The effect of heat-stable Escherichia coli enterotoxin, theophylline and forskolin on cyclic nucleotide levels and mucosal surface (acid microclimate) pH in rat proximal jejunum in vivo

The normally acidic mucosal surface pH of 6.24 +/- 0.02(30) in rat proximal jejunum in vivo is effectively neutralised by 30 min exposure to heat-stable Escherichia coli (STa) enterotoxin (14 micrograms/ml) to 6.80 +/- 0.07 (n = 5) or to a forskolin/theophylline combination (1 mM:20 mM) to 7.10 +/- 0.07(7) while perfusion with Krebs-phosphate buffer alone without glucose left the mucosal surface pH unchanged at a pH of 6.21 +/- 0.02(9). Forskolin alone had no effect, and 20 mM theophylline moderately elevated the surface pH to 6.52 +/- 0.03(5). Theophylline, forskolin and their combination all elevated cAMP levels per mg tissue DNA above control values while STa enterotoxin was without effect. In contrast, all agents elevated cGMP levels per mg tissue DNA above control levels. These findings indicate that surface pH is only moderately affected by changes in cAMP levels and is affected to a much greater extent by altered cGMP levels.     

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.     

Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.     

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.     

Effects of guanylin and uroguanylin on rat jejunal fluid and electrolyte transport: comparison with heat-stable enterotoxin

The effects of rat guanylin, human guanylin, human uroguanylin and STa on net fluid and electrolyte transport in the closed jejunal loop were compared in anesthetized rats. STa administered into the lumen caused a concentration-dependent (10(-8) to 10(-6) M) inhibition of net fluid and NaCl absorption in the jejunal loop. Uroguanylin had a similar but weaker effect than STa. Both rat and human guanylin inhibited fluid and NaCl absorption only at 10(-6) M. Their order of potency was STa > human uroguanylin > rat guanylin = human guanylin. Changing the luminal pH from 5 to 8 failed to affect the action of guanylin on fluid absorption. Both STa and uroguanylin, but not guanylin, increased the luminal pH by stimulating bicarbonate secretion. Pretreatment of the jejunal loop with guanylin (10(-6) M) 5 min before the instillation of STa (10(-7) M) significantly reduced the inhibitory effect of STa on fluid absorption. It is concluded that guanylin and uroguanylin administered into the rat jejunal lumen have an STa-like action on fluid and electrolyte transport. Guanylin may act as an endogenous antagonist of STa in the rat jejunum and prevent excessive fluid loss by STa.     

Effects of forskolin and cholera toxin on cyclic AMP release in a neurotensin-secreting rat C-cell line

The effects of forskolin and cholera toxin on the regulation of cAMP release were studied in a neurotensin-secreting rat C-cell line. The interaction of these agents with norepinephrine, a potent neurotensin secretagogue, was also investigated. Forskolin stimulated cAMP release 10(2)-10(3) fold while it increased neurotensin release 2-3 fold. Cholera toxin caused a 10(2)-10(3) fold increase in cAMP release and had no effect on neurotensin release. We conclude that the 44-2 C-cells provide a new model for studying the regulation of the concomitant (via forskolin) or independent (via cholera toxin) secretion of cyclic AMP and/or neurotensin.     

Human platelets are defective in processing of cholera toxin.

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.     

Relationship between endogenous cyclic AMP production and steroid hormone secretion in chick adrenal cells

Dispersed chick adrenal cells were incubated with either ACTH, cholera toxin or forskolin. All three agents stimulated cyclic AMP accumulation and secretion of corticosterone and aldosterone by the dispersed cells. The dose-response to ACTH was similar for cyclic AMP and corticosterone but aldosterone secretion appeared to be more sensitive to ACTH stimulation. Concentrations higher than 10(-8) M of ACTH caused suppression of corticosterone output but not of cyclic AMP accumulation or aldosterone secretion. A significant cyclic AMP accumulation occurred within 30 min of exposure to ACTH whereas significant increases in steroid secretion were observed only after 30 min. An early increase (within 30 min) in cyclic AMP accumulation with both cholera toxin and forskolin was not accompanied by any significant stimulation of steroid secretion, which occurred only after 120 min. The results with the avian adrenal cells are consistent with the thesis that steroid production in the adrenocortical cells is stimulated by cyclic AMP-dependent pathways, whereas steroid release may be modulated by others.     

Increase in Gs and cyclic AMP generation in HIT cells. Evidence that the 45-kDa alpha-subunit of Gs has greater functional activity than the 52-kDa alpha-subunit

Cyclic AMP accumulation in response to forskolin, cholera toxin, or isoproterenol is dramatically increased in HIT T-15 cells, a clonal cell line of Syrian hamster pancreatic islet beta cells, as a function of passage number. Forskolin and cholera toxin elevate cyclic AMP levels 5- to 10-fold higher in later passages (87-100) than in earlier passages (70-80). A similar phenomenon is observed with isoproterenol (10 microM) which increases cyclic AMP levels 56-fold in older HIT cells (passage 94), whereas only marginally stimulating cyclic AMP production in younger cells (passage 70-82). To determine whether a change in the stimulatory or inhibitory guanine nucleotide regulatory proteins, Gs or Gi, was responsible for these observations, ADP-ribosylation of HIT cell membranes with cholera toxin and pertussis toxin was examined. All passages contained two cholera toxin substrates at 52 and 45 kDa. The amount of 52 kDa did not appear to change with passage number, but the amount of 45 kDa increased in the later passages (89 and 94). The ratio of 45 to 52 kDa cholera toxin substrate, as determined by densitometric analysis, increased from 0.1 in passages 70, 75, and 82 to 0.45 at passage 89. No passage related changes in a 40-kDa pertussis toxin substrate were observed. An increase in the amount of the 45-kDa alpha-subunit of Gs was confirmed on immunoblots using antisera specific for the alpha-subunits of Gs. The amount of functional Gs present in various HIT cell passages was examined by determining the extent to which extracts from HIT cell membranes reconstituted guanine nucleotide-sensitive adenylyl cyclase in S49 cyc- membranes. Extracts derived from passage 94 reconstituted three to four times more adenylyl cyclase activity in cyc- membranes than extracts from passages 70, 75, and 82. These data indicate that an increase in functional Gs in later passages may be the underlying cause for the increased responsiveness to isoproterenol and forskolin in later passages. These data also suggest that functional differences exist between the Gs alpha-subunits, with the smaller 45-kDa subunit being more efficacious in coupling to cyclic AMP synthesis than the larger 52-kDa subunit. This is a departure from the commonly held view that the two subunits have similar efficacies in stimulating adenylyl cyclase.     

Potentiation of cholera toxin-stimulated cyclic AMP production in cultured cells by inhibitors of RNA and protein synthesis

Cyclic AMP increased 8- to 10-fold after a 3-h treatment with 6 nM cholera toxin in rat C6-2B astrocytoma cells. In the presence of cycloheximide, cholera toxin increased intracellular cyclic AMP about 50-fold. Qualitatively similar potentiation of cholera toxin action by cycloheximide was observed in isolated swine aortic vascular smooth muscle cells. Cycloheximide, by itself, had no effect upon cyclic AMP levels and did not alter the apparent Ka for cyclic AMP generation by cholera toxin in the cells. Also, cycloheximide did not appear to augment cholera toxin action via inhibition of cyclic nucleotide phosphodiesterase. Puromycin and actinomycin D also augmented cholera toxin action in C6-2B cells. Potentiation of cholera toxin-increased cyclic AMP formation by cycloheximide was correlated with the inhibition of [14C]leucine incorporation into protein. These results indicate that the ability of cholera toxin to stimulate cyclic AMP production in C6-2B astrocytoma and swine vascular smooth muscle cells is enhanced by inhibition of de novo protein synthesis.     

Increased Intracellular Cyclic AMP Differentially Modulates Nerve Growth Factor Induction of Three Neuronal Recognition Molecules Involved in Neurite Outgrowth

The relative expression of the immunoglobulin superfamily members Thy-1 and L1 and the neural cell adhesion molecule (N-CAM) in PC12 cells grown in the presence of nerve growth factor (NGF), cholera toxin, or both has been quantified. Whereas NGF treatment induced increases in the cell surface expression of all three glycoproteins, treatment with cholera toxin resulted in the specific induction of L1. During the first few days of culture, cholera toxin acted synergistically with NGF to promote increases in neuritic outgrowth and the synthesis and cell surface accumulation of the 140- and 180-kilodalton subunits of N-CAM. In contrast, over the same period of culture, cholera toxin inhibited the NGF induction of Thy-1 and L1. Over longer periods of culture (3-5 days), cholera toxin inhibited the NGF induction of N-CAM and neurite outgrowth. A similar pattern of synergistic and inhibitory responses was observed when differentiation was induced by fibroblast growth factor (FGF) rather than NGF or when cholera toxin was replaced with forskolin. These data suggest that intracellular cyclic AMP can differentially modulate cell surface glycoprotein expression induced by either NGF or FGF. Of the three cell surface glycoproteins we have studied, temporal changes in N-CAM expression correlate best with the morphological differentiation status of PC12 cells.     

Cyclic AMP agonists induce the phosphorylation of phospholipase C-tau and of a 76 kDa protein co-precipitated by anti-(phospholipase C-tau) monoclonal antibodies in BALB/c-3T3 cells. Relationship to inositol phosphate formation.

Previous studies have demonstrated enhanced phosphorylation of phospholipase C-tau (PLC-tau), a key regulatory enzyme in phosphoinositide metabolism, in cells treated with platelet-derived growth factor (PDGF) and epidermal growth factor, both of which act via specific receptor tyrosine kinases. Our studies on BALB/c-3T3 cells show that agents that promote cellular cyclic AMP accumulation also increase the phosphorylation, specifically the serine phosphorylation, of this enzyme. Increased phosphorylation of PLC-t (2-3-fold) was evident within 5-10 min of addition of isobutylmethylxanthine (IBMX) and either cholera toxin or forskolin to cells, and persisted for at least 3 h. Treatment of cells with cyclic AMP agonists also enhanced, with similar kinetics, the phosphorylation of a 76 kDa protein co-precipitated by anti-PLC-tau monoclonal antibodies. Brief exposure of cells to cholera toxin/IBMX or forskolin/IBMX decreased inositol phosphate formation induced by the GTP-binding protein (G-protein) activator aluminium fluoride by approx. 50%, but was without effect on PDGF-stimulated inositol phosphate formation. These findings suggest that PLC-tau, and perhaps the 76 kDa co-precipitated protein, are substrates of cyclic AMP-dependent protein kinase in BALB/c-3T3 cells: however, the lack of effect of cyclic AMP elevation on PDGF-stimulated inositol phosphate formation indicates that the intrinsic activity of PLC-tau is unaltered by cyclic AMP-mediated phosphorylation.     

Sodium-dependent phosphate transport inhibited by parathyroid hormone and cyclic AMP stimulation in an opossum kidney cell line

In the present study we investigated the characteristics of the transport of inorganic phosphate (Pi) in an opossum kidney cell line endowed with parathyroid hormone (PTH) receptors. In confluent epithelial cell culture, a Na-dependent Pi transport (NaPiT) was identified. Preincubation for 1 h with bovine (b)PTH(1-34) at 10(-7) M inhibited the NaPiT from 2.76 +/- 0.11 to 1.08 +/- 0.10 nmol/mg protein X 2 min-1 (p less than 0.001). This inhibition was already expressed 5 min after exposure to 10(-7) M bPTH. It was associated with a 4-fold increase in cellular cyclic AMP. The NaPiT was significantly inhibited at 10(-9) M bPTH, a hormonal concentration which stimulated the cellular cyclic AMP by only 30%. Kinetic analysis of the NaPiT inhibition by 10(-7) M bPTH revealed a decrease in Vmax (from 4.14 +/- 0.32 to 2.41 +/- 0.14 nmol/mg protein X 2 min-1) with no change in Km (0.093 +/- 0.016 versus 0.094 +/- 0.012 mM). The effect of bPTH on NaPiT was not associated with a change in the Na-dependent glucose methylglucopyranoside transport also present in the opossum kidney cell line. The inhibitory influence of bPTH on NaPiT was not affected by blockage of new protein synthesis by cycloheximide. Stimulation of cyclic AMP production by 10(-5) M forskolin, 10 micrograms/ml cholera toxin, 10(-5) M prostaglandin E2 or addition of 10(-5) M dibutyryl cyclic AMP mimicked the PTH-induced reduction in NaPiT. In conclusion, the present study indicates that the opossum epithelial cell line is endowed with a Na-dependent Pi transport system which is selectively inhibited by PTH and agents which increase cyclic AMP production.     

Cholera toxin-induced changes in force of contraction and cyclic AMP levels in canine ventricular myocardium: Inhibition by carbachol

Cholera toxin (1–10 μg/ml) had a biphasic inotropic action on the isolated canine ventricular muscle: it produced a transient negative and a long lasting positive inotropic effect. The negative effect reached a maximum 43 + 2 min (n = 12) after administration of the toxin, while it took 3–5 hrs for the positive effect to reach a steady level. The positive inotropic effect of cholera toxin was accompanied by a prominent abbreviation of the time to peak tension and the relaxation time of individual contractions. The level of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) of the tissue was elevated by cholera toxin in a time- and concentration-dependent manner. Carbachol (1 μmol/l) administered 3 or 5 hrs after the administration of cholera toxin (10 μg/ml) reversed the increase in force of contraction and the elevation of cyclic AMP levels induced by cholera toxin. These results indicate that cholera toxin exerts a cyclic AMP-dependent positive inotropic effect and a negative inotropic effect which is not related to cyclic AMP levels in canine ventricular myocardium.     

Influence of bacterial toxins and forskolin upon vasopressin-induced inositol phosphate accumulation in WRK 1 cells.

The accumulation of inositol phosphates in WRK 1 cells, stimulated with a range of vasopressin concentrations, was diminished by prior exposure to cholera toxin or forskolin, whilst that observed in the presence of maximal concentrations of the hormone was enhanced in pertussis-toxin-treated cells. In the presence of [32P]NAD+, both cholera toxin and pertussis toxin provoked the labelling of peptides with approximate Mrs of 45,000 and 41,000 respectively in the membranes of WRK 1 cells. Exposure to cholera toxin or forskolin for 15-18 h enhanced cyclic AMP accumulation in these cells. The concentrations of these agents which provoked half-maximal cyclic AMP accumulation were similar to those required to diminish receptor-mediated inositol phosphate accumulation by 50%. In contrast, half-maximal ADP-ribosylation of the 45,000Mr peptide needed 100-fold greater concentrations of the toxin than were effective in provoking half-maximal inhibition of inositol phosphate accumulation. Cholera toxin or forskolin also reduced the maximal specific binding, to intact WRK 1 cells, of both [3H][Arg8]vasopressin and the V1a antagonist [3H][beta-mercapto-beta,beta-cyclopentamethylenepropionic acid,O-methyl-Tyr2, Arg8]vasopressin. The kinetics for the loss of this binding capacity following cholera-toxin treatment were very similar to those describing the diminution of vasopressin-stimulated inositol phosphate accumulation in the same cells.     

A cholera toxin substrate regulates cyclic GMP content of rat pinealocytes

The adrenergic regulation of cyclic GMP in isolated pinealocytes was investigated. In this cell, norepinephrine stimulates cyclic GMP and cyclic AMP greater than 100-fold by activating both alpha 1- and beta-adrenoceptors. beta-Adrenergic activation is a requisite event and is potentiated by alpha 1-adrenergic activation (Vanecek, J., Sugden, D., Weller, J. L., and Klein, D. C. (1985) Endocrinology 116, 2167-2173). The current study found that cholera toxin could substitute for beta-adrenergic agonists in stimulating pinealocyte cyclic GMP content, as has been found to be the case for cyclic AMP. Treatment with cholera toxin alone (1 microgram/ml for 90 min) had a small effect (2- to 4-fold increase) on cyclic GMP; addition of the alpha 1-adrenergic agonists, phenylephrine, cirazoline, or methoxamine to cholera toxin-treated cells rapidly (peak at 5 min) caused a further 30- to 300-fold increase. The alpha 1-adrenergic agonists had little effect by themselves at concentrations which potentiated the effects of cholera toxin. The potentiating effect of phenylephrine was inhibited nearly completely by an alpha 1-adrenergic antagonist, but not by either an alpha 2- or beta-adrenergic antagonist. The purified cholera toxin subunits A and B did not stimulate cyclic GMP either alone or in the presence of phenylephrine. Furthermore, the potentiating action of phenylephrine was observed following 90 min but not 20 min of cholera toxin pretreatment. these results suggest that the regulation of cyclic GMP levels in the pineal gland involves an Ns-like GTP-binding regulatory protein. This is of interest because it is the first indication that cyclic GMP is regulated by such a GTP-binding protein in nonretinal tissue. It remains to be determined whether the mechanisms involved in the transmembrane regulation of cyclic AMP and cyclic GMP in any other tissue are similar.     

Na+-dependent elevation of the acidic cell surface pH (microclimate pH) of rat jejunal villus cells induced by cyclic nucleotides and phorbol ester: possible mediators of the regulation of the Na+/H+ antiporter

The effects of cyclic nucleotides and phorbol ester on the acidic cell surface pH of rat jejunal villi were studied by using single-barrelled pH-sensitive microelectrodes. Addition of dibutyryl cAMP (1 mM) to the mucosal bathing solution caused an elevation of the cell surface pH from 6.19 +/- 0.04 (n = 12 measurements from three animals) to 6.53 +/- 0.03 (12) in the presence of Na+ in the medium. However, dibutyryl cAMP had no significant effect in the absence of Na+ and presence of 1 mM amiloride. Dibutyryl cGMP (1 mM) also had an Na+-dependent inhibitory effect on the cell surface pH. A phorbol ester, phorbol 12-myristate 13-acetate, caused an elevation of the cell surface pH only in the presence of Na+ from 6.14 +/- 0.07 (12) to 6.46 +/- 0.08 (12). Phorbol and phorbol 13-acetate, which do not stimulate protein kinase C, were without significant effects. These results suggest that increased levels of the intracellular cyclic nucleotides and activation of protein kinase C raise the acidic cell surface pH by inhibiting the activity of the brush-border Na+/H+ antiporter in the rat jejunal villus cells.     

Elevated intracellular concentrations of cyclic AMP inhibited serum-stimulated, density-arrested BALB/c-3T3 cells in mid G1

The stimulation of DNA synthesis in quiescent, density-arrested BALB/c-3T3 cells by platelet-derived growth factor in plasma-supplemented medium was inhibited by the presence of isobutylmethylxanthine (IBMX) and cholera toxin, although neither IBMX or cholera toxin when used alone inhibited the stimulation of DNA synthesis. The cells were reversibly inhibited in mid G1 at a point 6 hr prior to the initiation of DNA synthesis. The inhibition of cell cycle traverse was associated with a 10-15 fold increase in cellular cyclic AMP concentration over basal levels. The reversal of this inhibition by removal of IBMX was correlated with a dramatic decrease in cyclic AMP levels. The traverse of G1 and the initiation of DNA synthesis after release from the cholera toxin and IBMX inhibition was dependent on the presence of plasma in the medium. Either somatomedin C (10-20 ng/ml) or insulin (10(-6)-10(-5) M) completely replaced the plasma requirement for late G1 progression and entry into S phase. Once the inhibited cells were released from the IBMX and cholera toxin block a subsequent increase in cyclic AMP did not prevent entry into S phase. The presence of cholera toxin alone inhibited the stimulation of human dermal fibroblasts. The elevation of intracellular cyclic AMP levels in the human dermal fibroblasts by cholera toxin was two to three fold greater than that found in the BALB/c-3T3 cells in the presence of cholera toxin and the IBMX.     

Effects of cholera toxin on thyroid cyclic AMP-dependent protein kinase and ornithine decarboxylase activities.

Cholera toxin activated beef thyroid cyclic AMP-dependent protein kinase in a dose (0.2 to 8 microgram/ml)-related fashion. Thus, when beef thyroid slices were incubated with toxin (8 microgram/ml) for 90 minutes and then assayed for protein kinase, the activity ratio (i.e. -cyclic AMP/+cyclic AMP) increased from 0.32 +/- 0.02 to 0.77 +/- 0.06. The toxin (5 microgram/ml)-induced increase was abolished by inclusion of ganglioside GM1 in the incubation medium (I50, 0.7 microgram/ml), whereas, gangliosides GD1a and GT1 were without effect. In contrast, TSH-activated protein kinase was unaffected by ganglioside addition. Cholera toxin increased rat thyroid ornithine decarboxylase (ODC) activity in-vitro in a dose (0.1 to 10 microgram/ml)-related fashion [basal, 100 cf cholera toxin (10 microgram/ml), 1500 pmol 14CO2/g tissue/30 min]. The toxin (1 microgram/ml)- (but not TSH-) induced increase in ODC was abolished by inclusion of ganglioside Ga and GT1 were without effect. Cholera toxin stimulation of ODC was inhibited by indomethacin or iodide as are the stimulatory effects of TSH or dibutyryl cyclic AMP. These results demonstrate that although there are differences in the TSH and cholera toxin responses with respect to receptor (ganglioside) interaction, they nevertheless elicit similar intracellular responses in thyroid.     

Induction of refractoriness to isoproterenol by prior treatment of C6-2B rat astrocytoma cells with cholera toxin.

Rat C6-2B astrocytoma cells responded to cholera toxin treatment with an 8-fold increase in intracellular cyclic AMP concentrations. Cyclic AMP levels began to rise 60--90 minutes after addition of the toxin and reached maximal concentrations in 3 hours. Cells exposed to cholera toxin and the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), displayed an increase in cyclic AMP of 15-fold. The peak isoproterenol response was reduced 80--90% in cells previously treated with cholera toxin. Cholera toxin-induced refractoriness was time dependent and was not altered by concurrent treatment with propranolol. Prolonged exposure of the cells to isoproterenol reduced the cyclic AMP response to cholera toxin by 80%. MIX augmented both cholera toxin-induced refractoriness and isoproterenol-induced refractoriness. Cycloheximide inhibited the full development of refractoriness to both cholera toxin and isoproterenol. These results indicate that C6-2B cell refractoriness to cholera toxin is mediated by cyclic AMP and requires new protein synthesis. Refractoriness in C6-2B cells does not appear to be agonist-specific and probably involves a common locus of action on adenylate cyclase beyond that of the membrane receptors for cholera toxin and isoproterenol.