3′,5′‐Cyclic adenosine monophosphate regulates expression of synaptotagmin in neonatal sympathetic ganglia in vitro

The expression of the synaptic vesicle protein, synaptotagmin, in developing rat superior cervical ganglia is influenced by transsynaptic factors associated with membrane depolarization. The present study examines the role of cyclic AMP in the regulation of synaptotagmin in neonatal superior cervical ganglia maintained in explant culture. Ganglia were treated for 48 h in vitro with the Na⁺‐channel ionophore, veratridine, or with pharmacological agents that alter cyclic AMP levels. Levels of cyclic AMP and synaptotagmin were determined by radioimmunoassay. Veratridine treatment significantly increased cyclic AMP in cultured ganglia, with a long time course, and also increased synaptotagmin levels. Drugs that elevate cyclic AMP levels significantly increased synaptotagmin levels, with similar magnitude to that produced by veratridine treatment. These pharmacological agents did not alter neuron survival or total ganglionic protein content. No additive effects were observed after combined treatment with veratridine and pharmacological agents that increased cyclic AMP. Agents that blocked adenylyl cyclase blocked the veratridine‐induced increase in synaptotagmin levels. The results suggest that regulation of expression of synaptotagmin in neonatal sympathetic neurons is mediated partially by cyclic AMP. © 2001 John Wiley & Sons, Inc. J Neurobiol 46: 281–288, 2001     

Depolarization regulates expression of a synaptic vesicle protein in rat superior cervical ganglia in vitro

The role of membrane depolarization in the regulation of expression of a neuron specific protein was evaluated by culturing superior cervical ganglia from neonatal rats in defined medium and manipulating neuronal activity by depolarizing agents. P65 is an integral membrane protein of synaptic vesicles and can be used as a marker for general neuronal maturation. P65 antigen levels were quantified by indirect radioimmunoassay, using monoclonal antibodies. The expression of p65 in ganglion explants increased by 40-100% when the cultures were treated with the depolarizing agents, veratridine or high potassium. The veratridine effect could be blocked by simultaneous treatment with the sodium channel blocker, tetrodotoxin (TTX). The rise in p65 was not evident until 36 h after depolarizing treatment had begun and reached peak levels after 48 h, with no further increases observed with sustained treatment. After removal of the depolarizing treatment, p65 levels returned to control values after 24 h. P65 joins a growing number of molecules whose expression is regulated by membrane depolarization.     

Methionine enkephalin and morphine alter monoamine and cyclic nucleotide levels in the cerebral ganglia of the freshwater bivalve Anodonta cygnea.

Methionine enkephalin and morphine increased dopamine levels in the cerebral ganglia of $\text{Anodonta cygnea}$. Both agents increased the levels of C'GMP and depressed the levels of C'AMP. The pharmacological effect on dopamine and cyclic nucleotide levels were blocked by prior treatments of the cerebral ganglia with naloxone. The study demonstrates pharmacologically the possible existence of an opiate receptor mechanism.     

Stimulation of DOPA Synthesis in the Superior Cervical Ganglion by Veratridine

We have investigated the effect of veratridine on DOPA (3,4-dihydroxyphenylalanine) accumulation by the superior cervical ganglion of the rat. Incubation of the ganglion with veratridine (50 microM) causes a 10-fold increase in the rate of DOPA accumulation. Veratridine-stimulated DOPA accumulation is blocked by tetrodotoxin, but not by cholinergic or adrenergic antagonists or by decentralization of the ganglion. The cyclic nucleotide 8-bromo cyclic GMP does not increase DOPA accumulation, and 8-bromo cyclic AMP causes only a 2-fold increase in DOPA accumulation, which is additive with the effect of veratridine. Thus, the action of veratridine appears to be independent of these cyclic nucleotides. The effect of veratridine on DOPA accumulation is probably due to a stable modification of tyrosine hydroxylase, since an increase in tyrosine hydroxylase activity can be measured in cell-free extracts of veratridine-treated ganglia. Both the increase in DOPA accumulation and the stable activation of tyrosine hydroxylase are dependent upon extracellular Ca2+. The activation of tyrosine hydroxylase by veratridine may be mediated by the depolarization of, and the subsequent entry of Ca2+ into, ganglionic neurons.     

EFFECT OF DIBUTYRYL-CYCLIC AMP and DEXAMETHASONE ON NORADRENALINE SYNTHESIS IN ISOLATED SUPERIOR CERVICAL GANGLIA

Abstract— Incubation with dibutyryl-cyclic AMP increased levels of both noradrenaline and dopamine- β -hydroxylase in isolated rat superior cervical ganglia. Dexamethasone also increased the dopamine- β -hydroxylase content but did not affect noradrenaline levels. Cycloheximide blocked the effect of dibutyryl-cyclic AMP on ganglion dopamine- β -hydroxylase but did not affect the rise in noradrenaline content.
Dibutyryl-cyclic AMP increased the synthesis of noradrenaline from [¹⁴C]tyrosine but not from [³H]DOPA.
The results are discussed in terms of a possible role for cyclic AMP in the control of noradrenaline synthesis in sympathetic ganglia.     

Muscarinic cholinergic and preganglionic physiological stimulation increase cyclic GMP levels in guinea-pig superior cervical ganglia.

Abstract— Incubation of guinea-pig superior cervical ganglia in 500μ4mUm -carbachol for 2min increased cyclic GMP levels 530% over control values. The increase was blocked by prior incubation in 300μm atropine. No increase in cyclic GMP levels after incubation in 100 μm -l -norepinephrine was observed. Preganglionic physiological stimulation for 8 min at 10 Hz increased cyclic GMP levels 180% over control values. We conclude that both muscarinic cholinergic and preganglionic physiological stimulation increase cyclic GMP levels in guinea pig superior cervical ganglia, while norepinephrine has no effect.     

Regulation of Cyclic AMP Accumulation in a Rat Sympathetic Ganglion: Effects of Vasoactive Intestinal Polypeptide

Cyclic AMP accumulation in rat superior cervical ganglia during synaptic activity occurs by a noncholinergic, nonadrenergic process. Both preganglionic nerve stimulation and 4-aminopyridine increase ganglion cyclic AMP levels in the presence of atropine or phentolamine. Of the polypeptides tested as putative transmitters, vasoactive intestinal polypeptide (10(-6) M) causes ganglion cyclic AMP accumulation comparable to that produced by preganglionic nerve stimulation.     

EFFECTS OF DIAMINOPROPIONATE, DEOXYADENOSINE, AND THEOPHYLLINE ON STIMULATED FORMATION OF CYCLIC AMP AND GMP BY DEPOLARIZING AGENTS IN SLICES OF GUINEA-PIG CEREBRAL CORTEX

In incubated slices of guinea-pig cerebral cortex depolarizing agents such as veratridine and high potassium ions caused 50 to 80-fold increases of adenosine 3', 5'-cyclic monophosphate (cyclic AMP) levels and these responses were inhibited about 50% by 2, 3-diaminopropionate and 2'-deoxyadenosine: the former is a specific antagonist for glutamate-elicited accumulation of cyclic AMP and the latter selectively for adenosine-elicited accumulation. Methylxanthines were powerful ‘inhibitors’toward the responses not only to depolarizing agents but also to glutamate and adenosine. These findings are consistent with the hypothesis that releases of both glutamate and adenosine are involved in the depolarization-elicited increases of cyclic AMP levels. Guanosine 3', 5'-cyclic monophosphate (cyclic GMP) levels in the slices were also elevated by veratridine as well as by glutamate, but always to a lesser extent (8 ～ 12 times the control value) than cyclic AMP levels were. The responses for cyclic GMP both to veratridine and glutamate were ‘augmented’by methylxanthines and were not inhibited by 2, 3-diaminopropionate. Thus, glutamate appears to cause the increase of cyclic GMP levels through a different mechanism or site of action from that for cyclic AMP.     

INTERRELATIONSHIPS AMONG THE LEVELS OF ATP, ADENOSINE AND CYCLIC AMP IN INCUBATED SLICES OF GUINEA-PIG CEREBRAL CORTEX: EFFECTS OF DEPOLARIZING AGENTS, PSYCHOTROPIC DRUGS AND METABOLIC INHIBITORS

—Adenine nucleotides of guinea-pig cerebral cortical slices were labelled during a 40 min incubation with [¹⁴C]adenine. Subsequent incubation of cortical slices with depolarizing agents, such as veratridine, ouabain, batrachotoxin and high concentrations of potassium ions, or with certain psychotropic drugs such as chlorpromazine, chlorimipramine or prenylamine resulted in a reduction in both endogenous and radioactive ATP, accompanied by a marked increase in levels of both endogenous and radioactive cyclic AMP. Reduction of ATP levels during incubation with depolarizing agents, such as veratridine, is probably associated with increased activity of membranal Na⁺-K⁺-activated ATPase, while the reduction elicited by psychotropic drugs is proposed to be due to inhibition of mitochondrial synthesis of ATP. With both classes of compounds reduction of ATP levels results in enhanced formation and efflux of adenosine which stimulates formation of cyclic AMP from intracellular ATP in the compartments of brain slices which contain the cyclic AMP-generating systems. Certain classical metabolic inhibitors such as 2,4-dinitrophenol, azide, 1,2-naphthoquinone-8-sulfonate and cyanide also reduce ATP levels and in the case of 2,4-dinitrophenol, cyanide, and azide elicit small but significant accumulations of cyclic AMP. With certain metabolic inhibitors reduction of ATP within the cyclic AMP generating compartments would appear to prevent or reduce the accumulation of cyclic AMP elicited by amines, adenosine or veratridine.     

Regulation of adenosine 3' :5'-monophosphate efflux from rat glioma cells in culture*.

Rat glioma cells grown in culture secrete cyclic adenosine 3':5'-monophosphate (cyclic AMP) into the culture medium following stimulation by beta-agonistic catecholamines. Agents which reduced cellular ATP levels such as valinomycin, oligomycin, and uncouplers of oxidative phosphorylation, inhibited cyclic AMP efflux. Secretion of cyclic AMP was also prevented by prostaglandin A-1 and pharmacological agents including probenecid and papaverine. Of the latter agents, only papaverine reduced ATP levels. These results suggest that the transport of cyclic AMP across animal cell membranes is energy-dependent and subject to regulation.     

Activation of Tyrosine Hydroxylase in the Superior Cervical Ganglion by Nicotinic and Muscarinic Agonists

Both dimethylphenylpiperazinium (DMPP), a nicotinic agonist, and bethanechol, a muscarinic agonist, increase 3,4-dihydroxyphenylalanine (DOPA) synthesis in the superior cervical ganglion of the rat. DMPP causes approximately a fivefold increase in DOPA accumulation in intact ganglia whereas bethanechol causes about a two-fold increase in DOPA accumulation. These effects are additive with each other and with the increase in DOPA accumulation produced by 8-bromo cyclic AMP. The action of DMPP is dependent on extracellular Ca2+ while the actions of bethanechol and 8-bromo cyclic AMP are not dependent on extracellular Ca2+. Cholinergic agonists and cyclic nucleotides produce a stable activation of tyrosine hydroxylase (TH) in the ganglion. The activation of TH by nicotinic and muscarinic agonists can be detected after 5 min of incubation of the ganglia with these agents. The nicotinic response disappears after 30 min of incubation, whereas the muscarinic response persists for at least 30 min. The Ca2+ dependence of the TH activation produced by these agents is similar to the Ca2+ dependence of their effects on DOPA accumulation in intact ganglia. These data are consistent with the hypothesis that nicotinic agonists, muscarinic agonists, and cyclic AMP analogues increase TH activity by three distinct mechanisms. The activation of TH presumably underlies the increase in DOPA synthesis produced by these agents.     

Depolarizing agent-induced cyclic AMP accumulation in isolated rat spinal cord

The stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by the depolarizing agents K+, ouabain and veratridine, was studied in rat and guinea pig spinal cord tissue slices. Significantly increased accumulation of cyclic AMP was produced by each of the agents in a concentration-dependent manner. Veratridine and ouabain were equipotent (EC50 = 5 x 10(-5)M) and approximately 500 fold more potent than K+ (EC50 = 10(-2)M). Depolarizing agent-induced cyclic AMP accumulation in slices from guinea pig spinal cord was approximately double the response in rat spinal cord. Maximum stimulation occurred within 2.5 min of incubation with these agents and lasted for at least 30 min. Regional studies demonstrated that the maximal accumulation of cyclic AMP occurred to a greater degree in tissue slices from the dorsal section of spinal cord from both rat and guinea pig. Whereas the ouabain and veratridine stimulatory responses are completely dependent on extracellular Ca++, the K+ response is only partially dependent. Stimulation due to ouabain and veratridine is dependent, and K+ is independent, of release of neurohumoral substances such as norepinephrine or adenosine from spinal neurons. These experiments indicate the possible modulatory role of depolarization-linked events in regulating the spinal cord cyclic AMP system.     

Regulation of albumin expression in fetal rat hepatocytes cultured under proliferative conditions: role of epidermal growth factor and hormones.

Sustained production of plasma proteins, notably albumin, is a reliable indicator of the differentiated state of hepatocytes. In this work, we have developed a fetal hepatocyte culture system where studying the regulation of albumin expression in proliferating liver cells. Our results show that under proliferative conditions (i.e., in the presence of EGF) fetal hepatocytes maintain albumin production above control quiescent non-treated cells. Glucagon and noradrenaline have no effect on the proliferation induced by EGF in cultured fetal hepatocytes; however, they act synergistically with the growth factor, increasing intracellular albumin levels. The maximum response is obtained by treatment of cells with EGF and noradrenaline. The stimulatory noradrenergic effect is mimicked by agents that increase cyclic AMP levels (forskolin plus IBMX). However, vasopressin or phorbol esters have no effect on albumin production, neither alone nor in combination with EGF. Dexamethasone, which does not alter the proliferative induction of EGF, increases albumin content. This effect is independent of the proliferative status of the cells and is not enhanced by glucagon, noradrenaline, or cyclic AMP increasing agents. The hormonal changes observed in albumin production partially correlate with changes in mRNA levels. This is the first time that cyclic AMP increasing agents are shown to act synergistically with EGF, increasing the expression of this liver specific gene.     

Effect of dibutyryl cyclic AMP and dexamethasone on glutamine synthetase gene expression in rat astrocytes in culture

Astrocytes are the primary site of glutamate conversion to glutamine in the brain. We examined the effects of treatment with either dibutyryl cyclic AMP and/or the synthetic glucocorticoid dexamethasone on glutamine synthetase enzyme activity and steady-state mRNA levels in cultured neonatal rat astrocytes. Treatment of cultures with dibutyryl cyclic AMP alone (0.25 mM–1.0 mM) increased glutamine synthetase activity and steady state mRNA levels in a dose-dependent manner. Similarly, treatment with dexamethasone alone (10^–7–10^–5 M) increased glutamine synthetase mRNA levels and enzyme activity. When astrocytes were treated with both effectors, additive increases in glutamine synthetase activity and mRNA were obtained. However, the additive effects were observed only when the effect of dibutyryl cyclic AMP alone was not maximal. These findings suggest that the actions of these effectors are mediated at the level of mRNA accumulation. The induction of glutamine synthetase mRNA by dibutyryl cyclic AMP was dependent on protein synthesis while the dexamethasone effect was not. Glucocorticoids and cyclic AMP are known to exert their effects on gene expression by different molecular mechanisms. Possible crosstalk between these effector pathways may occur in regulation of astrocyte glutamine synthetase expression.Abbreviations used GS glutamine synthetase - dbcAMP dibutyryl cyclic AMP - MEM minimal essential medium - cyx cycloheximide - GRE glucocorticoid response element - CRE cyclic AMP response element     

MODULATION OF CYCLIC AMP LEVELS IN THE BOVINE SUPERIOR CERVICAL GANGLION BY PROSTAGLANDIN E, AND DOPAMINE

Abstract— Dopamine, norepinephrine, carbamylcholine and PGE₁ (prostaglandin E₁). increased cyclic AMP concentrations in slices of bovine superior cervical ganglia. PGF_1α was less effective and neither PGE₂ nor PGF_2α had any effect. Dopamine and PGE, alone or in combination, did not modify low K _m cyclic AMP phosphodiesterase activity. Combinations of dopamine and PGE, showed a marked synergistic effect, increasing ganglionic cyclic AMP to a much greater extent than that observed when the two compounds were tested alone. Norepinephrine (10 μ M) , which increased cyclic AMP as much as 10 μ m -dopamine, showed no synergistic effect when tested in the presence of PGE₁ or other PGs. Phentolamine, fluphenazine and triflupromazine blocked the dopamine effect without suppressing its synergism with PGE₁ Adenylate cyclase of synaptosomes isolated from the ganglia under a variety of experimental conditions appeared to be as responsive to PGE₁ as the slices, but it was poorly stimulated by dopamine and was not synergistically modulated by dopamine in the presence of PGE₁
These and other data are interpreted as indicating the presence of both a PGE₁-sensitive and a PGE₁-modulated dopamine-sensitive adenylate cyclase in the cervical ganglion. These adenylate cyclases are tentatively assigned to pre- and post-synaptic structures respectively.     

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.