Regulation of Depolarization-Dependent Release of Neurotransmitters by Adenosine: Cyclic AMP-Dependent Enhancement of Release from PC12 Cells

We have used pheochromocytoma cells, clone PC12, as a model system for studying the effects of adenosine on neurosecretion. Exposure of the cells to adenosine or 2-chloroadenosine caused immediate activation of adenylate cyclase, increases in cellular cyclic AMP content, and inhibition of SAM-dependent phospholipid N-methylation and protein carboxymethylation. However, the effects on methylation were only observed with concentrations of adenosine 100 times greater than those that elevated cyclic AMP. Exposure of the cells to adenosine and 2-chloroadenosine did not alter the release of [3H]norepinephrine [(3H]NE) in the absence of depolarization. However, depolarization-dependent release of [3H]NE was markedly elevated by short (1-20 min) pretreatments with adenosine or 2-chloroadenosine. The enhancement of release was observed irrespective of the nature of the depolarizing stimulus (elevated K+, carbamylcholine, or veratridine). Release of [3H]acetylcholine in response to elevated K+ also was increased by adenosine pretreatment. These effects of adenosine and 2-chloroadenosine on neurotransmitter release closely paralleled elevation of cellular cyclic AMP but not inhibition of methylation. Taken together, the results show that adenosine, probably acting through adenosine receptors coupled to stimulation of adenylate cyclase, is able to modulate the neurosecretory process in PC12 cells. Furthermore, the enhancement of release occurred even though the extent of depolarization (measured as 86Rb+ flux through the acetylcholine receptor channel) and the amount of 45Ca2+ which entered upon depolarization were unchanged. Therefore, the enhancement of release produced by elevated cyclic AMP appeared to reflect increased efficiency of the stimulus-secretion coupling process.     

Comparison of the secretory processes in the parotid and sublingual glands of the mouse. 1. Regulation of the secretory processes.

1. Secretion from the mucous sublingual gland of the mouse has been investigated and compared with the serous parotid gland. The influence of acetylcholine, noradrenalin and adrenalin on the secretion of glycoproteins (e.g. mucins) and proteins (e.g. amylase) from these glands in vitro, and the involvement of cyclic AMP and Ca2+ has been studied. 2. Secretion from the parotid gland could be stimulated by both acetylcholine and the catecholamines. It appears that cyclic AMP plays an important role in the adrenergic secretory process, but not in the cholinergic-induced secretion. In the latter case, exogenous Ca2+ strongly increased the secretion. 3. Mucin secretion from the sublingual gland could be affected by acetylcholine in the presence of exogenous Ca2+. Noradrenalin and adrenalin induced only a slow mucin secretion and, for this secretory process, exogenous Ca2+ is also required. Though cyclic AMP is present in the sublingual gland, no influence on its level could be detected in this gland after stimulation of the adrenergic beta-receptor, whereas, in contrast to the parotid gland, dibutyryl cyclic AMP induced only a slow secretion. Because it was observed that the sublingual gland of the mouse is not innervated sympathetically, it seems reasonable to suppose that the catecholamines stimulate the mucin secretion from this gland via hormonal receptors and not via the adrenergic beta-receptor. 4. The protein secretion from the sublingual gland could be stimulated by both acetylcholine and the catecholamines. An involvement of cyclic AMP in this process was not observed. Addition of exogenous Ca2+ is less important, as was found for the mucin secretion. So it has been concluded that protein and mucin secretion from the sublingual gland are regulated via different pathways.     

Control of endogenous triglyceride breakdown in the mouse diaphragm

The control of endogenous triglyceride breakdown was studied in vitro, in the incubated intact mouse diaphragm. Isoproterenol (2 microgram/ml) produced parallel increases in glycerol and free fatty acid release, and in tissue cyclic AMP levels, suggesting that cyclic AMP mediates the action of the catecholamine on triglyceride mobilization. In addition to cyclic AMP, calcium seems to be involved in the action of isoproterenol because preincubation of hemidiaphragms in the presence of the calcium ionophore A23187 decreased the lipolytic effect of the drug. Insulin (12.5 mU/ml) antagonized the action of isoproterenol on triglyceride breakdown (it decreased glycerol and free fatty acid release) without altering its stimulatory effect on cyclic AMP levels. On the other hand, no detectable effect on lipolysis was observed with carbachol in control and denervated hemidiaphragms, although the latter possess acetylcholine receptors over the entire surface area of the muscle. It was concluded that catecholamines control triglyceride breakdown in muscle while the cholinergic system does not seem to be involved. Cyclic AMP, calcium, and insulin all affect lipolysis in muscle and the interrelationships remain to be elucidated.     

Receptor-specific threshold effects of cyclic AMP are involved in the regulation of enzyme release and superoxide production from human neutrophils

We have investigated the sequence of events leading from the activation of adenylate cyclase and increases in intracellular cyclic AMP to the modulation of enzyme release and superoxide production in human neutrophils. In the isolated plasma membrane, adenylate cyclase is activated by both prostaglandin E1 and isoproterenol. In the whole cell only a small increase in cyclic AMP is observed, though in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine a substantial amplification in intracellular cyclic AMP is observed with both isoproterenol and prostaglandin E1. These conditions are relevant to the regulation of cell function, since fMet-Leu-Phe-stimulated superoxide production is inhibited by either prostaglandin E1 or isoproterenol in the absence of methylisobutylxanthine, while enzyme release is inhibited only via the prostaglandin E1 receptor and then only in the presence of methylisobutylxanthine. For enzyme release and superoxide production, the order of potency for three prostaglandins tested was prostaglandin E1 greater than prostaglandin D2 much greater than prostaglandin F2 alpha. Our results suggest that (a) superoxide production is more sensitive to regulation by cyclic AMP than enzyme release, (b) the type of receptor occupied as well as the threshold level of cyclic AMP attained are important to the regulation of enzyme release, and (c) although elevation in cyclic AMP is inhibitory to neutrophil function, phosphodiesterase inhibition is required in addition to adenylate cyclase activation to effect maximal inhibition.     

Biochemical analysis of triggering signals induced by bridging of IgE receptors

Bridging of IgE receptors on rat mast cell plasma membranes induces phospholipid methylation and a monophasic increase in cyclic AMP. The stimulation of phospholipid methylation in the plasma membrane appears to be intrinsic to the processes leading to Ca2+ influx and histamine release. Evidence was obtained that IgE receptors are closely associated with methyltransferases and adenylate cyclase in the plasma membranes. The activation of one enzyme is regulated by the other. An increase in the cyclic AMP level before receptor bridging suppressed phospholipid methylation. On the other hand, inhibition of phospholipid methylation may affect the initial rise in cyclic AMP. Our experiments also indicated that bridging the receptor activates a membrane-associated proteolytic enzyme. Inasmuch as the inhibition of the enzyme activation results in the suppression of both phospholipid methylation and initial rise in cyclic AMP induced by receptor bridging, the proteolytic enzyme may be involved in the activation of methyltransferases and adenylate cyclase.     

Correlation between cyclic AMP and LH release following prostaglandin E2 administration.

Five min following a single iv injection of PGE2 into ovariectomized mature rats pretreated with estrogen and progesterone, plasma LH and plasma and pituitary cyclic AMP levels were raised significantly. A close correlation was observed between increased pituitary cyclic AMP contents and release of plasma LH. The average level of cyclic AMP in the anterior pituitary and plasma cyclic AMP increased significantly, while the circulating plasma LH level was not changed at 1 min after PGE2 injection. Plasma LH le-el increased at 2 min after PGE2 and reached a maximum level at the above-mentioned time. This is consistent with hypothesis that increased release of hormone is a consequence of increased pituitary cyclic AMP content.     

Transient Increase of Cyclic AMP Induced by Glutamate in Cultured Neurons from Rat Spinal Cord

Abstract: We demonstrated that glutamate increased the cyclic AMP level in cultured neurons from rat spinal cord. A bath application of glutamate (300 µ M ) elicited a rapid increase of the cyclic AMP concentration reaching a level three times as high as the basal level in ∼3 min, and its content then decreased to the control level in 15 min. The increase was not observed in a Ca²⁺-free medium and was inhibited by an antagonist of NMDA receptors or a voltage-sensitive Ca²⁺ channel blocker. Preincubation with W7 also inhibited the glutamate-evoked cyclic AMP increase. NMDA, aspartate, and high-K⁺ conditions also induced a cyclic AMP increase; however, a decreasing phase did not follow. The decreasing phase was observed when (2 S ,1' S ,2' S )-2-(carboxycyclopropyl)-glycine, a potent agonist for metabotropic glutamate receptors, was combined with NMDA. These results suggest that the cyclic AMP increase is mediated by a Ca²⁺ influx via both NMDA receptors and voltage-sensitive Ca²⁺ channels followed by an activation of the Ca²⁺/calmodulin system, and the decreasing phase observed in the case of glutamate exposure is due to the activation of the metabotropic glutamate receptors.     

Activation of β-Adrenergic Receptors Stimulates Release of an Inhibitory Transmitter from Astrocytes

Activation of beta-adrenergic receptors on astrocytes in primary cell culture results in the release of taurine, an inhibitory transmitter. Taurine release occurs via a cyclic AMP-mediated intracellular pathway, because (a) taurine release and intracellular cyclic AMP accumulation have similar pharmacologies and time courses of activation and (b) N6,O2'-dibutyryl cyclic AMP stimulates release with a time course similar to that observed with the beta-adrenergic agonist isoproterenol. These results describe a previously unrecognized physiological function of astrocytes in the CNS-receptor-mediated release of the neuroactive amino acid taurine. This observation indicates that astrocytes may function as local regulators of neuronal activity.     

Coupling of two endothelin receptor subtypes to differing signal transduction in transfected Chinese hamster ovary cells.

We examined the intracellular signal transduction of two endothelin receptor subtypes (ETA and ETB) by transfection and stable expression of individual receptor cDNAs in Chinese hamster ovary cells. Both receptors showed a rapid and marked stimulation of phosphatidylinositol hydrolysis and arachidonic acid release in response to agonist interaction. The two receptors, however, exhibited different responses in the cyclic AMP transduction cascades. ETA mediated the accumulation of cyclic AMP formation, whereas ETB displayed an inhibitory action on the forskolin-stimulated cyclic AMP accumulation. In both receptors, the responses of phosphatidylinositol hydrolysis, arachidonic acid release, and cyclic AMP formation were induced in complete agreement with the endothelin-binding selectivity of each receptor subtype. Endothelin, added together with GTP, activated the adenylate cyclase activity in membrane preparations of ETA-expressing cells, indicating the direct linkage of ETA to the adenylate cyclase system. Pertussis toxin treatment of ETA-expressing cells resulted in partial inhibition of the endothelin-induced cyclic AMP accumulation, whereas the same treatment of ETB-expressing cells completely abolished the endothelin-induced inhibition of cyclic AMP formation. Thus, the two endothelin receptor subtypes are coupled to multiple but distinct signal transduction cascades through different G proteins.     

Somatostatin promotes glucose generation of Ca2+oscillations in pancreatic islets both in the absence and presence of tolbutamide

Many cellular processes, including pulsatile release of insulin, are triggered by increase of cytoplasmic Ca²⁺. This study examines how somatostatin affects glucose generation of cytoplasmic Ca²⁺ oscillations in mouse islets in absence and presence of tolbutamide blockade of the K_ATP channels. Ca²⁺ was measured with dual wavelength microflurometry in isolated islets loaded with the indicator Fura-2. Rise of glucose from 3 to 20 mM evoked introductory lowering of Ca²⁺ prolonged by activation of somatostatin receptors. During continued superfusion exposure to somatostatin triggered oscillations mediated by periodic increase from the basal level (absence of tolbutamide) or by periodic interruption of an elevated level (presence of tolbutamide). In the latter situation the oscillations were transformed into sustained elevation by activation of muscarinic receptors (acetylcholine) or increase of cyclic AMP (IBMX, 8-bromo-cyclic AMP, forskolin). The observed effect of cyclic AMP raises the question whether high proportions of the glucagon-producing α-cells promote steady-state elevation of Ca²⁺. In support for this idea somatostatin was found to trigger glucose-induced Ca²⁺ oscillations essentially in small islets that contain very few α-cells. The results indicate that somatostatin promotes glucose generation of Ca²⁺oscillations with similar characteristics both in the absence and presence of functional K_ATP channels.     

Correlation between cyclic AMP and LH release following prostaglandin E2 administration

Five min following a single iv injection of PGE₂ into ovariectomized mature rats pretreated with estrogen and progesterone, plasma LH and plasma and pituitary cyclic AMP levels were raised significantly. A close correlation was observed between increased pituitary cyclic AMP contents and release of plasma LH. The average level of cyclic AMP in the anterior pituitary and plasma cyclic AMP increased significantly, while the circulating plasma LH level was not changed at 1 min after PGE₂ injection. Plasma LH level increased at 2 min after PGE₂ and reached a maximum level at the above-mentioned time. This is consistent with hypothesis that increased release of hormone is a consequence of increased pituitary cyclic AMP content.     

Regulation of Noradrenaline Release from Rat Occipital Cortex Tissue Chops by α2-Adrenergic Agonists

Noradrenaline (NA) and the alpha 2-adrenergic agonists clonidine, BHT-920, and UK 14304-18 inhibit potassium-evoked release of [3H]NA from rat occipital cortex tissue chops with similar potencies. NA (10(-5) M) was most effective as up to 85% inhibition could be observed compared with 75%, 55%, and 35% for UK 14304-18, clonidine, and BHT-920, respectively, all at 10(-5) M. Potassium-evoked release was enhanced by both forskolin (10(-5) M) and 1 mM dibutyryl cyclic AMP. Pretreatment of tissue chops with 1 mM dibutyryl cyclic AMP in the presence of 3-isobutyl-1-methylxanthine partially reversed the alpha 2-adrenergic agonist inhibition of NA release. No reversal of inhibition was observed following pretreatment with 10(-5) M forskolin. The effects of clonidine, BHT-920, UK-14308-18, and NA on cyclic AMP formation stimulated by (a) forskolin, (b) isoprenaline, (c) adenosine, (d) potassium, and (e) NA were examined. Only cAMP formation stimulated by NA was inhibited by these alpha 2-adrenergic agonists. These results suggest that only a small fraction of adenylate cyclase in rat occipital cortex is coupled to alpha 2-adrenergic receptors. These results are discussed in relation to recent findings that several alpha 2-adrenergic receptor subtypes occur, not all of which are coupled to the inhibition of adenylate cyclase, and that alpha 2-adrenergic receptors inhibit NA release in rat occipital cortex by a mechanism that does not involve decreasing cyclic AMP levels.     

Muscarine Receptors Regulating Electrically Evoked Release of Acetylcholine in Hippocampus Are Linked to Pertussis Toxin-Sensitive G Proteins but Not to Adenylate Cyclase

Abstract: [³H]Acetylcholine release elicited with 360 pulses/3 Hz from slices of rabbit hippocampus is facilitated in the presence of the muscarine (M) receptor antagonist atropine (indicating the existence of autoinhibition) and diminished by the M receptor agonists carbachol and oxotremorine. W-Ethylmaleimide (30 μM ) and pertussis toxin (8 μg/ml) counteracted antagonist-induced facilitation and agonist-induced inhibition of release, suggesting that a pertussis toxin-sensitive GTP-binding protein is involved in the chain of events mediating activation of M receptors to inhibition of release. Neither 8-bromo-cyclic AMP (300 μM ), a membrane analogue of cyclic AMP, nor rolipram (10 μM ), a phosphodiesterase inhibitor, affected electrically evoked release of [³H]acetylcholine. They also did not influence the oxotremorine-induced inhibition of transmitter release. In conclusion, no evidence was found for the assumption that activation of M autoreceptors is linked to inhibition of adenylate cyclase.     

Cyclic AMP and platelet prostaglandin synthesis.

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3'5'-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of 14C-arachidonic acid by washed platelets. Prostaglandin E1 (PGE1), PGE1 with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B2. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE1 with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of 14C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

Physiological roles of adenosine derivatives which are released during neurotransmission in mammalian brain.

1. Experiments using synaptosome beds suggested that ATP was released from presynaptic sites and degraded to adenosine in the synaptic cleft and that the resulting adenosine was taken up again into nerve endings where it was re-phosphorylated to ATP. 2. Adenosine derivatives in the synaptic cleft inhibited the postsynaptic potentials in olfactory cortex slices in vitro, presumably by the inhibition of Ca2+ influx into nerve endings which resulted in the reduction of transmitter release. 3. The adenosine derivatives also increased the level of cyclic AMP in the slices under the same conditions as above. 4. Although the nature of the "adenosine receptors" for both functions was remarkably similar, the increase of cyclic AMP did not mediate the inhibitory action, but the presynaptic increase of cyclic AMP induced by adenosine derivatives might mediate the facilitation observed in the olfactory cortex. 5. Possible physiological roles of extracellular adenosine derivatives in mammalian brain were classified, at different sites of action around the synapses, with different time courses and modes of action, directly or via the increase of intracellular cyclic AMP.     

The effects of norepinephrine on the release of cyclic AMP by the isolated perfused heart of the guinea pig.

In the isolated perfused heart preparation of the guinea pig, continuous infusions of norepinephrine in doses of 0.06, 0.2 and 0.6 μg/min resulted in a dose-dependent increase in the heart rate, augmentation of the heart contractility measured as increments of dp/dt max. and an increase in the coronary flow. Simultaneous with these changes, a dose-dependent release of substantial quantities of cyclic AMP into the perfusate was observed, and there was a close moment to moment correlation between the increments of dp/dt max. and the increases in the amount of released cyclic AMP produced by norepinephrine. A linear correlation, though less marked than that described above, was also observed between the positive chronotropic and coronary vasodilatatory effects and the increases in the amount of released cyclic AMP, while there was no dynamic correlation between the tissue cyclic AMP level and the augmentation of the mechanical performances of the heart; Increases in the tissue cyclic AMP level were observed only with the highest dose and were transient and a significant decrease was noted with the lowest dose. The decrease was abolished by phentolamine. It is concluded that cyclic AMP exists in different ntracellular compartments in the heart, only one of which -- comprising a small fraction of the total cellular cyclic AMP, -- is functionally-relevant, and that much of this fraction can pass relatively freely across the cell membrane.     

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTP gamma S, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to release IP3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not by GDP beta S. Enhanced IP3 synthesis was observed with a combination of GTP gamma S and carbachol. Exogenous IP3, as well as carbachol and GTP gamma S, provoked the release of sequestered 45Ca2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the beta-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini-the pertussis toxin-insensitive Gp of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin-sensitive Gi inhibitory protein of the adenylate cyclase complex.     

Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3',5'-cyclic monophosphate and protein kinase.

The events involved in platelet shape change, aggregation, the release reaction and contraction are thought to be mediated by the availability of Ca2+. Increased cytoplasmic calcium, released from intracellular stores, triggers platelet activity, and increased concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) inhibits platelet alterations. We have studied the hypothesis that cyclic AMP may regulate the level of platelet cytoplasmic calcium by stimulating calcium removal by a membrane system. Such a hypothesis would be consistent with the reversibility of most manifestations of platelet activation. Human platelets were sonicated and unlysed platelets, mitochondria and granules were removed by centrifugation at 19 000 X g. Electron microscopy shows that the sediment, after centrifugation of the supernatant at 40 000 X g consists to a large extent of membrane vesicles. Such preparations actively concentrate calcium, as measured by the uptake of 45Ca, and also have the maximal calcium-stimulated ATPase activity. Optimal calcium uptake requires ATP and oxalate, and release of calcium from loaded vesicles was stimulated by the calcium ionophore A23187 and inhibited by LaCl3. These data indicate that calcium was being actively concentrated within membrane vesicles. After washing of such preparations in the absence of ATP, their capacity to take up Ca2+ is reduced to an initial value of 2.8 nmol/mg protein per min. In the presence of 2 - 10(6) M cyclic AMP to which was added a protein kinase preparation from human platelets, up to a 3-fold increase of this rate of uptake was observed. These results suggest that in platelets, as in muscle, cyclic AMP is a regulatory factor in the control of cytoplasmic calcium. Although the cyclic nucleotide may have still other functions, it appears likely that the well-known inhibition of many platelet activities by high intracellular cyclic AMP concentrations is directly linked to the stimulation of the removal of Ca2+ from the cytoplasm.     

Glyceraldehyde, but not cyclic AMP-stimulated insulin release is preceded by a rise in cytosolic free Ca2+

The dynamics of changes in membrane potential, cytosolic free Ca2+, [Ca2+]i and immunoreactive insulin release were assessed in RINm5F cells. Membrane depolarization and a rise in [Ca2+]i preceded the stimulation of insulin release by D-glyceraldehyde. Forskolin, which raised the cellular cyclic AMP levels, stimulated insulin release without changing membrane potential or [Ca2+]i. It is concluded that cyclic AMP acts on insulin release not by mobilizing Ca2+ but by another, as yet undefined, mechanism.     

Stimulation-Evoked Ca2+ Fluxes in Cultured Bovine Adrenal Chromaffin Cells Are Enhanced by Forskolin

Forskolin, 1 microM, increased acetylcholine (ACh)-stimulated 45Ca uptake by chromaffin cells. The stimulatory effects of forskolin decreased with increasing concentration of ACh. The attenuation of the effect of forskolin on 45Ca uptake as a function of ACh concentration correlated well with changes in the forskolin effect on ACh-evoked catecholamine (CA) release. Forskolin increased excess KCl- and veratrine-evoked CA release and 45Ca uptake. Forskolin by itself stimulated 45Ca efflux and enhanced ACh-, excess KCl-, and veratrine-stimulated 45Ca efflux. High doses of forskolin inhibited both ACh-evoked 45Ca uptake and CA release. The inhibitory action of forskolin was specific to receptor-mediated response because excess KCl- and veratrine-stimulated 45Ca uptake and CA release were not inhibited. Forskolin, 0.3-30 microM, dose-dependently increased caffeine-stimulated CA release and 45Ca efflux in the absence of Ca2+ in the medium, and the effects were mimicked by dibutyryl cyclic AMP. These results suggest that cyclic AMP increases stimulation-induced CA release by enhancing calcium uptake across the plasma membrane and/or altering calcium flux in an intracellular calcium store.