Immunodetection of aldose reductase in normal and diseased human liver.

Aldose reductase is an NADPH-dependent aldo-keto reductase best known as the rate-limiting enzyme of the polyol pathway that is implicated in the complications of diabetes. Aldose reductase appears to be involved in a variety of disease states other than diabetes, presumably due to its ability to catalyze the reduction of a broad spectrum of aldehydes, including some cytotoxic products of lipid peroxidation. Although the data regarding expression of aldose reductase in normal liver are conflicting, prior studies have suggested that the enzyme may be induced in diseased liver. The goal of these studies was to characterize expression of aldose reductase in normal and diseased human liver, using RT-PCR, Western analysis and immunohistochemistry. Aldose reductase transcripts and protein were detected at low levels in control human livers. In contrast, levels of aldose reductase mRNA and protein were increased in chronically diseased human livers. Immunohistochemistry demonstrated localization of aldose reductase in sinusoidal lining cells; dual immunofluorescence confocal microscopy with the macrophage marker, CD68, confirmed that the aldose reductase-positive sinusoidal lining cells were Kupffer cells. Abundant aldose reductase-positive, CD68-positive cells were present in the fibrous septa of cirrhotic livers, accounting for the increase in immunoreactive aldose reductase in diseased livers. Immunostaining of human lung, spleen and lymph node revealed that macrophages in those tissues also express aldose reductase. These data are the first to demonstrate that aldose reductase is expressed by human macrophages in various tissues and suggest that this enzyme may play a role in immune or inflammatory processes.

GP73, a novel Golgi-localized protein upregulated by viral infection.

We report the isolation and characterization of GP73, a novel 73kDa human Golgi protein. The GP73 cDNA was cloned by differential screening of a cDNA library derived from the liver of a patient with adult giant-cell hepatitis (GCH), a rare form of hepatitis with presumed viral etiology. In vitro transcription-translation studies indicate that GP73 is an integral membrane protein, and immunolocalization experiments using epitope-tagged GP73 demonstrate that the protein is localized to the Golgi apparatus. Northern blot analysis of RNA from multiple human tissues reveals a single GP73 mRNA transcript with a size of approximately 3.0kb. Immunohistochemical studies using rabbit polyclonal antisera directed against recombinant GP73 demonstrate that the protein is preferentially expressed by epithelial cells in many human tissues. In normal livers, GP73 is consistently present in biliary epithelial cells, whereas hepatocytes show little or no signal. In contrast, livers of patients with GCH display strong GP73 immunoreactivity in multinucleated hepatocytes. GP73 mRNA and protein are expressed in highly differentiated HepG2 hepatoma cells after infection with adenovirus in vitro. We conclude that GP73 represents a novel, epithelial cell-specific integral membrane Golgi protein that can be upregulated in response to viral infection.

Effect of a slow-release formula of trimoprostil on intragastric acidity in healthy volunteers.

We investigated the effect of a slow-release formula of trimoprostil, a prostaglandin E2 analogue, at a dose of 3 mg b.d. on circadian intragastric acidity in nine healthy volunteers using ambulatory pH-metry in a placebo-controlled study. The effect of trimoprostil was long lasting (8 hours during the night). However, it lowered gastric pH on average only by 0.4 pH units. In four of the six women severe side-effects occurred in the form of abdominal cramping, metrorrhagia, and/or diarrhoea. These disadvantages may limit the clinical use of this drug.

Relationship of spinal cord blood flow to vascular anatomy during thoracic aortic cross-clamping and shunting.

No satisfactory explanation exists as to why paraplegia occurs despite distal aortic perfusion during thoracic aortic operations. We studied the hemodynamics, paraplegia rate, and spinal cord blood flow with radioactive microspheres in 17 male adult baboons, with particular reference to the arteria radicularis magna. The groups consisted of control animals, subjected to cross-clamping for 60 minutes, and animals with aorto-aortic shunts operational for 60 minutes. There were no significant left ventricular hemodynamic advantages with shunting. Shunting significantly increased lumbar spinal cord blood flow (p = 0.0009), which correlated with the distal aortic mean pressure (r = 0.59, p = 0.008). However, lower thoracic spinal cord blood flow did not increase during shunting (p = 0.2) and did not correlate with the distal aortic pressure (r = 0.11, p = 0.64). This is due to the vascular anatomy of the anterior spinal artery, which was, as in man, smaller above (0.278 mm) than below (0.744 mm) the entry of the arteria radicularis magna. Resistance to flow, as calculated by Poiseuille's equation, was 51.7 times greater up the anterior spinal artery as compared with down this artery. The vascular anatomy explains the absence of paraplegia in one baboon in the cross-clamp group and paraplegia in one baboon in the shunt group. Thus, distal aortic perfusion protects the spinal cord below the arteria radicularis magna but not above it.

Failure of 16,16-dimethyl-PGE2 to protect the human stomach from taurocholate induced damage.

This study examined the question as to whether 16,16-dimethyl prostaglandin E2 prevents bile-induced gastric mucosal damage in man. Damage was induced by intragastric instillation of a 10 millimolar taurocholate solution. DNA-shedding, gastric transmucosal electrical potential difference, and the outputs of potassium and urea in gastric washings were measured as indices of mucosal damage. DNA-shedding during gastric washings with saline was 0.24 mg/15 min +/- 0.05 SEM and rose to 0.46 +/- 0.08 (p less than 0.025) during washings with taurocholate. Potential difference dropped from -35.6 mV +/- 1.6 to -21.9 +/- 2.3 (p less than 0.005). Potassium output was increased from 0.21 mmol/15 min +/- 0.01 to 0.50 +/- 0.03 (p less than 0.005), and urea output was increased from 0.45 mmol/15 min +/- 0.09 to 1.33 +/- 0.21 (p less than 0.05). These changes could be prevented neither by 20 micrograms/100 ml of 16,16-dimethyl PGE2 instilled intragastrically 45 minutes prior to the start of taurocholate administration, nor by 10 micrograms/100 ml of 16,16-dimethyl PGE2 instilled prior to taurocholate plus 0.7 micrograms/100 ml given together with taurocholate. It is concluded that "cytoprotection" appears to depend on the nature of the damaging agent, and may not occur under some conditions which play an important role in the pathophysiology of gastric mucosal diseases.

[Substituted benzimidazoles--a new dimension in ulcer therapy?]. / Substituierte Benzimidazole--eine neue Dimension der Ulkustherapie?

Substituted benzimidazoles represent a new class of gastric secretory inhibitors, which suppress acid secretion via direct inhibition of the parietal cell H+/K+-ATPase. The most potent derivative so far is omeprazole, which inhibits basal acid secretion and prevents the stimulatory effect of all classical acid secretory stimulants. In man, a single dose of 80 mg leads to almost complete achlorhydria over 24 hours. The results of the first duodenal ulcer trial with omeprazole are encouraging. No definite side effects have been attributed to the drug so far. Omeprazole may possibly initiate a new era in the medical treatment of peptic ulcer disease.

Pepsin secretion: neurohumoral regulation and drug effects.

The regulation of pepsin secretion was studied in the in vitro perfused mouse stomach. In contrast to acid secretion, basal pepsin release was not inhibited by 10(-4) M carbonyl cyanide m-chlorophenylhydrazine (CCCP) and by N2-induced hypoxia. Both secretions were not affected by 10(-3) M cimetidine, 10(-3) M atropine or cycloheximide (2 mg i.p. + 10(-5) M). Secretory responses to classical stimulants were similar to those obtained under in vivo conditions: carbamylcholine (CCH) and histamine stimulated acid and pepsin secretion in parallel, with a maximal pepsin/acid ration of 34 +/- 4 (mean +/- SEM) and 40 +/- 5, respectively. CCH-induced pepsin secretion was inhibited by atropine and pirenzepine. Dibutyrylic cyclic AMP(db-cAMP) strongly stimulated pepsin release. This stimulation was partially inhibited by trifluoperazine. Pentagastrin was a weak stimulant of pepsin secretion (pepsin/acid ratio: 10 +/- 3), whereas 10(-4) M bombesin and 10(-6) M salmon calcitonin had no effect. Omeprazole (H168/68) strongly inhibited basal acid secretion and stimulated pepsin release in a dose-and energy-dependent fashion. In contrast to acid, basal pepsin release probably represents an 'overflow secretion'. Although pepsin and acid are usually stimulated in parallel, dissociated responses are obtained under in vitro conditions, indicating that separate regulatory pathways exist.

Bile salt-induced, acute gastric mucosal damage in man: time course and effect of misoprostol, a PGE1-analogue.

A model is presented that allows the assessment of acute mucosal damage in man. Damage is revealed by changes of gastric mucosal potential difference (PD), urea net fluxes and DNA shedding. Sodium taurocholate (TC) damages the mucosa in a dose-dependent fashion: 1 mM TC leads to a transient drop of PD with a subsequent rise of DNA-, but not of urea net fluxes. With 5 mM TC, the drop of PD drop lasts for more than 15 min, urea net fluxes are increased, and the rise in DNA is more pronounced. We examined in this model, whether misoprostol (SC 29333), a prostaglandin E1 derivative, could prevent the TC-induced damage. Misoprostol in an antisecretory dose lead to an overall increase of PD even in presence of TC, but did neither prevent the TC-induced drop of PD, nor the changes in urea and DNA net fluxes. Similar results were obtained, when a non-antisecretory ('cytoprotective') dose of misoprostol was given prior to TC. Misoprostol has gastrin antisecretory properties, but no cytoprotective effects against acute, taurocholate-induced mucosal damage in man.

Dissociated response of acid and pepsin secretion to omeprazole in an in vitro perfused mouse stomach.

The effect of omeprazole, an inhibitor of the parietal cell H+-K+-ATPase, on pepsin and acid secretion was studied in an in vitro perfused whole mouse stomach model. Omeprazole inhibited basal and dibutyryl cAMP (DBcAMP)- and histamine-stimulated acid secretion in a dose-dependent fashion with a maximally effective dose of 10(-4) M. At the same time, omeprazole induced a dose-dependent increase of unstimulated pepsin release. This increase was not affected by pretreatment with 10(-3) M atropine or 10(-4) M cimetidine. It was, however, inhibited by preincubation with 10(-4) M carbonyl cyanide m-chlorophenylhydrazone (CCCP). Pepsin secretion after maximally effective doses of histamine or DBcAMP was not affected by 10(-4) M omeprazole. In a concentration of 10(-5) M, the effect of omeprazole was additive to the effect of submaximal concentrations of carbachol and histamine. NaSCN and imidazole mimicked the effect of omeprazole on acid secretion, but pepsin release was only stimulated with 10(-2) M imidazole. Another weak base, benzylamine, stimulated acid and pepsin in parallel. Luminal perfusion with solutions of high K+ concentration did not enhance basal pepsin release. The dissociated response of acid and pepsin secretion indicates that omeprazole does not act selectively on the parietal cell. The stimulation of pepsin secretion might be related to the weak base properties of the compound.

SecY, SecE, and band 1 form the membrane-embedded domain of Escherichia coli preprotein translocase.

The preprotein translocase of Escherichia coli is a multisubunit enzyme with two domains, the peripheral membrane protein SecA and the membrane-embedded SecY/E protein. SecY/E has been isolated as a complex of three polypeptides, SecY, SecE, and band 1. We now present four lines of evidence that the active species of SecY/E is composed of a tightly associated complex of these three subunits: 1) antibodies to SecY efficiently precipitate SecY/E activity as well as all three polypeptides; 2) the proportions of SecY, SecE, and band 1 in the immunoprecipitates are the same as in the starting fraction; 3) the immunoprecipitable complex is not disrupted by treatment with either high salt or urea but is disrupted by brief incubation at 20 degrees C, and the kinetics of dissociation of both band 1 and SecE from SecY at 20 degrees C parallel the loss of translocation ATPase activity; 4) upon immunoprecipitation of similar units of activity of translocase from detergent solutions from either wild-type membranes or a SecY and SecE overproducer strain, the SecE and band 1 subunits are recovered in the same proportions. These data establish that the subunits of SecY/E are firmly associated and that it is the associated complex which is active for translocation.

Induction of anacidity restores gastric aminopyrine clearance in canine hemorrhagic shock.

The determinants of gastric aminopyrine clearance were studied in a canine hemorrhagic shock model. Adult mongrel dogs were anesthetized, and their stomachs perfused with 0.1 N HCl through an esophageal and a duodenal cannula. Blood flow to the serosal layer of the anterior gastric wall was measured by a laser flowmeter. Hemorrhagic shock was induced by controlled arterial bleeding to a mean systolic blood pressure of 40 +/- 5 mm Hg (n = 8), resulting in a 36 +/- 8% drop of gastric wall blood flow. In contrast, the aminopyrine clearance did not reveal the expected drop and remained unchanged during shock. When acid secretion was abolished by intravenous omeprazole (1.3 mg/kg bolus plus 0.75 mg/kg/h infusion), aminopyrine concentrations dropped in the gastric perfusate and rose in the serum during shock, resulting in a similar decrease in the clearance (53 +/- 25%) as compared to the flowmeter readings. In control experiments without hemorrhagic shock, omeprazole did not affect the concentrations of aminopyrine in serum and in the perfusate, or the recovery of 14C-labeled aminopyrine in the mucosa at the end of the experiment. These studies indicate that the aminopyrine clearance is impaired in hemorrhagic shock, and that complete inhibition of acid secretion by omeprazole restores the apparent aminopyrine clearance by divergent effects on blood and gastric juice concentrations of aminopyrine.

Polyenylphosphatidylcholine inhibits PDGF-induced proliferation in rat hepatic stellate cells.

Polyenylphosphatidylcholine (PPC), a polyunsaturated phospholipid extract from soy beans, prevents the development of liver cirrhosis in animal models. Its mechanism of action is unknown. Based on the hypothesis that PPC might act by decreasing hepatic stellate cell proliferation, we studied the effect of PPC and its main components, dilinoleoylphosphatidylcholine (DLPC) and palmitoyl-linoleoylphosphatidylcholine (PLPC), on PDGF-induced stellate cell proliferation and intracellular signal transduction. Normal rat hepatic stellate cells in tissue culture were serumstarved, and incubated with 10ng/ml PDGF in the absence or presence of phospholipids. Cell proliferation was measured by 3H-thymidine incorporation. P44MAPK activation was determined by kinase assay, and AP-1 binding by electrophoretic mobility shift assay. PPC (200 ng/ml) significantly inhibited PDGF-induced proliferation (p < 0.05; ANOVA, n = 3) and antagonized PDGF-induced P44MAPK activation and AP-1 binding. This effect was mimicked by DLPC but not by PLPC. Neither DLPC nor PLPC prevented PDGF receptor activation. We conclude that PPC exerts a previously unrecognized effect on mitogen-induced stellate cell proliferation which may be mediated by DLPC. Inhibition of this cascade represents a potential mechanism for the inhibitory effect of PPC on hepatic fibrogenesis.

Activation of the endoplasmic reticulum Ca2+ pump of pancreatic acini by Ca2+ mobilizing hormones.

Stimulation of the pancreatic acinar cells with Ca2+ mobilizing hormones increased the ATP-dependent Ca2+ uptake into the ER of permeabilized cells. Activation of the ER Ca2+ pump resulted in increased apparent affinity for Ca2+ from 0.26 to 0.09 uM and Vmax from 2.68 to 5.74 nmoles/mg prot./min. The apparent affinity of the pump for VO4 = was dependent on [Ca2+]. Activation of the pump also decreased apparent affinity for VO4 = from 12 to 32 uM at [Ca2+] of 0.138 uM. These findings suggest that pump activation is due to acceleration of the rate of the conformational transition between the VO4 = (E2) and Ca2+ (E1) sensitive forms of the pump.

Regulation of free cytosolic Ca2+ in the peptic and parietal cells of the rabbit gastric gland.

Quin 2-loaded isolated rabbit gastric glands and purified peptic cells were used to measure free cytosolic Ca2+ ([Ca2+]i) during hormone stimulation. Rabbit gastric glands are composed of peptic and parietal cells with less than 1% endocrine cells. Although both cell types responded to the same hormones, they may be distinguished in terms of the source of Ca2+ bringing about the change in [Ca2+]i. Experiments were designed to assign changes in [Ca2+]i to either the peptic or parietal cells and to attempt to maintain these distinctions in the mixed cell population of gastric glands. It was shown that the peptide cholecystokinin octapeptide induced a rapid and transient increase in [Ca2+]i of isolated peptic cells. This signal was independent of medium Ca2+ and insensitive to the Ca2+ channel blockers La3+ and nifedipine. In gastric glands, the Ca2+ outdependent increase in (Ca2+)i (the secondary transient) was slower and dose dependently blocked by La3+ and nifedipine. This allowed [Ca2+]i levels in the physiologically more intact rabbit gastric glands to be dissected and correlated with fluorescence changes of quin 2 in either cell type. The transient increase in [Ca2+]i coincided with a burst of pepsin but not acid secretion. A subsequent slower phase of pepsin secretion took place while the cells restored near resting [Ca2+]i. Using a combination of the Ca2+ ionophore A23187 and the protein kinase C activating phorbol ester 12-O-tetra-decanoylphorbol 13-acetate, the hormone response pattern of pepsin secretion could be mimicked. The intracellular Ca2+ stores of the peptic cells in the gastric gland remained depleted of Ca2+ until specific antagonists were added. The reloading of intracellular stores required medium Ca2+ although [Ca2+]i was maintained at resting level during the entire reloading period. Hence, a specialized pathway of Ca2+ reloading is postulated.

Evaluation of pirenzepine on gastric acidity in healthy volunteers using ambulatory 24 hour intragastric pH-monitoring.

The effect of pirenzepine on 24 hour intragastric acidity was studied in 10 healthy volunteers using ambulatory 24 hour intragastric pH-monitoring in a double blind crossover study. Tests were performed on the seventh day of ingestion of either placebo, 75 mg pirenzepine or 150 mg pirenzepine per day. The drugs were given at two doses at 8.30 am and 8.30 pm. Mean nocturnal hydrogen ion activity during placebo treatment was 68 mmol/l +/- 9 SEM and was reduced by 75 mg (26%, p less than 0.01) and 150 mg of pirenzepine (36%, p less than 0.01), respectively. Mean diurnal hydrogen ion activity was 32 mmol/l +/- 6 SEM and was not significantly reduced (p greater than 0.1) by either dose of pirenzepine (4% and 12% respectively). Thus, the effect of pirenzepine on intragastric acidity is small, even with high doses of the drug, and becomes apparent only during the night.

Agonist-sensitive calcium pool in the pancreatic acinar cell. I. Permeability properties.

45Ca2+ fluxes and free cytosolic Ca2+ [( Ca2+]i) were used to describe the Ca2+ permeability and Ca2+ reloading of the agonist-sensitive pool at rest, during stimulation, and at termination of stimulation. A sequence of stimulation with carbachol, inhibition with atropine (cycling), and restimulation with cholecystokinin octapeptide (CCK-8) was used to follow Ca2+ reloading. Reloading of the pool required extracellular Ca2+ and was measured as an increased rate and extent of 45Ca2+ uptake into the acini. The 45Ca2+ incorporated into cycled acini could be completely released with CCK-8. The dose-response curves for 45Ca uptake and release were identical to those of the hormonally evoked [Ca2+]i increase. The increased 45Ca2+ uptake during reloading was not due to an expansion of any intracellular pool size but reflects the labeling of the pool to isotopic equilibrium in cycled acini. The rate constant of Ca2+ efflux from the pool of resting cells was approximately 0.67 +/- 0.01/h. With stimulation, the Ca2+ permeability of the pool membrane rapidly increased, resulting in Ca2+ release into the cytosol and an increase in [Ca2+]i. With termination of stimulation, the Ca2+ permeability of the pool membrane rapidly decreased while the pool continued to reload with extracellular Ca2+. Labeling of the pool to isotopic equilibrium allowed determination of the amount of Ca2+ released from the pool, which was 2.94 +/- 0.06 nmol/mg protein. This indicates that total Ca2+ concentration in the pool is in the millimolar range.

Agonist-sensitive calcium pool in the pancreatic acinar cell. II. Characterization of reloading.

45Ca2+ fluxes and free cytosolic Ca2+ measurements in guinea pig pancreatic acini indicated that after agonist stimulation and the release of Ca2+ from the agonist-sensitive pool at least part of the Ca2+ is extruded from the cell, resulting in 45Ca2+ efflux. In the continued presence of agonist, the pool remains permeable to Ca2+ but partially refills with Ca2+. This reloading is dependent on the concentration of extracellular Ca2+. In the absence of extracellular Ca2+, the pool is completely depleted of Ca2+. However, with increasing concentrations of CaCl2 in the incubation solution (from 0.5 to 2.0 mM) there is increasing repletion of the pool with Ca2+ during agonist stimulation. With termination of agonist stimulation, the Ca2+ permeability of the agonist-sensitive pool is rapidly reduced to that measured in the unstimulated cell. As a result, the Ca2+ incorporated into the pool during the stimulation period is rapidly trapped within the pool and exchanges poorly with medium Ca2+. Subsequently, the pool completely refills with Ca2+. The rate of Ca2+ reloading at the termination of agonist stimulation is slower than the conversion of the pool to the impermeable state. In incubation media containing 1.3 mM CaCl2, the half-time for reloading at the termination of stimulation is 5 min. These observations demonstrate the characteristics of Ca2+ reloading of the agonist-sensitive pool both during stimulation and at the termination of stimulation.

Complement C4 protein expression by rat hepatic stellate cells.

Stellate cells play an important role in the production and turnover of the normal extracellular matrix of the liver and are key effector cells in the hepatic fibrogenesis that occurs in response to liver injury. In the present study, we used a rat model of long term dietary iron supplementation to identify stellate cell genes that are expressed during chronic hepatic iron overload. Using a subtraction cloning strategy, we identified a rat isoform of the complement C4 protein gene whose expression was strongly induced in stellate cells after iron overload. Highly purified, cultured stellate cells synthesized the C4 precursor protein and released its subunits into the culture medium. The C4 protein secreted in vitro was biologically active in a C4-specific hemolytic assay. C4 mRNA expression was minimal in freshly isolated stellate cells and increased between days 3 and 7 of primary culture, coincident with the expression of smooth muscle alpha-actin (alpha-SMA), a marker of cellular activation. C4 expression was absent in strongly alpha-SMA-positive, passaged cells, but was induced by IFN-gamma, which simultaneously inhibited alpha-SMA expression. Our studies establish hepatic stellate cells as a previously unrecognized source of C4 and raise the possibility that complement protein expression by the cells plays a role in the hepatic injury response and in fibrogenesis. Our in vitro data point to the presence of two distinct stimulatory pathways for C4 expression in stellate cells that differ with regard to their sensitivity to IFN-gamma and their relationship to cellular activation.

The agonist-sensitive calcium pool in the pancreatic acinar cell. Activation of plasma membrane Ca2+ influx mechanism.

The purposes of the present study were to investigate the characteristics and regulation of Ca2+ influx across the plasma membrane in pancreatic acini and to demonstrate the role of this Ca2+ influx in the mechanism of reloading of the agonist-sensitive Ca2+ pool. In pancreatic acini, depleted of intracellular Ca2+ by stimulation with carbachol in the absence of extracellular Ca2+, 25 microM LaCl3 inhibited the increase in free cytosolic Ca2+ ([Ca2+]i) and reloading of the agonist-sensitive pool that occurred with the addition of extracellular CaCl2 to the medium. LaCl3 also inhibited the increase in cellular 45Ca2+ uptake that occurred during agonist stimulation and its termination but not cellular 45Ca2+ uptake into unstimulated acini. In acini depleted of intracellular Ca2+, increased cellular Ca2+ influx and reloading of the agonist-sensitive pool occurred even if extracellular CaCl2 was added 10 min after the termination of agonist action. Maximal reloading was independent of the extracellular Ca2+ concentration between 0.5 and 2.0 mM CaCl2. However, the time to maximal reloading was longer at lower extracellular Ca2+ concentrations. These results demonstrate a plasma membrane Ca2+ influx mechanism in the pancreatic acinar cell that is activated during cell stimulation. This transport remains activated as long as the agonist-sensitive pool is not completely loaded with Ca2+ suggesting that the Ca2+ influx mechanism is regulated by the quantity of Ca2+ in the agonist-sensitive pool. The activation of this Ca2+ transport mechanism functions to allow Ca2+ influx across the plasma membrane and Ca2+ reloading of the agonist-sensitive pool. Furthermore, these results suggest that during reloading Ca2+ crosses the plasma membrane into the cytosol before entering the agonist-sensitive pool.