BACKGROUND: G protein-coupled receptor kinase 2 (GRK2) could participate in the regulation of diverse cells via interacting with non-G-protein-coupled receptors. In the present work, we explored how paroxetine, a GRK2 inhibitor, modulates the differentiation and activation of immune cells in rheumatoid arthritis (RA). METHODS: The blood samples of healthy individuals and RA patients were collected between July 2021 and March 2022 from the First Affiliated Hospital of Anhui Medical University. C57BL/6 mice were used to induce the collagen-induced arthritis (CIA) model. Flow cytometry analysis was used to characterize the differentiation and function of dendritic cells (DCs)/T cells. Co-immunoprecipitation was used to explore the specific molecular mechanism. RESULTS: In patients with RA, high expression of GRK2 in peripheral blood lymphocytes, accompanied by the increases of phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In animal model, a decrease in regulatory T cells (T regs ), an increase in the cluster of differentiation 8 positive (CD8 + ) T cells, and maturation of DCs were observed. Paroxetine, when used in vitro and in CIA mice, restrained the maturation of DCs and the differentiation of CD8 + T cells, and induced the proportion of T regs . Paroxetine inhibited the secretion of pro-inflammatory cytokines, the expression of C-C motif chemokine receptor 7 in DCs and T cells. Simultaneously, paroxetine upregulated the expression of programmed death ligand 1, and anti-inflammatory cytokines. Additionally, paroxetine inhibited the PI3K-AKT-mTOR metabolic pathway in both DCs and T cells. This was associated with a reduction in mitochondrial membrane potential and changes in the utilization of glucose and lipids, particularly in DCs. Paroxetine reversed PI3K-AKT pathway activation induced by 740 Y-P (a PI3K agonist) through inhibiting the interaction between GRK2 and PI3K in DCs and T cells. CONCLUSION Paroxetine exerts an immunosuppressive effect by targeting GRK2, which subsequently inhibits the metabolism-related PI3K-AKT-mTOR pathway of DCs and T cells in RA.
G-Protein-Coupled Receptor Kinase 2/metabolism* ; Arthritis, Rheumatoid/immunology* ; Animals ; Dendritic Cells/metabolism* ; Paroxetine/therapeutic use* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mice ; Humans ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Male ; Phosphatidylinositol 3-Kinases/metabolism* ; Lymphocyte Activation/drug effects* ; Female ; T-Lymphocytes/metabolism* ; Middle Aged

Lingguizhugan Decoction (LGZG) demonstrates significant efficacy in treating various cardiovascular diseases clinically, yet its precise mechanism of action remains elusive. This study aimed to elucidate the potential mechanisms and effects of LGZG on isoproterenol (ISO) continuous stimulation-induced chronic heart failure (CHF) in mice, providing direct experimental evidence for further clinical applications. In vivo, continuous ISO infusion was administered to mice, and ventricular myocytes were utilized to explore LGZG?s potential mechanism of action on the ?1-adrenergic receptor (?1-AR)/Gs/G protein-coupled receptor kinases (GRKs)/?-arrestin signaling deflection system in the heart. The findings reveal that LGZG significantly reduced the messenger ribonucleic acid (mRNA) expression of hypertrophy-related biomarkers [atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP)] and improved cardiac remodeling and left ventricular diastolic function in mice with ISO-induced CHF. Furthermore, LGZG inhibited the overactivation of Gs/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling and downregulated the downstream transcriptional activity of cAMP-response element binding protein (CREB) and the expression of the coactivator CBP/P300. Notably, LGZG downregulated the expression of ?-arrestin1 and GRK 2/3/5 while upregulating the expression of ?1-AR and ?-arrestin2. These results suggest that LGZG inhibits Gs/cAMP/PKA signaling and ?-arrestin/GRK-mediated desensitization and internalization of ?1-AR, potentially exerting cardioprotective effects through the synergistic regulation of the ?1-AR/Gs/GRKs/?-arrestin signaling deflection system via multiple pathways.
Animals ; Heart Failure/genetics* ; Signal Transduction/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Male ; G-Protein-Coupled Receptor Kinases/genetics* ; Mice, Inbred C57BL ; Humans ; Isoproterenol ; Arrestins/genetics* ; Chronic Disease

BACKGROUND/AIMS: Several disorders of the gastrointestinal tract are associated with abnormal serotonin (5-HT) signaling or metabolism where the 5-HT3 and 5-HT4 receptors are clinically relevant. The aim was to examine the distribution of 5-HT3, 5-HT4, and 5-HT7 receptors in the normal human colon and how this is associated with receptor interacting chaperone 3, G protein coupled receptor kinases, and protein LIN-7 homologs to extend previous observations limited to the sigmoid colon or the upper intestine. METHODS: Samples from ascending, transverse, descending, and sigmoid human colon were dissected into 3 separate layers (mucosa, longitudinal, and circular muscles) and ileum samples were dissected into mucosa and muscle layers (n = 20). Complementary DNA was synthesized by reverse transcription from extracted RNA and expression was determined by quantitative or end point polymerase chain reaction. RESULTS: The 5-HT3 receptor subunits were found in all tissues throughout the colon and ileum. The A subunit was detected in all samples and the C subunit was expressed at similar levels while the B subunit was expressed at lower levels and less frequently. The 5-HT3 receptor E subunit was mainly found in the mucosa layers. All splice variants of the 5-HT4 and 5-HT7 receptors were expressed throughout the colon although the 5-HT4 receptor d, g, and i variants were expressed less often. CONCLUSIONS: The major differences in 5-HT receptor distribution within the human colon are in relation to the mucosa and muscular tissue layers where the 5-HT3 receptor E subunit is predominantly found in the mucosal layer which may be of therapeutic relevance.
Colon* ; Colon, Sigmoid ; DNA, Complementary ; G-Protein-Coupled Receptor Kinases ; Gastrointestinal Tract ; Humans ; Ileum ; Intestines ; Metabolism ; Mucous Membrane ; Polymerase Chain Reaction ; Receptors, Serotonin ; Receptors, Serotonin, 5-HT3 ; Receptors, Serotonin, 5-HT4 ; Reverse Transcription ; RNA ; Serotonin

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 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

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 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

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