Phosphorylation of cytosolic phospholipase A2 and the release of arachidonic acid in human neutrophils.

Kinases mediating phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in intact cells remain to be fully characterized. Platelet-activating factor stimulation of human neutrophils increases cPLA2 phosphorylation. This increase is inhibited by PD 98059, a mitogen-activated protein (MAP)/extracellular signal-regulating kinase (erk) 1 inhibitor, but not by SB 203580, a p38 MAP kinase inhibitor, indicating that this action is mediated through activation of the p42 MAP kinase (erk2). However, platelet-activating factor-induced arachidonic acid release is inhibited by both PD 98059 and SB 203580. Stimulation by TNF-alpha increases cPLA2 phosphorylation, which is inhibited by SB 203580, but not PD 98059, suggesting a role for p38 MAP kinase. LPS increases cPLA2 phosphorylation and arachidonic acid release. However, neither of these actions is inhibited by either PD 98059 or SB 203580. PMA increases cPLA2 phosphorylation. This action is inhibited by PD 98059 but not SB 203580. Finally, FMLP increases cPLA2 phosphorylation and arachidonic acid release. Interestingly, while the FMLP-induced phosphorylation of cPLA2 is not affected by the inhibitors of the p38 MAP kinase or erk cascades, both inhibitors significantly decrease arachidonic acid release stimulated by FMLP. SB 203580 or PD 98059 has no inhibitory effects on the activity of coenzyme A-independent transacylase.

Ethyl propionate is more effective and less cytotoxic than methyl tert-butyl ether for topical gallstone dissolution.

BACKGROUND & AIMS: Ethyl propionate and isopropyl acetate were identified as gallstone solvents with more favorable physicochemical properties than the currently used solvent methyl tert-butyl ether (MTBE). In this study, their efficacy and toxicity were compared. METHODS: To compare efficacy, matched stones from 33 patients were subjected to dissolution with each solvent. To evaluate cytotoxicity, jejunal segments of the anesthetized rat were exposed to each solvent or saline; the segments were then perfused with markers for active absorption and passive permeability. RESULTS: For 23 gallstone sets that dissolved completely with all three solvents, the average dissolution time was shorter with ethyl propionate (38 +/- 8 minutes) than with MTBE (60 +/- 13 minutes) (P = 0.03) or isopropyl acetate (55 +/- 12 minutes) (P < 0.001). Four stones did not dissolve with ethyl propionate, seven with MTBE, and eight with isopropyl acetate. After 2 minutes of exposure to the solvents, the dry weight of the segments decreased by 36% after MTBE but was unchanged after the other two solvents (P < 0.001). MTBE caused more inhibition of active absorption than the other solvents (P < 0.001) and a greater increase in passive permeation (P < 0.03). CONCLUSIONS: Ethyl propionate and isopropyl acetate are less toxic to the intestinal mucosa than MTBE, and ethyl propionate is more effective for gallstone dissolution.

Transforming growth factor-alpha and epidermal growth factor activate mitogen-activated protein kinase and its substrates in intestinal epithelial cells.

The signal transduction pathways of mitogenic stimuli in intestinal epithelial cells are not clearly understood. We report here a possible signaling pathway of two closely related agonists, transforming growth factor-alpha (TGF alpha) and epidermal growth factor (EGF). Both increase thymidine incorporation in the intestinal epithelial cell (IEC) line IEC-6. This increase is dose dependent and inhibited by the tyrosine kinase inhibitors genistein and tyrphostin. The addition of either TGF alpha or EGF to IEC-6 cells also stimulates the activities of the two forms of mitogen-activated protein kinase, p42erk2 MAPK and p44erk1 MAPK, as evidenced by increased incorporation of radiolabeled phosphate in myelin basic protein. The main difference between the MAPK activity levels induced by the two agonists is in the intensity of the response. Maximum TGF alpha-induced stimulation of p42erk2 MAPK activity is 9-fold at 2 ng/ml, while maximum EGF stimulation is only 4.5-fold at 25 ng/ml. These doses correlated closely with the dose required for maximum thymidine incorporation. The activity of the 90-kDa ribosomal S6 kinase, a downstream substrate for activated MAPK, is also enhanced as evidenced by increased incorporation of radiolabeled phosphate in the rsk kinase substrate peptide in IEC-6 cells following stimulation with either TGF alpha or EGF. This increase correlates closely with the stimulus-induced increase in MAPK activity with respect to dose, but the time of increased activity is more prolonged, especially after EGF stimulation. TGF alpha induced the synthesis of both c-Fos and c-Myc, two nuclear substrates for MAPK, and increased c-fos and c-myc message levels as well. However, c-Jun protein and c-jun mRNA were not induced. The increase in IEC-6 cell proliferation in response to TGF alpha and EGF stimulation may then be due, in part, to an increase in immediate early gene expression as a direct result of MAPK and RSK activation.

Stimulation of intestinal Na+/H+ exchange by cell volume changes during fasting and refeeding in rats.

Intestinal cell water was studied in rats that have been fasted then refed, two conditions that are known to decrease and increase, respectively, proliferation of the intestinal epithelium. Cell water decreased (15%) during fasting and returned to normal with refeeding. The amiloride-sensitive sodium uptake, which estimates uptake through the Na+/H+ exchange was higher in the ileum than the jejunum, but the jejunal uptake, unlike the ileal, was significantly increased in fasted/refed rats than in normally fed or fasted ones. Osmotic shrinkage of intestinal cells followed by restitution of their cell volume stimulated the Na+/H+ exchange in all of the three groups of animals, but the increase was most prominent in the fasted and the fasted/refed groups. Also, shrinkage of cultured jejunal crypt cells (IEC-6) by a hypertonic solution increased intracellular alkalinization that was inhibited by amiloride. The results provide evidence for a relationship between the change in intestinal cell size, such as that which occurs during fasting/refeeding, and the activation of the Na+/H+ antiport system. This may represent one of the signals that initiates intestinal proliferation in the fasting/refeeding state.

A method to quantitatively compare in vivo the effects of gallstone solvents on intestinal mucosal function: a controlled study comparing mono-octanoin with methyl tert-butyl ether in the rat.

During contact dissolution of gallstones, solvents may escape from the gallbladder and damage the intestinal mucosa. In order to compare the extent of this potential injury, we developed a method to objectively quantify the effects of two commonly used cholesterol solvents, methyl tert-butyl ether and mono-octanoin, on mucosal transport function in the rat intestine. Two intestinal segments in each of 184 anesthetized rats were cannulated. Three milliliters of either solvent were instilled in one segment and left for varying periods of time, while saline was instilled in the other as control. The segments were then washed and perfused for 45 min with an isotonic solution containing [3H]polyethylene glycol 4000 (a nonabsorbable reference marker) and either [14C]alpha-aminoisobutyric acid (a marker for active absorption) or [14C]mannitol (a marker for passive permeability). Methyl tert-butyl ether caused more inhibition of alpha-aminoisobutyric acid absorption (64%) than mono-octanoin (48%) and a greater reduction of dry weight per centimeter of the perfused segment (22%) compared with mono-octanoin (10%). Such effects appeared after only 1 min of solvent exposure and did not appreciably increase with longer exposures. Permeation of mannitol increased by 26% after 1 min of exposure to mono-octanoin and by 54% after a similar period of exposure to methyl tert-butyl ether. Longer exposures to both solvents did not seem to cause progressive increases in mannitol permeation. The results indicate that brief exposure of the rat jejunum to either of the two solvents causes a reduction in active transport ([14C]alpha-aminoisobutyric acid absorption), an increase in passive permeability (mannitol permeation), and a loss of mucosal constituents. We conclude that the intestinal mucosa is susceptible to solvent damage and may be used as a selectively sensitive model that can characterize the biological injury of gallstone solvents. The study also suggests that escape of the currently available solvents into the small intestine in patients undergoing contact dissolution of gallbladder stones may cause injury to the small intestine.

Transforming growth factor-alpha increases tyrosine phosphorylation of microtubule-associated protein kinase in a small intestinal crypt cell line (IEC-6).

The small intestinal crypt cell line (IEC-6) is an undifferentiated, untransformed, mitotically active cell used in this study to determine the effect of transforming growth factor-alpha (TGF-alpha) on tyrosine phosphorylation levels of cellular proteins. Thymidine incorporation increased maximally after addition of 2 ng/ml TGF-alpha for 24 h. At the same dose, TGF-alpha induced the tyrosine phosphorylation of proteins with approximate molecular masses of 42, 44, 52, 80, 150 and 175 kDa as shown by Western blots treated with anti-phosphotyrosine antibody. The most intense phosphorylation was seen in the 42 kDa (p42) and 44 kDa (p44) proteins, which were identified as two isoforms of microtubule-associated protein kinase (MAPK). This phosphorylation was seen as early as 5 min post stimulation and was dose dependent. Both p42 and p44 were found in the nucleus after stimulation, although a basal level of unphosphorylated protein was present before stimulation. The observed tyrosine phosphorylation of p42 and p44 was inhibited by genistein, a tyrosine kinase inhibitor, and tyrphostin 23, an epidermal growth factor receptor tyrosine kinase inhibitor. We conclude that MAPK is tyrosine phosphorylated in response to TGF-alpha stimulation of IEC-6 cells.

Role of tyrosine kinase and phosphotyrosine phosphatase in growth of the intestinal crypt cell (IEC-6) line.

The roles of gastrin and sodium vanadate in proliferation were examined in cultured IEC-6 cells that are mitotically active and derived originally from jejunal crypts of the rat intestine. Incubation of the cells in the presence of gastrin at a concentration of 250 ng/ml or of sodium vanadate at a concentration of 0.2 mM leads to a 60% increase in cell growth in 24 hr. The stimulated growth in both cases was inhibited by genistein, a tyrosine kinase inhibitor. Incubation in the presence of gastrin and sodium vanadate together produced a small, albeit significant, potentiation of growth of the cells. Gastrin as well as sodium vanadate also promoted the phosphorylation on tyrosine of a similar group of proteins with molecular masses of 42, 45, 52, 60, 78, and 120 kDa. The phosphorylations were rapidly occurring as early as 5 min and lasted for only 15 min. Several proteins were detected in normal IEC-6 cells, including GTPase activating protein, raf1 kinase, phospholipase C gamma-1, and phosphoinositide 3-kinase. The results suggest that gastrin and sodium vanadate induce growth of IEC-6 cells by stimulation of tyrosine kinase and/or inhibition of tyrosine phosphatase. The gastrin and sodium vanadate effects also involve the phosphorylation of a number of proteins, the identities of which are not known at present but may include some of the kinases that are frequently associated with cell growth, such as mitogen-activated protein kinase, raf1 kinase, phosphoinositide 3-kinase, and others.

Enhancement of rat intestinal calcium absorption by vanadate.

Vanadate alters intestinal transport and may have a role in regulating cell function. To determine whether it influences calcium absorption, we tested the effects of acute and chronic vanadate administration on calcium absorption using single-pass perfusion of jejunal and ileal segments of the in vivo rat intestine. Acute vanadate administration increased the lumen-to-mucosa and net fluxes of calcium in both the jejunum and ileum. The increase was largely due to an enhancement of the saturable fluxes of calcium and was observed at 10(-4) M concentration of vanadate, but not at higher or lower concentrations of the oxyanion, except at the highest concentration used, 10(-2) M, where calcium absorption was inhibited. Chronic vanadate administration caused, on the other hand, no changes in calcium absorption. We have demonstrated previously that rat intestinal (Na+ + K+)-ATPase is inhibited by vanadate, an effect that could raise cell sodium and increase the efflux of sodium across the brush border membrane. The results suggest that the vanadate enhancement of calcium absorption may be related to an increased entry of calcium into the mucosa, possibly as a result of an augmented exchange through the Na+/Ca+ antiport system. Alternatively, vanadate may influence access to a calcium channel in the mucosal membrane of the intestinal epithelium, leading to the observed increase in absorption.

Reversal by vanadate of the effect of diabetes on intestinal growth and transport.

Diabetic changes in small intestinal growth and transport were studied in the rat during the administration of the pentavalent vanadium oxyanion, vanadate. This treatment reversed the effect of diabetes on growth of the intestinal mucosa. It eliminated hyperglycemia and restored the normal rate of absorption of AIB and 3-o-methyl glucose by the intestine. The changes are probably caused by an insulin-like action of vanadate on the intestine.

Effect of chronic vanadate ingestion on amino acid and water absorption in rat intestine.

Chronic administration of a relatively low concentration of vanadate to rats causes inhibition of water, alpha-aminoisobutyric acid (AIB) and L-alanine absorption. The mechanism responsible for this inhibition was tested by studying the uptake of alanine in isolated rat intestinal cells. The studies suggest that the vanadate inhibition of amino acid transport is primarily caused by a decreased activity of the Na+-K+ pump, an action that is similar to what is observed when the rat intestine is acutely exposed to vanadate. Vanadate appeared to have no direct effect on the entry of amino acids into the intestinal cell. This was evident by the fact that amino acid uptake by enterocytes of control rats was not different from the uptake by cells of vanadate-treated animals that have an inwardly directed Na gradient artificially created across them. Furthermore, 86RB influx and efflux into and out of intestinal tissues of the vanadate-treated animals were, respectively, decreased and increased as compared to normal control tissues and they were similar to what is observed when the intestine is acutely exposed to ouabain, a known specific inhibitor of the Na+-K+ pump.

Effect of acute and chronic vanadate administration on sugar transport in rat jejunum.

Vanadate is known to have an insulin-like action which stimulates sugar transport in some systems like adipocytes and muscle cells, but in other systems it inhibits sugar transport by decreasing the activity of (Na+ +K+)-ATPase. To evaluate whether these two opposing actions may influence sugar transport across the intestine, we studied the effects of acute and chronic vanadate administration on the uptake of glucose, galactose, and 3-O-methylglucose in isolated rat intestinal cells. The sugar uptake measurements were also coupled by determinations of rubidium-86 uptake as a measure of the activity of the Na-K pump. Both acute and chronic vanadate administration reduced rubidium uptake by the cells but the reduction did not uniformly influence the uptake of the three sugars in question which were stimulated by the acute exposure of the cells to vanadate. Glucose uptake was also stimulated by chronic vanadate administration, but the uptakes of galactose and 3-O-methylglucose were respectively unaffected or inhibited by chronic vanadate. The findings suggest that the effect of vanadate on sugar transport is dependent on the net difference between two actions of vanadate: (i) stimulation of a receptor site (possibly an insulin receptor site) in the intestinal cell membrane and (ii) inhibition of the Na-K pump. During acute vanadate exposure, the stimulation of the receptor site was very likely a dominant feature which overwhelms the inhibition of the pump. Chronic exposure to vanadate led, on the other hand, to only a limited degree of stimulation of the receptor site and the inhibition of the Na-K pump became evident in the uptake measurements of galactose and 3-O-methyl-glucose. Glucose uptake, however, was stimulated by chronic vanadate ingestion due, very likely, to an increase in the metabolism of this sugar which occurred only with prolonged exposure of the rat intestine to vanadate.

The nonspecific nature of the vanadate inhibition of rat ileal (NA,K)-ATPase.

Vanadate has been suggested as an intracellular regulator of (Na+ + K+)-ATPase. To test this hypothesis we examined the stimulatory and inhibitory effects of vanadate on 86--Rb efflux and influx (measurements of the activity of the Na-pump) in rat ileum under conditions of normal, reduced and increased (Na+ + K+)-ATPase activity. The half maximal stimulation of the Rb efflux and the half maximal inhibition of the Rb influx were not different in the three conditions tested. This suggests that vanadate does not have a regulatory effect on the activity of the Na-K-transport enzyme. The vanadate effect seem rather, to be nonspecific in terms of being unrelated, on a mole per mole basis, to the activity of the (Na+ + K+)-ATPase enzyme.

Effect of vanadate on amino acid transport in rat jejunum.

Vanadate has been reported to inhibit (Na+ + K+)-ATPase of many cells and in some systems to stimulate adenylate cyclase. Since intestinal transport is influenced by these enzymes, we studied the effects of varying concentrations of orthovanadate (VO-4) on alanine transport in the in vitro rat jejunum. At the higher concentrations tested (10(-3) and 10(-2) M) vanadate had a ouabainlike action on alanine transport. It decreased the mucosal-to-serosal flux and the influx of alanine into the intestinal epithelium and it caused a reduction of (Na+ + K+)-ATPase activity of basolateral membranes. The relatively lower vanadate concentration of 10(-4) M increased the influx and the efflux of alanine across the mucosal border of the jejunum. The increase was associated with elevation of cyclic AMP in the intestinal mucosa. The studies suggest the presence of a dual action of vanadate on amino acid transport, a stimulatory effect at low concentration, due to increased adenylate cyclase activity, and an inhibitory effect at higher concentrations, due to a decreased activity of (Na+ + K+)-ATPase.

Effects of vanadate on water and electrolyte transport in rat jejunum.

The effect of vanadate (orthovanadate, VO4-) on water and ion transport was studied in rat jejunum. Water transport was tested by single-pass perfusion in vivo and ion fluxes by the Ussing-chamber technique in vitro. The results suggest that vanadate has two actions on ion and water transport: At low concentrations (10(-4) M) it causes Cl-, Na+ and water secretion by stimulation of adenylate cyclase; At higher concentrations (10(-3) and 10(-2) M) it decreases net absorption of Na+ and Cl- by inhibition of (Na+ + K+)-ATPase.

Intestinal absorption in the mechanically obstructed rat intestine: protection by prostaglandins.

Intestinal obstruction inhibits amino acid absorption. The inhibition, being dependent on the pathological changes of the absorptive epithelium, was considered as an index of injury and measured after varying periods of obstruction and after pretreatment with clindamycin, indomethacin, 16,16-dimethyl-PGE2 or arachidonic acid. A reduction in amino acid uptake was apparent after 2h of obstruction and was increasingly evident after 4, 6 and 18 h. During the late phase (after 6 h), inhibition was partly prevented by pretreatment with clindamycin, but the antibiotic was ineffective during the early phase (within the first 2 h). Bacterial colony counts of luminal contents of rats obstructed for 2 h, were not different from counts obtained in controls, but significantly lower than counts in rats that have been obstructed for 6 h. Pretreatment of rats with 16,16-dimethyl-PGE2 or with arachidonic acid prevented the early inhibitory effects of the obstruction. The findings suggest that the early inhibition in amino acid uptake may be related to metabolic changes that are correctable by the administration of 16,16-dimethyl-PGE2 or of arachidonic acid. The inhibition, during the late phase, is mainly related to an overgrowth of the enteric bacteria.

Effect of ethanol on choline transport in rat jejunum.

The effect of ethanol on choline transport across the rat jejunum was studied by intraluminal perfusion in vivo and by influx measurement across the brush-border membrane in vitro. Acute ethanol administration (4 g/kg) through a gastric tube caused an increase in net choline absorption within 1 h. The increase was prevented by pretreatment with pyrazole, an inhibitor of ethanol metabolism. Chronic ethanol administration also caused an increase in choline absorption, the effect being unrelated to the nutritional changes that occur with ethanol ingestion. In contrast, direct instillation of 0.65 M ethanol through the perfusate caused no changes in choline absorption, and the perfusion of a 1.14 M solution even decreased absorption. The in vitro influx of choline across the mucosal membrane of the isolated rat jejunum was also enhanced by pretreatment with ethanol given by gavage 1 h prior to experimentation. Similarly, the ethanol-related increase in the influx rate was inhibited by pyrazole but was unaffected by acetaldehyde or acetate. Like ethanol, pretreatment of rats with methanol stimulated the choline influx rate. The results suggest that ethanol metabolism, rather than the direct effect of ethanol by itself, stimulates the absorption and influx of choline into the rat jejunum. The effect is not produced by the primary metabolites of ethanol, acetaldehyde, or acetate but is very likely related to stimulation by other products of ethanol metabolism.

The effect of stirring of the luminal solution on the protection of the rat intestinal mucosa during ischemic injury.

Segments of rat ileum made ischemic for 15 min by occlusion of their mesenteric vessels were intraluminally perfused at slow and rapid rates with Krebs buffer containing O2, O2 + glucose or nitrogen. Mucosal leucine influx was measured at the end of the ischemic period. O2 and O2 with glucose provided protection against the ischemic injury, particularly when the stirring of the luminal solution was increased. Rapid perfusion in the absence of oxygen or glucose also protected the intestine probably by rinsing the ischemic gut of noxious agents.

Amino acid accumulation in mouse-human parental hybrid cells.

Cell membrane function during hybridization was assessed by examining the accumulation of four representative amino acids and a sugar in mouse-human hybrid cells, parental mouse cells and human cells. Qualitatively, accumulation in the hybrids was found to resemble the accumulation in parental mouse more than the human cells. In particular, the steady-state uptake of leucine and glycine in hybrids was similar to that in parental mouse cells and different than that observed in the human cells. The findings suggest that hybrids retain transport properties of mouse rather than human cells. These results are in keeping with our previous observation that the human mouse hybrids conserve most of the mouse chromosomes and the membrane proteins of the hybrids are very likely of mouse parental origin.

Leucine absorption after mechanical obstruction of the rat small intestine.

Leucine absorption across the mechanically obstructed rat intestine was studied by in vivo and in vitro techniques. It varied between the early (after 2 h) and late (after 18 h) phases of the obstruction. During the early phase there was a reduction in mucosal uptake and in net absorption of the amino acid. At 18 h the inhibition in mucosal uptake was more prominent both above and below the occlusion site but net absorption was only reduced below the obstruction and was relatively unaffected above it. Mannitol and nonmediated leucine absorption were also increased above the obstruction. The findings during the late phase suggest the presence of an increase in leucine permeability across the intestine above the occlusion site. The observed normal rate of net leucine absorption across this segment is thought to be due to enhancement in intestinal diffusion which could be masking the depression in mucosal uptake.