Acute effects of methcathinone and manganese in mice: A dose response study

An intravenously injectable illicit drug made by mixing pseudoephedrine, potassium permanganate, vinegar and water, yielding methcathinone (Mcat) and manganese (Mn), induces an extrapyramidal syndrome with parkinsonism, dystonia, gait and balance disorders similar to manganism. Although the cause of the syndrome is largely attributed to Mn, the interaction of the drug's individual components is not known and the role of Mcat is possibly underestimated. Aim of the present study was to analyze dose-dependent behavioral effects of the mixture and its two main active components Mcat and Mn in an acute setting and determine the lethal doses of each substance. Three groups of C57BL/6 mice were injected intraperitoneally with (1) the drug mixture containing 10, 25, 50, 100 or 150 mg of Mcat and respectively 1.6, 3.8, 6.9, 17.1 and 22.6 mg of Mn per kilogram of body weight; (2) 10, 25, 50, 100, 150, 200 or 300 mg of racemic Mcat/kg of body weight; (3) MnCl2 10, 25 or 50 mg/kg of body weight. Locomotor activity of the animals, various signs and time of death were recorded. Lower doses (10 and 25 mg/kg) of Mcat had a clear motor activity stimulating effect and this was clearly dose-dependent. High doses of Mcat produced epileptic seizures in 74% of the animals and became lethal with the highest doses. Similarly, the mixture had a clear dose-dependent stimulating effect and the higher doses became lethal. The LD50 of the pseudoephedrine mixture was 110.2 mg of Mcat/kg and for pure Mcat 201.7 mg/kg. Mn did not prove to be lethal in doses up to 50 mg/kg, but had a strong dose dependent inhibitory effect on the animals’ behavior. Our data reveal that both Mn and Mcat have a significant role in the toxicity of the mixture

Coaggregation, cointernalization, and codesensitization of adenosine A2A receptors and dopamine D2 receptors

Antagonistic and reciprocal interactions are known to exist between adenosine and dopamine receptors in the striatum. In the present study, double immunofluorescence experiments with confocal laser microscopy showed a high degree of colocalization of adenosine A(2A) receptors (A(2A)R) and dopamine D(2) receptors (D(2)R) in cell membranes of SH-SY5Y human neuroblastoma cells stably transfected with human D(2)R and in cultured striatal cells. A(2A)R/D(2)R heteromeric complexes were demonstrated in coimmunoprecipitation experiments in membrane preparations from D(2)R-transfected SH-SY5Y cells and from mouse fibroblast Ltk(-) cells stably transfected with human D(2)R (long form) and transiently cotransfected with the A(2A)R double-tagged with hemagglutinin. Long term exposure to A(2A)R and D(2)R agonists in D(2)R-cotransfected SH-SY5Y cells resulted in coaggregation, cointernalization and codesensitization of A(2A)R and D(2)R. These results give a molecular basis for adenosine-dopamine antagonism at the membrane level and have implications for treatment of Parkinson's disease and schizophrenia, in which D(2)R are involved

Controlling complexity: the clinical relevance of mouse complex genetics.

Experimental animal models are essential to obtain basic knowledge of the underlying biological mechanisms in human diseases. Here, we review major contributions to biomedical research and discoveries that were obtained in the mouse model by using forward genetics approaches and that provided key insights into the biology of human diseases and paved the way for the development of novel therapeutic approaches

Dopamine D2 receptor G protein coupling and it´s regulation

Dopamine (DA) receptors belong to the superfamily of G protein coupled receptors. The D2 DA receptor is negatively coupled with adenylate cyclase via pertussis toxin sensitive (Gi/o) G proteins. In the brain the D2 DA receptor is mainly expressed in the caudate-putamen, the nucleus accumbens and the olfactory tubercle but also in the substantia nigra and the ventral tegmental area. The nigrostriatal dopaminergic system innervates the dorsal striatum. Most of the dorsal striatal D2 DA receptors are localized on the cell bodies of striatopallidal inhibitory GABAergic neurons that send their projections to the globus pallidus. DA D2 agonists inhibit striatopallidal GABA-ergic neurons thereby increasing motor activity since in this way the indirect pathway of the basal ganglia mediating motor inhibition will be inhibited. Degeneration of the nigrostriatal DA neurons causes a marked decrease in striatal DA levels and inhibition of motor functions. These changes are associated with a marked supersensitivity development in the striatal D2 receptors. In a hemiparkinsonian rat model with 6-hydroxydopamine (6-OHDA) induced unilateral lesions of the nigrastriatal DA system D2 agonists induce a strong contralateral rotational behaviour in very low doses due to preferential activation of the supersensitive D2 receptors on the DA denervated side. The aim of this study was to characterize in the above model potential changes in the coupling of the D2 receptor to the Gi/o protein that may play a role in the D2 receptor supersensitivity development. [35S]-guanosine 5'-0-(gamma-thio) triphosphate ([35S]GTP-gamma-S) binding as a method for direct visualization of G protein activation by receptors was adapted to study D2short receptors and their signalling in CHO cells transfected with the D2 receptor. Pharmacological characterization of 18 dopaminergic ligands revealed a good correlation of potencies of all ligands to modulate [35S]GTP-gamma-S binding with their potencies to inhibit [3H]raclopride binding. Also efficacies of dopaminergic ligands at D2 receptor were characterized. It was found that serotonin and other serotoninergic agonists have partial agonistic activity at the D2 receptor expressed in CHO cells. Studies on [35 [35S]GTP-gamma-S, [3 H]DA and [3H]-raclopride binding were used to investigate the cross regulation between G proteins and D2 DA receptors expressed in CHO cells. The obtained results indicate that not only analogues of GTP but a] so GDP and GMP turned D2 DA receptors into a low affinity state for DA. On the other hand, activation of the D2 receptor by DA caused a decrease in the binding affinity for GDP, but not for analogues of GTP. According to these results, the high-affinity state of agonist binding can be achieved only when no nucleotides are bound in the agonist receptor-G protein complex. The role of G-proteins in D2 receptor supersensitivity was studied in striatal membranes from rats with unilateral 6OHDA induced lesions of the nigrostriatal DA cells. The number of [ 3 H]raclopride binding sites was increased in the DA denervated striatum, but no changes in ligand binding affinities and in proportion of high-affinity agonist binding sites could be detected. The number and the affinity of [35S]GTP-gamma-S binding sites was unaltered after the striatal DA denervation, whereas the binding affinity of GDP was decreased in the DA denervated versus the intact striatum. It is proposed that the decrease in GDP binding affinity to D2 DA receptor-coupled G proteins is an important factor in D2 receptor supersensitivity appearing after degeneration of the striatal DA terminals. DSP4 induced lesions of locus coeruleus (LC) noradrenergic neurons influence the ascending mesencephalic DA systems by reducing striatal DA turnover and inducing behavioural supersensitivity to dopaminergic drugs, the latter effect being similar to that observed after the loss of striatal DA terminals. The density of striatal D2 receptors was increased following DSP4 treatment as also is the case after DA denervation of the striatum. In contrast, such NA lesions had no effect on D2 receptor G protein coupling as found after DA denervation. It is known that Ca2+/calmodulin suppresses the D2 receptor signaling by interacting with the calmodulin binding motif of the D2 receptor located in the N-terminal part of the third intracellular loop of the D2 receptor. This motif is also part of the A2A/D2 heterodimer interface and A2A strongly antagonizes the D2 signalling within the A2A/D2 heterodimer. It is demonstrated that in the A2A/D2 cotransfected, but not in the D2 alone transfected CHO cells, expressing endogenous calmodulin, Ca2+ substantially increases the basal and DA stimulated [35S]GTP-gamma-S binding. These results may be explained on the basis of a competition between calmodulin and A2A for their overlapping binding motifs at the D2 receptor. The results illustrate the dynamic interplay of A2A/D2 heterodimers and the D2 interacting protein in control of the D2 signalling

Hypothalamic gene expression profile indicates a reduction in G protein signaling in the <i>Wfs1</i> mutant mice

The Wfs1 gene codes for a protein with unknown function, but deficiency in this protein results in a range of neuropsychiatric and neuroendocrine syndromes. In the present study we aimed to find the functional networks influenced by Wfs1 in the hypothalamus. We performed gene expression profiling (Mouse Gene 1.0 ST Arrays) in Wfs1-deficient mice; 305 genes were differentially expressed with nominal P value &lt; 0.01. FDR (false discovery rate)-adjusted P values were significant (0.007) only for two genes: C4b (t=9.66) and Wfs1 ( t = −9.03). However, several genes related to G protein signaling were very close to the FDR-adjusted significance level, such as Rgs4 (regulator of G protein signaling 4) that was downregulated (−0.34, t = −5.4) in Wfs1-deficient mice. Changes in Rgs4 and C4b expression were confirmed by QRT-PCR. In humans, Rgs4 is in the locus for bipolar disease (BPD), and its expression is downregulated in BPD. C4b is a gene related to the neurodegenerative diseases. Functional analysis including the entire data set revealed significant alterations in the canonical pathway “G protein-coupled receptor signaling.” The gene expression profile in the hypothalami of the Wfs1 mutant mice was significantly similar to the profiles of following biological functions: psychological disorders, bipolar disorder, mood disorder. In conclusion, hypothalamic gene expression profile resembles with some molecular pathways functionally related to the clinical syndromes in the Wolfram syndrome patients. </jats:p

Effect of Chronic Valproic Acid Treatment on Hepatic Gene Expression Profile in Wfs1 Knockout Mouse

Valproic acid (VPA) is a widely used anticonvulsant and mood-stabilizing drug whose use is often associated with drug-induced weight gain. Treatment with VPA has been shown to upregulate Wfs1 expression in vitro. Aim of the present study was to compare the effect of chronic VPA treatment in wild type (WT) and Wfs1 knockout (KO) mice on hepatic gene expression profile. Wild type, Wfs1 heterozygous, and homozygous mice were treated with VPA for three months (300 mg/kg i.p. daily) and gene expression profiles in liver were evaluated using Affymetrix Mouse GeneChip 1.0 ST array. We identified 42 genes affected by Wfs1 genotype, 10 genes regulated by VPA treatment, and 9 genes whose regulation by VPA was dependent on genotype. Among the genes that were regulated differentially by VPA depending on genotype was peroxisome proliferator-activated receptor delta (Ppard), whose expression was upregulated in response to VPA treatment in WT, but not in Wfs1 KO mice. Thus, regulation of Ppard by VPA is dependent on Wfs1 genotype

The Expression of RAAS Key Receptors, Agtr2 and Bdkrb1, Is Downregulated at an Early Stage in a Rat Model of Wolfram Syndrome

Wolfram syndrome (WS) 1 is a rare monogenic neurodegenerative disorder caused by mutations in the gene encoding WFS1. Knowledge of the pathophysiology of WS is incomplete and to date, there is no treatment available. Here, we describe early deviations in the renin-angiotensin-aldosterone system (RAAS) and bradykinin pathway (kallikrein kinin system, KKS) observed in a rat model of WS (Wfs1 KO) and the modulative effect of glucagon-like peptide-1 receptor agonist liraglutide (LIR) and anti-epileptic drug valproate (VPA), which have been proven effective in delaying WS progression in WS animal models. We found that the expression of key receptors of the RAAS and KKS, Agtr2 and Bdkrb1, were drastically downregulated both in vitro and in vivo at an early stage in a rat model of WS. Moreover, in Wfs1, KO serum aldosterone levels were substantially decreased and bradykinin levels increased compared to WT animals. Neither treatment nor their combination affected the gene expression levels seen in the Wfs1 KO animals. However, all the treatments elevated serum aldosterone and decreased bradykinin in the Wfs1 KO rats, as well as increasing angiotensin II levels independent of genotype. Altogether, our results indicate that Wfs1 deficiency might disturb the normal functioning of RAAS and KKS and that LIR and VPA have the ability to modulate these systems

Energy Metabolic Plasticity of Colorectal Cancer Cells as a Determinant of Tumor Growth and Metastasis

Metabolic plasticity is the ability of the cell to adjust its metabolism to changes in environmental conditions. Increased metabolic plasticity is a defining characteristic of cancer cells, which gives them the advantage of survival and a higher proliferative capacity. Here we review some functional features of metabolic plasticity of colorectal cancer cells (CRC). Metabolic plasticity is characterized by changes in adenine nucleotide transport across the outer mitochondrial membrane. Voltage-dependent anion channel (VDAC) is the main protein involved in the transport of adenine nucleotides, and its regulation is impaired in CRC cells. Apparent affinity for ADP is a functional parameter that characterizes VDAC permeability and provides an integrated assessment of cell metabolic state. VDAC permeability can be adjuste

Early Intervention and Lifelong Treatment with GLP1 Receptor Agonist Liraglutide in a Wolfram Syndrome Rat Model with an Emphasis on Visual Neurodegeneration, Sensorineural Hearing Loss and Diabetic Phenotype

Wolfram syndrome (WS), also known as a DIDMOAD (diabetes insipidus, early-onset diabetes mellitus, optic nerve atrophy and deafness) is a rare autosomal disorder caused by mutations in the Wolframin1 (WFS1) gene. Previous studies have revealed that glucagon-like peptide-1 receptor agonist (GLP1 RA) are effective in delaying and restoring blood glucose control in WS animal models and patients. The GLP1 RA liraglutide has also been shown to have neuroprotective properties in aged WS rats. WS is an early-onset, chronic condition. Therefore, early diagnosis and lifelong pharmacological treatment is the best solution to control disease progression. Hence, the aim of this study was to evaluate the efficacy of the long-term liraglutide treatment on the progression of WS symptoms. For this purpose, 2-month-old WS rats were treated with liraglutide up to the age of 18 months and changes in diabetes markers, visual acuity, and hearing sensitivity were monitored over the course of the treatment period. We found that treatment with liraglutide delayed the onset of diabetes and protected against vision loss in a rat model of WS. Therefore, early diagnosis and prophylactic treatment with the liraglutide may also prove to be a promising treatment option for WS patients by increasing the quality of life