Association in normal human fibroblasts of elevated levels of adenosine 3':5'-monophosphate with a selective decrease in collagen production.

To evaluate the hypothesis that extracellular mediators may affect collagen production by mesenchymal cells via a cyclic AMP coordinated mechanism, normal human fibroblasts were exposed to a variety of agents (prostaglandin E1, isoproterenol, cholera toxin) which independently elevated intracellular cyclic AMP during a 6-h incubation. Concomitantly, each agent caused an average 47% reduction in the percentage of total protein synthesis represented by collagen, yet little change in other major extracellular proteins. Since no active collagenase was found in these cultures, these findings suggest cyclic AMP levels may modulate the differentiated state of normal fibroblasts with respect to collagen production.     

Cyclic AMP-induced inhibition of collagen lattice contraction by fibroblasts may be attenuated by both cyclic AMP dependent and independent mechanisms

The contraction of collagen lattices made with forskin fibroblasts in medium containing 1% fetal bovine serum was inhibited by intracelluar cyclic AMP raising drugs including cholera toxin (CT), forskolin, and dibutyryl-cAMP. The inhibition by CT was attenuated by insulin, acidic fibroblast growth factor (aFGF), and transforming growth factor-β (TGF-β). All three peptide factors have previously been reported to promote collagen lattice contraction by arterial smooth muscle cells and/or fibroblasts. Incubation of cells suspended in collagen gels with CT and forskolin resulted in a transient rise of the intracellular cyclic AMP levels, which peaked at 2 hr and 30 min, respectively, after drug exposure. Cholera toxin-induced intracellular cyclic AMP increase was attenuated by TGF-β, but not by aFGF and insulin, when added simultaneously. Thus, TGF-β may attenuate CT's inhibition on collagen lattice contraction by attenuating CTinduced intracellualr cyclic AMP increse, whereas the attenuation by insulin and aFGF on the inhibition of lattice contraction may be mediated by a cyclic AMPindependent mechannism. © 1993 Wiley-Liss, Inc.     

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

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

Action of cholera toxin on dispersed acini from guinea pig pancreas

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

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.     

Cholera toxin and analogues of cyclic AMP stimulate the growth of cultured human mammary epithelial cells

The growth of human epithelial cells is stimulated by cholera toxin and analogues of cyclic AMP, while the growth of breast derived fibroblasts is inhibited. These compounds have little effect on DNA synthesis in the absence of other mitogens but show a synergistic effect with serum and/or EGF. The results suggest that high intracellular levels of cyclic AMP in human mammary epithelial cells increase the growth response of the cell to mitogens.     

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.     

Hexose transport stimulation and membrane redistribution of glucose transporter isoforms in response to cholera toxin, dibutyryl cyclic AMP, and insulin in 3T3-L1 adipocytes

Exposure of 3T3-L1 adipocytes to 100 ng/ml of cholera toxin or 1 mM dibutyryl cyclic AMP caused a marked stimulation of deoxyglucose transport. A maximal increase of 10- to 15-fold was observed after 12-24 h of exposure, while 100 nM insulin elicited an increase of similar magnitude within 30 min. A short term exposure (4 h) of cells to cholera toxin or dibutyryl cyclic AMP resulted in a 3- to 4-fold increase in deoxyglucose transport which was associated with significant redistribution of both the HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) glucose transporters from low density microsomes to the plasma membrane fraction. Total cellular amounts of both transporter proteins remained constant. In contrast, cells exposed to cholera toxin or dibutyryl cyclic AMP for 12 h exhibited elevations in total cellular contents of GLUT1 (but not GLUT4) protein to about 1.5- and 2.5-fold above controls, respectively. Although such treatments of cells with cholera toxin (12 h) versus insulin (30 min) caused similar 10-fold enhancements of deoxyglucose transport, a striking discrepancy was observed with respect to the content of glucose transporter proteins in the plasma membrane fraction. While insulin elicited a 2.6-fold increase in the levels of GLUT4 protein in the plasma membrane fraction, cholera toxin increased the amount of this transporter by only 30%. Insulin or cholera toxin increased the levels of GLUT1 protein in the plasma membrane fraction equally (1.6-fold). Thus, a greater number of glucose transporters in the plasma membrane fraction is associated with transport stimulation by insulin compared to cholera toxin. We conclude that: 1) at early times (4 h) after the addition of cholera toxin or dibutyryl cyclic AMP to 3T3-L1 adipocytes, redistribution of glucose transporters to the plasma membrane appears to contribute to elevated deoxyglucose uptake rates, and 2) the stimulation of hexose uptake after prolonged treatment (12-18 h) of cells with cholera toxin may involve an additional increase in the intrinsic activity of one or both glucose transporter isoforms.     

Hormones and Neurotransmitters Control Cyclic AMP Metabolism in Choroid Plexus Epithelial Cells

The choroid plexus is a major site of CSF production. When primary cultures of bovine choroid plexus epithelial cells were exposed to 1 micrograms/ml cholera toxin, a 50-fold increase of intracellular cyclic AMP was found 1 h later. Exposure of cells to 10(-5) M isoproterenol, 10(-4) M prostaglandin E1, 10(-5) M histamine, and 10(-5) M serotonin caused increases of intracellular cyclic concentrations of 100-, 50-, 20-, and 4-fold, respectively. From 5 to 15 min were required for these maximal responses to occur. Many other molecules including prolactin, vasopressin, and corticotropin did not alter cellular cyclic AMP levels. The accumulation of cyclic AMP could be inhibited by specific antagonists: propranolol inhibited the isoproterenol-mediated stimulation while diphenhydramine and metiamide inhibited the histamine response. In addition, diphenhydramine inhibited serotonin-dependent cyclic AMP accumulation. Combinations of isoproterenol, prostaglandin E1, histamine, and serotonin elicited additive responses as measured by cyclic AMP accumulation with one exception, i.e., serotonin inhibited the histamine response. Our findings suggest that distinct receptor sites on choroid plexus epithelia exist for isoproterenol, prostaglandin E1, and histamine. Efflux of cyclic AMP into the extracellular medium was found to be a function of the intracellular cyclic AMP levels over a wide range of concentrations. Our studies provide direct evidence for hormonal regulation of cyclic AMP metabolism in epithelial cells of the choroid plexus.     

Cyclic 3',5'-adenosine monophosphate in the choroid plexus: stimulation by cholera toxin.

Cholera toxin was found to induce high accumulations of cyclic AMP in the isolated choroid plexus of the rabbit and in the incubation medium. The accumulation showed a characteristic lag phase of at least 30 min and continued for at least 3 hours. Inactivated cholera toxin was unable to increase cyclic AMP levels. There was only a moderate effect of cholera toxin on cyclic AMP “low K_m” phosphodiesterase activity in homogenates. The effect of cholera toxin on cyclic AMP levels confirms the existance of a potent cyclic AMP generating system in the choroid plexus which is activated also by β-adrenergic agonists, histamine and prostaglandin E₁.     

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.     

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.     

Effect of in vitro cultivation and Mycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures.

Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains of Mycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels.     

Effect of in vitro cultivation andMycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures

Summary Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains ofMycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels. This study was supported in part by Grant AI 12559 from the National Institutes of Health. The supply of cholera toxin from Dr. R. A. Finkelstein is most appreciated as are the assistance and advice of J. A. Engelhardt and Y. D. B. Stahl.     

Temperature-dependent alteration of cellular morphology by cholera toxin in rat liver epithelial cells which are ts for maintenance of transformed properties

Cholera toxin via its ability to increase intracellular cyclic AMP levels can induce drastic changes in cell morphology. This report describes a temperature sensitive mutant of chemically transformed rat liver epithelial cells which only display cell shape alterations in response to cholera toxin at the permissive temperature. Shift up-shift down experiments indicate that the change in the response occurs fairly rapidly, i.e., within 2 hours at the new temperature. The behavior of the temperature sensitive cells at the nonpermissive temperature mimics that of the untransformed rat liver epithelial cells (i.e., no morphological change in response to cholera toxin) while at the permissive temperature the positive cell shape change is identical to that exhibited by chemically transformed rat liver epithelial cells. The temperature sensitive response to cholera toxin is not a function of cyclic AMP production, since the amount of cyclic AMP found as a function of either time or concentration of cholera toxin is quite similar in cells treated at either temperature.     

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.     

On the mechanism of action of cholera toxin on isolated rat adrenocortical cells Comparison with the effects of adrenocorticotropin on steroidogenesis and cyclic AMP output

The effects of cholera toxin on isolated rat adrenocortical cells have been investigated. Both steroid and cyclic AMP output from adrenal cells were increased by the toxin in a dose dependent fashion. The concentration of toxin for half maximal stimulation for both of these responses was about 40 ng/ml. Maximal steroidogenesis and cyclic AMP output was obtained with similar concentrations of the toxin. A correlation was observed between the low amounts of cyclic AMP produced in response to all doses of cholera toxin and to physiologically significant concentrations of adrenocorticotropin (ACTH) (< 0.1 munit/ml; i.e. submaximal for steroidogenesis in this system). This was in direct contrast to the much higher levels of cyclic AMP generated by concentrations of ACTH greater than 1 munits/ml. Time course studies demonstrated a time-lag between toxin addition and steroid response of at least 40 min. Binding of cholera toxin to adrenal cells was rapid and was 90% complete within 15 min at both 37 and 0°C. These data indicate that most of the delay in response to cholera toxin is due to processes subsequent to the initial binding interaction. Following the initial delay the subsequent maximal rate of steroidogenesis brought about by cholera toxin was very similar to that obtained with a concentration of ACTH that was maximal for steroidogenesis. Significant increases in cyclic AMP levels were detected about 20 min before increased steroidogenesis was apparent. Possible explanations for this result are considered. The results presented indicate great potential use for cholera toxin in the study of adrenal steroidogenic control mechanisms, particularly at the level of receptor mechanisms and the role of cyclic AMP.     

Chronic Exposure to Pertussis Toxin Alters Muscarinic Receptor-Mediated Regulation of Cyclic AMP Metabolism in Neuroblastoma × Glioma NG108–15 Hybrid Cells

Chronic pertussis toxin treatment (5 days) of NG108-15 neuroblastoma X glioma hybrid cells had no significant effect on basal cyclic AMP levels whereas it effectively blocked the inhibitory action of acute (10 min) exposure of carbachol (10(-4)M) on intracellular cyclic AMP accumulation, stimulated by prostaglandin E1. This action of pertussis toxin was found to be long lasting: exposure of the cells to pertussis toxin (100 ng/ml) for only 24 h followed by a 5-day withdrawal period still was shown effective on day 7 in abolishing the inhibitory action of carbachol on prostaglandin E1-stimulated cyclic AMP production. Chronic exposure (5 days) of NG108-15 cells to carbachol (10(-5)M) causes an increase in basal cyclic AMP levels by 98%, and a desensitization of the muscarinic inhibition of cyclic AMP accumulation, assessed after a 24-h withdrawal period. When carbachol treatment is carried out in the presence of pertussis toxin (100 ng/ml) both of these effects of carbachol are abolished.     

MSH stimulates adenylate cyclase and tyrosinase in cultivated melanoma cells in the presence of cytochalasin B

MSH and cholera toxin increase intracellular levels of cyclic AMP (cAMP) and tyrosinase activity in cultivated mouse melanoma cells in the presence of cytochalasin B (CB) at a concentration sufficient to prevent pinocytosis and cytokinesis. The data suggest that MSH and cholera toxin exert their effects without entering the cell.