Effects Of Cyclic AMP Analogues Research Articles

The effect of cyclic AMP analogues on cholesteryl ester synthesis and hydrolysis in cultured rat peritoneal macrophages.

The accumulation of large amounts of cholesteryl ester in macrophages which have invaded the artery wall leads to the formation of foam cells, and is one of the early events in the development of atherosclerosis. The activities of both acyl Coenzyme A: cholesterol acyltransferase (ACAT), which is responsible for cholesterol esterification, and cholesteryl ester hydrolase (CEH) have been shown to be increased in foam cells ( 1 ), so that the cholesteryl ester undergoes a continual cycle of formation and hydrolysis ( l,2). Cyclic AMP (CAMP) has been reported to both stimulate (3) and inhibit (4) ACAT activity in the murine macrophage cell line 5774, while in a recent study we were unable to demonstrate any effect (5). CEH activity, on the other hand, has been shown to be increased by dibutyryl cAMP (Bt2cAMP) in two other murine cell lines, P388DI and WEHl (6.7). In the present work, we have investigated the role of cAMP in the regulation of cholesteryl ester metabolism and foam cell formation in cultured rat peritoneal macrophages using cellpermeable cAMP analogues. Macrophages were collected from male Wistar rats (250300g) by peritoneal washing (8) 2 days after intraperitoneal injection of 3ml thioglycollate (3%). Cells from 3-4 rats were pooled for each preparation. After washing, the cells were cultured as monolayers in supplemented Dulbecco's modified Eagle's medium at 37OC in 95% air l 5% CO?. Rat lipoprotein of density < 1.050glml was isolated from rat serum by ultracentrifugation and acetylated by incubation with acetic anhydride (9). Cholesterol esterification was assayed by the incorporation of radioactivity from potassium ('HI =!ca:c in:o cholesteryl esters during a 5h incubation period. Lipids were extracted with chloroform: methanol (2: 1, v:v) and separated by thin layer chromatography. For the determination of CEH activity, the cells were removed from the plates with a plastic scraper, washed with phosphate-buffered saline and disrupted by sonication. The release of I I-'%]oleate from cholesterol I I '%)oleate was then measured in the resulting homogenate (10). The cAMP analogues used were N6 benzoyl cAMP (BEAMP), B-(4-chlorophenylthio) cAMP (CPTcAMP), 8 chloro cAMP (ClcAMP), N6 monobutyryl cAMP (MBcAMP), 8-(6-amino hexyl) amino cAMP (AHA CAMP), 8-piperidino cAMP (PIP CAMP) and dibutyryl cAMP (BtZcAMP). Significance limits were calculated using Student's t test. Cholesterol esterification in the cultured macrophages was unaffected by a range of cAMP analogues at a concentration of

Comparison of the effects of cyclic AMP analogues on cholesterol metabolism in cultured rat and hamster hepatocytes

The effects of two cell-permeable cyclic AMP analogues, 8-chloro cyclic AMP (8-Cl cAMP) and 8-(4-chlorophenylthio) cyclic AMP (8-CPT cAMP), on cholesterol esterification, cholesteryl ester hydrolysis and bile acid synthesis were compared in cultured rat and hamster hepatocytes. Cholesterol esterification, as measured by the incorporation of [ 3H]oleate into cholesteryl ester, was increased by 58–88% by the analogues in rat hepatocytes and by 33–43% in hamster cells. The response in rat hepatocytes, however, was observed after a relatively short incubation time (28% increase after 1 hr), whereas that in hamster cells required a longer period (36% after 12 hr) to become apparent. The activity of the cytosolic neutral cholesteryl ester hydrolase in rat.hepatocytes was also stimulated by both cyclic AMP analogues (31–37%), but the microsomal activity was unaffected. In hamster hepatocytes, however, microsomal cholesteryl ester hydrolase activity was increased (47–80%) in the presence of 8-C1 cAMP or 8-CPT cAMP. Bile acid synthesis was increased by 8-CPT cyclic AMP in rat cells (-25%) but was unchanged by both analogues in hamster hepatocytes. These results indicate significant differences in the way in which cholesterol metabolism responds to cyclic AMP incultured rat and hamster hepatocytes.

Effects of cyclic AMP and analogues on neurogenic transmission in the rat tail artery

1 The effects of two 8-substituted analogues of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were compared with those of forskolin and isoprenaline on [3H]-noradrenaline release and vasoconstriction induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery, in the absence and in the presence of protein kinase inhibitors. 2 8-Bromo-adenosine 3':5'-cyclic monophosphate (8-bromo-cyclic AMP, 10-300 microM), 8-(4-chlorophenyl-thio)-adenosine 3':5' cyclic monophosphate (8-pCPT-cyclic AMP, 3-300 microM), forskolin (0.3-10 microM) and isoprenaline (1 nM-1 microM) all concentration-dependently enhanced stimulation-induced [3H]-noradrenaline release. The effect of cyclic AMP analogues was larger (2.5 fold at 300 microM) than those of cyclic AMP elevating drugs (1.6 fold at 10 microM for forskolin and 1.5 fold at 30 nM for isoprenaline). 3 At concentrations active at the prejunctional level, the four drugs had differential effects on stimulation-induced vasoconstriction, which was enhanced by the two cyclic AMP analogues, decreased by forskolin and not significantly altered by isoprenaline. 4 The [3H]-noradrenaline release-enhancing effects of 8-bromo-cyclic AMP, forskolin and isoprenaline were significantly decreased by the cyclic AMP-dependent protein kinase (PKA) inhibitor (N-[2-((3-(4-bromophenyl)-2-propenyl)-amino)-ethyl]-5- isoquinolinesulphonamide, di-hydrochloride) (H-89; 100 nM). By contrast they were unaffected by the cyclic GMP-dependent protein kinase (PKG) inhibitor, 8-bromo-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-bromo-cyclic GMPS; 10 microM). By contrast they were unaffected by the cyclic GMP-dependent protein kinase (PKG) inhibitor,8-bromo-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-bromo-cyclic GMPS; 10 MicroM).At the same concentrations the PKA inhibitor attenuated only the nerve-induced vasoconstrictor responses obtained in the presence of 8-bromo-cyclic AMP, whereas the PKG inhibitor did not modify that obtained in the presence of 8-bromo-cycic AMP or forskolin.5. Exposure to the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (1 MicroM) enhanced nerve-evoked [3H]-noradrenaline release, and this effect was decreased by the PKC inhibitor, 2-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(-indol-3-yl)-maleimide (GF 109203X; 100 nM). However, the latter drug did not modify the enhancing effect of 8-bromo-cyclic AMP on [3H]-noradrenaline release.6. It is concluded that activation of cyclic AMP-dependent protein kinase is involved in the enhancing effect of cyclic AMP-elevating compounds on prejunctional release of noradrenaline. In addition the results provide no clear-cut evidence for a vasodilator role of PKA.

The effect of cyclic AMP analogues on cholesterol metabolism in cultured rat hepatocytes.

The effect of cyclic AMP analogues on cholesterol metabolism in cultured rat hepatocytes.

The effect of cyclic AMP analogues and glucagon on cholesteryl ester synthesis and hydrolysis in cultured hamster hepatocytes

Two cyclic AMP analogues, 8-chloro cyclic AMP and 8-(4 ehlorophenylthio) cyclic AMP, were found to increase the incorporation of [ 3H]oleate into cholereryl ester in cultured hamster hapatocytes (30–40%), while incorporation into triacylglycerol was unaffected. An increase of a similar magnitude was observed in the presence of glucagon and the phosphodiesterase inhibitor, theophylline. The cyclic AMP analogues also stimulated the activity of neutral cholesteryl ester hydrolase in the cells, and this effect was mimicked by glucagon and theophylline. These results show that cyclic AMP can affect the cholesteryl ester cycle in hamster hepatocytes, and support the idea that the enzymes involved may be co-ordinately regulated.

Influence of phosphodiesterase inhibition and of carbachol on inotropic effects of 8-substituted cyclic AMP analogues

The influence of phosphodiesterase inhibitors and of carbachol on the positive inotropic effect of 8-substituted cyclic AMP analogues was studied on isometrically contracting guinea-pig papillary muscles driven at a rate of 0.2 Hz. In muscles from reserpine-pretreated animals, the phosphodiesterase inhibitors 3-isobutyl, 1-methyl xanthine (IBMX; 20 mumol/l) and papaverine (10 mumol/l) shifted the concentration-effect curves of 8-substituted cyclic AMP benzyl esters to the left, decreasing the EC50 by a factor of 10 to 25. In the presence of IBMX (5 and 20 mumol/l) or papaverine (10 mumol/l), the slopes of the concentration-effect curves of 8-substituted cyclic AMP benzyl esters became flatter. The positive inotropic effect and the increase in Vmax, overshoot and duration of slow action potentials induced by cyclic AMP analogues were not affected by carbachol (0.1-10 mumol/l). In the presence of IBMX (20 mumol/l), however, carbachol (3 mumol/l) antagonized the positive inotropic effect of 8-substituted cyclic AMP derivatives, shifting the EC50-values by a factor of 3 to the right. Cyclic AMP content determined by radioimmunoassay in individual papillary muscles was raised 1.22 and 1.63-fold in the presence of 3 and 20 mumol/l IBMX. Isoprenaline (0.1 mumol/l) induced an increase in cyclic AMP content which was not significantly different from that produced by 20 mumol/l IBMX, but in contrast to the phosphodiesterase inhibitor enhanced force of contraction by 17.7 mN as compared to 1.5 mN obtained with 20 mumol/l IBMX. The findings are consistent with a model that describes the interaction between IBMX and cyclic AMP analogues as an additive effect with only endogenously accumulated cyclic AMP (due to phosphodiesterase inhibition) being involved in the negative inotropic effect of carbachol. From the failure of carbachol to affect the positive inotropic effect of cyclic AMP analogues, it is concluded, that cyclic AMP derivatives do not act as phosphodiesterase inhibitors, and that the well-known negative inotropic effect of carbachol in the presence of cyclic AMP-elevating drugs does not occur at a step beyond cyclic AMP accumulation.

In Vitro Phosphorylation of Microtubule‐Associated Protein 2: Differential Effects of Cyclic AMP Analogues

Microtubules purified from brain tissue contain endogenous cyclic AMP (cAMP)-dependent protein kinase activity, and microtubule-associated protein 2 (MAP2) is the major substrate. Beef brain microtubules were prepared and used as a model system to study the differential effects of rationally selected cyclic nucleotide analogues on microtubule receptor protein kinase. Data are presented to indicate that the following molecular interactions are essential for activation of the phosphorylation of MAP2: (a) hydrogen bond formation toward the 2', 3', or 5' position, (b) interaction with phosphorus, and (c) no hydrogen bonds but hydrophobic interactions at the base moiety. Thus, the activation mechanism of the type II protein kinase associated with brain microtubules resembles the mechanism found in protein kinases of other systems. In addition, we have studied the effect of the two diastereomers of adenosine 3',5'-monophosphorothioate (cAMPS). The (Sp)-cAMPS isomer was found to activate MAP2 protein kinase, whereas the (Rp)-cAMPS isomer had no activating effect. In contrast, this compound was able to inhibit cAMP-stimulated MAP2 phosphorylation and thus acts as an antagonist of the Sp diastereomer and cAMP. Hence, this analogue provides a useful means to clarify further the effect of cAMP-dependent phosphorylation on functional properties in microtubules in general.

Effects of cyclic AMP analogues and phosphodiesterase inhibitors on phospholipid biosynthesis in fetal type II pneumocytes

Purified type II pneumocytes grown in monolayer cultures after isolation from fetal rabbit lung organotypic cultures were employed to investigate effects of cAMP analogues and phosphodiesterase inhibitors on [ methyl- 14C]choline and [9–10(n) 3H]palmitate incorporation into cell lipids. After 24 h exposure to 0.5 mM N 6,O 2-dibutyryl-cAMP or 8-bromo-cAMP, a significant increase was found in the rate of incorporation of choline into phospholipids. Addition of 1 mM 1-methyl-3-isobutylxanthine or aminophylline also increased incorporation of choline into phospholipids but did not significantly change the incorporation of choline into sphingomyelin. These effects were not due to increased uptake of choline or changes in the pool size of the precursor. Cyclic AMP analogues also stimulated the rate of incorporation of palmitate into most lipid fractions but did not alter the relative percentages of incorporation of either precursor into any of the phospholipids. Phosphodiesterase inhibitors did not significantly change the rate of incorporation of palmitate into neutral lipids and most phospholipids, except for a decrease into sphingomyelin, phosphatidylinositol and phosphatidylethanolamine. However, they increased the percentage of incorporation of palmitate into phosphatidylcholine and decreased the percentage of incorporation into most other phospholipids. These data clearly indicate that cAMP can stimulate the synthesis of phospholipids within the type II pneumocytes. This effect is probably a general stimulation effect for the cAMP analogues but methylxanthines may selectively increase the synthesis of surfactant lipids such as phosphatidylcholine while decreasing that of other membrane-associated phospholipids.

Maturation of neurotransmission at cholinergic synapses formed in culture by rat retinal neurons: Regulation by cyclic AMP

The objective of this study was to investigate how the maturation of neurotransmission is regulated. We used a retina-muscle cell culture system to explore the effects of cyclic AMP analogues on the developmental step in which a presynaptic neuron acquires the ability to transmit excitatory information across a synapse. Cholinergic neurons dissociated from the perinatal rat retina form synapses in culture with rat striated muscle cells. Early in the functional maturation of these retina-muscle synapses, there is a period in which the release of acetylcholine occurs spontaneously, but cannot be evoked. This stage is followed by the emergence of transmitter release that is stimulus-evoked. We report here that exposure of cultured embryonic neurons of the rat retina to 8-bromo-cyclic AMP precociously induced in these neurons the ability to release acetylcholine at synapses in response to excitatory stimulation. This effect on synaptic development could be mimicked by an inhibitor of phosphodiesterase, isobutylmethylxanthine. The results of a variety of experiments lead us to propose that 8-bromo-cyclic AMP may accelerate the development of neurotransmission by influencing pre-synaptic events linking neuronal depolarization with acetylcholine release. Our data support the hypothesis that cyclic AMP may be an intracellular mediator for developmental signals which regulate the emergence of effective neurotransmission across nascent synapses.

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.

Prolonged effects of cyclic AMP analogues of phosphatidylcholine biosynthesis in cultured rat hepatocytes

The short- and long-term effects of cyclic AMP analogues and phosphodiesterase inhibitors on phosphatidylcholine biosynthesis in monolayer cultures of rat hepatocytes were investigated. All the compounds tested produced an inhibition of phosphatidylcholine biosynthesis for up to 6 h after addition to the hepatocyte medium. The reduced rate of lipid synthesis was a function of the concentration of cyclic AMP analogue and was independent of the concentration of choline in the medium. The proportion of choline oxidized to betaine was relatively unaffected. Choline was incorporated into hepatocytes by saturable and non-saturable mechanisms. Although the various cAMP analogues had different effects on choline uptake, chlorophenylthio-cAMP reduced uptake of choline by 28% for cells treated for 1.5–15 h. This analogue lowered the V max of the saturable component of choline transport by 3.6-fold. Prolonged incubation of the hepatocytes with cAMP analogues resulted in a reversal of the inhibition of phosphatidylcholine synthesis. After 15 h all the compounds tested stimulated the relative incorporation of [ methyl- 3H]choline into phosphatidylcholine. For hepatocytes incubated with chlorophenylthio-cAMP for 14–16 h, there was a 2.8-fold stimulation of the rate of phosphatidylcholine synthesis. The enzymes responsible for the conversion of choline into phosphatidylcholine were examined at various times after addition of the chlorophenylthio-cAMP to the hepatocyte medium. The reduced synthesis of phosphatidylcholine strongly correlated with inhibition of CTP:phosphocholine cytidylyltransferase activity. After 12 h of treatment with the analogue, the relative inhibition of the cytidylyltransferase activity was reversed.

Effects of cyclic AMP analogues and phosphodiesterase inhibitors on K+-induced [3H]noradrenaline release from rat brain slices and on its presynaptic alpha-adrenergic modulation.

The possible role of cyclic AMP in the presynaptic alpha-adrenoceptor-mediated modulation of [3H]noradrenaline (NA) release induced by 13 mM K+ from superfused rat cerebral cortex slices was investigated. Both dibutyryl-cyclic AMP (db-cAMP) and 8-bromo-cyclic AMP (8-Br-cAMP) dose-dependently (10(-4) - 10(-2) M) enhanced K+-induced (3H]NA release, maximally to about 160% of control. In contrast, db-cAMP had no effect on calcium-induced [3H]NA release in the presence of the calcium ionophore A 23187. Surprisingly, the phosphodiesterase (PDE) inhibitors 3-isobutyl-1-methylxanthine (IBMX). 7-benzyl-IBMX, 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771), and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) appeared to inhibit K+-induced [3H]NA release in a dose-dependent (10(-5) - 10(-3) M) manner. At a concentration of 10(-4) M, AK 62771 caused an inhibition of [3H]NA release by 30%, and this inhibitory effect was not affected by 10(-6) M phentolamine nor by 10(-3) M db-cAMP or 10(-4) M theophylline. Theophylline by itself enhanced [3H]NA release to about 135% of control. The inhibitor effect of the alpha-adrenoceptor agonist oxymetazoline (1 micro M) and the enhancing effect of the antagonist phentolamine (1 micro M) on [3H]NA release were significantly decreased in the presence of 10(-3) M db-cAMP or 8-Br-cAMP, whereas 10(-4) M ZK 62771 had no effect. In the presence of 10(-2) M NaF, a potent activator of adenylate cyclase, the inhibitory effect of oxymetazoline (1 micro M) on [3H]NA release was significantly decreased. The data obtained with the cyclic AMP analogues support the hypothesis that activation of presynaptic alpha-receptors modulating NA release results in an inhibition of a presynaptic adenylate cyclase. Possible causes for the anomalous effects of th PDE inhibitors are discussed.

Analogues of cyclic AMP inhibit phosphatidylethanolamine N-methylation by cultured rat hepatocytes

The effect of cyclic AMP analogues on the methylation of phosphatidylethanolamine has been examined. Chloro-phenylthio-cyclic AMP (0.5 mM) in the culture medium of rat hepatocytes inhibited by approximately 50% the conversion of phosphatidylethanolamine to phosphatidylcholine in the intact cells. In contrast, the microsomal activity of phosphatidylethanolamine N-methyltransferase was stimulated 2-fold. The mechanisms of these cyclic AMP effects are unknown.

Concomitant elevation of tyrosine hydroxylase and dopamine beta-hydroxylase by cyclic AMP in cultured mouse neuroblastoma cells.

Abstract—The effects of cyclic AMP analogues and of phosphodiesterase inhibitors were investigated in neuroblastoma cells (NBD‐2) cloned from the C‐1300 tumor. 8Br‐cAMP and phosphodiesterase inhibitors that elevated cAMP induced large (greater than 15 fold) and specific increases in tyrosine hydroxylase and dopamine beta‐hydroxylase activity. In contrast, catechol O‐methyltransferase, monoamine oxidase and aromatic‐l‐amino‐acid decarboxylase were unaffected by the cAMP altering drugs. Similarly, AChE was unaffected and only a small increase in choline acetyltransferase (3 fold) was observed. The increases in tyrosine hydroxylase and dopamine beta‐hydroxylase were similar with respect to dose response relationships and with respect to time course of onset.Only those phosphodiesterase inhibitors that elevated cAMP (papaverine and Ro20‐1724 as opposed to theophylline) were effective in elevating tyrosine hydroxylase and dopamine beta‐hydroxylase. Further, the doses optimal for elevating cAMP coincided with the optimal doses for elevating the two enzymes. Theophylline had no influence either upon NBD‐2 cell cAMP levels or upon tyrosine hydroxylase and dopamine beta‐hydroxylase activity.The changes in protein synthesis rates produced by the cAMP altering drugs were temporally distinct from the changes in either tyrosine hydroxylase or dopamine beta‐hydroxylase. These results suggest that the intracellular messenger compound cAMP is involved in the specific regulation of both tyrosine hydroxylase and dopamine beta‐hydroxylase in adrenergic cells.

Cyclic adenosine monophosphate receptor: Effect of cyclic AMP analogues on DNA binding and proteolytic inactivation

The cyclic AMP receptor protein of Escherichia coli in the presence of cyclic AMP undergoes a conformational change resulting in an increased affinity for DNA and an increased susceptibility to attack by proteolytic enzymes resulting in loss of DNA binding capacity. Of several cyclic nucleotides tested only cyclic AMP and cyclic tubercidin monophosphate are able to effect the conformational transition in cyclic AMP receptor protein, prerequisite to proteolytic inactivation or DNA binding. Other analogues such as cyclic GMP or cyclic IMP which are competitive inhibitors of cyclic AMP do not support DNA binding or proteolytic inactivation.