Injection Of Dibutyryl Cyclic AMP Research Articles

Actions of neuropoietic cytokines and cyclic AMP in regenerative conditioning of rat primary sensory neurons

A conditioning lesion to peripheral axons of primary sensory neurons accelerates regeneration of their central axons in vivo or neurite outgrowth if the neurons are grown in vitro. Previous evidence has implicated neuropoietic cytokines and also cyclic AMP in regenerative conditioning. In experiments reported here, delivery through a lentivirus vector of ciliary neurotrophic factor to the appropriate dorsal root ganglion in rats was sufficient to mimic the conditioning effect of peripheral nerve injury on the regeneration of dorsal spinal nerve root axons. Regeneration in this experimental preparation was also stimulated by intraganglionic injection of dibutyryl cyclic AMP but the effects of ciliary neurotrophic factor and dibutyryl cyclic AMP were not additive. Dibutyryl cyclic AMP injection into the dorsal root ganglion induced mRNAs for two other neuropoietic cytokines, interleukin-6 and leukemia inhibitory factor and increased the accumulation of phosphorylated STAT3 in neuronal nuclei. The in vitro conditioning action of dibutyryl cyclic AMP was partially blocked by a pharmacological inhibitor of Janus kinase 2, a neuropoietic cytokine signaling molecule. We suggest that the beneficial actions of increased cyclic AMP activity on axonal regeneration of primary sensory neurons are mediated, at least in part, through the induction of neuropoietic cytokine synthesis within the dorsal root ganglion.

Intraocular injection of dibutyryl cyclic AMP promotes axon regeneration in rat optic nerve

The optic nerve is a CNS pathway containing molecules capable of inhibiting axon elongation. The growth program in embryonic retinal ganglion cell (RGC) neurons enables axons to regenerate in the optic nerve through at least two mechanisms. Namely, high cyclic AMP (cAMP) levels abrogate the ability of CNS molecules to inhibit elongation, and the pattern of gene expression enables axons to undergo rapid, sustained, and lengthy elongation. In adult mammals, recovery of visual function after optic nerve injury is limited by both the death of most RGC neurons and the inability of surviving axons to regenerate. We now report that a single intraocular injection of the membrane-permeable cAMP analogue dibutyryl cAMP (db cAMP) promotes the regeneration of RGC axons in the optic nerves of adult rats, but does not prevent the death of RGC neurons. This regeneration in optic nerves crushed within the orbit (2 mm from the eye) was equally effective either 1 day before or 1 day after db cAMP injection. The number of regenerating axons, which was maximal 14 days after crush, declined with increasing time after injury (i.e., 28, 56, and 112 days) and distance beyond the crush site (i.e., 0.25, 0.5, and 1.0 mm). Thus, db cAMP promotes optic nerve regeneration without increasing the survival of axotomized RGC neurons. Furthermore, since db cAMP does not enable axons to undergo rapid, sustained, and lengthy elongation, strategies that increase survival and promote these changes in elongation may critically complement the ability of db cAMP to promote regeneration.

Effect of reduced maternal inspired oxygen on hepatic glucose metabolism in the rat fetus.

Perturbations in glucose metabolism in the fetus and in the neonate are a consistent finding in several different animal models of intrauterine growth retardation (IUGR) as well as in humans. Studies in rats who have undergone IUGR have shown decreased hepatic glycogen stores in the fetus and delayed induction of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) at birth. Hepatic transcription factors CCAAT enhancer binding protein (C/EBP)alpha and C/EBPbeta and the increase in cyclic AMP at birth have been implicated in the initial appearance of PEPCK-C. We have examined the effect of IUGR induced by reduced maternal inspired oxygen (fractional inspired oxygen concentration 0.14) on a) the expression of genes for hepatic C/EBPalpha, C/EBPbeta, PEPCK-C and glycogen synthase; and b) transcription of the genes for C/EBPbeta and PEPCK-C by dibutyryl cyclic AMP in the fetus. Three days (d 18-21) of decrease in maternal inspired oxygen resulted in lower maternal arterial PO(2) and a lower birth weight of the pups (p < 0.01). Fetuses that underwent IUGR had significantly lower concentrations of plasma glucose, hepatic glycogen, and glycogen synthase mRNA and a higher hepatic lactate:pyruvate ratio. They also had lower levels of hepatic PEPCK-C mRNA at birth. The concentration of hepatic mRNA for C/EBPalpha and C/EBPbeta as well as the transcription factors themselves were not affected by the decreased maternal inspired oxygen. Fetal injection of dibutyryl cyclic AMP after 24 h of decreased maternal inspired oxygen (d 18-19) had no effect on the expression of C/EBPbeta. However, it resulted in an attenuated induction of PEPCK-C in the fetuses with IUGR. We speculate that a decrease in maternal inspired oxygen induced certain mediators, either in the mother or in the placenta, that caused lower fetal glucose concentration and affected the transcription of genes involved in fetal hepatic glucose metabolism. IUGR, as a result of decreased fractional inspired oxygen concentration may also be the consequence of pH-mediated changes in uterine blood flow. However, these remain to be examined in this experimental model.

Dibutyryl cAMP stimulates analgesia in rats bearing a ventricular adrenal medulla transplant

In the present study, a significant increase in pain threshold (current to elicit vocalization to tail shock) was found 15 and 60 min after injection of dibutyryl cyclic AMP (db cAMP) (30 μg) into the lateral ventricle in rats bearing a transplant of fetal adrenal medulla (AM). By contrast, no effect on pain threshold was observed in rats bearing an AM transplant but receiving no db cAMP, or in rats receiving db cAMP but not bearing an AM transplant. In primary cultures of rat fetal chromaffin cells, db cAMP increased the number of neuron-like cells that showed both vasoactive intestinal polypeptide (VIP)- and tyrosine hydroxylase (TH)-like immunoreactivity. These findings indicate that db cAMP exerts a pharmacological modulation of the functional activity (i.e. elevation in pain thresholds) of fetal adrenal AM transplants, and induces phenotypic changes in cultured chromaffin cells with expression of a peptide that elevates pain threshold.

Different behavioral and electrographic effects of acoustic stimulation and dibutyryl cyclic AMP injection into the inferior colliculus in normal and in genetically epilepsy-prone rats

The behavioral and electrographic effects of acoustic stimulation (100 dB) and injection of dibutyryl cyclic AMP (cAMP, 10 nmol) into the inferior colliculus were studied in normal and genetically epilepsy-prone (GEPR-9) rats. Acoustic stimulations induced behavioral seizures only in GEPR-9 rats; the seizures were associated with electrographic epileptiform discharges recorded from the inferior colliculus. Injections of dibutyryl cAMP into the inferior colliculus caused wild running episodes resembling the initial phase of audiogenic seizures in both groups. However, in GEPR-9 rats these episodes progressed to significantly more severe seizures than in normal rats and the convulsions culminated into status epilepticus. During drug-induced seizures, epileptiform activity was present in the inferior colliculus in both groups. The seizure generalization latency was markedly shorter in GEPR-9 rats than in normals. Furthermore, in GEPR-9 rats, the seizure generalization latency was in the same range with either acoustic stimulation-induced or dibutyryl cAMP-induced seizures. The data suggest that the increased susceptibility of genetically epilepsy-prone rats to acoustic stimuli may be related to a malfunction of the cyclic AMP system within the inferior colliculus.

P-6-7 Effect of neural blockers of autonomic nervous system on hyperglycemic action induced by intracranial injection of dibutyryl cyclic AMP (DBcAMP)

P-6-7 Effect of neural blockers of autonomic nervous system on hyperglycemic action induced by intracranial injection of dibutyryl cyclic AMP (DBcAMP)

Both dopaminergic and adrenergic receptors in the brain are involved in the behavioural excitation induced by dibutyryl 3',5'-adenosine monophosphate and aminophylline in the rat

Following administration of dibutyryl cyclic-AMP or aminophylline, but not vehicle solution, into the lateral cerebral ventricle of rats produced locomotor stimulation, head and body rearing, circling, (as indicated by an enhancement of gross movement), increased grooming, head swaying and scratching (as indicated by an enhancement of fine movement), tail elevation, piloerection and convulsion. The behavioural excitation produced by either dibutyryl cyclic-AMP or aminophylline was antagonized by pretreatment with intraventricular injection of either two alpha-adrenergic antagonists (phentolamine and yohimbine) or a dopaminergic antagonist (haloperidol), but not with either a beta-adrenergic antagonist (propranolol) or a narcotic antagonist (naloxone). In addition, direct administration of dibutyryl cyclic-AMP into either the anterior hypothalamus, the nucleus accumbens or the caudate-putamen complex of rats produced the same behavioural responses as those produced by the intraventricular injection. Again, the behavioural responses induced by intracerebral injection of dibutyryl cyclic-AMP was antagonized by pretreatment with either alpha-adrenergic antagonists or a dopaminergic antagonist. The present data indicate that both the dopaminergic and the adrenergic receptors in the brain are involved in the behavioural excitation induced by dibutyryl cyclic-AMP and aminophylline in the rat.

Evidence for a dual effect of dibutyryl cyclic AMP on the synthesis of tyrosine aminotransferase in rat liver.

A single injection of dibutyryl cyclic AMP (Bt2cAMP) into adrenalectomized rats results in rapid and proportionate increases in hepatic tyrosine aminotransferase catalytic activity and in the amount of functional mRNA coding for this enzyme. This effect is transient in that mRNATAT peaks at 0.065% of total poly(A)+RNA activity at 1 h and is back to the basal level of 0.012% in 2.5 h. Enzyme activity peaks at 2.5 h and is back to the basal level by 5 h. If Bt2cAMP is repeatedly injected (0, 1, 2.5, and 4 h), enzyme activity remains at maximal levels for 4 to 5 h, whereas changes in mRNATAT activity are identical with those observed in the single injected rats. The rate of tyrosine aminotransferase synthesis at 5.5 h in the multiply injected rats, a time when mRNATAT has already returned to the basal level, is 3 to 4 times greater than that in either control or singly injected rats at the same time (0.3% of total protein versus 0.07%) and is equivalent to the maximal rate seen 1 h after the initial injection of the cyclic nucleotide. Since the rate of synthesis is increased in proportion to the increase in enzyme catalytic activity, stabilization of the enzyme against degradation is excluded as an induction mechanism at this late time point. These responses are not due to differences in the metabolism of Bt2cAMP, and the effect depends on the presence of metabolically active derivatives of this nucleotide. It thus appears that Bt2cAMP induces the synthesis of tyrosine aminotransferase in rat liver in two distinct ways. One is pretranslational and involves a transient and rapid increase in mRNATAT activity. The second appears to involve a delayed but sustained increase in translation of a basal level of mRNATAT.

A possible role for cyclic AMP in the initiation of DNA synthesis by isoproterenol-activated parotid gland cells.

AbstractAn intraperitoneal injection of the β‐adrenergic agonist dl‐isoproterenol hydrochloride (100 mg/Kg body weight) into a rat caused an early, very large (400‐fold) cyclic AMP surge (peaking at 10 minutes) in the parotid gland which was followed by a second, much smaller (two‐fold) surge 12 to 16 hours later. DNA synthesis began about 16 to 20 hours after the isoproterenol injection and peaked between 24 and 28 hours. The maximum level of DNA‐synthetic activity at 24 hours was correlated positively to the magnitude of the small cyclic AMP surge at 12 hours, but not to the size of the much larger cyclic AMP surge at 10 minutes. An intraperitoneal injection of dl‐propranolol hydrochloride (59 mg/Kg body weight) at 8 hours after isoproterenol injection abolished the second cyclic AMP surge at 12 hours and markedly (65‐75%) reduced the incorporation of [3H]‐thymidine into DNA. Injection of dibutyryl cyclic AMP (6.3 mg/Kg body weight) and theophylline (25 mg/Kg body weight) at 8 hours prevented propranolol from inhibiting DNA synthesis. Propranolol appeared specifically to affect the cyclic AMP‐dependent pre‐DNA‐synthetic step because it did not reduce [3H]‐thymidine incorporation when injected after the second cyclic AMP surge had passed and DNA synthesis had just begun. Thus, the initial, large cyclic AMP surge following β‐adrenergic stimulation may not be necessary for the initiation of prereplicative development, while the much smaller second surge may be needed for the initiation of DNA synthesis.

Catecholamine and 5-hydroxytryptamine synthesis and metabolism following intracerebroventricular injection of dibutyryl cyclic AMP

Two to seven days after bilateral implantation of polyethylene tubings into the lateral ventricles rats were injected intracerebroventricularly with dibutyryl cAMP or dibutyryl cGMP. The accumulation of dopa and 5-hydroxytryptophan after inhibition of the aromatic amino acid decarboxylase with 3-hydroxybenzylhyrazine HCl, served as a measure of catecholamine and 5-hydroxytryptamine synthesis in vivo. A dose of 100 microgram dibutyryl cAMP per rat but not dibutyryl cGMP increased the accumulation of dopa in all brain regions by 70 to 130%. Both nucleotides stimulated the formation of 5-hydroxytryptophan in the dopamine-rich part of the limbic system and diencephalon and in addition dibutyryl cAMP increased 5-hydroxytryptophan in C. striatum and cerebellum. Dibutyryl cAMP (25-200 microgram per rat) did not change the dopamine levels in the dopamine-rich parts of the brain but decreased the noradrenaline level of the C. striatum in a dose-dependent manner. The same doses of dibutyryl cAMP elevated the 5-hydroxytryptamine level in brain stem and the level of 5-hydroxyindole acetic acid in diencephalon and the limbic system. The disappearance of noradrenaline but not of dopamine after inhibition of catecholamine synthesis with alpha-methyl-p-tyrosine methylester HCl was accelerated in most brain regions. The data are compatible with the view that dibutyryl cAMP stimulates tyrosine and tryptophan hydroxylase directly. In addition, dibutyryl cAMP appears to enhance the utilization of noradrenaline but not that of dopamine. The increased utilization of 5-hydroxytryptamine may be restricted to the diencephalon and the limbic system.

Hormonal Regulation of Three Urea Cycle Enzymes in Rat Fetal Liver

The purpose of such studies is to identify the hormones that act on the developmental formation of individual urea cycle enzymes, namely argininosuccinate synthetase, argininosuccinate lyase and arginase in the rat fetal liver. The development of argininosuccinate synthetase activity which is completely blocked by the suppression of glucocorticoids in the fetal liver is affected by neither glucagon nor thyroxine. The activity of argininosuccinate lyase which is never changed by the suppression or addition of glucocorticoids, is under the influences of glucagon and thyroxine at a late fetal period and the enzyme activity can be precociously induced in utero by injection of dibutyryl cyclic AMP to fetuses. Cortisol has been shown to precociously stimulate fetal liver arginase activity after an intraperitoneal injection, but the rise in activity at the late fetal period is not completely prevented by suppression of glucocorticoid and it seems that glucagon and thyroxine may also promote its developmental formation just before birth.

Cyclic AMP is a likely mediator of ovulation in the tsetse fly

Ovulation in tsetse flies is normally induced by mating, but virgins can be stimulated to ovulate with an injection of dibutyryl cyclic AMP, cholera toxin (a cyclic AMP generator), or aminophylline (a phosphodiesterase inhibitor). Thus, elevation of cyclic AMP is a likely link in the events leading to ovulation.

Changes in hepatic messenger RNA for phospho enol pyruvate carboxykinase (GTP) during development

Phosphoenolpyruvate carboxykinase (GTP) [GTP;oxaloacetate carboxy-lyase(transphosphorylating); EC 4.1.1.32] is absent in rat liver cytosol during fetal life and is synthesized initially at birth. De novo synthesis of the enzyme can be induced prematurely by injection of dibutyryl cyclic AMP or glucagon into fetal animals in utero. In this study a wheat germ translation assay was used to quantitate the level of total functional mRNA for phosphoenolpyruvate carboxykinase in the liver of fetal rats at 21 days of pregnancy under different induction situations. The translatable mRNA for the enzyme was marginally detectable in fetal rat liver. Administration of either glucagon or dibutyryl cyclic AMP to fetal rats in utero caused a marked induction of functional mRNA for this enzyme. Three hours after administration of dibutyryl cyclic AMP, the level of translatable mRNA increased almost 23-fold, but by 6 hr the level dropped approximately 60%. Administration of actinomycin D prior to dibutyryl cyclic AMP in 21-day fetal rats prevented the appearance of newly synthesized poly(A)-containing RNA in the cytoplasm as well as the induction of translatable mRNA for phosphoenolpyruvate carboxykinase. In animals delivered prematurely and maintained for varying periods, the translatable mRNA for the enzyme accumulated in the liver at a rate comparable to that observed for enzyme synthesis.

Studies on the locomotor activity produced by injection of dibutyryl cyclic 3 5 AMP into the nucleus accumbens of rats

Bilateral injection of dibutyryl cyclic AMP (5–25 μg) intracerebrally into the nucleus accumbens of rats produces an increase in co-ordinated locomotor activity lasting about 90 min, and behavioural changes similar to those produced by injection of dopamine. Haloperidol injection (2.5 μg bilaterally) into the n. accumbens 15 min before dibutyryl cyclic AMP (8 μg bilaterally) had no effect on the locomotor response but totally abolished the locomotor response to dopamine (5 μg bilaterally) in rats pretreated with tranylcypromine (10 mg/kg i.p., 30 min before dopamine). The observations are compatible with the hypothesis that dibutyryl cyclic AMP acts “beyond” the dopamine receptor. Haloperidol (0.05 mg/kg i.p.) inhibited the locomotor response produced by dibutyryl cyclic AMP (12.5 μg bilaterally) injected into the n. accumbens 15 min later. 2-Methyl- p-tyrosine (200 mg/kg i.p.) 3hr before dibutyryl cyclic AMP (12.5 μg bilaterally) also inhibited the locomotor response. These experiments suggest the existence of a dopaminergic system post-synaptic to the n. accumbens. Injection of p-chlorophenylalanine (200 mg/kg i.p. daily for 2 days) had no effect on the locomotor response to dibutyryl cyclic AMP 24 hr later. Amino-oxyacetic acid (25 mg/kg) produced an inhibition of the dibutyryl cyclic AMP locomotor activity 90 min later, in agreement with other reports suggesting a possible inhibitory influence of GABA on brain dopaminergic systems.

Increase in hepatic tyrosine aminotransferase mRNA during enzyme induction by N6,O2'-dibutyryl cyclic AMP.

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.

The phosphorylation of salivary gland chromatin proteins following treatment of rats with dibutyryl cyclic AMP and dibutyryl cyclic GMP

1. The phosphorylation and protein content of salivary gland chromatin were determined in rats 2 hr after injection of theophylline, dibutyryl cyclic AMP and dibutyryl cyclic GMP. 2. The non-histone protein/DNA ratio was decreased by about 40% in the parotid gland after dibutyryl cyclic AMP injection, but no change was observed in the submaxillary. 3. Both dibutyryl cyclic AMP and dibutyryl cyclic GMP stimulate the phosphorylation of protein above the theophylline control values. However, it appears from combined treatment experiments that both nucleotides exert their action independently.

The relation of cyclic AMP levels to phagocytosis and enzyme release in acute inflammation in vivo

The effect of altering endogenous leucocyte cyclic AMP levels on phagocytosis and lysosomal enzyme release was studied in vivo. Acute pleural exudates were produced in rats using either calcium pyrophosphate dihydrate crystals or rat serum. Despite marked increases in leucocyte cyclic AMP concentration produced by injection of dibutyryl cyclic AMP and theophylline, there was no reduction in crystal phagocytosis or in enzyme discharge.

In vivo reversal of thyroxine induction of DNA synthesis by dibutyryl cyclic AMP in developing rat cerebellum

Thyroxine, dibutyryl cyclic AMP, and a combination of both drugs were administered daily from birth to 2, 2 and 3 pups, respectively from each of 5 litters of Sprague-Dawley rats. Body weight, brain weight, cerebellar weight, and cerebellar DNA were measured in each animal at age 5 days and compared with values from a pair of controls from each litter. Cerebellar weight and DNA content were affected more severely than body weight in cyclic AMP-treated animals, with cerebellar DNA reduced significantly to 88% of control values. Cerebellar DNA was significantly elevated to 117% of control values in thyroxine-treated animals. This augmentation of cerebellar DNA synthesis by thyroxine was negated by administration of dibutyryl cyclic AMP 10 min prior to the thyroxine injection. These results support an hypothesis that the enhancement of cerebellar cell division by thyroxine involves an increase in the ratio of intracellular cyclic guanosine monophosphate to cyclic adenosine monophosphate. The reversal of the thyroxine-induced increase in cerebellar DNA synthesis by a prior injection of dibutyryl cyclic AMP suggests that the early stimulation of cell division by thyroxine may be mediated by cyclic AMP, and that the intracellular balance between cerebellar cyclic AMP and cyclic GMP was distorted by in vivo elevation of intracellular cyclic AMP levels.

Inhibitory effect of somatostatin on dibutyryl cyclic AMP-induced insulin and growth hormone release in human subjects

The effect of somatostatin on the responses of blood glucose, plasma immunoreactive insulin (IRI), growth hormone (GH), and free fatty acids (FFA) to the injection of dibutyryl cyclic AMP (DBC) was studied in six normal volunteers. DBC, when injected alone, induced a rapid increase in blood glucose and plasma IRI levels, while GH concentrations showed a less marked and more delayed increase and plasma FFA showed a clear downtrend. Somatostatin infusion suppressed the GH and IRI release induced by DBC, potentiated its hyperglycemic effect and changed the pattern of FFA. These results suggest that somatostatin inhibits hormone secretion distal to the generation of cyclic AMP.

Glucocorticoids and the regulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in the rat

The effect glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP)(EC4.1.1.32) in rat liver and kidney in vivo was studied immunochemically. The glucocorticoid analogue triamcinolone (9alpha-fluoro-11beta, 21-dihydroxy-16alpha,17alpha-isopropylidenedioxypregna-1,4-diene-3,20-dione) increased the synthesis rate of the kidney enzyme in starved animals. Both triamcinolone and cortisol decreased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP) in fed and starved rats, but were without effect on the degradation rate of the enzyme. This effect of triamcinolone in liver was reversed by injection of dibutyryl cyclic AMP. However, in diabetic animals glucocorticoids increased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP). Triamcinolone administration to starved rats in vivo is shown to cause an increase in the portal blood concentrations of insulin and glucose. Since the physiological de-inducer of liver phosphoenolpyruvate carboxykinase (GTP) is insulin, this is the probable cause of the decrease in the synthesis rate of the hepatic enzyme noted when glucocorticoids are administered to non-diabetic animals.