Dibutyryl Cyclic Guanosine Research Articles

Cyclic nucleotide metabolism in mouse brain during seizures induced by bicuculline or dibutyryl cyclic guanosine monophosphate

In adult male C57 black mice, intracortical injection of bicuculline (10 μg) elevated cGMP concentrations and produced contralateral clonic seizures. After intraperitoneal injection, bicuculline (10 mg/kg) elevated cGMP concentrations at the earliest time at which paroxysmal EEG activity could be detected. Changes in cAMP concentration followed those events. Further elevations of cGMP concentrations (and now of cAMP as well) were observed during clonic and tonic seizures. Intracortical injections of dibutyryl cGMP produced behavioral and EEG seizures. These data suggest that cGMP plays an important role in bicuculline seizures.

Cholinergic agonists and dibutyryl cyclic guanosine monophosphate inhibit the norepinephrine-induced accumulation of cyclic adenosine monophosphate in the rat cerebral cortex

Addition of cholinergic agonists, namely carbamylcholine (carbachol), acetylcholine, eserine, eserine plus acetylcholine and eserine plus choline chloride, and dibutyryl cyclic guanosine moue-phosphate inhibited the norepinephrine-induced accumulation of cyclic adenosine monophosphate in incubated slices of rat cerebral cortex. Methacholine was ineffective and atropine did not overcome the action of carbachol on the stimulation of cyclic adenosine monophosphate synthesis by norepinephrine. Carbachol stimulated the production of cyclic guanosine monophosphate in the tissue slices white norepinephrine did not influence cyclic guanosine monophosphate levels to a significant degree. The data are in keeping with the ‘Yin-Yang’ hypothesis in which under particular situations the intracellular levels of cyclic guanosine monophosphate may modulate cyclic adenosine monophosphate concentrations.

Dibutyryl cyclic guanosine monophosphate (Bt 2cGMP) inhibits the action of cholecystokinin octapeptide (CCK8) on the guinea pig ileum longitudinal muscle

Dibutyryl cyclic guanosine monophosphate (Bt 2cGMP) inhibits the action of cholecystokinin octapeptide (CCK8) on the guinea pig ileum longitudinal muscle

Separate activation sites for cholecystokinin and bombesin on pancreatic acini

The effects of dibutyryl cyclic guanosine 3':5' monophosphate (dbcGMP) on the electrical responses of in vitro mouse pancreatic acinar cells to caerulein, bombesin and acetylcholine, applied by microionophoresis, were investigated using intracellular microelectrode recordings. Exposure to dbcGMP (10(-3) mol . 1-1) quickly abolished the depolarization response to caerulein ionophoresis, while the bombesin-nonapeptide and acetylcholine-induced depolarizations were retained. Exposing acinar cells to 2 x 10(-4) mol . 1-1 dbcGMP reduced their sensitivity such that a 10-fold increase in the applied caerulein ionophoresis current was required to restore the responses to the same magnitude as those obtained in a dbcGMP-free environment. The response to topical applications of pentagastrin and CCK-33 were also abolished by dbcGMP. The results provide direct evidence that functional ACh, bombesin and CCK receptors are all present within the same acinar cell unit. The dbcGMP-induced inhibition of responses evoked by CCK-like peptides is immediate, specific and easily reversible. DbcGMP acts as a competitive antagonist of the CCK receptor on pancreatic acinar cells.

Studies on the oxidation of muscle triglycerides.

To study factors which might influence oxidation of muscle triglycerides, pieces of rat diaphragms were prelabelled with palmitate-1-14C for one hour and the subsequent changes in tissue lipids and 14CO2 production were measured. Muscle lipids were separated on silicic acid columns and the amount and specific activities of triglycerides, phospholipids and free fatty acids were determined before and after a second hour of incubation in the absence of glucose and radioactive palmitate. Changes in these parameters strongly suggested that over 80% of the 14CO2 produced during the second hour of incubation was derived from diaphragm triglyceride. Therefore, changes in 14CO2 production under these conditions were an index of triglyceride oxidation. The following agents had no effect on this pathway: cosyntropin (Cortrosyn®), nicotinic acid, insulin, acetylcholine, carbamylcholine chloride (Carbachol®), thyrotropin stimulating hormone, glucagon, prostaglandin E1 prostaglandin E2, dexamethasone, growth hormone, adenosine, di-butyryl cyclic guanosine monophosphate, imidazole, norepinephrine, and methoxamine. 14CO2 production was decreased by epinephrine (via its β-adrenergic action), isoproterenol, isobutyl-methylxanthine, and dibutyryl cyclic adenosine monophosphate. However, this effect was due to enhanced glycogenosis which diluted out lipid-derived carbon with a carbohydrate source. Aminophylline and theophylline increased lipid oxidation to 14CO2. Further studies revealed that this effect occurred distal to lipolysis (i.e., on free fatty acid oxidation to CO2). Since isobutylmethylxanthine did not affect 14CO2 production from lipid, aminophylline may influence free fatty acid oxidation by a mechanism other than inhibition of phosphodiesterase. The inability to find agents which modify oxidation of triglycerides in rat diaphragm suggests that rapid mobilization of lipid is not important as an energy source for muscle.

Membrane Fluidity in Human and Mouse Chediak-Higashi Leukocytes

Polymorphonuclear leukocytes from humans and mice with the Chediak-Higashi syndrome were characterized by spin label electron spin resonance spectrometry. Our results suggest that cells from afflicted mice and humans have membranes more fluid than controls. Order parameters for a spin label that probes near the membrane surface were 0.652 for normals and 0.645 for two Chediak-Higashi patients. Cells from Chediak-Higashi mice showed similar differences, as did isolated plasma membrane fractions. An increased membrane fluidity was also detected with a spin label that probes deeper in the bilayer. In vitro treatment of Chediak-Higashi mouse cells with 0.01 M ascorbate increased the order parameter to normal levels. In vitro incubation of mouse Chediak-Higashi cells with glucose oxidase increased the order parameter, similar to the effect of ascorbate. This increase was abolished when catalase was added to the incubation medium. In vitro incubation with dibutyryl cyclic guanosine monophosphate (1 muM to 0.1 mM) did not normalize order parameters. These results indicate that fluidity of Chediak-Higashi cell membranes was affected by treatments expected to alter the oxidation: reduction potential of the environment but was not affected by treatments expected to alter the ratio of intracellular cyclic nucleotides. The latter treatment would affect microtubule assembly. Therefore, it appears that the membrane fluidity abnormalities as demonstrated by electron spin resonance and the earlier demonstrated microtubule dysfunctions characteristic of Chediak-Higashi cells are coexisting defects and are probably not directly related.

Effect of cyclic adenosine 3',5'-monophosphate antagonists on endotoxin-induced inhibition of human neutrophil chemotaxis

We reported previously that Escherichia coli endotoxin inhibited human neutrophil chemotaxis toward C5a. This effect of endotoxin was antagonized by anti-inflammatory steroids. We now report that dibutyryl cyclic adenosine 3',5'-monophosphate, prostaglandin E1, isoproterenol, and cholera toxin also antagonize the suppression of chemotaxis by endotoxin. Each compound inhibited the effect of endotoxin in a dose-dependent fashion. To be effective, each compound except cholera toxin had to be present at the time of endotoxin challenge. Furthermore, propranolol blocked the protective effect of isoproterenol against endotoxin but not the protective effect of dibutyrl cyclic adenosine 3',5'-monophosphate or prostaglandin E1. Dibutyryl cyclic guanosine 3',5'-monophosphate, adenosine 5'-monophosphate, phenylephrine, prostaglandin F2 alpha, and carbachol did not modify the suppression of chemotaxis by endotoxin. Anti-inflammatory steroids and dibutyryl cyclic adenosine 3',5'-monophosphate are thought to stabilize phospholipids in certain cell membranes. This phospholipid-stabilizing action may contribute, at least in part, to the protective effect against endotoxin-mediated suppression of neutrophil chemotaxis.

EFFECTS OF CONCANAVALIN A ON 5‐HYDROXYTRYPTAMINE UPTAKE BY RABBIT BLOOD PLATELETS AND ON THEIR ULTRASTRUCTURE

1. Effects of concanavalin A (Con A) and other lectins on 5-hydroxytryptamine (5-HT) uptake by rabbit blood platelets and on their ultrastructure were studied. 2. Uptake of [3H]-5-HT by platelets was decreased by application of Con A, E-PHA (lectin from Phaseolus vulgaris) and lentil-PHA (lectin from Lens culinaris), but not by wheat germ agglutinin (WGA). Con A induced specific changes in the ultrastructure of platelets, causing (i) a change in external appearance from a discoid to an irregularly spherical shape, (ii) re-arrangement of the canalicular system and formation of a concentric structure. These effects of Con A on platelets were antagonized by pretreatment with alpha-methyl-D-mannoside (alpha-MM), a specific inhibitor of Con A binding to glycoprotein. 3. The inhibition of 5-HT uptake by Con A was antagonized by colchicine, vinblastine and sodium nitroprusside (SNP), but not by cytochalasin B. 4. Theophylline, papaverine and dibutyryl cyclic adenosine 3',5'-monophosphate (db cyclic AMP) antagonized the effect of Con A on 5-HT uptake, but dibutyryl cyclic guanosine 3',5'-monophosphate had no effect. Theophylline and db cyclic AMP did not influence the effect of Con A on the ultrastructure of platelets. 5. It is suggested that binding of Con A to specific receptor glycoproteins can inhibit the 5-HT uptake system of platelets. Microtubules, contractile protein and the membrane adenylate cyclase system of platelets may also be regulatory factors in this mechanism.

Gonadotropin stimulation of the expansion of cumulus oophori isolated from mice: General condition for expansion in vitro

AbstractGonadotropin Stimulation of the Expansion of Cumulus Oophori Isolated from Mice: General Conditions for Expansion In VitroThe hormonal stimulation of cumulus expansion (mucification) was studied using oocyte‐cumulus cell complexes isolated from Graafian follicles of C57BL/6J mice. Cumulus expansion was stimulated in vitro with human chorionic gonadotropin (HCG) and highly purified rat follicle stimulating hormone (FSH) in Whitten's medium containing fetal bovine serum (FBS). Highly purified rat luteinizing hormone failed to stimulate cumulus expansion. The 50% maximal stimulating concentration of FSH was found to be about 3–4 ng/ ml. Cumulus expansion was also stimulated by dibutyryl cyclic adenosine monophosphate (DBcAMP), but not by dibutyryl cyclic guanosine monophosphate or butyrate. Neither progesterone, testosterone, nor 17β‐estradiol stimulated cumulus expansion. Expanded cumuli were dispersed by hyaluronidase.The stimulation of cumulus expansion by FSH, HCG or DBcAMP required the presence of FBS. The active component of serum had a molecular weight greater than 10,000 daltons and did not require the association of a steroid for activity. Since DBcAMP does not stimulate cumulus expansion in the absence of FBS, it is suggested that the serum factor(s) is involved after the step of FSH stimulation of cAMP production by the complex.The cumulus does not expand if FSH is added to cultures after seven hours of spontaneous oocyte maturation, when the oocytes are at late metaphase I or early anaphase I of meiosis. Therefore, the changes which occur in the oocytecumulus cell complex during spontaneous oocyte maturation affect the ability of the complex to respond to FSH and bring about the expansion of the cumulus.

Effect of concanavalin A on tyrosine aminotransferase in rat hepatoma tissue culture cells. Rapid reversible inactivation of soluble enzyme.

Concanavalin A added to intact cells at 37 degrees caused rapid and reversible inactivation of a soluble enzyme, tyrosine aminotransferase, in two lines of rat hepatoma tissue culture cells grown in monolayer culture. This temperature-dependent process was independent of de novo protein and RNA synthesis and independent of increased uptake of Ca2+ and Mg2+ or glucose. The inactivation could be reversed by adding alpha-methyl-D-mannopyranoside a competing sugar for concanavalin A binding. Other lectins known to bind to different sugars did not bring about the inactivation of tyrosine aminotransferase. Addition of concanavalin A did not result in the inactivation of another soluble enzyme, lactic dehydrogenase. The maintenance of tyrosine aminotransferase in an inactive form after the binding of concanavalin A to the cells required the continued presence of concanavalin A. This effect of concanavalin A could not be mimicked either by dibutyryl cyclic adenosine or guanosine monophosphoric acid. Incubation of cell extracts with concanavalin A did not result in inactivation nor did mixing of extracts from concanavalin A-treated cells with extracts from untreated cells. On the basis of these results we conclude that the following are the essential requirements for concanavalin A to bring about the inactivation of tyrosine aminotransferase: (a) the binding of native concanavalin A to the cells; (b) integrity of certain structural elements of the cells.

Calcium ion uptake induced by cholinergic and ?-adrenergic stimulation in isolated cells of rat salivary glands

Adrenaline (10(-5) M) and carbamylcholine (10(-4) M) stimulate 45Ca2+ uptake into isolated cells of rat submandibular galnd and parotid glands. In the presence of the alpha-adrenoreceptor blocking agent phentolamine, adrenaline stimulation of 45Ca2+ uptake is abolished. The beta-adrenergic stimulant isoproterenol has no effect on 45Ca2+ uptake. Carbamylcholine induced 45Ca2+ uptake is inhibited by atropine. The Ca2+ ionophore A23187 stimulates 45Ca2+ uptake, whereas dibutyryl cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic guanosine 3',5'-monophosphate have no effect on 45Ca2+ uptake. A graphical analysis of the 45Ca2+ uptake curves reveals at least two phases: a fast phase and a slow phase, both of which are stimulated by adrenaline and carbamylcholine. The 45Ca-exchangeable pool size is increased by adrenaline and carbamylcholine in both the fast and the slow phases. These results suggest that alpha-adrenergic and cholinergic agonists act by increasing the rate of Ca2+ transfer into the cells of the parotid and submandibular salivary glands most probably through an increase of the cell membrane permeability for Ca2+.

Ca++ fluxes in isolated cells of rat pancreas. effect of secretagogues and different Ca++ concentrations.

Secretagogues of pancreatic enzyme secretion, the hormones pancreozymin, carbamylcholine, gastrin I, the octapeptide of pancreozymin, and caerulein as well as the Ca++ -ionophore A 23187 stimulate 45Ca efflux from isolated pancreatic cells. The non-secretagogic hormones adrenaline, isoproterenol, secretion, as well as dibutyryl cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic guanosine 3',5'-monophosphate have no effect on 45Ca efflux. Atropine blocks the stimulatory effect of carbamylcholine on 45Ca efflux complately, but not that of pancreozymin. A graphical analysis of the Ca++ efflux curves reveals at least three phases: a first phase, probably derived from Ca++ bound to the plasma membrane; a second phase, possibly representing Ca++ efflux from cytosol of the cells; and a third phase, probably from mitochondria or other cellular particles. The Ca++ efflux of all phases is stimulated by pancreozymin and carbamylcholine. Ca++ efflux is not significantly effected by the presence or absence of Ca++ in the incubation medium. Metabolic inhibitors of ATP production. Antimycin A and dinitrophenol, which inhibit Ca++ uptake into mitochondria, stimulate Ca++ efflux from the isolated cells remarkably, but inhibit the slow phase of Ca++ influx, indicating the role of mitochondria as an intracellular Ca++ compartment. Measurements of the 45Ca++ influx at different Ca++ concentrations in the medium reveal saturation type kinetics, which are compatible with a carrier or channel model. The hormones mentioned above stimulate the rate of Ca++ translocation. The data suggest that secretagogues of pancreatic enzyme secretion act by increasing the rate of Ca++ transport most likely at the level of the cell membrane and that Ca++ exchange diffusion does not contribute to the 45Ca++ fluxes.

Dibutyryl Cyclic AMP, Adenosine and Guanosine Blockade of the Dopamine, Ergocryptine and Apomorphine Inhibition of Prolactin Releasein Vitro1

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.

Effects of drugs affecting endogenous amines or cyclic nucleotides on ethanol withdrawal head twitches in mice.

1 Twenty-four hours after ethanol withdrawal, dependent mice exhibited frequent head twitching. Naive mice exhibited similar twitching 15 min after treatment with 5-hydroxytryptophan (5-HTP) or 6 h after alpha-methyl-p-tyrosine (AMPT). Ethanol lessened the incidence of head twitches induced by any of these treatments. 5-HTP and AMPT each increased the incidence of head twitches induced by withdrawal of ethanol from dependent mice. 2 Drugs that affect the amount or activity of endogenous amines or cyclic nucleotides modified the incidence of head twitches. Nearly all drugs acted in the same direction on twitching elicited by any of these three treatments. 3 The incidence was lessened by: (a) methysergide, methergoline, MA 1420, p-chlorophenylalanine and p-chloroamphetamine; (b) dopamine, noradrenaline, L-DOPA, amphetamine and apomorphine; (c) hyoscine and nicotine; and (d) adenosine triphosphate, dibutyryl cyclic adenosine-3',5'-monophosphate (db cyclic AMP) and prostaglandins E1 and E2. 4 The incidence was increased by: (a) acetylcholine, carbachol and physostigmine; and (b) guanosine triphosphate, dibutyryl cyclic guanosine monophosphate (db cyclic GMP), theophylline and 3-isobutyl-1-methyl-xanthine. 5 These findings suggest that head twitching induced by these three treatments arises from a common biochemical mechanism, which may ultimately be a change in favour of cyclic GMP of the balance between this nucleotide and cyclic AMP within appropriate neurones. This imbalance appears to be elicited or increased by 5-hydroxytryptamine and acetylcholine and to be decreased by dopamine, noradrenaline and E prostaglandins. 6 Neither actinomycin D nor cycloheximide, given during the induction of ethanol dependence, altered the incidence of head twitches after ethanol withdrawal.

An in vitro model of phagocytosis in bovine and human retinal pigment epithelium

We have developed an in vitro system for studying phagocytosis in retinal pigment epithelium (RPE). Explants of bovine and human RPE and choroid have been maintained in organ culture, and phagocytosis of 1·1 μm diameter latex microspheres by the RPE has been studied over 24-hr periods. After a latent period of about 8 hr latex was rapidly phagocytized from the apical surface of the cell by more than 90% of the cells in the explant. Each latex bead was surrounded by a membrane derived from apical plasma membrane. Morphometric analysis of organellar area in electron micrographs of phagocytizing vs. fresh RPE cells showed that microvillus membrane is internalized during phagocytosis. A web of microfilaments was apparent near the plasma membrane where initial engulfment occurs, and many microtubules surrounded latex phagosomes deeper in the cytoplasm. Lysosomes fused with many latex phagosomes, but the number of lysosomes in bovine RPE cultured 22 hr exceeded that of fresh RPE. This suggests that either more lysosomes are assembled during acute phagocytic challenge, or that degradation o phagolysosomes occurs less rapidly when inert particles are phagocytized. Phagocytosis by bovine RPE was not affected by continual light or darkness. Dibutyryl cyclic adenosine monophosphate enhanced the total incorporation of latex, but dibutyryl cyclic guanosine monophosphate so altered the shape of cells that phagocytosis could not be assessed. Phagocytosis by human RPE in vitro is reported for the first time. Explants from rod-rich and cone-rich areas of human retina did not differ significantly in their phagocytic ability. The potential use of this explants preparation in the study of various aspects of the phagocytic mechanism in RPE is discussed.

Inhibition of the noradrenergic induction of pineal N-acetyltransferase by dibutyryl cyclic guanosine monophosphate and by ionophore X-537A

Utilizing rat pineals in vitro, it can be demonstrated that dibutyryl cyclic GMP and the calcium ionophore X-537A are both able to reversibly inhibit the elevation of pineal N-acetyltransferase induced by the β-agonists norepinephrine or isoproterenol. These results are discussed in terms of the known hyposensitivy of the pineal gland to overstimulation by β-agonists.

Absence of effects of dibutyryl cyclic guanosine 3′,5′-monophosphate on release of α-amylase,45Ca efflux, and protein synthesis in rat pancreas in vitro

Dibutyryl cyclic GMP did not affect basal, or carbachol stimulated secretion of alpha-amylase from rat pancreas. The nucleotide did not have a significant effect on 45Ca release from the pancreas nor did it alter the response to carbachol. The dibutyryl analogue of cyclic GMP did not duplicate or alter the inhibitory effect of carbachol on 3H-leucine incorporation into pancreatic trichloroacetic acid-precipitable protein.

Comparative effects of adenosine and adenine and guanine nucleotides on drug biotransformation in rats

The effect of various nucleotides and adenosine on the hepatic biotransformation of hexobarbital sodium (HB) and p-chloro-N-methylaniline (PCMA) was determined in male rats. Intraperitoneal administration of dibutyryl cyclic adenosine 3',5'-monophosphate (DBcAMP), alone and in combination with theophylline, and the cyclic adenosine 3',5'-monophosphate (cAMP)-theophylline combination prolonged HB sleeping time by more than 70%. cAMP or dibutyryl cyclic guanosine 3',5'-monophosphate (DBcGMP), alone or in combination with theophylline, failed to significantly alter the duration of HB-induced hypnosis. Plasma levels of HB upon awakening suggested that the increase in sleeping time was apparently not due to an altered sensitivity of the brain to the barbiturate. The effect appears to be related to an impairment of HB metabolism since only those compounds that inhibited HB oxidation when added to liver slices prolonged hypnosis. In addition, 5'-AMP, adenosine, and cyclic guanosine 3',5'-monophosphate (cGMP) failed to alter HB biotransformation by liver slices. A similar pattern of impairment of metabolism by liver slices was observed when PCMA was used as the substrate. The inhibitory effect of DBcAMP was not altered by concurrent addition of either cGMP or DBcGMP. No impairment in the rate of microsomal PCMA biotransformation resulted from addition of adenosine or any of the nucleotides used in this study.

Promotion of conidia aggregation in aspergillus niger by cyclic AMP and 5′-GMP

Conidia of A. niger aggregated into clumps, and then germinated to form masses of mycelia (pellets) when grown in agitated, submerged, liquid cultures. Cyclic AMP and 5′-GMP promoted the aggregation of conidia, while adenosine, AMP, ADP, dibutyryl cyclic AMP, cyclic GMP, guanosine, GDP, GTP and theophylline were ineffective. ATP and 2′, 3′-cyclic AMP elicited a weak response. Conidia in the center of clumps did not germinate. The increase in “adhesiveness” of conidia by cyclic AMP and GMP may have implications in the regulation of germination, as well as the eventual growth and development of hyphae.