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PubMed2005

Anti-lipolytic action of AMP-activated protein kinase in rodent adipocytes.

Despite its importance in terms of energy homeostasis, the role of AMP-activated protein kinase in adipose tissue remains controversial. Initial studies have described an anti-lipolytic role for AMP-activated protein kinase, whereas more recent studies have suggested the converse. Thus we have addressed the role of AMP-activated protein kinase in adipose tissue by modulating AMP-activated protein kinase activity in primary rodent adipocytes using pharmacological activators or by adenoviral expression of dominant negative or constitutively active forms of the kinase. We then studied the effects of AMP-activated protein kinase activity modulation on lipolytic mechanisms. Finally, we analyzed the consequences of a genetic deletion of AMP-activated protein kinase in mouse adipocytes. AMP-activated protein kinase activity in adipocytes is represented mainly by the alpha(1) isoform and is induced by all of the stimuli that increase cAMP in adipocytes, including fasting. When AMP-activated protein kinase activity is increased by 5-aminoimidazole-4-carboxamide-riboside, phenformin, or by the expression of a constitutively active form, isoproterenol-induced lipolysis is strongly reduced. Conversely, when AMP-activated protein kinase activity is decreased either by a dominant negative form or in AMP-activated protein kinase alpha(1) knock-out mice, lipolysis is increased. We present data suggesting that AMP-activated protein kinase acts on hormone-sensitive lipase by blocking its translocation to the lipid droplet. We conclude that, in mature adipocytes, AMP-activated protein kinase activation has a clear anti-lipolytic effect.

3T3-L1 Cells↗
PubMed1984

PGE1-independent MDCK cells have elevated intracellular cyclic AMP but retain the growth stimulatory effects of glucagon and epidermal growth factor in serum-free medium.

Prostaglandin E1 (PGE1), a component in the hormone-supplemented, serum-free medium for the Madin Darby canine kidney (MDCK) cell line, has been proposed to increase MDCK cell growth by increasing intracellular cyclic AMP levels. The association between increased intracellular cyclic AMP and the growth stimulatory effect of PGE1 has been examined in normal MDCK cells and in PGE1-independent variants of MDCK. These variant cells have lost the PGE1 requirement for long term growth in defined medium. Normal MDCK cells had almost twofold higher intracellular cyclic AMP levels during growth in Medium K-1 (9.0 pmol/mg protein) than in Medium K-1 minus PGE1. Furthermore, PGE1-independent clone 1 had higher intracellular cyclic AMP levels in Medium K-1 minus PGE1 than normal MDCK cells in Medium K-1. This latter observation suggests that the PGE1 requirement for MDCK cell growth is associated with the low intracellular cyclic AMP levels of this cell line. An involvement of cyclic AMP in the growth response to PGE1 is supported by these observations, as well as by the growth stimulatory effects of other agents that affect cyclic AMP metabolism in MDCK cells. These agents include glucagon, isobutyl methylxanthine (IBMX), and dibutyryl cyclic AMP. The growth of PGE1-independent clone 1 was inhibited rather than stimulated by PGE1. Similarly, PGE1-independent cell growth was inhibited by IBMX and dibutyryl cyclic AMP. However, the growth response to one agent which increases cyclic AMP (glucagon) was retained in PGE1-independent clone 1. This result suggests that the effect of glucagon is not associated with increases in intracellular cyclic AMP. The growth stimulatory effect of epidermal growth factor (EGF) on normal MDCK cells was also studied. Although EGF does not act via a cyclic AMP-mediated mechanism, EGF increased normal MDCK cell growth and substituted for PGE1 in Medium K-1. Thus, EGF and PGE1 could possibly affect similar growth-related functions in MDCK cells, although by different pathways. This possibility was examined further, using PGE1-independent clone 1. EGF, like glucagon, was still growth stimulatory to the PGE1-independent cells. Consequently, the biochemical pathways by which EGF and PGE1 increase MDCK cell growth probably do not converge.

1-Methyl-3-isobutylxanthine↗
PubMed1975

Mode of action of antirheumatic drugs on the cyclic 3',5'-AMP regulated glycosaminoglycan secretion in fibroblasts.

The effect non-steroidal anti-inflammatory drugs on formation and release of glycosaminoglycans (GAG) and cyclic 3',5'-AMP levels was studied in embryonic mouse fibroblasts. The results were compared and correlated with the action of these drugs on cyclic 3',5'-AMP-dependent as well as independent protein kinase obtained from bovine diaphragm. 1. Phenylbutazone dose-dependently decreased cyclic 3',5'-AMP levels and GAG secretion both in unstimulated and PGE1 stimulated cells. 2. Indometacin decreased cyclic 3',5'-AMP levels and GAG secretion only in cells with elevated cyclic 3',5'-AMP levels after stimulation by PGE1. 3. Sodium salicylate decreased cyclic 3',5'-AMP levels in the presence and absence of PGE1. However, GAG secretion was reduced only in cells with elevated cyclic 3',5'-AMP levels, since the drug activated cyclic 3',5'-AMP-independent protein kinase activity, thus presumably precluding changes in GAG formation at low levels of cyclic 3',5'-AMP. 4. Mefenamic acid decreased cyclic 3',5'-AMP levels in cells stimulated by PGE1, whereas GAG secretion was increased both in the absence and presence of PGE1. This increase in GAG secretion was closely correlated to an enhanced cyclic 3',5'-AMP-dependent and independent protein kinase activity. The results indicate that non-steroidal anti-inflammatory drugs may exert their effects on GAG formation by interfering with cyclic 3',-5'-AMP formation or function.

Animals↗
PubMed1976

[Cyclic AMP and plasma renin activity in renal vein blood after amitryptiline, theophylline, furosemide and beta adrenergic blocking substances (author's transl)].

The influence of amitryptiline, theophylline and furosemide on the concentration of cyclic-AMP and plasma renin activity (PRA) was investigated in renal vein plasma. Additionally, the stimulating effect of furosemide on the PRA after application of the beta-adrenergic receptor antagonists propranolol and practolol and the cyclic AMP concentration in the plasma were measured. All drugs were given intravenously. After amitryptiline cyclic-AMP concentration increased about 1.5-fold compared with the basal value,PRA was not altered. After theophylline cyclic-AMP concentration increased about 1.2-fold, PRA 2.0-fold compared with the basal value, PRA was not altered. After theophylline cyclic-AMP concentration increased about 1.2-fold, PRA 2.0-fold coa increased within 7 min and no further increase was observed till the 15th min. After practolol cyclic-AMP concentration and PRA decreased about 20% compared with the basal value within 10 min. The stimulating effect of subsequently applied furosemide on PRA was not altered, but the cyclic-AMP concentration was not changed in this time by furosemide. After propranolol cyclic-AMP concentration and PRA decreased about 20% compared with the basal value. The cyclic-AMP concentration was not influenced by the following furosemide application, in agreement with the findings after practolol, however, PRA could be stimulated only in 36% of these patients under beta-receptor blockade. Our results show that changes of the concentrations of cyclic-AMP and of PRA are independent of each other. An elevated intracellular cyclic-AMP level due to the inhibition of phosphodiesterase is as a single factor unable to stimulate renin release. Our results give no evidence of a direct involvement of the adenylcyclase-system in the mechanism of renin release. The effect of propranolol and practolol on the basal value of PRA and cyclic-AMP is equal. The different influence of 10 mg propranolol and 20 mg practolol on the stimulating effect of 40 mg furosemide on the PRA can be interpreted as a dosage problem.

Adrenergic beta-Antagonists↗
PubMed1982

Regulation of rat brain (Na+ +K+)-ATPase activity by cyclic AMP.

The interaction between the (Na+ +K+)-ATPase and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5'-AMP, cyclic GMP or 5'-GMP, could inhibit the (Na+ +K+)-ATPase enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of (Na+ +K+)-ATPase enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854-3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in (Na+ +K+)-ATPase activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in (Na+ +K+)-ATPase enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of (Na+ +K+)-ATPase, resulted in a decrease in overall (Na+ +K+)-ATPase activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the (Na+ +K+)-ATPase has no effect on (Na+ +K+)-ATPase activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the (Na+ +K+)-ATPase enzyme, there appeared to be a decrease in the Na+-dependent phosphorylation of the Na+-ATPase enzyme, while the K+-dependent dephosphorylation of the (Na+ +K+)-ATPase was unaffected.

Adenylyl Cyclases↗
PubMed1975

Cyclic 3',5'-AMP phosphodiesterase of rabbit aorta.

Cyclic AMP and cyclic GMP phosphodiesterase activities (3' : 5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca2+ in the presence of the activator. However, the activity of the cyclic AMP phosphodiesterase was not significantly stimulated by Ca2+ up to 100 muM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60-70%) of the media enzyme was found in 105 000 times g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 times g supernatant the main kinetic parameters were specified as follows: 1) The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg2+ was achieved at 4mM of MgC12. 2) High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3) Activity-substrate concentration relationship revealed a high affinity (Km equals 1.0 muM) and low affinity (Km equals 45 muM) for cyclic AMP. The homogenate and 105 000 times g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent Km values for cyclic AMP hydrolysis were 1.2 muM and 36-40 muM and an enzyme extracted by sonication from 105 000 times g precipitate also exhibited non-linear kinetics (Km equals 5.1 muM and 70 muM). 4) Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I5 o at 0.62 muM, I5 o of the media at 0.62 muM and I5 o of the adventitia at 1.0 muM) than on the brain (I5 o at 8.5 muM) and serum (I5 o at 20 muM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.

Animals↗
PubMed1992

Regulation of cyclic AMP metabolism in bovine adrenal medullary cells.

The capacity of cultured bovine adrenal medullary cells to metabolize and export cyclic AMP has been studied. Basal cellular cyclic AMP levels were increased 50% by 100 microM 3-isobutyl-1-methylxanthine (IBMX) and rolipram, a class IV (cyclic AMP-specific) phosphodiesterase (PDE) inhibitor. They were not affected by inhibition of class I (Ca2+/calmodulin-dependent), class III (cyclic GMP-inhibited) or class V PDE (cyclic GMP-specific) with vinpocetine or 3-isobutyl-8-methoxymethyl-1-methylxanthine (8-methoxymethyl-IBMX), SK&F 94120, or MB 22,948, respectively, all at 100 microM. Furthermore, only IBMX and rolipram enhanced the cyclic AMP response to 0.3 microM forskolin. Rolipram had an EC50 of < or = 1 microM and was equally effective at 100 microM and 1 mM. IBMX enhanced cyclic AMP levels significantly more at 1 mM than at 100 microM. Neither vinpocetine nor 8-methoxymethyl-IBMX (100 microM) enhanced the Ca(2+)-dependent cyclic AMP response to K+ depolarization. Elevation of cyclic GMP levels with sodium nitroprusside (10 or 100 microM), to activate any cyclic GMP-stimulated class II PDE and to inhibit any cyclic GMP-inhibited class III PDE, also had no effect on basal or forskolin-stimulated cyclic AMP levels. In the presence of IBMX (1 mM), forskolin (5 microM) caused a rapid and large increase in cellular cyclic AMP levels which was maximal after about 5 min and declined slightly over 3 hr. Over this period, extracellular cyclic AMP levels rose almost linearly reaching levels 2-3 times those in the cells. The results indicate bovine adrenal medullary cells have a high capacity for sustained cyclic AMP export. Furthermore, two PDE isozymes appear to degrade cyclic AMP in these cells, a rolipram-sensitive, cyclic AMP-specific, class IV isozyme and a rolipram-insensitive isoform.

Adrenal Medulla↗
PubMed1985

Instability of succinyl ester linkages in O2'-monosuccinyl cyclic AMP-protein conjugates at neutral pH.

Chromatographic and immunological evidence is presented regarding the hydrolysis of the ester linkage of O2'-monosuccinyl cyclic AMP in neutral solutions. Such hydrolysis occurs whether the nucleotide derivative is present in free form in solution or conjugated through its succinyl carboxyl group via an amide bond to proteins. The latter process apparently occurs when succinyl cyclic AMP is conjugated to human serum albumin for use as an immunogen in the production of anti-cyclic AMP antibodies and when the derivative is coupled to the enzyme glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49). The enzyme conjugate has been used in developing a homogeneous enzyme immunoassay for cyclic AMP. Inhibition of the catalytic activity of enzyme-cyclic AMP conjugates by anti-cyclic AMP antibody decreases with time, apparently due to the loss of cyclic AMP from enzyme-cyclic AMP conjugates stored in neutral solutions. In addition, the ability of free cyclic AMP to completely reverse the inhibition process decreases with time because of the presence of antibodies in the anti-cyclic AMP sera that apparently inhibit enzyme activity because of their binding specificity for the residual succinate-protein determinant sites of the enzyme conjugates. Lyophilization of the conjugates immediately after preparation helps to overcome the problem; however, in vivo hydrolysis of immunogens prepared with the succinyl cyclic AMP derivative may always occur. The consequence of this hydrolysis reaction and the subsequent formation of anti-succinyl-protein antibodies will be discussed with regard to existing RIAs for cyclic AMP and a new homogeneous enzyme immunoassay for the nucleotide.

Antibody Specificity↗
PubMed1981

Enzyme immunoassay of cyclic adenosine 5', 5' - monophosphate (AMP) using beta-D-galactosidase as label.

Succinyl cyclic adenosine monophosphate (AMP) was synthesized and coupled to beta-D-galactosidase using 1-ethyl-3-(3-dimethylaminopropyl)carbodimide. An antiserum to cyclic AMP was raised in rabbits by immunization with a succinyl cyclic AMP-human serum albumin conjugate. An enzyme immunoassay of cyclic AMP was successfully performed via the competitive binding procedure using the succinyl cyclic AMP-enzyme conjugate and the antiserum to cyclic AMP. The sensitivity of this assay has been increased several hundredfold by prior 2'-O-succinylation of cyclic AMP as already described by Cailla et al. in the radioimmunoassay of cyclic nucleotides. The assay system makes it possible to ascertain values as low as 6 fmol of cyclic AMP/tube. Human plasma cyclic AMP could be accurately determined by this method without requiring a deproteinizing reagent as the first step of assay. The concentration of TCA-extracted cyclic AMP from various tissues of mice was determined by both enzyme immunoassay and radioimmunoassay. There was a good correlation between the values for cyclic AMP determined by the two methods (Y = 1.01X - 0.15, r = 0.996, n = 39). Succinylated nucleotides and nucleosides such as cyclic GMP, ATP, ADP, and AMP, adenosine, adenine, GTP, GDP, GMP, guanosine, and guanine had no effect on the immunoassay of cyclic AMP.

Animals↗
PubMed1979

Parathyroid hormone-induced calcium efflux from isolated renal cortical tubules: evidence for cyclic AMP mediation.

Effects of parathyroid hormone (PTH) upon cyclic AMP and calcium efflux in isolated renal cortical tubules from hamsters were investigated. PTH caused a rapid rise in cyclic AMP levels, temporally preceding an increase in calcium efflux. Increases in both cyclic AMP levels and calcium efflux were noted over an identical PTH concentration range 0.007--0.7 U/ml). Other peptide hormones tested which had no effect upon cyclic AMP levels did not enhance efflux of calcium. The phosphodiesterase inhibitor methyl isobutylxanthine (MIX) was utilized in other studies to potentiate the cyclic AMP response, and produce a range of cyclic AMP concentrations in response to PTH. In these experiments a range of calcium efflux responses was noted which closely paralleled changes in cyclic AMP. Direct addition of cyclic AMP or dibutyryl cyclic AMP to isolated renal tubules caused increased efflux of calcium, while addition of 5'-AMP did not. These results indicate a role for cyclic AMP as a mediator of PTH-induced calcium efflux in this system and suggest that cyclic AMP may mediate the action of this hormone in enhancing renal conservation of calcium in vivo.

1-Methyl-3-isobutylxanthine↗
PubMed1975

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat.

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.

Aging↗
PubMed1995

Haloperidol-induced Fos expression in striatum is dependent upon transcription factor cyclic AMP response element binding protein.

Haloperidol has been shown to induce rapid and transient expression of c-fos messenger RNA and Fos protein in striatal neurons via dopamine D2 receptors. Regulation of the c-fos gene by cyclic AMP and Ca2+ has been shown to be dependent on a DNA regulatory element within its promoter that binds the constitutively expressed transcription factor cyclic AMP response element binding protein. Cyclic AMP response element binding protein binds to an oligonucleotide containing the calcium/cyclic AMP response element of the c-fos promoter sequence in striatal cell extracts; the amount of binding is not regulated by haloperidol treatment. We have previously shown that haloperidol induces cyclic AMP response element binding protein phosphorylation in the striatum. Here we show by intrastriatal injection of antisense oligonucleotides that haloperidol-induced Fos expression is dependent on cyclic AMP response element binding protein. Intrastriatal injections of phosphorothioate oligonucleotides, in antisense orientation to cyclic AMP response element binding protein messenger RNA, reduce levels of cyclic AMP response element binding protein and completely prevent haloperidol-mediated induction of Fos. Oligonucleotides in sense orientation have no such effect. We observed a markedly different time course of the Fos protein inhibition by cyclic AMP response element binding protein antisense oligonucleotides compared to c-fos antisense oligonucleotides. This most likely reflects the different half-lives of c-fos and cyclic AMP response element binding protein messenger RNA and proteins. Neither cyclic AMP response element binding protein nor c-fos antisense oligonucleotide injection reduced c-Jun immunostaining in the striatum. We conclude that haloperidol induces Fos via transcription factor cyclic AMP response element binding protein.

Activating Transcription Factor 2↗
PubMed1985

Effect of cyclic AMP on acidification in the isolated turtle bladder.

Cyclic AMP (10 mM) has been demonstrated to inhibit hydrogen ion secretion in the isolated turtle bladder. These experiments were designed to study the effect of cyclic AMP on hydrogen ion secretion in the isolated turtle bladder using both the pH stat and the reverse short circuit current techniques. Sodium transport was measured as the short circuit current. Studies were carried out at 0% CO2 and 1% CO2 at pH 7.4. Cyclic AMP (10 mM) was added to either the serosal or mucosal solutions, and hydrogen ion secretion was measured from 0 to 120 min. In the presence or absence of carbon dioxide, cyclic AMP and dibutyryl cyclic AMP had no effect on hydrogen ion secretion in fasted turtles. The addition of theophylline (10 mM) to the serosal solution, with or without cyclic AMP had no effect on proton secretion. Sodium transport was unchanged from control following serosal or mucosal addition of 10 mM cyclic AMP in the presence or absence of carbon dioxide. In chronically bicarbonate-loaded turtles proton secretion was the same as control fasted turtles. In these animals, however, serosal administration of 10 mM cyclic AMP significantly stimulated bicarbonate secretion. Stimulation of bicarbonate secretion occurred in the presence of a 20 mM bicarbonate gradient. When there was no bicarbonate gradient, cyclic AMP was without effect; cyclic AMP had no effect on bicarbonate permeability when measured in the presence of acetazolamide. These results indicate that cyclic AMP has no effect on hydrogen ion secretion or sodium transport in the isolated turtle bladder when studied at two different rates of acidification (0 and 1% CO2). Cyclic AMP appears to stimulate active bicarbonate secretion.

Acetazolamide↗
PubMed1981

Effects of dopamine on prolactin secretion and cyclic AMP accumulation in the rat anterior pituitary gland.

The effects of dopamine on pituitary prolactin secretion and pituitary cyclic AMP accumulation were studied by using anterior pituitary glands from adult female rats, incubated in vitro. During 2h incubations, significant inhibition of prolactin secretion was achieved at concentrations between 1 and 10nm-dopamine. However, 0.1-1mum-dopamine was required before a significant decrease in pituitary cyclic AMP content was observed. In the presence of 1mum-dopamine, pituitary cyclic AMP content decreased rapidly to reach about 75% of the control value within 20min and there was no further decrease for at least 2h. Incubation with the phosphodiesterase inhibitors theophylline (8mm) or isobutylmethylxanthine (2mm) increased pituitary cyclic AMP concentrations 3- and 6-fold respectively. Dopamine (1mum) had no effect on the cyclic AMP accumulation measured in the presence of theophylline, but inhibited the isobutylmethylxanthine-induced increase by 50%. The dopamine inhibition of prolactin secretion was not affected by either inhibitor. Two derivatives of cyclic AMP (dibutyryl cyclic AMP and 8-bromo cyclic AMP) were unable to block the dopamine (1mum) inhibition of prolactin secretion, although 8-bromo cyclic AMP (2mm) significantly stimulated prolactin secretion and both compounds increased somatotropin (growth hormone) release. Cholera toxin (3mug/ml for 4h) increased pituitary cyclic AMP concentrations 4-5-fold, but had no effect on prolactin secretion. The inhibition of prolactin secretion by dopamine was unaffected by cholera toxin, despite the fact that dopamine had no effect on the raised pituitary cyclic AMP concentration caused by this factor. Dopamine had no significant effect on either basal or stimulated somatotropin secretion under any of the conditions tested. We conclude that the inhibitory effects of dopamine on prolactin secretion are probably not mediated by lowering of cyclic AMP concentration, although modulation of the concentration of this nucleotide in some other circumstances may alter the secretion of the hormone.

1-Methyl-3-isobutylxanthine↗
PubMed1974

Activation of protein kinase by physiological concentrations of cyclic AMP.

When determined under the usual conditions of an excess of ligand over protein, the concentration of cyclic AMP necessary to activate pure preparations of cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:-protein protein phosphotransferase) half-maximally is in the range of 0.2-0.3 muM when casein or glycogen synthetase is used as the substrate, i.e., essentially the same as the concentration of the nucleotide that is found in resting skeletal muscle. The apparent dissociation constant for cyclic AMP bound to the protein kinase is also about 0.2-0.3 muM when measured under similar conditions. The concentration of the protein kinase in muscle is relatively high (0.23 muM), however, and under these conditions the apparent activation constant of the enzyme for cyclic AMP is raised so that an increase in cyclic AMP levels in the tissue would cause a concomitant increase in protein kinase activity over a wide range of nucleotide concentration. As a result, it is unnecessary to invoke compartmentalization of cyclic AMP to explain how it can control protein kinase activity in vivo. Another factor that may increase the effectiveness of changes in cyclic AMP concentration is the heat-stable protein inhibitor of protein kinase that may function to inhibit the activity of nearly all the protein kinase catalytic subunit dissociated by basal concentrations of cyclic AMP. Finally, the near equity between the concentration of cyclic AMP binding sites and the ligand itself provides a potential mechanism whereby agents can affect the total cyclic AMP content without directly affecting adenylate cyclase, cyclic AMP phosphodiesterase, or cyclic AMP transport.

Animals↗
PubMed1983

Cyclic AMP, glucocorticoid, and retinoid modulation of in vitro keratinocyte growth.

The hypothesis that an imbalanced cyclic AMP and cyclic GMP ratio was central to the cutaneous expression of psoriasis prompted the design of a series of in vitro experiments. The aim of these studies was to describe the functional effects of increased intracellular cyclic AMP and of drugs therapeutic in psoriasis on epidermal keratinocyte growth. Epidermal basal cells trypsinized from neonatal mouse and adult and neonatal human skin were grown on plastic or on gelled collagen surfaces. These were used to study the effect of cyclic AMP analogues and cholera toxin (an irreversible stimulator of cyclic AMP synthesis) on keratinocyte growth. Greatly increased intracellular cyclic AMP levels, that is, 60-fold to 70-fold, stimulated neonatal mouse keratinocyte proliferation and differentiation; these same doses were cytotoxic to both neonatal and adult human cells. However, modest increases in intracellular cyclic AMP did stimulate adult human keratinocyte proliferation. The glucocorticoid triamcinolone acetonide inhibited neonatal mouse keratinocyte proliferation for approximately 1 week; the cells then became refractory to the triamcinolone acetonide effect. Triamcinolone acetonide did not apparently act through cyclic AMP-mediated events. In fact, this glucocorticoid inhibited cyclic AMP-stimulated epidermal keratinocyte proliferation. Likewise, vitamin A analogues, including the psoriasis therapy drug Ro 10-9359 inhibited neonatal mouse keratinocyte proliferation and specific differentiation events; the retinoids therapeutic in psoriasis apparently did not act via cyclic AMP-mediated events and inhibited cyclic AMP-stimulated functions. Our results indicated that cyclic AMP is a mitogenic signal for epidermal keratinocytes. This cyclic nucleotide may be important in regulating epidermal hyperproliferation. A central role for cyclic AMP in the cutaneous expression of psoriasis, however, is yet to be proven.

Animals↗
PubMed1973

The relation of adenyl cyclase to the activity of other ATP utilizing enzymes and phosphodiesterase in preparations of rat brain; mechanism of stimulation of cyclic AMP accumulation by adrenaline, ouabain and Mn++.

1. The mechanism of stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by adrenaline and ouabain and the effect of Mn(++) substitution for Mg(++) as the metal ion requirement of this system was studied in cell-free preparations of adenyl cyclase from rat brain.2. In the rat cerebral cortex preparation, substitution of Mn(++) for Mg(++) significantly increased cyclic AMP accumulation while significantly inhibiting adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis and adenosine 5'-monophosphate (AMP) accumulation. In the synaptic membrane preparation, in the absence of NaF, the highest amount of ATP hydrolysis was obtained in tissue prepared with Mn(++) and incubated with Mg(++); under these conditions cyclic AMP accumulation was equal to that produced under any other condition and significantly higher than that observed in the presence of Mg(++) prepared and Mg(++) incubated tissue.3. Preparation and/or incubation of tissue with Mn(++) significantly reduced phosphodiesterase (PDE) activity compared to that observed in Mg(++) prepared tissue.4. Adrenaline and ouabain both significantly increased cyclic AMP accumulation in the rat cerebral cortex preparation but did not inhibit ATP or ADP hydrolysis. In the synaptic membrane preparation, in the presence of 0.01 mM Ca(++), adrenaline but not ouabain significantly increased cyclic AMP accumulation. Phenoxybenzamine (0.1 mM) and pronethalol (0.1 mM) significantly inhibited adrenaline-induced cyclic AMP accumulation in both these preparations.5. Ouabain and adrenaline both failed to stimulate cyclic AMP accumulation in the presence of Mn(++) prepared and/or incubated tissue.6. Ouabain and adrenaline had no effect on PDE activity in either of these preparations.7. It was concluded that Mn(++) increased cyclic AMP accumulation in part by indirect inhibition of ATP and ADP hydrolysis which provides inhibitors of cyclic AMP destruction, by direct stimulation of adenyl cyclase and by inhibition of cyclic AMP destruction in a way unrelated to nucleotide inhibition of PDE. Adrenaline and ouabain appeared tp stimulate cyclic AMP accumulation in a more direct manner.

Adenosine Diphosphate↗
PubMedDate not supplied

The role of alpha-MSH, its agonists, and C-AMP in in vitro avian melanocytes.

Little is known about the effect of alpha-MSH and other melanogenic stimulators on avian melanocytes. Tissue cultures of Barred Plymouth Rock regenerating feather melanocytes were established and the culture medium contained selected concentrations of alpha-MSH and other melanogenic stimulators in Ham's F-10 medium supplemented with antibiotics and 10% new born calf serum. Cultures were maintained at 37 degrees C in 95% air/5% CO2. No increase in melanogenesis over control levels due to the addition of 10(-5) M Forskolin, 10(-4) M IBMX, 10(-3) M c-GMP, and 10(-3) M db-c-AMP was observed in the cultures on days 5 and 7. However, 2.5 (optimum), 5, and 10 micrograms/ml alpha-MSH and 10(-3) M 8-bromo-c-AMP significantly increased melanogenesis over control levels on days 5 and 7. The stimulation of melanogenesis was detectable by a significantly increased number of melanocytes containing numerous stage IV melanosomes. No increase in melanocyte cell number was observed in any of the experimental cultures. The addition of 1, 2 (optimum), or 3 mM calcium did enhance the increased pigmentation effect of 2.5 micrograms/ml alpha-MSH. Two very convincing experiments showed that c-AMP was the second messenger for alpha-MSH in these birds. First, the c-AMP inhibitor, 10(-3) M Rp-c-AMPS, completely inhibited the stimulatory effect of alpha-MSH in these in vitro melanocytes. Second, direct measurements of c-AMP levels in feather tissue showed a significant increase in c-AMP levels 10.min after alpha-MSH treatment. Controls received no alpha-MSH. The results showed that these avian melanocytes have alpha-MSH receptors and were able to respond to the hormone. C-AMP was the second messenger in this system. Apparently db-c-AMP was not able to enter these mature, highly-differentiated cells and c-AMP agonists, Forskolin and IBMX, were also either unable to enter these older cells or, if they did enter the cells, were unable to stimulate c-AMP production. Evidently the more lipophilic 8-bromo-c-AMP was able to enter these cells and stimulate melanogenesis.

1-Methyl-3-isobutylxanthine↗