Absence Of Cholera Toxin Research Articles

ARF-Protein Antisense Potato Displays Stable ADP-Ribosylation of 40 kDa Protein

Summary GTP binding and ADP-ribosylation activities in transgenic ARF antisense potato tuber were analyzed. Of proteins that bind GTP, only that with a molecular mass of 20.5 kDa is ADP-ribosylated when potato extract is incubated with [32P] NAD either in the presence or absence of cholera toxin. The decrease in ARF content resulted in an increase of the 40kDa protein ADP-ribosylation and a decreasein the modification of the 42 kDA polypeptide.

Potentiation by cholera toxin of bradykinin-induced inositol phosphate production in the osteoblast-like cell line MC3T3-E1.

Cells of the osteoblastic cell line MC3T3-E1 were shown to contain at least three phosphatidylinositol-specific phospholipase C (PI-PLC) isoenzymes (PLC-beta, PLC-gamma and PLC-delta) by Western blotting analysis with various anti-PLC antibodies. Stimulation of inositol phosphate production in MC3T3-E1 cells by bradykinin (BK) occurred via a GTP-binding protein. Inositol phosphate formation on stimulation by BK was not affected by pretreatment with pertussis toxin, whereas it was potentiated by cholera toxin pretreatment. Elevation of cellular cyclic AMP levels by brief pretreatment with dibutyryl cyclic AMP or forskolin failed to enhance the BK-mediated generation of inositol phosphates, but long-term preincubation with these agents partially mimicked the action of the cholera toxin. Cholera toxin also caused an increase in BK receptor number. Cycloheximide, a protein biosynthesis inhibitor, prevented the potentiating actions of the cholera toxin and the cyclic AMP-elevating agents on BK-induced inositol phosphate production, and also inhibited the increase in BK receptor number. The specific binding of [3H]BK to the whole MC3T3-E1 cells in the presence or absence of cholera toxin was completely inhibited by the B2 BK receptor antagonist D-Arg[Hyp3,Thi5,8,D-Phe7]BK, but not by the B1 BK receptor agonist des-Arg9-BK. These data suggest that the activation of PI-PLC induced by cholera toxin in BK-stimulated MC3T3-E1 cells was caused by an enhancement of the synthesis of BK receptor protein(s), at least part of which was mediated by a sustained increase in the intracellular level of cyclic AMP.

Cholera toxin enhances alloantigen presentation by cultured intestinal epithelial cells.

In the present study we show that cholera toxin (CT) strongly potentiates antigen presentation by intestinal epithelial cells, probably by enhancing co-stimulation. This was demonstrated in an allogeneic system using cells from the IEC-17 rat epithelial cell line as antigen presenting cells (APC). These cells were induced by optimal concentrations of IFN-gamma to express good amounts of Ia antigen and cultured for 24-48 h in the presence or absence of CT. Thereafter the cells were thoroughly washed and added to cultures containing MHC-incompatible spleen cells as responder cells. Epithelial cells exposed to CT demonstrated greatly enhanced ability to trigger allogen-specific T-cell proliferation as compared with IEC-17 cells treated with IFN-gamma alone. The mechanism for the enhanced APC function was investigated by analysing CT-treated IEC-17 cells for increased class II MHC antigen expression or enhanced production of cytokines with known co-stimulatory function. We found no significant increase in class II MHC antigen expression. By contrast, CT strongly promoted, in a dose-dependent fashion, the production of both IL-1 and IL-6 cytokines by IEC-17 cells as compared with untreated epithelial cells. This effect of CT was specific and not due to contaminating endotoxin because excess amounts of soluble toxin receptor, ganglioside GM1, added to the IEC-17 cultures completely abrogated the cytokine response to CT. These results together with our previous findings of enhanced antigen presentation by macrophages stimulated by CT suggest that the potent adjuvant function of CT for induction of mucosal immune responses might be attributed to an enhanced co-stimulating ability of several putative APC in the mucosal immune system: macrophages, B cells and epithelial cells.

Mitogenic, melanogenic, and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens.

The following studies have been undertaken to compare and correlate the effects of 12-O-tetradecanoylphorbol acetate (TPA), basic fibroblast growth factor (bFGF), cholera toxin (CT), and isobutyl methylxanthine (IBMX) on neonatal human melanocyte (NHM) proliferation, tyrosinase activity, and cyclic adenosine monophosphate (cAMP) concentration. NHM proliferated at a maximal rate in medium containing 8 nM TPA, 200 ng/ml CT, and 10(-4) M IBMX. TPA alone did not result in optimal melanocyte proliferation, and, as previously shown, its mitogenic effect was greatly enhanced by the addition of CT and IBMX individually or concomitantly. Human recombinant (hr) bFGF could replace TPA in the NHM growth medium. Maximal proliferation was achieved using 3 ng/ml hrbFGF, 20 ng/ml CT, and 10(-4) M IBMX. The mitogenic effect of 1.2 ng/ml hrbFGF was potentiated in the concomitant but not individual presence of CT and IBMX. TPA alone in the absence of CT and IBMX caused a dose-dependent stimulation of tyrosinase activity. Maximal tyrosinase activity was obtained in the presence of 0.8 nM TPA, 20 ng/ml CT, and 10(-4) M IBMX. Unlike TPA, hrbFGF alone resulted in inhibition of tyrosinase activity. In the presence of hrbFGF, tyrosinase activity was potentiated by CT and IBMX, but not by CT alone. Neither TPA nor hrbFGF alone could increase intracellular cAMP levels. The effects of CT and IBMX on intracellular cAMP concentration were enhanced to a greater extent by TPA than by hrbFGF. Under our experimental conditions, in the presence of hrbFGF, CT but not IBMX resulted in a dose-dependent increase in cAMP concentration. Further studies on NHM will be aimed at determining the exact role of protein kinase C (PKC) in regulating proliferation and melanogenesis and the mechanism(s) activated by hrbFGF.

Modification of the function of pertussis toxin substrate GTP-binding protein by cholera toxin-catalyzed ADP-ribosylation.

The alpha-subunit of Gi-2, in addition to that of Gs (GTP-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in HL-60 cell membranes when a chemotactic receptor was stimulated by formyl-Met-Leu-Phe (fMLP), and the sites modified by cholera and pertussis toxins on the alpha-subunit of Gi-2 were different (Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 21394-21400). In order to investigate how the functions of Gi-2 were modified by cholera toxin, the ADP-ribosylated and unmodified proteins were purified from HL-60 cell membranes that had been incubated in the presence and absence of cholera toxin, respectively. The modified Gi-2 displayed unique properties as follows. 1) The ADP-ribosylated alpha-subunit had a more acidic pI than the unmodified one, leading to a partial resolution of the modified Gir2 trimer from the unmodified protein by an anion column chromatography. 2) When the purified proteins were incubated with [gamma-32P]GTP, the radioactivity was more greatly retained in the modified Gi-2 than in the unmodified protein. 3) The actual catalytic rate (kcat) of GTP hydrolysis was, indeed, markedly inhibited by cholera toxin-induced modification. 4) There was an increase in the apparent affinity of Gi-2 for GDP by cholera toxin-induced modification. 5) The modified Gi-2 exhibited a low substrate activity for pertussis toxin-catalyzed ADP-ribosylation. 6) A high-affinity fMLP binding to HL-60 cell membranes was more effectively reconstituted with the ADP-ribosylated Gi-2 than with the unmodified protein. These results suggested that the agonist-fMLP receptor complex was effectively coupled with the ADP-ribosylated Gi-2, resulting in the GTP-bound form, and that the hydrolysis of GTP on the modified alpha-subunit was selectively attenuated. Thus, cholera toxin ADP-ribosylated Gi-2 appeared to be not only a less sensitive pertussis toxin substrate but also an efficient signal transducer between receptors and effectors.

Enhancement of cyclic AMP metabolism in a B cell line by protein kinase C

In a human B cell line in which we previously demonstrated an inverse relationship between cyclic adenosine monophosphate (cAMP) content and immunoglobulin secretion, the phorbol ester, phorbol myristate acetate, (PMA), was shown to augment the cAMP elevating ability of cholera toxin (CT), suggesting a regulatory linkage between the two transmembrane signaling pathways, cAMP and phospholipid ( J. Immunol. 141, 1678–1686, 1988). We now extend these studies and provide additional evidence that activated protein kinase C, a principal product of the activation of the hydrolytic phospholipid pathway, plays a direct role in the augmentation of cAMP levels in cells stimulated by diverse cAMP-elevating ligands. Prostaglandin E 1 (PGE 1), forskolin (FSK) and CT, all of which demonstrated a concentration and time-dependent elevation of intracellular cAMP, produced even greater (up to twofold) elevations of cAMP in the presence of PMA or the diacylglycerol analogs, 1, 2-dioctanoylglycerol (DiC 8), and 1-oleoyl-2-acetylglycerol (OAG). In the absence of CT, PGE 1, or FSK, these protein kinase C activators produced only small increases in cAMP content of the cells. Several tests of protein kinase C specificity in these PMA-, DiC 8-, and OAG-induced augmentations were made: (i) only phorbol esters known to activate protein kinase C worked, (ii) PMA augmentation was abolished by down-regulation of protein kinase C, (iii) Staurosporine (a known inhibitor of protein kinase C) selectively inhibited the effects of PMA on cAMP generation and on immunoglobulin secretion in the LA350 cell line.

Influence of dietary omega-3 fatty acids on transmembrane signalling in rat submandibular salivary gland

We have previously shown the incorporation of dietary ω-3 and ω-6 fatty acids from menhaden oil and corn oil, respectively, into membrane phospholipids of submandibular sllivary gland and (SMSG) of rat [Alam S. Q. and Alam B. S. (1988) Arch. Oral Biol. 33, 295–299]. We now demonstrate the influence of such incorporation on the regulation of G proteins and adenylate cyclase activity. Cholera toxin ribosylated three protein peptides (M r 42,000, 44,000 and 46,000) to different extents in the two groups. We found 4.9- and 2.6-fold higher and 0.4-fold lower ribosylation of M r 42,000, 44,000 and 46,000 peptides, respectively, in SMSG membranes of rats fed a diet containing 10% menhaden oil (group II) compared to those fed 10% corn oil (group I). Functional distinctions between different forms of these peptides are not known. Cholera toxin also exhibited radiolabelling of three peptides in the SMSG membranes from normal or fasting rats. In these membranes inhibitory G proteins were not detected by pertussis toxin dependent ADP ribosylation or by a low concentration of guanylyl 5-imidodiphosphate (10 −8 M), which selectively activates inhibitory G proteins which inhibit forskolin stimulated activity of adenylate cyclase. In group II membranes both basal and fluoride stimulated activities of adenylate cyclase were found to be significantly higher than the corresponding values in group I ( P < 0.02). In cholera toxin dependent ribosylated membranes of group I, basal and fluoride stimulated activities of adenylate cyclase were significantly higher than those obtained in the absence of cholera toxin ( P < 0.02). Surprisingly, corresponding values were found to be lower in ribosylated membranes of group II. This could be due either to conformational changes in heavily ribosylated G proteins, which alters coupling with the catalytic subunit of adenylate cyclase, or due to dissociation of excessive inhibitory βγ complex from αβγ complex upon the activation of G proteins.

Variable responsiveness of rat tracheal epithelial cells to bovine serum albumin in serum-free culture.

The colony-forming efficiency of rat tracheal epithelial (RTE) cells was determined in serum-free media containing different types of commercially available bovine serum albumin (BSA): crude fraction V, essentially globulin-free, essentially fatty-acid-free, and essentially globulin- and fatty-acid-free BSA. RTE cells exhibited a concentration-dependent increase in colony-forming efficiency in response to crude fraction V BSA. Similar results were obtained using essentially globulin-free BSA. However, deletion of cholera toxin from the medium resulted in a decrease in the colony-forming efficiency for cells plated in high concentrations (greater than 2 mg/ml) of globulin-free, but not one type of fraction V, BSA. Essentially fatty-acid-free or essentially fatty-acid- and globulin-free BSA stimulated RTE cell colony formation at low concentrations (less than 2.5 to 5 mg BSA/ml) but resulted in concentration-dependent decreases in colony-forming efficiency at higher concentrations. The response of cells to these BSAs was not dependent on cholera toxin. Finally, commercially available fraction V BSA prepared by heat shock, dialysis, charcoal treatment, and deionization was stimulatory at low concentrations but inhibitory at high concentrations. These data suggest that impure preparations of BSA can, under different conditions, stimulate or inhibit cell proliferation and that the expression of these activities is affected by the method of BSA preparation, the concentration of BSA used, and, in some cases, by the presence or absence of cholera toxin.

Effects of cholera toxin on gene expression in brown preadipocytes differentiating in culture.

To investigate the cellular control of the recruitment process in brown adipose tissue, the ability of cholera toxin to influence the differentiation of brown preadipocytes developing in culture was investigated. Stromalvascular cells obtained from the brown adipose tissue of 3-wk-old rats were grown in culture for 6-7 days in the presence or absence of cholera toxin. It was found that cholera toxin treatment decreased the expression of the actin gene (indicating an increased degree of differentiation), while at the same time promoting the expression of the genes coding for the mitochondriogenesis marker cytochrome-c oxidase and for the adipocyte conversion marker lipoprotein lipase (all followed at the mRNA level). Chronic cholera toxin treatment also increased the total amount of protein per cell in culture, and a specific cholera toxin-induced 35-kDa protein was identified. It was concluded that (in contrast to the case suggested for white preadipocytes) cholera toxin treatment of brown preadipocytes may not only affect the activity of catabolic enzymes but may also directly promote the differentiation process, indicating that this process is under beta-adrenergic control in the adapting animal.

Studies concerning a GTP regulatory subunit of rat luteal adenylate cyclase

The ADP-ribosylation of rat luteal membrane proteins has been investigated. In the presence of cholera toxin two membrane proteins, M r 115,000 and 46,000, incorporated [ 32P]ADP-ribose from [ 32P]NAD +. The larger protein also incorporated [ 32P]ADP-ribose in the absence of cholera toxin. The smaller protein was identified as the guanine nucleotide regulatory protein of adenylate cyclase. To facilitate studies concerning this species, a simple and convenient method of measuring ADP-ribose incorporation into the protein was developed. Levels of the protein were found to be approximately equal to those of the hCG receptor and 7- to 10-fold higher than those of the β-adrenergic receptor. Luteinization of rat ovaries by injection of hCG indicated that G F concentrations increased approximately 2-fold over a 5- to 11-day period and correlated significantly with increased β-adrenergic receptors and β-adrenergic and NaF-stimulated adenylate cyclase, but not with hCG receptors or hCG-stimulated adenylate cyclase. The distribution of the G F protein in purified luteal membrane preparations mimicked the distribution of adenylate cyclase activity. No evidence could be found for hormonally induced alterations in ADP-ribose incorporation into this protein under either ribosylation or adenylate cyclase conditions. The exact role of the protein in the activation of rat luteal adenylate cyclase has yet to be determined.

Cholera toxin and dibutyrl cyclic-AMP stimulate the growth of epithelial cells derived from epithelial rests from porcine periodontal ligament

The epithelial cells (E-cells) grew best at high (> 5 per cent) concentrations of fetal bovine serum (FBS). Growth could be obtained at low concentrations of dialysed FBS (DFBS) if the medium (α-MEM) was modified so that the levels of Ca 2+ and K + were reduced to 0.1 and 1.0 mM, respectively (β-MEM). The addition of 0.5 per cent DFBS to the β-MEM did not initiate good growth but was sufficiently supportive to enable the effects of various growth promoters to be tested. Cholera toxin and dibutyrl cyclic-3′5′-adenosine monophosphate (Bt 2cAMP), which are known to increase intracellular cAMP levels, at concentrations of 1 ng/ml and 0.5 mM, respectively increased cell number. Cholera toxin caused the E-cells to be more flattened when viewed by phase-contrast; this appeared to be due to spread of the cells. No difference in cell-size distributions obtained between the trypsinized E-cells grown in the presence or absence of cholera toxin was observed. Epithelial proliferation that occurs in dental cysts could result from a rise in intracellular cAMP levels in epithelial cell rests.

Cholera toxin does not prevent neurite outgrowth from adult human chromaffin cells in culture

Adult human adrenal medullary cells were dissociated and cultured for 14 days in the presence or absence of cholera toxin (CT), an activator of adenyl

Cyclic AMP-mediated control of meiosis: effects of progesterone, cholera toxin, and membrane-active drugs in Xenopus laevis oocytes.

Progesterone depressed rapidly (50% at 1 min) and persistently cyclic AMP (cAMP) concentration that had been elevated by cholera toxin in Xenopus laevis oocytes. cAMP remained below 1 pmol per oocyte (mean basal level) for approximately 1 hr and thereafter rose to approximately 120% of control values, while germinal vesicle (nucleus) breakdown did not occur. In the absence of cholera toxin, progesterone treatment for 6 hr maintained cAMP concentration below the basal level (but not lower than 80%), and germinal vesicle breakdown occurred. Experiments in the presence of phosphodiesterase inhibitors suggested that progesterone modulates adenylate cyclase activity. The maturation promoting factor, which is formed after 3-5 hr of progesterone treatment and provokes germinal vesicle breakdown after its injection into untreated oocytes, also decreased cAMP concentration, an observation that may explain its "autoamplification." Nonsteroidal inducers of meiosis reinitiation (e.g., propranolol, methoxyverapamil, mersalyl) diminished the cholera toxin-mediated accumulation of cAMP, in contrast to compounds devoid of meiotic-inducing capacity and antagonist to progesterone action, such as gammexane (an inositol analogue) and 5'-deoxy-S-(2-methylpropyl)-5'-thioadenosine (a methylase inhibitor), that increased the nucleotide level. The fine control, suggested by the effects of small changes in cAMP levels, gives evidence of great sensitivity to a critical determinant governing meiotic cell division.

Regulatory states of adenylate cyclase in RL-PR-C cloned rat hepatocytes

The adenylate cyclase of cloned differentiated rat hepatocytes (RL-PR-C) is regulated by cholera toxin, guanine nucleotides and fluoride. The activation of hepatic adenylate cyclase by cholera toxin was additive with that by GTP and synergistic with that by epinephrine. In contrast, when membranes were exposed to cholera toxin in the presence of Gpp(NH)p or fluoride, the response was the same as to these agents in the absence of cholera toxin. Cholera toxin-activated membranes were responsive only to epinephrine and GTP, while fluoride-activated membranes responded somewhat to all other agents, and Gpp(NH)p-activated membranes responded to no other agents. These data suggest that responsiveness of hepatic adenylate cyclase to cholera toxin, fluoride and Gpp(NH)p cannot be expressed simultaneously. A model is presented to explain these observations which invokes multiple state of adenylate cyclase, each being sensitive to, or brought about by, a different regulatory agent.