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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 (M2, M3 and M4) 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 D1 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 Dt 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 D1 expression in K562 cells. Conclusions: Our results suggest that muscarinic receptors regulate expression of c-Fos and cyclin D, genes in K562 cells via different signaling pathways. [ABSTRACT FROM AUTHOR]

Objectives: Muscarinic acetylcholine receptors play key roles in regulating many diverse physiological processes. Recent studies suggest that muscarinic receptors mediate some cellular events in hematopoietic cells. Muscarinic receptors are expressed in different human cells. cAMP an intracellular signaling molecule, is involved in a wide variety of physiological functions and stimulation of muscarinic receptors leads to the alteration of intracellular cAMP levels. The present study investigated muscarinic receptors mediated cAMP level in K562 chronic myelogenous leukemic cells. Methods: Muscarinic receptor mediated cAMP formation was measured by using the cAMP system colorimetric kit. Results: Carbachol stimulated intracellular cAMP accumulation in K562 chronic myelogenous leukemic cells. The stimulatory effect of carbachol was abolished by atropine and tropicamide, while gallamine had little effect. The calcium chelator 1,2-bis(oaminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester inhibited effects of carbachol on the production of cAMP in K562 cells. Conclusions: These results suggest that muscarinic receptor activation is linked to adenylate cyclase stimulation in K562 chronic myelogenous leukemic cells. [ABSTRACT FROM AUTHOR]

Abstract 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 M 1 and M 2 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 M 1 receptor antagonist pirenzepine (10 mg/kg; 20% and 60 mg/kg; 70%) and given food. Fasting increased expression of M 1 receptors in the frontal cortex and M 2 receptors in the hippocampus, but produced no change in the expression of both receptors in the amygdaloid complex. Food intake after fasting decreased M 1 receptor expression in the frontal cortex and M 1 and M 2 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 M 1 muscarinic receptor antagonism and fasting-induced increases in M 1 and M 2 expression possible underlying mechanism in the occurrence of convulsions in fasted animals. [ABSTRACT FROM AUTHOR]

[Display omitted] Post-traumatic stress disorder (PTSD) presents distinct sex-specific differences in both symptom expression and treatment outcomes, with the underlying biological mechanisms still remain unclear. Epigenetic modifications, particularly histone acetylation, have been increasingly recognized as critical factors in the pathophysiology of PTSD. Valproic acid (VPA), a potent histone deacetylase (HDAC) inhibitor, has shown promise in modulating epigenetic responses and improving therapeutic outcomes is PTSD, though its effect may differ between sexes. This study aimed to explore the sex-specific epigenetic changes in response to trauma and the impact of VPA treatment in a rat model of PTSD induced by predator scent stress. Sprague-Dawley rats of both sexes were randomly assigned to stressed and non-stressed groups and treated with either VPA (100 mg/kg) or vehicle. Anxiety levels were assessed using the elevated plus maze, followed by analysis of histone H3 and H4 acetylation, HDAC activity, and c-fos expression in the hippocampus. Our findings revealed that traumatic stress led to increased freezing time and anxiety levels, with more pronounced effects observed in females. Additionally, we have identified sex-specific differences in hippocampal epigenetic modifications; stressed females exhibited higher H3 acetylation, and VPA-treated stressed males showed increased H4 acetylation. These results highlight the importance of considering sex differences in the epigenetic mechanism underlying PTSD and suggest that personalized therapeutic approaches may be necessary to address these complexities. [ABSTRACT FROM AUTHOR]

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 72 h 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. [ABSTRACT FROM AUTHOR]

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<0.0001), but not in the dorsal hippocampus. Although the fluoxetine treatment reversed the apoptotic changes but propranolol failed and caused proapoptotic proteins to increase. Conclusion: These results may suggest a neuroprotective role for fluoxetine but not for propranolol. [ABSTRACT FROM AUTHOR]

In many studies on breast, skin and intestinal cancers, β‐adrenergic receptor antagonists have been shown to inhibit cell proliferation and angiogenesis and increase apoptosis in cancers. Carbachol inhibits chronic myeloid leukaemia K562 cell proliferation. Beta‐blockers are known to inhibit cell progression. The aim of this study is to explain the mechanism of action of β‐adrenergic receptors agonists and antagonists on apoptosis in chronic myeloid leukaemia cells. We tried to determine the effect of combined treatment of β‐adrenergic and cholinergic drugs on adrenergic β1 and β2 gene expression, cell proliferation and apoptosis in chronic myeloid leukaemia K562 cells. Cell proliferation was evaluated by the 5‐bromo‐2‐deoxy‐uridine (BrdU) incorporation kit. Caspase 3, 8, 9 activities were measured by the caspase assay kit. Protein expression level was detected by western blotting. We found that exposure to propranolol either by combination with carbachol facilitates additive effects on inhibition of caspase 3 and 8 expression in chronic myeloid leukaemia K562 cells. However, caspase 9 expression level was increased by propranolol alone or with propranolol and carbachol combination. The combined therapy of cholinergic and adrenergic receptor drugs will decrease cell proliferation in K562 cells. This decrease in cell proliferation may be mediated by the mitochondrial‐dependent intrinsic apoptosis pathway. [ABSTRACT FROM AUTHOR]

Abstract: 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 10min and the protocol was repeated 1 week later with a trauma reminder (clean litter). The rats also received intraperitoneal fluoxetine (2.5, 5 or 10mg/kg/day), propranolol (10mg/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 M1, M2, M3, M4 and M5 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±12s, 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 M2 and M5 expression in the frontal cortex. Expression of M1 receptors increased and M4 subtype decreased in the hippocampus. In the amygdaloid complex of rats, we also detected a down-regulation of M4 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objectives: Activation of neuronal nitric oxide synthase (nNOS) and interrelated alterations of calmodulin and ionotropic glutamate receptor (GluN1) levels are unknown in post traumatic stress disorder (PTSD). Materials and Methods: Sprague-Dawley rats of both sexes were exposed to dirty cat litter, and then placed on an elevated plus maze. An anxiety index was calculated and tissue samples from hippocampus and amygdala were prepared in order to detect calmodulin, NOS and GluN1 by immunoblotting. Results: The anxiety indices of the traumatized rats were markedly higher than those of the controls (p<0.05). GluN1 and calmodulin levels were decreased in the dorsal hippocampus and amygdaloid complex of the traumatized rats. NOS expression increased significantly in both the amygdaloid complex and dorsal hippocampus where the increase was statistically more prominent in the amygdaloid complex (p< 0.001) than in the dorsal hippocampus of the traumatized rats (p<0.05). Conclusion: Predator exposure in rats causes long-lasting anxiogenic effects associated with increases in NOS and decreases in GluN1 expressions in brain areas related to PTSD symptoms and excitotoxicity. The results suggest that excitotoxicity occurs through other mechanisms rather than GluN1 receptors. [ABSTRACT FROM AUTHOR]

The dysregulation of hypothalamic-pituitary-adrenal axis and noradrenergic, serotonergic and glutamatergic systems are thought to be involved in the pathophysiology of post-traumatic stress disorder. The effect of selective M1 muscarinic receptor antagonist, pirenzepine on anxiety indices was investigated by using elevated plus maze, following exposure to trauma reminder. Upon receiving the approval of ethics committee, Sprague-Dawley rats were exposed to dirty cat litter (trauma) for 10 min and 1 week later, the rats confronted to a trauma reminder (clean litter). The rats also received intraperitoneal pirenzepine (1 or 2 mg/kg/day) or saline for 8 days. Noradrenaline (NA) concentration in the rostral pons was analyzed by HPLC with electrochemical detection. The anxiety indices of the rats subjected to the trauma reminder were increased when compared to control rats ( p < 0.05). Pirenzepine treatment in traumatized rats displayed similar anxiety indices of non-traumatized rats treated with physiological saline. Although freezing time was prolonged with pirenzepine in traumatized groups the change was not found statistically significant. The NA level was 1.5 ± 0.1 pg/mg in non-traumatized rats and increased to 2.4 ± 0.2 pg/mg in traumatized rats. Bonferroni post hoc test revealed that the NA content of the rostral pons of the traumatized rats treated with physiological saline was significantly higher than the content of other groups ( p < 0.01). We conclude that NA content in the rostral pons increases in respect to confrontation to a trauma reminder which can be reversed by M1 antagonist pirenzepine indicating the roles of M1 receptors. [ABSTRACT FROM AUTHOR]