Ignaz Wessler

Acetylcholine beyond neurons: the non-neuronal cholinergic system in humans.

Animal life is controlled by neurons and in this setting cholinergic neurons play an important role. Cholinergic neurons release ACh, which via nicotinic and muscarinic receptors (n‐ and mAChRs) mediate chemical neurotransmission, a highly integrative process. Thus, the organism responds to external and internal stimuli to maintain and optimize survival and mood. Blockade of cholinergic neurotransmission is followed by immediate death. However, cholinergic communication has been established from the beginning of life in primitive organisms such as bacteria, algae, protozoa, sponge and primitive plants and fungi, irrespective of neurons. Tubocurarine‐ and atropine‐sensitive effects are observed in plants indicating functional significance. All components of the cholinergic system (ChAT, ACh, n‐ and mAChRs, high‐affinity choline uptake, esterase) have been demonstrated in mammalian non‐neuronal cells, including those of humans. Embryonic stem cells (mice), epithelial, endothelial and immune cells synthesize ACh, which via differently expressed patterns of n‐ and mAChRs modulates cell activities to respond to internal or external stimuli. This helps to maintain and optimize cell function, such as proliferation, differentiation, formation of a physical barrier, migration, and ion and water movements. Blockade of n‐ and mACHRs on non‐innervated cells causes cellular dysfunction and/or cell death. Thus, cholinergic signalling in non‐neuronal cells is comparable to cholinergic neurotransmission. Dysfunction of the non‐neuronal cholinergic system is involved in the pathogenesis of diseases. Alterations have been detected in inflammatory processes and a pathobiologic role of non‐neuronal ACh in different diseases is discussed. The present article reviews recent findings about the non‐neuronal cholinergic system in humans.

NON-NEURONAL ACETYLCHOLINE, A LOCALLY ACTING MOLECULE, WIDELY DISTRIBUTED IN BIOLOGICAL SYSTEMS : EXPRESSION AND FUNCTION IN HUMANS

Acetylcholine acts as a neurotransmitter in the central and peripheral nervous systems in humans. However, recent experiments demonstrate a widespread expression of the cholinergic system in non-neuronal cells in humans. The synthesizing enzyme choline acetyltransferase, the signalling molecule acetylcholine, and the respective receptors (nicotinic or muscarinic) are expressed in epithelial cells (human airways, alimentary tract, epidermis). Acetylcholine is also found in mesothelial, endothelial, glial, and circulating blood cells (platelets, mononuclear cells), as well as in alveolar macrophages. The existence of non-neuronal acetylcholine explains the widespread expression of muscarinic and nicotinic receptors in cells not innervated by cholinergic neurons. Non-neuronal acetylcholine appears to be involved in the regulation of important cell functions, such as mitosis, trophic functions, automaticity, locomotion, ciliary activity, cell-cell contact, cytoskeleton, as well as barrier and immune functions. The most important tasks for the future will be to clarify the multiple biological roles of non-neuronal acetylcholine in detail and to identify pathological conditions in which this system is up- or down-regulated. This could provide the basis for the development of new therapeutic strategies to target the non-neuronal cholinergic system.

The non-neuronal cholinergic system in humans: Expression, function and pathophysiology

Acetylcholine, a prime example of a neurotransmitter, has been detected in bacteria, algae, protozoa, and primitive plants, indicating an extremely early appearance in the evolutionary process (about 3 billion years). In humans, acetylcholine and/or the synthesizing enzyme, choline acetyltransferase (ChAT), have been found in epithelial cells (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium), endothelial, muscle and immune cells (mononuclear cells, granulocytes, alveolar macrophages, mast cells). The widespread expression of non-neuronal acetylcholine is accompanied by the ubiquitous presence of cholinesterase and receptors (nicotinic, muscarinic). Thus, the non-neuronal cholinergic system and non-neuronal acetylcholine, acting as a local cellular signaling molecule, has to be discriminated from the neuronal cholinergic system and neuronal acetylcholine, acting as neurotransmitter. In the human placenta anti-ChAT immunoreactivity is found in multiple subcellular compartments like the cell membrane (microvilli, coated pits), endosomes, cytoskeleton, mitochondria and in the cell nucleus. These locations correspond with the results of experiments where possible functions of non-neuronal acetylcholine have been identified (proliferation, differentiation, organization of the cytoskeleton and the cell-cell contact, locomotion, migration, ciliary activity, immune functions). In the human placenta acetylcholine release is mediated by organic cation transporters. Thus, structural and functional differences are evident between the non-neuronal and neuronal cholinergic system. Enhanced levels of acetylcholine are detected in inflammatory diseases. In conclusion, it is time to revise the role of acetylcholine in humans. Its biological and pathobiological roles have to be elucidated in more detail and possibly, new therapeutical targets may become available.

Non-neuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man

Abstract Acetylcholine acts as a prominent transmitter in the central and peripheral nervous system. The aim of the present study was to investigate whether mammalian non-neuronal cells can synthesize and store acetylcholine. A cotton tipped applicator (Q-tip) was used to collect surface cells from airways and alimentary tract. Histological inspection indicated that rubbing of the luminal surface of human bronchi did not penetrate the basal membrane. Acetylcholine was measured by an HPLC-method using substrate-specific enzyme reactor-columns.Non-neuronal acetylcholine was found in cells covering inner and outer surfaces of rat and man. For example, acetylcholine was detected in the surface epithelium of human bronchi (33 pmol/g), mouth (female 0.7 and male 8 pmol/sample), small and large intestine (800 and 16 pmol/g, respectively), gall bladder (12 pmol/g), vagina (6 pmol/sample), skin 1000 (pmol/g) and in pulmonary pleura (5 pmol/sample). Somewhat higher amounts of acetylcholine were found in rat tracheal and intestinal epithelium and in rat skin. The synthesizing enzyme choline acetyltransferase (ChAT) was demonstrated in human surface epithelium by immunohistochemistry and by Western blot analysis. Enzymatic ChAT activity was demonstrated in isolated epithelial cells of human bronchi and small intestine (3.5 and 28 nmol/mg protein/h, respectively). Applied acetylcholine (in nM concentrations) increased, whereas inhibition of ChAT activity by bromoacetylcholine (10 μM) reduced the growth of cultured human bronchial epithelial cells. Inhibition of cell growth occurred also in the presence of atropine (1 μM) together with (±)-tubocurarine (30 μM).In conclusion, the present experiments demonstrate a widespread existence of non-neuronal acetylcholine in surface cells of man. Non-neuronal acetylcholine may act as a local signalling molecule.

Control of transmitter release from the motor nerve by presynaptic nicotinic and muscarinic autoreceptors

Until recently, release studies have failed to indicate the existence of autoreceptors on motor nerves. Ignaz Wessler now reports on a refinement of the technique - the measurement of newly synthesized [3H]acetylcholine released from the phrenic nerve - which provides clear evidence in support of release-modulating autoreceptors. Presynaptic nicotinic receptors mediate a positive feedback mechanism, can rapidly be desensitized and appear to differ in their pharmacological profile from the postsynaptic receptors. In addition, inhibitory and facilitatory muscarinic receptors appear to be involved in the presynaptic control of transmitter release from the phrenic nerve.

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

The release of acetylcholine was investigated in the human placenta villus, a useful model for the characterization of the non‐neuronal cholinergic system. Quinine, an inhibitor of organic cation transporters (OCT), reduced acetylcholine release in a reversible and concentration‐dependent manner with an IC50 value of 5 μM. The maximal effect, inhibition by 99%, occurred at a concentration of 300 μM. Procaine (100 μM), a sodium channel blocker, and vesamicol (10 μM), an inhibitor of the vesicular acetylcholine transporter, were ineffective. Corticosterone, an inhibitor of OCT subtype 1, 2 and 3 reduced acetylcholine in a concentration‐dependent manner with an IC50 value of 2 μM. 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. Long term exposure (48 and 72 h) of villus strips to anti‐sense oligonucleotides (5 μM) directed against transcription of OCT1 and OCT3 reduced the release of acetylcholine, whereas OCT2 anti‐sense oliogonucleotides were ineffective. 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.

Presynaptic nicotine receptors mediating a positive feed-back on transmitter release from the rat phrenic nerve.

SummaryThe effects of 1,1-dimethyl-4-phenylpiperazinium (DMPP) and of nicotine receptor antagonists on [3H]acetylcholine release from the rat phrenic nerve preincubated with [3H]choline were investigated in the absence and presence of cholinesterase inhibitors (presynaptic effects). Additionally, the effects of hexamethonium and tubocurarine on the muscle contraction of the indirectly stimulated diaphragm were examined (postsynaptic effects).DMPP (1–30 μM) increased (76–92%), whereas hexamethonium (0.001–1 mM) and tubocurarine (1–10 μM) decreased (52–60%) the release of [3H]acetylcholine following a train of 100 pulses at 5 Hz. The release caused by a longer train (750 pulses at 5 Hz) was only slightly affected by DMPP and tubocurarine. In the presence of neostigmine (10 μM) neither tubocurarine nor DMPP significantly modulated the evoked [3H]acetylcholine release. High DMPP concentrations (10 and 30 μM) enhanced the evoked release only when the pretreatment interval was reduced from 15 min to 20 s.Tubocurarine and hexamethonium concentration-dependently inhibited the end-organ response. Hexamethonium was 250-fold more potent on presynaptic than on postsynaptic nicotine receptors.It is concluded that the motor nerve terminals are endowed with presynaptic nicotine receptors. These autoreceptors mediate a positive feed-back mechanism that can be triggered by previously released endogenous acetylcholine. Receptor desensitization can be produced by high agonist concentrations (endogenous or exogenous agonists) and is probably one mechanism to limit the autofacilitatory process. The presynaptic receptors appear to differ in their pharmacological properties from the post-synaptic receptors.

Alternative mechanisms for tiotropium

Tiotropium is commonly used in the treatment of chronic obstructive pulmonary disease. Although largely considered to be a long-acting bronchodilator, its demonstrated efficacy in reducing the frequency of exacerbations and preliminary evidence from early studies indicating that it might slow the rate of decline in lung function suggested mechanisms of action in addition to simple bronchodilation. This hypothesis was examined in the recently published UPLIFT study and, although spirometric and other clinical benefits of tiotropium treatment extended to four years, the rate of decline in lung function did not appear to be reduced by the addition of tiotropium in this study. This article summarizes data from a variety of investigations that provide insights into possible mechanisms to account for the effects of tiotropium. The report summarizes the discussion on basic and clinical research in this field.

Inhibition of arginase in rat and rabbit alveolar macrophages by Nω-hydroxy-D,L-indospicine, effects on L-arginine utilization by nitric oxide synthase

Alveolar macrophages (AMΦ) exhibit arginase activity and may, in addition, express an inducible form of nitric oxide (NO) synthase (iNOS). Both pathways may compete for the substrate, L‐arginine. The present study tested whether two recently described potent inhibitors of liver arginase (Nω‐hydroxy‐D,L‐indospicine and 4‐hydroxyamidino‐D,L‐phenylalanine) might also inhibit arginase in AMΦ and whether inhibition of arginase might affect L‐arginine utilization by iNOS. AMΦ obtained by broncho‐alveolar lavage of rat and rabbit isolated lungs were disseminated (2.5 or 3×106 cells per well) and allowed to adhere for 2 h. Thereafter, they were either used to study [*H]‐L‐arginine uptake (37 kBq, 0.1 μM, 2 min) or cultured for 20 h in the absence or presence of bacterial lipopolysaccharide (LPS). Cultured AMΦ were incubated for 1 h with [*H]‐L‐arginine (37 kBq, 0.1 μM) and the accumulation of [*H]‐L‐citrulline (NOS activity) and [*H]‐L‐ornithine (arginase activity) was determined. During 1 h incubation of rabbit AMΦ with [*H]‐L‐arginine, no [*H]‐L‐citrulline, but significant amounts of [*H]‐L‐ornithine (150 d.p.m.×1000) were formed. Nω‐hydroxy‐D,L‐indospicine and 4‐hydroxyamidino‐D,L‐phenylalanine, present during incubation, concentration‐dependently reduced [*H]‐L‐ornithine formation (IC50: 2 and 45 μM, respectively). Nω‐hydroxy‐D,L‐indospicine (up to 100 μM) had no effect on [*H]‐L‐arginine uptake into rabbit AMΦ, whereas 4‐hydroxyamidino‐D,L‐phenylalanine caused a concentration‐dependent inhibition (IC50: 300 μM). Rat AMΦ, cultured in the absence of LPS, formed significant amounts of [*H]‐L‐citrulline and [*H]‐L‐ornithine (133 and 212 d.p.m.×1000, respectively) when incubated for 1 h with [*H]‐L‐arginine. When AMΦ had been cultured in the presence of 0.1 or 1 μg ml−1 LPS, the formation of [*H]‐L‐citrulline was enhanced by 37±8.3 and 99±12% and that of [*H]‐L‐ornithine reduced by 21±8.7 and 70±2.5%, respectively. In rat AMΦ, cultured in the absence or presence of LPS, Nω‐hydroxy‐D,L‐indospicine (10 and 30 μM) greatly reduced formation of [*H]‐L‐ornithine (by 80–95%) and this was accompanied by increased formation of [*H]‐L‐citrulline. However, only 20–30% of the [*H]‐L‐arginine not metabolized to [*H]‐L‐ornithine after inhibition of arginase was metabolized to [*H]‐L‐citrulline, when the AMΦ had been cultured in the absence of LPS (i.e. low level of iNOS). On the other hand, when the AMΦ had been cultured in the presence of LPS (i.e. high level of iNOS), all the [*H]‐L‐arginine not metabolized by the inhibited arginase was metabolized to [*H]‐L‐citrulline. In conclusion, Nω‐hydroxy‐D,L‐indospicine is a potent and specific inhibitor of arginase in AMΦ. In cells in which, in addition to arginase, iNOS is expressed, inhibition of arginase can cause a shift of L‐arginine metabolism to the NOS pathway. However, the extent of this shift appears to depend in a complex manner on the level of iNOS.

Release of [3H]acetylcholine from a modified rat phrenic nerve-hemidiaphragm preparation

SummaryTwo different preparations of the rat phrenic nerve-hemidiaphragm (whole nerve-muscle preparation, end-plate preparation) were used for studying synthesis and release of radioactive acetylcholine in the absence and presence of cholinesterase inhibitors.When the whole nerve-muscle preparation (110–180 mg) was incubated with [3H]choline, only small amounts of radioactive acetylcholine were synthesized within the tissue. Electrical nerve stimulation of the whole nerve-muscle preparation produced no increase in tritium outflow.Incubation of the end-plate preparation (16–29 mg) which was obtained after removal of most of the muscle mass led to the formation of large amounts of [3H]acetylcholine. Synthesis depended on nerve activity and increased 13-fold during a high loading stimulation (50 Hz), as compared to the synthesis at rest. In a denervated end-plate preparation the formation of [3H]acetylcholine was reduced to 4% of the control preparation. Electrical nerve stimulation of the end-plate preparation produced a release of tritium that could be attributed entirely to the release of [3H]acetylcholine. The stimulated tritium efflux was completely suppressed in a calcium-free medium or in the presence of tetrodotoxin (300 nM). Release could even be detected during a short train of 50 pulses (5 Hz) with a fractional release of about 0.04% of the [3H]acetylcholine tissue content per pulse.It is concluded that the large muscle mass interferes with nerve labelling by a reduction of the [3H]choline supply to the nerve terminals when the whole nerve-muscle preparation is used. Removal of most of the muscle fibres reduces the possibility for [3H]choline to be captured by them and then more radioactive choline can enter the end-plate region. From this end-plate preparation a calcium-dependent release of radioactive transmitter can be measured in the absence of cholinesterase inhibitors.