Involvement of PI3K, Akt and RhoA in Oestradiol Regulation of Cardiac iNOS Expression

Author(s): Sonja Zafirovic*, Emina Sudar-Milovanovic, Milan Obradovic, Jelena Djordjevic, Nebojsa Jasnic, Milica Labudovic Borovic, Esma R. Isenovic.

Journal Name: Current Vascular Pharmacology

Volume 17 , Issue 3 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


Abstract:

Background: Oestradiol is an important regulatory factor with several positive effects on the cardiovascular (CV) system. We evaluated the molecular mechanism of the in vivo effects of oestradiol on the regulation of cardiac inducible nitric oxide (NO) synthase (iNOS) expression and activity.

Methods: Male Wistar rats were treated with oestradiol (40 mg/kg, intraperitoneally) and after 24 h the animals were sacrificed. The concentrations of NO and L-Arginine (L-Arg) were determined spectrophotometrically. For protein expressions of iNOS, p65 subunit of nuclear factor-κB (NFκB-p65), Ras homolog gene family-member A (RhoA), angiotensin II receptor type 1 (AT1R), insulin receptor substrate 1 (IRS-1), p85, p110 and protein kinase B (Akt), Western blot method was used. Coimmunoprecipitation was used for measuring the association of IRS-1 with the p85 subunit of phosphatidylinositol- 3-kinase (PI3K). The expression of iNOS messenger ribonucleic acid (mRNA) was measured with the quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical analysis of the tissue was used to detect localization and expression of iNOS in heart tissue.

Results: Oestradiol treatment reduced L-Arg concentration (p<0.01), iNOS mRNA (p<0.01) and protein (p<0.001) expression, level of RhoA (p<0.05) and AT1R (p<0.001) protein. In contrast, plasma NO (p<0.05), Akt phosphorylation at Thr308 (p<0.05) and protein level of p85 (p<0.001) increased after oestradiol treatment.

Conclusion: Our results suggest that oestradiol in vivo regulates cardiac iNOS expression via the PI3K/Akt signaling pathway, through attenuation of RhoA and AT1R.

Keywords: Cardioprotection, oestradiol, rat heart, inducible nitric oxide synthase, eNOS, CVS.

[1]
Lee HR, Kim TH, Choi KC. Functions and physiological roles of two types of estrogen receptors, ERalpha and ERbeta, identified by estrogen receptor knockout mouse. Lab Anim Res 2012; 28: 71-6.
[2]
Masood DE, Roach EC, Beauregard KG, Khalil RA. Impact of sex hormone metabolism on the vascular effects of menopausal hormone therapy in cardiovascular disease. Curr Drug Metab 2010; 11: 693-714.
[3]
Kauser K, Sonnenberg D, Diel P, Rubanyi GM. Effect of 17beta-oestradiol on cytokine-induced nitric oxide production in rat isolated aorta. Br J Pharmacol 1998; 123: 1089-96.
[4]
Nuedling S, Karas RH, Mendelsohn ME, et al. Activation of estrogen receptor β is a prerequisite for estrogen-dependent upregulation of nitric oxide synthases in neonatal rat cardiac myocytes. FEBS Lett 2001; 502: 103-8.
[5]
Kypreos KE, Zafirovic S, Petropoulou PI, et al. Regulation of endothelial nitric oxide synthase and high-density lipoprotein quality by estradiol in cardiovascular pathology. J Cardiovasc Pharmacol Ther 2014; 19: 256-68.
[6]
Rhodes P, Leone AM, Francis PL, Struthers AD, Moncada S, Rhodes PM. The L-arginine:nitric oxide pathway is the major source of plasma nitrite in fasted humans. Biochem Biophys Res Commun 1995; 209: 590-6.
[7]
Ikeda U, Shimada K. Nitric oxide and cardiac failure. Clin Cardiol 1997; 20: 837-41.
[8]
Moncada S, Higgs EA. Molecular mechanisms and therapeutic strategies related to nitric oxide. FASEB J 1995; 9: 1319-30.
[9]
Obradovic M, Stewart AJ, Pitt SJ, et al. In vivo effects of 17beta-estradiol on cardiac Na(+)/K(+)-ATPase expression and activity in rat heart. Mol Cell Endocrinol 2014; 388: 58-68.
[10]
Andjelkovic M, Maira SM, Cron P, Parker PJ, Hemmings BA. Domain swapping used to investigate the mechanism of protein kinase B regulation by 3-phosphoinositide-dependent protein kinase 1 and Ser473 kinase. Mol Cell Biol 1999; 19: 5061-72.
[11]
Alessi DR, Cohen P. Mechanism of activation and function of protein kinase B. Curr Opin Genet Dev 1998; 8: 55-62.
[12]
Hattori Y, Hattori S, Kasai K. Lipopolysaccharide activates Akt in vascular smooth muscle cells resulting in induction of inducible nitric oxide synthase through nuclear factor-kappa B activation. Eur J Pharmacol 2003; 481: 153-8.
[13]
Karpuzoglu E, Ahmed SA. Estrogen regulation of nitric oxide and inducible nitric oxide synthase (iNOS) in immune cells: implications for immunity, autoimmune diseases, and apoptosis. Nitric Oxide 2006; 15: 177-86.
[14]
Fischer M, Baessler A, Schunkert H. Renin angiotensin system and gender differences in the cardiovascular system. Cardiovasc Res 2002; 53: 672-7.
[15]
Hoeg JM, Willis LR, Weinberger MH. Estrogen attenuation of the development of hypertension in spontaneously hypertensive rats. Am J Physiol 1977; 233: H369-73.
[16]
Sudhir K, Chou TM, Mullen WL, et al. Mechanisms of estrogen-induced vasodilation: in vivo studies in canine coronary conductance and resistance arteries. J Am Coll Cardiol 1995; 26: 807-14.
[17]
Silva-Antonialli MM, Tostes RC, Fernandes L, et al. A lower ratio of AT1/AT2 receptors of angiotensin II is found in female than in male spontaneously hypertensive rats. Cardiovasc Res 2004; 62: 587-93.
[18]
Ertemi H, Mumtaz FH, Howie AJ, Mikhailidis DP, Thompson CS. Effect of angiotensin II and its receptor antagonists on human corpus cavernous contractility and oxidative stress: modulation of nitric oxide mediated relaxation. J Urol 2011; 185: 2414-20.
[19]
Mehta PK, Griendling KK. Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol 2007; 292: C82-97.
[20]
Kimura K, Eguchi S. Angiotensin II type-1 receptor regulates RhoA and Rho-kinase/ROCK activation via multiple mechanisms. Focus on Angiotensin II induces RhoA activation through SHP2-dependent dephosphorylation of the RhoGAP p190A in vascular smooth muscle cells. Am J Physiol Cell Physiol 2009; 297: C1059-61.
[21]
Peters SL, Michel MC. The RhoA/Rho kinase pathway in the myocardium. Cardiovasc Res 2007; 75: 3-4.
[22]
Lin G, Craig GP, Zhang L, et al. Acute inhibition of Rho-kinase improves cardiac contractile function in streptozotocin-diabetic rats. Cardiovasc Res 2007; 75: 51-8.
[23]
Sah VP, Minamisawa S, Tam SP, et al. Cardiac-specific overexpression of RhoA results in sinus and atrioventricular nodal dysfunction and contractile failure. J Clin Invest 1999; 103: 1627-34.
[24]
Oviedo PJ, Sobrino A, Laguna-Fernandez A, et al. Estradiol induces endothelial cell migration and proliferation through estrogen receptor-enhanced RhoA/ROCK pathway. Mol Cell Endocrinol 2011; 335: 96-103.
[25]
Gayard M, Guilluy C, Rousselle A, et al. AMPK alpha 1-induced RhoA phosphorylation mediates vasoprotective effect of estradiol. Arterioscler Thromb Vasc Biol 2011; 31: 2634-42.
[26]
Sudar E, Velebit J, Gluvic Z, et al. Hypothetical mechanism of sodium pump regulation by estradiol under primary hypertension. J Theor Biol 2008; 251: 584-92.
[27]
Fabris B, Candido R, Bortoletto M, et al. Stimulation of cardiac apoptosis in ovariectomized hypertensive rats: potential role of the renin-angiotensin system. J Hypertens 2011; 29: 273-81.
[28]
Dean SA, Tan J, O’Brien ER, Leenen FH. 17beta-estradiol downregulates tissue angiotensin-converting enzyme and ANG II type 1 receptor in female rats. Am J Physiol Regul Integr Comp Physiol 2005; 288: 759-66.
[29]
Fischer M, Baessler A, Schunkert H. Renin angiotensin system and gender differences in the cardiovascular system. Cardiovasc Res 2002; 53: 672-7.
[30]
Nuedling S, Kahlert S, Loebbert K, et al. 17β-Estradiol stimulates expression of endothelial and inducible NO synthase in rat myocardium in-vitro and in-vivo. Cardiovasc Res 1999; 43: 666-74.
[31]
Maggi A, Cignarella A, Brusadelli A, Bolego C, Pinna C, Puglisi L. Diabetes undermines estrogen control of inducible nitric oxide synthase function in rat aortic smooth muscle cells through overexpression of estrogen receptor-beta. Circulation 2003; 108: 211-7.
[32]
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-75.
[33]
Luiken JJ, Koonen DP, Willems J, et al. Insulin stimulates long-chain fatty acid utilization by rat cardiac myocytes through cellular redistribution of FAT/CD36. Diabetes 2002; 51: 3113-9.
[34]
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
[35]
Kowalczuk D, Pietraś R, Baran Annales J. Spectrophotometric analysis of cefepime and L-arginine the pharmaceutical preparation. Universitatis Mariae Curie-Sklodowska, Lublin 2007; pp. 83-7.
[36]
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25: 402-8.
[37]
Liaudet L, Gnaegi A, Rosselet A, et al. Effect of L-lysine on nitric oxide overproduction in endotoxic shock. Br J Pharmacol 1997; 122: 742-8.
[38]
Sari AN, Kacan M, Unsal D, et al. Contribution of RhoA/Rho-kinase/MEK1/ERK1/2/iNOS pathway to ischemia/reperfusion-induced oxidative/nitrosative stress and inflammation leading to distant and target organ injury in rats. Eur J Pharmacol 2014; 723: 234-45.
[39]
Peluffo RD. L-Arginine currents in rat cardiac ventricular myocytes. J Physiol 2007; 580: 925-36.
[40]
Palmer RM, Rees DD, Ashton DS, Moncada S. L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun 1988; 153: 1251-6.
[41]
Chang KA, Lin IC, Sheen JM, et al. Sex differences of oxidative stress to cholestatic liver and kidney injury in young rats. Pediatr Neonatol 2013; 54: 95-101.
[42]
Yang S, Bae L, Zhang L. Estrogen increases eNOS and NOx release in human coronary artery endothelium. J Cardiovasc Pharmacol 2000; 36: 242-7.
[43]
McNeill AM, Zhang C, Stanczyk FZ, Duckles SP, Krause DN. Estrogen increases endothelial nitric oxide synthase via estrogen receptors in rat cerebral blood vessels: effect preserved after concurrent treatment with medroxyprogesterone acetate or progesterone. Stroke 2002; 33: 1685-91.
[44]
Isenovic ER, Divald A, Milivojevic N, Grgurevic T, Fisher SE, Sowers JR. Interactive effects of insulin-like growth factor-1 and beta-estradiol on endothelial nitric oxide synthase activity in rat aortic endothelial cells. Metabolism 2003; 52: 482-7.
[45]
Guetta V, Quyyumi AA, Prasad A, Panza JA, Waclawiw M, Cannon RO 3rd. The role of nitric oxide in coronary vascular effects of estrogen in postmenopausal women. Circulation 1997; 96: 2795-801.
[46]
Datta B, Tufnell-Barrett T, Bleasdale RA, et al. Red blood cell nitric oxide as an endocrine vasoregulator: a potential role in congestive heart failure. Circulation 2004; 109: 1339-42.
[47]
Hishikawa K, Nakaki T, Marumo T, Suzuki H, Kato R, Saruta T. Up-regulation of nitric oxide synthase by estradiol in human aortic endothelial cells. FEBS Lett 1995; 360: 291-3.
[48]
MacRitchie AN, Jun SS, Chen Z, et al. Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium. Circ Res 1997; 81: 355-62.
[49]
Cirino G, Wheeler-Jones CP, Wallace JL, Del Soldato P, Baydoun AR. Inhibition of inducible nitric oxide synthase expression by novel nonsteroidal anti-inflammatory derivatives with gastrointestinal-sparing properties. Br J Pharmacol 1996; 117: 1421-6.
[50]
Park SK, Lin HL, Murphy S. Nitric oxide regulates nitric oxide synthase-2 gene expression by inhibiting NF-kappaB binding to DNA. Biochem J 1997; 322(Pt 2): 609-13.
[51]
Colasanti M, Suzuki H. The dual personality of NO. Trends Pharmacol Sci 2000; 21: 249-52.
[52]
Nweze IC, Smith JW, Zhang B, Klinge CM, Lakshmanan J, Harbrecht BG. 17beta-Estradiol attenuates cytokine-induced nitric oxide production in rat hepatocyte. J Trauma Acute Care Surg 2012; 73: 408-12.
[53]
Sunday L, Tran MM, Krause DN, Duckles SP. Estrogen and progestagens differentially modulate vascular proinflammatory factors. Am J Physiol Endocrinol Metab 2006; 291: 261-7.
[54]
Ganster RW, Taylor BS, Shao L, Geller DA. Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-kappa B. Proc Natl Acad Sci USA 2001; 98: 8638-43.
[55]
Ellerhorst JA, Ekmekcioglu S, Johnson MK, Cooke CP, Johnson MM, Grimm EA. Regulation of iNOS by the p44/42 mitogen-activated protein kinase pathway in human melanoma. Oncogene 2006; 25: 3956-62.
[56]
Paech K, Webb P, Kuiper GG, et al. Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites. Science 1997; 277: 1508-10.
[57]
Tsutsumi S, Zhang X, Takata K, et al. Differential regulation of the inducible nitric oxide synthase gene by estrogen receptors 1 and 2. J Endocrinol 2008; 199: 267-73.
[58]
Menazza S, Murphy E. The expanding complexity of estrogen receptor signaling in the cardiovascular system. Circ Res 2016; 118: 994-1007.
[59]
Kim JK, Levin ER. Estrogen signaling in the cardiovascular system. Nucl Recept Signal 2006; 4: e013.
[60]
Grohé C, Meyer R, Vetter H. Crosstalk between the estrogen receptor and the insulin-like growth factor (IGF-1) receptor. Implications for cardiac disease. In: Doevendans PA, Reneman RS, van Bilsen M, Eds. Cardiovascular specific gene expression developments in cardiovascular medicine. Springer, Dordrecht 1999.
[61]
Gual P, Le Marchand-Brustel Y, Tanti JF. Positive and negative regulation of insulin signaling through IRS-1 phosphorylation. Biochimie 2005; 87: 99-109.
[62]
Craparo A, Freund R, Gustafson TA. 14-3-3 (epsilon) interacts with the insulin-like growth factor I receptor and insulin receptor substrate I in a phosphoserine-dependent manner. J Biol Chem 1997; 272: 11663-9.
[63]
Sun XJ, Liu F. Phosphorylation of IRS proteins: YinYang regulation of insulin signaling. In: Litwack G, Ed. Vitamins and the immune System. Academic Press 2009; pp. 351-87.
[64]
Sykiotis GP, Papavassiliou AG. Serine phosphorylation of insulin receptor substrate-1: a novel target for the reversal of insulin resistance. Mol Endocrinol 2001; 15: 1864-9.
[65]
Sun XJ, Goldberg JL, Qiao LY, Mitchell JJ. Insulin-induced insulin receptor substrate-1 degradation is mediated by the proteasome degradation pathway. Diabetes 1999; 48: 1359-64.
[66]
Koricanac G, Milosavljevic T, Stojiljkovic M, et al. Impact of estradiol on insulin signaling in the rat heart. Cell Biochem Funct 2009; 27: 102-10.
[67]
Pederson TM, Kramer DL, Rondinone CM. Serine/threonine phosphorylation of IRS-1 triggers its degradation: possible regulation by tyrosine phosphorylation. Diabetes 2001; 50: 24-31.
[68]
Isenovic E, Walsh MF, Muniyappa R, Bard M, Diglio CA, Sowers JR. Phosphatidylinositol 3-kinase may mediate isoproterenol-induced vascular relaxation in part through nitric oxide production. Metabolism 2002; 51: 380-6.
[69]
Chan TW, Pollak M, Huynh H. Inhibition of insulin-like growth factor signaling pathways in mammary gland by pure antiestrogen ICI 182,780. Clin Cancer Res 2001; 7: 2545-54.
[70]
Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 2000; 407: 538-41.
[71]
Ren J, Hintz KK, Roughead ZK, et al. Impact of estrogen replacement on ventricular myocyte contractile function and protein kinase B/Akt activation. Am J Physiol Heart Circ Physiol 2003; 284: H1800-7.
[72]
Sovershaev MA, Egorina EM, Andreasen TV, Jonassen AK, Ytrehus K. Preconditioning by 17beta-estradiol in isolated rat heart depends on PI3-K/PKB pathway, PKC, and ROS. Am J Physiol Heart Circ Physiol 2006; 291: 1554-62.
[73]
Almeida-Pereira G, Coletti R, Mecawi AS, Reis LC, Elias LL, Antunes-Rodrigues J. Estradiol and angiotensin II crosstalk in hydromineral balance: Role of the ERK1/2 and JNK signaling pathways. Neuroscience 2016; 322: 525-38.
[74]
Xue Q, Xiao D, Zhang L. Estrogen regulates angiotensin ii receptor expression patterns and protects the heart from ischemic injury in female rats. Biol Reprod 2015; 93: 6.
[75]
Schunkert H, Danser AH, Hense HW, Derkx FH, Kurzinger S, Riegger GA. Effects of estrogen replacement therapy on the renin-angiotensin system in postmenopausal women. Circulation 1997; 95: 39-45.
[76]
de Oliveira-Sales EB, Nishi EE, Boim MA, Dolnikoff MS, Bergamaschi CT, Campos RR. Upregulation of AT1R and iNOS in the rostral ventrolateral medulla (RVLM) is essential for the sympathetic hyperactivity and hypertension in the 2K-1C Wistar rat model. Am J Hypertens 2010; 23: 708-15.
[77]
Nickenig G, Baumer AT, Grohe C, et al. Estrogen modulates AT1 receptor gene expression in vitro and in vivo. Circulation 1998; 97: 2197-201.
[78]
Zhou H, Li YJ, Wang M, et al. Involvement of RhoA/ROCK in myocardial fibrosis in a rat model of type 2 diabetes. Acta Pharmacol Sin 2011; 32: 999-1008.
[79]
Handgraaf S, Riant E, Fabre A, et al. Prevention of obesity and insulin resistance by estrogens requires ERalpha activation function-2 (ERalphaAF-2), whereas ERalphaAF-1 is dispensable. Diabetes 2013; 62(12): 4098-108.
[80]
Hao L, Wang Y, Duan Y, Bu S. Effects of treadmill exercise training on liver fat accumulation and estrogen receptor alpha expression in intact and ovariectomized rats with or without estrogen replacement treatment. Eur J Appl Physiol 2010; 109: 879-86.
[81]
Kumagai S, Holmang A, Bjorntorp P. The effects of oestrogen and progesterone on insulin sensitivity in female rats. Acta Physiol Scand 1993; 149: 91-7.
[82]
Stubbins RE, Najjar K, Holcomb VB, Hong J, Nunez NP. Oestrogen alters adipocyte biology and protects female mice from adipocyte inflammation and insulin resistance. Diabetes Obes Metab 2012; 14: 58-66.
[83]
Wagner JD, Thomas MJ, Williams JK, Zhang L, Greaves KA, Cefalu WT. Insulin sensitivity and cardiovascular risk factors in ovariectomized monkeys with estradiol alone or combined with nomegestrol acetate. J Clin Endocrinol Metab 1998; 83: 896-901.
[84]
Zhu L, Brown WC, Cai Q, et al. Estrogen treatment after ovariectomy protects against fatty liver and may improve pathway-selective insulin resistance. Diabetes 2013; 62: 424-34.
[85]
Barrett-Connor E, Slone S, Greendale G, et al. The postmenopausal estrogen/progestin interventions study: primary outcomes in adherent women. Maturitas 1997; 27: 261-74.
[86]
Colditz GA, Manson JE, Hankinson SE. The nurses’ health study: 20-year contribution to the understanding of health among women. J Womens Health 1997; 6: 49-62.
[87]
Harman SM, Brinton EA, Cedars M, et al. KEEPS: The kronos early estrogen prevention study. Climacteric 2005; 8: 3-12.
[88]
Hulley S, Furberg C, Barrett-Connor E, et al. Noncardiovascular disease outcomes during 6.8 years of hormone therapy: Heart and estrogen/progestin replacement study follow-up (HERS II). JAMA 2002; 288: 58-66.
[89]
Hulley S, Grady D, Bush T, et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/progestin Replacement Study (HERS) Research Group. JAMA 1998; 280: 605-13.
[90]
Grady D, Herrington D, Bittner V, et al. Cardiovascular disease outcomes during 6.8 years of hormone therapy: Heart and Estrogen/progestin Replacement Study follow-up (HERS II). JAMA 2002; 288: 49-57.
[91]
Prelevic GM, Kwong P, Byrne DJ, Jagroop IA, Ginsburg J, Mikhailidis DP. A cross-sectional study of the effects of hormon replacement therapy on the cardiovascular disease risk profile in healthy postmenopausal women. Fertil Steril 2002; 77: 945-51.
[92]
Rosano GM, Vitale C, Fini M. Cardiovascular aspects of menopausal hormone replacement therapy. Climacteric 2009; 12(Suppl. 1): 41-6.


Rights & PermissionsPrintExport Cite as


Article Details

VOLUME: 17
ISSUE: 3
Year: 2019
Page: [307 - 318]
Pages: 12
DOI: 10.2174/1570161116666180212142414
Price: $58

Article Metrics

PDF: 38
HTML: 2
EPUB: 1
PRC: 1