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.

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.

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.