antral pressure waves (p < 0.05), altered the organisation of antropyloric pressure waves (p < 0.05), stimulated isolated pyloric pressure waves (p < 0.05), and increased electrogastrogram frequency (p < 0.05) when compared with the 37 degrees C drink. These changes were greatest in the first 30 minutes after ingestion and greater (p < 0.05) with the 4 degrees C drink. Temperature has major effects on postprandial antropyloroduodenal motility in normal subjects. Both cold and warm drinks stimulate a pattern of motility associated with retardation of transpyloric flow.

Adolescent↗

PubMed1995

Evaluation of antral motility in humans using manometry and scintigraphy.

Recent studies suggest that scintigraphy can be used to evaluate non-invasively antral motility in humans, although scintigraphic techniques have not yet been compared with more conventional measurements of intraluminal pressures by manometry. Simultaneous scintigraphic and manometric measurements of antral motility were performed in nine healthy volunteers. After intubation with a sleeve/sidehole catheter which incorporated five pressure sideholes located at 1.5 cm intervals spanning the antrum, each subject ingested 100 g minced beef labelled with 100 MBq 99mTc-chicken liver and 150 ml water. Between 40-43, 60-63, 80-83, and 100-103 minutes after meal ingestion, radioisotopic data were acquired in two second frames. Time-activity curves showing antral 'contractions' resulting from wall motion were derived by drawing small regions of interest over the antrum to coincide with the position of the antral manometric sideholes. Scintigraphic contraction rates approximated 3/minute, whereas antral pressure waves that occluded the lumen were less frequent (p < 0.01 for all), particularly in the proximal antrum. The amplitude of wall motion, evaluated scintigraphically, and the amplitude of pressure waves were both inversely related to the distance from the pylorus (r > -0.32, p < 0.05) and antral volume r > -0.29 (p < 0.05). There were significant relationships between the amplitude of contractions assessed scintigraphically and the number of lumen-occlusive antral pressure waves in the distal antrum (r -0.48, p < 0.05) but not in the more proximal antral regions. It is concluded that scintigraphy can detect antral wall motion with greater sensitivity than manometry, particularly in the proximal antrum. As manometry gives information on the amplitude as well as the temporal and spatial organisation of those contractions which result in lumen occlusion, the combination of scintigraphic and manometric techniques in the evaluation of antral motility shows considerable promise.

Adult↗

PubMed1995

Relationship between surface electrogastrography and antropyloric pressures.

The cutaneous electrogastrogram (EGG) and intraluminal antropyloroduodenal pressures were recorded in 12 healthy volunteers for 30-min periods during phase II of the interdigestive motor complex, during intraduodenal infusion of 10% triglyceride, and after intravenous erythromycin (3 mg/kg). During phase II, the frequency of the EGG was relatively constant in each individual, with a median frequency of 0.046 Hz [2.8 counts per minute (cpm)]. EGG frequency was greater (P < 0.05) than the median rate of antral pressure waves (1.8 cpm). The suppression of antral pressure waves (P < 0.05) and stimulation of isolated pyloric pressure waves (IPPWs) (P < 0.05) produced by triglyceride infusion were not associated with changes in EGG frequency compared with phase II. The frequency of the EGG and the rate of IPPWs were comparable. After erythromycin, EGG frequency was 0.03 Hz (1.8 cpm), less than during both phase II and triglyceride infusion (P < 0.05) and almost identical to the rate of antral pressure waves. Pressure waves were nearly always associated with an EGG signal. In contrast, the temporal relationship between the EGG signal and pressure waves was variable. During triglyceride infusion (r = 0.83, P < 0.001) and after erythromycin (r = 0.83, P < 0.001) there was a close (approximately 1:1) relationship between the rate of pressure waves and EGG frequency. However, there was no significant relationship (r = 0.32, not significant) between the number of pressure waves and EGG frequency frequency during pase II.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1995

Gastric emptying of oil and aqueous meal components in pancreatic insufficiency: effects of posture and on appetite.

The aims of this study were to evaluate the effects of posture on gastric emptying, intragastric distribution, and satiation after a meal containing oil and aqueous phases in patients with exocrine pancreatic insufficiency. Five patients with cystic fibrosis (CF) consumed 60 ml 99mTc-labeled (V)-thiocyanate olive oil and 290 ml 113mIn-labeled diethylenetriaminepentaacetic acid soup while sitting and while lying in the left lateral decubitus position. Hunger and fullness before and after the meal were recorded. Results were compared with those obtained in 11 normal volunteers. In both postures emptying of oil was faster (P < 0.01) in CF patients. Emptying of the aqueous phase was faster (P < 0.01) in CF patients in the decubitus position. In normal subjects there was no overall difference in emptying of oil between the two postures, whereas emptying of the aqueous phase was delayed (P < 0.01) in the decubitus position. In CF patients emptying of oil was faster (P < 0.01) in the decubitus position, and emptying of the aqueous phase was only slightly faster (P < 0.05) in the sitting position. For both postures there was greater retention (P < 0.05) of oil in the proximal stomach in normal subjects than CF patients. Hunger decreased (P < 0.05) after the meal in the control subjects, but there was no change in CF patients. These results indicate that in CF patients with pancreatic exocrine insufficiency 1) gastric emptying of nonhomogenized fat is faster than normal, 2) gravity affects gastric emptying of oil, and 3) effects of a fatty meal on hunger are reduced.

Adult↗

PubMed1995

Pharmacokinetic considerations in gastrointestinal motor disorders.

Although it has been recognised that alterations in gastrointestinal motility, whether induced by physiological or pathological processes, have significant effects on the pharmacokinetics of orally administered drugs, this subject has received inappropriately little attention. Studies relating to this topic have focused on healthy volunteers and animals and have largely been confined to the effects of single drug doses. There is limited information about the effects of disease on pharmacokinetics under steady-state conditions. Changes in gastrointestinal motility may affect the pharmacokinetics of orally administered drugs by altering the rate of delivery, bioavailability or mucosal absorption of the drug. In general the rate of absorption and time taken to achieve maximal plasma concentrations for well absorbed drugs may be modified by changes in gastrointestinal motility, but overall bioavailability is not usually affected. In these cases the therapeutic and clinical effects of the alteration in pharmacokinetics will, therefore, depend on which parameters are important for the action of the drug. For poorly absorbed drugs both the rate of absorption and bioavailability are likely to be altered by changes in gastrointestinal motility. However, the complex effects of food and disease, as well as the properties and formulation of any drug (solubility, ease of dispersion, delayed release formulation) often make the prediction of the magnitude, or even the direction, of any effect difficult to predict. Drugs with direct effects on gastrointestinal motility may influence their own patterns of absorption. In patients with gastrointestinal motility disorders, drugs administered in a controlled release formulation, or those with poor bioavailability, are most likely to have a poorly predictable therapeutic effect. Care should be taken to ensure that the formulation of the drug, its timing of administration in relation to meals and the use of coadministered drugs optimise, or at least ensure consistent absorption.

Administration, Oral↗

PubMed1995

Bidirectional glutathione transport by cultured human retinal pigment epithelial cells.

PURPOSE: To characterize glutathione (GSH) transport by cultured human retinal pigment epithelial (HRPE) cells. METHODS: Cultured HRPE cells were pretreated with acivicin for GSH efflux and with buthionine sulfoximine for GSH uptake to prevent the breakdown and resynthesis of GSH. Efflux was measured by the linear rate of accumulation of GSH in the supernatant; uptake was measured using [35S] GSH plus varying concentrations of GSH. Molecular forms were verified by high-performance liquid chromatography. HRPE cell mRNA was probed for the presence of the two recently cloned rat sinusoidal and canalicular GSH transporters, (RsGshT and RcGshT), by Northern blot analysis. RESULTS: Glutathione efflux was temperature dependent (undetectable at 4 degrees C), and its averaged 23 +/- 3.3 pmol/10(6) cells/minute or 10% of the total GSH effluxed per hour (total cell GSH = 13.6 +/- 1.5 nmol/10(6) cells). Efflux was not influenced by dithiothreitol or sulfobromophthalein-reduced GSH adduct, agents known to affect liver sinusoidal GSH transport. Glutathione uptake was linear up to 45 minutes and was temperature dependent. The difference between 37 degrees C and 4 degrees C uptake values represented true uptake. Glutathione uptake (2 microCi/ml + 1 mM mass) was Na independent and was inhibited significantly by phenol-3,6-dibromphthalein disulfonate. The kinetics of GSH uptake was assessed by measuring uptake with 35S-GSH and 0.05 to 40 mM extracellular GSH for 30 minutes. Uptake was saturable with Vmax = 18.7 +/- 1.7 nmol/10(6) cells/30 minutes, Km = 12.1 +/- 1.9 mM, n (binding site) = 1. On Northern blot analysis, HRPE cells express mRNA for RcGshT but not for RsGshT. CONCLUSIONS: The similarities in functional characteristics of GSH transport and the presence of RcGshT-like mRNA suggest GSH transport in HRPE cells is mediated by a RcGshT homolog. Although the transporter can operate bidirectionally, it is expected to be a net efflux pump under normal physiologic conditions because the intracellular GSH concentration is much higher.

Biological Transport↗

PubMed1995

Hormonal and cell density regulation of hepatic gamma-glutamylcysteine synthetase gene expression.

We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis, is under both hormonal and cell density regulation in cultured rat hepatocytes. Specifically, the addition of insulin or hydrocortisone to culture media or the lowering of the initial plating cell density increased cell GSH by increasing the activity of GCS. In the present study, we examined the molecular mechanism of these effects. To determine whether the increase in GCS activity is associated with an increase in GCS heavy subunit (GCS-HS) mRNA expression, the steady state mRNA levels of GCS-HS were examined with the use of Northern blots. After 24-hr treatment of high density (0.6 x 10(5) cells/cm2) cultured rat hepatocytes with insulin (1 micrograms/ml) or hydrocortisone (50 nM), the steady state GCS-HS mRNA level increased by approximately 1-2 fold. When the plating density was decreased to 0.1 x 10(5) cells/cm2, the steady state GCS-HS mRNA level also increased by 1-2 fold 24 hr later. An increase in the steady state GCS-HS mRNA level was found within 4 hr of either hormonal treatment or cell density manipulation. The increase in steady state GCS-HS mRNA level resulted from increased gene transcription, as the transcriptional rates of GCS-HS after hormonal or cell density manipulation were increased by 2-3-fold, whereas the rates of GCS-HS mRNA degradation remained unchanged. Western blotting confirmed the increase in GCS-HS protein level after hormone treatment or lowering of plating cell density. When examined in vivo, the steady state GCS-HS mRNA level decreased by 50% in a rat in which diabetes had been induced with streptozotocin for 1 week; this was prevented with insulin replacement. In summary, GCS-HS gene expression is under both hormonal and cell density regulation.

Amino Acid Sequence↗