Dexamethasone and 8-bromo-cyclic AMP depress the incorporation of [3H]thymidine into mouse condylar cartilage by different pathways

We examined whether cyclic AMP (cAMP) affects the incorporation of [3H]thymidine into cartilage cells and, if so, whether this action could be related to the inhibitory effect of glucocorticoid hormones on the growth of ossifying cartilage. Incorporation of [3H]thymidine into trichloroacetic acid-precipitable material by mouse cartilage was measured concomitantly with the concentration of cAMP. Dexamethasone (1 mumol/l) significantly (P less than 0.05) depressed the incorporation of [3H]thymidine. The cAMP analogue 8-bromo-cAMP (0.01-1 mmol/l) also depressed the incorporation of the radionucleotide in a dose-dependent fashion. When various concentrations of 8-bromo-cAMP were added with dexamethasone (1 mumol/l), no apparent changes took place compared with the effect of dexamethasone alone. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2-1 mmol/l) elicited an inhibitory effect on [3H]thymidine incorporation and a stimulatory influence on cartilage cAMP concentrations. Dexamethasone, at doses (0.01-1 mumol/l) causing significant inhibition of [3H]thymidine incorporation, failed to increase cartilage levels of cAMP. It seems, therefore, that the depressive effect of dexamethasone on [3H]thymidine incorporation in condylar cartilage is not mediated through an increase of cAMP in the tissue.     

Regulation of Y-organ ecdysteroidogenesis by molt-inhibiting hormone in crabs: involvement of cyclic AMP-mediated protein synthesis

The crustacean neuropeptide, molt-inhibiting hormone (MIH), directly inhibits Y-organ ecdysteroidogenesis, an effect mediated by cyclic AMP (cAMP) and antagonized by calcium-calmodulin. We investigated regulation of Y-organ protein. RNA, and DNA syntheses by MIH, cAMP, and calcium in relation to steroidogenesis in vitro. Ecdysteroid production and [3H]leucine incorporation into protein were inhibited 50-60 and 80-90%, respectively, by MIH activity in eyestalk extracts (4 eyestalk equivalents), 10(-6) M forskolin, or a combination of 10(-2) M dibutyryl cAMP and 10(-4) M 3-isobutyl-1-methylxanthine (dbcAMP-IBMX). Calcium ionophore A23187 (10(-4) M) stimulated ecdysteroidogenesis two-fold, did not affect the relatively high basal (control) rate of protein synthesis, and reduced the inhibitory effects of forskolin on steroidogenesis and protein synthesis. Incorporation of [3H]uridine into RNA was unaffected by MIH, forskolin, or A23187 but was reduced 50% by dbcAMP-IBMX. Basal rates of [3H]thymidine incorporation into DNA were low and were not affected by treatments. The effects of MIH were specific; extracts of brain or muscle did not alter Y-organ steroidogenesis or protein synthesis, while muscle extract increased precursor incorporation into RNA. Eyestalk extract did not affect [3H]leucine incorporation into protein of brain, muscle, or gill. Cycloheximide (5 micrograms/ml) depressed protein synthesis 90% and steroidogenesis 60%, enhanced the inhibition induced by MIH, and blocked the stimulation of steroidogenesis induced by A23187; effects on basal steroidogenesis were evident after 1 hr. Actinomycin D (1 microgram/ml) depressed RNA synthesis 86% but did not alter basal, MIH-inhibited, or A23187-stimulated ecdysteroidogenesis during incubations. These results indicate that MIH suppresses Y-organ steroidogenesis in part by inhibiting protein synthesis at the translational level; the effect is mediated by cAMP. The stimulation of steroidogenesis by calcium, mediated by lowering cAMP, also appears to depend in part upon protein synthesis.     

Alteration of Nucleoside Transport of Chinese Hamster Cells by Dibutyryl Adenosine 3′:5′-Cyclic Monophosphate

Cultured Chinese hamster ovary cells showed no significant change in generation time or fraction in the S-phase in the presence of 1 mM N(6),O(2')-dibutyryl adenosine 3':5'-cyclic monophosphate. Growth continued for at least two generations after expression of the morphological transformation induced by this cyclic AMP analog. Despite identical growth rates, apparent rates of DNA and RNA synthesis (incorporation of [(3)H]-thymidine or [(3)H]uridine) were reduced up to 15-fold in log phase by 1 mM cyclic nucleotide. [(3)H]Deoxycytidine incorporation was much less sensitive to dibutyryl cyclic AMP. Uptake studies with [(3)H]thymidine demonstrated an inhibition of transport rate dependent on the concentration of dibutyryl cyclic AMP in the growth medium. The rate of thymidine uptake at 1 degrees was decreased 21-fold by 1 mM cyclic nucleotide; half-maximal inhibition occurred at 6 muM. At 37 degrees , the pool size of acid-soluble thymidylate was strongly reduced by 1 mM cyclic nucleotide, and synergistic reduction of the pool size was found with 0.5 mM aminophylline. Phosphorylation of the acid-soluble intracellular label was unaffected by dibutyryl cyclic AMP. Inhibition of thymidine uptake is attributed to an observed decrease in thymidine kinase activity caused by growth in 1 mM dibutyryl cyclic AMP, and possibly to a simultaneous alteration in membrane permeability. Kinase-facilitated uptake of other metabolites may be regulated in a similar fashion by cyclic AMP.     

A growth stimulatory effect of iodide is suggested by its effects on c-myc messenger ribonucleic acid levels, [3H]thymidine incorporation, and mitotic activity of porcine follicle cells in suspension culture

In the present study the effect of iodide on thyroid cell growth was investigated in primary suspension cultures of porcine thyroid cells capable of organifying iodide. The addition of a high dose of iodide (10(-4) M) to such cultures caused a marked increase in c-myc mRNA levels, [3H]thymidine incorporation, and mitotic activity. The incorporation of [3H]thymidine started 30-36 h after the addition of iodide. The stimulatory effect was abolished by a simultaneous incubation with methimazole. The concentration dependence of the iodide-induced stimulation of [3H]thymidine incorporation was similar to that of an inhibitory effect on adenylate cyclase activity. W-7, an inhibitor of calmodulin activity, as well as epinephrine, agents that reduce cAMP levels, also stimulated [3H]thymidine incorporation. Moreover, the stimulatory effect of iodide was reduced in the presence of forskolin. The results suggest that an organic form of iodine stimulates thyroid cell growth by reducing cAMP levels and demonstrate the presence of a growth stimulatory pathway in porcine thyroid cells that is independent of exogenous polypeptide growth factors or hormones.     

Inhibition of Replication in Functional Mouse Adrenal Tumor Cells by Adrenocorticotropic Hormone Mediated by Adenosine 3′:5′-Cyclic Monophosphate

Adrenocorticotropic hormone (ACTH) inhibited replication in functional adrenal tumor cells with a concomitant stimulation of steroidogenesis and a characteristic change of morphology from a flattened to a spherical type. [(3)H]Thymidine incorporation into DNA was inhibited by about 50% 6 hours after ACTH treatment. Both cyclic AMP and dibutyryl cyclic AMP inhibited [(3)H]thymidine incorporation and caused the characteristic morphological change noted with ACTH. The extent of stimulation of steroidogenesis and the amount of inhibition of [(3)H]thymidine incorporation in response to various doses of ACTH were closely related and both were in parallel with the concentration of cyclic AMP in the cells. Cyclic GMP and cyclic IMP did not inhibit [(3)H]thymidine incorporation significantly, and did not change the morphology of the cells. AMP inhibited [(3)H]thymidine incorporation into DNA and caused the characteristic morphological change. However, AMP did not increase the cyclic AMP content of the cells. CMP, GMP, and UMP showed a significant inhibition of [(3)H]thymidine incorporation into DNA, but the extent of the inhibition was much less than that with AMP. These nucleotides did not change the morphology of the cells.     

Inhibition of replication of normal adrenocortical cells in culture by adrenocorticotropin.

Adrenocorticotropic hormone (ACTH) inhibited [3H]thymidine incorporation in normal adrenocortical cells of adult rats in culture, with a concomitant increase in corticosterone production and a characteristic retraction of cells. Both dibutyryl cyclic AMP and an analog of ACTH, which produces virtually no cyclic AMP, inhibited DNA synthesis and stimulated steroid production. ACTH inhibited the proliferation of adrenocortical cells obtained from suckling rats as well as the cells obtained from the capsular tissue of adult rat adrenal glands, whereas insulin caused a stimulation of DNA synthesis. These results suggest that the major role of ACTH is to induce the transformation of the undifferentiated cells of the adrenal gland into functional fasciculata cells and that the proliferation of adrenocortical cells may be under control of factors other than ACTH.     

Cyclic AMP: a mitogenic signal for Swiss 3T3 cells.

Addition of cholera toxin (100 ng/ml) to quiescent cultures of Swiss 3T3 cells acts synergistically with serum (2-4%), insulin, phorbol esters, epidermal growth factor, and fibroblast-derived growth factor to stimulate DNA synthesis. In the presence of insulin, cholera toxin caused a dose-dependent increase in cumulative [3H]thymidine incorporation into acid-insoluble material and in the intracellular cyclic AMP (cAMP) level. The dose--response curves for the two processes were similar. Furthermore, addition of 1-methyl-3-isobutylxanthine (15--500 microM) or of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (5--100 microM), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase which are potent inhibitors of cyclic nucleotide phosphodiesterase activity, stimulated DNA synthesis and increased cAMP levels in Swiss 3T3 cells. These compounds strikingly potentiated the effect of cholera toxin on DNA synthesis and on cAMP levels. When quiescent Swiss 3T3 cells were exposed to cholera toxin (100 ng/ml) and insulin at 10 micrograms/ml (4- to 7-fold increase in cAMP level) or to these agents and 1-methyl-3-isobutyl xanthine at 50 microM (35-fold increase in cAMP level), DNA synthesis began after a lag of 16 hr. These results indicate that cAMP acts as a mitogenic signal for Swiss 3T3 cells and differ from the widely held view that cyclic AMP inhibits the proliferation of fibroblast cells.     

Mitogenic effects of thyrotropin and adenosine 3',5'-monophosphate in differentiated normal human thyroid cells in vitro

Previous studies of human thyroid cells in culture (mostly from pathological tissues) failed to demonstrate a mitogenic effect of TSH, leading to the proposal that the growth effect of TSH in vivo might be indirect. To reexamine the influence of TSH on DNA synthesis and cell proliferation, we established primary cultures of normal thyroid tissue from nine subjects. When seeded in a 1% serum-supplemented medium, thyroid follicles released by collagenase/dispase digestion developed as a cell monolayer that responded to TSH by rounding up and by cytoplasmic retraction. When seeded in serum-free medium, the cells remained associated in dense aggregates surrounded by few slowly spreading cells. In the latter condition, the cells responded to TSH and other stimulators of cAMP production, such as cholera toxin and forskolin, by displaying very high iodide-trapping levels. Exposure to serum irreversibly abolished this differentiated function. TSH stimulated the proliferation (as shown by DNA content per culture dish) of 1% serum cultured cells (doubling times were reduced from 106 to 76 h) and increased by 100% the [3H]thymidine labeling indices. In serum-free cultured cells (dense aggregates or cell monolayers after initial seeding with serum), control levels of DNA synthesis were lower, and up to 8-fold stimulation of DNA synthesis occurred in response to 100 mU/L TSH (stimulation was consistently detected with 20 mU/L), based on measurements of [3H]thymidine incorporation into acid-precipitable material and counts of labeled nuclei on autoradiographs (up to 40% labeled nuclei within 24 h). The mitogenic effect of TSH required a high insulin concentration (8.3 X 10(-7) mol/L) or a low insulin-like growth factor I concentration. The mitogenic effects of TSH were mimicked in part by cholera toxin, forskolin, and dibutyryl cAMP. Epidermal growth factor and phorbol myristate ester also stimulated thyroid cell proliferation and DNA synthesis, but they potently inhibited TSH-stimulated iodide transport. We conclude that TSH, acting at least in part through cAMP, is a potent growth factor for human thyroid cells and thus provide an experimental basis in vitro for the well established in vivo goitrogenic action of TSH.     

Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture.

We have previously reported that serotonin (5-hydroxytryptamine [5HT]) alters cultured bovine pulmonary artery smooth muscle cell (SMC) configuration through two different regulatory mechanisms. We now report that 5HT also regulates SMC growth through these same two mechanisms--a stimulatory event initiated intracellularly and inhibition of growth resulting from a cell surface action. 5HT (1 microM) plus 0.1 mM iproniazid (a 5HT metabolic inhibitor) produced a severalfold stimulation of DNA synthesis (as measured by [3H]thymidine incorporation) of SMCs after a 17-24-hour incubation with only a slight elevation of cellular cAMP. This stimulatory effect responded synergistically with other growth factors including platelet-derived growth factor, fibroblast growth factor, and epidermal growth factor and was effectively reversed by 5HT uptake inhibition. It was not produced by 5-hydroxyindoleacetic acid, a metabolite of 5HT. In the presence of 1 microM 5HT plus 0.1 mM isobutylmethylxanthine (IBMX), cAMP was elevated eightfold, dendritic formation occurred, and [3H]thymidine labeling of SMCs was inhibited. Inhibition of labeling by [3H]thymidine was mimicked by other agents that elevated cellular cAMP (10 microM histamine, 1 microM isoproterenol plus 0.1 mM IBMX, and 10 microM forskolin) and by 1 mM dibutyryl cAMP. This inhibitory effect was not blocked by either inhibition of 5HT uptake or 5HT-receptor antagonists ketanserin (5HT2); methiothepin, spiperone, and mianserin (5HT1/5HT2); and 3-tropanyl-indole-3-carboxylate and 3-tropanyl-3,5-dichlorobenzoate (5HT3). However, similar to 5HT, the 5HT1A agonist, (+/-)-8-hydroxy-(+/-)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalenehydrobromide, in association with IBMX, produced an elevation in cAMP and inhibition of labeling by [3H]thymidine. 5HT, in the presence of either iproniazid or IBMX, did not alter [Ca2+]i, indicating that [Ca2+]i was not a signal for either of these actions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP and adrenergic receptors in Gambusia affinis melanophore response in vitro

In vitro responses of Gambusia affinis caudal fin melanophores to norepinephrine, epinephrine, and isoproterenol after incubation in Ringer solution and after alpha- and beta-adrenergic blockade are consistent with the presence of both alpha- and beta-adrenergic receptors on these cells. Mediation of aggregation is regulated through alpha receptors and dispersion through beta-adrenergic stimulation.Dispersion of pigment by cyclic AMP (cAMP), dibutyryl cAMP, and caffeine and acceleration of dispersion by simultaneous administration of either caffeine + cAMP or caffeine + dibutyryl cAMP are interpreted as evidence for mediation of dispersion by the cyclic nucleotide. Dispersion of pigment by ACTH and enhanced dispersion by ACTH + cAMP and by ACTH + caffeine suggest participation of the hormone in increasing cAMP levels.An increase in cAMP levels in response to beta-adrenergic stimulation is indicated by (1) dispersing action of 10^?6M isoproterenol, which is blocked by simultaneous administration of the beta-adrenergic antagonist dichloroisoproterenol (DCI), (2) significant enhancement of the dispersing action of cyclic nucleotides, caffeine, and ACTH by simultaneous treatment with 10^?6M isoproterenol or by previous exposure to 10^?4M isoproterenol, which weakly aggregates pigment, and (3) abolition of enhancement of dispersion when cells are exposed simultaneously to 10^?4M isoproterenol and DCI prior to treatment with dispersing agents.Prior exposure to norepinephrine prevents dispersion by both ACTH and exogenous cAMP, and inhibitory effects of norepinephrine are abolished when cells are pretreated simultaneously with the catecholamine and the alpha-adrenergic blocking agent dibenamine. These observations are interpreted as evidence supporting the view that alpha-adrenergic stimulation decreases cAMP levels and suggesting that, in these cells, it does so by enhancing phosphodiesterase activity rather than by adenyl cyclase inhibition.     

Effects of interleukin 1 on growth and adenosine 3',5'-monophosphate generation of the rat thyroid cell line, FRTL-5 cells.

We investigated the effects of IL-1, IL-2, IL-6, interferon-gamma and tumour necrosis factor-alpha on growth and cAMP generation of FRTL-5 cells. IL-1 produced a significant stimulation of [3H]thymidine incorporation into FRTL-5 cells without TSH, whereas IL-1 caused significant reductions in [3H]thymidine incorporation induced by TSH or forskolin, which is known as an adenylate cyclase activator. Intracellular cAMP generation of FRTL-5 cells was stimulated by IL-1, whereas TSH-stimulated cAMP generation was inhibited by IL-1. These effects of IL-1 was neutralized by addition of anti-IL-1 antibody. The studies suggested that IL-1 blocks the effects of TSH on proliferation and cAMP generation of FRTL-5 cells on a post-receptor site of TSH.     

Growth promoting effect of head activator in cultured chick embryo brain cells

To investigate the regulatory role of head activator (HA) and its synthetic analogue, (Arg1,Phe5)-HA(AHA) on brain cell growth, we measured serial uptakes of [3H]thymidine, [3H]uridine and [3H]leucine and changes in cyclic AMP content in cultured chick embryo brain cells. HA stimulated all of these uptakes at a concentration of 10(-10) M, while 10(-9) M AHA suppressed them. The stimulatory effect of HA on [3H]thymidine uptake was observed after 4 h of the treatment, reached a maximum of 200% of the initial value at 8 h and declined to the pre-treatment level at 14 h. [3H]uridine uptake began to increase after 6 h of HA treatment, and the effect lasted for 4 h. Increase in [3H]leucine followed after 12 h and sustained for 4 h. Prior to the initiation of HA stimulation, cyclic AMP also began to rise, reaching 170% of the pre-treatment level at 6 h. In contrast, depression of [3H]thymidine uptake by AHA was noted at 6 h and continued for 8 h. Uptake of [3H]uridine and [3H]leucine showed similar tendency. Cyclic AMP in AHA-treated cells at 6 h was significantly lower than that in non-treated cells. These results indicate that HA stimulates DNA, RNA and protein synthesis in an early stage of growing brain cells, in which cAMP may be involved as a regulator of nerve cell growth.     

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.     

Role of protein kinase a in human hepatocyte DNA synthesis

The cellular mechanisms associated with the replicative response of hepatocytes to growth factor stimulation is incompletely understood. Murine hepatocyte DNA synthesis is altered by cyclic AMP, suggesting that protein kinase A is involved in the cellular mechanisms associated with liver growth. The purpose of this study was to evaluate the role of protein kinase A in human hepatocyte DNA synthesis. Human hepatocytes were isolated and maintained in primary culture on rat tail collagen. DNA synthesis was evaluated by determining [³H]thymidine incorporation. Human hepatocytes between 24 and 96 hr following harvest increased DNA synthesis in response to epidermal growth factor but not in response to glucagon, a stimulant of adenyl cyclase, or dibutyryl cyclic AMP. Mitogen-stimulated DNA synthesis was decreased by dibutyryl cyclic AMP. Cyclic AMP isomers that block or stimulate the effect of cyclic AMP on protein kinase A did not significantly alter resting or mitogen-stimulated human hepatocyte DNA synthesis. The results suggest that increased protein kinase A activity does not produce human hepatocyte replicative DNA synthesis.Supported in part by USPHS DK27695.     

Effects of cyclic nucleotides on deoxyribonucleic acid synthesis in hypophysectomized rat cartilage: stimulation of thymidine incorporation and potentiation of the action of somatomedin by analogs of adenosine 3',5'-monophosphate or a cyclic nucleotide phosphodiesterase inhibitor

The actions of cyclic nucleotides on basal and somatomedin-stimulated thymidine incorporation into DNA by costal cartilage from hypophysectomized rats were investigated. Three analogs of cAMP (dibutyryl, 8-bromo, and 8-dimethylamino derivatives, which are alternate activators of cAMP-dependent protein kinase and resistant to degradation by cAMP phosphodiesterase but represent a wide difference in potency as phosphodiesterase inhibitors) in range of concentrations from about 10(-5) to 3 X 10(-4) M enhanced basal and somatomedin-stimulated thymidine incorporation. Each cAMP analog at optimal concentration produced combined effects with a suboptimal concentration of somatomedin which were additive or greater. cAMP itself, 5'-AMP, adenosine, 8-Br-5'-AMP, 8-Br-AMPT, and cGMP at concentrations from 10(-7)--10(-3) M or dibutyryl cGMP at concentrations from 10(-10)--10(-3) M did not reproduce the effects of the cAMP analogs. A phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine) at concentrations of 100 or 500 microM also potentiated the effects of somatomedin. At 100- or 500-microM concentrations, the phosphodiesterase inhibitor increased cartilage levels of cAMP and cGMP. These results suggest a role for cAMP in DNA synthesis in rat cartilage. However, they fail to support the hypothesis that all effects of somatomedin on that process are mediated by cAMP, since stimulation of thymidine incorporation by the hormone can be demonstrated in cartilage maximally stimulated by analogs of cAMP.     

Control of deoxyribonucleic acid synthesis in normal rabbit colonic mucosa.

Although cyclic adenosine 3':5'-monophosphate (cyclic AMP, cAMP) is known to suppress DNA synthesis is cultured cells and experimental tumors, its role in normal intact tissue has been little explored. This study helps to define the influence of modifiers of cyclic AMP levels on DNA synthesis in rabbit colonic mucosa maintained in short term organ culture system. Base line studies showed that incorporation of [3H]thymidine into DNA was linear for 24 hr and predominantly in mucosal cells, as shown by autoradiography. Colon from a normal fed rabbit showed a gradient of DNA synthesis, lowest in the cecum and increasing to a maximum, 3-fold greater, at the splenic flexure. This pattern was obliterated by fasting, at which time no formed stool remained in the colon, and all colon mucosa incorporated thymidine at the lower level of the right colon. Known modifiers of intracellular cAMP were found to depress colonic DNA synthesis. Theophylline inhibited DNA synthesis by 35% at 0.5 mM concentration and increased intracellular cAMP levels. This inhibition took 10 hr to be manifest and was at least partly reversible. It was by far the most active of the methylxanthines, consistent with its potency as a phosphodiesterase inhibitor. N6,02-dibutyryl cyclic AMP inhibited DNA synthesis at concentrations as low as 0.025 mM, whereas adenosine and sodium butyrate were ineffective up to 1.0 mM. 5'-AMP did inhibit DNA synthesis, but only at 0.1 mM or higher and did not elevate intracellular cAMP levels. Other modifiers of cAMP which were effective included prostaglandins E1, E2, and F2alpha (2 times 10(-6) M) and papaverine (1 muM). Thymidine uptake was not affected by any of these drugs. The intrinsic thymidine pool was estimated at 20 muM by isotope dilution, and was not altered by theophylline. DNA synthesis in rabbit colon can be suppressed by increased cAMP levels within the time period allowed by organ culture. Thus, these drugs that elevated cAMP levels did not seem to suppress DNA synthesis by decreasing intracellular thymidine concentrations.     

Dibutyryl cyclic AMP, adenosine and guanosine blockade of the dopamine, ergocryptine and apomorphine inhibition of prolactin release in vitro.

Rat anterior hemipituitaries were incubated in Krebs-Ringer bicarbonate containing [3H]leucine. Newly synthesized [3H]prolactin and [3H]GH in the pituitary and incubation medium were assayed, as was the radioimmunoassayable prolactin released into the medium during a 5-h incubation. Dopamine (7.5 X 10(-8)M), ergocryptine (4 X 10(-10) M) and apomorphine (6 X 10(-8)M) all significantly inhibited both radioimmunoassayable prolactin release and newly-labeled [3H]prolactin release without affecting [3H]GH release. Conversely, dibutyryl cyclic AMP (2.5 mM) stimulated radioimmunoassayable prolactin release as well as [3H]prolactin and [3H]GH release. The addition of 2.5 mM dibutyryl cyclic AMP to media containing dopamine, ergocryptine or apomorphine completely restored both radioimmunoassayable prolactin release and [3H]prolactin release to at least control levels. Dopamine, ergocryptine and apomorphine all inhibited incorporation of [3H]leucine into prolactin but not into GH, whereas 2.5 mM dibltyryl cyclic AMP with any one of the inhibitors restored total incorporation into [3H]prolactin to levels insignificantly lower than the nucleotide-stimulated incorporation. Adenosine and guanosine at 2.5 mM also stimulated incorporation into [3H]prolactin and blocked the inhibitory effects of apomorphine upon [3H]prolactin synthesis and release. These nucleosides also stimulated [3H]GH release; and guanosine, but not adenosine, stimulated incorporation into [3H]GH. The ability of dibutryl cyclic AMP to block the effects of dopamine, ergocryptine and apomorphine upon prolactin release is consistent with these three inhibitors acting by a common mechanism. Cyclic AMP could be hypothesized as a second messenger for prolactin release, but the ability of adenosine and guanosine to mimic almost perfectly the effects of this cyclic nucleotide does not allow any conclusive interpretation.     

Evidence of a Stimulatory Effect of Cyclic AMP on Pancreatic Lipase and Colipase Synthesis in Rats

The effects of endogenous and exogenous cyclic AMP on the synthesis of pancreatic lipase, colipase, and amylase were studied. Pancreatic lobules were prepared and incubated with forskolin, dibutyryl cyclic AMP (dbcAMP), and dibutyryl cyclic GMP (dbcGMP), respectively, in the presence of “S-cysteine”. The individual pancreatic enzymes were isolated by polyacrylamide gel electrophoresis, and the incorporation of radioactive cysteine into lipase, colipase, and amylase was determined. Incubation with forskolin (25 uM) rapidly increased lipase synthesis rate within 30 min, followed by an increase in colipase synthesis rate after 60 min of incubation. Amylase synthesis rate did not change during the 1st h of incubation but decreased slightly when incubated for 2h. Incubation of pancreatic lobules with dbcAMP (1 mM) for 1 h also stimulated the incorporation of cysteine into lipase and colipase by 21% and 25%, respectively, whereas incubation with dbcGMP had no effect on the synthesis rates of lipase and colipase. Neither dbcAMP nor dbcGMP had any effect on synthesis rate of amylase. It is concluded that cyclic AMP might be an important intracellular signal for the synthesis of pancreatic lipase and colipase in the rat     

Evidence of a stimulatory effect of cyclic AMP on pancreatic lipase and colipase synthesis in rats.

The effects of endogenous and exogenous cyclic AMP on the synthesis of pancreatic lipase, colipase, and amylase were studied. Pancreatic lobules were prepared and incubated with forskolin, dibutyryl cyclic AMP (dbcAMP), and dibutyryl cyclic GMP (dbcGMP), respectively, in the presence of 35S-cystine. The individual pancreatic enzymes were isolated by polyacrylamide gel electrophoresis, and the incorporation of radioactive cystine into lipase, colipase, and amylase was determined. Incubation with forskolin (25 microM) rapidly increased lipase synthesis rate within 30 min, followed by an increase in colipase synthesis rate after 60 min of incubation. Amylase synthesis rate did not change during the 1st h of incubation but decreased slightly when incubated for 2 h. Incubation of pancreatic lobules with dbcAMP (1 mM) for 1 h also stimulated the incorporation of cysteine into lipase and colipase by 21% and 25%, respectively, whereas incubation with dbcGMP had no effect on the synthesis rates of lipase and colipase. Neither dbcAMP nor dbcGMP had any effect on synthesis rate of amylase. It is concluded that cyclic AMP might be an important intracellular signal for the synthesis of pancreatic lipase and colipase in the rat.