Publications by authors named "Xiaoxu Zheng"

26 Publications

  • Page 1 of 1

Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

Cell Death Dis 2021 05 20;12(6):518. Epub 2021 May 20.

Center for Molecular and Translational Medicine, Georgia State University, 157 Decatur St SE, Atlanta, GA, 30303, USA.

Oncogenic KRAS mutations combined with the loss of the LKB1 tumor-suppressor gene (KL) are strongly associated with aggressive forms of lung cancer. N6-methyladenosine (m6A) in mRNA is a crucial epigenetic modification that controls cancer self-renewal and progression. However, the regulation and role of m6A modification in this cancer are unclear. We found that decreased m6A levels correlated with the disease progression and poor survival for KL patients. The correlation was mediated by a special increase in ALKBH5 (AlkB family member 5) levels, an m6A demethylase. ALKBH5 gain- or loss-of function could effectively reverse LKB1 regulated cell proliferation, colony formation, and migration of KRAS-mutated lung cancer cells. Mechanistically, LKB1 loss upregulated ALKBH5 expression by DNA hypermethylation of the CTCF-binding motif on the ALKBH5 promoter, which inhibited CTCF binding but enhanced histone modifications, including H3K4me3, H3K9ac, and H3K27ac. This effect could successfully be rescued by LKB1 expression. ALKBH5 demethylation of m6A stabilized oncogenic drivers, such as SOX2, SMAD7, and MYC, through a pathway dependent on YTHDF2, an m6A reader protein. The above findings were confirmed in clinical KRAS-mutated lung cancer patients. We conclude that loss of LKB1 promotes ALKBH5 transcription by a DNA methylation mechanism, reduces m6A modification, and increases the stability of m6A target oncogenes, thus contributing to aggressive phenotypes of KRAS-mutated lung cancer.
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http://dx.doi.org/10.1038/s41419-021-03793-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137886PMC
May 2021

Deletion of inhibits neointima formation by enhancing KAT2A/GCN5-mediated acetylation of TUBA/α-tubulin .

Autophagy 2021 May 14:1-18. Epub 2021 May 14.

Center of Molecular and Translational Medicine, Georgia State University, Atlanta, Georgia.

ULK1 (unc-51 like autophagy activating kinase) has a central role in initiating macroautophagy/autophagy, a process that contributes to atherosclerosis and neointima hyperplasia, or excessive tissue growth that leads to vessel dysfunction. However, the role of ULK1 in neointima formation remains unclear. We aimed to determine how deletion affected neointima formation and to investigate the underlying mechanisms. We measured autophagy activity, vascular smooth muscle cell (VSMC) migration and neointima hyperplasia in cultured VSMCs and ligation-injured mouse carotid arteries from male wild-type (WT, C57BL/6 J) and VSMC-specific knockout ( KO) mice. Carotid artery ligation in WT mice increased ULK1 protein expression, and concurrently increased autophagic flux and neointima formation. Treating human aortic smooth muscle cells (HASMCs) with PDGF (platelet derived growth factor) increased ULK1 expression, activated autophagy, and promoted cell migration. Further, smooth muscle cell-specific deletion of suppressed autophagy, inhibited VSMC migration, and impeded neointima hyperplasia. Mechanistically, deletion inhibited autophagic degradation of histone acetyltransferase protein KAT2A/GCN5 (K[lysine] acetyltransferase 2A), resulting in accumulation of KAT2A that directly acetylated TUBA/α-tubulin and subsequently increased protein levels of acetylated TUBA. The acetylation of TUBA increased microtubule stability and inhibited VSMC directional migration and neointima formation. Finally, local transfection of siRNA decreased TUBA acetylation and prevented the attenuation of vascular injury-induced neointima formation in KO mice. These findings suggest that deletion inhibits neointima formation by reducing autophagic degradation of KAT2A and increasing TUBA acetylation in VSMCs. ACTA2/α-SMA: actin, alpha 2, smooth muscle, aorta; ACTB: actin beta; ATAT1: alpha tubulin acetyltransferase 1; ATG: autophagy related; BECN1: beclin 1; BP: blood pressure; CAL: carotid artery ligation; CQ: chloroquine diphosphate; EC: endothelial cells; EEL: external elastic layer; FBS: fetal bovine serum; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HASMCs: human aortic smooth muscle cells; HAT1: histone acetyltransferase 1; HDAC: histone deacetylase; IEL: inner elastic layer; IP: immunoprecipitation; KAT2A/GCN5: K(lysine) acetyltransferase 2A; KAT8/hMOF: lysine acetyltransferase 8; MAP1LC3: microtubule associated protein 1 light chain 3; MYH11: myosin heavy chain 11; PBS: phosphate-buffered saline; PDGF: platelet derived growth factor; PECAM1/CD31: platelet and endothelial cell adhesion molecule 1; RAC3: Rac family small GTPase 3; SIRT2: sirtuin 2; SPP1/OPN: secreted phosphoprotein 1; SQSTM1/p62: sequestosome 1; TAGLN/SM22: transgelin; TUBA: tubulin alpha; ULK1: unc-51 like autophagy activating kinase; VSMC: vascular smooth muscle cell; VVG: Verhoeff Van Gieson; WT: wild type.
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http://dx.doi.org/10.1080/15548627.2021.1911018DOI Listing
May 2021

Lipid rafts are required for effective renal D dopamine receptor function.

FASEB J 2020 05 7;34(5):6999-7017. Epub 2020 Apr 7.

Division of Renal Diseases & Hypertension, Department of Medicine, The George Washington University School of Medicine & Health Sciences, Washington, DC, USA.

Effective receptor signaling is anchored on the preferential localization of the receptor in lipid rafts, which are plasma membrane platforms replete with cholesterol and sphingolipids. We hypothesized that the dopamine D receptor (D R) contains structural features that allow it to reside in lipid rafts for its activity. Mutation of C347 palmitoylation site and Y218 of a newly identified Cholesterol Recognition Amino Acid Consensus motif resulted in the exclusion of D R from lipid rafts, blunted cAMP response, impaired sodium transport, and increased oxidative stress in renal proximal tubule cells (RPTCs). Kidney-restricted silencing of Drd1 in C57BL/6J mice increased blood pressure (BP) that was normalized by renal tubule-restricted rescue with D R-wild-type but not the mutant D R 347A that lacks a palmitoylation site. Kidney-restricted disruption of lipid rafts by β-MCD jettisoned the D R from the brush border, decreased sodium excretion, and increased oxidative stress and BP in C57BL/6J mice. Deletion of the PX domain of the novel D R-binding partner sorting nexin 19 (SNX19) resulted in D R partitioning solely to non-raft domains, while silencing of SNX19 impaired D R function in RPTCs. Kidney-restricted silencing of Snx19 resulted in hypertension in C57BL/6J mice. Our results highlight the essential role of lipid rafts for effective D R signaling.
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http://dx.doi.org/10.1096/fj.201902710RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200283PMC
May 2020

Maize (Zea mays L. Sp.) varieties significantly influence bacterial and fungal community in bulk soil, rhizosphere soil and phyllosphere.

FEMS Microbiol Ecol 2020 03;96(3)

Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.

The plant-microbe interaction can affect ecosystem function, and many studies have demonstrated that plant species influence relevant microorganisms. In this study, microbial communities in bulk soil, rhizosphere soil and phyllosphere from different maize varieties were investigated using high-throughput sequencing method. Results demonstrated that cultivar Gaoneng 1 (G1) showed higher bacterial diversity in soil (both bulk and rhizosphere soils) and lower bacterial diversity in the phyllosphere, while cultivar Gaoneng 2 (G2) had lower fungal diversity in both the soil and phyllosphere compare to the other cultivars. The bacterial community structure of soils among the three varieties was significantly different; however, no significant differences were found in the soil fungal community and phyllosphere bacterial and fungal community. The soil networks from cultivar G1 and phyllosphere networks from cultivar Zhengdan (ZD) have the highest complexity in contrast to the other two cultivars. In conclusion, the bacterial community structure in bulk soil of different cultivars was significantly different, so do the co-occurrence ecological networks of phyllosphere bacterial community. This study comprehensively analyzed the microbial community among different maize cultivars and could be useful for guiding practices, such as evaluation of new plant cultivars and quality predictions of these varieties at the microbial level.
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http://dx.doi.org/10.1093/femsec/fiaa020DOI Listing
March 2020

Altered DNA methylation of TRIM13 in diabetic nephropathy suppresses mesangial collagen synthesis by promoting ubiquitination of CHOP.

EBioMedicine 2020 Jan 2;51:102582. Epub 2020 Jan 2.

Department of Nephrology, The Second Affiliated Hospital of Nanchang University, No.1 Minde Road, Donghu District, Nanchang, Jiangxi 330006, China. Electronic address:

Background: Mesangial collagen synthesis in renal glomeruli contributes to the pathogenesis of diabetic nephropathy (DN) which is one of the most serious complications of diabetes mellitus. However, the underlying mechanism of mesangial collagen synthesis is largely unknown.

Methods: The differential expression of CHOP and TRIM13 which is a well-defined E3 ubiquitin ligase was compared in renal biopsy samples from DN/normal renal tissues, in isolated glomeruli of diabetic/control mice, as well as in high glucose (HG) or TGF-β1-stimulated renal mesangial cells. Then the relationship between TRIM13 and CHOP was explored using the ubiquitination assay.

Findings: We found that the expression of TRIM13 was downregulated in renal biopsies, isolated glomeruli of diabetic mice, and HG/TGF-β1-stimulated renal mesangial cells, while the expression of CHOP was upregulated. An increased level of TRIM13 promoter methylation contributed to the deregulation of TRIM13 in renal glomeruli of DN. The ubiquitination assay confirmed that TRIM13 promoted ubiquitination and degradation of CHOP. Meanwhile, overexpressing TRIM13 attenuated DN-induced collagen synthesis and restored renal function in vitro and in vivo via downregulating CHOP.

Interpretation: Our findings demonstrated that overexpressed TRIM13 suppresses mesangial collagen synthesis in DN by promoting ubiquitination of CHOP, suggesting TRIM13 as a potential therapeutic target in treating DN.
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http://dx.doi.org/10.1016/j.ebiom.2019.11.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940716PMC
January 2020

Dopamine D receptor modulates Wnt expression and control of cell proliferation.

Sci Rep 2019 11 14;9(1):16861. Epub 2019 Nov 14.

Department of Medicine, School of Medicine and Health Sciences, The George Washington University, Washington, DC, 20052, USA.

The Wnt/β-catenin pathway is one of the most conserved signaling pathways across species with essential roles in development, cell proliferation, and disease. Wnt signaling occurs at the protein level and via β-catenin-mediated transcription of target genes. However, little is known about the underlying mechanisms regulating the expression of the key Wnt ligand Wnt3a or the modulation of its activity. Here, we provide evidence that there is significant cross-talk between the dopamine D receptor (D2R) and Wnt/β-catenin signaling pathways. Our data suggest that D2R-dependent cross-talk modulates Wnt3a expression via an evolutionarily-conserved TCF/LEF site within the WNT3A promoter. Moreover, D2R signaling also modulates cell proliferation and modifies the pathology in a renal ischemia/reperfusion-injury disease model, via its effects on Wnt/β-catenin signaling. Together, our results suggest that D2R is a transcriptional modulator of Wnt/β-catenin signal transduction with broad implications for health and development of new therapeutics.
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http://dx.doi.org/10.1038/s41598-019-52528-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856370PMC
November 2019

G protein-coupled receptor 37L1 regulates renal sodium transport and blood pressure.

Am J Physiol Renal Physiol 2019 03 19;316(3):F506-F516. Epub 2018 Dec 19.

Department of Medicine, The George Washington University , Washington, District of Columbia.

G protein-coupled receptors (GPCRs) in the kidney regulate the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Abnormalities in renal epithelial ion transport play important roles in the pathogenesis of essential hypertension. The orphan G protein-coupled receptor 37L1 (GPR37L1), also known as endothelin receptor type B-like protein (ETBR-LP2), is expressed in several regions in the brain, but its expression profile and function in peripheral tissues are poorly understood. We found that GPR37L1 mRNA expression is highest in the brain, followed by the stomach, heart, testis, and ovary, with moderate expression in the kidney, pancreas, skeletal muscle, liver, lung, and spleen. Immunofluorescence analyses revealed the expression of GPR37L1 in specific regions within some organs. In the kidney, GPR37L1 is expressed in the apical membrane of renal proximal tubule cells. In human renal proximal tubule cells, the transient expression of GPR37LI increased intracellular sodium, whereas the silencing of GPR37LI decreased intracellular sodium. Inhibition of Na/H exchanger isoform 3 (NHE3) activity abrogated the GPR37L1-mediated increase in intracellular sodium. Renal-selective silencing of Gpr37l1 in mice increased urine output and sodium excretion and decreased systolic and diastolic blood pressures. The renal-selective silencing of GPR37L1 decreased the protein expression of NHE3 but not the expression of Na-K-ATPase or sodium-glucose cotransporter 2. Our findings show that in the kidney, GPR37L1 participates in renal proximal tubule luminal sodium transport and regulation of blood pressure by increasing the renal expression and function of NHE3 by decreasing cAMP production. The role of GPR37L1, expressed in specific cell types in organs other than the kidney, remains to be determined.
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http://dx.doi.org/10.1152/ajprenal.00289.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459304PMC
March 2019

Increased renal oxidative stress in salt-sensitive human GRK4γ486V transgenic mice.

Free Radic Biol Med 2017 05 9;106:80-90. Epub 2017 Feb 9.

Department of Obstetrics & Gynecology, Nanjing Drum Tower Hospital, Nanjing University, Nanjing, Jiangsu, China; Division of Renal Diseases & Hypertension, Department of Medicine, The George Washington University, Washington, DC, USA. Electronic address:

We tested the hypothesis that salt-sensitive hypertension is caused by renal oxidative stress by measuring the blood pressure and reactive oxygen species-related proteins in the kidneys of human G protein-coupled receptor kinase 4γ (hGRK4γ) 486V transgenic mice and non-transgenic (Non-T) littermates on normal and high salt diets. High salt diet increased the blood pressure, associated with impaired sodium excretion, in hGRK4γ486V mice. Renal expressions of NOX isoforms were similar in both strains on normal salt diet but NOX2 was decreased by high salt diet to a greater extent in Non-T than hGRK4γ486V mice. Renal HO-2, but not HO-1, protein was greater in hGRK4γ486V than Non-T mice on normal salt diet and normalized by high salt diet. On normal salt diet, renal CuZnSOD and ECSOD proteins were similar but renal MnSOD was lower in hGRK4γ486V than Non-T mice and remained low on high salt diet. High salt diet decreased renal CuZnSOD in hGRK4γ486V but not Non-T mice and decreased renal ECSOD to a greater extent in hGRK4γ486V than Non-T mice. Renal SOD activity, superoxide production, and NOS3 protein were similar in two strains on normal salt diet. However, high salt diet decreased SOD activity and NOS3 protein and increased superoxide production in hGRK4γ486V mice but not in Non-T mice. High salt diet also increased urinary 8-isoprostane and 8-hydroxydeoxyguanosine to a greater extent in hGRK4γ486V than Non-T mice. hGRK4γwild-type mice were normotensive and hGRK4γ142V mice were hypertensive but both were salt-resistant and in normal redox balance. Chronic tempol treatment partially prevented the salt-sensitivity of hGRK4γ486V mice. Thus, hGRK4γ486V causes salt-sensitive hypertension due, in part, to defective renal antioxidant mechanisms.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376361PMC
May 2017

Renal rescue of dopamine D2 receptor function reverses renal injury and high blood pressure.

JCI Insight 2016 Jun;1(8)

Department of Medicine, The George Washington University, Washington, DC, USA, and Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.

Dopamine D2 receptor (DRD2) deficiency increases renal inflammation and blood pressure in mice. We show here that long-term renal-selective silencing of using siRNA increases renal expression of proinflammatory and profibrotic factors and blood pressure in mice. To determine the effects of renal-selective rescue of expression in mice, the renal expression of DRD2 was first silenced using siRNA and 14 days later rescued by retrograde renal infusion of adeno-associated virus (AAV) vector with . Renal siRNA treatment decreased the renal expression of DRD2 protein by 55%, and AAV treatment increased the renal expression of DRD2 protein by 7.5- to 10-fold. Renal-selective rescue reduced the expression of proinflammatory factors and kidney injury, preserved renal function, and normalized systolic and diastolic blood pressure. These results demonstrate that the deleterious effects of renal-selective silencing on renal function and blood pressure were rescued by renal-selective overexpression of . Moreover, the deleterious effects of 45-minute bilateral ischemia/reperfusion on renal function and blood pressure in mice were ameliorated by a renal-selective increase in DRD2 expression by the retrograde ureteral infusion of AAV immediately after the induction of ischemia/reperfusion injury. Thus, 14 days after ischemia/reperfusion injury, the renal expression of profibrotic factors, serum creatinine, and blood pressure were lower in mice infused with AAV than in those infused with control AAV. These results indicate an important role of renal DRD2 in limiting renal injury and preserving normal renal function and blood pressure.
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http://dx.doi.org/10.1172/jci.insight.85888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922424PMC
June 2016

Localization and signaling of GPCRs in lipid rafts.

Methods Cell Biol 2016 10;132:3-23. Epub 2016 Feb 10.

Division of Renal Diseases & Hypertension, Department of Medicine, The George Washington University School of Medicine and Health Sciences, WA, USA.

The understanding of how biological membranes are organized and how they function has evolved. Instead of just serving as a medium in which certain proteins are found, portions of the lipid bilayer have been demonstrated to form specialized platforms that foster the assembly of signaling complexes by providing a microenvironment that is conducive for effective protein-protein interactions. G protein-coupled receptors (GPCRs) and relevant signaling molecules, including the heterotrimeric G proteins, key enzymes such as kinases and phosphatases, trafficking proteins, and secondary messengers, preferentially partition to these highly organized cell membrane microdomains, called lipid rafts. As such, lipid rafts are crucial for the trafficking and signaling of GPCRs. The study of GPCR biology in the context of lipid rafts involves the localization of the GPCR of interest in lipid rafts, at the basal state and upon receptor agonism, and the evaluation of the biological functions of the GPCR in appropriate cell lines. The lack of standardized methodology to study lipid rafts, in general, and of the workings of GPCRs in lipid rafts, in particular, and the inherent drawbacks of current methods have hampered the complete understanding of the underlying molecular mechanisms. Newer methodologies that allow the study of GPCRs in their native form are needed. The use of complementary approaches that produce mutually supportive results appear to be the best way for drawing conclusions with regards to the distribution and activity of GPCRs in lipid rafts.
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http://dx.doi.org/10.1016/bs.mcb.2015.11.008DOI Listing
December 2016

Redox signaling and splicing dependent change in myosin phosphatase underlie early versus late changes in NO vasodilator reserve in a mouse LPS model of sepsis.

Am J Physiol Heart Circ Physiol 2015 May 27;308(9):H1039-50. Epub 2015 Feb 27.

Department of Medicine, Divisions of Cardiovascular Medicine and Nephrology, University of Maryland, Baltimore, Maryland

Microcirculatory dysfunction may cause tissue malperfusion and progression to organ failure in the later stages of sepsis, but the role of smooth muscle contractile dysfunction is uncertain. Mice were given intraperitoneal LPS, and mesenteric arteries were harvested at 6-h intervals for analyses of gene expression and contractile function by wire myography. Contractile (myosin and actin) and regulatory [myosin light chain kinase and phosphatase subunits (Mypt1, CPI-17)] mRNAs and proteins were decreased in mesenteric arteries at 24 h concordant with reduced force generation to depolarization, Ca(2+), and phenylephrine. Vasodilator sensitivity to DEA/nitric oxide (NO) and cGMP under Ca(2+) clamp were increased at 24 h after LPS concordant with a switch to Mypt1 exon 24- splice variant coding for a leucine zipper (LZ) motif required for PKG-1α activation of myosin phosphatase. This was reproduced by smooth muscle-specific deletion of Mypt1 exon 24, causing a shift to the Mypt1 LZ+ isoform. These mice had significantly lower resting blood pressure than control mice but similar hypotensive responses to LPS. The vasodilator sensitivity of wild-type mice to DEA/NO, but not cGMP, was increased at 6 h after LPS. This was abrogated in mice with a redox dead version of PKG-1α (Cys42Ser). Enhanced vasorelaxation in early endotoxemia is mediated by redox signaling through PKG-1α but in later endotoxemia by myosin phosphatase isoform shifts enhancing sensitivity to NO/cGMP as well as smooth muscle atrophy. Muscle atrophy and modulation may be a novel target to suppress microcirculatory dysfunction; however, inactivation of inducible NO synthase, treatment with the IL-1 antagonist IL-1ra, or early activation of α-adrenergic signaling did not suppressed this response.
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http://dx.doi.org/10.1152/ajpheart.00912.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551119PMC
May 2015

TRA2β controls Mypt1 exon 24 splicing in the developmental maturation of mouse mesenteric artery smooth muscle.

Am J Physiol Cell Physiol 2015 Feb 26;308(4):C289-96. Epub 2014 Nov 26.

Division of Cardiovascular Medicine, School of Medicine, University of Maryland, Baltimore, Maryland;

Diversity of smooth muscle within the vascular system is generated by alternative splicing of exons, yet there is limited understanding of its timing or control mechanisms. We examined splicing of myosin phosphatase regulatory subunit (Mypt1) exon 24 (E24) in relation to smooth muscle myosin heavy chain (Smmhc) and smoothelin (Smtn) alternative exons (Smmhc E6 and Smtn E20) during maturation of mouse mesenteric artery (MA) smooth muscle. The role of transformer 2β (Tra2β), a master regulator of splicing in flies, in maturation of arterial smooth muscle was tested through gene inactivation. Splicing of alternative exons in bladder smooth muscle was examined for comparative purposes. MA smooth muscle maturation began after postnatal week 2 and was complete at maturity, as indicated by switching to Mypt1 E24+ and Smtn E20- splice variants and 11-fold induction of Smmhc. Similar changes in bladder were complete by postnatal day 3. Splicing of Smmhc E6 was temporally dissociated from Mypt1 E24 and Smtn E20 and discordant between arteries and bladder. Tamoxifen-induced smooth muscle-specific inactivation of Tra2β within the first week of life but not in maturity reduced splicing of Mypt1 E24 in MAs. Inactivation of Tra2β causing a switch to the isoform of MYPT1 containing the COOH-terminal leucine zipper motif (E24-) increased arterial sensitivity to cGMP-mediated relaxation. In conclusion, maturation of mouse MA smooth muscle begins postnatally and continues until sexual maturity. TRA2β is required for specification during this period of maturation, and its inactivation alters the contractile properties of mature arterial smooth muscle.
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http://dx.doi.org/10.1152/ajpcell.00304.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329427PMC
February 2015

Ischemic preconditioning inhibits mitochondrial permeability transition pore opening through the PTEN/PDE4 signaling pathway.

Cardiology 2014 4;129(3):163-73. Epub 2014 Oct 4.

Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Md., USA.

Objectives: Ischemic preconditioning (IPC) induces cardioprotection against ischemia-reperfusion (IR) injury by inhibiting the mitochondrial permeability transition pore (mPTP). Here, we tested the hypothesis that IPC-induced cardioprotection is mediated by the phosphatase PTEN and PDE4 (phosphodiesterase 4).

Methods: Isolated hearts from wild-type mice (WT, n = 110) and myocyte-specific PTEN-knockout mice (PKO, n = 94) were exposed to IPC or control conditions followed by IR. Subcellular fractionation was performed by sucrose gradient ultracentrifugation.

Results: IPC limited myocardial infarct size (IS) in WT mice. The PDE4 inhibitor rolipram abolished the protective effect of IPC. However, small IS was found in PKO hearts after IR, and IPC did not decrease IS but enlarged it in PKO hearts. IPC promoted PDE4D localization to caveolin-3-enriched fractions in WT mice by increasing Akt levels at the caveolae. In PKO hearts, basal PDE4D levels were elevated at the caveolae, and IPC decreased PDE4D levels. Consistent with the subcellular PDE4D protein levels and its activity, elevation in intracellular Ca(2+) levels in the ischemic heart and opening of mPTP after IR were inhibited by IPC in WT mice, but not by IPC in PKO mice.

Conclusions: IPC inhibits mPTP opening by regulating the PTEN/PDE4 signaling pathway.
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http://dx.doi.org/10.1159/000363646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240802PMC
July 2015

Neural programming of mesenteric and renal arteries.

Am J Physiol Heart Circ Physiol 2014 Aug;307(4):H563-73

There is evidence for developmental origins of vascular dysfunction yet little understanding of maturation of vascular smooth muscle (VSM) of regional circulations. We measured maturational changes in expression of myosin phosphatase (MP) and the broader VSM gene program in relation to mesenteric small resistance artery (SRA) function. We then tested the role of the sympathetic nervous system (SNS) in programming of SRAs and used genetically engineered mice to define the role of MP isoforms in the functional maturation of the mesenteric circulation. Maturation of rat mesenteric SRAs as measured by qPCR and immunoblotting begins after the second postnatal week and is not complete until maturity. It is characterized by induction of markers of VSM differentiation (smMHC, γ-, α-actin), CPI-17, an inhibitory subunit of MP and a key target of α-adrenergic vasoconstriction, α1-adrenergic, purinergic X1, and neuropeptide Y1 receptors of sympathetic signaling. Functional correlates include maturational increases in α-adrenergic-mediated force and calcium sensitization of force production (MP inhibition) measured in first-order mesenteric arteries ex vivo. The MP regulatory subunit Mypt1 E24+/LZ- isoform is specifically upregulated in SRAs during maturation. Conditional deletion of mouse Mypt1 E24 demonstrates that splicing of E24 causes the maturational reduction in sensitivity to cGMP-mediated vasorelaxation (MP activation). Neonatal chemical sympathectomy (6-hydroxydopamine) suppresses maturation of SRAs with minimal effect on a conduit artery. Mechanical denervation of the mature rat renal artery causes a reversion to the immature gene program. We conclude that the SNS captures control of the mesenteric circulation by programming maturation of the SRA smooth muscle.
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http://dx.doi.org/10.1152/ajpheart.00250.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4137124PMC
August 2014

Myosin phosphatase isoforms and related transcripts in the pig coronary circulation and effects of exercise and chronic occlusion.

Microvasc Res 2015 Mar 15;98:166-71. Epub 2014 Feb 15.

Division of Cardiology, School of Medicine, University of Maryland, Baltimore, MD 21201, USA. Electronic address:

Myosin phosphatase (MP) is a key target of signaling pathways that regulate smooth muscle tone and blood flow. Alternative splicing of MP targeting subunit (MYPT1) exon 24 (E24) generates isoforms with variable presence of a C-terminal leucine zipper (LZ) required for activation of MP by NO/cGMP. Here we examined the expression of MP and associated genes in a disease model in the coronary circulation. Female Yucatan miniature swine remained sedentary or were exercise-trained beginning eight weeks after placement of an ameroid constrictor around the left circumflex (LCX) artery. Fourteen weeks later epicardial arteries (~1mm) and resistance arterioles (~125 μm) were harvested and assayed for gene expression. MYPT1 isoforms were distinct in the epicardial arteries (E24-/LZ+) and resistance arterioles (E24+/LZ-) and unchanged by exercise training or coronary occlusion. MYPT1, CPI-17 and PDE5 mRNA levels were not different between arteries and arterioles while Kir2.1 and eNOS were 6.6-fold and 3.9-fold higher in the arterioles. There were no significant changes in transcript abundance in epicardial arteries of the collateralized (LCX) vs. non-occluded left anterior descending (LAD) territories, or in exercise-trained vs. sedentary pigs. There was a significant 1.2 fold increase in CPI-17 in collateral-dependent arterioles, independent of exercise, and a significant 1.7 fold increase in PDE5 in arterioles from exercise-trained pigs, independent of occlusion. We conclude that differences in MYPT1 E24 (LZ) isoforms, eNOS, and Kir2.1 distinguish epicardial arteries and resistance coronary arterioles. Up-regulation of coronary arteriolar PDE5 by exercise and CPI-17 by chronic occlusion could contribute to altered vasomotor responses and requires further study.
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http://dx.doi.org/10.1016/j.mvr.2014.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134433PMC
March 2015

Smooth muscle contractile diversity in the control of regional circulations.

Am J Physiol Heart Circ Physiol 2014 Jan 1;306(2):H163-72. Epub 2013 Nov 1.

Division of Cardiology, School of Medicine, University of Maryland, Baltimore, Maryland.

Each regional circulation has unique requirements for blood flow and thus unique mechanisms by which it is regulated. In this review we consider the role of smooth muscle contractile diversity in determining the unique properties of selected regional circulations and its potential influence on drug targeting in disease. Functionally smooth muscle diversity can be dichotomized into fast versus slow contractile gene programs, giving rise to phasic versus tonic smooth muscle phenotypes, respectively. Large conduit vessel smooth muscle is of the tonic phenotype; in contrast, there is great smooth muscle contractile diversity in the other parts of the vascular system. In the renal circulation, afferent and efferent arterioles are arranged in series and determine glomerular filtration rate. The afferent arteriole has features of phasic smooth muscle, whereas the efferent arteriole has features of tonic smooth muscle. In the splanchnic circulation, the portal vein and hepatic artery are arranged in parallel and supply blood for detoxification and metabolism to the liver. Unique features of this circulation include the hepatic-arterial buffer response to regulate blood flow and the phasic contractile properties of the portal vein. Unique features of the pulmonary circulation include the low vascular resistance and hypoxic pulmonary vasoconstriction, the latter attribute inherent to the smooth muscle cells but the mechanism uncertain. We consider how these unique properties may allow for selective drug targeting of regional circulations for therapeutic benefit and point out gaps in our knowledge and areas in need of further investigation.
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http://dx.doi.org/10.1152/ajpheart.00493.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920130PMC
January 2014

Sulfhydryl-dependent dimerization and cGMP-mediated vasodilatation.

J Cardiovasc Pharmacol 2013 Jul;62(1):1-5

Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China.

Sulfhydryl-dependent formation of interprotein disulfide bonds in response to physiological oxidative stimuli is emerging as an important mechanism in the regulation of various biological activities. Soluble guanylyl cyclase (sGC) and cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) are key enzymes for actions caused by cGMP-elevating agents, including nitric oxide (NO). Both sGC and PKG are dimers. The dimerization of sGC is obligatory for its activity, whereas the dimerization of PKG improving its signaling efficacy. sGC dimerization is decreased by endogenous and exogenous thiol reductants, associated with reduced cGMP elevation and attenuated vasodilatation to NO. The dimerization of PKG Iα is increased by oxidative stress, coincident with improved PKG signaling and augmented vasodilatation to NO. In coronary arteries, the dimerizations and activities of sGC and PKG are increased by hypoxia, accompanied by enhanced relaxation induced by NO. In contrast, the dimerizations and activities of these enzymes and NO-induced relaxation of pulmonary arteries are reduced by hypoxia. These opposite effects may result from divergent changes in the redox status of cytoplasmic reduced nicotinamide adenine dinucleotide phosphate between coronary and pulmonary arteries in response to hypoxia.
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http://dx.doi.org/10.1097/FJC.0b013e3182813865DOI Listing
July 2013

Remote ischemic preconditioning confers late protection against myocardial ischemia-reperfusion injury in mice by upregulating interleukin-10.

Basic Res Cardiol 2012 Jul 1;107(4):277. Epub 2012 Jul 1.

Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Remote ischemic preconditioning (RIPC) induces a prolonged late phase of multi-organ protection against ischemia-reperfusion (IR) injury. In the present study, we tested the hypothesis that RIPC confers late protection against myocardial IR injury by upregulating expression of interleukin (IL)-10. Mice were exposed to lower limb RIPC or sham ischemia. After 24 h, mice with RIPC demonstrated decreased myocardial infarct size and improved cardiac contractility following 30-min ischemia and 120-min reperfusion (I-30/R-120). These effects of RIPC were completely blocked by anti-IL-10 receptor antibodies. In IL-10 knockout mice, RIPC cardioprotection was lost, but it was mimicked by exogenous IL-10. Administration of IL-10 to isolated perfused hearts increased phosphorylation of the protein kinase Akt and limited infarct size after I-30/R-120. In wild-type mice, RIPC increased plasma and cardiac IL-10 protein levels and caused activation of Akt and endothelial nitric oxide synthase in the heart at 24 h, which was also blocked by anti-IL-10 receptor antibodies. In the gastrocnemius muscle, RIPC resulted in immediate inactivation of the phosphatase PTEN and activation of Stat3, with increased IL-10 expression 24 h later. Myocyte-specific PTEN inactivation led to increased Stat3 phosphorylation and IL-10 protein expression in the gastrocnemius muscle. Taken together, these results suggest that RIPC induces late protection against myocardial IR injury by increasing expression of IL-10 in the remote muscle, followed by release of IL-10 into the circulation, and activation of protective signaling pathways in the heart. This study provides a scientific basis for the use of RIPC to confer systemic protection against IR injury.
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http://dx.doi.org/10.1007/s00395-012-0277-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596418PMC
July 2012

Hydrogen peroxide enhances vasodilatation by increasing dimerization of cGMP-dependent protein kinase type Iα.

Circ J 2012 11;76(7):1792-8. Epub 2012 Apr 11.

Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.

Background: cGMP-dependent protein kinase type I (PKG I) plays a key role in vasodilatation caused by cGMP-elevating agents. It is a homodimer in mammalian cells, existing as 2 isoforms, Iα and Iβ. The aim of the present study was both to determine whether PKG I dimerization and activity are modulated by hydrogen peroxide (H(2)O(2)) and its influence on vasodilatation.

Methods And Results: The dimers and monomers of total PKG I and PKG Iβ were analyzed by Western blotting. PKG I activity was assayed by measuring the incorporation of (32)P into BPDEtide. Changes in vessels tension were determined by organ chamber technique. In isolated porcine coronary arteries, H(2)O(2) increased the dimers of total PKG I in a concentration-dependent manner, but had no effect on dimerization of PKG Iβ. The dimerization of PKG I caused by H(2)O(2) was prevented by catalase but not by deferoxamine and tiron. H(2)O(2) promoted the translocation of PKG I from cytoplasm to membrane. H(2)O(2) enhanced the activity of PKG I and relaxations of porcine coronary arteries to the nitric oxide donor and 8-Br-cGMP. Inhibition of catalase under in vivo conditions significantly decreased rat mean arterial pressure, which was associated with increased dimerization of PKG I.

Conclusions: The present study suggests that H(2)O(2) may enhance the activity of PKG Iα-and PKG I-dependent vasodilatation via increased dimerization of the enzyme.
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http://dx.doi.org/10.1253/circj.cj-11-1368DOI Listing
October 2012

Phosphatase PTEN is critically involved in post-myocardial infarction remodeling through the Akt/interleukin-10 signaling pathway.

Basic Res Cardiol 2012 Mar 2;107(2):248. Epub 2012 Feb 2.

Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

The inflammatory cytokines interleukin (IL)-10 and tumor necrosis factor (TNF)-α play an important role in left ventricular (LV) remodeling after myocardial infarction (MI). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) inactivates protein kinase Akt and promotes cell death in the heart. However, it is not known whether PTEN promotes post-MI remodeling by regulating IL-10 and TNF-α. MI was induced in wild-type (WT) mice and Pten heterozygous mutant (HET) mice. Pten adenoviruses (adPten) or empty vectors (adNull) were injected into the peri-infarct area of WT mice. LV dilation was attenuated and fractional shortening was increased in HET mice compared to WT mice. Survival rate and fractional shortening were decreased in adPten mice compared to adNull mice. Leukocyte infiltration into the peri-infarct area was attenuated in HET mice and worsened in adPten mice. PTEN expression was upregulated in the infarcted heart of WT mice. Partial inactivation of PTEN increased the production of IL-10 and decreased the expression of TNF-α and matrix metalloproteinase (MMP)-2 and -9 after MI in HET mice. PTEN overexpression caused opposite effects in the infarcted heart. Moreover in the infarcted heart of HET mice, Akt inhibition decreased Stat3 phosphorylation and IL-10 expression, and blockade of the IL-10 receptor increased TNF-α and MMP-2 expression. Both Akt inhibition and IL-10 receptor blockade abolished the attenuation of post-MI remodeling in HET mice. In conclusion, PTEN is critically involved in post-MI remodeling through the Akt/IL-10 signaling pathway. Therefore, targeting PTEN may be an effective approach to post-MI remodeling.
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http://dx.doi.org/10.1007/s00395-012-0248-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366430PMC
March 2012

Ischemic preconditioning attenuates mitochondrial localization of PTEN induced by ischemia-reperfusion.

Am J Physiol Heart Circ Physiol 2011 Jun 18;300(6):H2177-86. Epub 2011 Mar 18.

720 Rutland Ave., Ross 333, Baltimore, MD 21205, USA.

Although the induction of myocyte apoptosis by ischemia-reperfusion (I/R) is attenuated by ischemic preconditioning (IPC), the underlying mechanism is not fully understood. Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) promotes apoptosis through Akt-dependent and -independent mechanisms. We tested the hypothesis that IPC attenuates the mitochondrial localization of PTEN in the myocardium induced by I/R. Isolated hearts from wild-type mice were exposed to IPC or normal perfusion followed by 30 min of ischemia and reperfusion. IPC attenuated myocardial infarct size and apoptosis after I/R. Heart fractionation showed that mitochondrial PTEN and Bax protein levels and the physical association between them were increased by 30 min of I/R and that IPC attenuated all of these effects of I/R. Muscle-specific PTEN knockout decreased mitochondrial Bax protein levels in the reperfused myocardium and increased cell survival. To determine whether PTEN relocalization to mitochondria was influenced by I/R-induced production of ROS, hearts were perfused with N-acetylcysteine (NAC) to scavenge ROS or H(2)O(2) to mimic I/R-induced ROS. Mitochondrial PTEN protein levels were decreased by NAC and increased by H(2)O(2). PTEN protein overexpression was generated in mouse hearts by adenoviral gene transfer. PTEN overexpression increased mitochondrial PTEN and Bax protein levels and ROS production, whereas muscle-specific PTEN knockout produced the opposite effects. In conclusion, myocardial I/R causes PTEN localization to the mitochondria, related to the generation of ROS; IPC attenuates the mitochondrial localization of PTEN after I/R, potentially inhibiting the translocation of Bax to the mitochondria and resulting in improved cell viability.
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http://dx.doi.org/10.1152/ajpheart.01138.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3119092PMC
June 2011

Role of sulfhydryl-dependent dimerization of soluble guanylyl cyclase in relaxation of porcine coronary artery to nitric oxide.

Cardiovasc Res 2011 Jun 19;90(3):565-72. Epub 2011 Jan 19.

Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xue Yuan Road, Beijing 100191, China.

Aims: Soluble guanylyl cyclase (sGC) is a heterodimer. The dimerization of the enzyme is obligatory for its function in mediating actions caused by agents that elevate cyclic guanosine monophosphate (cGMP). The present study aimed to determine whether sGC dimerization is modulated by thiol-reducing agents and whether its dimerization influences relaxations in response to nitric oxide (NO).

Methods And Results: The dimers and monomers of sGC and cGMP-dependent protein kinase (PKG) were analysed by western blotting. The intracellular cGMP content was measured by enzyme-linked immunosorbent assay. Changes in isometric tension were determined in organ chambers. In isolated porcine coronary arteries, the protein levels of sGC dimer were decreased by the thiol reductants dithiothreitol, l-cysteine, reduced l-glutathione and tris(2-carboxyethyl) phosphine. The effect was associated with reduced cGMP elevation and attenuated relaxations in response to nitric oxide donors. The dimerization of sGC and activation of the enzyme were also decreased by dihydrolipoic acid, an endogenous thiol antioxidant. Dithiothreitol at concentrations markedly affecting the dimerization of sGC had no significant effect on the dimerization of PKG or relaxation in response to 8-Br-cGMP. Relaxation of the coronary artery in response to a NO donor was potentiated by hypoxia when sGC was partly inhibited, coincident with an increase in sGC dimer and enhanced cGMP production. These effects were prevented by dithiothreitol and tris(2-carboxyethyl) phosphine.

Conclusion: These results demonstrate that the dimerization of sGC is exquisitely sensitive to thiol reductants compared with that of PKG, which may provide a novel mechanism for thiol-dependent modulation of NO-mediated vasodilatation in conditions such as hypoxia.
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http://dx.doi.org/10.1093/cvr/cvr016DOI Listing
June 2011

Increased degradation of MYPT1 contributes to the development of tolerance to nitric oxide in porcine pulmonary artery.

Am J Physiol Lung Cell Mol Physiol 2010 Jul 23;299(1):L117-23. Epub 2010 Apr 23.

Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China.

Myosin phosphatase target subunit 1 (MYPT1) is the regulatory subunit of myosin light chain phosphatase (MLCP). It plays a critical role in vasodilatation induced by cGMP-elevating agents such as nitric oxide (NO). The present study was performed to determine the role of MYPT1 in the development of tolerance of the pulmonary artery to NO. Incubation of isolated porcine pulmonary arteries for 24 or 48 h with DETA NONOate (DETA NO) significantly reduced protein levels of MYPT1 and the leucine zipper-positive (LZ+) isoform of MYPT1 but not that of PP1cdelta. The extent of reduction in total MYPT1 protein level was comparable to that of MYPT1 (LZ+). The decrease in MYPT1 protein caused by 48-h DETA NO incubation was prevented by ODQ, an inhibitor of guanylyl cyclase, and by inhibitors of proteasomes (MG-132 and lactacystin) but was not affected by the inhibitor of protein synthesis, cycloheximide. A reduction in MYPT1 protein was also obtained with 8-bromo-cGMP, but this was prevented by Rp-8-bromo-PET-cGMP [inhibitor of cGMP-dependent protein kinase (PKG)]. Incubation for 48 h with DETA NO also reduced dephosphorylation of myosin light chain and relaxation of the artery in response to DETA NO, which was prevented by MG-132. These results suggest that the reduction in MYPT1 protein contributes to the development of tolerance of pulmonary arteries to NO. This may result from increased degradation of MYPT1 after prolonged PKG activation.
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http://dx.doi.org/10.1152/ajplung.00340.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904097PMC
July 2010

Degradation of leucine zipper-positive isoform of MYPT1 may contribute to development of nitrate tolerance.

Cardiovasc Res 2010 Apr 25;86(1):151-9. Epub 2009 Nov 25.

Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xue Yuan Road, Beijing 100191, People's Republic of China.

Aims: A depressed cGMP-dependent protein kinase (PKG) activity is implicated in nitrate tolerance. The present study determines whether the leucine zipper-positive (LZ+) isoform of myosin phosphatase target subunit 1 (MYPT1), a key target protein for PKG actions, is involved in the development of nitrate tolerance.

Methods And Results: Nitrate tolerance in in vitro preparations was obtained by a 24 h incubation with nitroglycerin (NTG). Nitrate tolerance in in vivo preparations was obtained by subcutaneous injection of mice with NTG, and the aortas were used. Protein levels of total MYPT1, MYPT1 (LZ+), PP1Cdelta, myosin light chain (MLC), and phosphorylated MLC were determined by Western blot analysis. Isometric vessel tension was determined by an organ chamber technique. Protein levels of MYPT1 (LZ+), but not of PP1Cdelta, were significantly reduced in in vitro and in vivo nitrate-tolerant arteries. The decrease in the MYPT1 (LZ+) protein level of coronary artery was also induced by a nitric oxide donor and a cGMP analogue, which was prevented by the inhibitors of soluble guanylyl cyclase and PKG. The decrease in MYPT1 (LZ+) protein levels was not affected by the inhibitor of protein synthesis, but was prevented by the inhibitors of proteasomes. The diminished inhibition of dephosphorylation of MLC as well as the attenuated relaxation of porcine coronary artery and mouse aorta to NTG was improved by proteasome inhibitors.

Conclusion: This study demonstrates that a reduction in the protein level of MYPT1 (LZ+) is involved in nitrate tolerance. This may result in part from a proteasome-dependent degradation of MYPT1 (LZ+).
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http://dx.doi.org/10.1093/cvr/cvp376DOI Listing
April 2010

cGMP-dependent protein kinase in regulation of basal tone and in nitroglycerin- and nitric-oxide-induced relaxation in porcine coronary artery.

Pflugers Arch 2007 Sep 22;454(6):913-23. Epub 2007 Mar 22.

Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xue Yuan Road, Beijing, 100083, China.

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) may act as a critical enzyme for nitric-oxide-induced vasodilation. In this study, the role of PKG in regulation of basal tension and in relaxation induced by nitrovasodilators in coronary arteries was determined. Under basal conditions, Rp-8-Br-PET-cGMPS, a specific PKG inhibitor, evoked a significant contraction of isolated porcine coronary arteries, which was prevented by nitro-L: -arginine or the removal of the endothelium. Relaxation to nitroglycerin and (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) in vessels preconstricted with U46619 was largely abolished by 1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one (ODQ) and inhibited by 48 to 79% by Rp-8-Br-PET-cGMPS. Relaxation of the vessels to 8-Br-cGMP was inhibited by 56% by Rp-8-Br-PET-cGMPS. The basal activity of PKG but not that of cyclic adenosine monophosphate-dependent protein kinase (PKA) was inhibited by nitro-L: -arginine, ODQ, or Rp-8-Br-PET-cGMPS. The activity of PKG but not that of PKA was increased by nitroglycerin and DETA NONOate in intact vessels and increased by cGMP in the tissue homogenates. These effects were abolished by Rp-8-Br-PET-cGMPS but not by myristoylated PKI, a specific inhibitor of PKA. These results suggest that in porcine coronary arteries, PKG is involved in the regulation of basal tension and plays a primary role in relaxation induced by nitrovasodilators, whereas PKA may play a minor role.
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http://dx.doi.org/10.1007/s00424-007-0249-8DOI Listing
September 2007
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