Acute and chronic acidosis influence on antioxidant equipment and transport proteins of rat jejunal enterocyte.

Acidosis elicits the formation of oxidants and, in turn, ROS (reactive oxygen species)-induced intestinal diseases cause acidosis. This research investigated whether both acute and chronic acidosis influence the antioxidant enzymatic equipment of rat jejunocyte, including γ-GT activity, involved in GSH (glutathione) homoeostasis. Lipid peroxidation level and the expressions of (Na+, K+)-ATPase and GLUT2 were also investigated. The possible influence of acidosis on ROS action was tested. Isolated apical membranes, everted sac preparations and homogenates from acidotic rats were used. γ-GT activity is inhibited after incubation of isolated membranes at acidic pH, but using the whole intestinal tract this inhibition disappears, while SOD (superoxide dismutase) and GR (glutathione reductase) activities are enhanced. Also, in conditions of chronic acidosis, γ-GT activity is unaffected, but no variations of antioxidant activities are apparent. (Na+, K+)-ATPase expression increases, while GLUT2 decreases in acidotic animals. Lipid peroxidation level is unaffected by acidosis. H2O2 inhibits γ-GT activity only in isolated membranes; in the whole tissue, it enhances CAT (catalase) and SOD activities and reduces GLUT2 expression. The pattern of responses to oxidant agents is unaffected by acidosis. Although jejunum seems quite resistant to acidosis, results, suggesting specific responses to this condition, may direct further research on antioxidant supplementation.

Endogenous lactate transport in Xenopus laevis oocyte: dependence on cytoskeleton and regulation by protein kinases.

Carbon flux in Xenopus laevis oocyte is glycogenic and an endogenous monocarboxylate transporter is responsible for intracellular lactate uptake. The aim of the present study was to determine if direct activation of protein kinases C and A modulates the activity of lactate transporter, as well as to investigate the possible role of cytoskeleton in these regulatory phenomena. The modulation was studied in isolated Xenopus oocytes of stage V-VI by measuring (14)C-lactate uptake, both in the absence and in the presence of cytoskeletal-perturbing toxins. We found that the basal lactate transporter activity depends on the integrity of the cytoskeleton since it is partially inhibited by cytoskeleton disorganisation. Both PKA and PKC activation caused a significant decrease in transport activity and this decrease could be blocked by specific protein kinase inhibitors. The evidenced effects were not additive. Transport inhibition was annulled by agents that destabilize actin filaments or microtubules. We conclude that both protein kinases A and C, whose effects are mediated by cytoskeleton, negatively regulate the endogenous lactate transporter of Xenopus oocyte, suggesting that these kinases may have a role in the control of cytosolic pyruvate/lactate pool in the oocyte.

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions.

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.

Oxidative stress reduces transintestinal transports and (Na+, K+) -ATPase activity in rat jejunum.

Because oxidative stress is a component of gastrointestinal injury, we investigated the effect of H(2)O(2) on transintestinal transport using isolated rat jejunum incubated in vitro. Millimolar concentrations of H(2)O(2) inhibited all the tested parameters without inducing any cytotoxic effect. Electrophysiological experiments indicated that H(2)O(2) decreases significantly both short circuit current and transepithelial electrical potential difference without affecting transepithelial resistance. The possibility that H(2)O(2) could influence (Na+, K+) -ATPase activity was explored using isolated basolateral membranes. Besides H(2)O(2), free radicals (O(2)(*-), HO*) were generated using different iron-dependent and independent systems; (Na+, K+) -ATPase activity was inhibited after membrane exposure to all ROS tested. The inhibition was prevented by allopurinol, superoxide dismutase or desferrioxamine. Western blot analysis showed a decreased expression of the alpha(1)-subunit of (Na+, K+) -ATPase. We conclude that H(2)O(2) may be a modulator of jejunal ion and water transport by multiple mechanisms, among which a significant inhibition of the basolateral (Na+, K+) -ATPase.

PKA regulation of bicarbonate and lactate movements across rat jejunal plasma membranes.

BACKGROUND/AIMS: Evidence was previously given that the mechanisms involved in bicarbonate and lactate movements across rat jejunal enterocyte are modulated by PKC and Ca2+/CaM. Aim of this study was to investigate the possible role of PKA on bicarbonate and lactate transports. METHODS: Enzymatic assays in isolated plasma membranes were performed. Moreover membrane vesicles, transiently opened and resealed, were treated with a phosphorylating solution (leading to PKA activation) and were used after that to perform uptake studies. RESULTS: Enzymatic assays give evidence for the presence of PKA in plasma membranes from rat jejunum. Uptake experiments suggest that PKA stimulates the two systems that accomplish basolateral HCO3- efflux from the enterocyte, namely Cl-/ HCO3- exchanger and HCO3- conductance, without affecting HCO3- influx from the lumen mediated by Na+/H+ exchanger activity. Moreover basolateral H+-lactate symporter is stimulated by PKA, as well as the brush border isoform of Na+-glucose cotransporter SGLT1. CONCLUSION: PKA activation evokes individual responses that could be coordinated through cellular metabolism.

Protein kinase C regulation of rat jejunal transport system: mechanisms involved in lactate movement.

We examined whether protein kinase C (PKC) modulates the transport systems involved in lactate movements across the plasma membranes of rat jejunum. In vitro phosphorylated membrane vesicles were used to perform uptake studies, the results of which suggested that PKC activation exerts an inhibitory effect on basolateral H+-lactate symport, as well as on apical N-+glucose cotransport. The specificity of the response to PKC was confirmed by using staurosporine, chelerythrine or 4-alpha-PMA. Experiments performed using the whole tissue incubated in vitro confirmed the reduction of lactate transport elicited by PKC and gave evidence for an associated inhibition of fluid transport. Na+K+-ATPase activity seems to be unaffected by the kinase and inhibited by Ca2+. Taken together, our results suggest that the overall action of PKC results from the simultaneous modulation of multiple pathways, targeted to a reduction of both lactate and bicarbonate transports without altering cell pH homeostasis.

Calmodulin-mediated regulation of bicarbonate and lactate transports in rat jejunum.

BACKGROUND/AIMS: Ca(2+)/CaM is known to modulate the activity of several transport systems and its regulation can be accomplished either directly or via the involvement of specific protein kinases. Aim of this study was to investigate the possible role of Ca(2+)/CaM on bicarbonate and lactate transports in rat jejunal enterocyte. METHODS: Enzymatic assays in isolated plasma membranes were performed. Moreover membrane vesicles, transiently opened and resealed, were loaded with Ca(2+) and calmodulin, both in the absence and in the presence of ATP, and were used after that to perform uptake studies. RESULTS: Enzymatic assays gave evidence for the presence of Ca(2+)/CaM-dependent protein kinase II (CaMKII) in plasma membranes from rat jejunum. However, uptake experiments suggest that Ca(2+)/CaM, and not CaMKII, inhibits both basolateral Cl(-)/HCO(3)(-) exchange and H(+)-lactate symport, whilst HCO(3)(-) and Cl(-) conductances are unaffected. Neither Ca(2+)/CaM nor CaMKII seem to regulate brush border Na(+)/H(+) exchanger activity. CONCLUSION: These data are consistent with a Ca(2+)/CaM-mediated reduction of bicarbonate and lactate exit from jejunal enterocyte.

Protein kinase C regulation of rat jejunal transport systems: mechanisms involved in bicarbonate absorption.

We examined whether protein kinase C (PKC) modulates the transport systems involved in bicarbonate movements across the plasma membranes of rat jejunum. Results of enzymatic assays provide evidence that under basal conditions conventional PKC (cPKC) is present in both basolateral membranes (BLMs) and apical (brush border) membranes (BBMs) of the enterocyte. In BLMs the basal expression of the kinase is low compared to expression in BBMs; however, treatment with Ca(2+) and phorbol 12-myristate 13-acetate (PMA) causes a significant increase, thus suggesting an asymmetrical kinase translocation. To explore the effect of PKC activation on membrane-bound transport mechanisms, 'in vitro' phosphorylated membrane vesicles were used to perform uptake studies. Results suggest that PKC activation exerts an inhibitory effect on the basolateral Cl(-)-HCO(3)(-) antiporter, whereas the basolateral HCO(3)(-) conductive pathway seems to be stimulated and Cl(-) conductance unaffected. The apical, but not basolateral, Na(+)-H(+) exchanger is inhibited by PKC activation. The specificity of the response to PKC was confirmed by using the kinase inhibitor staurosporine or the inactive phorbol ester 4-alpha-PMA. The inhibition of both apical Na(+)-H(+) and basolateral Cl(-)-HCO(3)(-) exchange activities suggests that the overall action of PKC causes a reduction of transepithelial bicarbonate transport.

PYY-Tag transgenic mice displaying abnormal (H+-K+)ATPase activity and gastric mucosal barrier impairment.

The mechanism by which the gastrointestinal hormones peptide YY and glucagon inhibit gastric acid secretion is largely unknown. PYY-Tag transgenic mice develop endocrine tumors in the colon that are composed mainly of peptide YY/enteroglucagon-producing L type cells. Therefore we studied the functional activity of such tumors and the gastric functions of PYY-Tag mice. Fasting and fed PYY-Tag transgenic mice and CD1 controls were assayed for circulating levels of peptide YY, glucagon, insulin, and gastrin. The gastric pH was determined and gastric samples were examined for (a) histologic appearance; (b) K(+)-stimulated p-nitrophenylphosphatase activity and [(14)C]aminopyrine accumulation of apical and tubulovesicle membranes; (c) adherent mucus determination by Alcian blue recovery; and (d) DNA/RNA/protein epithelial content and in vivo incorporation of [(3)H]thymidine into DNA. Transgenic mice showed high serum levels of peptide YY and glucagon, increased gastric pH, and a high incidence of gastric ulcers after fasting. p-Nitrophenylphosphatase activity, [(14)C] aminopyrine accumulation, and proton pump redistribution from cytoplasmic tubulovesicles to apical membranes were significantly lower in the gastric mucosa of transgenic mice compared with the controls. In addition, the adherent mucus was thinner, and [(3)H]thymidine incorporation into the DNA was decreased. The abnormal and unregulated levels of circulating peptide YY and glucagon led to gastric acid inhibition and an impairment of gastric barrier function as a result of a striking reduction in epithelial proliferation.