Hülya Cabadak

The change in muscarinic receptor subtypes in different brain regions of rats treated with fluoxetine or propranolol in a model of post-traumatic stress disorder

This study shows the possible contribution of muscarinic receptors in the pathophysiology of post-traumatic stress disorder. Sprague-Dawley rats of both sexes were exposed to dirty cat litter (trauma) for 10 min and the protocol was repeated 1 week later with a trauma reminder (clean litter). The rats also received intraperitoneal fluoxetine (2.5, 5 or 10 mg/kg/day), propranolol (10 mg/kg/day) or saline for 7 days between two exposure sessions. Functional behavioral experiments were performed using elevated plus maze, following exposure to trauma reminder. Western blot analyses for M(1), M(2), M(3), M(4) and M(5) receptor proteins were employed in the homogenates of the hippocampus, the frontal cortex and the amygdaloid complex. The anxiety indices increased from 0.63±0.02 to 0.89±0.04 in rats exposed to the trauma reminder. The freezing times were also recorded as 47±6 and 133±12 s, in control and test animals respectively. Fluoxetine or propranolol treatments restored the increases in the anxiety indices and the freezing times. Female rats had higher anxiety indices compared to males. Western blot data showed increases in M(2) and M(5) expression in the frontal cortex. Expression of M(1) receptors increased and M(4) subtype decreased in the hippocampus. In the amygdaloid complex of rats, we also detected a down-regulation of M(4) receptors. Fluoxetine and propranolol only corrected the changes occurred in the frontal cortex. These results may imply that muscarinic receptors are involved in this experimental model of post-traumatic stress disorder.

Regulation of M2, M3, and M4 muscarinic receptor expression in K562 chronic myelogenous leukemic cells by carbachol

Context: Muscarinic receptors mediate a variety of cellular responses to acetylcholine, including inhibition of adenylate cyclase, breakdown of phosphoinositide and modulation of ion channels. These receptors are relatively abundant in the central nervous system and peripheral parasympathetic nervous system. Many cells express a mixture of muscarinic receptor transcripts. Changes in muscarinic M2 and M3 receptor mRNA levels in response to agonist treatment have been reported in cerebellar granule cells, Chinese hamster ovary cells, lymphocytes and in the human neuroblastoma cell line SH-SY5Y. Objective: In this study, we investigated the effects of agonist stimulation on cell proliferation and on the levels of muscarinic receptor expression in K562 chronic myelogenous leukemia cells. Methods: Total RNA and crude membrane fractions were prepared from K562 cells challenged with carbachol (CCh). Muscarinic receptor subtype expression was determined by RT-PCR and western blot analysis. Proliferation and cell viability were evaluated by the trypan blue exclusion test and BrDU labeling. Results: We showed that CCh-treatment leads to changes in muscarinic M2, M3, and M4 receptor transcripts as well as M2 and M3 protein levels. We also found that CCh decreased proliferation of K562 cells in a time dependent manner, an effect prevented by atropine. These results suggest that CCh modulates K562 chronic myelogenous leukemic cells proliferation through muscarinic acetylcholine receptors.

D-Cycloserine acts via increasing the GluN1 protein expressions in the frontal cortex and decreases the avoidance and risk assessment behaviors in a rat traumatic stress model.

D-cycloserine (DCS), an FDA approved anti-tuberculosis drug has extensively been studied for its cognitive enhancer effects in psychiatric disorders. DCS may enhance the effects of fear extinction trainings in animals during exposure therapy and hence we investigated the effects of DCS on distinct behavioral parameters in a predator odor stress model and tested the optimal duration for repeated daily administrations of the agent. Cat fur odor blocks were used to produce stress and avoidance and risk assessment behavioral parameters were used where DCS or saline were used as treatments in adjunct to extinction trainings. We observed that DCS facilitated extinction training by providing further extinction of avoidance responses, risk assessment behaviors and increased the contact with the cue in a setting where DCS was administered before extinction trainings for 3 days without producing a significant tolerance. In amygdala and hippocampus, GluN1 protein expressions decreased 72h after the fear conditioning in the traumatic stress group suggesting a possible down-regulation of NMDARs. We observed that extinction learning increased GluN1 proteins both in the amygdaloid complex and the dorsal hippocampus of the rats receiving extinction training or extinction training with DCS. Our findings also indicate that DCS with extinction training increased GluN1 protein levels in the frontal cortex. We may suggest that action of DCS relies on enhancement of the consolidation of fear extinction in the frontal cortex.

Muscarinic receptor-mediated nitric oxide release in a K562 erythroleukaemia cell line

1 In the present study we have investigated the expression of muscarinic receptors in K562 erythroleukaemic cells and the effects of muscarinic agonist and antagonists on extracellular citrulline levels in these cells, as a marker of nitric oxide (NO) generation. 2 Muscarinic acetylcholine receptors (M(1)-M(5)) play key roles in regulating many diverse physiological processes. Recent studies suggest that muscarinic receptors mediate some cellular events in haematopoietic cells. Multiple subtypes of muscarinic receptors are expressed in different human cells. NO, a free radical and a signaling molecule, is involved in the regulation of many physiological functions and derived from certain nitric oxide synthases (NOS), which are related to muscarinic receptors. 3 In this study, the presence of M(2), M(3) and M(4) subtypes in K562, an erythroleukaemic cell line, was demonstrated by using the reverse transcriptase-polymerase chain reaction. Moreover, the generation of NO induced by carbachol, a non-selective muscarinic agonist, was investigated by using high-performance liquid chromatography to measure changes in extracellular l-citrulline levels. 4 We found that carbachol enhanced l-citrulline production in K562 erythroleukaemic cells. The effect of carbachol on l-citrulline production was antagonized by atropine and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), while tropicamide had little effect. These results suggest that the muscarinic receptor M(3) subtype may mediate NO signaling in K562 erythroleukaemic cells.

Investigation of the Association Between Dopamine D1 Receptor Gene Polymorphisms and Essential Hypertension in a Group of Turkish Subjects

Dopamine has been shown to influence blood pressure by regulating renal sodium excretion through direct interaction with the dopamine receptors, especially with the Dopamine D1 receptor (DRD1). To better understand the role of polymorphisms in those effects, we investigated the association between two polymorphic sites in the DRD1 promoter region (A–48G, G–94A) and essential hypertension in the Turkish population. The DRD1 variants were genotyped by restriction fragment length polymorphism (RFLP) analysis. A total of 205 unrelated individuals were enrolled in the study. We found that genotype distributions and allele frequencies of the control and hypertensive subjects were very similar and did not show any significant difference with respect to blood pressure (BP) and hypertension. Contribution of the gene variances in BP or hypertension by sex differences and dependence on body mass index (BMI) were also evaluated. Distribution of genotypes and allele frequencies were found to be in line with previous reports. However, increments detected in hypertensive subjects were far from being statistically significant.

The role of intracellular pathways in the proliferation of human K562 cells mediated by muscarinic receptors.

Muscarinic acetylcholine receptors (mAChRs) are members of the superfamily of G protein coupled receptors (GPCRs). Muscarinic receptors are relatively abundant in the central nervous system and in the peripheral parasympathetic nervous system. Several studies have suggested that muscarinic receptors also mediate some cellular events in hematopoietic cells. K562 erythroleukemia cells contain muscarinic receptors M2, M3 and M4, and activation of muscarinic receptors changes cell proliferation. We examined the effects of several compounds on cell proliferation in K562 erythroleukemia cells. These included a muscarinic receptor agonist carbachol (CCh), a protein kinase inhibitor staurosporine; the phospholipase C inhibitor U73122, the MEK 1-2 inhibitor UO126, the PI3-kinase inhibitor wortmannin, the Ca(2+) chelators BAPTA/AM and 2-aminoethoxy-diphenylborate (2APB). In addition, we also investigated muscarinic receptor mediated protein kinase C (PKC) expression in K562 cells. CCh caused a decrease in DNA synthesis in K562 cells supplemented with 1% fetal bovine serum after starvation. Pre-treatment of K562 cells with U73122 and BAPTA/AM antagonized the inhibitory effect of CCh, suggesting that phospholipase C and intracellular calcium are involved in CCh-mediated inhibition of proliferation in K562 cells. Our data also suggest that the regulatory roles of protein kinase C and the MAPK/ERK pathways in K562 cell proliferation are independent of cholinergic activation.

Contribution of M1 and M2 muscarinic receptor subtypes to convulsions in fasted mice treated with scopolamine and given food

&NA; Treatment of fasted mice and rats with the nonselective muscarinic antagonist, scopolamine or atropine, causes convulsions after food intake. This study evaluated the effect of fasting on the expression of M1 and M2 muscarinic receptors in the brain regions, the relationship between receptor expression and seizure stages, and the muscarinic receptor subtype which plays a role in the occurrence of convulsions. Mice were grouped as allowed to eat ad lib (fed) and deprived of food for 24 h (fasted). Fasted animals developed convulsions after being treated with scopolamine (60%) or the selective M1 receptor antagonist pirenzepine (10 mg/kg; 20% and 60 mg/kg; 70%) and given food. Fasting increased expression of M1 receptors in the frontal cortex and M2 receptors in the hippocampus, but produced no change in the expression of both receptors in the amygdaloid complex. Food intake after fasting decreased M1 receptor expression in the frontal cortex and M1 and M2 receptor expression in the hippocampus. Seizure severity was uncorrelated with muscarinic receptor expression in the brain regions. Taken together, these findings provide evidence for the role of M1 muscarinic receptor antagonism and fasting‐induced increases in M1 and M2 expression possible underlying mechanism in the occurrence of convulsions in fasted animals.

M2, M3, and M4 muscarinic receptors are expressed in the guinea pig gallbladder

Aim: The identity of muscarinic acetylcholine receptors (mAchR) involved in cholinergic-mediated contraction of the guinea pig gallbladder has been a matter of debate. Different groups have suggested the involvement of M1, M2, M3, or M4 receptor subtypes in the contraction of this tissue. The objective of this study was to identify the mAchR subtypes expressed in the guinea pig gallbladder by RT-PCR. Methods: Total RNA prepared from frozen guinea pig gallbladder tissue was amplified by using specific primers for the M1–M4 receptor subtypes. Results: M2, M3, and M4 transcripts were detected in the following rank order: M4 > M2 > M3. We were unable to demonstrate the expression of the M1 receptor subtype in this tissue. Conclusions: Our results are in agreement with our previous binding and functional data.

MP58-15 M2 AND M3 MUSCARINIC RECEPTOR EXPRESSION IN BLADDER TUMOR

INTRODUCTION AND OBJECTIVES: High T cell receptor (TCR) repertoire clonality is associated with clinical response to immune checkpoint blockade in bladder cancer (Funt et al ASCO 2016). We hypothesized that T cell repertoire is more clonal in tumors than in benign inflammation. METHODS: After obtaining IRB approval, we prospectively identified n1⁄412 patients with bladder lesions at Montefiore Medical Center undergoing transurethral resection. Specimens were collected at time of transurethral resection and frozen and stored at -80C. DNA was extracted and high throughput DNA sequencing of the CDR3 region of the TCR beta chain using the immunoSEQ assay (Adaptive Biotechnologies) was performed. T cell fraction, clonal dominance, and maximum frequency of TCR clone were assessed. RESULTS: There was an even distribution of specimens across all pathologic stages: 3/12 were T0, 3/12 were Ta, 3/12 were T1, and 3/ 12 were T2 or greater. The median number of productive TCR rearrangements sequenced in each sample in urothelial carcinoma specimens (UCþ) and benign (UC-) specimens was 5,569 and 25,872 respectively. The median number of unique TCR rearrangements sequenced in UCþ and UCspecimens was 3,069 and 9,680, respectively. The median tumor infiltrating lymphocyte (TIL) percentage in UCþ and UCspecimens was 2% and 12%, respectively. The UCþ specimens demonstrated clonality as evidenced by identification of a specific T cell clone being present in up to 17% of the total TIL pool; in contrast, the frequency of the max T cell clone was 2% in UCspecimens (Figure 1). CONCLUSIONS: Primary urothelial tumors contain clonally expanded T cell populations that are not present in benign urothelial inflammation. These data support the hypothesis that bladder tumors induce an antigen driven immunogenic host response, in contrast to the benign inflammatory response, which does not appear to demonstrate any T cell clonal dominance. Future studies should be aimed to validate these results and to identify which tumor derived antigens are of clinical significance.

Investigation of the roles of non-neuronal acetylcholine in chronic myeloid leukemic cells and their erythroid or megakaryocytic differentiated lines

BACKGROUND Many studies suggested that Acetylcholine (ACh) might serve as an autocrine/ paracrine growth factor in several types of tumors or tumor cell lines. High levels of Acetylcholinesterase (AChE) activity have been reported in primary brain tumors, ovarian, colon and lung tumors. OBJECTIVES The role of cholinergic signaling needs to be clarified in in leukemia. METHOD K562 cells were derived from a chronic myelogenous leukemia patient during blast crisis serving as pluripotent hematopoietic stem cells. K562 cells were incubated with various cholinergic agonists or antagonists to investigate the role of ACh in different differentiated cell lines. RESULTS Our experiments showed that AChE activity was increased in response to ACh in undifferentiated K562 cells, but in the erythroid differentiated K562 cells a high concentration of ACh (1 mM) decreased the AChE activity. ACh failed to elevate the AChE activity in the megakaryocytic differentiated K562 cells. An AChE inhibitor, eserine, also suppressed the AChE activity in a concentration-dependent manner. Choline uptake inhibition by hemicholinium did increase the AChE activity but not in the erythroid differentiated K562 cell line. Likewise, megakaryocytic differentiated K562 cells also displayed a similar pattern. Vesamicole, a vesicular choline uptake inhibitor, produced similar results. Curare, a nicotinic antagonist, elevated the cell counts of the megakaryocytic differentiated cells. CONCLUSION Our findings may suggest excess extracellular ACh will decrease the cell growth in undifferentiated and megakaryocytic differentiated K562 cell lines through nicotinic type cholinoceptors.