Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells.

Vanadyl sulfate and sodium orthovanadate in the concentration range between 5 and 50 microM are shown to be mitogenic for quiescent cultures of Swiss mouse 3T3 and 3T6 cells. The compounds caused a striking shift in the dose-response for the effect of serum on [3H]thymidine incorporation and DNA synthesis. In the absence of serum the effect of vanadium was greatly potentiated by insulin. Vanadium ions produced no more than additive increases in [3H]thymidine incorporation when combined with epidermal growth factor, cholera toxin, or phorbol 12-myristate 13-acetate. Both vanadium compounds stimulated ouabain-inhibitable 86Rb+ uptake, indicating that the vanadium ions increase, rather than inhibit, Na+/K+ pump activity in the intact cell. Neither vanadium compound had any effect on cellular cAMP under a variety of different conditions. The mitogenic effect of the vanadium compounds was similar to that of colchicine. Taxol, which stabilizes cytoplasmic microtubules, prevented the stimulation of DNA synthesis by vanadium.     

Cholera toxin stimulates secretion of saturated phosphatidylcholine and increases cellular cyclic AMP in isolated rat alveolar type II cells

We compared the time course of saturated phosphatidylcholine secretion and cellular cyclic AMP concentrations in isolated rat alveolar type II cells following stimulation by cholera toxin, terbutaline, and 12-0-tetradecanoyl-13-phorbol acetate. Secretion of saturated phosphatidylcholine was stimulated by cholera toxin at concentrations from 1.2 X 10(-11) M to 5.0 X 10(-7) M. In time course experiments there was no significant stimulation with cholera toxin before 1 hr; all subsequent points between 90 and 180 min were significantly different from controls. Secretion stimulated by 10 microM terbutaline was similar in magnitude to stimulation by 1.2 X 10(-9) M cholera toxin; stimulation following either of these agonists was higher than control secretion and lower than secretion stimulated by 10 nM 12-0-tetradecanoyl-13-phorbol acetate. Terbutaline caused an early rise in cellular cyclic AMP that peaked within 5 min and then returned to basal level by 60 min. In contrast, cholera toxin did not increase cellular cAMP levels until 60 min after addition, but then produced a sustained increase in cyclic AMP levels for up to 3 hr. In conclusion, we have presented evidence that more than one mechanism exists by which secretion can be stimulated in type II cells. It is likely that both terbutaline and cholera toxin act by stimulating cellular cyclic AMP and that 12-0-tetradecanoyl-13-phorbol acetate acts by a mechanism different from terbutaline or cholera toxin.     

Effect of cyclic AMP on lipid accumulation and metabolism in human atherosclerotic aortic cells

The effects of dibutyryl cyclic AMP (db cAMP), cholera toxin, and methylisobutylxanthine on the content and metabolism of lipids in smooth muscle cells cultured from normal and atherosclerotic intima of human aorta have been studied. Db cAMP (0.1 mM) decreased the levels of triglycerides and esterified sterols 1.5-3-fold in cells cultured from fatty streaks and atherosclerotic plaques. Cholera toxin (100 micrograms/ml) and methylisobutylxanthine (0.1 mM) stimulated by 2-fold the cyclic AMP level in aortic smooth muscle cells and decreased the content of triglycerides and esterified sterols by 2-3-fold. Prolonged treatment with db cAMP and methylisobutylxanthine resulted in a fall in the intracellular phospholipids and free sterols. Using the labelled precursors, [3H]acetate and [14C]oleate, it was established that the agents increasing the intracellular cyclic AMP level inhibit the formation of sterols and fatty acids, impede the synthesis of phospholipids, triglycerides and esterified sterols, and stimulate their hydrolysis. The data demonstrate that cyclic AMP facilitates the regression of cellular lipoidosis by altering the intracellular lipid metabolism.     

Facilitative actions of the protein kinase-C effector system on hormonally stimulated adenosine 3',5'-monophosphate production by swine luteal cells

The exact nature of the interaction(s) between cAMP and calcium-sensitive phospholipid-dependent protein kinase-C effector pathways is not well understood in many tissues, including the ovary. In the present work we have evaluated the ability of protein kinase-C to modulate receptor-and nonreceptor-mediated cAMP generation in acute suspension cultures of swine luteal cells. Cells were exposed to LH (1 micrograms/ml), forskolin (100 microM), cholera toxin (1 microgram/ml), pertussis toxin (100 ng/ml), and/or phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] for 0-90 min. TPA had no effect on basal cAMP accumulation, but increased (P less than 0.05) LH-, forskolin-, and cholera toxin-activated cAMP formation, with maximal facilitation at 30, 45, and 60 min, respectively. This facilitative effect was robust, as it could be demonstrated in both the presence and absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). TPA increased dose-dependent LH (0.1-1 microgram/ml)-, forskolin (3-300 microM-, and cholera toxin (0.3-10 microgram/ml)-stimulated cAMP accumulation. TPA induced a dose-dependent (0.3-30 ng/ml) increase in cAMP accumulation when incubated with the half-maximally effective (ED50) and maximally effective doses of LH (0.8 and 1 microgram/ml, respectively), forskolin (10 and 300 microM), and cholera toxin (0.2 and 3 micrograms/ml). TPA had an ED50 for this functional activation of 6.1 (67% confidence interval, 4.4-9.7) nM. The stimulatory effect of TPA could be mimicked by two synthetic diacylglycerols, 1,2-Dioctanoylglycerol and 1-oleoyl-2-acetylglycerol, but not by inactive phorbol esters. In addition, TPA augmented the stimulatory effect of pertussis toxin when combined with maximally effective doses of LH, forskolin, and cholera toxin. The stimulatory action of TPA on cAMP production was limited to endogenous cellular adenylyl cyclase. Bacterially derived adenylyl cyclase toxin isolated from Bordetella pertussis resulted in a dose-dependent increase in cAMP formation over 60 min, which was not facilitated by phorbol ester. We conclude that stimulatory coupling exists between the calcium-dependent protein kinase-C and cAMP-generating systems in swine luteal cells. This stimulatory coupling is enacted in part at the levels of both the guanine binding and the catalytic subunits of adenylyl cyclase.     

Cyclic AMP as a negative regulator of hormonally induced lactogenesis in mouse mammary gland organ culture.

In organ cultures of mammary glands from mice in midpregnancy, addition of both insulin and prolactin induces a marked accumulation of alpha-lactalbumin, whereas the augmentation of casein synthesis requires the presence of insulin, prolactin, and cortisol. Addition of 0.5 mM dibutyryl cyclic AMP resulted in complete inhibition of alpha-lactalbumin accumulation and partial inhibition of casein synthesis. Furthermore, either cholera toxin at 0.1-1.0 microgram/ml (a stimulator of adenylate cyclase) or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) in combination with 2 mM cyclic AMP, produced a similar pattern of inhibition of alpha-lactalbumin and casein synthesis in cultured tissue. During culture of mammary explants in medium containing no hormone, or insulin alone, or insulin, prolactin, and cortisol, the tissue content of cyclic AMP decreased rapidly, reaching half the initial level in 24-48 hr. These results indicate that cyclic AMP plays "negative" regulatory function in hormonal induction of milk protein synthesis during the development of the mammary gland.     

Antilipolytic effects of insulin and adenylate cyclase inhibitors on isolated human fat cells

Antilipolysis induced by insulin by adenylate cyclase inhibitors was compared in isolated human fat cells when lipolysis was activated at well-defined steps in the cyclic AMP system. The latter was achieved with isoprenaline (beta-adrenoreceptor agonist), cholera toxin and pertussis toxin (acting on the GTP-sensitive coupling proteins), forskolin (stimulating the catalytic component of adenylate cyclase), enprofylline (selective phosphodiesterase inhibitor) and N6-monobutyryl-cyclic-AMP or 8-bromo cyclic-AMP (cyclic AMP analogues which are resistant or sensitive to phosphodiesterase, respectively). Clonidine (alpha 2-adrenoreceptor agonist), prostaglandin E2 and N6-(phenylisopropyl) adenosine (adenosine analogue) failed to inhibit lipolysis stimulated by cholera toxin or pertussis toxin, but were effective under all other conditions. Insulin failed to inhibit lipolysis stimulated by enprofylline or N6-monobutyryl cyclic AMP, but was effective under all other circumstances. In conclusion, insulin and adenylate cyclase inhibitors are antilipolytic in human fat cells through different mechanisms. Adenylate cyclase inhibitors act predominantly on the GTP-sensitive coupling proteins and, to a minor extent, at some yet unidentified distal step in the lipolytic machinery. As regards insulin, the major site of the antilipolytic action is phosphodiesterase.     

Role of cyclic nucleotides in the inhibition of growth-hormone secretion by somatostatin

The effects of somatostatin on GH secretion, cyclic AMP and cyclic GMp concentrations in dispersed bovine anterior pituitary cells were studied following activation of adenylate cyclase with cholera toxin and inhibition of phosphodiesterase with isobutylmethylxanthine (IBMX). Cholera toxin (10(-5)M) increased intracellular cyclic AMP concentration 10-fold and cyclic GMP concentration 3-fold relative to control, and stimulated the secretion of GH. IBMX (10(-4) M) als increased intracellular concentrations of both cyclic AMP and cyclic GMP and the secretion of GH and potentiated the actions of cholera toxin particularly in raising intracellular cyclic AMP concentrations which were elevated 40-fold in the presence of cholera toxin and IBMX. Somatostatin (5 X 10(-7) M) completely prevented GH secretion elicited by cholera toxin and/or IBMX. Somatostatin was without effect on control cyclic AMP and cyclic GMp concentrations and on the increases in both cyclic AMP and cyclic GMP caused by cholera toxin and by IBMX alone, or in combination. The data suggest that bovine GH secretion is increased when concentrations of either or both cyclic nucleotides are elevated within the cells, although incubation of cells with extracellular concentrations of cyclic AMP and cyclic GMP derivatives up to 2 x 10(-3) M caused only small changes in GH release. We suggest that somatostatin inhibits cholera toxin-induced bovine GH secretion by preventing activation of the secretory process by either cyclic AMP or cyclic GMP.     

Response to cholera toxin of 2 epithelial intestinal cell lines. Effect of Saccharomyces boulardii

Cholera toxin acts in vivo by activating intestinal adenylate cyclase. This study was designed to determine (1) whether normal rat epithelial intestinal cell lines (IRD 98 and IEC 17) respond to cholera toxin (CT) by an increased concentration of cyclic AMP and (2) whether the yeast Saccharomyces boulardii, which reduced CT-induced secretion of water and electrolytes using the isolated jejunal loop technique, has an effect on these models. The cAMP concentration evaluated in cells exposed to Saccharomyces boulardii and to cholera toxin (1 microgram/ml for 90 min) was compared to the concentration of cAMP obtained in control cells without yeast. Prior exposure of IRD 98 and IEC 17 cells to Saccharomyces boulardii, reduced CT-induced cAMP by 50 p. 100. This effect disappeared after destruction of the yeast by heating. Results show that the IRD 98 and IEC 17 cells are good models for in vitro investigation of the effects of cholera toxin. Our results suggests that Saccharomyces boulardii prevents the water and electrolyte secretion induced by cholera toxin.     

Studies on the mechanisms of somatostatin action on insulin release. IV. effect of somatostatin on cyclic AMP levels and phosphodiesterase activity in isolated rat pancreatic islets.

The effects of somatostatin on insulin release and cyclic AMP metabolism were studied in collagenase-isolated islets of Langerhans from the rat. Ceoncentrations from 500 to 2000 ng/ml significantly inhibited glucose stimulated insulin release, while 100 and 200 ng/ml were ineffective. Somatostatin (2000 ng/ml) inhibited insulin release and [3H]-cyclic AMP accumulation induced by 16.7 mM glucose after 10 and 30 min of incubation. In dose-response studies, the inhibition by somatostatin of the effect of glucose on [3H]cyclic AMP and insulin release could be overcome by a high concentration of the hexose (44.9 mM), suggesting competitive inhibition. In the absence of glucose, somatostatin inhibited [3H]cyclic AMP accumulation induced by the phosphodiesterase inhibitor, IBMX, while no inhibition was seen, again in the absence of hexose, when the [3H]cyclic AMP levels had been raised by the adenyl cyclase stimulator, cholera toxin. Somatostatin did not affect phosphodiesterase activity when added to islet homogenates, but preincubation of the islets with the peptide before homogenization decreased the activity by about 30%. It is suggested that somatostatin-induced inhibition of insulin release is, at least partially, mediated by cyclic AMP, probably through an action on islet adenyl cyclase.     

Conversion of 3T3 fibroblasts into adipose cells: triggering of differentiation by prostaglandin F2alpha and 1-methyl-3-isobutyl xanthine.

Green and Kehinde [(1974) cell 1, 113-116] have isolated clones of Swiss 3T3 fibriblasts that are able to convert to adipose cells. In this paper we report on two chemicals (prostaglandin F2alpha, 0.1 mug ml, and 1-methyl-3-isobutyl xanthine, 0.5 mM) that are able to rapidly and irreversibly program the fibroblasts to differentiate into adipose cells. Confluent cultures treated with prostaglandin F2alpha and insulin for 3-5 days, followed by insulin alone for 7-48 hr, yield numerous adipocyte colonies compared to control dishes and dishes treated with insulin alone. Cells treated with prostaglandin F2alpha or 1-methyl-3-isobutyl xanthine alone, rinsed, and then exposed to insulin gave similar results, indicating that the continuous presence of the triggering agent is not required to elicit the conversion of the fibroblasts to adipocytes. Agents that raise intracellular levels of 3':5'-cyclic AMP. 1.0 mM; 8-bromo-cyclic AMP,0.5 mM; and prostaglandin E1, 0.1 mug/ml) do not trigger the conversion process, suggesting that cyclic AMP may not be the mediator of differentiation in these cells. 8-Bromo-cyclic AMP, however, does induce thase; 3':5'-nucleotidohydrolase; EC 3.1.4.17) in these cells; the induction appears to be mediated by increases in intracellular cyclic AMP levels. These results indicate that this cell line might offer a system for studying the regulation of a type of cellular differentiation.     

Adenosine 3',5'-monophosphate-binding capacity in small and large ovine luteal cells

The photoaffinity analog [32P]8-azidoadenosine cAMP ([32P]8-N3cAMP) was used to study available cAMP-binding sites on cAMP-dependent protein kinases in homogenates of ovine small (12-22 microns) and large (greater than 22 microns) luteal cells. Binding of the analogs to specific proteins was detected by autoradiography after sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and was quantitated by liquid scintillation counting. In homogenates of untreated small and large cells, only the type I isoenzyme of the regulatory subunit (R1) of protein kinase had a measurable number of available cAMP-binding sites. Small luteal cells were incubated for 2 h with increasing concentrations of ovine LH (oLH), cholera toxin, or forskolin, and media were collected for quantification of cAMP and progesterone. Cells were harvested and homogenized, and intracellular cAMP content and photoincorporation of [32P]8-N3cAMP into RI were measured. Treatment of small luteal cells with oLH, cholera toxin, and forskolin resulted in dose-dependent increases in cAMP in both the cells and the incubation media and in the progesterone content of the media. These increases were accompanied by a dose-dependent decrease in photoincorporation of [32P]8-N3cAMP into RI of small cells, which was correlated (P less than 0.05) with the increase in progesterone secretion. A time-dependent decrease (P less than 0.05) in photoincorporation in small cells incubated with oLH (100 ng/ml), cholera toxin (1000 ng/ml), or forskolin (5 microM) preceded an increase (P less than 0.05) in progesterone secretion by these cells. Large ovine luteal cells incubated with increasing concentrations of cholera toxin or forskolin demonstrated a dose-dependent decrease in photoincorporation of [32P]8-N3cAMP into RI. The time-dependent decrease (P less than 0.05) in photoincorporation in large cells incubated with cholera toxin (1000 ng/ml) or forskolin (5 microM) was not followed by enhanced progesterone secretion. These observations are consistent with a role for cAMP involvement in progesterone secretion by ovine small luteal cells and suggest a lack of cAMP involvement in progesterone synthesis by large cells.     

Angiotensin converting enzyme induction by cyclic AMP and analogues in cultured endothelial cells

The role of cyclic AMP in regulating the production of angiotensin converting enzyme (ACE) was investigated using cultured bovine aortic endothelial cells. Addition of dibutyryl cAMP [Bu)2cAMP) at 100 microM increased the ACE activity to 126% of control (P less than 0.005). This effect was blocked by either actinomycin D (0.1 microgram/ml) or cycloheximide (1.7 microM) indicating that RNA as well as protein synthesis was required for induction of the enzyme. After addition of (Bu)2cAMP, a lag period of 8 h was observed before increased ACE activity was detected. The stable analogues, 8-bromo cAMP (100 microM) and N6-monobutyryl cAMP (100 microM) also increased ACE activity but cAMP (100 microM) and O2-monobutyryl cAMP (100 microM) had no effect, in keeping with their susceptibility to phosphodiesterase in this system. Sodium butyrate (100 microM) was also inactive. The effect of (Bu)2cAMP on ACE was still observed in the presence of a maximal dose of dexamethasone, indicating that (Bu)2cAMP stimulates by mechanism(s) independent of the previously observed action of glucocorticoids on these cells. The phosphodiesterase inhibitor IBMX caused a dose-related increase in ACE activity with a threshold at 30 microM (P less than 0.05) and produced a 4-fold increase above control at 1 mM IBMX.     

Effects of cyclic AMP elevation on the levels of insulin receptors in glial C6 cells

The regulation of the insulin receptor by cAMP has been examined in glial C6 cells. Incubation for 48 h with dibutyryl cyclic AMP produced a dose-dependent inhibition of 125I-insulin binding to the cells. Other agents that elevate intracellular cAMP levels such as forskolin and cholera toxin mimicked the effect of this cyclic AMP analog. With all compounds the maximal decrease of binding was found between 24 and 48 h and normally varied between 40 and 60%. Forskolin, cholera toxin and dibutyryl cyclic AMP also affected C6 cell proliferation, and the dose-response for decreasing the receptor was very similar to that observed for the inhibition of cell growth, suggesting a relationship between both phenomena. Scatchard analysis showed that cAMP did not produce major changes in the affinity of the receptor for insulin, but rather decreased receptor number. An accumulation of receptors at the cell surface was observed in the absence of de novo protein synthesis, since cycloheximide caused a significant increase in insulin binding to the cells. This inhibitor almost totally blocked the cAMP effect both when added simultaneously to the cells with the agents which increase cAMP and when added to cells pre-treated for 48 h with the same compounds.     

Proliferative response to cyclic AMP elevation of thyroid epithelium in suspension culture

The role of cyclic AMP (cAMP) as an intracellular growth signal in thyroid epithelial (follicular) cells has been studied using primary suspension cultures of rat thyroid follicles closely resembling the in vivo state. TSH at 0.1 mU/ml in the presence of insulin (0.08 microgram/ml) induced a 9.6-fold increase in [3H]thymidine incorporation with a peak after 48-72 h. The response to the cAMP elevating agent cholera toxin (10 ng/ml) was identical both in timing, magnitude and dependence on insulin. A lower amplitude response occurred with forskolin. The data further support the conclusion that elevation of intracellular cAMP concentration is a major, if not the only, signal required for the proliferative response of the follicular cell to its physiological mitogen, TSH.     

Actions of dopamine on prolactin secretion and cyclic AMP metabolism in ovine pituitary cells

Prolactin secretion from cultured sheep pituitary cells was inhibited by low concentrations of dopamine (0.1 nM-0.1 microM) with a half-maximal effect at 3 nM. At a maximally effective dose (0.1 microM) dopamine significantly inhibited prolactin secretion within 5 min. with an 80% inhibition of basal secretion over 2 h. Basal prolactin secretion was stimulated by the addition of methylisobutylxanthine (MIX) (0.3-1.0 mM) and 8-bromo-cyclic AMP (2 mM), but cholera toxin (3 micrograms/ml) and prostaglandin E2 (0.1-1.0 microM), which also raised cellular cyclic AMP levels, had no effect on prolactin release. The inhibition of prolactin release by dopamine (0.1 microM) was not affected by any of these compounds. Dopamine inhibited MIX-induced cyclic AMP accumulation over a similar concentration range to the inhibition of secretion, but had no effect on the changes in cyclic AMP concentration produced by cholera toxin and prostaglandin E2. Overall the results with sheep pituitary cells suggest that lowered cyclic AMP levels do not mediate the inhibitory effects of dopamine on basal prolactin secretion, but that changes in cellular cyclic AMP levels may alter the secretion of this hormone, and dopamine may affect pituitary cell cyclic AMP concentrations in some circumstances.     

Desensitization of the cAMP system in mouse Leydig cells by hCG, cholera toxin, dibutyryl cAMP and cAMP: localization of the 'lesion' to the guanine nucleotide regulatory protein-adenylate cyclase complex

The mechanism of hCG-induced desensitization of the cAMP system was studied in Percoll-purified mouse Leydig cells. Pretreatment of Leydig cells with hCG resulted in a time- and dose-dependent decrease in the capacity of hCG-induced cAMP formation. Maximal desensitization (approximately 90%) was induced by only partial prior stimulation. Desensitization, however, was not observed without a prior increase in cAMP or testosterone production. Pretreatment of the cells with N6,O2'-dibutyryl cAMP (DBcAMP) also induced a dose- and time-dependent densensitization. cAMP was only effective in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX). Cholera toxin desensitized the hormone-induced cAMP response as drastically as hCG. Cholera toxin was unable to reverse the refractory state induced by one of the agonists. hCG-induced desensitization was not associated with a loss in [125I]hCG binding or an increase in maximal phosphodiesterase activity, and appeared not to be dependent on protein synthesis. Membranes from hCG, cholera toxin of DBcAMP-desensitized cells showed an impaired adenylate cyclase activity in response to hCG, hCG plus beta-gamma-imidoguanosine 5'-triphosphate (GPPNP) and NaF. In conclusion, hCG-induced desensitization of the adenylate cyclase system in mouse Leydig cells can be mimicked by cholera toxin, DBcAMP and cAMP, indicating a cAMP-mediated process. The site of the 'lesion' has to be localized to the guanine nucleotide regulatory protein-adenylate cyclase complex rather than to its uncoupling from the hormone receptor.     

Activation of early events of the mitogenic response by a P2Y purinoceptor with covalently bound 3''-O-(4-benzoyl)-benzoyladenosine 5''-triphosphate.

3'-O-(4-Benzoyl)benzoyl-ATP (BzATP), a photoaffinity analog of ATP, was used as a ligand for a P2Y purinoceptor (adenine nucleotide receptor) present in intact Swiss 3T3 and 3T6 cells and A-431 epidermoid carcinoma cells. Photolysis of serum-starved cells in the presence of 10-50 microM BzATP, followed by extensive washing to remove unincorporated BzATP, induced the release of arachidonic acid. A trace (less than 0.01%) of photoincorporated BzATP was as effective as when 50 microM BzATP or ATP was contained in the incubation medium during the assay. Photoincorporated BzATP also stimulated the production of prostaglandin E2 and the accumulation of cyclic AMP. In previous studies, we demonstrated that these three events are obligatory early steps in a pathway leading to DNA synthesis in the above cell lines. The evidence indicated that the purinoceptor activated by extracellular ATP or BzATP was linked to a pertussis toxin-sensitive GTP-binding protein. Consistent with these observations, we now find that pertussis toxin inhibits the effect of photoincorporated BzATP on arachidonic acid release. These results indicate that BzATP is an effective agonist for the P2Y purinoceptor concerned with stimulation of DNA synthesis in 3T3, 3T6, and A-431 cells. Furthermore, after photolysis it becomes irreversibly associated with intact cells and promotes the activation of early events required for DNA synthesis.     

Effect of dexamethasone on adenosine 3',5'-monophosphate content and phosphodiesterase activities in 3T3-L1 adipocytes

After incubation with 0.5 mM isobutylmethylxanthine, 1 microM dexamethasone, and 1 microM insulin for 72 h, 3T3-L1 cells acquire the phenotypic characteristics of mature adipocytes, including a hormone-sensitive particulate cAMP phosphodiesterase activity. In addition, adipocytes contain soluble cAMP and calmodulin-sensitive and -insensitive cGMP phosphodiesterase activities. After exposure of the differentiated cells to 1 microM epinephrine, cAMP content increased, reaching a maximum in 2-4 min, and then declined to the control level by 20 min. After incubation of adipocytes with 10 nM dexamethasone for 72 h, the initial increment in cAMP produced by epinephrine was not altered, but the decline in cellular cAMP to basal levels was delayed. Treatment with 10 nM dexamethasone prevented hormonal activation of particulate cAMP phosphodiesterase activity without altering basal activity (11). Soluble cAMP and calmodulin-sensitive and -insensitive cGMP phosphodiesterase activities were also reduced by exposure to 10 nM dexamethasone; higher concentrations were required to decrease basal particulate phosphodiesterase activities. Estradiol did not alter phosphodiesterase activities. Incubation of either undifferentiated (fibroblasts) or differentiated (adipocytes) 3T3-L1 cells with 1 microM dexamethasone for 48 h reduced cAMP and cGMP phosphodiesterase activities. After removal of dexamethasone, phosphodiesterase activities were restored to control levels in 4-6 days. The effects of dexamethasone on phosphodiesterase activities could in part account for the observed alterations in hormone-induced accumulation of cAMP in steroid-treated cells and for the permissive effects of glucocorticoids on certain cAMP-mediated processes.     

Activation of adenylate cyclase in rat fat cells promotes an increase in GTP content which controls the enzyme activity

In previous studies, we demonstrated that the treatment of adipocytes with cholera toxin or Bordetella pertussis toxin (IAP) promoted an increase in the total guanosine triphosphate (GTP) content of the cells concomitant with the increase in cyclic adenosine monophosphate (AMP) level and the resulting lipolysis. In the present studies, we show that the acute challenge of fat cells with 1 microM isoproterenol (IPNE) is associated with a transient increase in GTP level (3-fold in 6 min). This increase may be attributed to an inhibition of the disposal of GTP or to a stimulation of its synthesis. To evaluate the actual role of GTP, we used virazole, an antitumor agent which inhibits inosinic acid dehydrogenase. After 2 h preincubation of the cells with 1 mM virazole, the effect of a 6 min challenge with 1 microM IPNE is decreased by 59% at the GTP level and by 42% in cyclic AMP production. One hour later, the resulting lipolytic efficiency is reduced by 57%. IAP treatment (10 micrograms/ml) produced its maximal effect on GTP and cyclic AMP levels and on lipolysis after 90 min incubation. The antilipolytic effect of 1 microM phenylisopropyladenosine (PIA) is almost abolished. When 1 mM virazole is added to the cell suspension to deplete the guanyl nucleotide pool, the resulting lipolysis due to IAP treatment is decreased by 85%, whereas GTP and cyclic AMP levels were decreased by 80 and 70%, respectively. We can conclude that the cyclic AMP synthesis in intact cells is accompanied by a parallel increase of their GTP content, whether the stimulation results from the activation of Gs or the inhibition of Gi. The reduction of the guanyl nucleotide pool under virazole results in a relatively less important inhibition of lipolysis when Gs is stimulated than when it is Gi.     

Epidermal Growth Factor: Receptors in Human Fibroblasts and Modulation of Action by Cholera Toxin

Epidermal growth factor (EGF) stimulates both DNA and RNA synthesis in contact-inhibited human fibroblasts. Stimulation of DNA synthesis is observed at concentrations as low as 3 pM, is half-maximal at 70 pM, and is maximal at 300 pM EGF. The action of EGF is similar to that of fetal-calf serum, but is distinguished by the time-course of stimulation and by the ability of serum to stimulate further those cells maximally stimulated by EGF. Cells that synthesize DNA in response to physiological concentrations of EGF (10(-11) to 10(-10) M) are insensitive to physiological concentrations of insulin (10(-11) to 10(-10) M) and respond only minimally to very high concentrations of this hormone (10(-6) M). The biological activity of EGF is paralleled by binding of this peptide to fibroblasts in a specific and saturable manner; the dissociation constant is about 800 pM. The binding of EGF is unaffected by either insulin or cholera toxin. Cholera toxin inhibits the action of both EGF and serum. Suppression of DNA synthesis is observed at 0.02 pM toxin, and is maximal at about 2 pM. Cells treated with cholera toxin at these concentrations appear to be otherwise viable by several criteria. The stimulatory effects of EGF are also inhibited by theophylline and dibutyryl cyclic AMP separately or in combination. These observations indicate that fibroblasts possess receptors for EGF by biological and physicochemical criteria, and suggest that a similar if not identical peptide may be amongst those factors in sera which stimulate cell growth. The possibility is considered that EGF and cholera toxin modulate the ability of a cell to initiate polynucleotide synthesis by way of specific cell-surface interactions which in turn alter the levels of intracellular cyclic AMP.