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Results for “Functional characterization”

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At least 199 records · Page 11Linked to original sources

PubMed2005

Functional characterization and expression of endothelin receptors in rat carotid artery: involvement of nitric oxide, a vasodilator prostanoid and the opening of K+ channels in ETB-induced relaxation.

We aimed to functionally characterize endothelin (ET) receptors in the rat carotid artery. mRNA and protein expressions of both ETA and ETB receptors, evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western immunoblotting, were detected in carotid segments. Immunohistochemical assays showed that ETB receptors are expressed in the endothelium and smooth muscle cells, while ETA receptors are expressed only in the smooth muscle cells. In endothelium-denuded vessels, levels of ETB receptor mRNA were reduced. Vascular reactivity experiments, using standard muscle bath procedures, showed that ET-1 induces contraction in endothelium-intact and -denuded carotid rings in a concentration-dependent manner. Endothelial removal enhanced ET-1-induced contraction. BQ123 and BQ788, selective antagonists for ETA and ETB receptors, respectively, produced concentration-dependent rightward displacements of the ET-1 concentration-response curves. IRL1620, a selective agonist for ETB receptors, induced a slight vasoconstriction that was abolished by BQ788, but not affected by BQ123. IRL1620-induced contraction was augmented after endothelium removal. ET-1 concentration dependently relaxed phenylephrine-precontracted rings with intact endothelium. The relaxation was augmented in the presence of BQ123, reduced in the presence of BQ788 and completely abolished after endothelium removal. IRL1620 induced vasorelaxation that was abolished by BQ788 and endothelium removal, but not affected by BQ123. Preincubation of intact rings with N(G)-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), indomethacin or tetraethylammonium (TEA) reduced IRL1620-induced relaxation. The combination of L-NAME, indomethacin and TEA completely abolished IRL1620-induced relaxation while sulfaphenazole did not affect this response. 4-aminopyridine (4-AP), but not apamin, glibenclamide or charybdotoxin, reduced IRL1620-induced relaxation. The major finding of this work is that it firstly demonstrated functionally the existence of both ETA and ETB vasoconstrictor receptors located on the smooth muscle of rat carotid arteries and endothelial ETB receptors that mediated vasorelaxation via NO-cGMP pathway, vasodilator cyclooxygenase product(s) and the activation of voltage-dependent K+ channels.

Animals↗

PubMed2026

Identification and functional characterization of a novel antiviral chicken interferon-υ.

Interferons are critical mediators of antiviral immunity in vertebrates. While type IV interferon (IFN-υ) has been identified in fish and amphibians, its existence and function in chickens remained unknown. Through systematic genomic screening, we identified and cloned a novel chicken interferon gene, designated ChIFN-υ. Phylogenetic analysis placed ChIFN-υ within a distinct clade alongside zebrafish and clawed frog IFN-υ, confirming its identity as a type IV interferon, with minimal homology to classical type I, II, or III IFNs. Expression profiling revealed constitutive ChIFN-υ expression in mucosal and immune tissues of healthy chickens, exhibiting a distinct developmental shift: highest in trachea and small intestine in 1-day-old chicks, shifting to spleen and lung in 4-week-old chickens. ChIFN-υ expression was strongly upregulated following H9N2 AIV infection. Functionally, recombinant ChIFN-υ protein activated the interferon-stimulated response element (ISRE) and Mx promoter in a dose-dependent manner and significantly inhibited the replication of both vesicular stomatitis virus (VSV) and H9N2 AIV in DF-1 cells. In vivo, early treatment with exogenous ChIFN-υ significantly reduced pulmonary and tracheal viral loads and decreased oropharyngeal and cloacal virus shedding in H9N2-infected chickens. In conclusion, this study identifies and functionally characterizes the type IV interferon in chickens, elucidating the evolutionary status, regulated expression, and antiviral efficacy of ChIFN-υ. These findings highlight its potential as a candidate for developing interferon-based therapies against avian viral diseases.

Animals↗

PubMed2006

Phenotypic and functional characterization of bone marrow mesenchymal stem cells derived from patients with multiple myeloma.

Multiple myeloma (MM) is a B-cell neoplasia caused by the proliferation of clonal plasma cells, primarily in the bone marrow (BM). The role of the BM microenvironment in the pathogenesis of the disease has been demonstrated, especially for the survival and growth of the myeloma plasma cells. Functional characterization of the major component of the BM microenvironment, namely the recently characterized mesenchymal stem cells (MSCs), was never performed in MM. Based on a series of 61 consecutive patients, we evaluated the ability of MSCs derived from myeloma patients to differentiate into adipocytes and osteocytes, inhibit T-cell functions, and support normal hematopoiesis. MSCs phenotypic characterization and quantification of interleukin-6 (IL-6) secretion were also performed. As compared to normal MSCs, MSCs from MM patients exhibited normal phenotype, differentiation capacity and long-term hematopoietic support, but showed reduced efficiency to inhibit T-cell proliferation and produced abnormally high amounts of IL-6. Importantly, these characteristics were observed in the absence of any detectable tumor plasma cell. Chromosomal analysis revealed that MM patients MSCs were devoid of chromosomal clonal markers identified in plasma cells. MM MSCs present abnormal features that may participate in the pathogenesis of MM.

Adipocytes↗

PubMed2000

Cloning and functional characterization of the 5' regulatory region of the human mitochondrial glycerol-3-phosphate dehydrogenase gene. Lack of 3,5,3'-triiodothyronine responsiveness in adipose tissue.

We report data on the structural and functional characterization of the 5' flanking region of the human mitochondrial glycerol-3-phosphate dehydrogenase (mtGPDH) gene. We found two regions upstream of 5'-untranslated sequences exhibiting promoter activity in transient transfection assays. Transcription start sites and potential regulatory sites in both promoter regions were defined. The proximal promoter was approximately sevenfold more active than the distal one in most cell lines, but it was only twice as active in a neuroblastoma cell line. These observations seem to indicate that the rate of transcription, as well as the tissue-specific expression of the human mtGPDH gene, is the result of a combinatorial effect of transcription factors on at least two promoters. 3,5,3'-Triiodothyronine failed to alter the transcriptional activity of human mtGPDH promoter(s) constructs in transient transfection assays. Although this finding seems to be in conflict with the reported effect of 3,5,3'-triiodothyronine in rodents, it is consistent with our observation of 3,5, 3'-triiodothyronine stimulation of mtGPDH activity in primary cultures of rat adipocytes, but not human cultured adipocytes, suggesting distinct regulation of this gene in both species.

Adipose Tissue↗

PubMed2000

The Na+-driven Cl-/HCO3- exchanger. Cloning, tissue distribution, and functional characterization.

The Na(+)-driven Cl(-)/HCO(3)(-) exchanger is an important regulator of intracellular pH in various cells, but its molecular basis has not been determined. We show here the primary structure, tissue distribution, and functional characterization of Na(+)-driven chloride/bicarbonate exchanger (designated NCBE) cloned from the insulin-secreting cell line MIN6 cDNA library. The NCBE protein consists of 1088 amino acids having 74, 72, and 55% amino acid identity to the human skeletal muscle, rat smooth muscle, and human kidney sodium bicarbonate cotransporter, respectively. The protein has 10 putative membrane-spanning regions. NCBE mRNA is expressed at high levels in the brain and the mouse insulinoma cell line MIN6 and at low levels in the pituitary, testis, kidney, and ileum. Functional analyses of the NCBE protein expressed in Xenopus laevis oocytes and HEK293 cells demonstrate that it transports extracellular Na(+) and HCO(3)(-) into cells in exchange for intracellular Cl(-) and H(+), thus raising the intracellular pH. Thus, we conclude that NCBE is a Na(+)-driven Cl(-)/HCO(3)(-) exchanger that regulates intracellular pH in native cells.

Animals↗

PubMed2000

Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom.

BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom, was structurally and functionally characterized. Several biological activities were assayed and compared with those of the chemically modified toxin involving specific amino acid residues. The cDNA produced from the total RNA by RT-PCR contained approximately 400 bp which codified its 121 amino acid residues with a calculated pI and molecular weight of 8.9 and 13,727, respectively. Its amino acid sequence showed strong similarities with several Lys49 phospholipase A(2) homologues from other Bothrops sp. venoms. By affinity chromatography and gel diffusion, it was demonstrated that heparin formed a complex with BnSP-7, held at least in part by electrostatic interactions. BnSP-7 displayed bactericidal activity and promoted the blockage of the neuromuscular contraction of the chick biventer cervicis muscle. In addition to its in vivo myotoxic and edema-inducing activity, it disrupted artificial membranes. Both BnSP-7 and the crude venom released creatine kinase from the mouse gastrocnemius muscle and induced the development of a dose-dependent edema. His, Tyr, and Lys residues of the toxin were chemically modified by 4-bromophenacyl bromide (BPB), 2-nitrobenzenesulfonyl fluoride (NBSF), and acetic anhydride (AA), respectively. Cleavage of its N-terminal octapeptide was achieved with cyanogen bromide (CNBr). The bactericidal action of BnSP-7 on Escherichia coli was almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The neuromuscular effect induced by BnSP-7 was completely inhibited by heparin, BPB, acetylation, and CNBr treatment. The creatine kinase releasing and edema-inducing effects were partially inhibited by heparin or modification by BPB and almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The rupture of liposomes by BnSP-7 and crude venom was dose and temperature dependent. Incubation of BnSP-7 with EDTA did not change this effect, suggesting a Ca(2+)-independent membrane lytic activity. BnSP-7 cross-reacted with antibodies raised against B. moojeni (MjTX-II), B. jararacussu (BthTX-I), and B. asper (Basp-II) myotoxins as well as against the C-terminal peptide (residues 115-129) from Basp-II.

Amino Acid Sequence↗

PubMed2001

Functional characterization of the splicing variants of human norepinephrine transporter.

Human norepinephrine transporter (NET) displays three splicing variants having different carboxy terminals, hNET, hNET C-t var1 and hNET C-t var2. Functional characterization of these isoforms was performed with transient expression system in COS-7 cells. Cells transfected with hNET C-t var2, but not hNET C-t var1, revealed a significant increase in [(3)H]norepinephrine (NE) uptake and [(3)H]nisoxetine binding as well as hNET, in association with their different cellular localization indicated by immunostaining using NET-specific antisera. Kinetic and pharmacological analyses of [(3)H]NE uptake revealed different characteristics between hNET and hNET C-t var2. These results suggest that hNET C-t var2 may participate in NE transport in a manner different from hNET at noradrenergic synapses or in other tissues including placenta where NET variants were found to exist.

Adrenergic Uptake Inhibitors↗

PubMed1996

Functional characterization of mouse syndecan-1 promoter.

The members of the syndecan family are temporally and spatially expressed heparan sulfate proteoglycans of various tissues, where they mediate extracellular influences on cell morphology and behavior. Functional characterization of the mouse syndecan-1 promoter was carried out in order to elucidate the mechanisms involved in the maintenance of the high transcription levels of syndecan-1 gene in various epithelia. For that 9.5 kilobase pairs of the upstream region of mouse syndecan-1 gene were cloned, sequenced, and used to prepare chimaeric constructs with a reporter gene followed by transient or stable transfections into NMuMG cells, cultured either in the presence or absence of serum, the 2.5-kilobase pair promoter region resulted in the constitutive transcription activity, whereas in 3T3 cells the serum depletion decreased the promoter activity significantly. Deletion of the upstream sequences to -437 base pairs relative to the translation initiation site had little effect on this promoter activity. Further deletion to -365 base pairs removed three GT boxes and slightly increased the promoter activity, whereas the deletion of the next two GC boxes (to -326 base pair) reduced the promoter activity dramatically. All of the GC or GT box sequences bound the same set of Sp1-like nuclear protein in gel shift assays. Nuclear protein binding was also demonstrated around both of the most intense transcription initiation sites. Mutation of these regions separately resulted in total loss of transcription initiation from the deleted site and decreased the promoter activity in relation to the intensity of the abolished start site. This indicates that the transcription initiation of the syndecan-1 gene is directed through initiator-like elements directly overlapping the start sites, as shown for several TATA-less housekeeping and growth regulated genes. We assume that the constitutive high level gene expression in epithelial cells is achieved by the proximal promoter, which is controlled by members of Sp1 transcription factor family.

3T3 Cells↗

PubMed2001

Functional characterization of heme proteins encapsulated in wet nanoporous silica gels.

Myoglobin and hemoglobin were encapsulated in wet, nanoporous silica gels. A rigorous evaluation of the effect of the encapsulation on protein dynamics and function was carried out by measuring the kinetics of carbon monoxide rebinding after nanosecond laser flash-photolysis with transient absorption detection, and the oxygen affinity with absorption microspectrophotometry. The time course of carbon monoxide binding to myoglobin evidenced a strongly enhanced geminate recombination and a faster bimolecular rebinding with respect to solution, whereas T and R quaternary states of hemoglobin exhibited a geminate phase and a bimolecular binding rate very similar to those observed in solution. Oxygen affinity of T-state hemoglobin was found to be close to that observed for the binding of the first oxygen to T-state hemoglobin in solution. Results indicate that some conformational transitions are kinetically restricted, allowing to isolate distinct tertiary and quaternary states. This opens the way to their detailed functional characterization and application to biodevices.

Absorption↗

PubMed2003

Pulmonary vein stenosis after radiofrequency ablation of atrial fibrillation: functional characterization, evolution, and influence of the ablation strategy.

BACKGROUND: Pulmonary vein (PV) stenosis is a complication of ablation for atrial fibrillation. The impact of different ablation strategies on the incidence of PV stenosis and its functional characterization has not been described. METHODS AND RESULTS: PV isolation was performed in 608 patients. An electroanatomic approach was used in 71 and circular mapping in 537 (distal isolation, 25; ostial isolation based on PV angiography, 102; guided by intracardiac echocardiography, 140; with energy delivery based on visualization of microbubbles, 270). Severe (> or =70%) narrowing was detected in 21 patients (3.4%), and moderate (50% to 69%) and mild (<50%) narrowing occurred in 27 (4.4%) and 47 (7.7%), respectively. Severe stenosis occurred in 15.5%, 20%, 2.9%, 1.4%, and 0%, respectively. Development of symptoms was correlated with involvement of >1 PV with severe narrowing (P=0.01), whereas all patients with mild and moderate narrowing were asymptomatic. In the latter group, lung perfusion (V/Q) scans were normal in all but 4 patients. All patients with severe stenosis had abnormal perfusion scans. CONCLUSIONS: V/Q scans are useful to assess the functional significance of PV stenosis. Mild and moderate degrees of PV narrowing are not associated with development of symptoms and seem to have no or minimal detrimental effect on pulmonary flow. The incidence of severe PV stenosis seems to be declining with better imaging techniques to ensure ostial isolation and to guide power titration. Mild narrowing 3 months after ablation does not preclude future development of severe stenosis and should be assessed with repeat imaging studies.

Atrial Fibrillation↗

PubMed1990

The cloning and functional characterization of the nifH gene of Rhodospirillum rubrum.

Dinitrogenase reductase (the nifH product) from Rhodospirillum rubrum is regulated by a post-translational modification system encoded by draTG. As demonstrated in this report, the cloning, sequencing, and functional characterization of the nifH gene provides a basis for further analysis as well as revealing interesting features of gene organization. The coding regions of nifH and draT are separated by only 400 bp, though the genes are divergently transcribed and differentially regulated. The construction of a nifH insertion caused a Nif- phenotype and destroyed the mutant's ability to synthesize both dinitrogenase and dinitrogenase reductase, verifying functionality and transcriptional organization of the nifHDK genes.

ADP Ribose Transferases↗

PubMed2002

Cloning and functional characterization of two glycine receptor alpha-subunits from the perch retina.

Glycine receptors are ligand-gated ion channel proteins mediating synaptic inhibition in the spinal cord, retina and brain of vertebrates. We have cloned and functionally characterized two glycine receptor alpha-subunits from the perch (Roccus americana) retina. Based on sequence homology with the mammalian counterparts, we termed these subunits alpha 1 and alpha 3. RT-PCR revealed the presence of both subunits in retina and brain, whereas alpha1 was predominant in spinal cord. A short splice variant of alpha1 was detected in the brain but not in the retina. Functional expression of the perch subunits in HEK-293 cells yielded robust glycine-gated currents sensitive to strychnine. The perch receptors displayed a high efficacy for taurine and GABA and thus differ from the mammalian counterparts. Because the retina is a rich source for taurine, this finding could be of physiological importance. The structural features of the ligand binding domain strongly support the notion of increased glycine/GABA discrimination in higher vertebrates.

Amino Acid Sequence↗

PubMed1997

Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action.

ATP-dependent DNA ligases are essential enzymes in both DNA replication and DNA repair processes. Here we report a functional characterization of the T4 DNA ligase. One N-terminal and two C-terminal deletion mutants were expressed in Escherichia coli as histidine- tagged proteins. An additional mutant bore a substitution of Lys159 in the active site that abolished ATP binding. All the proteins were tested in biochemical assays for ATP-dependent self-adenylation, DNA binding, nick joining, blunt-end ligation and AMP- dependent DNA relaxation. From this analysis we conclude that binding to DNA is mediated by sequences at both protein ends and plays a key role in the reaction. The enzyme establishes two different complexes with DNA: (i) a transient complex (T.complex) involving the adenylated enzyme; (ii) a stable complex (S.complex) requiring the deadenylated T4 DNA ligase. The formation of an S. complex seems to be relevant during both blunt-end ligation and DNA relaxation. Moreover the inactive His-K159L substitution mutant, although unable to self-adenylate, still possesses AMP-dependent DNA nicking activity.

Adenosine Monophosphate↗

PubMed2003

Molecular and functional characterization of an octopamine receptor from honeybee (Apis mellifera) brain.

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera. The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis. Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.

Amino Acid Sequence↗

PubMed2006

Genomic organization and functional characterization of the alcohol dehydrogenase locus of Ceratitis capitata (Medfly).

Approximately 30 kb of genomic DNA enclosing the Adh locus from the medfly, Ceratitis capitata have been cloned and about 15 kb has been structurally and functionally characterized. The locus consists of two genes, Adh-1 and Adh-2, separated by an intergenic region, which is polymorphic in size ranging from approximately 6.4 kb to 8.1 kb. Both genes consist of three exons and two introns. The introns are below 200 bp in size, except the 1st intron of Adh-1, which is unexpectedly long, variable in size and contains a deleted mariner-like element (postdoc). The two genes are transcribed in different orientations. The Adh-2 gene shows the typical pattern of transcription seen in the homologous genes of Drosophilidae presenting high levels of expression in the fat body, gut and ovaries. The Adh-1 gene is only expressed in the body muscle tissues of embryos, larvae and adult flies, raising the question of what its biological function may be. A DNA fragment containing bases -102 to -1666 relative to the first base of the initiating ATG of Adh-1 is sufficient to drive the expression of a reporter gene in body muscles of Drosophila melanogaster embryos, larvae and adult flies. The study provides further insights into the evolution of the Adh genes of higher diptera.

Alcohol Dehydrogenase↗

PubMed1999

Functional characterization of a piscine retroviral promoter.

Walleye dermal sarcoma virus (WDSV) is a retrovirus aetiologically associated with a multifocal skin tumour of walleye. Tumours synchronously develop on 27% of fish and regress seasonally; their severity is influenced by water temperature. To functionally characterize the LTR of WDSV, the LTR was fused to the luciferase reporter gene. WDSV LTR was found to be transcriptionally active in both fish and mammalian cells. WDSV LTR deletion mutants were constructed to identify specific regions that were functionally important in modulating viral gene expression and in temperature responsiveness. The 5' end 60 bp, which contain a putative ecdysone-response element also present in another fish retrovirus, positively modulated transcription from the WDSV LTR at 25 degrees C, but not at lower temperatures. A 13 bp region (nt -288 to -275) comprising a putative activator protein-1 element was necessary for maintaining WDSV LTR activity at all temperatures. In marked contrast to the short direct repeats found in mammalian retroviral LTRs, five 5 bp direct repeats (nt -336 and -272) were found to negatively regulate transcription from the WDSV LTR. A region spanning nt -440 to -218 stimulated the activity of a heterologous mammalian promoter in an orientation-dependent manner, modestly in fish cells (1.3- to 2-fold), but markedly (3.7- to 5.1-fold) in mammalian cells. Our results strongly suggest that the putative promoter elements present in the WDSV LTR function differentially in a temperature-specific manner and that complex interactions between these elements modulate WDSV LTR activity in response to temperature changes.

Animals↗

PubMed1998

The human LGALS3 (galectin-3) gene: determination of the gene structure and functional characterization of the promoter.

Galectin-3 is a beta-galactoside-specific lectin that is a pre-mRNA splicing factor. Here we report the genomic organization of the human LGALS3 (galectin-3) gene and functional characterization of the promoter. Southern blot analysis of genomic DNA revealed that galectin-3 is coded by a single gene in the human genome. The gene is composed of six exons and five introns, spanning a total of approximately 17 kilobases (kb). Based on primer extension and ribonuclease protection analyses, there are two transcription initiation sites located 52 and 50 nucleotides (nt) upstream of the exon I-intron 1 border, and defined here as +1a and +1b, respectively. The translation start site is in exon II. The ribonucleoprotein-like N-terminal domain, containing the proline-glycine-alanine-tyrosine (PGAY) repeat motif, is found entirely within exon III. The carbohydrate recognition sequence is found entirely within exon V. Genomic fragments encompassing -836 to +141 nt (relative to +1a) have significant promoter activity when linked to the luciferase reporter gene and transiently transfected into HeLa cells or human diploid fibroblasts. Quiescent fibroblasts have low promoter activity but the activity increases 100-fold following serum addition. Serum responsive activation regions in the promoter are located between -513 and -339 nt and between -339 and -229 nt; an additional activation region may be located between -105 and -15 nt. Because galectin-3 is an immediate-early gene whose expression is dependent on the proliferative state of the cell, this study provides the basis for determining the molecular mechanisms of transcriptional regulation in neoplasia or cellular senescence.

Antigens, Differentiation↗

PubMed2000

Functional characterization of the human high-affinity choline transporter.

Na(+)-dependent, high-affinity choline uptake in cholinergic neurons is the rate-limiting step in acetylcholine synthesis. Here we report the molecular cloning and functional characterization of the human high-affinity choline transporter (hCHT1). The hCHT1 exhibits significant homology with known members of the Na(+)-dependent glucose transporter family, but not with members of the neurotransmitter transporter family. The human CHT1 gene is 25 kb in length with 9 exons and was assigned to chromosome II at position IIq11-12. Northern blot analysis showed that a 5.4 kb hCHT1 transcript was expressed exclusively in tissues containing cholinergic neurons. When expressed in Xenopus oocytes, the human clone induced Na(+)- and Cl(-)-dependent, high-affinity choline uptake, which was sensitive to the specific inhibitor hemicholinium-3, with a K(i) of 1.3 nM. The hCHT1-mediated choline uptake increased with increasing concentrations of choline, Na(+) and Cl(-), with EC(50) values of 2.0 microM, 76 mM, and 48 mM, and with apparent Hill coefficients of 1, 2.5 and 2.3, respectively.

Acetylcholine↗