Cyclic AMP-Elevating Agents Prevent Oligodendroglial Excitotoxicity

Abstract: Previously, we have demonstrated that cells of the oligodendroglial lineage express non-NMDA glutamate receptor genes and are damaged by kainate-induced Ca²⁺ influx via non-NMDA glutamate receptor channels, representing oligodendroglial excitotoxicity. We find in the present study that agents that elevate intracellular cyclic AMP prevent oligodendroglial excitotoxicity. After oligodendrocyte-like cells, differentiated from the CG-4 cell line established from rat oligodendrocyte type-2 astrocyte progenitor cells, were exposed to 2 mM kainate for 24 h, cell death was evaluated by measuring activity of lactate dehydrogenase released into the culture medium. Released lactate dehydrogenase increased about threefold when exposed to 2 mM kainate. Kainate-induced cell death was prevented by one of the following agents: adenylate cyclase activator (forskolin), cyclic AMP analogues (dibutyryl cyclic AMP and 8-bromo-cyclic AMP), and cyclic AMP phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine, pentoxifylline, propentofylline, and ibudilast). Simultaneous addition of both forskolin and phosphodiesterase inhibitors prevented the kainate-induced cell death in an additive manner. A remarkable increase in Ca²⁺ influx (～5.5-fold) also was induced by kainate. The cyclic AMP-elevating agents caused a partial suppression of the kainate-induced increase in Ca²⁺ influx, leading to a less prominent response of intracellular Ca²⁺ concentration to kainate. The suppressing effect of forskolin on the kainate-induced Ca²⁺ influx was partially reversed by H-89, an inhibitor of cyclic AMP-dependent protein kinase. In contrast to this, okadaic acid, an inhibitor of protein phosphatases 1 and 2A, brought about a decrease in the kainate-induced Ca²⁺ influx. We therefore concluded that cyclic AMP-elevating agents prevented oligodendroglial excitotoxicity by cyclic AMP-dependent protein kinase-dependent protein phosphorylation, resulting in decreased kainate-induced Ca²⁺ influx.     

The effect of cyclic AMP on neuritic outgrowth in explant cultures of developing chick olfactory epithelium

The effect of cyclic AMP (cAMP) analogs and phosphodiesterase (PDE) inhibitors on neurite outgrowth was studied in explant cultures of olfactory neurons. Nasal pits from 5- or 6-day-old chick embryos were minced, explanted into culture dishes, and grown in a serum-free medium. One of the cyclic AMP analogs, dibutyryl cyclic AMP (dbcAMP) or 8-bromo-cyclic AMP (8-Br-cAMP), or one of the PDE inhibitors, theophylline or isobutylmethylxanthine (IBMX), was added to the culture medium. The explants were examined for neurite outgrowth after 2 days in vitro. Db-cAMP increased the number of explants expressing neurites by 25-35% over control cultures, whereas 8-Br-cAMP had essentially no effect at the same concentrations. Addition of dibutyryl cyclic GMP (dbcGMP) gave no increase in neurite outgrowth, thus indicating that the effect of enhancing neuritic growth is specific to cAMP and not cyclic nucleotides in general. The resulting increase in neurite outgrowth is due to the cyclic nucleotide component of dbcAMP, since both IBMX and theophylline, which elevate intracellular cAMP, also increased neurite outgrowth significantly. When forskolin was added to the culture medium, there was a trend to increased neurite outgrowth; this was significantly enhanced when a subthreshold concentration of theophylline was added in addition to the forskolin.     

Dual control of pupal diapause by cyclic nucleotides in the bertha armyworm,Mamestra configurata Wlk.

Treatment of diapausing pupae of M. configurata with dibutyryl cyclic AMP or 8-(4-chlorophenylthio) cyclic AMP (CPT cyclic AMP) reduced the incidence of eclosion to zero compared to about 15% for controls, whereas treatment with cyclic GMP increased eclosion to more than 90%. Treatment with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) resulted in a high incidence (79.8%) of eclosion, but treatment with dibutyryl cyclic AMP + IMBX or CPT cyclic AMP + IBMX gave low incidences (<9.1%) of eclosion. Other methylxanthines (theophylline, 8-phenyltheophylline, caffeine) and papaverine had relatively little effect on eclosion even at high doses.Treatment of post-diapause pupae with dibutyryl cyclic AMP or CPT cyclic AMP resulted in a low incidence (<5.0%) of eclosion compared to 98.8% eclosion in controls. Suppression of eclosion was more effective if dibutyryl cyclic AMP was given within the first 2 days of pupal-adult development at 20°C and became less effective as development progressed, indicating that dibutyryl cyclic AMP inhibits endocrine events initiating development rather than inhibiting subsequent metamorphic development. Treatment of post-diapausing pupae with cyclic GMP, IBMX, other methylxanthines or papaverine did not affect eclosion. These results are consistent with a dual control of pupal diapause in M. configurata by cyclic nucleotides, with cyclic AMP acting to maintain diapause and cyclic GMP acting to terminate it.     

Inhibition of the Chick Pineal Rhythm in Rate of Thymidine Incorporation by Cyclic AMP

Chick pineal glands in organ culture showed a circadian rhythm in the rate of thymidine incorporation. Thymidine incorporation was very markedly inhibited when 3-isobutyl-l-methylxanthine (IBMX) was continuously present. When IBMX was added to cultures in control medium during the photoperiod of the second day in culture, the extent of inhibition of incorporation during that photoperiod increased with the increase in length of the photoperiod remaining. Incorporation did not resume at the start of a second photoperiod if IBMX was added within the first 10 h of the first photoperiod. Corresponding results were obtained with glands continuously cultured in constant darkness. Similar results were also obtained using glands treated with 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), 7 beta-acetoxy-8,13-epoxy-1 alpha, 6 beta, 9 alpha-trihydroxy-1abd-14-ene-11-one (forskolin), or 8-bromo-cyclic AMP, but not with 8-bromo-cyclic GMP. When glands cultured with IBMX were transferred to control medium, incorporation remained inhibited until the start of the next photoperiod. We conclude that the increase in the rate of thymidine incorporation at the start of each new photoperiod is dependent on a "switch" process that is inhibited by elevated concentrations of cyclic AMP.     

Cyclic AMP increases the concentration of insulin receptors in cultured fibroblasts and lymphocytes.

Incubation of SV40 transformed fibroblasts with dibutyryl cyclic AMP, 8-bromo-cyclic AMP, or 1-methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, produced a two-fold increase in insulin receptor concentration without an effect on receptor affinity. The increase was dose-dependent, was observed after 8 hrs of treatment, and reached a maximum level by 12 to 24 hours. Upon removal of the nucleotide, receptor number decreased towards basal level.Incubation of cultured human lymphocytes (IM-9 line) with cyclic AMP derivatives or MIX also increased the number of insulin receptors without an alteration in receptor affinity. This effect was partially blocked by inhibition of protein synthesis and was independent of changes in cell cycle. The increase in insulin receptors was a specific response to cyclic AMP as the number of receptors for human growth hormone was unaltered. Incubation with 8-bromo-cyclic GMP did not alter the level of insulin binding.     

Regulation by Dibutyryl Cyclic AMP of Carnosine Synthesis in Astroglia-Rich Primary Cultures Kept in Serum-Free Medium

The synthesis of carnosine (beta-Ala-His) by astroglia-rich primary cultures was much higher if the cells were cultivated in Ham's nutrient mixture F-12 than if they were grown in Dulbecco's modified Eagle's medium. Carnosine synthesis was not affected by the presence of insulin, transferrin, phorbol myristate acetate, or dexamethasone. However, dibutyryl cyclic AMP and other agents that can, directly or indirectly, activate cyclic AMP-dependent protein kinases strongly lower the rate of carnosine synthesis. The depression of carnosine synthesis was dependent on the concentration of dibutyryl cyclic AMP. The effect was maximal (approximately 80% inhibition) in cultures preincubated with 1 mM dibutyryl cyclic AMP for 4 days. The adenylate cyclase activator forskolin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, and 8-bromo-cyclic AMP caused the same depression as dibutyryl cyclic AMP, whereas neither butyrate nor dibutyryl cyclic GMP elicited any effect.     

Striatal Dopamine Release In Vitro: A β-Adrenoceptor-Regulated Response Not Mediated Through Cyclic AMP

The effects of (-)isoproterenol (10(-6) M), dibutyryl cyclic AMP (10(-3) M), and the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) (10(-4) M) on in vitro [3H]dopamine ([3H]DA) efflux and synthesis were studied in rat striatal slices continuously superfused with [3H]tyrosine. The beta-adrenoceptor agonist (-)isoproterenol induced an immediate and significant facilitation of [3H]DA efflux but did not alter [3H]DA synthesis as measured by [3H]H2O formation. In contrast, both dibutyryl cyclic AMP and IBMX enhanced [3H]DA synthesis as well as efflux. The presence of IBMX in the superfusing medium did not potentiate the augmentation of [3H]DA efflux caused by (-)isoproterenol. Additionally, the blockade of [3H]DA synthesis by alpha-methyl-p-tyrosine (10(-4) M) completely prevented the action of dibutyryl cyclic AMP on [3H]DA efflux. However, under similar conditions, (-)isoproterenol was still able to increase [3H]DA efflux. The results suggest that (-)isoproterenol can modify striatal DA release through a mechanism not involving cyclic AMP.     

Inhibition of cardiac slow action potentials by 8-bromo-cyclic GMP occurs independent of changes in cyclic AMP levels

Cyclic GMP inhibits the slow inward Ca current of cardiac cells. This effect could be due to a cyclic GMP-mediated phosphorylation of the Ca channel (or some protein modifying Ca channel activity), or alternatively, to enhanced degradation of cyclic AMP owing to stimulation of a phosphodiesterase by cyclic GMP. To test the latter possibility, we examined the effect of extracellular 8-bromo-cyclic GMP on cyclic AMP levels in guinea pig papillary muscles, in parallel with electrophysiological experiments. Isoproterenol (10(-6) M) significantly increased the cyclic AMP levels and induced Ca-dependent slow action potentials. Superfusion with 8-bromo-cyclic GMP (10(-3) M) inhibited the slow action potentials induced by isoproterenol. However, muscles superfused with 8-bromo-cyclic GMP had cyclic AMP levels identical to those of muscles superfused with isoproterenol alone. Similarly, 8-bromo-cyclic GMP had no effect on the increase in cyclic AMP levels of muscles treated with forskolin (10(-6) M) or histamine (10(-6) M). We conclude that the inhibitory effect of cyclic GMP on slow Ca channels in guinea pig ventricular cells is not due to a decrease in the cyclic AMP levels. We hypothesize that a cyclic GMP-mediated phosphorylation is the most likely explanation for the Ca channel inhibition observed in this preparation.     

Forskolin and phosphodiesterase inhibitors release adenosine but inhibit morphine-evoked release of adenosine from spinal cord synaptosomes.

The effects of forskolin, Ro 20-1724, rolipram, and 3-isobutyl-1-methylxanthine (IBMX) on morphine-evoked release of adenosine from dorsal spinal cord synaptosomes were evaluated to examine the potential involvement of cyclic AMP in this action of morphine. Ro 20-1724 (1-100 microM), rolipram (1-100 microM), and forskolin (1-10 microM) increased basal release of adenosine, and at 1 microM inhibited morphine-evoked release of adenosine. Release of adenosine by Ro 20-1724, rolipram, and forskolin was reduced 42-77% in the presence of alpha,beta-methylene ADP and GMP, which inhibits ecto-5'-nucleotidase activity by 81%, indicating that this adenosine originated predominantly as nucleotide(s). Significant amounts of adenosine also were released from the ventral spinal cord by these agents. Ro 20-1724 and rolipram did not significantly alter the uptake of adenosine into synaptosomes. Although Ro 20-1724 and rolipram had only limited effects on the extrasynaptosomal conversion of added cyclic AMP to adenosine, IBMX, a phosphodiesterase inhibitor with a broader spectrum of inhibitory activity for phosphodiesterase isoenzymes, significantly inhibited the conversion of cyclic AMP to adenosine and resulted in recovery of a substantial amount of cyclic AMP. As with the non-xanthine phosphodiesterase inhibitors, IBMX increased basal release of adenosine and reduced morphine-evoked release of adenosine. Adenosine released by IBMX was reduced 70% in the presence of alpha,beta-methylene ADP and GMP, and release from the ventral spinal cord was 61% of that from the dorsal spinal cord. Collectively, these results indicate that forskolin and phosphodiesterase inhibitors release nucleotide(s) which is (are) converted extrasynaptosomally to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the developing neuromuscular junction

During formation of the neuromuscular junction (NMJ), agrin secreted by motor axons signals the embryonic muscle cells to organize a postsynaptic apparatus including a dense aggregate of acetylcholine receptors (AChRs). Agrin signaling at the embryonic NMJ requires the activity of nitric oxide synthase (NOS). Common downstream effectors of NOS are guanylate cyclase (GC), which synthesizes cyclic GMP, and cyclic GMP-dependent protein kinase (PKG). Here we show that GC and PKG are important for agrin signaling at the embryonic NMJ of the frog, Xenopus laevis. Inhibitors of both GC and PKG reduced endogenous AChR aggregation in embryonic muscles by 50-85%, and blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. A cyclic GMP analog, 8-bromo-cyclic GMP, increased endogenous AChR aggregation in embryonic muscles to 3- to 4-fold control levels. Overexpression of either GC or PKG in embryos increased AChR aggregate area by 60-170%, whereas expression of a dominant negative form of GC inhibited endogenous aggregation by 50%. These results indicate that agrin signaling in embryonic muscle cells requires the activity of GC and PKG as well as NOS.     

In vitro studies of the testis: the effect of LH on cyclic nucleotides.

This study evaluated the relationship between LH, cyclic AMP, cyclic GMP, and testosterone using in vitro incubation of decapsulated rat testes and sampling incubation medium. With added LH (1.0, 5.0, 100, and 500 mIU/ml) there were statistically significant increases in cyclic AMP at 5 mIU/ml or more LH, and progressively greater titers of this nucleotide were produced as LH was increased. For cyclic GMP all levels of added LH caused significant increments in titers of nucleotide; however, peak cyclic GMP concentrations occurred with 5 mIU/ml of LH. The addition of 10(-3) and 10-(4)M 8-bromo-cyclic AMP caused significant increases in testosterone production, while no changes in production of this androgen were found with 10(-3), 10(-4), or 10(-5)M 8-bromo-cyclic GMP. Neither cyclic AMP nor cyclic GMP titers were altered by the addition of 1 to 50 micrograms/ml of testosterone to medium bathing the rat testes. The dose response curves of cyclic AMP and cyclic GMP to LH are different. Progressive increments in added LH cause parallel increases of cyclic AMP and a biphasic change of cyclic GMP, 8-bromo-cyclic GMP does not cause testosterone generation, suggesting that cyclic GMP does not result in androgen synthesis. However, cyclic GMP may be involved in other Leydig cell functions.     

8-Bromo-cyclic GMP inhibits the calcium channel current in embryonic chick ventricular myocytes

Superfusion with 8-bromo-cyclic GMP or intracellular injection of cyclic GMP inhibits calcium-dependent slow action potentials in embryonic chick or guinea pig ventricular cells, suggesting that cyclic GMP inhibits calcium currents. Recently, cyclic GMP has been shown to reduce cyclic AMP-stimulated calcium currents in voltage-clamped ventricular myocytes. Since earlier results in intact cells had suggested that cyclic GMP might inhibit basal (i.e., unstimulated by cyclic AMP) calcium currents, we directly investigated the effect of 8-bromo-cyclic GMP on basal calcium channel currents (using barium as the charge carrier) in voltage-clamped ventricular myocytes isolated from embryonic chick hearts. Superfusion with 1 mM 8-bromo-cyclic GMP (without prior cyclic AMP elevation) progressively decreased peak calcium channel currents (-68% at 15 min after the onset of drug exposure). In contrast, the currents were unchanged during 15 min superfusion with control solution, or 1 mM 8-bromo-GMP (the noncyclic inactive analog of 8-bromo-cyclic GMP). The present results in voltage-clamped embryonic chick heart cells indicate that cyclic GMP can inhibit basal calcium channel currents, apparently through a cyclic AMP-independent mechanism.     

Mode of action of heat-stable Escherichia coli enterotoxin Tissue and subcellular specificities and role of cyclic GMP

Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E. coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2). We have exmined the tissue specificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP. Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP oncentration throughout the small and large intestine. It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon. This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP. However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments. The enterotoxin failed to stimulate guanylate cyclase in liver, lung, pancreas or gastric antral mucosa. In the intestines, it stimulated only the particulate and not the soluble form of the enzyme. Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase. Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase. This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.     

Cyclic GMP-Dependent Protein Kinase and Phosphorylation of the Endogenous Substrate Proteins in the Rabbit Superior Cervical Ganglion

When the homogenate of rabbit superior cervical ganglia (SCG) was incubated in the presence of [gamma-32P]ATP and Mg2+, two specific proteins were strongly labeled. Their apparent molecular weights were 90,000 and 54,000, respectively. The phosphorylation of the latter was significantly stimulated by 10-50 nM cyclic GMP but to a lesser extent by cyclic AMP, whereas that of the former was not stimulated significantly by either of the cyclic nucleotides. The purified protein kinase inhibitor from rabbit skeletal muscle did not inhibit the phosphorylation. These results indicated that the observed phosphorylation of 54K protein was dependent on cyclic GMP but not on cyclic AMP. When intact SCG was incubated in the presence of 32Pi, phosphorylation of 90K protein was stimulated by cyclic GMP, dibutyryl cyclic GMP, and 8-bromo-cyclic GMP (10 microM), whereas phosphorylation of 54K protein was not significantly stimulated by any of these substances. The present demonstration of endogenous cyclic GMP-dependent protein kinase activity and its endogenous substrate proteins raises a possibility that the physiological actions of cyclic GMP in SCG are mediated by the phosphorylation of these proteins.     

Insulin modifies the properties of glucose transporters in rat brown adipose tissue.

The influence of cyclic AMP on cartilage degradation was investigated by using phosphodiesterase inhibitors [theophylline and 3-isobutyl-1-methylxanthine (IBMX)], forskolin (which activates the catalytic subunit of adenylate cyclase) and cyclic AMP analogues (dibutyryl and 8-bromo). Breakdown was assessed by quantification of proteoglycans released into the media of 8-day bovine nasal-septum cartilage cultures. Theophylline (1-20 mM), IBMX (0.01-2 mM) and dibutyryl cyclic AMP (0.1-2 mM) had little or no influence on the rate of proteoglycan release from unstimulated (no-endotoxin) cartilages. A small but detectable increase in breakdown was observed with 8-bromo cyclic AMP (0.5-2 mM) and forskolin (50-75 micrograms/ml). To examine potential inhibitory influences of these agents, the cyclic AMP modulators were added to cultures simultaneously treated with Salmonella typhosa endotoxin (12-25 micrograms/ml), a potent stimulator of cartilage degradation. The 3-4-fold stimulation of breakdown by endotoxin was strikingly inhibited by all three classes of cyclic AMP regulators. Optimal inhibition was found at 10-20 mM-theophylline, 1-2 mM-IBMX, 50-75 micrograms of forskolin/ml, 2 mM-dibutyryl cyclic AMP and 2 mM-8-bromo cyclic AMP. Inhibition was shown to be reversible, indicating that cartilages were viable after treatment. Sepharose CL-2B chromatography of proteoglycan products released from treated cartilages showed that the endotoxin-stimulated shift to lower average Mr was significantly prevented by cyclic AMP analogues and phosphodiesterase inhibitors. Together, these results show that agents which increase cyclic AMP inhibit both quantitative and qualitative aspects of endotoxin-mediated cartilage degradation.     

Natriuretic peptides inhibit G protein activation. Mediation through cross-talk between cyclic GMP-dependent protein kinase and regulators of G protein-signaling proteins

Atrial natriuretic peptide (ANP) inhibits the proliferation of many cells, in part through interfering with signal transduction enacted by G protein-coupled growth factor receptors. Signaling interactions between ANP and the G protein-coupled growth factor receptor ligand, endothelin-3 (ET-3), regulate astrocyte proliferation at a very proximal but undefined point. Here, we find that ANP inhibits the ability of ET-3 to activate Galpha(q) and Galpha(i) in these cells. ANP stimulated the translocation of endogenous regulators of G protein-signaling (RGS) proteins 3 and 4 from the cytosol to the cell membrane, and enhanced their association with Galpha(q) and Galpha(i). ANP effects were significantly blocked by HS-142-1, an inhibitor of guanylate cyclase activation, or by ET-3. KT5823, an inhibitor of cyclic GMP-dependent protein kinase (PKG) reversed the RGS translocation induced by ANP; conversely, expression of an active catalytic subunit of PKG-I, or 8-bromo-cyclic GMP stimulated RGS translocation. ANP caused the phosphorylation of both RGS proteins in a PKG-dependent fashion, and the expressed PKG (in the absence of ANP) also stimulated RGS phosphorylation. A novel cross-talk between PKG and RGS proteins is stimulated by ANP and leads to the increased translocation and association of RGS proteins with Galpha. The rapid inactivation of G proteins provides a mechanism by which ANP inhibits downstream signaling to the cell proliferation program.     

Insulin and glucagon regulate the activation of two distinct membrane-bound cyclic AMP phosphodiesterases in hepatocytes.

Glucagon (10 nM) caused a transient elevation of intracellular cyclic AMP concentrations, which reached a peak in around 5 min, and slowly returned to basal values in around 30 min. When 1 mM-3-isobutyl-1-methylxanthine (IBMX) was present, this process yielded a Ka of 1 nM for glucagon. The addition of insulin (10 nM) after 5 min exposure to glucagon (10 nM) caused intracellular cyclic AMP concentrations to fall dramatically, attaining basal values within 10 min. The regulation of this process was dose-dependent, exhibiting a Ka of 0.4 nM for insulin. If insulin and glucagon were added together to hepatocytes, then insulin decreased the magnitude of the cyclic AMP response to glucagon. IBMX (1 mM) prevented insulin antagonizing the action of glucagon in both of these instances. A gentle homogenization procedure followed by a rapid subcellular fractionation of hepatocytes on a Percoll gradient was developed. This was used to resolve subcellular membrane fractions and to identify cyclic AMP phosphodiesterase activity in both membrane and cytosol fractions. Glucagon and insulin only affected the activity of two distinct membrane-bound species, a plasma-membrane enzyme and a 'dense vesicle' enzyme. Glucagon (10 nM), insulin (10 nM), IBMX (1 mM), dibutyryl cyclic AMP (10 microM) and cholera toxin (1 microgram/ml) all elicited the activation of the 'dense vesicle' enzyme. The plasma-membrane enzyme was not activated by glucagon, IBMX or dibutyryl cyclic AMP, although insulin and cholera toxin both led to its activation. The degree of activation of the plasma-membrane enzyme produced by insulin was increased in the presence of IBMX or dibutyryl cyclic AMP. Glucagon pretreatment (5 min) of hepatocytes blocked the ability of insulin to activate the plasma-membrane enzyme. The activity state of these phosphodiesterases is discussed in relation to the observed changes in intracellular cyclic AMP concentrations. It is suggested that insulin exerts its action on the plasma-membrane phosphodiesterase through a mechanism involving a guanine nucleotide-regulatory protein.     

Independence of the insulin effect from the guanosine-3',5'-monophosphate level in muscle tissue

The role of cyclic GMP in the insulin effect was investigated using isolated frog sartorii. A study was made of the effect of exogenous cyclic GMP, dibutyryl cyclic GMP, 8-bromo-cyclic GMP on xylose transport, glycogen synthesis and muscle respiration. Only dibutyryl cyclic GMP (1.10(-6) - 10(-4) M) alone was observed to have a stimulating effect on glycogen synthesis and respiration. The xylose transport was but slightly accelerated only following a 20 hours incubation of muscles in the cyclic GMP solution. Cyclic GMP was shown to penetrate the muscle fibres. The cyclic GMP content in muscles was equal to 22.7 +/- 2.0 pM per gram of wet weight. Insulin exerted no effect on cyclic GMP concentration in muscles. The data obtained do not allow to conclude that cyclic GMP may serve as a mediator in realization of the insulin effect on membrane and intracellular processes.     

Forskolin, phosphodiesterase inhibitors, and cyclic AMP analogs inhibit proliferation of cultured bovine aortic endothelial cells

The role of cyclic AMP on endothelial cell proliferation was investigated, since these cells can be exposed to high concentrations of physiological and pharmacological agents that alter cyclic AMP metabolism. Cloned bovine aortic endothelial cells were plated at 25,000 cells/35mm dish and grown for 5 days in the presence of phosphodiesterase (PDE) inhibitors, forskolin, or cyclic AMP analogs. The PDE inhibitors dipyridamole, ZK 62 711, isobutylmethylxanthine (IBMX) and theophylline inhibited cell growth in a concentration-dependent manner. Dipyridamole produced a 30% and a 50% inhibition at 5 microM and 12.5 microM, while higher concentrations were cytotoxic. At its therapeutic plasma concentration range (50-100 microM) theophylline inhibited cell proliferation by 15-25%, while IBMX and the highly specific cyclic AMP phosphodiesterase inhibitor, ZK 62 711 inhibited growth by 60-80% and 40-50%, respectively. Forskolin (5 microM) increased cyclic AMP levels and cyclic AMP-kinase activity ratios by 2.5-fold and 2-fold. In the absence of PDE inhibitors forskolin produced a 20% growth inhibition at 0.5 microM and a 60% inhibition at 10 microM. The forskolin dose-response curve was not altered by theophylline, but was shifted to the left by approximately 10-fold with dipyridamole and ZK 62 711 and 5-fold with IBMX. Forskolin (5 microM), by itself produced a 1.8-fold increase in cyclic AMP. In the presence of 5 microM theophylline, dipyridamole, IBMX, and ZK 62 711, cyclic AMP was increased by forskolin 2.0, 2.6, 3.5, and 6.6-fold, respectively. 8-Bromo cyclic AMP and dibutyryl cyclic AMP produced a 55% and 60% growth inhibition at 100 microM. The cyclic GMP analogs were less effective inhibitors of growth (15-30%). Our results demonstrate that cyclic AMP analogs and pharmacological agents that elevate intracellular cyclic AMP levels inhibit cell growth and suggest that cyclic AMP may be an important endogenous regulator of endothelial cell proliferation.     

Relaxation of Mytilus catch muscle by 8-bromo-cyclic GMP and related compounds

Relaxation of catch tension by 8-bromo-cyclic GMP in the ABRM of Mytilus was blocked in the presence of mersalyl and was markedly reduced after treatment of the muscle with alpha-methyldopa. In the muscle depolarized by 540 mM KCl + 5 mM EGTA solution, 8-bromo-cyclic GMP could not relax Ca-contracture. Hexylamine and phenylethylamine, which are assumed to relax the catch acting on relaxing nerve terminals, could not relax the contracture either. Serotonin and dopamine, which are known to relax the catch acting directly on the muscle fibre membrane, could relax it. In the muscle depolarized by 250 mM KCl + 5 mM EGTA solution, all of the cyclic nucleotides tested (cyclic AMP, cyclic GMP and their analogues), serotonin and dopamine relaxed Ca-contracture, but hexylamine and phenylethylamine did not relax the contracture. The possibilities of the involvement of cyclic GMP in the presynaptic and postsynaptic relaxing mechanisms in the ABRM are discussed.