Distinct effects of the C-terminal octapeptide of cholecystokinin and of a cholera toxin pretreatment on the kinetics of rat pancreatic adenylate cyclase activity

(1) The kinetic parameters of rat pancreatic adenylate cyclase were evaluated, using GTP, p[NH]ppG or GTPγS as nucleotide activator, cholecystokinin as peptide hormone, and GDPβS and dibutyryl cyclic GMP as inhibitors of guanosine triphosphate and CCK-8, respectively. The time courses of activation and the degree of activation at steady state (E_A/E_TOT) were compatible with a simple two-state model of activation-deactivation based on a pseudo-monomolecular activation process (rate constant k₊₂, and a deactivation process (rate constant k_off) that included, depending on the activating nucleotide, the hydrolysis of GTP (rate constant k₂) and/or the dissociation of the intact nucleotide (rate constant k_?1), so that E_A/E_TOT = k₊₁/(k₊₁ + k₂ + k_?a). (2) The hormone CCK-8 increased the value of k₊₁ with GTP dose-dependently, from 0.2 to 10.9 min^?1. The value of k_?1 increased 0.01 to 0.3 min^?1 but the value of k₂ was unaltered at 7 min^?1, so that E_A/E_TOT increased 15-fold, from 4% to 61%. (3) A cholera toxin pretreatment at 30 μg/ml allowed also a large increase in E_A/E_TOT with GTP (up to 51%) but the underlying mechanism was different. It consisted of a 14-fold decrease in the k_off value of the GTP-activated enzyme (from 7 min^? to 0.5 min^?1) that corresponded to a reduction in GTPase activity. When testing the system with p[NH]ppG, two added effects of the cholera toxin pretreatment were observed: a 4-fold increase in the value of k₊₁ (from 0.2 to 0.8 min^?1) and the occurrence of a significant 0.3 min^?1 value for k_?1.     

A second guanyl nucleotide-binding site associated with adenylate cyclase. Distinct nucleotides activate adenylate cyclase and permit ADP-ribosylation by cholera toxin

There are two functionally and physically distinct types of guanyl nucleotide site associated with the adenylate cyclase system of pigeon erythrocytes. One is on the well known regulatory protein, N, that mediates the adenylate cyclase response to hormones, guanyl nucleotides and fluoride, and is the substrate for ADP-ribosylation by cholera toxin. We now describe a second site that must be occupied by GTP or an analog of GTP before N can be ADP-ribosylated. We call this second site S. It differs from the site on N in many respects. GTP appears to be rapidly hydrolyzed when it is bound to N but not when bound at S. GTP analogs such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) bind stably to both sites but the binding of GTP gamma S to N is more sensitive to EDTA and is more easily prevented by guanosine 5'-O-(2-thiodiphosphate). The nucleotide binding only to S is promoted by the cytosolic protein required by cholera toxin. Isoproterenol decreases GTP gamma S binding to S while indirectly increasing GTP gamma S binding to N. By adjusting the binding conditions, the nucleotides bound functionally to N and S can be varied independently and then the effect of ADP-ribosylation upon the adenylate cyclase activity can be seen to depend on the type of nucleotide bound to N. This activity rises, falls slightly, or remains at zero, if N is occupied by GTP, GTP gamma S, or guanosine 5'-O-(2-thiodiphosphate, respectively.     

Activation by cholera toxin of adenylate cyclase solubilized from rat liver.

Cholera toxin, or peptide A1 from the toxin, activates adenylate cyclase solubilized from rat liver with Lubrol PX, provided that cell sap, NAD+, ATP and thiol-group-containing compounds are present. The activation is abolished by antisera to whole toxin, but not to subunit B.     

Characterization of the C-terminal domain essential for toxic activity of adenylate cyclase toxin

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the RTX family of toxins. These toxins are characterized by a series of glycine- and aspartate-rich nonapeptide repeats located at the C-terminal half of the toxin molecules. For activity, RTX toxins require Ca²⁺, which is bound through the repeat region. Here, we identified a stretch of 15 amino acids (block A) that is located C-terminally to the repeat region and is essential for the toxic activity of CyaA. Block A is required for the insertion of CyaA into the plasma membranes of host cells. Mixing of a short polypeptide composed of block A and eight Ca²⁺ binding repeats with a mutant of CyaA lacking block A restores toxic activity fully. This in vitro interpolypeptide complementation is achieved only when block A is present together with the Ca²⁺ binding repeats on the same polypeptide. Neither a short polypeptide composed of block A only nor a polypeptide consisting of eight Ca²⁺ binding repeats, or a mixture of these two polypeptides, complement toxic activity. It is suggested that functional complementation occurs because of binding between the Ca²⁺ binding repeats of the short C-terminal polypeptide and the Ca²⁺ binding repeats of the CyaA mutant lacking block A.     

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.     

Activation of rat liver adenylate cyclase by cholera toxin requires toxin internalization and processing in endosomes

Involvement of acidic cell compartments in processing and action of cholera toxin (CT) in rat liver has been examined using subcellular fractionation. Liver cell fractions prepared various times after CT injection display, after a lag phase, a progressive increase in adenylate cyclase activity, detectable earlier in Golgi-endosomal fractions (20 min) than in plasma membrane fractions (30 min), with a maximum (3-fold basal activity) achieved by 60-90 min. Endosomes containing in vivo internalized CT display a time-dependent increase in their ability to bind anti-A-subunit antibodies and to stimulate exogenous adenylate cyclase, which kinetically parallels the generation of A1 peptide, suggesting a translocation of A-subunit (or A1 peptide) across the endosomal membrane. In vivo chloroquine treatment inhibits endocytosis of CT taken up into the liver, lengthens the lag phase for adenylate cyclase activation by CT, and reduces by 3- to 10-fold the apparent affinity of the toxin for the enzyme. Incubation of endosomes containing internalized toxin at 37 degrees C under isotonic conditions results in a pH-dependent increase in generation of A1 peptide, membrane translocation of A-subunit (or A1 peptide), and degradation of toxin, with a maximum at pH 5. Addition of ATP, by decreasing the internal endosomal pH, stimulates both generation of the A1 peptide and degradation of toxin at pH 6-8. It is concluded that activation of adenylate cyclase by CT in intact liver requires association and subsequent processing of toxin in an acidic cell compartment, presumably endosomal.     

Differential effects of Ca2+-calmodulin on adenylate cyclase activity cyclase activity in mouse and rat pancreatic islets.

The subcellular localization of the beta-galactoside-binding protein, or lectin, from rat lung was investigated by the specific binding of anti-lectin immunoglobulin G to subcellular fractions. We used both adult and immature (12-day-old) rats; the immature rat lungs have an 8-10-fold greater concentration than adult rat lungs [Powell & Whitney (1980) Biochem. J. 188, 1-8]. In both groups of animals we observed greater specific binding of anti-lectin immunoglobulin G to intracellular membrane (mitochondrial and microsomal fractions) than to plasma membranes. Pre-incubation of membrane fractions with lactose resulted in a marked diminution of anti-lectin immunoglobulin G binding. In the adult rat lung most (approx. 80%) of the lectin activity was membrane-associated. In the immature rat lung only approx. 30% of the lectin activity was membrane associated and most of the beta-galactoside-binding protein appeared to be a soluble cytoplasmic component. The rat lung beta-galactoside-binding protein appeared to have a broad but predominantly intracellular location, being associated with membranes through one of its galactoside-binding sites.     

Essential role of GTP in the expression of adenylate cyclase activity after cholera toxin treatment.

Expression of activation of rat liver adenylate cyclase by the A1 peptide of cholera toxin and NAD is dependent on GTP. The nucleotide is effective either when added to the assay medium or during toxin (and NAD) treatment. Toxin treatment increases the Vmax for activation by GTP and the effect of GTP persists in toxin-treated membranes, a property seen in control membranes only with non-hydrolyzable analogs of GTP such as Gpp(NH)p. These observations could be explained by a recent report that cholera toxin acts to inhibit a GTPase associated with denylate cyclase. However, we have observed that one of the major effects of the toxin is to decrease the affinity of guanine nucleotides for the processes involved in the activation of adenylate cyclase and in the regulation of the binding of glucagon to its receptor. Moreover, the absence of lag time in the activation of adenylate cyclase by GTP, in contrast to by Gpp(NH)p, and the markedly reduced fluoride action after toxin treatment suggest that GTPase inhibition may not be the only action of cholera toxin on the adenylate cyclase system. We believe that the multiple effects of toxin action is a reflection of the recently revealed complexity of the regulation of adenylate cyclase by guanine nucleotides.     

Stimulation of intestinal adenylate cyclase by cholera toxin in malnourished rats

The stimulation of intestinal adenylate cyclase by cholera toxin (CT) was studied in normal and malnourished rats 4 to 24 hr after a 30-min incubation of intestinal loops with the toxin. Whereas in control rats the enzyme activity returned to basal levels after 12 hr of incubation, in malnourished rats the activity of the enzyme remained significantly elevated even after 24 hr of the initial incubation. Malnourished animals had a reduced turnover rate of intestinal cells as determined by thymidine kinase activity. The delayed turnover of intoxicated cells may account for continuous activation of mucosal adenylate cyclase and possibly for prolongation of diarrhea in malnutrition.     

Stimulation by cholera toxin of ADP-ribosylation of membrane proteins, adenylate cyclase and insulin release in pancreatic islets

In rat pancreatic islet membranes exposed to [alpha-32P]NAD, cholera toxin stimulated the labelling of three peptides with Mr close to 22 000, 42 000 and 48 000, respectively. In the islets, the toxin-stimulated ADP-ribosylation of the heavy form of the Ns alpha-subunit predominated over that of the light form, in mirror image of the situation found in the exocrine pancreas. When intact islets were preincubated with cholera toxin, the adenylate cyclase activity of a subcellular particulate fraction was increased. The responsiveness of adenylate cyclase to GTP was also augmented, but that to NaF was decreased. In intact islets, the production of cyclic AMP and the glucose-stimulated release of insulin were also enhanced after pretreatment with cholera toxin. These findings reveal the presence in pancreatic islets of the guanyl nucleotide regulatory protein of adenylate cyclase, with an unusual predominance of the heavy form of the Ns alpha-subunit.     

In vivo activities of peptide and pseudo-peptide analogs of the C-terminal octapeptide of cholecystokinin on pancreatic secretion in the rat

Most studies measuring the agonist and antagonist activities of CCK analogs and derivatives on the exocrine pancreas have been done with in vitro models. However, extrapolation to the in vivo situation may be sometimes hazardous, due to the catabolism of the peptides by circulating and tissue peptidases, and to their eventual interaction with various endogenous factors. The present experiments were organized to measure the efficacy and potency on pancreatic secretion of the rat in vivo of a series of CCK 8 analogs whose binding and activity had been previously measured on guinea-pig and rat isolated acini. The molecules tested were derivatives of Boc-(Nle 28-Nle 31)-CCK 26–33 (1), and comprised 2-phenylethylester derivatives, des-Phe derivatives, and a series of pseudo-peptides with a “reduced” bond CH2-NH replacing the peptide bond in position 28–29 to 32–33. They were perfused in anaesthetized rats, and the outputs of sodium, bicarbonate and total protein were measured. All of the derivatives studied had in vivo the same efficacy as (1) on the output of protein, and were 10 to 500 times less potent. For most compounds, the relative order of potencies measured in vivo was similar to that measured in vitro on amylase secretion by rat acini. However, the derivatives with reduced bonds in positions 28–29 and 29–30 were respectively 3 and 2 times less potent in vivo, relative to (1), while derivatives with reduced bonds in positions 30–31, 31–32 and 32–33 were 1.5 to 2.5 times more potent in vivo. The 2-phenylethylester derivatives were 7 and 9 times as potent in vitro as in vivo. The des-Phe derivative, which had in vitro antagonist properties on guinea-pig acini, and acted like a partial agonist on rat acini, was in vivo a complete agonist and was relatively 300 times as potent in vivo as in vitro. These results indicate that the metabolism of the peptides and/or their interaction with endogenous factors may change appreciably the effect of CCK derivatives on pancreatic secretion of the rat in vivo.     

Influence of cholera toxin on the regulation of adenylate cyclase by GTP.

In the presence of NAD⁺, cholera toxin activates adenylate cyclase in membranes of S49 mouse lymphoma cells. The following evidence supports the hypothesis that the toxin acts by inhibiting a specific GTPase associated with a guanyl nucleotide regulatory component of hormone-responsive cyclase: 1. GTP alone markedly stimulates cyclase activity in toxin-treated, but not in untreated membranes; 2. The poorly hydrolyzable GTP analog, guanosine 5′-(β,γ-imino) triphosphate (Gpp(NH)p), stimulates cyclase equally well in toxin-treated and untreated membranes; 3. Cyclase activation by isoproterenol plus GTP persists in toxin-treated membranes, but not in controls, after addition of propranolol; 4. GTP is a more potent competitive inhibitor of the irreversible activation of cyclase by Gpp(NH)p in toxin-treated than in untreated membranes.     

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.     

Requirement for guanosine triphosphate in the activation of adenylate cyclase by cholera toxin

The activation of adenylate cyclase in lysed pigeon erythrocytes requires, among several cofactors, a nucleotide which may be ATP, GTP, or many other triphosphates. However, after removal of endogenous nucleotides by gel filtration or by adsorption onto charcoal the requirement can be met only by GTP, or an analog of GTP. The GTP is required during the activation of the cyclase by toxin even if GTP is also included during the subsequent adenylate cyclase assay, conducted without toxin. In the presence of GTP it is possible to assay for the cytosolic protein that is also required for the action of cholera toxin. By gel filtration, its apparent molecular weight is 15,000–20,000.     

Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase.

Cholera toxin, using [32P]NAD+ as substrate, specifically radiolabels at least two proteins in plasma membranes of wild type S49 mouse lymphoma cells. The toxin-specific substrates are detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as bands corresponding to molecular weights of 45,000 and a doublet of 52,000 to 53,000. Membranes of two other cell types exhibit similar patterns of radiolabeled bands specifically produced by incubation with cholera toxin: the "uncoupled" variant S49 cell, which possesses adenylate cyclase activity unresponsive to hormones, and the HTC4 rat hepatoma cell, which lacks detectable catalytic adenylate cyclase activity but contains components of the cyclase system necessary for regulation by guanyl nucleotides and NaF. Little or no toxin-specific radiolabeling is observed in membranes of a fourth cell type, the adenylate cyclase activity-deficient S49 variant, which functionally lacks components of the cyclase system involved in cholera toxin action and regulation by guanyl nucleotides and NaF. The toxin-specific labeling pattern is not observed in membranes prepared from wild type S49 cells previously treated with cholera toxin in culture. One or both of the toxin substrates thus appears to be involved in regulation of adenylate cyclase by guanyl nucleotides and fluoride ion.     

Alkaline phosphatases and adenylate cyclase in the normal and desensitized rat small intestine after acute cholera toxin challenge: a histochemical study

The effect of cholera toxin (CT)-challenge on histochemically demonstrable activities of adenylatecyclase and alkaline phosphatase was investigated in rat small intestine, using an intestinal loop model. CT-challenge increased the activities of adenylatecyclase and alkaline phosphatases within 15 minutes, and the changes were confined to enterocytes in the upper third parts of the villi. There was no change in the staining of the crypt cells. There was an increased basal activity of both adenylatecyclase and alkaline phosphatases in animals desensitized to cholera toxin by multiple peroral exposures. CT-challenge in the desensitized rats did not further increase the enzyme activity. It is concluded that desensitization to secretagogues induces profound alterations in the cell systems responsible for fluid secretion.     

Effect of Bordetella pertussis toxin on ADP-ribosylation of membrane proteins, adenylate cyclase activity and insulin release in rat pancreatic islets

Exposure of rat pancreatic islet membranes to [alpha-32P]-NAD+ in the presence of Bordetella Pertussis toxin (islet-activating protein) reveals the ADP-ribosylation of a peptide with a Mr close to 41 kDa, which corresponds to the alpha-subunit of the guanine nucleotide regulatory protein Ni. Islets removed from rats pretreated with the Bordetella Pertussis toxin display a specific increase in adenylate cyclase responsiveness to GTP and are characterized by a resistance to the inhibitory action of alpha2-adrenergic agonists upon either adenylate cyclase activity or glucose-induced insulin release.