OBJECTIVETo explore the effect of organophosphorus insecticides (OPs) on G protein-coupled receptor kinase 2 mediated phosphorylation of M2 muscarinic receptors in vitro and to understand an alternative target of the OPs for human and other animals.

METHODSThe acetylcholine M2 muscarinic receptors (mAChR2) were purified from rat brain by single step affinity chromatography. In vitro experiments, the purified mAChR2, G-protein coupled receptor kinase 2 (GRK2) and the (gamma-p32) labeled ATP were incubated with paraoxon (PO), chlorpyrifos oxon (CPO) or chlorpyrifos (CPF) of varying concentrations. The proteins were separated by the polyacrylamide gel electrophoresis. The gels were dried and the phosphorylation of mAChR2 was detected with autoradiograms. Bands containing M2 receptor were excised and counted by liquid scintillation.

RESULTSCPO inhibited phosphorylation of M2 muscarinic receptors by GRK2 with a median inhibition concentration (IC(50)) at 70 micromol/L. CPF also inhibited M2 receptors phosphorylation, but was less potent and less efficacious than that of CPO. PO and parathion (PT) had little effect on the receptor phosphorylation under the same conditions. CPO and CPF didn't inhibit the beta2 Adrenalin (beta2-AR) receptor phosphorylation also mediated by GRK2.

CONCLUSIONCPO and CPF can selectively inhibit the GRK2 mediated mAChR2 phosphorylation while PO and PT have no this effect.

Animals ; Chlorpyrifos ; analogs & derivatives ; toxicity ; Cholinesterase Inhibitors ; toxicity ; G-Protein-Coupled Receptor Kinase 2 ; Paraoxon ; toxicity ; Phosphorylation ; Rats ; Receptor, Muscarinic M2 ; metabolism ; beta-Adrenergic Receptor Kinases ; metabolism ; physiology

OBJECTIVETo explore the correlation of lymphocyte G protein-coupled receptor kinases 2 (GRK2) expression of the very elderly with chronic heart failure (HF) and heart ejection fraction (EF).

METHODS16 elderly patients with chronic heart failure were divided into 2 groups as following: EF < 45% (n=7), EF > or = 45% (n=9); and health elderly as control (n=8). Lymphocytes were obtained from blood, reverse transcription polymerase chain reaction were used to measure GRK2 mRNA levels.

RESULTSLymphocyte GRK2 mRNA levels of EF < 45% group were higher than that of EF > 45% group, which were greater than that of control.

CONCLUSIONElevation of lymphocyte GRK2 levels in HF is associated with heart EF, lymphocytes may provide a surrogate for monitoring cardiac GRK2 in human HF.

Aged, 80 and over ; Chronic Disease ; G-Protein-Coupled Receptor Kinase 2 ; genetics ; metabolism ; Heart Failure ; blood ; physiopathology ; Humans ; Lymphocytes ; metabolism ; Male ; RNA, Messenger ; genetics ; metabolism ; Stroke Volume ; physiology

OBJECTIVETo investigate the effect of metoprolol on the expression of G protein-coupled receptor kinases 2 (GRK2) in lymphocyte of advanced elderly patients with chronic heart failure.

METHODS32 elderly patients with chronic heart failure were divided into control group and metoprolol group, 16 each. Conventional therapy was used in the control group, conventional therapy plua metoprolol was used in metoprolol group. The treatment courses were 8 weeks in both groups.

RESULTSLeft ventricular end-diastolic diameter and left ventricular ejection fraction were not different between the two groups. Lymphocyte GRK2 mRNA level in metoprolol group was lower than that in control group.

CONCLUSIONMetoprolol can inhibit the expression of GRK2 in lymphocyte of advanced elderly patients with chronic heart failure.

Aged, 80 and over ; Chronic Disease ; G-Protein-Coupled Receptor Kinase 2 ; blood ; genetics ; metabolism ; Heart Failure ; metabolism ; Humans ; Lymphocytes ; metabolism ; Metoprolol ; pharmacology

The effects of glucagon and epinephrine on gluconeogenesis in young (4 month) and old (24 month) Fisher 344 rat hepatocytes were compared. In contrast to glucagon, which had a similar effect on gluconeogenesis in both young and old cells, epinephrine caused a smaller increase in gluconeogenesis in old rat hepatocytes than in young hepatocytes. beta2 adrenergic receptor (beta2-AR) expression slightly decreased in aged rat liver, and there were differences between young and old hepatocytes in their patterns of G protein coupled receptor kinases, which are involved in the activation of beta2-AR receptor signal desensitization. The major isoform of the kinase changed from GRK2 to GRK3 and the expression of beta-arrestin, which is recruited by the phosphorylated beta2-AR for internalization and degradation, increased in aged rat liver. GRK3 overexpression also decreased the glucose output from young rat hepatocytes. We conclude that an age-associated reduction in epinephrine-induced gluconeogenesis occurs through the epinephrine receptor desensitizing system.
Adrenergic beta-Agonists/*pharmacology ; Aging/*drug effects ; Animals ; Epinephrine/*pharmacology ; G-Protein-Coupled Receptor Kinase 2/metabolism ; G-Protein-Coupled Receptor Kinase 3/metabolism ; Glucagon/pharmacology ; *Gluconeogenesis/drug effects ; Male ; Models, Biological ; Phosphorylation ; Rats ; Rats, Inbred F344 ; Receptors, Adrenergic, beta-2/agonists/metabolism

The effects of glucagon and epinephrine on gluconeogenesis in young (4 month) and old (24 month) Fisher 344 rat hepatocytes were compared. In contrast to glucagon, which had a similar effect on gluconeogenesis in both young and old cells, epinephrine caused a smaller increase in gluconeogenesis in old rat hepatocytes than in young hepatocytes. beta2 adrenergic receptor (beta2-AR) expression slightly decreased in aged rat liver, and there were differences between young and old hepatocytes in their patterns of G protein coupled receptor kinases, which are involved in the activation of beta2-AR receptor signal desensitization. The major isoform of the kinase changed from GRK2 to GRK3 and the expression of beta-arrestin, which is recruited by the phosphorylated beta2-AR for internalization and degradation, increased in aged rat liver. GRK3 overexpression also decreased the glucose output from young rat hepatocytes. We conclude that an age-associated reduction in epinephrine-induced gluconeogenesis occurs through the epinephrine receptor desensitizing system.
Adrenergic beta-Agonists/*pharmacology ; Aging/*drug effects ; Animals ; Epinephrine/*pharmacology ; G-Protein-Coupled Receptor Kinase 2/metabolism ; G-Protein-Coupled Receptor Kinase 3/metabolism ; Glucagon/pharmacology ; *Gluconeogenesis/drug effects ; Male ; Models, Biological ; Phosphorylation ; Rats ; Rats, Inbred F344 ; Receptors, Adrenergic, beta-2/agonists/metabolism

OBJECTIVETo study the effect of high glucose on GRK2 gene expression in H9C2 cardiomyoblasts in vitro.

METHODSH9C2 cardiomyoblasts were cultured for 72 h in the presence of 0, 5.5, 12.5, 25 or 33 mmol/L glucose (with the osmotic pressure adjusted with monnitol). Semi-quantitative detection of GRK2 gene expression in H9C2 cardiomyoblasts was carried out using RT-PCR and phosph-Akt (Ser473) protein level was measured by Western blotting.

RESULTSGlucose in the culture medium (5.5 to 33 mmol/L) concentration-dependently increased the mRNA expression of GRK2 concentration and decreased phosphorylation Akt (ser473) level in in H9C2 cardiomyoblasts.

CONCLUSIONIncreased GRK2 gene expression may play an important role in cardiac dysfunction in diabetes.

Cell Line ; Diabetes Complications ; metabolism ; G-Protein-Coupled Receptor Kinase 2 ; genetics ; metabolism ; Gene Expression Regulation ; Glucose ; pharmacology ; Humans ; Myocytes, Cardiac ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Up-Regulation

OBJECTIVETo observe the effect and mechanism of Huatuo Zaizao extractum (HTZZ) on focal ischemia/reperfusion (I/R) blood-brain barrier injury induced by middle cerebral artery occlusion.

METHODSixty healthy male adult Sprague-Dawley rats was randomly divided into the sham operation group, the MCAO model group, the Tanakan (20 mg x kg(-1)) group, and high, middle and low-dose HTZZ groups (5, 2.5, 1.25 g x kg(-1)), with 10 in each group and single-dose duodenal administration. Middle cerebral artery occlusion was adopted to establish the rat focal I/R model. After ischemia for 90 min and reperfusion for 24 h, the pathological injury at the ischemia side was observed by HE staining. The blood-brain barrier structure was observed under transmission electron microscope. Expressions of G protein-coupled receptor kinases 2 (GRK2), matrix metalloproteinases 2 (MMP-2) and MMP-9 were detected by western blotting technique.

RESULTAfter 90 min MCAO/24 h reperfusion, penumbra cerebral cortical micro-vessels showed edema, mitochondrial injury, vacuolation, membrane injury and reduction. Along with the changes, sub-cells of G protein-coupled receptor kinase 2 (GRK2) in cortical penumbra brain tissues transferred from cytoplasm to membrane, with increase in expressions of MMP-2 and MMP-9. HTZZ could effectively recover cerebral micro-vascular endothelial edemaand blood-brain barrier ultrastructure injury induced by I/R, reduce expression of functional (membrane coupling) GRK2, and inhibit expressions of MMP-2 and MMP-9.

CONCLUSIONCell membrane coupling GRK2 may be the effective target of Huatuo Zaizao extractum.

Animals ; Behavior, Animal ; drug effects ; physiology ; Blood-Brain Barrier ; drug effects ; injuries ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; G-Protein-Coupled Receptor Kinase 2 ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Infarction, Middle Cerebral Artery ; complications ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Microvessels ; drug effects ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; complications ; metabolism ; physiopathology

BACKGROUNDThe effect of impaired glucose tolerance (IGT) on cardiac function during the chronic prediabetes state is complicated and plays an important role in clinical outcome. However, the molecular mechanisms are not fully understood. This study was designed to observe cardiac dysfunction in prediabetic rats with IGT and to determine whether glucose metabolic abnormalities, inflammation and apoptosis are linked to it.

METHODSThe IGT rat models were induced by streptozocin, and the heart functions were assessed by echocardiography. Myocardial glucose metabolism was analyzed by glycogen periodic acid-Schiff staining, and the pro-apoptotic effect of IGT was evaluated by TUNEL staining. Additionally, caspase-3 activation, macrophage migration inhibitory factor (MIF) and G-protein coupled receptor kinase 2 (GRK2) were detected by Western blotting in cardiac tissue lysates.

RESULTSArea-under-the-curve of blood glucose in rats injected with streptozotocin was higher than that in controls, increased by 16.28%, 38.60% and 38.61% at 2, 4 and 6 weeks respectively (F = 15.370, P = 0.003). Abnormal cardiac functions and apoptotic cardiomyocytes were observed in the IGT rats, the ejection fraction (EF) being (68.59 ± 6.62)% in IGT rats vs. (81.07 ± 4.59)% in controls (t = 4.020, P = 0.002). There was more glucose which was converted to glycogen in the myocardial tissues of IGT rats, especially in cardiac perivascular tissues. Compared to controls, the cleaved caspase-3, MIF and GRK2 were expressed at higher levels in the myocardial tissues of IGT rats.

CONCLUSIONSIGT in the prediabetes period resulted in cardiac dysfunction linked to abnormal glycogen storage and apoptosis. Additionally, MIF and GRK2 may be involved in the pathogenesis of cardiac dysfunction in prediabetes and their regulation may contribute to the design of novel diagnostic and therapeutic strategies for those who have potential risks for diabetic cardiovascular complications.

Animals ; Apoptosis ; drug effects ; Blotting, Western ; Disease Models, Animal ; Echocardiography ; G-Protein-Coupled Receptor Kinase 2 ; metabolism ; Glucose Intolerance ; chemically induced ; physiopathology ; Glucose Tolerance Test ; In Situ Nick-End Labeling ; Intramolecular Oxidoreductases ; metabolism ; Macrophage Migration-Inhibitory Factors ; metabolism ; Myocardium ; metabolism ; pathology ; Myocytes, Cardiac ; pathology ; Rats ; Streptozocin ; toxicity

OBJECTIVETo study the effects of the Weinaokang (WNK), the active compounds extracted from Ginkgo, Ginseng, and saffron, on ischemia/reperfusion (I/R)-induced vascular injury to cerebral microvessels after global cerebral ischemia.

METHODSMale C57BL/6J mice were randomly divided into 5 groups (10 animals/group): the sham group (0.5% CMC-Na, 20 mL/kg), the I/R model group (0.5% CMCNa, 20 mL/kg), the I/R+Crocin control group (20 mg/kg), the I/R+high dose WNK group (20 mg/kg), and the I/R+low dose WNK group (10 mg/kg). Bilateral common carotid artery occlusion (BCCAO, 20 min) in mice, followed by 24 h reperfusion, was built. The generation of nitric oxide (NO), the activity of nitric oxide synthase (NOS), the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), and the expression of matrix metalloproteinases-9 (MMP-9) and G protein-coupled receptor kinase 2 (GRK2) in cortical microvascular homogenates were evaluated. The ultrastructural morphology of cortical microvascular endothelial cells (CMEC) was observed.

RESULTSThe transient global cerebral ischemia (20 min), followed by 24 h of reperfusion, significantly promoted the generation of NO and the activity of NOS. The reperfusion led to serious edema with mitochondrial injuries in the cortical CMEC, as well as enhanced membrane GRK2 expression and reduced cytosol GRK2 expression. Furthermore, enhanced phosphorylation of ERK1/2 and decreased expression of MMP-9 were detected in cortical microvessels after I/R (20 min/24 h). As well as the positive control Crocin (20 mg/kg, 21days), pre-treatment with WNK (20, 10 mg/kg, 21 days) markedly inhibited nitrative injury and modulated the ultrastructure of CMEC. Furthermore, WNK inhibited GRK2 translocation from cytosol to the membrane (at 20 mg/kg) and reduced ERK1/2 phosphorylation and MMP-9 expression in cortical microvessels.

CONCLUSIONWNK and its active compounds (Crocin) are effective to suppress I/R-induced vascular injury to cerebral microvessels after global cerebral ischemia with the target on GRK2 pathways.

Animals ; Brain Ischemia ; drug therapy ; metabolism ; Cerebral Cortex ; blood supply ; drug effects ; metabolism ; ultrastructure ; Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; G-Protein-Coupled Receptor Kinase 2 ; metabolism ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred C57BL ; Microvessels ; drug effects ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Phosphorylation ; Reperfusion Injury ; drug therapy ; metabolism ; Signal Transduction ; drug effects ; Tissue Distribution ; drug effects