Effects of crude and pure cholera toxin on prostaglandin release from the rabbit ileum

Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum. Perfusion of ileal loops with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE₂ equivalents). In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin. Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE₂/g tissue when incubated with crude toxin for 1–4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions.Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin. These results support the view that prostaglandins do not play an essential role in the action of cholera toxin.     

Effects of crude and pure cholera toxin on prostaglandin release from the rabbit ileum

Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum. Perfusion of ileal loops in vivo with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE₂ equivalents). In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin. Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE₂/g tissue when incubated with crude toxin for 1–4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions.Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin. These results support the view that prostaglandins do not play an essential role in the action of cholera toxin.     

Effect of cholera toxin on glutamine metabolism and transport in rabbit ileum

The aim of the present study was to evaluate the effect of cholera toxin on energy balance from intestinal glutamine metabolism and oxidation, glutamine-dependent sodium absorption, and cholera toxin-dependent ion flux. Cholera toxin-stimulated sodium and L-glutamine ileal transport and metabolism were studied in Ussing chambers. Glutamine (10 mM) transport and metabolism were simultaneously studied using (14)C flux and HPLC. In the same tissues, the flux of each amino acid was studied by HPLC, and glutamine metabolism and oxidation were studied by the determination of amino acid specific activity and (14)CO(2) production. In control tissues, glutamine stimulated sodium absorption and was mainly oxidized. The transepithelial flux of intact glutamine represented 45% of glutamine flux across the luminal membrane. The other metabolites were glutamate and, to a lesser degree, citrulline, ornithine, and proline. Cholera toxin did not alter glutamine-stimulated sodium absorption, glutamine oxidation, transport, and metabolism. In conclusion, the present results indicate that cholera toxin does not alter glutamine intestinal function and metabolism. In addition, approximately 95% of the energy provided by glutamine oxidation remains available to the enterocyte.     

Alanine and sodium fluxes across mucosal border of rabbit ileum

Unidirectional influxes of L-alanine and Na from the mucosal solution into the epithelium of in vitro rabbit ileum have been determined. In the presence of 140 mM Na, alanine influx is approximately 2.2 µmoles/hr cm², but is inhibited if the NaCl in the mucosal solution is replaced by choline Cl, Tris-Cl, mannitol, LiCl, or KCl. Although alanine influx is strongly dependent upon Na in the mucosal solution, it is uninfluenced by marked reduction of intracellular Na pools. In addition, alanine influx is unaffected by intracellular alanine concentration. Na influx is markedly inhibited by the presence of Li. Evidence is presented that Na transport across the mucosal border cannot be attributed to simple diffusion even though the net flux across this surface is in the direction of the electrochemical potential difference.     

Effects of cholera toxin, Escherichia coli heat stable toxin and sodium deoxycholate on neurotensin release from the ileum in vivo

Neurotensin (NT) is a biologically active peptide found in specialized epithelial cells (N-cells) in the distal small intestine. In this study we tested the hypothesis that NT may be released by luminal secretagogues, i.e., cholera toxin, Escherichia coli heat-stable toxin and sodium deoxycholate. Cholera toxin elicited net fluid secretion in anesthetized cats. This secretion was accompanied by an increased release of NT-like immunoreactivity (NTLI) into the mesenteric vein when NTLI was measured with either a C-terminally or a N-terminally directed antibody. An increasing plasma NTLI concentration (N-terminally directed antibody) was recorded in the mesenteric vein and femoral artery in cholera experiments. These results indicate that cholera toxin releases NT from the small intestine. Since neurotensin causes intestinal fluid secretion at least in part via an activation of enteric nerves we propose that the N-cell functions as a 'receptor cell' which activates an intramural secretory reflex upon luminal stimulation by cholera toxin. This study does not support a similar role for NT in the secretion elicited by the heat stable toxin of Escherichia coli or by sodium deoxycholate since we were unable to demonstrate any intestinal release of NTLI after exposing the intestine to these secretory agents.     

In vitro effects of tetrodotoxin and hexamethonium on electrolyte transport in rabbit ileum treated with cholera toxin.

To determine if there was a role for the submucosal nerves in cholera toxin (CT)-induced secretion, we studied the effects of serosal addition of two neurotoxins, the nerve conduction blocking agent, tetrodotoxin (TTX), and the nicotinic ganglionic blocking agent, hexamethonium (HXM), on electrolyte secretion in control isolated rabbit ileum and in that stimulated by CT. 1). In the absence of CT, the short circuit current (Isc) decreased after TTX (10(-7) M) (P less than 0.01) and was unaltered by HXM (10(-5) M). In the presence of CT, Isc increased but was not modified by 10(-7) M TTX or 10(-5) M HXM. 2) In control tissues the mean isotopic Na+ and Cl- fluxes were not significantly altered by TTX addition. Cl- absorption alone was significantly reduced by HXM (delta JCl- = 1.95 +/- 0.81 microEq.hr-1.cm-2; P less than 0.02). After stimulation with CT, TTX significantly inhibited Na+ and Cl- secretion (delta JNa+ = 2.15 +/- 0.61 and delta JCl- = 2.15 +/- 0.76 microEq.hr-1.cm-2; P less than 0.01). Similarly, HXM significantly inhibited CT-stimulated Na+ and Cl- secretion (delta JNa+ = 1.73 +/- 0.70 and delta JCl- = 1.46 +/- 0.62 microEq.hr-1.cm-2; P less than 0.02). 3) In TTX and HXM treated tissues there was no difference in the increase in Isc caused by cAMP (2 x 10(-3) M), calcium ionophore A 23187 (4 x 10(-6) M) and glucose (10(-3) M) compared to the untreated tissues in the presence or absence of CT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cholera toxin on vasoconstriction and cyclic AMP content of the isolated rabbit ear artery

We have studied the effect of cholera toxin on the constrictor responses of the isolated, perfused rabbit ear artery to nerve stimulation and to norepinephrine infusion. We found that when we perfussed arteries with cholera toxin (1–9 μg/ml) for five minutes or longer, the toxin gradually inhibited the responses to intermittent stimulation of the adrenergic nerves and to brief infusion of norepinephrine. The constrictor responses began to decrease between one and two hours after we added cholera toxin, and the responses were still depressed after 24 hours. Cholera toxin inhibited both the rapid, initial phase and the slower, sustained phase of the biphasic response of the ear artery to nerve stimulation. Propranolol and indomethacin did not block the effect of cholera toxin on vasoconstriction. However, when we mixed the toxin with antitoxin or G_M1 ganglioside, we prevented the inhibitory effect on vasoconstriction. Levels of adenosine 3′:5′-cyclic monophosphate (cyclic AMP) in arteries treated with cholera toxin were greater than levels of cyclic AMP in untreated arteries. The cyclic AMP content increased and the constrictor responses decreased with a similar time course after the arteries were exposed to the toxin. Thus an increase in cyclic AMP may be involved in the relaxation of vascular smooth muscle induced by cholera toxin.     

Membrane traffic and the cellular uptake of cholera toxin.

In nature, cholera toxin (CT) and the structurally related E. coli heat labile toxin type I (LTI) must breech the epithelial barrier of the intestine to cause the massive diarrhea seen in cholera. This requires endocytosis of toxin-receptor complexes into the apical endosome, retrograde transport into Golgi cisternae or endoplasmic reticulum (ER), and finally transport of toxin across the cell to its site of action on the basolateral membrane. Targeting into this pathway depends on toxin binding ganglioside GM1 and association with caveolae-like membrane domains. Thus to cause disease, both CT and LTI co-opt the molecular machinery used by the host cell to sort, move, and organize their cellular membranes and substituent components.     

Effects of crude and pure cholera toxin on prostaglandin.

Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum. Perfusion of ileal loops in vivo with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE2 equivalents). In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin. Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE2/g tissue when incubated with crude toxin for 1-4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions. Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin. These results support the view that prostaglandins do not play an essential role in the action of cholera toxin.     

Electron microscopic demonstration of adenylate cyclase in rabbit small intestine enterocytes doubly stimulated by cholera toxin and sodium fluoride]

Cytochemical investigations showed adenylate cyclase in the rabbit small intestine enterocytes to be activated both with cholera toxin and sodium fluoride. Following double stimulation of adenylate cyclase in the intestinal enterocytes by the mentioned two substances maximal critical levels of cAMP were attained resulting in self-inhibition of adenylate cyclase; in this case only a low adenylate cyclase activity, if any, could be demonstrated by electron microscopy.     

Electrophysiological properties of cellular and paracellular conductive pathways of the rabbit cortical collecting duct

Summary Microelectrode techniques were applied to the rabbit isolated perfused cortical collecting duct to provide an initial quantitation and characterization of the cell membrane and tight junction conductances. Initial studies demonstrated that the fractional resistance (ratio of the resistance of the apical cell membrane to the sum of the resistances of the apical and basolateral membranes) was usually independent of the point along the tubule of microelectrode impalement—implicating little cell-to-cell coupling—supporting the application of quantitative techniques to the cortical collecting duct. It was demonstrated that in the presence of amiloride, either reduction in the luminal pH or the addition of barium to the perfusate selectively reduced the apical membrane potassium conductance. From the changes inG ^te and fractional resistance upon reducing the luminal pH or addition of barium to the perfusate, the transepithelial, apical membrane, basolateral membrane and tight junction conductances were estimated to be 9.3, 6.7, 8.1 and 6.0 mS cm^–2, respectively. Ninety to ninety-five percent of the apical membrane conductance reflected the barium-sensitive potassium conductance in the presence of amiloride with an estimated potassium permeability of 1.1×10^–4 cm sec^–1. Reduction in the perfusate pH to 4.0 caused a 70% decrease in the apical membrane potassium conductance, implying a blocking site with an acidic group having a pK _a near 4.4. It is concluded that both the transcellular and paracellular pathways of the cortical collecting tubule have high ionic conductances, and that the apical membrane conductance primarily reffects a high potassium conductance. Furthermore, both reduction in the perfusate pH and addition of barium to the perfusate selectively block the apical potassium channels, although the site of inhibition likely differs since the two ions display markedly different voltage-dependent blocks of the channel.     

Effects of inhibitors on 3-O-methylglycose transport in rabbit ileum

Summary Previous studies (Goldner, Schultz & Curran,J. Gen. Physiol. 1969,53:362) have suggested a direct coupling between influxes of sugars and Na across the brush border membrane of rabbit ileum. Effects of several inhibitors, ouabain, cyanide, dinitrophenol and iodoacetate on 3-O-methylglucose fluxes were examined in an effort to obtain information about coupling of sugar transport to metabolism. The inhibitors virtually abolished net active sugar transport across the whole tissue but had less striking effects on sugar influx across the brush border membrane, particularly when the cells were prevented from gaining Na as a result of inhibitor action. However, substantial but incomplete inhibition of influx was observed when the cells were permitted to gain Na. Mucosal strips incubated with ouabain to elevate cellular Na extruded sugar against a concentration gradient when cell Na concentration exceeded that in the medium. Conversely, a small extrusion of Na from ouabain-poisoned cells was observed in the presence of an outwardly directed concentration gradient for sugar. These results provide further evidence of coupling between Na and sugar movement. Additional direct coupling of sugar movement to metabolism cannot be ruled out.     

Effects of monovalent cations on the sodium-alanine interaction in rabbit ileum. Implication of anionic groups in sodium binding

H, K, Rb, and Li inhibit Na-dependent alanine influx across the brush border of rabbit ileum. Kinetic analysis indicates that H and K behave as competitive inhibitors of influx so that increasing the concentration of H or K in the mucosal solution is kinetically indistinguishable from decreasing the Na concentration. In addition the coupling between alanine and Na influxes is markedly reduced at pH 2.5. With the exception of H and Li, none of these monovalent cations significantly affects carrier-mediated alanine influx in the absence of Na indicating that their inhibitory effects are largely restricted to the Na-dependent fraction of influx. Increasing H concentration from 0.03 to 3 mM does not affect influx in the absence of Na but markedly inhibits influx in the presence of Na. Li significantly enhances alanine influx in the absence of Na. Ag, UO₂, and La also inhibit the Na-dependent fraction of alanine influx. These findings suggest that anionic groups having a pK_a of approximately 4 are involved in the interaction between Na and the alanine-carrier complex; present evidence implicates carboxylate groups however, phosphoryl residues cannot be ruled out. The previously proposed kinetic model for the Na-alanine interaction has been extended to accommodate these effects of H and other monovalent cations. The mechanistic and physiological implications of these findings are discussed.     

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.     

ADP-ribosylation by cholera toxin: functional analysis of a cellular system that stimulates the enzymic activity of cholera toxin fragment A1

We have clarified relationships between cholera toxin, cholera toxin substrates, a membrane protein S that is required for toxin activity, and a soluble protein CF that is needed for the function of S. The toxin has little intrinsic ability to catalyze ADP-ribosylations unless it encounters the active form of the S protein, which is S liganded to GTP or to a GTP analogue. In the presence of CF, S.GTP forms readily, though reversibly, but a more permanent active species, S-guanosine 5'-O-(3-thiotriphosphate) (S.GTP gamma S), forms over a period of 10-15 min at 37 degrees C. Both guanosine 5'-O-(2-thiodiphosphate) and GTP block this quasi-permanent activation. Some S.GTP gamma S forms in membranes that are exposed to CF alone and then to GTP gamma S, with a wash in between, and it is possible that CF facilitates a G nucleotide exchange. S.GTP gamma S dissolved by nonionic detergents persists in solution and can be used to support the ADP-ribosylation of nucleotide-free substrates. In this circumstance, added guanyl nucleotides have no further effect. This active form of S is unstable, especially when heated, but the thermal inactivation above 45 degrees C is decreased by GTP gamma S. Active S is required equally for the ADP-ribosylation of all of cholera toxin's protein substrates, regardless of whether they bind GTP or not. We suggest that active S interacts directly with the enzymic A1 fragment of cholera toxin and not with any toxin substrate.(ABSTRACT TRUNCATED AT 250 WORDS)