Chunjiang Fu

Inhibitory effect of D1-like and D3 dopamine receptors on norepinephrine-induced proliferation in vascular smooth muscle cells

The sympathetic nervous system plays an important role in the regulation of blood pressure. There is increasing evidence for positive and negative interactions between dopamine and adrenergic receptors; the activation of the alpha-adrenergic receptor induces vasoconstriction, whereas the activation of dopamine receptor induces vasorelaxation. We hypothesize that the D1-like receptor and/or D3 receptor also inhibit alpha1-adrenergic receptor-mediated proliferation in vascular smooth muscle cells (VSMCs). In this study, VSMC proliferation was determined by measuring [3H]thymidine incorporation, cell number, and uptake of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT). Norepinephrine increased VSMC number and MTT uptake, as well as [3H]thymidine incorporation via the alpha1-adrenergic receptor in aortic VSMCs from Sprague-Dawley rats. The proliferative effects of norepinephrine were attenuated by the activation of D1-like receptors or D3 receptors, although a D1-like receptor agonist, fenoldopam, and a D3 receptor agonist, PD-128907, by themselves, at low concentrations, had no effect on VSMC proliferation. Simultaneous stimulation of both D1-like and D3 receptors had an additive inhibitory effect. The inhibitory effect of D3 receptor was via protein kinase A, whereas the D1-like receptor effect was via protein kinase C-zeta. The interaction between alpha1-adrenergic and dopamine receptors, especially D1-like and D3 receptors in VSMCs, could be involved in the pathogenesis of hypertension.

Role of GRK4 in the Regulation of Arterial AT1 Receptor in Hypertension

G-protein–coupled receptor kinase 4 (GRK4) gene variants, via impairment of renal dopamine receptor and enhancement of renin–angiotensin system functions, cause sodium retention and increase blood pressure. Whether GRK4 and the angiotensin type 1 receptor (AT1R) interact in the aorta is not known. We report that GRK4 is expressed in vascular smooth muscle cells of the aorta. Heterologous expression of the GRK4&ggr; variant 142V in A10 cells increased AT1R protein expression and AT1R-mediated increase in intracellular calcium concentration. The increase in AT1R expression was related to an increase in AT1R mRNA expression via the NF-&kgr;B pathway. As compared with control, cells expressing GRK4&ggr; 142V had greater NF-&kgr;B activity with more NF-&kgr;B bound to the AT1R promoter. The increased AT1R expression in cells expressing GRK4&ggr; 142V was also associated with decreased AT1R degradation, which may be ascribed to lower AT1R phosphorylation. There was a direct interaction between GRK4&ggr; and AT1R that was decreased by GRK4&ggr; 142V. The regulation of AT1R expression by GRK4&ggr; 142V in A10 cells was confirmed in GRK4&ggr; 142V transgenic mice; AT1R expression was higher in the aorta of GRK4&ggr; 142V transgenic mice than control GRK4&ggr; wild-type mice. Angiotensin II–mediated vasoconstriction of the aorta was also higher in GRK4&ggr; 142V than in wild-type transgenic mice. This study provides a mechanism by which GRK4, via regulation of arterial AT1R expression and function, participates in the pathogenesis of conduit vessel abnormalities in hypertension.

Insulin Increases D5 Dopamine Receptor Expression and Function in Renal Proximal Tubule Cells From Wistar-Kyoto Rats

BACKGROUND Ion transport in the renal proximal tubule (RPT) is regulated by numerous hormones and humoral factors, including insulin and dopamine. Previous studies show an interaction between insulin and the D(1) receptor. Because both D(1) and D(5) receptors belong to the D(1)-like receptor subfamily, it is possible that an interaction between insulin and the D(5) dopamine receptor exists in RPT cells from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). METHODS D(5) receptor expression in immortalized RPT cells from WKY and SHRs was quantified by immunoblotting and D(5) receptor function by measuring Na(+)-K(+) ATPase activity. RESULTS Insulin increased the expression of the D(5) receptor. Stimulation with insulin (10(-7) mol/l) for 24 h increased D(5) receptor expression in RPT cells from WKY rats. This effect of insulin on D(5) receptor expression was aberrant in RPT cells from SHRs. The stimulatory effect of insulin on D(5) receptor expression in RPT cells from WKY rats was inhibited by a protein kinase C (PKC) inhibitor (PKC inhibitor peptide 19-31, 10(-6) mol/l) or a phosphatidylinositol 3 (PI3) kinase inhibitor (wortmannin, 10(-6) mol/l), indicating that both PKC and PI3 kinase were involved in the signaling pathway. Stimulation of the D(5) receptor heterologously expressed in HEK293 cells with fenoldopam (10(-7) mol/l/15 min) inhibited Na(+)-K(+) ATPase activity, whereas pretreatment with insulin (10(-7) mol/l/24 h) increased the D(5) receptor-mediated inhibition. CONCLUSIONS Insulin and D(5) receptors interact to regulate renal sodium transport; an aberrant interaction between insulin and D(5) receptor may participate in the pathogenesis of hypertension.

Relaxant effect of all-trans-retinoic acid via NO-sGC-cGMP pathway and calcium-activated potassium channels in rat mesenteric artery.

Intraperitoneal injection of all-trans-retinoic acid (ATRA) results in a reduction of blood pressure in spontaneously hypertensive rats. However, the mechanisms involved in this effect are not clear. We hypothesized that ATRA may relax resistance arteries. In this study, we found that ATRA relaxed phenylephrine-preconstricted mesenteric arterial rings, which were abrogated by the removal of the endothelium. Pretreatment of endothelium-intact arterial rings with an inhibitor of endothelial nitric oxide (NO) synthase, N(G)-nitro-l-arginine methyl ester (l-NAME), or soluble guanylyl cyclase, 1H-[1,2,4]-oxadiazole-[4,3-α]-quinoxaline-1-one, reduced the vasorelaxant effect of ATRA. Incubation of mesenteric arterial rings with ATRA increased the production of NO and cGMP, which were blocked by N(G)-nitro-l-arginine methyl ester. The vasorelaxant effect of ATRA was markedly attenuated in the presence of an inhibitor of big conductance calcium-activated potassium channels (charybdotoxin), but not with an inhibitor of voltage-dependent potassium channel (4-aminopyridine) or ATP-sensitive potassium channel (glibenclamide). Activation of retinoic acid receptors (RARs) with CH55 or retinoic X receptors (RXRs) with LGD1069 induced the vasorelaxation of phenylephrine-preconstricted mesenteric arterial rings. The RAR (BMS493) and RXR (UVI3003) antagonists blocked the ATRA-induced vasorelaxation. The vasorelaxant effect ATRA is physiologically relevant because the intravenous infusion of ATRA decreased blood pressure in normotensive rats. We conclude that ATRA relaxes resistance vessels via both RARs and RXRs receptors that are mediated by the endothelium-dependent NO-cGMP pathway, which may participate in the control of blood pressure.

Impaired Stimulatory Effect of ETB Receptor on D3 Receptor in Immortalized Renal Proximal Tubule Cells of Spontaneously Hypertensive Rats

Background: Activation of renal D₃ receptor induces natriuresis and diuresis in Wistar-Kyoto (WKY) rats; in the presence of ETB receptor antagonist, the natriuretic effect of D₃ receptor in WKY rats is reduced. We hypothesize that ETB receptor activation may regulate D₃ receptor expression in renal proximal tubule (RPT) cells from WKY rats, which is impaired in RPT cells from spontaneously hypertensive rats (SHRs). Methods: D₃ receptor expression was determined by immunoblotting; the D₃/ETB receptor linkage was checked by coimmunoprecipitation; Na⁺-K⁺-ATPase activity was determined as the rate of inorganic phosphate released in the presence or absence of ouabain. Results: In RPT cells from WKY rats, the ETB receptor agonist BQ3020 increased D₃ receptor protein. In contrast, in RPT cells from SHRs, BQ3020 did not increase D₃ receptor. There was coimmunoprecipitation between D₃ and ETB receptors in RPT cells from WKY and SHRs. Activation of ETB receptor increased D₃/ETB coimmunoprecipitation in RPT cells from WKY rats, but not from SHRs. The basal levels of D₃/ETB receptor coimmunoprecipitation were greater in RPT cells from WKY rats than in those from SHRs. Stimulation of D₃ receptor inhibited Na⁺-K⁺-ATPase activity, which was augmented by the pretreatment with the ETB receptor agonist BQ3020 in WKY RPT cells, but not in SHR RPT cells. Conclusion: ETB receptors regulate and physically interact with D₃ receptors differently in WKY rats and SHRs. The impaired natriuretic effect in SHRs may be, in part, related to impaired ETB and D₃ receptor interactions.

Role of Gα 12 - and Gα 13 -protein subunit linkage of D 3 dopamine receptors in the natriuretic effect of D 3 dopamine receptor in kidney

The D3 dopamine receptor is the major D2-like receptor that regulates sodium transport in the renal proximal tubule (RPT) and helps maintain blood pressure in the normal range. In Wistar–Kyoto (WKY) rats chronically fed high-salt diet, the intrarenal arterial infusion of a D3 receptor agonist, PD128907, increased absolute and fractional sodium excretion. We have reported that Gα12 and Gα13, which participate in the signal transduction of the D5 receptor, are expressed in RPTs. As the D3 receptor is also expressed in RPTs, we hypothesized that it may also interact with Gα12/Gα13 in RPTs from WKY rats. There were co-localization and co-immunoprecipitation of D3 receptor and Gα12/Gα13 in renal brush border membranes (BBMs) and RPT cells. The intrarenal infusion of PD128907 (1 μg kg−1 min−1) that increased sodium excretion also increased the co-immunoprecipitations of D3/Gα12 and D3/Gα13 in renal BBMs; their co-immunoprecipitation was confirmed in RPT cells. As Gα12 and Gα13 increase sodium pump and transporter activity (for example, Na+–K+–ATPase, NHE3), an increased association of D3 receptors with Gα12/Gα13 receptors after D3 receptor activation may be a mechanism to prevent Gα12/Gα13-mediated stimulation of sodium transport (and thus enhance natriuresis). We conclude that a D3 receptor interaction with Gα12/Gα13 that increases sodium excretion may have a role in the regulation of blood pressure.

Circulating "LncPPARδ" From Monocytes as a Novel Biomarker for Coronary Artery Diseases.

AbstractTo investigate long noncoding RNA NONHSAT112178 (LncPPAR&dgr;) as a biomarker for coronary artery disease (CAD) in peripheral blood monocyte cells, RT-qPCR was performed to validate the microarray results, receiver operating characteristic curve was applied to study the potential of LncPPAR&dgr; as a biomarker. Diagnostic models from LncPPAR&dgr; alone or combination of risk factors were constructed by Fisher criteria. The expression of genes neighboring the LncPPAR&dgr; gene was examined with RT-qPCR in THP-1 cell line treated with LncPPAR&dgr; siRNA. Using a diagnostic model by Fisher criteria, the consideration of risk factors increased the optimal sensitivity from 70.00% to 82.00% and decreased the specificity from 94.00% to 78.00%. The consideration of risk factors also increased area under the receiver operating characteristic curve from 0.727 to 0.785 (P = 0.001), from 0.712 to 0.768 (P = 0.01), and from 0.769 to 0.835 (P = 0.07), in the original, training, and test sets, respectively. Finally, we found that the expression of peroxisome proliferator-activated receptor &dgr; (PPAR&dgr;), Adipose Differentiation-Related Protein (ADRP), and Angiopoietin-like 4 (ANGPTL4) were affected by LncPPAR&dgr; silencing.Our present study indicated that LncPPAR&dgr;, especially combined with risk factors, can be a good biomarker for CAD. LncPPAR&dgr; regulates the expression of neighboring protein-coding genes, PPAR&dgr; and its direct target genes ADRP and ANGPTL4.

Correlation between single nucleotide polymorphisms of the ACTA2 gene and coronary artery stenosis in patients with type 2 diabetes mellitus

This study aimed to analyze the correlation between single nucleotide polymorphisms (SNPs) of the actin, aortic smooth muscle (ACTA2) gene and coronary artery stenosis in patients with type 2 diabetes mellitus (T2DM). Eight SNPs from the promoter region of the ACTA2 gene were screened. Patients with T2DM (n=251) were divided into two groups, those with severe coronary stenosis (SCS+ group; n=168) and those without severe coronary stenosis (SCS− group; n=83). Patients were also divided according to lesion branching into those whose lesions involved less than three branches (LCA− group) and those whose lesions involved at least three branches (LCA+ group). The clinical and laboratory data of the patients were collected, and the genotyping of eight SNPs was conducted followed by statistical analysis. Of the eight SNPs, only the rs1324551 SNP was identified to be significantly different between the SCS+ and SCS− groups (P<0.05). The frequency of the rs1324551 G allele and GG genotype in the SCS+ group was significantly higher than that of the SCS− group (P=0.044 and P=0.001, respectively). No significant difference was observed between the LCA− and LCA+ groups. Following the deduction of age, gender and traditional risk factors, the odds ratios of the GG genotype in additive and recessive models were 2.93 [95% confidence interval (CI), 1.05–8.19; P=0.04] and 2.34 (95% CI, 1.09–5.02; P=0.03), respectively, and this relevancy was represented only in patients with low insulin levels. Age and smoking were also found to increase the risk of coronary artery lesions. In conclusion, the rs1324551 SNP in the promoter region of the ACTA2 gene was identified to be independently correlated with the degree of coronary artery stenosis in patients with T2DM and plasma insulin may inhibit coronary artery stenosis during the pathogenic process.

CYP4F2 genetic polymorphisms are associated with coronary heart disease in a Chinese population

BackgroundTo explore the relationship between CYP4F2 gene polymorphism and coronary heart disease (CHD) in a Chinese Han population.MethodsWe selected 440 CHD patients and 440 control subjects to perform a case - control study. Four SNPs (rs2108622, rs3093100, rs3093105 and rs3093135) in CYP4F2 gene were genotyped using polymerase chain reaction - restriction fragment length polymorphism (PCR - RFLP) methods. The genotype and haplotype distributions were compared between the case and the control group.ResultsWe found both rs2108622 and rs3093105 in CYP4F2 gene were associated with the risk for CHD (P <0.01). Haplotype analysis indicated that GGGT haplotype consisted by rs2108622-rs3093100-rs3093105-rs3093135 was associated with CHD risk (OR = 4.367, 95% CI: 2.241 ~ 8.510; P < 0.001), but GGTA haplotype was associated with decreased risk for CHD (OR = 0.450, 95% CI: 0.111 ~ 0.777; P <0.001).ConclusionCYP4F2 gene polymorphisms were associated with the risk of CHD in Chinese population.

Identification of the differentially expressed genes associated with familial combined hyperlipidemia using bioinformatics analysis

The aim of the present study was to screen the differentially expressed genes (DEGs) associated with familial combined hyperlipidemia (FCHL) and examine the changing patterns. The transcription profile of GSE18965 was obtained from the NCBI Gene Expression Omnibus database, including 12 FCHL samples and 12 control specimens. The DEGs were identified using a linear models for microarray data package in the R programming language. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was also performed. Protein-protein interaction (PPI) networks of the DEGs were constructed using the EnrichNet online tool. In addition, cluster analysis of the genes in networks was performed using ClusterONE. A total of 879 DEGs were screened, including 394 upregulated and 485 downregulated genes. Enrichment analysis identified four important KEGG pathways associated with FCHL: One carbon pool by folate, α-linolenic acid metabolism, asthma and the glycosphingolipid biosynthesis-globo series. GO annotation identified 12 enriched biological processes, including one associated with hematopoiesis and four associated with bone cell differentiation. This identification was in accordance with clinical data and experiments into hyperlipidemia and bone lesions. Based on PPI networks, these DEGs had a close association with immune responses, hormone responses and cytokine-cytokine receptors. In conclusion, these DEGs may be used as specific therapeutic molecular targets in the treatment of FCHL. The present findings may provide the basis for understanding the pathogenesis of FCHL in future studies. However, further experiments are required to confirm these results.