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© 2023, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.Pilocarpine is a selective M1/M3 agonist of muscarinic acetylcholine receptor subtypes. Muscarinic acetylcholine receptors are G protein-coupled receptors. These receptors are different drug targets. The aim of the present work was to investigate the effect of pilocarpine on the expression of M3 muscarinic acetylcholine receptor, the AChE activity, IL-8 release response, and proliferation in K562 cells, via muscarinic receptor activation. Human chronic myeloid leukemic cell cultures were incubated with drugs. Proliferation assays were performed by BrdU assay. Expression of M3 muscarinic acetylcholine receptor and apoptosis proteins such as bcl, bax, cyt C, and caspases was assessed with the semiquantitative Western blotting method. Pilocarpine inhibits chronic myeloid cell proliferation and M3 muscarinic acetylcholine receptor protein expression. Pilocarpine increases caspase-8 and -9 expression levels, upregulating the proapoptotic protein Bax and downregulating the expression levels of the antiapoptotic protein Bcl-2. The apoptotic activity of pilocarpine is associated with an increase in AChE activity. M3 muscarinic acetylcholine receptors can activate multiple signal transduction systems and mediate inhibitory effects on chronic myeloid K562 cell proliferation depending on the presence of 1% FBS conditions. This apoptotic effect of pilocarpine may be due to the concentration of pilocarpine and the increase in AChE level. Our results suggest that inhibition of cell proliferation by inducing apoptosis of pilocarpine in K562 cells may be one of the targets. M3 selective agonist may have therapeutic potential in chronic myeloid leukemia. Graphical Abstract: [Figure not available: see fulltext.].

OBJECTIVE: Mechanisms contributing to the post-traumatic stress disorder (PTSD) that involve several physiological sys- tems, and the activation of the hypothalamic-pituitary-adrenal axis (HPA) is one of the most known systems in the PTSD pathophysiology. The present study investigates the potential effects of methylprednisolone, metyrapone and their association with the noradrenergic system within the rostral pons, a region containing the locus coeruleus (LC) in a rat model of PTSD induced with predator scent. METHODS: In this study, Sprague-Dawley rats were exposed to the stress by exposure to the scent of dirty cat litter, which is a natural stressor of a predator. One week later, the rats were re-exposed to a situational reminder (clean cat litter). The rats were treated using either methylprednisolone, metyrapone or physiological saline before exposure to a situational reminder (n=8 in each group). Noradrenaline (NA) levels in the rostral pons homogenates were analysed using ELISA. RESULTS: The anxiety indices of the rats exposed to the trauma were found to be significantly higher than the anxiety indices of the control rats. Metyrapone produced a significant increase in the anxiety indices of the non-stressed rats, and methylprednisolone did not produce a change in the anxiety indices of the non-stressed rats. Methylprednisolone treatment suppressed the anxiety in the stressed rats. Metyrapone treatment increased the anxiety indices in the stressed rats but still being lower than that of the saline-treated stressed rats. Significant decrease in the freezing time was observed following the methylprednisolone treatment both in the stressed and non-stressed rats. NA content in the rostral pons of the stressed rats was significantly higher than that of the non-stressed rats. Methylprednisolone or metyrapone treatments decreased the NA content in the non-stressed rats as compared to the saline treatment. However, these decreases were not significant. CONCLUSION: In this study, findings suggest that stress may give rise to endocrine, autonomic and behavioural responses. The anxiety indices and NA levels in the rostral pons increased with the traumatic event. The methylprednisolone treatment may suppress anxiety through interactions between the LC and the HPA axis.

Objective The timing of administration of pharmacologic agents is crucial in traumatic stress since they can either potentiate the original fear memory or may cause fear extinction depending on the phase of fear conditioning. Brain noradrenergic system has a role in fear conditioning. Data regarding the role of prazosin in traumatic stress are controversial. Methods In this study, we examined the effects of prazosin and the noradrenergic system in fear conditioning in a predator stress rat model. We evaluated the direct or indirect effects of stress and prazosin on noradrenaline (NA), gamma-aminobuytyric acid (GABA), glutamate, glycine levels and choline esterase activity in the amygdaloid complex, the dorsal hippocampus, the prefrontal cortex and the rostral pons. Results Our results demonstrated that prazosin might alleviate defensive behaviors and traumatic stress symptoms when given during the traumatic cue presentation in the stressed rats. However prazosin administration resulted in higher anxiety levels in non stressed rats when the neutral cue was presented. Conclusion Prazosin should be used in PTSD with caution because prazosin might exacerbate anxiety in non-traumatized subjects. However prazosin might as well alleviate stress responses very effectively. Stress induced changes included increased NA and GABA levels in the amygdaloid complex in our study, attributing noradrenaline a possible inhibitory role on fear acquisition. Acetylcholine also has a role in memory modulation in the brain. We also demonstrated increased choline esterase acitivity. Cholinergic modulation might be another target for indirect prazosin action which needs to be further studied.

Amaç: Muskarinik reseptörler merkezi sinir sistemindeasetilkolinin çeşitli etkilerine aracılık ettiği gibi, parasempatik sinirsistemi ile etkileşen sinirsel olmayan dokulara da aracılık ederler.Çalışmamızda, M3 muskarinik reseptör alttipinin K562 kanserhücre çoğalması ve ölümündeki rolünü belirlemeye çalıştık.Gereçler ve Yöntemler: Hücre çoğalması bromodeoksiüridin(BrDU) yöntemi ile belirlenmiştir. Hücre ölümü, erken ve geçapoptoz Anneksin V ve propidyum iodid (PI) varlığında akışsitometrisi yöntemi ile gösterilmiştir. Nuklear dış sinyal düzenleyicikinaz (ERK/fosfo-ERK) ekspresyonu western emdirimi yöntemiile belirlenmiştir.Bulgular: Çalışmamızda 48 saat karbakol(CCh) ile muameleedilen K562 hücre sayısında azalma belirlenmiştir. Hücreler24 saat CCh ile muamele edildiklerinde erken apoptotik hücresayısında azalma gözlemlenirken geç apoptoz ve nekrotik hücresayısında değişim olmamıştır. Bununla birlikte, 48 saat boyuncaCCh ile muamele olan hücrelerde, erken ve geç apoptotik hücresayısı azalırken, nekrotik hücrelerin sayısı artmıştır. CCh ilemuamele edilen hücrelerde nüklear ERK ekspresyonu artarkenbu etki 1,1-dimetil-4-difenilasetoksipiperidinium iodid (4DAMP)ile geri çevrilmiştir. Aynı koşullarda CCh ile muamele edilenhücrelerde nuklear pERK ekspresyonu azalmış, bu etki 4DAMPile geri çevrilmemiştir.Sonuç: Bulgularımız, K562 hücre proliferasyonundakikolinerjik etkinin yalnızca muskarinik mekanizma ile değildiğer kolinerjik reseptörlerin de katkısıyla gerçekleştiğinidüşündürmektedir., Objectives: Muscarinic receptors mediate diverse actions ofacetylcholine in the central nervous system and in non-nervoustissues innervated by the parasympathetic nervous system.Our study aims to evaluate the potential association of theM3 muscarinic receptor with K562 cell proliferation and death.Materials and Methods: Cell proliferation was evaluatedby bromodeoxyuridine (BrDU) incorporation. To show early,late apoptosis and cell death, cells were labelled with AnnexinV, propidium iodide (PI) and analyzed by flow cytometry. Nuclearextracellular signal-regulated kinase (ERK/pERK) expressionwas measured by western blot analysis.Results: Treatment with carbachol (CCh) for 48h decreased cellnumber. Exposing K562 cells to CCh for 24h decreased the number ofearly apoptotic cells but did not change the number of late apoptotic andnecrotic cells. CCh treatment for 48h increased the number of necroticcells, but decreased the number of early and late apoptotic cells. Inresponse to CCh, nuclear ERK expression was increased and this effectwas reversed by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide(4DAMP). Nuclear pERK expression was decreased in CCh treatedcells, 4DAMP did not reverse the effect.Conclusion: Our data suggest that cholinergic agonist CChaffects cell proliferation in K562 cells not only through muscarinicreceptors but also through other cholinergic receptors.

Objective: The purpose of this study was to investigate ifstimulating auto-paracrine muscarinic receptor signalling pathwaycould change human erythroleukemia K562 cell proliferation andcaspase 3, 8 and 9 expression levels. To better understand the role ofmuscarinic receptors in cell signalling mechanism, we investigatedthe effects of several compounds on human erythroleukemiaK562 cell proliferation and caspase 3, 8 and 9 expression. Thesecompounds were M3 muscarinic receptor agonist, pilocarpine, proinflammatorycytokine, tumor necrosis factor (TNF)-alpha, andthe wortmannin which is a phosphoinositide 3-kinase inhibitor.Materials and Methods: Cell proliferation and cell viabilitywere evaluated by the trypan blue exclusion test and 5-Bromo-2-deoxy-uridine (BrdU) Labelling and Detection Kits. Caspase 3, 8and 9 expression levels were determined by immunoblot analysis.Results: Both pilocarpine and TNF-alpha caused a small increasein human erythroleukemia K562 cell proliferation. However, whenall the compounds were treated together, proliferation of humanerythroleukemia K562 cells increased significantly when compared tountreated control cells. TNF-alpha and wortmannin treatment increasedcaspase 3 and caspase 8 expression patterns significantly in humanerythroleukemia K562 cells. TNF-alpha and wortmannin treatmentincreased caspase 9 expression level (P>0.05) but it was not significant.Conclusion: These findings partly demonstrated that M3muscarinic receptor mediated an increase in K562 cell proliferation.Pilocarpine prevented TNF-alpha and wortmannin inducedcaspase 3 and 8 expression and indirectly showed apoptosis inhuman erythroleukemia K562 cells., Amaç: Bu çalışmanın amacı, otoparakrin M3 muskarinikreseptör sinyal yolağının uyarılmasının, insan eritrolösemi K562hücrelerinin çoğalmasında ve kaspaz 3, 8 ve 9 ekspresyon seviyeleriüzerinde etkisi olup olmadığını araştırmaktır. Hücre sinyalileti mekanizmasında muskarinik reseptörlerin rolünü daha iyianlamak üzere, çeşitli bileşiklerin insan eritrolösemisi K562 hücreproliferasyonu ve kaspaz 3, 8 ve 9 ekspresyon seviyeleri üzerindekietkilerini araştırdık. Bu bileşikler, M3 muskarinik reseptör agonisti,pilokarpin, pro-enflamatuvar sitokin, tümör nekroz faktör (TNF)-alfa ve fosfoinositid 3-kinaz inhibitörü wortmanindir.Gereçler ve Yöntemler: Hücre çoğalması ve hücre canlılığı,tripan mavisi testi ve 5-Bromo-2-deoxy-uridine (BrdU) İşaretlemeve Belirleme kitleri ile değerlendirildi. Kaspaz 3, 8 ve 9 ekspresyonseviyeleri immunoblot analizi ile belirlendi.Bulgular: Hem pilokarpin, hem de TNF-alfa, insan eritrolösemiK562 hücre çoğalmasında çok az artışa neden oldu. Ancak, tümbileşikler birlikte muamele edildiğinde, insan eritrolösemi K562hücrelerinin çoğalması, muamele edilmeyen kontrol hücrelerinegöre anlamlı olarak arttı. TNF-alfa ve wortmanin ile muameleedilen K562 hücrelerinde kaspaz 3 ve kaspaz 8 ekspresyonuseviyelerinde anlamlı değişim belirlendi. TNF-alfa ve wortmanninmuamelesi kaspaz 9 ekspresyon seviyesini arttırdı (P> 0.05) ancakanlamlı değildi.Sonuç: Bu bulgular, kısmen M3 muskarinik reseptör aracılıK562 hücre çoğalmasında artış olduğunu göstermektedir.Pilokarpin, insan eritrolösemi K562 hücrelerinde, TNF-alfa vewortmanin ile uyarılan kaspaz 3 ve 8 ekspresyonunu önledi vedolaylı olarak apoptozu gösterdi.

Objective: The B-cell lymphoma/leukemia-2 (Bcl-2) family of proteins governs mitochondrial membrane permeability where the programmed apoptotic process is controlled by the balance between proapoptotic (Bax) and antiapoptotic (Bcl-2) proteins. We aimed to investigate the [Bcl-2]/[Bax] in different brain regions in a post-traumatic stress disorder rat model. Methods: Female Sprague-Dawley rats were exposed to dirty cat litter (trauma) for 10 min and the protocol was repeated 1 week later with a trauma reminder (clean litter) in reversed 12 h light/dark cycle. The rats received intraperitoneal saline, fluoxetine (2.5 mg/kg/day) or propranolol (10 mg/kg/day) for 7 days between exposure sessions. Following exposure to the trauma reminder, elevated plus maze experiments were done. Immunoblotting was used to quantify [Bcl-2] and [Bax] proteins in the homogenates of the dorsal hippocampus, the frontal cortex and the amygdaloid complex. Results: Fluoxetine reversed the increases in the anxiety indices and the freezing times. In the amygdaloid complex and the frontal cortex, the [Bcl-2]/[Bax] decreased in the traumatized control rats significantly (p

Programlanmış hücre ölümü olan apoptoz birçok fizyolojik süreçte aktif rol oynamaktadır. Apoptozun, büyüme faktörlerinin eksikliği, DNA hasarı ve birden fazla faktörü içeren çeşitli hücresel stresle aktive olan ‘hücre içi’ ve ölüm reseptörlerine ligandın bağlanmasıyla kaspazların aktive olduğu ‘hücre dışı’ olmak üzere iki yolağı vardır. Apoptotik hücre sayısı ile organizmanın sağlıklı olup olmadığı belirlenir. Apoptoz oranının azalması hücre sayısını arttırırken, apoptoz oranının artması hücre sayısını azaltarak dokularda tahribata neden olmaktadır. Apoptotik sinyallemede düzensizlik çeşitli hastalıklarda/bozukluklarda primer ya da sekonder rol oynamaktadır. Son yıllarda apoptozun nörodejeneratif hastalıklarla ilgili çalışmaları ön plana çıkmaya başlamıştır. Apoptotik sinyal yolaklarının daha iyi tanımlanması, pro- ve anti-apoptotik genlerin belirlenmesiyle, çalışmalar hız kazanmıştır. Travma Sonrası Stres Bozukluğu gibi nörodejeneratif bozukluklarda beyindeki yapısal ve fonksiyonel değişiklikler mitokondriyal stres ile ilişkilidir. Fizyolojik koşullarda hayati öneme sahip olan apoptoz, patolojik koşullarda mekanizmanın tetiklenmesine ve kontrolsüz hücre çoğalmasına yol açmaktadır. Hücre ölümünü engelleyen terapötik ilaçların geliştirilmesiyle apoptoz aracılı nörodejenaratif bozuklukların tedavisine yeni umutlar oluşacaktır., Apoptosis is programmed cell death, which actively occurs in many physiological processes. It can be triggered in two ways: (i) defects in growth factor, DNA damage, and other many factors that can cause cellular stress, which is an intracellular pathway, and (ii) ligand binding to death receptors and activation of caspases. The apoptotic cell count can be determined by the health of the whole organism. A higher apoptotic ratio can indicate a decrease in the number of cells and tissue damage, while a lower apoptotic ratio can indicate an increase in the number of cells. Irregularity in apoptotic signals can play primary or secondary roles in various diseases/disorders. Research on apoptosis depends on neurodegeneration has been initiated in the past few years. Definition of apoptotic signal pathways and apoptotic regulation and determination of pro- and anti-apoptotic genes are the main topics that have accelerated research on apoptosis. Neurodegenerative disorders such as post-traumatic stress disorder neuronal damage associated with changes in brain structure and function may be related to the mitochondrial stresses. In physiological conditions, apoptosis is crucial for the organism, while in pathological conditions, apoptosis can cause uncontrolled cell division. Development of therapeutic medicine that inhibits the cell death may be the new choice of treatment for neurodegenerative diseases/disorders.

Objectives: Muscarinic acetylcholine receptors (mAChR) belong to a family of G protein coupled receptors (GPCRs). These mAChRs regulate several important physiological functions by activating a wide variety of cellular signaling pathways. We have previously shown that muscarinic acetylcholine (M-2, M-3 and M-4) receptors are expressed in K562 cells. In this study, we investigated the effect of muscarinic agonist, antagonists and different signaling pathway inhibitors on c-Fos and cyclin D-1 transcripts, using reverse transcriptase polymerase chain reaction (RT-PCR) that allows changes of very rare transcripts to be monitored. Material and Methods: Total RNA was prepared from K562 cells challenged with muscarinic agonist, antagonists and inhibitors. c-Fos and cyclin D-1 expression were determined by RT-PCR. Results: We showed that treatment with muscarinic agonist, antagonists and inhibitors leads to changes in c-Fos and cyclin D-1 expression in K562 cells. Conclusions: Our results suggest that muscarinic receptors regulate expression of c-Fos and cyclin D-1 genes in K562 cells via different signaling pathways.

Amac: Post travmatik stres bozuklugunda (PTSD) iyonotropik glutamat reseptor (GluN1)’lerinin (GluN1) ve kalmodulinin birlikte etki ederek nitrik asit sentaz (NOS) aktivasyonu yaptigi bilinmemektedir.Gerec ve Yontem: Her iki cinsiyetten Sprague-Dawley sicanlari kirli kedi kumuyla saldirgan hayvan kokusuna maruz birakildiktan sonra, sicanlara yukseltilmis t labirenti deneyleri uygulanmistir. Deney sonrasi sicanlarin anksiyete endeksleri hesaplanmis ve kalmodulin, NOS ve GluN1 olcumleri icin hipokampus ve amigdala doku ornekleri Western Blot yontemi icin hazirlanmistir.Bulgular: Travmatize edilen sicanlarin anksiyete indeksleri kontrol grubuna gore anlamli olarak yuksek bulunmustur (p < 0.05). Dorsal hipokampus ve amygdaloid komplekste travmatize sicanlarin GluN1 ve kalmodulin duzeyleri azalmistir. NOS ekspresyonu her iki bolgede artis gostermesine karsin, amigdaloid kompleksteki artis (p < 0.001) dorsal hipokampuse gore (p < 0.05) istatiksel olarak daha anlamli bulunmustur.Sonuc: Saldirgan hayvan kokusu maruziyeti sicanlarda, PTSD semptomlari ve eksitotoksisite ile iliskili beyin alanlarinda nNOS ekspresyonunda artma ve GluN1 reseptorlerinde azalma ile birlikte uzun sure devam eden anksiyete iliskili etkilere neden olmaktadir. Sonuclarimiz, NOS aktivitesinin GluN1 disindaki nedenlerle arttigini gostermektedir