Effects of various kinds of stimulation on ornithine decarboxylase activity in superior cervical sympathetic and nodose ganglia of rats

Levels of cyclic nucleotides and ornithine decarboxylase (ODC) activity were examined following the application of various kinds of stimuli to superior cervical sympathetic ganglia (SCG), nodose ganglia, and vagus nerve fibers excised from the rat. The level of cyclic GMP in the SCG rose rapidly to about 4.5- to 7.5-fold the unstimulated control with 10 min of incubation after applications of preganglionic electrical stimulation (10 Hz), acetylcholine (ACh; 1 mM), or high extracellular K+ ( [K+]0, 70 mM). The cyclic GMP level in nodose ganglia was increased less than in the SCG by either ACh or high [K+]0 but was not affected by ACh in vagus fibers. Cyclic AMP in the SCG was also increased about 4- to 5.5-fold over the control within 10 min with the addition of ACh, norepinephrine (NE; 0.05 mM), or high [K+]0. Although NE caused a small increase in cyclic AMP, neither ACh nor high [K+]0 produced any appreciable change in nodose ganglia or vagus fibers. The ODC activity in the SCG was increased by preganglionic stimulation of 3- to 4-hr duration but not by a shorter period. A similar change in ODC activity was caused by the addition of oxotremorine (1 mM), isoproterenol (0.1 mM), NE, cyclic AMP (1 mM), or dibutyryl cyclic GMP (1 mM). The effect was exaggerated by the further addition of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. The increase in ODC activity caused by ACh was abolished by a muscarinic cholinergic antagonist, atropine (0.01 mM), and following axotomy for a week, but not by a nicotinic antagonist or by denervation in the SCG. A similar increase in ganglionic ODC activity by NE was inhibited by an adrenergic blocker, propranolol (0.01 mM), and following axtotomy for a week, but not by denervation. Cholinergic or adrenergic stimulation did not cause an increase in ODC activity in nodose ganglia or vagus fibers. These results suggest that the stimulation-induced increase in ODC activity occurs in postganglionic neurons rather than in satellite glial cells and is mediated by muscarinic cholinergic or adrenergic receptors. The process appears to involve cyclic nucleotide-mediated protein biosynthesis in the SCG.     

The effect of Ca++ on cyclic nucleotide phosphodiesterases of superior cervical ganglion.

Cyclic nucleotide phosphodiesterase was examined in canine and bovine superior cervical ganglia. Activity in crude supernatant fractions was only slightly stimulated by Ca++ despite the presence of protein activating factor. Three forms of phosphodiesterase were resolved from bovine ganglia supernatant extracts by chromatography on DEAE-cellulose. The first enzyme eluted, (DI), was almost completely specific for cyclic GMP, while the other two (DII and DIII), hydrolyzed both cyclic AMP and cyclic GMP; all were free of heat-stable protein activator. Each enzyme was inhibited by low concentrations of Ca++ in the assay medium. Inhibition by Ca++ was reversed by addition of protein activator, but activity did not increase above the control level. Cyclic AMP hydrolysis by enzyme DII was stimulated by micromolar concentrations of cyclic GMP. This stimulation was reduced by Ca++ unless protein activator was present.     

Stimulation of Transglutaminase Activity by GM1-Ganglioside and α-Sialylcholesterol in Superior Cervical and Nodose Ganglia Excised from Adult Rat

Changes in transglutaminase (TG) activity in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined following application of selected membrane transport-altering agents, including GM1-ganglioside (GM1) and alpha-sialylcholesterol (alpha-SC). Although TG activity of freshly dissected SCG and NG was relatively low, it increased gradually during 30 min of incubation, and it stayed at this elevated level for 2 h. Addition of alpha-SC at its maximal effective concentration, 20 microM, stimulated TG activity more than eightfold in SCG and more than twofold in NG by 30 min. Addition of GM1 at its most effective concentration, 5 nM, had similar effects, but of lesser magnitude. Cycloheximide, a potent inhibitor of protein biosynthesis, did not affect the GM1- or alpha-SC-evoked increases in ganglionic TG activity, suggesting that enzyme activation rather than synthesis of new enzyme was occurring. The stimulation of TG activity in both ganglia caused by either GM1 or alpha-SC was associated with a decrease in Km and an increase in Vmax values. Addition of cholera toxin B, which specifically masks the oligosaccharide chain of GM1, reduced the GM1-induced increase in TG activity by approximately 60% in SCG and 88% in NG. The alpha-SC-induced increase in TG activity was only partially mimicked by free cholesterol. Although application of either dibutyryl cyclic AMP or dibutyryl cyclic GMP produced little change in TG activity of either ganglion, phorbol ester clearly inhibited the enzymic activity. Because TG is a calcium-dependent enzyme, we measured 45Ca2+ influx into either ganglion, and found that it was reduced by GM1 and alpha-SC in SCG and by alpha-SC in NG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic factor stimulates luteal guanylate cyclase

Effect of a synthetic atrial natriuretic peptide, rat atriopeptin II (rAP-II) on the formation of cyclic nucleotides and progesterone production in Percoll-purified rat luteal cells was investigated. Incubation of luteal cells with varying concentrations of rAP-II resulted in a dose-related stimulation of intracellular cyclic GMP content; maximum stimulation being achieved with 10 nM rAP-II. The increase in cyclic GMP formation was extremely rapid and a 12-fold increase in the cyclic GMP content over basal level was attained within 5 min of incubation of the cells with 10 nM rAP-II. In the presence of phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine, both basal and rAP-II-stimulated levels of cyclic GMP were increased approximately 10 times, but the magnitude of stimulation remained similar in the presence or absence of the inhibitor. The atrial peptide at the concentration of 1-100 nM, however, had no effect on either basal or gonadotropin-stimulated progesterone production and cyclic AMP formation by the luteal cells. Furthermore, the increase in the level of cellular cyclic GMP content of rAP-II was demonstrated to result from a selective activation of particulate guanylate cyclase.     

Effects of parathyroid hormone on cyclic AMP, cyclic GMP, and efflux of calcium in isolated renal tubules.

The effects of parathyroid hormone (PTH) on concentrations of cyclic AMP and cyclic GMP were investigated in isolated renal cortical tubules from hamsters. Efflux of 45Ca from tubules was compared to temporal changes in both cyclic nucleotide concentrations. A rapid increase in cyclic AMP occurred following addition of PTH which was maximal by 1 min but decreased over the next 4 min period. Cyclic GMP concentrations were not significantly altered at 1 min but increased between 1 and 5 min from basal levels. Concentrations of both nucleotides remained significantly elevated from basal levels between 5 and 15 min following PTH. Efflux of 45Ca was increased by PTH with time-course changes closely paralleling changes in cyclic GMP concentrations. Changes in both cyclic AMP and cyclic GMP were related to PTH concentrations of the incubation media and were increased by addition of theophylline. Increasing the calcium concentration from 1 to 3 mM did not significantly alter the effect of PTH on cyclic AMP, however, cyclic GMP concentrations were further increased.     

Action of cholecystokinin, cholinergic agents, and A-23187 on accumulation of guanosine 3':5'-monophosphate in dispersed guinea pig pancreatic acinar cells.

The COOH-terminal octapeptide of cholecystokinin (CCK-OP) and carbamylcholine each increased calcium outflux, cellular cyclic GMP and amylase secretion in dispersed guinea pig pancreatic acinar cells. Following addition of CCK-OP or carbamylcholine, cellular cyclic GMP increased as early as 15 s, became maximal after 1 to 2 min, and then decreased steadily during the subsequent incubation. For both CCK-OP and carbamylcholine there was close agreement between the dose-response curve for stimulation of calcium outflux and that for increase of cellular cyclic GMP. With CCK-OP an effect on both functions could be detected at 10(-10) M and maximal stimulation occurred at 3 X 10(-8) M. With carbamylcholine an effect on both functions could be detected at 10(-5) M and maximal stimulation occurred at 3 X 10(-3) M. Atropine inhibited stimulation of both cyclic GMP and calcium outflux by carbamylcholine but not by CCK-OP. Stimulation of calcium outflux or cellular cyclic GMP by CCK-OP or carbamylcholine did not require extracellular calcium since stimulation occurred in a calcium-free, ethylene glycol bis(beta, beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA)-containing solution. The divalent cation ionophore A-23187 increased bidirectional fluxes of calcium, cellular cyclic GMP and secretion of amylase from dispersed pancreatic acinar cells. Like CCK-OP and carbamylcholine, the ionophore stimulated calcium outflux and cellular cyclic GMP in a calcium-free, EGTA-containing solution. These results suggest that in pancreatic acinar cells the initial step in the sequence of events mediating the action of ionophore as well as that of CCK-OP and carbamylcholine is stimulation of calcium outflux, and that this stimulation then increases cellular cyclic GMP.     

Effect of Increasing Choline, In Vivo and In Vitro, on the Synthesis of Acetylcholine in a Sympathetic Ganglion

Abstract: The experiments described in this paper were designed to test whether increasing choline availability over normal physiological levels increases acetylcholine synthesis in the cat's superior cervical ganglion. When ganglia were perfused with Krebs solution, an increase in the medium's choline concentration over physiological (10⁻³M) levels increased tissue choline but did not increase tissue acetylcholine or the release of acetylcholine from stimulated ganglia. However, increasing plasma choline in the whole animal increased ganglionic acetylcholine levels. The basis for this difference in the effects of in vivo and in Vitro exposure to elevated choline levels on the tissue acetylcholine content was found to involve plasma factor(s), rather than indirect actions of choline, and the acetylcholine content of isolated ganglia was increased when the tissue was perfused with plasma, instead of Krebs solution, containing 10⁻³M-choline. The extra acetylcholine generated by this procedure was associated with a subsequent transient increase in transmitter release during short intervals of stimulation, but most of the extra acetylcholine was not readily available for release from stimulated ganglia. It is concluded that increasing choline available to sympathetic ganglia over physiological concentration does not have a sustained effect on the turnover of releasable transmitter under the conditions of these experiments.     

Phorbol 12,13-Dibutyrate Increases Tyrosine Hydroxylase Activity in the Superior Cervical Ganglion of the Rat

Phorbol 12,13-dibutyrate (PDBu) increased the production of 3,4-dihydroxyphenylalanine (DOPA) in the superior cervical ganglion of the rat. This effect occurred without a detectable lag and persisted for at least 90 min of incubation. The action of PDBu was half-maximal at a concentration of approximately 0.1 microM; at high concentrations, PDBu produced about a twofold increase in DOPA accumulation. PDBu increased DOPA production in decentralized ganglia and in ganglia incubated in a Ca2+-free medium. The action of PDBu was additive with the actions of dimethylphenylpiperazinium, muscarine, and 8-Br-cyclic AMP, all of which also increase DOPA accumulation, and was not inhibited by the cholinergic antagonists hexamethonium (3 mM) and atropine (6 microM). Finally, PDBu did not increase the content of cyclic AMP in the ganglion. Thus, the action of PDBu does not appear to be mediated by the release of neurotransmitters from preganglionic nerve terminals, by the stimulation of cholinergic receptors in the ganglion, or by an increase in ganglionic cyclic AMP. PDBu also increased the incorporation of 32Pi into tyrosine hydroxylase. PDBu activates protein kinase C, which in turn may phosphorylate tyrosine hydroxylase and increase the rate of DOPA synthesis in the ganglion.     

Atrial natriuretic peptide receptors in sympathetic ganglia: biochemical response and alterations in genetically hypertensive rats

High concentration of atrial natriuretic peptide (99-126) (ANP) receptors were localized by quantitative autoradiography in superior cervical and stellate ganglia from young and adult Wistar Kyoto (WKY) rats. ANP increased cyclic GMP formation in stellate ganglia from adult rats. Both young and adult spontaneously hypertensive rats (SHR) had a much lower number of ANP receptors in the sympathetic ganglia. In spite of low receptor concentration, the cyclic GMP response to ANP in SHR was unchanged. These results suggest the existence of physiologically active ANP receptors in the rat sympathetic ganglia. These receptors may also be involved in the pathophysiology of spontaneous hypertension.     

Effect of Yersinia enterocolitica ST on cyclic guanosine 3',5'-monophosphate levels in mouse intestines and cultured cells

We determined the effect of heat-stable enterotoxin produced by Yersinia enterocolitica (Y. enterocolitica ST) on cyclic nucleotide levels in the intestines of 6-day-old mice and in cultured cell line cells. The concentration of cyclic guanosine 3',5'-monophosphate (cyclic GMP) in homogenates of the intestines increased four- to fivefold by 3 min after intragastric administration of 10 units of purified Y. enterocolitica ST. This increase continued for 60 min, and then the concentration of cyclic GMP fell toward the levels of the controls. On the other hand, fluid accumulation in the intestines was not evident until 60 min after administration of the toxin. Thus, the increase in intestinal cyclic GMP concentration preceded measurable fluid accumulation. The effect on both cyclic GMP levels and fluid accumulation was abolished by treatment of the ST with either alkali solution (pH 10.7) or 2-mercaptoethanol. Likewise, cyclic GMP levels in cultured cells (CCL-6, HeLa, L, and Mm-1 cells) increased dose-dependently by 10 min after incubation of the cells with the ST. Cyclic adenosine 3',5'-monophosphate levels in both intestines and cultured cells were not affected by the toxin.     

Effect of serotonin on cyclic nucleotides of human platelets.

Serotonin produced a 6 to 10 fold increase of cyclic GMP over baseline levels of this nucleotide in platelets. Maximum stimulation was reached within 30 sec to 1 min after addition of serotonin and was dependent upon its concentration in the medium. Inhibition of serotonin uptake by methysergide, dihydroergotamine and chloroimipramine did not influence the serotonin-induced stimulation of cyclic GMP but glutaraldehyde and formaldehyde blocked it completely. Cyclic AMP levels in platelets were not affected by serotonin. The serotonin-induced stimulation of cyclic GMP is independent of the uptake of this biogenic amine by platelets and is not due to platelet aggregation.     

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.     

Biosynthesis of prostaglandin E by rat superior cervical ganglia.

Abstract— —The biosynthesis of immunoreactive prostaglandin E (iPGE) was examined in homogenates of rat superior cervical ganglia and in isolated intact ganglia incubated in vitro. Ganglia homogenates produced iPGE from exogenous arachidonic acid. Prostaglandin synthesis by the homogenates was inhibited by the prostaglandin synthetase inhibitors, eicosatetraynoic acid, indomethacin and sodium meclofenamate and was stimulated by norepinephrine and dopamine. Whole ganglia incubated in Krebs-bicarbonate solution also synthesized iPGE which was released into the incubation bath in a time-dependent manner. As observed in the homogenates, norepinephrine and dopamine enhanced iPGE formation by the intact tissue. Phospholipase A also stimulated iPGE synthesis by the whole ganglia. The effect of phospholipase A was antagonized by dibutyryl cyclic AMP but not by dibutyryl cyclic GMP. The results suggest that neuronally synthesized prostaglandins may be available for modulating adrenergic neuron function and that endogenous neuronal constituents such as catecholamines and cyclic AMP may influence the activity of the prostaglandin synthetase system.     

The accumulation of cyclic AMP and cyclic GMP in guinea pig brain slices: Effect of calcium ions,norepinephrine and adenosine

The effect of Ca²⁺ and putative neurotransmitters on formation of cyclic AMP and cyclic GMP has been studied in incubated slices of brain tissue. Cyclic AMP levels in cerebellar slices after about 90 min of incubation ranged from 10 pmol/mg protein in rabbit, to 25 in guinea pig, to 50 in mouse and 200 in rat. Cyclic GMP levels in the same four species showed no correlation with cyclic AMP levels and were, respectively, 1.3, 20, 5 and 30 pmol/mg protein. The absence of calcium during the prolonged incubation of cerebellar slices had little effect on final levels of cyclic AMP, while markedly decreasing final levels of cyclic GMP. Reintroduction of Ca²⁺ resulted in a rapid increase in cerebellar levels of cyclic GMP which was most pronounced for guinea pig where levels increased nearly 7-fold within 5 min. Prolonged incubation of guinea pig cerebral cortical slices in calcium-free medium greatly elevated cyclic AMP levels apparently through enhanced formation of adenosine, while having little effect on final levels of cyclic GMP. Norepinephrine and adenosine elicited accumulations of cyclic AMP and cyclic GMP in both guinea pig cerebral cortical and cerebellar slices. Glutamate, γ-aminobutyrate, glycine, carbachol, and phenylephrine at concentrations of 1 mM or less had little or noe effect on cyclic nucleotide levels in guinea pig cerebellar slices. Prostaglandin E₁ and histamine slightly increased cerebellar levels of cyclic AMP. Isoproterenol increased both cyclic AMP and cyclic GMP. The accumulation of cyclic AMP and cyclic GMP elicited by norepinephrine in cerebellar slices appeared, baed on dose vs. response curves, agonist-antaganonist relationships and calcium dependency, to involve in both cases activation of a similar set of ß-adrenergic receptors. In cerebellar slices accumulations of cyclic AMP and cyclic GMP elicted by norepinephrine and by a depolarizing agent, veratridine, were strongly dependent on the presence of calcium. The stimulatory effects of adenosine on cyclic AMP and cyclic GMP formation were antagonized by theophylline. The lack of correlations between levels of cyclic AMP and cyclic GMP under the various conditions suggested independent activation of cyclic AMP- and cyclic GMP-generating systems in guinea pig cerebellar slices by interactions with Ca²⁺, norephinephrine and adenosine.     

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.     

Effects of electrical stimulation of the parasympathetic nerve on the levels of cholinergic muscarinic and beta-adrenergic receptors and cyclic nucleotides in rat salivary glands

Density of muscarinic receptors of rat parotid gland, although unchanged after 5 or 10 min of stimulation of the parasympathetic nerve to the gland, showed a decrease of 23% following a 15-min period of stimulation. After 30 min the decrease was 19% but by 60 min density of receptors returned to within 5% of receptor density of the unstimulated gland; there was virtually no change in density of beta adrenoceptors at any time during the 60 min of stimulation. Markedly elevated (30-fold increase) levels of cyclic GMP appeared within 5 min after initiation of nerve stimulation and remained at this level at 10 min, but dropped from 90 to 46 pmol/mg total protein by 15 min, the time at which a decrease in muscarinic receptors first was evident. GMP levels continued to decrease but were still four times basal levels after 60 min of stimulation and did not return to normal concentration until 120 min. Cyclic AMP was generally unchanged. These changes in muscarinic receptors and cyclic GMP are apparently closely related to the kind of neural stimulation, unlike the condition when stimulation of the sympathetic nerve was employed.     

Brain natriuretic peptide receptors in the rat peripheral sympathetic ganglia

High affinity binding sites for brain natriuretic peptide were characterized in the rat superior cervical ganglia by quantitative autoradiography. In addition, the peptide increased the formation of cyclic GMP in the ganglia in vitro. Brain natriuretic peptide displaced atrial natriuretic peptide from its binding sites. Our results suggest that brain natriuretic peptide and atrial natriuretic peptide may share physiologically active receptors in sympathetic ganglia. Brain natriuretic peptide may modulate the synaptic transmission in sympathetic ganglia, in addition or in conjunction with atrial natriuretic peptide.     

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.     

The Receptor-Mediated Activation of Tyrosine Hydroxylation in the Superior Cervical Ganglion of the Rat

The addition of carbachol to superior cervical ganglia causes a rapid increase in tyrosine hydroxylation in situ. The increase occurs in ganglia from both newborn and adult animals, and in ganglia from animals pretreated with reserpine. The increase is not due to increased transport of the substrate. The increase is dependent upon the presence of calcium, and is additive to the stimulation produced by dibutyryl cyclic AMP. The stimulation seems specific for tyrosine hydroxylation; dopamine beta-hydroxylation is not increased. Preincubation experiments suggest that the carbachol-induced stimulation is due to a change in the availability of, or the affinity of the enzyme for, reduced pterin cofactor. The stimulation is inhibited by atropine and also by low concentrations of phenoxybenzamine or haloperidol, which suggests that it is caused by an action of carbachol on the interneurons in the ganglia.     

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.