Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase.

Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca(2+) responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-gamma, regulates Ca(2+) responses and the Ca(2+)-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3-kinase involvement in Ca(2+) responses induced in these cells by CCK and carbachol. The PI3-kinase inhibitors inhibited both Ca(2+) influx and mobilization from intracellular stores induced by stimulation of acini with physiological and pathological concentrations of CCK, as well as with carbachol. PI3-kinase inhibition facilitated the decay of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) oscillations observed in individual acinar cells. The PI3-kinase inhibitors decreased neither CCK-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] production nor Ins(1,4,5)P(3)-induced Ca(2+) mobilization, suggesting that the effect of PI3-kinase inhibition is not through Ins(1,4,5)P(3) or Ins(1,4,5)P(3) receptors. PI3-kinase inhibition did not affect Ca(2+) mobilization induced by thapsigargin, a specific inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacological and genetic PI3-kinase inhibition on [Ca(2+)](i) signals, suggesting SERCA as a downstream target of PI3-kinase. Both pharmacological PI3-kinase inhibition and genetic deletion of PI3-kinase-gamma increased the amount of Ca(2+) in intracellular stores during CCK stimulation. Finally, addition of the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate to permeabilized acini significantly attenuated Ca(2+) reloading into the endoplasmic reticulum. The results indicate that PI3-kinase regulates Ca(2+) signaling in pancreatic acinar cells through its inhibitory effect on SERCA.

Epidermal growth factor increases surface hydrophobicity and resistance to acid in the rat duodenum.

Epidermal growth factor (EGF) is produced in Brunner's glands and plays a role in healing and repair of duodenal ulcers. We examined the participation of zwitterionic phospholipids of mucus in the effects of EGF. Under anesthesia, groups of rats received an intraduodenal bolus of either saline or EGF. Some rats received subcutaneous indomethacin followed by EGF or EGF followed by a detergent (5% Brij 35, a nonionic detergent that solubilizes luminal phospholipids). Thirty minutes after treatment, mucosal surface hydrophobicity and phospholipid concentration in the mucus layer were measured. Matched groups of rats were challenged with 0.5 M HCl, instilled intraduodenally 30 min after treatment, and mucosal damage was assessed 1 h after acid challenge. Exogenous EGF significantly increased surface hydrophobicity and phosphatidylcholine concentration in the mucus layer. EGF treatment also reduced mucosal damage induced by acid. However, indomethacin pretreatment or detergent administration after EGF abolished both protection against acid and changes in the mucus layer. These data suggest that EGF increases duodenal resistance to luminal acid via stimulation of mucosal zwitterionic phospholipids.

Surface hydrophobicity of the rat colonic mucosa is a defensive barrier against macromolecules and toxins.

BACKGROUND: Mucosal surface hydrophobicity is a key factor of the gastric acid defence barrier. In the colon, surface hydrophobicity is high but its biological function remains unexplored. AIMS: To investigate the functional changes of the barrier due to removal of the surface active phospholipid layer by a detergent, or to reinforcement of the surface active phospholipid by local application of a suspension of lipids. METHODS: Surface hydrophobicity (contact angle measurement), colonic permeability (lumen to blood clearance of mannitol and dextran), and mucosal resistance against luminal aggression (distal colitis induced by dextran sodium sulphate, DSS) were investigated in three study groups: (a) rats pretreated with a detergent (Brij 35) known to remove surfactant lipids; (b) rats pretreated with a suspension of surface active lipids (tripalmitin and dipalmitoyl-phosphatidylcholine); and (c) control rats pretreated with the corresponding vehicles. RESULTS: In controls, surface hydrophobicity was low on the caecal mucosa and high in colon and rectum. Detergent treatment reduced surface hydrophobicity, and increased colonic permeability to mannitol and dextran. Conversely, treatment with lipids increased surface hydrophobicity, and reduced colonic permeability. Administration of DSS induced a progressive loss of colonic surface hydrophobicity, and an increase in permeability to mannitol and dextran. Detergent treatment increased susceptibility to epithelial damage and mucosal inflammation by DSS. Treatment with lipids reduced susceptibility to DSS colitis. CONCLUSION: Colonic surface hydrophobicity modulates permeability to hydrophilic molecules and protects against toxins.

Deranged hydrophobic barrier of the rat gastroduodenal mucosa after parenteral nonsteroidal anti-inflammatory drugs.

BACKGROUND & AIMS: Parenteral administration of nonsteroidal anti-inflammatory drugs (NSAIDs) may cause gastrointestinal mucosal lesions. The aim of this study was to investigate whether parenteral NSAIDs alter surface hydrophobicity of the gastroduodenal mucosa. METHODS: Conscious rats received indomethacin or diclofenac subcutaneously at different doses (0.5-10 mg/kg). Surface hydrophobicity of gastric and duodenal mucosa was determined by contact angle measurement at various time points; mucosal prostaglandin synthesis and mucus phospholipid content were measured. Also, the effects of NSAIDs were studied in bile duct-ligated rats. RESULTS: A single 1-2-mg/kg dose significantly decreased hydrophobicity in the stomach and duodenum. The decrease was associated with a reduction in mucus phosphatidylcholine. In the duodenum, mucosal prostaglandin synthesis was restored 24 hours after NSAID dosing, but hydrophobicity was still decreased. There was no adaptation to long-term treatment. In bile duct-ligated rats, NSAIDs did not decrease gastric or duodenal hydrophobicity. Moreover, oral administration of bile from rats pretreated with parenteral NSAIDs significantly decreased mucosal hydrophobicity in untreated rats. CONCLUSIONS: Low-dose NSAIDs by parenteral route impair the physicochemical barrier against luminal acidity and render the mucosa susceptible to injury. Excretion of NSAIDs in bile seems to play a key role in this effect.

Effect of zinc on D-galactose and L-phenylalanine uptake in rat intestine in vitro.

The effect of zinc on galactose and phenylalanine uptake was studied using rat everted jejunal rings. The rings were incubated for 2 min in oxygenated Krebs-Ringer-Tris (KRT) solution. Galactose and phenylalanine uptake was reduced by zinc in a dose-dependent manner, but not in a time-dependent way. One mM Zn2+ but not 0.5 mM Zn2+ inhibited galactose transport without modifying sugar diffusion. Na(+)-dependent phenylalanine transport was reduced by 0.5 mM and 1 mM Zn2+. However, the metal did not change phenylalanine diffusion obtained in the presence of 40 mM L-methionine or Na(2+)-independent phenylalanine transport. Therefore, zinc seems to interact only with the sodium-galactose or sodium-phenylalanine cotransporters. Zinc inhibited sugar and amino acid transport in a non-competitive way, without a significant change in the affinity of the transporters for their substrates and with a Vmax decrease. The inhibitory effect of Zn2+ on galactose and phenylalanine uptake was reversed by washing intestinal rings for 5 min with KRT solution. These results suggest that zinc might exert its inhibitory action by a weak binding to chemical groups related with sodium-substrate cotransporters and located in the luminal membrane of the enterocytes.

Phosphatidylcholines as mediators of adaptive cytoprotection of the rat duodenum.

BACKGROUND/AIMS: Surfactant phospholipids impede diffusion of acid through the gastric mucus, but their relevance in the defense of the duodenum against luminal acid is not known. METHODS: Duodenal resistance to acid was tested in anesthetized rats by instillation of HCl using a tube implanted in the proximal duodenum. The effects of a detergent (Brij 35; Sigma, St. Louis, MO) and a lipid mixture flushed through the luminal surface on duodenal resistance to acid were studied. The lipid content in the mucus and the effects of acid, prostaglandins, and indomethacin on the lipid layer were also analyzed. RESULTS: Instillation of 100 mumol HCl or 5 micrograms/kg 16,16-dimethyl prostaglandin E2 increased resistance to acid, preventing duodenal lesions induced by 500 mumol HCl. However, 100 mumol HCl or 16,16-dimethyl prostaglandin E2 did not prevent lesions induced by 500 mumol HCl in rats undergoing perfusions with 5% Brij 35. Indomethacin suppressed acid-induced protection. A mixture of tripalmitin and dipalmitoyl-phosphatidylcholine protected against 500 mumol HCl, and the effect was also observed in rats receiving indomethacin. Finally, 100 mumol HCl increased the phosphatidylcholine content in the duodenal mucus but not in rats receiving 5% Brij 35 or indomethacin. CONCLUSIONS: Surface-active phospholipids are critical for adaptive cytoprotection to acid in the rat duodenum.

Inhibition of D-galactose and L-phenylalanine transport by HgCl2 in rat intestine in vitro.

The effect of Hg2+ on galactose and phenylalanine uptake has been studied in rat everted intestinal rings incubated for 2 minutes. The presence of 0.5 mM Hg2+ in the incubation medium inhibited the total galactose uptake from 30% to 40% and that of the phenylalanine about 70%. The inhibition was due to a reduction of galactose transport and Na(+)-dependent phenylalanine transport. Hg2+ inhibited the galactose transport in a non-competitive way, with a Vmax diminution without Km modification. The Na(+)-dependent phenylalanine transport was totally blocked in the presence of 1 mM Hg2+. The washing of the intestinal rings with 5 mM EDTA slightly decreased the inhibition produced by 0.5 mM Hg2+ on phenylalanine uptake whereas it did not modify the inhibition of galactose uptake. However, the inhibition of galactose uptake was completely reversed after washing with 10 mM cysteine. Therefore, phenylalanine transport seems to be more sensitive to HgCl2 than galactose transport. The inhibition of these intestinal transport systems by Hg2+ might be due to its interaction with ligands of the transport proteins located in the luminal membrane of enterocytes.

Influence of dietary fat on duodenal resistance to acid.

Three experimental diets were prepared from a standard formula plus pure oleic, linoleic, or eicosapentaenoic acid (2% by weight). Mucosal resistance to acid was tested in anaesthetised rats fed the experimental diets for at least four weeks (60 rats per diet) by duodenal infusion of HCl (200 to 700 mumol) 30 minutes after pretreatment with either saline or 100 mumol HCl (used as a mild irritant). Rats were killed one hour after the test and the duodenal damage was assessed 'blindly' using a combined macroscopic and histological score. Differences were tested by analysis of covariance of the dose-response curves. Mucosal resistance was similar in the three groups when the acid challenge was given after saline pretreatment. Resistance to acid in all three groups was significantly increased by previous exposure to 100 mumol HCl (p < 0.01). Interestingly, rats fed a linoleic or eicosapentaenoic supplemented diet after pretreatment with HCl developed significantly higher resistance to acid than those fed the diet with oleic acid (p < 0.05). Pretreatment with indomethacin suppressed the difference between diets. In conclusion, dietary polyunsaturated fatty acids enhance duodenal resistance to acid by potentiation of adaptive cytoprotection.

Adaptive cytoprotection of the rat duodenum is not dependent on nitric oxide-induced changes in blood flow.

Mild irritation of the rat duodenum enhances mucosal resistance to acid via a prostaglandin-mediated mechanism. Prostaglandins increase mucosal blood flow, but it is not known whether such changes in blood flow significantly contribute to adaptive cytoprotection. We induced blood flow changes by manipulating the arginine-nitric oxide pathway, which is independent from prostaglandin synthesis, and determined the resulting effects on the cytoprotective response. In anesthetized rats, we tested the effects of intraduodenal infusion of mild acid on duodenal blood flow and on the prevention of mucosal damage by subsequent strong acid in control, indomethacin-pretreated, and NG-nitro-L-arginine-pretreated rats (NG-nitro-L-arginine inhibits the nitric oxide synthase). Additional experiments tested the effects of the prostaglandin analogue 16,16-dimethyl-prostaglandin (PG) E2 or sodium nitroprusside (nitric oxide donor) on duodenal blood flow and on mucosal protection against acid. Exposure of the duodenal mucosa to mild acid increased duodenal blood flow and mucosal resistance to acid. Instillation of 16,16-dimethyl-PGE2 also increased blood flow and mucosal resistance to acid. However, sodium nitroprusside increased blood flow without increasing mucosal resistance to acid, and NG-nitro-L-arginine inhibited the change in blood flow induced by acid while preserving the adaptive cytoprotection phenomenon intact. In conclusion, adaptive cytoprotection of the duodenal mucosa appears to be independent of changes in blood flow.

Duodenal mucosal resistance to intraluminal acid in the rat: role of adaptive cytoprotection.

The duodenal mucosa is normally challenged by intermittent exposure to acid because of periodic gastric emptying. We studied the mechanisms of duodenal adaptation to acid in anesthetized rats. A polyvinyl chloride tube passing through a ligated pylorus was used for duodenal pulse instillations of 1 mL of saline or acid (100 or 400 mumols HCl) at 30-minute intervals. Duodenal lesions were blindly assessed using a combined macroscopic and histological score. Mucosal damage after exposure to saline or 100 mumols HCl was negligible in intact, vagotomized, and indomethacin-pretreated rats, whereas 400 mumols induced noticeable macroscopic and microscopic lesions. Interestingly, in intact and vagotomized rats, previous exposure to a 100-mumols HCl bolus significantly prevented mucosal damage by a subsequent 400-mumols bolus. This effect was not observed in indomethacin-pretreated rats. In these rats, however, intraduodenal instillation of exogenous 16,16-dimethyl prostaglandin E2 (16,16-dm-PGE2) prevented the damage induced by 400 mumols of HCl. A second protocol investigated the luminal release of bicarbonate and PGE2 in response to intraduodenal perfusion with 100 mumols of HCl. Duodenal bicarbonate release was stimulated by acid in all groups, whereas the release of PGE2 increased in intact and vagotomized rats but not in the indomethacin-pretreated group. In summary, these data suggest that adaptive cytoprotection plays a significant role in protecting the duodenal mucosa from acid. Vagal innervation and bicarbonate release do not appear to be as critical as cyclo-oxygenase activity for this mechanism.

Inhibition of sugar and amino acid transport across rat jejunum by cadmium, copper and mercury.

Cd2+, Cu2+ and Hg2+ (0.5 mM) inhibit the absorption of D-galactose (1 mM) across rat jejunum in vivo. The inhibition increases by 3-5 min previous exposure of the mucosa to the heavy metals, and is appreciably reversed by washing the intestinal lumen with 10 mM EDTA, and in higher proportion with 10 mM dithioerythritol. The metals do not affect the passive absorption of galactose (sugar transport blocked by 0.5 mM phlorizin), or that of L-sorbose. In vitro accumulation of D-galactose by jejunum rings is also inhibited by Cd and Cu. This inhibition is reversed by washing more readily than in vivo. The metals do not affect sugar entry into the tissues in the presence of 0.5 mM phlorizin. The inhibition of the transport component follows a non-competitive kinetics. Cd and Cu inhibit also the L-phenylalanine accumulation by the jejunum rings, but they do not modify the passive entry (in the presence of 40 mM methionine) or that in the absence of Na+. Heavy metals appear to inhibit sugar and amino acid intestinal absorption by their binding to proteins (prevailing to thiol groups) of the luminal membrane of enterocytes, which pertain or are functionally related to the corresponding Na(+)-substrate cotransport system.

Inhibition by cadmium of D-galactose and L-phenylalanine transport by rat intestine in vitro.

The effect of cadmium (CdCl2) on galactose and phenylalanine uptake by rat everted intestinal rings has been studied. The rings were preincubated (15 min) and incubated (5 min) in the presence of Cd. Galactose uptake (from 0.5 mM to 10 mM) was inhibited by 0.5 mM Cd about 25%. Only the phlorizin-dependent galactose transport was affected by cadmium, being a non-competitive type inhibition. A 15 min washing with saline solution significantly reduced the cadmium induced inhibition, which was practically reversed by washing with 5 mM EDTA. The uptake of 0.5 mM phenylalanine was not affected by 0.5 mM Cd but it was depressed by 1 mM Cd. Such inhibition was exerted on the sodium-dependent phenylaline transport. Washing with 5 mM EDTA diminished only slightly the inhibition of the transport by cadmium. It is suggested that the inhibition of intestinal transport of galactose and phenylalanine by cadmium may be due to its reversible interaction with metal-binding ligands, possibly sulfhydryl groups, related to the luminal transport systems.