Introduction: the non-neuronal cholinergic system in humans.
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Acetylcholine↗
Biomedical subjects
Koichiro Kawashima
Publications and source records attributed to Koichiro Kawashima.
The lymphocytic cholinergic system and its biological function.
Lymphocytes are now known to possess the essential components for a non-neuronal cholinergic system. These include acetylcholine (ACh); choline acetyltransferase (ChAT), its synthesizing enzyme; and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Stimulating lymphocytes with phytohemagglutinin, a T-cell activator; Staphylococcus aureus Cowan I, a B-cell activator; or cell surface molecules enhances the synthesis and release of ACh and up-regulates expression of ChAT and M(5) mAChR mRNAs. Activation of mAChRs and nAChRs on lymphocytes elicits increases in the intracellular Ca(2+) concentration and stimulates c-fos gene expression and nitric oxide synthesis. On the other hand, long-term exposure to nicotine down-regulates expression of nAChR mRNA. Abnormalities in the lymphocytic cholinergic system have been detected in spontaneously hypertensive rats and MRL-lpr mice, two animal models of immune disorders. Taken together, these data present a compelling picture in which immune function is, at least in part, under the control of an independent non-neuronal lymphocytic cholinergic system.
Acetylcholine↗
Expression of multiple mRNA species for choline acetyltransferase in human T-lymphocytes.
Acetylcholine (ACh) is synthesized by choline acetyltransferase (ChAT) in cholinergic neurons. However, both ACh and mRNA for ChAT are expressed in mononuclear leukocytes and various human leukemic T-cell lines. Multiple ChAT mRNA species (R-, N0-, N1-, N2-, and M-types) having an identical coding region and different 5'-noncoding regions have been discovered in human brain and spinal cord. These mRNAs are transcribed by a combination of use of different promoter regions and alternative splicing. However, which types of ChAT mRNA species are expressed in T-lymphocytes remains to be elucidated. In the present study, we used two human leukemic T-cell lines, CCRF-CEM (CEM) and MOLT-3, which express the same ChAT mRNA as that in the nervous system. Major mRNA species in CEM were N2- and M-type, and to a lesser extent N1-type, while MOLT-3 expressed only N2-type. Neither CEM nor MOLT-3 expressed R-type mRNA. We previously found a lack of mRNA expression encoding vesicular acetylcholine transporter (VAChT) in CEM and MOLT-3, which mediates ACh transport to synaptic vesicles in cholinergic neurons. These findings suggest that the mechanisms regulating ChAT mRNA expression in T-lymphocytes differ from those in cholinergic neurons.
Carrier Proteins↗
Detection of the high-affinity choline transporter in the MOLT-3 human leukemic T-cell line.
We previously showed that lymphocytes possess the necessary components to constitute an independent, non-neuronal cholinergic system; these include acetylcholine (ACh) itself, choline acetyltransferase (the ACh-synthesizing enzyme), and both muscarinic and nicotinic ACh receptors (AChRs). In addition, we showed that stimulation of AChRs with their respective agonists elicits a variety of biochemical and functional effects, suggesting that lymphocytic cholinergic system is involved in the regulation of immune function. In nerve terminals, choline taken up via the high-affinity choline transporter (CHT1) is exclusively utilized for ACh synthesis. In the present study, therefore, we investigated the expression of CHT1 in T-lymphocytes. Reverse transcription-polymerase chain reaction analysis revealed that MOLT-3 cells, a human leukemic T-cell line used as a T-lymphocyte model, expressed CHT1 mRNA, but that the CEM and Jurkat T-cell lines did not. Consistent with that finding, specific binding of [3H]hemicholinium-3 (HC-3), an inhibitor of CHT1, and HC-3-sensitive [3H]choline uptake were also detected in MOLT-3 cells. These results suggest that CHT1 plays a role in mediating choline uptake in T-lymphocytes and provides further evidence for the presence of an independent lymphocytic cholinergic system.
Cell Line↗
Acetylcholine increase in amniotic fluid of experimental rats for intrauterine growth retardation.
Previous reports from this laboratory have demonstrated evidence for synthesis and release of acetylcholine (ACh) and catecholamines (CAs) by human amniotic epithelial cells (HAEC) and the presence of ACh and CAs in amniotic fluid. To study the physiological role of amniotic ACh, we used an experimental pregnant rat model for intrauterine growth retardation. Prior to this experiment, we confirmed the presence of choline acetyltransferase in the HAEC by immunocytochemical staining. Amniotic fluid was collected at 48 and 72 h after a transient ligation of the uterine vessels near the lower and upper ends of the right horn of the pregnant rat. The ACh concentration in the amniotic fluid from rats received intrauterine ischemia increased with time to a greater degree compared with the control rat, although the increase was not statistically significant. These results suggest that intrauterine hypoxic conditions cause a tendency to increase ACh concentrations in the amniotic fluid.
Acetylcholine↗
Nitric oxide (NO) synthase mRNA expression and NO production via muscarinic acetylcholine receptor-mediated pathways in the CEM, human leukemic T-cell line.
Nitric oxide (NO) is synthesized from L-arginine by neuronal, endothelial and inducible isoforms of NO synthase (nNOS, eNOS and iNOS, respectively) and is involved in the regulation of a variety of physiological functions, including immune activity. In vascular endothelial cells, stimulation of M(3) subtype of muscarinic acetylcholine receptors (mAChRs) triggers NO synthesis by eNOS. Human lymphocytes express several mAChR subtypes and their stimulation increases the intracellular free Ca(2+) concentration and up-regulates c-fos gene expression. While the above findings suggest involvement of the lymphocytic cholinergic system in the regulation of immune function, little is known on NOS expression and NO synthesis in T-lymphocytes. In the present study, using reverse transcription-polymerase chain reaction, we found that CEM cells express mRNAs encoding iNOS and nNOS, but not for eNOS. In addition, using quantitative fluorescence microscopy and a novel NO-sensitive fluorescent indicator, DAF-2, we found that oxotremorine-M (Oxo-M) (100 microM), a non-selective mAChR agonist, enhances NO production in the cells. This effect of Oxo-M was antagonized by pirenzepine (10 microM), an antagonist acting preferentially at M(1) mAChR and by atropine (10 microM). Also 4-DAMP (10 microM), an antagonist acting preferentially at M(3) mAChR, reduced significantly the effect of Oxo-M, while AFDX-116 (10 microM), an antagonist acting preferentially at M(2) mAChR, was ineffective. These findings suggest that T-lymphocytes express functional mAChRs linked to NO synthesis by nNOS and/or iNOS.
Humans↗
Nicotine-induced Ca2+ signaling and down-regulation of nicotinic acetylcholine receptor subunit expression in the CEM human leukemic T-cell line.
We previously showed that T- and B-lymphocytes express both muscarinic and nicotinic acetylcholine (ACh) receptors (mAChR and nAChR, respectively), and that stimulation of M(3) mAChRs on lymphocytes increases the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and up-regulates c-fos gene expression. Little is known about the effects of nicotinic stimulation on lymphocyte function, however. We therefore investigated the acute effect of nicotine on [Ca(2+)](i) in CEM cells, a model of T-lymphocytes, using confocal laser scanning microscopy with fluo-3, a Ca(2+)-sensitive fluorescent indicator. In addition, we examined the long-term effect of nicotine on the expression of selected nAChR subunits using semiquantitative reverse transcription-polymerase chain reaction analysis. In the presence of extracellular Ca(2+), nicotine (30 microM) evoked rapid, transient increases in [Ca(2+)](i). This effect was concentration-dependently inhibited by the alpha7 nAChR subunit antagonists, alpha-bungarotoxin (0.01-10 microM) and methyllycaconitine (0.01-10 mM), suggesting that the alpha7 nAChR subunit mediates Ca(2+) signaling in T-lymphocytes. Nicotine (0.01-10 microM) also concentration-dependently down-regulated expression of mRNAs for all the nAChR subunits tested: expression of the alpha6 and alpha7 subunits was down-regulated within 1 week, while expression of the alpha3 and alpha5 subunits declined gradually throughout the 8-week experimental period. These findings indicate that nicotine--and therefore likely smoking--affects immune function by suppressing expression of the neuronal nAChR subtype involved in Ca(2+) signaling in lymphocytes.
Aniline Compounds↗
Evolutional study on acetylcholine expression.
Acetylcholine (ACh) is a well-known neurotransmitter in the cholinergic nervous systems of vertebrates and insects; however, there is only indirect evidence for its presence in lower invertebrates, such as plants and fungi. We therefore investigated the expression of ACh in invertebrates (sea squirt, sea urchin, trepang, squid, abalone, nereis, sea anemone, coral and sponge), plants (arabidopsis, eggplant, bamboo shoot, cedar, hinoki, pine, podcarp, fern, horsetail and moss), fungi (yeast and mushroom) and bacteria by assaying ACh content and synthesis, focusing on the presence of two synthetic enzymes, choline acetyltransferase (ChAT) and carnitine acetyltransferase (CarAT). Using a specific radioimmunoassay, ACh was detected in all samples tested. The levels varied considerably, however, with the upper portion of bamboo shoots having the highest content (2.9 micromol/g). ACh synthesis was also detected in all samples tested; moreover, the activity in most samples from the animal kingdom, as well as bamboo shoots and the stem of the shiitake mushroom, were sensitive to both ChAT and CarAT inhibitors. Levels of ACh synthesis were lower in samples from other plants, fungi and bacteria and were insensitive to ChAT and CarAT inhibitors. These findings demonstrate the presence of ACh and ACh-synthesizing activity in evolutionally primitive life as well as in more complex multicellular organisms. In the context of the recent discovery of non-neuronal ACh in various mammalian species, these findings suggest that ACh been expressed in organisms from the beginning of life, functioning as a local mediator as well as a neurotransmitter.
Acetylcholine↗
Upregulation of mRNA encoding the M5 muscarinic acetylcholine receptor in human T- and B-lymphocytes during immunological responses.
Lymphocytes possess an independent, non-neuronal cholinergic system. Moreover, both T- and B-lymphocytes express multiple muscarinic acetylcholine receptors (mAChR). To obtain a better understanding of the regulatory mechanisms governing mAChR gene expression in the lymphocytic cholinergic system, we examined the effects of lymphocyte activation on expression of mAChR mRNA. Stimulation of T- and B-lymphocytes, respectively, with T-cell activator phytohemagglutinin and B-cell activator Staphylococcus aureus Cowan I upregulated M5 mAChR mRNA expression in the CEM human leukemic T-cell line and in the Daudi B-cell line, which served as models of lymphocytes. In striking contrast, M3 and M4 mAChR mRNA expression was not affected in either cell line. Nonetheless, stimulating lymphocytes with phorbol 12-myristate 13-acetate, a protein kinase C activator, plus ionomycin, a calcium ionophore, upregulated expression of both M3 and M5 mAChR mRNA. This represents the first demonstration that immunological stimulation leads to M5 mAChR gene expression in lymphocytes.
Antibody Formation↗
Analysis of flexural mode focusing by a semianalytical finite element method.
Focusing is one of the most promising techniques for the detection of small defects in pipe works, in which guided waves including longitudinal and flexural modes are tuned so that ultrasonic energy can be focused at a target point in a pipe, and analysis of reflected waves gives information on defects such as location and size. In this paper, the focusing technique is discussed by way of a simulation of guided wave propagation in pipe by a semianalytical finite element method (SAFE). Experiments and SAFE calculations were compared for waveforms transmitted by a single transducer and received at different circumferential positions initially, and then the focusing phenomena were experimentally observed using focusing parameters obtained by calculations. Calculation and visualization were conducted to clarify focusing phenomena in pipe in investigating the potential of the focusing technique. These results show that the time-reversal idea helps in understanding focusing and that resolution of focusing is strongly affected by incident waveforms as well as the number of channels available in an experiment.
Journal Article↗
Nicotine-induced NO-mediated increase in cortical cerebral blood flow is blocked by beta2-adrenoceptor antagonists in the anesthetized rats.
Involvement of nitric oxide (NO) and beta-adrenoceptors in an increase in the cortical cerebral blood flow (CBF) following an intravenous (i.v.) injection of a small dose of nicotine which did not affect the systemic blood pressure in the rats was investigated. I.v. injection of nicotine (30 microg/kg) for 1 min produced a significant increase in CBF lasting for more than 20 min without a significant effect on the systemic blood pressure. I.v. injection of L-N(G)-nitroarginine methylester (30 mg/kg) significantly attenuated nicotine-induced increase in the cortical CBF. The attenuation was reversed by i.v. injection of L-arginine (300 mg/kg), suggesting an intimate role of nitric oxide (NO) in nicotine-induced increase in the cortical CBF. The nicotine-induced increase in the cortical CBF was significantly attenuated by propranolol (10 mg/kg, i.v.) and ICI 118,551 (a beta2-adrenoceptor antagonist, 10 mg/kg, i.v.) but not by metoprolol (a beta1-adrenoceptor antagonist, 10 mg/kg, i.v.). Beta2-adrenoceptors on presynaptic nitrergic nerves may be involved in nicotine-induced NO-mediated increase in the cortical CBF.
Adrenergic beta-Antagonists↗
Multiple reflections of Lamb waves at a delamination.
Wave propagation in laminated plates with delaminations was calculated using the semi-analytical finite element method. The visualization results and deeper numerical analyses revealed the following phenomena on the fundamental Lamb modes at delamination regions of laminated plates. First, Lamb wave propagates toward the delamination, and then splits into two independent waves at the "Entrance" of the delamination with no significant reflections. These two waves reach the "Exit" of the delamination with the different phases and arrival time. Thus reflected and transmitted waves are excited at the Exit. The repetition of such reflections at the Exit causes the multiple reflections at regular intervals corresponding to the delamination size.
Journal Article↗
Nonlinear acoustic response through minute surface cracks: FEM simulation and experimentation.
The second harmonic of a Rayleigh wave passing through a minute surface crack has been numerically analyzed by semi-explicit FEM including special elements which account for a nonlinear stress-strain relation at crack surfaces. Minute cracks perpendicular to a free, flat surface close under compressive stress when width of the crack opening is less than the longitudinal amplitude of the Rayleigh wave. Thereafter, compressive and shear stresses are partially transmitted through the closed cracks, whereas tensile and shear stresses are not transmitted through cracks that remain open. This leads to marked nonlinear ultrasonic response. Calculation was performed for an aluminum block having a surface crack. The transverse component of the Rayleigh wave propagating through the cracks shows distorted waveforms, making the second harmonic amplitude clearly noticeable. In an experiment, the second harmonic component of the leaky Rayleigh wave was detected for a simple crack model consisting of two aluminum blocks, by use of a PVDF line-focused transducer. The results of the experiment show that the second harmonic amplitude is a second-order function of the fundamental wave amplitude, and is more pronounced for low compressive stress applied to close the crack surfaces.
Journal Article↗
Effects of human antithymocyte globulin on acetylcholine synthesis, its release and choline acetyltransferase transcription in a human leukemic T-cell line.
Lymphocytes possess an independent, nonneuronal cholinergic system. In the present study, we investigated the short- and long-term effects of antithymocyte globulin (ATG)-Fresenius (ATG-F), a human antithymocyte globulin that binds to CD2, CD7 and CD11a, on acetylcholine (ACh) synthesis and transcription of choline acetyltransferase (ChAT) in CCRF-CEM cells, a human leukemic T-cell line. In the short-term (6 h), ATG-F enhanced ACh release, likely through transient increases in intracellular Ca(2+) ([Ca(2+)](i)) mediated by CD7, which led to declines in intracellular ACh content. By 48 h, however, the ACh content had increased as compared to control due to up-regulation of ChAT expression mediated by CD11a.
Acetylcholine↗
Induction of choline acetyltransferase mRNA in human mononuclear leukocytes stimulated by phytohemagglutinin, a T-cell activator.
The induction of mRNA for choline acetyltransferase (ChAT), which catalyzes acetylcholine (ACh) synthesis was investigated in human mononuclear leukocytes (MNL) stimulated by phytohemagglutinin (PHA), a T-cell activator, using the reverse transcription-polymerase chain reaction. Stimulation of MNL by PHA induced the expression of ChAT mRNA, and potentiated ACh synthesis. ChAT mRNA induction required more time than the induction of interleukin-2 mRNA. Expression of the gene encoding the vesicular ACh transporter, which mediates ACh transport in cholinergic neurons, was not observed in PHA-stimulated MNL, suggesting that the mechanisms controlling ACh release from T-lymphocytes differ from those in cholinergic neurons. These findings demonstrate that activation of T-lymphocytes up-regulates ACh synthesis in the blood, and suggest that ACh plays an important role as a neuroimmunomodulator besides its role as a neurotransmitter.
Blotting, Southern↗