The epigenetic changes are affected by sex and valproic acid treatment in a rat model of post-traumatic stress disorder.

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.

The effect of acute topiramate administration on morphine withdrawal syndrome and brain-derived neurotrophic factor in central nervous system.

OBJECTIVES: Nucleus accumbens plays an important role in opioid addiction. Topiramate, increases postsynaptic gamma-aminobutyric acid receptor activity and antagonizes glutamatergic activity. Brain-derived neurotrophic factor (BDNF), which plays a key role in synaptic plasticity, is produced from proBDNF. The aim of this study is to investigate the effects of 100 µM topiramate applied into the lateral ventricle or nucleus accumbens on naloxone-induced morphine withdrawal and the BDNF/proBDNF ratio in the frontal cortex. METHODS: In the study, 36 adult male Wistar rats weighing 250-350 g were used. Morphine dependence was created with morphine pellets following guide cannula implantations. Withdrawal findings were evaluated in naloxone-induced morphine withdrawal syndrome following topiramate administration, and locomotor activity measurements were performed simultaneously. The brains of sacrificed animals were removed for determination of BDNF/proBDNF ratio. RESULTS: Topiramate administered by either route significantly suppressed the number of jumps in morphine withdrawal. Topiramate applied into the nucleus accumbens significantly reduced stereotypical behavior in morphine withdrawal, but did not cause any changes in other locomotor activity behaviors. Topiramate applied into the lateral ventricle significantly decreased the BDNF/proBDNF ratio, whereas administered into the nucleus accumbens significantly increased this ratio. CONCLUSION: The findings of this study indicate that topiramate administered into the lateral ventricle and nucleus accumbens reduces naloxone-induced morphine withdrawal symptoms, stereotypical locomotor activity, and changes the BDNF/proBDNF ratio.

Potential antiproliferative and apoptotic effects of pilocarpine combined with TNF alpha in chronic myeloid leukemia cells.

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.

The Effect of Metformin on Ethanol- and IndomethacinInduced Gastric Ulcers in Rats.

BACKGROUND: Previous studies found metformin as an effective agent to suppress oxidative stress, inflammation, and apoptosis in various inflammatory diseases. The present study investigated the effect of metformin against 2 experimental gastric injury models in rats, using macroscopical, histopathological, biochemical, and immunostaining studies. METHODS: After 24 hours of fasting, male Sprague-Dawley rats (280-400 g) (n = 8 per group) received indomethacin (80 mg/kg; indo ulcer group) or absolute ethanol (5 mL/kg; ethanol ulcer group) or vehicle orally by gavage. Metformin (500 mg/kg) was given orally for 3 days prior to indomethacin or ethanol challenge. Ranitidine (50 mg/kg) was given orally for 3 days before indomethacin or ethanol administration as a positive control. On day 3, the animals were euthanized 6 hours after indo or 1 hour after ethanol challenge. Gastric samples were used for macroscopic scoring, histopathological examinations, and biochemical assays. Trunk blood was collected for the assessment of interleukin-1ß level. RESULTS: In both ethanol ulcer and indo ulcer groups, metformin decreased the extent of gastric lesions macroscopically and microscopically, improved the high chemiluminescence levels, and the percentage of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells compared with untreated ulcer groups. Gastric blood flow analysis revealed significant increases in both metformin-treated ulcer groups compared to untreated ulcer groups. CONCLUSION: The findings of the present work demonstrated the gastroprotective effect of metformin against the development of gastric mucosal lesions induced by ethanol and indomethacin in non-diabetic, normoglycemic rats via its antioxidant and anti-apoptotic properties and partly from its ability to restore blood flow.

Cross-talk of cholinergic and ß-adrenergic receptor signalling in chronic myeloid leukemia K562 cells.

In many studies on breast, skin and intestinal cancers, ß-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 ß-adrenergic receptors agonists and antagonists on apoptosis in chronic myeloid leukaemia cells. We tried to determine the effect of combined treatment of ß-adrenergic and cholinergic drugs on adrenergic ß1 and ß2 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.

Studies on the role of alpha 7 nicotinic acetylcholine receptors in K562 cell proliferation and signaling.

The results we obtained from this study gave information about the determination of alpha 7 nicotinic acetylcholine receptor (α7-nACh) expression in human erythroleukemia cells, as well as whether it has a role in calcium release and cell proliferation in the presence of nicotinic agonist, antagonists. Determining the roles of α7 nicotinic receptors in erythroleukemia cells will also contribute to leukemia-related signal transduction studies. This study is primarily to determine the role of nicotinic agonists and antagonists in cell proliferation, α7 nicotinic acetylcholine receptor expression, and calcium release. The aim of this study, which is a continuation and an important part of our previous studies on the cholinergic system, has contributed to the literature on the human erythroleukemia cell signaling mechanism. Cell viability was evaluated by the trypan blue exclusion test and Bromodeoxyuridine/5-Bromo-2'-deoxyuridine (BrdU) labeling. Acetylcholine, nicotinic alpha 7 receptor antagonist methyllycaconitine citrate, and cholinergic antagonist atropine were used to determine the role of α7-nACh in K562 cell proliferation. In our experiments, the fluorescence spectrophotometer was used in Ca2+ measurements. The expression of nicotinic alpha 7 receptor was evaluated by western blot. The stimulating effect of acetylcholine in K562 cell proliferation was reversed by both the α7 nicotinic antagonist methyllycaconitine citrate and the cholinergic antagonist, atropine. Methyllycaconitine citrate inhibited K562 cell proliferation partially explained the roles of nicotinic receptors in signal transduction. While ACh caused an increase in intracellular Ca2+, methyllycaconitine citrate decreased intracellular Ca2+ level in K562 cell. The effects of nicotinic agonists and/or antagonists on erythroleukemic cells on proliferation, calcium level contributed to the interaction of nicotinic receptors with different signaling pathways. Proliferation mechanisms in erythroleukemic cells are under the control of the α7 nicotinic acetylcholine receptor via calcium influx and different signalling pathway.

The Neurochemical Effects of Prazosin Treatment on Fear Circuitry in a Rat Traumatic Stress Model.

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.

Role of cholinergic agents in proliferation and caspase activity of hemin-induced erythroid differentiated K562 cells.

Background: Muscarinic receptors have many functions in the cells and tissues. Acetylcholine (ACh) plays an important role in cellular physiology. ACh also acts at the different parts of the central nervous system and nonneuronal cells. Cholinergic receptors also have different functions in many cell types and tissues. Caspases (cysteine aspartic proteases and cysteine aspartases) are cysteine dependent aspartate-specific proteases. They are an important role in necrosis and cell death and inflammation signaling pathways. They are also the primary mediators of apoptosis. During apoptosis, different caspase types participate in different functions. We have previously shown that carbachol (CCh) inhibits K562 cell proliferation. This study was performed to investigate the anti-tumor efficacy of cholinergic drugs in hemin-induced erythroid differentiated K562 cells. The aim of this study was to address the mechanism of cholinergic drugs on hemin-induced erythroid differentiated K562 cell proliferation and caspase activities. We detected M3 muscarinic receptor expression in erythroid differentiated K562 cell line.Methods: K562 cells were differentiated with hemin (50 µM). The expression of the M3 muscarinic receptor was detected by the western blotting technique. Erythroid differentiated K562 cells treated with CCh (100 µM). After 24 and 48 h, cells were counted by BrdU cell proliferation kit. Caspase 3,8, and 9 activities were measured by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions.Results: Erythroid differentiated K562 cell proliferation was not significantly increased after CCh treatment. In the meantime, caspases 8 and 9 activities in erythroid differentiated K562 cell line was significantly higher than undifferentiated K562 cells (p < .05).

The behavioral and neurochemical effects of methylprednisolone or metyrapone in a post-traumatic stress disorder rat model.

OBJECTIVE: Mechanisms contributing to the post-traumatic stress disorder (PTSD) that involve several physiological systems, 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.

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

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 24h (fasted). Fasted animals developed convulsions after being treated with scopolamine (60%) or the selective M1 receptor antagonist pirenzepine (10mg/kg; 20% and 60mg/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.

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.

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.

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.

Increased noradrenaline levels in the rostral pons can be reversed by M1 antagonist in a rat model of post-traumatic stress disorder.

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.

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.

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 G protein ß3 subunit polymorphisms C825T, C1429T, and G5177A in Turkish subjects with essential hypertension.

Hypertension is a multifactorial disorder that constitutes a major risk factor for the cardiovascular system. Heterotrimeric G-proteins, which couple receptors for diverse extracellular enzymes or ion channels, are correlated with disease mechanisms. Several studies have demonstrated an association between G protein polymorphisms and essential hypertension in some populations, although contradictive results also exist. In this study, we have investigated the potential role of the C825T, C1429T, and G5177A polymorphisms of the ß3 subunit of G-proteins in essential hypertension in a group of Turkish subjects. Genomic DNA from 106 normotensive individuals (117.4 ± 13.1, 75.2 ± 10.5; systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels, respectively) and 101 hypertensive subjects (152.3 ± 18.0, 92.5 ± 11.6; SBP and DBP levels, respectively) were studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing methods for these polymorphisms. Allele frequencies of the polymorphisms were consistent with Hardy Weinberg equilibrium, except for the C825T polymorphism (χ(2) = 7.8). The frequencies of the 825T and 1429T variants were higher in hypertensive subjects compared to those of controls. Differences between hypertensives and controls were not statistically significant, though difference was very close to significance for C825T (p = 0.056 and 0.099 for 825T and 1429T, respectively). T allele frequency in overall population showed significant association with hypertension for C825T (0.0134). The prevalence of the 5177A-variant was very low and all subjects carrying it were heterozygotes in both groups.

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 M(2) and M(3) 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 M(2), M(3), and M(4) receptor transcripts as well as M(2) and M(3) 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.

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 M(1), M(2), M(3), or M(4) 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 M(1)-M(4) receptor subtypes. RESULTS: M(2), M(3), and M(4) transcripts were detected in the following rank order: M(4) > M(2) > M(3). We were unable to demonstrate the expression of the M(1) receptor subtype in this tissue. CONCLUSIONS: Our results are in agreement with our previous binding and functional data.

Methoctramine and gallamine inhibit PI hydrolysis in guinea-pig gallbladder.

The present study aimed to determine the effect of two M2/M4-selective muscarinic receptor antagonists on blocking the hydrolysis of carbachol (CCh) stimulated phospho-inositide (PI) breakdown in order to address the possibility that a muscarinic receptor other than the M(3) receptor is involved in PI hydrolysis in this tissue. Gallbladder tissue slices labeled with myo-[2-3H] inositol were incubated with increasing concentrations of antagonists and agonist. After the reactions were terminated by the addition of chloroform/methanol, labeled inositol phosphates were separated using anion exchange chromatography. Muscarinic M2 antagonists methoctramine and gallamine both inhibited carbachol-induced PI breakdown at high concentrations, with log IC50 values of -5.145 and -6.049, respectively. Gallamine at 10(-5)M concentration failed to displace the dose-response curve for carbachol-induced accumulation of inositol triphosphate (IP3). Our data suggest that M(3) receptors play a major role in stimulation of PI hydrolysis in the guinea-pig gallbladder.