Mode of action of heat-stable Escherichia coli enterotoxin. Tissue and subcellular specificities and role of cyclic GMP

Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E. coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2). We have examined the tissue spcificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP. Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP concentration throughout the small and large intestine. It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon. This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP. However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments. The enterotoxin failed to stimulate guanylate cyclase inliver, lung, pancreas or gastric antral mucosa. In the intestines, it stimulated only the particulate and not the soluble form of the enzyme. Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase. Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase. This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.     

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.     

Mode of action of heat-stable Escherichia coli enterotoxin Tissue and subcellular specificities and role of cyclic GMP

Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E. coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2). We have exmined the tissue specificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP. Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP oncentration throughout the small and large intestine. It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon. This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP. However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments. The enterotoxin failed to stimulate guanylate cyclase in liver, lung, pancreas or gastric antral mucosa. In the intestines, it stimulated only the particulate and not the soluble form of the enzyme. Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase. Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase. This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.     

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.     

Effect of cholera enterotoxin on carbohydrate metabolism of the liver and small intestine mucosa in the rabbit

Changes in carbohydrate metabolism were studied in the isolated intestinal loops of rabbits during secretory diarrhea, induced by cholera enterotoxin. Glucose synthesis level in the small intestinal mucosa and liver was measured by isotope technique, using L-alanine as a precursor. Intestinal gluconeogenesis, calculated per mg of protein, appeared to be twice higher than in the liver of fasting rabbits. Cholera enterotoxin administration enhanced gluconeogenesis in the liver by 60%, as compared to the control. The rate of glucose synthesis and glucose-6-phosphatase activity in the intestinal mucosa remained unchanged, whereas glucose-6-phosphatase in the liver was slightly inhibited. It is suggested that gluconeogenesis in the liver supplies glucose as a convenient energy source for the secretory process induced by cholera enterotoxin in the rabbit small intestine.     

Mechanism of action of ATP on intestinal epithelial cells. Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH2)p. ATP also increased the formation of cyclic [14C]AMP in enterocytes that had been preincubated with [14C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and augmenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the Isc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

Mechanism of action of ATP on intestinal epithelial cells: Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH₂)p. ATP also increased the formation of cyclic [¹⁴C]AMP in enterocytes that had been preincubated with [¹⁴C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and agumenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the I_sc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

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.     

Some comparative properties and localization of porcine jejunal adenylate cyclase.

Cholera toxin is thought to cause intestinal secretion by activating adenylate cyclase and increasing intracellular 3',5'-cyclic AMP concentrations in intestinal mucosa. Cholera toxin causes profuse secretion of fluid into ligated intestinal loops of both pigs and rabbits, but cholera toxin-induced increases in 3',5'-cyclic AMP concentration are much lower in the pig than in the rabbit. Porcine jejunal adenylate cyclase was examined for unusual properties which might account for a lack of 3'-5'-cyclic AMP accumulation after treatment with cholera toxin. The divalent cation requirements, the pH optimum, and the stimulation by fluoride ion were unremarkable. The Km for ATP was 0.11 mM with negative cooperativity indicated by a Hill coefficient of 0.83. Triton X-100 was inhibitory and guanosine diphosphate methylenephosphate stimulated enzyme activity. Adenylate cyclase activity was highest in the basal and lateral membrane fractions of jejunal mucosa and relatively low in brush-border preparations. Pretreatment of pig jejunum with cholera toxin caused a 30-40% activation of the crude and of the partly purified enzyme. A relatively low activation of adenylase cyclase in pig jejunal mucosa, compared with rabbit, may account for the absence of 3',5'-cyclic AMP accumulation after cholera-toxin treatment in the pig.     

Inhibition of guanylate cyclase and cyclic GMP phosphodiesterase by cholera toxin.

Results of cholera toxin exposure in rabbit small intestinal epithelial cells, following 4 to 6 hours of incubation, indicate that there is simultaneous dose-dependent activation of adenylate cyclase and deactivation of guanylate cyclase. In addition, cyclic GMP phosphodiesterase activity is repressed. These data indicate that cholera toxin interacts with a binding site of dissociation constant K_d=3.8±1.3 × 10^?9M to produce multiple coordinated events in the cells.     

Evidence that the mitochondrial activator of phosphorylated branched-chain 2-oxoacid dehydrogenase complex is the dissociated E1 component of the complex

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate). The half-maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon-stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 microgram/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon-stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.     

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.     

Development of the rat oocyte in vitro: inhibition and induction of maturation in the presence or absence of the cumulus oophorus

The effect in vitro of oocyte maturation inhibitors and the ability of preparations of luteinizing hormone to relieve the arrest induced by these substances was studied in preparations of cumulus-free (naked) rat oocytes, and compared to previously obtained results from oocytes enclosed by their cumulus cells. The development of both the cumulus-oocyte complex and naked oocyte is arrested in vitro by cyclic AMP derivatives or cyclic nucleotide phosphodiesterase inhibitors. While gonadotropins can overcome the effect of these substances in the cumulus oocyte complex, they have no effect on naked oocytes. Cholera enterotoxin, an irreversible activator of adenylate cyclase, maintains developmental arrest in cultured cumulus oocyte complexes but not in naked oocytes. Preparations of luteinizing hormone can partially overcome the effect of cholera enterotoxin in the complexes. Furthermore, the acceleration of oocyte maturation in vitro observed in the presence of gonadotropins, which is seen in cumulus oocyte complexes, can be mimicked by stripping the oocyte of its associated cumulus cells. The results of these and other studies suggest that: (1) the cytoplasmic levels of cyclic AMP in the isolated oocyte are high enough to maintain meiotic arrest if a phosphodiesterase inhibitor is present; (2) the oocyte contains an active phosphodiesterase; (3) the oolemma may lack the adenylate cyclase system and; (4) gonadotropins seem to affect the oocyte indirectly, via the cumulus cells, possibly by interrupting communication between the two cell types.     

Somatostatin acts through G-proteins on dopaminergic adenylate cyclase in the caudate-putamen of the rat

Somatostatin was incubated in an adenylate cyclase assay of a particulate fraction of caudateputamen tissue of the rat in order to examine the effect of the peptide on D-1 receptor coupled adenylate cyclase in vitro. Somatostatin was able to enhance cyclic AMP formation in the presence of guanylylimidodiphosphate and guanosine-triphosphate. In contrast to this, somatostatin inhibited both dopamine and forskolin-stimulated cyclic AMP accumulation. Pertussis toxin and cholera toxin also depressed forskolin-induced stimulation. Somatostatin was found to antagonize these inhibitory effects of pertussis toxin and cholera toxin. The results suggest that somatostatin acts through a stimulatory as well as an inhibitory guanine nucleotide regulatory protein subtype to affect dopaminergic adenylate cyclase activity.     

Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin. Relations to the mode of activation by hormones.

The influence of Vibrio cholerae enterotoxin (choleragen) on the response of adenylate cyclase to hormones and GTP, and on the binding of 125I-labeled glucagon to membranes, has been examined primarily in rat adipocytes, but also in guinea pig ileal mucosa and rat liver. Incubation of fat cells with choleragen converts adenylate cyclase to a GTP-responsive state; (-)-isoproterenol has a similar effect when added directly to membranes. Choleragen also increases by two- to fivefold the apparent affinity of (-)-isoproterenol, ACTH, glucagon, and vasoactive intestinal polypeptide for the activation of adenylate cyclase. This effect on vasoactive intestinal polypeptide action is also seen with the enzyme of guinea pig ileal mucosa; the toxin-induced sensitivity to VIP may be relevant in the pathogenesis of cholera diarrhea. The apparent affinity of binding of 125I-labeled glucagon is increased about 1.5- to twofold in choleragen-treated liver and fat cell membranes. The effects of choleragen on the response of adenylate cyclase to hormones are independent of protein synthesis, and they are not simply a consequence to protracted stimulation of the enzyme in vivo or during preparation of the membranes. Activation of cyclase in rat erythrocytes by choleragen is not impaired by agents which disrupt microtubules or microfilaments, and it is still observed in cultured fibroblasts after completely suppressing protein synthesis with diphtheria toxin. Choleragen does not interact directly with hormone receptor sites. Simple occupation of the choleragen binding sites with the analog, choleragenoid, does not lead to any of the biological effects of the toxin.     

Autoradiographic distribution of vasoactive intestinal polypeptide receptors in rabbit and rat small intestine

Vasoactive intestinal peptide (VIP) is found in the enteric nervous system of all layers of the small intestine. In the gastrointestinal tract, VIP receptors coupled to adenylate cyclase are present on epithelial, smooth muscle and possibly mononuclear cells. This study analyzes the distribution of VIP binding using in vitro autoradiographic techniques. VIP binding was present in high density in the mucosal layer of rabbit duodenum, jejunum and ileum. Low VIP binding was noted over the smooth muscle layers or the lymphoid follicles. Similar results were obtained in rat small intestine. The density of VIP binding was greatest in duodenal mucosa but was present in lower density in jejunal and ileal mucosa. Again, low VIP binding was noted in the smooth muscle layers or lymphoid follicles. Thus, autoradiographic maps of small intestine indicate that VIP receptors are found primarily in the small intestinal mucosa.     

Regulation of GH3 pituitary tumour-cell adenylate cyclase activity by activators of protein kinase C.

The influence of protein kinase C (PKC) activation on cyclic AMP production in GH3 cells has been studied. The stimulation of cyclic AMP accumulation induced by forskolin and cholera toxin was potentiated by 4 beta-phorbol 12,13-dibutyrate (PDBu). Moreover, PDBu, which causes attenuation of the maximal response to vasoactive intestinal polypeptide (VIP), also induced a small right shift in the dose-response curve for VIP-induced cyclic AMP accumulation. PDBu-stimulated cyclic AMP accumulation was unaffected by pretreatment of cells with pertussis toxin or the inhibitory muscarinic agonist, oxotremorine. PDBu stimulation of adenylate cyclase activity required the presence of a cytosolic factor which appeared to translocate to the plasma membrane in response to the phorbol ester. The diacylglycerol-generating agents thyroliberin, bombesin and bacterial phospholipase C each stimulated cyclic AMP accumulation, but, unlike PDBu, did not attenuate the stimulation induced by VIP. These results suggest that PKC affects at least two components of the adenylate cyclase complex. Stimulation of cyclic AMP accumulation is probably due to modification of the catalytic subunit, whereas attenuation of VIP-stimulated cyclic AMP accumulation appears to be due to the phosphorylation of a different site, which may be the VIP receptor.     

Enhanced GTP-dependent activities of the adenylate cyclase system: basis for increased hormonal responsiveness.

Normal rat kidney (NRK) cells growth arrested by picolinic acid and isoleucine deprivation exhibit an increased response to certain agents (i.e., prostaglandin E₁, (?)-isoproterenol, and cholera toxin) which elevate intracellular cyclic AMP levels. The enhanced hormonal response is apparently due, at least in part, to increased adenylate cyclase activity. Adenylate cyclase activities measured in the presence of GTP, GTP plus prostaglandin E₁, and GTP plus (?)-isoproterenol are increased two- to threefold in membranes prepared from treated cells. In contrast, basal activity is potentiated only 20 to 50% and activity determined in the presence of fluoride is only marginally altered. Also of interest is the increase in cholera toxin activation of cyclase activity in the treated cells. Lower concentrations of cholera toxin (5 ng/ml) are required to achieve maximal stimulation of cyclase activity from picolinic acid-treated and isoleucine-deprived cells; maximal stimulation of control cell adenylate cyclase is attained with 25 to 50 ng/ml cholera toxin. Picolinic acid treatment and isoleucine deficiency both have been shown to arrest NRK cell growth in the G₁ phase of the cell cycle. However, results with cells arrested in G₁ by serum starvation and by growth to high cell population density indicate that G₁ specific growth arrest does not appear to account for the increase in hormonal responsiveness. Chelation of inhibitory metals and proteolytic activation also do not appear to be involved in the mechanism by which picolinic acid enhances cyclic AMP formation. Rather, the results suggest that the treated cells have an increased amount of an active GTP-dependent function required for hormone and cholera toxin stimulation of adenylate cyclase. Thus, picolinic acid treatment and isoleucine deprivation may provide a useful means of modulating the GTP-dependent step required to potentiate hormonal responsiveness.     

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.     

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.