Membrane phospholipid polar heads influence the coupling of M2 muscarinic receptors to G proteins

Treating membranes from rat heart with phospholipase C (phosphatidylcholine choline-phosphohydrolase) fromClostridium perfringens increased the affinity of muscarinic acetylcholine receptors (M₂) for the agonists carbachol and oxotremorine. The affinity for antagonists was not affected. Phospholipase C activity, i.e., the cleavage of polar heads of membrane phospholipids, led to the disappearance of the guanine nucleotide-dependent rightward shift of the isotherm for agonist binding. The treatment of tracheal smooth muscle with phospholipase C led to a decrease in the maximum contractile effect of muscarinic (M₂) stimulation with no modification of the agonist EC₅₀, i.e., to the uncoupling of the stimulation-contraction process. These results demonstrate that when phospholipid polar heads are hydrolysed by phospholipase C, M₂ receptors are uncoupled from G proteins, which enhances their affinity for agonists but prevents information transfer.     

Acetylcholine stimulates cyclic ADP-ribose formation via M1 muscarinic receptors in rat superior cervical ganglion

The role of cyclic ADP-ribose (cADPR) as the downstream signal of neuronal muscarinic acetylcholine receptors (mAChRs) and the enzyme responsible for its synthesis, ADP-ribosyl cyclase, were examined in the rat superior cervical ganglion (SCG). Application of acetylcholine or other mAChR agonists increased the ADP-ribosyl cyclase activity by about 250-300% in crude membrane fractions from the SCG of 14-day-old rats. This effect was inhibited by atropine or by the M1-mAChR antagonist, pirenzepine, and was mimicked by GTP. These results indicate that the M1 mAChRs couple to the membrane-bound form of ADP-ribosyl cyclase and suggest that cADPR is a second messenger of M1 mAChR signaling in nervous tissue.     

Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast

The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.     

Iron increases liver injury through oxidative/nitrative stress in diabetic rats: Involvement of nitrotyrosination of glucokinase

Excessive tissue iron levels are associated with the increase of oxidative/nitrative stress which contributes to tissue damage that may elevate the risk of diabetes. Therefore, we investigated the effects of iron on diabetes-associated liver injury and whether iron-related tyrosine nitration participated in this process. Rats were randomly divided into four groups: control, iron overload (300 mg/kg iron dextran, i.p.), diabetic (35 mg/kg of streptozotocin i.p. after administration of a high-fat diet) and diabetic simultaneously treated with iron. Iron supplement markedly increased diabetes-mediated liver damage and hepatic dysfunction by increasing liver/body weight ratio, serum levels of aspartate and alanine aminotransferase, and histological examination, which were correlated with elevated levels of lipid peroxidation, protein carbonyls and tyrosine nitration, oxidative metabolism of nitric oxide, and reduced antioxidant capacity. Consequently, the extent of oxidized/nitrated glucokinase was markedly increased in the iron-treated diabetic rats that contribute to a decrease in its expression and activity. Further studies revealed a significant contribution of iron-induced specific glucokinase nitration sites to its inactivation. In conclusion, iron facilitates diabetes-mediated elevation of oxidative/nitrative stress, simultaneously impairs liver GK, and can be a link between enzymatic changes and hepatic dysfunction. These findings may provide new insight on the role of iron in the pathogenesis of diabetes mellitus.     

No change in cortical muscarinic M2, M3 receptors or [35S]GTPgammaS binding in schizophrenia

Muscarinic M1, but not M4, receptors have been shown to be decreased in Brodmann's area (BA) 9 obtained postmortem from subjects with schizophrenia. This study extends that data by measuring levels of muscarinic M2 and M3 receptor protein and mRNAs in BA 9 and BA 40 from the same cohorts of subjects used in the study of M1 and M4 receptors. In addition, the ability of carbachol to stimulate muscarinic receptors that signal through the Gi/o G-proteins was measured in BA 9 from the same cohorts of subjects. There were no changes in levels of muscarinic M2 or M3 protein or M3 mRNA with diagnosis in either CNS region. M2 receptor mRNA could not be detected in BA 9 or BA 40. Finally, carbachol-stimulated GTPgammaS binding did not differ between the diagnostic cohorts in BA 9 (p = 0.64). These data add considerable weight to the argument that the muscarinic M1 receptor is the muscarinic receptor predominantly affected in BA 9 by the pathology of schizophrenia. Given the widespread changes in muscarinic receptors identified in the CNS of subjects of schizophrenia using functional neuroimaging it remains possible that receptors other than the M1 receptor may be altered in different CNS regions.     

Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol

Recent studies indicate that membrane cholesterol can associate with G protein-coupled receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 cholecystokinin receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 cholecystokinin receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wild-type and CCK2R/CCK1R chimeric receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs.     

Resveratrol, a red wine polyphenol, attenuates ethanol-induced oxidative stress in rat liver

The involvement of oxidative stress in the pathogenesis of alcoholic diseases in the liver has been repeatedly confirmed. Resveratrol, a natural phytoalexin present in grape skin and red wine possesses a variety of biological activities including antioxidant. This study was conducted to evaluate whether resveratrol has a preventive effect on the main indicators of hepatic oxidative status as an expression of the cellular damage caused by free radicals, and on antioxidant defence mechanism during chronic ethanol treatment. Wistar rats were treated daily with 35% ethanol solution (3 g/kg/day i.p.) during 6 weeks and fed basal diet or basal diet containing 5 g/kg resveratrol. Control rats were treated with i.p. saline and fed basal diet. Experimentally, chronic ethanol administration leads to hepatotoxicity as monitored by the increase in the level of hepatic marker enzymes and the appearance of fatty change, necrosis, fibrosis and inflammation in liver sections. Ethanol also enhanced the formation of MDA in the liver indicating an increase in lipid peroxidation, a major end-point of oxidative damage, and caused drastic alterations in antioxidant defence systems. Particularly the activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were found reduced by ethanol treatment while glutathione reductase (GR) activity was unchanged. Dietary supplementation with resveratrol during ethanol treatment inhibited hepatic lipid peroxidation and ameliorated SOD, GPx and CAT activities in the liver. Conclusively, we can suggest that resveratrol could have a beneficial effect in inhibiting the oxidative damage induced by chronic ethanol administration, which was proved by the experiments that we conducted on rats.     

P2X7 receptor deletion attenuates oxidative stress and liver damage in sepsis

Purinergic Signalling - Sepsis is a severe disease characterized by an uncontrolled systemic inflammation and consequent organ dysfunction generated in response to an infection. Extracellular ATP...     

Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation

Knowledge of early molecular events occurring upon ischemia/reperfusion (I/R) during liver transplantation (LT) is of great importance to improve the therapeutic intervention of surgical treatment. However, nowadays, few data are available on early protein targets of I/R injury. To identify these proteins, we used a differential proteomics approach in the characterization human liver biopsies during I/R upon LT. Analyses were performed on nine donor livers during LT. By using 2-DE and MALDI-TOF MS, we identified 36 proteins which resulted significantly altered upon I/R injury. The majority of these proteins are functionally involved in lipid and energy metabolism, in different metabolic pathways, in redox signalling and in oxidative-stress response. Our data represent the first global approach in the study of I/R injury in liver.     

The oxidative stress response in Bacillus subtilis

Abstract Bacillus subtilis undergoes a typical bacterial stress response when exposed to low concentrations (0.1 mM) of hydrogen peroxide. Protection is thereby induced against otherwise lethal, challenge concentrations (10 mM) of this oxidant and a number of proteins are induced including the scavenging enzymes, catalase and alkyl hydroperoxide reductase, and a putative DNA binding and protecting protein. Induced protection against higher concentrations (10–30 mM) of hydrogen peroxide is eliminated in a catalase-deficient mutant. Both RecA and Spo0A influence the basal but not the induced resistance to hydrogen peroxide. A regulatory mutation has been characterized that affects the inducible phenotype and is constitutively resistant to high concentrations of hydrogen peroxide. This mutant constitutively overexpresses the proteins induced by hydrogen peroxide in the wild-type. The resistance of spores to hydrogen peroxide is partly attributable to binding of small acid soluble proteins by the spore DNA and partly to a second step which coincides with the depletion of the NADH pool, which may inhibit the generation of hydroxyl radicals from hydrogen peroxide.     

Neuronal oxidative stress in acute ischemic stroke: Sources and contribution to cell injury

Oxidative stress has emerged as a key deleterious factor in brain ischemia and reperfusion. Malfunction of the oxidative respiratory chain in mitochondria combines with the activation of cytoplasmic oxidases to generate a burst of reactive oxygen species that cannot be neutralised by the cell’s antioxidant mechanisms. As a result, oxidative stress contributes directly to necrosis and apoptosis through a number of pathways in ischemic tissue. Pharmacological intervention with antioxidants or enhancers of endogenous antioxidant molecules is proving to be difficult due to the speed and scope of the oxidative impact. Additionally, the knowledge that neuronal fate in ischemic stroke is tightly linked to other brain cells like endothelial cells and astrocytes has shifted the focus of study from isolated neurons to the neurovascular unit. For this reason, recent efforts have been directed towards understanding the sources of oxidative stress in ischemic stroke and attempting to block the generation of oxygen radicals.     

Melatonin reduces oxidative stress induced by ochratoxin A in rat liver and kidney

Melatonin (MEL) displays antioxidant and free radical scavenger properties. In the present study, the effect of MEL on the oxidative stress induced by ochratoxin A (OTA) administration in rats was investigated. Four groups of 15 rats each were used: controls, MEL-treated rats (5 mg/kg body mass), OTA-treated rats (250 μg/kg) and MEL+OTA-treated rats. After 4 weeks of treatment, the levels of malondialdehyde (MDA), a lipid peroxidation product (LPO) were measured in serum and homogenates of liver and kidney. Also, the levels of glutathione (GSH), and activities of glutathione reductase (GR), glutathione peroxidase (GSPx), superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) in liver and kidney were determined. In OTA-treated rats, the levels of LPO in serum and in both liver and kidney were significantly increased compared to levels in controls. Concomitantly, the levels of GSH and enzyme activities of SOD, CAT, GSPx and GR in both liver and kidney were significantly decreased in comparison with controls. In rats received MEL+OTA, the changes in the levels of LPO in serum and in liver and kidney were not statistically significant compared to controls. Concomitantly, the levels of GSPx, GR and GST activities in both liver and kidney tissues were significantly increased in comparison with controls. Similar increases in GSPx, GR and GST activities were also observed in MEL-treated rats when compared with controls. In conclusion, the oxidative stress may be a major mechanism for the toxicity of OTA. MEL has a protective effect against OTA toxicity through an inhibition of the oxidative damage and stimulation of GST activities. Thus, clinical application of melatonin as therapy should be considered in cases of ochratoxicosis.     

The C-Terminal Domain of the mGluR1 Metabotropic Glutamate Receptor Affects Sensitivity to Agonists

Abstract: The metabotropic glutamate receptor (mGluR) subtype 1 exists as at least three variants (−1a, −1b, and −1c) generated by alternative splicing at the C-terminal domain. Fluorometric Ca²⁺ measurements were used to compare the concentration dependency of agonist-induced rises in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in human embryonic HEK 293 cells transiently expressing rat mGluR1a, mGluR1b, or mGluR1c. The rank order of agonist potencies was quisqualate ≫ (2 S, 1' S, 2' S )-2-(carboxycyclopropyl)glycine (L-CCG-I) > (1 S, 3 R )-1-aminocyclopentane-1,3-dicarboxylic acid [(1 S, 3 R )-ACPD] and did not differ among the splice variants. However, agonists were consistently more potent at mGluR1a than at mGluR1c and mGluR1b. In the same system, we characterized the agonist pharmacology of two chimeric rat mGluR3/1 receptors where the first and/or the second intracellular loop(s) and the C-terminal domain were exchanged with the corresponding mGluR1a or mGluR1c sequences and that were previously shown to mediate elevations in [Ca²⁺]_i in response to agonists. The potency of agonists was higher at the chimera having the C-terminus of mGluR1a as compared with those having the mGluR1c C-terminus. Both chimeric mGluR3/1 receptors had the same rank order of agonist potencies: L-CCG-I ≫ (1 S, 3 R )-ACPD ∼ quisqualate. These data support the hypothesis that the C-terminal domain of mGluRs plays a role in determining the potency of agonists for inducing mGluR-mediated functional responses.     

Deoxamuscaroneoxime derivatives as useful muscarinic agonists to explore the muscarinic subsite: demox, a modulator of orthosteric and allosteric sites at cardiac muscarinic M2 receptors

A series of muscarinic agonists, straight chained, branched, cyclic alkyl and aromatic derivatives of the oxime 1 (demox) was designed with the aim of investigating their activity on muscarinic receptor subtypes. Effects on M1 receptor were assessed functionally by a microphysiometer apparatus, while M2, M3, and M4 receptor potency and affinity were studied on isolated preparations of guinea pig heart, ileum, and lung, respectively. The results suggest that the substitution of a hydrogen with a long side-chain or bulky group generally induces a decrease in potency at M1 and M3 subtypes, while a general increase in this parameter is obtained at M2 subtype. Among the agonists 2-18, compound 4 behaves as a full agonist with a preference for M3 subtype. Moreover, compound 12 is inactive at M1 and M4 receptors while it displays a full agonist activity at M2 and M3 subtypes. Since demox displays a variable response on cardiac M2 receptors regulating heart force, an in-depth inquiry of the functional behaviour of this compound was carried out at M2 receptors. In presence of 10(-11) and 10(-10) M demox, the binding of [3H]-NMS was increased by approximately 30% as a consequence of an increase of the association of [3H]-NMS to membranes; this effect was not observed in presence of a higher concentration of [3H]-NMS. Higher concentrations of demox decreased the binding of [3H]-NMS to heart atrial membranes but significantly retarded the dissociation of this radioligand. Our results suggest that demox may interact with orthosteric and allosteric sites of atrial M2 muscarinic receptor.     

Comparative study of oxidative stress parameters and acetylcholinesterase activity in the liver of Pelophylax esculentus complex frogs

Comparative activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), the phase II biotransformation enzyme glutathione-S-transferase (GST), the concentrations of total glutathione (GSH), sulfhydryl groups (–SH) and the activity of the neurotoxicity biomarker acetylcholinesterase (AChE) were investigated in the livers of species belonging to the Pelophylax esculentus “complex” (parental species Pelophylax ridibundus, Pelophylax lessonae, and their hybrid Pelophylax kl. esculentus) from the wetland, Obedska bara in Serbia. The condition factor (CF) and hepato somatic index (HSI) were also calculated. All three species were caught at same locality and were exposed to the same environmental conditions. Liver SOD activity was lower in P. ridibundus than in P. kl. esculentus and P. lessonae; higher activities of CAT, GR and GST were observed in P. kl. esculentus frogs as compared to their parental species. The activity of GSH-Px was significantly lower in P. kl. esculentus. The activity of AChE was increased in P. lessonae as compared to P. kl. esculentus and P. ridibundus. Similar concentrations of GSH and SH groups were observed in all investigated species. P. kl. esculentus had a higher CF, while the HSI was lower when compared to the parental species. Our findings suggest that the parental species (P. ridibundus and P. lessonae) possess more similar antioxidative responses to environmental conditions than the hybrid species P. kl. esculentus. The obtained results improve our understanding of the biology and physiology of these three closely related species.     

The Ccz1 mediates the autophagic clearance of damaged mitochondria in response to oxidative stress in Candida albicans

Autophagy plays a critical role in response to numerous cellular stresses, such as nutrient deprivation, hypoxia, starvation and organelle damage. The disruption of autophagy pathway affects multiple aspects of cellular stress response. Here we for the first time identified Ccz1 as an essential component for autophagy in Candida albicans. Our experiments demonstrated that loss of CCZ1 gene led to vacuolar fragmentation and disruption of the autophagy pathway. Our results also suggested that Ccz1 functioned in oxidative stress. In the ccz1Δ/Δ mutant, the levels of reactive oxidative species (ROS) sharply increased under H₂O₂ treatment. Further studies demonstrated that breakdown of the autophagic clearance pathway led to the accumulation of oxidative stress-damaged mitochondria, and consequently elevated cellular ROS levels in the ccz1Δ/Δ mutant. Furthermore, deletion of CCZ1 led to a significant defect in filamentous development at both 30 °C and 37 °C. The disruption of CCZ1 gene led to decreased capacity of macrophage killing and increased sensitivity to the macrophages. In addition, the ccz1Δ/Δ mutant exhibited attenuated virulence and decreased fungal burdens in the mouse systemic infection model, indicating that CCZ1 might provide a promising target for antifungal drugs development. In summary, our findings provide new insights into the understanding of autophagy-related gene in C. albicans.