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 Na+–glucose cotransport. The specificity of the response to PKC was confirmed by using staurosporine, chelerythrine or 4-α-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 simultananeous modulation of multiple pathways, targeted to a reduction of both lactate and bicarbonate transports without altering cell pH homeostasis.
Experiments were performed with everted sacs of jejunum and ileum and brush-border membrane vesicles from four different age groups of rats: very young, young, adult, and old. The changes with age in ...several parameters, such as cell electrolyte and D-glucose concentrations, cell volume, net D-glucose, Na+ and water transport, and D-glucose uptake into vesicles, were studied. Differences in transport activities in the four age groups of animals were observed. In the jejunum the greatest transport was found in young rats and less in adult, very young, and old rats. In the ileum, the greatest transport activity was found in very young and young rats, while in adult and old animals transport activity was negligible. The cell volume of the enterocyte was significantly smaller in old rats. Changes in cell electrolytes (increased Na+ and decreased K+) were observed only in the jejunum of old rats. The overshoot of D-glucose in brush-border membrane vesicles varied considerably in the four different age groups of animals. In other words, the capacity for sugar uptake into vesicles is greater in young rats than in very young or adult animals. In old rats there was no overshoot, and a slow linear increase of sugar entry was observed. The results indicate that the changes in D-glucose transport with age might be explained in part by the changes in the amount of sugar that crosses the brush-border membrane of the enterocyte.
Abstract only
21070
Background: In current clinical practice Cetuximab represents a standard therapy for Irinotecan resistant pts with ACC, where EGFR-immunohistochemistry (IHC) evaluation is ...positive. In absence of reliable molecular predictors of the clinical benefit to anti-EGFR therapy, we have studied EGFR protein expression and gene status in order to correlate them to the clinical response. Methods: We investigated 40 primary colorectal carcinoma specimens. EGFR-protein expression was evaluated by Immunohistochemistry (IHC) using PharmDX-Kit DAKO Cytomation. IHC evaluation was performed using combined score based on the sum of percentage of positive cells and the staining of each sample. We identified three EGFR-score categories: 0–2, 3–5, 6–7 corresponding to low, intermediate and high expression respectively. EGFR-genetic status was conducted by Dual-Colour FISH (LSI EGFR-probe Vysis Inc).Gene and chromosome 7 copy numbers were identified by fluorescence in situ hybridization (FISH-LSI EGFR-Dual-colour-probe Vysis-Inc) as follows: tumor samples had a high EGFR-gene copy number if there was high polysomy ( 4-copies 40% of cells) or gene amplification (gene-clusters, gene/chromosome ratio per cell of 2, 15 copies of EGFR/ cell in 10% of cells) . The analysis of clinical data from all patients treated with Cetuximab (400–250mg/m
2
/w) and Irinotecan (300mg/m
2
/d1q21) was then performed. Results: EGFR-(IHC) high score was observed in 7pts from which only 2 had an objective response (OR). Intermediate (13pts) with 3 OR, low (12pts) with 1 OR and negative (8pts) with 2OR. Main FISH patterns were: high polisomy/amplification in 8pts with no response on cetuximab therapy. Low polisomy described in 19pts with 3 cases of disease remission. Disomy in 13pts with 5OR. No relationship was detected between IHC-score evaluation and FISH pattern. The clinical benefit (OR+stable disease =6months) was 65% in score-group 0–2; whereas 46% and 57% in groups 3–5/6–7 respectively, without statistical significance. Conclusion: According to our experience, IHC and FISH are unable to provide adequate selection criteria for the patients with different EGFR-status expression, who can benefit from Cetuximab therapy.
No significant financial relationships to disclose.
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.
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 stimulates Na
+ uptake at both 5 μM and 5 mM concentrations. The proton gradient effect can be inhibited completely by 2 mM amiloride and partially by either FCCP or NH
4Cl (NH
3 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.
Under normal conditions the jejunal tract of the rat intestine absorbs HCO3. A basolateral Cl/HCO3 exchange, evidenced by means of membrane vesicles, could be involved in this process. Aim of this ...study was to investigate the anion exchange activity in the whole jejunal tract, where various transport systems could interact.
In the jejunal tract of rat intestine everted and incubated in vitro, the experimental conditions set up minimized loss of CO2 from the serosal solution, where pH and pCO2 were determined together with fluid and electrolyte transintestinal transport.
The serosal pCO2 increase and pH decrease, evident during the experiment, could be antagonized by enhancing the 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) concentrations in the serosal fluid. Moreover high DIDS concentration affected fluid, sodium, lactate, bicarbonate and, although in the opposite direction, chloride transport, whilst they were ineffective on K flux.
These results give evidence that in the basolateral membrane the inhibition of Cl/HCO3 antiport causes a diminution of lactic acid movement. Therefore we can hypothesize that Cl/HCO3 antiport facilitates basolateral H-lactate symport in order to carry endogenous lactic acid towards the blood stream.
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 (P
f
) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg
2+
, Cu
2+
, and Zn
2+
caused a significant decrease in the BBMV P
f
, whereas Ni
2+
and Li
+
had no effect. AQP8-transfected cells showed a reduction in P
f
in the presence of Hg
2+
and Cu
2+
, whereas AQP7-transfected cells were insensitive to all tested metals. The P
f
of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the P
f
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 Hg
2+
and Cu
2+
.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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 Ca2+ 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--HCO3- antiporter, whereas the basolateral HCO3- 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-α-PMA. The inhibition of both apical Na+-H+ and basolateral Cl--HCO3- exchange activities suggests that the overall action of PKC causes a reduction of transepithelial bicarbonate transport. Experimental Physiology (2002) 87.3, 299-309.
The mechanisms of HCO3- and Cl- transport across basolateral membranes from rat ileum were investigated in isolated vesicles by means of uptake experiments. Neither Cl-/HCO3- exchanger nor ...Na(+)-(HCO3-)n cotransport seem to be present in ileal basolateral membranes. Moreover Cl- uptake is unaffected by cis Na+ and/or K+ gradients, indicating the absence of Na(+)-Cl-, K(+)-Cl- and Na(+)-K(+)-2Cl- symport activity. An electrically conductive pathway seems to be responsible for both HCO3- and Cl- fluxes. Evidence is also given for the presence of a Na+/H+ exchanger at the basolateral pole of ileal enterocytes.
Among the numerous species of the family Euphorbiaceae the photosynthetic carbon fixation follows three pathways: C3, C4, CAM. The object of the present research was Euphorbia peplis L., grown and ...collected on a Mediterranean coast. Leaf anatomy and ultrastructure together with carbon isotope discrimination measurements indicated that E. peplis is ascribable to the NADP-malic enzyme C4 photosynthetic type.
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