Systematic arrangement within the family Clitocybaceae (Tricholomatineae, Agaricales): phylogenetic and phylogenomic evidence, morphological data and muscarine-producing innovation

The Clitocybaceae is a recently established family.Currently,the infrafamilial divisions and relationships within the family are vague due to limited sampling and genes employed for phylogenetic analysis.Some mushrooms of the family contain the neurotoxic muscarine,which has caused many severe and even deadly poisonings worldwide.However,the taxonomic distribution and evolution of the toxin within the family is largely unknown.In this study,phylogenetic analyses based on nucleotide sequences of ITS and of six molecular loci(ITS,LSU,TEF1,RPB1,RPB2 and ATP6),plus a phylogenomic analysis based on 485 single-copy orthologous genes,were performed to reconstruct the framework of Clitocybaceae.BEAST analysis was used to estimate the divergence times within the family.Additionally,biochemical analysis for muscarine was conducted of 32 representative species.Based on these analyses,an updated classification of Clitocybaceae into six genera(Clitocybe,Collybia,Dendrocollybia,Lepista,Pseudolyophyllum,and Singerocybe)is proposed.The genus Collybia is emended to accommodate four subgenera(Collybia,Crassicybe,Leucocalocybe,and Macrosporocybe).Seventeen new Chinese species and 15 new combinations are proposed.Keys to the genera of Clitocybaceae and the subgenera of Collybia,as well as to the known species of Clitocybe and Collybia subgen.Collybia in China,are presented.In addition,muscarine was detected in 18 species,and these muscarine-containing species formed a major monophyletic clade within Collybia subgen.Collybia.Finally,our phylogenetic,phylogenomic,chemotaxonomic and molecular dating results indicate that the Clitocybaceae is a natural group estimated to have arisen some 60 million years ago,and in this family,muscarine has evolved only once circa 20 million years ago without later losses.

Brain-derived neurotrophic factor signaling in the neuromuscular junction during developmental axonal competition and synapse elimination

During the development of the nervous system,there is an overproduction of neurons and synapses.Hebbian competition between neighboring nerve endings and synapses performing different activity levels leads to their elimination or strengthening.We have extensively studied the involvement of the brain-derived neurotrophic factor-Tropomyosin-related kinase B receptor neurotrophic retrograde pathway,at the neuromuscular junction,in the axonal development and synapse elimination process versus the synapse consolidation.The purpose of this review is to describe the neurotrophic influence on developmental synapse elimination,in relation to other molecular pathways that we and others have found to regulate this process.In particular,we summarize our published results based on transmitter release analysis and axonal counts to show the different involvement of the presynaptic acetylcholine muscarinic autoreceptors,coupled to downstream serine-threonine protein kinases A and C(PKA and PKC)and voltage-gated calcium channels,at different nerve endings in developmental competition.The dynamic changes that occur simultaneously in several nerve terminals and synapses converge across a postsynaptic site,influence each other,and require careful studies to individualize the mechanisms of specific endings.We describe an activity-dependent balance(related to the extent of transmitter release)between the presynaptic muscarinic subtypes and the neurotrophin-mediated TrkB/p75NTR pathways that can influence the timing and fate of the competitive interactions between the different axon terminals.The downstream displacement of the PKA/PKC activity ratio to lower values,both in competing nerve terminals and at postsynaptic sites,plays a relevant role in controlling the elimination of supernumerary synapses.Finally,calcium entry through L-and P/Q-subtypes of voltage-gated calcium channels(both channels are present,together with the N-type channel in developing nerve terminals)contributes to reduce transmitter release and promote withdrawal of the most unfavorable nerve terminals during elimination(the weakest in acetylcholine release and those that have already become silent).The main findings contribute to a better understanding of punishment-rewarding interactions between nerve endings during development.Identifying the molecular targets and signaling pathways that allow synapse consolidation or withdrawal of synapses in different situations is important for potential therapies in neurodegenerative diseases.

Potencies of Four Stereoisomers of Anisodamine on Muscarinic Receptor

用[3~H]-QNB配体受体结合实验,抗Ach对豚鼠回肠纵肌条收缩效应,抗毛果芸香碱流涎及扩瞳效应为指标,比较山莨菪碱四个立体异构体(A.6S,2′S;B.6S,2′R;C.6R,2′R;D.6R,2′S)与M受体相互作用的构效关系,结果A,B,C,D四个异构体效应分别为:(1)受体结合实验K_i(M)2.69×10^(-7),1.43×10^(-7),1.63×10^(-5),2.15×10^(-6);(2)抗Ach豚鼠回肠纵条收缩:K_6(M)2.98×10^(-8);1.38×10^(-8);1.52×10~5;1.0l×10^(-7);(3)扩瞳:ED_4(mol/kg)1.6×10^(-6),1.9×10^(-6),5.1×10^(-5),3.6×10^(-5);(4)抗毛果芸香碱流涎:ED_(50)(mol/kg)1.5×10^(-6);1.3×10^(-6);2.6×10^(-5);3.1×10^(-5)。以上结果表明四个异构体效应强度为6S,2′R≌6S,2′S>>6R,2′S>6R,2′R。

Microinjection of M_5 muscarinic receptor antisense oligonucleotide into VTA inhibits FosB expression in the NAc and the hippocampus of heroin sensitized rats

Objective To investigate the effect of M5 muscarinic receptor subtype on the locomotor sensitization induced by heroin priming, and it＇s effect on the FosB expression in the nucleus accumbens （NAc） and the hippocampus in the heroin sensitized rats. Methods Locomotor activity was measured every 10 min for 1 h after subcutaneous injection of heroin. FosB expression was assayed by immunohistochemistry, and the antisense oligonucleotides （AS-ONs） targeting M5 muscarinic receptor was transferred with the lipofectin. Results Microinjection of AS-ONs targeting M5 muscarinic receptor in the ventral tegmental area （VTA） blocked the expression of behavioral sensitization induced by heroin priming in rats. Meanwhile, the expression of FosB-positive neurons in either the NAc or the dentate gyrus （DG） of the hippocam- pus increased in heroin-induced locomotor sensitized rats. The enhancement of FosB-positive neurons in the NAc or DG could be inhibited by microinjection of M5 muscarinic receptor AS-ONs into the VTA before the heroin-induced locomotor sensitization was performed. In contrast, microinjection of M5 muscarinic receptor sense oligonucleotide （S-ONs） into the VTA did not block the expression of behavioral sensitization or the expression of FosB in the NAc or DG in the heroin sensitized rats. Conclusion Blocking M5 muscarinic receptor in the VTA inhibits the expression of heroin-induced locomotor sensitization, which is associated with the regulation of FosB expression in the NAc and hippocampus neurons. M5 muscarinic receptor may be a useful pharmacological target for the treatment of heroin addiction.

Acupuncture at heterotopic acupoints enhances jejunal motility in constipated and diarrheic rats

AIM: To investigate the effect and mechanism of acupuncture at heterotopic acupoints on jejunal motility, particularly in pathological conditions.

Mechanisms mediating cholinergic antral circular smooth muscle contraction in rats

AIM:To investigate the pathway(s)mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent,bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction. METHODS:Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer.Isometric tension was recorded.Cumulative concentration-response curves were obtained for(+)-cis- dioxolane(cD),a nonspecific muscarinic agonist,at 10^(-8)- 10^(-4)mol/L,in the presence of tetrodotoxin(TTX,10^(-7)mol/L). Results were normalized to cross sectional area.A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1(pirenzepine), M2(methoctramine)and M3(darifenadn)muscarinic receptor subtypes.The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment.The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol. RESULTS:A dose-dependent contractile response observed with bethanechol,was not affected by TTx.The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol.Lack of calcium as well as the presence of the L-type calcium channel blocker,nifedipine,also inhibited the cholinergic contraction,with a reduction in response from 2.5±0.4 g/mm^2 to 1.2±0.4 g/mm^2(P<0.05).The dose- response curves were shifted to the right by muscarinic antagonists in the following order of affinity:darifenacin (M_3)>methocramine(M_2)>pirenzepine(M_1). CONCLUSION:The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s)involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels.The presence of the residual contractile response after the treatment with nifedipine,suggests that an additional pathway could mediate the cholinergic contraction.The involvement of more than one muscarinic receptor(functionally predominant type 3 over type 2)also suggests more than one pathway mediating the cholinergic contraction in rat antrum.

Methanol extract of Desmodium gangeticum DC root mimetic post-conditioning effect in isolated perfused rat heart by stimulating muscarinic receptors

Objective:To evaluate pharmacological mimetic action of herbal extract Desmodium gangeticum (DC) roots on ischemia reperfusion injury.Methods:With the help of Langendroff perfusion technique,ischemic post condition(POC) mimetic action of DG methanol root extract was evaluated and compared by using standard drugs that acts as muscarinic receptor agonist and antagonist,namely acetylcholine(Ach) and atropine(Atr) respectively in an isolated rat heart. Results:The physiological parameters like left ventricular developed pressure,end diastolic pressure and working index of isolated rat heart showed significant recovery in DG root extract administrated rat heart,similar to the recovery by POC.Kymogram results showed muscarinic receptor agonist like action for DG methanol root extract,confirmed in rat heart by muscarnic receptor agonist(acetylcholine) and anatoginst(atropine).Administration of DC root extract prior to reperfusion showed better antioxidant status in myocardial tissue homogenate and mitochondrial,complemented by the levels of cardiac specific marker proteins in myocardial tissue and perfusate.Even though DG methanol root extract mimics its action similar to that of Ach,the myocardial protection mediated by the extract was superior to Ach,due to the presence of antioxidants in the crude extract.Conclusions:DG methanol root extract provides myocardial protection towards IRI by stimulating muscarinic receptors.

AduoLa Fuzhenglin Down-regulates Microwave-induced Expression of β_1-adrenergic Receptor and Muscarinic Type 2 Acetylcholine Receptor in Myocardial Cells of Rats

This paper is aimed to study the effect of ADL on expression of ~z-AR and Mz-AchR in myocardial cells of rats exposed to microwave radiation. Immunohistochemistry, Western blot and image analysis were used to detect the expression of ~I-AR and Mz-AchR in myocardial cells at 7 and 14 d after microwave exposure. The results show that the expression level was higher in microwave exposure group and 0.75 g/（kg.d） ADL group than in sham operation group and significantly lower in 1.5 and 3.0 g/（kg.d） ADL groups than in microwave group. So we have a conclusion that the expression of I^z-AR and Mz-AchR is down-regulated in myocardial cells of rats exposed to microwave radiation. ADL can protect rats against microwave-induced heart tissue injury.

Effect of green flickering light on myopia development and expression of M1 muscarinic acetylcholine receptor in guinea pigs

AIM： To investigate the effects of green flickering light on refractive development and expression of muscarinic acetylcholine receptor（mAChR） M1 in the eyes of guinea pigs.METHODS： Thirty guinea pigs（15-20 days old） were randomly divided into three groups（n=10/group）. Animals in group I were raised in a completely closed carton with green flickering light illumination. Those in group II were kept in the open top closed carton under normal natural light. Guinea pigs were raised in a sight-widen cage under normal natural light in group III. The refractive status and axial length were measured before and after 8 weeks＇ illumination. Moreover, total RNA extracted from retinal, choroidal, and scleral tissues were determined by real-time reverse transcription polymerase chain reaction（RT-PCR）. The expressions of the receptor M1 were also explored in the retina, choroid, and sclera using immunohistochemistry.RESULTS： There was a remarkable reduction in refractive error and increase in axial length after 8-weeks＇ green flickering light stimulation（P〈0.001）. The expression of M1 receptor mRNA in sclera and retina in myopia group were remarkably lower than that in group II and III（P〈0.01）. Significant reduced expression of M1 receptor stimulated by green flickering light in retina and sclera tissues were also observed（P〈0.05）. However, there was no M1 receptor expression in choroid in 3 groups.CONCLUSION： Myopia can be induced by 8 weeks＇ green flickering light exposure in the animal model. M1 receptor may be involved causally or protectively in myopia development.

The Regulatory Action of Radix Astragali on M-Cholinergic Receptor of the Brain of Senile Rats

The changes in density of M-cholinergic receptors in different areas of senile rats and the regulatory action of Huang Qi ([symbol: see text] Radix Astragali, a drug for warming yang and replenishing qi) were observed by autoradiography. The results showed that the gray scale displayed in brain sections was clear and mainly distributed in the cortex, hippocampus and striate body, while that due to nonspecific combination was negligible. The gray scale in the cortex, hippocampus and striate body of the experimental group was markedly lower than that in the young control rats, decreased respectively by 24.87%, 14.12% and 12.76% (all P

Allosteric modulation of cholinergic system:Potential approach to treating cognitive deficits of schizophrenia

Schizophrenia is a psychiatric disorder affecting approximately 1% of the population worldwide and is characterised by the presence of positive and negative symptoms and cognitive deficits. Whilst current therapeutics ameliorate positive symptoms, they are largely ineffective in improving negative symptoms and cognitive deficits. The cholinergic neurotransmitter system heavily influences cognitive function and there is evidence that implicates disruption of the central cholinergic system in schizophrenia. Historically, targeting the cholinergic system has been impeded by poor selectivity leading to intolerable side effects warranting the need to develop more targeted therapeutic compounds. In this review we will summarise evidence supporting the roles of the cholinergic system, particularly the muscarinic M1 receptor, in the pathophysiology of schizophrenia and discuss the potential of a promising new class of candidate compounds, allosteric ligands, for addressing the difficulties involved in targeting this system. The body of evidence presented here highlights the dysfunction of the cholinergic system in schizophrenia and that targeting this system by taking advantage of allosteric ligands is having clinically meaningful effect on cognitive deficits.

Beauty of the beast: anticholinergic tropane alkaloids in therapeutics

Tropane alkaloids(TAs)are among the most valued chemical compounds known since pre-historic times.Poisonous plants from Solanaceae family(Hyoscyamus niger,Datura,Atropa belladonna,Scopolia lurida,Mandragora officinarum,Duboisia)and Erythroxylaceae(Erythroxylum coca)are rich sources of tropane alkaloids.These compounds possess the anticholinergic properties as they could block the neurotransmitter acetylcholine action in the central and peripheral nervous system by binding at either muscarinic and/or nicotinic receptors.Hence,they are of great clinical impor-tance and are used as antiemetics,anesthetics,antispasmodics,bronchodilator and mydriatics.They also serve as the lead compounds to generate more effective drugs.Due to the important pharmacological action they are listed in the WHO list of essential medicines and are available in market with FDA approval.However,being anticholinergic in action,TA medication are under the suspicion of causing dementia and cognitive decline like other medications with anticholinergic action,interestingly which is incorrect.There are published reviews on chemistry,biosynthesis,phar-macology,safety concerns,biotechnological aspects of TAs but the detailed information on anticholinergic mecha-nism of action,clinical pharmacology,FDA approval and anticholinergic burden is lacking.Hence the present review tries to fill this lacuna by critically summarizing and discussing the above mentioned aspects.

The use of antimuscarinics,phosphodiesterase type Ⅴ inhibitors and phytotherapy for lower urinary tract symptoms in men

Besides the mainstay of α-blockers and 5α-reductase inhibitors,other forms of medical therapy complete the armamentarium in the treatment of lower urinary tract symptoms(LUTS)in men.These treatments can target specific symptoms as well as associated symptoms that would affect the quality of life of the patients.Many patients are bothered by storage symptoms,more so than the voiding symptoms.Antimuscarinics are efficacious and safe,provided the patients do not have high post void residual urine.Many patients with LUTS also have erectile dysfunction,and phosphodiesterase type Ⅴ inhibitors are effective in relieving both LUTS as well as erectile dysfunction for such patients.Phytotherapy provides a popular and safe treatment for LUTS,however,the efficacy of the treatment has not been proven in well conducted prospective randomized controlled studies.

Effects of Cyclopentolate on Form Deprivation Myopia in Guinea Pigs

Purpose: To investigate the effects of intravitreal injection of cyclopentolate on form deprivation myopia in guinea pigs. Methods: Thirty-five guinea pigs at age of 3 weeks were randomly divided into 5 groups (n = 7 for each group): deprived, deprived plus saline, deprived plus cyclopentolate, normal control, and cyclopentolate group. Form deprivation was only performed in right eyes with translucent membranes for 4 weeks. Physiological saline and cyclopentolate were intravitreally injected into deprived eyes at four-day intervals. All the left eyes remained untreated as group control. Refraction was measured by retinoscopy after cycloplegia. The axial dimensions were measured by A-scan ultrasound. Subsequently, retinal histology was observed by light microscopy. Results: After 4 weeks of treatment, intravitreal injection of cyclopentolate significantly reduced the degree of myopia in the deprived eyes (from -3.92 D to -0.86 D, P < 0.001), and retarded the increase of vitreous chamber depth (from 3.83 ± 0.06 mm to 3.70 ± 0.05 mm, P < 0.001) and axial length (from 8.42 ± 0.04 mm to 8.30 ± 0.05 mm, P < 0.001) in the deprived eyes. Histological ex-amination revealed no evidence of retinal damage of eyes injected with physiological saline or cy-clopentolate compared with normal control eyes. Conclusions: Intravitreal administration of cy-clopentolate reduces axial elongation of the deprived eyes in guinea pigs. Further investigations are required to identify the optimal dose.

Synthesis and Bioactivity of Arecoline Derivatives Containing Amino-s-triazole Coupled Oxadiazole

5-（5＂-Substituted phenyl-[1＂,3＂,4＂]oxadiazol-2＂-yl methylsulfanyl）-3-pyridin-3′-yl- [1,2,4] triazol-4-yl amines la-j were quaternarized with methyl iodide to afford the corresponding methyl pyridinium salts 2a-j. The reduction of compounds 2 with NaBH4 in methanol gave the target compounds 5-（5＂-substituted phenyl-[1＂,3＂,4＂]oxadiazol-2＂-yl methylsulfanyl）-3-（1′-methyl-1＇,2＇,5＇,6＇-tetrahydropyridin-3＇-yl）-[1,2,4]triazol-4-ylamines 3a-j. The endothelium vascular relaxing activity of the target compounds were screened.

Muscarinic receptor-mediated calcium changes in a rat model of facial nerve nucleus injury

The muscarinic receptor modulates intracellular free calcium ion levels in the facial nerve nucleus via different channels. In the present study, muscarinic receptor-mediated free calcium ions levels were detected by confocal laser microscopy in the facial nerve nucleus following facial nerve injury in rats. There was no significant difference in muscarinic receptor expression at the affected facial nerve nucleus compared with expression prior to injury, but muscarinic receptor-mediated free calcium ion levels increased in the affected side following facial nerve injury （P 〈 0.01）. At day 30 after facial nerve injury, 50 pmol/L muscarinic-mediated free calcium ion levels were significantly inhibited at the affected facial nerve nucleus in calcium-free artificial cerebrospinal fluid, and the change range was 82% of artificial cerebrospinal fluid （P 〈 0.05）. These results suggest that increased free calcium ion concentrations are achieved by intracellular calcium ion release, and that the transmembrane flow of calcium ions is also involved in this process.

Cardiac autonomic nerve distribution and arrhythmia

OBJECTIVE： To analyze the distribution characteristics of cardiac autonomic nerves and to explore the correlation between cardiac autonomic nerve distribution and arrhythmia. DATA RETRIEVAL： A computer-based retrieval was performed for papers examining the distribution of cardiac autonomic nerves, using “heart, autonomic nerve, sympathetic nerve, vagus nerve, nerve distribution, rhythm and atrial fibrillation” as the key words. SELECTION CRITERIA： A total of 165 studies examining the distribution of cardiac autonomic nerve were screened, and 46 of them were eventually included. MAIN OUTCOME MEASURES： The distribution and characteristics of cardiac autonomic nerves were observed, and immunohistochemical staining was applied to determine the levels of tyrosine hydroxylase and acetylcholine transferase （main markers of cardiac autonomic nerve distribution）. In addition, the correlation between cardiac autonomic nerve distribution and cardiac arrhythmia was investigated. RESULTS： Cardiac autonomic nerves were reported to exhibit a disordered distribution in different sites, mainly at the surface of the cardiac atrium and pulmonary vein, forming a ganglia plexus. The distribution of the pulmonary vein autonomic nerve was prominent at the proximal end rather than the distal end, at the upper left rather than the lower right, at the epicardial membrane rather than the endocardial membrane, at the left atrium rather than the right atrium, and at the posterior wall rather than the anterior wall. The main markers used for cardiac autonomic nerves were tyrosine hydroxylase and acetylcholine transferase. Protein gene product 9.5 was used to label the immunoreactive nerve distribution, and the distribution density of autonomic nerves was determined using a computer-aided morphometric analysis system. CONCLUSION： The uneven distribution of the cardiac autonomic nerves is the leading cause of the occurrence of arrhythmia, and the cardiac autonomic nerves play an important role in the occurrence, maintenance, and symptoms of arrhythmia.

M3 Muscarinic Acetylcholine Receptor Antagonist Darifenacin Protects against Pulmonary Fibrosis through ERK/NF-κB/miR-21 Pathway

Idiopathic pulmonary fibrosis is an untreatable lethal lung disease, which is related to the aberrant proliferation of fibroblasts. M3 muscarinic acetylcholine receptor (M3-mAChR) activation exerts proliferative effect on various kinds of cells. However, whether M3-mAChR inhibition has a protective effect on pulmonary fibrosis remains unexplored. A rat model of pulmonary fibrosis was established by intratracheal instillation of bleomycin. Darifenacin was used to block M3-mAChR. Histological changes were observed using Masson’s Trichrome and hematoxylin and eosin (HE) staining. Hydroxyproline was measured by Hydroxyproline detection kit. Transforming growth factor β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). In vitro, pulmonary fibroblasts were isolated from lungs of neonatal rat. After treatment, the cell viability, Hydroxyproline level was measured by MTT and Hydroxyproline detection kit respectively. The expression level of extracellular signal-regulated kinase (ERK), nuclear factor kappa-B (N-NF-κB), and microRNA-21 (miR-21) was detected by western blot or quantitative real-time PCR (qRT-PCR). Darifenacin relieved the fibrotic effects provoked by bleomycin. The expression level of hydroxyproline, TGF-β1 and TNF-α level was all downregulated after darifenacin treatment. In lung fibroblasts, darifenacin decreased cell viability and hydroxyproline level induced by bleomycin. Besides, phosphorylation-ERK and nuclear N-NF-κB protein level was downregulated, as well as miR-21 level. M3-mAChR antagonist darifenacin attenuates bleomycin-induced pulmonary fibrosis in rats, which may relate to the ERK/NF-κB/miRNA-21 signaling pathway.

METABOLIC KINETICS OF BRAIN MUSCARINIC CHOLINERGIC RECEPTORS IN NORMAL AND HYPOTHYROID MICE

A technique for studying in vivo the production rate and turnover rate constant of mouse brain M-receptors was established. A single injection of 25 mg / kg of Benzilylcholine Mustard to living mice resulted in 90 % irreversible block of brain M-receptors. The time course of the receptor density was then monitored by 3H-QNB binding assay and the production rate and turnover rate constant were calculated from the time course curve with a computer program. It was found that in normal mice the turnover rate constant was about 0.035 h-1 (half-life was about 20 h) and the production rate was 30-42 fmol / (h ·mg protein). Parallel experiments revealed a significant slow down of the turnover of brain M-receptors in hypothyroid mice (turnover rate constant was 0.0257±0.0012 h-1 in hypothyroid vs. 0.0356±0.0021 h-1 in normal) while the production rate was not changed significantly. The results suggest that thyroid hormones have a regulatory action on the turnover of brain M-receptors and the elevation of brain M-receptor density together with slow down of the turnover of brain M- receptors is probably one of the important mechanisms relevant to the brain dysfunction in hypothyroidism.

Synthesis of Tetrahydropyridinyltriazolothiadiazines as Possible Muscarinic Agonists

Amino-5-(pyridin-3-yl)-4H-1,2,4-triazole-3-thiol 1 were condensed with 2-bromo-1- (substituted phenyl)ethanone to give pyridinyltriazolothiadiazines 2a^c, which were quaternarized with methyl iodide and oxidized with 30 % hydrogen peroxide to afford the corresponding methyl pyridinium salts 3a^c and pyridine-1-oxides 4a^c, respectively. The reduction of compounds 3 and 4 with NaBH4 in methanol produced the target compounds 1-methyl-1, 2, 5, 6-tetrahydropyridin-3- yl)-6-aryl-s-triazolothiadiazines 5a^c and 3-(1-hydroxyl-1, 2, 5, 6-tetrahydropyridin -3-yl)-6-aryl- s-triazolothiadiazines 6a^c, respectively. The endothelium vascular relaxing activity of the target compounds was screened.