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.

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 and choline acetyltransferase in oviductal epithelial cells of cyclic and pregnant pigs.

Certain female reproductive tissues are known to express the non-neuronal cholinergic system. Using different experimental approaches, we tested the hypothesis that acetylcholine (ACh) in the porcine oviduct may also be derived from non-neuronal structures. Immunohistochemistry was performed to detect acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) in different segments of the oviduct of cyclic and pregnant sows. Immunohistochemical experiments revealed strong immunoexpression of ChAT in the entire oviductal epithelium at metoestrus. Thereby, a particular pronounced staining was found in the supranuclear region of almost all epithelial cells. Immunostaining of ChAT decreased markedly during dioestrus and prooestrus stages, respectively. At prooestrus, ChAT immunoreactivity was confined to ciliated cells. Furthermore, we found elevated level of staining intensity of ChAT in the pregnant oviduct at day 13. Using the same ChAT antibody for Western blot analyses, we detected immunoreactive bands of MW 69,000 and 46,000 mainly in ampulla, while MW 58,000 and 30,000 forms were present mainly in infundibulum and isthmus. Furthermore ACh was detected by HPLC and fluorimetric methods in oviductal epithelium. In conclusion, we show expression of ChAT in oviductal epithelial cells at different stages of the oestrus cycle and pregnancy, indicating that these cells can synthesize ACh in a cycle-dependent manner. These results suggest as yet unexplored roles of epithelial ACh in the oviduct.

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.

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.

Expression of muscarinic receptor types in the primate ovary and evidence for nonneuronal acetylcholine synthesis.

The presence of muscarinic receptors (MR) in the ovary of different species has been recognized, but the identity of these receptors as well as ovarian sources of their natural ligand, acetylcholine (ACh), have not been determined. Because luteinized human granulosa cells (GC) in culture express functional MR, we have determined whether the group of the related MR subtypes, M1R, M3R, and M5R, are present in vivo in human and rhesus monkey ovaries. To this end, ribonucleic acids (RNAs) of different human and monkey ovaries as well as RNAs from human GC and monkey oocytes were reverse transcribed and subjected to PCR amplification, followed by sequencing of the amplified complementary DNAs. Results obtained showed that M1R, M3R, and M5R messenger RNAs are present in adult human and monkey ovaries; oocytes express exclusively the M3R subtype, whereas GC express M1R and M5R. To determine the ovarian source(s) of the natural ligand of these ACh receptors, we attempted to localize the enzyme responsible for its synthesis with the help of a monoclonal antibody recognizing choline acetyltransferase for immunohistochemistry. In neither human nor monkey sections did we detect immunoreactive choline acetyltransferase-positive fibers or nerve cells, but, surprisingly, GC of antral follicles showed prominent staining. To determine whether GC can produce ACh, human cultured GC derived from preovulatory follicles were analyzed using a high pressure liquid chromatography technique. The results showed that these cells contained ACh in concentrations ranging from 4.2-11.5 pmol/10(6) cells. Samples of a rat granulosa cell line likewise contained ACh. Thus, the ovary contains multiple MR, and GC of antral follicles are able to synthesize ACh, the ligand of MR. We propose that ACh may serve as an as yet unrecognized factor involved in the complex regulation of ovarian function in the primate, e.g. regulation of cell proliferation or progesterone production.

Differential release of [3H]acetylcholine from the rat phrenic nerve-hemidiaphragm preparation by electrical nerve stimulation and by high potassium.

Neuronal transmitter stores of the phrenic nerve were labelled under different conditions. Subsequently, transmitter release evoked by electrical nerve stimulation and by a high potassium-low sodium solution was studied. Incubation of the end-plate preparation with [3H]choline at rest led to the synthesis of [3H]acetylcholine which could not be released by electrical nerve stimulation but it was released by high potassium-low sodium solution, independent of the presence of extracellular calcium. When the end-plate preparation was labelled during stimulation at 1 Hz, prolonged periods of electrical nerve stimulation released 83% of the total releasable [3H]transmitter pool in a completely calcium-dependent manner. After exhaustion of the electrically releasable pool, high potassium-low sodium solution still caused a significant outflow. Without a preceding exhaustion of the [3H]acetylcholine pool, high potassium-low sodium solution released a similar amount in the absence of extracellular calcium or after pretreatment with the intracellular calcium chelating substance, Quin-2. When evoked transmitter release was studied at different temperatures (36, 26 and 16 degrees C) Q 10 values of 1.6 and 1.0 were found for the release caused by electrical nerve stimulation and high potassium-low sodium solution (calcium-independent effect), respectively. After labelling during a short interval (2 min) but at a high stimulation rate (50 Hz), only 72% of the releasable [3H]transmitter could be released by electrical nerve stimulation, whereas the outflow due to the calcium-independent effect of high potassium-low sodium solution increased from 17 (labelling during stimulation at 1 Hz) to 28%. It is suggested that the calcium-independent effect of high potassium-low sodium solution reflects the release of acetylcholine from the cytoplasmic compartment, as this outflow occurred after labelling at rest and increased when cytoplasmic synthesis was enhanced by a high loading stimulation. In contrast to high potassium-low sodium solution, propagated nerve activity cannot release acetylcholine synthesized at rest (presumed to be cytoplasmic), but only [3H]acetylcholine synthesized during quantal release (presumed to be vesicular). The absolute requirement of extracellular calcium for electrically stimulated release suggests an exocytotic release mechanism. The low Q 10 value of 1.6 does not fit into the concept of a carrier- or channel-operated release mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Beta-adrenoceptor stimulation enhances transmitter output from the rat phrenic nerve.

1. Neurally-evoked output of newly synthesized [3H]-acetylcholine from the rat phrenic nerve was measured in the absence of cholinesterase inhibitors. 2. Noradrenaline and isoprenaline enhanced neurally-evoked transmitter output markedly. Moreover, immediately after the application of noradrenaline the basal tritium efflux increased significantly. 3. Pretreatment with propranolol (0.1 mumol l-1) or atenolol (0.3 mumol l-1) completely prevented the stimulatory effect of noradrenaline and isoprenaline on evoked transmitter output. 4. The facilitatory effect of isoprenaline declined, when the exposure time was increased. This observation supports the assumption that beta-adrenoceptors can be desensitized or inactivated during continued exposure to agonists. 5. It was shown for the first time that stimulation of beta-adrenoceptors enhances transmitter output from the motor nerve. It is proposed that these beta-adrenoceptors are of the beta 1-subtype and are localized on the endings of motor nerves. Circulating catecholamines may facilitate neuromuscular transmission by stimulation of presynaptic beta-adrenoceptors.

Adrenoceptor- and cholinoceptor-mediated mechanisms in the regulation of 5-hydroxytryptamine release from isolated tracheae of newborn rabbits.

1. Isolated tracheae of newborn rabbits were incubated in vitro and the outflow of 5-hydroxytryptamine (5-HT) was determined by h.p.l.c. with electrochemical detection. Evidence has previously been provided that this 5-HT outflow derives from neuroendocrine epithelial (NEE) cells of the airway mucosa. 2. Phenylephrine (1, 10 and 30 microM) enhanced the outflow of 5-HT by 80, 290 and 205%, respectively. 5-HT outflow evoked by 10 microM phenylephrine was not affected by the presence of the neurotoxin tetrodotoxin (1 microM). 3. Rauwolscine, ARC 239 (an alpha(2B)-adrenoceptor preferring antagonist), yohimbine and prazosin antagonized the effect of 10 microM phenylephrine in a concentration-dependent manner with IC50 values of 150, 295, 300 and 1,700 nM, respectively. Comparison of the ratios (between all antagonists) of the present IC50 values with the corresponding ratios of Ki values obtained in binding studies for the alpha(2A)-, alpha(2B)-, alpha(2C)- and alpha(2D)-adrenoceptor subtypes strongly suggests the involvement of an alpha(2B)-receptor. 4. 5-HT outflow evoked by 10 microM phenylephrine was inhibited by 65% in the presence of 1 microM forskolin and abolished in the presence of 10 microM forskolin. 5. 5-HT outflow evoked by 10 microM phenylephrine was inhibited by about 45 and 70% in the presence of 0.1 and 1 microM isoprenaline, respectively. The inhibitory effect of 1 microM isoprenaline was only marginally antagonized by 1 microM, but blocked by 10 microM propranolol. 6. 5-HT outflow was not affected by the muscarine receptor agonist oxotremorine (10 microM), but was enhanced by 175% by 100 microM nicotine. The effect of nicotine was blocked by 100 microM hexamethonium and prevented by 1 microM tetrodotoxin or 1 microM yohimbine. 7. In conclusion, 5-HT release from NEE cells of the rabbit trachea is stimulated via alpha-adrenoceptors most likely of the alpha(2B)-subtype localized directly at the NEE cells. Activation of beta-adrenoceptors as well as direct activation of adenylyl cyclase by forskolin exert inhibitory effects on 5-HT release. Activation of nicotinic, but not of muscarinic receptors, also evokes the release of 5-HT. However, the effect of nicotine appears to be mediated indirectly via the release of noradrenaline.

Endogenous noradrenaline release from guinea-pig isolated trachea is inhibited by activation of M2 receptors.

Overflow of endogenous noradrenaline (NA) from guinea-pig isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses). The muscarinic receptor agonist oxotremorine inhibited the evoked overflow of NA in a concentration-dependent manner (EC50 84 nM). Methoctramine, pirenzepine and p-fluoro-hexahydrosiladiphenidol (each 1 microM) shifted the concentration-response curves of oxotremorine to the right with apparent pA2 values of 7.60, 6.74 and 6.18, respectively. It is concluded that sympathetic nerve terminals in the guinea-pig trachea are endowed with inhibitory muscarinic M2 receptors.

Mucosa-dependent muscarinic liberation of prostaglandins from rat isolated trachea.

1. The present study examined whether cholinoceptor stimulation modulates the release of arachidonic acid-derived mediators from rat isolate tracheae. 2. Tracheae were preincubated with [3H]-arachidonic acid and the outflow of 3H-compounds was determined. Acetylcholine and the muscarinic agonist, carbachol but not nicotine, increased the rate of tritium outflow maximally by about 30%. The M3 receptor-preferring antagonist rho-fluoro-hexahydrosiladiphenidol was more effective than pirenzepine and methoctramine in antagonizing the effect of acetylcholine. 3. High performance liquid chromatography analysis (methanol gradient) of the released 3H-compounds showed that one peak, co-eluting with [14C]-prostaglandin E2([14C]-PGE2) and [3H]-PGD2, was enhanced almost 10 fold following muscarinic receptor activation, whereas the outflow of [3H]-arachidonic acid remained unaffected. 4. Using an acetonitril gradient separation it was shown that [3H]-PGE2, [3H]-PGD2 and [3H]-PGF2alpha are released spontaneously, but [3H]-PGE2 represented the major fraction of 3H-prostaglandins. Acetylcholine enhanced the release of all three 3H-prostaglandins, but the effect on PGE2 was most pronounced and most consistent. 5. After removal of the mucosa the muscarinic effect of acetylcholine on total tritium and on that of the 3H-prostaglandins ([3H]-PGE2/PGD2 peak) was abolished. 6. Acetylcholine also enhanced the outflow of radioimmunologically determined PGE2 in a mucosa-dependent manner. 7. After inhibition of cyclo-oxygenase by 3 microM indomethacin, the outflow of 3H-prostaglandins was reduced to almost undetectable levels and acetylcholine evoked a marked release [3H]-arachidonic acid. The phospholipase A2 inhibitor, quinacrine (up to 100 microM) also blocked the effect of acetylcholine on the outflow of 3H-prostaglandins, but this was not followed by a compensatory increase in the outflow of [3H]-arachidonic acid. 8. In conclusion, activation of muscarinic receptors which have characteristics of the M3 subtype can evoke release of prostaglandins from the airway mucosa.

Prostanoid receptors of the EP3 subtype mediate inhibition of evoked [3H]acetylcholine release from isolated human bronchi.

1. The release of neuronal [3H]acetylcholine (ACh) from isolated human bronchi after labelling with [3H]choline was measured to investigate the effects of prostanoids. 2. A first period of electrical field stimulation (S1) caused a [3H]ACh release of 320+/-70 and 200+/-40 Becquerel (Bq) g(-1) in epithelium-denuded and epithelium-containing bronchi respectively (P>0.05). Subsequent periods of electrical stimulation (Sn, n=2, 3, and 4) released less [3H]ACh, i.e. decreasing Sn/ S1 values were obtained (0.76+/-0.09, 0.68+/-0.07 and 0.40+/-0.04, respectively). 3. Cumulative concentrations (1-1000 nM) of EP-receptor agonists like prostaglandin E2, nocloprost, and sulprostone (EP1 and EP3 selective) inhibited evoked [3H]ACh release in a concentration dependent manner with IC50 values between 4- 14 nM and maximal inhibition of about 70%. 4. The inhibition of evoked [3H]ACh release by prostaglandin E2, nocloprost and sulprostone was not affected by the DP-, EP1- and EP2-receptor antagonist AH6809 at a concentration of 3 microM, i.e. a 3-30 times greater concentration than its affinity (pA2 values) at the respective receptors. 5. Circaprost (IP-receptor agonist; 1-100 nM), iloprost (IP- and EP1-receptor agonist; 10-1000 nM) and U-46619 (TP-receptor agonist; 100-1000 nM) did not significantly affect [3H]ACh release. 6. Blockade of cyclooxygenase by 3 microM indomethacin did not significantly modulate evoked [3H]ACh release in epithelium-containing and epithelium-denuded bronchi. Likewise, the combined cyclo- and lipoxygenase inhibitor BW-755C (20 microM) did not affect evoked [3H]ACh release. 7. In conclusion, applied prostanoids appear to inhibit [3H]ACh release in epithelium-denuded human bronchi under the present in vitro conditions, most likely via prejunctional prostanoid receptors of the EP3 subtype.

Characterization of endogenous noradrenaline release from intact and epithelium-denuded rat isolated trachea.

1. Overflow of endogenous noradrenaline (NA) from the in vitro incubated rat trachea evoked by two periods of electrical field stimulation (S1, S2 at 3 or 15 Hz) or by high potassium (60 mM) was determined by high performance liquid chromatography (h.p.l.c.) with electrochemical detection. 2. In the presence of the neuronal uptake inhibitor desipramine, the alpha 2-adrenoceptor antagonist, yohimbine, enhanced the overflow of NA evoked by stimulation at 3 Hz by about 100% suggesting the presence of presynaptic inhibitory autoreceptors on the sympathetic nerves innervating the trachea. 3. When desipramine and yohimbine were present throughout the experiments, the overflow of NA evoked by the second period of electrical stimulation (S2) was significantly smaller than that evoked by the first (S1). This decline of overflow was prevented when the NA precursor, tyrosine, was additionally present throughout the experiments. 4. After removal of the epithelium, the tissue content of NA was reduced by about 30%, suggesting that part of the NA may be present and released within the epithelium. However, the overflow of NA evoked by stimulation at 3 Hz or 15 Hz was reduced by 70-80%, indicating that the epithelium may additionally exert a permissive role on the release of NA within the airways, possibly by suppressing inhibitory factors. 5. Stimulation by high potassium (60 mM for 10 min) caused a large overflow of NA (about 45% of the tissue NA), both from epithelium-free and epithelium-denuded tracheae. Thus the 'endogenous inhibition' of NA release after removal of the epithelium is surmountable when a high potassium stimulus is applied.

Beta-adrenoceptors mediate inhibition of [3H]-acetylcholine release from the isolated rat and guinea-pig trachea: role of the airway mucosa and prostaglandins.

1. Rat or guinea pig isolated tracheae were labelled with [3H]-choline to measure evoked tritium outflow, which reflects neuronal release of [3H]-acetylcholine. Tritium outflow was evoked either by electrical stimulation of the extrinsic vagal nerve (rat tracheae) or by 27 mM potassium (guinea pig tracheae). 2. In rat tracheae isoprenaline (0.01, 0.1 microM) inhibited evoked [3H]-acetylcholine release, whereas beta 2-adrenoceptor-selective agonists (fenoterol, formoterol, salbutamol) were ineffective. 3. The inhibitory effect of isoprenaline was abolished under the following conditions: (i) presence of propranolol (1 microM) or of the beta 1-selective antagonist CGP 20712 A (0.1 microM); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 microM indomethacin. 4. In rat isolated tracheae prelabelled with [3H]-arachidonic acid, isoprenaline (0.1 microM) but not formoterol (0.01 microM) enhanced the outflow of [3H]-prostaglandins (PGD2, PGE2). This effect was blocked by 0.1 microM CGP 20712 A. 5. In guinea pig tracheae electrical stimulation of the extrinsic vagal nerve did not cause a constant release of [3H]-acetylcholine, but 27 mM potassium elicited a reproducible release of [3H]-acetylcholine. In this species both isoprenaline (0.1 microM) and formoterol (0.01 microM) inhibited evoked [3H]-acetylcholine release. Inhibition was abolished under the following conditions: (i) presence of propranolol (1 microM) or of the beta 2-selective antagonist ICI 118551 (0.3 microM); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 microM indomethacin. 6. In conclusion, the present experiments have demonstrated that activation of beta-adrenoceptors localized in the mucosa mediates inhibition of [3H]-acetylcholine release from the neuroeffector junctions of the pulmonary, parasympathetic nerves most probably by the liberation of inhibitory prostaglandins from the airway mucosa. The adrenoceptor subtype involved differs in rat (beta 1 subtype) and guinea pig (beta 2 subtype) airways.

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

1. Alveolar macrophages (AM phi) 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 (N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine) might also inhibit arginase in AM phi and whether inhibition of arginase might affect L-arginine utilization by iNOS. 2. AM phi obtained by broncho-alveolar lavage of rat and rabbit isolated lungs were disseminated (2.5 or 3 x 10(6) cells per well) and allowed to adhere for 2 h. Thereafter, they were either used to study [3H]-L-arginine uptake (37 kBq, 0.1 microM, 2 min) or cultured for 20 h in the absence or presence of bacterial lipopolysaccharide (LPS). Cultured AM phi were incubated for 1 h with [3H]-L-arginine (37 kBq, 0.1 microM) and the accumulation of [3H]-L-citrulline (NOS activity) and [3H]-L-ornithine (arginase activity) was determined. 3. During 1 h incubation of rabbit AM phi with [3H]-L-arginine, no [3H]-L-citrulline, but significant amounts of [3H]-L-ornithine (150 d.p.m x 1000) were formed. N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine, present during incubation, concentration-dependently reduced [3H]-L-ornithine formation (IC50: 2 and 45 microM, respectively). 4. N omega-hydroxy-D,L-indospicine (up to 100 microM) had no effect on [3H]-L-arginine uptake into rabbit AM phi, whereas 4-hydroxyamidino-D,L-phenylalanine caused a concentration-dependent inhibition (IC50: 300 microM). 5. Rat AM phi, cultured in the absence of LPS, formed significant amounts of [3H]-L-citrulline and [3H]-L-ornithine (133 and 212 d.p.m x 1000, respectively) when incubated for 1 h with [3H]-L-arginine. When AM phi had been cultured in the presence of 0.1 or 1 microgram ml-1 LPS, the formation of [3H]-L-citrulline was enhanced by 37 +/- 8.3 and 99 +/- 12% and that of [3H]-L-ornithine reduced by 21 +/- 8.7 and 70 +/- 2.5%, respectively. 6. In rat AM phi, cultured in the absence or presence of LPS, N omega-hydroxy-D,L-indospicine (10 and 30 microM) greatly reduced formation of [3H]-L-ornithine (by 80-95%) and this was accompanied by increased formation of [3H]-L-citrulline. However, only 20-30% of the [3H]-L-arginine not metabolized to [3H]-L-ornithine after inhibition of arginase was metabolized to [3H]-L-citrulline, when the AM phi had been cultured in the absence of LPS (i.e. low level of iNOS). On the other hand, when the AM phi had been cultured in the presence of LPS (i.e. high level of iNOS), all the [3H]-L-arginine not metabolized by the inhibited arginase was metabolized to [3H]-L-citrulline. 7. In conclusion, N omega-hydroxy-D,L-indospicine is a potent and specific inhibitor of arginase in AM phi. 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 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.

delta 1-Opioid receptor-mediated control of acetylcholine (ACh) release in human neocortex slices.

In slices of human neocortex, prelabelled with [3H]-choline, the release of [3H]-acetylcholine reflects the evoked release of endogenous acetylcholine which was elicited by the same electrical stimulation paradigm. [3H]-Acetylcholine release was depressed by the delta-opioid receptor agonist D-Pen2-D-Pen5-enkephalin. When the nerve endings were depolarized by elevating extracellular potassium the evoked [3H]-acetylcholine release was similarly depressed by D-Pen2-D-Pen5-enkephalin in the absence, but not in the presence, of tetrodotoxin which blocks action potential propagation. Therefore, the delta-opioid receptor inhibiting [3H]-acetylcholine release should not be located to cholinergic nerve terminals, but rather to interneurons. The somatostatin2 receptor partial agonist octreotide per se did not influence action potential-evoked [3H]-acetylcholine release, but prevented the inhibition of release of [3H]-acetylcholine by D-Pen2-D-Pen5-enkephalin. Similarly, the delta 1-opioid receptor antagonist 7-benzylidenenaltrexon per se did not influence [3H]-acetylcholine release, but prevented of the inhibition of release by D-Pen2-D-Pen5-enkephalin. From the present findings we conclude: (1) The evoked release of [3H]-acetylcholine from human neocortex slices reflects the release of endogenous acetylcholine. (2) It is inhibited in an indirect manner by opioid receptors of the delta 1-subtype, which (3) are not localized on cholinergic axon terminals but on soma and dendrites of somatostatin-containing interneurons, where they inhibit somatostatin release. (4) These interneurons innervate cholinergic nerve endings in the human neocortex and appear to facilitate acetylcholine release via somatostatin2 receptors.

Increase by tubocurarine of [3H]acetylcholine release from the rat phrenic nerve after desensitization of nicotine receptors.

Tubocurarine reduced the neurally evoked [3H]acetylcholine release in the absence of nicotine, indicating positive nicotinic feedback. Tubocurarine, however, enhanced [3H]acetylcholine release after pretreatment with nicotine, a condition which causes desensitization of nicotine autoreceptors. After a nicotine-free period (15-60 min) during which the facilitatory autoreceptors could recover from desensitization, tubocurarine again reduced [3H]acetylcholine release. Protection of facilitatory nicotine autoreceptors from desensitization could explain the tubocurarine-induced increase in transmitter release.

[3H]acetylcholine release from the phrenic nerve is increased or decreased by activation or desensitization of nicotine receptors.

Nicotinic agonists (nicotine, 1,1-dimethyl-4-phenylpiperazinium (DMPP) increased [3H]acetylcholine release when the pre-exposure time was short (20 s). This effect was antagonized by pretreatment with d-tubocurarine (d-TC), which indicates a receptor-mediated effect. After a longer pre-exposure time (3 min) 10 microM nicotine significantly decreased the evoked [3H]acetylcholine release, probably because desensitization of the nicotine autoreceptors had abolished the nicotinic autofacilitation of transmitter release.

Stimulation of alpha 1-adrenoceptors increases electrically evoked [3H]acetylcholine release from the rat phrenic nerve.

The modulation by sympathomimetic amines of the electrically evoked [3H]acetylcholine release from the motor nerve was investigated. Phenylephrine (10 mumol/l), alpha-methylnoradrenaline (10 mumol/l) and adrenaline (1 mumol/l) enhanced the electrically evoked [3H]acetylcholine release from the rat phrenic nerve. The enhancing effect of phenylephrine was completely prevented by low concentrations of prazosin (0.1 or 0.01 mumol/l) whereas a high concentration of yohimbine (1 mumol/l) was necessary to abolish the effect of phenylephrine. The simultaneous blockade of alpha- and beta-adrenoceptors, i.e. propranolol (0.1 mumol/l) together with prazosin (0.01 mumol/l) or yohimbine (1 mumol/l), was required to abolish the enhancing effect of alpha-methylnoradrenaline. Likewise, the enhancing effect of adrenaline could be abolished only by a combination of two antagonists (propranolol together with yohimbine or prazosin). Neither clonidine nor oxymetazoline (1 or 10 mumol/l) modulated the evoked [3H]acetylcholine release. The experiments showed the existence of alpha-adrenoceptors which are present on the motor nerve and whose stimulation mediates an increase in evoked transmitter release. The different potencies found for prazosin and yohimbine indicate that an alpha 1-subtype of the receptors was involved. Increased sympathetic activity may facilitate neuromuscular transmission by stimulation of presynaptic alpha- and beta-adrenoceptors.