Liming Jin

Publications (22)84.98 Total impact

• Article: Serum biomarker measurements of endothelial function and oxidative stress after daily dosing of sildenafil in type 2 diabetic men with erectile dysfunction.

  Arthur L Burnett, Travis D Strong, Bruce J Trock, Liming Jin, Trinity J Bivalacqua, Biljana Musicki
  [show abstract] [hide abstract]
  ABSTRACT: We investigated changes in serum biomarkers of vascular function after short-term, continuous sildenafil dosing in men with type 2 diabetes with erectile dysfunction. Men with erectile dysfunction associated with type 2 diabetes mellitus were randomized to receive continuous, daily sildenafil (50 mg for 1 week run-in and 100 mg for 3 weeks) (148), or placebo (144) for 4 weeks (phase I) and then sildenafil (25, 50 or 100 mg) on demand for 12 weeks (phase II). Blood draws at baseline and after phases I and II were analyzed for cyclic guanosine monophosphate (endothelial function marker), 8-isoprostane (oxidative stress marker), and interleukin-6 and interleukin-8 (inflammatory cytokines). Primary and secondary erectile function outcome variables were affirmative responses on Sexual Encounter Profile question 3 (ability to maintain erection sufficient for sexual intercourse) and Erection Hardness Score, respectively. Serum cyclic guanosine monophosphate levels were increased in the sildenafil group relative to the placebo group at 4 (p <0.01) and 16 (p <0.05) weeks, correlating with affirmative responses to Sexual Encounter Profile question 3 at the 4-week interval only (p <0.05). Serum 8-isoprostane levels were decreased to a nonsignificant degree in the sildenafil group at 4 weeks with no further change at 16 weeks, whereas interleukin-6 and interleukin-8 levels were unchanged at either interval, and these levels were unassociated with erectile function outcomes. These data suggest that short-term, continuous sildenafil treatment causes systemic endothelial function to be enhanced and remain so for a duration after its discontinuation. However, they do not indicate any influence of this treatment on systemic oxidative stress or inflammation, or an effect on long-term erectile function improvement.
  The Journal of urology 11/2008; 181(1):245-51. · 4.02 Impact Factor
• Article: Comparison of the involvement of protein kinase C in agonist-induced contractions in mouse aorta and corpus cavernosum.

  Liming Jin, Cleber E Teixeira, R Clinton Webb, Romulo Leite
  [show abstract] [hide abstract]
  ABSTRACT: Protein kinase C (PKC) is involved in the regulation of vascular smooth muscle contraction. However, the role of PKC in erectile function is poorly understood. This study investigated whether PKC mediates agonist-induced contractions in mouse penile tissue (corpora cavernosa). We also compared the effects of PKC activators and inhibitors on contractile responses in mouse corpus cavernosum with those in mouse aorta. Aortic rings and corpus cavernosal strips from C57BL/6J mice were mounted in the organ bath for isometric tension recording. Our data showed that a PKC(alpha/beta) selective inhibitor, G(ö)6976 (10 microM), inhibited phenylephrine and 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha) (U46619, a thromboxane mimetic)-induced contractions in mouse aorta, reducing the maximum contraction by 94% and 17%, respectively. A non-selective PKC inhibitor, chelerythrine (30 microM), also significantly reduced phenylephrine- and U46619-induced maximum contractions in mouse aorta. However, G(ö)6976 and chelerythrine had no significant effects on phenylephrine- and U46619-induced contractions in corpus cavernosum. Furthermore, a PKC activator, phorbol-12,13-dibutyrate (0.1 microM), significantly increased contractions in aorta (208+/-14% of KCl-induced maximum contraction) but failed to cause contractions in corpus cavernosum at 1 and 10 microM. Western blot analysis data suggested that protein expression of PKC was similar in aorta and corpus cavernosum. Taken together, our data indicate that PKC does not have a significant role in agonist-induced contractions in mouse corpus cavernosum, whereas it mediates the contractile response to agonists in the aorta.
  European Journal of Pharmacology 07/2008; 590(1-3):363-8. · 2.52 Impact Factor
• Article: NADPH oxidase activation: a mechanism of hypertension-associated erectile dysfunction.

  Liming Jin, Gwen Lagoda, Romulo Leite, R Clinton Webb, Arthur L Burnett
  [show abstract] [hide abstract]
  ABSTRACT: Hypertension is a risk factor for erectile dysfunction (ED). The pathophysiologic basis of ED in hypertension remains largely unknown. The goal of this study was to test the hypothesis that increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity contributes to the development of hypertension-associated ED. Male Sprague-Dawley rats were implanted with osmotic pumps containing saline or angiotensin II (Ang II, 70 ng/min) for 28 days and treated with or without the NADPH oxidase inhibitor apocynin (10 mM) in the drinking water. Erectile function was examined by measuring the mean arterial blood pressure (MAP) and intracavernosal pressure (ICP) upon electrical stimulation of the cavernous nerve. Protein expression levels of NADPH oxidase subunits were analyzed by Western blot. Reactive oxygen species production was determined by dihydroethidium (DHE) staining and thiobarbituric acid reactive substances (TBARS) assay. Maximum ICP (MaxICP) and ICP area under the curve, which were normalized by MAP, were significantly reduced in Ang II-infused hypertensive rats compared to that of normotensive rats (P < 0.05). Protein expression of NADPH oxidase subunit p47(phox) was significantly increased by 30% in Ang II-infused hypertensive rat penes along with increased DHE staining and TBARS levels (P < 0.05) when compared to that of controls. There were no significant changes in p67(phox) or gp91(phox) protein expression. Apocynin reduced NADPH oxidase protein expression and TBARS levels as well as improved MaxICP and ICP area under curve in Ang II-infused hypertensive rats (P < 0.05). These data suggest that activation of NADPH oxidase is a molecular mechanism for hypertension-associated ED. Apocynin treatment exerted protective effects on erectile function through inhibition of NADPH oxidase activity, thereby reducing oxidative stress in Ang II-infused hypertensive rats. This is the first study to identify the importance of NADPH oxidase in the regulation of erectile function in vivo.
  Journal of Sexual Medicine 04/2008; 5(3):544-51. · 3.55 Impact Factor
• Article: Low-fat diet and exercise preserve eNOS regulation and endothelial function in the penis of early atherosclerotic pigs: a molecular analysis.

  Biljana Musicki, Tongyun Liu, Travis Strong, Liming Jin, M Harold Laughlin, James R Turk, Arthur L Burnett
  [show abstract] [hide abstract]
  ABSTRACT: Diet and exercise affect endothelial function in the penis, but the molecular mechanisms underlying their effects are not understood. We evaluated endothelial nitric oxide synthase (eNOS) interaction with its negative regulator caveolin-1 and eNOS uncoupling as molecular targets in the penis associated with the beneficial effects of low-fat diet and chronic exercise. The penes were obtained from adult male Yucatan pigs fed a normal-fat or high-fat diet on exercised or sedentary regimen for 24 weeks. Markers of endothelial function (guanosine 3',5'-monophosphate [cGMP] production), endothelial dysfunction (eNOS uncoupling and eNOS interaction with caveolin-1), and oxidative stress (thiobarbituric acid reactive substances [TBARS]) were measured in the penes. The concentrations of cGMP and TBARS were determined using commercial kits. eNOS uncoupling was determined by low-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis. eNOS binding to caveolin-1, eNOS phosphorylation (Ser-1177), and protein expression of eNOS and caveolin-1 were measured by Western blot analysis in penes purified for NOS and in homogenates, respectively. Molecular parameters of endothelial function including eNOS regulatory function. Relative to normal-fat diet, high-fat diet significantly (P < 0.05) reduced cGMP levels and significantly (P < 0.05) increased eNOS uncoupling, eNOS binding to caveolin-1, and TBARS production in the penis of sedentary pigs. Exercise of pigs on high-fat diet reversed (P < 0.05) the abnormalities in cGMP levels, eNOS uncoupling, and eNOS binding to caveolin-1, but not TBARS levels. Exercise of pigs on normal-fat diet did not affect any of these parameters. Protein expressions of caveolin-1, phosphorylated (Ser-1177), and total eNOS were unaffected by diet or exercise. Low-fat diet and chronic exercise preserve endothelial function in the pig penis by sustaining active eNOS in its dimeric form and by limiting eNOS interaction with its negative regulator caveolin-1.
  Journal of Sexual Medicine 03/2008; 5(3):552-61. · 3.55 Impact Factor
• Article: NADPH oxidase: recent evidence for its role in erectile dysfunction.

  Liming Jin, Arthur L Burnett
  [show abstract] [hide abstract]
  ABSTRACT: Important roles for reactive oxygen species (ROS) in physiology and pathophysiology have been increasingly recognized. Under normal conditions, ROS serve as signaling molecules in the regulation of cellular functions. However, enhanced ROS production as a result of the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contributes significantly to the pathogeneses of vascular diseases. Although it has become evident that increased ROS is associated with erectile dysfunction (ED), the sources of ROS in the penis remain largely unknown. In recent years, emergent evidence suggests the possible role of NADPH oxidase in inducing ED. In this review, we examine the relationship between ROS and ED in different disease models and discuss the current evidence basis for NADPH oxidase-derived ROS in ED.
  Asian Journal of Andrology 02/2008; 10(1):6-13. · 1.52 Impact Factor
• Article: FK506 and sildenafil promote erectile function recovery after cavernous nerve injury through antioxidative mechanisms.

  Gwen Lagoda, Liming Jin, Todd J Lehrfeld, Tongyun Liu, Arthur L Burnett
  [show abstract] [hide abstract]
  ABSTRACT: Immunophilin ligands and phosphodiesterase type 5 (PDE5) inhibitors are touted to promote erectile function recovery after cavernous nerve (CN) injury. However, the mechanisms for their effects remain unclear. To compare the erection recovery effects of the immunophilin ligand FK506 and the PDE5 inhibitor sildenafil after CN injury and determine whether they involve antioxidative and/or antiapoptotic mechanisms. Initial experiments established conditions of our CN injury model in adult male Sprague-Dawley rats. Subsequently, we evaluated treatment effects 14 days after: (i) unilateral CN injury (UNI) + saline (vehicle control); (ii) UNI + FK506 (5 mg/kg once daily, subcutaneous x 5 days); (iii) UNI + sildenafil (20 mg/kg every 8 hours, subcutaneous x 7 days); (iv) UNI + FK506/sildenafil; and (v) sham surgery. Intracavernous pressure (ICP) measurement after CN electrical stimulation to assess erectile function and Western blot analysis of expressions of glutathione peroxidase (GPX; antioxidant enzyme), nitrotyrosine (NT; oxidative stress marker), and phosphorylated and total Akt (antiapoptotic factor) in penes. In the UNI model, GPX expression was increased at Days 1 and 7, while p-Akt expression decreased at Day 1 and returned to baseline at Day 7. GPX expression was significantly higher in the UNI + FK506 group compared with the saline-treated group (P < 0.05). ICP increased in all treatment groups compared with that of the saline-treated group (P < 0.05). NT levels were increased after saline treatment (P < 0.05) but not after FK506 and sildenafil treatment, alone or in combination. GPX was localized to nerves coursing through the penis and to smooth muscle and endothelium of the dorsal vein and arteries. Both FK506 and sildenafil protect erectile function after CN injury by decreasing oxidative stress-associated tissue damage. FK506 may act through increased GPX activity. Further research is required to elucidate mechanisms associated with the beneficial effect of sildenafil.
  Journal of Sexual Medicine 08/2007; 4(4 Pt 1):908-16. · 3.55 Impact Factor
• Article: Comparative pharmacological analysis of Rho-kinase inhibitors and identification of molecular components of Ca2+ sensitization in the rat lower urinary tract.

  Cleber E Teixeira, Liming Jin, Fernanda B M Priviero, Zhekang Ying, R Clinton Webb
  [show abstract] [hide abstract]
  ABSTRACT: We aimed to compare the expression and function of molecular components of the RhoA/Rho-kinase signaling pathway in the contractile responses of detrusor, trigonal and urethral smooth muscle, using selective Rho-kinase inhibitors. Contractility studies and molecular approaches were employed to demonstrate the expression patterns and functional activity of the RhoA/Rho-kinase signaling pathway in the lower urinary tract. Frequency-response curves (1-32 Hz) and concentration-response curves (CRC) to carbachol (CCh, 0.01-30 microM), phenylephrine (PE, 0.01-300 microM) and endothelin-1 (ET-1, 0.01-100 nM) were significantly attenuated (p<0.01) following incubation with the Rho-kinase inhibitors H-1152 (0.1-1 microM), Y-27632 (1-10 microM) or HA-1077 (10 microM). Addition of Rho-kinase inhibitors also markedly reduced (p<0.01) the contractions evoked by either KCl (80 mM) or alpha,beta-methylene ATP (alpha,beta-mATP, 10 microM). Among the Rho-kinase inhibitors tested, H-1152 was approximately 9-16 times more potent than Y-27632 or HA-1077. In addition, basal tone of detrusor and trigonal strips was reduced following addition of Y-27632 (10 microM), H-1152 (1 microM) and HA-1077 (10 microM). The expression of RhoA, RhoGDI, leukemia-associated RhoGEF (LARG) and p115RhoGEF was similar among the detrusor, trigone and urethra, whereas Rho-kinase alpha, Rho-kinase beta and PDZ-RhoGEF protein levels were significantly lower in the urethra. Components of the RhoA/Rho-kinase signaling are expressed in detrusor, trigonal and urethral smooth muscle and dynamically regulate contraction and tone. Manipulation of RhoGEF expression may provide further understanding of mechanisms involving Ca(2+) sensitization in the lower urinary tract.
  Biochemical Pharmacology 08/2007; 74(4):647-58. · 4.70 Impact Factor
• Article: Increased RhoA/Rho-kinase signaling mediates spontaneous tone in aorta from angiotensin II-induced hypertensive rats.

  Liming Jin, Zhekang Ying, Rob H P Hilgers, Jia Yin, Xueying Zhao, John D Imig, R Clinton Webb
  [show abstract] [hide abstract]
  ABSTRACT: Spontaneous tone in large arteries may contribute to the pathogenesis of hypertension. Reactive oxygen species and Ca2+ influx have been shown to stimulate the development of spontaneous tone in isolated aortic rings in several models of hypertensive rats. The aim of this study was to investigate the role of the RhoA/Rho-kinase signaling pathway in the development of spontaneous tone in angiotensin II-induced hypertension and to explore the underlying mechanisms of RhoA/Rho-kinase activation. Our results showed that spontaneous tone was greatly enhanced in endothelium-denuded aortic rings from angiotensin II-induced hypertensive rats compared with their normotensive counterparts (73+/-5 versus 7+/-3% of phenylephrine-induced maximal contraction, respectively). The Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) (0.1-10 microM) concentration dependently inhibited spontaneous tone in aortic rings from angiotensin II-treated rats. NADPH oxidase inhibitors diphenylene iodonium and apocynin also significantly reduced spontaneous tone. Chronic angiotensin II treatment markedly increased RhoA protein expression (57%) but had no effect on Rho guanine nucleotide exchange factor mRNA or Rho-kinase protein expression levels. In endothelium-denuded rings from normotensive rats, angiotensin II (100 nM) increased RhoA membrane translocation and phosphorylation of the myosin light chain phosphatase target subunit, which were both blocked by the NADPH oxidase inhibitor diphenylene iodonium (10 microM). In conclusion, these data suggest that chronic treatment with angiotensin II leads to up-regulation of the RhoA/Rho-kinase pathway, contributing to spontaneous tone development in rat aorta. Increased NADPH oxidase-dependent reactive oxygen species may be one of the mechanisms mediating the RhoA/Rho-kinase activation.
  Journal of Pharmacology and Experimental Therapeutics 08/2006; 318(1):288-95. · 3.83 Impact Factor
• Article: Nitric oxide/redox-based signalling as a therapeutic target for penile disorders.

  Arthur L Burnett, Biljana Musicki, Liming Jin, Trinity J Bivalacqua
  [show abstract] [hide abstract]
  ABSTRACT: Oxidative and/or nitrosative stress is implicated in the pathogeneses of assorted penile disorders of clinical significance, notably erectile dysfunction, priapism and penile fibrosis. It is becoming increasingly recognised that the generation and activity of reactive oxygen and nitrogen species in the penis influence vascular homeostasis of this organ, with adverse effects exerted at cellular and molecular levels. Furthermore, these elements may interact with molecular signalling pathways operating in the penis, modulating their functional roles. This interaction in particular suggests that by accessing molecular targets associated with oxidative/nitrosative stress in the penis, new pharmacotherapeutic approaches may be developed to promote normal erectile ability and preserve erectile tissue health. This notion pertains to, but also extends beyond, interventions which predictably target components of the nitric oxide-based signal transduction pathway for the on-demand treatment of erectile dysfunction. The next line of pharmaceuticals for disorders of the penis, in general, may well spawn from an integrative understanding of the complex regulatory interactions influenced by, as well as influencing nitric oxide signalling in this organ.
  Expert opinion on therapeutic targets 07/2006; 10(3):445-57. · 3.72 Impact Factor
• Article: Angiotensin II up-regulates the leukemia-associated Rho guanine nucleotide exchange factor (RhoGEF), a regulator of G protein signaling domain-containing RhoGEF, in vascular smooth muscle cells.

  Zhekang Ying, Liming Jin, Trenis Palmer, R Clinton Webb
  [show abstract] [hide abstract]
  ABSTRACT: In vascular smooth muscle, stimulation of heterotrimeric G protein-coupled receptors (GPCRs) by various contractile agonists activates intracellular signaling molecules to result in an increase in cytosolic Ca2+ and the subsequent phosphorylation of myosin light chain (MLC) by Ca2+/calmodulin-dependent MLC kinase. In addition, a portion of agonist-induced contraction is partially mediated by the Ca2+-independent activation of the small G protein RhoA and a downstream target, Rho-kinase. The activation of RhoA is controlled by several regulatory proteins, including guanine nucleotide exchange factors (GEFs). GEFs activate RhoA by promoting the release of GDP and then facilitating the binding of GTP. There are three Rho-specific GEFs (RhoGEFs) in vascular smooth muscle that contain a binding domain [regulator of G protein signaling (RGS) domain] capable of linking GPCRs to RhoA activation: PDZ-RhoGEF, leukemia-associated RhoGEF (LARG), and p115RhoGEF. We hypothesized that RGS domain-containing RhoGEFs, especially LARG, participate in linking GPCR to RhoA activation in vascular smooth muscle. We observed that angiotensin II up-regulates LARG via the AT1 receptor, and this up-regulation is signaled via the phosphatidylinositol 3-kinase pathway. Furthermore, angiotensin II treatment caused a small, but significant, increase in the component of contractile responses sensitive to Rho-kinase antagonism. These observations support the hypothesis that RhoGEFs, particularly LARG, participate in linking GPCR to RhoA activation in vascular smooth muscle.
  Molecular Pharmacology 04/2006; 69(3):932-40. · 4.88 Impact Factor
• Article: Cytochrome P450 epoxygenases provide a novel mechanism for penile erection.

  Liming Jin, Clare E Foss, Xueying Zhao, Thomas M Mills, Mong-Heng Wang, Lynnette P McCluskey, Ganesh S S Yaddanapud, John R Falck, John D Imig, R Clinton Webb
  [show abstract] [hide abstract]
  ABSTRACT: Erectile dysfunction (ED) is estimated to affect more than 30 million American men and 152 million men worldwide. Therapeutic agents targeting the nitric oxide/cyclic GMP signaling pathway have successfully treated patients with ED; however, the efficacies of these treatments are significantly lower in specific populations such as patients with diabetes. The goal of this study was to discover and identify new endothelium-derived relaxing factors involved in the regulation of erectile function, providing alternative therapeutic targets for treatment of ED. Immunoblotting results showed that protein expressions of epoxygenases from cytochrome P450 (CYP)2B, 2C and 2J subfamilies, as well as NADPH CYP reductase were present in rat corpora cavernosa, which was confirmed by immunohistochemical analysis. Furthermore, CYP2C was localized in cavernosal endothelial cells using double immunolabeling. CYP epoxygenase activity was analyzed by reverse-phase high-pressure liquid chromatography; and the results showed that 11,12- epoxyeicosatrienoic acid (EET) was the major product metabolized by CYP epoxygenases in rat corpora cavernosa. Inhibition of EETs function by injection of an EETs antagonist into rat penis significantly decreased intracavernosal pressure-induced by electrical stimulation of the major pelvic ganglion in vivo. In conclusion, our results suggest that EETs, produced by CYP epoxygenases, in penile endothelial cells serve as vasodilators. Inhibition of this pathway attenuated erectile function, suggesting that EETs are required for normal erection.
  The FASEB Journal 04/2006; 20(3):539-41. · 5.71 Impact Factor
• Article: RhoA/Rho-kinase in erectile tissue: mechanisms of disease and therapeutic insights.

  Liming Jin, Arthur L Burnett
  [show abstract] [hide abstract]
  ABSTRACT: Penile erection is a complicated event involving the regulation of corpus cavernosal smooth muscle tone. Recently, the small monomeric G-protein RhoA and its downstream effector Rho-kinase have been proposed to be important players for mediating vasoconstriction in the penis. RhoA/Rho-kinase increases MLC (myosin light chain) phosphorylation through inhibition of MLCP (MLC phosphatase) thereby increasing Ca2+ sensitivity. This review will outline the RhoA/Rho-kinase signalling pathway, including the upstream regulators, guanine nucleotide exchange factors, GDP dissociation inhibitors and GTPase-activating proteins. We also summarize the current knowledge about the physiological roles of RhoA/Rho-kinase in both male and female erectile tissues and its aberrations contributing to erectile dysfunction in several disease states. Understanding the RhoA/Rho-kinase signalling pathway in the regulation of erection is important for the development of therapeutic interventions for erectile dysfunction.
  Clinical Science 03/2006; 110(2):153-65. · 4.61 Impact Factor
• Article: Elevated RhoA/Rho-kinase activity in the aged rat penis: mechanism for age-associated erectile dysfunction.

  Liming Jin, Tongyun Liu, Gwen A Lagoda, Hunter C Champion, Trinity J Bivalacqua, Arthur L Burnett
  [show abstract] [hide abstract]
  ABSTRACT: Epidemiologic studies have shown that aging accounts significantly for the prevalence of erectile dysfunction (ED). The pathophysiology of ED during aging and its underlying molecular mechanisms are largely unknown. We hypothesized that increased RhoA/Rho-kinase signaling is a major factor in the pathogenesis of age-associated ED and the mechanism involves increased penile smooth muscle contractility through inhibition of myosin light chain phosphatase. Male Fischer 344 young (4 month old) and aged (20-22 month old) rats underwent erectile function testing in vivo by measuring intracavernosal pressure (ICP) and mean arterial blood pressure (MAP) upon electrical stimulation of the cavernous nerve. The data demonstrated that erectile function was significantly lower in aged rats than that in young rats at all voltages tested (P<0.05). Western blot analysis results showed that there were no significant changes in protein expressions of RhoA, Rho-kinase-alpha and -beta isoforms, and myosin light chain phosphatase target subunit (MYPT1); however, membrane-bound RhoA and phosphorylated MYPT1 were increased in aged rat penes by 95 +/- 15 and 56 +/- 8% (P<0.05), respectively, indicating enhanced RhoA and Rho-kinase activity. Inhibition of Rho-kinase with Y27632 maximally increased ICP/MAP to 0.72 +/- 0.05 in aged rats vs. 0.47 +/- 0.06 in young rats (P<0.05). Gene transfer of adeno-associated virus (AAV) encoding dominant negative RhoA (T19NRhoA) to penes of aged and young rats for 7 days markedly improved erectile function in aged rats when compared with that in young rats (P<0.05). These observations were also supported by Rho-kinase activity assay results showing that basal Rho-kinase activity in aged rat penes receiving AAV vehicle treatment was twofold greater than that in young rat penes receiving AAV vehicle treatment, while it was reduced to a level similar to that in young rat penes after gene therapy of T19NRhoA (P<0.05). Taken together, these data suggest that impaired erectile function during the aging process involves increased RhoA/Rho-kinase signaling, and this pathway may be exploited for the treatment of age-associated ED.
  The FASEB Journal 03/2006; 20(3):536-8. · 5.71 Impact Factor
• Article: Hyperglycemia and reactive oxygen species mediate apoptosis in aortic endothelial cells through Janus kinase 2.

  Amany Tawfik, Liming Jin, Amy K L Banes-Berceli, Ruth B Caldwell, Safia Ogbi, Amanda Shirley, Dwayne Barber, John D Catravas, David M Stern, David Fulton, R William Caldwell, Mario B Marrero
  [show abstract] [hide abstract]
  ABSTRACT: The generation of reactive oxygen species (ROS) has been implicated in the perturbation of endothelial function and cell death. However, the specific signaling pathways which mediate and modifying this response have not been fully elucidated. Therefore, in this study we tested the hypothesis that activation of JAK2 is involved in the aortic endothelial cell (EC) response to ROS. When ECs were exposed to HG (25 mM) for 6 h or ROS (i.e., H(2)O(2) (100 microM)) for 1 h and returned to normal medium we found a decrease in cell density and morphologic signs of apoptosis. Furthermore, incubation of ECs with HG and H(2)O(2) also resulted in the tyrosine phosphorylation of JAK2. In addition, pretreatment of ECs with AG-490, an inhibitor of JAK2, prevented nuclear fragmentation, whereas inhibitors of Jun kinase (SP 600125), MAP kinase (PD 98059), Src kinase (PP2) or PI-3 kinase (wortmannin) were without effect. Finally, immunoblot analysis of caspase-3 and PARP cleavage confirmed a role for activation of JAK2 in both HG- or ROS-induced apoptosis, based on inhibition by either AG-490 or adenoviral transfection with a dominant-negative JAK2 mutant. In conclusion the activation of JAK2 plays a pivotal role in oxidant stress-induced commitment of ECs to apoptosis, based on studies with HG and H(2)O(2).
  Vascular Pharmacology 12/2005; 43(5):320-6. · 1.99 Impact Factor
• Article: Expression and functional role of the RhoA/Rho-kinase pathway in rat coeliac artery.

  Cleber E Teixeira, Liming Jin, Zhekang Ying, Trenis Palmer, Fernanda B M Priviero, R Clinton Webb
  [show abstract] [hide abstract]
  ABSTRACT: 1. Rho-kinase (ROK) stimulation represents a key step in the maintenance of agonist-induced contraction, an effect counteracted by nitric oxide (NO) released from the endothelium. The aim of the present study was to characterize the involvement of ROK in smooth muscle contraction of the rat coeliac artery using functional and expression studies. 2. Rings of rat coeliac artery were mounted in 5 mL myographs containing warmed and oxygenated Krebs' solution. Rings were connected to isometric transducers and data were recorded in a PowerLab system (ADInstruments, Colorado Springs, CO, USA). After a 60 min equilibration period, preparations were precontracted with phenylephrine (1 micromol/L). Endothelial integrity was assessed by treating the vessels with acetylcholine (1 micromol/L). Expression of ROKalpha, ROKbeta and RhoA was analysed using western blot, whereas Rho guanine nucleotide exchange factors (RhoGEF) were measured at the mRNA level. 3. The addition of Y-27632 (0.01-30 micromol/L) caused sustained relaxation of rings contracted with phenylephrine (PE; 1 micromol/L), with intact or denuded endothelium (pEC50 = 6.38 +/- 0.03 and 5.65 +/- 0.02, respectively). NG-Nitro-L-arginine methyl ester (100 micromol/L) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 micromol/L), but not indomethacin (10 micromol/L), caused marked rightward shifts of the concentration-response curves to Y-27632. The contractile response to KCl (80 mmol/L) was significantly reduced by Y-27632, with a maximal inhibition of 57 +/- 6%. Nifedipine (0.1-100 nmol/L) fully blocked KCl-evoked contractions, but only marginally affected those in response to PE (27 +/- 2% maximal inhibition). At 1 micromol/L, Y-27632 also significantly enhanced relaxations to sodium nitroprusside (SNP; 0.0001-1 micromol/L). 4. At 1 micromol/L, SNP (but not 1 micromol/L Y-27632) significantly elevated the cGMP content above basal levels. Coincubation with SNP and Y-27632 increased cGMP levels, but the results were not significantly different from those in the presence of SNP alone. 5. Western blot analysis revealed the protein expression of RhoA, ROKalpha and ROKbeta. The PDZ-RhoGEF, p115RhoGEF and leukaemia-associated RhoGEF (LARG) mRNA expression in coeliac artery was visualized by electrophoresis on agarose gels. 6. The results clearly demonstrate a role for the RhoA/ROK signalling pathway in the regulation of rat coeliac artery smooth muscle contraction. The findings of the present study suggest that endogenous nitric oxide-induced relaxation is mediated, in part, by inhibition of RhoA/ROK signalling in this tissue.
  Clinical and Experimental Pharmacology and Physiology 11/2005; 32(10):817-24. · 1.85 Impact Factor
• Article: Ca2+ sensitization and the regulation of contractility in rat anococcygeus and retractor penis muscle.

  Cleber E Teixeira, Liming Jin, Zhekang Ying, Trenis Palmer, R Clinton Webb
  [show abstract] [hide abstract]
  ABSTRACT: Stimulation of the RhoA/Rho-kinase (ROK) signaling represents a key step in the maintenance of agonist-induced contraction of smooth muscle. We aimed to demonstrate Ca(2+) sensitization in rat anococcygeus and retractor penis muscles and to identify the molecular expression of major components of this pathway. Both anococcygeus and retractor penis showed a similar expression of RhoA, ROKalpha, and ROKbeta at the protein level as well as the mRNA for RhoGEFs. Cumulative addition of the ROK inhibitors H-1152 (0.001-3 microM), Y-27632 (0.01-30 microM) or HA-1077 (0.01-30 microM) caused sustained relaxations of precontracted smooth muscle strips. Ca(2+) sensitization induced by phenylephrine, norepinephrine and carbachol was markedly antagonized by all three ROK inhibitors. In addition, the contractile response to KCl-induced depolarization was highly sensitive to these ROK inhibitors. H-1152 was approximately 8-20 more potent than Y-27632 and HA-1077 to inhibit contraction. Electrical field stimulation (EFS, 1-32 Hz) caused transient contractions in both anococcygeus and retractor penis muscle, which were blocked by tetrodotoxin (1 microM), phentolamine (1 microM) or bretylium tosylate (30 microM). Similarly, H-1152 (0.1-1 microM), Y-27632 (1-10 microM) or HA-1077 (1-10 microM) significantly reduced EFS-evoked contractions in a concentration-dependent manner. The results indicate that the RhoA/ROK-mediated Ca(2+) sensitization pathway is expressed in anococcygeus and retractor penis muscles and enhances contractions produced by receptor-dependent and independent mechanisms.
  Biochemical Pharmacology 06/2005; 69(10):1483-92. · 4.70 Impact Factor
• Article: Decreased epoxygenase and increased epoxide hydrolase expression in the mesenteric artery of obese Zucker rats.

  Xueying Zhao, Aparajita Dey, Olga P Romanko, David W Stepp, Mong-Heng Wang, Yiqing Zhou, Liming Jin, Jennifer S Pollock, R Clinton Webb, John D Imig
  [show abstract] [hide abstract]
  ABSTRACT: Previous studies suggest that epoxyeicosatrienoic acids (EETs) are vasodilators of the mesenteric artery; however, the production and regulation of EETs in the mesenteric artery remain unclear. The present study was designed 1) to determine which epoxygenase isoform may contribute to formation of EETs in mesenteric arteries and 2) to determine the regulation of mesenteric artery cytochrome P-450 (CYP) enzymes in obese Zucker rats. Microvessels were incubated with arachidonic acid, and CYP enzyme activity was determined. Mesenteric arteries demonstrate detectable epoxygenase and hydroxylase activities. Next, protein and mRNA expressions were determined in microvessels. Although renal microvessels express CYP2C23 mRNA and protein, mesenteric arteries lacked CYP2C23 expression. CYP2C11 and CYP2J mRNA and protein were expressed in mesenteric arteries and renal microvessels. In addition, mesenteric artery protein expression was evaluated in lean and obese Zucker rats. Compared with lean Zucker rats, mesenteric arterial CYP2C11 and CYP2J proteins were decreased by 38 and 43%, respectively, in obese Zucker rats. In contrast, soluble epoxide hydrolase mRNA and protein expressions were significantly increased in obese Zucker rat mesenteric arteries. In addition, nitric oxide-independent dilation evoked by acetylcholine was significantly attenuated in mesenteric arteries of obese Zucker rats. These data suggest that the main epoxygenase isoforms expressed in mesenteric arteries are different from those expressed in renal microvessels and that decreased epoxygenases and increased soluble epoxide hydrolase are associated with impaired mesenteric artery dilator function in obese Zucker rats.
  AJP Regulatory Integrative and Comparative Physiology 02/2005; 288(1):R188-96. · 3.34 Impact Factor
• Article: Hypertension and RhoA/Rho-kinase signaling in the vasculature: highlights from the recent literature.

  Dexter L Lee, R Clinton Webb, Liming Jin
  [show abstract] [hide abstract]
  ABSTRACT: Under normal conditions, contractile activity in vascular smooth muscle is initiated by either receptor activation (norepinephrine, angiotensin II, etc.) or by a stretch-activated mechanism. After this activation, several signaling pathways can initiate a Ca2+-calmodulin interaction to stimulate phosphorylation of the light chain of myosin. Ca2+ sensitization of the contractile proteins is signaled by the RhoA/Rho-kinase pathway to inhibit the dephosphorylation of the light chain by myosin phosphatase thereby maintaining force generation. In opposition to force generation, NO is released from endothelial cells and causes vasodilation through inhibition of the RhoA/Rho-kinase signaling pathway. This brief review will highlight recent studies demonstrating a role for the RhoA/Rho-kinase signaling pathway in the increased vasoconstriction characteristic of hypertension.
  Hypertension 01/2005; 44(6):796-9. · 6.21 Impact Factor
• Article: Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta.

  Liming Jin, Zhekang Ying, R Clinton Webb
  [show abstract] [hide abstract]
  ABSTRACT: Evidence indicates that both the Rho/Rho kinase signaling pathway and reactive oxygen species (ROS) such as superoxide and H(2)O(2) are involved in the pathogenesis of hypertension. This study aimed to determine whether ROS-induced vascular contraction is mediated through activation of Rho/Rho kinase. Rat aortic rings (endothelium denuded) were isolated and placed in organ chambers for measurement of isometric force development. ROS were generated by a xanthine (X)-xanthine oxidase (XO) mixture. The antioxidants tempol (3 mM) and catalase (1,200 U/ml) or the XO inhibitor allopurinol (400 microM) significantly reduced X/XO-induced contraction. A Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632), decreased the contraction in a concentration-dependent manner; however, the Ca(2+)-independent protein kinase C inhibitor rottlerin did not have an effect on X/XO-induced contraction. Phosphorylation of the myosin light chain phosphatase target subunit (MYPT1) was increased by ROS, and preincubation with Y-27632 blocked this increased phosphorylation. Western blotting for cytosolic and membrane-bound fractions of Rho showed that Rho was increased in the membrane fraction by ROS, suggesting activation of Rho. These observations demonstrate that ROS-induced Ca(2+) sensitization is through activation of Rho and a subsequent increase in Rho kinase activity but not Ca(2+)-independent PKC.
  AJP Heart and Circulatory Physiology 11/2004; 287(4):H1495-500. · 3.71 Impact Factor
• Article: Increaseed expression of mRNA for regulator of G protein signaling domain-containing Rho guanine nucleotide exchange factors in aorta from stroke-prone spontaneously hypertensive rats.

  Zhekang Ying, Liming Jin, Anne M Dorrance, R Clinton Webb
  [show abstract] [hide abstract]
  ABSTRACT: Rho guanine nucleotide exchange factors (RhoGEF) link activation of G protein-coupled receptors (GPCR) to RhoA/Rho-kinase signaling. This cellular signaling pathway regulates vascular tone and is implicated in hypertension. The RhoGEF, providing this coupling, contain the regulator of G protein signaling (RGS) domain. We hypothesized higher mRNA expression levels for these RhoGEF in aorta from stroke-prone spontaneously hypertensive rats (SHRSP). We measured mRNA expression by semiquantitative reverse transcriptase-polymerase chain reaction in the aorta of Wistar-Kyoto rats (WKY) and SHRSP. The protein expression level of p115RhoGEF was examined by Western Blot, too. Our results showed that mRNA for RGS domain containing RhoGEF is higher in SHRSP. The results of this study indicate that RhoGEF may contribute to increased activation of RhoA/Rho-kinase pathway in hypertension.
  American Journal of Hypertension 11/2004; 17(10):981-5. · 3.18 Impact Factor