Galactose transport inhibition by cytochalasin E in rat intestine in vitro

1999 ◽  
Vol 77 (2) ◽  
pp. 96-101 ◽  
Author(s):  
Ana Díez-Sampedro ◽  
Elena Urdaneta ◽  
M Pilar Lostao ◽  
Ana Barber

Cytochalasins are cytoskeleton disrupters, and cytochalasin E has been reported to increase intestinal paracellular permeability. In this study, the cytochalasin E effect on galactose transport has been investigated. Ussing-type chamber experiments show an inhibitory effect of 20 µM cytochalasin E on unidirectional mucosal to serosal flux of galactose. On the contrary, the opposite unidirectional flux is not modified by the inhibitor. Results using intestinal everted sacs and rings confirm that galactose uptake by the tissue is diminished by cytochalasin E. The effect appears already after 5 min incubation, depends on cytochalasin E concentration, and does not occur in the absence of Na+. The inhibition is accompanied by an increase in the apparent Km of the active sugar transport (11.5 vs.15.8 mM) without significant change in the Vmax (10.6 vs. 9.1 µmol·g-1 wet weight·5 min-1). Cytochalasin E does not modify either galactose uptake by brush border membrane vesicles or Na+-K+ ATPase activity in the enterocytes, indicating that the inhibitory effect on the Na+-dependent sugar transport cannot be explained as a direct effect on SGLT1 activity or as an indirect effect through the Na+-K+ ATPase. Thus, our results suggest that cytochalasin E decreases SGLT1 activity indirectly through cytoskeleton disruption.Key words: cytochalasin E, intestinal absorption, cytoskeleton.

1987 ◽  
Vol 65 (4) ◽  
pp. 579-586 ◽  
Author(s):  
P. K. Dinda ◽  
I. T. Beck

We reported previously that when jejunal transmural glucose transport was inhibited by phlorizin the ratio of Na:giucose transport increased from 2.0:1 (in controls) to 3.3:1. To elucidate the mechanism of this increased ratio of Na:glucose transport, in the present study we have investigated the effect of phlorizin on Na uptake by brush border membrane vesicles and by everted sacs of hamster jejunum. In experiments on membrane vesicles the following observations were made. The time course of Na uptake showed that the control vesicles were in complete equilibrium with a Na-containing (100 mM) medium between 30 and 90 min incubation. In these periods of incubation, the vesicles incubated with phlorizin presumably also equilibrated with the medium, but lost their intravesicular Na during Millipore filtration and washing, and consequently the residual Na content was lower than that of controls. This effect of phlorizin was concentration dependent, and appeared to be unrelated to Na-coupled glucose transport, because it was also observed in the absence of glucose. This loss of Na during Millipore filtration and washing was also observed (i) when vesicles were equilibrated in a Na-containing solution in the absence of phlorizin and then exposed to a similar solution containing phlorizin, or (ii) when vesicles were equilibrated in a Na-containing solution in the presence of phlorizin and then washed repeatedly following Millipore filtration. Preincubation of vesicles for 10 min in a Na- and glucose-free solution containing phlorizin followed by incubation for 30–90 s in solutions containing 1 mM glucose and various concentrations of Na (from 10 to 100 mM) caused an increase in Na uptake from all concentrations of Na. After similar preincubation, when jejunal everted sacs were incubated for 15 s in a Na- and glucose-containing medium, Na uptake by the sacs increased. These findings suggest that phlorizin causes an increase in permeability of mucosal membrane of the enterocyte to Na. This may cause a rapid dissipation of Na gradient and an increase in the ratio of Na:glucose transport. The dissipation of Na gradient may be an additional mechanism for phlorizin-induced inhibition of intestinal sugar transport.


2000 ◽  
Vol 74 (20) ◽  
pp. 9464-9470 ◽  
Author(s):  
Nabil Halaihel ◽  
Vanessa Liévin ◽  
Judith M. Ball ◽  
Mary K. Estes ◽  
Francisco Alvarado ◽  
...  

ABSTRACT The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport ofd-glucose and of l-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na+-d-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na+-l-leucine symport about equally and partially via a different transport mechanism, in that Na+behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.


1987 ◽  
Vol 252 (3) ◽  
pp. G320-G324
Author(s):  
P. Brachet ◽  
F. Alvarado ◽  
A. Puigserver

The kinetics of D- and L-methionine uptake by rings of everted intestine in vitro are consistent with a saturable Michaelis-Menten component (Km = 11.7 and 1.7 mM; Vmax = 0.53 and 0.74 mumol X g-1 X min-1 for D- and L-methionine, respectively) plus a linear, diffusional one. All the data could be fit with a diffusion constant (Kd = 3.2 microliters X g-1 X min-1), which was essentially the same, independent of whether it was estimated by iteration or by using the extracellular marker, inulin. Similar results were obtained from in vivo perfusion experiments, except that the diffusional term was negligible. D-Methionine was found to inhibit L-methionine uptake by intestinal rings according to fully noncompetitive kinetics (Ki = 45 mM). Another set of experiments with jejunal brush-border membrane vesicles showed that D-methionine uptake is dependent on a Na+ gradient and is significantly inhibited by L-methionine and L-proline, but not by beta-alanine and alpha-methylaminoisobutyric acid. Our results indicate that, in rat jejunum, D-methionine is taken up through a Na+-dependent pathway distinct from the neutral amino acid (L-methionine) carrier and from the imino acid (L-proline, alpha-methylaminoisobutyric acid, beta-alanine) carrier.


1986 ◽  
Vol 64 (5) ◽  
pp. 568-574 ◽  
Author(s):  
David D. Maenz ◽  
G. W. Forsyth

Cholera toxin is very well characterized in terms of the activation of adenylate cyclase. In some systems, however, this cyclase activation does not seem to account for all of the physiological responses to the toxin. On the premise that cholera toxin may also exert effects through other second messenger compounds we have studied the effect of cholera toxin on the rate of Ca2+ movement across the membrane of intestinal brush border vesicles. Increasing concentrations of cholera toxin progressively accelerated the passive uptake of Ca2+ into, and the efflux of Ca2+ from, an osmotically active space in brush border membrane vesicles. This effect of cholera toxin was saturable by excess Ca2+ and was relatively specific, as the toxin did not affect vesicle permeability to an uncharged polar solute. The toxin had two high affinity Ca2+ binding sites on the A subunit as measured by equilibrium dialysis. Ca2+ transport facilitated by cholera toxin was temperature dependent, required the holotoxin, and could be inhibited by preincubation of the toxin with excess free ganglioside GM1.This increased rate of Ca2+ influx caused by the in vitro addition of cholera toxin to brush border membrane vesicles may have physiological significance as it was comparable to rates observed with the Ca ionophore A23187. Similar effects occurring in vivo could permit cholera toxin to increase cytoplasmic Ca2+ concentrations and to produce accompanying second messenger effects.


1993 ◽  
Vol 290 (1) ◽  
pp. 237-240 ◽  
Author(s):  
H M Said ◽  
R Mohammadkhani

We examined the possible existence of histidine residue(s) in the folate transporter of rabbit intestine. This was done with use of the histidine-specific reagent diethyl pyrocarbonate (DEPC) and purified intestinal brush-border-membrane vesicles. DEPC caused significant concentration- and time-dependent inhibition of folic acid transport. The inhibition was only seen when transport was examined in vesicles incubated in buffer at pH 5.2 and not in those incubated in buffer at pH 7.4. The addition of unlabelled folic acid to vesicle suspension before treatment with DEPC (2.5 mM) led to a significant (P < 0.01) protection (84%) against the inhibition of folic acid transport. Treating vesicles pretreated with DEPC (2.5 mM) with reducing reagents (dithiothreitol, 2-mercaptoethanol and 2,3-dimercaptopropanol, all at a final concentration of 10 mM) did not reverse the inhibitory effect of DEPC on folic acid transport. On the other hand, treating the DEPC-pretreated vesicles with hydroxylamine (140 mM) led to a significant reversal (P < 0.01) (54%) of the inhibition of folic acid transport. The inhibitory effect of DEPC on carrier-mediated folic acid transport was found to be mediated through a decrease in the Vmax. (i.e. a decrease in the number and/or activity) of the carriers and an increase in the apparent Km (i.e. a decrease in their affinity), classifying the effect as a mixed-type inhibition. These results demonstrate the existence of critical histidine residue(s) in the intestinal brush-border-membrane folate transporter which is essential for its interaction with, and transport of, the vitamin. These findings also suggest that the histidine residue(s) is located at (or near) the substrate-binding site.


1968 ◽  
Vol 52 (3) ◽  
pp. 482-494 ◽  
Author(s):  
Robert G. Faust ◽  
Mary G. Leadbetter ◽  
Regina K. Plenge ◽  
Alston J. McCaslin

Tris-disrupted and intact brush border membrane preparations from mucosa of hamster jejunum were capable of preferentially binding actively transported D-glucose in a similar manner. Density gradient centrifugation of the Tris-disrupted brush borders indicated that D-glucose was bound to a fraction containing the cores or inner material of the microvilli. The properties of this binding were examined with the Tris-disrupted brush border preparation. Actively transported sugars competitively inhibited preferential D-glucose binding, whereas no effect was observed with nonactively transported sugars. Neither actively nor nonactively transported amino acids affected D-glucose binding. D-Glucosamine, which is not actively transported, was inhibitory to preferential D-glucose binding as well as to the active transport of D-glucose by everted sacs of hamster jejunum. No inhibitory effect was observed with the same concentration of D-galactosamine. Preferential D-glucose binding was also inhibited by sulfhydryl-reacting compounds, Ca2+, and Li+ ions. On the other hand, Mg2+ was shown to be stimulatory and Na+, NH4+, and K+ had no effect on this phenomenon. The results of these experiments suggest that preferential D-glucose binding to brush borders is related to the initial step in active sugar transport by the small intestine.


1981 ◽  
Vol 97 (4) ◽  
pp. 491-495 ◽  
Author(s):  
S. Matsuzaki ◽  
M. Suzuki

Abstract. The effect of sodium perchlorate (NaClO4) on the methylthiouracil-induced increase in the activity of thyroid glucose 6-phosphate dehydrogenase (G6PDH), ornithine decarboxylase (ODC) and polyamine contents was studied in the rat. The G6PDH activity was increased nearly three-fold by methylthiouracil (MTU) but not by ClO4- at 7 days of treatment. Perchlorate lowered the MTU-induced enzyme activity to nearly the control level, without changing circulating thyrotrophin (TSH). The anion had no inhibitory effect on G6PDH activity in vitro. The possibility that an inhibitor specific for G6PDH was generated in ClO4- treated rat thyroids was excluded. The activity of ODC was greatly increased by both ClO4- and MTU, the increase being significant as early as on the second day of treatment. Perchlorate had no inhibitory effect on MTU-induced ODC activity in vivo but decreased total contents of spermidine and spermine in the thyroid, without affecting the concentration (nmoles/ g wet weight) of the polyamines. These results suggest that ClO4- acts directly on the thyroid to suppress specifically the stimulatory effect of TSH on G6PDH activity and possibly on polyamine accumulation.


1984 ◽  
Vol 108 (1) ◽  
pp. 329-339
Author(s):  
V. F. SACCHI ◽  
G. M. HANOZET ◽  
B. GIORDANA

1. A net absorption of α-aminoisobutyric acid (AIB) takes place in vitro in the midgut of two lepidopteran larvae, Philosamia cynthia Drury and Bombyx mori L. 2. InP. cynthia the midgut epithelium accumulates AIB from the lumen, while in the same conditions AIB accumulation is not observed in B. mori midgut cells. 3. In P. cynthia, when the lumen is bathed with a low K solution, the net absorption of AIB is reduced and the intracellular accumulation from the lumen is abolished. 4. Brush border membrane vesicles, prepared from the midgut of both species, show a transient K-dependent concentrative uptake of AIB. 5. The relationship between AIB uptake and AIB concentration in the presence of a transmembrane K gradient was studied in B. mori vesicles and the kinetic constants were calculated. 6. These results confirm that there is a K-amino acid co-transport system on the brush border of columnar cells in the midgut of both larvae.


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