Choline analogues: their use in studies of acetylcholine synthesis, storage, and release

The objective of this article is to illustrate how choline analogues might provide insight into mechanisms that regulate the synthesis, storage, and release of acetylcholine (ACh). Studies with false neurotransmitters provide information about the origin of releasable transmitter. Thus, false esters that distribute like ACh to vesicle-bound stores are as releasable as is ACh, but esters that poorly localize to synaptic vesicles are poorly releasable. Studies of choline analogue uptake provide information about the structural specificity of that transport process and, also, show that choline uptake is regulated in response to activity. Thus, stimuli that normally release transmitter increase the rate of choline transport, presumably to provide more precursor for ACh synthesis. However, the relationship between precursor delivery and product formed can be dissociated, suggesting that some factor in addition to choline delivery is involved in ACh synthesis regulation. Studies with a compound (AH5183), which inhibits ACh uptake by synaptic vesicles, provide information about the relationship of ACh stores and releasable transmitter. In the presence of AH5183 some 15% of nerve terminal ACh is released in response to nerve impulses, suggesting the existence of a small population of vesicles that contain readily releasable ACh. In presence of AH5183, ACh synthesis is activated even when ACh release is depressed, showing that transmitter synthesis can be regulated by some factor other than nerve terminal ACh levels.     

A proposed model for the synthesis,storage and release of acetylcholine at the neuromuscular junction

Sythesis, storage and release of acetylcholine at the neuromuscular junction are still area of speculation and controversy. This paper seeks to introduce a lumped parameter mathematical model which may be used to describe and quantify some of the various processes involved. The construction of the model is fully discussed, compared with previous models, and its shortcomings noted.     

Atriopeptin release from the isolated perfused rabbit heart

Mammalian atrial extracts have been shown to contain bioactive peptides which exert natruiretic, diuretic, and smooth muscle relaxant effects. These extracts include several low molecular weight (< 5,000 Mr) atrial peptides (atriopeptins) which exhibit identical sequences over a central core region which are derived from the high molecular weight peptide (atriopeptigen) precursor which has been purified and sequenced. In the current study we found that extracts of rabbit atria possess both high and low molecular weight bioactive atrial peptides, however, the coronary venous effluent obtained from the isolated perfused rabbit heart only contained the low molecular weight peptide. This trypsin labile activity causes a dose-dependent relaxation of rabbit aorta and chicken rectum assay strips. Separation of the bioactivity with gel filtration chromatography and reversed phase HPLC indicates the heart releases a single substance similar to atriopeptin III. There was no evidence that atriopeptigen was released from the isolated perfused rabbit heart. We suggest that atriopeptigen is proteolytically processed in the atria to an atriopeptin which is subsequently the released form of the atrial peptide.     

Increased acetylcholine synthesis and release following presynaptic activity in a sympathetic ganglion

Abstract: The acetylcholine (ACh) content of sympathetic ganglia increases above its normal level following a period of preganglionic nerve stimulation. In the present experiments, this extra ACh that accumulates following activity was labeled radioactively from [³H]choline and its specific activity was compared with that of ACh subsequently released during preganglionic nerve stimulation. The specific activity of the released ACh was similar to that of the total tissue ACh, suggesting that the extra ACh mixes fully with endogenous stores. The present experiments also show that transmitter release during neuronal stimulation is necessary for the poststimulation increase in transmitter store. However, the increase was not evident when transmitter release was induced by K⁺. It is concluded that both transmitter release and impulse invasion of the nerve terminals are necessary for the adaptive phenomenon to manifest itself. The role of choline delivery and choline acetyltransferase activity in generating the poststimulation increase in transmitter store was tested. When choline transport activity measured as choline analogue (homocholine) accumulation increased, ACh synthesis was increased and when transport activity was not increased, neither was ACh synthesis. There was no poststimulation increase in measured choline acetyltransferase activity.     

Neurotensin stimulates histamine release from the isolated, spontaneously beating heart of rats

Neurotensin (NT) evoked a transient, dose-dependent histamine release (ED50 170 ng ml-1) from the rat perfused heart. Histamine release by NT occurred within seconds and lasted less than 2 min. The histamine releasing effect of NT was followed by a dose-dependent increase of the perfusion pressure and a slight tachycardia. The histamine releasing effect of NT was completely abolished in hearts derived from rats pretreated for 3 days with high doses of compound 48/80. The coronary vasoconstrictor effect of NT was increased in hearts derived from compound 48/80-pretreated rats. The mast cell inhibitor cromoglycate markedly inhibited NT-induced histamine release without affecting the coronary vasoconstrictor effect of NT. The histamine releasing effect of NT was inhibited, while its coronary vasoconstrictor effect was markedly potentiated, in hearts derived from rats pretreated with the antiallergic and antiinflammatory steroid dexamethasone. The increase of perfusion pressure evoked by NT was not modified by antihistamine drugs. Infusions of exogenous histamine (10(-6)-10(-5) g ml-1) caused a dose-dependent coronary vasodilation in the rat perfused heart. The results suggest that NT stimulates histamine release from cardiac mast cells. These results together with those obtained in previous studies suggest that mast cell mediators (particularly histamine and serotonin) are unlikely to be responsible for the coronary vasoconstrictor effect of NT in the rat perfused heart.     

Effects of choline augmentation on acetylcholine synthesis and release in rat atrial minces

The uptake and acetylation of [3H]-choline, as well as the calcium-dependent release of a newly synthesized [3H]-ACh, was studied in a new rat atrial mince preparation. The hemicholinium-3-sensitive uptake and acetylation of [3H]-choline increased as [3H]-choline concentrations were elevated to 100 microM in atrial minces. In contrast, hemicholinium-3-sensitive [3H]-choline uptake was saturated with 15 microM [3H]-choline in brain synaptosomes. The increased atrial [3H]-ACh synthesized in the presence of [3H]-choline augmentation was releasable by 50 mM K+-depolarization in a 1 mM cobalt-sensitive manner. These results suggest that atrial parasympathetic activity may be more sensitive to circulating choline concentrations than brain cholinergic neurons are.     

Nuclear columns, Kinetics of RNA synthesis and release in isolated rat liver nuclei

By continuous perfusion of columns containing isolated immobilized rat liver nuclei with media containing labeled RNA precursors, the in vitro synthesis and release of RNA was studied. The combined reaction of synthesis and release could be adjusted to proceed at a constant rate. The reaction rate responded to variation of termperature, ionic conditions, nucleoside triphosphate concentration and to the addition of RNA polymerase inhibitors. During 60 min perfusion approximately equal amounts of radioactive low molecular weight RNA and of ribonucleoproteins were released. Pulse-chase experiments showed that the low molecular weight RNA was synthesized throughout the perfusion and released immediately after formation. The ribonucleoproteins were primarly labeled during the first period of perfusion and were gradually released. Synthesis of RNA contained in the ribonucleoproteins was inhibited by low alpha-amanitin concentrations, indicating that it was catalyzed by RNA polymerase II. The in vitro labeled ribonucleoproteins exhibited properties of the stable nuclear particles which can be extracted from isolated nuclei after rapid in vivo labeling of RNA. They had a buoyant density of 1.41--1.43 in CsCl, were partially unstable in 1% deoxycholate, but stable in 0.1% deoxycholate, in 100 mM NaCl and in 10 mM EDTA. Due to the dilution by the perfusion medium, the ribonucleoproteins sedimented with a peak at 22--27 S, and not at 30--45 S. The RNA synthesized in the immobilized nuclei was not degraded during the perfusion. Less than 20% was gradually released, whereby the 20--30 S peak zone was reduced. While the properties of the in vitro labeled ribonucleoproteins and of rapidly in vivo labeled ribonucleoproteins were the same, the kinetics of their release differed.     

Naloxone reduces release of creatine kinase in the isolated ischemic rat heart

The effects of naloxone, propranolol, or both on the release of creatine kinase (CK) from the isolated ischemic rat heart were studied. Naloxone at concentrations of 1.1 and 3.6 mmole liter-1 in the perfusate at a rate of 1-2 ml min-1 reduced the release of CK from the isolated ischemic rat heart during myocardial ischemia in a dose-dependent manner. Propranolol at a concentration of 7 mumole liter-1 in the perfusate also reduced the release of CK. Addition of naloxone (1.1 mmole liter-1) to propranolol further reduced the release of CK. The effect of the joint administration of the two drugs seemed to be additive.     

Influence of prostaglandins upon adenosine release and of adenosine upon prostaglandin release in the isolated rabbit heart.

Prostaglandin (PG) E1 with its high effect of coronary dilation causes a distinct increase in the adenosine release from the isolated rabbit heart (Langendorff technique). Compared to this, an equal dose of PGF2alpha increases the release of adenosine only to a small extent. Conversely, application of adenosine results in a considerable release of PG-like substances from the rabbit heart in vitro. The present investigations support the hypothesis that, apart from adenosine, PG's too, are involved in coronary regulation as modulators.     

Inhibition of choline efflux results in enhanced acetylcholine synthesis and release in the guinea-pig corticocerebral synaptosomes.

Synthesis and release of [3H]acetylcholine ([3H]ACh) were measured in synaptosomes from the guinea pig cerebral cortex after preloading with [3H]choline ([3H]Ch). We demonstrate here that inhibition of choline (Ch) efflux results in an increase in acetylcholine (ACh) synthesis and release. Our findings are as follows: (1) inhibition of [3H]Ch efflux by hemicholinium-3 (HC-3) (100 microM), increased the levels of both the released (116% of control) and the residing (115% of control) [3H]ACh. (2) The muscarinic agonist, McN-A-343 (100 microM), which was previously shown to inhibit Ch efflux, also increased the released (121% of control) and the residing (109% of control) [3H]ACh. (3) Omission of Na+ ions (which are required for Ch transport) from the incubation medium had similar effects to those observed with McN-A-343 and HC-3. These results suggest inverse relationships between Ch efflux on one hand, and ACh synthesis and release on the other hand. (4) Depolarization with 50 mM K+, or with the K+ channel blocker, 4-aminopyridine (100 microM), also increased the total level of [3H]ACh (113 and 107% of nondepolarized synaptosomes, respectively). However, whereas conditions that inhibit Ch transport such as HC-3, McN-A-343 and "no sodium" increased both the residing and the released [3H]ACh depolarization with high K+ or 4-aminopyridine reduced the residing (79 and 87% of control, respectively) and increased only the released [3H]ACh (182 and 148% of control, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP-stimulated transmitter release and cyclic amp synthesis in isolated chromaffin granules

ATP stimulates chromaffin granules from the bovine adrenal medulla to release epinephrine and specific soluble proteins. ATP analogs substituted in the β-γ-position with either nitrogen or carbon were also found to be effective at inducing release from isolated chromaffin granules. However, an ATP analog substituted at the α-β position with carbon was strongly inhibitory. Cyclic AMP was also found to be synthesized by isolated chromaffin granules under release conditions. ATP analogs were effective as substrates for adenylate cyclase in the same order as their efficiency for inducing release from vesicles. Hydrolysis at the β-γ linkage of ATP therefore is probably not necessary for release; however, hydrolysis at the α-β position may be important in the release process. Cyclic AMP may be produced and play a regulatory role in this event.     

Down-regulation of the non-neuronal acetylcholine synthesis and release machinery in acute allergic airway inflammation of rat and mouse

Acetylcholine (ACh), derived both from nerve fibres and from non-neuronal sources such as epithelial cells, is a major regulator of airway function. There is evidence that dysfunction of the neuronal cholinergic system is involved in the pathogenesis of asthma. Here, we asked whether the pulmonary non-neuronal ACh-synthesis and release machinery is altered in a rat and a mouse model of allergic airway disease. Animals were sensitized against ovalbumin, challenged by allergen inhalation, and sacrificed 24 or 48 h later. Targets of investigation were the high-affinity choline transporter-1 (CHT1), that mediates cellular uptake of choline, the ACh-synthesizing enzyme choline acetyltransferase (ChAT), the vesicular ACh transporter (VAChT), and the polyspecific organic cation transporters (OCT1-3), which are able to translocate choline and ACh across the plasma membrane. With cell-type specific distribution patterns, immunohistochemistry identified these proteins in airway epithelial cells and alveolar macrophages. Real-time RT-PCR revealed significant decreases in ChAT-, CHT1-, VAChT-, OCT-mRNA in the lung of sensitized and allergen challenged animals. These data were supported by immunohistochemistry, demonstrating reduced labeling intensity of airway epithelial cells. ChAT-, CHT1-, VAChT-, and OCT1-mRNA were also significantly reduced in cells recovered by bronchoalveolar lavage from sensitized and challenged rats. In conclusion, the pulmonary non-neuronal cholinergic system is down-regulated in acute allergic airway inflammation. In view of the role of ACh in maintenance of cell-cell-contacts, stimulation of fluid-secretion and of ciliary beat frequency, this down-regulation may contribute to epithelial shedding and ciliated cell dysfunction that occur in this pathological condition.     

Refinement and validation of a model describing synthesis 'storage and release of acetylcholine at the neuromuscular junction

This paper seeks to provide a refinement and validation for a lumped parameter quasi time independent model which defines synthesis, storage and release of acetylcholine at the neuromuscular junction. Model results are shown to be compatible with data produced during physiological experiments carried out under normal electrolytic conditions.