Non-neuronal Acetylcholine Release Research Articles

Expression of cholinesterases and their anchoring proteins in rat heart.

Acetylcholine (ACh)-mediated vagal transmission as well as nonneuronal ACh release are considered cardioprotective in pathological situations with increased sympathetic drive such as ischemia-reperfusion and cardiac remodeling. ACh action is terminated by hydrolysis by the cholinesterases (ChEs), acetylcholinesterase, and butyrylcholinesterase. Both ChEs exist in multiple molecular variants either soluble or anchored by specific anchoring proteins like collagen Q (ColQ) anchoring protein and proline-rich membrane anchoring protein (PRiMA). Here we assessed the expression of specific ChE molecular forms in different heart compartments using RT-qPCR. We show that both ChEs are expressed in all heart compartments but display different expression patterns. The acetylcholinesterase-T variant together with PRiMA and ColQ is predominantly expressed in rat atria. Butylcholinesterase is found in all heart compartments and is accompanied by both PRiMA and ColQ anchors. Its expression in the ventricular system suggests involvement in the nonneuronal cholinergic system. Additionally, two PRiMA variants are detected throughout the rat heart.

P2 The action of endothelial–derived acetylcholine is impaired in diabetes

The endothelium is highly sensitive to acetylcholine and responds to the neurotransmitter by releasing vasodilators that control smooth muscle tone. However, acetylcholine is not expected to reach the endothelium through the vessel wall or via blood. Here, by studying calcium signalling in large areas of the endothelium of intact arteries, we show: (1) the endothelium itself is a source of acetylcholine, (2) acetylcholine release from the endothelium underlies flow-mediated vascular responses; and (3) flow-mediated responses are all but abolished in a rat model of diabetes. Calcium signalling was studied in large fields of intact endothelia (~150 cells) of carotid and mesenteric arteries from healthy and diabetic rats (Sprague-Dawley, 150–250 g). In both carotid and mesenteric arteries, flow evoked repeatable endothelial calcium signalling. Importantly, flow-mediated calcium responses could be manipulated by modulating cholinergic signalling pathways: the muscarinic receptor blocker, atropine (100 nM), and the acetylcholine hydrolase, acetylcholinesterase (4 U/ml), each abolished flow-induced calcium activity, whilst the acetylcholinesterase inhibitor, neostigmine (10 µM), enhanced calcium activity, and the choline acetyltransferase inhibitor, bromoacetylcholine (50?µM), attenuated responses. Significantly, responses were reduced in diabetic, compared to control, endothelia. Indeed, the number of diabetic endothelial cells responding to flow was 45% of control, and the peak and time-averaged magnitude of calcium signals was reduced to 54% and 12% of control, respectively. These results identify the endothelium as a source of non-neuronal acetylcholine release and thus demonstrate the reason for the endothelium’s sensitivity to acetylcholine. Significantly, reduced acetylcholine release may underlie the pathophysiology and endothelial dysfunction that occurs in diabetes.

Sodium ion transport participates in non-neuronal acetylcholine release in the renal cortex of anesthetized rabbits.

This study examined the mechanism of release of endogenous acetylcholine (ACh) in rabbit renal cortex by applying a microdialysis technique. In anesthetized rabbits, a microdialysis probe was implanted into the renal cortex and perfused with Ringer's solution containing high potassium concentration, high sodium concentration, a Na+/K+-ATPase inhibitor (ouabain), or an epithelial Na+ channel blocker (benzamil). Dialysate samples were collected at baseline and during exposure to each agent, and ACh concentrations in the samples were measured by high-performance liquid chromatography. High potassium had no effect on renal ACh release. High sodium increased dialysate ACh concentrations significantly. Ouabain increased dialysate ACh concentration significantly. Benzamil decreased dialysate ACh concentrations significantly both at baseline and under high sodium. The finding that high potassium-induced depolarization does not increase ACh release suggests that endogenous ACh is released in renal cortex mainly by non-neuronal mechanism. Sodium ion transport may be involved in the non-neuronal ACh release.

Expression of choline and acetylcholine transporters in synovial tissue and cartilage of patients with rheumatoid arthritis and osteoarthritis.

Increasing evidence is showing that the non-neuronal cholinergic system plays an important role in the pathology of rheumatoid arthritis (RA). Choline transport into the cell is the rate-limiting step for the synthesis of acetylcholine (ACh), which can be released directly or in vesicles from the cell. However, in the human joint little is known about choline import or the release of ACh from the cell. Thus, we analyze the expression of members of the organic cation transporter (OCT), of the newly discovered choline transporter-like (CTL) family and of classical neuronal components such as the high-affinity choline transporter (CHT1) and the vesicular ACh transporter (VAChT) in the synovium and cartilage of the human hip joint from patients with osteoarthritis (OA) and RA. OCT1, OCT3 and OCTN1 and all members of the CTL family were expressed in synovial and cartilage samples. The expression of CTL1 and CTL2 was localized in synovial macrophages and fibroblasts. CHT1 mRNA expression was detectable only in the synovium, whereas VAChT was completely absent in all samples. Therefore, in the human joint, choline transport into the cell and the release of ACh seems to be mediated mainly by members of the OCT and CTL family. Expression of transporters appears not to be influenced by the pathological state, as no differences have been detected between joints from OA or RA patients. Importantly, however, all necessary components for choline import and the release of non-neuronal ACh are present in the human joint.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-014-2036-0) contains supplementary material, which is available to authorized users.

Cholinergic receptors as target for cancer therapy in a systems medicine perspective.

Epithelial cells not innervated by cholinergic neurons express nicotinic and muscarinic acetylcholine (ACh) receptors (nAChR, mAChR). nAChR and mAChR are components of the auto-/paracrine-regulatory loop of non-neuronal ACh release. The cholinergic control of non-neuronal cells may be mediated by different effects (synergistic, additive, or reciprocal) triggered by these receptors. The ionic events (Ca(+2) influx) are generated by the ACh-opening of nAChR channels, while the metabolic events by ACh-binding to G-proteincoupled mAChR. Effective inter- and intracellular signaling is crucial for valuable cancer cells proliferation and survival. Depending on cancer cell type, different AChR have been identified. The proliferation of airways epithelial cancer cells and pancreatic cancer cells may be under the control of α7-nAChR and M3-mAChR, while breast cancer cells and colon cancer cells are regulated by α9-nAChR, and M3-mAChR, respectively. In turn, these receptors may activate different pathways (Ras-Raf-1-Erk-AKT) as well as other receptors (β- adrenergicR). nAChR or mAChR antagonists may inhibit cancer growth. Inhibition of M3 by antisense or antagonists (Darifenacin, Tiotropium) reduces lung or colon cancer proliferation, as well as inhibition of α9- nAChR [polyphenol (-)-epigallocatechin-3-gallate] diminishes breast cancer cells growth. α7-nAChR silencing inhibits lung cancer proliferation. Moreover, inhibition of the nAChR-β-adrenergicR pathway (β-blockers) could be also useful. This review will describe the future translational perspectives of cholinergic receptors druginhibition in a complex disease such as cancer that poses compelling treatment challenges. Cancer happens as consequence of disease-perturbed molecular networks in relevant organ cells that change during progression. The framework for approaching these challenges is a systems approach.

369 Release of Non-Neuronal Acetylcholine From Biliary Epithelial Cells During Extrahepatic Cholestasis Induced by Bile Duct Ligation

369 Release of Non-Neuronal Acetylcholine From Biliary Epithelial Cells During Extrahepatic Cholestasis Induced by Bile Duct Ligation

Epithelial acetylcholine – a new paradigm for cholinergic regulation of intestinal fluid and electrolyte transport

Epithelial acetylcholine – a new paradigm for cholinergic regulation of intestinal fluid and electrolyte transport

T1848 Measurement of Non-Neuronal Acetylcholine Release By a Colon Cancer Cell Line

T1848 Measurement of Non-Neuronal Acetylcholine Release By a Colon Cancer Cell Line

Basic and Clinical Aspects of Non-neuronal Acetylcholine: Expression of Non-neuronal Acetylcholine in Urothelium and Its Clinical Significance

Recently, several reports demonstrate that non-neuronal acetylcholine (ACh) release may contribute to various pathophysiological conditions. In this review, we presented our experiments designed to evaluate the non-neuronal cholinergic system in human bladder. After insertion of the microdialysis probe, human bladder strips were suspended in an organ bath filled with Krebs-Henseleit solution, and Ringer solution was perfused into the probe. ACh release was measured by microdialysis and HPLC. The contribution of urothelium and the effects of age and stretch of bladder strips on non-neuronal ACh release were evaluated. Choline acetyl-transferase (ChAT) immunohistochemical staining of bladder was also performed. Immunohistochemistry showed marked ChAT-positive staining in the urothelium. There was tetrodotoxin-insensitive non-neuronal ACh release and this was significantly higher in strips with urothelium than in strips without urothelium. The non-neuronal ACh release was increased with age. Stretch of bladder strips caused increases in non-neuronal ACh release. The stretch-induced release of non-neuronal ACh was increased with age. Our data demonstrate that there is a non-neuronal cholinergic system in human bladder and that urothelium contributes to non-neuronal ACh release. There was significant age-related and stretch-induced increase in non-neuronal ACh release. It is suggested that the non-neuronal cholinergic system may contribute to the physiology and pathophysiology of human bladder. We also discussed the clinical significance of the nonneuronal cholinergic system in human bladder.

In vivo release of non-neuronal acetylcholine from human skin by dermal microdialysis: Effects of sunlight, UV-A and tactile stimulus

Non-neuronal acetylcholine (ACh) is expressed in epithelial, endothelial and immune cells. For example, the in vivo release of ACh from the human skin pretreated with botulinum toxin has recently been demonstrated. In the present experiments the effects of light (sunlight and solar radiation by a commercial UV-A applier) and of a tactile stimulus on the release of non-neuronal ACh were investigated. Release of ACh from the proximal and distal shin, i.e. anterior tibial region, was measured by dermal microdialysis in 20 min samples over a time period of at least 140 min. Control experiments were performed in a dark room throughout. In some experiments volunteers were exposed to sunshine (80–140 min) or the shin region was illuminated (80–95 min) by a commercial UV-A lamp (400 W at a distance of 50 cm). In control experiments ACh release between 20 and 80 min (B1) amounted to 118±32 pmol (n=17) and gradually declined between 80 and 140 min (B2) to 112±34 pmol, resulting in a B2/B1 ratio of 0.95. When the skin was exposed to sunlight ACh release increased from 205±58 pmol (B1) to 349±122 pmol resulting in a B2/B1 ratio of 1.70. UV-A radiation, however, had no significant effect on the B2/B1 ratio. When very smooth tactile stimuli were applied by a cotton wool tip for 20 min to the skin close to the microdialysis membranes in a dark room, ACh release was increased from 9±2 pmol/20 min to 52±36 (n=7). In conclusion, the in vivo release of ACh from the human skin appears to be regulated by external stimuli like sunlight and tactile stimuli.

Release of non-neuronal acetylcholine from the isolated human placenta is affected by antidepressants

Non-neuronal acetylcholine (ACh) is released from the human placenta into the extracellular space via organic cation transporters (OCTs). The present experiments investigated whether ACh release from epithelial cells is affected by drugs which are substrates of OCTs. The antidepressant drugs amitriptyline and doxepine were tested as both substances are not approved for pregnant women but frequently used. Release of ACh was measured in 10 min intervals over a period of 100 min. Test substances were added from t=50 min of incubation onwards. The effect was calculated by comparing the ACh release of the last three samples (t=70–100 min; B2) with that immediately before the application of the test substances (t=20–50 min; B1). The baseline ACh release amounted to 2.07±0.17 nmol/10 min (n=29; villus). Under control conditions a B2/B1 ratio of 0.78±0.02 was obtained. The following B2/B1 ratios were found, when therapeutic drugs were added: 0.54±0.04 (n=7; P<0.05) in the presence of 10 μM amitriptyline; 0.44±0.04 (10; P<0.01) in the presence of 10 μM doxepin; 0.73±0.04 (13) in the presence of 10 μM metformin; 0.76±0.06 (7) in the presence of 10 μM minoxidil; 0.63±0.03 (10) in the presence of 1 μM theophylline. The results demonstrate that antidepressants reduce the release of non-neuronal ACh at least in the human placenta, most likely by intracellular substrate competition at the polyspecific organic cation transporters (OCTs) but only at concentrations roughly 30-fold above the therapeutic range. Theophylline may also interfere with the release of non-neuronal ACh.

Management of detrusor dysfunction in the elderly: changes in acetylcholine and adenosine triphosphate release during aging

Numerous studies have detailed age-related changes in the structure and function of the bladder that may contribute to the high prevalence of overactive bladder (OAB) in the elderly population, but the relation of these changes to OAB symptoms remains unclear. Physiologic and neurochemical studies have been conducted in human detrusor strips obtained from people of different ages, focusing on potential changes in cholinergic and purinergic neurotransmission, as well as the release and actions of acetylcholine (ACh) from nonneuronal bladder cells. Results from physiologic and microdialysis experiments indicate that purinergic transmission increases with age, whereas cholinergic transmission decreases. These effects are most likely because of decreased release of ACh and increased release of adenosine triphosphate (ATP) from postganglionic parasympathetic axons innervating the bladder. Immunohistochemical experiments showed that choline acetyltransferase in the human detrusor is contained not only in parasympathetic axons, but also in cells of the urothelium. The release of nonneuronal ACh increases with age and detrusor stretch. The age-related increase in purinergic transmission in the detrusor and other data indicating that responses to ATP are increased in detrusor overactivity suggest that purinergic receptor antagonists may provide a useful complement to muscarinic receptor antagonists in the treatment of older patients with OAB. Nonneuronal ACh release may play a key role in the storage phase of the micturition reflex, and this may explain, at least in part, the effectiveness of antimuscarinic agents for the treatment of OAB.

Release of non-neuronal acetylcholine from the isolated human placenta is mediated by organic cation transporters.

1. The release of acetylcholine was investigated in the human placenta villus, a useful model for the characterization of the non-neuronal cholinergic system. 2. Quinine, an inhibitor of organic cation transporters (OCT), reduced acetylcholine release in a reversible and concentration-dependent manner with an IC(50) value of 5 microM. The maximal effect, inhibition by 99%, occurred at a concentration of 300 microM. 3. Procaine (100 microM), a sodium channel blocker, and vesamicol (10 microM), an inhibitor of the vesicular acetylcholine transporter, were ineffective. 4. Corticosterone, an inhibitor of OCT subtype 1, 2 and 3 reduced acetylcholine in a concentration-dependent manner with an IC(50) value of 2 microM. 5. Substrates of OCT subtype 1, 2 and 3 (amiloride, cimetidine, guanidine, noradrenaline, verapamil) inhibited acetylcholine release, whereas carnitine, a substrate of subtype OCTN2, exerted no effect. 6. Long term exposure (48 and 72 h) of villus strips to anti-sense oligonucleotides (5 microM) directed against transcription of OCT1 and OCT3 reduced the release of acetylcholine, whereas OCT2 anti-sense oliogonucleotides were ineffective. 7. It is concluded that the release of non-neuronal acetylcholine from the human placenta is mediated via organic cation transporters of the OCT1 and OCT3 subtype.

Release of non-neuronal acetylcholine from the human placenta: difference to neuronal acetylcholine.

The synthesis and release of non-neuronal acetylcholine, a widely expressed signaling molecule, were investigated in the human placenta. This tissue is free of cholinergic neurons, i.e. a contamination of neuronal acetylcholine can be excluded. The villus showed a choline acetyltransferase (ChAT) activity of 0.65 nmol/mg protein per h and contained 500 nmol acetylcholine/g dry weight. In the absence of cholinesterase inhibitors the release of acetylcholine from isolated villus pieces amounted to 1.3 nmol/g wet weight per 10 min corresponding to a fractional release rate of 0.13% per min. The following substances did not significantly modify the release of acetylcholine: oxotremorine (1 microM), scopolamine (1 microM), (+)-tubocurarine (30 microM), forskolin (30 microM), ouabain (10 microM), 4alpha-phorbol 12,13-didecanoate (1 microM) and tetrodotoxin (1 microM). Removal of extracellular calcium, phorbol 12,13-dibutyrate (1 microM) and colchicine (100 microM) reduced the acetylcholine release between 30% and 50%. High potassium chloride (54 mM and 108 mM) increased the acetylcholine release slightly (by about 30%). A concentration of 10 microM nicotine was ineffective, but 100 microM nicotine enhanced acetylcholine release gradually over a 50-min period without desensitization of the response. The facilitatory effect of nicotine was prevented by 30 microM (+)-tubocurarine. Inhibitors of cholinesterase (physostigmine, neostigmine; 3 microM) facilitated the efflux of acetylcholine about sixfold, and a combination of both (+)-tubocurarine (30 microM) and scopolamine (1 microM) halved the enhancing effect. In conclusion, release mechanisms differ between non-neuronal and neuronal acetylcholine. Facilitatory nicotine receptors are present which are activated by applied nicotine or by blocking cholinesterase. Thus, cholinesterase inhibitors increase assayed acetylcholine by two mechanisms, protection of hydrolysis and stimulation of facilitatory nicotine receptors.