Role of glucose metabolism in acid formation by isolated gastric glands

The role played by glucose in providing energy for acid formation was studied in isolated gastric glands from rabbit. The widely-used inhibitors of glycolysis, iodoacetic acid and iodoacetamide were found to inhibit glucose oxidation as well as the indicators of acid formation, respiration and accumulation of aminopyrine. However, the potent inhibition of acid formation was found to involve a nonspecific mechanism other than the simple inhibition of glycolysis. An alternative approach involved use of the glucose transport inhibitor, phloretin. Phloretin blocked glucose oxidation and also inhibited functional responses. Acid formation was restored easily by the addition of pyruvate or various other oxidizable substrates. Measurement of lactate formation in the absence of exogenous glucose showed that the gastric glands contain very little glycogen. Addition of external glucose resulted in a 10-fold increase in lactate formation and this rate was stimulated further by histamine and rotenone. Rotenone also inhibited both respiration and aminopyrine accumulation; however, the inhibition was not complete. Phloretin treatment resulted in total inhibition of the residual aminopyrine accumulation after rotenone treatment. The results are interpreted to indicate that gastric glands are dependent almost totally on external substrate supply to support acid formation; and, that while anaerobic glucose metabolism can sustain a very low level of acid formation, the major role of glucose is to yield pyruvate equivalents for subsequent oxidation.     

Effects of common inhibitors of gastric acid secretion on secretagogue-induced respiration and aminopyrine accumulation in isolated gastric glands.

1. The effects of three inhibitors of gastric acid secretion, atropine, burimamide and thiocyanate, have been studied in isolated glands from the rabbit gastric mucosa. The glands were either resting or stimulated by carbachol, histamine or dibutyryl cyclic AMP. The effects were determined from changes in oxygen consumption and accumulation of the weak base aminopyrine. The latter gives an indirect measurement of the acid production in the glands. 2. Atropine (10 (-6) M) almost totally inhibited the transient response induced by carbachol (10 (-4) M) in both measured parameters. The histamine-induced increase in respiration was inhibited when the atropine concentration was raised to 10 (-4) M. To a lesser extent also, histamine-induced aminopyrine accumulation was reduced. The dibutyryl cyclic AMP stimulated oxygen consumption was not affected by atropine. 3. Burimamide competitively inhibited the histamine responses but was without effect on those of carbachol and dibutyryl cyclic AMP. 4. Thiocyanate (10 (-2) M) inhibited the increase in oxygen consumption induced by all three secretagogues but not down the prestimulatory level, in spite of total abolishment of the aminopyrine accumulation. 5. In unstimulated glands, burimamide (10 (-3) M) or atropine (10 (-4) M) did not alter the normal aminopyrine ratio (aminopyrine in intraglandular water/ aminopyrine in extraglandular water) of approximately 50. This may indicate the existence of preformed acid in resting parietal cells. Thiocyanate, on the other hand, lowered the aminopyrine ratio in unstimulated glands from 46 to 2. Possible mechanisms for the thiocyanate effect are discussed in terms of an inability to separate acid and base in the secreting membrane.     

Evaluation of (14C)aminopyrine clearance for determination of gastric mucosal blood flow.

This study has demonstrated high correlation between the radiometric and spectrophotometric determinations of gastric mucosal aminopyrine clearance. The radiometric method is technically easier, allows a larger number of samples to be determined and is safer to the subject. The clearance of small amounts of [14C]aminopyrine was unaffected by large doses of unlabeled aminopyrine showing that mucosal extraction is not concentration limited. Small amounts of [14C]aminopyrine may provide an excellent tool for examining the role of mucosal blood flow in the pathogenesis of gastric disease in man.     

Perturbation of hepatic glutathione level and glutathione-related enzyme activities by repeated administration of aminopyrine in rats

The influence of repeated administration of aminopyrine on the tissue glutathione level and related enzyme activities was investigated in rats. Reduced glutathione level in the liver was not changed after 5 days of treatment but a significant increase was seen after 15 days of aminopyrine treatment. Oxidized glutathione level was unaltered throughout the experiment. Repeated administration of aminopyrine for 5 days caused a marked increase in gamma-glutamyl transpeptidase activities in liver whole homogenates as well as in the nuclear fraction, but not in liver microsomes. These results suggest that gamma-glutamyl transpeptidase located in plasma membrane may be induced by repeated administration of aminopyrine for 5 days. The activities of cytosolic glutathione peroxidase, which modulates glutathione level, were also significantly increased by aminopyrine treatment. Under the same conditions, glutathione peroxidase activity with H2O2 as a substrate was unaltered, while a time-dependent increase in the activity was found when cumene hydroperoxide was used as a substrate, even after a single administration of aminopyrine. The intracellular cysteine level was increased accompanying the increased gamma-glutamyl transpeptidase activities. Therefore, induced gamma-glutamyl transpeptidase may play a role in the reclamation of extracellular oxidized glutathione.     

Effects of cholecystokinin on acid formation in glands and cells isolated from rabbit and rat gastric mucosa

Isolated gastric glands and isolated cells prepared from rabbit and rat were studied to analyse the influence of cholecystokinin octapeptide (CCK 8) on histamine stimulated parietal cell acid formation as assessed by [14C]aminopyrine sequestered in acid tissue compartments. In rabbit gastric glands, CCK 8 evoked 32+/-6% (P<0. 01) inhibition of histamine stimulated acid formation, whereas in glands prepared from rat no inhibition was recorded. Instead, CCK 8 seemed to induce a variable increase of the histamine stimulation in rat gastric glands as the aminopyrine accumulation was increased by 110+/-46% (P<0.1). Further studies on cell preparations derived from rabbit gastric mucosa revealed dual properties of CCK 8, eliciting either inhibition or stimulation of the parietal cell depending on the presence of endocrine cells. The results show that paracrine communication may be effective in glandular preparations, but seems to vary depending on species.     

Calcium involvement in the muscarinic response of the gastric parietal cell

The influence of extracellular Ca2+ on the mediation of carbachol stimulation in isolated rabbit gastric parietal cells was studied. Removing Ca2+ from extracellular medium caused a 42% decrease of the aminopyrine accumulation due to carbachol with the same EC50 value (approximately 5 microM). A short time depletion in extracellular calcium suppressed the carbachol-dependent Ca2+ influx without affecting Ca2+ release from internal stores (fura-2 measurements). Similarly, the production of inositol phosphates under cholinergic stimulation was reduced by 29%. A rapid increase in Ins(1,4,5)P3 was obtained 5 s after carbachol stimulation, and this increase was not changed in Ca2(+)-depleted medium. In contrast, a 20 min incubation with carbachol caused a 50% reduction in both basal and carbachol-stimulated inositol phosphate accumulations. In conclusion, phospholipase C activation, intracellular Ca2+ release and aminopyrine accumulation were sequentially observed following carbachol stimulation of the isolated gastric parietal cell and extracellular calcium contributed to sustain this acid secretory response.     

Involvement of calcium-sensitive phospholipid-dependent protein kinase in control of acid secretion by isolated rat parietal cells.

The relative potency with which phorbol esters inhibited histamine-stimulated aminopyrine accumulation (an index of acid secretion) paralleled that which has been established for the activation of purified protein kinase C. The inhibitory effect of 1-oleoyl-2-acetylglycerol on aminopyrine accumulation stimulated by various secretagogues was similar to that of 12-O-tetradecanoylphorbol 13-acetate. Protein kinase C activity was present in a parietal-cell-enriched fraction. In conclusion, protein kinase C could be involved in mechanisms regulating gastric acid secretion.     

Aminopyrine uptake by guinea pig gastric mucosal cells. Mediation by cyclic AMP and interactions among secretagogues

The role of cyclic nucleotides in regulating acid secretion by dispersed mucosal cells from guinea-pig stomach was examined by measuring first the ability of histamine and carbachol to stimulate [dimethylamine-14C]aminopyrine uptake and cyclic nucleotide metabolism and secondly, the effect of exogenous cyclic nucleotides on basal and stimulated [14C]aminopyrine uptake. The [14C]aminopyrine was found in an acidic, osmotically sensitive compartment, probably associated with the initial steps in acid secretion by these cells. Although histamine increased [14C]aminopyrine uptake and cyclic AMP synthesis as expected, histamine was approx. 10-fold more potent in inducing [14C]aminopyrine uptake. This dissociation of [14C]aminopyrine uptake and cyclic AMP metabolism process was further manifested by the observation that prostaglandin E1 failed to increase [14C]aminopyrine uptake, although it did cause a rise in cellular cyclic AMP. Furthermore, prostaglandin E1 did not alter the [14C]-aminopyrine uptake caused by histamine. Carbachol was found to increase the [14C]aminopyrine uptake and also to potentiate the ability of histamine to increase [14C]aminopyrine uptake. Carbachol, however, affected neither the histamine-induced increase in cyclic AMP nor the binding of [3H]histamine to the cells. Cimetidine, a histamine H2 receptor antagonist, blocked the [14C]aminopyrine uptake induced either by histamine alone or by the potentiating combination of histamine plus carbachol. These results suggest that cyclic AMP is mediating the action of histamine on [14C]aminopyrine uptake but changes in cyclic AMP per se are not necessarily the cause for the potentiated increase in [14C]aminopyrine uptake. Furthermore, the potentiated response observed with histamine plus carbachol on [14C]aminopyrine uptake occurs at a biochemical step distal to and not obviously related to cyclic AMP generation.     

Action of glucagon and atropine on mucosal blood flow of resitng fundic pouches of dogs.

Clearance of plasma aminopyrine (an index of mucosal blood flow, MBF) into acid (0.1 N HCl) instilled into Heidenhain pouches was about 200 ml per 30 min under basal conditions. Glucagon 50 μg/kg s.c. significantly decreased the MBF from 200 to 132 ml per 30 min at one hr; the decrease lasted about 60 min. Infusion of glucagon 50 μg/kg i.v. for 1 hr produced a delayed reduction of MBF lasting for more than 90 min. Under the same experimental conditions, atropine 50 μg/kg reduced MBF from 200 to 166 ml per 30 min upon subcutaneous administration and showed no significant effect by i.v. infusion. The increase in residual volume of the pouch caused by glucagon could not account for all the decrease in clearance of aminopyrine. We conclude that glucagon reduces gastric mucosal blood flow under basal conditions.     

Metabolism and gastric acid secretion. Substrate-dependency of aminopyrine accumulation in isolated rat parietal cells.

The substrate-dependency of gastric acid secretion was investigated in isolated rat parietal cells by using the accumulation of the weak base aminopyrine as an index of acid secretion. Exogenous substrates enhanced accumulation of aminopyrine in rat parietal cells stimulated by secretagogues, and this effect was probably directly related to the provision of energy for acid secretion. At physiological concentrations, certain of the substrates (glucose, oleate, lactate, D-3-hydroxybutyrate, L-isoleucine, L-valine and acetoacetate) could support acid secretion, with glucose being the most effective. L-Leucine and acetate were only effective stimulators of parietal-cell aminopyrine accumulation at high concentrations (5mM). L-Glutamine was unable to stimulate aminopyrine accumulation even at high concentrations, and glutaminase activity in parietal cells was estimated to be low by comparison with small-intestinal epithelial cells. Variation in the concentrations of D-3-hydroxybutyrate and L-isoleucine, but not of glucose, within the physiological range affected their ability to support aminopyrine accumulation. The presence of 5 mM-L-isoleucine, 5 mM-lactate and combinations of certain substrates at physiological concentrations produced aminopyrine accumulation in stimulated parietal cells that was greater than that obtained in cells incubated with 5 mM-glucose alone. In conclusion, fulfillment of the metabolic requirements of the acid-secreting parietal cell under physiological circumstances requires a combination of substrates, and integration of the results with previous data [Anderson & Hanson (1983) Biochem. J. 210, 451-455; 212, 875-879] suggests that after overnight starvation in vivo metabolism of glucose, D-3-hydroxybutyrate and L-isoleucine may be of particular importance.     

Close arterial infusion of calcitonin gene-related peptide into the rat stomach inhibits aspirin- and ethanol-induced hemorrhagic damage

Afferent neuron-mediated gastric mucosal protection has been suggested to result from the local release of vasodilator peptides such as calcitonin gene-related peptide (CGRP) from afferent nerve endings within the stomach. The present study, therefore, examined whether rat alpha-CGRP, administered via different routes, is able to protect against mucosal injury induced by gastric perfusion with 25% ethanol or acidified aspirin (25 mM, pH 1.5) in urethane-anesthetized rats. Close arterial infusion of CGRP (15 pmol/min) to the stomach, via a catheter placed in the abdominal aorta proximal to the celiac artery, significantly reduced gross mucosal damage caused by ethanol and aspirin whereas mean arterial blood pressure (BP) was not altered. Intravenous infusion of CGRP (50 pmol/min) did not affect aspirin-induced mucosal injury but significantly enhanced ethanol-induced lesion formation. Intravenous CGRP (50 pmol/min) also lowered BP and increased the gastric clearance of [14C]aminopyrine, an indirect measure of gastric mucosal blood flow while basal gastric output of acid and bicarbonate was not altered. Intragastric administration of CGRP (260 nM) significantly inhibited aspirin-induced mucosal damage but did not influence damage in response to ethanol. BP, gastric clearance of [14C]aminopyrine, and gastric output of acid and bicarbonate remained unaltered by intragastric CGRP. These data indicate that only close arterial administration of CGRP to the rat stomach, at doses devoid of a systemic hypotensive effect, is able to protect against both ethanol- and aspirin-induced mucosal damage. As this route of administration closely resembles local release of the peptide in the stomach, CGRP may be considered as a candidate mediator of afferent nerve-induced gastric mucosal protection.     

Pancreastatin: a novel peptide inhibitor of parietal cell signal transduction

The direct inhibition of secretion by pancreastatin was investigated in rabbit isolated parietal cells. Pancreastatin exerted no influence on basal aminopyrine uptake. Pancreastatin inhibited histamine stimulated aminopyrine uptake through a decrease in intracellular cAMP. Pancreastatin inhibition of histamine stimulated uptake was blocked in the presence of pertussis toxin. Pancreastatin also inhibited the carbachol stimulated increase in aminopyrine accumulation. However, the effects of pancreastatin on carbachol stimulation were not reversed by pertussis toxin. Pancreastatin did not alter the carbachol induced increase in cytosolic free calcium. Thus, pancreastatin appears to inhibit parietal cell signal transduction at multiple points along the second messenger pathways.     

Interrelationship between drug oxidation ureogenesis and gluconeogenesis in isolated hepatocytes

1. The effect of increased ureogenesis--provoked by NH4Cl and ornithine--on gluconeogenesis and aminopyrine oxidation was studied in isolated hepatocytes prepared from 24 hr starved mice; lactate or fructose was used as gluconeogenic precursor. 2. Increased ureogenesis caused about 40% inhibition both on aminopyrine oxidation and gluconeogenesis when lactate was added as gluconeogenic substrate. 3. On the other hand, only 10% inhibition of aminopyrine oxidation and about 15% inhibition of gluconeogenesis were observed when fructose was used as gluconeogenic precursor. 4. Aminopyrine has been reported to inhibit gluconeogenesis from fructose by 30% and from lactate by 85%. The inhibitory effect of the combined addition of aminopyrine, NH4Cl and ornithine on gluconeogenesis was also dependent on the applied gluconeogenic precursor. 5. The provoked ureogenesis by ammonia and ornithine was not inhibited by aminopyrine. N6, O2-dibutyryl cAMP known to cause an increase of gluconeogenesis a decrease of aminopyrine oxidation enhanced the inhibitory action of increased ureogenesis on aminopyrine oxidation and on gluconeogenesis further. 6. The role of NADPH in the regulation of drug oxidation and ureogenesis is underlined.     

Characterization of cytochrome P450 2E1 activity by the [14C]nitrosodimethylamine breath test

The objective of this study was to measure the rate of demethylation of nitrosodimethylamine in vivo in the rat and determine its value to assess CYP2E1 activity in intact animals. Nitrosodimethylamine labeled with 14C on both methyl groups was administered to rats and exhaled 14CO2 was collected during 2-3 h. The nitrosodimethylamine breath test was increased by inducers of CYP2E1, such as ethanol (+139%) and 4-methylpyrazole (+115%), and decreased by the inhibitor diallyl sulfide (-53%). In addition, the nitrosodimethylamine breath test was not changed significantly by inducers specific for other cytochrome P450 such as beta-naphthoflavone, dexamethasone, and phenobarbital. The specificity of the induction by 4-methylpyrazole and of the inhibition by diallyl sulfide for CYP2E1 was determined using the [14C]caffeine (CYP1A2), [14C]aminopyrine (CYP2C11), and [14C]erythromycin (CYP3A2) breath tests. 4-Methylpyrazole treatment caused a small increase of the caffeine (+33%) and aminopyrine (+9%) breath tests and no change of the erythromycin breath test. Diallyl sulfide treatment led to a small decrease of the caffeine breath test (-33%) and of the aminopyrine breath test (-13%) but a 23% increase of the erythromycin breath test. It is concluded that the [14C]nitrosodimethylamine breath test is useful to assess CYP2E1 activity in vivo in the rat.     

Direct hyposmotic stimulation of gastric acid secretion

Gastric glands isolated from rabbit stomach were incubated in isosmotic medium or media made hyposmotic by 50-100 mOsm/kg. As indicated by radiolabeled aminopyrine accumulation, acid secretion was nearly 3 times greater in 200 mOsm/kg hyposmotic than in isosmotic medium after a 30-min incubation. The hyposmotic stimulation appeared within 2 min, peaked at 10-15 min and declined almost to the isosmotic control by 45 min. As estimated by the wet weight corrected for inulin extracellular space, the intracellular water of the glands also peaked at 15 min and returned to the isosmotic norm by 45 min. Hyposmotic stimulation of acid secretion directly involved the parietal cell, since parietal cells obtained from gastric glands were also stimulated. That the hyposmotic response was direct was indicated by omeprazole inhibition of aminopyrine accumulation in hyposmotic medium.     

ATP dependence of H+ secretion

Cells in isolated rabbit gastric gland were made permeable to ATP by high voltage discharge across a gland suspension. In both normal (5.4 mM K+) and high K+ (108 mM) medium, this electrical shock resulted in a marked reduction in the ability of the parietal cell to produce and accumulate acid. Acid production was monitored both microscopically by acridine orange accumulation in the secretory canaliculus and by accumulation of the weak base [14C]aminopyrine. In 108 mM K+ solutions but not in 5.4 mM K+ solutions 5, mM ATP was able to restore the accumulation of these probes to control (unshocked) levels. When shocked glands had been previously stimulated by secretagogues, the aminopyrine accumulation ratio was only partly restored by ATP. Inhibition of mitochondrial respiration by cyanide, azide, or Amytal abolished acid secretion; the subsequent addition of ATP to shocked glands increased the aminopyrine accumulation ratio to 47 and resulted in an acridine orange fluorescence indistinguishable from that of histamine-stimulated, unshocked glands. We conclude that ATP can act as a substrate for H+ secretion in the parietal cell, and that perhaps no additional energy source is necessary.     

The effect of ascorbic acid on the amine-nitrite and nitrosamine mutagenicity in bacteria injected into mice

Ascorbic acid was tested for its ability to increase or decrease the induction of bacterial mutations by dimethylnitrosamine (DMN) or aminopyrine plus nitrite within intact mice. No evidence was found of the mutagenicity of ascorbic acid itself when tested alone or in the presence of copper ions. Similarly, no increase or decrease in the DMN-induced mutation frequency was observed. However, ascorbic acid was found to decrease the aminopyrine/ nitrite-induced mutation frequency to an extent which was dependent on the experimental conditions used.     

Specific inhibition by ATP of histamine-stimulated acid secretion in the gastric glands of the rabbit]

The influence of adenosine 5'-triphosphate on gastric acid secretion stimulated by histamine, carbachol, dibutyryl-cAMP and the phosphodiesterase inhibitors 8-phenyl-theophylline and rolipram in isolated rabbit gastric glands was studied. Changes oi gastric acid secretion were measured by the aminopyrine accumulation method. Histamine-stimulated acid secretion was significantly inhibited by ATP 1 mM, whereas the secretory responses elicited by carbachol, dibutyryl-cAMP, 8-phenyl-theophylline or rolipram were not. Assays with indomethacin, a well known prostaglandin synthesis inhibitor, showed that this agent significantly reduced the inhibitory effect of ATP on histamine responses. The results indicate that the antisecretory effect of ATP was specific for histamine and that it was mediated, at least in part, via stimulation of endogenous prostaglandin production.     

Alterations of liver ergastoplasmic membranes during DL-ethionine hepatocarcinogenesis. Aminopyrine demethylase activity and ribosome-membrane interaction

The activity of microsomal aminopyrine demethylase and the degree of association between ribosomes and endoplasmic membranes were studied in liver of ethionine-fed rats, as well as in hyperplastic nodules and in hepatoma. In the course of the ethionine-feeding, inhibition of aminopyrine demethylase and increase in the relative amounts of membrane-free ribosomes occured in liver cells. After 1–3 months from the end of the ethionine feeding, when hyperplastic nodules and hepatoma develop in the liver, a sharp inhibition of aminopyrine demethylase was found in the latter tissues, but not in the surrounding nonnodular liver. At the same time an increase of membrane-free ribosomes occured in surrounding nonnodular liver, in nodules and in hepatoma. The extent of this alteration, however, was significantly lower in surrounding nonnodular liver than in nodules and in hepatoma. These results are discussed in relation to the problem of the cellular precursors of hepatoma.