Effects of creatine in a rat intestinal model of ischemia/reperfusion injury.

PURPOSE: Creatine belongs to a buffering system of cellular ATP level and has been reported to display direct antioxidant activity. Aim of this work was to investigate whether creatine treatment could ameliorate the antioxidant response of intestinal cells and limit the oxidative injury induced by anoxia and subsequent reoxygenation. METHODS: Jejunal and ileal tracts of rat intestine were everted and incubated in vitro under normoxic, anoxic and reoxygenation conditions in the absence and in the presence of 10 mM creatine. (Na+, K+)-ATPase, γ-GT and antioxidant enzymes activities were determined in mucosal homogenate, as well as malondialdehyde production and HSP70 expression. RESULTS: Both in jejunum and ileum, creatine treatment increases (Na+, K+)-ATPase activity; γ-GT is unaffected in jejunum but stimulated in ileum. In both tissues, creatine does not alter the antioxidant activities or malondialdehyde level. HSP70 expression is increased only in jejunum. Anoxic conditions stimulate antioxidant activities to a greater extent in jejunum compared to ileum; reoxygenation does not evoke further effects, but enhances malondialdehyde production in both tracts. The protective action of creatine, in reoxygenation, is more marked in jejunum as for its stimulation of antioxidant activities; however, in jejunum, a prooxidant action of creatine is suggested, since malondialdehyde production is enhanced by its presence; on the contrary in ileum, where HSP70 is overexpressed in reoxygenation, peroxidation level is significantly reduced. CONCLUSIONS: The presence of creatine seems to potentiate the defensive response of both tissues, in jejunum by means of cell antioxidant equipment, in ileum by the involvement of HSP70.

Role of EGFR family receptors in proliferation of squamous carcinoma cells induced by wound healing fluids of head and neck cancer patients.

BACKGROUND: Mounting evidence suggests that recurrence of resected head and neck squamous cell carcinomas (HNSCCs) is due to the outgrowth of unrecognized residual tumor cells as well as to the premalignant and/or precursor-field epithelial cells. We studied the impact of processes triggered by HNSCC surgery in stimulating both residual tumor cells [demonstrated to overexpress epidermal growth factor receptor (EGFR)], and premalignant cells surrounding the resected lesion. PATIENTS AND METHODS: EGFR expression/activation by immunohistochemistry/biochemistry and gene status by FISH were investigated in 23 primary HNSCCs and surrounding tissues. The ability to induce cell proliferation of wound healing drainages collected from 12 relapsed and 11 not relapsed patients was evaluated by a colorimetric assay in squamous cell carcinoma cell lines A431 (carrying EGFR amplification) and CAL27 (carrying three EGFR copies) in the presence/absence of EGFR therapeutic inhibitors. RESULTS: Primary tumors showed intermediate/high EGFR expression (91%), EGFR phosphorylation and EGFR-positive FISH (35%). Normal, metaplastic and dysplastic epithelium surrounding the resected tumor displayed EGFR overexpression. EGFR activation and gene amplification were observed in normal and dysplastic epithelium, respectively. Each tested wound healing drainage induced the cells to proliferate and the proliferation was significantly higher in relapsed compared with not relapsed HNSCC patients (P = 0.02 and P = 0.03). Anti-EGFR treatments inhibited the drainage-induced proliferation, with the highest inhibitory efficiency by cetuximab on A431 cells, while CAL27 cell growth was more efficiently inhibited by tyrosine kinase inhibitors. CONCLUSIONS: Surgery could favor the proliferation of cells showing EGFR overexpression/activation/amplification such as residual tumor cells and/or precursor-field epithelial cells already present after surgery. Treatment with anti-EGFR reagents inhibits wound-induced stimulation, according to the EGFR family status.

PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients.

BACKGROUND: It has been reported that KRAS mutations (and to a lesser extent KRAS mutations with the BRAF V600E mutation) negatively affect response to anti-epidermal growth factor receptor (EGFR) mAbs in metastatic colorectal cancer (mCRC) patients, while the biological impact of the EGFR pathway represented by PI3K/PTEN/AKT on anti-EGFR treatment is still not clear. PATIENTS AND METHODS: We analysed formalin-fixed samples from a cohort of 32 mCRC patients treated with cetuximab by means of EGFR immunohistochemistry, EGFR and PTEN FISH analysis, and KRAS, BRAF, PI3KCA, and PTEN genomic sequencing. RESULTS: Ten (31%) of 32 patients showed a partial response to cetuximab and 22 (69%) did not [nonresponder (NR)]. EGFR immunophenotype and FISH-based gene status did not predict an anti-EGFR mAb response, whereas KRAS mutations (24%) and PI3K pathway activation, by means of PI3KCA mutations (13%) or PTEN mutation (10%)/loss (13%), were significantly restricted to, respectively, 41% and 37% of NRs. CONCLUSION: These findings suggested that KRAS mutations and PI3KCA/PTEN deregulation significantly correlate with resistance to cetuximab. In line with this, patients carrying KRAS mutations or with activated PI3K profiles can benefit from targeted treatments only by switching off molecules belonging to the downstream signalling of activated EGFR, such as mammalian target of rapamycin.

Basolateral Cl-/HCO3- exchange in rat jejunum: evidence from H14CO3- uptake in membrane vesicles.

Bicarbonate transport across basolateral membrane vesicles from rat jejunal enterocyte was studied at 28 degrees C and pH 8.2. These experimental conditions make possible the determination of [14C]bicarbonate uptake. Inward gradients of Na+, K+, and Li+ did not stimulate HCO3- uptake, suggesting that a cotransport mechanism with these cations does not occur. On the contrary a countertransport of bicarbonate driven by a Cl- gradient was evidenced. The ability of other inorganic anions to exchange with HCO3- was examined and results indicate that Cl- can be substituted by NO3-, Br- and SCN-. The Cl(-)-dependent HCO3- uptake was strongly inhibited by SITS and DIDS, whereas acetazolamide was ineffective: thus transfer of labelled CO2 is eliminated as a possible mode of HCO3- permeation. HCO3- uptake was also affected by the presence of superimposed membrane potentials, suggesting that a HCO3- conductive pathway is present in the jejunal basolateral membrane. These results show that there are no fundamental differences between data obtained performing H14CO3- and 36Cl- (previously reported) uptake experiments.

Characterization of basolateral membrane Na/H antiport in rat jejunum.

Na uptake studies were performed in order to examine the activity of a Na/H exchanger in basolateral membrane vesicles isolated from rat jejunum. Experiments were carried out under voltage-clamped conditions in order to avoid electrodiffusional ionic movements. 1 mM Na uptake was found to be enhanced by an outward proton gradient and its initial rate was further increased by the presence of monensin or nigericin. The pH gradient-driven Na uptake was inhibited by 2 mM amiloride and unaffected by 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The initial rate of the proton gradient-induced Na uptake was saturable with respect to external Na, with a Km of 13.6 +/- 1.4 mM and a Vmax of 35.4 +/- 2.2 nmol/mg protein per min. Li competed with Na for the exchange process, whereas K, Rb, Cs, tetramethylammonium had no effect. We conclude that rat jejunal basolateral membrane contains a Na/H exchanger whose properties are similar to those of the antiporter identified in the brush-border membrane.

Na+/H+ exchange mechanism in the basolateral membrane of the rat enterocyte.

Basolateral membrane vesicles from rat jejunal enterocytes, especially purified of brush-border contamination, were used for Na+ uptake. The basolateral membrane vesicles are osmotically active and under our experimental conditions Na+ binding is much lower than transport. An outwardly directed proton gradient stimulates Na+ uptake at both 5 microM and 5 mM concentrations. The proton gradient effect can be inhibited completely by 2 mM amiloride and partially by either FCCP or NH4Cl (NH3 diffusion). Membrane potential effects can be excluded by having valinomycin plus K+ on both sides of the vesicles. These results suggest that there is an Na+/H+ exchanger in the basolateral membrane of rat enterocytes.

pH dependence of Cl/HCO3 exchanger in the rat jejunal enterocyte.

During bicarbonate absorption in rat jejunum, a Cl/HCO3 exchanger mediates bicarbonate extrusion across the basolateral membrane of the enterocyte. Previous studies demonstrated that anion antiport exhibits a particular behaviour: its activity is positively affected by the presence of sodium, but the cation is not translocated by the carrier protein. In view of the particular features of the jejunal Cl/HCO3 antiporter, first we performed a pharmacological characterisation of the transport protein using various Cl channels blockers. Then, since it is well known that anion exchangers play a substantial role in cell pH regulation, we investigated the possible involvement of jejunal basolateral Cl/HCO3 antiporter in intracellular pH maintenance. The sensitivity of the exchanger to pH was investigated by measuring 36Cl uptake into basolateral membrane vesicles either varying simultaneously intra- and extravesicular pH, or presetting at 7.4 external pH and varying only the internal one. Experiments were performed both in the absence and in the presence of Na. In all the tested conditions, uptake peaked at pH of about 7. 3-7.4 and then decreased, suggesting that the main function of Cl/HCO3 exchanger is related to HCO3 absorption rather than to intracellular pH control. Since pH-regulating mechanisms counteracting acidification are well known in the jejunal enterocyte, we investigated how it regulates pH after alkalinisation of the cytosol. We tested both basolateral and brush border membrane vesicles for the presence of a K/H exchanger, but we could not give evidence for its presence by means of 86Rb uptake experiments. In conclusion, the jejunal enterocyte seems to lack a mechanism counteracting cellular alkalinisation: the main purpose of pH homeostasis might be to hinder acidification of the cytosol due to influx of protons and production of acid by the metabolism.

In vivo and in vitro sugar transport in frog intestine.

In the frog intestine, both in vitro and in vivo, experiments were carried out in order to increase knowledge of the mechanism of sugar exit across the basolateral membrane of the enterocyte. The frog intestine was chosen because it lacks crypt cells and, consequently, any external fluid circuit mechanism during sugar transport can be avoided. Therefore, the sugar concentration in the absorbate collected on the serosal side is likely to be similar to that present underneath the basolateral membrane of the enterocyte. Under this condition, cell and absorbate sugar concentrations are similar; yet there is a concomitant net transintestinal sugar transport. Moreover, in in vivo experiments a net transintestinal sugar transport takes place even against a concentration difference. These results suggest that sugar exit across the basolateral membrane is not simply due to a chemically facilitated diffusion.

D-glucose transport systems in rat jejunal brush border membrane: influence of ageing.

Jejunal brush border membranes were isolated from rats of different ages (very young, young, adult and old); the gamma-GT specific activity and the vesicle volumes were unaffected by ageing, whilst protein content was significantly reduced in brush border from old rats. Vesicles were used to investigate the kinetics of Na-glucose cotransport under voltage-clamped and zero-trans conditions over a wide range of D-glucose concentrations (0.005-70 mM). Results provide evidence that in all the ages tested D-glucose can cross the brush border membrane both by a passive diffusional component and by two Na-dependent saturable transport systems, namely one with high-affinity and low-capacity and the other with low-affinity and high-capacity. However, in some old rats only one saturable and a very small passive component occur. The two Na-dependent transport systems were analyzed to define the stoichiometry of coupling between Na and glucose fluxes. In all the ages tested the Na:glucose ratio is higher in the high-affinity system than in the low-affinity one. Accordingly the effect of a superimposed membrane potential is more evident for the high-affinity transport mechanism. In conclusion, D-glucose transport systems seem to be unaffected by ageing from very young to adult rats; only in old animals age-related alterations can be observed.

Aging and ATPase activities in rat jejunum.

In addition to the well-known (Na,K)-ATPase activity, an ouabain-insensitive Na-ATPase has been evidenced in the basolateral membrane of intestinal and renal cells from different mammals. Basolateral membranes of jejunal enterocytes from rats of different ages, i.e., very young, young, adult and old were separated by self-orienting, Percoll-gradient centrifugation. The total protein content and both Na- and (Na,K)-ATPase activities in initial homogenate and final pellets were analyzed. The dry weight of homogenate and pellet was also determined. The two ATPase activities and the protein content of the basolateral membrane fraction decrease with age when referred to the dry weight of the pellet. This diminution is also evident in the initial homogenate. The activation curve of Na-ATPase, hyperbolic in shape, gives Km and Vmax values unaffected by aging. The same behaviour is true for the kinetic parameters of (Na,K)-ATPase, which has a sigmoidal velocity curve. From these results, it seems that both Na- and (Na,K)-ATPase have the same characteristics in the basolateral membrane of the enterocyte throughout the life span of the animal, but they decrease quantitatively with aging.

Proton-lactate cotransport in basolateral membrane vesicles from rat jejunum.

Proton-coupled lactate transport across the basolateral membrane of rat jejunal enterocyte was studied using well purified membrane vesicles. L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP and by pCMBS; unlabelled L-lactate causes a strong inhibition, whilst furosemide is uneffective. The H+ gradient-dependent stimulation of L-lactate uptake is significantly inhibited also by SCN-: this finding could explain results recently reported in the literature in which H(+)-lactate symport was not evidenced in basolateral membranes from rat jejunum.

A method for the in situ measurement of the electrical quantities in frog skin.

A method allowing the measurement of the electrical quantities related to the physiological functions of the frog skin in situ is presented. The method allows the performance of several experiments on the same pithed animal, which remains alive for a number of days. The preparation is very stable, and the electrical potential difference and short-circuit current values are higher than in isolated skin. The theory of measurement and the possible systematic errors are discussed. The possibilities of the method are evaluated on comparing the pH and temperature dependence of the electrical quantities in situ with previous measurements on isolated skin.

Cetuximab in recurrent and/or metastatic salivary gland carcinomas: A phase II study.

EGFR overexpression in salivary gland carcinomas provides the rational for the investigation of anti-EGFR treatments in recurrent and/or metastatic salivary gland cancers (RMSGCs). The activity of cetuximab in terms of clinical benefit rate (CBR) defined as the occurrence of objective response (CR or PR) or stable disease (SD) for >or=6months was investigated. From April to December 2005, 30 patients [23 adenoid cystic carcinoma (ACC) and 7 non-ACC] were treated with cetuximab at 400mg/m(2)/week followed by 250mg/m(2)/week until progression, major toxicity or voluntary discontinuation. EGFR expression and gene status were retrospectively analyzed by immunocytochemistry and fluorescence in situ hybridization, respectively. A median of 14 courses of cetuximab (range 5-54) were infused. Skin toxicity was the main adverse event. Cetuximab provides a CBR in 50% (95% CL, 31 to 69%) of cases. None tumor sample showed EGFR gene amplification and an increased EGFR copy number was observed in 12% of samples, all ACC. Skin rash >or=G2, EGFR overexpression and EGFR copy number were not statistically correlated to CB. In RMSGCs further evaluations of EGFR targeting agents are advisable and should take place by appropriate tumor biological selection, differentiating ACC from non-ACC.

Analysis of potential receptor tyrosine kinase targets in intimal and mural sarcomas.

Primary sarcomas of the great vessels are very rare neoplasms and only a few cases have been reported. They are divided into the two broad categories of intimal or luminal and mural sarcomas. We analysed eight advanced high-grade sarcomas originating from major vessels (seven intimal and one mural sarcoma) by means of immunohistochemistry and FISH analysis for PDGFRA, PDGFRB, EGFR and KIT receptor tyrosine kinases (RTKs), together with immunoprecipitation/western blotting, sequencing of the corresponding genes, and the search for cognate ligands. The intimal sarcomas showed a wide spectrum of morphologies and immunophenotypes, whereas the mural sarcoma had common leiomyosarcomatous features. Regardless of their category, all of the cases had a PDGFRA-deregulated cytogenetic profile mainly consisting of an amplification cluster; five were also polysomic for PDGFRB, whereas three showed disomy. Six cases had a deregulated EGFR gene, and c-Kit gene status was similar to that of PDGFRA. In one case, biochemical analysis revealed the presence of activated and highly expressed PDGFRA, PDGFRB and EGFR, whereas KIT was expressed at reference level. Sequencing of the corresponding genes revealed no activating mutations in any of the analysed receptors. The cognate ligands were detected in all cases. In predictive terms, the evidence of gene amplification/high polysomy of several RTKs, together with PDGFRA, PDGFRB and EGFR expression and phosphorylation, suggests that these tumours may be sensitive to RTK-inhibiting treatments.

Jejunal creatine absorption: what is the role of the basolateral membrane?

The mechanism of the intestinal creatine absorption is not well understood. Previous studies have established the involvement of a CT1 carrier system in jejunal apical membrane. The current research was aimed at completing the picture of creatine absorption. To investigate the process supporting creatine exit from enterocyte, basolateral membrane vesicles isolated from rat jejunum were used. The presence of various symport and antiport mechanisms was searched and a NaCl-dependent electrogenic transport system for creatine was evidenced, which shares some functional and kinetic features with the apical CT1. However, Western blot and immunohistochemical experiments ruled out the presence of a CT1 transporter in the basolateral membrane. Further studies are required to identify the basolateral transport mechanism. However, in the in vivo conditions, the NaCl gradient is inwardly directed, therefore such a mechanism cannot energetically mediate the exit of creatine from the cell into the blood during the absorptive process, but rather it may drive creatine into the enterocyte. To shed more light on the creatine absorption process, a possible creatine movement through the paracellular pathway has been examined using the jejunal tract everted and incubated in vitro. A linear relationship between creatine transport and concentration was apparent both in the mucosa-to-serosa and serosa-to-mucosa directions and the difference between the two slopes suggests that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. As a matter of fact, when transepithelial water flux is reduced by means of a mucosal hypertonic solution, the opposite creatine fluxes tend to overlap. The findings of the present study suggest that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption.

Chloroplast fine structure in the japonica-2 maize mutant exposed to continuous illumination. 1. The green tissues.

Exposure to continuous illumination causes the appearance of numerous plastoglobuli in the stroma of both the mesophyll and bundle sheath chloroplasts of the green tissues of the leaves of the japonica-2 mutant of maize. In the pale green tissues the thylakoids have markedly swollen membranes. Another feature of the plastids exposed to continuous illumination is the heavy accumulation of starch. The japonica-2 chloroplasts show a different sensitivity to light, the chloroplasts of the pale green tissues being affected more markedly than the ones of the dark green tissues, and the bundle sheath chloroplasts more than those of the mesophyll. The effects of continuous illumination may be interpreted as an acceleration of chloroplast ontogenesis.

Chloroplast fine structure in the japonica-2 maize mutant exposed to continuous illumination. 2. The white tissues.

Exposure to continuous illumination causes an enhancement of thylakoid swelling in the mesophyll chloroplasts of the white tissues of the japonica-2 maize mutant. In the bundle sheath plastids the effects of continuous illumination are striking and intriguing, because a regular honeycomb-like fret of membranes is formed from a provesicular body. No interpretation of this fret of membranes is, at present, possible.

Cl/HCO3 exchange in the basolateral membrane domain of rat jejunal enterocyte.

Basolateral membrane vesicles isolated from rat jejunal enterocyte and well purified from brush border contamination were tested to examine Cl and HCO3 movements. Uptake experiments provided no evidence for a coupling between Na and HCO3 fluxes; K-HCO3 and K-Cl cotransports also could be excluded. Transport studies revealed the presence of a Cl/HCO3 exchanger accepting other anions and inhibitable by the disulfonic stilbenes SITS and DIDS. We can exclude that the evidenced HCO3-dependent Cl uptake is due to brush border contamination, since in jejunal brush border membranes this mechanism, if present, has a very low transport rate. Besides the Cl/HCO3 antiporter, a Cl-conductive pathway seems to exist in jejunal basolateral membranes.

Ouabain-insensitive transintestinal transport in the rat jejunum incubated in vitro.

The jejunal tract of rat intestine, everted and incubated in vitro at 28 degrees C for 2 hr in Krebs-Ringer bicarbonate solution, was used to test the existence of a ouabain-insensitive sodium pump. Cell water, Na, and K together with Na, fluid, K, and lactate transported into the serosal compartment were determined and, under control conditions, the tested parameters were found constant in time. By blocking the Na-K pump with 20 mM ouabain in the serosal compartment, the enterocyte lost K and gained Na, but the cell volume did not vary. Moreover, the transport of Na, fluid, and lactate, although lower, was constant for 2 hr. When ethacrynate was added or when the ATP supply was blocked by adding 2,4-dinitrophenol plus iodoacetate, the cell swelled and the transport of Na and fluid stopped. These results are interpreted as suggesting the existence of a ouabain-insensitive Na pump, in addition to the well-known Na-K pump.

Mechanisms of Na transport at the basolateral pole of rat enterocyte.

Basolateral membranes isolated from rat jejunal enterocytes and enriched 14 times were found present as unsealed and sealed right side out (RSO) or inside out (IO) vesicles in the ratio 2:2:1. Entrance of 1 mM Na (JNa) into basolateral membrane vesicles was measured in the presence and in the absence of 5 mM ATP by a rapid filtration technique, under different experimental conditions. JNa across the basolateral membrane is cis-inhibited by K, transstimulated by K and further stimulated by ATP (activation of the Na pump). Both K- and ATP-positive effects are also obtained by other cations: the relative stimulation sequence is K greater than Rb = NH4 greater than Cs. The ATP effect can be suppressed by vanadate and strophanthidin; moreover the K effect on JNa is not influenced by blocking the mechanism of the Na pump with ouabain, strophanthidin and adenosine 5'-[beta,gamma-imido]triphosphate. The transmembrane potential does not influence the K-stimulated JNa. In addition to the Na pump this study demonstrates the existence of an exchange mechanism between Na and K, which seems to be different from the Na pump. There appears to be no Na-K cotransport.