Failure to reverse cholera toxin induced intestinal secretion by agents which decrease mucosal cAMP.

The feasibility of reducing intestinal secretion by the use of agents which decrease intestinal mucosal cAMP concentration has been investigated in the weanling pig and the rabbit. Three different agents for decreasing mucosal cAMP concentration were studied. The cyclic nucleotide phosphodiesterase activator, imidazole, significantly reduced mucosal cAMP concentrations only in the weanling pig. Intraluminal 2'-deoxyadenosine-3'AMP inhibited adenylate cyclase and caused a decrease in mucosal cAMP concentration in both the pig and the rabbit. The introduction of the heat-stable enterotoxin of Escherichia coli into pig jejunal segments also gave lowered mucosal cAMP concentrations. While these three agents effectively reduced cAMP concentrations in intestinal mucosa, they were ineffective in reducing the net fluid secretory effects of cholera toxin. Secretion caused by cholera toxin apparently persists independent of the temporary changes in cAMP concentration which can be induced by pharmacological agents.     

Inhibition of cholera toxin-stimulated intestinal epithelial cell adenylate cyclase by adenosine analogs.

The ability of various adenosine analogs to inhibit cholera toxin activation of the intestinal epithelial cell adenylate cyclase-cyclic AMP system was investigated. After incubation of cells with cholera toxin for 6 hr, large increases in cellular cyclic AMP content were observed. Addition of 2', 5'-dideoxyadenosine during the last 30 min of this 6-hr incubation resulted in 70% reduction in elevated cyclic AMP content. Other analogs were not effective inhibitors. 2', 5'-Dideoxyadenosine was also a potent inhibitor of cholera toxin-activated intestinal cell adenylate cyclase activity with half-maximal inhibition occuring at 16 muM. NaF-stimulated cyclase was less susceptible to inhibition. The data suggest that inhibition by 2', 5'-dideoxyadenosine is due at least in part to direct inhibition of the cholera toxin-activated intestinal adenylate cyclase activity.     

The activation of adenylate cyclase from small intestinal epithelium by cholera toxin

ADP-ribosylation of membrane proteins from rabbit small intestinal epithelium was investigated following incubation of membranes with [32P]NAD and cholera toxin. Cholera toxin catalyzes incorporation of 32P into three proteins of 40 kDA, 45 kDa and 47 kDa located in the brush-border membrane. In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and [32P]NAD. The modification of membrane proteins from brush border occurred in spite of the virtual absence in these membranes of adenylate cyclase activatable either by cholera toxin, vasoactive intestinal peptide (VIP) or fluoride. The three agents activated adenylate cyclase when crude plasma membrane were used. Cholera toxin activated fivefold at 10 micrograms/ml. Vasoactive intestinal peptide activated at concentrations from 10-300 nM, the maximal stimulation being sixfold. Fluoride activated 10-fold at 10 mM. When basal lateral membranes were assayed for adenylate cyclase it was found that, with respect to the crude membranes, the specific activity of fluoride-activated enzyme was 3.3-fold higher, VIP stimulated enzyme was maintained while cholera-toxin-stimulated enzyme showed half specific activity. Moreover, while fluoride stimulated ninefold and VIP stimulated fivefold, cholera toxin only stimulated twofold at the highest concentration. The results suggest that the activation by cholera toxin of adenylate cyclase located at the basal lateral membrane requires ADPribosylation of proteins in the brush border membrane.     

Action of cholera toxin on dispersed acini from guinea pig pancreas.

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Action of cholera toxin on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of ⁴⁵Ca or cellular cyclic AMP. Binding of ¹²⁵I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of bindind of ¹²⁵I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in ⁴⁵Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretion or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Inhibitory effect of ibuprofen on the cholera toxin-induced cyclic AMP response in Chinese hamster ovary cells

Abstract Ibuprofen, an inhibitor of prostaglandin synthesis in eukaryotic cells, was shown to inhibit the accumulation of 3',5'-cyclic adenosine monophosphate (cyclic AMP) in Chinese hamster ovary (CHO) cells exposed to cholera toxin. The inhibition was dose dependent, with a dose of 100 μg/ml reducing the cholera toxin response by approximately 50%, and maximal inhibition was observed when the drug was applied to the cells simulataneously with or 1 h before the toxin. Although ibuprofen also inhibited adenylate cyclase stimulation by forskolin, suggesting a nonspecific effect, the drug had no effect on cholera toxin-induced cyclic AMP accumulation when added to the culture medium 15 min or more after the toxin.     

The immunologic control mechanism against cholera toxin: II. Stimulation of adenylate cyclase without fluid secretion

Cholera toxin-stimulated fluid secretion by the ileum is believed to be mediated by an adenylate cyclase-cyclic AMP mechanism. Immunization against cholera toxin (CT) reduces CT binding to microvillus membranes and suppresses fluid secretion following CT challenge. The present study disclosed no alteration in the fluid absorption rates. However, suppression of fluid secretion occurred despite maximal stimulation of mucosal adenylate cyclase in immunized animals.     

Dissociation by cooling of hormone and cholera toxin activation of adenylate cyclase in intact cells.

Cholera toxin, through adenylate cyclase activation reproduced cyclic AMP-mediated effects of thyroid-stimulating hormone (TSH) in dog thyroid slices, i.e. protein iodination, [1-14C]glucose-oxidation and hormone secretion. Iodide and carbamylcholine decreased the cyclic AMP accumulation induced by cholera toxin as well as by TSH, which supports the hypothesis of an action of these agents beyond the steps of hormone-receptor and receptor-adenylate cyclase interaction. Cooling to 20 degrees C did not impair the TSH induced cyclic AMP accumulation in thyroid slices, but completely suppressed the cholera toxin effect. This observation has been extended to other hormones and target tissues, such as the parathyroid hormone (PTH) (kidney cortex), adrenocorticotropic hormone (ACTH) (adrenal cortex) and luteinizing hormone (LH) (ovary systems). As in thyroid, cooling dissociated the cholera toxin and hormonal effects on cyclic AMP accumulation. In homogenate, cooling decreased cyclic AMP generation in the presence of cholera toxin but at 20 degrees C and 16 degrees C a cholera toxin stimulation was still observed. These results bear strongly against the hypothesis that the glycoprotein hormones TSH and LH acetivate adenylate cyclase by a mechanism identical to cholera toxin.     

Inhibition of plasminogen activator secretion by cyclic AMP in a macrophage-like cell line.

The continuous cell line, J774.2, exhibits many macrophage-like functions such as latex and Fc-mediated phagocytosis, antibody mediated phagocytosis, antibody mediated cytotoxicity, chemotaxis, and lysozyme secretion. Cyclic AMP stimulates Fc-mediated phagocytosis and inhibits the growth of J774.2. To further evaluate the relationship between cyclic AMP and the specialized functions exhibited by these cells. Variants deficient in phagocytosis, adenylate cyclase and cyclic AMP-dependent protein kinase were derived. We have now shown that J774.2 also secretes plasminogen activator and that this secretion is rapidly and specifically inhibited by 8-bromoadenosine 3':5'-cyclic monophosphoric acid (8 Br--cAMP) or cholera toxin under conditions where lysozyme secretion is unaltered. Utilizing protein kinase-deficient variants, the ability of cyclic AMP to inhibit plasminogen activator secretion was shown to be mediated by a cyclic AMP-dependent protein kinase. We conclude that cyclic AMP has diametrically opposing effects on two macrophage-like functions: Fc-mediated phagocytosis and plasminogen activator secretion.     

Properties of protein kinase and adenylate cyclase-deficient variants of a macrophage-like cell line.

Stable variants of the macrophage-like cell line J774.2, defective in adenylate cyclase and protein kinase activities, were selected by cloning cells resistant to the growth-inhibitory effect of cholera toxin and 8-bromo-adenosine 3':5' cyclic monophosphoric acid (8 Br-cAMP), respectively. These variants were analyzed for their ability to respond to cyclic AMP-mediated enhancement of phagocytosis and cyclic AMP-mediated inhibition of plasminogen activator secretion and growtn. The adenylate cyclase variants were unaffected by cholera toxin but were sensitive to 8 Br-cAMP-mediated inhibition of plasminogen activator secretion and growth. One of these variants exhibited a defect in phagocytosis that could be corrected by 8 Br-cAMP. The protein kinase variants exhibited normal basal phagocytosis that could not be stimulated by either 8 Br-cAMP or cholera toxin; they were also insensitive to cyclic AMP-mediated inhibition of plasminogen activator secretion and growth. The studies demonstrate that the three effects of cyclic AMP in J774.2--inhibition of growth and plasminogen activator secretion, and enhancement of basal Fc-mediated phagocytosis--are mediated by a cyclic AMP-dependent portein kinase. The results support the usefulness of variants in cyclic nucleotide metabolism in understanding the regulation of differentiated cell function by cyclic AMP.     

Dissociation by cooling of hormone and cholera toxin activation of adenylate cyclase in intact cells

Cholera toxin, through adenylate cyclase activation reproduced cyclic AMP-mediated effects of thyroid-stimulating hormone (TSH) in dog thyroid slices, i.e protein iodination, [1-¹⁴C]glucose-oxidation and hormone secretion. Iodide and carbamylcholine decreased the cyclic AMP accumulation induced by cholera toxin as well as by TSH, which supports the hypothesis of an action of these agents beyond the steps of hormone-receptor and receptor-adenylate cyclase interaction. Cooling to 20°C did not impair the TSH induced cyclic AMP accumulation in thyroid slices, but completely suppressed the cholera toxin effect.This observation has been extended to other hormones and target tissues, such as the parathyroid hormone (PTH) (kidney cortex), adrenocorticotropic hormone (ACTH) (adrenal cortex)_and luteinizing hormone (LH) (ovary systems). As in thyroid, cooling dissociated the cholera toxin and hormonal effects on cyclic AMP accumulation. In homogenate, cooling decreased cyclic AMP generation in the presence of cholera toxin but at 20°C and 16°C a cholera toxin stimulation was still observed. These results bear strongly against the hypothesis that the glycoprotein hormones TSH and LH activate adenylate cyclase by a mechanism identical to cholera toxin.     

Temporal desensitization of rat uteri for the decidual cell reaction is abolished by cholera toxin acting by a mechanism apparently not involving adenosine 3':5'-cyclic monophosphate

To determine if increased endometrial vascular permeability (a response which precedes decidualization) could be obtained in temporally nonsensitized uteri by treatments designed to increase endometrial adenosine 3':5'-cyclic monophosphate (cAMP) concentrations, cholera toxin (an activator of adenylate cyclase) was injected into the uterine lumen of immature rats treated to be at the equivalent of day 4, 5, or 6 of pseudopregnancy. In all experiments, the rats were pretreated with indomethacin to inhibit endogenous prostaglandin (PG) synthesis. Endometrial vascular permeability, determined using 125I-labeled bovine serum albumin, was assessed 8 h later. Cholera toxin increased endometrial vascular permeability to the same level in all groups. As determined by uterine weights 5 days after the intrauterine administration of cholera toxin or its vehicle, the toxin produced the same extent of decidualization in all groups. Cholera toxin had no detectable effect on uterine cAMP concentrations in rats sacrificed 15 min after intrauterine treatment. In contrast, intrauterine administration of PGE2 increased uterine cAMP concentrations at 15 min in all groups. These data suggest that the effects of cholera toxin and of PGE2 on endometrial vascular permeability and decidualization are not mediated by cAMP.     

The antisecretory factors: inducible proteins which modulate secretion in the small intestine

1. Cholera toxin and glucose induce the synthesis of antisecretory factors (ASF) of isoelectric points 5.0 and 4.3, respectively, and of a molecular mass of ca 60,000. 2. ASF, in nanogram amounts, inhibit intestinal secretion induced by cholera toxin, Campylobacter toxin, E. coli heat-stable toxin, C. difficile toxin A, and Dinophysis toxin. 3. Intraspinal injection of cholera toxin and glucose induces the synthesis of pituitary ASF much more effectively than does either peroral or intranasal administration. 4. Cholera toxin and glucose seem to act synergistically while inducing ASF. 5. Vagotomy abolishes both the intestinal effects of ASF and the peroral, but not the intraspinal induction of pituitary ASF. 6. ASF has no effect on ion transport across isolated intestinal mucosa from either pig or hen. 7. The results suggest that both the induction and the intestinal effects of ASF are mediated via the central and intestinal nervous system.     

Adenylate cyclase from rabbit small intestine: activation by cholera toxin and interaction with calcium

The stimulation of adenylate cyclase in various fractions of plasma membranes from rabbit small intestinal epithelium has been studied. In crude plasma membranes cholera toxin activated 5-fold at 10 micrograms/ml; vasoactive intestinal peptide (VIP) activated at concentration from 10(-8) to 10(-7) M, the maximal stimulation being 6-fold. Fluoride activated 10-fold at 10 mM. VIP-stimulated enzyme was inhibited by Ca2+ concentrations in the micromolar range. In the presence of calmodulin a biphasic response was obtained. At low Ca2+ concentration (4 x 10(-9)-6 x 10(-8) M) the enzyme was activated. As the Ca2+ concentration was increased the enzyme was concomitantly inhibited. We have investigated the mechanism by which cholera toxin activates intestinal adenylate cyclase. We have found that cholera toxin catalyzed incorporation of 32P into proteins located in the brush-border membrane whose molecular weights are in the range of 40-45kDa. These membranes bind [3H]GTP with a Kd of 1.8 x 10(-7) M. In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and [32P]NAD. The modification of brush-border membrane protein occurred in spite of the absence of adenylate cyclase in these membranes. Adenylate cyclase in basal lateral membranes was poorly activated by cholera toxin as compared to crude plasma membranes. On the other hand, the ability of VIP and fluoride to activate the enzyme was enhanced in basal lateral membranes with respect to crude membranes. The results are discussed in relation to the mechanism by which cholera toxin activates adenylate cyclase in intact intestinal cells.     

Sodium 2,3-dithiopropanesulfate blockade of the effect of cholera enterotoxin on adenylate cyclase and the concentration of cyclic 3',5'-adenosine monophosphate in the small intestine mucosa of the rabbit

Activation of adenylate cyclase in membrane preparations of the rabbit small intestine mucosa by cholera enterotoxin in the presence of sodium 2,3-dithiopropanesulfate (DTPS) is similar to that in the presence of dithiothreitol (DTT). Both DTT and DTPS do not influence the activity of adenylate cyclase without cholera enterotoxin. DTPS activates cyclic 3,5-AMP phosphodiesterase of mucosa homogenates (K0.5 = 10(-5) M). Combined administration of cholera enterotoxin and DTPS in situ into isolated loops of the rabbit small intestine decreases the activating effect of cholera enterotoxin on adenylate cyclase and diminishes the content of cyclic AMP in the mucosa. The destruction of disulfide bonds of cholera enterotoxin by DTPS is assumed to lower its ability to penetrate the mucosal cells of the small intestine.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.     

Determination of the turn-off reaction for the epinephrine-inhibited human platelet adenylate cyclase

Epinephrine inhibits human platelet adenylate cyclase by an alpha 2-adrenoceptor-mediated and GTP-dependent process. The turn-off reaction for this epinephrine-inhibited enzyme was studied by measuring the rate of cyclic AMP formation upon addition of the alpha2-adrenoceptor antagonist, yohimbine, or upon addition of an excess of the stable GDP analog, guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which competitively inhibited the action of GTP in the epinephrine-induced inhibition. The decay of the inhibited state of the adenylate cyclase was used to calculate the rate constant of the turn-off reaction. With both methods, almost identical koff values of 0.6-0.7 min-1 at 25 degrees C were obtained for the epinephrine-inhibited platelet enzyme. Cholera toxin, which does not inhibit the epinephrine-induced GTPase stimulation in platelet membranes, did not affect this turn-off reaction. In contrast, the turn-off rate of the prostaglandin-E1-stimulated human platelet adenylate cyclase, measured with GDP beta S, was reduced from about 9 min-1 to 2 min-1 at 25 degrees C by pretreatment of the membranes with cholera toxin, which inhibits the prostaglandin-E1-stimulated GTPase activity. The data strongly suggest that the guanine nucleotide regulatory site, mediating epinephrine-induced adenylate cyclase inhibition, is activated and inactivated by similar mechanisms as is the site mediating adenylate cyclase stimulation, and that cholera toxin affects only the stimulatory site. The findings furthermore suggest that the activity states of these two regulatory sites control the activity of the adenylate cyclase.     

Rat pancreas adenylate cyclase V. Its presence in isolated rat pancreatic acinar cells.

(1) In order to determine the cellular localization of the secretin- and pancreozymin-sensitive adenylate cyclase in rat pancreas, the occurence of this enzyme system has been investigated in isolated pancreatic cells. (2) Digestion of rat pancreatic lobules with collagenase yields a preparation of isolated cells which upon differential morphological analysis appears to consist for 97% of acinar cells and to contain for fewer centro-acinar and ductal cells than undissociated lobules. (3) Expressed per mg protein, the isolated cells contain the same amount of DNA, chymotrypsin and lactic dehydrogenase as the undissociated tissue. The stimulated adenylate cyclase activity is nearly entirely recovered in the isolated acinar cells, as is also the case for the low Km adenosine 3',5-cyclic monophosphate phosphodiesterase activity and the adenosine 3',5'-cyclic monophosphate (cyclic AMP) content. Marked losses are noted for the basal adenylate cyclase and the high Km cyclic AMP phosphodiesterase activities. (4) Washing the isolated acinar cells in Krebs-Ringer bicarbonate medium containing 10 mM 1-methyl-3-isobutylxanthine causes a cyclic AMP level 2.6 times that in cells washed in Krebs-Ringer bicarbonate alone. The cyclic AMP level is further increased by subsequently incubating the cells for 10 min in the presence of 3-10(-7) M pancreozymin-C-octapeptide or secretin to values 1.7 or 4.7 times the control level in cells incubated for 10 min with 1-methyl-3-isobutylxanthine alone. (5) It is suggested that the adenylate cyclase of the acinar cells may be involved, with another factor, in the stimulation of enzyme secretion, whereas a ductular cyclase would function in the regulation of the bicarbonate-dependent fluid secretion.     

Effects of cholera toxin and 5'-guanylylimidodiphosphate on human spermatozoal adenylate cyclase activity

The effects of cholera toxin and 5′-guanylylimidodiphosphate (Gpp(NH)p) on human spermatozoal adenylate cyclase activity were tested. Cholera toxin had no demonstrable effect on adenylate cyclase activity in human spermatozoa at concentrations between 5 and 20 μg/ml, whether the toxin was preincubated with intact spermatozoa between 5 min and 5 h prior the adenylate cyclase assay, or was added to lysed spermatozoa, where the adenylate cyclase would be accessible to the toxin. In contrast, Gpp(NH)p at concentrations between 10 and 100 μM was effective in activating human spermatozoal adenylate cyclase activity.