A historic perspective on the current progress in elucidation of the biologic significance of non-neuronal acetylcholine.

The "5th International Symposium on Non-neuronal Acetylcholine: from bench to bedside" was held on September 27-29, 2019 in Hyatt Regency, Long Beach, CA, USA. Approximately 50 scientists from 11 countries over 6 continents participated in this meeting. The major topics included an overall biologic significance of non-neuronal acetylcholine (ACh) and the roles of the non-neuronal cholinergic systems in mucocutaneous, respiratory, digestive, immunologic, endocrine, cardiovascular, musculoskeletal and kidney diseases, and cancer. This meeting facilitated continued work to advance the fundamental science and translational aspects of the interdisciplinary studies on non-neuronal ACh. The progress made has opened a new chapter in the field of cholinergic pharmacology, and advanced our knowledge beyond regulation of individual cell- and tissue-types, defining a new paradigm of selective pharmacological regulation of vital function of practically all types of non-neuronal cells. It is now clear that the autocrine and paracrine control of non-neuronal cells by non-neuronal ACh is implemented through synergistic, additive, and reciprocal effects triggered by two different cholinergic receptor classes. Each biologic effect of ACh is determined by a unique combination of cholinergic receptors subtype expressed at each stage of cell development and differentiation. The plasticity of the non-neuronal cholinergic system helps adjust homeostasis to new environmental conditions.

Cholinergic signaling controls immune functions and promotes homeostasis.

Acetylcholine (ACh) was created by nature as one of the first signaling molecules, expressed already in procaryotes. Based on the positively charged nitrogen, ACh could initially mediate signaling in the absence of receptors. When evolution established more and more complex organisms the new emerging organs systems, like the smooth and skeletal muscle systems, energy-generating systems, sexual reproductive system, immune system and the nervous system have further optimized the cholinergic signaling machinery. Thus, it is not surprising that ACh and the cholinergic system are expressed in the vast majority of cells. Consequently, multiple common interfaces exist, for example, between the nervous and the immune system. Research of the last 20 years has unmasked these multiple regulating mechanisms mediated by cholinergic signaling and thus, the biological role of ACh has been revised. The present article summarizes new findings and describes the role of both non-neuronal and neuronal ACh in protecting the organism from external and internal health threats, in providing energy for the whole organism and for the individual cell, controling immune functions to prevent inflammatory dysbalance, and finally, the involvement in critical brain functions, such as learning and memory. All these capacities of ACh enable the organism to attain and maintain homeostasis under changing external conditions. However, the existence of identical interfaces between all these different organ systems complicates the research for new therapeutic interventions, making it essential that every effort should be undertaken to find out more specific targets to modulate cholinergic signaling in different diseases.

[Joint pilot project of the German higher federal authorities and ethics committees on implementing the EU Regulation on clinical trials]. / Gemeinsames Pilotprojekt von Bundesoberbehörden und Ethikkommissionen zur Umsetzung der EU-Verordnung zu klinischen Prüfungen.

BACKGROUND: The European Clinical Trials Regulation 536/2014 and the corresponding national legal transitions will require close cooperation between the federal higher authorities and ethics committees in the assessment of clinical trial applications involving medicinal products in humans. In preparation for this, a pilot project was launched to simulate the future processes of the regulation in line with current legal requirements and in order to give applicants, authorities and ethics committees the opportunity to familiarise themselves with the new procedures. OBJECTIVES: The aim of this paper is to examine all pilot project procedures of the first year since starting the pilot project at the end of 2015. MATERIALS AND METHODS: All 20 pilot projects completed in the first year were analysed for adherence to deadlines and results of the assessments. RESULTS: Within the time limits specified in the EU regulation, 17 of 20 procedures were fully completed. In two cases, the sponsors slightly exceeded the additional delivery period. In one case, the sponsor withdrew the application within the pilot procedure. All 20 applications were processed jointly by the federal authorities and ethics committees, and in all cases a coordinated assessment report was successfully compiled on time. All 20 applications were approved, five of which were subject to suspensive conditions. CONCLUSIONS: Compliance with the deadlines set by federal authorities and ethics committee shows that the technical infrastructures and processes established in the pilot procedure are fully functional. The cooperation between the federal higher authorities and ethics committees was very successful from the perspective of the parties involved.

Non-neuronal acetylcholine involved in reproduction in mammals and honeybees.

Bacteria and archaea synthesize acetylcholine (ACh). Thus, it can be postulated that ACh was created by nature roughly three billion years ago. Therefore, the wide expression of ACh in nature (i.e., in bacteria, archaea, unicellular organisms, plants, fungi, non-vertebrates and vertebrates and in the abundance of non-neuronal cells of mammals) is not surprising. The term non-neuronal ACh and non-neuronal cholinergic system have been introduced to describe the auto- and paracrine, that is, local regulatory actions of ACh in cells not innervated by neuronal cholinergic fibers and to communicate among themselves. In this way non-neuronal ACh binds to the nicotinic or muscarinic receptors expressed on these local and migrating cells and modulates basic cells functions such as proliferation, differentiation, migration and the transport of ions and water. The present article is focused to the effects of non-neuronal ACh linked to reproduction; data on the expression and function of the non-neuronal cholinergic system in the following topics are summarized: (i) Sperm, granulosa cells, oocytes; (ii) Auxiliary systems (ovary, oviduct, placenta); (iii) Embryonic stem cells as first step for reproduction of a new individual after fertilization; (iv) Larval food as an example of reproduction in insects (honeybees) and adverse effects of the neonicotinoids, a class of world-wide applied insecticides. The review article will show that non-neuronal ACh is substantially involved in the regulation of reproduction in mammals and also non-mammals like insects (honeybees). There is a need to learn more about this biological role of ACh. In particular, we have to consider that insecticides like the neonicotinoids, but also carbamates and organophosphorus pesticides, interfere with the non-neuronal cholinergic system thus compromising for example the breeding of honeybees. But it is possible that other species may also be adversely affected as well, a mechanism which may contribute to the observed decline in biodiversity. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Honeybees Produce Millimolar Concentrations of Non-Neuronal Acetylcholine for Breeding: Possible Adverse Effects of Neonicotinoids.

The worldwide use of neonicotinoid pesticides has caused concern on account of their involvement in the decline of bee populations, which are key pollinators in most ecosystems. Here we describe a role of non-neuronal acetylcholine (ACh) for breeding of Apis mellifera carnica and a so far unknown effect of neonicotinoids on non-target insects. Royal jelly or larval food are produced by the hypopharyngeal gland of nursing bees and contain unusually high ACh concentrations (4-8 mM). ACh is extremely well conserved in royal jelly or brood food because of the acidic pH of 4.0. This condition protects ACh from degradation thus ensuring delivery of intact ACh to larvae. Raising the pH to ≥5.5 and applying cholinesterase reduced the content of ACh substantially (by 75-90%) in larval food. When this manipulated brood was tested in artificial larval breeding experiments, the survival rate was higher with food supplemented by 100% with ACh (6 mM) than with food not supplemented with ACh. ACh release from the hypopharyngeal gland and its content in brood food declined by 80%, when honeybee colonies were exposed for 4 weeks to high concentrations of the neonicotinoids clothianidin (100 parts per billion [ppb]) or thiacloprid (8,800 ppb). Under these conditions the secretory cells of the gland were markedly damaged and brood development was severely compromised. Even field-relevant low concentrations of thiacloprid (200 ppb) or clothianidin (1 and 10 ppb) reduced ACh level in the brood food and showed initial adverse effects on brood development. Our findings indicate a hitherto unknown target of neonicotinoids to induce adverse effects on non-neuronal ACh which should be considered when re-assessing the environmental risks of these compounds. To our knowledge this is a new biological mechanism, and we suggest that, in addition to their well documented neurotoxic effects, neonicotinoids may contribute to honeybee colony losses consecutive to a reduction of the ACh content in the brood food.

Recent progress in revealing the biological and medical significance of the non-neuronal cholinergic system.

This special issue of International Immunopharmacology is the proceedings of the Fourth International Symposium on Non-neuronal Acetylcholine that was held on August 28-30, 2014 at the Justus Liebig University of Giessen in Germany. It contains original contributions of meeting participants covering the significant progress in understanding of the biological and medical significance of the non-neuronal cholinergic system extending from exciting insights into molecular mechanisms regulating this system via miRNAs over the discovery of novel cholinergic cellular signaling circuitries to clinical implications in cancer, wound healing, immunity and inflammation, cardiovascular, respiratory and other diseases.

Murine embryonic stem cell line CGR8 expresses all subtypes of muscarinic receptors and multiple nicotinic receptor subunits: Down-regulation of α4- and ß4-subunits during early differentiation.

Non-neuronal acetylcholine mediates its cellular effects via stimulation of the G-protein-coupled muscarinic receptors and the ligand-gated ion channel nicotinic receptors. The murine embryonic stem cell line CGR8 synthesizes and releases non-neuronal acetylcholine. In the present study a systematic investigation of the expression of nicotinic receptor subunits and muscarinic receptors was performed, when the stem cells were grown in the presence or absence of LIF, as the latter condition induces early differentiation. CGR8 cells expressed multiple nicotinic receptor subtypes (α3, α4, α7, α9, α10, ß1, ß2, ß3, ß4, γ, δ, ε) and muscarinic receptors (M1, M3, M4, M5); M2 was detected only in 2 out of 8 cultures. LIF removal caused a down-regulation only of the α4- and ß4-subunit. In conclusion, more or less the whole repertoire of cholinergic receptors is expressed on the murine embryonic stem cell line CGR8 for mediating cellular signaling of non-neuronal acetylcholine which acts via auto- and paracrine pathways. During early differentiation of the murine CGR8 stem cell signaling via nicotinic receptors containing α4- or ß4 subunits is reduced. Thus, the so-called neuronal α4 nicotine receptor composed of these subunits may be involved in the regulation of pluripotency in this murine stem cell line.

pH-dependent hydrolysis of acetylcholine: Consequences for non-neuronal acetylcholine.

Acetylcholine is inactivated by acetylcholinesterase and butyrylcholinesterase and thereby its cellular signalling is stopped. One distinguishing difference between the neuronal and non-neuronal cholinergic system is the high expression level of the esterase activity within the former and a considerably lower level within the latter system. Thus, any situation which limits the activity of both esterases will affect the non-neuronal cholinergic system to a much greater extent than the neuronal one. Both esterases are pH-dependent with an optimum at pH above 7, whereas at pH values below 6 particularly the specific acetylcholinesterase is more or less inactive. Thus, acetylcholine is prevented from hydrolysis at such low pH values. The pH of the surface of the human skin is around 5 and therefore non-neuronal acetylcholine released from keratinocytes can be detected in a non-invasive manner. Several clinical conditions like metabolic acidosis, inflammation, fracture-related haematomas, cardiac ischemia and malignant tumours are associated with local or systemic pH values below 7. Thus, the present article describes some consequences of an impaired inactivation of extracellular non-neuronal acetylcholine.

Effect of LIF-withdrawal on acetylcholine synthesis in the embryonic stem cell line CGR8 is not mediated by STAT3, PI3Ks or cAMP/PKA pathways.

Acetylcholine (ACh) acts as a local cellular signaling molecule and is widely expressed in nature, including mammalian cells and embryonic stem cells. The murine embryonic stem cell line CGR8 synthesizes and releases substantial amounts of ACh. Particularly during early differentiation - a period associated with multiple alterations in geno-/phenotype functions - synthesis and release of ACh are increased by 10-fold. In murine stem cells second messengers of the STAT-3, PI3K and cAMP/PKA pathways are involved in maintaining self-renewal and pluripotency. The present experiments were designed to test whether blockers of these signaling pathways enhance ACh cell content in the presence of LIF, i.e. when CGR8 is pluripotent. NSC74859, an inhibitor of STAT-3, affected neither the proliferation rate nor ACh cell content, whereas the more sensitive STAT-3 inhibitor FLLL31 reduced the proliferation rate and increased ACh cell content by about 3-fold. The PI3K inhibitor LY294002 reduced the proliferation rate but did not modify the ACh cell content, whereas the PKA inhibitor H89 produced effects comparable to FLLL31. Interestingly, in control experiments a strong inverse correlation was found between cell density and ACh cell content, which could explain the 3-fold increase in the ACh cell content observed in the presence of FLLL31 and H89. Forskolin, a PKA activator, had no effect. In conclusion, it appears unlikely that the 10-fold increase in ACh cell content induced by LIF removal, i.e. during early differentiation, is mediated by second messengers of the STAT-3, PI3K and cAMP/PKA pathways. However, the PI3K pathway appears to be involved in control of the inverse relation between cell density and ACh cell content, because this correlation was significantly attenuated in the presence of LY294002.

Upregulated acetylcholine synthesis during early differentiation in the embryonic stem cell line CGR8.

Stem cells are used to generate differentiated somatic cells including neuronal cells. Synthesis and release of acetylcholine, a neurotransmitter and widely expressed signaling molecule, were investigated in the murine embryonic stem cell line CGR8 during early differentiation, i.e. in the presence of leukemia inhibitory factor (LIF) to maintain pluripotency and in the absence of LIF to induce early differentiation. CGR8 cells express choline acetyltransferase (ChAT) as demonstrated by measurement of enzyme activity and substantial inhibition by bromoacetylcholine. Pluripotent CGR8 cells showed a ChAT activity of 250 pmol acetylcholine/mg/h, contained 1.1 pmol acetylcholine/106 cells and released about 12.00 pmol acetylcholine/1 x 106 cells/6 h. Removal of LIF induced early differentiation as evidenced by reduced transcription factors Oct-4 and Nanog and a substantial slowing of the proliferation rate. Under this condition acetylcholine synthesis increased to 1640 pmol/mg/h; related to the pluripotent state the content of acetylcholine increased 10-fold and the release to about 32 pmol acetylcholine/1 x 106 cells/6 h. Enzyme kinetic analysis showed a significant increase of the K(m) for the precursor acetyl-CoA and of V(max) without a change of the K(m) for the precursor choline. In conclusion, early differentiation of the stem cell line CGR8 is associated with a substantial increase in ChAT activity and acetylcholine release.

Cholinergic modulation of epithelial integrity in the proximal colon of pigs.

BACKGROUND: Within the gut, acetylcholine (ACh) is synthesised by enteric neurons, as well as by 'non-neuronal' epithelial cells. In studies of non-intestinal epithelia, ACh was involved in the generation of an intact epithelial barrier. In the present study, primary cultured porcine colonocytes were used to determine whether treatment with exogenous ACh or expression of endogenous epithelium-derived ACh may modulate epithelial tightness in the gastrointestinal tract. METHODS: Piglet colonocytes were cultured on filter membranes for 8 days. The tightness of the growing epithelial cell layer was evaluated by measuring transepithelial electrical resistance (TEER). To determine whether ACh modulates the tightness of the cell layer, cells were treated with cholinergic, muscarinic and/or nicotinic agonists and antagonists. Choline acetyltransferase (ChAT), cholinergic receptors and ACh were determined by immunohistochemistry, RT-PCR and HPLC, respectively. RESULTS: Application of the cholinergic agonist carbachol (10 µm) and the muscarinic agonist oxotremorine (10 µM) resulted in significantly higher TEER values compared to controls. The effect was completely inhibited by the muscarinic antagonist atropine. Application of atropine alone (without any agonist) led to significantly lower TEER values compared to controls. Synthesis of ACh by epithelial cells was proven by detection of muscarinic and nicotinic receptor mRNAs, immunohistochemical detection of ChAT and detection of ACh by HPLC. CONCLUSION: ACh is strongly involved in the regulation of epithelial tightness in the proximal colon of pigs via muscarinic pathways. Non-neuronal ACh seems to be of particular importance for epithelial cells forming a tight barrier.

Subcellular distribution of choline acetyltransferase by immunogold electron microscopy in non-neuronal cells: placenta, airways and murine embryonic stem cells.

AIMS: Acetylcholine is synthesized in more or less all mammalian cells. However, little is known about the subcellular location of acetylcholine synthesis. Therefore, in the present experiments the subcellular location of the synthesizing enzyme choline acetyltransferase (ChAT) was investigated by anti-ChAT immunogold electron microscopy in human placenta and airways as well as in a murine embryonic stem cell line (CGR8 cell line). MAIN METHODS: Human tissue was obtained as so-called surplus tissue (after delivery/surgical removal because of lung tumor); the CGR8 stem cell line was cultured under standard conditions. For human tissue a monoclonal mouse anti-ChAT antibody (ab) was used and for the CGR8 cell line a polyclonal goat anti-ChAT ab. Immunogold electron microscopy was applied to identify the subcellular location of ChAT. KEY FINDINGS: In trophoblast cells (placenta) specific anti-ChAT immunogold deposition was found within the cell membrane, microvilli, and caveolae but also within the cytosol, for example associated with intermediate filaments. In addition, immunogold deposition was identified within mitochondria and the nuclear membrane. In airway epithelial cells anti-ChAT immunogold was found particularly within the apical cell membrane, cilia, submucosa, cytosol and nuclear membrane. Likewise alveolar macrophages showed positive anti-ChAT immunogold within the nucleus, nuclear membrane and granula. Also in the CGR8 cell line positive anti-ChAT immunogold was identified within the cell nucleus and cytosol. SIGNIFICANCE: The present experiments demonstrate a wide subcellular distribution of ChAT with particular preference of the cell membrane in human epithelial cells.

Release of acetylcholine from murine embryonic stem cells: effect of nicotinic and muscarinic receptors and blockade of organic cation transporter.

AIMS: The non-neuronal cholinergic system is widely expressed in nature. The present experiments were performed to characterize the non-neuronal cholinergic system in murine embryonic stem cells (CGR8 cell line). MAIN METHODS: CGR8 cells were cultured in gelatinized flasks with Glasgow's buffered minimal essential medium (Gibco, Germany). Acetylcholine was measured by HPLC combined with bioreactor and electrochemical detection. KEY FINDINGS: CGR8 cells contained 1.08±0.12 pmol acetylcholine/10(6) cells (n=7) which was reduced to 0.50±0.06 pmol/10(6) cells (n=6; p<0.05) in the presence (4h) of 30µM bromoacetylcholine to block choline acetyltransferase. A time-dependent release of acetylcholine into the incubation medium was demonstrated, when cholinesterase activity was blocked by 10 µM physostigmine, with 97±13, 180±15 and 216±14 pmol being released from 65×10(6) cells after incubation periods of 2, 4 and 6h, respectively. The cumulative release corresponds to a fractional release rate of 2%/min. Blockade of nicotine or muscarine receptors did not significantly modulate the release of acetylcholine which was substantially reduced by 300 µM quinine (inhibitor of organic cation transporters). This inhibition showed considerable fading over the incubation period, indicating additional release mechanisms activated upon inhibition of organic cation transporters. SIGNIFICANCE: Murine embryonic stem cells contain and release significant amounts of acetylcholine. The high fractional release rate and the compensation for blocked organic cation transporters indicate that non-neuronal acetylcholine may play a functional role in the homeostasis of murine embryonic stem cells.

Blockade of nicotinic and muscarinic receptors facilitates spontaneous migration of human peripheral granulocytes: failure in cystic fibrosis.

AIMS: Circulating leucocytes express muscarinic (m) and nicotinic (n) receptors and synthesize acetylcholine (ACh) regulating various cell functions. Leucocytes from patients with cystic fibrosis contain less ACh; therefore it was tested whether the regulation of cellular functions like migration differed from healthy volunteers. MAIN METHODS: Peripheral blood (10-20 ml) was used, leucocytes were isolated by Ficoll® gradient and the commercial MIGRATEST® combined with flow cytometric analysis was applied (pore size 3 µm). KEY FINDINGS: In the absence of test substances 4900±1800 (n=10) leucocytes migrated within a time period of 2 h. In the presence of tubocurarine (TC, 30 µM) the cell number increased to 7500±2700 [n=10] corresponding to an increase of 162±20% (mean of individual experiments; p<0.02). Atropine (1 µM) was not effective (120±17%, n=7). Simultaneous application of atropine and TC produced a slightly higher effect than TC alone (185±23%; n=8); a 10-fold increase of TC and atropine resulted to a somewhat stronger effect (248±39%; n=8). When migration time was reduced to 30 min or the chemoattractant fMLP (0.05 µM) present neither atropine nor TC affected migration. Granulocytes isolated from patients with cystic fibrosis did not respond (2h migration) to 30 µM TC (control: 5180±1400 cells [n=10]; TC: 5800±1400 [n=10]). Also in the presence of atropine (1 µM) and TC (30 µM) a significant effect was not detected (5800±1300 [n=10]). SIGNIFICANCE: Auto-paracrine acetylcholine limits the migration of unstimulated peripheral granulocytes. This effect is impaired in cystic fibrosis most likely because of a reduced endogenous cholinergic tone.

Activation of muscarinic receptors by non-neuronal acetylcholine.

The biological role of acetylcholine and the cholinergic system is revisited based particularly on scientific research early and late in the last century. On the one hand, acetylcholine represents the classical neurotransmitter, whereas on the other hand, acetylcholine and the pivotal components of the cholinergic system (high-affinity choline uptake, choline acetyltransferase and its end product acetylcholine, muscarinic and nicotinic receptors and esterase) are expressed by more or less all mammalian cells, i.e. by the majority of cells not innervated by neurons at all. Moreover, it has been demonstrated that acetylcholine and "cholinergic receptors" are expressed in non-neuronal organisms such as plants and protists. Acetylcholine is even synthesized by bacteria and algae representing an extremely old signalling molecule on the evolutionary timescale. The following article summarizes examples, in which non-neuronal acetylcholine is released from primitive organisms as well as from mammalian non-neuronal cells and binds to muscarinic receptors to modulate/regulate phenotypic cell functions via auto-/paracrine pathways. The examples demonstrate that non-neuronal acetylcholine and the non-neuronal cholinergic system are vital for various types of cells such as epithelial, endothelial and immune cells.

Acetylcholine and chronic vasculopathy in rat renal allografts.

BACKGROUND: Chronic allograft vasculopathy (CAV) is an important aspect of chronic allograft injury, which limits the long-term success of renal transplantation. The pathogenesis of CAV is ill defined, and no effective therapies exist. Acute rejection episodes are a major risk factor for CAV. Recently, we demonstrated that leukocytes, which strongly accumulate in allograft blood vessels during fatal acute rejection, produce acetylcholine (ACh), which has the potential to provoke CAV. Herein, we test the hypothesis that ACh is also produced by leukocytes during the development of CAV. METHODS: Kidneys were transplanted in the Fischer 344 to Lewis rat strain combination, an established experimental model for CAV. Isografts were performed in Lewis rats. The capacity of intravascular graft leukocytes to synthesize ACh was investigated during reversible acute rejection on day 9 posttransplantation and during the process of vascular remodeling on day 42. Furthermore, allograft recipients were treated with rivastigmine, which blocks enzymatic degradation of ACh. RESULTS: The protein expression of the high-affinity choline transporter-1 and choline acetyltransferase was increased in leukocytes from allografts on day 9 and 42 posttransplantation. In addition, leukocytes accumulating in the lumina of allograft blood vessels were by far more numerous compared with isografts. In line with our hypothesis, ACh itself was detected by high-pressure liquid chromatography in graft leukocytes but not in leukocytes from untreated kidneys. Treatment with rivastigmine drastically exacerbated CAV compared with placebo. CONCLUSION: We suggest that endogenous ACh contributes to the pathogenesis of CAV and may be a promising target for novel therapies preventing CAV.

Pivotal Advance: Up-regulation of acetylcholine synthesis and paracrine cholinergic signaling in intravascular transplant leukocytes during rejection of rat renal allografts.

During acute rejection, large numbers of leukocytes accumulate in the blood vessels of experimental renal allografts. About 70% of them are activated, cytotoxic monocytes that appear to be involved in allograft destruction. ACh exerts anti-inflammatory effects upon monocytes/macrophages and has been proposed to be a key player in neuroimmunological interactions. Its short half-life, however, makes it unlikely that neuronal ACh affects blood leukocytes. Renal transplantation was performed in the allogeneic DA to LEW and in the isogeneic LEW to LEW rat strain combination. Intravascular leukocytes were harvested after 4 days, and the expression of CHT1, cChAT, pChAT, and nAChR subunits was investigated by RT-PCR, immunoblotting, and immunohistochemistry. Monocytes were identified by double-labeling with ED1-antibody, directed to a CD68-like antigen. ACh content was measured by HPLC. [Ca(2+)](i) was monitored by Fura-2. Intravascular graft leukocytes express CHT1 and cChAT mRNA and protein and pChAT protein. Their expression is strongly up-regulated in vivo during acute allograft rejection. Immunohistochemistry revealed CHT1, cChAT, and pChAT protein in ED1-positive monocytes. The ACh content of allograft intravascular leukocytes was sixfold higher than that of isografts. Intravascular leukocytes express nAChR subunits, and an ATP-induced increase in [Ca(2+)](i) was augmented in vitro by a nAChR inhibitor in allograft but not isograft leukocytes. Intravascular graft leukocytes, among them monocytes, up-regulate non-neuronal ACh synthesis and develop auto-/paracrine cholinergic attenuation of ATP signaling during acute allograft rejection.